APP Profile

Autonomic Pharmacodynamic Pharmacogenomics

Abstract (July, 2003 Update)

Goals

We developed a center devoted to uncovering how allelic variation in genetic loci encoding autonomic junctional receptors, and their post-receptor signal-transducing molecules, determines cardiovascular drug responses in humans. We will focus on drug responses which influence pre- and post-synaptic receptor-mediated autonomic events in the cardiovascular system, including regulation of catecholamine storage and release, heart rate, and regional vascular tone. The disease targets, in which these autonomic drugs are used therapeutically, include systemic and pulmonary hypertension, heart failure, arrhythmias, renal failure, and edema (sodium and water retention).

Determinants of drug action can be broadly divided into two areas: pharmacokinetics (disposition of the drug and access to the target site) and pharmacodynamics (mode and intensity of drug action at the target site). Our NIGMS pharmacogenomics center will probe pharmacodynamic determinants of human drug responses. Our focus is human autonomic cardiovascular drug responses, in both the systemic and pulmonary circulations. To direct this focus to pharmacodynamics of autonomic cardiovascular responses, our initial phenotyping strategy will emphasize regional (rather than systemic) vascular responses, so as to remove pharmacokinetic and baroreflex variables from their confounding influence, and thereby to isolate and focus upon pharmacodynamic (receptor, post-receptor, and effector) determinants of drug responses. Phenotyping projects 1-4 generate information and samples which flow systematically through 7 cores (AàG), to achieve center goals. The regional responses are characterized in the pulmonary (Phenotyping project 1), renal (Phenotyping project 2), forearm (Phenotyping project 3), and hand (Phenotyping project 4) circulatory beds. Both pre-synaptic (Phenotyping project 3) and post-synaptic (Phenotyping projects 2&4) responses are studied. This center will evaluate both diagnostic (Phenotyping projects 1-4) and therapeutic (Phenotyping projects 1&2; Core G) drug responses. Diagnostic drug responses will allow us to more effectively and "cleanly" probe receptor and post-receptor signaling, thereby providing rigorous pharmacodynamic parameters to be applied later to therapeutic drug responses in humans. Variation in responses to exogenous agonist should be specific to variation (either qualitative or quantitative) in receptor, post-receptor signaling, or effector components; by contrast, variation in antagonist responses could represent changes in the amount of the endogenous agonist present. Diagnostic responses should allow definition of which SNP alleles contribute to variation in signaling in a particular pathway. We hypothesize that agonist responses, obtained under carefully defined circumstances in regional circulatory beds (pulmonary, renal, forearm, hand), will lead to the discovery of associated SNP alleles which will ultimately yield diagnostic tools to predict therapeutic drug responses (in particular, for antagonists at the same receptors). The functional significance of discovered SNP alleles will be verified in vitro (Cores D&E) and in experimental organisms (Core F). Finally, such SNP alleles will then be tested in human trials of therapeutic drug responses (Core G) to answer the question: Does SNP stratification predict therapeutic drug responses in large, prospective, randomized, controlled clinical trials?

Progress

Sequential strategy: an integrated, multidisciplinary approach. Our sequential strategy will provide a systematic approach to ascertaining the influence of polymorphic alleles on drug responses. Our strategic flow of information and samples is

• Autonomic pharmacodynamic phenotyping (Phenotyping projects 1-4): We begin by careful, systematic autonomic pharmacologic phenotyping in human subjects. Drug targets are carefully selected, to represent determinants of vascular smooth muscle responses to neural and local influences. Human subjects are
  elected to represent individuals at risk for two vascular diseases: either patients with pulmonary hypertension (Phenotyping project 1), or the still-normotensive offspring of patients with systemic hypertension (Phenotyping projects 2&3), a group noted already to display alterations in autonomic control of the systemic circulation (Dao et al, 1998. O'Connor et al, 2000). Whenever practical, careful regional phenotyping will be performed: in the pulmonary circulation (Phenotyping project 1), the renal circulation (Phenotyping project 2), the isolated forearm circulation (Phenotyping project 3), or the isolated hand circulation (Phenotyping project 4).

• Genomics: re-sequencing and SNP genotyping at candidate genetic loci (Core B: Genomics). Because heritable determinants of drug action are likely to be complex, and since determination of the genetic underpinnings of autonomic drug responses is a major undertaking involving the influence of multiple genes, UCSD has undertaken systematic SNP discovery by resequencing genomic DNAs. At candidate genetic loci likely to influence drug responses, especially those which are not yet well-studied, we will re-sequence the locus (each exon and splice junctions, as well as ~1000 bp of 5'-flanking promoter) in 100 individuals (200 chromosomes), to discover not only common SNPs, but also less common allelic variants (sequencing 200 chromosomes allows the 50% likelihood of discovering SNPs with allele frequencies as low as ~0.5%).

• Genetic (drug response phenotype/genotype) analysis (Core C: Bioinformatics and statistical genetics):
  The drug responses to be tested for association with SNP alleles include both potency (drug EC50 determined from the log10 concentration (dose)/response pattern) and agonist efficacy (maximal or ceiling drug effect). Several analytic methods will be employed to discern the effects of particular alleles or genotypes on drug responses. Analyses will be headed by Nicholas J. Schork, Ph.D., a statistical geneticist with expertise in complex trait linkage. Allelic association tests will determine whether particular SNP alleles are associated with drug responses. In two of the phenotyping projects (Phenotyping projects 2&3), the availability of parental DNA will allow intra-familial controls for allelic association, such as the transmission disequilibrium test (TDT), or the TDT for quantitative traits (QTDT); such controls guard against population admixture as a spurious explanation for allelic association. In addition, the sibling pairs phenotyped in projects 2&3 will allow a sib-pair linkage approach to discovery of loci determining drug responses; the analyses will be by SIBPAL in SAGE (SAGE - Statistical Analysis for Genetic Epidemiology), as well as Genehunter; availability of parental DNAs in projects 2&3 will allow more exact IBD (identity-by-descent) analyses.

• Verification of functional significance of any SNP: 3-fold strategy (Cores D&G). Once a SNP has been statistically associated with a pharmacologic phenotype (by either allelic association, or sib-pair linkage), we will proceed to verification of the biological impact of the SNP. Three general approaches are taken, progressing from in vitro (studies in isolated cells), to in vivo in experimental animals (transgenic mice), and in humans (predictive studies in human therapeutic drug trials).

• In vitro: receptor signaling and sympathochromaffin consequences (Cores D&E). Using transfection of either vascular smooth muscle cells or neutral control cells (such as Cos-7) in Core D (Receptors and signal transduction), we will evaluate the post-synaptic functional consequences of alterations in G protein coupled receptor (GPCR) alterations, in both the ORFs (open reading frames) as well as in non-coding regions. Using transfected chromaffin cells and sympathetic neurons in Core E (Sympathochromaffin biochemistry and cell biology), we will test whether polymorphic variants alter catecholamine or neurotransmitter biosynthesis, vesicular storage, or exocytotic release.

• In vivo: experimental animals (Core F: Transgenic mice). When in vitro studies indicate altered expression or responsiveness, Core F will allow us to examine in vivo the function of particular SNP variant, coding or non-coding. The strategy will be to introduce the variant (versus the wild-type) on a human BAC, into a mouse in which the locus has been ablated by previous homologous recombination ("knockout").

• In vivo: human clinical trials (Core G: Human systemic responses in disease). Here, we will answer the question: Does SNP stratification predict systemic therapeutic drug responses in large, prospective, randomized, controlled USA clinical trials? The trials to which we have arranged access include "AASK" (NIDDK "African-American Study of Kidney Disease and Hypertension").

APP Team