Takahiro Murai

Predictability of Idiosyncratic Drug Toxicity Risk for Carboxylic Acid-Containing Drugs Based on the Chemical Stability of Acyl Glucuronide

Acyl glucuronides (AGs) formed from carboxylic acid-containing drugs have been considered to be a cause of idiosyncratic drug toxicity (IDT). Chemical stability of AGs is supposed to relate to their reactivity. In this study, the half-lives of 21 AGs of carboxylic drugs in potassium phosphate buffer (KPB), human serum albumin (HSA) solution, and human fresh plasma were analyzed in relation to the IDT risk derived from these drugs. The carboxylic drugs were classified into three safety categories of “safe,” “warning,” and “withdrawn” in terms of their IDT risk. As for the results, the half-lives of AGs in KPB correlated with the IDT risk better than those in HSA solution or in human fresh plasma with regard to the separation of the safe drugs from the warning drugs or the withdrawn drugs. In KPB, whereas the half-lives in the safe category were 7.2 h or longer, those in the withdrawn category were 1.7 h or shorter. The classification value of the half-life in KPB, which separated the safe drugs from the withdrawn drugs was calculated to be 3.6 h by regression analysis. In conclusion, this is the first report that clearly shows the relationship between the IDT risk and chemical stability of AGs in several in vitro systems. The KPB system was considered to be the best for evaluating the stability of AGs, and the classification value of the half-life in KPB serves as a useful key predictor for the IDT risk.

Metabolic Pathways of Inhaled Glucocorticoids by the CYP3A Enzymes

Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and ∆6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of ∆6-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis.

Mechanism for covalent binding of rofecoxib to elastin of rat aorta.

We have previously reported that oral administration of [14C]rofecoxib to rats resulted in the long retention of radioactivity by the aorta as a consequence of covalent binding to elastin. Treatment of rats with α-phenyl-α-propylbenzeneacetic acid 2-[diethylamino]-ethyl ester hydrochloride (SKF-525A), a cytochrome P450 inhibitor, significantly decreased the systemic exposure of 5-hydroxyrofecoxib, one of the main metabolites of rofecoxib, whereas there was no statistically significant change in the retention of radioactivity from [14C]rofecoxib in the aorta. On the other hand, the aortic retention of radioactivity closely correlated to the systemic exposure of unchanged rofecoxib in the dose range between 2 and 10 mg/kg. A covalent binding study of [14C]rofecoxib in vitro using rat aorta homogenate in the presence of d-penicillamine, hydralazine, β-aminopropionitrile, and sodium borohydride suggested that the aldehyde group of allysine in elastin was relevant to the covalent binding. In a model reaction using benzaldehyde, rofecoxib but not 5-hydroxyrofecoxib reacted with the aldehyde group of benzaldehyde in a manner of condensation reaction under a physiological pH condition. A histopathological examination using an electron microscope demonstrated that multiple oral administration of rofecoxib to rats caused marked degradation of the elastic fiber system of the aorta. These results suggested that rofecoxib as such is reactive in vivo, undergoing a condensation reaction with allysine, thereby preventing the formation of cross-linkages in elastin, i.e., desmosine and isodesmosine, and causing the degradation of the elastic fibers.

HUMAN UDP-GLUCURONOSYLTRANSFERASE, UGT1A8, GLUCURONIDATES DIHYDROTESTOSTERONE TO A MONOGLUCURONIDE AND FURTHER TO A STRUCTURALLY NOVEL DIGLUCURONIDE

We identified human UDP-glucuronosyltransferase (UGT) isoforms responsible for producing dihydrotestosterone (DHT) diglucuronide, a novel glucuronide in which the second glucuronosyl moiety is attached at the C2′ position of the first glucuronosyl moiety, leading to diglucuronosyl conjugation of a single hydroxyl group of DHT at the C17 position. Incubation of the DHT monoglucuronide with 12 cDNA-expressed recombinant human UGT isoforms and uridine 5′-diphosphoglucuronic acid resulted in a low but measurable DHT diglucuronidation activity primarily with UGT1A8, a gastrointestinal UGT, and to a lesser extent with UGT1A1 and UGT1A9. In contrast, the activity of DHT monoglucuronidation was high and was found in UGT2B17, UGT2B15, UGT1A8, and UGT1A4 in descending order. Among the 12 UGT isoforms tested, only UGT1A8 was capable of producing DHT diglucuronide from DHT. The kinetics of DHT diglucuronidation by microsomes from human liver and intestine fitted the Michaelis-Menten model, and the Vmax/Km value for the intestinal microsomes was approximately 4 times greater than that for the liver microsomes.

Covalent binding of rofecoxib, but not other cyclooxygenase-2 inhibitors, to allysine aldehyde in elastin of human aorta.

In rats, it has been reported that rofecoxib, a cyclooxygenase-2 (COX-2) inhibitor, reacts with the aldehyde group of allysine in elastin to give a condensation covalent adduct, thereby preventing the formation of cross-linkages in the elastin and causing degradation of the elastic fibers in aortas in vivo. Acid, organic solvent, and proteolytic enzyme treatments of human aortic homogenate after incubation with [14C]rofecoxib demonstrated that most of the radioactivity is covalently bound to elastin. The in vitro covalent binding was inhibited in the presence of β-aminopropionitrile, d-penicillamine, and hydralazine, which suggested that the aldehyde group of allysine in human elastin was relevant to the covalent binding. The in vitro covalent binding of [14C]rofecoxib was significantly decreased by the addition of only nonradiolabeled rofecoxib but not the other COX-2 inhibitors, celecoxib, valdecoxib, etoricoxib, and CS-706 [2-(4-ethoxyphenyl)-4-methyl 1-(4-sulfamoylphenyl)-1H-pyrrole], a novel selective COX-2 inhibitor. All the above COX-2 inhibitors except for rofecoxib had no reactivity with the aldehyde group of benzaldehyde used as a model compound of allysine aldehyde under a physiological pH condition. On the other hand, no retention of the radioactivity of [14C]rofecoxib was observed in human aortic endothelial cells in vitro, suggesting that rofecoxib is not retained in aortic endothelial cells in vivo. These results suggest that rofecoxib, but not other COX-2 inhibitors, is capable of covalently binding to the aldehyde group of allysine in human elastin. This might be one of the main causes of cardiovascular events by rofecoxib in clinical situations.

The inhaled glucocorticoid fluticasone propionate efficiently inactivates cytochrome P450 3A5, a predominant lung P450 enzyme.

Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5, and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with K(I), k(inact), and partition ratio of 16 muM, 0.027 min(-1), and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 muM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs.

Isolation and Identification of Diglucuronides of Some Endogenous Steroids in Dogs

Diglucuronidation is a novel glucuronidation reaction where the second glucuronosyl moiety is attached at the C2′ position of the first glucuronosyl moiety. To examine whether diglucuronidation takes place in endogenous substrates in vivo, control urine and bile samples were collected from male Crl:CD(SD) IGS rats, beagle dogs, and cynomolgus monkeys and analyzed by liquid chromatography-mass spectrometry (LC-MS) after solid phase extraction. Several diglucuronides of C19 steroids, including M1 (C31H46O14) and M2 (C31H44O14), were detected in the urine and bile of the dogs but not in the excreta of the rats and monkeys. A milligram quantity of M1 was successfully isolated from the pooled dog urine and analyzed by nuclear magnetic resonance (NMR) spectroscopy. M1 was unambiguously identified as epiandrosterone 3-O-diglucuronide by comparing the LC-MS and two-dimensional NMR data of M1 with those of the biosynthesized epiandrosterone 3-O-diglucuronide. M2 was identified as dehydroepiandrosterone 3-O-diglucuronide. According to these findings, the diglucuronidation reaction was proven to be occurring on steroid hormones in vivo in dogs.

CYP2D6-Mediated Metabolism of a Novel Acyl Coenzyme A:Cholesterol Acyltransferase Inhibitor, Pactimibe, and Its Unique Plasma Metabolite, R-125528

Pactimibe sulfate is a novel acyl coenzyme A:cholesterol acyltransferase inhibitor. We conducted metabolic studies of pactimibe and its plasma metabolite, R-125528. Pactimibe had multiple metabolic pathways including indolin oxidation to form R-125528, ω-1 oxidation, N-dealkylation, and glucuronidation. Among them, the indolin oxidation and the ω-1 oxidation were dominant and were mainly catalyzed by CYP3A4 and CYP2D6, respectively. The intrinsic clearance (CLint) values for these pathways in human hepatic microsomes were 0.63 and 0.76 μl/min/mg-protein, respectively. On the other hand, the metabolic reaction for R-125528 was restricted. It was demonstrated that ω-1 oxidation was the only pathway that could eliminate R-125528 from the systemic circulation. To our surprise, only CYP2D6-expressing microsomes could catalyze the reaction, and ω-1 oxidation was strongly correlated with the CYP2D6 marker reaction, dextromethorphan O-demethylation (r2 = 0.90), in human hepatic microsomes. Although R-125528 is an atypical substrate for CYP2D6 because of its acidity, the Km value was 1.8 μM for the reaction in human hepatic microsomes and the CLint value was as high as 75.0 μl/min/mg-protein. These results suggested that the systemic clearance of R-125528 was highly dependent on CYP2D6 activity and that several studies with CYP2D6 including drug-drug interaction and polymorphism sensitivity should be performed during development from the viewpoint of metabolite safety assessment. The finding that R-125528, an acidic compound devoid of basic nitrogen, was a good substrate for CYP2D6 raised a question about previously reported CYP2D6 models based on a critical electrostatic interaction with Asp301 and/or Glu216.