Mechanism-Based Inactivators as Probes of Cytochrome P450
Structure and Function Pp. 215-243
Cytochrome P450 in the Brain; A Review Pp. 245-263
Cytochrome P450 4A Fatty Acid Omega Hydroxylases Pp. 265-281
Human UDP-Glucuronosyltransferase 2B7 Pp. 283-298
The Use of In Vitro Methods to Predict In Vivo Pharmacokinetics
and Drug Interactions Pp. 299-314
Modulation of Inducible Nitric Oxide Synthase Activation by
Immuno-suppressive Drugs Pp. 315-329
Agents for the Treatment of Glycosphingolipid Storage
D-isorders Pp. 331-338
[Back
to top] Mechanism-Based Inactivators as Probes of Cytochrome P450
Structure and Function
The
cytochromes P450 superfamily of enzymes is a group of hemeproteinsthat catalyze
the metabolism of an extensive series of compounds including drugs,chemical
carcinogens, fatty acids, and steroids. They oxidize substrates ranging in
sizefrom ethylene to cyclosporin. Although significant efforts have been made
to obtainstructural information on the active sites of the microbial P450s,
relatively little iscurrently known regarding the identities of the critical
amino acid residues in the P450active sites that are involved in substrate
binding and catalysis. Since information on thecrystal structures of the
eukaryotic P450s has been relatively limited, investigators have used a variety
ofother techniques in attempts to elucide the structural features that play a
role in the catalytic properties andsubstrate specificity at the enzyme active
site. These include site-directed mutagenesis, natural mutations,homology
modeling, mapping with aryl-iron complexes, affinity and photoaffinity
labeling, and mechanism-basedinactivators. A variety of different
mechanism-based inactivators have proven to be useful inidentifiying active
site amino acid residues involved in substrate binding and catalysis. In this
review wepresent a sampling of the types of studies that can be conducted using
mechanism-based inactivators andhighlight studies with several classes of
compounds including acetylenes, isothiocyanates, xanthates,aminobenzotriazoles,
phencyclidine, and furanocoumarins. Labeled peptides isolated from the
inactivatedproteins have been analyzed by N-terminal amino acid sequencing in
conjunction with mass spectrometry todetermine the sites of covalent
modification. Mechanistic studies aimed at identifying the basis for
theinactivation following adduct formation are also presented.
[Back
to top] Cytochrome P450 in the Brain; A Review
After
many frustrating decades of unsuccessful attempts to characterize the isoforms
of P450 in thebrain, several scientific breakthroughs in the 80s and 90s have
resulted in major advances in ourunderstanding of cytochromes P450 (CYP) in
brain. We now know that classical CYP inducers, e.g.phenobarbital and pregnenolone
16a-carbonitrile, which regulate drug-metabolizing enzymes in the liver,
arespecific ligands for ligand-activated transcription factors, and that the
brain content of many of thesetranscription factors is low. This explains why
these inducers have little effect on brain CYP content. Themost effective
inducers of brain P450 are some of the CNS active drugs and solvents.
The level of CYPs in brain, approximately 0.5-2% of that in liver, is too low to significantly influence theoverall pharmacokinetics of drugs and hormones in the body. Instead CYPs appear to have specific functionsin brain, e.g. regulation of the levels of endogenous GABAA receptor agonists maintenance of braincholesterol homeostasis and elimination of retinoids The novel CYPs which catalyse these reactions haverecently been charaterized. They are abundantly expressed in the brain confirming what has been previouslyfound, i.e. that the major hepatic, adrenal and gonadal CYP isozymes contribute very little to the overallcontent of CYP in brain. It is not clear what fraction of brain CYP has been characterized, although acomplete characterization of constitutive and induced CYPs in brain is essential for understanding the role ofthese enzymes in brain physiology as well as in age-related and xenobiotic-induced neurotoxicity.
[Back
to top] Cytochrome P450 4A
Fatty Acid Omega Hydroxylases
The
Cytochrome P450 4A subfamily is one of eighteen subfamilies in the CYP4 family
andpresently consists of twenty individual forms in nine different mammalian
species. The major substrates forCYP4A forms are fatty acids, but recent
studies have shown other non-fatty acid substrates may bemetabolized by
specific CYP4A forms. The physiological and metabolic functions of the CYP4A
subfamilyhave not been elucidated, but the ability of CYP4A forms to metabolize
medium and long chain length fattyacids at their omega (w)-carbon
atom has generated significant interest because of the possible role that w-hydroxylatedfatty
acids may have in cell signalling processes and as an alternative pathway for
fatty acidmetabolism. A number of different compounds or physiological
conditions have been shown to regulate the expression of CYP4A forms in liver
and/or kidney. Several CYP4A forms may serve as a marker for theexposure to
compounds that are classified as peroxisome proliferators. There is also
considerable interest whymultiple CYP4A forms exist in different tissues.
Recent studies in the rat and human indicate that other CYP4forms besides CYP4A
forms may be responsible for the metabolism of arachidonic acid to its w-hydroxyproduct.
The focus of this review will be to summarize recent studies that have
characterized the substrate specificity of rat, rabbit and human CYP4A forms
and discuss the significance of CYP4A-mediated hydroxylation of fatty acids. In
addition, dietary effects or novel compounds that have been reported to
regulate CYP4A expression in the rat and mouse will be discussed
[Back
to top] Human UDP-Glucuronosyltransferase 2B7
UDP-Glucuronosyltransferases (UGTs) are glycoproteins, localized in endoplasmic reticulum (ER) and nuclear membranes, which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GlcUA) as the sugar donor. The major function of glucuronidation is to change hydrophobic compounds into hydrophilic derivatives, a process which facilitates their detoxification and excretion. However, it is also widely recognized that glucuronidation can result in compounds, which are biologically active or demonstrate increased toxicity. UGTs, like other drug-metabolizing enzymes, have been postulated to be involved in controlling the steady state concentrations of nuclear receptor ligands for interactions with nuclear receptors [1,2]. One of the isoforms from the UGT2B subfamily, UGT2B7, has been found to be a major human UGT2B isoform, involved in the glucuronidation of a variety of endogenous compounds and xenobiotics.
In this review, we included all available information from our studies and those of other investigators on a) the history of the identification and expression of UGT2B7 in human tissues, b) the substrate specificity of UGT2B7, c) the extrahepatic localization of UGT2B7 d) the nuclear localization of UGT2B7 and e) characterization of the UGT2B7 gene and promoter.
[Back
to top] The Use of In Vitro Methods to Predict In Vivo Pharmacokinetics and
Drug Interactions
With the dramatic change underway in the process of drug discovery and development it has become increasingly important to define, both qualitatively and quantitatively, the dispositional features of new chemical entities (NCEs) as early in the process as possible. To that end strategies have emerged that are designed to enable reasonable predictions about a NCE’s absorption from the gastrointestinal tract, systemic bioavailability and likelihood for significant pre-systemic clearance, character of metabolic processing both within the gastrointestinal tract and the liver, in vivo pharmacokinetics (PK), and likelihood for clinically significant interactions with other drugs. To some extent these strategies have embraced interspecies allometric scaling in which findings in animals are extrapolated to predict outcomes in humans. However, a greater emphasis in recent years has been placed on predicting human PK and the likelihood of clinically significant drug-drug interactions for NCEs solely from in vitro experiments. These general strategies have been methodologically streamlined so that hundreds or even thousands of experiments on a given NCE can be conducted within several days. Dispositional data from these pre-clinical experiments is useful for rapidly identifying potential marketing advantages for NCEs, and for screening out those substances that should not be placed into more expensive and labor-intensive animal experiments or brought to clinical trial. The key issue in these strategies is the accuracy with which pre-clinical findings predict clinical outcomes. Based largely on retrospective analyses the current state of the art exhibits a high percentage of useful predictions. However, there are many examples in which the prediction of either human PK or clinical drug-drug interactions from pre-clinical data has failed. The reasons for inaccurate predictions are manifold, and may include the actual in vitro methodology used, inappropriate model selection, and errant scale-up factors. Additionally, in vitro methods may fail to account for complex hepatobiliary processing including transport phenomena and Phase II metabolism. Progress has been made in establishing humanized methodologies that accurately describe these processes, with a view toward reconstituting the contributions of each into a more complex and accurate depiction and prediction of in vivo PK and drug-interaction potential.
[Back
to top] Modulation of Inducible Nitric Oxide Synthase Activation by
Immuno-suppressive Drugs
The activation of inducible form of nitric oxide (NO) synthase (iNOS, type II, or macrophage NOS) and subsequent production of free radical gas NO is an important anti-infectious and anti-tumor mechanism of innate immunity. On the other hand, high amounts of iNOS-derived NO have been implicated in self-tissue destruction during autoimmune diseases, allograft rejection, sepsis, and other disorders accompanied by excessive activation of the immune system. It is generally accepted that beneficial effects of some recently designed immunosuppressive agents primarily stem from their ability to interfere with the function of T and/or B cells, thus preventing deleterious consequences of specific immunity-innate immunity positive feedback, with high NO production being one of them. However, it has been recently observed that drugs like cyclosporin A, FK506, leflunomide, mycophenolate mofetil, pentoxifylline, and linomide can directly modulate cytokine and/or LPS-induced NO production in various cell types in vitro, probably by interfering with iNOS gene transcription or catalytic activity of iNOS enzyme. Interestingly, some of these drugs exhibited cell-specific pattern of iNOS modulation, thus indirectly revealing distinct requirements for iNOS induction in different cell types. Possible impact of this direct and cell-selective interference with iNOS activation on the therapeutic effectiveness of immunosuppressive drugs is discussed.
[Back
to top] Agents for the Treatment of Glycosphingolipid Storage Disorders
We have developed a series of inhibitors of glucosylceramide synthase that are structurally based on the parent compound D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol (PDMP). These inhibitors provide useful tools for manipulating glycosphingolipid levels in cells and for elucidating questions associated with sphingolipid signaling. Recently, two highly active glucosylceramide synthase inhibitors, D-threo-3’,4’-ethylenedioxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol and D-threo-4’-hydroxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol, were designed, synthesized, and studied. These inhibitors markedly reduced glycosphingolipid levels in MDCK cells without any accumulation of intracellular ceramide and associated growth inhibition. Subsequently, each inhibitor was evaluated for its ability to lower glycolipid levels in virally transformed lymphoblasts from a patient with a-galactosidase A deficiency. Both compounds significantly reduced neutral glycosphingolipid levels in the lymphoblasts without any morphological changes and growth inhibition. Furthermore, the inhibitors were applied to a mouse knockout model of Fabry disease. Inhibitor treatment blocked accumulation of globotriaosylceramide (Gb3) in the kidney, liver and heart of mice. In contrast to another glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin, this treatment was not associated with any significant change in body weight or organ weight and without immunodepletion. These results suggest that these newest PDMP homologues are promising as therapeutic agents for the treatment of glycosphingolipid storage disorders.