Current Drug Metabolism - Volume 3, Issue 5, 2002

In the drug discovery process the pharmacokinetic screening, drug stability studies, evaluation of metabolites, CYP involvement, enzyme induction and inhibition, and excretion studies play a major role. The use of more sensitive and novel detection systems have made the discovery process less cumbersome than in previous years. In particular, the use of whole-body autoradiography (WBA) for tissue distribution, which was once considered an impractical tool, owing to the long turn around time (4-10 weeks), is coming to the forefront for rapidly resolving issues encountered in discovery. In today's research environment early lead compounds can be radio-labeled and whole-body sections imaged quickly (3-5 days) using new techniques, which has made 14C- and 3H-WBA a viable tool. The technique has been used in vivo in species from mice to monkeys, and ex vivo and / or in vitro in larger animals and humans. WBA has considerable merit in identifying ‘pharmacodefficient’ compounds and providing insight on mechanistic questions. WBA data can provide information related to tissue pharmacokinetics, routes of elimination, CYP or Pgp mediated drug-drug interactions, tissue distribution, site specific drug localization and retention, metabolism, clearance, compound solubility issues, routes of administration, penetration into specific targets (e.g., tumors), tissue binding (e.g., melanin), and interspecies kinetics. Thus, WBA is quickly becoming part of the battery of studies conducted during the lead optimization process to select optimal drug candidates. Examples of the use of the WBA tool in early discovery are reviewed.

There is an increasing awareness that the metabolites of anticancer drugs can contribute to the pharmacodynamic effects that are observed, which has stimulated a much greater emphasis on metabolic and pharmacokinetic issues. This has coincided with the development of electrospray and related atmospheric pressure ionization mass spectrometry techniques such as ionspray (nebulizer assisted electrospray), turboionspray (heated nebulizer assisted electrospray) and atmospheric pressure chemical ionization (nebulization coupled with corona discharge). The combination of collision induced dissociation and tandem mass spectrometry coupled with a softionization process that produces abundant molecular species provides very powerful methodology for the trace analysis of drugs and their metabolites. The present review has emphasized the more rigorous quantitative applications that have appeared in the literature over the last five years. It is evident that modern techniques of liquid chromatography tandem mass spectrometry coupled with stable isotope dilution methodology have had a profound effect on our ability to analyze anticancer drugs and their metabolites. As new drugs emerge into the clinic, this methodology will clearly be the method of choice, particularly when many samples have to be analyzed over a short time. This approach was beautifully demonstrated in the study of the novel signal transduction inhibitor, Gleevec where thousands of clinical samples were analyzed for drug and metabolites over a relatively short period of time. The need to analyze anticancer drugs and their metabolites with such prompt turn around times has stimulated even more rapid approaches to analysis using roboticbased purification methodology and short LC chromatographic run times.

Drug or xenobiotics metabolizing enzymes (DMEs or XMEs) play central roles in the biotransformation, metabolism and / or detoxification of xenobiotics or foreign compounds, that are introduced to the human body. In general, DMEs protect or defened the body against the potential harmful insults from the environment. Once in the body, many xenobiotics may induce signal transduction events either specifically or non-specifically leading to various cellular, physiological and pharmacological responses including homeostasis, proliferation, differentiation, apoptosis, or necrosis. For the body to minimize the insults caused by these xenobiotics, various tissues / organs are well equipped with diverse DMEs including various Phase I and Phase II enzymes, which are present in abundance either at the basal level and / or increased / induced after exposure. To better understand the pharmacogenomic / gene expression profile of DMEs and the underlying molecular mechanisms after exposure to xenobiotics or drugs, we will review our current knowledge on DNA microarray technology in gene expression profiling and the signal transduction events elicited by various xenobiotics mediated by either specific receptors or non-specific signal transduction pathways. Pharmacogenomics is the study of genes and the gene products (proteins) essential for pharmacological or toxicological responses to pharmaceutical agents. In order to assess the battery of genes that are induced or repressed by xenobiotics and pharmaceutical agents, cDNA microarray or oligonucleotide-based DNA chip technology can be a powerful tool to analyze, simultaneously, the gene expression profiles that are induced or repressed by xenobiotics. The regulation of gene expression of the various phase I DMEs such as the cytochrome P450 (CYP) as well as phase II DMEs generally depends on the interaction of the xenobiotics with the receptors. For instance, the expression of CYP1 genes can be induced via the aryl hydrocarbon receptor (AhR) which dimerizes with the AhR nuclear translocator (ARNT), in response to many polycyclic aromatic hydrocarbon (PAHs). Similarly, the steroid family of orphan receptors, the constitutive androstane receptor (CAR) and pregnane X receptors (PXR), heterodimerize with the retinoid X receptor (RXR), transcriptionally activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such as phenobarbital-like compounds (CAR) and dexamethasone and rifampin-type of agents (PXR). The peroxisome proliferator activated receptor (PPAR) which is one of the first characterized members of the nuclear hormone receptor, also dimerizes with RXR and it has been shown to be activated by lipid lowering agent fibrate-type of compounds leading to transcriptional activation of the promoters on the CYP4A genes. The transcriptional activation of these promoters generally leads to the induction of their mRNA. The physiological and the pharmacological implications of common partner of RXR for CAR, PXR, and PPAR receptors largely remain unknown and are under intense investigations.For the phase II DMEs, phase II gene inducers such as phenolic compounds butylated hydroxyanisol (BHA), tertbutylhydroquinone (tBHQ), green tea polyphenol (GTP), (-)-epicatechin-3-gallate (EGCG) and the isothiocyanates (PEITC, sulforaphane) generally appear to be electrophiles. They can activate the mitogen-activated protein kinase (MAPK) pathway via electrophilic-mediated stress response, resulting in the activation of bZIP transcription factors Nrf2 which dimerizes with Mafs and binds to the antioxidant / electrophile response element (ARE / EpRE) enhancers which are found in many phase II DMEs as well as many cellular defensive enzymes such as thioredoxins, γGCS and HO-1, with the subsequent induction of gene expression of these genes. It appears that in general, exposure to phase I or phase II gene inducers or xenobiotics may trigger a cellular ‘stress’ response leading to the increase in the gene expression of these DMEs, which ultimately enhance the elimination and clearance of the xenobiotics and / or the ‘cellular stresses’ including harmful reactive intermediates such as reactive oxygen species (ROS), so that the body will remove the ‘stress’expeditiously. Consequently, this homeostatic response of the body plays a central role in the protection of the organism against environmental insults such as xenobiotics.Advances in DNA microarray technologies and mammalian genome sequencing will soon allow quantitative assessment of expression profiles of all genes in the selected tissues. The ability to predict phenotypic outcomes from gene expression profiles is currently in its infancy, however, and will require additional bioinformatic tools. Such tools will facilitate information gathering from literature and gene databases as well as integration of expression data with animal physiolog

The antitumor ether lipid ET-18-OCH3 (edelfosine) is the prototype of a new class of antineoplastic agents, synthetic analogues of lysophosphatidylcholine, that shows a high metabolic stability, does not interact with DNA and shows a selective apoptotic response in tumor cells, sparing normal cells. Unlike currently used antitumor drugs, ET-18- OCH3 does not act directly on the formation and function of the replication machinery, and thereby its effects are independent of the proliferative state of target cells. Because of its capacity to modulate cellular regulatory and signalingevents, including those failing in cancer cells, like defective apoptosis, ET-18-OCH3, beyond its putative clinical importance, is an interesting model compound for the development of more selective drugs for cancer therapy. Although ET-18-OCH3 enhances host defense mechanisms against tumors, its major antitumor action lies in a direct effect on cancer cells, inhibiting phosphatidylcholine biosynthesis and inducing apoptosis in tumor cells. Recent progress has allowed unraveling the molecular mechanism underlying the apoptotic action of ET-18-OCH3, leading to the notion that ET-18-OCH3 is selectively incorporated into tumor cells and induces cell death by intracellular activation of the cell death receptor Fas / CD95. This intracellular Fas / CD95 activation is a novel mechanism of action for an antitumor drug and represents a new way to target tumor cells in cancer chemotherapy that can be of interest as a new framework in designing novel antitumor drugs. ET-18-OCH3 and some analogues are pleiotropic agents that affect additional biomedical important diseases, including parasitic and autoimmune diseases, suggesting new therapeutic indications for these compounds.

In response to the challenge laid down by advances in other drug discovery functions, DMPK has now established an array of automated, miniaturised in vitro screens, rapid bioanalytical methodologies and in silico tools with which to optimise or predict passive absorption, metabolic clearance and minimise drug-drug interaction potential. The awareness of the pivotal role that physicochemical properties play in the control of many of these processes has been key. This review highlights some of these structure-activity relationships with emphasis on drug absorption, clearance, protein binding and distribution. However, some fundamental processes remain to be elucidated fully, including the in vivo impact of non-specific or futile binding in in vitro screens and the functional significance of intestinal and hepatobiliary transporter proteins. Transgenic animals should soon add value to our understanding of the contribution of transporter proteins to drug bioavailability (intestinal and hepatic drug uptake / efflux) and drug interactions and in validating projections for Man. Future studies should also focus on the evaluation of the various in vitro human CYP induction screens available, with particular emphasis on their predictive value for the clinical scenario.

The purpose of this study was to evaluate the permeability characteristics of endocrine disrupting chemicals utilizing epithelial monolayers of Caco-2 cells. The drugs tested in this study were bisphenol A (BPA), tert-octylphenol (tOP), tert-butylphenol (tBP), di(2-ethylhexyl)phthalate (DOP), dibutylphthalate (DBP), and butylbenzylphthalate (BBP), all of which are used in plastic materials. The Caco-2 cell line was grown on cell culture inserts with polyethylene terephthalate membranes, and Hank's balanced salt solution (HBSS, pH 7.4) was used for the transport experiments. The barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a volt ohmmeter, and transport of these endocrine disrupting chemicals was examined in both directions. The permeated amounts of these chemicals within 180 min in the apical to basolateral (A-to-B) and the basolateral to apical (B-to-A) directions without verapamil, a P-glycoprotein (P-gp) inhibitor, were in the rank order of tBP > tOP > BPA > DOP > DBP > BBP and BPA >> tBP > tOP > DOP > DBP > BBP, respectively. In the presence of 100 mM verapamil, the permeated amounts of BPA, tOP and tBP within 180 min in the B-to-A direction decreased by 12-, 2.6- and 3.1-fold, respectively. In the case of phthalate esters, the permeated amount of DOP within 180 min in the B-to-A direction decreased by 1.6-fold, while that of DBP and BBP showed no significant changes. The ratios of apparent permeability coefficient of B-to-A against A-to-B, Papp ratios, for BPA, tOP and tBP were markedly decreased in the presence of 100 μM verapamil. These findings indicated that both BPA and alkyl phenols are substrates of the P-gp located in the apical side of Caco-2 cells, and suggested that the P-gp in the small intestine may act as an organic barrier against BPA and alkyl phenols.