Current Drug Metabolism - Volume 9, Issue 2, 2008

The sulfotransferase (SULTs) catalyzes the sulfonation of a multitude of xenobiotics, hormones and neurotransmitters. This review has summarised the SULT family in detail, the structure of the twelve known enzymes, in their four known groups (SULT1, SULT2, SULT4, and SULT6) and the substrates for each respective SULT. Hepatic sulfonation is a common phase II metabolic mechanism for increasing molecular hydrophilicity in preparation for biliary excretion or efflux across the hepatic basolateral membrane for subsequent renal clearance. To date, a total of 13 human cytosolic SULT genes have been identified which spread across four families; SULT1, SULT2, SULT4, and SULT6. The established structures of SULTs provide evidence for both enzyme/substrate and enzyme/ cofactor binary complexes, consistent with a random bi-bi mechanism and ruling out an ordered mechanism in which binding of substrate requires binding of cofactor (or vice versa). Members of the SULT1 family have demonstrated the ability to sulfonate simple (small planar) phenols including estradiol, thyroid hormones, environmental xenobiotics and drugs. The SULT2 family members catalyze sulfonation of hydroxyl groups of steroids, such as androsterone, allopregnanolone, and dehydroepiandrosterone. As yet, no known substrate or function has been identified for the SULT4 family, and the SULT6B1 gene, expressed in the testis of primates, has neither the protein nor its enzymatic activity characterized. The extent of nucleotide variation found in members of the SULT gene family is similar to that observed for other groups of human genes. Substrate inhibition was observed for most substrates with a trend in maximum velocity (Vmax) of *1>*3>*2. There does appear to be an inter-ethnic/inter-racial difference in the incidence of the various SULT1A1 alleles also. There is mounting evidence to suggest that further research and understanding in the area of phase II metabolism and the SULT enzyme will have a great benefit in a clinical setting. Already research in the field is finding links with cancer and sulfonation-related disease, promising to deliver great advances in clinical practice in the future.

The placenta is a unique organ that is essential to a healthy and normal pregnancy. A number of phase I and II metabolizing enzymes are expressed at moderate levels in the placenta, and have been proven to have the ability to metabolize certain xenobiotics. Depending on the substrate, this metabolic action may have significant clinical implications on how it affects the fetus. A wide variety of transporters including P-glycoprotein, breast cancer resistance protein, and multidrug resistance associated proteins have also been discovered in the placenta, and while most are found to have mainly physiological substrates, there are a number of xenobiotics which are also able to gain access to the fetus through transport across the placenta. Depending on the xenobiotics and its intended action, drug transport across the placenta may be desired, acceptable or undesirable. Medications administered to the mother but designed to work on the fetus are now being used increasingly, and demonstrates an important clinical implication in which drug transport across the placenta is desirable. However, medications designed to treat the mother but are also able to cross the placenta carry potential risks to damage the developing fetus, and it is therefore essential that the effects of different drugs on the fetus are known before they are administered during pregnancy. There is still much unknown about drug transport and drug metabolism in the placenta, and it is vital that in the future further research is done to discover the clinical implications of these activities in the placenta. This research is often complicated by the fact that it is unethical to run studies in pregnant women, and so research is often carried out in pregnant animals. These results are not always accurate, however, as the human's placental structure is different from the placenta in other animals. Drug metabolism and drug transport across the placenta should continue to be researched, and guidelines need to be developed to ensure that any medications used during pregnancy are safe to both the mother and the fetus, and that successful treatment of the medical condition is carried out.

Heart failure is a very serious cardiovascular disease that affects more than five million people in North America. The role of cytochrome P450 (CYP) in cardiovascular health and disease is well established. Many CYP enzymes have been identified in the heart and their levels have been reported to be altered during cardiac hypertrophy and heart failure. There is a great deal of discrepancy between various reports on CYP alterations during heart failure, likely due to differences in disease severity, species in question and other underlying conditions. In general, however, cardiac CYP1B and CYP2A, CYP2B, CYP2E, CYP2J, CYP4A and CYP11 mRNA levels and related enzyme activities are usually increased. Moreover, there is a strong correlation between CYP-mediated endogenous metabolites and the pathogenesis of cardiac hypertrophy and heart failure. Some of these metabolites confer cardioprotective effect such as estradiol, dehydroepiandrosterone, epoxyeicosatrienoic acids, and prostaglandin I2; whereas, other metabolites may be harmful to the heart such as androgens, aldosterone, hydroxyeicosatetraenoic acids, and thromboxane A2. On the other hand, heart failure plays an important role in the down-regulation of hepatic CYP involved in drug metabolism through several mechanisms which include hepatocellular damage, hypoxia, elevated levels of pro-inflammatory cytokines, and increased production of heme oxygenase-1. Therefore, more research is needed to elucidate the mechanisms by which CYP affect the development and/or progression of heart failure and also the mechanism by which heart failure alters cardiac and hepatic CYP enzymes.

Numerous members of the cytochrome P450 (CYP) superfamily are induced after exposure to a variety of xenobiotics in human liver. We have gained considerable mechanistic insights into these processes in hepatocytes and multiple ligand-activated transcription factors have been identified over the past two decades. Families CYP1, CYP2 and CYP3 involved in xenobiotic metabolism are also expressed in a range of extrahepatic tissues (e.g. intestine, brain, kidney, placenta, lung, adrenal gland, pancreas, skin, mammary gland, uterus, ovary, testes and prostate). Since the expression of the majority of the isoforms appears to be very low in the extrahepatic tissues in comparison with predominant expression in adult liver, the role of the enzymes in overall biotransformation and total body clearance is minor. However, basal expression and up-regulation of extrahepatic CYP enzymes can significantly affect local disposition of xenobiotics or endogenous compounds in peripheral tissues and thus modify their pharmacological/toxicological effects or affect absorption of xenobiotics into systemic circulation. The goal of this review is to critically examine our current understanding of molecular mechanisms involved in induction of xenobiotic metabolizing CYP genes of human families CYP1, CYP2 and CYP3 by exogenous chemicals in extrahepatic tissues. We concentrate on organs such as the intestine, kidney, lung, placenta and skin, which are involved in drug distribution and clearance or are in direct contact with environmental xenobiotics. We also discuss single nucleotide polymorphisms (SNPs) of key CYPs, which at the level of transcription affect expression of the genes in the extrahepatic tissues or are associated with some pathophysiological stages or disorders in the organs.

The anti-N-methyl-D-aspartate (NMDA) effect of dextromethorphan (DEM) seems to be mainly related to the unchanged drug rather than to its more potent metabolite dextrorphan (DOR). The aim of our study was to assess the involvement of P-glycoprotein (Pgp) and pH conditions in the transmembranal transport of these two NMDA antagonists, using a human in vitro Caco-2 cell monolayer model. Transmission electron microscopy, transepithelial electrical resistance, [3H]-mannitol permeability, Western blot analysis and the bidirectional transport of the positive controls, rhodamine and digoxine were used to confirm model's integrity and validity. The bidirectional transport of DEM and DOR (1 to 100μM) across the monolayers was investigated in the presence and absence of the P-gp inhibitor cyclosporine A (10μM) at two pH conditions (pH 6.8/7.7-pH 7.4/7.4) and assessed with the specific and more potent P-gp inhibitor GF120918 (4μM). Analytical quantification was achieved using high performance liquid chromatography. At a pH gradient, DEM and DOR were subject to a significant active efflux transport (Papp(B-A) > 2-3x Papp(A-B); p<0.01). However, neither the influx nor the efflux was affected by P-gp inhibitors. At physiological pH, we observed no more efflux of the drugs and no influence of the inhibitors. In conclusion, dextromethorphan and dextrorphan are not P-gp substrates. However, pH-mediated efflux mechanisms seem to be involved in limiting DEM gastrointestinal absorption. The preferential anti-NMDA central effect of DEM appears to be P-gp independent.

Drug in vivo pharmacokinetic performances in nature consist of sequential membrane transporting processes and are based on the entry into and exit of drugs from cell, even for metabolism process requiring parent drugs delivered into and metabolites effluxed from the metabolizing cells. Efficient and reliable high throughput screen of membrane permeability properties as early as possible in drug discovery and development program is accordingly desirable. Biopartitioning chromatography (BPC) introduces biomembrane-mimetic structures (such as liposome, phospholipid monolayer, micelle, microemulsion, vesicle and bicelle, etc) into chromatographic system, i.e. liquid system or capillary electrophoresis, and thereby emulates drug-membrane interactions difficult to study in the liquid state by well reproducible, rapid, sensitive and adequately designed chromatographic technique. And recently BPC has been becoming a high-throughput screening platform for drug membrane permeability and biological activity. The theoretical basis, classification and application of BPC were summarized based on the latest advances and our recent works. The development potential and perspectives of this field were also discussed.

P-glycoprotein (P-gp, ABCB1, MDR1) was recognized as a drug-exporting protein from cancer cells three decade ago. Apart from the multidrug transporter side effects of P-gp, normal physiological functions of P-gp have been reported. P-gp could be responsible for translocating platelet-activating factor (PAF) across the plasma membrane and PAF inhibited drug transport mediated by P-gp in cancer cells. P-gp regulated the translocation of sphingomyelin (SM) and GlcCer, and short chain C6-NBD-GlcCer was found in the apical medium of P-gp cells exclusively and not in the basolateral membrane. SM plays an important role in the esterification of cholesterol. High expression of P-gp prevents stem-cell differentiation, leading to the proliferation and amplification of this cell repertoire, and functional P-gp plays a fundamental role in regulating programmed cell death, apoptosis. The transporter function of P-gp is therefore necessary to protect cells from death. P-gp can translocate both C6-NBD-PC and C6-NBD-PE across the apical membrane. This PC translocation was also confirmed with [3H]choline radioactivity. Progesterone is not transported by P-gp, but blocks P-gp-mediated efflux of other drugs and P-gp can mediate the transport of a variety of steroids. Cells transfected with human P-gp esterified more cholesterol. P-gp might also be involved in the transport of cytokines, particularly IL-1β, IL-2, IL-4 and IFNα, out of activated normal lymphocytes into the surrounding medium. P-gp expression is also associated with a volume-activated chloride channel, thus P-gp is bifunctional with both transport and channel regulators. We also present information about P-gp polymorphism and new structural concepts, “gate” and “twist”, of the P-gp structure.

Metabolism of xenobiotics can sometimes generate cyclic metabolites. Such metabolites are usually the result of intramolecular reactions occurring within a primary or secondary metabolite and this chemistry may lead to unexpected structures. Intramolecular chemistry is often driven by nucleophilic groups reacting with electrophilic atoms, often carbon, although radical processes also occur. Conjugation of xenobiotics or their metabolites with endogenous thiols, such as glutathione or cysteine, introduce a reactive amino group that can lead to the formation of cyclic structures. Less common than chemically driven cyclizations are enzymatically mediated ringclosures, although this may reflect our incomplete recognition of enzymatic involvement in this step of cyclic metabolite formation. While some cyclic metabolites are biologically inactive, others are biologically active. Thus, a cyclic metabolite may display desirable pharmacology, or, contribute to toxicology. When a cyclic metabolite is identified, it is important to consider the possibility that it is an artifact, i.e. metabonate, that was formed during processing of the sample, for example, through degradation or by chemical reactions with other components present in the matrix. From a medicinal chemistry perspective, a cyclic metabolite with a different chemical scaffold from the parent structure may lead to a new series of structurally novel, biologically active molecules with the same, or different, pharmacology from the parent. This review will cover a selection of cyclic metabolites from a mechanistic point of view, and when possible, discuss their biological relevance.