G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 ...has 99.6% sequence identity to pXO1 carried by
and encodes the tripartite anthrax toxin genes and
a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of
G9241 using a transcriptomic analysis and by studying spore formation, an important part of the
lifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37°C in comparison to 25°C. At 37°C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showed
G9241 sporulated rapidly in comparison to the
type strain ATCC 14579, particularly at 37°C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37°C in comparison to 25°C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant of
G9241. This study provides an insight on how the extrachromosomal genetic elements in
G9241 has an influence in bacterial phenotypes.
Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and ...the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4β,25-dihydroxyvitamin D(3) 4β,25(OH)(2)D(3) dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4β,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4β,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4β,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.
Characterization of S-linked conjugates of the endogenous tripeptide glutathione (γ-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approach for the identification of chemically ...reactive, electrophilic intermediates formed during the metabolism of both foreign compounds and endogenous substances. In most cases, GSH adducts generated in vitro or excreted in the bile of animals are detected by the use of liquid chromatography−tandem mass spectrometry (LC−MS/MS), employing survey scans based on characteristic fragmentations of this class of conjugates. However, a limitation of current LC−MS/MS approaches, which typically employ electrospray ionization with analysis of positive ions, is that no single survey scan exhibits broad utility in the detection of unknown GSH adducts, since different structural classes of conjugate (aromatic, benzylic, aliphatic, thioester, etc.) behave differently upon collision-induced dissociation (CID) of the respective M + H+ parent ions. In the present study, we evaluated MS/MS in the negative ion mode as an alternative approach and report herein that the spectra obtained by CID of the M − H- ions of a number of representative GSH adducts, as well as GSH itself, are dominated by fragments originating from the glutathionyl moiety of the tripeptide. In particular, the anion at m/z 272, corresponding nominally to deprotonated γ-glutamyl-dehydroalanyl-glycine, was abundant in the negative ion spectra of free GSH and all GSH conjugates examined, suggesting that scanning for precursors of this ion may provide a generally applicable technique for the detection of adducts of unknown structure. The utility of this novel detection strategy was demonstrated in a series of in vitro and in vivo experiments where compounds known to undergo metabolic activation were examined for their propensity to form conjugates with GSH. In all cases, scanning for precursors of m/z 272 in the negative ion mode revealed the presence of the expected adducts and in some instances revealed additional conjugates that had not been reported previously. Positive ion MS/MS, on the other hand, was more useful than the corresponding negative ion scans in providing information on the molecular structure of GSH conjugates.
Organic Chemistry in Drug Discovery MacCoss, Malcolm; Baillie, Thomas A.
Science (American Association for the Advancement of Science),
03/2004, Letnik:
303, Številka:
5665
Journal Article
Recenzirano
The role played by organic chemistry in the pharmaceutical industry continues to be one of the main drivers in the drug discovery process. However, the precise nature of that role is undergoing a ...visible change, not only because of the new synthetic methods and technologies now available to the synthetic and medicinal chemist, but also in several key areas, particularly in drug metabolism and chemical toxicology, as chemists deal with the ever more rapid turnaround of testing data that influences their day-to-day decisions.
The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently ...attributed to the generation of protein‐reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug–drug interactions that may ensue. One important class of protein‐reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.
The active forms of all marketed hydroxymethylglutaryl (HMG)-CoA reductase inhibitors share a common dihydroxy heptanoic or heptenoic acid side chain. In this study, we present evidence for the ...formation of acyl glucuronide conjugates of the hydroxy acid forms of simvastatin (SVA), atorvastatin (AVA), and cerivastatin (CVA) in rat, dog, and human liver preparations in vitro and for the excretion of the acyl glucuronide of SVA in dog bile and urine. Upon incubation of each statin (SVA, CVA or AVA) with liver microsomal preparations supplemented with UDP-glucuronic acid, two major products were detected. Based on analysis by high-pressure liquid chromatography, UV spectroscopy, and/or liquid chromatography (LC)-mass spectrometry analysis, these metabolites were identified as a glucuronide conjugate of the hydroxy acid form of the statin and the corresponding delta-lactone. By means of an LC-NMR technique, the glucuronide structure was established to be a 1-O-acyl-beta-D-glucuronide conjugate of the statin acid. The formation of statin glucuronide and statin lactone in human liver microsomes exhibited modest intersubject variability (3- to 6-fold; n = 10). Studies with expressed UDP glucuronosyltransferases (UGTs) revealed that both UGT1A1 and UGT1A3 were capable of forming the glucuronide conjugates and the corresponding lactones for all three statins. Kinetic studies of statin glucuronidation and lactonization in liver microsomes revealed marked species differences in intrinsic clearance (CL(int)) values for SVA (but not for AVA or CVA), with the highest CL(int) observed in dogs, followed by rats and humans. Of the statins studied, SVA underwent glucuronidation and lactonization in human liver microsomes, with the lowest CL(int) (0.4 microl/min/mg of protein for SVA versus approximately 3 microl/min/mg of protein for AVA and CVA). Consistent with the present in vitro findings, substantial levels of the glucuronide conjugate (approximately 20% of dose) and the lactone form of SVA simvastatin (SV); approximately 10% of dose were detected in bile following i.v. administration of (14)CSVA to dogs. The acyl glucuronide conjugate of SVA, upon isolation from an in vitro incubation, underwent spontaneous cyclization to SV. Since the rate of this lactonization was high under conditions of physiological pH, the present results suggest that the statin lactones detected previously in bile and/or plasma following administration of SVA to animals or of AVA or CVA to animals and humans, might originate, at least in part, from the corresponding acyl glucuronide conjugates. Thus, acyl glucuronide formation, which seems to be a common metabolic pathway for the hydroxy acid forms of statins, may play an important, albeit previously unrecognized, role in the conversion of active HMG-CoA reductase inhibitors to their latent delta-lactone forms.
Izencitinib is a novel, orally administered pan-JAK inhibitor designed as a gut-selective therapy that was under development for the treatment of inflammatory bowel disease. The objectives of this ...study were to define the mass balance, routes and rates of excretion, and metabolic fate of izencitinib after oral administration of
14
C-izencitinib in humans. Six healthy adult male subjects were administered a single 100 mg (~300 μCi) oral dose of
14
C-izencitinib. Fecal excretion was the dominant route of elimination with >90% of the administered dose recovered in the feces. As expected by design, plasma concentrations of total radioactivity and izencitinib were low with the mean terminal half-life of total radioactivity (138 h) exceeding that of izencitinib (32.4 h). Izencitinib represented approximately 17% of the total circulating radioactivity, suggesting the presence of multiple circulating plasma metabolites. However, no individual metabolite exceeded 10% of total drug-related material in plasma. The major metabolites in feces, M18 and M9, were found to have unusual structures that reflected the presence of a nucleophilic carbon center in the naphthyridine ring of izencitinib. Proposed mechanisms for the formation of these metabolites involved oxidation and rearrangement (M18) and a one-carbon addition, potentially occurring through reaction with endogenous formaldehyde. Given the gut-selective properties of izencitinib, it is proposed that these novel fecal metabolites are the most relevant for evaluating the impact of metabolism on the pharmacological and toxicological properties of izencitinib, and that the circulating plasma metabolite profile is of little consequence in the assessment of the safety characteristics of izencitinib metabolites.
A series of studies were conducted to explore the mechanism of the pharmacokinetic interaction between simvastatin (SV) and gemfibrozil (GFZ) reported recently in human subjects. After administration ...of a single dose of SV (4 mg/kg p.o.) to dogs pretreated with GFZ (75 mg/kg p.o., twice daily for 5 days), there was an increase (approximately 4-fold) in systemic exposure to simvastatin hydroxy acid (SVA), but not to SV, similar to the observation in humans. GFZ pretreatment did not increase the ex vivo hydrolysis of SV to SVA in dog plasma. In dog and human liver microsomes, GFZ exerted a minimal inhibitory effect on CYP3A-mediated SVA oxidation, but did inhibit SVA glucuronidation. After i.v. administration of (14)CSVA to dogs, GFZ treatment significantly reduced (2-3-fold) the plasma clearance of SVA and the biliary excretion of SVA glucuronide (together with its cyclization product SV), but not the excretion of a major oxidative metabolite of SVA, consistent with the in vitro findings in dogs. Among six human UGT isozymes tested, UGT1A1 and 1A3 were capable of catalyzing the glucuronidation of both GFZ and SVA. Further studies conducted in human liver microsomes with atorvastatin (AVA) showed that, as with SVA, GFZ was a less potent inhibitor of the CYP3A4-mediated oxidation of this drug than its glucuronidation. However, with cerivastatin (CVA), the glucuronidation as well as the CYP2C8- and CYP3A4-mediated oxidation pathways were much more susceptible to inhibition by GFZ than was observed with SVA or AVA. Collectively, the results of these studies provide metabolic insight into the nature of drug-drug interaction between GFZ and statins, and a possible explanation for the enhanced susceptibility of CVA to interactions with GFZ.
Therapy with the oral antidiabetic agent troglitazone (Rezulin) has been associated with cases of severe hepatotoxicity and drug-induced liver failure, which led to the recent withdrawal of the ...product from the U.S. market. While the mechanism of this toxicity remains unknown, it is possible that chemically reactive metabolites of the drug play a causative role. In an effort to address this possibility, this study was undertaken to determine whether troglitazone undergoes metabolism in human liver microsomal preparations to electrophilic intermediates. Following incubation of troglitazone with human liver microsomes and with cDNA-expressed cytochrome P450 isoforms in the presence of glutathione (GSH), a total of five GSH conjugates (M1−M5) were detected and identified tentatively by LC−MS/MS analysis. In two cases (M1 and M5), the structures of the adducts were confirmed by NMR spectroscopy and/or by comparison with an authentic standard prepared by synthesis. The formation of GSH conjugates M1−M5 revealed the operation of two distinct metabolic activation pathways for troglitazone, one of which involves oxidation of the substituted chromane ring system to a reactive o-quinone methide derivative, while the second involves a novel oxidative cleavage of the thiazolidinedione (TZD) ring, potentially generating highly electrophilic α-ketoisocyanate and sulfenic acid intermediates. When troglitazone was administered orally to a rat, samples of bile were found to contain GSH conjugates which reflected the operation of these same metabolic pathways in vivo. The finding that metabolism of the TZD ring of troglitazone was catalyzed selectively by P450 3A enzymes is significant in light of the recent report that troglitazone is an inducer of this isoform in human hepatocytes. The implications of these results are discussed in the context of the potential for troglitazone to covalently modify hepatic proteins and to cause oxidative stress through redox cycling processes, either of which may play a role in drug-induced liver injury.
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