Population pharmacokinetic modeling for enterohepatic recirculation in Rhesus monkey.

Enterohepatic recirculation (EHR) occurs via biliary excretion and intestinal reabsorption of a drug. Drug recycling through EHR can lead to a change in pharmacokinetic (PK) properties, such as reduced clearance (CL), extended half-life (T(1/2)) and increased plasma exposure (AUC). As a result, EHR may prolong the pharmacological effect of drugs. In the present study, the compound (Cpd A) was found to exhibit EHR in Rhesus monkeys associated with a reduction in CL (from 3.8 to 0.33 Lh(-1), IV; from 2.3 to 0.4 Lh(-1), PO), and an increase in T(1/2) (from 0.9 to 18 h, IV) and in AUC (from 1.5 to 17.4 microg h/mL, IV; from 2.8 to 16.3 microg h/mL, PO), by comparing the PK in the monkeys via the interruption of EHR (bile-duct cannulation) with that in the intact monkeys. A population four-compartment model was constructed based on recirculation loops incorporating all possible inputs (bile secretion, a lag-time model for gall bladder emptying, routes and amounts of a single dose administration) to fully evaluate the EHR of Cpd A. The plasma concentrations versus time profiles predicted from the model had a good fit to the values observed in the subjects and were further simulated with 90% confidence interval to demonstrate its utility. Thus, the model could be applied as a useful tool to evaluate the drugs or compounds that undergo EHR in different species.     

In vitro and in vivo metabolism of a potent and selective integrin alpha v beta 3 antagonist in rats, dogs, and monkeys.

Compound A (3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyrindin-2-yl)propyl]-imidazolidin-1-yl]-3(S)-(6-methoxy-pyridin-3-yl)-propionic acid), a potent and selective antagonist of integrin alpha(v)beta(3) receptor, is under development for treatment of osteoporosis. This study describes metabolism and excretion of A in vivo in rats, dogs, and monkeys, and metabolism of A in vitro in primary hepatocytes from rats, dogs, monkeys, and humans. In all three animal species studied, A was primarily excreted as unchanged drug and, to a lesser degree, as phase I and phase II metabolites. Major biotransformation pathways of A included glucuronidation/glucosylation on the carboxylic group to form acyl-linked glucuronides/glucosides; and oxidation on the tetrahydronaphthyridine moiety to generate a carbinolamine and its further metabolized products. Minor pathways involved O-demethylation and hydroxylations on the alkyl chain. Only in rats, a glutathione adduct of A was also observed, and its formation is proposed to be via an iminium intermediate on the tetrahydronaphthyridine ring. Similar metabolic pathways were observed in the incubates of hepatocytes from the corresponding animals as well as from humans. CYP 3A and 2D subfamilies were capable of metabolizing A to its oxidative products. Overall, these in vitro and in vivo findings should provide useful insight on possible biotransformation pathways of A in humans.     

Metabolism and Renal Elimination of Gaboxadol in Humans: Role of UDP-Glucuronosyltransferases and Transporters

Purpose  Gaboxadol, a selective extrasynaptic agonist of the delta-containing γ-aminobutyric acid type A (GABA_A) receptor, is excreted in humans into the urine as parent drug and glucuronide conjugate. The goal of this study was to identify the UDP-Glucuronosyltransferase (UGT) enzymes and the transporters involved in the metabolism and active renal secretion of gaboxadol and its metabolite in humans.Methods. The structure of the glucuronide conjugate of gaboxadol in human urine was identified by LC/MS/MS. Human recombinant UGT isoforms were used to identify the enzymes responsible for the glucuronidation of gaboxadol. Transport of gaboxadol and its glucuronide was evaluated using cell lines and membrane vesicles expressing human organic anion transporters hOAT1 and hOAT3, organic cation transporter hOCT2, and the multidrug resistance proteins MRP2 and MRP4.Results. Our study indicated that the gaboxadol-O-glucuronide was the major metabolite excreted in human urine. UGT1A9, and to a lesser extent UGT1A6, UGT1A7 and UGT1A8, catalyzed the O-glucuronidation of gaboxadol in vitro. Gaboxadol was transported by hOAT1, but not by hOCT2, hOAT3, MRP2, and MRP4. Gaboxadol-O-glucuronide was transported by MRP4, but not MRP2.Conlusion. Gaboxadol could be taken up into the kidney by hOAT1 followed by glucuronidation and efflux of the conjugate into urine via MRP4.     

Validation of (-)-N-3-benzyl-phenobarbital as a selective inhibitor of CYP2C19 in human liver microsomes.

(-)-N-3-Benzyl-phenobarbital (NBPB) was reported to be a potent and selective inhibitor of CYP2C19. To validate the selectivity of NBPB toward CYP2C19 in human liver microsomes, the inhibitory effects on major cytochrome P450 isoform-specific reactions were evaluated in the present study. In human liver microsomes, NBPB showed potent competitive inhibition on CYP2C19-mediated S-mephenytoin 4'-hydroxylation with an IC(50) value of 0.25 microM and K(i) value of 0.12 microM, whereas weak inhibition was observed for CYP1A2-, CYP2A6-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6-, and CYP3A4-mediated reactions with IC(50) values >100, >100, 62, 34, 19, >100, and 89 microM, respectively. Importantly, its selectivity toward CYP2C19 among the CYP2C subfamily was demonstrated. Therefore, NBPB can be used as a potent and selective inhibitor to establish the relative contribution of CYP2C19 for in vitro reaction phenotyping studies. This compound can also serve as a positive control inhibitor of CYP2C19 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.     

Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz[a]anthracene

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s. 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0–2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 281 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA ≥ DMBA trans-5,6-dihydrodiol ≥ 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

9,10-Dihydroxy-9,10-dihydro-3-methylcholanthrene-2-one: a principal matabolite of the potent carcinogen 3-methylcholanthrene-2-one by rat liver microsomes

A principal metabolite, formed in the metabolism of the potentcarcinogen 3-methylcholanthrene-2-one (3MC-2-one) by liver microsomesfrom either untreated, or phenobarbitaltreated or 3-methylcholanthrene(3MC)-treated rats, was isolated by reversed-phase HPLC. Thismetabolite has been identified as a 9,10-dihydrodiol with a(9R,10R):(9S,10S) enantiomer ratio of -84:16 by all three ratliver microsomal preparations. The 9,10-dihydrodiol metaboliteand its NaBM₄ reduction products [a pair of diastereomeric 9,10-dihydroxy-9,10-dihydro-2-OH-3MC(2-OH-3MC 9,10-dihydrodiols)] were characterized by UV-visibleabsorption, mass, and circular dichroic spectral, and chiralstationary phase HPLC analyses. Identification of 9,10-dihydroxy-9,10-dihydro-3MC-2-one(3MC-2-one 9,10-dihydrodiol) as the predominant metabolite ofthe potent carcinogen 3MC-2-one suggests that 3MC-2-one maybe metabolically activated to a bay region 9,10-diol-7,8-epoxide,similar to the previously established metabolic activation pathwaysof 3MC and 1-hydroxy-3-methylcholanthrene (1-OH-3MC).     

Characterization of the rhesus monkey CYP3A64 enzyme: species comparisons of CYP3A substrate specificity and kinetics using baculovirus-expressed recombinant enzymes.

The rhesus monkey (Macaca mulatta) is a primate species used extensively as a preclinical safety species in drug development. In this report, we describe the cloning, expression, and characterization of CYP3A64 (AY334551), a CYP3A4 homolog expressed in rhesus liver. The deduced amino acid sequence was found to be 93% similar to human CYP3A4, 83% similar to human CYP3A5, and identical to the previously reported cynomolgus monkey CYP3A8 (Komori et al., 1992). The substrate specificity of CYP3A64 for testosterone (0-250 microM), midazolam (0-200 microM), nifedipine (0-200 microM), and 7-benzoxy-4-trifluoromethylcoumarin (0-200 microM) were compared with recombinant enzymes from rat (CYP3A1, CYP3A2), dog (CYP3A12, CYP3A26), rabbit (CYP3A6), and human (CYP3A4, CYP3A5). Immunoinhibition and chemical inhibition of CYP3A64 was demonstrated using the inhibitory monoclonal antibody (MAb) 10-1-1 (anti-3A4) and ketoconazole (0-10 microM). The utility of CYP3A64 to be used as a standard in monkey induction assays was shown and the concentration of CYP3A64 protein in rhesus liver microsomes was estimated to be 72 pmol/mg protein. In summary, these results support the utilization of rhesus monkey CYP3A64 for in vitro drug metabolism studies and provide a more complete understanding of CYP3A substrate specificities and species differences in metabolic capabilities.     

Impact of pH on plasma protein binding in equilibrium dialysis

Many pharmacokinetic analyses require unbound plasma concentrations, including prediction of clearance, volume of distribution, drug-drug interactions, brain uptake analysis, etc. It is most often more convenient to measure the total drug concentration in plasma rather than the unbound drug concentration. To arrive at unbound plasma concentrations, separate in vitro determinations of the plasma protein binding of a drug are usually carried out in serum or in plasma, and the plasma pharmacokinetic results are then mathematically adjusted by this fraction unbound ( f u,p). Plasma protein binding or the drug fraction unbound in plasma ( f u,p) is known to be affected by protein, drug, free fatty acid concentrations, lipoprotein partitioning, temperature, pH, and the presence or absence of other drugs/displacing agents within plasma samples. Errors in f u,p determination caused by lack of adequate pH control in newer assay formats for plasma protein binding (e.g., 96-well equilibrium thin walled polypropylene dialysis plates) will have significant drug-specific impact on these pharmacokinetic calculations. Using a diverse set of 55 drugs and a 96-well equilibrium dialysis plate format, the effect of variable pH during equilibrium dialysis experiments on measured values of f u,p was examined. Equilibrium dialysis of human plasma against Dulbecco's phosphate buffered saline at 37 degrees C under an air or 10% CO 2 atmosphere for 22 h resulted in a final pH of approximately 8.7 and 7.4, respectively. The ratio of f u,p at pH 7.4 (10% CO 2) vs pH 8.7 (air) was >or=2.0 for 40% of the 55 compounds tested. Only one of the 55 compounds tested had a ratio <0.9. Select compounds were further examined in rat and dog plasma. In addition, physicochemical properties were calculated for all compounds using ACD/Labs software or Merck in-house software and compared to plasma protein binding results. Changes in plasma protein binding due to pH increases which occurred during the equilibrium dialysis experiment were not species specific but were drug-specific, though nonpolar, cationic compounds had a higher likely hood of displaying pH-dependent binding. These studies underscore the importance of effectively controlling pH in plasma protein binding studies.     

The role of P-glycoprotein in the bioactivation of raloxifene.

Drug transporters have been shown to alter drug metabolism. Similarly, bioactivation of drugs may also be altered by drug transporters. The aim of this work was to examine the role of P-glycoprotein (Pgp) in the bioactivation of a Pgp substrate, raloxifene, and a non-Pgp substrate, naphthalene. To evaluate the extent of bioactivation, covalent binding was measured. In both freshly isolated and cryopreserved hepatocytes, the extent of raloxifene covalent binding increased significantly (p < 0.05) in the presence of verapamil, whereas no change was observed with the covalent binding of naphthalene. To ascertain that the change was a Pgp effect, covalent binding was examined in microsomes in which raloxifene and naphthalene covalent binding was not altered in the presence of verapamil. In addition, the measure of raloxifene-glutathione adducts in the cryopreserved hepatocytes showed that the formation of the adducts increased in the presence of verapamil, which supports the idea that blocking Pgp in the liver increases metabolism and, therefore, the bioactivation of raloxifene. Because raloxifene and naphthalene are known to undergo bioactivation mediated by CYP3A4, covalent binding in the presence of ketoconazole was examined. In both hepatocytes and microsomes, raloxifene covalent binding decreased significantly (p < 0.01). It is interesting that naphthalene covalent binding was not affected. In the presence of the CYP2E inhibitor 4-methylpyrazole, a decrease in naphthalene covalent binding was observed, suggesting that the formation of the 1,2-epoxide may be the main culprit contributing to naphthalene covalent binding. In conclusion, these data suggest that in addition to other "protective" mechanisms, Pgp may attenuate bioactivation of drugs.     

珊瑚／骨髓复合人工骨的实验研究

目的： 评价珊瑚／ 骨髓复合人工骨（ 简称复合骨） 的成骨效应。方法： 将复合骨植入兔背部肌袋和颅骨缺损, 以单纯珊瑚植入作对照。取材后通过组织学观察和计算机图像分析, 检测其成骨情况。结果： 复合骨植入肌袋后４ 周, 局部有骨组织形成, 而单纯珊瑚植入区未见骨组织成分。复合骨植入骨缺损后６ 周, 局部形成的骨组织量明显多于同期的珊瑚植入区（ Ｐ＜ ０－０１） 。术后１２ 周, 复合骨完全被成熟的骨组织取代, 而单纯珊瑚植入区则表现为不完全性骨修复。结论： 复合骨具有良好的骨再生和骨修复能力, 是一种较理想的骨移植替代材料。