Current Drug Metabolism - Volume 1, Issue 2, 2000

Drug-metabolizing enzymes are called mixed-function oxidase or monooxygenase and containing many enzymes including cytochrome P450, cytochrome b5 , and NADPH-cytochrome P450 reductase and other components. The hepatic cytochrome P450s (Cyp) are a multigene family of enzymes that play a critical role in the metabolism of many drugs and xenobiotics with each cytochrome isozyme responding differently to exogenous chemicals in terms of its induction and inhibition. For example, Cyp 1A1 is particularly active towards polycyclic aromatic hydrocarbons (PAHs), activating them into reactive intermediates those covalently bind to DNA, a key event in the initiation of carcinogenesis. Likewise, Cyp 1A2 activates a variety of bladder carcinogens, such as aromatic amines and amides. Also, some forms of cytochrome P450 isozymes such as Cyp 3A and 2E1 activate the naturally occurring carcinogens (e.g. aflatoxin B1 ) and N-nitrosamines respectively into highly mutagenic and carcinogenic agents. The carcinogenic potency of PAHs, and other carcinogens and the extent of binding of their ultimate metabolites to DNA and proteins are correlated with the induction of cytochrome P450 isozymes.Phase II drug-metabolizing enzymes such as glutathione S-transferase, aryl sulfatase and UDP-glucuronyl transferase inactivate chemical carcinogens into less toxic or inactive metabolites. Many drugs change the rate of activation or detoxification of carcinogens by changing the activities of phases I and II drug-metabolizing enzymes. The balance of detoxification and activation reactions depends on the chemical structure of the agents, and is subjected to many variables that are a function of this structure, or genetic background, sex, endocrine status, age, diet, and the presence of other chemicals. It is important to realize that the enzymes involved in carcinogen metabolism are also involved in the metabolism of a variety of substrates, and thus the introduction of specific xenobiotics may change the operating level and the existence of other chemicals. The mechanisms of modification of drug-metabolizing enzyme activities and their role in the activation and detoxification of xenobiotics and carcinogens have been discussed in the text.

The unexpected occurrence of idiosyncratic drug reactions during late clinical trials or after a drug has been released can lead to a severe restriction in its use or failure to release/withdrawal. This leads to considerable uncertainty in drug development and has led to attempts to try to predict a drugs potential to cause such reactions. It appears that most idiosyncratic drug reactions are due to reactive metabolites however, many drugs that form reactive metabolites are associated with a very low incidence of idiosyncratic drug reactions. Therefore, screening drugs for their ability to generate reactive metabolites is likely to cause the rejection of many good drug candidates. There is evidence to suggest that an idiosyncratic drug reaction is more likely if there is some danger signal. Thus drugs that cause some degree of cell stress or damage may be more likely to lead to a high incidence of idiosyncratic drug reactions. The exact nature of the putative danger signals is unknown. However, a screen of the effects of drugs known to be associated with a high incidence of idiosyncratic reactions using expression genomics and proteomics may reveal a pattern or patterns of mRNA and protein expression that predict which drugs will cause a high incidence of idiosyncratic drug reactions. Although idiosyncratic drug reactions are not usually detected in animal tests because they are just as idiosyncratic in animals as they are in humans, it is likely that drug reactive metabolites would also cause similar cell stress in animals. It is more likely that in most cases it is differences in the immune response to the reactive metabolites that determine which individuals will develop an idiosyncratic reaction. If the expression of certain proteins in animals treated with a drug candidate could be used as a screening method to predict a drugs potential to cause a high incidence of idiosyncratic drug reactions, it would greatly facilitate the development of safer drugs.

Glucuronidation represents a major pathway which enhances the elimination of many lipophilic xenobiotics and endobiotics to more water-soluble compounds. The UDP-glucuronosyltransferase (UGT) family catalyzes the glucuronidation of the glycosyl group of a nucleotide sugar to an acceptor compound (aglycone) at a nucleophilic functional group of oxygen (eg, hydroxyl or carboxylic acid groups), nitrogen (eg, amines), sulfur (eg, thiols), and carbon, with the formation of a b-D-glucuronide product. At this time, over 35 different UGT gene products have been described from several different species. UGTs have been divided into two distinct subfamilies based on sequence identities, UGT1 and UGT2. The UGT1 gene subfamily consists of a number of UGTs that result from alternate splicing of multiple first exons and share common exons 2-5. The substrate specificities of the various isoforms have been examined in cultured cell experiments, and include bilirubin, amines, and planar and bulky phenol. The UGT2 gene family is different in that the UGT2 mRNAs are transcribed from individual genes. The UGT2 subfamily consists of numerous enzymes which catalyze the glucuronidation of a diverse chemical base including steroids, bile acids, and opioids.Until recently, the liver has been the major focus for studying the metabolism of xenobiotics and endobiotics. Several groups have identified extrahepatic tissues that express UGT isoforms including the kidney, gastrointestinal tract and brain. This review discusses the two UGT gene families, substrate specificities, and the recent discoveries of UGTs in extrahepatic tissues.

Acyl glucuronides are a unique class of electrophilic metabolites, capable of non-enzymatic reactions including acylation and/or glycation of endogenous macromolecules, hydrolysis to reform the parent aglycone, and intra-molecular rearrangement. Three human UDP-glucuronosyltransferases (UGTs) catalyzing the hepatic glucuronidation of carboxylic acid drugs have been identified, UGT1A3, UGT1A9 and a UGT2B7 variant. Within the liver, acyl glucuronides also undergo enzymatic hydrolysis by b-glucuronidase and esterases which, like the UGTs, are located in the endoplasmic reticulum. In addition, the liver also transports acyl glucuronides between the sinusoidal circulation and bile. Due to their polarity, membrane transport of acyl glucuronides is carrier-mediated, resulting in the establishment of significant concentration gradients between sinusoidal circulation, hepatocyte and bile, in the order of 150 5000 in these compartments, respectively. As a result of exposure to high acyl glucuronide concentrations, the liver is a major target of protein adduct formation. Dipeptidylpeptidase IV, UGTs and tubulin have been identified as intra-hepatic targets of adduct formation by acyl glucuronides. Adduct formation results in altered protein activity and potentially contributes to hepatotoxicity. Hepatic protein adducts are also immunogenic and may cause immune mediated cytotoxicity. Both intra- and extra-hepatic exposure to acyl glucuronides depends not only on the efficiency of glucuronidation and hydrolysis by the liver, but also on the efficiency of the hepatic membrane transport systems. Thus, changes in membrane transporter activities, as may occur due to saturation or drug-drug interactions, can significantly affect acyl glucuronide disposition, adduct formation and the disposition of parent aglycone, thereby affecting clinical efficacy and toxicity of acyl glucuronide forming drugs.

The human flavin-containing monooxygenase (FMO3) is a prominent enzyme system that converts nucleophilic heteroatom-containing chemicals, drugs and xenobiotics to more polar materials that are more efficiently excreted in the urine. The substrate specificity for FMO 3 is distinct from that of FMO1. Human FMO3 N-oxygenates primary, secondary and tertiary amines whereas human FMO1 is only highly efficient at N-oxygenating tertiary amines. Both human FMO1 and FMO3 S-oxygenate a number of nucleophilic sulfur-containing substrates and in some cases, does so with great stereoselectivity. Human FMO3 is sensitive to steric features of the substrate and aliphatic amines with linkages between the nitrogen atom and a large aromatic group such as a phenothiazine of at least five carbons are N-oxygenated significantly more efficiently than those substrates with two or three carbons. For amines with smaller aromatic substituents such as phenethylamines, often these compounds are efficiently N-oxygenated by human FMO3. Currently, the most promising non-invasive probe of in vivo human FMO3 functional activity is the formation of trimethylamine N-oxide from trimethylamine that comes from dietary choline. (S)-Nicotine N-1 -oxide formation can also be used as a highly stereoselective probe of human FMO3 function for adult humans that smoke cigarettes. Finally, cimetidine S-oxygenation or ranitidine N-oxidation can also be used as a functional probe of human FMO3. With the recent observation of human FMO3 genetic polymorphism and poor metabolism phenotype in certain human populations, variant human FMO3 may contribute to adverse drug reactions or exaggerated clinical response to certain medications. Knowledge of the substrate specificity for human FMO3 may aid in the future design of more efficacious and less toxic drugs.

Tryptophan is a constituent of proteins and in parallel it represents a source for mainly two pivotal biochemical pathways the generation of 5-hydroxytryptamine (serotonin), and the formation of kynurenine by the enzymes tryptophan pyrrolase (TP) and indoleamine 2,3-dioxygenase (IDO). IDO is induced by interferon-g (IFN-g ) in a broad variety of cells. Therefore, enhanced tryptophan degradation is observed in diseases and disorders concomitant with cellular immune activation, e.g. infectious diseases, autoimmune diseases, malignant diseases as well as in pregnancy. IFN-g -derived tryptophan degradation may represent an effector mechanism within in the comprehensive network of immune stimulation. In addition, the cytostatic and, respectively, antiproliferative properties on e.g., T-lymphocytes may contribute to the immunomodulatory function of IFN-g . However, especially in states of persistent immune activation increased tryptophan catabolism leads to the depletion of free serum tryptophan and to the accumulation of neuroactive kynurenine metabolites. As a consequence, serotonergic functions may be affected, and the neurotoxic properties of kynurenine derivatives may lead to neuronal disorders evoking neurological/psychiatric symptoms. This notion provides a basis for the better understanding of mood disorders and related syptoms in chronic diseases. Moreover, IDO could represent a link between the immunological network and neuroendocrine functions with far reaching consequences regarding to the psychological status of patients.

Accelerator mass spectrometry (AMS) permits the measurement of elemental isotopes at the individual atom level. The main application of AMS in drug discovery and development will be in the analysis of 14-carbon ( 14 C). The principle behind AMS is the separation of individual positively charged atoms through mass, charge and momentum differences. In order to obtain the high-energy charge state required for separation, negative atoms are accelerated through a high voltage field (up to 10 million volts) generated by a tandem Van de Graaff accelerator. In the middle of the accelerator, the outer valency electrons are stripped from the atom and the resulting charged species are separated and counted. For 14 C, AMS counts the number of individual atoms rather than measuring radioactive decays. The result is that AMS is up to one million times more sensitive than decay counting. Radioactivity levels as low 0.0001 dpm can be detected using AMS. The exquisite sensitivity of AMS analysis means that much lower amounts of 14 C can be used than for conventional counting methods. This makes it easier to use 14 C for in vitro , preclinical and clinical research programmes. As 14 C poses both a biological and environmental hazard, AMS permits much lower doses to be used. Human drug mass balance studies have been conducted with doses of 50 nanoCuries and below. Radioactive HPLC metabolite profiles of plasma extracts from subjects given nanoCurie doses of 14 C-labelled drug have been obtained by injecting as little as 0.25 dpm onto an HPLC column. In studies of biologics, biosynthetically 14 C-labelled recombinant protein has been produced with a specific radioactivity sufficient to conduct human clinical studies with AMS analysis. For one human recombinant protein an increase in sensitivity of 2000-fold over ELISA was obtained with AMS measurement. AMS is an enabling technology that should prove of value in increasing human and environmental safety as well as allowing new research directions to be followed.