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Showing papers in "Current Drug Metabolism in 2001"


Journal Article•DOI•
TL;DR: Mechanistic studies of CYP1A1 induction have provided insights into P450 induction, PAH carcinogenesis, dioxin action, AhR function, and receptor-mediated mammalian gene expression.
Abstract: The CYP1A1 gene encodes microsomal cytochrome P4501A1 that catalyzes the metabolism of many xenobiotics, including the oxygenation of polycyclic aromatic hydrocarbons (PAH). Induction of CYP1A1 enhances the metabolism of PAHs, and therefore, represents an adaptive response to chemical exposure in mammalian cells. Mechanistic studies reveal an AhR/DRE paradigm for the induction, which involves activation of the aryl hydrocarbon receptor (AhR) by an agonist, dimerization of AhR with the Ah recceptor nuclear translocator (Arnt), followed by binding of the AhR/Arnt heterodimer to the dioxin-responsive enhancer (DRE) and transcription of the gene. The AhR mediated transcription is tightly regulated through, at least, two mechanisms: (a) the cytoplasmic AhR interacts with hsp90 and an immunophilin chaperone AIP for proper folding and receptivity, and (b) the agonist-activated, nuclear AhR is degraded through the ubiquitin-26S proteasome mediated protein turnover, such that the transcription by AhR is controlled at a physiologically adequate level. In addition to CYP1A1 induction, AhR mediates a broad range of biological responses to CYP1A1 inducers, typified by the environmental contaminant dioxin, via modulating gene expression. Thus, mechanistic studies of CYP1A1 induction have provided insights into P450 induction, PAH carcinogenesis, dioxin action, AhR function, and receptor-mediated mammalian gene expression.

213 citations


Journal Article•DOI•
Wei Tang1, Ralph Stearns•
TL;DR: Effector-induced increases in CYP3A4 activity were observed during the 1'-hydroxylation of midazolam and 4'- and 10-hydroxYLation of warfarin in human hepatocyte systems, implying that CYP cooperativity has the potential to cause in vivo drug-drug interactions.
Abstract: Cytochromes P450 (CYP) 3A4 is the most abundant human hepatic CYP isoform catalyzing the metabolism of approximately 50% of therapeutic agents. In addition to inhibition or induction, CYP3A4 is subject to stimulation, termed homotropic (substrate stimulation) and heterotropic (stimulation by effectors) cooperativity. The heterotropic cooperativity of CYP3A4 may result from an increase in Vmax, a decrease in Km or a combination of the two and sometimes exhibits regio-selectivity when the enzyme is involved in two or more metabolic pathways for a single substrate. An effector of CYP3A4 can also be a substrate; its metabolism may or may not be inhibited by another substrate. These characteristics of heterotropic cooperativity of CYP3A4 have been interpreted in the context of two binding domains in the active site of the enzyme, two substrate binding plus a distinct allosteric binding site, multiple enzyme conformations or multiple binding sites accompanied by conformational changes. Examples of in vivo CYP cooperativity are rare; representative cases include flavone-dependent stimulation of zoxazolamine metabolism in rats and enhancement of CYP3A-mediated hepatic clearance of diclofenac by quinidine in monkeys. Effector-induced increases in CYP3A4 activity were observed during the 1'-hydroxylation of midazolam and 4'- and 10-hydroxylation of warfarin in human hepatocyte systems. These data imply that CYP cooperativity has the potential to cause in vivo drug-drug interactions. Because cooperative and inhibitory responses from CYP3A4 are known to be substrate-dependent, projection of the pharmacokinetics of an investigational drug and CYP-associated risks of drug-drug interactions in humans can be very complex. Further investigation of CYP cooperativity is warranted.

150 citations


Journal Article•DOI•
TL;DR: It is proposed that this family members have been diverged from the prototype neutral amino acid transporter such as LAT1 by acquiring the mechanisms for the recognition of electric charges on the substrate amino acid side chains.
Abstract: In the last decade, a lot of amino acid transporters were identified by molecular cloning and assigned to the classically characterized amino acid transport systems. Among them, ones which belong to the heterodimeric amino acid transporter family are unique because of their broad substrate selectivity and their pathological implications as well as their structural features. The heterodimeric amino acid transporter family is a subfamily of SLC7 solute transporter family which includes 14-transmembrane cationic amino acid transporters as well as 12-transmembrane heterodimeric amino acid transporters. The members of heterodimeric amino acid transporter family are linked via a disulfide bond to single membrane spanning type II membrane glycoproteins such as 4F2hc (4F2 heavy chain) and rBAT (related to b(0,+)-amino acid transporter). Six members are associated with 4F2hc and one is linked to rBAT. The neutral amino acid transporter of this family seems to rely on the hydrophobic interactions for their substrate recognition which can explain their broad substrate selectivity. Because of this characteristic, they can permeate amino-acid-related drugs and contribute to the pharmacokinetics of these drugs. A neutral amino acid transporter LAT1 (L-type amino acid transporter 1) has actually been shown to be present at the blood-brain-barrier. Because the members of the heterodimeric amino acid transporter family exhibit variety of substrate selectivity, it is proposed that this family members have been diverged from the prototype neutral amino acid transporter such as LAT1 by acquiring the mechanisms for the recognition of electric charges on the substrate amino acid side chains. The dysfunction or hyperfunction of the members of the heterodimeric amino acid transporter family are involved in some diseases and pathologic conditions. The genetic defects of the renal and intestinal transporters BAT1/b(0,+) AT (b(0,+)-type amino acid transporter 1/b(0,+)-type amino acid transporter) and y+ LAT1 (y+ L-type amino acid transporter 1) result in the amino aciduria with sever clinical symptoms such as cystinuria and lysinuric protein intolerance, respectively. LAT1 is proposed to be involved in the progression of malignant tumor. xCT (x- C-type transporter) functions to protect cells against oxidative stress, while its over-function may be damaging neurons leading to the exacerbation of brain damage after, brain ischemia. Therefore, these transporters would be candidates for therapeutic targets based on new strategies. Through the interaction with the associating proteins, the transporters of this family would be endowed with more possibility to be regulated via intracellular and extracellular signalling pathways, which is critical to tune the transporter functions to meet the metabolic requirements of cells.

149 citations


Journal Article•DOI•
TL;DR: In this article, site-directed mutagenesis has been used to identify residues that do not lie within substrate recognition sites and to predict more precise functions for specific residues, making it possible to test these predictions.
Abstract: Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

137 citations


Journal Article•DOI•
TL;DR: It is not clear what fraction of brain CYP has been characterized, although a complete characterization of constitutive and induced CYPs in brain is essential for understanding the role of these enzymes in brain physiology as well as in age-related and xenobiotic-induced neurotoxicity.
Abstract: After many frustrating decades of unsuccessful attempts to characterize the isoforms of P450 in the brain, several scientific breakthroughs in the 80s and 90s have resulted in major advances in our understanding of cytochromes P450 (CYP) in brain. We now know that classical CYP inducers, e.g. phenobarbital and pregnenolone 16alpha-carbonitrile, which regulate drug-metabolizing enzymes in the liver, are specific ligands for ligand-activated transcription factors, and that the brain content of many of these transcription factors is low. This explains why these inducers have little effect on brain CYP content. The most 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 the overall pharmacokinetics of drugs and hormones in the body. Instead CYPs appear to have specific functions in brain, e.g. regulation of the levels of endogenous GABAA receptor agonists maintenance of brain cholesterol homeostasis and elimination of retinoids The novel CYPs which catalyse these reactions have recently been characterized. They are abundantly expressed in the brain confirming what has been previously found, i.e. that the major hepatic, adrenal and gonadal CYP isozymes contribute very little to the overall content of CYP in brain. It is not clear what fraction of brain CYP has been characterized, although a complete characterization of constitutive and induced CYPs in brain is essential for understanding the role of these enzymes in brain physiology as well as in age-related and xenobiotic-induced neurotoxicity.

132 citations


Journal Article•DOI•
TL;DR: A sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins are presented.
Abstract: The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate 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-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.

116 citations


Journal Article•DOI•
TL;DR: This review will concentrate on regulation of P450s in diabetes, obesity and infectious and inflammatory disease, conditions that affect millions of people worldwide every day.
Abstract: Hepatic cytochrome P450 (P450) enzyme activities and gene expression can be profoundly altered in disease states. In general the levels of affected hepatic P450 enzymes are depressed by diseases, causing potential and documented impairment of drug clearance and clinical drug toxicity. However, modulation of P450s is enzyme selective and this selectivity differs among different diseases. This review will concentrate on regulation of P450s in diabetes, obesity and infectious and inflammatory disease, conditions that affect millions of people worldwide every day.

107 citations


Journal Article•DOI•
TL;DR: Differences on inversion rate between compounds and species will be discussed as well as its modification by different patho-physiologic processes such as, inflammation.
Abstract: Molecules exist as three dimensional structures. Therefore they can exist in symmetrical and asymmetrical forms. Molecules with an asymmetric centre are chiral. If the molecule and its mirror image are non-superimposable, the relationship between the two molecules is enantiomeric and the two stereoisomers are enantiomers. Since enantiomers have very similar or identical physicochemical properties, it is very difficult to distinguish between them in an achiral environment. However, once in a chiral environment, as in the body, they exhibit clear differences. In fact, most of the physiological processes in nature are stereospecific. Stereospecificity can occur in pharmacokinetic processes, in particular that utilise a carrier protein, receptor or enzyme. In addition, stereoselectivity occurs in pharmacodynamic processes and the differences between enantiomers can be either qualitative and quantitative. 2-arylpropionic acid derivatives (2APAs - profens) are an important subgroup within the class of NSAIDs. These are chiral compounds marketed as racemic mixtures. Some members of the group in an species-dependent manner undergo a special type of metabolic transformation leading to partial inversion to the optical antipode through a specific conjugation with CoA (coenzyme A) and subsequent epimerization. This metabolic inversion has not only pharmacological consequences (related to clinical effect) but also toxicological consequences such as, formation of hybrid triglycerides and even inhibition of fatty acid beta-oxidation. Differences on inversion rate between compounds and species will be discussed as well as its modification by different patho-physiologic processes such as, inflammation.

107 citations


Journal Article•DOI•
Okita Rt1, Okita•
TL;DR: 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 CYP 4A-mediated hydroxylation of fatty acids.
Abstract: The Cytochrome P450 4A subfamily is one of eighteen subfamilies in the CYP4 family and presently consists of twenty individual forms in nine different mammalian species. The major substrates for CYP4A forms are fatty acids, but recent studies have shown other non-fatty acid substrates may be metabolized by specific CYP4A forms. The physiological and metabolic functions of the CYP4A subfamily have not been elucidated, but the ability of CYP4A forms to metabolize medium and long chain length fatty acids at their omega (omega)-carbon atom has generated significant interest because of the possible role that omega-hydroxylated fatty acids may have in cell signalling processes and as an alternative pathway for fatty acid metabolism. 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 the exposure to compounds that are classified as peroxisome proliferators. There is also considerable interest why multiple CYP4A forms exist in different tissues. Recent studies in the rat and human indicate that other CYP4 forms besides CYP4A forms may be responsible for the metabolism of arachidonic acid to its omega-hydroxy product. 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.

102 citations


Journal Article•DOI•
TL;DR: Aminopeptidase N emerges as the major target for the effects of bestatin on the immune system and some of its effects on tumor growth and the endometrium, and bestatin-sensitive LTA4 hydrolase generates the potent chemotactic agent, LTB4.
Abstract: Bestatin, an antibiotic of microbial origin, is a potent inhibitor of some, but not all aminopeptidases. It can be administered, with low toxicity, to cultured cells, intact animals and humans. It has become a useful tool in elucidating the physiological role of some mammalian exopeptidases in the regulation of the immune system, in the growth of tumors and their invasion of surrounding tissues, and in the degradation of cellular proteins. Bestatin-sensitive enzymes play important roles in the digestion and absorption of peptides in the brush border of the intestine and the kidney, in the reproductive system, and in the metabolism of opioid peptides and leukotrienes. Aminopeptidase N emerges as the major target for the effects of bestatin on the immune system and some of its effects on tumor growth and the endometrium. It is also the major bestatin-sensitive enzyme involved in the degradation of oligopeptides on the surface of intestine and kidney brush borders, and the inactivation of enkephalins in the brain. Bestatin-sensitive cytosolic exopeptidases are important in the degradation to amino acids of di- and tripeptides generated in most cells by cellular protein degradation, as well as those absorbed through the brush border of intestine and kidney. Inhibition of one of these exopeptidases, cytosol alanine aminopeptidase, results in apoptosis. Bestatin-sensitive cystinyl aminopeptidase is abundant in placenta. Two bestatin-sensitive enzymes, aminopeptidase B and nardilysin, are particularly abundant in late spermatids. Finally bestatin-sensitive LTA4 hydrolase generates the potent chemotactic agent, LTB4.

99 citations


Journal Article•DOI•
F. P. Guengerich1•
TL;DR: Reactions discussed here include mechanism-based heme inactivation, mechanism- based protein modification, 1,2-shifts, 1- and 2-electron reductions, 1 -electron oxidation, oxidative cleavage of carboxylic acid esters, desaturation, deformylation of aldehydes, ring formation, ipso mechanisms for aryl dehalogenation and O- and N-dearylation.
Abstract: Cytochrome P450 (P450) enzymes play major roles in the metabolism of drugs, carcinogens, steroids, eicosanoids, alkaloids, pesticides, and other important xenobiotics, as well as chemicals normally endogenous to the body. P450s are generally considered in a classical catalytic reduction-oxidation cycle and an odd-electron abstraction/rebound chemical mechanism that can be used to rationalize carbon hydroxylation, dealkylation of heteroatomic substrates, heteroatom oxygenation, and the oxidation of unsaturated compounds to epoxides and phenols. However, many other reactions are catalyzed by P450s but not generally appreciated. The classical catalytic mechanism requires some expansion to explain all of these reactions. Reactions discussed here include mechanism-based heme inactivation, mechanism-based protein modification, 1,2-shifts, 1- and 2-electron reductions, 1-electron oxidation, oxidative cleavage of carboxylic acid esters, desaturation, deformylation of aldehydes, ring formation, ipso mechanisms for aryl dehalogenation and O- and N-dearylation, cis-trans bond isomerization, several rearrangements of oxidized eicosanoids, aldoxime dehydration, and hydrolysis of phosphatidylcholine.

Journal Article•DOI•
TL;DR: An overview of the MRPs substrates recently clarified, mainly about anticancer drugs is described, and it is expected that the anticancer agents transporter, moreover anti cancer drug resistance mechanisms, can be clarified.
Abstract: Acquirement of drug resistance by tumor cells is a major chemotherapeutic problem. It is well known that typical multidrug resistance is caused by P-glycoprotein and multidrug resistance related protein (MRP1) which belong to the ATP binding cassette (ABC) transporter family. Ishikawa proposed that the ATP-dependent glutathione-S-conjugate export pump (GS-X pump) and phase III detoxification system are essential to drug metabolism, and this constituted a new concept in drug metabolism and the detoxification of xenobiotics. The GS-X pump has been revealed to belong to the ABC transporter family and suggested to the contribution to anticancer drug resistance. The GS-X pump actively effluxes the glutathione S-platinum (GS-Pt) complex. We cloned novel ABC transporter cDNA from the PC-14/CDDP cell line, and the cloned cDNA was designated as a short-type MRP homologue, SMRP. Further investigation suggested that SMRP is a splicing variant of MRP5. The MRP5 mRNA levels in tumors from lung cancer patients treated with platinum regimen were significantly higher than in tumors from patients treated with non-platinum regimens, and the MRP5 expression levels were correlate with the GCS expression levels that is the rate-limiting step enzyme in glutathione biosynthesis. These results suggested that MRP5 take part in the function of GS-X pump. Recently many transporter molecules belong to the ABC transporter family such as MRP family have been identified, and appear to express in various human tissues. It can be presumed that their molecules are affected by the disposition and metabolism of drugs, but their substrates are still unclear. If the substrate specificity is revealed in the future, it is expected that the anticancer agents transporter, moreover anti cancer drug resistance mechanisms, can be clarified. This review is cited in the cisplatin resistance and the GS-X pump, and finally describes an overview of the MRPs substrates recently clarified, mainly about anticancer drugs.

Journal Article•DOI•
TL;DR: The interaction of surface-modified liposomes, which are designed to avoid interactions with blood proteins and/or cells and to actively target specific cells or tissues are briefly touched on.
Abstract: A number of studies have appeared recently on the underlying mechanisms of liposome-cell interactions under in vitro conditions, in which isolated cell populations or cell lines were used. However, our knowledge of how liposomes interact with cells and the parameters that influence this in vivo is limited. We will summarize and discuss the relevant studies on this matter in this article. In addition, researchers in this field have long been aware of the interaction of liposomes with blood (or serum/plasma) proteins in vivo and their potential role in the process of the clearance of liposomes from the circulation. Some of the 'opsonizing' proteins, such as complement components, immunoglobulins, which enhance the interactions of liposomes with 'phagocytic cells' have been identified. However, the issue of which types of opsonins determine the fate of liposomes in vivo and how liposomal physicochemical properties such as size, charge and fluidity play an important role in the process of liposome clearance is not clear. Our own observations of one of opsonins, complement component are reviewed herein. As opposed to the fate of conventional liposomes, we briefly touch on the interaction of surface-modified liposomes, which are designed to avoid interactions with blood proteins and/or cells (sterically stabilized liposomes, long-circulating liposomes) and to actively target specific cells or tissues (targeted liposomes: immunoliposomes). Blood proteins such as opsonins are not usually thought to play an important role in the clearance of such liposomes.

Journal Article•DOI•
TL;DR: The major function of glucuronidation is to change hydrophobic compounds into hydrophilic derivatives, a process which facilitates their detoxification and excretion, but it is also widely recognized that glucuronisation can result in compounds which are biologically active or demonstrate increased toxicity.
Abstract: UDP-Glucuronosyltransferases (UGTs) are glycoproteins, localized in endoplasmic reticulum (ER) and nuclear membranes, which catalyze the confugation 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.

Journal Article•DOI•
TL;DR: M mammals have evolved mechanisms to induce proteins involved in xenobiotic detoxification, andabolism by Phase I enzymes, particularly the heme containing monooxygenases cytochromes P450 is frequently the first line of defense against such xenobiotics.
Abstract: Humans and rodents are exposed to many foreign compounds in their diet (e.g., herbal supplements such as St. John's wart), in their environment (e.g., organochlorine pesticides and polychorinated biphenyls), and as clinically prescribed drugs (e.g., rifampin and phenobarbital). In response to these exposures mammals have evolved mechanisms to induce proteins involved in xenobiotic detoxification. Metabolism by Phase I enzymes, particularly the heme containing monooxygenases cytochromes P450 is frequently the first line of defense against such xenobiotics.

Journal Article•DOI•
TL;DR: Using in situ hybridization and immunohistochemical techniques, many cytochrome P450 enzymes have been demonstrated to be present in brain and to have a regional rather than universal distribution.
Abstract: The presence and activity of cytochromes P450 in brain regions and various brain cells have been extended and advanced over the last five years covered by this review. Using in situ hybridization and immunohistochemical techniques, many cytochrome P450 enzymes have been demonstrated to be present in brain and to have a regional rather than universal distribution. Many of these various cytochromes P450 have been shown to catalyze the metabolism of neurosteroids as well as other biologically significant compounds in brain. In addition, many cytochrome P450 enzymes have been implicated in the metabolism of psychoactive drugs such as neuroleptics and antidepressants. The regulation of cytochrome P450 expression has been studied at greater detail, the regulation of aromatase being a prominent example during the last five years.

Journal Article•DOI•
TL;DR: 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 theintestinal tract and the liver, in vivo pharmacokinetics (PK), and likelihood of clinically significant interactions with other drugs.
Abstract: 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.

Journal Article•DOI•
TL;DR: 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) provide useful tools for manipulating glycosphingolipid levels in cells and for elucidating questions associated with sphingoline signaling.
Abstract: 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 alpha-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.

Journal Article•DOI•
TL;DR: With the modification of an anthracycline molecule as a substrate for the nucleoside transport system, the anthrACYcline could be delivered selectively to tumor cells.
Abstract: Anthracycline antibiotics are very effective neoplastic agents widely used clinically. However, because of their many adverse effects (e.g. cardiotoxicity, leukopenia and alopecia), their clinical use has been limited. In order to minimize their adverse effects in clinical cancer chemotherapy, anthracyclines must be selectively transported into tumor cells. If there are differences in transport characteristics between tumor and normal cells, it should be possible to establish a strategy for selectively delivering anthracyclines to tumor cells on the basis of the differences. In human cultured leukemia HL60 cells, as tumor cells, and human fresh mononuclear cells, as normal cells, doxorubicin, pirarubicin, daunorubicin and idarubicin were incorporated via a common carrier-mediated system, but the carriers were different in the two cell types. In HL60 cells, it was indicated that a nucleoside transport system contributed, at least in part, to the transport of doxorubicin and pirarubicin, but not daunorubicin and idarubicin, and its contribution to pirarubicin transport was found in other tumor cells, i.e. mouse ovarian sarcoma M5076 and Ehrlich ascites carcinoma cells. On the other hand, in mononuclear cells, there was no involvement of a nucleoside transport system for the four anthracyclines examined. Therefore, we thought that with the modification of an anthracycline molecule as a substrate for the nucleoside transport system, the anthracycline could be delivered selectively to tumor cells.

Journal Article•DOI•
Kakuyama A1, Sadzuka Y•
TL;DR: The results suggested that caffeine, which is one of xanthine derivatives will be useful for biochemical modulator and the increase of antitumor activity of doxorubicin by caffeine is suggested.
Abstract: Biochemical modulation, which is more effective with the use of antitumor agents, has recently played very important role in cancer chemotherapy. In this review, it was reported that some of the methylxanthine derivatives, e.g. caffeine, were useful for modulator and attempted to defined the relation between the effect of methylxanthine derivatives on the doxorubicin transport and antitumor activity. Caffeine and theobromine inhibited the doxorubicin efflux from tumor cells, increased the doxorubicin concentration in a tumor, and enhanced the antitumor effect of doxorubicin. However, the caffeine metabolites, which had no effect on the doxorubicin efflux, did not increase antitumor activity. Moreover, caffeine and theobromine did not enhance the side toxicity of doxorubicin on the lipid peroxide level, DNA biosynthesis and the doxorubicin concentrations in normal tissues. Moreover, we investigated the effect of the combination of doxorubicin with caffeine or theobromine on the change in cyclic adenosine 3',5'-monophosphate (cyclic AMP) in tissues in vivo, and the effect of cyclic AMP on doxorubicin efflux in vitro, and measured the distribution of caffeine and theobromine in normal and tumor tissues. In Ehrlich ascites carcinoma bearing mice, the level of cyclic AMP in a tumor was decreased by doxorubicin. With the combination of caffeine or theobromine and doxorubicin, the cyclic AMP level recovered to the control level. This tendency was not seen in normal tissues (heart and liver). Moreover, the doxorubicin efflux from the Ehrlich cells was inhibited on the addition of cyclic AMP in vitro. And the caffeine concentration in the tumors was the same as that in the heart, and was increased in combination with doxorubicin compared with that in the caffeine-only group during the 4 hr after caffeine treatment. Furthermore, the doxorubicin efflux was promoted by the supply of energy (addition of glucose), influx was decreased relatively, doxorubicin efflux needs the existence of glucose and the inhibition of energy related drug export pump by caffeine induced inhibition of doxorubicin efflux. The treatment of doxorubicin nor caffeine, and any treatment schedule did not change the amount and appearance of GLUT 1 as glucose transporter on Ehrlich ascites carcinoma cell. For the mentioned above, we thought as concerns the increase of antitumor activity of doxorubicin by caffeine which is xanthine derivatives as follows. Caffeine distributes, high level in tumor, keeps the cyclic AMP level, and effects glucose transport or doxorubicin transport depend on energy and inhibits doxorubicin efflux. And then DNA synthesis was increased with the maintenance the concentration of doxorubicin in tumor. These action did not show in normal tissues, caffeine did not influence the side toxicity of doxorubicin. These results suggested that caffeine which is one of xanthine derivatives will be useful for biochemical modulator.

Journal Article•DOI•
V Trajkovic1•
TL;DR: 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 iN OS enzyme.
Abstract: 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 froin 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.

Journal Article•DOI•
Alfin D. N. Vaz1•
TL;DR: The extent of current knowledge on the roles proposed for the heme- peroxo, hydroperoxO, and superoxo complexes in various reactions is reviewed to establish such species in the reactions they are proposed to effect.
Abstract: The activation of molecular oxygen by Cytochromes P450 to the ultimate mono-oxygen oxidant species involves three distinct dioxygen species coordinated to the heme iron These intermediates have different chemical properties, and have recently been proposed to participate in some Cytochrome P450-catalyzed oxidation reactions This article reviews the extent of our current knowledge on the roles proposed for the heme- peroxo, hydroperoxo, and superoxo complexes in various reactions The extent to which such species contribute to the breadth of reactions catalyzed by Cytochrome P450 has yet to be defined, and more definitive experiments are needed to establish such species in the reactions they are proposed to effect

Journal Article•DOI•
TL;DR: The results from the successful application of the in vivo brain microdialysis technique to BBB drug transport studies are reviewed, which include novel and CNS-active peptides, some agents that are actively removed from the brain ISF across the BBB, and a brain-directed prodrug.
Abstract: There is a wide range of methods available for studying the transport of drugs across the blood-brain barrier (BBB) which is equipped with several systems to transport drugs as well as endogenous nutrients and waste products. The in vivo brain microdialysis technique, which allows direct sampling of the brain interstitial fluid (ISF), is a powerful means of characterizing influx and efflux transport across the BBB. In this paper, we review our results from the successful application of this technique to BBB drug transport studies. The drugs investigated include novel and CNS-active peptides, some agents that are actively removed from the brain ISF across the BBB, and a brain-directed prodrug.

Journal Article•DOI•
TL;DR: This review will analyze the current knowledge of identified pharmacodynamic correlations in high-dose chemotherapy on drugs employed at high doses, such as cyclophosphamide, melphalan, busulfan, carmustine, paclitaxel, or docetaxel.
Abstract: There is usually considerable variability in anticancer drug plasma levels when delivered at high doses requiringstem-cell support. Given their narrow therapeuticwindows and wide interpatient pharmacokinetic variability, drug monitoring and pharmacokinetic-directed dosing representan attractive strategy in this setting. A major previous requirement to successful application of therapeuticdrug monitoringis identificationof a significant and clinically meaningful pharmacodynamic correlation between a pharmacokinetic parameter and a toxic or therapeutic outcome, or preferably, both. In this review, we will analyze the current knowledge of identified pharmacodynamic correlations in high-dose chemotherapy. We will summarize the observations from other authors and our own, on drugs employed at high doses, such as cyclophosphamide, melphalan, busulfan, carmustine, paclitaxel, or docetaxel.