Gen-T nº4 by Javier Masoliver

DICIEMBRE 2009 | Nº 4 | P.V.P 5,00€

FARMACOGENÉTICA DE LOS AINEs Personalizar el Tratamiento

PERSONALIZED MEDICINE OF DEMENTIA

EFFECTS OF FR-91 ON HUMAN TUMOR CELL LINES

Pharmacogenomics of Alzheimer´s Disease www.gen-t.es

Pídanos cita: +34 + 902 154 476 + 981 780 505 +34

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Centro Médico EuroEspes: Santa Marta de Babío s/n, 15165 Bergondo, La Coruña

EDITORIAL Ramón Cacabelos rcacabelos@gen-t.es

Paso a la Cultura del Conocimiento

A CIENCIA no puede esconderse ni permanecer ajena a la crisis económica mundial; porque la ciencia tiene que vivir inmersa en el mundo real. La ciencia, de hecho, no ignora la crisis porque gran parte de la ciencia oficial es mercenaria de presupuestos públicos y prebendas privadas, que se desmoronan con la crisis. Los oficialistas de la ciencia contribuyen a la burbuja, llamada del ladrillo, por su falta de compromiso con las necesidades de la sociedad. La ortodoxia científica no puede vivir agazapada en su atalaya subterránea y sólo sacar la cabeza cuando se le tocan los presupuestos de supervivencia. Los obsesos de la trampa estadística son cómplices de la crisis; porque la crisis no es sólo económica. La crisis que nos afecta a todos es el resultado del fracaso del modelo de sociedad que hemos estado creando sobre la base de un crecimiento insostenible, asimétrico e injusto, en el cual se ignora al 80% y en el cual un ciudadano que trabaja mantiene a cinco que no lo hacen, entre los cuales abundan caraduras y vagonetas impertérritos. La crisis de valores arrastra a la educación, la justicia, la sanidad, el trabajo, la familia, la política, la moral… y acaba midiéndose en parámetros económicos. Si educación, justicia, sanidad, trabajo, familia, política y moral se desvían hacia principios erráticos, es lógico que se venga abajo la economía; después de todo, como diría mi amigo Manuel Gago, la moderna “neuroeconomía”, basada en los estudios pioneros de Camerer, Loewenstein y Prelec, recientemente revisada por Kenning y Plassmann (o George Soros), y ferozmente criticada por Ariel Rubinstein o Faruk Gul y Wolfgang Pesendorfer, no es otra cosa que neurociencia, economía y psicología aplicadas a entender las tomas de decisiones humanas. Si fallan los valores que gobiernan nuestra conducta cotidiana, tarde o temprano se nos vendrá abajo el andamio. Las crisis no aparecen por casualidad; ocurren porque o se las provoca o porque revienta el modelo que las alimenta. Resulta paradójico que las aparentemente privilegiadas mentes de los que dirigen el destino de los pueblos no se hayan dado cuenta de que en el último siglo el mundo se ha transformado, la pirámide de la población se ha invertido, las patologías humanas han pasado de ser infecciosas a degenerativas (excepto en el mundo subdesarrollado) y los ciudadanos hoy tienen libre acceso al mundo del conocimiento del que se veían privados antes de la era Internet. Han cambiado tantas cosas que pretender sostener el modelo sociopolítico y socioeconómico de

comienzos del siglo XX en plena efervescencia del XXI parece propio de dirigentes muy limitados. El modelo no sirve y hay que cambiarlo, pero nadie quiere poner el cascabel al gato. La responsabilidad (o irresponsabilidad) de científicos e intelectuales radica en que han dejado en manos de “opinadores” a sueldo el futuro de la sociedad y se han escondido en los despachos y laboratorios que mantiene el dueño público, confundiendo que quien paga su silencio cómplice no es el partido gobernante sino la ciudadanía a la que debieran servir con lealtad. Los sostenedores de la ciencia, la tecnología y la medicina actuales son cómplices cuando anteponen el salario personal a la responsabilidad profesional, cuando la miseria de la supervivencia está por encima de los principios, cuando la individualidad mata el interés de la colectividad, cuando se utiliza lo público para el beneficio privado, cuando se sacrifica la colaboración por mantener un liderazgo vacío. En medio de esta trama de putrefacción colectiva está la clase política, preocupada por la próxima legislatura, por sus intrigas internas, por el enemigo externo, por desenterrar muertos y cultivar odios, por las tramas de corrupción institucional encubiertas y por el aplauso de los adeptos, más que por el análisis de los críticos bienpensantes; todo ello cuidadosamente retroalimentado por los núcleos de poder mediático al servicio del color que más nutre. En medio está la población desorientada, adoctrinada por los predicadores diarios, confundida por la contradicción informativa, atónita ante el descalabro social, la desaparición de puestos de trabajo, la huida de los inversores hacia cloacas más favorables, la hipocresía de sus dirigentes, la debilidad del principio de autoridad en la escuela, el silencio de la universidad, la pasividad comprada de los sindicatos, el hedor a estiércol de las instituciones. Este mundo en crisis viene a recordarnos que el dinero no lo es todo; que el beneficio económico debe ser el resultado justo del esfuerzo, del sacrificio, del trabajo bien hecho, de la honradez, de la corresponsabilidad, de la profesionalidad, de la correcta gestión de lo propio y de lo ajeno, del respeto a lo público y a lo privado, de garantizar los lindes de la libertad individual sin violar las fronteras que delimitan el derecho de los otros, de mensurar el crecimiento sostenible, de garantizar el progreso personal y colectivo mediante la colaboración, de entender que el valor de las ideas y los principios está por encima del precio de las cosas.

Este mundo en crisis nos viene a recordar que el tornillo del progreso a fuerza de golpes y talonario, prevaricación, chantaje o fraude, se pasó de rosca, y hay que reponerlo; hay que taladrar la plataforma del progreso y fortalecerla con puntales más sólidos para soportar el peso del desarrollo futuro. Hay que empezar a crear un nuevo modelo de sociedad donde el peso de las ideas y de los principios suplanten al hormigón del inmovilismo anacrónico, al pladur de las asimetrías ficticias y a las ventanas de cristal oscuro que adulteran la transparencia. Hay que empezar a creer en el cambio de las estructuras de poder, sin perder de vista que la rueda ya está inventada y que la historia de la humanidad evoluciona en ciclos. Hay que dar paso a la cultura del conocimiento. Los cimientos sobre los que asienta el edificio del conocimiento son el sentido común y la experiencia. Lo que distingue al sabio es su capacidad de discernimiento y su habilidad para no instalarse en falsos postulados de verdades inquebrantables. Dice un proverbio americano que la prosperidad descubre el vicio y la adversidad la virtud. En las escuelas anglosajonas se enseña que el peor enemigo del conocimiento es la ignorancia; y en la cultura hindú se conserva el dicho de que el conocimiento inútil es como una antorcha en las manos de un ciego. En los pasillos de Wall Street y de la CIA es frecuente oír que knowledge is power. Pero no basta con adquirir sabiduría, como decía Cicerón; es preciso además saber usarla; y ser humilde. Confucio predicaba: “El sabio sabe que ignora. El sabio teme la bonanza; pero cuando descarga la tempestad camina sobre las olas y desafía los vientos. Lo que quiere el sabio lo busca en sí mismo; el vulgo lo busca en los demás”. Para Lao-Tsé saber creyendo no saber era algo excelso y no saber creyendo saber era una enfermedad. Y en esta aldea global, hasta Demócrito está de moda: “Toda la tierra está al alcance del sabio, ya que la patria de un alma elevada es el universo”. La megalomanía científica también tiene crítica en los pensamientos de Remy de Gourmont: “Saber lo que todo el mundo conoce es como no saber nada. El saber comienza allí donde el mundo comienza a ignorar. La verdadera ciencia está más allá de la verdadera ciencia” porque las verdades absolutas de hoy son sólo verdades relativas de mañana. A los que perdieron el rumbo, Sócrates les dice que el saber es la parte principal de la felicidad; y a los que se mueven en las ciénagas de la envidia ibérica, Solón les diría “guárdate bien de decir todo lo que sabes”.

diciembre 2009

A modo de Presentación

por Ciprián Rivas

Gen-T Número 4 Diciembre 2009 Editorial

Ciencia y divulgación

EuroEspes Publishing info@euroespespublishing.com en-T, The EuroEspes Journal, acude de nuevo a la cita con sus lectores. Y lo hace coincidiendo con la

Patrocina Fundación EuroEspes

Editor-Jefe Ramón Cacabelos

Dirección Ciprián Rivas

Dirección administrativa Javier Sánchez Gladys Bahamonde administración@gen-t.es

Dirección de Producción Antonio Bermo

Producción Gráfica EuroEspes Publishing

Gen-T Santa Marta de Babío s/n 15165 Bergondo Coruña, España Teléfono: 981 780 505 ISSN 1888-7937 Depósito Legal C 713-2007 Copyright 2009 Gen-T no se responsabiliza de las opiniones y criterios emitidos por los autores, reservándose la propiedad de los trabajos publicados. Queda expresamente prohibida la reproducción parcial, literaria o iconográfica de cualquier contenido sin previa autorización del editor.

celebración, el 19 de diciembre, de la IV Conferencia Anual EuroEspes. Este año, está dedicada a los avances en medicina genómica. Expertos de diversas especialidades disertarán ante un público con ganas de intercambiar, madurar y aumentar su conocimiento en una materia de vanguardia y también de enorme actualidad. El esfuerzo realizado para la puesta en marcha de esta actividad, de alto nivel científico, por el Comité Organizador, es la respuesta al interés de los asistentes de las conferencias que previamente se han celebrado. La III Conferencia realizada el pasado año es objeto de un breve análisis en nuestras páginas. Uno de los objetivos marcados por el Comité Organizador de la IV Conferencia, que organiza la Fundación EuroEspes, es educar y difundir -entre todo el colectivo sanitario - los beneficios de la medicina genómica. Hoy, la utilidad clínica de la farmacogenómica es ya una realidad en la optimización del uso de medicamentos y en la personalización del tratamiento farmacológico. En este número de Gen-T pueden leer a algunos de los ponentes. Se deben destacar todos los trabajos publicados, realizados con gran rigurosidad científica, pero nos gustaría incidir en dos de ellos. Uno lo firma el director de la Conferencia, el doctor Ramón Cacabelos y su equipo de investigación: “Personalized medicine of Dementia. Pharmacogenomics of Alzheimer’s disease”. El otro ha sido elaborado por el profesor J.A. García-Agúndez de la Universidad de Extremadura. Se titula “Farmacogenómica de los AINEs. Personalizar el tratamiento”. En este trabajo nos informa que más de 30 millones de personas son tratadas diariamente con antiinflamatorios no esteroideos (AINEs) y cerca del 25 por ciento de la población ha experimentado alguna vez en su vida reacciones adversas causadas por AINEs que han requerido tratamiento médico. Gen-T, por primera vez, se convierte en un medio de comunicación bilingüe, vertiendo en sus páginas artículos en lengua inglesa, como el ya mencionado, escrito por el editor de la revista y los pertenecientes a los liderados por el Dr. Andreas Pfützner y el Dr. Valter Lombardi. En otro orden de cosas, el Grupo EuroEspes celebra con su presidente el recién creado Máster en Biotecnología de la Salud de la Universidad Camilo José Cela de Madrid. La Agencia Estatal de Evaluación de la Calidad y Acreditación (ANECA) ha informado favorablemente sobre este Plan de Estudios que se impartirá a partir del curso académico 2010/11. Ramón Cacabelos dirigirá también el doctorado en Medicina Genómica de la Universidad Camilo José Cela. Este programa fue aprobado recientemente por el Ministerio de Educación. Son dos actividades educativas de alto nivel que se imparten desde la Cátedra EuroEspes que hace años viene apostando por la innovación en el campo de la medicina. Gen-T es una revista con clara vocación científica para uso de científicos e investigadores. Es un compromiso adquirido convertirse en un medio que tiene como misión integrar la medicina genómica en la sociedad a través de estudios rigurosos que se pueden ver en formato papel o bien a través de la web de la propia publicación, amplificando de esta manera su difusión y aproximación a la ciudadanía. Somos una revista única en su género en el campo editorial español y, por ello, nos obligamos a ser críticos con la obra que tiene en sus manos en este momento. Conocemos de antemano que estamos ante una labor difícil, pero apostamos por este reto con la fuerza que da un grupo empresarial que tiene como misión la innovación y el desarrollo en aras de la salud y del bienestar del ser humano.

Optimize sus defensas naturales

DefenVid ® (E-JUR-94013 ®) es el primer nutracéutico biomarino con estructura lipoproteica natural que presenta propiedades de inmunopotenciación y regulación metabólica e inmunoglobulínica

19 de Diciembre de 2009, Bergondo, La Coruña

8:30

RECEPCIÓN

14:00 COMIDA

9:00

Acto inaugural

9:30

Sesión Plenaria-I Farmacogenómica de los trastornos metabólicos Prof. Dr. Andreas Pfützner PharmGenomics, Mainz, Alemania

16:00 Sesión Plenaria-III Farmacogenética de los AINEs Prof. Dr. J.A. García-Agúndez Departamento de Farmacología y Psiquiatría Facultad de Medicina, Universidad de Extremadura, Badajoz

10:15 Estrategias terapéuticas en la enfermedad de Alzheimer Dr. X. Antón Álvarez Departamento de Farmacología Clínica y Experimental Centro de Investigación Biomédica EuroEspes, Coruña

16:45 Modelos transgénicos en enfermedades neurodegenerativas Dr. Iván Carrera Departamento de Neurociencias EuroEspes Biotecnología (Ebiotec), Coruña

10:45 Genómica clínica de los trastornos del movimiento Dra. Lucía Fernández-Novoa Departamento de Genómica Médica EuroEspes Biotecnología (Ebiotec), Coruña

17:15 Neurodegeneración y cáncer Dr. Salvador Harguindey Instituto de Biología Clínica y Metabolismo Vitoria, Álava

11:15 DESCANSO

17:45 DESCANSO

11:30 Sesión Plenaria-II Medicina personalizada en el abordaje clínico de la demencia: Farmacogenómica de la enfermedad de Alzheimer Prof. Dr. Ramón Cacabelos Departamento de Neurociencias Clínicas Centro de Investigación Biomédica EuroEspes, Coruña

18:00 Aplicaciones actuales de la farmacogenética en terapias del cáncer Dr. Stefan Prause PharmGenomics GmbH Mainz, Alemania

12:15 Farmacogenética de los tratamientos anticoagulantes Dra. Ruth Llovo Departamento de Farmacogenética EuroEspes Biotecnología (Ebiotec), Coruña 12:45 Genómica de la patología cerebrovascular Dr. Juan Carlos Carril Departamento de Genómica e Identificación Humana EuroEspes Biotecnología (Ebiotec), Coruña

18:30 Fibronectina en la enfermedad de Alzheimer Prof. Dr. Jerzy Leszek Medical University of Wroclaw Wroclaw, Polonia 19:00 Acto de clausura Presidido por D. Augusto Silva Director General Terapias Avanzadas y Trasplantes Ministerio de Sanidad y Consumo, Madrid

EUROESPES

FUNDACIÓN

SEDE: Centro de Investigación Biomédica EuroEspes, Bergondo, Coruña, España | ORGANIZA: Fundación EuroEspes

SUMARIO

Opinión

03 Editorial 04 A modo de presentación

Personalized medicine of dementia Pharmacogenomics of Alzheimer´s disease

18-48

Ciencia

08 Pharmacogenomics of Metabolic Disease 18 Personalized medicine of dementia: Pharmacogenomics of Alzheimer´s disease 50 Effects of FR-91on human tumor cell lines 62 Farmacogenómica de los AINEs

Farmacogenómica de los AINEs

62-68

70 Genómica de la patología cerebrovascular

Sociedad

86 Cooperación multisectorial para impulsar el desarrollo de Galicia 88 III conferencia anual EuroEspes Effects of FR-91 on human tumor cell lines

50-60

Noticias

94 Noticias EuroEspes

Suscripción

98 Boletín de suscripción

Genómica de la patología cerebrovascular

70-84 diciembre 2009

Pharmacogenomics of Metabolic Disease Andreas Pf端tzner1,2, Stefan Prause2, Moritz Eidens2, Alexander Weise1, Thomas Forst1 1. IKFE - Institute for Clinical Research and Development, Parcusstr. 8, D-55116 Mainz, Germany 2. PharmGenomics GmbH, Parcusstr. 8, D-55116 Mainz, Germany

Ciencia

Sumary

he prevalence of metabolic diseases, such as

atherosclerosis, diabetes mellitus and cardiometabolic syndrome, has reached pandemic dimensions. The increasing incidence in emerging countries, which is linked to an improved access to processed food, will make these diseases the major burden for the affected health care systems. Orchestrated by a complex interaction of the joint underlying pathophysiological deteriorations (insulin resistance, Ă&#x;-cell dysfunction, visceral adipogenesis, and chronic systemic inflammation), the disease presents with a variety of clinical phenotypes, characterized by different compositions and severities of the most common symptoms: hypertension, dyslipidemia, hyperglycemia and atherosclerosis. The disease diagnosis is usually defined by the major symptoms and (e.g.) many patients with normoglycemic vascular insulin resistance die from a macrovascular event without ever being treated with a drug affecting insulin resistance. These drugs are normally only prescribed in case of overt diabetes with hyperglycemia. On the contrary, even well controlled patients with diabetes die from macrovascular events because the internationally accepted therapy guidelines target high blood glucose levels and HbA1c only and disregard the macrovascular risk induced by insulin resistance and the inflammatory activity of the visceral lipid tissue. All these findings underline the immediate necessity to develop diagnostic approaches for individualized assessment of the diverse contributions of the underlying disease components to the patient risk profile. Such approaches would help to improve the major challenges in this indication: identifying patients at risk of disease development, monitoring the efficacy of preventive measures, identifying the most optimal therapeutic approaches and monitoring their effectiveness in patients with overt disease, and identifying patients at a very high risk for macrovascular events justifying intense and expensive treatment interventions.

and will help to avoid the otherwise unavoidable progression of metabolic syndrome, cardiometabolic syndrome or type 2 diabetes mellitus to finally end in macrovascular death.

Introduction

Type 2 diabetes mellitus is one of the most frequent diseases with a worldwide prevalence of 4-5 % and a 10 % annual incidence rate. The major pathophysiological drivers are a hereditary or metabolic insulin resistance in combination with the inability of the pancreas to augment insulin secretion to the required amount1. About 40 % of the US population are overweight and develop insulin resistance. In the majority of these cases, the pancreas is able to counterbalance the increasing insulin need by an appropriately increased secretion. However, in a significant minority of about a third of these cases, a concomitant Ă&#x;-cell dysfunction leads to the development of a metabolic syndrome and to diabetes mellitus2. Insulin resistance, however, is also associated with an increased cardiovascular risk and the majority of the patients finally die from myocardial infarction or other macrovascular complications3, 4. The disease is commonly regarded as chronic progressive and general treatment guidelines are available to guide physicians how to best reach normoglycemia, which is commonly defined via a hemoglobin A1c value in the target range of < 6.5 % (Europe) or < 7.0 % (USA)5, 6. However, recent outcome studies have demonstrated that HbA1c is only a very moderate surrogate marker with limited or almost no prognostic value for the prediction of cardiovascular outcome.

Appropriate diagnostic options can be identified at all levels of cellular activity starting with DNA markers for risk identification based on determination of candidate gene mutations (usually linked to Ă&#x;-cell function or atherosclerosis), assessment of increased mRNA expression (e.g. as a measure of macrophage activation), and determination of plasma protein levels of biomarkers specifically associated with the related disorders. Modern laboratory platforms, such as the MutaChip technology, allow for economic and specific determination of DNA, mRNA, and protein biomarker panels to increase the efficacy of individually selected therapeutic interventions, diciembre 2009

Pharmacogenomics of Metabolic Disease

anti-insuline hormones

adiponectin

insulin resistance

insulin requirement

adipogenesis

ß-Cell dysfuntion

insulin proinsulin

Fig. 1 5 The pathophysiological link between insulin resistance, ß-cell dysfunction and visceral adipogenesis.

In these trials, achievement and maintenance of the target HbA1c range for several years had no pronounced influence on the incidence and outcome of cardiovascular events7, 8. While elevated blood glucose is certainly a contributor to the increased cardiovascular risk, even normalization of HbA1c leaves the patient with substantial further event risk9. It has to be concluded that current treatment guidelines address the underlying pathophysiology only in a suboptimal way and that new and more individualized treatment targets may be required to effectively improve the vascular prognosis of the affected patients. A closer investigation of the pathophysiology offers surprising insights into this complex disease and offers attractive ways for identification of suitable biomarkers for more effective and individualized interventions.

Pathophysiology of the cardiometabolic syndrome

The close relation between insulin resistance and ß-cell dysfunction has long been established and confirmed in large epidemiological studies10-13. Insulin resistance may occur based on

genetic predisposition or other environmental factors including excess food uptake and development of obesity. Increased insulin resistance demands for increased insulin secretion, which is normally provided by the ß-cells in a compensatory manner to lower blood glucose levels into the normal range. However, insulin is the only physiological hormone known to induce growth of lipid tissue, and an increase in body weight will most likely occur in the presence of sufficient food supply. It has recently been shown that growth of visceral adipose tissue results in differentiation of mesenchymal stem cells to preadipocytes, which finally become mature lipid cells. The pre-adipocyte, however, is the source of a substantial number of different cytokines and hormones (referred to as “adipokines”) known to support insulin resistance and thus a circle is closed leading to constantly increasing insulin resistance and obesity (Fig. 1)14. As long as the ß-cells are not compromised by a ß-cell dysfunction, the situation may be metabolically under control and can be reversed by reduction of food intake and increased physical exercise. However, in patients developing type 2 diabetes, ß-cell dysfunction will occur and will further accelerate the disease progression. Dysfunction of the ß-cell in type 2 diabetes is comprised of three components: secretion timing disorder, quantitative disorder and qualitative disorder. An indicator for the secretion timing disorder in early stages of type 2 diabetes is the loss of first phase insulin response, an important inhibitory signal for hepatic glucose release15. In later stages, loss of pulsatile insulin release can also be observed as additional failure in secretion timing16. The quantitative disorder starts when the ß-cell increases the volume of insulin release based on the increased external demand. In later stages, increasing exhaustion of the production capacity may result in complete loss of insulin secretion17. An increased quantitative secretion of proinsulin, and - in parallel - also of other pro-hormones, like pro-islet amyloid polypeptide (which is processed by the same conversion enzymes as proinsulin18) can finally lead to a deterioration of the secretion product composition. When new assays for assessment of unprocessed intact proinsulin became available, they helped to understand previous findings regarding elevated fasting proinsulin levels in the plasma of non-diabetic patients19. In the natural development of type 2 diabetes, proinsulin may only be elevated in the case of a significant insulin resistance, and we were thus able to demonstrate that elevated fasting morning intact proinsulin is indeed a highly specific indicator for insulin resistance20. Proinsulin is able to lower glucose levels but shows only 10-20 % of the efficacy of

In many patients, proinsulin action is sufficient to maintain blood glucose at normal levels, while the other pathophysiological drivers are still deteriorating. This may explain the findings from several groups that normoglycemic patients with elevated fasting proinsulin levels have an excess mortality from stroke and myocardial infarction23-25. In our opinion, the ultimate proof of a harmful action of proinsulin was provided when an interventional phase II trial testing subcutaneous injection of proinsulin vs. insulin for preprandial treatment of diabetes had to be stopped prematurely due to an elevated number of cardiovascular events which happened exclusively in the proinsulin arm21. In the presence of sufficient food supply, the metabolic situation deteriorates when insulin and proinsulin induce a major visceral adipogenesis, which leads to an increase in body weight. This process, based on the differentiation of mesenchymal stem cells to adipocytes26 is usually accompanied by an elevation in the tissue expression and plasma levels of proinflammatory cytokines, such as tumor necrosis factor-α (TNFα)27, interleukin-6 (IL-6)28, 29, plasminogen activator-inhibitor-1 (PAI-1)30 and others31. This protein expression profile indicates the prevalence of a chronic systemic inflammation. It has been demonstrated by Ghanim and coworkers that circulating mononuclear cells in obese patients are in a proinflammatory state with an in-

Ciencia

insulin21. Unfortunately, it has similar adipogenetic effects and, therefore, a five- to ten-fold higher driving force for development of obesity is prevalent in the affected patients22.

crease in intranuclear NF-κB binding, a decrease in IκB-ß and an increase in the transcription of proinflammatory genes regulated by NF-κB, including migration inhibitory factor (MIF), IL-6, TNFα, and matrix metalloproteinase 9 (MMP9)9. It is believed that the crosstalk between the pre-adipocytes and other tissues contributes to a general up-regulation of the immune system, including an activation of circulating monocytes and macrophages, resulting in an increased risk for atherosclerosis and vascular disease32, 33. The integrated knowledge about these complex pathophysiological conditions and relations enable the introduction of new diagnostic tools, which may help to identify the underlying genetic risk, may be helpful to describe the acute proinflammatory risk and may also allow identification of the most optimal therapeutic interventions by introducing concepts leading to individualized medicine approaches. These diagnostic opportunities can be found on the DNA, mRNA and protein level.

In many patients, proinsulin action is sufficient to maintain blood glucose at normal levels

diciembre 2009

Pharmacogenomics of Metabolic Disease

DNA Markers for diabetes prediction and cardiovascular risk assessment

There is strong evidence that genetic factors play an important role in the development of insulin resistance and ß-cell dysfunction. The majority of the single nucleotide polymorphisms (SNPs) of genes associated with an increased risk of type 2 diabetes is hypothesized to influence B-cell function34. It has been concluded from linkage studies, candidate gene approaches, and genomewide association studies that single nucleotide polymorphisms (SNPs) within up to 10 genes are associated with an increased risk of type 2 diabetes35-38. The suspected SNPs are located in the regions encoding the following proteins: Transcription factor 7-like 2 (TCF7L2), Cyclindependent kinase inhibitor-2A (CDKN2A) and CDKN2B, Human hematopoietically expressed homeobox (HHEX), CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1), Solute carrier family 30 (zinc transporter) member 8 (SLC30A8), and Potassium inwardly-rectifying channel subfamily J member 11 (KCNJ11)34. Each of these proteins plays a major role in insulin processing or insulin secretion. A list of the candidate mutations is provided in Table 1. Table 1

However, it has been suggested that the SNPs within or near these genes most likely do not alter their function or expression and emphasize a lack of influencing pancreatic B-cell development, regeneration, and function in the etiology of type 2 diabetes. It is also possible that there is an inherited reduction of the mitochondrial content in skeletal muscle of the insulinresistant pre-diabetic offspring of parents with type 2 diabetes. This reduction may be responsible for decreased oxidative phosphorylation in skeletal muscle39. In addition, a recent study suggests a link between mitochondrial function and glucose transporter trafficking. Impairment of respiratory chain function leads to impaired insulin-stimulated glucose transport in adipose cells40. It was demonstrated that the insulin sensitizer pioglitazone stabilizes the outer mitochondrial membrane protein mitoNEET, which is expressed in many insulin-responsive tissues and plays a key role in regulating the maximal capacity for electron transport and oxidative phosphorylation41. It is therefore highly possible that a hereditary mitochondrial defect, and not an inherited B-cell defect, plays the critical role in the onset of type 2 diabetes. Further work is required to elucidate the genetic origin of the observed reduction in mitochondrial ATP synthesis, which may add to existing concepts to explore the genetic basis of type 2 diabetes.

rs10946398

20,769,013

rs13266634

118,253,964

rs10811661

10q

Reference

CDKAL1

Mutation

20,769,229

Position bp

Gene

rs7754840

SNP

Nucleotide number

Chromosome

Confirmed loci for risk of type 2 diabetes development 34

INTRON 20,757,58720,847,729

35,37

SLC30A8

EXON-10 Arg325Trp 118,253,956– 114,789,774

35, 37

22,124,094

CDKN2B

21,992,902– 21,999,280

35-37

rs1111875 rs5015480

94,452,862 94,455,539

HHEX

94,439,690– 94,445,383

35, 37 36

10q

rs7903146 114,748,339

114,748,339

TCF7L2

INTRON-3 114,714,373– 114,789,774

35, 37

rs5219 KCNJ11

17,366,148

KCNJ11

EXON 17,365,042– 17,366,214

Glu23Lys

35, 37

Use of mRNA for individual cardiovascular risk assessment in patients with type 2 diabetes

As mentioned above, it has been demonstrated by Ghanim and coworkers that circulating mononuclear cells in obese patients are in a proinflammatory state with increased expression of intranuclear NF-κB protein, and subsequent up-regulation in the transcription of proinflammatory genes regulated by NF-κB9. The same group was able to demonstrate that an increased plasma concentration of MIF and an increased transcription of MIF mRNA in mononuclear cells, which was related to the body-mass index and hsCRP concentrations, could be reduced by a six week treatment with metformin in eight non-diabetic patients with obesity. The authors concluded that metformin might have beneficial effects on cardiovascular mortality in patients with type 2 diabetes42, which is in part confirmed by the few currently existing larger outcome trials on this topic43, 44. It has been shown in randomized prospective trials that treatment with pioglitazone, an agonist to the peroxisome proliferators-activated receptor γ, may improve clinical and laboratory surrogate markers for atherosclerosis and car-

In conclusion, mRNA quantification of distinct mRNA markers in peripheral macrophages may be a way to determine the acute macrophage activation, which is an indicator of acute atherogenic action in the vasculature. Assessment of these markers by means of more efficacious and less costly diagnostic tools, e.g. macro-arrays, may allow determination of an acute atherogenic activity in the vasculature and may become a future tool to monitor the efficacy of anti-inflammatory therapeutic interventions.

Protein biomarkers for selection and monitoring of individualized treatment interventions

The determination of clinical and laboratory routine markers (glucose, HbA1c, lipids, BMI, blood pressure) provides only insufficient information regarding the severity of the underlying pathophysiology, but new laboratory markers may provide the means to classify the acute individual metabolic and cardiovascular risk situation. The determination of ß-cell dysfunction may provide additional information with regard to disease stage and may be helpful for the selec-

Characteristics of the marker panel for assessment of metabolic and cardiovascular risk in patients with type 2 diabetes mellitus Marker Intact Proinsulin

Adiponectin

hsCRP

Ranges ≤ 11 pmol/l

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Table 2 diovascular risk, such as intima-media thickness, hsCRP, or MMP-9 independent from glycemic control45-47, and that it may even improve macrovascular outcome in type 2 diabetic patients when used in secondary prevention48-50. The anti-inflammatory and anti-thrombotic effects of glitazones occur very rapidly and significantly earlier as compared to the metabolic and glycemic effects of the drugs51, 52. In a recent study, we explored the short-term effects of the addition of pioglitazone (vs. placebo) to an existing effective oral anti-diabetic therapy with metformin and/or sulfonylurea on the proinflammatory activation of circulating mononuclear cells in well controlled patients with type 2 diabetes mellitus and elevated risk for atherosclerosis. For this purpose, we investigated the mRNA expression of the inhibitors to NF-κB (IκB-α and IκB-β)53, p105 (precursor to the p50 subunit) and Rel-A (p65 subunit) as measures for of the quantity of intranuclear NF-κB54, and several proinflammatory mediators and markers known to be modulated by NF-κB, such as TNFα, IL-6, MIF, and MMP-928, 55, 56 before and after four weeks of treatment in relation to a housekeeping gene. We found significant reduction of NF-κB expression, and the expression of the modulated cytokines by pioglitazone, while no change occurred with placebo. We concluded that the TZD downregulated macrophage activation, which could be visualized by quantification of macrophage mRNA57, 58.

Interpretation

> 11 pmol/l

normal value, no severe insulin resistance, ß-cell dysfunction stage I-II elevated, clinically relevant insulin resistance, stage III ß-cell dysfunction, elevated cardiovascular risk

10 - 12 mg/l 8 – 10 mg/l

normal reference range in women normal reference range in men

> 10 mg/l 7 – 10 mg/l < 7 mg/l

no elevated risk unclear result, no risk determination possible elevated risk, insulin resistance

0 - 1 mg/l >1 – 3 mg/l >3 – 10 mg/l > 10 mg/l

low cardiovascular risk moderate cardiovascular risk high cardiovascular risk unspecific inflammation, no conclusion regarding cardiovascular risk elevation possible

tion of appropriate therapeutic interventions. Recent studies have indicated that “intact proinsulin” measured with newly developed assays for the complete protein [19] may serve as an excellent indicator of a clinically relevant later stage ß-cell dysfunction and as a highly specific laboratory marker for insulin resistance20. Fasting intact proinsulin levels and the HOMA-score for insulin resistance can be used for a clinically useful staging of ß-cell dysfunction, which has been measured and validated in multiple large cross-sectional and prospective controlled interventional studies47, 60, 61. The characteristics and interpretation of fasting intact proinsulin as a marker for ß-cell dysfunction and insulin resistance are provided in Table 2.

New laboratory markers may provide the means to classify the acute individual metabolic and cardiovascular risk situation diciembre 2009

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Pharmacogenomics of Metabolic Disease Another laboratory indication for the actual metabolic situation is provided by the total plasma adiponectin concentrations. Values below 7 mg/dl have been associated with an increased cardiovascular risk in controlled clinical trials62, 63 . While adiponectin may have less value for the initial diagnosis of insulin resistance than intact proinsulin64, it appears to provide substantial information regarding the overall metabolic situation that reacts very sensitively to successful therapeutic measures. Different high and low molecular weight isoforms of adiponectin have been detected. However, their distinct determination does not seem to provide additional information in the context of metabolic risk screening. An increase in total adiponectin concentrations after intervention indicates an improvement in the metabolic and cardiovascular risk profile (Table 2)62, 63.

Dr. Andreas Pfützner andreasp@ikfe.de

A detailed analysis of the Framingham study cohort by Ridker et al. showed that CRP concentrations in the near normal range (> 10 mg/ dl) allow for an independent stratification of the cardiovascular risk into three risk groups, when measured with a highly sensitive assay method (hsCRP, see Table 2)65, 66. This marker has been globally accepted and has become part of the risk assessment guidelines of many scientific associations, including the American Heart Association and the American Diabetes Association. Values below 1 mg/l describe a low cardiovascular risk, 1 – 3 mg/l indicate a moderate cardiovascular risk, and 3 – 10 mg/l describe a high risk population. Values above

10 mg/l may arise due to other unspecific infections and inflammations and cannot be used for assessment of the chronic systemic vascular inflammatory process causing atherosclerosis. A reduction in the hsCRP concentrations is associated with the reduction of the cardiovascular risk profile67.

Perspectives

As of now, the laboratory determination of HbA1c, glucose, cholesterol, and triglycerides and the clinical assessment of blood pressure and the body-mass-index are used to obtain a basic and crude understanding of the degree and severity of insulin resistance, ß-cell dysfunction and cardiovascular risk. Currently multiple studies investigate the effects of different therapies on chronic systemic inflammation and on protein biomarkers, such as intact proinsulin, adiponectin and hsCRP concentrations. However, assessment of multimarker panel is still a costly undertaking. Modern laboratory platforms, such as the MutaChip technology (PharmGenomics, Mainz, Germany), allow for specific but economically sound determination of DNA, mRNA, and protein biomarker panels to increase the efficacy of individually selected therapeutic interventions, and will help to avoid the otherwise unavoidable progression of metabolic syndrome, cardiometabolic syndrome or type 2 diabetes mellitus to finally end in macrovascular death. 

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Abstract:

ecent advances in genomic medicine can contribute to accelerating our understanding on the pathogenesis of dementia, improving diagnostic accuracy with the introduction of novel biomarkers, and personalizing therapeutics with the incorporation of pharmacogenetic and pharmacogenomic procedures to drug development and clinical practice. Most neurodegenerative disorders, including Alzheimer’s disease (AD), share some common features, such as a genomic background in which hundreds of genes might be involved, genome-environment interactions, complex pathogenic pathways,

poor therapeutic outcomes and chronic disability. The main aim of a cost-effective treatment is to halt disease progression via a modification of the functional cascade involving AD genomics, transcriptomics, proteomics and metabolomics. Unfortunately, the drugs available for the treatment of dementia are not cost-effective. The pharmacological treatment of dementia accounts for 10-20% of direct costs, and less than 20% of the patients are moderate responders to conventional drugs, some of which may cause important adverse drug reactions. Future anti-dementia drugs must address the complex pathogenic niche of the disease from a multifactorial perspective. Pharmacogenetic and phar-

macogenomic factors may account for 60-90% of drug variability in drug disposition and pharmacodynamics. In addition to anti-dementia drugs, patients with AD, or with other forms of dementia, need concomitant medications for the treatment of diverse disorders of the central nervous system (CNS) associated with progressive brain dysfunction. Approximately 60-80% of drugs acting on the CNS are metabolised via enzymes of the CYP gene superfamily, and 10-20% of Caucasians are carriers of defective CYP2D6 polymorphic variants that alter the metabolism of many psychotropic agents. Only 26% of the patients are pure extensive metabolisers for the trigenic cluster integrated by allel-

ic variants of the CYP2D6, CYP2C19, CYP2C9 in combination. Although many genes have been suggested to be associated with AD, with the exception of APOE, most polymorphic variants of potential risk exhibit a very weak association with AD. APOE-4/4 carriers exhibit a dramatic biological disadvantage in comparison with other genotypes, and AD patients harbouring this homozygous condition are the worst responders to conventional drugs. The incorporation of pharmacogenetic/pharmacogenomic protocols into AD research and clinical practice can foster the optimisation of therapeutics by helping to develop cost-effective biopharmaceuticals and improving drug efficacy and safety. diciembre 2009

Personalized Medicine of Dementia

Introduction

The lack of accurate diagnostic markers for early prediction and of effective therapy of dementia are the two most important problems to efficient diagnosis and halting of progression of the disease. About 10-20% of the costs in dementia are attributed to pharmacological treatment, including anti-dementia drugs, psychotropics (antidepressants, neuroleptics, anxiolytics), and other drugs currently prescribed in the elderly (antiparkinsonians, anticonvulsants, vasoactive compounds, anti-inflammatory drugs, etc)1. During the past 25 years, over 300 drugs have been partially or totally developed for Alzheimerâ&#x20AC;&#x2122;s disease (AD) with poor repercussion in public health. Despite a considerable research effort and high expenditure over more than two decades, only 5 drugs (tacrine, donepezil, rivastigmine, galantamine, memantine) with moderate-to-poor efficacy and questionable cost-effectiveness have been approved in developed countries, and less than 20% of the patients can benefit from current anti-dementia drugs1-3. Fig.1 6

Common features in CNS disorders include the following: (a) polygenic/complex disorders in which genomic and environmental factors are involved; (b) deterioration of higher activities of the CNS; (c) multifactorial dysfunctions in several brain circuits; and (d) accumulation of toxic proteins in the nervous tissue in cases of neurodegeneration. For instance, the neuropathological hallmark of Alzheimerâ&#x20AC;&#x2122;s disease (AD) (amyloid deposition in senile plaques, neurofibrillary tangle formation, and neuronal loss) is but the phenotypic expression of a pathogenic process in which different gene clusters and their products are potentially involved4. Drug metabolism, and the mechanisms underlying drug efficacy and safety, are also genetically regulated complex traits in which hundreds of genes cooperatively participate. Structural and functional genomics studies demonstrate that genomic factors, probably induced by environmental factors, cerebrovascular dysfunction, and epigenetic phenomena, might be responsible for pathogenic events leading to premature neuronal dysfunction and/ or death.

The process of pharmacogenomics intervention in CNS disorders and dementia

Therapeutic Intervention Functional Genomics Transcriptomics Proteomics Metabolomics

Genotype Genomic Profile

Ethnic background Family History Disease Genotype Pharmacogenetic Genotype Pharmacogenomic Genotype Nutrigenetic Genotype Nutrigenomic Genotype

Genotype-Related Drug Metabolism Pharmacokinetics Pharmacodynamics

Phenotype CNS Disorder

Disease Phenotype Age and Gender Age at onset Disease Stage and Severity Concomitant Pathology Genotype-Phenotype Correlations Nutritional Conditions

Pharmacogenomic outcome

Disease Phenotype Modification Biochemical changes Neurochemical changes Neuropsychological changes Mood Behavior Cognition Functioning Neuroimaging changes Brain Function Cerebrovascular changes Gene Expression Profile Transcriptomics Proteomics Metabolomics

Pharmacogenetic and pharmacogenomic factors may account for 60-90% of drug variability in drug disposition and pharmacodynamics. About 10-20% of Caucasians are carriers of defective CYP2D6 polymorphic variants which alter the metabolism of many psychotropic agents. The incorporation of pharmacogenetic/pharmacogenomic protocols into dementia research and clinical practice can foster the optimization of therapeutics by helping to develop cost-effective pharmaceuticals and improving drug efficacy and safety5-11.

Dementia Phenotype and Biomarkers

Alzheimerâ&#x20AC;&#x2122;s disease is the most common form of dementia (50-60%), followed by vascular dementia (20-30%) and mixed dementia (10-20%), which has become the most prevalent type of dementia in individuals older than 75 years of age. There are over 100 different types of dementia with a common phenotypic denominator composed of cognitive and mental deterioration, psychomotor dysfunction, behavioural changes, and progressive functional decline1. AD is a complex disorder in which multiple pathogenic mechanisms may be involved giving rise to a common phenotype. From a didactic point of view, it has been established that primary pathogenic events in AD are represented by genetic factors (mutations, susceptibility SNPs) and programmed neuronal death, since neurons start to die 30-40 years before the onset of the disease. Secondary pathogenic events are associated with the phenotypic expression of senile plaques (amyloid deposition) and neurofibrillary tangles (NFT), together with synaptic loss, dendritic desarborization, and neuronal death, as the major hallmarks of AD pathology. Tertiary and quaternary pathogenic events are reflected by neurotransmitter deficits, neuroinflammatory reactions, oxidative stress phenomena and free radical formation, excitotoxic reactions, alterations in calcium homeostasis, deficit of neurotrophic factors, and cerebrovascular perturbations, among many other neurochemical phenotypes12. All these pathogenic elements configure the AD phenotype which differs from that of the healthy elderly population. The phenotypic features of the disease represent the biomarkers to be modified with an effective therapeutic intervention (Fig.1). Important differences have been

found in the AD population as compared with healthy subjects in different biological parameters, including blood pressure, glucose, cholesterol and triglyceride levels, transaminase activity, haematological parameters, metabolic factors, thyroid function, brain haemodynamic parameters, and brain mapping activity (Table 1; Fig.2) 7-10,12-14 . Blood pressure values, glucose levels and cholesterol levels are higher in AD than in healthy elderly subjects (Table 1). Approximately 20% of AD patients are hypertensive, 25% are diabetics, 50% are hypercholesterolemic (Fig.2a), and 23% are hypertriglyceridemic. Over 25% of the patients exhibit high GGT activity (Fig.2b), 5-10% show anaemic conditions, 30-50% show an abnormal cerebrovascular function characterized by poor brain perfusion (Fig.2c), and over 60% have an abnormal electroencephalographic pattern, especially in frontal, temporal, and parietal regions, as revealed by quantitative EEG (qEEG) or computerized mapping (Table 1; Fig.2d) 14-16. Significant differences are currently seen between females and males, indicating the effect of gender on the phenotypic expression of the disease. In fact, the prevalence of dementia is 1015% higher in females than in males from 65 to 85 years of age. All these parameters are highly relevant when treating AD patients because some of them reflect a concomitant pathology which also needs therapeutic consideration. On the other hand, they can also represent general biomarkers together with regional brain atrophy and perfusion and cognitive function, which may serve as therapeutic outcome measures. Other biomarkers of potential interest include cerebrospinal fluid (CSF) and peripheral levels of Aß42, protein tau, histamine, interleukins, and some other candidate markers4,13-16. The molecular mechanisms underlying β-amyloid deposition in brain tissue and blood vessels, as well as abnormalities in tau protein leading to NFT formation, have been elucidated over the past 20 years by a number of groups all over the world defining the fundamentals for promising therapeutic strategies oriented towards inhibiting the formation of amyloid deposits or reducing senile plaque burden. Notwithstanding, the complexity of the pathogenic cascade in AD invites the prediction that many other genetic factors and pathogenic mechanisms may be involved in the etiology of AD, together with epigenetic phenomena, cerebrovascular dysfunction, and environmental events12(Fig.3).

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Pharmacogenomics of Alzheimer’s disease Genomics

Structural Genomics: Approximately 5% of the human genome is structurally variant in the normal population, involving more than 800 genes17. There are roughly 7-10 million positions in the human genome that can show variability among individuals, and differences in the DNA sequence are the genetic basis of human variability and complex traits. The spectrum of variation in the human genome includes: (a) single changes (single nucleotide polymorphisms (SNPs), point mutations)(1 bp), (b) small insertions/deletions (binary insertion/deletion events of short sequences)(1-50 bp), (c) short tandem repeats (microsatellites) (1-500 bp), (d) fine-scale structural variation (deletions, duplications, tandem repeats, inversions)(50 bp – 5 kb), (e) retroelement insertions (SINEs, LINEs, LTRs, ERVs)(300 bp – 10 kb), (f) intermediatescale structural variations (deletions, duplications, tandem repeats, inversions) (5 kb – 50 kb), (g) large-scale structural variation (deletions, duplications, large tandem repeats)(50 kb – 5 Mb), and (h) chromosomal variations (euchromatic variations, cytogenetic deletions, duplications, translocations, inversions, and aneuploidy)(>5 Mb)17,18. Segmental duplications of low copy repeats are blocks of DNA ranging from 1-400 kb in length which occur at multiple sites within the genome and typically share a high level (>95%) of sequence identity17. Segmental duplications frequently mediate polymorphic rearrangements of intervening sequences via non-allelic homologous recombination (NAHR) with major implications for human disease. SNPs and insertion (I)/deletion (D) events are the most frequent types of structural variation. I/D polymorphisms of several genes with functions in enzymatic pathways or in drug metabolising enzymes (e.g. CYP2D6) may drastically influence a variety of common phenotypes with pathogenic and/ or pharmacogenetic relevance. The differential expression of common variants is a major source of genetic variation with important repercussions in human diversity and disease heterogeneity. Prior to the completion of the Human Genome Project and the emergence of dense genetic maps, scientists used linkage studies and positional cloning to identify DNA mutations in rare diseases, but in the past two decades association study designs became more powerful compared with linkage

Fig.2 5 Comparison of biological parameters between patients with Alzheimer’s disease and the general population. a. Total cholesterol, LDL-cholesterol, and HDL-cholesterol. b. Transaminase activity: GOT/AST, GPT/ALT, GGT. c. Brain hemodynamic parameters: Systolic velocity (Sv), Diastolic velocity (Dv) and Mean velocity (Mv) d. Brain mapping activity AD: Alzheimer’s disease; FD: Frontal Delta; FT: Frontal Theta; GP: General population; PD: Parietal Delta; PT: Parietal Theta; TD: Temporal Delta; TT: Temporal Theta; OA: Occipital Alpha. Adapted from Cacabelos42

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Fig.3 5 Pathogenetic mechanisms potentially involved in Alzheimerâ&#x20AC;&#x2122;s disease. (Adapted from http://www.genome.jp/. Kyoto University Bioinformatics Center, Kyoto, Japan; Kanehisa Laboratories).

study designs in identifying susceptibility loci and SNP variation18. Currently, over 10 million DNA sequence variations have been uncovered in the human genome18. It has been observed that the genetic variation rate (GVR) is higher in AD patients than in the general population12,14,19. Karyotype anomalies appear in less than 3% of the cases, with no differences as compared with the general population. The variability of bigenic, trigenic, tetragenic and polygenic genotypes of AD-related genes is currently higher in AD than in controls, with an absolute genetic variation (AGV) of 40-

60% and a relative genetic variation (RGV) of 0.85-1.89% depending on the number of genes included in the haplotype-like cluster. Approximately, 40% of AD cases exhibit a GVR higher than 1% as compared to controls when a trigenic cluster integrated by combinations of APOE+PSEN1+PSEN2 polymorphic variants is examined14. Increased GVR in AD might indicate that the over-representation of a series of genes involved in brain maturation and in the maintenance of higher activities of the CNS has surpassed a natural selection threshold (excessive genome complexity, genomic overdiversification), constituting a Darwinian disadvantage which shortens life span in humans12,19. Recent observations support the contention that serial segmental duplication events might have orchestrated primate evolution by the generation of novel fusion/fission genes as well as potentially

by genomic inversions associated with decreased recombination rates facilitating gene divergence20. Recent studies with alignments of 10,238 human genes have identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a tendency to contain clusters of AT-to-GC biased substitutions. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of non-synonymous substitutions relative to the genomic average. These findings might indicate that a recombination-associated process (biased gene conversion) is driving fixation of CG alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replacement substitutions, thereby contributing to positive or negative selection21. Genes that

Table 1 have undergone positive selection during species evolution are indicative of functional adaptations which drive differences between species. In has been shown that genes predicted to have been subject to positive selection pressure during human evolution are implicated in diseases such as epithelial cancers, schizophrenia, autoimmune diseases and AD22. Extensive molecular genetics studies carried out during the past two decades have demonstrated that most CNS disorders are multifactorial, polygenic/complex disorders in which hundreds of genes distributed across the human genome might be involved12,23. Many of these genetic associations could not be replicated in different settings and different populations due to a number of complex (methodological, technological) factors12,24. Furthermore, the same genomic defect can give rise to apparent diverse phenotypes, and different genomic defects can converge in an apparently common phenotype, this increasing the complexity of genomic studies. The genetic defects identified in AD can be classified into 3 main categories: a. Mendelian or mutational defects in genes directly linked to AD, including (i) 32 mutations in the amyloid beta (Aβ)(ABP) precursor protein (APP) gene (21q21) (AD1); (ii) 165 mutations in the presenilin 1 (PSEN1) gene (14q24.3)(AD3); and (iii) 12 mutations in the presenilin 2 (PSEN2) gene (1q31-q42)(AD4)12,23,25. PSEN1 and PSEN2 are important determinants of γ-secretase activity responsible for proteolytic cleavage of APP and NOTCH receptor proteins. Mendelian mutations are very rare in AD (1:1000). Mutations in exons 16 and 17 of the APP gene appear with a frequency of 0.30% and 0.78%, respectively, in AD patients. Likewise, PSEN1, PSEN2, and microtubule-associated protein Tau (MAPT)(17q21.1) mutations are present in less than 2% of the cases. Mutations in these genes confer specific phenotypic profiles to patients with dementia: amyloidogeneic pathology associated with APP, PSEN1 and PSEN2 mutations; and tauopathy associated with MATP mutations, representing the two major pathogenic hypotheses for AD12,26-28.

Biological parameters in patients with Alzheimer’s disease versus general population. Parameter

General population

Alzheimer’s disease

P value

Abnormal Rate in AD

Blood Pressure Systolic (SBP) (mm Hg) Diastolic (DBP) (mm Hg)

127.46 ± 21.60 76.49 ± 10.81

138.42 ± 20.46 79.47 ± 10.34

<0.001 <0.001

SBP>160: 17.92% DBP>85: 28.52

Glucose (mg/dL)

94.97 ± 23.10

101.02 ± 27.75

<0.001

Glucose>105: 25.89%

Cholesterol Total-Cholesterol (mg/dL) HDL-Cholesterol (mg/dL) LDL-Cholesterol (mg/dL)

210.19 ± 46.48 52.76 ± 17.03 136.54 ± 40.23

220.26 ± 45.54 53.32 ± 14.22 144.31 ± 40.02

<0.001 <0.001 <0.001

T-CHO>220: 50.15% HDL-CHO<45: 29.69% LDL-CHO>140: 47.18%

Triglycerides (mg/dL)

106.07 ± 72.70

114.09 ± 65.23

<0.001

TG>140: 23.25%

Transaminase acivity GOT/AST (IU/L) GPT/ALT (IU/L) GGT (IU/L)

22.15 ± 15.89 24.49 ± 20.14 28.74 ± 36.11

22.21 ± 17.92 23.66 ± 18.80 30.84 ± 37.64

0.59 0.46 <0.001

GOT/AST>30: 9.09% GPT/ALT>30: 18.91% GGT>30: 28.55%

Red Blod Cells (RBC) (x106/mm3)

4.66 ± 0.46

4.61 ± 0.44

<0.001

RBC<4: 7.58%

Hematocrit (Ht)(%)

42.01 ± 4.26

41.93 ± 4.22

0.60

Ht<35: 3.94% Ht>45: 21.04%

Hemoglobin (Hb)(g/dL)

14.07 ± 2.03

13.98 ± 1.38

0.24

Hb<12: 6.47% Hb>15: 22.45%

Iron (Fe)(µg/dL)

87.82 ± 40.81

86.79 ± 43.66

0.44

Fe<50: 12.04%

Ferritin (Fer)(ng/mL)

106.58 ± 126.64

127.11 ± 147.74

<0.001

Fer<30: 15.26% Fer>300: 8.09%

Folate (Fol)(ng/mL)

7.14 ± 4.20

7.03 ± 3.99

<0.01

Fol<3: 4.98%

Vitamin B12 (pg/mL)

493.62 ± 254.88

505.81 ± 289.74

0.89

B12<300: 19.17%

TSH (µIU/mL)

1.55 ± 1.99

1.50 ± 2.63

<0.001

TSH<1: 41.05% Tsh>5: 1.59%

Tiroxin (T4)

0.88 ± 0.35

0.91 ± 0.44

<0.001

T4<0.6: 3.05% T4>1.5: 1.14%

51.58 ± 16.44

42.77 ± 12.86

<0.001

Mv<40: 30.05%

81.33 ± 24.82

69.11 ± 19.52

<0.001

Sv>60: 43.28%

33.70 ± 12.10

26.66 ± 9.03

<0.001

Dv<25: 37.45%

0.94 ± 0.23 0.58 ± 0.07

1.01 ± 0.24 0.61 ± 0.08

<0.001 <0.001

PI>1: 63.60% RI>0.5>72.04%

4.30 ± 2.05 3.69 ± 1.80 3.55 ± 2.07 2.82 ± 1.48 2.60 ± 1.39 2.36 ± 1.44 3.54 ± 3.68

5.21 ± 3.65 4.38 ± 3.28 4.43 ± 3.74 2.97 ± 1.89 2.78 ± 1.89 2.56 ± 1.89 2.72 ± 1.59

<0.001 <0.001 <0.001 0.11 <0.05 <0.002 <0.001

68.30% 73.56% 75.82% 40.24% 69.54% 71.65% 83.12%

Hemodynamic Parameters in the Left Middle Cerebral Artery (LMCA) Mean blood velocity (Mv) (cm/sec) Systolic blood velocity (Sv) (cm/sec) Diastolic blood velocity (Dv) (cm/sec) Pulsatility Index (Units) Resistance Index (Units) Brain Mapping Activity Frontal Delta (%) Parietal Delta (%) Temporal Delta (%) Frontal Theta (%) Parietal Theta (%) Temporal Theta (%) Occipital Alpha (%)

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Source: R. Cacabelos, EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine (2009) General Population: N=3301; Alzheimer’s disease: N=1364 (Females: 777; Males: 587); Race: Caucasians. Adapted from Cacabelos42

b. Multiple polymorphic variants of risk characterized in more than 200 different genes can increase neuronal vulnerability to premature death12 (Table 1). Among these genes of susceptibility, the apolipoprotein E (APOE) gene (19q13.2) diciembre 2009

Personalized Medicine of Dementia

Genetic Polymorphisms Pharmacokinetics

Pharmacodynamics

Absortion Distribution Metabolism Excretion

Receptors Ion channels Enzymes Proteins

Pathogenic mechanisms

Safety

METABOLISM

Transcriptomics Proteomics Metabolomics

DRUGS DRUGS

Efficacy

DISEASE

Fig.4 5 Different polymorphic variants in genes distributed across the human genome are responsible for pharmacokinetic and pharmacodynamic variability through pharmacogenetic / pharmacogenomic mechanisms influencing drug efficacy and safety.

(AD2) is the most prevalent as a risk factor for AD, especially in those subjects harbouring the APOE-4 allele, whereas carriers of the APOE-2 allele might be protected against dementia12. APOE-related pathogenic mechanisms are also associated with brain aging and with the neuropathological hallmarks of AD. Other genes of this category include the following: AD5 (Alzheimer’s disease 5)( 12p11.23-q13.12), AD6 (10q24), AD7 (10p13), AD8 (20p), AD9 (19p13.2), AD10 (7q36), APBB2 (Amyloid beta A4 precursor protein-binding, family B, member 2, FE65L1)(4p14), HFE (Hemochromatosis gene, HLA-H, HFE1, MVCD7)(6p21.3), eNOS3 (Nitric oxide synthase 3, endothelial cell)(7q36), PACIP1 (PAX transcription activation domain-interacting protein 1, PAXIP1L, PTIP)(7q36), PLAU (Plasminogen activator, urokinase, URK)(10q24), SORL1 (Sortilin-related receptor, L(DLR class) A repeats-containing, LR11, SORLA) (11q23.2-q24.2), A2M (Alpha-2-macroglobulin)(12p13.3-p12.3), BLMH (Bleomycin hydrolase, BMH)(17q11.2), ACE

(Angiotensin I converting enzyme, dipeptidyl carboxypeptidase-1, DCP1, ACE1, MVCD3)(17q23), MPO (Myeloperoxidase) (17q23.1), FOS (V-Fos FBJ murine osteosarcma viral oncogene homolog)(14q24.3), MTHFR (Methylenetetrahydrofolate reductase)(1p36.3), CETP (Cholesteryl ester transfer protein, Lipid transfer protein 1) (16q21), AGT (Angiotensinogen, Serpina 8)(1q42-q43), BDNF (Brain-derived neurotrophic factor)(11p13), CH25H (Cholesterol 25-hydroxylase)(10q23), CHRNB2 (Cholinergic receptor, nicotinic, beta polypeptide-2, EFNL3)(1q21), CST3 (Cystatin C, ARMD11)(20p11.2), CTSD (Cathepsin D, lysosomal aspartyl protease, CPSD, CLN10)(11p15.5), DAPK1 (Death-associated protein kinase-1)(9q34.1), DHCR24 (24-dehydrocholesterol reductase, KIAA0018)(1p33-p31.1), IL1B (Interleukin-1, beta)(2q14), LMNA (Lamin A/C, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B)(1q21.2), MYH13 (Myosin, heavy polypeptide 13, skeletal muscle)(17p13.1p12), PCK1 (Phosphoenolpyruvate carboxykinase-1)(20q13.31), PRNP (Prion protein)(20pter-p12), SORCS1 (SORCS receptor 1)(10q23.3), TFAM (Transcription factor A, mitochondrial, TCF6L2, TCF6L1, TCF6L3, MTTF1, TCF6)(10q21), TNK1 (Tyrosine kinase, nonreceptor, 1) (17p13.1), CALHM1 (Calcium homeostasis modulator 1, FAM26C)(10q24.33), and

some other candidate genes recently incorporated to the AD-related gene pipeline, such as CLU (Clusterin (complement lysis inhibitor, SP-40,40; sulfated glycoprotein 2; testosterone-repressed prostate message-2; apolipoprotein J))(8p21-p12) and CR1 (Complement component (3b/4b) receptor-1)(1q32) and PICALM (Phosphatidylinositol-binding clathrin assembly protein)(11q14)12,23,25,29,30,31 (Table 1). One of the newest members of the AD-gene family is SORL1, a gene which encodes a mosaic protein with a domain structure which suggests it is a member of both the vacuolar protein sorting-10 (Vps10) domain-containing receptor family and the low density lipoprotein receptor (LDLR). Inherited variants of the SORL1 neuronal sorting receptor are associated with late-onset AD. Polymorphisms in two different clusters of intronic sequences within the SORL1 gene may regulate tissue-specific expression of SORL1, which directs trafficking of APP into recycling pathways. When SORL1 is underexpressed, APP is sorted into Aß-generating compartments leading to amyloid accumulation in neuronal tissues32. As with many other potential AD-related genes, the association of SORL1 with AD32,33 could not be replicated in other studies34. Another interesting gene is DHCR24 (3ß-hydroxysterol-δ-24-reductase) or Seladin-1, a key element in the cholesterologenic pathway in which the DHCR24 enzyme catalyses the transformation of desmosterol into cholesterol35,36. Seladin-1 was originally identified as a gene whose expression was down-regulated in the AD brain, demonstrating a neuroprotective effect against neurodegeneration. Recent studies indicate that Seladin-1/DHCR24 is an LXR (liver X nuclear hormone receptor) target gene potentially involved in the regulation of lipid raft formation35. Another gene, with potential therapeutic interest as a tau kinase, might be the GSK3 gene. Analysis of the promoter and all 12 exons revealed that an intronic polymorphism (IVS2-68G>A) occurred at more than twice the frequency among patients with frontotemporal dementia (10.8%) and patients with AD (14.6%) than in aged healthy subjects (4.1%). This is the first evidence that a gene known to be involved in tau phosphorylation is associated with risk for primary neurodegenerative dementias37. Promoter polymorphisms modulating HSPA5 expression might also increase susceptibility to AD. Endoplasmic reticulum chaperone heat shock 70 kDa protein 5 (HSPA5/ GRP78) is known to be involved in APP metabolism and neuronal death in AD.

Table 2 Selected human genes investigated as potential candidate genes associated with dementia and age-related neurodegenerative disorders Locus

Symbol

Title/Gene

MIM

ZFYVE9 SARA MADHIP

Zinc finger, FYVE domain containing 9 SMAD anchor for receptor activation MADH-interacting protein

603755

1p32

PRNPIP, PINT1

Prion protein-interacting protein

609917

1p33-p31.1

DHCR24, KIAA0018

24-dehydrocholesterol reductase

606418

1p34

LRP8 APOER2

Low-density lipoprotein receptor-related protein 8

602600

1p36

AD7CNTP

Alzheimer disease neuronal thread protein (ADNTP)

607413

1p36.3

MTHFR

Methylenetetrahydrofolate reductase

1q21

S100A

1q21

CHRNB2, EFNL3

1p32

Locus

Symbol

Title/Gene

MIM

5q15-q21

CAST

Calpastatin

114090

5q31

APBB3 FE65L2

Amyloid beta A4 precursor proteinbinding, family B, member 3

602711

5q35.3

DBN1

Drebrin E

12660

6p21.3

AGER RAGE

Advance glycosylation end productspecific receptor

600214

6p21.3

HFE, HLA-H, HFE1, MVCD7

Hemochromatosis gene

235200

6p21.3

TNFA

Tumor necrosis factor-a Cachectin

191160

236253 104300

7p21

IL-6 IFNB2

Interleukin-6 Beta-2 interferon

147620

S100 Calcium-binding protein A1

176940

7q36

AD10

Alzheimer disease-10

609636

Cholinergic receptor, nicotinic, beta polypeptide-2

118507

7q36

NOS3

Nitric Oxide Synthase-3

163729

LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B

Lamin A/C

150330

7q36

PACIP1, PAXIP1L, PTIP

PAX transcription activation domaininteracting protein 1

608254

8p21-p12

CLU, CLI, SGP2, TRPM2

185430

1q21-q23

APCS

Serum amyloid P component

104770

Clusterin (complement lysis inhibitor, SP-40,40; sulfated glycoprotein 2; testosterone-repressed prostate message-2; apolipoprotein J)

1q23

NCSTN APH2

Nicastrin

605254

8p22

CTSB CPSB

Cathepsin B Amyloid precursor protein secretase

116810

1q25

SOAT1 STAT ACAT

Acyl-CoA:Cholesterol acyltransferase Sterol O-acyltransferase 1

102642

9q13

Amyloid beta-A4 precursor proteinbinding, family A, member 1

602414

1q31-q42

AD4 PSEN2 STM2

Presenilin-2

600759 104300

APBA1 X11 MINT1 LIN10

9q34

HSPA5, GRP78

Heat-shock 70kD protein-5 (glucoseregulated protein, 78kD)

138120

1q32

CR1, C3BR

Complement component (3b/4b) receptor-1

120620

9q34.1

DAPK1

Death-associated protein kinase-1

600831

10p13

AD7

Alzheimer disease-7

606187

10q21

TFAM, TCF6L2, TCF6L1, TCF6L3, MTTF1, TCF6

Transcription factor A, mitochondrial

600438

10q23

CH25H

Cholesterol 25-hydroxylase

604551

10q23.3

SORCS1

SORCS receptor 1

606283

10q23-q25

IDE

Insulin-degrading enzyme

146680

1q21.2

1q42-q43

AGT, SERPINA8

Angiotensinogen

106150

Chr. 1

APH1A

C. elegans anterior pharynx defective homolog

607629

2p14-p13

RTN4 NOGO

Neurite outgrowth inhibitor (reticulon 4)

604475

2p25

ADAM17 TACE

A desintegrin and metalloproteinase domain 17 Tumor necrosis factor-alpha converting enzyme

603639

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2q14

IL1A

Interleukin-1-Alpha

147760

10q24

AD6

Alzheimer disease-6

6,05526E+11

2q21.1

CSEN DREAM KCNIP3

Calsenilin

604662

10q24

PLAU URK

Plasminogen activator, urokinase

191840

10q24.33

612234

LRP1B

Low density lipoprotein receptor-related protein 1B

608766

CALHM1, FAM26C

Calcium homeostasis modulator 1

2q21.2

11p13

BDNF

Brain-derived neurotrophic factor

113505

3q13.3

GSK3B

Glycogen synthase kinase 3-beta

605004

11p15

BCHE

Butyrylcholinesterase

177400

Amyloid beta-A4 precursor proteinbinding, family B, member 1

602709

3q26.1q26.2

APBB1 F65

11p15.1

SAA1

Serum amyloid A1

104750

3q26.2-qter

APOD

Apolipoprotein D

107740

11p15.5

116840

CREB1

cAMP response element-binding protein

123810

CTSD, CPSD, CLN10

Cathepsin D (lysosomal aspartyl protease)

3q32.3-q34 4p14

APBB2 FE65L1

Amyloid beta-A4 precursor proteinbinding, family B, member 2

602710

11q14

PICALM, CALM, CLTH, LAP

Phosphatidylinositol-binding clathrin assembly protein

603025

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Personalized Medicine of Dementia Continued, Table 2 Locus

Symbol

Title/Gene

MIM

11q23.2q24.2

SORL1, LR11, SORLA

Sortilin-related receptor, L(DLR class) A repeats-containing

602005

11q23.3

BACE1 BACE

Beta-site amyloid beta A4 precursor protein-cleaving enzyme Beta-secretase Memapsin-2

604252

11q24

APLP2

Amyloid beta-A4 precursor-like protein 2

104776

12p11.23q13.12

AD5

Familial AD-5

602096

12p12.3p12.1

IAPP IAP DAP

Islet amyloid polypeptide Amylin Diabetes-associated peptide

147940

12p13.3p12.3

A2M

Alpha-2-Macroglobulin

103950

12q13.1q13.3

LRP1 A2MR

Low density lipoprotein-related protein-1 Alpha-2-macroglobulin receptor

107770

14q24.3

FOS

FBJ murine osteosarcoma viral (v-fos) oncogene homolog Oncogene Fos

14q24.3

AD3 PSEN1

14q32.1

Locus

Symbol

Title/Gene

MIM

17q21-q22

GPSC

Familial progressive subcortical gliosis

221820

17q22-q23

APPBP2 PAT1

Amyloid beta precursor protein-binding protein 2

605324

17q23

ACE ACE1 DCP1

Angiotensin I converting enzyme Dipeptidyl carboxipeptidase-1

106180 104300

17q23.1

MPO

Myeloperoxidase

254600

17q24

FALZ FAC1

Fetal Alzheimer antigen

601819

18q11.2q12.2

TTR PALB

Transthyretin Prealbumin

176300

19p13.2

NOTCH3 CADASIL CASIL

Drosophila Notch 3 homolog

600276

19p13.2

AD9

Alzheimer disease-9

608907

164810

19p13.3p13.2

ICAM CD54 BB2

Intercellular adhesion molecule 1

147840

Presenilin-1

104311

19p13.3

APBA3 X11L2

Amyloid beta-A4 precursor protein binding, family A, member 3

604262

SERPINA3 AACT ACT

Alpha-1-antichymotrypsin

107280

19q13.12

PEN2

Presenilin enhancer 2

607632

19q13.2

APOE

Apolipoprotein E

107741

CYP46 CYP46A1

Cytochrome P450 Family 46, Subfamily A Polypeptide 1 Cholesterol 24-hydrolase

604087

19q13.2

APOC1

Apolipoprotein C-I

107710

19cenq13.2

AD2

Alzheimer disease-2

104310

Chr. 15

APH1B

Homolog of C. elegans anterior pharynx defective 1B

607630

19cenq13.2

APLP1

Amyloid beta-A4 precursor-like protein 1

104775

15q11-q12

APBA2 X11L

Amyloid beta-A4 precursor proteinbinding, family A, member 2

602712

19q31-qter

APPL1

Amyloid beta-A4 precursor protein-like 1

104740

20p

AD8

Alzheimer disease-8

607116

16q21

CETP, HDLCQ10

Cholesteryl ester transfer protein, plasma

118470

20p11.2

CST3

Cystatin 3

604312

20p11.2

CST3

Cystatin C

604312

20pter-p12

PRNP, PRIP

Prion protein (p27-30)

176640

20q13.31

PCK1

Phosphoenolpyruvate carboxykinase-1 (soluble)

261680

21q21

AD1 APP AAA CVAP

Amyloid beta (A4) precursor protein Amyloid of aging and Alzheimer disease Cerebrovascular amyloid peptide Protease nexin II

104760

21q22.3

BACE2 ALP56 DRAP

Beta-site amyloid beta A4 precursor protein-cleaving enzyme 2 Down syndrome-region aspartic protease

605668

22q11

RTN4R, NOGOR HN

NOGO receptor (reticulon 4 receptor) Humanin

605566 606120

14q32.1

16q22

APPBP1

Amyloid beta precursor protein-binding protein 1

603385

17p13.1

TNK1

Tyrosine kinase, nonreceptor, 1

608076

17p13.1p12

MYH13

Myosin, heavy polypeptide 13, skeletal muscle

603487

17q11.2

BLMH BMH

Bleomycin hydrolase

602403

17q21

STH

Saitohin

607067

17q21.1

MAPT MTBT1 DDPAC MST

Macrotubule-associated protein tau

157140 600274 168610 172700 601104

Adapted from Cacabelos12,42, Cacabelos and Takeda9 and OMIM23

Of the 3 major polymorphisms (-415G/ A (rs391957), -370C/T (rs17840761), -180del/G (rs3216733)), the HSPA5415G/A and -180del/G variants showed significant differences between AD cases and controls. Subjects harbouring the -415AA/180GG genotype or the -415A/-180G allele might be less susceptible to develop AD37.

The rs5952C and rs1568566T alleles of the APOD rs5952T/C and rs1568566C/T variants increase the risk for AD, whereas the rs5952T-rs1568566C haplotype reduces it38. ApoD is a lipoprotein associated glycoprotein which is increased in the hippocampus and CSF of AD patients39. CALHM1 encodes a multipass transmembrane glyco-

protein that controls cytosolic Ca2+ concentrations and Aβ levels. The CALHM1 P86L polymorphism (rs2986017) has been associated with AD40, but this association could not be replicated in other studies. Harold et al31 undertook a two-stage genome-wide association study (GWAS)

Table 3 Distribution and frequency of polymorphic variants of selected Alzheimer’s disease-associated genes in the general population, in adults (>45 years) with no family history of dementia, and in patients with Alzheimer’s disease. Gene

Polymorphism

General Population

APOE

APOE-2/2

0,21

0,13

0,11

APOE-2/3

228

7,94

7,37

A2MV100I

ACE

Polymorphism

AGT-235

AGT-M/M

230

10,1

10,45

8,97

7,68

AGT-M/T

1560

68,48

454

67,76

600

69,03

AGT-T/T

488

21,42

146

21,79

191

0,94

1,47

APOE-3/3

1938

67,51

534

71,58

577

60,74

APOE-3/4

600

20,9

136

18,24

250

26,32

AGT-174

100

869

100

0,9

0,46

477

20,93

137

20,45

194

22,32

1789

78,5

527

78,65

671

77,22

2279

100

670

100

869

100

eNOS3-G/A

0,24

0,58

eNOS3-G/G

647

39,33

198

38,37

241

38,94

776

47,17

250

48,45

299

48,3

218

13,26

12,6

12,76

1645

100

516

100

619

100

eNOS3-A/A

2,74

2,72

1,94

eNOS3-A/B

408

24,82

123

23,88

164

26,54

eNOS3-B/B

1191

72,44

378

73,4

442

71,52

3,68

AGT-T/T

746

100

950

100

139

25,27

213

27,95

PSEN1-1/2

974

59,14

345

62,73

435

57,09

PSEN1-2/2

224

13,6

114

14,96

eNOS3E298D

1647

100

550

100

762

100

eNOS3-G/T

A2M-A/A

769

46,89

251

46,48

359

48,32

eNOS3-T/T

A2M-A/G

728

44,39

238

44,07

325

43,74

143

8,72

9,45

7,94

1640

100

540

100

743

100

A2M-D/D

1,81

2,22

1,33

A2M-I/D

434

26,24

143

26,38

201

26,8

A2M-I/I

1190

71,95

387

71,4

539

71,87

eNOS327bpTR

CETP

1644

100

515

100

618

100

CETP-B1/B1

361

36,54

108

40,6

116

34,32

497

50,3

124

46,62

173

51,18

130

13,16

12,78

14,5

988

100

266

100

338

100

MTHFR-C/C

407

42,48

103

40,71

133

40,43

MTHFR-C/T

417

43,53

109

43,08

149

45,29

MTHFR-T/T

134

13,99

16,21

14,28

958

100

253

100

329

100

1654

100

542

100

750

100

CETP-B1/B2

ACE-D/D

839

35,96

261

38,33

304

34,35

CETP-B2/B2

ACE-I/D

1137

48,74

333

48,9

430

48,59

357

15,3

12,77

151

17,06

2333

100

681

100

885

100

FOS-A/A

733

71,1

252

314

71,85

FOS-A/B

259

25,12

26,11

107

24,49

FOS-B/B

3,78

3,89

3,66

1031

100

360

100

437

100

c. Diverse mutations located in mitochondrial DNA (mtDNA) through heteroplasmic transmission can influence aging and oxidative stress conditions, conferring phenotypic heterogeneity12,41.

MTHFR

670

27,26

0,57

1,74

100

449

2,12

2871

Alzheimer’s disease

2278

PSEN1-1/1

Adults>45 yrs NFHD

AGT-M/M AGT-M/T

of AD involving over 16,000 individuals, and found association with SNPs at two loci not previously associated with the disease, at the CLU (Clusterine, APOJ) gene (rs11136000) and 5’ to the PICALM gene (rs3851179). In another GWAS with patients from France, Belgium, Finland, Italy and Spain, Lambert et al30 found association with CLU and with the CR1 gene, encoding the complement component (3b/4b) receptor 1, on chromosome 1 (rs6656401).

General Population

1,32

ACE-I/I

FOS

Gene

A2M-G/G

A2M-I/D

Alzheimer’s disease

APOE-2/4

APOE-4/4

PSEN1

Adults>45 yrs NFHD

Although APP and PSEN mutations are considered causative factors for AD, the total number of mutations identified in the APP, PSEN1 and PSEN2 genes account for less than 3% of the cases with AD, clearly indicating that neurodegeneration associated with AD pathogenesis cannot be exclusively attributed to APP/PSEN-related cascades (amyloid hypothesis). Alterations in the ubiquitin-proteasome system and biochemical disarray in the chaperone machinery are alternative and/or complementary pathogenic events potentially leading to defects in protein synthesis, folding, and degradation with subsequent conformational changes, aggregation, and accumulation in cytotoxic deposits10,12. A more plausible explanation would seem to

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Pharmacogenomics of Alzheimer’s disease

Source: R. Cacabelos 42

be that multiple susceptibility SNPs with a very subtle genetic variation cooperatively contribute, in concert with environmental factors and concomitant CNS vulnerability, to premature neurodegeneration in dementia. We have compared the distribution and frequency of major polymorphic variants of different genes potentially associated with AD (i.e. APOE, PSEN1, A2M-V1001, A2M-I/D, ACE, FOS, AGT-235, AGT-174, eNOS3-E298D, eNOS3-27bpTR, CETP, MTHRF) in the general population, in adults (>45 years) with no family history of dementia, and in patients with dementia, and could not find any significant differences among the three groups except diciembre 2009

Personalized Medicine of Dementia 100

(%)

have robust influences on the hippocampal transcriptome of middle-aged animals. Prominent functional groups of age- and energy-sensitive genes are those encoding proteins involved in DNA damage responses, mitochondrial and proteasome functions, cell fate determination and synaptic vesicle trafficking. The systematic transcriptome dataset provides a window into mechanisms of neuropathogenesis and CNS vulnerability44.

Antidepressants Neuroleptics Benzodiazepines

CYP1A2

CYP2B6

Fig.5 5 Psychotropic drugs acting as major substrates for enzymatic products of CYP genes. (Adapted from Cacabelos 42)

in the case of the APOE gene which exhibits a clear accumulation of APOE-3/4 and APOE-4/4 genotypes (overload of the APOE-4 allele) in AD cases42 (Table 3). If we consider that a genetic variation higher than 2% could be of significant value, then several polymorphisms clearly differ in AD as compared with the other two population clusters, including the PSEN1-1/2, ACED/D, ACE-I/I, CEPT-B1/B1, and MTHFRT/T polymorphisms42 (Table 3). It is also likely that defective functions of genes associated with longevity may influence premature neuronal survival, since neurons are potential pacemakers defining life span in mammals9,12. All these genetic factors may interact in genetic networks which are still unknown, leading to a cascade of pathogenic events characterized by abnormal protein processing and misfolding with a subsequent accumulation of abnormal proteins (conformational changes), ubiquitin-proteasome system dysfunction, excitotoxic reactions, oxidative and nitrosative stress, mitochondrial injury, synaptic failure, altered metal homeostasis, dysfunction of axonal and dendritic transport, and chaperone misoperation6-10,12. These pathogenic events may exert an additive effect, converging in final pathways leading to premature neuronal death. Some of these mechanisms are common to several neurodegenerative disorders which differ depending upon

CYP2C19

CYP2D6

CYP3A4

the gene(s) affected and the involvement of specific genetic networks, together with cerebrovascular factors, epigenetic factors (DNA methylation) and environmental conditions (nutrition, toxicity, social factors, etc)6-13 (Fig.3). The higher the number of genes involved in AD pathogenesis, the earlier the onset of the disease, the faster its clinical course, and the poorer its therapeutic outcome6-13.

Functional genomics It is very likely that over 80% of the genes which conform the structural architecture of the human genome are expressed in the brain in a time-dependent manner along the lifespan. High throughput microarray gene expression profiling is an effective approach for the identification of candidate genes and associated molecular pathways implicated in a wide variety of biological processes or disease states. The cellular complexity of the CNS (with 103 different cell types) and synapses (with each of the 1011 neurons in the brain having around 103-104 synapses with a complex multiprotein structure integrated by 103 different proteins) requires a very powerful technology for gene expression profiling, which is still in its very early stages and is not devoid of technical obstacles and limitations43. Transcripts of 16,896 genes have been measured in different CNS regions. Each region possesses its own unique transcriptome fingerprint which is independent of age, gender and energy intake. Less than 10% of genes are affected by age, diet or gender, with most of these changes occurring between middle and old age. Gender and energy restriction

Functional genomics studies have demonstrated the influence of many genes on AD pathogenesis and phenotype expression. The study of genotype-phenotype correlations is essential for the evaluation of the actual impact of specific polymorphic variants of a particular gene on the clinical manifestation of the disease and/or biological markers reflecting the disease condition or different biological states of the individual. It has been demonstrated that mutations in the APP, PSEN1, PSEN2, and MAPT genes give rise to well-characterized differential neuropathological and clinical phenotypes of dementia12,23,25. APP mutations are associated with AD1, early-onset progressive autosomal recessive dementia, early-onset AD with cerebral amyloid angiopathy, and hereditary amyloidosis with cerebral haemorrhage Dutch type, Italian type, or Iowa type. PSEN1 mutations are associated with the phenotypes of familial AD3, familial AD3 with unusual plaques, familial AD with spastic paraparesis and unusual plaques, familial AD with paraparesias and apraxia, frontotemporal dementia, Pickâ&#x20AC;&#x2122;s disease, and dilated cardiomyopathy. MAPT mutations are associated with frontotemporal dementia, frontotemporal dementia with parkinsonism, Pickâ&#x20AC;&#x2122;s disease, progressive supranuclear palsy, progressive atypical supranuclear palsy, and tauopathy and respiratory failure12,23. Transgenic animals also reproduce to some extent the neuropathological hallmarks of AD in a sequential manner. The triple transgenic mouse model of AD (3xTg-AD) harbours 3 AD-related loci: human PS1M146V, human APPswe, and human MAPTP301L. These animals develop both amyloid plaques and NFT-like pathology in a progressive and age-dependent manner in hippocampus, amygdala, and cerebral cortex, the main foci of human AD neuropathology. The evolution of AD-related transgene expression, amyloid deposition, tau phosphorylation, astrogliosis, and microglia activation throughout the hippocampus, entorhinal cortex, primary motor cortex, and

amygdala over a 26-month period has been immunohistochemically documented. Intracellular Aß accumulation is the earliest of AD-related pathologies to be detectable, followed temporally by phospho-tau, extracellular Aß, and finally paired helical filament and NFT pathology45. In the same model, a decrease in neurogenesis directly associated with the presence of amyloid plaques and an increase in the number of Aß containing neurons in the hippocampus has been demonstrated46. Different APOE genotypes also confer specific phenotypic profiles to AD patients. Some of these profiles may add risk or benefit when the patients are treated with conventional drugs, and in many instances the clinical phenotype demands the administration of additional drugs which increase the complexity of therapeutic protocols. From studies designed to define APOErelated AD phenotypes6-16,28,42,47-52, several confirmed conclusions can be drawn: (i) the age-at-onset is 5-10 years earlier in approximately 80% of AD cases harbouring the APOE-4/4 genotype; (ii) the serum levels of ApoE are lowest in APOE-4/4, intermediate in APOE-3/3 and APOE-3/4, and highest in APOE-2/3 and APOE-2/4; (iii) serum cholesterol levels are higher in APOE-4/4 than in the other genotypes; (iv) HDL-cholesterol levels tend to be lower in APOE-3 homozygotes than in APOE4 allele carriers; (v) LDL-cholesterol levels are systematically higher in APOE-4/4 than in any other genotype; (vi) triglyceride levels are significantly lower in APOE4/4; (vii) nitric oxide levels are slightly lower in APOE-4/4; (viii) serum Aß levels do not differ between APOE-4/4 and the other most frequent genotypes (APOE3/3, APOE-3/4); (ix) blood histamine levels are dramatically reduced in APOE-4/4 as compared with the other genotypes; (x) brain atrophy is markedly increased in APOE-4/4>APOE-3/4>APOE-3/3; (xi) brain mapping activity shows a significant increase in slow wave activity in APOE-4/4 from early stages of the disease; (xii) brain haemodynamics, as reflected by reduced brain blood flow velocity and increased pulsatility and resistance indices, is significantly worse in APOE-4/4 (and in APOE4 carriers, in general, as compared with APOE-3 carriers); (xiii) lymphocyte apoptosis is markedly enhanced in APOE-4 carriers; (xiv) cognitive deterioration is faster in APOE-4/4 patients than in carriers of any other APOE genotype; (xv) occasionally, in approximately 3-8% of the AD cases, the presence of some dementia-

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Pharmacogenomics of Alzheimer’s disease

Table 4 Potential therapeutic strategies in Alzheimer disease and dementia. Pathogenic Mechanism Genomic disarray Monogenic-related Polygenic-related β-amyloid deposition

Tau pathology

Apoptosis Neurotransmission deficits Acetylcholine Enzymes Muscarinic receptors

Nicotinic receptors GABA Glutamate NMDA AMPA Dopamine Noradrenaline Histamine Serotonin

Therapeutic Strategy

Pathogenic Mechanism Neuronal loss

MOP inhibitors GSK-3 inhibitors JNK inhibitors P38 inhibitors

Neuroinflammation

Cyclooxygenase-1 inhibitors Cyclooxygenase-2 inhibitors Complement activation inhibitors P38 inhibitors Caspase-1 inhibitors eNOS inhibitors PPARα agonists PPARγ agonists Novel NSAIDs Cytokine inhibitors

Oxidative stress

Antioxidants Caspase inhibitors Antioxidating enzyme enhancers

Excitotoxic reactions

NMDA antagonists Ampakines Modulators of glutamate transporters

Calcium dysmetabolism

Calcium channel blockers

Neuronal hypometabolism

PPARγ agonists GSK-3 inhibitors

Lipid dysfunction

HMG-CoA reductase inhibitors PPARγ agonists Novel biomarine lipoproteins

Cerebrovascular dysfunction

Vasoactive substances NO inhibitors HIF inhibitors Dandrolene-related agents Novel lipoproteins Liver X receptor agonists

Neuronal dysfunction associated with nutritional deficiency

Nutrigenomics Nutraceuticals Brain metabolic enhancers

Other pathogenic mechanisms

Estrogen agonists MAO-B inhibitors Somatostatin stimulants Insulin sensitizers Immunostimulants MAP kinase inhibitors Prolyl-endopeptidase inhibitors Anti-neurodegenerative agents Immunotrophins Endogenous nucleotides Neurotrophic Antibiotherapy Benzodiazepine partial inverse agonist Others

Gene therapy RNAi β-secretase inhibitors γ-secretase inhibitors α-secretase activators Aβ-fibrillization and aggregation inhibitors Amyloid Immunotherapy Copper chelating agents Solubilizers of Aβ aggregates APP Production inhibitors Aβ selective regulators Phosphatase activators GSK-3 inhibitors Cdk5 inhibitors P38 inhibitors JNK inhibitors Caspase inhibitors Neurotrophic agents

Acetylcholine-release stimulant Acetylcholine reuptake inhibitor Cholinesterase inhibitors Choline-acetyl-transferase stimulant Muscarinic agonists Muscarinic antagonists Nicotinic agonists GABA modulators Inverse GABA-receptor agonist Glutamate agonists NMDA antagonists Ampakines Dopamine reuptake inhibitors Adrenoreceptor modulators Histamine H3 antagonists 5HT3 receptor agonist 5HT1A receptor agonist 5HT6 antagonist Serotonin stimulant

Neurotrophic deficit

Neurotrophic agents NGF agonists Growth factors Synthetic neuropeptides

Neuronal loss

Neuronal stem cells Growth factors Neurite outgrowth activators Synaptogenesis activators Nogo inhibitors

Therapeutic Strategy

Adapted from Cacabelos42

diciembre 2009

Personalized Medicine of Dementia

*1/*1 *1xN/*1 *1/*2 *1/*3 *1/*4 *1/*5 *1/*6 *1xN/*2 *1xN/*4 *1xN/*5 *3/*3 *3/*4 *3/*6 *4/*4 *4/*5 *4/*6 *5/*5 *5/*6

General Population Alzheimer's Disease

CYP2D6 *1/*1 *1xN/*1 *1/*2 *1/*3 *1/*4 *1/*5 *1/*6 *1xN/*2 *1xN/*4 *1xN/*5 *3/*3 *3/*4 *3/*6 *4/*4 *4/*5 *4/*6 *5/*5 *5/*6

N 947 100 1 33 373 59 19 1 27 1 1 6 1 42 12 5 3 6 1637

% AD 57,84972511 6,108735492 0,061087355 2,015882712 22,78558338 3,60415394 1,160659743 0,061087355 1,649358583 0,061087355 0,061087355 0,36652413 0,061087355 2,565668907 0,733048259 0,305436775 0,183262065 0,36652413 100

358 39 0 14 153 19 13 0 13 1 1 1 0 24 4 1 2 1 644

CYP2D6 Genotypes General Population vs Alzheimer’s disease

% 55,5900621 6,05590062 0 2,17391304 23,757764 2,95031056 2,01863354 0 2,01863354 0,1552795 0,1552795 0,1552795 0 3,72670807 0,62111801 0,1552795 0,31055901 0,1552795 100

Frequency (%)

60 50 40

CYP2D6 Phenotypes Metabolizer N EM IM PM UM

Metabolizer AD EM IM PM UM

372 200 34 38 644

% 57,7639752 31,0559006 5,27950311 5,90062112 100

Extensive Metabolizer Intermediate Metabolizer Poor Metabolizer Ultra-Rapid Metabolizer

20 10 0

% 973 59,5107034 487 29,7859327 73 4,4648318 102 6,23853211 1635 100

General Population

Alzheimer's Disease

F(%) 30

Fig.6 5 Distribution and frequency of CYP2D6 genotypes and phenotypes in Alzheimer’s disease and in the general population. Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos42)

related metabolic dysfunctions (e.g. iron, folic acid, vitamin B12 deficiencies) accumulate more in APOE-4 carriers than in APOE-3 carriers; (xvi) some behavioral disturbances (bizarre behaviors, psychotic symptoms), alterations in circadian rhythm patterns (e.g., sleep disorders), and mood disorders (anxiety, depression) are slightly more frequent in APOE-4 carriers; (xvii) aortic and systemic atherosclerosis is also more frequent in APOE-4 carriers; (xviii) liver metabolism and transaminase activity also differ in APOE-4/4 with respect to other genotypes; (xix) blood pressure (hypertension) and other cardiovascular risk factors also accumulate in APOE-4; and (xx) APOE-4/4 are the poorest responders to conventional drugs. These 20 major phenotypic features clearly illustrate the biological disadvantage of APOE-4 homozygotes and the potential consequences that these patients may experience when they receive pharmacological treatment6-16,28,42,47-54.

1*/1* 1*xN/1* 1*/2* 1*/3* 1*/4* 1*/5* 1*/6* 1*xN/2* 1*xN/4* 1*xN/5* 3*/3* 3*/4* 3*/6* 4*/4* 4*/5* 4*/6* 5*/5* 5*/6*

GP-F GP-M AD-F AD-M

CYP2D6 Genotypes: Gender-Related Differences General Population vs Alzheimer’s disease

*1/*1 *1xN/*1 *1/*2 *1/*3 *1/*4 *1/*5 *1/*6 *1xN/*2 *1xN/*4 *1xN/*5 *3/*3 *3/*4 *3/*6 *4/*4 *4/*5 *4/*6 *5/*5 *5/*6

Females 488 50 1 18 196 32 10 0 12 1 1 3 1 21 4 1 3 3 845

% Males 57,75147929 5,917159763 0,118343195 2,130177515 23,19526627 3,786982249 1,183431953 0 1,420118343 0,118343195 0,118343195 0,355029586 0,118343195 2,485207101 0,473372781 0,118343195 0,355029586 0,355029586 100

459 50 0 15 177 27 9 1 15 0 0 3 0 21 8 4 3 0 792

% AD-F 57,9545455 6,31313131 0 1,89393939 22,3484848 3,40909091 1,13636364 0,12626263 1,89393939 0 0 0,37878788 0 2,65151515 1,01010101 0,50505051 0,37878788 0 100

208 20 0 9 87 11 6 0 8 1 1 1 0 12 2 0 2 0 368

% AD-M 56,5217391 5,43478261 0 2,44565217 23,6413043 2,98913043 1,63043478 0 2,17391304 0,27173913 0,27173913 0,27173913 0 3,26086957 0,54347826 0 0,54347826 0 100

150 19 0 5 66 8 7 0 5 0 0 0 0 12 2 1 0 1 276

% 54,3478261 6,88405797 0 1,8115942 23,9130435 2,89855072 2,53623188 0 1,8115942 0 0 0 0 4,34782609 0,72463768 0,36231884 0 0,36231884 100

F(%) 0

Fig.7 5 Sex-Related differences in the distribution and frequency of CYP2D6 genotypes in patients with Alzheimer’s disease and in the general population. Source: R. Cacabelos. EuroEspes Biomedical Research Center Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos42)

Therapeutic Strategies

Modern therapeutic strategies in AD are addressed to interfering with the main pathogenic mechanisms potentially involved in AD (Table 4). Major pathogenic events (drug targets) and their respective therapeutic alternatives include the following: genetic defects, β-amyloid deposition, tau-related pathology, apoptosis, neurotransmitter deficits, neurotrophic deficits, neuronal loss, neuroinflammation, oxidative stress, calcium dysmetabolism, neuronal hypometabolism, lipid metabolism dysfunction, cerebrovascular dysfunction, neuronal dysfunction associated with nutritional and/or metabolic deficits, and a miscellany of pathogenic mechanisms potentially manageable with diverse classes of chemicals or biopharmaceuticals6-16,28,42,47,52(Table 4). Since the early 1980s, the neuropharmacology of AD was dominated by the acetylcholinesterase inhibitors, represented by tacrine, donepezil, rivastigmine, and galantamine2,3,55. Memantine, a partial NMDA antagonist, was introduced in the 2000s for the treatment of severe dementia56; and the first clinical trials with immunotherapy, to reduce amyloid burden in senile plaques, were withdrawn due to severe ADRs57. During the past few years no relevant drug candidates have been postulated for the treatment of AD, despite the initial promises of β- and γ-secretase inhibitors7,10,42 (Table 4).

Pharmacogenomics

Pharmacogenetics/Pharmacogenomics relates to the application of genomic technologies, such as genotyping, gene sequencing, gene expression, genetic epidemiology, transcriptomics, proteomics, metabolomics and bioinformatics, to drugs in clinical development and on the market, applying the large-scale systematic approaches of genomics to speed up the discovery of drug response markers, whether they act at the level of drug target, drug metabolism, or disease pathways8-11,42,58.

pathways leading to disease phenotype expression).

The potential implications of pharmacogenomics in clinical trials and molecular therapeutics is that a particular disease could be treated according to genomic and biological markers, selecting medications and diseases which are optimised for individual patients or clusters of patients with a similar genomic profile. For many medications, interindividual differences are mainly due to SNPs in genes encoding drug metabolising enzymes, drug transporters, and/or drug targets (e.g., genome-related defective enzymes, receptors and proteins, which alter metabolic

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Pharmacogenomics of Alzheimer’s disease

The application of these procedures to CNS disorders is an extremely difficult task, since most neuropsychiatric diseases are complex disorders in which many different genes might be involved4. In addition, it is very unlikely that a single drug be able to reverse the multifactorial mechanisms associated with neuronal dysfunction in most CNS processes with a complex phenotype affecting mood, personality, behaviour, cognition, and functioning. This heterogeneous Table 5

Antidepressants metabolized via enzymes of the CYP gene family and other gene-related enzymes. Drugs

Pharmacological Category

Major Substrate

Minor Substrate

Inhibitors

Other Genes

Amitriptyline

Tricyclic antidepressant Tertiary amine Benzodiazepine

CYP2D6

CYP1A2,CYP2B6, CYP2C8/9,CYP2C19, CYP3A4

CYP1A2,CYP2C8/9, CYP2C19,CYP2D6 CYP2E1

ABCB1,ADRA1,GNB3,GNAS1, KCNE2,SCN5A,TNF-A

Amoxapine

Tricyclic antidepressant Secondary amine

CYP2D6

ADRA1, GnB3 GNAS1

Bupropion

Antidepressant Dopamine-reuptake inhibitor

CYP2B6

CYP1A2,CYP2A6, CYP2C8/9,CYP2D6, CYP2E1,CYP3A4

CYP2D6

Citalopram

Antidepressant Selective serotonin reuptake inhibitor

CYP2C19 CYP3A4

CYP2D6

CYP1A2,CYP2B6, CYP2C19,CYP2D6

GNB3,GNAS1,HTR2A,MAOA, SLC6A4

Clomipramine

Tricyclic Antidepressant Tertiary amine

CYP1A2 CYP2C19 CYP2D6

CYP3A4

CYP2D6

GNB3 GNAS1

Desipramine

Tricyclic Antidepressant Secondary amine

CYP2D6

CYP1A2

CYP2A6,CYP2B6, CYP2D6,CYP2E1, CYP3A4

Doxepin

Tricyclic Antidepressant Tertiary amine

CYP1A2 CYP2D6 CYP3A4

ABCB1 GNB3 GNAS1

Duloxetin

Antidepressant Serotonin/Norepinephrine Reuptake Inhibitor

CYP1A2 CYP2D6

CYP2D6

Escitalopram

Antidepressant Selective Serotonin Reuptake Inhibitor

CYP2D6 CYP3A4

CYP2D6

GNB3,GNAS1,,HTR2A,SLC6A4

Fluoxetine

Antidepressant Selective Srotonin Reuptake Inhibitor

CYP2C8/9 CYP2D6

CYP1A2,CYP2B2, CYP2C19,CYP2E1 CYP3A4

CYP1A2,CYP2B2, CYP2C8/9,CYP2C19, CYP2D6,CYP3A4

GNB3,GNAS1,HTR2A, SLC6A4,MAOA

Fluvoxamine

Antidepressant Selective Serotonin Reuptake Inhibitor

CYP1A2 CYP2D6

CYP1A2,CYP2B6 CYP2C8/9,CYP2C19 CYP2D6,CYP3A4

Imipramine

Tricyclic Antidepressant Tertiary Amine

CYP2C19 CYP2D6

CYP1A2,CYP2B6 CYP3A4

CYP1A2,CYP2C19 CYP2D6,CYP2E1

ABCB1,ADRA1,GNB3,GNAS1 KCNE2,SCN5A

Maprotiline

Tetracyclic Antidepressant

CYP2D6

ABCB1

Mirtazapine

Antidepressant Alpha-2 antagonist

CYP1A2 CYP2D6 CYP3A4

CYP2C8/9

CYP1A2 CYP3A4

ADRA1 GNB3 GNAS1

Continue 6

diciembre 2009

Personalized Medicine of Dementia Continued, Table 5 Drugs

Pharmacological Category

Major Substrate

Minor Substrate

Other Genes

Inhibitors

Moclobemide

Antidepressant Reversible MAO inhibitor

CYP2C19 CYP2D6

CYP1A2,CYP2C19 CYP2D6

MAOA

Nefazodone

Antidepressant Serotonin Reuptake Inhibitor/Antagonist

CYP2C8/9 CYP3A4

CYP1A2,CYP2B6 CYP2D6,CYP3A4

ABCB1,ADRA1,GNB3,GNAS1

Nortriptyline

Tricyclic Antidepressant Secondary Amine

CYP2D6

CYP1A2,CYP2C19 CYP3A4

CYP2D6 CYP2E1

ABCB1,ADRA1,GNB3 GNAS1

Paroxetine

Antidepressant Selective Serotonin Reuptake Inhbitor

CYP2D6

CYP1A2,CYP2B6 CYP2C8/9,CYP2C19 CYP2D6,CYP3A4

DRD2,DRD4,GNB3,GNAS1 HTR2A,MAOA,SLC6A4 TNF-A,TPH2

Protriptyline

Tricyclic Antidepressant, Secondary Amine

CYP2D6

Sertraline

Antidepressant Selective Serotonin Reuptake Inhibitor

CYP2C19 CYP2D6

CYP2B6,CYP2C8/9 CYP3A4

CYP1A2,CYP2B6 CYP2C8/9,CYP2C19 CYP2D6,CYP3A4

Trazodone

Antidepressant Serotonin Reuptake Inhibitor/Antagonist

CYP3A4

CYP2D6

ADRA1,GNB3,GNAS1

Trimipramine

Tricyclic Antidepressant Tertiary Amine

CYP2C19 CYP2D6 CYP3A4

ABCB1 ADRA1 GNB3 GNAS1

Venlafaxine

Antidepressant Norepinephrine/Serotonin Reuptake Inhibitor

CYP2D6 CYP3A4

CYP2C8/9 CYP2C19

CYP2B6,CYP2D6 CYP3A4

Symbols: ABCB1: ATP-Binding Cassette, Subfamily B, Member 1;ACHE: Acetylcholinesterase; ADRA1: Alpha-1-Adrenergic Receptor; ADRB1: Beta-1-Adrenergic Receptor; ADRB3: Beta-3-Adrenergic Receptor; APOE: Apolipoprotein E; CHRNA2: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 2; CHRNA3: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 3; CHRNA4: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 4; CHRNA5: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 5; CHRNA9: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 9; CHRNA10: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 10; CHRNB2: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 2; CHRNA3: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 3; CHRNA4: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 4; CHRNA7: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 7; COMT: Catechol-O-Methyl Transferase; CYP: Cytochrome P450 Family Genes; DRD2: Dopamine Receptor D2; DRD3: Dopamine Receptor D3; DRD4: Dopamine Receptor D4; GABAR: Gamma-Aminobutyric Acid Receptors; G6PD: Glucose-6-Phosphate Dehydrogenase; GNB3: G-Protein Beta-3 Subunit; GNAS1: Gs Protein Alpha-Subunit; GPIIIA: Glycoprotein IIIa Receptor; HLA-A1: Minor Histocompatibility Antigen HA-1; HRH1: Histamine Receptor H1; HRH2: Histamine Receptor H2; HTR1A: Serotonin Receptor 1A; HTR1B: Serotonin Receptor 1B; HTR1D: Serotonin Receptor 1D; HTR2A: Serotonin Receptor 2A; HTR2C: Serotonin Receptor 2C; HTR6: Serotonin Receptor 6; INPP1: Inositol Polyphosphate 1-Phosphatase; KCNE2: Cardiac Potassium Ion Channel; LTC4S: Leukotriene C4 Synthase; MAOA: Monoamine Oxidase A; MAOB: Monoamine Oxidase B; RGS2: Regulator of G-Protein Signaling 2; SCN5A: Cardiac Sodium Channel; SLC6A2: Solute Carrier Family 6 (Neurotransmitter Transporter, Noradrenaline), Member 2; SLC6A3: Solute Carrier Family 6 (Neurotransmitter Transporter, Dopamine), Member 3; SLC6A4: Solute Carrier Family 6 (Neurotransmitter Transporter, Serotonin), Member 4; TNF-A: Tumor Necrosis Factor-Alpha; TPH2: Tryptophan Hydroxylase. Adapted from Cacabelos42

clinical picture usually requires the utilization of different drugs administered simultaneously. This is particularly important in the elderly population. In fact, the average number of drugs taken by patients with dementia ranges from 6 to over 10 per day depending upon their physical and mental conditions. Nursing home residents receive, on average, 7-8 medications each month, and over 30% of residents have monthly drug regimes of 9 or more medications, including (in descending order) analgesics, antipyretics, gastrointestinal agents, electrolytic and caloric preparations, CNS agents, anti-infective agents, and cardiovascular agents59. In population-based studies over 35% of patients older than 85 years are moderate or chronic antidepressant users60. Polypharmacy, drug-drug interactions, adverse reactions, and non-compliance are substantial therapeutic problems

in the pharmacological management of elderly patients61, adding further complications and costs to the patients and their caregivers. More than 25% of elderly individuals receive at least one of more than 30 potentially inappropriate medications in 10 health maintenance organizations (HMOs) of the USA62. Although drug effect is a complex phenotype which depends on many factors, it is estimated that genetics accounts for 20% to 95% of variability in drug disposition and pharmacodynamics63. Under these circumstances, therapeutics optimisation is a major goal in neuropsychiatric disorders and in the elderly population, and novel pharmacogenetic and pharmacogenomic procedures may help in this endeavour4-10,28,42,47-52,64. The pharmacogenomic outcome depends upon many different determinant factors

including (i) genomic profile, (ii) disease phenotype, (iii) concomitant pathology, (iv) genotype-phenotype correlations, (v) nutritional conditions, (vi) age and gender, (vii) pharmacological profile of the drugs, (viii) drug-drug interactions, (ix) gene expression profile, (x) transcriptomic cascade, (xi) proteomic profile, and (xii) metabolomic networking (Fig.1). The dissection and further integration of all these factors is of paramount importance for the assessment of the pharmacogenomic outcome in terms of safety and efficacy (Fig.4). The vast majority of drugs in current use and many psychotropics (Tables 5-7; Fig.5) are metabolised by enzymes known to be genetically variable, including: (a) esterases: butyrylcholinesterase, paraoxonase/arylesterase; (b) transferases: Nacetyltransferase, sulfotransferase, thiol

Table 6 Neuroleptics metabolized via enzymes of the CYP gene family and other gene-related enzymes Drugs

Pharmacological Category

Major Substrate

Minor Substrate

Inhibitors

Other Genes

Aripiprazole

Atypical antipsychotic

CYP2D6,CYP3A4

ADRA1,DRD2,DRD3, HTR1A,HTR2A,HTR2C

Chlorpromazine

Phenothiazine Antipsychotic

CYP2D6

CYP1A2,CYP3A4

CYP2D6,CYP2E1

ABCB1,ADRA1,DRD2, KCNE2,SCN5A

Clozapine

Atypical antipsychotic

CYP1A2

CYP2A6,CYP2C8/9, CYP2C19,CYP2D6, CYP3A4

CYP1A2,CYP2C8/9 CYP2C19,CYP2D6 CYP2E1,CYP3A4

ADRA1,ADRB3,DRD2, DRD3,DRD4,GNB3, GNAS1,RGS2, HLAA1,HRH1,HRH2, HTR1A,HTR2A, HTR2C,HTR6,SLC6A2, SLC6A4, TNF-A

Droperidol

Atypical Antipsychotic

ADRA1,DRD2,KCNE2, SCN5A

Haloperidol

Typical Antipsychotic

CYP2D6,CYP3A4

CYP1A2

CYP2D6,CYP3A4

ABCB1,ADR1A,DRD2, DRD3,DRD4,KCNE2, SCN5A

Loxapine

Typical Antipsychotic

ADR1A,DRD2,KCNE2, SCN5A

Mesoridazine

Typical Antipsychotic

ADR1A,DRD2,KCNE2, SCN5A

Molindone

Typical Antipsychotic

ADRA1,DRD2

Olanzapine

Atypical Antipsychotic

CYP1A2,CYP2D6

CYP1A2,CYP2C8/9 CYP2C19,CYP2D6 CYP3A4

ADRA1,DRD2,DRD3, HRH1,HRH2,HTR2A HTR2C,HTR6,RGS2, TNF-A

Perphenazine

Typical Antipsychotic Phenothiazine

CYP2D6

CYP1A2,CYP2C8/9, CYP2C19,CYP3A4

CYP1A2 CYP2D6

ADRA1 DRD3

Pimozide

Typical Antipsychotic

CYP1A2 CYP3A4

CYP2C19,CYP2D6 CYP2E1,CYP3A4

ADRA1,DRD2,KCNE2, SCN5A

Pipotiazine

Typical Antipsychotic

CYP2D6,CYP3A4

Prochlorperazine

Typical Antipsychotic

Quetiapine

Atypical Antipsychotic

CYP3A4

CYP2D6

ADRA1,DRD2,KCNE2, SCN5A

Risperidone

Atypical Antipsychotic

CYP2D6

CYP2A4

CYP2D6 CYP3A4

ABCB1,ADRA1,DRD2,DRD3,DRD4,H TR1A,HTR2A HTR2C,KCNE2,RGS2,SLC6A2SCN5A

Thioridazine

Typical Antipsychotic Phenothiazine

CYP2D6

CYP2C19

CYP1A2,CYP2C8/9 CYP2D6,CYP2E1

ADRA1,DRD2,KCNE2 SCN5A

Thiothixene

Typical Antipsychotic

CYP1A2

CYP2D6

ADRA1,DRD2,KCNE2, SCN5A

Trifluoperazine

Typical Antipsychotic Phenothiazine

CYP1A2

ADRA1 DRD2

Ziprasidone

Atypical Antipsychotic

CYP1A2 CYP3A4

CYP2D6 CYP3A4

ADRA1,DRD2,DRD3, HTR1A,HTR2A,HTR2C KCNE2,SCN5A

Zonisamide

Anticonvulsant

CYP3A4

Zuclopenthixol

Typical Antipsychotic

CYP2D6

ADRA1,DRD2,KCNE2, SCN5A

Ciencia

Pharmacogenomics of Alzheimer’s disease

ABCB1,ADRA1,DRD2

Symbols: as in table 5 Adapted from Cacabelos42

diciembre 2009

Personalized Medicine of Dementia Distribution and Frequency of CYP2C9 Genotypes/Phenotypes

*1/*1-EM

Frequency (%)

*1/*2-IM D i stri b u ti o n a n d F re q u e n c y o f C Y P 2 C 9 G e n o ty p e N % *1 / *1 -E M 957 60,8778626 *1 / *2 -I M 377 23,9821883 *1 / *3 -I M 160 10,178117 *2 / *2 -P M 40 2,54452926 *2 / *3 -P M 34 2,16284987 *3 / *3 -P M 4 0,25445293 1572 100

*1/*3-IM *2/*2-PM *2/*3-PM *3/*3-PM 0

Fig.85 Distribution and frequency of CYP2C9 genotypes/ phenotypes in the Spanish population. Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, CoruĂąa, Spain.

methyltransferase, thiopurine methyltransferase, catechol-O-methyltransferase, glutathione-S-transferases, UDP-glucuronosyltransferases, glucosyltransferase, histamine methyltransferase; (c) Reductases: NADPH:quinine oxidoreductase, glucose-6-phosphate dehydrogenase; (d) oxidases: alcohol dehydrogenase, aldehydehydrogenase, monoamine oxidase B, catalase, superoxide dismutase, trimethylamine N-oxidase, dihydropyrimidine dehydrogenase; and (e) cytochrome P450 enzymes, such as CYP1A1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A5 and many others4,9,10,42. Polymorphic variants in these genes can induce alterations in drug metabolism modifying the efficacy and safety of the prescribed drugs. Drug metabolism includes phase I reactions (i.e. oxidation, reduction, hydrolysis) and phase II conjugation reactions (i.e., acetylation, glucuronidation, sulphation, methylation). The principal enzymes with polymorphic variants involved in phase I reactions are the following: CYP3A4/5/7, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1B1, CYP1A1/2, epoxide hydrolase, esterases, NQO1 (NADPH-quinone oxidoreductase), DPD (dihydropyrimidine dehydrogenase), ADH (alcohol dehydrogenase), and ALDH (aldehyde dehydrogenase). Major enzymes involved in phase II reactions include the

following: UGTs (uridine 5â&#x20AC;&#x2122;-triphosphate glucuronosyl transferases), TPMT (thiopurine methyltransferase), COMT (catecholO-methyltransferase), HMT (histamine methyl-transferase), STs (sulfotransferases), GST-A (glutathion S-transferase A), GST-P, GST-T, GST-M, NAT2 (N-acetyl transferase), NAT1, and others4,9,10,42.

Pharmacogenetics of Psychotropic Drugs The typical paradigm for the pharmacogenetics of phase I drug metabolism is represented by the cytochrome P-450 enzymes, a superfamily of microsomal drug-metabolising enzymes. P450 enzymes comprise a superfamily of heme-thiolate proteins widely distributed in bacteria, fungi, plants and animals. The P450 enzymes are encoded in genes of the CYP superfamily and act as terminal oxidases in multicomponent electron transfer chains which are called P450-containing monooxigenase systems. Some of the enzymatic products of the CYP gene superfamily can share substrates, inhibitors and inducers whereas others are quite specific for their substrates and interacting drugs. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. About 60-80% of the psychotropic agents currently used for the treatment of neuropsychiatric disorders are metabolised via enzymes of the CYP family, especially CYP1A2, CYP2B6, CYP2C8/9, CYP2C19, CYP2D6 and CYP3A4 (Tables 5-7; Fig.5). CYP3A4 metabolises more drug molecules than all

other isoforms together. Most of these polymorphisms exhibit geographic and ethnic differences65-68. These differences influence drug metabolism in different ethnic groups in which drug dosage should be adjusted according to their enzymatic capacity, differentiating normal or extensive metabolisers (EMs), intermediate metabolisers (IMs), poor metabolisers (PMs) and ultrarapid metabolisers (UMs). Most drugs act as substrates, inhibitors or inducers of CYP enzymes. Enzyme induction enables some xenobiotics to accelerate their own biotransformation (autoinduction) or the biotransformation and elimination of other drugs. A number of P450 enzymes in the human liver are inducible. Induction of the majority of P450 enzymes occurs by an increase in the rate of gene transcription and involves ligandactivated transcription factors, aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor (PXR)69,70. In general, binding of the appropriate ligand to the receptor initiates the induction process that cascades through a dimerisation of the receptors, their translocation to the nucleus and binding to specific regions in the promoters of CYPs70. CYPs are also expressed in the CNS, and a complete characterization of constitutive and induced CYPs in the brain is essential for understanding the role of these enzymes in neurobiological functions and in age-related and xenobiotic-induced neurotoxicity71. CYP2D6 mRNA expression is detected in all regions of the human brain where it may be involved in the metabolism of amines and steroids and in the regulation of diverse CNS activities72. There are substantial differences between individuals in the effects of psychotropic drugs in the treatment of neuropsychiatric disorders. Pharmacogenetic studies of psychotropic drug response have focused on determining the relationship between variation in specific candidate genes and the positive and adverse effects of drug treatment73-75. Approximately, 18% of neuroleptics are major substrates of CYP1A2 enzymes, 40% of CYP2D6, and 23% of CYP3A4 (Table 6); 24% of antidepressants are major substrates of CYP1A2 enzymes, 5% of CYP2B6, 38% of CYP2C19, 85% of CYP2D6, and 38% of CYP3A4 (Table 5); 7% of benzodiazepines are major substrates of CYP2C19 enzymes, 20% of CYP2D6, and 95% of CYP3A4 (Table 7; Fig.5) 4,42. About 80% of patients with resistant depression, 60% of patients non-responsive to neuro-

Table 7 leptics, and 50-70% of patients with paradoxical responses to benzodiazepines are carriers of mutant variants of the CYP2D6, CYP2C9 and CYP3A4 genes, falling within the categories of poor or ultra-rapid metabolisers42.

Benzodiazepines metabolized via enzymes of the CYP gene family and other generelated enzymes. Drugs Alprazolam

CYPs in Dementia

In dementia, as in any other CNS disorder, CYP genomics is a very important issue since in practice over 90% of patients with dementia are daily consumers of psychotropics. Furthermore, some acetylcholinesterase inhibitors (the most prescribed anti-dementia drugs worldwide) are metabolised via CYP enzymes4,9,10,28,42,49,50. CYP2D6, CYP2C19, CYP2C9 and CYP3A4/5 deserve special consideration. The CYP2D6 enzyme, encoded by a gene that maps on 22q13.1-13.2, catalyses the oxidative metabolism of over 100 clinically important and commonly prescribed drugs such as cholinesterase inhibitors, antidepressants, neuroleptics, opioids, some β-blockers, class I antiarrhythmics, analgesics and many other drug categories, acting as substrates, inhibitors or inducers with which many other drugs may potentially interact, this leading to the outcome of ADRs. The CYP2D6 locus is highly polymorphic, with over 100 different CYP2D6 alleles identified in the general population showing deficient (PM), normal (EM), intermediate (IM) or increased enzymatic activity (UM)76,77. Most individuals (>80%) are EMs; however, remarkable interethnic differences exist in the frequency of the PM and UM phenotypes among different societies all over the world9,68,78. On the average, approximately 6.28% of the world population belongs to the PM category. Europeans (7.86%), Polynesians (7.27%), and Africans (6.73%) exhibit the highest rate of PMs, whereas Orientals (0.94%) show the lowest rate. The frequency of PMs among Middle Eastern populations, Asians, and Americans is in the range of 2-3%. CYP2D6 gene duplications are relatively infrequent among Northern Europeans, but in East Africa the frequency of alleles with duplication of CYP2D6 is as high as 29%79. The most frequent CYP2D6 alleles in the European population are as follows: CYP2D6*1 (wild-type) (normal), CYP2D6*2 (2850C>T) (normal), CYP2D6*3 (2549A>del)

Major Substrate CYP3A4

Minor Substrate

Inhibitors

Other Genes

Bromazepam

CYP3A4

CYP2E1

Chlordiazepoxide

CYP3A4

Clobazam

CYP2D6, CYP3A4

Clonazepam

CYP3A4

Clorazepate

CYP3A4

Diazepam

CYP2C19, CYP3A4

CYP1A2, CYP2B6, CYP2C8/9

CYP2C19,CYP3A4

Estazolam

CYP3A4

Flurazepam

CYP3A4

CYP2E1

Midazolam

CYP3A4

CYP2B6

CYP2C9,CYP3A4

ABCB1

Oxacepam

Pinazepam

CYP3A4

Prazepam

CYP3A4

Quazepam

CYP3A4

CYP2B6,CYP2C8/9, CYP2D6,CYP3A4

CYP2C8/9

Temazepam

Triazolam

CYP3A4

Ciencia

Pharmacogenomics of Alzheimer’s disease

Symbols: as in table 5. Adapted from Cacabelos42

(inactive), CYP2D6*4 (1846G>A)(inactive), CYP2D6*5 (gene deletion) (inactive), CYP2D6*6 (1707T>del) (inactive), CYP2D6*7 (2935A>C)(inactive), CYP2D6*8 (1758G>T)(inactive), CYP2D6*9 (2613-2615 delAGA)(partially active), CYP2D6*10 (100C>T)(partially active), CYP2D6*11 (883G>C)(inactive), CYP2D6*12 (124G>A)(inactive), CYP2D6*17 (1023C>T)(partially active), and CYP2D6 gene duplications (with increased or decreased enzymatic activity depending upon the alleles involved)4,9,10,77. In the Spanish population, where the mixture of ancestral cultures has occurred for centuries, the distribution of the CYP2D6 genotypes differentiates 4 major categories of CYP2D6-related metabolyzer types: (i) Extensive Metabolisers (EM)(*1/*1, *1/*2,*1/*10); (ii) Intermediate Metabolisers (IM)(*1/*3, *1/*4, *1/*5, *1/*6, *1/*7, *10/*10, *4/*10, *6/*10, *7/*10); (iii) Poor Metabolisers (PM)(*4/*4, *5/*5); and (iv) Ultra-rapid Metabolisers (UM)(*1xN/*1, *1xN/*4, Dupl). In this sample we have found 51.61% EMs, 32.26%

IMs, 9.03% PMs, and 7.10% UMs10,18,28,4750,52 . In a more recent study with 1637 subjects and 644 patients with AD (Fig.6) we did not find any significant difference between AD cases and the general population (GP)42 (Fig.6). A variation rate higher than 2% was only found in the EM-*1/*1 genotype which is more frequent in the GP than in AD. The proportion of EMs was 59.51% in GP and 57.76% in AD; IMs were 29% in GP and 31% in AD; PMs were 4.46% in GP and 5.27% in AD; and UMs were 6.23% in GP and 5.9% in AD42 (Fig.6). No major differences between females and males were found in the GP group; however, in AD, EMs are more frequent in females than in males, and PMs are more frequent in males than in females, indicating that males might be at higher risk for developing ADRs42 (Fig.7).

Association of CYP2D6 Variants with Alzheimer’s Disease-Related Genes We have also investigated the association of CYP2D6 genotypes with AD-related genes, such as APP, MAPT, APOE, diciembre 2009

Personalized Medicine of Dementia PSEN1, PSEN2, A2M, ACE, AGT, FOS, and PRNP variants10,42,47-52. Homozygous APOE-2/2 (12.56%) and APOE4/4 (12.50%) accumulate in UMs, and APOE-4/4 cases were also more frequent in PMs (6.66%) than in EMs (3.95%) or IMs (0%). PS1-1/1 genotypes were more frequent in EMs (45%), whereas PS-1/2 genotypes were over-represented in IMs (63.16%) and UMs (60%). The presence of the PS1-2/2 genotype was especially high in PMs (38.46%) and UMs (20%). A mutation in the PS2 gene exon 5 (PS2E5+) was markedly present in UMs (66.67%). About 100% of UMs were A2M-V100I-A/A, and the A2M-V100IG/G genotype was absent in PMs and UMs. The A2M-I/I genotype was absent in UMs, and 100% of UMs were A2M-I/D and ACE-D/D. Homozygous mutations in the FOS gene (B/B) were also only present in UMs. AGT-T235T cases were absent in PMs, and the AGT-M174M genotype appeared in 100% of PMs. Likewise, the PRNP-M129M variant was present in 100% of PMs and UMs. These association studies clearly show that in PMs and UMs there is an accumulation of AD-related polymorphic variants of risk which might be responsible for the defective therapeutic responses currently seen in these AD clusters10,47-52.

CYP2D6-Related Biochemical and Haemodynamic Phenotypes in Alzheimerâ&#x20AC;&#x2122;s Disease It appears that different CYP2D6 variants, expressing EMs, IMs, PMs, and UMs, influence to some extent several biochemical parameters, liver function,

and vascular haemodynamic parameters which might affect drug efficacy and safety. Blood glucose levels are found to be elevated in EMs (*1/*1 vs *4/*10) and in some IMs (*4/*10 vs *1xN/*4), whereas other IMs (*1/*5 vs *4/*4) tend to show lower levels of glucose compared with PMs (*4/*4) or UMs (*1xN/*4). The highest levels of total-cholesterol are detected in the EMs with the CYP2D6*1/*10 genotype (vs *1/*1, *1/*4 and *1xN/*1). The same pattern has been observed with regard to LDL-cholesterol levels, which are significantly higher in the EM*1/*10. In general, both total cholesterol levels and LDL-cholesterol levels are higher in EMs (with a significant difference between *1/*1 and *1/*10), intermediate levels are seen in IMs, and much lower levels in PMs and UMs; and the opposite occurs with HDL-cholesterol levels, which on average appear much lower in EMs than in IMs, PMs, and UMs, with the highest levels detected in *1/*3 and *1xN/*4. The levels of triglycerides are highly variable among different CYP2D6 polymorphisms, with the highest levels present in IMs (*4/*10 vs *4/*5 and *1xN/*1)10,50,52. These data clearly indicate that lipid metabolism can be influenced by CYP2D6 variants or that specific phenotypes determined by multiple lipid-related genomic clusters are necessary to confer the character of EMs and IMs. Another possibility might be that some lipid metabolism genotypes interact with CYP2D6-related enzyme products leading to the definition of the pheno-genotype of PMs and UMs. No significant changes in blood pressure values have been found among CYP2D6 genotypes;

however, important differences became apparent in brain cerebrovascular haemodynamics. In general terms, the best cerebrovascular haemodynamic pattern is observed in EMs and PMs, with higher brain blood flow velocities and lower resistance and pulsatility indices, but differential phenotypic profiles are detectable among CYP2D6 genotypes. For instance, systolic blood flow velocities (Sv) in the left middle cerebral arteries (LMCA) of AD patients are significantly lower in *1/*10 EMs, with high total cholesterol and LDL-cholesterol levels, than in IMs (*4/*10); and diastolic velocities (Dv) also tend to be much lower in *1/*10 and especially in PMs (*4/*4) and UMs (*1xN/*4), whereas the best Dv is measured in *1/*5 IMs. More striking are the results of both the pulsatility index (PI=(Sv-Dv)/Mv) and resistance index (RI=(Sv-Dv)/Sv), which are worse in IMs and PMs than in EMs and UMs. These data taken together seem to indicate that CYP2D6-related AD PMs exhibit a poorer cerebrovascular function which might affect drug penetration into the brain with the consequent therapeutic implications10,47-52.

Influence of CYP2D6 Genotypes on Liver Transaminase Activity In order to elucidate whether or not CYP2D6-related variants may influence transaminase activity, we have studied the association of GOT, GPT, and GGT activity with the most prevalent CYP2D6 genotypes in AD10,48-50. Globally, UMs and PMs tend to show the highest GOT activity and IMs the lowest. Significant differences appear among different IM-related genotypes. The *10/*10 genotype exhibited the lowest GOT activity with marked differences as compared to UMs. GPT activity was significantly higher in PMs (*4/*4) than in EMs (*1/*10) or IMs (*1/*4, *1/*5). The lowest GPT activity was found in EMs and IMs. Striking differences have been found in GGT activity between PMs (*4/*4), which showed the highest levels, and EMs (*1/*1; *1/*10), IMs (*1/*5), or UMs (*1xN/*1)50. Interesting enough, the *10/*10 genotype, with the lowest values of GOT and GPT, exhibited the second highest levels of GGT after *4/*4, probably indicating that CYP2D6-related enzymes differentially regulate drug metabolism and transaminase activity in the liver. These results are also clear in demonstrating the direct effect of CYP2D6 variants on transaminase activity10,49,50.

Table 8 The EuroEspes Protocol for Genomic Medicine of CNS Disorders Procedure

Technology

Parametric Data

Procedure

Technology

Parametric Data

Clinical history

Anamnesis. Pedigree. Physical, neurologic and psychiatric examination

Present conditions Family history Personal history Physical, neurological and psychiatric information

Functional Genomics

Microarray Technology Genotype-Phenotype Correlations Transcriptomics Proteomics Metabolomics

Genotype-associated defects

Laboratory tests

Conventional Test-specific

Blood, urine, cerebrospinal fluid

Pharmacogenetics

Genotyping of genes associated with drug metabolism

Neuropsychological Assessment

Neuropsychological tests Batteries

Mood. Behavior. Cognition. Functioning

Prediction of therapeutic response Drug toxicity ADRs Safety issues

Cardiovascular Evaluation

Electrocardiogram Ecocardiogram Functional tests

Heart function Circulatory function

Pharmacogenomics

Genotyping of genes associated with disease phenotype High Throughput Screening

Drug-induced gene(s) expression and disease phenotype modification Efficacy issues

Imaging

Conventional X-Ray

Chest, neck, other structures or organs

Nutrigenetics

Genotyping of genes associated with nutrients metabolism

Structural Neuroimaging

Computerized Tomography (CT-Scan) Magnetic Resonance Imaging (MRI)

Brain structure

Nutrition-related effects Nutrition benefits Nutrition toxicity Safety issues

Nutrigenomics

Single Photon Emission Computerized Tomography (PECT) Positron Emission Tomography (PET) CT-Brain Perfusion, Brain Digital Topography

Brain Function Cerebrovascular function Brain oxygenation

Genotyping of genes associated with disease induced by nutritional factors High Throughput Screening

Nutrition-related disease analysis Nutrition-induced gene expression and disease phenotype modification Efficacy issues

Data Integration

Bioinformatics

Brain Electrophysiology

EEG, qEEG, EMG, EP

Brain mapping Neuromuscular transmission Evoked Potentials

Data Management Correlation Analysis

Intelligent Assignments

Artificial Intelligence

Cerebrovascular Assessment

SPECT CT-Brain Perfusion Brain Digital Topography Transcranial Doppler Ultrasonography

Brain Perfusion Brain Oxygeneation Cerebrovascular Hemodynamics

Probabilistic Diagnosis Therapeutic Optimization Nutritional Optimization Predictive Analysis Individual Preventive Options Risk Evaluation Genetic Counselling

Structural Genomics

Gene mapping Linkage analysis Association studies DNA Microarrays

Mutations Disease-associated genotypes SNPs

Functional Neuroimaging

CYP2D6-Related Therapeutic Response to a Multifactorial Treatment in Dementia No clinical trials had been performed to elucidate the influence of CYP2D6 variants on the therapeutic outcome in AD in response to cholinesterase inhibitors or other anti-dementia drugs. We have performed the first prospective study in AD patients who received a combination therapy with (a) an endogenous nucleotide and choline donor, CDP-choline (500 mg/day), (b) a nootropic substance, piracetam (1600 mg/ day), (c) a vasoactive compound, 1,6 dimethyl 8β-(5-bromonicotinoyl-oxymethyl)10α-methoxyergoline (nicergoline)(5 mg/day), and (d) a cholinesterase inhibitor, donepezil (5 mg/day), for one year. With this multifactorial therapeutic intervention, EMs improved their cognitive function (MMSE score) from 21.58±9.02

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Pharmacogenomics of Alzheimer’s disease

Adapted from Cacabelos4

at baseline to 23.78±5.81 after 1-year treatment. IMs also improved from 21.40±6.28 to 22.50±5.07 (r=+0.96), whereas PMs and UMs deteriorate from 20.74±6.72 to 18.07±5.52 (r=-0.97), and from 22.65±6.76 to 21.28±7.75 (r=-0.92), respectively. According to these results, PMs and UMs were the worst responders, showing a progressive cognitive decline with no therapeutic effect, and EMs and IMs were the best responders, with a clear improvement in cognition after one year of treatment. Among EMs, AD patients harbouring the *1/*10 genotype responded better than patients with the *1/*1 genotype. The best responders among IMs were the *1/*3, *1/*6 and *1/*5 genotypes, whereas the *1/*4, *10/*10, and *4/*10 genotypes were poor responders. Among PMs and UMs, the poorest responders were carriers of the *4/*4 and *1xN/*1 genotypes,

respectively10,28,42,47-50. In a recent study, Pilotto et al80 have confirmed the influence of CYP2D6 variants (rs1080985) on the efficacy of donepezil in AD. From all these data we can conclude the following: (i) The most frequent CYP2D6 variants in the Southern European population (Iberian peninsula) are the *1/*1 (57.84%), *1/*4 (22.78%), *1xN/*1 (6.10%), *4/*4 (2.56%), and *1/*3 (2.01%) genotypes, accounting for more than 80% of the population; (ii) the frequency of EMs, IMs, PMs, and UMs is about 59.51%, 29,78%, 4.46%, and 6.23%, respectively, in the general population, and 57.76, 31.05%, 5.27%, and 5.90%, respectively in AD cases (Fig.6); (iii) EMs are more prevalent in GP (59.51%) than in AD (57.76%); IMs are more frequent in AD (31.05%) than in GP (29.78%); diciembre 2009

Personalized Medicine of Dementia 30

Number

F(%) 25

CYP2D6 1 *1/*1 2 *1/*1 3 *1/*1 4 *1/*1 5 *1/*1 6 *1/*1 7 *1/*1 8 *1/*1 9 *1/*1 10 *1/*1 11 *1/*1 12 *1/*2 13 *1/*3 14 *1/*3 15 *1/*3 16 *1/*3 17 *1/*3 18 *1/*3 19 *1/*4 20 *1/*4 21 *1/*4 22 *1/*4 23 *1/*4 24 *1/*4 25 *1/*4 26 *1/*4 27 *1/*5 28 *1/*5 29 *1/*5 30 *1/*5 31 *1/*5 32 *1/*5 33 *1/*5 34 *1/*6 35 *1/*6 36 *1/*6 37 *1/*6 38 *1xN/*1 39 *1xN/*1 40 *1xN/*1

CYP2C19 *1/*1 *1/*2 *1/*3 *1/*4 *1/*5 *1/*6 *1/*2 *1/*2 *1/*2 *1/*2 *2/*2 *1/*2 *1/*1 *1/*1 *1/*1 *1/*1 *1/*2 *1/*2 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*2 *1/*2 *1/*2 *1/*1 *1/*1 *1/*1 *1/*2 *1/*2 *1/*2 *2/*2 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1

CYP2C9 *1/*1 *1/*2 *1/*3 *2/*2 *2/*3 *3/*3 *1/*1 *1/*2 *1/*3 *2/*3 *1/*1 *1/*2 *1/*1 *1/*2 *1/*3 *2/*3 *1/*1 *1/*2 *1/*1 *1/*2 *1/*3 *2/*2 *2/*3 *1/*1 *1/*2 *1/*3 *1/*1 *1/*2 *1/*3 *1/*1 *1/*2 *1/*3 *1/*1 *1/*1 *1/*2 *1/*3 *1/*1 *1/*1 *1/*2 *1/*3

N 403 167 68 27 19 3 164 39 13 1 10 1 16 4 2 1 6 3 127 73 35 7 7 77 20 8 18 15 3 15 1 4 1 9 1 2 6 42 16 15

% 25,7015306 10,6505102 4,33673469 1,72193878 1,21173469 0,19132653 10,4591837 2,4872449 0,82908163 0,06377551 0,6377551 0,06377551 1,02040816 0,25510204 0,12755102 0,06377551 0,38265306 0,19132653 8,0994898 4,65561224 2,23214286 0,44642857 0,44642857 4,91071429 1,2755102 0,51020408 1,14795918 0,95663265 0,19132653 0,95663265 0,06377551 0,25510204 0,06377551 0,57397959 0,06377551 0,12755102 0,38265306 2,67857143 1,02040816 0,95663265

Phenotype EM+EM+EM EM+EM+IM EM+EM+IM EM+EM+PM EM+EM+PM EM+EM+PM EM+IM+EM EM+IM+IM EM+IM+IM EM+IM+PM EM+PM+EM EM+IM+IM IM+EM+EM IM+EM+IM IM+EM+IM IM+EM+PM IM+IM+EM IM+IM+IM IM+EM+EM IM+EM+IM IM+EM+IM IM+EM+PM IM+EM+PM IM+IM+EM IM+IM+IM IM+IM+IM IM+EM+EM IM+EM+IM IM+EM+IM IM+IM+EM IM+IM+IM IM+IM+IM IM+PM+EM IM+EM+EM IM+EM+IM IM+EM+IM IM+IM+EM UM+EM+EM UM+EM+IM UM+EM+IM

Number 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

CYP2D6

CYP2C19

CYP2C9

*1xN/*1 *1xN/*1 *1xN/*1 *1xN/*1 *1xN/*1 *1xN/*4 *1xN/*4 *1xN/*4 *1xN/*4 *1xN/*4 *1xN/*4 *3/*3 *3/*4 *3/*4 *3/*4 *3/*4 *3/*6 *4/*4 *4/*4 *4/*4 *4/*4 *4/*4 *4*/4 *4/*4 *4/*4 *4/*4 *4/*5 *4/*5 *4/*5 *4/*5 *4/*5 *4/*5 *4/*6 *4/*6 *4/*6 *4/*6 *5/*5 *5/*5 *5/*5 *5/*6 *5/*6 *5/*6

*1/*1 *1/*1 *1/*2 *1/*2 *1/*2 *1/*1 *1/*1 *1/*1 *1/*1 *1/*2 *1/*2 *1/*1 *1/*1 *1/*1 *1/*1 *1/*2 *1/*2 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*2 *1/*2 *1/*2 *1/*1 *1/*1 *1/*1 *1/*2 *1/*2 *2/*2 *1/*1 *1/*1 *1/*1 *1/*2 *1/*1 *1/*2 *2/*2 *1/*1 *1/*1 *1/*2

*2/*2 *2/*3 *1/*1 *1/*2 *1/*3 *1/*1 *1/*2 *1/*3 *2/*3 *1/*1 *1/*2 *1/*2 *1/*1 *1/*2 *2/*3 *1/*1 *1/*2 *1/*1 *1/*2 *1/*3 *2/*2 *2/*3 *3/*3 *1/*1 *1/*2 *1/*3 *1/*1 *1/*2 *1/*3 *1/*1 *1/*3 *1/*1 *1/*1 *1/*2 *2/*2 *1/*1 *1/*1 *1/*1 *1/*1 *1/*1 *1/*2 *1/*1

% 3 1 14 5 1 9 6 4 1 5 2 1 2 1 2 1 1 13 10 1 2 2 1 2 6 1 5 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1568

0,19132653 0,06377551 0,89285714 0,31887755 0,06377551 0,57397959 0,38265306 0,25510204 0,06377551 0,31887755 0,12755102 0,06377551 0,12755102 0,06377551 0,12755102 0,06377551 0,06377551 0,82908163 0,6377551 0,06377551 0,12755102 0,12755102 0,06377551 0,12755102 0,38265306 0,06377551 0,31887755 0,12755102 0,06377551 0,06377551 0,06377551 0,06377551 0,06377551 0,12755102 0,06377551 0,06377551 0,06377551 0,06377551 0,06377551 0,06377551 0,06377551 0,06377551 100

Phenotype UM+EM+PM UM+EM+PM UM+IM+EM UM+IM+IM UM+IM+IM EM+EM+EM EM+EM+IM EM+EM+IM EM+EM+PM EM+IM+EM EM+IM+IM PM+EM+IM PM+EM+EM PM+EM+IM PM+EM+IM PM+IM+EM PM+IM+EM PM+EM+EM PM+EM+IM PM+EM+IM PM+EM+PM PM+EM+PM PM+EM+PM PM+IM+EM PM+IM+IM PM+IM+IM PM+EM+EM PM+EM+IM PM+EM+IM PM+IM+EM PM+IM+IM PM+PM+IM PM+EM+EM PM+EM+IM PM+EM+PM PM+IM+EM PM+EM+EM PM+IM+EM PM+PM+EM PM+EM+EM PM+EM+IM PM+IM+EM

1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383840414243444546474849505152535455565758596061626364656667686970717273747576777879808182

Fig.9 5 Distribution and frequency of trigenic clusters integrated by combination of CYP2D6, CYP2C19, and CYP2C9 genotypes in patients with Alzheimer’s disease. Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

substances; and (viii) the pharmacogenetic response in AD appears to be dependent upon the networking activity of genes involved in drug metabolism and genes involved in AD pathogenesis10,28,42,47-50.

CYP Clustering in Alzheimer’s disease the frequency of PMs is slightly higher in AD (5.27%%) than in GP (4.46%); and UMs are more frequent in GP (6.23%) than in AD (5.90%)(all these differences are not significant)(Fig.6); (iv) there are differences between females and males in the distribution and frequency of CYP2D6 genotypes which might be of relevance in therapeutic terms and risk of ADRs (Fig.7); (v) there is an accumulation of AD-related genes of risk in PMs and UMs; (vi) PMs and UMs tend to show higher transaminase activities than EMs and IMs; (vii) EMs and IMs are the best responders, and PMs and UMs are the worst responders to a combination therapy with cholinesterase inhibitors, neuroprotectants, and vasoactive

Since more than half of the available drugs are metabolised via different CYP enzymes and other metabolic pathways, it is convenient to understand the networking activity of CYP genes and the genomic profiles of these genes in particular groups of risk. In the case of dementia, 73.71% of AD patients are CYP2C19-EMs, 25.12% IMs, and 1.16% PMs. The distribution and frequency of CYP2C9 genotypes is as follows: *1/*1-EM 60.87%, *1/*2-IM 23.98%, *1/*3-IM 10.17%, *2/*2-PM 2.54%, *2/*3-PM 2.16%, and *3/*3-PM 0.25%, globally representing 60.87% CYP2C9EMs, 34.16% IMs, and 4.97% PMs42(Fig.8). This is especially important because the CYP2C9-Ile359Leu (CYP2C9*3 allele) and CYP2C9-Arg144Cys (CYP2C9*2 allele) vari-

ants are associated with warfarin sensitivity. Clustering together CYP2C9 and VKORC1 variants, we can estimate that approximately 30% of the elderly population is sensitive to warfarin anticoagulants. Concerning CYP3A4/5 polymorphisms, 82.75% of AD cases are EMs (CYP3A5*3/*3), 15.88% are IMs (CYP3A5*1/*3), and 1.37% are UMs (CYP3A5*1/*1)42. The construction of a genetic map integrating the most prevalent CYP2D6+CYP2C19+CYP2C9 polymorphic variants in a trigenic cluster yields 82 different haplotype-like profiles (Fig.9). The most frequent trigenic genotypes in the AD population are *1*1-*1*1-*1*1 (25.70%), *1*1-*1*2-*1*2 (10.66%), *1*1-*1*1*1*1 (10.45%), *1*4-*1*1-*1*1 (8.09%), *1*4-*1*2-*1*1 (4.91%), *1*4-*1*1-*1*2 (4.65%), and *1*1-*1*3-*1*3 (4.33%) (Fig.9). These 82 trigenic genotypes represent 36 different pharmacogenetic phenotypes (Fig.10). According to these trigenic clusters, only 26.51% of the patients show a pure 3EM phenotype, 15.29% are 2EM1IM,

EM+EM+EM EM+EM+IM EM+EM+PM EM+IM+EM EM+IM+IM EM+IM+PM EM+PM+EM EM+PM+IM EM+PM+PM IM+EM+EM IM+EM+IM IM+EM+PM IM+IM+EE IM+IM+IM IM+IM+PM IM+PM+EM IM+PM+IM IM+PM+PM PM+EM+EM PM+EM+IM PM+EM+PM PM+IM+EM PM+IM+IM PM+IM+PM PM+PM+EM PM+PM+IM PM+PM+PM UM+EM+EM UM+EM+IM UM+EM+PM UM+IM+EM UM+IM+IM UM+IM+PM UM+PM+EM UM+PM+IM UM+PM+PM

Frequency (%)

CYP2D6+CYP2C19+CYP2C9 Clustered Phenotypes Alzheimer´s Disease 5

Phenotype N EM+EM+EM EM+EM+IM EM+EM+PM EM+IM+EM EM+IM+IM EM+IM+PM EM+PM+EM EM+PM+IM EM+PM+PM IM+EM+EM IM+EM+IM IM+EM+PM IM+IM+EE IM+IM+IM IM+IM+PM IM+PM+EM IM+PM+IM IM+PM+PM PM+EM+EM PM+EM+IM PM+EM+PM PM+IM+EM PM+IM+IM PM+IM+PM PM+PM+EM PM+PM+IM PM+PM+PM UM+EM+EM UM+EM+IM UM+EM+PM UM+IM+EM UM+IM+IM UM+IM+PM UM+PM+EM UM+PM+IM UM+PM+PM

% 416 240 49 167 56 1 10 0 0 171 135 16 105 32 0 1 5 0 25 18 7 8 8 0 2 0 0 43 30 4 14 6 0 0 0 0 1569

26,513703 15,2963671 3,12300829 10,6437221 3,56915233 0,06373486 0,63734863 0 0 10,8986616 8,6042065 1,01975781 6,69216061 2,03951562 0 0,06373486 0,31867431 0 1,59337157 1,14722753 0,44614404 0,5098789 0,5098789 0 0,12746973 0 0 2,74059911 1,91204589 0,25493945 0,89228808 0,38240918 0 0 0 0 100

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Pharmacogenomics of Alzheimer’s disease

Fig.10 5 2.04% are pure 3IM, 0% are pure 3PM, and 0% are 1UM2PM (the worst possible phenotype) 42 (Fig.10). Taking into consideration the data available, it might be inferred that at least 1015% of the AD population may exhibit an abnormal metabolism of cholinesterase inhibitors and/or other drugs which undergo oxidation via CYP2D6-related enzymes. Approximately 50% of this population cluster would show an ultrarapid metabolism, requiring higher doses of cholinesterase inhibitors in order to reach a therapeutic threshold, whereas the other 50% of the cluster would exhibit a poor metabolism, displaying potential adverse events at low doses. If we take into account that approximately 60-70% of therapeutic outcomes depend upon pharmacogenomic criteria (e.g. pathogenic mechanisms associated with AD-related genes), it can be postulated that pharmacogenetic and pharmacogenomic factors are responsible for 7585% of the therapeutic response (efficacy) in AD patients treated with conventional drugs10,42,47-52. Of particular interest are the potential interactions of cholinesterase inhibitors with other drugs of current use in patients with AD, such as antidepressants,

neuroleptics, antiarrhythmics, analgesics, and antiemetics which are metabolised by the cytochrome P450 CYP2D6 enzyme. Although most studies predict the safety of donepezil and galantamine, as the two principal cholinesterase inhibitors metabolised by CYP2D6-related enzymes few pharmacogenetic studies have been performed so far on an individual basis to personalize the treatment, and most studies reporting safety issues are the result of pooling together pharmacological and clinical information obtained with routine procedures. In certain cases, genetic polymorphism in the expression of CYP2D6 is not expected to affect the pharmacodynamics of some cholinesterase inhibitors because major metabolic pathways are glucuronidation, O-demethylation, N-demethylation, N-oxidation, and epimerization. However, excretion rates are substantially different in EMs and PMs. For instance, in EMs, urinary metabolites resulting from O-demethylation of galantamine represent 33.2% of the dose compared with 5.2% in PMs, which show correspondingly higher urinary excretion of unchanged galantamine and its N-oxide81. Therefore, there are still many unanswered questions regarding the metabolism of cholinesterase inhibitors and

Frequency of trigenic phenotypes resulting from the combination of CYP2D6, CYP2C19 and CYP2C9 genotypes in patients with Alzheimer’s disease. Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

their interaction with other drugs (potentially leading to ADRs) which require pharmacogenetic elucidation. It is also worth mentioning that dose titration (a common practice in AD patients treated with cholinesterase inhibitors; e.g., tacrine, donepezil) is an unwise strategy, since approximately 30-60% of drug failure or lack of therapeutic efficacy (and/or ADR manifestation) is not a matter of drug dosage but a problem of poor metabolising capacity in PMs. Additionally, inappropriate drug use is one of the risk factors for adverse drug reactions (ADRs) in the elderly. The prevalence of use of potentially inappropriate medications in patients older than 65 years of age admitted to a general medical or geriatric ward ranges from 16% to 20%82, and these numbers may double in ambulatory patients. Overall, the most prevalent inappropriate drugs currently prescribed to the elderly are amiodarone, long-acting diciembre 2009

Personalized Medicine of Dementia benzodiazepines and anticholinergic antispasmodics; however, the list of drugs with potential risk also include antidepressant, antihistaminics, NSAIDs, amphetamines, laxatives, clonidine, indomethacin, and several neuroleptics82, most of which are processed via CYP2D6 and CYP3A5 enzymes. Therefore, pre-treatment CYP screening might be of great help in order to rationalize and optimise therapeutics in the elderly, by avoiding medications of risk in PMs and UMs.

APOE Pharmacogenomics APOE-Related Therapeutic Response to a Multifactorial Treatment in patients with Alzheimer’s Disease MMSE Score

APOE-2/3

24 �

�

APOE-3/4

' !19,55 , 22,37 !20,86

APOE-3/3

21,9

Maximum value

23,1

23,36

23,79

Average

22,53

23,39 0,22

0,89

1,2

-0,52

0,37

-0,24

-0,84

Correlation coefficient

& 0,65

APOE-3/4

APOE-4/4

� Total

18,26

! 22,13

21,56

22,78

19,8

22,55

R-squared

,0,27

0,14

0,05

0,72

0,03

23,23

23,21

21,89

22,63

b Coefficient N Females Males Age

-0,2 52 30 22 64.36±11,47

0,05 379 220 159 65,55±10,60

&21,31

-0,59 26 18 8 65,88±9,26

-0,02 595 351 244 66,23±10,58

�

0,2

a Coefficient

-0,12 138 83 55 68,63±9,87

! � �

-0,19

�

Total �

�

APOE-2/3 Minimum value

Standard deviation

APOE-3/3

� ♦

The pharmacogenomics of AD is still in a very primitive stage. In over 100 clinical trials for dementia, APOE has been used as the only gene of reference for the pharmacogenomics of AD10,42,47-52,83,84. Several studies indicate that the presence of the APOE-4 allele differentially affects the quality and extent of drug responsiveness in AD patients treated with cholinergic enhancers (tacrine, donepezil, galantamine, rivastigmine), neuroprotective compounds (nootropics), endogenous nucleotides (CDP-choline), immunotrophins (anapsos), neurotrophic factors (cerebrolysin), rosiglitazone or combination therapies10,42,47-52,83-85; however, controversial results are frequently found due to methodological problems, study design, and patient recruitment in clinical trials.

♦

12M

Treatment Period

Fig.11 5 APOE-Related therapeutic response to a multifactorial treatment in patients with Alzheimer’s disease. Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

Presenilin-1 Pharmacogenomics PSEN1-Related Therapeutic Response to a Multifactorial Treatment in patients with Alzheimer’s Disease MMSE Score

PSEN1-1/1

PSEN1-1/2

PSEN1-1/1 Minimum value Maximum value

Average

� ♦

�

Standard deviation

' & ! ,

& '21,7 23,44 !

22,72

PSEN1-2/2

Total

PSEN1-1/2

PSEN1-2/2

Total

22,35

21,44

22,03

�

24,8

23,75

23,19

23,08

♦ �

23,66 23,23

0,65

0,47

0,09

0,69

! & ,'

1,16

0,54

0,51

0,52

0,48

0,26

0,27

22,56

21,95

22,5

0,19

0,35

0,16

198 108 90

403 232 171

104 47 57

705 387 318

66.09±11,03

65,55±10,98

66,58±12,02

65,85±11,14

Correlation coefficient R-squared

0,009

a Coefficient

22,59

b Coefficient

0,03

N Females Males Age

�

Treatment Period Fig.12 5 PSEN1-Related therapeutic response to a multifactorial treatment in patients with Alzheimer’s disease. Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

Pharmacogenomics of AD-Related Genes

12M

In long-term open clinical trials with a multifactorial treatment, APOE-4/4 carriers are the worst responders (Fig.11)10,42,47-52. With a similar therapeutic protocol, PSEN1-1/1 homozygotes are the worst responders and PSEN1-2/2 carriers are the best responders (Fig.11)42. Significant ACE-related therapeutic responses to multifactorial treatments have also been reported10,42,48,50. Among ACE-I/D variants, ACE-D/D patients were the worst responders (r=-0.58), and ACEI/D carriers were the best responders (r=+0.26), with ACE-I/I showing an intermediate positive response (r=+0.01)10. ACE-related biochemical and haemodynamic phenotypes have been studied in patients with AD9,10,12. ACE-I/I patients tend to be younger than ACE-I/D or ACE-D/D patients at the time of diagnosis and also to show a more severe cognitive deterioration. Serum ApoE, total cholesterol, LDLcholesterol, HDL-cholesterol, nitric oxide, histamine, and ACE levels are higher in ACE-I/I carriers than in patients with the

other genotypes; in contrast, serum triglyceride and VLDL levels are notably lower in ACE-I/I patients compared to patients harbouring the ACE-I/D or ACE-D/D genotypes, whereas Aß levels do not show any clear difference among ACE-related genotypes. Cerebrovascular function tends to be worse in ACE-D/D, with lower brain blood flow velocities and higher pulsatility and resistance indices, than in ACE-I/D (intermediate cerebrovascular haemodynamics) or ACE-I/I (almost normal cerebrovascular function)9,10,12,50. The correlation between lipid levels and brain haemodynamics is very similar in this study to data observed in that of CYP2D6-related metaboliser profiles in which EM patients with moderate cholesterol and lipoprotein levels (as well as relatively high nitric oxide, histamine, ACE, and ApoE levels) tend to show a better cerebrovascular haemodynamic profile than AD patients with lower cholesterol and lipoprotein levels50. This apparently paradoxical correlation appears to indicate that major influences in cerebrovascular homeostasis and haemodynamic brain blood flow are cholesterol, lipoproteins, nitric oxide, ACE, and histamine, among many other factors, in AD, and that peripheral levels of Aß are indifferent in this concern. On the other hand, it seems likely that low triglyceride levels may facilitate cerebrovascular function. It is also worth mentioning that ACE-I/I patients with the highest cholesterol levels are the worst in mental performance. Other interpretation of these data might suggest an association between poor cerebrovascular function with ACE-D/D and ACE-I/D, and an association between alterations in lipid metabolism in ACE-I/I10,50. Both APOE and ACE variants also affect behaviour and the modification of behavioural changes (mood, anxiety) in demen-

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Pharmacogenomics of Alzheimer’s disease APOE-CYP2D6 Association Distribution of APOE Genotypes in CYP2D6-Related EM, IM, PM and UM Genotypes 100% APOE-4/4 - CYP2D6 Association

80%

Presence of the APOE-4/4 genotype in CYP2D6-related EMs, IMs, PMs and IMs

F(%)

60%

8 6

40%

4 APOE-4/4 (%)

20% 0% APOE-4/4 APOE-3/4 APOE-3/3 APOE-2/4 APOE-2/3 APOE-2/2

0 APOE-4/4 (%)

1,96

2,65

7,14

8,69

APOE-4/4 APOE-3/4 APOE-3/3 APOE-2/4 APOE-2/3 APOE-2/2

1,96 16,67 70,26 0,65 10,13 0,33

2,65 19,08 68,42 0,66 9,21 0

7,14 32,15 57,14 0 3,57 0

8,69 13,05 78,26 0 0 0

Fig.13 5 Distribution and frequency of APOE genotypes in CYP2D6-related extensive (EM), intermediate (IM), poor (PM), and ultra-rapid metabolizers (UM). Accumulation of APOE-4/4 in PMs and UMs. (Adapted from Cacabelos42)

tia after non-psychotropic pharmacological treatment4,10,48,50,52. At baseline, all APOE variants show similar anxiety and depression rates, except the APOE-4/4 carriers who differed from the rest in significantly lower rates of anxiety and depression. Remarkable changes in anxiety were found among different APOE genotypes. Practically, all APOE variants responded with a significant diminution of anxiogenic symptoms, except patients with the APOE4/4 genotype who only showed a slight improvement. The best responders were APOE-2/4 ( r=-0.87) > APOE-2/3 (r=-0.77) > APOE-3/3 (r=-0.69) > APOE-3/4 carriers (r=-0.45). The potential influence of APOE

variants on anxiety and cognition in AD does not show a clear parallelism, suggesting that other more complex mechanisms are involved in the onset of anxiety in dementia. Concerning depression, all APOE genotypes improved their depressive symptoms with treatment except those with the APOE-4/4 genotype which worsen along the treatment period. The best responders were APOE-2/4 (r=-0.85) > APOE-2/3 (r=-0.77) > APOE-3/3 (r=-0.73) > APOE3/4 (r=-0.16), and the worst responder was APOE-4/4 (r=+0.31)10,48,50,52. Patients with each one of the 3 ACE-I/D indel variants are equally anxiogenic and depressive at baseline and all of them respond favourably to the multifactorial protocol by gradually reducing anxiety and depressive symptoms over the 12-month treatment period. The best responders were ACE-I/D (r=-0.89) > ACE-D/D (r=-0.68) > ACE-I/I (r=-0.08). Depressive symptoms were also similarly improved in all ACE-I/D variants. The best responders were ACE-I/D (r=-0.88) > ACED/D (r=-0.55) > ACE-I/I (r=-0.13). Comparatively, the worst responders among ACE-I/D variants were carriers of the ACEI/I genotype which were also the poorest responders in anxiety and cognition10,50,52. The combination of APOE and ACE polymorphic variants in bigenic clusters yields different anxiety and depression patterns at baseline and after one year of treatdiciembre 2009

Personalized Medicine of Dementia Efficacy

Pharmacogenomics

ACE/DCP1 A2M APOE BLMH/BMH BACE1 BACE2 LRP1/A2MR FOS CTSB/CPSB IL1A TNFA NOS3 BCHE CST3 MTHFR SORT1

ABCBs ABCCs/MD Rs CACNAs CYPs GSTAs GSTMs GSTTs KCNs

NATs PAPSSs PPARs PRKs PTKs SCNs SLCs SULTs UGTs

Drug MetabolismRelated Genes

Locus

CYP1A1

15q22-q24

CYP1A2

15q22-qter

CYP1B1

2p22-p21

CYP2A3/6

19q13.2

CYP2A7

19q13.2

CYP2A13

1913.2

CYP2B6

19q13.2

CYP2C8

10q24

CYP2C9

10q24

CYP2C19

10q24.1-q24.3

CYP2D6

22q13.1

CYP2E

10q24.3-qter

CYP2F1

19q13.2

CYP2J2

1p31.3-p31.2

CYP2R1

11p15.2

CYP3A4

7q22.1

CYP3A5

7q22.1

CYP3A7

7q22.1

CYP3A43

7q22.1

CYP4B1

1p34-p12

CYP5A1

7q34

CYP8A1

20q13.11-q13.13

CYP19A1

15q21.1

CYP21

6p21.3

CYP33

1p32

CYP40

4q31.3

CYP51A1

7q21.2-q21.3

Disease Phenotype

Outcome Measures

Brain Neuropathology Amyloid Deposition Senile plaques Amyloid angiopathy NFT/PHF Synaptic Loss Neuronal Loss Brain Atrophy Brain Activity Neurotransmission Bioelectrical activity Neurotrophic function Brain Function Cognition Behavior Language Psychomotor Activity Cerebrovascular Funtion Brain perfusion Neuroendocrine Function Metabolic Function Haematology Blood Biochemistry Lipid Metabolism Proteasome Function Chaperone Function Immune Funtion Apoptosis Age of Onset Concomitant Pathology

Pharmacogenetics

Therapeutic effects Disease-modifying effects

17q23 12p13.3-p12.3 19q13.2 17q11.2 11q23.3 21q22.3 12q13.1-q13.3 14q24.3 8p22 2q14 6p21.3 7q36 3q26.1-q26.2 20p11.2 1p36.3 1p21.3-p13.1

CYP Gene

Side-effects Adverse Drug Reactions (ADRs)

AD1/APP AD2 AD3/PSEN1 AD4/PSEN2 AD5 AD6 AD7

Transcriptomics Proteomics Metabolomics

21q21 19cen-q13.2 14q24.3 1q31-q42 12p11.23-q13.12 10q24 10p13

Genomics

Pharmacological Treatment

AD PathogenesisRelated Genes

Cognitive symptoms Memory Behavior Mood Disorders Circadian changes Functioning (ADLs) Brain imaging Structural Functional Biological markers Body fluids Molecular markers Gene expression Genotype-phenotype Correlations

Pharmacodynamics Receptors Ion channels Enzymes Proteins

Pharmacokinetics Absortion Distribution Metabolism Excretion

Safety

Fig.14 5 Pharmacogenetic and pharmacogenomic components of the therapeutic process in dementia. (Adapted from Cacabelos42)

ment. The most anxiogenic patients at baseline are those with the 23DD, 44ID, and 34II genotypes, and the least anxiogenic patients are those harbouring the 23II, 44DD, and 23ID genotypes. The most depressive clusters at baseline are those harbouring the 23DD, 33ID, and 33II genotypes, with a clear accumulation of APOE-3/3 carriers in these groups, and the least depressive clusters are those represented by carriers of the 23II, 44ID, and 23ID genotypes. All bigenic clusters show a positive anxiolytic and anti-depressive response to the multifactorial treatment, except 44DD carriers who exhibited the worst response10,47,50,52. APOE influences liver function and CYP2D6-related enzyme activity probably via regulation of hepatic lipid metabolism. It has been observed that APOE may influence liver function and drug metabolism by modifying hepatic steatosis and transaminase activity. There is a clear correlation between APOE-related TG levels and GOT,

GPT, and GGT activities in AD10,50. Both plasma TG levels and transaminase activity are significantly lower in AD patients harbouring the APOE-4/4 genotype, probably indicating (a) that low TG levels protect against liver steatosis, and (b) that the presence of the APOE-4 allele influences TG levels, liver steatosis, and transaminase activity. Consequently, it is very likely that APOE influences drug metabolism in the liver through different mechanisms, including interactions with enzymes such as transaminases and/or cytochrome P450related enzymes encoded in genes of the CYP superfamily10,48,50,52. When APOE and CYP2D6 genotypes are integrated in bigenic clusters and the APOE+CYP2D6-related therapeutic response to a combination therapy is analysed in AD patients, it becomes clear that the presence of the APOE-4/4 genotype is able to convert pure CYP2D6*1/*1 EMs into full PMs, indicating the existence of a powerful influence of the APOE-4 homozygous genotype on the drug metabolising capacity of pure CYP2D6-EMs. In addition, a clear accumulation of APOE-4/4

genotypes is observed among CYP2D6 PMs and UMs42 (Fig.13). From these studies we can conclude the following: (i) Most studies with acetylcholinesterase inhibitors indicate that the presence or absence of the APOE-4 allele influences the therapeutic outcome in patients with AD. (ii) Multifactorial treatments combining neuroprotectants, endogenous nucleotides, nootropic agents, vasoactive substances, cholinesterase inhibitors, and NMDA antagonists associated with metabolic supplementation on an individual basis adapted to the phenotype of the patient may be useful to improve cognition and slow-down disease progression in AD. (iii) The therapeutic response in AD seems to be genotype-specific under different pharmacogenomic conditions. (iv) In monogenic-related studies, patients harbouring the APOE-4/4 genotype are the worst responders (Fig.11). (v) APP, PSEN1 (Fig.12) and PSEN2 mutations influence the therapeutic response in AD (vi) In trigenicrelated studies (APOE+PSEN1+PSEN2) the best responders are those patients carrying the 331222-, 341122-, 341222-, and

441112- genomic clusters. (vii) The worst responders in all genomic clusters are patients with the 441122+ genotype. (viii) The interaction of several AD-related genes seems to be determinant for drug efficacy and safety. (ix) APOE-CYP2D6 interactions might influence the therapeutic response in AD via changes in lipid metabolism and liver function. (x) APOE may also interact with PSEN1, ACE, A2M and other genes to regulate the effect of drugs on cognition and behavioural changes in dementia. (xi) The APOE-4/4 genotype seems to accelerate neurodegeneration anticipating the onset of the disease by 5-10 years; and, in general, APOE-4/4 carriers show a faster disease progression and a poorer therapeutic response to all available treatments than any other polymorphic variant. (xii) Pharmacogenomic studies using monogenic, bigenic, trigenic, tetragenic or polygenic clusters as a harmonization procedure to reduce genomic heterogeneity in clinical trials are very useful in order to widen the therapeutic scope of limited pharmacological resources3-15,28,47-52.

Practical Considerations

The great variability in the therapeutic response of AD patients to conventional treatments (<20% effective responders), the heterogeneity of the disease and its complex pathogenesis, the occurrence of neuropsychiatric disorders associated with cognitive deterioration, as well as the presence of other age-related disorders in patients with dementia, seem to suggest that: (a) it is very unlikely that a single drug may be able to halt disease progression after the onset of the disease; (b) multifactorial interventions (as in other complex disorders, such as cardiovascular disease, cancer, AIDS, etc) might be an alternative strategy; however, drug-drug interactions in elderly patients who receive over 6 different drugs per day can represent a serious drawback in terms of safety; (c) the co-administration of many different drugs in patients with concomitant pathologies (i.e. coronary disease, hypertension, atherosclerosis, hyperlipidemia, dementia) may represent an obstacle for an effective pharmacological management of dementia since some drugs effective for a peripheral medical condition can exert a deleterious effect on brain function and brain perfusion with severe effects on cognition, behaviour and psychomotor function; (d) the fact that approximately 50-60% of patients with dementia exhibit

a marked cerebrovascular dysfunction recommends that cerebrovascular protection should not be neglected in the treatment of AD; (e) the co-administration of psychotropic drugs should be carried out with extreme care as most psychotropics deteriorate cognitive function, psychomotor activity, and cerebrovascular function; (f) the conventional procedures currently used in drug development (i.e. trial-and-error) and serendipity are not cost-effective nowadays; (g) the bimodal fashion of the amyloid-tau hypothesis of AD as a major target for future drug developments is a focus of controversy with unpredictable consequences for the industry and the public; (h) the reluctant attitude of the medical community to incorporate genomic procedures as diagnostic aids and disease biomarkers is not contributing to accelerating our understanding of the dementia syndrome and its biological diversity; and (i) the underdeveloped field of pharmacogenetics and pharmacogenomics is delaying the possibility of optimising our limited therapeutic resources for the treatment of AD42.. The introduction of novel procedures into an integral genomic medicine protocol for CNS disorders is an imperative requirement in drug development and in the clinical practice in order to improve diagnostic accuracy and to optimise therapeutics. This kind of protocol should integrate the following components: (i) clinical history, (ii) laboratory tests, (iii) neuropsychological assessment, (iv) cardiovascular evaluation, (v) conventional X-ray technology, (vi) structural neuroimaging, (vii) functional neuroimaging, (viii) computerized brain electrophysiology, (ix) cerebrovascular evaluation, (x) structural genomics, (xi) functional genomics, (xii) pharmacogenetics, (xiii) pharmacogenomics, (ix) nutrigenetics, (x) nutrigenomics, (xi) bioinformatics for data management, and (xii) artificial intelligence procedures for diagnostic assignments and probabilistic therapeutic options4 (Table 8). All these procedures, under personalized strategies adapted to the complexity of each case, are essential in order to depict a clinical profile based on specific biomarkers correlating with individual genomic profiles. Our understanding of the pathophysiology of CNS disorders has advanced dramatically during the last 30 years, especially in terms of their molecular pathogenesis and genetics. The drug treatment of CNS disorders has also made remarkable strides, with the introduction of many new drugs for the

Ciencia

Pharmacogenomics of Alzheimerâ&#x20AC;&#x2122;s disease

The human genome contains about 20,00030,000 genes. At the present time only 0.31% of commercial drugs have been assigned to corresponding genes treatment of schizophrenia, depression, anxiety, epilepsy, Parkinsonâ&#x20AC;&#x2122;s disease, and Alzheimerâ&#x20AC;&#x2122;s disease, among many other quantitatively and qualitatively important neuropsychiatric disorders. Improvement in terms of clinical outcome, however, has fallen short of expectations, with up to one third of the patients continuing to experience clinical relapse or unacceptable medication-related side effects in spite of efforts to identify optimal treatment regimes with one or more drugs. Potential reasons to explain this historical setback might be that: (a) the molecular pathology of most CNS disorders is still poorly understood; (b) drug targets are inappropriate, not fitting into the real etiology of the disease; (c) most treatments are symptomatic, but not anti-pathogenic; (d) the genetic component of most CNS disorders is poorly defined; and (e) the understanding of genome-drug interactions is very limited4,42. Assuming that the human genome contains about 20,000-30,000 genes, at the present time only 0.31% of commercial drugs have been assigned to corresponding genes whose gene products might diciembre 2009

Personalized Medicine of Dementia be involved in pharmacokinetic and pharmacodynamic activities of a given drug; and only 4% of the human genes have been assigned to a particular drug metabolic pathway. Supposing a theoretical number of 100,000 chemicals in current use worldwide, and assuming that practically all human genes can interact with drugs taken by human beings, each gene in the human genome should be involved in the metabolism and/or biopharmacological effect of 30-40 drugs; however, assuming that most xenobiotic substances in contact with our organism can influence genomic function, it might be possible that for 1,000,000 xenobiotics in daily contact with humans, an average of 350-500 xenobiotics have to be assigned to each one of the genes potentially involved in drug metabolism and/or the processing of xenobiotics. To fulfil this task a single gene has to possess the capacity of metabolizing many different xenobiotic substances and at the same time many different genes have to cooperate in orchestrated networks in order to metabolise a particular drug or xenobiotic under sequential biotransformation steps (Fig.14). Numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolising enzymes, such as CYPs, transferases and transporters. Many natural and artificial substances induce the hepatic CYP subfamilies in humans, and these inductions might lead to clinically important drug-drug interactions. Some of the key cellular receptors which mediate such inductions have been recently identified, including nuclear receptors, such as the constitutive androstane receptor (CAR, NR1I3), the retinoid X receptor (RXR, NR2B1), the pregnane X receptor (PXR, NR1I3), and the vitamin D receptor (VDR, NR1I1) and steroid receptors such as the glucocorticoid receptor (GR, NR3C1)86. There is a wide promiscuity of these receptors in the induction of CYPs in response to xenobiotics. Indeed, this adaptive system acts as an effective network where receptors share partners, ligands, DNA response elements and target genes, influencing their mutual relative expression86,87. The optimisation of CNS therapeutics requires the establishment of new postulates regarding (a) the costs of medicines, (b) the assessment of protocols for multifactorial treatment in chronic disorders, (c) the implementation of novel therapeutics addressing causative factors, and (d) the setting-up of pharmacogenetic/pharmacogenomic strategies for drug development42.

The cost of medicines is a very important issue in many countries due to (i) the growing of the aging population (>5% disability), (ii) neuropsychiatric and demented patients (>5% of the population) belonging to an unproductive sector with low income, and (iii) the high cost of health care systems and new health technologies in developed countries. Despite the effort of the pharmaceutical industry to demonstrate the benefits and cost-effectiveness of available drugs, the general impression in the medical community and in some governments is that some psychotropics and most anti-dementia drugs present in the market are not cost-effective2. Conventional drugs for neuropsychiatric disorders are relatively simple compounds with unreasonable prices. Some new products are not superior to conventional antidepressants, neuroleptics, and anxiolytics. There is an urgent need to assess the costs of new trials with pharmacogenetic and pharmacogenomic strategies, and to implement pharmacogenetic procedures for the prediction of drug-related adverse events. Pharmacogenomics can also help to reduce costs in drug development as well as the number of patients in clinical trials with high risk of toxicity. It has been suggested that the two critical strategies for pipeline genetics must make use of

fewer patients: (i) the early identification of efficacy signals so that they can be applied early in development for targeted therapies, and (ii) identification of safety signals which can subsequently be validated prospectively during development using the least number of patients with adverse responses83. Cost-effectiveness analysis has been the most commonly applied framework for evaluating pharmacogenetics. Pharmacogenetic testing is potentially relevant to large populations which incur in high costs. For instance, the most common drugs metabolised by CYP2D6 account for 189 million prescriptions and US$12.8 billion annually in expenditures in the US, which represent 5-10% of total utilization and expenditures for outpatient prescription drugs88. Pharmacogenomics offer great potential to improve patientsâ&#x20AC;&#x2122; health in a cost-effective manner; however, pharmacogenetics/pharmacogenomics will not be applied to all drugs available in the market, and careful evaluations should be made prior to investing resources in R&D of pharmacogenomic-based therapeutics and making reimbursement decisions89. In performing pharmacogenomic studies in dementia, it is necessary to rethink the therapeutic expectations of novel drugs,

(v) vascular factors, (vi) social factors, and (vii) genomic factors (nutrigenetics, nutrigenomics, pharmacogenetics, pharmacogenomics). Among genomic factors, nutrigenetics/nutrigenomics and pharmacogenetics/pharmacogenomics account for more than 80% of efficacy-safety outcomes in current therapeutics9,10,50,52.

Ciencia

Pharmacogenomics of Alzheimerâ&#x20AC;&#x2122;s disease

To achieve a mature discipline of pharmacogenetics and pharmacogenomics in CNS disorders and dementia it would be convenient to accelerate the following processes: (a) to educate physicians and the public on the use of genetic/genomic screening in the daily clinical practice; (b) to standardize genetic testing for major categories of drugs; (c) to validate pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) to regulate ethical, social, and economic issues; and (e) to incorporate pharmacogenetic and pharmacogenomic procedures to both drugs in development and drugs on the market in order to optimise therapeutics4,9,10,42,47-52.

redesign the protocols for drug clinical trials, and incorporate biological markers as assessable parameters of efficacy and prevention. In addition to the characterization of genomic profiles, phenotypic profiling of responders and non-responders to conventional drugs is also important (and currently neglected). An important issue in AD therapeutics is that anti-dementia drugs should be effective in covering the clinical spectrum of dementia symptoms represented by memory deficits, behavioural changes, and functional decline. It is difficult (or impossible) for a single drug to be able to fulfil these criteria. A potential solution to this problem is the implementation of cost-effective, multifactorial (combination) treatments integrating several drugs, taking into consideration that traditional neuroleptics and novel antipsychotics (and many other psychotropics) deteriorate both cognitive and psychomotor functions in the elderly and may also increase the risk of stroke90. Few studies with combination treatments have been reported and most of them are poorly designed. We also have to realize that the vast majority of dementia cases in people older than 75-80% are of a mixed type, in which the cerebrovascular component associated with neurodegeneration

cannot be therapeutically neglected. In most cases of dementia, the multifactorial (combination) therapy appears to be the most effective strategy10,42,47-52. The combination of several drugs increases the direct costs (e.g. medication) by 5-10%, but in turn, annual global costs are reduced by approximately 18-20% and the average survival rate increases about 30% (from 8 to 12 years post-diagnosis)4,42. There are major concerns regarding the validity of clinical trials in patients with severe dementia. If we assume that AD is a complex disorder where genomic and environmental factors interact to induce the premature death of neurons (which begins 30 years prior to the onset of the disease), it seems clear that future therapeutic strategies must be addressed towards the prevention of neurodegeneration because when the first symptoms appear thousands of millions of neurons have already died, and under these circumstances the possibility of being therapeutically effective is very remote. Major impact factors associated with drug efficacy and safety include the following: (i) the mechanisms of action of drugs, (ii) drug-specific adverse reactions, (iii) drugdrug interactions, (iv) nutritional factors,

Future Trends

The globalisation of the present economic crisis will negatively affect future investments in dementia research and drug development; however, for the first half of the coming decade, after an initial period with some programmes put on standby, it is expected that progress in AD pathogenesis, molecular diagnosis, and therapeutics will evolve favourably. Genome-wide familybased association studies, using single SNPs or haplotypes, will help to identify associations with genome-wide significance30,31,91,92; similarly, genome-wide expression analysis will be useful for the discovery of new drug targets. Some studies will try to elucidate the weight of genome-environment interactions in the pathogenesis and clinical course of dementia, and also the emerging role of epigenetics, as well. The validation of protocols for genomic screening will contribute to introducing structural genomics (genotyping, genome-wide analysis), functional genomics (genotypephenotype correlations), and proteomics as diagnostic aids and therapeutic targets93. New initiatives for the prevention of dementia will also emerge94, together with new insights into the role of nutrition and nutrigenomics in brain function and neurodegeneration95.

diciembre 2009

Personalized Medicine of Dementia

Some studies will try to elucidate the weight of genome-environment interactions in the pathogenesis and clinical course of dementia Priority areas for pharmacogenetic research are to predict serious adverse reactions (ADRs) and to establish variation in efficacy96. Both requirements are necessary in dementia to cope with efficacy and safety issues associated with both current anti-dementia drugs and new drugs. Since drug response is a complex trait, genomewide approaches (oligonucleotide microarrays, proteomic profiling) may provide new insights into drug metabolism and drug response. Of paramount importance is the identification of polymorphisms affecting gene regulation and mRNA processing in genes encoding cytochrome P450s and other drug-metabolising enzymes, drug transporters, and drug targets and receptors, with broad implication in pharmacogenetics since functional polymorphisms which alter gene expression and mRNA processing appear to play a critical role in shaping human phenotypic variability97. It is also most relevant, from a practical point of view, to understand the pharmacogenomics of drug transporters, especially ABCB1 (P-glycoprotein/MDR1) variants, due to the pleiotropic activity of this gene on a large number of drugs98. There are over 170 human solute carrier transporters which transport a variety of substrates, including amino acids, lipids, inorganic ions, peptides, saccharides, metals, drugs, toxic xenobiotics, chemical compounds, and proteins99.

Prof. Dr. Ramón Cacabelos rcacabelos@gen-t.es

In approximately 3-5 years novel data on clinical trials with anti-amyloid vaccines will be delivered and AD immunotherapy will face new vaccine models (active and passive immunization) and new therapeutic challenges regarding the amyloid burden in AD100. Other expected developments in AD therapeutics include γ-secretase inhibitors, ß-secretase inhibitors, ß-sheet breakers and chaperone inhibitors, regulators of the

ubiquitin-proteasome system, small molecule activators (non-peptide neurotrophic factors) of the Trk receptors, p38α mitogen-activated protein kinase (MAPK) regulators, ADNP (activitydependent neuroprotective protein) derivatives (NAP peptides), GSK-3ß modulators, phospholipase A2 inhibitors, the medium-chain triglyceride AC-1202, inhibitors of insulin-regulated aminopeptidase, amphiphilic pyridinium salts, and some other novel compounds, still in a preclinical stage, most of which are intended to be Aß lowering agents. There will be some initiatives for nanotechnology approaches to crossing the blood-brain barrier and drug delivery to the CNS, as well as for new transdermal and intranasal delivery systems. Another important issue in the pathogenesis and therapeutics of CNS disorders is the role of microRNAs (miRNAs). New inventories of miRNA expression profiles from CNS regions must be reported. These inventories of CNS miRNA profiles will provide an important step toward further elucidation of miRNA function and miRNA-related gene regulatory networks in the mammalian CNS. RNA interference (RNAi) has led in recent years to powerful approaches to silencing targeted genes in a sequence-specific manner with potential therapeutic applications in neurodegenerative diseases. RNAi procedures for gene selective inhibition must improve (a) cytoplasmic delivery of short sdRNA oligonucleotides (siRNA), which mimics an active intermediate of an endogenous RNAi mechanism, and (b) nuclear delivery of gene expression cassettes which express a short hairpin RNA (shRNA), which mimics the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism. These technologies, complemented by non-viral gene delivery systems and ligand-targeted plasmidbased nanoparticles for RNAi agents, will bring new hopes for the treatment of different complex disorders101,102. We need more information about the feasibility of targeting AD genes (e.g. APP-London mutation, APP-Swedish mutation, PS1, APLP1, APLP2, PEN-2, APH-1a, Nicastrin, BACE, MAPT-V337M) with RNAi and making sure that gene silence in CNS disorders does not affect proteomic and/or metabolomic networks which are fundamental for a correct brain function103. Another area of growing interest is the role of adult neurogenesis and stem cells in AD. Stem cell therapy has been suggested as a possible strategy for replacing damaged circuitry and restoring learning and memory abilities in patients with AD; however, there is a long path ahead from the promising investigations which are raising hopes, and the challenges behind translating underlying stem cell biology into an effective therapy for AD104. 

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Effects of FR-91 on human tumor cell lines VRM Lombardi1, E. Martínez2, R. Chacon2, I. Etcheverría1, R. Cacabelos1 1. EBIOTEC, Department of Cellular Immunology , La Coruña, Spain 2. Georgian Alternative Medicine, Madrid, Spain

A 50

poptosis has been an intensive research area, which involves the study of compounds that trigger or inhibit this mechanism of death. Being an important process implicated in many pathological diseases, including cancer, a number of new natural compounds has been investigated in the attempt to inhibit or trigger this fundamental cellular process making apoptosis amenable to new biopharmaceutical interventions. The aim of this study was to investigate the antitumor effect of FR-91, a standardized lysate of microbial cells belonging to the Bacillus genus, against SW872 (human adipose cells), SW982 (human synovial sarcoma line),

HL-60 (promyelocytic cells), HS 274.T (breast adenocarcinoma), HS 313.T (lymphoma), H2126 (lung adenocarcinoma), TOV 21G (epithelial ovarian cancer), WM 115 (melanoma), and HS 281T (breast adenocarcinoma) human tumor cell lines. We used the MTT (3-(4,5-dymethylthiazol-2-y1)2,5-diphenyltetrazolium bromide) assay to study the growth inhibition activity of FR-91 and apoptosis. We report the potential apoptogenic activity after 24 hours incubation with 10, 25 and 50 μl/ml of FR-91. Apoptosis, determined by DNA cellular fragmentation, was observed in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines treated

with 25 and 50 Îźl/ml of FR-91. The highest level of growth inhibition, cytotoxicity assay, was observed in SW872 (55, 78, 87%), SW982 (50, 70, 87%) and TOV-21G (42, 66, 85%), with respect to untreated cells, while the results of the expression of genes associated with apoptosis indicated a down-regulation of Bcl-2 in all cell lines. Taken together these results suggest that FR-91 contains small peptides which can contribute to the induction of apoptosis. Further investigations will be required to clarify the nature of these bioactive constituents and the in vivo effects of FR-91 on tumor-induced in experimental animal models.

Introduction

Dysregulated proliferation appears to be a hallmark of increased susceptibility to neoplasia. Cancer prevention is generally associated with inhibition, reversion, or retardation of cellular hyperproliferation. Both epidemiological and experimental studies have shown that a variety of food and food components, by exhibiting several biological properties that are able to modulate mammalian immune system, could be used for the prevention of many degenerative diseases1,2,3, and would be more economical and less painful, representing a new rational

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Effects of FR-91 on human tumor cell lines approach for cancer control. It is well known that dietary flavonoids and isoflavonoids behave as general cell growth inhibitors. One of their biological properties in plants, in fact, is providing resistance to fungal or bacterial growth, and although most flavonoids and isoflavonoids appear nontoxic to humans and animals, they have demonstrated to inhibit proliferation in many kinds of cultured human cancer cell lines4,5,6,7. Antiproliferative effects of quercetin8, taxifolin9, nobiletin10, and tangretin11 at 2-8 Îźg/ml for 3-7 day on squamous cell carcinoma, meningioma, colon carcinoma, leukaemia, and lung carcinoma tumor cell lines have been reported. Studies on mice, rats, and chickens have shown that many animals carry latent oncogenic viruses that usually remain harmless but occasionally become activated to cause the development of tumors or leukaemia12. The activation process can be triggered by several external or internal factors, such as radiation, certain hormones, or chemicals. However, the activation of the latent tumor-inducing viruses can be substantially delayed, or even prevented, by avoiding or reducing exposure to tumor-inducing factors, and also by the use of natural products13. The bacteria that reside in the intestinal tract generally have a symbiotic relationship with their host. Beneficial bacteria produce natural antibiotics14 to keep pathogenic bugs in check (preventing diarrhea and infections) and produce some B vitamins in the small intestine where they can be utilized. Beneficial bacteria help a) with food digestion15,16,17 by providing extra enzymes, such as lactase, in the small intestine; b) strengthen the immune system18,19 right in the gut where much of the interaction between the outside world and the body goes on; and c) prevent food allergies20,21,22. In addition, they can help to prevent cancer at various stages of development23. These good bacteria can improve mineral absorption, maximizing food utilization. However, the balance of beneficial and

potentially pathogenic bacteria in the gut is dependent on the diet. Studies in rats have shown that probiotics can inhibit the formation of aberrant crypt foci, thought to be a pre-cancerous lesion in the colon. Some of the best results were obtained with a probiotic strain consumed with inulin, a type of fructooligosaccharide. Total aberrant crypt foci, chemically induced, were reduced 74% by the treatment of rats with inulin and B. longum, but only 29 and 21% by B. longum and inulin alone, respectively24. There was a synergistic effect in using both products together. Similar synergy was seen in rats with azoxymethane-induced colon cancer in another study. Rats fed Raftilose, a mixture of inulin and oligofructose, or Raftilose with Lactobacilli rhamnosus (LGG) and Bifidobacterium lactis (Bb12) had a significantly lower number of tumors compared to the control group25. A probiotic mixture, without any prebiotic, given to rats fed azoxymethane reduced colon tumors compared to the control (50% vs 90%), and also reduced the number of tumors per tumor-bearing rat26. In the present study, different concentrations of FR-91, a standardized lysate of microbial cells belonging to the Bacillus genus, have been used to investigate potential antitumor activity against SW872, SW982, HL-60, HS 274.T, HS 313.T, H2126, TOV 21G, WM 115, and HS 281T human tumor cell lines.

Materials and methods

Cell lines and cell cultures Human adipose cells (SW872) and human synovial sarcoma line (SW 982) were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). Cells were maintained in L-15 (Leibovitz-15 medium with l-glutamine [PAA,

Prior to use in experimental assays, the cells growing in monolayer were released from the culture flask with 0.25% trypsin (PAA), washed twice with fresh medium, and seeded onto 96well microculture flat plates. Viable cell counts were confirmed prior to each experiment using trypan exclusion.

Chemicals HEPES-buffered RPMI 1640 medium, DMEM, DMEM/F12, L-15, MEM, FBS, trypsin, G5 supplement, and CryoMaxx S were obtained from PAA. Glutamine and antibiotics (penicillin and streptomycin) were purchased from Sigma-Aldrich (Madrid, Spain).

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The Cell Culture Company, Pasching, Austria]), supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA) and sodium bicarbonate [1.5 g/liter; Gibco]) and incubated at 37°C and 5% CO2. HL-60 (promyelocytic cells) cell line was obtained from the American Type Culture Collection. Cells were grown in RPMI 1640 medium supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. HS 274.T (breast adenocarcinoma) cell line was obtained from the American Type Culture Collection. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. HS 313.T (lymphoma) cell line was obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. H2126 hypotriploid cell line from metastatic site, pleural effusion adenocarcinoma, was obtained from the American Type Culture Collection. Cells were grown in DMEM/F12 supplemented with 5% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin, G5 supplement, and 2 mM L-glutamine. TOV 21G epithelial ovarian cancer cell line was obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. WM 115 (melanoma) cell line was obtained from the American Type Culture Collection. Cells were grown in Minimal essential Medium Eagle (MEM), supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. HS 281T (breast adenocarcinoma) cell line was obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Cell stocks were maintained in liquid nitrogen.

MTT reduction assay Cell proliferation assay was determined using MTT assay. Cell lines (1 x 105 cells/ml) were incubated in 96 well plates with different doses of FR-91 (10, 25 and 50 µl/w) for 24 hours. Ten µl of MTT (10 mg/ml) was added to each well and incubated further at 37ºC for 4 hours. After incubation, MTT-formazan precipitate was dissolved in 100 µl of DMSO and absorbance was recorded at 570 nm in an automatic plate reader (BioRAD instrument). Data are presented as percentage of cytotoxicity of treated versus untreated cells.

DNA ladder assay DNA ladder assay was carried out as per standard method. This method prevents the contamination of entire genomic DNA with fragmented DNA. Briefly, after treatment with FR-91, cells were harvested, washed twice with cold PBS and lysed for 30 min at 4ºC in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2% Triton X-100) using zirconium beads and automatic cell lyser. After centrifugation at 15,000 x g for 20 min, the supernatants was treated with protease inhibitor cocktail and 0.5% SDS for 1 hour at 37ºC. DNA was extracted twice with phenol and precipitated with 150 mM NaCl and two volumes of ethanol at -20ºC. DNA precipitate was washed twice with cold 70% ethanol, dissolved in TE buffer and treated for 1 hour with Rnase at 37ºC. Finally, DNA precipitates were stained with propidium iodide, electrophoresed on 2% agarose gel and visualized in an automatic gel documentation system (BioRAD system).

The balance of beneficial and potentially pathogenic bacteria in the gut is dependent on the diet Quantitative estimation of DNA fragmentation using an enzyme-linked immunosorbent assay (ELISA) After trypsin treatment, cells collected by brief centrifugation were plated to a density of 1104 cells per well on flat-bottomed 96-well plates (Costar) and incubated overnight in complete medium at 37°C under a 5% CO2 atmosphere. The next day, the culture medium was exchanged for 100 µl of complete medium with or without the indicated concentration of 10, 25 and 50 µl of FR-91 and incubated for an additional diciembre 2009

Effects of FR-91 on human tumor cell lines Table 1 Oligonucleotides used in the RT-PCR p53

sense: antisense:

5’-AAAACTTACCAAGGCAACTA-3’ 5’-TGAAATATTCTCCATCGAGT-3’

p21

sense: antisense:

5’CATGTCCGATCCTGGTGATG-3’ 5’-AGTGCAAGACAGCGACAAGG-3’

Bcl-2

sense: antisense:

5’-TGCACCTGACGCCCTTCAC-3’ 5’-AGACAGCCAGGAGAAATCAAACAG-3’

Bax

sense: antisense:

5’-ACCAAGAAGCTGAGCGAGTGTC-3’ 5’-ACAAAGATGGTCACGGTCTGCC-3’

24 h. Insome experiments, cells were preincubated with z-VAD-fmk (Promega) or the adenosine kinase inhibitor 5’-iodotubercidin (IT; Sigma) prior to incubation with FR-91. Thereafter, DNA fragmentation was quantitatively estimated in the remaining attached cells using an ELISA (Cell Detection ELISAPLUS, Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s protocol. Absorbance at 405 nm (reference at 492 nm) was measured in each well, and the means±SD were plotted as a function of the concentration of the indicated reagent. All control incubations contained the maximal concentration of DMSO, which was typically <0.1%; DMSO concentrations of up to 1.25% did not induce significant DNA fragmentation (data not shown). Each independent experiment was carried out in triplicate using all the different tumor cell lines.

RNA isolation and RT-PCR Total cellular RNA was isolated by lysis in a guanidinium isothiocyanate buffer followed by single step phenol-chloroform-isoamyl alcohol extraction27. Briefly, cells were harvested and lysed in a solution containing 4M guanidium isothiocyanate, 25mM sodium citrate (pH 7.0), 0.5% sodium sarkosine and 0.1M β-mercaptoethanol. Sequentially, 1/10 volume of 2M sodium acetate (pH 4.9), one volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49:1, v:v) were added to the homogenate. After vigorous shaking for 30 seconds, the solution was centrifuged at 10,000 x g for 15 minutes at 4ºC. RNA in the aqueous phase was precipitated by the addition of 0.5 ml isopropanol. One μg of total RNA was reverse-transcribed into cDNA by incubating with 200 units of reverse transcriptase in 20 μl of reaction buffer containing 0.25 μg of random primers and 0.8 mM dNTPs at 42ºC for one hour. Two μl of the cDNA was used for the PCR reaction as templates. The PCR was performed in buffer containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 μM of each primer and 5 units Taq DNA poly-

merase for 30 cycles of denaturing at 94ºC for 1 min, annealing at 55ºC for 1 min and extension at 72ºC for 2 min. The resulting PCR products were analyzed by 1.5% agarose gel electrophoresis. Sequences for the specific primers used in the PCR were summarized in Table 1.

Statistical analysis All statistical tests were performed with the use of SPSS (version 11.0; SPSS Inc, Chicago) and a P value <0.05 indicated statistical significance. Data were analyzed by using a two-factor repeated-measures analysis of variance (ANOVA) followed by a post hoc analysis where relevant (one-factor repeated-measures ANOVA, followed by Tukey’s tests for a significant effect of dose and paired t tests for a significant effect of FR-91 treatment).

Results

FR-91 growth inhibition in tumor cell lines To examine the possible antineoplastic effect of FR-91 in tumor cells, we first determined its effects on cell growth by the MTT assay, which measures the metabolically live cells based on their mitochondrial dehydrogenase activity. As shown in Fig 1, FR-91 caused growth inhibition in a dose-dependent manner. The cytotoxicity was not restricted to a specific cell line, since five different cell lines were sensible to the effects of FR-91. The highest level of growth inhibition was observed in SW872 (55%, 78%, 87%), SW982 (50%, 70%, 87%) and TOV-21G (42%, 66%, 85%), while HL-60 (6%, 33%, 48%) and HS 313.T (5%, 50%, 60%) showed a slight, but significant, cytotoxic effect with respect to untreated cells. At the lowest concentration of FR-91 (10µl/ml) a significant difference between controls and treated cells was observed in SW 872, SW 982, and TOV-21G cell lines. No cytotoxic effects were observed with HS 274.T, H2126, WM 115, and HS281.T tumor cell lines. To determine if the antiproliferative effect was reversible, SW872, SW982, and TOV-21G cells were treated with 25 μl/ml FR-91 or culture medium for 48 hours. The medium-treated and FR91-treated cells were then incubated in complete medium for 0, 6, 24, 48, and 72 hours followed by trypsinization and counting of the cells. After 48 hours of treatment with FR-91, the cell number had decreased by 50% in all cell lines. Once FR-91 was removed, the cell number did not increase but rather showed a small further decrease, whereas the cell number in the absence of treatment, as expected, increased with time (Fig. 2). These data indicate that the FR-91 effect was not reversible.

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FR-91 induction of apoptosis in tumor cell lines FR-91 10 _l

100

FR-91 25 _l

FR-91 50 _l

% of growth inhibition

80 70 60 50 40 30 20 10 0 HL-60

H.S. 274.T H.S. 313.T SW 872

SW982

H2126

TOV-21G

WM 115 H.S. 281.T

5 Figure 1 Growth inhibition of SW872, SW982, HL-60, HS 274.T, HS 313.T, H2126, TOV 21G, WM 115, and HS 281T human tumor cell lines. Cells were plated on 96 well plates and exposed to three 10, 25 and 50 μl/ml of FR-91. The growth inhibition is expressed as percentage from cells exposed to culture medium. The mean shown was calculated from five independent experiments performed by triplicate.

350

SW 872 UNTREATED SW 872 TREATED SW982 UNTREATED

300

SW982 TREATED TOV-21G UNTREATED TOV-21G TREATED

Cell number (% of control)

Next, we tested wether the administration of FR-91 induced any cytotoxic effects on SW872, SW982, HL-60, HS 274.T, HS 313.T, H2126, TOV 21G, WM 115, and HS 281T human tumor cell lines. When cells were treated with 10, 25, and 50 µl/ml of FR-91 for 24 hours and then examined morphologically by light microscopy, a portion of the cells exhibited condensation (arrow-head) and cleavage (arrow) of the nuclei, findings that are typical of apoptosis (Fig.3). No such images were observed in control, untreated cells. The following results, expressed as absorbances (570 nm), clearly show that the treatment with FR-91 indeed induced apoptosis in HL-60 (FR-91 25 μl/ml: 0.96, p<0.001 vs negative, 0.12, and positive, 1.47, controls); FR-91 50 μl/ml: 1.1, p<0.001 vs negative, 0.18, and positive, 1.72, controls); HS 313T (FR-91 25 μl/ml: 0.77, p<0.001 vs negative, 0.18, and positive, 1.72, controls; FR91 50 μl/ml: 0.68, p<0.001 vs negative, 0.18, and positive, 1.72, controls ); SW872 (FR-91 10 μl/ ml: 0.58, p<0.001 vs negative, 0.17, and positive, 1.64, controls; FR-91 25 μl/ml: 0.76, p<0.001 vs negative, 0.17, and positive, 1.64, controls; FR91 50 μl/ml: 1.55, p<0.001 vs negative, 0.17, and positive, 1.64, controls); SW982 (FR-91 10 μl/ ml: 0.77, p<0.002 vs negative, 0.24, and positive, 1.8, controls; FR-91 25 μl/ml: 0.99, p<0.001 vs negative, 0.17, and positive, 1.64, controls; FR91 50 μl/ml: 1.48, p<0.001 vs negative, 0.17, and positive, 1.64, controls); and TOV-21G (FR-91 25 μl/ml: 0.58, p<0.001 vs negative, 0.17, and positive, 1.8, controls; FR-91 50 μl/ml: 1.6, p<0.001 vs negative, 0.17, and positive, 1.8, controls), cell lines (Fig. 4). DNA prepared from HL-60, HS 313T, SW872, SW982 and TOV-21G cells treated with FR-91 for 24 hours showed oligonucleosomal ladder fragmentation on agarose gel electrophoresis (data not shown). No signs of apoptosis were observed on HS 274.T (FR-91 10 μl/ml: 0.22, p=0.28 vs negative, 0.2, and positive, 1.65, controls; FR-91 25 μl/ml: 0.24, p=0.08 vs negative, 0.22, and positive, 1.65, controls; FR91 50 μl/ml: 0.2, p=0.33 vs negative, 0.22, and positive, 1.65, controls), H2126 (FR-91 10 μl/ ml: 0.18, p=0.32 vs negative, 0.2, and positive, 1.75, controls; FR-91 25 μl/ml: 0.23, p=0.78 vs negative, 0.2, and positive, 1.75, controls; FR91 50 μl/ml: 0.22, p=0.52 vs negative, 0.2, and positive, 1.75, controls), WM 115 (FR-91 10 μl/ ml: 0.23, p=0.25 vs negative, 0.14, and positive, 1.69, controls; FR-91 25 μl/ml: 0.22, p=0.27 vs negative, 0.14, and positive, 1.69, controls; FR91 50 μl/ml: 0.24, p=0.07 vs negative, 0.14, and positive, 1.69, controls), and HS 281T (FR-91 10 μl/ml: 0.2, p=0.25 vs negative, 0.24, and positive, 1.81, controls; FR-91 25 μl/ml: 0.22, p=0.27 vs negative, 0.24, and positive, 1.81, controls; FR91 50 μl/ml: 0.24, p=0.07 vs negative, 0.24, and positive, 1.81, controls) (Fig. 5).

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5 Figure 2 Antiproliferative effects of FR-91 in SW 872, SW 982, and TOV-21G cells. Cells were treated for 48 hours with 25 µl/ml of FR-91 or complete medium. The medium was replaced with only medium, and after various times (0-72 hours) the cells were trypsinized and counted. Results are expressed relative to control (48 hours incubation with medium). All values are mean of triplicate cultures in three independent experiments.

diciembre 2009

Effects of FR-91 on human tumor cell lines

Discussion

In the attempt of understanding and treating diseases, natural components have been discovered, by trial and error, and used for thousands of years by a significant fraction of the population in many countries or regions of the world. It is estimated that approximately 25% of the drugs prescribed worldwile at present derive from plants and 60% of antitumor/antiinfectious drugs already on the marker or under clinical investigations are of natural origin, and extracts from plants such as Taxol28, curcumin29, phenolic acids30 and flavonoids31 are reported to inhibit tumor growth in many types of cancer. Tumor growth is generally associated with marked changes in hematopoiesis and immune response, myelosuppression and anemia. The immune system has several potential means to limit or even prevent tumor growth. These include: a) specific T cell-mediated immunity against tumor associated transplantation antigens (TATAs) on tumor cells; b) production of antibodies against TATAs and/or other antigenic structures associated with the tumor cells; c) natural cell-mediated immunity against tumors, which consists mainly of natural killer (NK) cells and activated macrophages; and d) natural antitumor antibodies.

5 Figure 3 Morphological examination of FR-91 treated SW 982 cells. SW 982 cells were cultured in the presence or absence of either 25 Âľl/ml of FR-91 and 1 Âľl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide), as a positive control of apoptosis induction, for 24 hours as indicated in the materials and methods section. After Giemsa-staining, the morphological appearance of the cells was examined using light microscopy. The black arrows indicate nuclear condensation. Typical apoptotic cells, characterized by cleaved nuclei, are indicated by the white arrows. Magnification 400x.

Down-regulation of Bcl-2 gene expression in FR-91-treated tumor cell lines To further investigate the molecular mechanisms responsible for the FR-91 induced apoptosis in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines, the gene expression of some apoptosis-related genes such as p53, p21, Bax and Bcl-2 was analyzed by RT-PCR. In Fig. 6, FR-91 treatment caused the down-regulation of Bcl-2 gene expression, while other genes were not affected, which therefore resulted in the relative increase of Bax/Bcl-2 ratio.

Although there is some evidence for substantial anti-tumor effects of specifically induced or natural antibodies, particularly if they are cytotoxic in the presence of complement or in combination with lymphocytes or macrophages that express receptors for the Fc portion of immunoglobulins (and hence can mediate antibodydependent cell-mediated cytotoxicity (ADCC), most attention and experimental data have been focused on cell-mediated immunity as the basis for possible resistance of the host against progressive growth of tumor. There has been much interest and controversy for many years over the possible role of each of these immunological mechanisms in prevention of the initial development of detectable tumors. Considerations along these lines are encompassed in the hypothesis of immunological surveillance against tumors. An updated version of this hypothesis, which includes the potential involvement of NK cells and other aspects of natural immunity as well as specific T-cell mediated immunity has been reported32,33,34. The most compelling support for this hypothesis has come from observations, in clinical situations as well as in experimental animal models, of substantially more frequent tumor development in immunodeficient individuals as compared to individuals with normally functioning immune system. For example, the frequent

exp2 exp3 exp4

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313.

exp2 exp3

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3 Figure 4 Detection of DNA fragmentation in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines after culturing 24 hours in the presence of the indicated concentrations of FR-91 and 1 Âľl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide), as a positive control of apoptosis induction. Experiments were repeated five times and the results are expressed as optical density.

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development of lymphoproliferative disease in immunosuppressed organ transplant recipients, patients with infection by the human immunodeficiency virus (HIV), or in children with congenital immunodeficiencies points strongly to a

FR-91

key role of the immune system in preventing this type of cancer35,36. However, it has been very difficult to determine clearly which components of the immune system are most critical for effective immunological surveillance. diciembre 2009

Effects of FR-91 on human tumor cell lines Figure 5 4 Detection of DNA fragmentation in HS 274.T, H2126, WM 115, and HS 281T cell lines after culturing 24 hours in the presence of the indicated concentrations of FR-91 and 1 Âľl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide), as a positive control of apoptosis induction. Experiments were repeated five times and the results are expressed as optical density.

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It remains quite possible that several different arms of the immune system contribute to resistance against primary tumor development, with the appearance of overt malignant disease possibly representing failure of more than one line of defense. Although at least 120 chemical substances useful as antineoplastic drugs, and among them Paclitaxel (Taxol) isolated from three species

FR-91 10_l

FR-91 25 _l

FR-91 50 _l

of the genus Taxus, are still isolated from plants throughout the world, research regarding the role of natural compounds in the treatment of different types of tumors still remains controversial. Epidemiological studies assessing dietary intake of natural compounds, although firmly based on biologically plausible hypotheses, have provided support for the association between reduced risk and bioactive intake, in most, but not all, studies published to date. Although the

The results of this study suggest that FR-91 inhibited proliferation of tumor cells by a mechanism that involves cytotoxicity. The predominant form of cell death is likely apoptosis since evidence of apoptotic cell death was seen initially. However, at longer times of incubations, necrotic cell death was also observed. It is likely that necrotic cell death occurred as a secondary event and is a phenomen seen in vitro due to the lack of white cell phagocytosis. The possibility cannot be excluded, however, that FR-91 causes both forms of cell death and the incidence of these forms of death depends on the concentration that is used and the length of incubation. Apoptosis is regulated and executed by different interplay of many genes responsive to various stimuli. There are two central patways that lead to apoptosis: 1) positive induction by ligand binding to a plasma membrane receptor and 2) negative induction by loss of a suppressor activity. Positive-induction involves ligands related to TNF, while negative induction of apoptosis by loss of a suppressor activity involves the mitochondria. The study of apoptosis in cancer therapy is very important37. It has been proved that occurrence of cancers is due to the loss of control of normal apoptosis and the disturbance of balance between cell apoptosis and cell proliferation38. The apoptotis related genes (bcl-2 family) are divided in two categories: apoptotic repressor and apoptotic promoter. Bcl-2 is an important apoptotic repressor, while Bax is one of the most important apoptotic promoters. The protein it encodes can combine with Bcl2 to form compounds which resist the action of repressing apoptosis. But it has a positive regulatory action39. Recent studies indicate that the regulation of apoptosis by Bcl-2 and Bax is not only based on the level of either of the two

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p53 p21

Bax 70

% of gene expression

FR-91 extract has demonstrated significant in vitro and in vivo antineoplastic activities against different tumor cell lines, the mechanism of this effect has not been fully examined. In this study, FR-91 demonstrated selective cytotoxicity in vitro for human tumor cells tested (liposarcoma, sarcoma, promyelocitic, ovarian, and lymphoma cancer cell lines) when compared to untreated cells. Moreover, FR-91 showed the greatest cytotoxicity towards HS 313.T cells that are resistant to chemotherapy. This compound’s ability to effectively kill several types of tumors without significant cytotoxicity to normal cells indicates that this compound may be a potentially chemotherapeutic agent. The FR-91 induced apoptosis occurred in a dose-dependent manner and was accompanied by the disruption of the mitochondrial transmembrane potential (data not shown) and the activation of caspase-3 and perhaps caspase-8.

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5 Figure 6 FR-91 induced the Bcl-2 down-regulation in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines. Cells were treated with 25µl of FR-91 as indicated for 24 hours and cells were harvested for RNA isolation and RT-PCR. Positive controls were treated with 1 µl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide). Experiments were repeated three times and the results are expressed as % of gene expression compared to positive controls.

regulatory proteins but also based on the ratio of them. If the ratio is high, the cells go to apoptosis40. All cell lines used in the presented study have been induced by a variety of chemical reagents to undergo apoptosis through different pathways such as p53-dependent pathway or Bcl-2 family-related pathway. To clarify the molecular mechanism of apoptosis mediated by different concentrations of FR-91, we examined the expression of genes including p53, p21, Bax and Bcl-2 family by RT-PCR. Results indicated that apoptosis occurred in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines treated with 25 μl/ml of FR-91 accompanied by the dose-dependent down-regulation of Bcl-2 gene expression, while others were not significantly changed (Fig. 6). In the present study, we demonstrated that the relative increase of apoptotic Bax/Bcl-2 ratio correlated well with FR-91induced apoptosis in different human cell lines. It is possible that FR-91, through an appropriate signal, induces a conformational change in the Bax which moves to the mitochondrial membrane where it causes release of mitochondrial cytochrome c into the cytosol. In conclusion, the use of FR-91 extracts exhibited an apoptosis-inducing effect in various human tumor cell lines. Although further studies must be performed to elucidate the mechanisms by which FR-91 induces apoptosis in tumor cell lines, the present data indicate that FR-91 might be a useful chemotherapeutic compound for patients with different types of tumors. 

Dr. Valter R.M. Lombardi biotecnologia@ebotec.com

diciembre 2009

Effects of FR-91 on human tumor cell lines 1.

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Personalizar el Tratamiento

a farmacogenómica se encuentra en pleno desarrollo. Pero 20 años de investigación han sido suficientes para desmostrar que los métodos basados en la farmacogenómica permiten detectar factores hereditarios que causan alteraciones en el metabolismo de fármacos y en la predisposición a padecer reacciones adversas. Y estos factores hereditarios pueden ser detectados con sencillos análisis genéticos. El resultado se conoce en horas. Uno de los objetivos de la farmacogenómica es ajustar la dosis del fármaco a la capacidad metabólica del paciente.

Los métodos basados en la farmacogenómica muestran su mayor utilidad con fármacos cuyo

metabolismo predominante es llevado a cabo por una enzima polimórfica, cuando las mutaciones que se analizan son frecuentes en la población de estudio y tienen un claro efecto sobre los parámetros farmacocinéticos y/o la respuesta clínica a ese fármaco. Con la mayor parte de los antiinflamatorios no esteroideos (AINEs), se dan las tres situaciones anteriores, y, por este motivo, estos fármacos constituyen un excelente ejemplo de cómo la determinación de mutaciones en determinados genes puede ser de utilidad en la aplicación personalizada de los tratamientos más adecuados y seguros para cada paciente según su ge-

Prof. Dr. J.A. García-Agúndez Departamento de Farmacología y Psiquiatría, Facultad de Medicina Universidad de Extremadura, Badajoz, España

notipo. Con la mayor parte de AINEs, los genes más relevantes son los que codifican las enzimas citocromos P450 CYP2C8 y CYP2C9. En estos genes existen determinadas mutaciones y, lo que es más importante aún, determinadas combinaciones de ellas (haplotipos) que se asocian a la aparición de reacciones adversas severas. Actualmente, disponemos de test genéticos fundamentales en la detección de pacientes con alto riesgo de desarrollar reacciones adversas con estos medicamentos y con cuyo uso se pueden seleccionar, para cada paciente, los AINEs que comporten un menor riesgo de provocar reacciones adversas.

La variabilidad interindividual en la respuesta a los fármacos es una importante causa de efectos adversos. En muchos casos, esta variabilidad está ligada al polimorfismo de genes que codifican las enzimas responsables del metabolismo de dichos fármacos. La mayoría de enzimas que metabolizan fármacos es polimórfica, debido a la presencia de mutaciones en los genes que las codifican. Estas mutaciones, que consisten en la ausencia completa del gen, polimorfismos de un solo nucleótido, aislados o combinados, y duplicaciones génicas, causan ausencia, reducción, alteración o incremento de la actividad enzimática 1, 2.

diciembre 2009

Farmacogenómica de los AINEs Los portadores de mutaciones en genes codificadores de enzimas metabolizadoras de fármacos, cuando son tratados con dosis estándar de un fármaco que sea sustrato de la enzima afectada, suelen presentar niveles plasmáticos más elevados, cifras de aclaramiento más bajas 3, 4, y un incremento en la frecuencia y severidad de reacciones adversas secundarias al uso de dicho fármaco 5, 6.

La utilidad clínica de la farmacogenética es ya un hecho en el metabolismo de los fármacos La farmacogenómica es un área de la farmacología que se encuentra en pleno desarrollo y que estudia la contribución de factores genéticos a las diferencias interindividuales en la respuesta a fármacos. Aunque el ámbito de estudio de la farmacogenómica implica también la variabilidad farmacodinámica - por ejemplo, a través del estudio de polimorfismos en genes que codifican receptores -, es en el ámbito de la farmacocinética, y, en particular, en el metabolismo de fármacos donde la farmacogenómica ha adquirido un desarrollo pleno y donde se están obteniendo los primeros resultados de utilidad clínica.

Las enzimas citocromo P450 2C8 (CYP2C8) y 2C9 (CYP2C9) pertenecen a una de las principales familias de enzimas implicadas en el metabolismo de fármacos. Los genes que codifican estas dos enzimas, junto a los que codifican los otros componentes de CYP2C (denominados CYP2C18 y CYP2C19) se agrupan en dos clusters consecutivos en el cromosoma 10 y muestran un alto grado de asociación, de modo que la presencia de mutaciones en uno de los genes suele coincidir con mutaciones en otros genes del cluster 7. La importancia clínica de los polimorfismos de CYP2C8 y CYP2C9 radica en la concurrencia de dos factores: ambas enzimas están implicadas en el metabolismo de numerosos fármacos de uso clínico, algunos de los cuales tienen un margen terapéutico muy estrecho, y, además, un porcentaje elevado de la población española es portadora de mutaciones en los genes que codifican estas enzimas. Más de 30 millones de personas son tratadas diariamente con antiinflamatorios no esteroideos (AINEs) y cerca del 25% de la población ha experimentado alguna vez en su vida reacciones adversas causadas por AINEs que han requerido tratamiento médico 8, 9. Entre estas reacciones adversas, las que tienen una mayor relevancia clínica, por su severidad y frecuencia, son las relacionadas con las hemorragias digestivas altas (HDA) que, solamente en los estados Unidos causan más de 30.000 hospitalizaciones anuales10. Se define como hemorragia digestiva

alta (HDA) la que se origina en una lesión situada por encima del ángulo de Treitz. Es una causa frecuente de consulta en servicios de urgencias y de ingreso hospitalario. En España, presenta una incidencia de entre 85 y 106 casos por 100.000 habitantes/año 11. La mortalidad asociada a la HDA ha permanecido invariable en las últimas décadas 12 , debido, fundamentalmente, al incremento de la edad de los pacientes con HDA y a la coexistencia de enfermedades asociadas 13.

Tabla 1: Principales enzimas implicadas en el metabolismo de AINEs Fármaco

Relevancia de CYP2C

Enzima principal

Enzima secundaria

Aceclofenaco

parcial

CYP2C9

Esterasas plasmáticas

Aspirina

secundario

UGT1A6

CYP2C9

Celecoxib

predominante

CYP2C9

Diclofenaco

parcial

CYP2C9

UGT2B7, y diversos CYPs

Dipirona

secundario

CYP2C19, CYP2C8

CYPs

Etoricoxib

secundario

CYP3A4

Flurbiprofeno

parcial

CYP2C9

Ibuprofeno

predominante

CYP2C8, CYP2C9

Además del peligro que pueden suponer las reacciones adversas a AINEs, las implicaciones económicas para el sistema sanitario son también muy relevantes. El coste de los efectos adversos gastrointestinales a veces supera el propio coste de los AINEs 15, 16. No sorprende, por lo tanto, el creciente interés de las autoridades sanitarias en la identificación de pacientes con alto riesgo de desarrollar hemorragias digestivas con el uso de AINEs.

Indometacina

parcial

CYP2C9

Carboxil esterasas

Ketoprofeno

secundario

UGTs

CYP

Lornoxicam

predominante

CYP2C9

Meloxicam

parcial

CYP2C9

CYP3A4

Naproxeno

secundario

UGT2B7

CYP2C9, CYP1A2

Parecoxib

secundario

Hidrolisis a valdecoxib

CYP3A4, CYP2C9

En los últimos años se han realizado considerables avances en el conocimiento de las enzimas implicadas en el metabolismo de los AINEs y en la identificación de metabolitos de estos fármacos. Adicionalmente, se han desarrollado procedimientos para identificar pacientes con una alteración en el metabolismo de estos fármacos determinada genéticamente. Aunque los AINEs constituyen un grupo de fármacos químicamente heterogéneo, la mayor parte de ellos comparte las principales enzimas implicadas en su metabolismo, que son CYP2C8 y CYP2C9.

Piroxicam

predominante

CYP2C9

Rofecoxib

secundario

UGT2B7, UGT2B15

CYP2C9, CYP3A4

Sulindac

secundario

UGTs

CYP2C9

Tenoxicam

parcial

CYP2C9

Valdecoxib

secundario

CYP3A4

La tasa de mortalidad por HDA secundaria a AINEs en España es de más del 5% 11. En estudios realizados en otros países, se ha estimado que uno de cada 1.200 pacientes tratado con AINEs por vía oral durante al menos 2 meses morirá, debido a complicaciones gastroduodenales directamente relacionadas con el uso de AINEs 14.

Sin embargo, el papel relativo de estas enzimas difiere entre diferentes AINEs. La Tabla 1 resume las principales enzimas implicadas en el metabolismo de AINEs. Los polimorfismos en los genes que codifican estas enzimas causan importantes cambios en la farmacocinética de algunos de estos AINEs, de los cuales los más importantes son aquellos cuyo metabolismo predominante es a través de estas enzimas: celecoxib, ibuprofeno, lornoxicam y piroxicam son metabolizados en más de un 90% por estas enzimas, mientras que aceclofenaco, diclofenaco, flurbiprofeno, indometacina, meloxicam y tenoxicam son metabolizados en más de un 50% por CYP2C8 y/o CYP2C9. Dado que existen numerosas mutaciones en los genes que codifican estas enzimas que pue-

Ciencia

Personalizar el tratamiento

CYP2C9

Relevancia de las enzimas CYP2C en el metabolismo primario de AINEs. Predominante: más del 90% del fármaco es metabolizado por CYP2C8 o CYP2C9. Parcial: del 50% al 90% del fármaco es metabolizado por CYP2C8 o CYP2C9. Secundario: Menos del 50% del fármaco es metabolizado por CYP2C8 o CYP2C9.

30 millones de personas son tratadas diariamente con AINEs diciembre 2009

Farmacogenómica de los AINEs den modificar la actividad metabólica, y, por ende, la farmacocinética de estos AINEs, los portadores de estas mutaciones pueden tener un mayor riesgo de desarrollar reacciones adversas cuando sean tratados con estos fármacos. La Tabla 2 resume las principales mutaciones en el gen CYP2C8, y la Tabla 3 resume las principales mutaciones en el gen CYP2C9. Por razones de espacio, solamente se han incluido aquellas en las que la variante causa una sustitución de aminoácido. Las variantes CYP2C8*2, *3, *5, *7 y *8 conducen a una alteración de la actividad enzimática. Las más frecuentes son CYP2C8*2 en individuos de origen africano, CYP2C8*3 y CYP2C8*4 en individuos de origen caucásico (europeo) y CYP2C8*5 en individuos de origen oriental. El resto aparece con frecuencias muy bajas.

Ajustar la dosis a la capacidad metabólica del paciente es uno de los objetivos de la Farmacogenética Tabla 2: Variantes del gen CYP2C8 que causan sustituciones de aminoácidos. Nombre del alelo

Cambio de aminoácido

CYP2C8*2

I269F

CYP2C8*3

R139K; K399R

Efecto en la actividad

Identificación del SNP.

Aumento en la Km

rs11572103

Disminución

rs11572080; rs10509681

CYP2C8*4

I264M

No concluyente

rs1058930

CYP2C8*5

159 Frame shift

Ausente

Sin designación

CYP2C8*7

R186X

Ausente

Sin designación

CYP2C8*8

R186G

Desconocido

Sin designación

CYP2C8*9

K247R

Desconocido

Sin designación

CYP2C8*10

K383N

Desconocido

Sin designación

CYP2C8*12

V461deletion

Desconocido

Sin designación

CYP2C8*13

I223M

Desconocido

Sin designación

CYP2C8*14

A238P

Desconocido

Sin designación

A82S

Desconocido

rs17851796

Sin designación

I244V

Desconocido

rs11572102

Sin designación

L361F

Desconocido

rs45438799

En cuanto a CYP2C9, existen numerosas variantes que conducen a una baja actividad enzimática, pero la mayor parte de ellas son muy infrecuentes. Entre las más frecuentes, se encuentran, para individuos de origen caucásico, CYP2C9*2 y CYP2C9*3. En individuos orientales, son muy infrecuentes y se limitan a CYP2C9*3 y en individuos de origen africano aparecen diversas variantes con frecuencias entre el 1% y el 3% que incluyen CYP2C9*2, *3, *5, *6 y *11. Además se han demostrado variaciones en las frecuencias entre distintas poblaciones del mismo origen étnico. Por ejemplo, la variante CYP2C9*3 aparece con una frecuencia del 11% en Españoles y menos de la mitad de esta frecuencia en Suecos 7, 17. Se ha descrito una clara asociación entre la presencia de variantes de CYP2C8 y/o de CYP2C9 y la capacidad de metabolizar diversos AINEs. La farmacocinética de celecoxib, diclofenaco, flurbiprofeno, ibuprofeno, lornoxicam, piroxicam y tenoxicam se ve alterada en portadores de estas variantes alélicas que, comparados con los no portadores, presentan una farmacocinética más lenta y, por lo tanto, pueden presentar efectos adversos con más facilidad que los no portadores. Recientes estudios llevados a cabo por nuestro grupo – y, posteriormente, confirmados por varios grupos independientes 18-23 - indican de forma inequívoca que los portadores de variantes de CYP2C8 y/o CYP2C9 presentan un mayor riesgo de desarrollar hemorragias digestivas altas (HDA) asociadas al tratamiento con AINEs. En nuestra población, la frecuencia de portadores de al menos una de estas mutaciones es de más del 40% 3, 4. Estas personas tienen un mayor riesgo de presentar HDA cuando son tratados con AINEs que son sustratos de CYP2C8 y CYP2C9. Sin embargo, el grupo más llamativo es el de portadores de varias de estas mutaciones simultáneamente, especialmente si son homocigotos 20-23 . Cerca del 10% de la población española pertenece a este grupo que tiene un riesgo global de desarrollar hemorragias digestivas cuando son tratados con AINEs que puede ser 4 veces superior al de la población general, especialmente cuando concurren variantes de CYP2C8 y CYP2C9 en el mismo paciente. Por otra parte, el incremento del riesgo en este grupo de portadores de variantes genéticas varía para cada fármaco. Así, fármacos considerados seguros para la mayor parte de la población, como el ibuprofeno, pueden convertirse en fármacos inseguros para este 10% de personas que son portadores homocigotos. En estos pacientes, es más seguro el uso de aspirina o diclofenaco, que son AINEs teóricamente menos seguros

Ciencia

Personalizar el tratamiento Tabla 3: Variantes del gen CYP2C9 que causan sustituciones de aminoazidos. Nombre del alelo

Cambio de aminoácido

Efecto en la actividad

Identificación del SNP.

Nombre del alelo

Cambio de aminoácido

Efecto en la actividad

Identificación del SNP.

CYP2C9*2

R144C

Disminución

rs1799853

CYP2C9*22

N41D

Desconocido

Sin designación

CYP2C9*3

I359L

Disminución

rs1057910

CYP2C9*23

V76M

Desconocido

Sin designación

CYP2C9*4

I359T

Desconocido

rs56165452

CYP2C9*24

E354K

Desconocido

Sin designación

CYP2C9*5

D360E

Disminución

rs28371686

CYP2C9*25

118 Frame shift

Ausente

Sin designación

CYP2C9*6

273 Frame shift

Ausente

rs9332131

CYP2C9*26

T130R

Disminución

Sin designación

CYP2C9*7

L19I

Desconocido

Sin designación

CYP2C9*27

R150L

Desconocido

Sin designación

CYP2C9*8

R150H

No concluyente

rs7900194

CYP2C9*28

Q214L

Disminución

Sin designación

CYP2C9*9

H251R

Desconocido

rs2256871

CYP2C9*29

P279T

Desconocido

Sin designación

CYP2C9*10

E272G

Desconocido

rs9332130

CYP2C9*30

A477T

Disminución

Sin designación

CYP2C9*11

R335W

Disminución

rs28371685

CYP2C9*31

I327T

Desconocido

rs57505750

CYP2C9*12

P489S

Disminución

rs9332239

CYP2C9*32

V490F

Desconocido

Sin designación

CYP2C9*13

L90P

Disminución

Sin designación

CYP2C9*33

R132Q

Disminución

Sin designación

CYP2C9*14

R125H

Disminución

Sin designación

CYP2C9*15

S162X

Ausente

Sin designación

CYP2C9*34

R335Q

Desconocido

Sin designación

CYP2C9*16

T299A

Disminución

Sin designación

I112L

Desconocido

rs5030781

CYP2C9*17

P382S

Desconocido

Sin designación

R124Q

Desconocido

rs12414460

CYP2C9*18

I359L; D397A

Disminución

rs1057910; sin designación

Sin designación

R150C

Desconocido

rs17847037

Sin designación

P337L

Desconocido

rs58368927

CYP2C9*19

Q454H

Desconocido

Sin designación

Y358C

Desconocido

rs1057909

CYP2C9*20

G70R

Desconocido

Sin designación

L413P

Desconocido

rs28371687

CYP2C9*21

P30L

Desconocido

Sin designación

L447F

Desconocido

rs59485260

para la población general que el ibuprofeno. Con el caso del naproxeno, el efecto es aún más llamativo. El naproxeno está considerado como un AINE de riesgo intermedio para el desarrollo de HDA; sin embargo, en este 10% de personas que son portadoras de variantes de CYP2C8 y/o CYP2C9 en homocigosidad, el riesgo de presentar HDA con naproxeno se multiplica en un factor de casi 5, lo que convierte a este fármaco en uno de los AINEs con mayor riesgo 20-23. Por los motivos expuestos, resulta de extraordinario interés saber si un paciente pertenece a ese 40% de portadores de mutaciones, y especialmente si pertenece a ese 10% de portadores homocigotos, antes de iniciar un tratamiento con AINEs. Existen varios AINEs alternativos en los que las variantes de CYP2C8 y CYP2C9 tienen una escasa relevancia, como la aspirina, el paracetamol, los coxibs etoricoxib, rofecoxib o parecoxib, el ketoprofeno o la dipirona (metamizol). En los pacientes con alteraciones en los genes CYP2C8 y/o CYP2C9, deberían utilizarse estos AINEs en lugar de aquellos cuya farmacocinética o sus efectos adversos se asocian a las variaciones genéticas de CYP2C8 y CYP2C9.

Una buena predicción del riesgo genético puede incrementar la calidad de vida y la seguridad de estos pacientes. Uno de los primeros objetivos de la farmacogenómica es el ajuste de la dosis de acuerdo a la capacidad metabólica del paciente. Este objetivo es especialmente relevante cuando hay pocos tratamientos alternativos, por ejemplo, con determinados antineoplásicos. Pero, afortunadamente, la farmacología actual dispone de un amplio rango de AINEs que nos permite simplemente seleccionar otros tratamientos más seguros para los pacientes con un factor genético de riesgo. Es previsible, y deseable, que en breve, antes de iniciar un tratamiento con AINEs, los médicos sepamos a qué tipo genético de paciente estamos tratando para seleccionar no sólo la dosis más adecuada, sino también el fármaco más seguro para este paciente en concreto. De este modo, la farmacogenómica, con el concurso de otras disciplinas que ahora están en sus inicios, como la toxicogenómica y la metabolómica, nos ayudará a llegar al objetivo de una medicina lo más personalizada, segura y eficaz posible. 

Prof. Dr. J.A. García-Agúndez jagundez@unex.es

diciembre 2009

Farmacogen贸mica de los AINEs 1.

Meyer UA. Pharmacogenetics - five decades of therapeutic lessons from genetic diversity. Nat Rev Genet 2004;5(9):669-76.

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Tramer MR, Moore RA, Reynolds DJ, McQuay HJ. Quantitative estimation of rare adverse events which follow a biological progression: a new model applied to chronic NSAID use. Pain 2000;85(1-2):169-82.

Martinez C, Garcia-Martin E, Blanco G, Gamito FJ, Ladero JM, Agundez JA. The effect of the cytochrome P450 CYP2C8 polymorphism on the disposition of (R)-ibuprofen enantiomer in healthy subjects. Br J Clin Pharmacol 2005;59(1):62-9.

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Walan A, Wahlqvist P. Pharmacoeconomic aspects of non-steroidal anti-inflammatory drug gastropathy. Ital J Gastroenterol Hepatol 1999;31 Suppl 1:S79-88.

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Rahme E, Joseph L, Kong SX, Watson DJ, LeLorier J. Cost of prescribed NSAID-related gastrointestinal adverse events in elderly patients. Br J Clin Pharmacol 2001;52(2):185-92.

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Garcia-Martin E, Martinez C, Ladero JM, Gamito FJ, Agundez JA. High frequency of mutations related to impaired CYP2C9 metabolism in a Caucasian population. Eur J Clin Pharmacol 2001;57(1):47-9.

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Pilotto A, Seripa D, Franceschi M, et al. Genetic susceptibility to NSAID-related gastroduodenal bleeding: Role of cytochrome P450 (CYP) 2C9 polymorphisms. Gastroenterology 2007;doi:10.1053/j.gastro.2007.05.025.

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Vonkeman HE, van de Laar MA, van der Palen J, Brouwers JR, Vermes I. Allele variants of the cytochrome P450 2C9 genotype in white subjects from The Netherlands with serious gastroduodenal ulcers attributable to the use of NSAIDs. Clin Ther 2006;28(10):1670-6.

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Blanco G, Martinez C, Ladero JM, et al. Interaction of CYP2C8 and CYP2C9 genotypes modifies the risk for nonsteroidal anti-inflammatory drugsrelated acute gastrointestinal bleeding. Pharmacogenet Genomics 2008;18(1):37-43.

21.

Agundez JA, Garcia-Martin E, Martinez C. Genetically based impairment in CYP2C8- and CYP2C9dependent NSAID metabolism as a risk factor for gastrointestinal bleeding: is a combination of pharmacogenomics and metabolomics required to improve personalized medicine? Expert Opin Drug Metab Toxicol 2009;5(6):607-20.

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Agundez JA, Martinez C, Garcia-Martin E, Ladero JM. Cytochrome P450 CYP2C9 polymorphism and NSAID-related acute gastrointestinal bleeding. Gastroenterology 2007;133(6):2071-2.

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Martinez C, Blanco G, Ladero JM, et al. Genetic predisposition to acute gastrointestinal bleeding after NSAIDs use. Br J Pharmacol 2004.

Garcia-Martin E, Martinez C, Tabares B, Frias J, Agundez JA. Interindividual variability in ibuprofen pharmacokinetics is related to interaction of cytochrome P450 2C8 and 2C9 amino acid polymorphisms. Clin Pharmacol Ther 2004;76(2):119-27.

Martinez C, Blanco G, Ladero JM, et al. Genetic predisposition to acute gastrointestinal bleeding after NSAIDs use. Br J Pharmacol 2004;141(2):205-8.

Sanderson S, Emery J, Higgins J. CYP2C9 gene variants, drug dose, and bleeding risk in warfarintreated patients: a HuGEnet systematic review and meta-analysis. Genet Med 2005;7(2):97104.

Garcia-Martin E, Martinez C, Ladero JM, Agundez JA. Interethnic and intraethnic variability of CYP2C8 and CYP2C9 polymorphisms in healthy individuals. Mol Diagn Ther 2006;10(1):29-40.

Singh G, Triadafilopoulos G. Epidemiology of NSAID induced gastrointestinal complications. J Rheumatol Suppl 1999;56:18-24.

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Tarone RE, Blot WJ, McLaughlin JK. Nonselective nonaspirin nonsteroidal anti-inflammatory drugs and gastrointestinal bleeding: relative and absolute risk estimates from recent epidemiologic studies. Am J Ther 2004;11(1):17-25.

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Referencias Bibliogr谩ficas

Lanas A, Perez-Aisa MA, Feu F, et al. A nationwide study of mortality associated with hospital admission due to severe gastrointestinal events and those associated with nonsteroidal antiinflammatory drug use. Am J Gastroenterol 2005;100(8):1685-93. Olsen KM. Use of acid-suppression therapy for treatment of non-variceal upper gastrointestinal bleeding. Am J Health Syst Pharm 2005;62(10 Suppl 2):S18-23.

HepatoSar庐 es el primer nutrac茅utico compuesto por un 57% de una estructura lipoproteica natural de origen marino (S. pilchardus) y un extracto vegetal de Cynara scolymus L. estandarizado al 5%, que aporta propiedades muy beneficiosas sobre la funci贸n hepatobiliar y la digesti贸n de las grasas.

Genómica de la patología cerebrovascular Dr. Juan Carlos Carril Departamento de Genómica e Identificación Humana Euroespes Biotecnología, Bergondo, Coruña, España

E 70

l accidente cerebrovascular (ACV) se pro-

duce por la interacción de múltiples factores ambientales y mutaciones en diferentes genes. Estos polimorfismos genéticos determinan la susceptibilidad o la resistencia a la enfermedad y la respuesta al tratamiento. Debido a la interacción entre genes y ambiente, las enfermedades complejas se pueden prevenir a través de la actuación sobre

los factores ambientales con un plan de prevención adecuado, siempre y cuando se tenga conocimiento del riesgo genético relativo de padecer la enfermedad, lo que se consigue con un buen panel genético de susceptibilidad. La definición del panel de riesgo genético cerebrovascular pasa por abordar el estudio

de genes implicados en los diferentes eventos que desencadenan el proceso aterogénico: metabolismo lipídico, respuesta inmunitaria y estabilidad de la placa de ateroma. La determinación de los factores genéticos y sus interacciones con factores ambientales que ocasionan el desarrollo de la enfermedad, tan sólo es la mitad del camino hacia la obtención

de un test genético predictivo de utilidad en la práctica diagnóstica. La validación del panel genético, la determinación de diferencias entre individuos enfermos y controles sanos, así como la elección del modelo estadístico adecuado, es la clave para obtener una herramienta predictiva de utilidad real en la práctica médica. diciembre 2009

Genómica de la patología cerebrovascular

Accidente Cerebrovascular

Un accidente cerebrovascular (ACV), ictus o infarto cerebral consiste en la alteración permanente o transitoria de la función cerebral que aparece como consecuencia de un trastorno circulatorio, bien de los vasos cerebrales o bien de alteraciones hemáticas. La incidencia del accidente cerebrovascular, igual que otras enfermedades, es variable en diferentes países y tiene relación con factores genéticos, edad de la población y factores ambientales asociados1. La incidencia de nuevos casos en España se sitúa alrededor de 156 por 100.000

habitantes, aunque es presumible que estén alrededor de los 200 casos anuales2. Existen muy pocos datos sobre la prevalencia de ictus en España, con frecuencias que oscilan entre el 2.1% en la población mayor de 20 años hasta el 8.5% en la población mayor de 65 años3. La mortalidad por ictus en España oscila entre un 10% y un 34% en las estadísticas hospitalarias, siendo mucho más elevada en los casos de hemorragia cerebral4. El accidente cerebrovascular es un episodio neurológico agudo, con afectación de las funciones del sistema nervioso central. Según su etiología se suelen clasificar en:

Accidentes isquémicos:

También se llaman infartos cerebrales y se deben a la oclusión de alguna de las arterias que irrigan la masa encefálica, generalmente por aterosclerosis. Podemos distinguir: a.

Accidente isquémico transitorio.- Episodio de déficit focal de la circulación cerebral, de comienzo brusco, con alteraciones que duran generalmente unos 2-10 minutos pero que pueden persistir hasta las 24 horas.

Déficit neurológico isquémico reversible.- La duración del cuadro deficitario es superior a las 24 horas, pero los síntomas y signos clínicos desaparecen de forma total durante las tres semanas siguientes al episodio.

Infarto cerebral.- Como consecuencia de la falta de aporte circulatorio a un territorio cerebral se presenta un déficit neurológico, de duración superior a 24 horas. El infarto puede ser silente, pero generalmente da manifestaciones clínicas neurológicas según el territorio afectado.

Infarto cerebral de tipo aterotrombótico.- La lesión de la pared del vaso determina una estenosis u oclusión de la luz arterial y se produce una lesión dentro de su territorio de irrigación que puede ser total o parcial, dependiendo de la posible compensación de la circulación colateral.

Accidentes hemorrágicos:

También se denominan hemorragias cerebrales o apoplejías y se deben a la ruptura de un vaso sanguíneo encefálico debido a un pico hipertensivo o a un aneurisma congénito. Tipos de hemorragias cerebrales:

Infarto cerebral por embolismo de origen cardíaco.- La lesión de las válvulas cardíacas, del miocardio y/o los trastornos del ritmo cardíaco dan origen a trombos que llegan a las arterias cerebrales.

Infarto hemorrágico cerebral.- Sobre la lesión isquémica se produce un fondo hemorrágico por alteración de la barrera hematoencefálica en una zona de reperfusión, generalmente tras la lisis del émbolo.

Infarto lacunar.- Es un infarto pequeño, de menos de 15 mm, situado en las áreas profundas del cerebro o del tronco cerebral, que se produce por la oclusión de las ramas perforantes de las arterias cerebrales.

Hemorragia intracerebral.- Es una colección hemática dentro del parénquima encefálico debido a la rotura de un vaso encefálico.

Aterogénesis

La aterosclerosis es un síndrome caracterizado por el depósito de sustancias lipídicas, llamado placa de ateroma, en las paredes de las arterias de mediano y grueso calibre. El término “aterosclerosis” proviene de los vocablos griegos athero (pasta) y skleros (duro/piedra). No debe confundirse con arterioesclerosis, ya que esta última se refiere al endurecimiento de las paredes arteriales - arterio de arteria, esclerosis de endurecimiento - y en todo caso, el término arterioesclerosis abarca varias afecciones que llevan al endurecimiento, incluyendo la aterosclerosis.

Fig. 1 5 Fases iniciales de la lesión aterosclerótica: Oxidación de LDL, migración celular y formación de células espumosas.

La aterosclerosis puede ser considerada como una forma de inflamación crónica resultado de la interacción entre lipoproteínas modificadas, macrófagos derivados de monocitos, células T, y los elementos celulares normales de la pared arterial. Este proceso inflamatorio puede dar lugar a la formación de lesiones complejas, o placas, que resaltan en el lumen arterial. La ruptura de la placa y la trombosis dan lugar a complicaciones clínicas agudas como el infarto de miocardio y el ictus5. A mediados del siglo XIX, Virchow6 describió el engrosamiento de la íntima debido al acúmulo de cristales de colesterol extracelular, así como en el interior de las llamadas “células espumosas”, como característica histológica clave en la aterosclerosis. De este modo, formuló la denominada como “Hipótesis de Infiltración”, en la que se establece la aterosclerosis como el resultado de la infiltración de lípidos y células inflamatorias procedentes de la sangre.

Fig. 2 5 Evolución de la lesión aterosclerótica: Respuesta inmunitaria, migración de células de músculo liso y formación de la capa fibrosa.

En 1972, Ross y Glomset7 establecieron la denominada “Hipótesis de Respuesta a la Lesión”, según la cual la pared vascular responde, por disfunción endotelial y proliferación de células vasculares de músculo liso, cuando es dañada mecánicamente por flujo anormal o por otros agentes nocivos como el tabaquismo o las proteínas glicoxiladas de pacientes con diabetes. Basándose en el trabajo sobre los mecanismos de incorporación celular del colesterol de Goldstein y Brown8 (por el que recibieron el Premio Nobel), Steinberg et al.9 postularon la denominada “Hipótesis del LDL modificado”, según la cual las modificaciones de LDL-colesterol, incluida la oxidación, aumentan su captación por los macrófagos dando lugar a las células espumosas.

Fig. 3 5 Fases tardías de la lesión aterosclerótica: Muerte celular programada, formación de las “gachas” de lípido extracelular y agregación plaquetaria.

diciembre 2009

Factores de riesgo de enfermedad vascular

Entre los muchos factores de riesgo ambientales y genéticos, los niveles elevados de colesterol en suero son, por sí solos, suficientes para provocar el desarrollo aterosclerótico, incluso en ausencia de otros factores de riesgo. El estudio de los mecanismos moleculares que controlan la biosíntesis de colesterol y los niveles de colesterol en suero condujo al desarrollo de las “estatinas”, una potente clase de drogas que reducen el colesterol y que se han demostrado efectivas en la reducción de la mortalidad cardiovascular en pacientes con hipercolesterolemia. Otro factor de riesgo relevante es la hipertensión arterial, que provoca fuerzas de cizallamiento que rompen el frágil endotelio que recubre la superficie interior de las arterias. Por otra parte, se ha descrito que las hormonas masculinas son aterogénicas, mientras que los estrógenos protegen de la aterosclerosis, por eso las mujeres se afectan después de la menopausia. Este factor, junto con la edad, no depende del estilo de vida. En definitiva, podemos enumerar los principales factores de riesgo genéticos y su grado de heredabilidad como:

•

LDL y VLDL colesterol elevados (40%–60%) (Hipercolesterolemia).

•

HDL colesterol bajo (45%–75%).

•

Triglicéridos elevados (40%–80%).

•

Índice de Masa Corporal incrementado (25%–60%) (Obesidad).

•

Presión sistólica elevada (50%–70%) (Hipertensión arterial).

•

Presión diastólica elevada (50%–65%).

•

Lipoproteína (a) elevada (90%).

•

Homocisteína elevada (45%).

•

Diabetes mellitus tipo 2 (40%–80%).

•

Fibrinógeno elevado (20%–50%).

•

Proteína C-reactiva elevada (40%).

•

Sexo (Hormonas sexuales).

Entre los factores de riesgo modificables, relacionados con hábitos de vida, podemos destacar el tabaquismo y la vida sedentaria. Las sustancias tóxicas que contiene el tabaco, como la nicotina, tienen un efecto tóxico directo sobre la pared de las arterias, provocando una respuesta inflamatoria. Otros factores de riesgo no genético serían: la dieta, las infecciones víricas y bacterianas, el ambiente fetal y la polución ambiental.

Evolución de la lesión aterosclerótica

Las lesiones ateroscleróticas comienzan como una línea grasa bajo el endotelio de las grandes arterias. La llegada de macrófagos y colesterol derivado de LDL son los principales eventos celulares que contribuyen a la formación de la línea grasa. El primer evento de carácter aterogénico consiste en la oxidación del LDL-colesterol. Los lípidos y moléculas de ApoB que forman parte del LDL sufren modificaciones oxidativas mínimas y da lugar al mmLDL, que todavía es reconocido por los receptores de LDL (Fig. 1). Cuando estas modificaciones se hacen más extensivas, ApoB se fragmenta y los residuos de lisina se modifican covalentemente con reactivos producto de la fragmentación de lípidos oxidados, dando lugar a oxLDL, que ya no tiene capacidad para unirse a los receptores de LDL, y es capturado por los receptores limpiadores (“scavenger receptors”) que expresan en macrófagos y células musculares lisas. Las células endoteliales, en respuesta al estímulo inflamatorio que provoca el oxLDL, generan moléculas de adhesión celular en la superficie y tiene lugar el reclutamiento de monocitos a las regiones propensas a lesiones de las grandes arterias y su subsiguiente diferenciación en macrófagos. Este fenómeno de migración a la íntima, si bien puede cumplir inicialmente una función protectiva eliminando partículas oxLDL proinflamatorias y citotóxicas y células apoptóticas, finalmente contribuye en el desarrollo de lesiones ateroscleróticas. A continuación se desarrollan las denominadas “células espumosas” o macrófagos “foam cell”, que contienen cantidades masivas de ésteres de colesterol. La transición desde la relativamente simple línea grasa hasta la lesión más compleja se caracteriza por la inmigración de células de músculo liso desde la capa media de la pared arterial

Genes y polimorfismos relacionados con enfermedad vascular Símbolo

Gen

Locus

Polimorfismo

ABCA1

ATP-binding cassette, subfamily A, member 1

9q22-q31

T-477C

ABCA1

ATP-binding cassette, subfamily A, member 1

9q22-q31

G1051A (Arg219Lys)

rs2230806

ABCA1

ATP-binding cassette, subfamily A, member 1

9q22-q31

A2583G (Ile823Met)

rs4149313

ACE

Angiotensin I- converting enzyme

17q23

A-240T

rs4291

ACE

Angiotensin I- converting enzyme

17q23

Intron 16 Alu 287bp I/D

ADRB1

Beta-1-adrenergic receptor

10q24-q26

G1165C (Gly389Arg)

rs1801253

ADRB2

Beta-2-adrenergic receptorHA

5q32-q34

A46G (Arg16Gly)

rs1042713

ADRB2

Beta-2-adrenergic receptor

5q32-q34

C79G (Gln27Glu)

rs1042714

ADRB3

Beta-3-adrenergic receptor

8p12-p11.2

T190C (Trp64Arg)

rs4994

AGT

Angiotensinogen

1q42-q43

G-6A

rs5051

AGT

Angiotensinogen

1q42-q43

M235T

rs699

AGT

Angiotensinogen

1q42-q43

T174M

rs4762

AGTR1

Angiotensin receptor 1

3q21-q25

C-535T

rs1492078

AGTR1

Angiotensin receptor 1

3q21-q25

A1166C

rs5186

AGTR1

Angiotensin receptor 1

3q21-q25

G→A (Ala163Thr)

rs12721226

AGTR1

Angiotensin receptor 1

3q21-q25

G→T (Ala244Ser)

rs12721225

AGTR1

Angiotensin receptor 1

3q21-q25

A→C (Thr336Pro)

rs1801021

AGTR2

Angiotensin II receptor, type 2

Xq22-q23

G1675A

rs1403543

AGTR2

Angiotensin II receptor, type 2

Xq22-q23

C3123A

rs11091046

ALOX5

Arachidonate 5-lipoxygenase

10q11.2

C3175G

rs12762604

ALOX5

Arachidonate 5-lipoxygenase

10q11.2

Sp1 STR

ALOX5

Arachidonate 5-lipoxygenase

10q11.2

G→A (Phe42Leu)

rs12762604

ALOX5

Arachidonate 5-lipoxygenase

10q11.2

G→A (Glu254Lys)

rs2228065

ANXA5

Annexin A5

4q26-q28

C-1T

rs11575945

AP2M1

Adaptor-related protein complex 2, MU-1 subunit

3q28

G62T

rs1501299

APOA1

Apolipoprotein A-I

11q23

G-75A

rs670

APOA1

Apolipoprotein A-I

11q23

T84C

rs5070

APOA5

Apolipoprotein A-V

11q23

T-1131C

rs662799

APOB

Apolipoprotein B

2p24

C2488T (XbaI)

APOB

Apolipoprotein B

2p24

G10708A (Arg3500Gln)

APOB

Apolipoprotein B

2p24

C10800T (Arg3531Cys)

APOC3

Apolipoprotein C-III

11q23

C-482T

rs2854117

APOC3

Apolipoprotein C-III

11q23

C1100T

rs4520

APOC3

Apolipoprotein C-III

11q23

C3175G (S1/S2)

rs5128

Ciencia

Tabla 1

dbSNP

Continúa 6

diciembre 2009

Genómica de la patología cerebrovascular Continuación Tabla 1 Símbolo

Gen

Locus

Polimorfismo

dbSNP

APOE

Apolipoprotein E

19q13.2

G-219T

rs405509

APOE

Apolipoprotein E

19q13.2

T3932C (Cys112Arg)

rs429358

APOE

Apolipoprotein E

19q13.2

C4070T (Arg158Cys)

rs7412

CAPN10

Calpain 10

2q37.3

G4852A

rs3792267

CCL2

Chemokine, CC motif, ligand 2

17q11.2-q12

A-2518G

CCL5

Chemokine, CC motif, ligand 5

17q11.2-q12

C-28G

rs2280788

CCL5

Chemokine, CC motif, ligand 5

17q11.2-q12

G-403A

rs2107538

CCL11

Chemokine, CC motif, ligand 11

17q21.1-q21.2

G→A (Ala23Thr)

rs3744508

CCND1

Cyclin D1

11q13

G→T (Ala30Ser)

rs2220247

CCR2

Chemokine, CC motif, receptor 2

3p21

G190A (Val64Ile)

rs1799864

CCR5

Chemokine, CC motif, receptor 5

3p21

G59029A

rs1799987

CD14

Monocyte differentiation antigen CD14

5q31.1

C-260T

rs2569190

CD36

CD36 antigen

7q11.2

G30294C

rs1049673

CD36

CD36 antigen

7q11.2

C12293T (Pro90Ser)

CD40

CD40 antigen

20q12-q13.2

A455T

CETP

Cholesteryl ester transfer protein, plasma

16q21

G279A (TaqB1/B2)

CETP

Cholesteryl ester transfer protein, plasma

16q21

C-629A

rs1800775

CETP

Cholesteryl ester transfer protein, plasma

16q21

A1061G (Ile405Val)

rs5882

COL1A2

Collagen, type I, alpha-2

7q22.1

G→C (Ala459Pro)

rs42524

COL3A1

Collagen, type III, alpha-1

2q31

G2209A (Ala698Thr)

rs1800255

COL3A1

Collagen, type III, alpha-1

2q31

A3730G (Ile1205Val)

rs2271683

CRP

C-reactive protein, pentraxin-related

1q21-q23

C1444T

rs1130864

CX3CR1

Chemokine, CX3C motif, receptor 1

3pter-p21

C926T (Thr280Met)

rs3732378

CXCL16

Chemokine, CXC motif, ligand 16

17p13

C→T (Ala181Val)

rs2277680

ELN

Elastin

7q11.2

G1264A (Gly422Ser)

rs2071307

EPHX2

Epoxide hydrolase 2, cytosolic

8p21-p12

G→A (Arg287Gln)

rs751141

ESR1

Estrogen receptor 1

6q25.1

T-1989G

rs2071454

Coagulation factor III

1p22-p21

A-603G

rs1361600

Factor VII

13q34

G11496A (Arg353Gln)

rs6046

F12

Factor XII

5q33-qter

C46T

rs17876008

FABP2

Fatty acid-binding protein 2

4q28-q31

G2445A (Ala54Thr)

rs1799883

FBN1

Fibrillin 1

15q21.1

T1875C

rs25458

Símbolo

Gen

Locus

Polimorfismo

dbSNP

FGB

Fibrinogen, B beta polypeptide

4q28

G-455A

rs1800790

FGB

Fibrinogen, B beta polypeptide

4q28

G8059A (Arg448Lys)

rs4220 Continúa 6

Símbolo

Gen

Locus

Polimorfismo

dbSNP

HNF4A

Hepatocyte nuclear factor 4-alpha

20q12-q13.1

A→G

rs2425640

ICAM1

Intercellular adhesion molecule 1

19p13.3-p13.2

G1462A (Glu469Lys)

rs5498

IGF2R

Insulin-like growth factor II receptor

6q26

A5002G (Arg1619Gly)

rs629849

IL1B

Interleukin 1-beta

2q14

C-511T

rs16944

IL1RN

Interleukin 1 Receptor Antagonist

2q14.2

IL1RN*2 VNTR

IL6

Interleukin 6

7p21

G-174C

IL6

Interleukin 6

7p21

G-572C

IL10

Interleukin 10

1q31-q32

A-1082G

IL10

Interleukin 10

1q31-q32

T-819C

rs1800871

IL10

Interleukin 10

1q31-q32

A-592C

rs1800872

INS

Insulin

11p15.5

T-23A

rs689

INSR

Insulin receptor

19p13.2

C7067365A

rs2860172

IPF1

Insulin promoter factor 1

13q12.1

-108/3G→4G

(S82168)

IRS1

Insulin receptor substrate 1

2q36

A3694G (Ser892Gly)

rs1801277

IRS1

Insulin receptor substrate 1

2q36

G3931A (Gly972Arg)

rs1801278

ITGA2

Integrin, alpha-2

5q23-q31

A1648G (Lys505Glu)

rs10471371

ITGB2

Integrin, beta-2

21q22.3

C1323T

rs235326

LDLR

Low density lipoprotein receptor

19p13.2

G1184A (Ala370Thr)

rs11669576

LMNA

Lamin A/C

1q21.2

A→G (Glu2Gly)

rs11549669

LPL

Lipoprotein lipase

8p22

C1595G (Ser447Stop)

rs328

MMP1

Matrix metalloproteinase 1

11q22-q23

-1607/1G→2G

rs1799750

MMP1

Matrix metalloproteinase 1

11q22-q23

A-519G

MMP1

Matrix metalloproteinase 1

11q22-q23

T-340C

MMP2

Matrix metalloproteinase 2

16q13

C-1306T

rs243865

MMP3

Matrix metalloproteinase 3

11q23

-1171/5A→6A

rs3025058

MMP3

Matrix metalloproteinase 3

11q23

indel-1612A

MMP3

Matrix metalloproteinase 3

11q23

A-709G

MMP3

Matrix metalloproteinase 3

11q23

A→G (Lys45Glu)

rs679620

MMP3

Matrix metalloproteinase 3

11q23

A→C (His113Pro)

rs11606831

MMP9

Matrix metalloproteinase 9

20q11.2-q13.1

C-1562T

rs3918242

MMP9

Matrix metalloproteinase 9

20q11.2-q13.1

G855A (Arg279Gln)

rs2664538

MMP12

Matrix metalloproteinase 12

11q22.2-q22.3

A-82G

rs2276109

MPO

Myeloperoxidase

17q23.1

G-463A

(NT_035425)

MPO

Myeloperoxidase

17q23.1

G-129A

(AH002972)

MTHFR

5,10-Methylenetetrahydrofolate reductase

1p36.3

C677T (Ala222Val)

rs1801133

Ciencia

Continuación Tabla 1

rs1800796

Continúa 6

diciembre 2009

Genómica de la patología cerebrovascular Continuación Tabla 1 Símbolo

Gen

Locus

Polimorfismo

dbSNP

NFKB

Nuclear Factor Kappa B, subunit 1

4q23-q24

indel-94ATTG

NOS3

Nitric oxide synthase 3

7q36

G37498A

NOS3

Nitric oxide synthase 3

7q36

T-786C

NOS3

Nitric oxide synthase 3

7q36

4a/4b

NOS3

Nitric oxide synthase 3

7q36

G894T

NPY

Neuropeptide Y

7p15.1

T1128C (L7P)

OLR1

Low density lipoprotein, oxidized, receptor 1

12p13-p12

G501C (Lys167Asn)

rs11053646

P2RY12

Purinergic receptor P2Y, G protein-coupled, 12

3q24-q25

T744C

NC_000003

PAI1

Plasminogen activator inhibitor 1

7q21.3-q22

-668/4G→5G

rs1799768

PAI1

Plasminogen activator inhibitor 1

7q21.3-q22

A→C (His25Pro)

rs2227647

PAI1

Plasminogen activator inhibitor 1

7q21.3-q22

G→A (Arg209His)

rs2227669

PAI1

Plasminogen activator inhibitor 1

7q21.3-q22

A→G (Tyr243Cys)

rs13306846

PAX4

Paired box gene 4

7q32

C567T (Arg121Trp)

(AF043978)

PECAM1

Platelet-endothelial cell adhesion molecule 1

17q23

C1454G (Leu125Val)

rs668

PECAM1

Platelet-endothelial cell adhesion molecule 1

17q23

G2201A (Gly670Arg)

rs1131012

PIK3R1

Phosphatidylinositol 3-kinase, regulatory, 1

5q13

G1020A (Met326Ile)

rs3730089

PON1

Paraoxonase 1

7q21.3

G-162A

rs705381

PON1

Paraoxonase 1

7q21.3

A532G (Arg160Gly)

rs13306698

PON1

Paraoxonase 1

7q21.3

G584A (Gln192Arg)

rs662

PON2

Paraoxonase 2

7q21.3

C475G (Ala148Gly)

rs11545941

PPARD

Peroxisome proliferator-activated receptor-delta

6p21.2-p21.1

T294C

rs2016520

PPARG

Peroxisome proliferator-activated receptor-gamma

3p25

C-681G

rs10865710

PPARG

Peroxisome proliferator-activated receptor-gamma

3p25

C34G (Pro12Ala)

rs1801282

PPARGC1

Peroxisome proliferator-activated receptor-gamma, coactivator 1 4p15.1

G1564A (Gly482Ser)

rs8192678

SAH

Hypertension-associated SA, rat, homolog of

16p13.11

A→G (-7 from exon 13)

rs13306607

SELE

Selectin E

1q23-q25

A561C (Ser128Arg)

rs5361

SELP

Selectin P

1q23-q25

A37674C (Thr715Pro)

rs6136

SELP

Selectin P

1q23-q25

G→T (Val640Leu)

rs6133

SRA

Scavenger Receptor A1

8p22

C877T

SRB1

Scavenger Receptor B1

12q24.31

G2S

SREBF1

Sterol regulatory element-binding transcription factor 1

17p11.2

indel-36G

(AX977070)

TCF1

Transcription factor 1

12q24.2

C→T (Ala98Val)

rs1800574

TGFB1

Transforming growth factor, beta-1

19q13.1

C-509T

rs1800469

TGFBR2

Transforming growth factor-beta receptor, type II

3p22

C1167T (Asn389Asn)

rs2228048

THBD

Thrombomodulin

20p11.2

C2136T (Ala455Val)

rs1042579

rs2070744

Continúa 6

Símbolo

Gen

Locus

Polimorfismo

dbSNP

THBS2

Thrombospondin II

6q27

T3949G

rs8089

THBS4

Thrombospondin IV

5q13

G1186C (Ala387Pro)

rs1866389

THPO

Thrombopoietin

3q26.3-q27

A5713G

rs6141

TLR4

Toll-like receptor 4

9q32-q33

A896G

TLR4

Toll-like receptor 4

9q32-q33

A2326G (Asp299Gly)

rs4986790

TNF

Tumor necrosis factor

6p21.3

C-863A

rs1800630

TNF

Tumor necrosis factor

6p21.3

C-850T

rs1799724

TNF

Tumor necrosis factor

6p21.3

G-238A

rs361525

TNFRSF1A

Tumor necrosis factor receptor 1

12p13.2

R92Q

TNFSF4

Tumor necrosis factor ligand superfamily, member 4

1q25

A→G

VEGF

Vascular endothelial growth factor

6p12

A-2518G

VEGF

Vascular endothelial growth factor

6p12

C936T

pasando la lámina elástica interna hasta la íntima o el espacio subendotelial (Fig. 2). Las células de músculo liso de la íntima pueden proliferar y captar lipoproteínas modificadas contribuyendo a la formación de células espumosas, así como sintetizar proteínas de matriz extracelular para la formación de la capa fibrosa. Esta fase del desarrollo de la lesión está influenciada por interacciones entre monocitos/ macrófagos y células T dando lugar a un amplio rango de respuestas celulares y humorales y a la adquisición de muchas características del estado inflamatorio crónico.

Ciencia

Continuación Tabla 1

rs3850641

rs3025039

La llegada de macrófagos y colesterol derivado de LDL son los principales eventos celulares que contribuyen a la formación de la línea grasa

Aunque las lesiones ateroscleróticas avanzadas pueden dar lugar a síntomas isquémicos como resultado del progresivo angostamiento del lumen del vaso, los eventos cardiovasculares agudos que resultan en infarto de miocardio e ictus se achacan generalmente a la ruptura de la placa y trombosis. La ruptura de la placa expone los lípidos de la placa y factores tisulares a los componentes sanguíneos, iniciando la cascada de coagulación, adherencia de plaquetas y trombosis (Fig. 3).

tienen lugar en las regiones laterales de la placa y ocurren más probablemente en lesiones con capas fibrosas finas, una relativamente elevada concentración de células espumosas en las regiones laterales, y grandes núcleos necróticos. La apoptosis de macrófagos y células musculares lisas aparece como resultado de interacciones célula-célula y composición de citoquinas en la pared arterial, involucrando la acción de proteínas pro- y antiapoptóticas que incluyen receptores de muerte celular, proto-oncogenes y genes supresores tumorales.

El análisis de la aterosclerosis humana sugiere que la evolución de las placas avanzadas puede involucrar ciclos repetitivos de microhemorragias y trombosis. Las roturas de la placa asociadas con infartos de miocardio, generalmente

La liberación de lípidos oxidados e insolubles por parte de las células necróticas contribuye a la formación de las características “gachas” de las lesiones avanzadas. La apoptosis de macrófagos y células de músculo liso pueden no sólo ser diciembre 2009

Genómica de la patología cerebrovascular importantes para determinar la capacidad de las lesiones en sufrir una regresión, sino también pueden influir en la estabilidad de la placa, así como los lípidos del núcleo necrótico pueden incrementar el potencial de trombosis. Las metaloproteasas de la matriz segregadas por los macrófagos en regiones de ruptura de la placa influyen en la estabilidad de la misma degradando proteínas de matriz extracelulares. Adicionalmente, se observa un acúmulo extensivo de fibrina en las lesiones más complejas, y se ha propuesto un descenso de la actividad fibrinolítica como acelerador de la aterogénesis arterial facilitando la trombosis y la deposición de fibrina durante el desarrollo de las lesiones ateroscleróticas. Tabla 2 Panel de riego cerebrovascular: Metabolismo Lipídico. METABOLISMO LIPÍDICO

La neovascularización es frecuente en lesiones ateroscleróticas asociadas con ruptura de la placa, hemorragias o angina inestable (episodios progresivos de isquemia cardiaca temporal debidos a formación de trombos transitorios). La angiogénesis ocurre en asociación con la remodelación y la activación de proteasas en tejidos circundantes, sugiriendo que la neovascularización puede contribuir a la inestabilidad y ruptura de la placa.

Genes de susceptibilidad

Aunque la aterosclerosis ha sido objeto de intensivas investigaciones epidemiológicas y patofisiológicas, son considerables las evidencias de que importantes determinantes de la susceptibilidad a la enfermedad permanecen sin identificar. La historia familiar es un importante factor de riesgo tras eliminar otros factores independientes previamente identificados, implicando factores genéticos adicionales (Tabla 1). Estudios de raros trastornos mendelianos como la hipercolesterolemia familiar y la enfermedad de Tangier podrían seguir facilitando la identificación de nuevos genes que influyen en el desarrollo de la aterosclerosis.

Símbolo

Gen

Locus

Polimorfismo

dbSNP

APOC3

Apolipoprotein C-III

11q23

C3175G (S1/S2)

rs5128

APOE

Apolipoprotein E

19q13.2

T3932C (Cys112Arg)

rs429358

APOE

Apolipoprotein E

19q13.2

C4070T (Arg158Cys)

rs7412

APOB

Apolipoprotein B

2p24

C7673T (Thr2488Thr, XbaI)

rs693

CETP

Cholesteryl ester transfer protein, plasma

16q21

G279A (TaqB1/B2)

rs708272

LPL

Lipoprotein lipase

8p22

C1595G (Ser447Stop)

rs328

La definición del panel de riesgo genético cerebrovascular pasa por abordar el estudio de genes implicados en los diferentes eventos que desencadenan el proceso aterogénico, es decir, metabolismo lipídico (modificación de LDL-colesterol), función endotelial, respuesta inmunitaria (reclutamiento de macrófagos y formación de células espumosas) y estabilidad de la placa de ateroma (trombosis).

Tabla 3

Panel de riesgo genético cerebrovascular

Panel de riego cerebrovascular: Función Endotelial e Hipertensión.

Panel de Metabolismo Lipídico

Función Endotelial e Hipertensión

Símbolo

Gen

NOS3

Locus

Polimorfismo

dbSNP

Nitric oxide synthase 3 7q36

G894T

rs1799983

ACE

Angiotensin Iconverting enzyme

17q23

C547T

rs4332

ACE

Angiotensin Iconverting enzyme

17q23

Intron 16 Alu 287bp I/D

AGT

Angiotensinogen

1q42-q43 Met235Thr

rs699

AGT

Angiotensinogen

1q42-q43 Thr174Met

rs4762

El panel de metabolismo lipídico aborda el estudio de genes implicados en la modificación de los niveles colesterol en sus distintas formas y su contribución en el proceso aterogénico como factor de riesgo vascular (Tabla 2). El polimorfismo C3175G (rs5128), también conocido como Sst I, se encuentra en la región 3’UTR del mRNA de APOC3. La variante 3175G (S2) está relacionada con mayor estabilidad y mayores niveles de expresión de ApoCIII, por lo que se relaciona con riesgo incrementado de enfermedad vascular debido a su implicación en el metabolismo de triglicéridos10.

Los niveles aumentados de ApoB se asocian directamente con las lipoproteínas aterógenas, VLDL, IDL y LDL. Se sintetiza principalmente en hígado e intestino. El alelo 7673C está asociado a menores niveles de triglicéridos, colesterol y colesterol LDL. Sin embargo, individuos portadores del alelo 7673T responden mejor a una dieta baja en grasa y colesterol, con una disminución significativamente mayor de sus niveles de LDL y ApoB.

Panel de riego cerebrovascular: Respuesta Inmunitaria.

Ciencia

Tabla 4 ApoE interviene en el catabolismo de proteínas ricas en triglicéridos y en la homeostasis del colesterol. La presencia del alelo e4 del gen APOE está ligada a niveles altos de colesterol y de betalipoproteínas, así como a la propensión a sufrir enfermedades cardiovasculares11. La presencia del alelo e2 está ligada a riesgo incrementado de Hiperlipoproteinemia tipo III, niveles altos de colesterol, triglicéridos y beta-VLDL, así como al desarrollo de aterosclerosis e incremento del riesgo vascular12.

RESPUESTA INMUNITARIA Símbolo

Gen

Locus

Polimorfismo

dbSNP

IL1B

Interleukin 1-beta

2q14

T5887C

rs1143634

IL6

Interleukin 6

7p21

G-174C

rs1800795

IL6

Interleukin 6

7p21

G-573C

rs1800796

IL6R

Interleukin 6 receptor

1q21

A1510C

rs8192284

TNFA

Tumor necrosis factor alpha

6p21.3

G-308A

rs1800629

Tabla 5 Panel de riego cerebrovascular: Trombosis.

CETP codifica para la proteína de transferencia de ésteres de colesterol, que facilita el intercambio de triglicéridos y ésteres de colesterol estimulando la recuperación de colesterol. El polimorfismo G+279A (rs708272) del gen CETP (también denominado TaqIB) está asociado con niveles bajos de colesterol HDL y niveles altos de actividad CETP en plasma (presencia del alelo +279G o B1), que contribuyen a un incremento en el riesgo de enfermedades cardiovasculares. La lipoproteína lipasa (LPL) desempeña una función clave en el metabolismo lipoproteico, hidrolizando los triglicéridos que forman parte del VLDL y los quilomicrones, así como eliminando las lipoproteínas de la circulación13. LPL influye en la interacción de las lipoproteínas aterogénicas con la superficie de la célula y con los receptores de la pared vascular14. Estudios recientes relacionan el polimorfismo C1421G (rs328) [S447X] (proteína truncada de 446 aminoácidos en lugar de 448) con un menor riesgo de padecer CAD, debido a su relación con un aumento de HDL y una disminución de triglicéridos15. Por lo tanto, la variante 447X tiene una mayor actividad enzimática, por lo que debería tener un efecto protector contra el desarrollo de la aterosclerosis y CAD posterior.

Panel de Función Endotelial e Hipertensión La transición desde la relativamente simple línea grasa hasta la lesión más compleja se caracteriza por la inmigración de células de músculo liso desde la capa media de la pared arterial pasando la lámina elástica interna hasta la íntima o el espacio subendotelial (Tabla 3).

TROMBOSIS Símbolo

Gen

Locus

Polimorfismo

dbSNP

Coagulation factor II (thrombin)

11p11

G20210A

rs1799963

Coagulation factor V proaccelerin)

1q23

G1691A

rs6025

MTHFR

5,10-Methylenetetrahydrofolate reductase

1p36.3

A1298C

rs1801131

MTHFR

5,10-Methylenetetrahydrofolate reductase

1p36.3

C677T (Ala222Val) rs1801133

El polimorfismo G894T (rs1799983): E298D, y concretamente la presencia del alelo 894T está asociada a una menor actividad del enzima NOS3, lo que implica un mayor riesgo vascular y una mayor susceptibilidad de padecer patologías cardiovasculares16. La enzima convertidora de angiotensina (ACE), es una dipeptidil carboxipeptidasa que desempeña un papel importante en la regulación de la presión arterial y en el balance de electrolitos y la presión sanguínea hidrolizando la angiotensina I en angiotensina II, un potente vasopresor, y un péptido estimulante de aldosterona. La enzima también es capaz de inactivar la bradicinina, un potente vasodilatador. Los polimorfismos a estudiar son C547T (rs4332) y la presencia (inserción, I) o ausencia (delección, D) de una secuencia alu repetitiva de 287 pb en el intrón 16 del gen está asociada a niveles circulantes de la enzima y a patologías diciembre 2009

Genómica de la patología cerebrovascular cardiovasculares. El alelo D (delección) está asociado a una alta predisposición a desarrollar hipertensión arterial esencial lo que favorece el padecimiento de otras patologías cardiovasculares17. El gen AGT codifica el angiotensinógeno, el cual, mediante la renina, se transforma en angiotensina I. Los alelos 235T y 174M están asociados con un mayor riesgo de sufrir hipertensión arterial esencial18, 19.

Panel de Respuesta Inmunitaria Actualmente se conoce el papel relevante de la inflamación en el inicio y progresión de la aterosclerosis20 y se sospecha que influye en el desarrollo trombótico activando el proceso de coagulación21 (Tabla 4). Se han descrito niveles incrementados de marcadores de inflamación con enfermedad vascular isquémica22, 23. Se postula la influencia de polimorfismos en IL1 en la modulación del patrón inflamatorio involucrado en la formación de trombos que pudiera desencadenar procesos arteriales isquémicos24. La interleuquina 6 (IL-6) es una citoquina pleiotrópica implicada en la regulación de la reacción de fase aguda, la respuesta inmune, y la hematopoyesis, pudiendo jugar un papel en la megacariocitopoyesis y producción plaquetaria. El polimorfismo G-174C (rs1800795) en la región 5’ parece estar asociado con diferencias en los niveles plasmáticos de IL-6 en voluntarios sanos. Fernández-Real et al.25 encontraron que los portadores del alelo -174G, que se asocia con mayor secreción de IL-6, tienen niveles incrementados de triglicéridos plasmáticos, VLDL y ácidos gra-

sos libres, así como niveles más bajos de HDL-colesterol. Por otra parte, se ha descrito una fuerte asociación entre el genotipo -174CC y el infarto lacunar26. El polimorfismo G-573C (rs1800796) en la región 5’ está significativamente asociado con infarto cerebral aterotrombótico y hemorragia intracerebral27. El factor de necrosis tumoral (TNF-alfa) es una citoquina proinflamatoria secretada predominantemente por monocitos y macrófagos y que afecta al metabolismo lipídico, coagulación, resistencia a insulina y función endotelial. Se han encontrado evidencias in vivo de la implicación de TNF-alfa en la hidrólisis de esfingomielina, producción de ceramida y apoptosis mediada por ceramida28. El polimorfismo G308A (rs1800629) se ha relacionado con niveles incrementados de cortisol en saliva y obesidad en individuos homocigotos AA29. También se ha descrito una asociación entre la variante -308G en homocigosis y un riesgo incrementado de padecer migraña30, debido probablemente al efecto de este polimorfismo sobre el flujo sanguíneo cerebral.

Panel de Trombosis Aunque las lesiones ateroscleróticas avanzadas pueden dar lugar a síntomas isquémicos como resultado del progresivo angostamiento del lumen del vaso, los eventos vasculares agudos que resultan en infarto de miocardio e ictus se achacan generalmente a la ruptura de la placa y trombosis (Tabla 5). El Factor II de coagulación o protrombina está implicado en la coagulación sanguínea. Esta proteína plasmática es la precursora de la trombina, responsable de la formación del coágulo. El po-

El Factor V de Leiden es uno de los factores implicados en la coagulación sanguínea. La función del Factor V es inactivada por la Proteína C, que constituye uno de los mecanismos anticoagulantes más importantes. La trombina, cuando se une a la trombomodulina en la superficie endotelial, activa a la proteína C y ésta a su vez, inactiva a los factores V y VIII. La mutación G1691A (rs6025): Arg506Gln en el gen F5, presenta una alta prevalencia en caucasoides, entre un 5 y un 10%. La presencia de la mutación 1691A impide la inactivación del factor V por parte de la proteína C, provocando un estado de hipercoagulabilidad y un aumento del riesgo trombótico. Algunos estudios sugieren un aumento de 50 a 100 veces en el riesgo de trombosis venosa para los portadores en homocigosis del alelo 506Q y de 5 a 10 veces para los portadores heterocigotos R506Q33. La metilentetrahidrofolato reductasa (MTHFR) cataliza la conversión de 5,10-metilentetrahidrofolato a 5-metiltetrahidrofolato, un cosustrato para la remetilación de homocisteína a metionina. El polimorfismo C677T (rs1801133): A222V da lugar a una proteína con actividad enzimática reducida y termolabilidad incrementada cuando aparece la variante 222V en homocigosis o heterocigosis. Los individuos 677TT presentan niveles en plasma de homocisteína elevados y tienen niveles de riesgo de padecer una enfermedad cardiovascular prematura hasta tres veces superiores al resto34. Otra mutación también relacionada con una reducción en la actividad enzimática es la A1298C (rs1801131): E429A, aunque este descenso en la actividad no parece estar relacionado con niveles plasmáticos de homocisteína incrementados ni concentraciones menores de folato en plasma como ocurre con los 677T homocigotos. Aumentar la ingesta de folato (0,8 mg de ácido fólico) reduce en un 16% el riesgo de cardiopatía isquémica y en un 24% el de accidente cerebrovascular35.

La pregunta que pretendemos responder es simple: ¿Cuál es el riesgo objetivo de un individuo sano de padecer la enfermedad?

Ciencia

limorfismo G20210A (rs1799963) se encuentra en el 3% de la población del sur de Europa. Esta alteración está relacionada con un aumento de los niveles plasmáticos de protrombina. Las personas que llevan una copia de esta mutación (alelo 20210A) tienen 6 veces más probabilidades de sufrir una trombosis31. Las mujeres embarazadas o tratadas con anticonceptivos tienen un riesgo 16,3 veces mayor de sufrir trombosis si son portadoras de la mutación32.

Capacidad predictiva del panel genético

En las enfermedades multifactoriales, la herencia genética así como los factores ambientales, físicos y de estilo de vida son determinantes en el establecimiento de un perfil de riesgo de enfermedad. La ponderación inadecuada de dichos factores, así como la baja o incompleta penetrancia, provocan resultados de asociaciones débiles en los estudios epidemiológicos, con resultados, muchas veces, no reproducibles e incluso contradictorios entre estudios. La pregunta que pretendemos responder es simple, lo complejo es llegar a una respuesta con suficientes garantías: ¿Cuál es el riesgo objetivo de un individuo sano (del que conocemos su perfil genético, sus características físicas y su estilo de vida) de padecer la enfermedad? La respuesta pasa por la elección de las herramientas de detección adecuadas (polimorfismos de interés) y del modelo de tratamiento de los datos que mejor se ajuste a la enfermedad en cuestión. La determinación de los factores genéticos y de las interacciones entre ellos y con los factores ambientales que desencadenan el desarrollo de la enfermedad, tan sólo es la mitad del camino hacia la obtención de un test genético predictivo de utilidad en la práctica diagnóstica. La validación del panel genético, es decir, la determinación de que las diferencias entre casos y controles son causales y no espúreas, así como la elección del modelo estadístico adecuado, es la clave para obtener una herramienta predictiva de utilidad real en la práctica médica. 

Dr. Juan C. Carril geneticaforense@ebiotec.com

diciembre 2009

Genómica de la patología cerebrovascular

Referencias Bibliográficas

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20. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917):868-74. 21. Luther T, Mackman N. Tissue factor in the heart. Multiple roles in hemostasis, thrombosis, and inflammation. Trends Cardiovasc Med. 2001; 11(8):307-12. 22. Danesh J, Appleby P. Persistent infection and vascular disease: a systematic review. Expert Opin Investig Drugs. 1998; 7(5):691-713. 23. Rost NS, Wolf PA, Kase CS et al. Plasma concentration of C-reactive protein and risk of ischemic stroke and transient ischemic attack: the Framingham study. Stroke. 2001; 32(11):2575-9. 24. Iacoviello L, Di Castelnuovo A, Gattone M et al. Polymorphisms of the interleukin-1ß gene affect the risk of myocardial infarction and ischemic stroke at young age and the response of mononuclear cells to stimulation in vitro. 2004. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 222-227. 25. Fernández-Real JM, Broch M, Vendrell J et al. Interleukin-6 gene polymorphism and lipid abnormalities in healthy subjects. J Clin Endocrinol Metab. 2000; 85(3):1334-9. 26. Revilla M, Obach V, Cervera A et al. A -174G/C polymorphism of the interleukine-6 gene in patients with lacunar infarction. Neurosci Lett. 2002; 324(1):29-32.

10. Paré G, Serre D, Brisson D et al. Genetic analysis of 103 candidate genes for coronary artery disease and associated phenotypes in a founder population reveals a new association between endothelin-1 and high-density lipoprotein cholesterol. Am J Hum Genet. 2007; 80(4):673-82.

27. Yamada Y, Metoki N, Yoshida H et al. Genetic risk for ischemic and hemorrhagic stroke. Aterosclerosis, thrombosis, and vascular biology 2006; 26 (8): 1920-5.

11. Pedro-Botet J, Sentí M, Nogués X et al. Lipoprotein and apolipoprotein profile in men with ischemic stroke. Role of lipoprotein(a), triglyceride-rich lipoproteins, and apolipoprotein E polymorphism. Stroke. 1992; 23(11):1556-62.

29. Rosmond R, Chagnon M, Bouchard C et al. G-308A polymorphism of the tumor necrosis factor alpha gene promoter and salivary cortisol secretion. J Clin Endocrinol Metab. 2001; 86(5):2178-80.

12. Breslow JL, Zannis VI, SanGiacomo TR, et al. Studies of familial type III hyperlipoproteinemia using as a genetic marker the apoE phenotype E2/2. J Lipid Res. 1982; 23(8):1224-35.

30. Rainero I, Grimaldi LM, Salani G et al. Association between the tumor necrosis factor-alpha -308 G/A gene polymorphism and migraine. Neurology 2004; 62(1):141-3.

13. Eckel RH. Lipoprotein lipase. A multifunctional enzyme relevant to common metabolic diseases. N Engl J Med. 1989; 320(16):1060-8. 14. Beisiegel U, Weber W, Bengtsson-Olivecrona G. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc Natl Acad Sci USA. 1991; 88(19):8342-6. 15. Stocks J, Thorn JA, Galton DJ. Lipoprotein lipase genotypes for a common premature termination codon mutation detected by PCR-mediated site-directed mutagenesis and restriction digestion. J Lipid Res. 1992; 33(6):853-7. 16. Rankinen T, Rice T, Perusse L, et al. NOS3 Glu298Asp genotype and blood pressure response to endurance training: the HERITAGE family study. Hypertension 2000; 36:885–9.

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31. De Stefano V, Martinelli I, Mannucci PM et al. The risk of recurrent deep venous thrombosis among heterozygous carriers of both factor V Leiden and the G20210A prothrombin mutation. N Engl J Med. 1999; 341(11):801-6. 32. Gerhardt A, Scharf RE, Beckmann MW et al. Prothrombin and factor V mutations in women with a history of thrombosis during pregnancy and the puerperium. N Engl J Med. 2000; 342(6):374-80. 33. Casas JP, Hingorani AD, Bautista LE et al. Meta-analysis of genetic studies in ischemic stroke: thirty-two genes involving approximately 18,000 cases and 58,000 controls. Arch Neurol. 2004; 61(11):1652-61.

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cemento, FSC, se creó con el objetivo de establecer puentes de comunicación y colaboración permanente entre la empresa, la universidad y la administración de Galicia, en la búsqueda de un objetivo común: incrementar la prosperidad de Galicia y sus ciudadanos, a través del incremento de la competitividad del tejido empresarial gallego. La colaboración triple hélice es un modelo de cooperación esencial en el contexto actual, caracterizado por la cada vez mayor importancia estratégica del conocimiento, en sus distintas realizaciones (formación, talento, tecnologías, creación y combinación de conocimiento, etc), el cual tiene su mayor exponente en el mundo académico.

“

El mundo empresarial, verdadero agente creador de riqueza para una sociedad, pone en valor el conocimiento a través de la innovación y el emprendimiento, lo cual faculta a una sociedad la internacionalización de la misma. Y la Administración, como elemento dinamizador, que debe encauzar sus políticas de una manera eficiente, alineando los recursos y capacidades de una sociedad para el desarrollo de las necesidades reales de la empresa, así como establecer las pautas para favorecer un crecimiento equilibrado y sostenible en el tiempo de la sociedad en su conjunto.

El metadistrito es el punto de encuentro de los clusters gallegos La estructura de la FSC está diseñada en torno a cuatro Comités de expertos, formados por agentes de la triple hélice, con el objetivo de trabajar de forma coordinada en aquellos ámbitos clave que permitan incrementar la competitividad de Galicia. Los comités se centran en: cooperación, como elemento que facilita alcanzar las sinergias y masa crítica necesaria para competir; innovación, en sentido amplio, como elemento fundamental para impulsar la competitividad; nuevos negocios y emprendedores, como elemento para valorizar/comercializar las innovaciones que nos permitan dar un salto cualitativo

y, en cuarto lugar, la internacionalización, como resultado de los tres elementos anteriores y expresión de la competitividad de la empresa. En este entorno, cada vez más competitivo y globalizado, la interdependencia entre entidades y la creación de sinergias han hecho de la cooperación un elemento esencial. La Fundación Galega para a Sociedade do Coñecemento impulsa el modelo de cooperación Cluster, dado que facilita una perspectiva más significativa en cuanto al modo en que funciona una economía y cómo se interrelacionan las empresas; asimismo, ofrece la configuración y externalidades adecuadas para impulsar la innovación y el incremento de la productividad. Desde la FSC se promueve la figura del Metadistrito, como punto de encuentro y colaboración de los clusters gallegos para el impulso de la cooperación entre los mismos, que propicie el trabajo en red, abierto al conocimiento, la investigación aplicada y la innovación, desarrollando modelos de cooperación multisectorial para alcanzar la excelencia competitiva, a través de valores y estrategias comunes abiertas al mercado mundial. La Unión Europea está alentando y promoviendo entre sus miembros (países y regiones) el uso de los clusters y la mejora de la calidad del entorno empresarial como elementos para dinamizar la economía y marco de referencia para el incremento de la innovación y la productividad y, por ende, la competitividad. Prueba de ello es el nuevo marco Europeo de apoyo a los clusters, buscando la excelencia y cooperación entre los clusters de Europa. Asimismo, hay que mencionar que las regiones/países más desarrollados aplican la política cluster, desde hace algunos años, como elemento central de su política de crecimiento económico (Cataluña, País Vasco, Navarra, Baden-Württenberg, Ille de France, Dinamarca, Estados Unidos, Reino Unido, etc.). Entre los objetivos de la Fundación está el propiciar el incremento de la productividad, generar mejores empleos, difundiendo la tecnología y el conocimiento para, en definitiva, poder alcanzar un desarrollo sostenible de Galicia, a través de la excelencia empresarial y la innovación, teniendo en cuenta las características únicas de nuestra comunidad autónoma, sus recursos naturales, capacidades, localización y activos culturales e históricos.

Sociedad Desde la FSC hemos lanzado el Observatorio de Competitividad de Galicia (proyecto financiado por el IGAPE, Caixanova y Caixa Galicia), centro dinámico formado por agentes locales del conocimiento y entes públicos con el objetivo de potenciar y medir la competitividad de nuestra economía. En los trabajos de lanzamiento del Observatorio, se ha realizado un análisis y diagnostico de la situación competitiva de Galicia, con la utilización de herramientas avanzadas de benchmarking internacional. Asimismo, se ha articulado una Visión Galicia 2014 y un plan de acción concreto, utilizando como referencia las experiencias de éxito llevadas a cabo en otras economías avanzadas, pero adaptado a la realidad y circunstancias gallegas, con el fin último del incremento de la prosperidad socio-económica de Galicia. Para determinar la situación competitiva de partida, se han utilizado dos herramientas avanzadas que permiten la comparación de Galicia con otras localizaciones: Ranking de Competitividad de Galicia y Benchmarking competitivo basado en clusters. Dichas herramientas han sido facilitadas por las colaboraciones que ha establecido la Fundación Galega para a Sociedade do Coñecemento con dos entidades de reconocido prestigio internacional: Institute for Management Development (IMD) y Monitor Group. El IMD, con sede en Lausanne (Suiza), es una de las principales escuelas de negocios a nivel mundial que dirige y gestiona la elaboración de uno de los dos rankings de competitividad de referencia a nivel internacional (el “IMD World Competitiveness Yearbook”). Su programa

MBA se clasificó en el 1er puesto mundial en los rankings del Financial Times “Ranking of the Rankings”. Monitor Group, empresa de consultoría, fundada en 1983 por Michael Porter, con amplia experiencia en competitividad regional, dispone de bases de datos únicas en el mundo facilitando el benchmarking de la situación de Galicia en distintos aspectos. Recientemente, la Unión Europea, a través de la Comisión Europea, está trabajando con Monitor Group para aplicar su metodología basada en clusters a toda Europa.

“

Son objetivos de la FSC incrementar la productividad y difundir la tecnología y el conocimiento Felicito a la Fundación EuroEspes por el importante trabajo que llevan a cabo y, en concreto, por la revista Gen-T, que supone un paso más en la difusión del conocimiento científico, agradeciendo su amable invitación para dar a conocer a la sociedad las líneas de trabajo llevadas a cabo por la FSC. Les invito a visitar nuestro sitio web www.fgsc.es en el que podrán seguir de cerca toda la actividad desarrollada por la fundación. 

José Mª Martín Moreno jmartin@fgsc.es Director general de la Fundación Galega para a Sociedade do Coñecemento

diciembre 2009

www.euroespesannualconference.org

Ist Meeting of the World Association of Genomic Medicine

Genomic Medicine and PHARMACOGENOMICS Future Challenges for PERSONALIZED MEDICINE in the European Union

La III Conferencia Anual se celebró en A Coruña en diciembre de 2008 La III Conferencia Anual EuroEspes, celebrada en A Coruña los días 12 y 13 de diciembre de 2008, reunió a los más prestigiosos especialistas en medicina genómica de todo el mundo. La Fundación EuroEspes organizó, en el marco de la III Conferencia Anual, el Primer Encuentro de la Asociación Mundial de Medicina Genómica.

Sociedad

EUROESPES REÚNE A LOS MEJORES ESPECIALISTAS EN MEDICINA GENÓMICA DEL MUNDO

xpertos como los profesores Urs A. Meyer, Allen

D. Roses, Filipo Guglielmo de Braud, Francesco Marotta, Masatoshi Takeda, Christian A. Scerri, Valter Lombardi, John R. Cockcroft y Ramón Cacabelos, entre otros, analizaron y debatieron las posibilidades - y los retos - que ofrece la medicina genómica en el tratamiento de distintas enfermedades. Entre ellas, el cáncer, los trastornos neuropsiquiátricos y las patologías respiratorias y cardiovasculares. El nivel científico del encuentro fue altísimo. Las previsiones de la organización del evento (“Medicina Genómica y Farmacogenómica. Futuros desafíos para la Medicina Personalizada en la Unión Europea”) se superaron ampliamente. Una de las intervenciones más esperadas fue la del profesor de Farmacología del Biocentro de la Universidad Basilea ( Suiza ), Urs A. Meyer. En su ponencia, explicó que la “la farmacogenética permitirá mejorar los resultados clínicos a través de un diagnóstico preciso mediante la optimización de la elección del fármaco y de la dosis adecuada para cada paciente. Con ello, se producirán menos reacciones adversas y los tratamientos serán más efectivos”. El profesor Meyer repasó también los desarrollos en genómica más recientes, como el proyecto internacional HapMap y también los proyectos 1000 Genomas, Genoma Personal, Atlas del Genoma del Cáncer y las iniciativas de “consumer genomics” que genotipan más de 500.000 SNP por menos de 400 dólares. La ponencia plenaria de Allen D. Roses causó gran expectación. El profesor de Neurología de la Universidad de Duke, en California (Estados Unidos), explicó que “para que la farmacogenética sea integrada en la práctica clínica, el modelo de negocio farmacéutico debe evolucionar. Durante el desarrollo de fármacos, se debe aplicar, de forma rigurosa, la ciencia, de manera que se recopilen datos significativos que puedan ser empleados para demostrar los valores predictivos positivos y negativos para las necesidades médicas no cumplidas”.

Ramón Cacabelos, director del Centro de Investigación Biomédica EuroEspes, primer grupo genómico en Europa, habló de la importancia de la medicina genómica para conocer mejor las patologías que afectan al sistema nervioso central.

5 Acto de inauguración III Conferencia Anual EuroEspes. El Dr. Cacabelos, en primer término.

En su intervención, Ramón Cacabelos explicó que “los mecanismos patogénicos de la mayoría de los desórdenes del sistema nervioso central son poco conocidos aún. Los estudios genéticos realizados en las dos últimas décadas han demostrado que las patologías del Sistema Nervioso Central (SNC) son multifactoriales y poligénicas. De ahí que los recientes avances en medicina genómica puedan contribuir a acelerar nuestro conocimiento de la patogénesis de los desórdenes del SNC, mejorando la precisión del diagnóstico con la introducción de nuevos biomarcadores, y personalizando los tratamientos con la incorporación de procedimientos farmacogenéticos y farmacogenómicos al desarrollo farmacológico y la práctica clínica”. John R. Cockcroft, del Instituto de Investigación Cardiaca de la Universidad de Gales, en el Reino Unido, abordó el papel de la Medicina Genómica y de la Farmacogenómica en las patologías cardiacas. Según este especialista, “desde que las cardiopatías son una de las mayores causas de morbilidad y fallecimiento, se han convertido en un objetivo central de la investigación sobre la interacción entre el genoma y los fármacos cardiovasculares. Sin embargo, aunque la farmacogenómica cardiovascular es un campo prometedor, todavía estamos trabajando en la consecución de un test con verdadera utilidad clínica”.

La nutrigenómica ayudará a prevenir enfermedades como la diabetes Marvin Edeas diciembre 2009

III Conferencia Anual EuroEspes La ponencia sobre los biomarcadores en oncología corrió a cargo de Filippo Guglielmo de Braud, director de la Unidad de Farmacología Clínica y Nuevos Fármacos de la Facultad Europea de Oncología de Milán.

Los biomarcadores genómicos son la base de la medicina personalizada Filippo Guglielmo de Braud “Los biomarcadores genómicos”, explicó, “son la base de la medicina personalizada. Pueden ser utilizados para seleccionar poblaciones de pacientes adecuados para recibir tratamientos adecuados evitando la toxicidad, o seleccionar el fármaco óptimo para poblaciones de células cancerosas específicas. Sin embargo, la metodología de los ensayos clínicos y del desarrollo farmacológico debe cambiar para que la farmacogenética mejore los resultados del tratamiento, permitiendo la selección de los pacientes más adecuados para beneficiarse del tratamiento propuesto o para excluir aquellos tumores con menos probabilidades de ser sensibles al tratamiento. El futuro de la oncología pasa por la identificación de los perfiles químico-genéticos de diversos fármacos antitumorales”.

El Prof. Dr. Allen D. Roses (EE.UU.), durante su ponencia

El genetista maltés Christian A. Scerri abordó las oportunidades que una población insular puede ofrecer a la farmacogenética, que, en su opinión, son muchas. Por su parte, Gerardo Jimenez-Sánchez,

5 Vista del acto de inauguración celebrada en el Palacio de Exposiciones y Congresos de A Coruña (Palexco)

director del Instituto Nacional de Medicina Genómica de México, explicó cómo la medicina genómica se ha convertido en una prioridad para el Gobierno mexicano como un medio para encontrar nuevas estrategias para abordar patologías comunes: “Dicho compromiso - contó - se ha plasmado con la creación del Instituto Nacional de Medicina Genómica (INMEGEN) por el Congreso Mexicano en 2004. Un centro diseñado para desarrollar investigación transnacional centrada en los problemas de salud de la población mexicana, que tiene una estructura genómica ancestral particular, debido a su mezcla de orígenes”. Masatoshi Takeda, jefe del Departamento de Psiquiatría y Ciencia del Comportamiento de la Universidad de Osaka, centró su conferencia en la implantación de la Medicina Genética en su país, en Japón, y, en especial, en las patologías neurodegenerativas. Su conclusión es clara: “Los estudios genéticos han acelerado dramáticamente la investigación en Alzheimer, y no solo para el conocimiento de la enfermedad, también para el desarrollo del tratamiento y del diagnóstico”.

Sociedad

Para Francesco Marotta, especialista en Biogerontología de la Universidad de Pavia, en Italia, “los alimentos funcionales representan una oportunidad emergente y jugarán un papel importante en el futuro. La nutrigenómica no solo promoverá la nutrición, sino que, además, ayudará a prevenir enfermedades como la diabetes, la obesidad, las patologías neurodegenerativas y el envejecimiento”. Munir Pirmohamed, del Departamento de Farmacología de la Universidad de Liverpool, en el Reino Unido, fue el encargado de analizar el papel de la Medicina Genómica en las enfermedades metabólicas. Ian P. Hall, de la División de Medicina Molecular

La farmacogenética permite mejorar los resultados clínicos

Urs A. Meyer

y Terapéutica de la Universidad de Nottingham, repasó el valor del abordaje genético en las patologías respiratorias. “Puede -dijo- ser de gran utilidad, bien definiendo nuevos marcadores predictivos de eficacia, o respuestas adversas a fármacos, o bien identificando nuevos objetivos potenciales para el desarrollo farmacológico”.

El desarrollo de fármacos y la farmacogenómica en Estados Unidos fue otro de los asuntos tratados en la III Conferencia Anual EuroEspes.

Valter Lombardi, director del Departamento de Inmunología Molecular de EuroEspes Biotecnología, disertó sobre la medicina genómica y farmacogenómica en las patologías inmunológicas y las vacunas. Eugenio Luigi Dorio, nutricionista y bioquímico de la Universidad de Nápoles (Italia), abordó los retos de la redoxómica.

El Prof. Dr. Urs A. Meyer (Suiza) en plena intervención

Para hablar de este tema, viajó a A Coruña Carmen Vigo, directora de Diagnóstico y Transplante de Invitrogen (Estados Unidos). Vigo asegura que “uno de los retos más importantes en la definición de los rasgos farmacogenéticos es la necesidad de contar con pacientes bien caracterizados que hayan sido uniformemente tratados y sistemáticamente evaluados para hacer posible cuantificar, objetivamente, la respuesta farmacológica”.

Las ponencias sobre Nutrigenómica fueron también muy seguidas por los asistentes a la conferencia. Marvin Edeas, profesor de medicina preventiva en la Universidad de París XIII, sostiene que “el impacto futuro de la nutrigenómica en Europa aún no se comprende bien. Por ello, nuestro reto actual es unir las visiones científica, industrial y política de la nutrigenómica para discutir el modo de lograr el objetivo último, que es mejorar la salud del consumidor”.

diciembre 2009

III Conferencia Anual EuroEspes Premios Fundación EuroEspes La III Conferencia Anual EuroEspes y el Primer Encuentro Mundial de Medicina Genómica finalizaron con la entrega de la Primera Edición de los Premios Fundación EuroEspes. Se entregaron tres galardones. Uno de los premios correspondió al Cuerpo Nacional de Policía por la “creación del Laboratorio Territorial de Biología y ADN de la Jefatura Superior de Policía en Galicia”. Recogió el premio Luis García Mañá, jefe superior de policía en Galicia. 5 El Dr. Valter Lombardi, director del departamento de inmunología celular de Ebiotec, durante su ponencia.

Para Amalio Telenti, del Instituto de Microbiología de Lausanne, en Suiza, las “grandes diferencias en la susceptibilidad a patógenos como la tuberculosis, la malaria o el VIH y en la progresión de la enfermedad que muestran los humanos hacen necesario analizar la patogénesis de dichas enfermedades”. El cofundador y director ejecutivo de la empresa alemana FarmacoGenómica GmbH, Stefan Prause, fue el último de los ponentes de la III Conferencia. Su conclusión es que la “implementación de la farmacogenética en la medicina diaria es difícil por el consumo de tiempo y lo costoso de las tecnologías, que aún requieren equipamientos muy sofisticados. Los test farmacogenéticos aún están reservados a los laboratorios centralizados. Sin embargo, nosotros ya disponemos de un test farmacogenético que se dirige a ese vacío diagnóstico con un método rápido y coste efectivo que utiliza equipamiento poco costoso y que es adecuado para introducir la farmacogenética en la práctica clínica diaria y en las aplicaciones diagnósticas rutinarias”.

El presidente de Laboratorios PARGGON de Guadalajara ( México ), Rafael López Martínez, recibió el premio Fundación EuroEspes por su “apuesta empresarial por el desarrollo de la biotecnología aplicada a la salud, la nutracéutica médica y la farmacogenómica”. Otro de los galardones recayó en Eulalia Puig Colominas, presidenta de Genomax Iberplus. El patronato de la Fundación EuroEspes la premió por ser “la primera compañía española dedicada a la difusión del conocimiento genómico y a la implementación de instrumentos para el diagnóstico genómico y la personalización del tratamiento basado en la farmacogenómica”. El director general de Terapias Avanzadas y Transplantes del Ministerio de Sanidad y Consumo, Augusto Silva González, asistió a la entrega de premios. 

Para el director de la III Conferencia Anual EuroEspes, Ramón Cacabelos, la conclusión es clara: Seguir trabajando en la divulgación e implantación de la Medicina Genómica. “Esta conferencia”, recordó, “es sólo un pequeño ejemplo de hacia dónde debe ir en el futuro la medicina genómica”. Durante la III Conferencia, la nueva Asociación Mundial de Medicina Genómica (WAGEM) celebró su reunión inaugural. Los principales objetivos de esta asociación es educar y concienciar sobre los beneficios de la medicina geómica a la población general y al colectivo sanitario en particular.

5 Luis García Mañá, Jefe Superior de Policía de Galicia, recoge el galardón otorgado por la Fundación EuroEspes en presencia del Dr. Augusto Silva, Director General de Terapias Avanzadas y Transplantes del Ministerio de Sanidad. Entrega el Presidente de la Fundación, el Dr. Cacabelos.

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Noticias EuroEspes Conferencia en el Club Tomás de Mercado Psiquiatras japoneses se interesan por la Farmacogenética Una docena de psiquiatras japoneses, pertenecientes a su asociación nacional que engloba a 1.214 hospitales, visitaron las instalaciones de EuroEspes para conocer los avances en la medicina genómica y en especial mostraron un gran interés por la tarjeta farmacogenética que ha desarrollado la citada institución médica gallega que dirige el doctor Ramón Cacabelos. Yasuo Okuyama, jefe de la delegación, comentó que “el conocimiento de la genética del paciente”, es algo “fundamental” para “acertar con la medicación correcta”, y además darle “la dosis adecuada”. “La tarjeta farmacogenética viene a marcar el camino a seguir en un futuro próximo”, apostilló.

La Junta Directiva del Club Tomás de Mercado invitó al Dr. Ramón Cacabelos, Presidente del Grupo EuroEspes, a un encuentro en torno a una cena coloquio en la que debatió sobre “la importancia de la medicina predictiva en la salud de la empresa”. El Club Tomás de Mercado tiene la sede en Barcelona y allí fueron invitados ministros de diferentes gobiernos así como empresarios de importantes sociedades de origen multinacional. El número de socios está en 60, y su objetivo es que el debate que se produzca entre los asistentes sirva para incrementar su nivel de conocimiento en las materias de actualidad que se programan. 

Ebiotec comercializará productos nutracéuticos en Portugal La distribución de nutracéuticos en Portugal es posible gracias al acuerdo alcanzado entre Ebiotec y la distribuidora de productos farmacéuticos IA FARMA, con sede en Lisboa. El acuerdo de colaboración fue firmado por el presidente del Grupo EuroEspes, Ramón Cacabelos, y el director general de IA FARMA, Joao Alves. Para el doctor Ramón Cacabelos, la firma de este acuerdo “significa la entrada en un estado de la Península Ibérica con el que formamos una unidad geográfíca común con hábitos muy semejantes que nos llevan a compartir dietas y enfermedades semejantes”. 

Ebiotec lanza HepatoSar Nutracéutico,que ayuda a la digestión de las grasas y favorece la función hepatobiliar Este es un nutracéutico con lipoproteínas naturales de origen marino que se obtienen mediante procesos biotecnológicos no desnaturalizantes que preservan todas las propiedades biológicas de la especie marina original, S. pilchardus (E-SAR-94010®), un pescado azul de la familia de la sardina y extracto de alcachofa (cynara scolymus) estandarizado al 5%. Las excelentes propiedades de este pescado azul, junto con las del extracto de alcachofa, hacen que, HepatoSar Nutracéutico, ayude al funcionamiento fisiológico de nuestro organismo. 

Directivos de la Fundación Sociedade Galega para O Coñecemento visitan el Grupo EuroEspes José María Martín Moreno, Director General y Luis González, Secretario General de la Fundación Sociedade Galega para O Coñecemento realizaron una visita al Grupo EuroEspes. Era la primera toma de contacto entre ambas organizaciones y en ella se realizó un amplio recorrido por Ebiotec donde escucharon y vieron los diferentes proyectos y estudios en los que los investigadores de EuroEspes Biotecnología se encuentran trabajando en la actualidad. En el transcurso de la visita mostraron interés por los procesos de liofilización, la genética y por la nutracéutica así como por el proyecto Ebiosea, que les fue explicado detalladamente.

EuroEspes crea una unidad para tratar los problemas de la visión asociados a trastornos neurológicos (UNO) El nuevo departamento se denomina Unidad de Neuro-oftalmología (UNO). La Neuro-oftalmología es una rama de la oftalmología que se dedica al estudio anatomofisiológico y patológico así como de la estrecha relación que existe entre el ojo y el sistema nervioso central. En esta Unidad de Neuro-oftalmología se pueden tratar casos de visión doble, disminución del campo visual, pupilas con distinto tamaño, molestias visuales, disminución inexplicada de la visión, problemas del nervio óptico o molestias visuales. Con la puesta en funcionamiento de este servicio, EuroEspes se convierte en el primer centro de España con un programa integral que vincula la estrecha relación que existe entre la visión y el cerebro: el 70% del cerebro participa, de alguna manera, en la función visual. 

El Presidente del Grupo EuroEspes participa en el VI Congreso de Directivos CEDE El Dr. Ramón Cacabelos, Presidente del Grupo EuroEspes, participó en el programa del VI Congreso Directivos CEDE, los días 28 y 29 de octubre, en Pamplona, que se celebró bajo el lema “Liderando en positivo”. Su intervención tuvo lugar dentro de la sesión temática “Biotecnología”. En este evento, al que asistieron destacados profesionales de la gestión empresarial de nuestro país, EuroEspes participó como empresa colaboradora del mismo, con el fin de difundir la importancia de la predicción y la prevención en “la salud del cerebro del directivo”. El acto de clausura fue presidido por S.A.R. el Príncipe de Asturias. 

En el Centro de Investigación Biomédica EuroEspes (CIBE) fueron recibidos por el Dr. Cacabelos, quien les introdujo en la filosofía del Grupo y mostró los últimos avances tecnológicos para la detección y diagnóstico de enfermedades neurodegenerativas del sistema nervioso central pudiendo comprobar el alto grado de innovación e investigación que se está realizando en la actualidad. 

diciembre 2009

Noticias EuroEspes Anfaco Cecopesca, Fundación EuroEspes y Ebiotec organizaron una jornada de biotecnología aplicada La biotecnología aplicada a la nutrición humana en la industria de conservación de productos de la pesca y la acuicultura es el título de la Jornada que el pasado día 24 de junio tuvo lugar en las instalaciones de ANFACO CECOPESCA dirigida a los representantes del sector conservero gallego y a empresas de biotecnología. Con el enunciado que da titulo a la Jornada el Dr. Ramón Cacabelos, presidente del Grupo EuroEspes expuso que la biotecnología aplicada a la salud es un poderoso instrumento para la innovación tecnológica e industrial en el sector de la nutrición humana y la medicina.

Máster en Biotecnología de la Salud y Programa de Doctorado en Medicina Genómica La Universidad Camilo José Cela de Madrid crea, en colaboración con el Grupo EuroEspes, el Máster Universitario en Biotecnología de la Salud. Se impartirá a partir del curso académico 2010/11, y será dirigido por el profesor Ramón Cacabelos, presidente del Centro de Investigación Biomédica EuroEspes (CIBE) y Director de la Cátedra EuroEspes de Biotecnología y Genómica. La Agencia Estatal de Evaluación de la Calidad y la Acreditación (ANECA) ha informado favorablemente el Plan de Estudios presentado por la Universidad Camilo José Cela.

NOTA ACLARATORIA Sr. Director de la revista “Gen-T. The Euroespes Journal”: En relación con el artículo titulado “Sobre la ética de los análisis de ADN”, firmado por mí, Juan Carlos Carril, y publicado en la sección “Tribuna Abierta” de la revista “Gen-T The EuroEspes Journal”, número 2, y en respuesta al acto de conciliación entre la Universidad de Santiago de Compostela (USC) y el que suscribe celebrado en Bergondo el 27 de junio de 2008, y en interés de una mejor comprensión de lo expuesto en el citado artículo, solicito a la dirección de la revista la publicación de la siguiente nota aclaratoria: En el artículo mencionado, se hace referencia al Instituto de Medicina Legal de Santiago de Compostela (en adelante IML), únicamente como ejemplo de laboratorio serio y riguroso en sus actuaciones, y no como sospechoso de ningún tipo de actuación irregular: “(…) participan en rigurosos controles de calidad, cumplen normas internacionales de buenas prácticas de laboratorio y están avalados por sociedades científicas internacionales como la Sociedad Internacional de Genética Forense (ISFG). El Instituto de Medicina Legal de Santiago de Compostela es uno de estos laboratorios serios, pero no el único.” El autor no pretende afirmar, ni de la lectura del mencionado artículo se puede deducir, que exista demora por parte del IML en la realización de las pruebas solicitadas por los Juzgados y Tribunales. El autor alerta de la necesidad de que no tengan lugar

La Comisión de Emisión de Informes de Máster reconoce que la Universidad Camilo José Cela, en su informe, “ aporta diferentes evidencias” que ponen de manifiesto el “interés y la relevancia académica y científica” del Máster en Biotecnología de la Salud.

demoras más allá de las estrictamente derivadas de la complejidad de los análisis realizados. En ningún caso se afirma - y ni tan siquiera se insinúa - que se prioricen las pruebas privadas en detrimento de las solicitadas por los órganos judiciales. No existe ni una sola frase en toda la redacción

Desde la perspectiva de la investigación indicó las líneas de trabajo en las que se mueve el Grupo EuroEspes y, especialmente, Ebiotec. Participaron como representantes de Ebiotec y con sendas ponencias, el Dr. Valter Lombardi con el trabajo “Alimentos funcionales y productos nutracéuticos: una estrategia de futuro en nutrición humana”, por su parte Ramón Alejo, Director Técnico de Producción de Ebiotec habló de “Liofilización industrial: nuevas tecnologías para la conservación y preservación de propiedades de los productos de la pesca”. La jornada fue inaugurada por Juan Manuel Vieites Baptista de Sousa, secretario general de ANFACO y director general de CECOPESCA.

Así mismo el Ministerio de Educación, a través del Consejo de Universidades, aprobó la creación del Doctorado en Medicina Genómica de la Universidad Camilo José Cela.

del artículo que insinúe que los responsables o los

Ramón Cacabelos, será el responsable del Programa de Doctorado.

En ningún caso se hace referencia a las tarifas vi-

trabajadores, o la propia Universidad, se estén lucrando con la realización en el IML de pruebas de paternidad privadas.

gentes en el IML ya que no es sobre este laboratorio que el autor está dando la voz de alarma. Por todo lo anteriormente expuesto, el autor entiende que el artículo aquí mencionado no difama ni calumnia ni al Instituto de Medicina Legal, ni a sus trabajadores, ni a la Universidad de Santiago de Compostela, además de considerar fruto de una interpretación errónea la asunción de cualquier tipo de ataque o falsedad contra los antes mencionados. Es por ello que el autor considera oportuna la publicación de esta nota aclaratoria que, en esencia, considera que se corresponde con lo expuesto en el escrito propuesto por la USC en el acto de conciliación.

Fdo. Juan Carlos Carril Bergondo, 10 de septiembre de 2008

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