For some time, a major interest of our group has been the effect of nutritional state, aging and advanced disease states on drug metabolism. To this end we have utilized a rat model of protein calorie malnutrition (PCM), and a collection of clinical studies involving HIV+ and cancer patients have been carried out. PCM is a nutritional state frequently observed in aged populations, in particular frail individuals, and cancer patients. A group of drug metabolizing enzymes called cytochromes P450 (CYPs) are of major importance and thus of major interest. PCM leads to a down-regulation of a number of drug metabolizing enzymes, particularly CYPs. Clinical studies of HIV and cancer have involved the analysis of a selection of enzymes, for which discrete genetically determined subgroups are present in the human population. These genetic polymorphisms are generated by mutations in the genes coding for these enzymes, which cause decreased, increased or absent enzyme activity. In healthy individuals, such metabolic activity falls into two clearly defined and qualitatively different populations: individuals whose rate and extent of metabolism is poor (poor metabolizers, PMs) and those who have faster or more extensive metabolism (extensive metabolizers, EMs). Genetic polymorphisms exist for a number of the CYP enzymes. CYP2A6 is one such enzyme, with 15 to 20% of the population being slow metabolizers. The Phase II enzyme named acetyltransferase-2 (NAT2) is also polymorphic, with 50 to 60% of individuals being classified as slow metabolizers. Innocuous drugs are used to probe enzyme activity, and thus metabolic phenotype. This technique involves oral administration of the drug to a subject who will then provide a urine sample a few hours later. The sample is then analyzed for drug and its metabolites using HPLC. In this way, caffeine metabolite ratios are used to probe for the functional activity of CYP2A6 and NAT2. In healthy individuals, as long as there are no drug-drug interactions, metabolic genotype normally predicts metabolic phenotype. However, acute and chronic disease states and frailty can lead to changes in the relative levels and activities of metabolizing enzymes and this is our major focus. In a clinical study of disease effects on drug metabolism, NAT2 genotypes were compared to NAT2 phenotypes in a group of HIV+ patients. Unlike a healthy population, which can be genotypically and phenotypically divided into two subsets consisting of NAT2(fast)and NAT(poor) acetylators, the HIV+ population was phenotypically unimodally distributed, and skewed towards a slow acetylator status, despite the expected bimodal distribution of the genotype. In our second clinical study, sixteen patients with advanced cancer were genotyped and phenotyped. Although all 16 patients had an extensive metabolizer genotype, four patients displayed an apparent slow phenotype and, again, patients phenotypically displayed one population only, skewed towards the slow metabolizer status. In both studies described above, liver and renal function was assessed using routine hematological and biochemical markers, and was considered normal. Additionally, no metabolic drug interactions were apparent. Therefore, we have established that in certain advanced disease states such as AIDS and cancer, often involving wasting syndromes, genotype may not predict the corresponding metabolic phenotype and there is discordance between the two.The studies described above indicate that disease, and the nutritional status that often accompanies disease, has significant impact on the way in which the body handles drugs. Determination of the concordance or discordance between genotype and phenotype provides a means to thoroughly study the effect of disease state on drug metabolism in humans. It is an individual probe that can be used to study the general phenomenon or to optimize patient-specific clinical protocols, avoiding drug toxicity by predicting disturbances in drug metabolizing enzyme activities. The study of this phenomenon continues to be a primary objective in future clinical studies of disease effects on drug metabolism. In one such planned study, is the effect of frailty on the discordance between metabolic genotype and phenotype which will be conducted in subjects enrolled in the Baltimore Longitudinal Study of Aging using caffeine as the probe drug for CPY2A6 and NAT2 activity. We are also investigating the effect of calorie restriction on metabolic activity using rat models. We have seen significant changes in the ability of the calorie restricted animals to glucuronidate test compounds and to metabolize a probe drug, ketamine. These results will be reported. In a parallel study, patients with unresponsive chronic pain that is diagnosed as Complex Regional Pain Syndrome (CRPS) and patients suffering from treatment-resistant bipolar depression (BP) have responded to treatment with ketamine. The CRPS patients were treated with a five-day infusion of the drug produced pain relief in 75% of the patients and 67% of the BP responded after a single dose. The objective of the ongoing study is to identify the reasons why some patients respond and other do not, to be able to identify the responders before treatment begins and to individualize treatment to increase response. These initial pharmacokinetic and pharmacodynamic studies of ketamine in CRPS and BP patients are completed, the patients are being genotyped and pharmacogenetic studies initiated. The data indicates that the therapeutic activity of ketamine is due, in part, to hydroxynorketamine and dehydronorketamine and the pharmacological activities of the compounds are being investigated. In addition, metabolomic studies have determined that response and non-response to treatment with ketamine in BP patients is related to mitochodrial function. This relationship is under investigation. The laboratory is also involved in the development of a series of new therapeutic agents based upon the drug (R,R)-fenoterol. At the current time (R,R)-fenoterol is in initial clinical studies for use in congestive heart failure. The laboratory will analyze plasma and urine samples from these studies and determine the pharmacokinetics and pharmacodynamics profiles of the compound. In addition, second generation fenoterol derivatives have been developed and are undergoing preliminary pharmacological testing. It has been observed that some of the fenoterol analogs are inactive in the cardiovascular system but highly effect in the suppression of glioblastoma brain cancers which express the beta2-adrenoceptor. The compounds are being developed for clinical use and the molecular mechanisms underlying these differences is being explored.
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