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Animal species used in the preclinical studies for development of monoclonal antibody (mAb) drugs are surveyed in this review. Relevant animal species for preclinical studies of mAb candidates are those express desired epitope of mAb candidates. Cynomolgus monkeys cross-react with mAb drugs much higher than other animal species commonly used in preclinical studies such as absorption, distribution, metabolism and excretion (ADME), efficacy, and toxicity studies, for development of new drugs. Moreover, plasma exposure of the mAb drugs in humans is predicted well from the exposure in the monkeys, and the placental transfer of immunoglobulin G (IgG, all the mAb drugs contain IgG) from mother to fetus is similar between humans and the monkeys from a viewpoint of time course and plasma level of IgG transferred. These observed findings indicate that the monkeys are the most suitable animal species used in the ADME and toxicity studies for development of new mAb drugs.

Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans.

Neonatal Fc receptor (FcRn), a heterodimer of MHC class I-like protein and β2-microglobulin, encoded by FCGRT and B2M, respectively, is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of IgG. Cynomolgus macaques are important animal species used in the evaluation of therapeutic antibodies, largely due to sequence similarities of target proteins to those of humans. Because the function of FcRn could be modified by mutations in FCGRT or B2M, 71 cynomolgus and 24 rhesus macaques were analyzed in the present study. A total of 21 variants were identified, of which 4 were non-synonymous in FCGRT. Fifteen variants were unique to cynomolgus macaques, of which 3, 2, and 5 were unique to cynomolgus macaques bred in China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN), respectively. Five variants were shared by MacfaCHN and MacfaCAM, but not by MacfaIDN. In B2M, only 5 variants were found, including 2 non-synonymous variants. Tissue expression analysis showed that cynomolgus FCGRT and B2M were widely expressed in the 10 tissue types analyzed. None of the non-synonymous variants of FCGRT or B2M found changes in the amino acid residues known to be important for FcRn function, suggesting that substantial inter-animal variability of FcRn is not expected for the cynomolgus macaques analyzed.

Cynomolgus macaques, used in drug metabolism studies due to their evolutionary closeness to humans, are mainly bred in Asian countries, including Cambodia, China, and Indonesia. Cytochromes P450 (P450s) are important drug-metabolizing enzymes, present in the liver and small intestine, major drug metabolizing organs. Previously, our investigation did not find statistically significant differences in hepatic P450 metabolic activities measured in cynomolgus macaques bred in Cambodia (MacfaCAM) and China (MacfaCHN). In the present study, P450 metabolic activity was investigated in the small intestine of MacfaCAM and MacfaCHN, and cynomolgus macaques bred in Indonesia (MacfaIDN) using P450 substrates, including 7-ethoxyresorufin, coumarin, bupropion, paclitaxel, diclofenac, S-mephenytoin, bufuralol, chlorzoxazone, and testosterone. The results indicated that P450 metabolic activity of the small intestine was not statistically significantly different (2.0-fold) in MacfaCAM, MacfaCHN, and MacfaIDN. In addition, statistically significant sex differences were not observed (2.0-fold) in any P450 metabolic activity in MacfaCAM as supported by mRNA expression results. These results suggest that P450 metabolic activity of the small intestine does not significantly differ statistically among MacfaCAM, MacfaCHN, and MacfaIDN.

Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (

Small minipigs (Microminipig, registered as a novel variety of pig in Japan) were developed for use in non-clinical pharmacological/toxicological studies for new drug development. To assess the pharmacokinetics of selective substrates of human cytochrome P450s in Microminipigs, caffeine (human P450 1A2), warfarin (P450 2C9), omeprazole (P450 2C19), metoprolol (P450 2D6), and midazolam (P450 3A) were administered in combination, intravenously (0.20 mg kg(-1))( )or orally (1.0 mg kg(-1)). Plasma samples obtained, up to 24 hr after dosing, from four male and four female Microminipigs were analyzed by liquid chromatography tandem mass spectrometry to estimate typical pharmacokinetic parameters for each analyte. Bioavailabilities were approximately 80% for caffeine and warfarin, but less than 10% for omeprazole, metoprolol, and midazolam. No significant differences were noted, for the five probes, in area under the plasma concentration-time curve and peak plasma concentration values obtained from male and female Microminipigs. Clearance of caffeine, warfarin, omeprazole or midazolam in vivo, mediated mainly by cytochrome P450s 1A, 2C or 3A in Microminipigs, was similar to data reported for human. However, metoprolol metabolism, mediated by P450 2D enzymes in Microminipigs, was faster than reported for in vivo human kinetic parameters and in vitro in a human liver microsomal system. The results of this study suggest that the Microminipig is a suitable animal model for use in biological experiments for comparisons of pharmacokinetics of drugs in humans. The five-probes in combination used in this study demonstrate the disposition of typical P450 drugs in Microminipigs in vivo, with the aim of use in non-clinical pharmacological/toxicological studies.

Summary: Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1­4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs ( mfCYPs ) have been identified in the CYP1­4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs , confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders ( mfCYP2C43 , mfCYP2C75 , and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.

Cynomolgus monkeys [Macaca fascicularis (mf)] are widely used to determine pharmacokinetics and toxicological potential of many drug candidates as human models in the drug discovery and development. Cytochrome P450s (P450, CYP), one of the most important enzymes in drug metabolism, in monkey livers are generally similar to corresponding human P450s exhibiting high degrees of homologies in cDNAs and amino acid sequences. Species differences regarding important liver P450 3A and 2D function were examined between cynomolgus monkeys and humans using typical human P450 probe reactions using midazolam (a P450 3A marker), dextromethorphan and bufuralol (P450 2D markers). P450 3A-mediated midazolam 1'-hydroxylation activities in liver microsomes from individual monkey were highly correlated with midazolam 4'-hydroxylation activities but not correlated with dextromethorphan N-demethylation activities. Recombinant monkey CYP2D17 and CYP2D44 catalyzed dextromethorphan O- and N-demethylations as well as monkey mfCYP3A4 and mfCYP3A5 did. On the other hand, contributions of corresponding P450 2D6 and P450 3A4/5 to dextromethorphan N- and O-demethylations, in human liver microsomes were negligible under the present conditions. From these results, monkey P450 2D and 3A enzymes might have broader substrate specificity toward dextromethorphan oxidation than those in human livers. Special attention should be paid when enzymatic and pharmacokinetic data are extrapolated from monkeys to humans.

The cytochrome P450 (P450, CYP) enzyme superfamily is associated with the metabolism of drugs, environmental pollutants and endogenous substrates with broad overlapping substrate specificities. In addition, species differences between experimental animals and humans in the roles of P450 enzymes in drug metabolism are determinant factors in the drug discovery process. The induction and inhibition of P450-dependent metabolism, especially in the case of P450 3A enzyems, can cause serious problems in clinical practice and in the attrition of drug candidates because of adverse toxicology or pharmacokinetics. Recently, chimeric mice whose livers have been replaced to a level of 80% or more with human hepatocytes were established using urokinase-type plasminogen activator transgenic/severe combined immunodeficiency (uPA/SCID) mice. In the livers of these chimeric mice, hepatic cords and sinusoid-like structures were observed. Moreover, bile canaliculi associated with human hepatocytes were also detected. The mRNA expressions derived from humans of 52 phase I and 26 phase II enzymes (including 20 P450 s) and 21 transporters were ascertained in the chimeric mice liver. Taken together, these chimeric mice exhibited the glutathione conjugate form of a possible quinine-imine metabolite and responded to treatment with rifampicin or rifabutin by induction of P450 3A enzyems. This animal model should prove to be a good in vivo tool to assess the safety of drug candidates in terms of toxicity and drug-drug interactions caused by P450 induction.

Summary: Preclinical data of fetal, infant, and juvenile animals are important for the prediction of drug toxicity in fetuses and children. However, expression of drug-metabolizing enzymes, including cytochromes P450 (CYPs), have not been fully investigated in fetal, infant, or juvenile liver of the cynomolgus macaque, an animal species important for preclinical studies. In this study, hepatic expression of 20 cynomolgus macaque CYPs ( mfCYPs ) in the CYP1­4 subfamilies that are relevant to drug metabolism was measured in fetuses, infants, and juveniles using DNA microarrays. Expression of most mfCYPs , including those moderately or abundantly expressed in postnatal livers such as mfCYP2A23 , mfCYP2A24 , mfCYP2B6 , mfCYP2C9 , mfCYP2C19 , mfCYP2C76 , mfCYP2D17 , mfCYP2E1 mfCYP3A4 , and mfCYP3A5 , was much less abundant in fetal livers, but increased substantially after birth. In contrast, expression of mfCYP2C8 in fetal livers was not substantially different from postnatal livers. Since human CYP3A7 is expressed more abundantly in fetal livers than in adult livers, mfCYP3A7 , an ortholog of human CYP3A7 , was analyzed by quantitative polymerase chain reaction. Expression of mfCYP3A7 in fetal livers was much lower than that in postnatal livers, and greatly increased after birth, unlike the expression of human CYP3A7 . These results indicate that expression of most mfCYPs examined was low in fetal livers, but increased greatly in postnatal livers, with a few exceptions such as mfCYP2C8 .

Rationale Esophageal carcinosarcoma generally comprises 2 histological components: squamous cell carcinoma (SqCC) and sarcoma. Esophageal carcinosarcoma comprising 3 components is extremely rare and no reports have described therapeutic effects for this disease with metastasis. Patient concerns A 76-year-old man with dysphagia presented to a local clinic. Gastrointestinal endoscopy revealed a polypoid tumor in the middle esophagus and he was referred to our hospital. Diagnosis and interventions Thoracoscopic esophagectomy with super-extended (D3) nodal dissection and gastric tube reconstitution was performed, which resulted in carcinosarcoma comprising neuroendocrine carcinoma (NEC), SqCC, and sarcoma. Pathological stage was T1bN1M0 stage IIB according to the TNM Classification of Malignant Tumors-7th edition. The NEC component was observed in lymph node. At 47 days after surgery, lymph nodes, liver, and bone metastasis appeared, and tumor markers such as ProGRP and NSE were elevated. Combination chemotherapy with cisplatin and etoposide (EP) adapted to NEC was performed. Outcomes The patient showed complete response within 4 cycles of chemotherapy. However, the disease recurred 5.5 months after the final course of EP chemotherapy. Lessons A therapeutic strategy based on assessment of which component caused metastasis might be important for metastatic carcinosarcoma comprising 3 components, although more accumulation of data about the efficacy of chemotherapy is necessary. Moreover, elucidation of the mechanisms underlying generation of carcinosarcoma is expected in the future.

Drug metabolizing activities of cytochromes P450 (P450s, or CYPs) 3A4 and 3A5 in liver microsomes from the cynomolgus monkey [Macaca fascicularis (mf)] were investigated and compared with those of human P450 3A enzymes. Low activities for dealkylation of ethoxyresorufin and pentoxyresorufin were seen in recombinant monkey mfCYP3A4 and mfCYP3A5 and in recombinant human CYP3A4 and CYP3A5 expressed in bacterial membranes. Hydroxylation activities of mfCYP3A4 and mfCYP3A5 toward coumarin, paclitaxel, diclofenac, flurbiprofen, and S-mephenytoin were below detectable levels, as was also true for CYP3A4 and CYP3A5. Monkey mfCYP3A5 and mfCYP3A4 were highly active in bufuralol 1'-hydroxylation. mfCYP3A5 was efficient at dextromethorphan O-demethylation, although human CYP3A5 was unable to catalyze this reaction. Apparent bufuralol 1'-hydroxylation and dextromethorphan O-demethylation activities of monkey liver microsomes were higher than those of human liver microsomes, possibly because of contributions of mfCYP3A5 to these P450 2D-dependent drug oxidations. mfCYP3A5 and CYP3A5 catalyzed midazolam 1'-hydroxylation at a low substrate concentration more efficiently than the corresponding CYP3A4. mfCYP3A5 had higher testosterone 6beta-hydroxylase activity than mfCYP3A4, but the reverse relationship was observed in oxidation of nifedipine and hydroxylation of dexamethasone. These results demonstrate that monkey P450 3A enzymes have similar substrate selectivity to that of human P450 3A enzymes, but exhibit wider substrate selectivity toward P450 2D substrates.

The cynomolgus monkey is used to study drug metabolism because of its evolutionary closeness to humans. Despite their importance, regional distribution of cytochrome P450 (CYP) enzymes including CYP3As in the liver and small intestine, the major sites of drug metabolism, has not been fully investigated in cynomolgus monkeys. In this study, we measured mRNA expression levels of 14 CYPs in the CYP1, 2, and 3 subfamilies, including orthologs of human CYP3A4 and CYP3A5, in the liver and small intestine of cynomolgus monkeys. Expression levels of each CYP mRNA in various regions of the liver were quantified and comparisons were made between the right lobe, quadrate lobe, left medial lobe, left lateral lobe, and caudate lobe and with four different sections of the right lobe. In the small intestine, the same mRNAs were measured in the duodenum and six different sections from the proximal jejunum to the distal ileum. Expression levels of the CYP mRNAs were not substantially different between liver samples, but varied between the different sections of the small intestine, including CYP3A4. These results suggest that analysis of distinct sections is required for a better understanding of cynomolgus monkey CYPs in the small intestine.

A human hepatocyte-transplanted chimeric mouse has been established by transplantation of human hepatocytes to urokinase-type plasminogen activator transgenic/severe combined immunodeficiency (uPA+/+/SCID) mice. These chimeric mice have various amounts of human hepatocytes that proliferate extensively and progressively replace mouse hepatocytes. In the chimeric liver, hepatic cords and sinusoid-like structures were observed. The human hepatocytes expressed human albumin, human cytochrome P450 enzymes, and human transporter proteins. Furthermore, electron microscopic analysis demonstrated bile canaliculi associated with human hepatocytes in the chimeric mouse livers. These results indicate that the chimeric mouse livers contain functionally intact and differentiated human hepatocytes. Additionally, the toxicologic response of hepatocytes to acetaminophen (APAP) administration was compared in normal and chimeric mouse livers. Following 1,400 mg/kg APAP, mild hepatocellular degeneration was observed in the human hepatocyte areas in the chimeric mice, compared with severe centrilobular hepatocellular necrosis in the ICR mouse livers. In conclusion, these chimeric livers contain functionally differentiated human hepatocytes, and are less susceptible to APAP toxicity, compared to ICR mice.

Although the expenditure for new drug development by the pharmaceutical industry has been increasing in recent years, the number of new drugs approved has remained unchanged or has been gradually decreasing. For the effective development of new drugs, attrition rates of drug candidates should be reduced. In 1991, major causes of the attrition were ADME (absorption, distribution, metabolism and excretion) (about 40%), efficacy (about 30%), and safety issues (about 25%). In 2000, the causes were ADME (less than 10%), efficacy (about 30%), and safety issues (about 30%). Between 1991 and 2000, the attrition caused by ADME issues decreased drastically, but that caused by efficacy and safety issues did not change. To improve this situation, exploratory clinical studies including microdose and exploratory IND (Investigational New Drug) studies proposed in EU and USA, respectively, have been started at the late 1990s and the early 2000s. This commentary reviews the current situation of the exploratory clinical studies and the recent trend of pharmacokinetic studies in new drug development.

Tacrolimus (FK506), an immunosuppressive drug, is co-medicated with multiple drugs under clinical conditions. Tacrolimus is highly lipophilic and is excreted from the body after receiving extensive metabolism. Due to its narrow therapeutic window following organ transplantation, tacrolimus requires therapeutic drug monitoring by an enzyme immunoassay using the monoclonal antibody raised against tacrolimus. Therefore, metabolism studies including drug-drug interaction and metabolite identification studies are essential for the efficient development and clinically optimal usage of this drug. Tacrolimus was metabolized by the cytochrome P450 (CYP) 3A subfamily. Metabolic drug-drug interaction studies were conducted to provide information regarding the optimal usage of tacrolimus, and its metabolism was inhibited by known CYP3A inhibitors such as ketoconazole, cyclosporine A, and nifedipine. Recent reports on clinical pharmacokinetics indicate that dose levels of tacrolimus need to be adjusted in transplant patients with CYP3A5 polymorphism.

1-Aminobenzotriazole (ABT) is extensively used as a non-specific cytochrome P450 (CYP) inhibitor. In this study, the inhibitory effect of ABT on CYP-dependent drug oxidations was investigated in human liver microsomes (HLM) and compared with that of SKF-525A, another non-specific inhibitor. The following probe activities for human CYP isoforms were determined using pooled HLM: phenacetin O-deethylation (CYP1A2); diclofenac 4'-hydroxylation (CYP2C9); S-mephenytoin 4'-hydroxylation, (CYP2C19); bufuralol 1'-hydroxylation (CYP2D6); chlorzoxazone 6-hydroxylation (CYP2E1); midazolam 1'-hydroxylation, nifedipine oxidation, and testosterone 6beta-hydroxylation (CYP3A). ABT had the strongest inhibitory effect on the CYP3A-dependent drug oxidations and the weakest effect on the diclofenac 4'-hydroxylation. SKF-525A potently inhibited the bufuralol 1'-hydroxylation, but weakly inhibited chlorzoxazone 6-hydroxylation. The inhibitory effects of ABT and SKF-525A were increased by preincubation in some probe reactions, and this preincubation effect was greater in ABT than in SKF-525A. The remarkable IC50 shift (> 10 times) by preincubation with ABT was observed on the phenacetin O-deethylation, chlorzoxazone 6-hydroxylation, and midazolam 1'-hydroxylation. In conclusion, ABT and SKF-525A had a wide range of IC50 values in inhibiting the drug oxidations by HLM with and without preincubation.

Pharmacokenetic (PK) properties have been recognized as one of the most important attrition factors in the drug development. Recently, there has been a substantial increase in the variety and number of newly synthesized compounds in the drug discovery stage. Therefore, an improved capacity to evaluate such huge numbers of compounds is one of the most important needs for high-throughput screening. One key to successful analysis technology for PK studies is the LC/MS. However, there is a problem to establishing high-throughput LC/MS. It is important that a suitable LC system is selected from various instruments to analyze with accuracy. For example, short columns, parallel LC systems, unique types of columns, and high flow assays have been examined to find the most rapid and efficient analysis systems. Impurities from reaction mixture or medium should be separated from analytes to get stable detection, as those can alter the ion effect of analytes. In addition to developing these methods, keeping the LC/MS system a good working order is also important, as high-throughput LC/MS is used for continuous screening. High-throughput LC/MS will be indispensable technology to PK studies in drug discovery, but much work has to be done in order to determine which optimal condition will be developed.

1-Aminobenzotriazole (ABT) is widely used as a non-specific inhibitor of animal cytochrome P450 (CYP). In the present study, the inhibitory effect of ABT was investigated on drug oxidations catalyzed by human CYP isoforms. This inhibitory effect was compared with that of SKF-525A, another non-specific inhibitor, and ketoconazole, a potent inhibitor of CYP3A. Bacurovirus-expressed recombinant human CYP isoforms were used as an enzyme source. The specific activities for human CYP isoforms are: phenacetin O-deethylation, for CYP1A2; diclofenac 4'-hydroxylation, for CYP2C9; S-mephenytoin 4'-hydroxylation, for CYP2C19; bufuralol 1'-hydroxylation, for CYP2D6; chlorzoxazone 6-hydroxylation, for CYP2E1; testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 1'-hydroxylation, for CYP3A4. ABT inhibited both CYP1A2-dependent activity (Ki=330 microM) and CYP2E1-dependent activity (Ki=8.7 microM). In contrast, SKF-525A weakly inhibited CYP1A2-dependent activities (46% inhibition at 1200 microM) and CYP2E1-dependent activities (65% inhibition at 1000 microM). ABT exhibited the highest Ki value for CYP2C9-dependent diclofenac 4'-hydroxylation among those determined by this assay (Ki=3500 microM). Moreover, SKF-525A showed strong inhibition of CYP2D6-dependent bufuralol 1'-hydroxylation (Ki=0.043 microM). Ketoconazole inhibited all tested drug oxidations, however, its inhibitory effect on CYP1A2-dependent activities was very weak (50% inhibition at 120 microM). ABT, SKF-525A, and ketoconazole showed different selectivity and had a wide range of Ki values for the drug oxidations catalyzed by human CYP enzymes. Therefore, we conclude that inhibitory studies designed to predict the contribution of CYP enzymes to the metabolism of certain compounds should be performed using multiple CYP inhibitors, such as ABT, SKF-525A, and ketoconazole.

The absorption, distribution, and excretion of radioactivity were studied in the male rat after a single dermal application of 14C-labeled FK506 ointment at an FK506 dose level of 1.6 mg/kg. The application period of the ointment was 24 hours. 1. Urinary and fecal excretion of radioactivity were 0.5 and 5.1% of the administered dose, respectively, over 168 hours after application to the intact skin under the non-occlusive dressing condition, 0.4 and 4.2% to the intact skin under the occlusive condition, and 2.4 and 53.8% to the damaged skin under the occlusive condition. Radioactivity remaining at the application site and in the animal body were respectively 14.6 and 0.3% at 168 hours after application to the intact skin under the non-occlusive condition, and were 4.7 and 0.4% after application to the damaged skin under the occlusive condition. 2. Radioactivity was not detected in the whole blood and plasma at any sampling points after ointment application to the intact dorsal skin under occlusive dressing condition. After application to the damaged dorsal skin under the occlusive condition, the pharmacokinetic parameters of radioactivity in the whole blood and plasma were respectively as follows: maximal concentration (Cmax), 42.7 and 37.9 ng eq./mL; time to reach Cmax, both 2 hours; area under the concentration-time curve, 559 and 457 ng eq.·h/mL; and half life, 9.0 and 9.3 hours. 3. After ointment application to the damaged dorsal skin under the occlusive dressing condition, rank order of radioactivity detected at 30 minutes after application was as follows: lung and adrenal gland>brown fat, heart, thyroid gland, kidney, liver and spleen>plasma, urinary bladder and eyeball>cerebrum and testis. The maximal level of radioactivity was observed between 1/2 and 2 hours after application in most tissues. The proportion of radioactivity at 72 hours after application of the maximal level was less than 0.1 in all tissues except the urinary bladder where the proportion was 0.94.

The absorption, distribution and excretion of tacrolimus (FK506) was studied in the baboon after intravenous (i.v.) and oral administration of 14C-labeled FK506 (14C-FK506) at respective dose levels of 1 and 10 mg/kg. 1. After i.v. injection of 14C-FK506, both total radioactivity and FK506 were eliminated bi or tri-exponentially from the body. The whole blood levels of radioactivity were higher than those in the plasma, and were almost the same as the FK506 levels in the whole blood, which were much higher than those in the plasma. Model-independent pharmacokinetic parameters of FK506 in the whole blood were as follows: elimination half-life (T1/2), 12.3 hours; total body clearance, 0.265 L/h·kg; and volume of distribution at steady state, 3.93 L/kg. After oral administration, the parameters were as follows: T1/2, 10.4 hours; maximal blood concentration (Cmax), 190 ng/ml; time to reach Cmax, 2 hours; and area under the concentration-time curve (AUC0-∞), 2007 ng·h/ml. The bioavailability of radioactivity and FK506 was 7.0 and 5.1% respectively. The levels of radioactivity and FK506 were much lower in the plasma than those of the whole blood at FK506 level less than 250 ng/ml in the whole blood. 2. Tissue distribution of radioactivity was studied at 1, 24 and 120 hours after dosing with 14C-FK506. After i.v. injection, the radioactivity in all tissues collected was higher than that in the plasma 1 hour after injection, and was highest in the lungs, liver, pancreas, and small intestine. Radioactivity decreased gradually and was not detected in almost any tissues 120 hours after injection, except that maximal radioactivity in the large intestine was observed at 24 hours after injection. After oral administration, the radioactivity was detected in a limited number of the tissues such as thymus, liver, spleen, pancreas, bone marrow, and alimentary tract 1 hour after dosing and was not observed in almost any tissues 120 hours after dosing. The radioactivity in the body was mainly excreted in the bile to the feces after both administrations. 3. For 240 hours after administration of 14C-FK506, 5 and 86% of the dosed radioactivity were excreted in the urine and feces, respectively, after i.v. injection, and 4 and 79% after oral administration. The majority of radioactivity dosed was excreted in the urine and feces during the first 72 hours after both administrations.

The oxidative metabolism of tacrolimus (FK506) and its in vitro major metabolite was studied using rat liver microsomes. The microsomes were incubated with 14C-labeled (14C-) substrates and NADPH under aerobic condition, and the reaction products were analyzed by high-performance liquid chromatography. The 13-O-mono-demethylated metabolite (M-I) was detected as the major metabolite of 14-CFK506 at the early stage of incubation and many polar metabolites including di-demethylated and unidentified metabolites were detected at the late stage. The 13, 15 and 13, 31-O-di-demethylated metabolites (M-VII and M-VI, respectively) and more polar metabolites were produced from 14C-M-I. The oxidative metabolism of M-I by rat liver microsomes was higher in adult males than in females, was inhibited by the addition of polyclonal antibody against cytochrome P450 3A2, and was induced by the pretreatment of rats with phenobarbital and dexamethasone. These results indicate that FK506 is metabolized to polar metabolites via M-I, and the metabolism of M-I is catalyzed by rat cytochrome P450 3A subfamily, the same as in the case of FK506 metabolism.

The absorption, distribution, metabolism and excretion of tacrolimus (FK506) were studied in the rat after intravenous (i.v.) and oral administration of 14C-labeled FK506 (14C-FK506). 1. After i.v. injection at a dose level of 1.0 mg/kg, the pharmacokinetic parameters of FK506 in the whole blood were as follows: elimination half life, 6.4 hours; total body clearance, 1.59 l/h ·kg; and volume of distribution at steady state, 11.8 l/kg. After oral administration at a dose level of 3.2 mg/kg, the parameters in the whole blood were as follows: maximal blood concentration (Cmax), 41 ng/ml; time to reach Cmax, 0.25 hour; and area under the concentration-time curve (AUC0-24h), 255 ng·h/ml. Absorption of radioactivity and FK506 was 37 and 14%, respectively, calculated from the values of AUC0-24 h in the whole blood. Distribution of FK506 in the blood changed depending on its levels. The ratio of whole blood to plasma levels was more than 2.5 at less than the whole blood levels of 50 ng/ml but decreased above these levels. 2. Radioactivity was distributed throughout the body after i.v. injection of 14C-FK506 at a dose of 0.32 mg/kg. Radioactivity was highest in most of tissues at 5 minutes after injection, the first sampling point after injection, and the levels were higher than those in the plasma except that in the white fat, testis, cerebellum and cerebrum. At 72 hours after injection, radioactivity in most tissues decreased to less than 10% of the maximal concentrations except that in the cerebrum, testis and urinary bladder, in which about half of the concentration was detected. After oral administration at a dose of 1.0 mg/kg, radioactivity was distributed mainly in the gut and liver and was hardly detected in the other tissues. 3. During 72 hours after administration of 14C_ FK506 at a dose of 1.0 mg/kg, 8 and 95%of the dosed radioactivity were respectively excreted to the urine and feces after i.v. injection and 4 and 96% after oral administration. During 48 hours after i.v. injection to the bile duct-cannulated rats, 3, 8 and 82% of the dosed radioactivity were recovered in the urine, feces and bile, respectively, and 3, 6 and 35% during the same period after oral administration. 4. Less than 0.4% of the dosed radioactivity was excreted as unchanged FK506 in the urine, feces and bile, and the elution pattern of excreted radioactivity was very complex on high performance liquid chromatography and no major but many small radioactive peaks were observed. The major in vitro metabolite, 13-O-mono-demethylated metabolite, was detected in the urine, feces and bile but its portion to total radioactivity was very small.

The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, althoughin vitrostudies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The presentin vivostudy demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56–58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%. The temporal window for FK506 neuroprotection was defined in studies demonstrating efficacy using intravenous administration at 120 min, but not 180 min, after middle cerebral artery occlusion. The noncompetitive NMDA receptor antagonist MK801 reduced both ischemic and excitotoxic damage. Histopathological data concerning striatal quinolinate lesions were replicated in neurochemical experiments. MK801, but not FK506, attenuated the loss of glutamate decarboxylase and choline acetyltransferase activity induced by intrastriatal injection of quinolinate. The contrasting efficacy of FK506 in ischemic and excitotoxic lesion models cannot be explained by drug pharmacokinetics, because brain FK506 content rose rapidly using both treatment protocols and was sustained at a neuroprotective level for 3 d. Although these data indicate that an antiexcitotoxic mechanism is unlikely to mediate the neuroprotective action of FK506 in focal cerebral ischemia, the finding that intravenous cyclosporin A (20 mg/kg) reduced ischemic cortical damage is consistent with the proposed role of calcineurin.

Temporal and gender-related changes of sulfotransferase activities in the rat were studied using alcoholic, amine and phenolic compounds as substrates. Alcohol and amine sulfotransferase activities in the rat exhibited different temporal and sex-related changes of those for phenol. Formation of sulfoconjugates in vivo correlated well with in vitro sulfotransferase activities in the rat liver.

The distribution of FK506 in the blood was estimated in-vitro. At a drug level of 5 ng mL−1, FK506 mainly distributed in erythrocytes (95–98%) in dog, monkey and human blood, and its distribution was affected by drug concentration, temperature, and haematocrit values. In erythrocytes most of FK506 was distributed in cytoplasmic components and was bound strongly to a protein having a molecular weight of 10–11 kDa. The molecular weight of this protein agrees with FK506-binding protein found in various cells. Greater than 98·8% of FK506 was bound to the plasma proteins in all species studied. FK506 bound to various plasma proteins such as lipoproteins, globulins, α1-acid glycoprotein and albumin.

A nobel N-sulfoconjugate of alicyclic amine was identified in the metabolic study of tiaramide. Highly polar N-and O-sulfoconjugates were analyzed by secondary ion mass spectrometry (SIMS) and negative SIMS was an effective mode to detect the molecular ion peak of the conjugates. Sulfoconjugation of alicyclic, alkyl-and aryl-amines was detected in the hepatic soluble fractions in the presence of PAPS, a biologically active sulfate. Subcellular distribution, coenzyme requirement and tissue distribution of amine sulfoconjugations were similar to those in phenol and hydroxysteroid (alcohol) sulfoconjugations. Some gender-, age and species-related differences of N and O-sulfoconjugations were observed in animal species, but the differences were substrate-dependent. Analyses using purified enzyme proteins and genes of sulfotransferases will contribute to the understanding of multiplicity in N-and O-sulfoconjugations.

Four different forms of cytochrome P450 (P450) were purified from liver microsomes of untreated or phenobarbital (PB)-treated male beagle dogs using HPLC techniques, and designated as DUT-1, DPB-1, DPB-2 and DPB-3, respectively. Specific contents of the purified DUT-1, DPB-1, DPB-2 and DPB-3 were 13.3, 9.6, 15.6 and 12.2 nmol/mg protein, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the monomeric molecular weights of DUT-1, DPB-1, DPB-2 and DPB-3 were estimated to be 57.5, 50.0, 47.0 and 50.0 kDa, respectively. The absolute spectra of the oxidized forms indicated that they exist in the low-spin state of heme in their oxidized forms. The NH2-terminal amino acid sequence of DUT-1 was unique and different from that of any other P450 so far reported. DUT-1 was active in the omega-hydroxylation of lauric acid. The amino-terminal sequences of DPB-1, DPB-2 and DPB-3 suggested that they belong to the P450 3A, 2C and 2B gene families, respectively. DPB-3 was a major form of P450 in PB-treated dog liver microsomes. Purified DPB-1 catalyzed nifedipine and (+)- and (-)-nilvadipine oxidations, as well as testosterone 6 beta-hydroxylation in the reconstituted system. These activities were enhanced 3- to 5-fold by the addition of cytochrome b5. DPB-2 and DPB-3 catalyzed aminopyrine N-demethylation, 7-ethoxycoumarin O-deethylation, biphenyl 4-hydroxylation and testosterone 16 alpha-hydroxylation. We believe that DUT-1 is a new form not purified previously.

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.

Age- and gender-related changes of sulfoconjugations were studied in the rat. Alcohol and amine sulfoconjugating activities by rat hepatic soluble fractions were very low or negligible in the fetus. The activities increased after birth and attained maximal in 3-week-old males and females. Female rats maintained the maximal levels until the age of 2 years. In males, the activities lowered at 7 weeks old, were maintained at the low levels until the age of 1 year and increased thereafter but never reached the levels of 2-year-old females. Urinary excretion of sulfoconjugates of alcoholic and amine compound exhibited similar temporal changes to the in vitro activities. Phenol sulfoconjugating activity by the soluble fractions was very low in the fetus. The activity was about ten-fold higher in the neonates at birth than in the fetus and relatively constant until 3 weeks after birth. The activity in male 7-week-old rats attained the maximal level, and was about two-fold higher than females of the same age and about four-fold higher than the neonates. The activity in male and female rats at the age of 2 years were intermediate between the 7-week-old males and females, and no sex differences were observed. The results indicate that the substrate of sulfoconjugations could be divided into at least two groups, an alcohol and amine type, and a phenol type.

Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP) 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C93 cDNA contained an open reading frame of 490 amino acids approximately 84–86% identical to human CYP2Cs. CYP2C93 was located in the genomic region, which corresponded to the intergenic region in the human genome, indicating that CYP2C93 does not correspond to any human genes. CYP2C93 mRNA was expressed predominantly in the liver among 10 tissues analyzed. The CYP2C93 proteins heterologously expressed in Escherichia coli metabolized human CYP2C substrates, diclofenac, flurbiprofen, paclitaxel, S-mephenytoin, and tolbutamide. In addition to a normal transcript (SV1), an aberrantly spliced transcript (SV2) lacking exon 2 was identified, which did not give rise to a functional protein due to frameshift and a premature termination codon. Mini gene assay revealed that the genetic variant IVS2-1G>T at the splice site of intron 1, at least partly, accounted for the exon-2 skipping; therefore, this genotype would influence CYP2C93-mediated drug metabolism. SV1 was expressed in 6 of 11 rhesus monkeys and 1 of 8 cynomolgus monkeys, but the SV1 in the cynomolgus monkey was nonfunctional due to a rare null genotype (c.102T>del). These results suggest that CYP2C93 can play roles as a drug-metabolizing enzyme in rhesus monkeys (not in cynomolgus monkeys), although its relative contribution to drug metabolism has yet to be validated.

Summary: It is important to estimate the defective metabolism caused by genetic polymorphism of drug metabolizing enzymes before the clinical stage. We evaluated the utility of cryopreserved human hepatocytes of CYP2D6 poor metabolizer (PM) for the estimation of the metabolism in PM using dextromethorphan (DEX) as the probe drug for CYP2D6 substrate. The results of low formations of dextrorphan (DXO) and 3-hydroxymorphinan (3-HM) in CYP2D6 PM hepatocytes incubated with dextromethorphan reflected the clinical data. Formation of 3-methoxymorphinan (3-MEM) normalized by CYP3A4/5 activity in the PM hepatocytes reached about 2.8-fold higher than that in CYP2D6 extensive metabolizer (EM) hepatocytes, which clearly showed the compensatory metabolic pathway of O-demethylation catalyzed by CYP2D6 as seen in clinical study. On the contrary, in the condition of the EM hepatocytes with CYP2D6 inhibitors, the enhancement of 3-MEM formation was not observed. In phase II reaction, the glucuronide formation rate of DXO in the PM hepatocytes was lower than that in the EM hepatocytes, which was consistent with clinical data of DXO-glucuronide (DXO-glu) concentration. These results would suggest that CYP2D6 PM hepatocytes could be a good in vitro tool for estimating CYP2D6 PM pharmacokinetics.

We investigated the antioxidative effects of fluvastatin (FV or (+/-)-FV), each enantiomer ((+)-FV, (-)-FV) and its major metabolites on lipid peroxidation using rat and human liver microsomes. The extent of NADPH induced microsomal (Ms) lipid peroxidation was determined by thiobarbituric acid (TBA) assay. The antioxidative effect of each compound was shown as the percentage of inhibition on the formation of TBA reactive substance (TBARS) against vehicle control. The antioxidative effects of alpha-tocopherol (Toc), a potent antioxidative vitamin, probucol (PR), a potent antioxidative drug, pravastatin (PV) and simvastatin (SV), HMG-CoA reductase inhibitors, were also tested. The (+/-)-FV inhibit the formation of TBARS by 40 to 70% depending on Ms concentrations. The antioxidative effects of PR and TOC were comparable to those of FV. The inhibitory effects of PV and SV on the formation of TBARS were less potent than (+/-)-FV, PR and TOC. (+)-FV, (-)-FV, and (+/-)-FV inhibited the formation of TBARS by approximately 50% using rat hepatic microsomes. The antioxidative effects of (+)-FV was comparable to that of (-)-FV using human hepatic microsomes. These results indicated that the antioxidative effects of (+)-FV were comparable to those of (-)-FV, although the HMG-CoA reductase inhibitory activity of (+)-FV was 30-fold higher than that of (-)-FV.

An alicyclic amine N-sulfotransferase sulfonating 4-phenyl-1,2,3,6-tetrahydropyridine (PTHP) was purified from female rat liver cytosol and showed a homogenous band with a molecular weight of 30500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme, designated NST-1, catalyzed sulfonation not only of the alicyclic amine but also dehydroepiandrosterone, a typical substrate of hydroxysteroid sulfotransferase (STs), but had little sulfonating activity towards 2-naphthol, a typical substrate of aryl STs. The N-terminal amino acid sequence for the first 24 residues had a high homology with those of rat liver hydroxysteroid STs. Therefore, it is suggested that NST-1 is classified as a member of the hydroxysteroid ST. Immunoblot analysis of male and female rat liver cytosol, carried out by using rabbit antisera raised against NST-1, indicated that the female cytosol contained a higher level of the enzyme than that of male. The marked sex difference in the expression level of NST-1 was in good accordance with the previous demonstration that female rat liver cytosol catalyzed sulfonation of PTHP to a greater extent than that of male.

Hepatic constituents and the cytochrome contents in addition to activities of drugmetabolizing enzymes and δ-aminolevulinic acid (δ-ALA) synthetase were examined in rats treated with each isomer of dichlorobenzene (DCB), trichlorobenzene (TRCB) and tetrachlorobenzene (TECB) in an oral dose of 250 mg/kg once daily for 3 days. 1) In the studies on DCB-isomers, activities of aminopyrine demethylase and aniline hydroxylase were enhanced markedly by treatment with m-DCB, whereas cytochrome content was not altered significantly by treatment with any DCB-isomers. δ-ALA synthetase activity was enhanced 63, 32 and 42% by treatment with o-, m- and p-DCB, respectively, but these enhancement were not paralleled with the changes in cytochrome P-450 content. 2) In the administration of TRCB-isomers, enzyme activities were enhanced markedly by treatment with 1, 2, 4-TRCB. Cytochrome P-450 content was also increased by treatment with 1, 2, 4-TRCB, and this increase could be partly related with the enhancement of δ-ALA synthetase activity. 3) By treatment with all TECB-isomers, activity of aminopyrine demethylase was enhanced, whereas aniline hydroxylase was not altered. Cytochrome P-450 content was increased by treatment with all TECB-isomers. 4) Microsomal protein content was increased with all isomers of DCB, TRCB and TECB treatment. Microsomal Pi was increased markedly 36, 70 and 91% by treatment with m-DCB, 1, 2, 4-TRCB and 1, 2, 3, 5-TECB, respectively. Hepatic glycogen content was decreased only by 1, 2, 3, 5-TECB, but triglyceride content was not altered. 5) The spectral change induced by substrate-cytochrome P-450 binding was characterized by type I in each treatment, but conversion of the type occurred in treatment with 1, 2, 4, 5-TECB in high concentration of 1 mM.

To elucidate the relationship between chemical structure and their biological activities, the contents of cytochromes and hepatic constituents in addition to the activities of drug-metabolizing enzymes and δ-aminolevulinic acid (δ-ALA) synthetase were examined in rats treated with various chlorinated benzenes, i.e., monochlorobenzene (MCB), p-dichlorobenzene (p-DCB), 1, 3, 5-trichlorobenzene (1, 3, 5-TRCB), 1, 2, 4, 5-tetrachlorobenzene (1, 2, 4, 5-TECB), pentachlorobenzene (PECB) and hexachlorobenzene (HCB). 1) The content of cytochrome P-450 and activities of aminopyrine demethylase and aniline hydroxylase were increased by oral administration of all chlorobenzenes except MCB at daily dose of 250 mg/kg, once daily, for 3 days. The contents of microsomal protein and phospholipids also showed a similar tendency to those as described above. The activity of δ-ALA synthetase was increased by treatment with all compounds used. 2) The content of cytochrome P-450 and activity of aminopyrine demethylase were decreased in 24 hr after a single administration of MCB in doses of 125, 250, 500 and 1000 mg/kg, whereas the activity of δ-ALA synthetase was increased markedly by all doses used. The activity of aniline hydroxylase was increased by a dosing of 1000 mg/kg MCB. 3) In the time-course after a single administration of MCB in a dose of 250 mg/kg, the activity of δ-ALA synthetase was decreased in 6 hr after administration, and subsequently restored to normal levels in 12 hr, and then increased markedly in 24 hr. The opposite changes were noted in the content of cytochrome P-450.