Application Of Drug Metabolism And Pharmacokinetics Evaluation In Drug Discovery And Development

Drug research encompasses several diverse disciplines united by a common goal, namely the development of novel therapeutic agents. Since the early 1990s, several new forces in drug discovery, including chemistry, molecular biology, and robotics, have changed the pursuit of this endeavor. The recent developments in combinatorial chemistry synthesis and high-throughput screening in pharmaceutical research and development have driven the production of very large numbers of compounds with potential for pharmacological activity.It has been estimated that nearly 90% of drugs fail because of unacceptable efficacy, toxicity and poor drug metabolism and pharmacokinetics (DMPK) properties, although no single factor can be highlighted as the major cause. Moreover, the assignment of failure to a distinct category might itself be misleading, as these factors are all inter-related, and poor DMPK properties may be the reason of poor efficacy and toxicity. It is, therefore, becoming apparent that in addition to pharmacological properties, DMPK properties, including intestinal absorption, first-pass effect, serum plasma protein binding, blood-brain barrier permeability, metabolic stability, inhibition or induction of drug metabolism enzymes, drug-drug interactions, genetic polymorphism in metabolism, and toxicity, are crucial determinants of the ultimate clinical success of a drug. This realization has led to the early introduction of high-throughput absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) screening during the drug discovery process, in an effort to select against drugs with problematic ADME/Tox profiles. Preclinical ADME screening facilitates early elimination of weak candidates and directs the entire focus of the drug development program towards fewer potential lead candidates.Inhibition and induction of cytochrome P450 (CYP) enzymes are probably the most common causes for documented drug-drug interactions. Inhibition of drug metabolism by competition for the same enzyme may result in undesirable elevations in plasma concentrations of drugs, which can lead to serious adverse effects and toxicity. On the other hand, enzyme induction, which is defined as an increase in the amount and activity of a drug-metabolizing enzyme, may increase the drug's elimination and attenuate its pharmacological effect as a result of decreases in plasma concentrations. Therefore, drug interactions represent not only a medical problem for clinicians and patients, but also an economic loss. We established a high-throughput assay platform for cytochrome P450 induction and inhibition profiling of lead compounds. Moreover, new non-steroidal anti-inflammatory drugs—methyl salicylate glycosides were discovered in our lab by metabolism methods. We found methyl salicylate glycosides had significant analgesic and anti-inflammatory effects while causing no gastrointestinal toxicity.ChapterⅡHigh-throughput screening approaches for inductive activities of compounds toward cytochrome P450-mediated metabolismEach of us is confronted daily by an extensive array of environmental chemicals or xenobiotics. Members of the cytochrome P450 (CYP) family of haem-containing monooxygenases often catalyse the first step in the detoxification of lipophilic substances. Two closely related orphan nuclear hormone receptors—the pregnaneⅩreceptor (PXR) and the constitutive androstane receptor (CAR)—have recently emerged as transcriptional regulators of cytochrome P450 expression that couple xenobiotic exposure to oxidative metabolism. Although the induction of CYP gene expression represents an adaptive response to protect against chemical exposure, this physiological response is also the basis for an important class of drug-drug interactions. This phenomenon, can be a serious problem in these days of 'polypharmacy', in which a patient is likely to be taking several medications. Ideally, drugs should be developed that are neutral with respect to their effects on CYP expression.On agonist binding, nuclear receptors undergo a conformational change that increases their affinity for coactivators. Recruitment of coactivator to agonist-boundnuclear receptor is a critical step in the formation of an active transcription complex on DNA. Studies have demonstrated that coactivator fragments containing the motif LXXLL, where L is leucine and X is any amino acid, bind to nuclear receptors in an agonist-dependent manner. In this study, we synthesized a short synthetic FITC-labeled peptide containing an LXXLL motif and used it to develop a fluorescence polarization assay for agonist binding to PXR. In this homogeneous biochemical assay, the greater the extent of rhodamine-peptide binding to PXR, the greater the extent of fluorescence polarization observed. The development of high-throughput PXR binding assays for the identification of PXR ligands at the early stage of the drug discovery process will improve the likelihood that the undesired activity can be removed subsequently. The FP-based assay that can be used to identify the binding of test compounds to all metabolic nuclear receptors, including CAR, VDR, PPAR, LXR, FXR et al.ChapterⅢ. High-throughput screening approaches for inhibitory activities of compounds toward cytochrome P450-mediated metabolismMany drug-drug interactions are metabolism-based, and result from two or more drugs competing for the same enzyme with the majority of these interactions involving CYP. Thus, much higher plasma concentrations of a drug are attained if a second drug competes for the enzyme responsible for metabolic clearance of the first drug. For a drug with a narrow therapeutic index, this would lead to an adverse reaction. These problems have prompted the need to assess drug safety early during drug discovery/development, and to identify and eliminate compounds that may exhibit a potential for undesirable drug interactions. Assessing the safety of new drug candidates during drug discovery can save a considerable amount of time and money, and prevent the exposure of patients to unnecessary risk, especially if a drug must later be removed from the market because of safety issues.Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. Recombinant CYPs (rCYPs) often serve as the metabolizing system because, in addition to the more obvious selectivity advantages, their use keeps the total protein concentration low, minimising non-specific binding. In this study, we used genetically engineered yeast cells containing Helianthus tuberosus CPR gene to co-express human CYP450 & reductase. We developed microtiter plate-based, direct, fluorometric assays for the activities of the heterologously expressed principal human drug-metabolizing enzymes, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. This methodology is quantitative, rapid, reproducible, and compatible with common high throughput screening instrumentation.ChapterⅣ. Application of DMPK in drug discovery——Discovery of new NSAIDS, methyl salicylate glycosidesThe role of drug metabolism in drug discovery is more than just excluding compounds with poor DMPK properties. The prodrug concept was first proposed based on drug metabolism and pharmacokinetics. This approach has been widely used in drug design. Although there are many reasons to use prodrugs, improvement of gastrointestinal stimulations is one of the most common.Salicylic acid was identified in willow bark as an active analgesic and anti-inflammatory compound over a century ago. Because of its unpleasant taste, chemical derivatives of salicylic acid were synthesized and tested. Aspirin, acetylated form of salicylic acid, eliminated the unpleasant taste but retained the same pharmacological effects, and it was introduced for treating human maladies over 100 years ago. Aspirin is one of the most widely used drugs worldwide at present. It acetylates cyclooxygenases thereby irreversibly blocking the conversion of arachidonic acid to prostanoids. A major factor limiting its use is gastrointestinal toxicity produced by inhibiting cytoprotective COX-1. A new generation of NSAIDs, cyclooxygenase-1 sparing, cyclooxygenase-2 selective inhibitors, have been developed in an attempt to improve gastrointestinal tolerance. But cyclooxygenase-2 inhibitors could increase the risk of cardiovascular events by inhibiting synthesis of "protective" prostacyclin in the vascular wall but not platelet thromboxane formation.Gaultherin, a natural salicylate derivative from G. yunnanensis, exhibited similar analgesic and anti-inflammatory activity to aspirin. Orally-administered gaultherin did not act by being directly absorbed and metabolized to salicylate in the liver, but rather only after it was transformed into methyl salicylate by intestinal bacteria.and then the absorbed methyl salicylate was hydrolyzed rapidly by esterases in intestine, blood and liver to produce salicylate. Gaultherin released active ingredient, salicylate slowly and provided effective concentrations for an extended period without incurring the high concentrations that might provoke side effects. Sodium salicylate lacks ulcerogenicity for the reason that it doesn't inhibit the constitutive cyclooxygenase-1 and cyclooxygenase-1 has been shown to be the predominant source of cytoprotective prostaglidins in normal gastric mucosa. This contrasts with direct administration of aspirin, which inhibits cytoprotective cyclooxygenase-1 and releases high concentration of salicylate acid to gastric mucosa, which is the reason aspirin cause the side effect of gastric ulcer. Compared with aspirin, gaultherin causes none of the side effects associated with inhibition of cyclooxygenase-1, including gastric ulcer and bleeding.