Perspectives on non-clinical safety evaluation of drug metabolites through the JSOT workshop.

The prompt and appropriate safety assessment of drug metabolite(s) was mentioned in regulatory guidances such as an International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance, entitled "Guidance on Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals" (ICH M3(R2)) implemented in January 1 of 2011 in Japan, and has become a significant issue in the drug development. Upon release of ICH M3(R2) Step 4, a survey was conducted between March and April 2010 on the safety assessment of drug metabolites in 63 member companies of the Japan Pharmaceutical Manufacturers Association (JPMA). The Pharmacokinetics Team in the Non-Clinical Evaluation Expert Committee in JPMA conducted a questionnaire survey and compiled the results to comprehend how safety of drug metabolites are currently assessed at research-based pharmaceutical companies in Japan. The assessment of "Metabolites in Safety Testing" (MIST) can be divided into three stages based on the research purpose as follows: MIST 1 is a stage of estimating human drug metabolites and predicting their potential risks, MIST 2 is a stage of deciding the necessity for non-clinical safety studies, and MIST 3 is a stage of conducting non-clinical safety studies. In this paper, we propose typical approaches on safety assessment of metabolites that meet the purpose of each stage, considering the current level of scientific technology. Our proposals are based on the results from our survey and a symposium about the safety assessment of drug metabolites at the 37th annual meeting of the Japanese Society of Toxicology held in June 2010.

Acotiamide hydrochloride (Z-338), a new selective acetylcholinesterase inhibitor, enhances gastric motility without prolonging QT interval in dogs: comparison with cisapride, itopride, and mosapride.

Acotiamide hydrochloride (acotiamide; N-[2-[bis(1-methylethyl) amino]ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl) amino] thiazole-4-carboxamide monohydrochloride trihydrate, Z-338) has been reported to improve meal-related symptoms of functional dyspepsia in clinical studies. Here, we examined the gastroprokinetic effects of acotiamide and its antiacetylcholinesterase activity as a possible mechanism of action in conscious dogs. Acotiamide increased postprandial gastric motor activity in conscious dogs with chronically implanted force transducers and, like itopride, mosapride, and cisapride, exhibited gastroprokinetic activity in these dogs. Furthermore, acotiamide improved clonidine-induced hypomotility and delayed gastric emptying. Acotiamide-enhanced postprandial gastroduodenal motility was suppressed completely by pretreatment with atropine, a muscarinic receptor antagonist. In in vitro studies, acotiamide enhanced acetylcholine- but not carbachol-induced contractile responses of guinea pig gastric antrum strips. Moreover, like itopride and neostigmine, acotiamide inhibited recombinant human and canine stomach-derived acetylcholinesterase (AChE) activity in vitro. The mode of the AChE inhibitory action of acotiamide was selective and reversible. Unlike itopride or mosapride, acotiamide showed no affinity for dopamine D(2) or serotonin 5-HT(4) receptors. With regard to cardiovascular side effects, unlike cisapride, acotiamide did not affect myocardial monophasic action potential duration, QT interval, or corrected QT interval in anesthetized dogs. These results suggest that acotiamide stimulates gastric motility in vivo by inhibiting AChE activity without affecting QT interval. Acotiamide thus represents a beneficial new drug for the treatment of functional dyspepsia involving gastric motility dysfunction, with differences from other prokinetic agents.

Polaprezinc, a zinc compound, is distributed to the lingual epithelium and increases its zinc concentration in zinc-deficient rats.

AIMS: To clarify the mechanism underlying the effect of polaprezinc on hypogeusia, we investigated the uptake of polaprezinc by the tongue in rats. MAIN METHODS: Rats were fed a zinc-sufficient (Zn(+)) or zinc-deficient (Zn(-)) diet. After 4weeks on the Zn(-) diet, polaprezinc (1, 3, or 10mg/kg) or [(65)Zn] polaprezinc (10mg/kg) was administered orally once a day. The zinc concentration or the (65)Zn radioactivity of the tongue was measured by inductively-coupled plasma mass spectrometry or gamma counting, respectively. In addition, the distribution of (65)Zn in the tongue was analyzed by microautoradiography and the proliferative activity of taste bud cells was measured from the uptake of 5-bromo-2'-deoxyuridine. KEY FINDINGS: The zinc concentration of the lingual epithelium, but not the whole tongue, was markedly decreased in Zn(-) rats compared with Zn(+) rats. After administration of polaprezinc to Zn(-) rats at doses of 1, 3, and 10mg/kg, the zinc concentration in the lingual epithelium increased significantly from 85+/-4 to 105+/-7 (p<0.05), 120+/-3 (p<0.001), and 124+/-3 (p<0.001) microg/g, respectively. After administration of [(65)Zn] polaprezinc, the (65)Zn radioactivity of the tongue and serum were higher in Zn(-) rats than in Zn(+) rats. (65)Zn was mainly detected in the epithelium on microautoradiograms of the tongue in Zn(-) rats. In addition, polaprezinc (3 and 10mg/kg) improved the reduced proliferation of taste bud cells due to zinc deficiency. SIGNIFICANCE: Polaprezinc is distributed to the lingual epithelium and restores its zinc concentration in Zn(-) rats resulting in improvement of cellular functions, especially proliferation.

Pharmacokinetics and safety of single and multiple doses of Asacol tablets in Japanese healthy volunteers.

INTRODUCTION: The pharmacokinetics and safety of Asacol (Tillotts Pharma AG, Ziefen, Switzerland), which has been used worldwide to treat ulcerative colitis and Crohn's disease, were studied in Japanese healthy male volunteers. METHODS: Drug plasma concentrations and urinary and fecal excretions after a single dose (400-4800 mg) and multiple doses (3600 mg/day for 7 days) were investigated. RESULTS: All adverse events were "not serious." The peak plasma concentration (C max) was reached at 12.3-18.0 hours after a single dose, and the C max and area under the plasma concentration-time curve (AUC) of mesalazine and its N-acetyl metabolite were proportional to the doses. The C max and AUC in non-Japanese subjects reported in the literature were closely correlated to findings in Japanese subjects, and external excretions were also similar in the Japanese and non-Japanese subjects. CONCLUSIONS: Asacol was safe and well tolerated in this Japanese population, and the non-Japanese clinical data could be extrapolated to the Japanese population.

Current opinion: safety evaluation of drug metabolites in development of pharmaceuticals.

Safety assessment of drug metabolites in the development of pharmaceuticals was discussed in January 2007 at the kick-off meeting of a "Drug Evaluation Forum", with reference to the views of clinicians and other academic representatives. Safety evaluation of metabolites cannot readily be based on a single theoretical framework, and basically a case-by-case approach is called for. These evaluations should be performed precisely and an accurate profile secured taking into account adverse reactions that are unpredictable from the parent compound administered in clinical studies and any signs or symptoms that may be associated with the metabolites. In addition, elimination of scientifically meaningless metabolite safety assessment studies is essential for prompt supply of high-quality drugs to the medical frontline. Preparation of an outline concept paper would be useful for achievement of shared understanding of issues of this type. Collective viewpoints obtained in this fashion are also relevant to the discussion on the need for guidance, and given a degree of flexibility may also be helpful for drug development and, in turn, society at large.

Drug-drug interactions of Z-338, a novel gastroprokinetic agent, with terfenadine, comparison with cisapride, and involvement of UGT1A9 and 1A8 in the human metabolism of Z-338.

In the present study, the inhibitory properties of N-[2-(diisopropylamino)ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl)amino]-1,3-thiazole-4-carboxamide monohydrochloride trihydrate (Z-338), a novel gastroprokinetic agent, were investigated and compared with those of cisapride to establish its potential for drug-drug interactions. There was no notable inhibition of terfenadine metabolism or of any of the isoforms of cytochrome P450 (CYP1A1/2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) by Z-338 in in vitro studies using human liver microsomes. Z-338 was mainly metabolized to its glucuronide by UGT1A9 (UDP glucoronosyltransferase 1 family, polypeptide A9) and UGT1A8, and did not show marked inhibition of P-glycoprotein activity. On the other hand, cisapride strongly inhibited CYP3A4 and markedly inhibited CYP2C9. Furthermore, we used the whole-cell patch-clamp technique to investigate the effects of Z-338 and cisapride on potassium currents in human embryonic kidney (HEK) 293 cells transfected with the human ether-a-go-go-related gene (hERG). Z-338 had no significant effect on hERG-related current at the relatively high concentration of 10 microM. In contrast, the inhibition by Z-338 was very small compared with that of cisapride at 10 nM, which was a thousand-fold lower concentration. In the prediction method for the drug interaction between terfenadine and cisapride based on the K(i) and PK parameters, we suggest the possibility that terfenadine mainly affect the QT interval, since its plasma concentration would be markedly increased, but cisapride may not be changed. Thus, in contrast with cisapride, Z-338 did not inhibit CYP and the hERG channel, and is predominantly metabolized by glucuronide conjugation, Z-338 is considered unlikely to cause significant drug-drug interactions when coadministered with CYP substrates at clinically effective doses.

Suitability of the odor stick identification test for the Japanese in patients suffering from olfactory disturbance.

We studied the suitability of the Odor Stick Identification Test for the Japanese (OSIT-J) in patients suffering from olfactory disturbance. In 120 patients with olfactory disturbance (age range 12-85 years) there were statistically significant correlations between the odor identification rate on the OSIT-J, the results of the Japanese standardized olfactory test (T&T olfactometry) and subjective symptom scores. In every patient treated for olfactory disturbance, the OSIT-J reflected the grade of recovery from the olfactory disturbance as determined by means of T&T olfactometry. The odor identification rate on the OSIT-J also correlated significantly with the results of the i.v. Alinamin test. Regarding the rate of correct recognition of odors on the OSIT-J, menthol and curry odors were recognized with a high rate and orange and wood odors with a low rate. Although the OSIT-J includes 13 kinds of odorants, the number of odorants used can be reduced to a minimum of 5 as the results obtained with this reduced form of the OSIT-J also correlated with the results of T&T olfactometry and the subjective symptom scores as well as with the results obtained with the 13-odorant OSIT-J. We conclude that the OSIT-J is suitable not only as a screening test for olfactory disturbance but also for practical use in clinical otorhinolaryngology.

ATP-dependent transport of a novel thromboxane A2 receptor antagonist, [2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl]acetate (Z-335) and its xenobiotic taurine conjugate (Z-335-Tau) by rat bile canalicular membrane vesicles.

PURPOSE: The characteristics of bile canalicular transport processes for xenobiotic taurine conjugates have not yet been clarified. To elucidate the biliary excretion characteristics of xenobiotic taurine conjugates, we investigated the transport of a novel thromboxane A2 receptor antagonist, Z-335, and its taurine conjugate (Z-335-Tau) across the bile canalicular membrane. METHODS: We examined the uptake of Z-335 and Z-335-Tau by isolated bile canalicular membrane vesicles (CMVs) from Sprague Dawley and Eisai-hyperbilirubinemic rats (EHBRs) which EHBRs have a hereditary defect of canalicular multidrug resistance-associated protein 2 (Mrp2) function. Also, the in vitro and in vivo kinetics of Z-335-Tau uptake and excretion were compared. RESULTS: Z-335 uptake by CMVs from normal rats exhibited marked ATP-dependence, whereas ATP-dependent uptake of Z-335 into CMVs from EHBRs was not observed. In contrast, Z-335-Tau uptake into CMVs from both normal rats and EHBRs was ATP dependent. The initial uptake velocity was concentration-dependent, with an in vitro Michaelis constant for initial uptake of 189 microM, which was similar to the in vivo value. CONCLUSIONS: The biliary excretion of Z-335 involves Mrp2, whereas that of Z-335-Tau involves active transport systems that remain intact in EHBRs and show marked ATP dependence, which ATP-dependent transport is involved in the biliary excretion of Z-335-Tau in vivo.

Mechanism of hepatobiliary transport of a novel thromboxane A2 receptor antagonist, [2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl]acetate (Z-335), and its xenobiotic taurine conjugate (Z-335-Tau) in rats.

We investigated the mechanism of hepatobiliary transport of a novel thromboxane A(2) receptor antagonist, [2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl]acetate (Z-335), and its taurine conjugate (Z-335-Tau) in normal Sprague-Dawley rats (SDRs) and Eisai hyperbilirubinemic rats (EHBRs). The biliary excretion rate/unbound concentration in the cytosol (nu(bile)/C(u,cyt)) of Z-335 was markedly decreased in EHBRs, whereas nu(bile)/C(u,cyt) values for Z-335-Tau did not differ significantly between EHBRs and SDRs. These results suggest that biliary excretion of Z-335 involves mrp2, whereas Z-335-Tau is excreted by other transporters. The effects of inhibitors on the biliary excretion of Z-335 and Z-335-Tau were also examined in SDRs. After infusion of bromosulfophthalein (BSP), the nu(bile)/C(u,cyt) of Z-335 was significantly decreased, whereas that of Z-335-Tau decreased to 50% of control values by infusion of indocyanine green (ICG) or taurocholate. However, biliary excretion of Z-335-Tau was maintained at a highly concentrative. In conclusion, the biliary excretion of Z-335 involves mrp2, whereas Z-335-Tau is excreted into the bile by active transport systems that remain intact in EHBRs. The mdr2 and/or BSEP/spgp might contribute to a part of total biliary excretion of Z-335-Tau, however, these transporters have not played a major role in the biliary excretion of Z-335-Tau.

[Bacterial examination of sinusitis using antral puncture and irrigation].

Aerobic bacterial examination for sinusitis was conducted using specimens from maxillary sinuses collected by antral puncture in 540 patients--284 men and 256 women aged 6-89 years--between May 1999 and April 2000. We obtained 528 strains of bacteria. Our results were as follows: 1. We obtained 303 pathogens from 540 patients. In acute sinusitis, the most frequently found was Streptococcus pneumoniae (30.4%), followed by Hemophilus influenzae (27.7%). In chronic cases, the most frequently found was Streptococcus pneumoniae (16.0%), followed by Hemophilus influenzae (15.1%) and Staphylococcus epidermidis (12.6%). 2. We found an increase in bacteria resistant to multiple drugs, with 11.1% of the Staphylococcus aureus isolates methicillin-resistant in acute sinusitis and 40% methicillin-resistant in chronic sinusitis, and that 30.6% of Streptococcus pneumoniae isolates were penicillin-resistant. 3. Ciclacillin was effective against 64.7% of all pathogens isolated in this study, cefpodoxime proxetil effective against 6.5%, and cefixime effective against 2.4%. 4. In considering pathogens, we therefore choose antibiotics and make a maxillary aspiration puncture.

Involvement of CYP2C9 and UGT2B7 in the metabolism of zaltoprofen, a nonsteroidal anti-inflammatory drug, and its lack of clinically significant CYP inhibition potential.

AIMS: To identify the cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) isoforms responsible for the formation of the primary metabolite(s) of zaltoprofen, and to predict possible drug interactions by investigating the inhibition of CYP isoforms in vitro. METHODS: The metabolism of zaltoprofen was studied in vitro using recombinant CYP and UGT isoform cDNA-expression systems. The effects of selective isoform inhibitors on zaltoprofen metabolism were studied using human liver microsomes. The inhibitory effects of zaltoprofen on the metabolism of selective probe substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were also determined in human liver microsomes. RESULTS: Zaltoprofen was extensively metabolized by CYP2C9 and UGT2B7. CYP2C9 catalysed sulphoxidation but not hydroxylation of zaltoprofen. In the human liver microsomal metabolism study, zaltoprofen metabolism was markedly inhibited by sulphaphenazole, a selective inhibitor of CYP2C9. In the drug interaction study, negligible inhibition (< 15%) of the activities of CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 was apparent at 5 micro g ml(-1), the maximum plasma concentration observed in humans after oral administration of an 80 mg zaltoprofen tablet. However, zaltoprofen inhibited CYP2C9 by 26% at 5 micro g ml(-1). At higher concentrations, zaltoprofen produced some inhibition of CYP2C9 (IC50 = 19.2 micro g ml(-1); 64.4 micro m) and CYP3A4 (IC50 = 53.9 micro g ml(-1); 181 micro m). The free drug concentrations in plasma (0.02 micro g ml(-1), 67.0 nm) at the Cmax of the clinically effective doses are much lower than the IC50 values corrected for the nonspecific binding ratio of zaltoprofen to microsomal protein (15.5 micro g ml(-1) for CYP3A4, 49.5 micro g ml(-1) for CYP3A4). Furthermore, the maximum free drug concentrations in the hepatic intracellular was calculated to be 0.068 micro g ml(-1) and the increase in the AUC in the presence of zaltoprofen was estimated to be only 0.4% for CYP2C9 substrates and 0.1% for CYP3A4 substrates, respectively. CONCLUSIONS: Zaltoprofen is predominantly metabolized by CYP2C9 and UGT2B7, and is considered unlikely to cause significant drug interactions in vivo when coadministered with CYP substrates at clinically effective doses.

Carrier-mediated active transport of a novel thromboxane A(2) receptor antagonist [2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl]acetate (Z-335) into rat liver.

To elucidate the transport system by which [2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl]acetate (Z-335) is taken up into the liver, we investigated the uptake characteristics of Z-335 in isolated rat hepatocytes. In addition, we estimated the hepatic uptake of Z-335 in intact rats under steady-state conditions and compared it with the in vitro uptake clearance. Uptake of Z-335 is highly concentrative (cell-to-medium concentration ratios were 21.2 at 0.5 min and 71.7 at 5 min), temperature-dependent, and sensitive to metabolic inhibitors, indicating that uptake is mediated by energy-dependent uphill transport. In the presence of metabolic inhibitors [carbonyl cyanide p-trifluoromethoxyphenylhydrazone and rotenone], uptake remained at 37 and 49% of the control value, respectively, suggesting that ATP-independent uptake contributes to the total uptake of Z-335. The concentration dependence of the initial uptake velocity indicated a two-component process, one saturable component, with a K(m) value of 45.6 microM and a V(max) value of 4.1 nmol/min/mg of protein, and a nonspecific diffusion clearance, with a P(dif) value of 8.3 microl/min/mg of protein. The contribution of the carrier-mediated uptake to the total uptake in a linear range was estimated as 91%. The in vivo hepatic intrinsic clearance (CL(int, app)) was comparable with that in vitro uptake clearance (PS(influx)) and indicated that the CL(int, app) of Z-335 at steady state is rate-limited by the uptake process. In conclusion, hepatic intrinsic clearance of Z-335 at steady state is rate-limited by the uptake process since Z-335 is efficiently taken up by an active transport mechanism, followed by metabolism or biliary excretion.