Prediction of in vivo interaction between triazolam and erythromycin based on in vitro studies using human liver microsomes and recombinant human CYP3A4.

PURPOSE: To quantitatively predict the in vivo interaction between triazolam and erythromycin, which involves mechanism-based inhibition of CYP3A4, from in vitro studies using human liver microsomes (HLM) and recombinant human CYP3A4 (REC). METHODS: HLM or REC was preincubated with erythromycin in the presence of NADPH and then triazolam was added. alpha- and 4-hydroxy (OH) triazolam were quantified after a 3 min incubation and the kinetic parameters for enzyme inactivation (k(inact) and K('app)) were obtained. Drug-drug interaction in vivo was predicted based on a physiologically-based pharmacokinetic (PBPK) model, using triazolam and erythromycin pharmacokinetic parameters obtained from the literature and kinetic parameters for the enzyme inactivation obtained in the in vitro studies. RESULTS: Whichever enzyme was used, triazolam metabolism was not inhibited without preincubation, even if the erythromycin concentration was increased. The degree of inhibition depended on preincubation time and erythromycin concentration. The values obtained for k(inact) and K('app) were 0.062 min(-1) and 15.9 microM (alpha-OH, HLM), 0.055 min(-1) and 17.4 microM (4-OH, HLM), 0.173 min(-1) and 19.1 microM (alpha-OH, REC), and 0.097 min(-1) and 18.9 microM (4-OH, REC). Based on the kinetic parameters obtained using HLM and REC, the AUCpo of triazolam was predicted to increase 2.0- and 2.6-fold, respectively, following oral administration of erythromycin (333 mg t.i.d. for 3 days), which agreed well with the reported data. CONCLUSIONS: In vivo interaction between triazolam and erythromycin was successfully predicted from in vitro data based on a PBPK model involving a mechanism-based inhibition of CYP3A4.

Quantitative prediction of in vivo drug-drug interactions from in vitro data based on physiological pharmacokinetics: use of maximum unbound concentration of inhibitor at the inlet to the liver.

PURPOSE: To assess the degree to which the maximum unbound concentration of inhibitor at the inlet to the liver (I(inlet,u,max), used in the prediction of drug-drug interactions, overestimates the unbound concentration in the liver. METHODS: The estimated value of I(inlet,u,max) was compared with the unbound concentrations in systemic blood, liver, and inlet to the liver, obtained in a simulation study based on a physiological flow model. As an example, a tolbutamide/sulfaphenazole interaction was predicted taking the plasma concentration profile of the inhibitor into consideration. RESULTS: The value of I(inlet,u,max) differed from the concentration in each compartment, depending on the intrinsic metabolic clearance in the liver, first-order absorption rate constant, non-hepatic clearance and liver-to-blood concentration ratio (Kp) of the inhibitor. The AUC of tolbutamide was predicted to increase 4-fold when co-administered with sulfaphenazole, which agreed well with in vivo observations and was comparable with the predictions based on a fixed value of I(inlet,u,max). The blood concentration of tolbutamide was predicted to increase when it was co-administered with as little as 1/100 of the clinical dose of sulfaphenazole. CONCLUSIONS: Although I(inlet,u,max) overestimated the unbound concentration in the liver, the tolbutamide/sulfaphenazole interaction could be successfully predicted by using a fixed value of I(inlet,u,max) indicating that the unbound concentration of sulfaphenazole in the liver after its clinical dose is by far larger than the concentration to inhibit CYP2C9-mediated metabolism and that care should be taken when it is co-administered with drugs that are substrates of CYP2C9.

Prediction of in vivo drug-drug interactions based on mechanism-based inhibition from in vitro data: inhibition of 5-fluorouracil metabolism by (E)-5-(2-Bromovinyl)uracil.

The fatal drug-drug interaction between sorivudine, an antiviral drug, and 5-fluorouracil (5-FU) has been shown to be caused by a mechanism-based inhibition. In this interaction, sorivudine is converted by gut flora to (E)-5-(2-bromovinyl)uracil (BVU), which is metabolically activated by dihydropyrimidine dehydrogenase (DPD), and the activated BVU irreversibly binds to DPD itself, thereby inactivating it. In an attempt to predict this interaction in vivo from in vitro data, inhibition of 5-FU metabolism by BVU was investigated by using rat and human hepatic cytosol and human recombinant DPD. Whichever enzyme was used, increased inhibition was observed that depended on the preincubation time of BVU and enzyme in the presence of NADPH and BVU concentration. The kinetic parameters obtained for inactivation represented by k(inact) and K'(app) were 2.05 +/- 1.52 min(-1), 69.2 +/- 60.8 microM (rat hepatic cytosol), 2.39 +/- 0.13 min(-1), 48.6 +/- 11.8 microM (human hepatic cytosol), and 0.574 +/- 0.121 min(-1), 2.20 +/- 0.57 microM (human recombinant DPD). The drug-drug interaction in vivo was predicted quantitatively based on a physiologically based pharmacokinetic model, using pharmacokinetic parameters obtained from the literature and kinetic parameters for the enzyme inactivation obtained in the in vitro studies. In rats, DPD was predicted to be completely inactivated by administration of BVU and the area under the curve of 5-FU was predicted to increase 11-fold, which agreed well with the reported data. In humans, a 5-fold increase in the area under the curve of 5-FU was predicted after administration of sorivudine, 150 mg/day for 5 days. Mechanism-based inhibition of drug metabolism is supposed to be very dangerous. We propose that such in vitro studies should be carried out during the drug-developing phase so that in vivo drug-drug interactions can be predicted.

Prediction of in vivo drug-drug interactions between tolbutamide and various sulfonamides in humans based on in vitro experiments.

Drug-drug interactions between tolbutamide and sulfonamides have extensively been reported. We attempted to predict the in vivo interaction between tolbutamide and sulfonamides from the in vitro metabolic inhibition studies. The inhibition constant (K(i)) was derived from the inhibitory effects of eight sulfonamides (sulfaphenazole, sulfadiazine, sulfamethizole, sulfisoxazole, sulfamethoxazole, sulfapyridine, sulfadimethoxine, and sulfamonomethoxine) on tolbutamide metabolism. We found that the inhibitory effect of sulfaphenazole was greatest among the eight sulfonamides examined. Furthermore, the contribution of each P450 enzyme to tolbutamide metabolism was investigated by using recombinant P450 enzymes. Although cytochrome P450 (CYP) 2C8, 2C9, and 2C19 metabolized tolbutamide, the main enzyme involved was CYP2C9. The K(i) values of several sulfonamides were comparable between human liver microsomes and recombinant CYP2C9. The maximum unbound plasma concentration of sulfonamides in the portal vein was calculated from literature data on the pharmacokinetics of sulfonamides. Using the K(i) values obtained from in vitro inhibition studies, the degree of increase in tolbutamide area under the plasma concentration-time curve (AUC) was predicted. About 4.8- and 1.6-fold increases in tolbutamide AUC were predicted by coadministration of sulfaphenazole and sulfamethizole, respectively, which agreed well with the reported increases in humans. Furthermore, the increase in tolbutamide AUC by coadministration of sulfadiazine, sulfisoxazole, and sulfamethizole was predicted to be 1.5- to 2. 6-fold, although the corresponding in vivo effects have not been reported. It is concluded that some of these sulfonamides have to be carefully coadministered with CYP2C9 substrates such as tolbutamide although coadministration of sulfaphenazole needs the greatest care.

Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport.

It is of great importance to predict in vivo pharmacokinetics in humans based on in vitro data. We summarize recent findings of the quantitative prediction of the hepatic metabolic clearance from in vitro studies using human liver microsomes, hepatocytes, or P450 isozyme recombinant systems. Furthermore, we propose a method to predict pharmacokinetic alterations caused by drug-drug interactions that is based on in vitro metabolic inhibition studies using human liver microsomes or human enzyme expression systems. Although we attempt to avoid the false negative prediction, the inhibitory effect was underestimated in some cases, indicating the possible contribution of the active transport into hepatocytes and/or interactions at the processes other than the hepatic metabolism, such as the metabolism and transport processes during gastrointestinal absorption.

Induction of DNA recombination by activated 3-amino-1-methyl-5H-pyrido[4,3-b]indole.

To investigate the genotoxic properties of a food-derived carcinogen, 3-amino-1-methyl-5H-pyrido-[4,3-b]indole (Trp-P-2), we have tested whether Trp-P-2 and its metabolically transformed products can induce DNA recombinations. Trp-P-2 is a strong mutagen and its activated form, the N-hydroxylated derivative, Trp-P-2(NHOH), is known to form DNA adducts and cause DNA chain cleavage. Using a system in which phage lambda undergoes recombination inside host Escherichia coli, we have found that Trp-P-2(NHOH), but not Trp-P-2 itself, can induce recombination. A nitroso derivative of Trp-P-2, Trp-P-2(NO), which can be reduced intracellularly to form Trp-P-2(NHOH), also induced recombination. Active oxygens are implicated in this recombinogenic action, since Trp-P-2(NHOH) is known to undergo spontaneous oxidative degradation, generating active oxygen radicals which can cause DNA chain cleavages. 4-Hydroxyaminoquinoline N-oxide and phenyl-hydroxylamine also showed recombinogenic actions in this assay system; hence, it is suspected that aromatic amine-type carcinogens have this property in common.

Permanganate oxidation of nucleic acid components: a reinvestigation.

KMnO4 consumption in solution by nucleoside added at an excess was measured to explore any possible oxidative interactions between these molecules. Thymidine, 5-methyldeoxycytidine and deoxycytidine rapidly decomposed KMnO4 with the pseudo-first-order kinetics (thymidine greater than 5-methyl-deoxycytidine greater than deoxycytidine), while deoxyguanosine showed only a very slow consumption and deoxyadenosine did not decompose KMnO4 at all under the conditions employed (0.16 mM KMnO4, 0.80 mM nucleoside, at 27 degrees C and pH 4.3, 7.4 and 8.6, up to 60 min). UV absorption spectra of KMnO4-treated nucleosides also indicated modification of the pyrimidine nucleosides but not of the purine nucleosides. These results are consistent with the original report of Hayatsu and Ukita published in 1967 on the KMnO4 oxidation of nucleosides, and provide difficulty in understanding the mechanism of recently reported formation of 8-hydroxypurines on treatment of DNA with KMnO4.