Masashi Mise

Evaluation of the potential for drug-induced liver injury based on in vitro covalent binding to human liver proteins

Prediction of idiosyncratic drug-induced liver injury (DILI) is difficult, and the underlying mechanisms are not fully understood. However, many drugs causing DILI are considered to form reactive metabolites and covalently bind to cellular macromolecules in the liver. The objective of this study was to clarify whether the risk of idiosyncratic DILI can be estimated by comparing in vitro covalent binding (CB) levels among 12 positive compounds (acetaminophen, alpidem, bromfenac, carbamazepine, diclofenac, flutamide, imipramine, nefazodone, tacrine, ticlopidine, tienilic acid, and troglitazone) for DILI and 12 negative compounds (acetylsalicylic acid, caffeine, dexamethasone, losartan, ibuprofen, paroxetine, pioglitazone, rosiglitazone, sertraline, theophylline, venlafaxine, and zolpidem). After incubation with human liver microsomes in the presence of NADPH, there was a large overlap in the distribution of CB amounts between the positive and negative groups. On addition of UDP-glucuronic acid (UDPGA) as a cofactor for glucuronidation, the CB levels of bromfenac and diclofenac were increased. With addition of nucleophilic glutathione (GSH), values for most compounds were decreased. However, separation of the two groups on the basis of CB could not be improved by UDPGA or GSH. Furthermore, CB with human hepatocytes also failed to discriminate positive from negative compounds. Therefore, the CB amount alone is not sufficient for risk assessment of DILI. In contrast, when the CB amount was multiplied by the maximum daily dose, which may reflect maximum hepatic exposure, the two groups did become discriminated. Taken together, our findings suggest that the combination of CB amount and daily dose can estimate the risk of idiosyncratic DILI.

Identification of non-functional allelic variant of CYP1A2 in dogs.

OBJECTIVES Recently, we reported that AC-3933, a novel cognitive enhancer, is polymorphically hydroxylated in beagle dogs and that dogs could be phenotyped as extensive metabolizers (EM) or poor metabolizers (PM). AC-3933 polymorphic hydroxylation is caused by polymorphic expression of CYP1A2 protein in dog liver. METHODS In order to clarify the mechanism of polymorphic expression of CYP1A2 protein in beagle dogs, we investigated, in this study, the sequence of CYP1A2 cDNA in EM and PM dogs. RESULTS In PM dogs CYP1A2 gene, we discovered a nonsense mutation (C1117T) that induces a premature termination, and is associated with PM phenotype for AC-3933 hydroxylation. All PM dogs studied were homozygote of the mutant allele (m/m) and seemed to be CYP1A2-null phenotype as they lacked the heme-binding region in CYP1A2. These results indicate that the polymorphic expression of CYP1A2 protein observed in our previous study is caused by a single nucleotide polymorphism on CYP1A2 coding region. Furthermore, we developed a genotyping method for the mutant allele using a mismatch PCR-restriction fragment length polymorphism, and carried out frequency analysis in 149 beagle dogs. CONCLUSION Our results indicate that more than 10% of the dogs studied were CYP1A2-null. Because CYP1A2-null phenotype in dogs affects the results of pharmacokinetic, toxicological and pharmacological studies of drug candidates, these findings are important in the pharmaceutical and the veterinary fields.

Characterization of Substrate Specificity of Dog CYP1A2 Using CYP1A2-Deficient and Wild-Type Dog Liver Microsomes

Beagle dogs are commonly used for toxicological and pharmacological studies of drug candidates in the pharmaceutical industry. Recently, we reported a CYP1A2-deficient dog with a nonsense mutation (C1117T). In this study, using CYP1A2-deficient and wild-type dog liver microsomes, substrate specificity of dog CYP1A2 was investigated and compared with human CYP1A2. For this purpose, 11 cytochrome P450 assays were conducted in human or dog liver microsomes, genotyped for the CYP1A2 C1117T mutation. There was no statistical difference between C/C, C/T, and T/T dogs in activities of aminopyrine N-demethylase, aniline hydroxylase, bufuralol 1′-hydroxylase, and midazolam 1′-hydroxylase. On the other hand, activities of phenacetin O-deethylase, ethoxyresorufin O-deethylase, and tacrine 1-hydroxylase, which were catalyzed by human CYP1A2, were significantly lower in T/T dogs than C/C dogs, indicating that dog and human CYP1A2 was responsible for these activities. However, dog CYP1A2 was not involved in caffeine metabolism, a marker activity for human CYP1A2. As for endogenous substances, our results indicated that human CYP1A2, but not dog CYP1A2, is responsible for melatonin 6-hydroxylase, 9-cis-retinal oxidase, and estradiol 2-hydroxylase activity. In conclusion, tacrine, ethoxyresorufin, and phenacetin are probe substrates for CYP1A2 not only in humans but also in dogs. However, caffeine, melatonin, 9-cis-retinal, and estradiol, which are substrate for human CYP1A2, are not good substrates for dog CYP1A2. The finding that there are species differences in substrate specificity of CYP1A2 between humans and beagle dogs is an important issue and must be considered for preclinical studies using beagle dogs.