Disease Status Drug Metabolism and New Drug Discovery

一段时间以来，我们小组的主要兴趣一直是营养状况，年龄和晚期疾病状态对药物代谢的影响。为此，我们利用了蛋白质热量营养不良（PCM）大鼠模型，并开展了一系列涉及HIV+和癌症患者的临床研究。PCM是一种经常在老年人，特别是体弱个体和癌症患者中观察到的营养状态。一组称为细胞色素P450 （CYPs）的药物代谢酶非常重要，因此引起了人们的广泛关注。PCM导致一些药物代谢酶，特别是CYPs的下调。艾滋病毒和癌症的临床研究涉及对酶的选择进行分析，这些酶在人类群体中存在离散的遗传决定亚群。这些遗传多态性是由编码这些酶的基因突变产生的，这些突变导致酶活性降低、增加或缺失。在健康个体中，这种代谢活动分为两个明确定义且在质量上不同的人群：代谢速率和程度较差的个体（贫代谢者，PMs）和代谢更快或更广泛的个体（广泛代谢者，EMs）。遗传多态性存在于许多CYP酶。CYP2A6就是这样一种酶，15%到20%的人是慢代谢者。名为乙酰转移酶-2 （NAT2）的II期酶也是多态性的，50%至60%的个体被归类为慢代谢者。无害的药物被用来探测酶的活性，从而代谢表型。这项技术包括给受试者口服药物，几小时后受试者将提供尿液样本。然后用高效液相色谱法分析样品中的药物及其代谢物。通过这种方法，我们利用咖啡因代谢物比率来探测CYP2A6和NAT2的功能活性。在健康个体中，只要没有药物相互作用，代谢基因型通常可以预测代谢表型。然而，急性和慢性疾病状态和虚弱可以导致代谢酶的相对水平和活动的变化，这是我们的主要关注点。在一项疾病对药物代谢影响的临床研究中，在一组HIV+患者中比较了NAT2基因型和NAT2表型。与健康人群不同，健康人群可以被基因典型和表型上分为NAT2（快速）和NAT（差）乙酰化两个亚群，HIV+人群在表型上是单峰分布的，并且偏向于慢乙酰化状态，尽管预期的基因型是双峰分布。在我们的第二项临床研究中，16例晚期癌症患者进行了基因分型和表型分型。尽管所有16例患者都具有广泛的代谢基因型，但4例患者表现出明显的慢表型，并且患者表型上仅显示一个人群，倾向于慢代谢状态。在上述两项研究中，使用常规血液学和生化指标评估肝肾功能，并认为正常。此外，没有明显的代谢药物相互作用。因此，我们已经确定，在某些晚期疾病状态，如艾滋病和癌症，往往涉及消耗综合征，基因型可能无法预测相应的代谢表型，两者之间存在不一致。上述研究表明，疾病以及经常伴随疾病的营养状况对人体处理药物的方式有重大影响。确定基因型与表型之间的一致性或不一致性，为深入研究疾病状态对人体药物代谢的影响提供了手段。它是一种个体探针，可用于研究一般现象或优化患者特异性临床方案，通过预测药物代谢酶活性的紊乱来避免药物毒性。对这一现象的研究仍然是未来疾病对药物代谢影响的临床研究的主要目标。在一项这样的计划研究中，脆弱对代谢基因型和表型之间不一致的影响将在巴尔的摩衰老纵向研究中进行，该研究将使用咖啡因作为CPY2A6和NAT2活性的探测药物。我们也在用大鼠模型研究卡路里限制对代谢活动的影响。我们已经看到热量限制的动物对葡萄糖醛酸盐测试化合物的能力发生了重大变化，并代谢了一种探测药物，氯胺酮。这些结果将被报告。

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 drugg 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 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.