Search

The pharmacokinetics of cilostazol was investigated after oral and intravenous administration in both male and female rats. After oral administration, area under serum concentration-time curve (AUC) was about 35-fold higher in female rats than in male rats, and absolute bioavailability was about 5.8-fold higher in female rats than in male rats. Total body clearance (CL(total)) for female rats was around one-sixth of that for male rats. In vivo hepatic clearance (CL(h)) calculated based on isolated liver perfusion studies was even higher than or around 90% of the in vivo CL(total) of cilostazol for female and male rats, respectively, indicating that cilostazol is mainly eliminated by the liver in both male and female rats. In vitro metabolism studies utilizing hepatic microsomes and recombinant cytochrome (CYP) isoforms clearly indicated that major metabolites of cilostazol were generated extensively with hepatic microsomes of male rats and that male-predominant CYP3A2 and male-specific CYP2C11 were mainly responsible for the hepatic metabolism of cilostazol. Therefore, the great sex differences in the pharmacokinetics of cilostazol were mainly attributed to the large difference in hepatic metabolism. Our experimental results also suggested that the substantial metabolism of cilostazol in the small intestine and its possible saturation would be responsible for dose-dependent bioavailability in both male and female rats.

Specific antibody production is an important issue in crossover pharmacokinetic (PK) studies of protein-based formulations. We recently reported that intravenous co-administration of tacrolimus with multiple human interferon-alpha (h-IFN) administrations successfully suppressed the production of anti-h-IFN antibodies in rats. Since crossover PK studies are preferentially carried out using larger animals such as dogs or monkeys that are capable of accepting the same dosage formulations as those for clinical use, we extended our study of co-administration of tacrolimus with multiple h-IFN administrations to beagle dogs in the present study. Beagle dogs were subcutaneously administered 0.5 million IU/kg of h-IFN once a week for 4 weeks. In some experiments, tacrolimus at 0.01 or 0.1 mg/kg was intravenously co-administered at the same time as the h-IFN administration. Co-administration of the lower dose of tacrolimus (0.01 mg/kg) failed to suppress the anti-h-IFN IgG responses, while co-administration of the higher dose (0.1 mg/kg) successfully suppressed these responses. Moreover, co-administration of tacrolimus had little effect on the serum creatinine concentrations, suggesting that multiple administrations of tacrolimus at the concentrations examined did not cause severe renal disorders. Taken together, the present data confirm that co-administration of tacrolimus is a promising way to assess crossover PK studies of human or humanized proteinic formulations in beagle dogs.

Purpose: Repeated administration of exogenous proteinic compounds triggers the production of specific antibodies. This reaction is limits not only pharmacokinetic studies in animal but also development of human or humanized proteins as drugs. We investigated the effect of co-administration of tacrolimus on pharmacokinetic of human interferon-alpha (h-IFN) following multiple subcutaneous administration in rats., Methods: h-IFN was administered at a dose of 5 million IU/kg. For some experiments, tacrolimus was also either subcutaneously or intravenously injected in rats at a dose of 0.001 or 0.5 mg/kg as well as with administration of h-IFN., Results: Multiple administration of h-IFN without co-administration of tacrolimus induced IgG response at 2 or 3 weeks following first administration in the short dosing interval (daily, once per 3 days, or once per a week), irrespective of the dosing interval. Both intravenous and subcutaneous administration of tacrolimus (0.5 mg/kg) with multiple h-IFN injections successfully suppressed IgG response against h-IFN. Interestingly, in lower doses (0.001 mg/kg), intravenous co-administration of tacrolimus showed much stronger suppressive effect than subcutaneous co-administration., Conclusion: Intravenous co-administration of tacrolimus (0.001 mg/kg) may be a promising way to assess crossover pharmacokinetic study of human or humanized proteinic formulations with multiple dosing schedules in an experimental animal.

The purpose of the present study was to examine whether the intestinal absorption of low-permeability drugs could be improved by utilization of the intestinal influx transporter PEPT1. We investigated whether peptide derivatives of poorly absorbable nonamino acid-like drugs might be substrates of PEPT1, using rebamipide (Reb) as a model drug. We synthesized several peptide derivatives of rebamipide and examined their inhibitory effect on the uptake of [(3)H]Gly-Sar by PEPT1-expressing HeLa cells. Some of the peptide derivatives inhibited PEPT1-mediated uptake of [(3)H]Gly-Sar. Next, uptake of the inhibitory peptide derivatives was evaluated in PEPT1-expressing Xenopus oocytes and HeLa cells. Ser(Reb)-Gly exhibited significantly increased uptake by PEPT1-expressing cells in comparison with that by mock cells. The permeability of Ser(Reb)-Gly across a Caco-2 cell monolayer was significantly higher than that of rebamipide itself, and the transport was decreased in the presence of PEPT1 substrates. Further, a rat intestinal perfusion study revealed increased absorption of Ser(Reb)-Gly compared with rebamipide. These results demonstrate that the addition of a dipeptide moiety to a poorly absorbable nonpeptide/nonamino acid-like drug can result in absorption via the intestinal transporter PEPT1, though there is some selectivity as regards the structure of the added peptide moiety.

The combinatorial use of spermine (SPM), a typical polyamine, and sodium taurocholate (STC), a typical bile salt, was found to be a promising safe preparation for improving the oral absorption of poorly water-soluble and/or poorly absorbable drug in our previous studies utilizing rats and dogs. To clarify the mechanisms behind the synergistic enhancement effect of the polyamine and bile salt, the transport of rebamipide, which is classified into Biopharmaceutics Classification System Class IV, was investigated in Caco-2 cell monolayers. The synergistic enhancement of rebamipide transport by SPM and STC was certainly observed in Caco-2 cells as well, while the separate use of either SPM or STC did not significantly improve the transport of rebamipide. The combinatorial use of SPM and STC significantly decreased the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers, suggesting that the opening of paracellular pathway. On the other hand, it was also confirmed that the decrease in TEER was transient and reversible after removal of SPM and STC and that cell viability was maintained. Voltage-clamp study clearly showed that their combinatorial use improved rebamipide transport via both paracellular and transcellular pathways, and that the contribution of transcellular route could be larger than paracellular route.

The purpose of the present study was to investigate oral bioavailability of an immediate release tablet containing wet-milled crystals of a poorly water-soluble drug, cilostazol, and to establish in vitro-in vivo correlation. Sub-micron sized cilostazol (median diameter: 0.26 microm) was successfully prepared using a beads-mill in water in the presence of a hydrophilic polymer and an anionic surfactant. The milled suspension was solidified with a sugar alcohol as a water-soluble carrier by spray-drying method. The co-precipitate was compressed into an immediate release tablet with common excipients. Oral bioavailability of the wet-milled cilostazol tablet in male beagle dogs was 13-fold higher than the hammer-milled commercial tablet in fasted condition. Food did not increase the oral bioavailability of the wet-milled tablet, while 4-fold increase was found for the commercial tablet. Irrespective to the bioavailability enhancement, in vitro dissolution rate of the wet-milled tablet was even slower than the commercial tablet by the compendial method (USP Apparatus 2). On the other hand, a good correlation was found between the dissolution profiles obtained by a flow-through cell method (USP Apparatus 4, closed-loop system without outlet filter) using a large volume of water and sodium lauryl sulfate (SLS) solution at the concentration lower than the critical micellar concentration (cmc) as dissolution media corresponding to the fasted and fed conditions, respectively.

The synergetic improving effect of bile acids with spermine (SPM), a major polyamine, on the absorption of rebamipide, a poorly soluble and poorly absorbable drug (BCS Class IV), was evaluated in rats and beagle dogs. Although the absorption of rebamipide was improved by the addition of polyamines alone in normal rats, it was not improved in bile duct ligated (BDL) rats. The combinatorial use of sodium taurocholate (STC), a bile acid, with SPM improved the absorption of rebamipide even in BDL rats. In the beagle dogs, the oral administration of SPM alone did not enhance the absorption of rebamipide, but the combinatorial use of STC with SPM improved the absorption as well as in the BDL rats. These results indicate that bile acids are indispensable for the novel formulation containing SPM to improve the absorption of rebamipide after oral administration.

The purpose of the present study was to investigate the effects of particle size on the dissolution and oral absorption of cilostazol. Three types of suspensions having different particle size distributions were prepared of the hammer-milled, the jet-milled cilostazol crystals and the NanoCrystal spray-dried powder of cilostazol. In vitro dissolution rate of cilostazol was significantly increased by reducing the particle size. The dissolution curves of the cilostazol suspensions were in good agreement with the simulation based on the Noyes-Whitney equation. The bioavailability of cilostazol after oral administration to dogs was increased with reducing the particle size. While positive food effect on the absorption was observed for the suspensions made of the hammer-milled and the jet-milled crystals, no significant food effect was found for the suspension made of the NanoCrystal cilostazol spray-dried powder. These results could be qualitatively predicted from the in vitro dissolution data using the bio-relevant media, FaSSIF and FeSSIF. In conclusion, the NanoCrystal technology is found to be efficient to improve the oral bioavailability of cilostazol and to avoid the food effect on the absorption.

In order to develop a novel oral formulation that can safely improve the intestinal absorption of poorly absorbable drugs, polyamines such as spermine (SPM) and spermidine (SPD) was examined as an absorption enhancing adjuvant in rats. The absorption of rebamipide, classified into BCS Class IV, from colon was significantly improved by SPM or SPD, and the enhancing ability of SPM was larger than that of SPD. As a possible mixing and/or interaction of polyamines with bile acids were expected, the combinatorial use of sodium taurocholate (STC) with polyamines was also examined. The absorption of rebamipide was drastically improved by the combinatorial use of SPM or SPD with STC. As STC itself did not enhance the absorption of rebamipide so much, it was considered that polyamines and STC had a synergistic enhancing effect. In-vivo oral absorption study was also performed to investigate the effectiveness and safety of polyamines and their combinatorial use with STC in rats. Although the enhancing effect slightly attenuated comparing with the in-situ loop study, the absorption of rebamipide was significantly improved and the combinatorial use of 10 mM SPM with 25 mM STC showed the largest enhancing effect. Histopathological studies clearly showed that any significant change in stomach and duodenum was not caused by SPM (10 mM), SPD (10 mM) or their combinatorial use with STC (25 mM) at 1.5 or 8.0 h after oral administration. Taken all together, polyamines, especially SPM, and its combinatorial use with STC could improve the absorption of poorly absorbable drugs without any significant changes in gastrointestinal tract after oral administration in rats.

We previously reported that the fatty base suppository containing sodium laurate (C12) and taurine (Tau) (C12-Tau suppository) could enhance the colonic absorption of rebamipide, a poorly water-soluble and poorly absorbable drug, without any serious mucosal damages in rats. In the preset study, in order to make C12-Tau suppositories available for practical use, the scaling-up studies of animal and formulation size were performed, compared with the suppositories containing sodium caprate (C10) (C10 suppository) at the same amounts as those contained in the commercial products. Twenty-mg C12 improved the dissolution of rebamipide from suppository remarkably and the addition of 30-mg Tau only slightly decreased the dissolution rate. The absorption of rebamipide from rabbit rectum was more markedly improved by suppositories containing C12 than C10 suppositories. Although Tau tended to attenuate the absorption-enhancing effect of C12, several C12-Tau suppositories kept high bioavailability values, which were much higher than control. Histopathological studies showed that Tau exerted the cytoprotective action and that C12-Tau suppositories were better than C10 suppositories in safety. Considering the balance between efficacy and safety, the suppository containing 10- or 20-mg C12 with 30-mg Tau is better than C10 suppositories as commercial products and could be promising for practical use in human.

Effects of chitosan oligomers on pulmonary absorption of interferon-alpha (IFN) were examined by means of an in vivo pulmonary absorption experiment. Chitosan oligomers used in this study were chitosan dimer, tetramer, hexamer, and water-soluble (WS) chitosan. A significant increase in serum IFN concentrations was observed after intratracheal administration of IFN with these oligomers. Of these chitosan oligomers, 0.5% w/v chitosan hexamer appeared to be more effective in enhancing the pulmonary absorption of IFN than other oligomers at the same concentration, and the AUC value of IFN with chitosan hexamer increased 2.6-fold as compared with the control. On the other hand, chitosan polymers, which have relatively high molecular weights (22-96 kDa), were not effective in enhancing the pulmonary absorption of IFN due to their low solubility in water. Additionally, the effect of different concentrations (0.1%-1% w/v) of chitosan hexamer on the pulmonary absorption of IFN was studied. Of these different concentrations of chitosan hexamers, the highest AUC value of IFN was obtained in the presence of 0.5% w/v chitosan hexamer. Furthermore, chitosan oligomers did not cause any membrane damage to the rat pulmonary tissues, as determined by leakage of protein and lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid. Therefore, these findings indicated that the use of chitosan oligomers would be a promising approach for improving of the pulmonary absorption of biologically active peptides including IFN.

Effects of various viscous vehicles on pulmonary absorption of antiasthmatic drugs were examined by an in situ pulmonary absorption experiment. Theophylline and fluticasone propionate were used as antiasthmatic drugs. The serum concentration-time profile of theophylline without viscous vehicles was similar to that following the intravenous injection, indicating that pulmonary absorption of theophylline was rapid and absolute. The serum concentration of theophylline was not controlled in the presence of 5% gelatin or 2% sodium alginate. However, 1% iota-carrageenan could control and regulate the serum concentration of theophylline. In the pharmacokinetic analysis, the C(max) values of theophylline significantly decreased, and its T(max) values increased in the presence of 1% and 2% iota-carrageenan, 1% kappa-carrageenan, and 2% sodium alginate compared with the control. The MRT and MAT values of theophylline with 1% iota-carrageenan were significantly higher than those without viscous vehicles. The local concentration of theophylline in the lung at 1h after intratracheal administration increased five-fold with 1% iota-carrageenan compared with the control. On the other hand, the pulmonary absorption of fluticasone propionate was controlled and regulated in the presence of 0.5% kappa-carrageenan. Additionally, the pulmonary inflammation after the exposure of carrageenans administered to the lung was evaluated in rats. Iota- and kappa-carrageenans did not cause local serious damage and inflammation to the pulmonary tissue. Therefore, these findings indicated that the carrageenans were effective to regulate the absorption rate of antiasthmatic drugs including theophylline and fluticasone propionate.

To develop the safe formulation that can safely improve bioavailability of poorly absorbable drugs and that is practically available, we prepared the suppositories of rebamipide, a poorly soluble and poorly absorbable antiulcer drug, by employing the combinatorial use of sodium laurate (C12), an absorption enhancer, with taurine (Tau) or L-glutamine (L-Gln), an adjuvant exerting the cytoprotective action. Although the dissolution of rebamipide from fatty base (FB) suppository prepared using Witepsol H-15 was very slow, it was remarkably improved by the addition of C12 and L-Gln or Tau into the suppository. On the other hand, the dissolution of rebamipide from water-soluble base (WB) suppository prepared using polyethylene glycol was very rapid and the addition of adjuvants did not influence its dissolution so much. Rectal absorption of rebamipide examined in rats was remarkably improved by FB suppository containing C12 or both C12 and Tau, while the enhancing effect of C12 was relatively small in the case of WB suppositories. Biochemical and histopathological studies have confirmed that FB suppository containing both C12 and Tau or L-Gln did not cause any serious local damage, while FB suppository containing C12 only caused the erosion and shrinkage for a lot of rectal epithelial cells. In conclusion, FB suppository employing the combinatorial use of C12 with Tau could be a promising formulation that is effective and safe enough for poorly absorbable drugs to be practically administered.

An improved formulation of the enteral nutrient Aminoleban EN (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan), has been commercially available since Spring 2004. Like the previous formulation, the improved product contains branched-chain amino acids (BCAAs) L-isoleucine (L-Ile), L-leucine (L-Leu), and L-valine (L-Val), but the average particle size of these amino acids has been increased to 180 to 250 microm in the improved formulation, compared with 40 to 90 microm in the old product. The improved formulation has a significantly lower bitterness intensity score than the older formulation, as evaluated both in human gustatory tests and using the artificial taste sensor. We propose that this improved taste masking is due to the larger particle size of the BCAA crystals, due to which their release rates are reduced. The addition of improved flavours has also helped to reduce the bitterness of the improved Aminoleban EN formulation significantly. Analysis of the taste sensor data suggests that the sourness and sweetness of the added flavours were critical in diminishing the bitterness of Aminoleban EN.

An asthmatic patient with corticosteroid treatment for 45 years presented with slowly progressive limb muscle atrophy. His muscle symptoms were involved in four limbs and tongue, and deep tendon reflexes were exaggerated. Biopsied muscle pathology indicated the presence of neurogenic muscular atrophy in combination with corticosteroid myopathy. Furthermore, 8-hydroxy-deoxyguanosine (8-OH-dG) was prominently increased in mitochondrial and nuclear DNA. An aerobic exercise test demonstrated remarkable serum lactate elevation, which was attenuated by the administration of coenzyme Q10. These findings are consistent with the assumption that long-term corticosteroid administration potentially induces not only myopathy but also motor neuron involvement as in mitochondrial diseases.

We previously reported that the combinatorial use of sodium laurate (C12) with several amino acids such as taurine (Tau) and L-glutamine (L-Gln) enhanced the colonic absorption of phenol red with attenuating the local toxicity caused by C12. However, even these amino acids could not protect epithelial cells from being damaged if the mucosal damage got worse to the coagulation necrosis by an excessive dose of C12. Comparing C12 with sodium caprate (C10), used in drug products marketed, 100 micromol C10 was needed to exert the similar absorption-enhancement of rebamipide, a poorly absorbable antiulcer drug, to that by 10 micromol C12, and 100 micromol C10 was obviously more toxic to the mucosa than 10 micromol C12. The combinatorial use of C12 with Tau or L-Gln enhanced the colonic absorption of rebamipide four to nine times larger in AUC than the control. Histopathologic studies clearly showed that Tau and L-Gln exerted the cytoprotective action on epithelial cells suffering from slight damages such as shrinkage and exfoliation, more articulately at 6 h than at 1.5 h after dosing. In conclusion, the combinatorial use of C12 with Tau or L-Gln could lead to a novel formulation improving the bioavailability of poorly absorbable drugs without any serious local damages., (Copyright 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92: 911-921, 2003)

Two patients with long-term corticosteroid administration sporadically developed limb muscle wasting followed by ophthalmoplegia, and the skeletal muscle pathology revealed ragged-red fibers (RRFs) with abnormal mitochondria, in addition to the findings of corticosteroid myopathy. The oculoskeletal symptoms of the present cases resemble those of chronic progressive external ophthalmoplegia, a type of mitochondrial disease. The ocular muscles have more RRFs than limb muscles, and large multiple deletions of mitochondrial DNA was detected in ocular and limb muscles of the two patients by PCR but not by Southern blotting. Immunohistochemistry demonstrated that 8-hydroxy-deoxyguanosine (8-OH-dG) and 4-hydroxy-2-nonenal were intensely stained in skeletal muscles of these patients particularly in RRFs. High-performance liquid chromatography with electrochemical detection analysis revealed an increase in 8-OH-dG from mitochondrial DNA. These findings may suggest that long-term corticosteroid administration potentially induces oxidative stress-mediated mitochondrial damage, resulting in the development of the oculoskeletal symptoms in some patients.

Corticosteroid myopathy is a major clinical problem in patients undergoing chronic corticosteroid treatment and shows insidious and progressive muscle atrophy in proximal limbs. Although several mechanisms underlying the pathophysiology of muscle injury have been postulated, precise pathogenesis is still not clear. We evaluated the mitochondrial functions in patients receiving corticosteroids compared with those in healthy controls or patients not receiving corticosteroids. The serum levels and total production of lactate were investigated by an aerobic exercise test using a bicycle ergometer. Mitochondrial respiratory activities and oxidative damage in biopsied skeletal muscles were also studied. The results of aerobic exercise tests revealed a significant overproduction of lactate in patients treated with corticosteroids ( p < 0.005), which was positively correlated with total corticosteroid doses administered ( p < 0.0001). In these patients, mitochondrial enzyme activity in complex I was significantly decreased ( p < 0.05) and oxidative damage of biopsied skeletal muscle was remarkable both in mitochondrial and nuclear DNAs ( p < 0.001). The results suggest that chronic corticosteroid administration induces mitochondrial dysfunction and oxidative damage in skeletal muscles, which may be the pathogenesis, at least in part, of corticosteroid-induced myopathy.

The metabolism of 1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045), a new potent biguanide antiseptic, was investigated using rat and dog liver preparations to elucidate the mechanism of OPB-2045 metabolite formation, in which the octyl side chain is reduced to four, five, or six carbon atoms. Chemical structures of metabolites were characterized by 1H NMR, fast atom bombardment/mass spectrometry, and liquid chromatography/electrospray ionization-tandem mass spectrometry. Three main metabolites were observed during incubation of OPB-2045 with rat liver S9: 2-octanol (M-1), 3-octanol (M-2), and 4-octanol (M-3). In the incubation of OPB-2045 with dog liver S9, eight metabolites were observed, seven of which being M-1, M-2, M-3, 2-octanone (M-4), threo-2,3-octandiol (M-5), erythro-2,3-octandiol (M-6), and 1,2-octandiol (M-7). M-5 and M-6 were further biotransformed to a ketol derivative and C-C bond cleavage metabolite (hexanoic acid derivative), an in vivo end product, in the incubation with dog liver microsomes. The reactions required NADPH as a cofactor and were significantly inhibited by the various inhibitors of cytochrome P450 (i.e., CO, n-octylamine, SKF 525-A, metyrapone, and alpha-naphthoflavone). The results indicate that the degraded products of OPB-2045 are produced by C-C bond cleavage after monohydroxylation, dihydroxylation, and ketol formation at the site of the octyl side chain with possible involvement of cytochrome P450 systems. This aliphatic C-C bond cleavage by sequential oxidative reactions may play an important role in the metabolism of other drugs or endogenous compounds that possess aliphatic chains.

1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045) is a new biguanide antimicrobial agent currently in clinical use as a topical bactericidal antiseptic. A method combining high-performance liquid chromatography (HPLC) with electrospray ionization (triple and quadruple stage) tandem mass spectrometry (ESI-MS/MS) was developed to quantify OPB-2045 in human serum. Solid phase extraction was performed on 0.2 ml of sample to ensure a high level of sensitivity before HPLC-ESI-MS/MS analysis. The limit of quantitation for the method was set at 0.05 ng/ml. Intra-assay and interassay precision were less than 13.7%, with a deviation from the expected value of no greater than 10.5% at a concentration range of 0.05 ng/ml to 5 ng/ml. Decomposition of OPB-2045 in human serum did not occur after storage for 15 months at -20 degrees C, even after three repetitions of freezing and thawing. Application of this method was demonstrated in a pharmacokinetic study of OPB-2045 in healthy patient subjects after a single topical application of 5 g/L preparation of its liquid formulation.

The metabolism of toborinone, (+/-)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2(1H)-quin - olinone, a novel inotropic agent, was studied in rats and dogs after intravenous administration. Chemical structures of the 13 metabolites were characterized by direct-probe FAB/MS and field desorption/MS, LC/FAB/MS, and various NMR measurements. After intravenous dosing of 10 mg/kg [14C]toborinone, fecal and urinary recoveries of the 14C dose were approximately 70% and 26-30%, respectively, in both rats and dogs. The predominant component of radioactivity was the unchanged toborinone in every biological specimen in rats and dogs. Although unchanged toborinone was predominantly observed, toborinone underwent extensive conjugations with glucuronic acid, sulfate, and glutathione, either directly or following phase I reaction. Metabolites resulting from oxidative N-C cleavage were minor both in number and in quantity in every biological specimen in rats and dogs. In rats, toborinone underwent O-demethylation to form M-7 and successive phase it reaction to yield the glucuronide M-1 and the sulfoconjugate M-2, and deconjugation to yield M-7, which was a primary metabolite accounted for 35.67% of the radioactivity excreted in the feces by 48 hr. Conjugates M-1 and M-2 were the major metabolites in rat plasma. In dogs, toborinone was metabolized via mercapturic acid pathway to yield the primary metabolites, cysteine conjugates M-10 and M-11 that accounted for 19.10% and 6.70% of the radioactivity excreted in the feces by 48 hr and that were detected species specifically in dogs. The glutathione conjugate M-13, which was isolated from in vitro incubations using dog liver, led us to consider a possible mercapturic acid pathway from the parent compound to M-10. Metabolites in dog plasma and those in urine in both rats and dogs were minor in quantity. The metabolic pathways of toborinone in rats and dogs are proposed herein.

The forms of cytochrome P450 involved in the stereoselective S-oxidation of flosequinan [(+/-)-7-fluoro-1-methyl-3-methylsulfinyl-4-quinolone] were investigated in vitro using liver microsomes from rats and humans. Rat liver microsomes supplemented with NADPH catalyzed the four different S-oxidations, which were from flosequinan sulfide (FS; 7-fluoro-1-methyl-3-methylthio-4-quinolone) to R(+)- and S(-)-flosequinan (R-FSO and S-FSO, respectively) and from R-FSO and S-FSO to flosequinan sulfone (FSO2; 7-fluoro-1-methyl-3-methylsulfonyl-4-quinolone). The activities of all the S-oxidases in liver microsomes from male rats were higher than those from female rats. The activities of the S-oxidases measured at a high substrate concentration (1 mM) were induced by treatment of rats with phenobarbital and dexamethasone. Treatment of rats with 3-methylcholanthrene also induced the activities, but only at a low substrate concentration (50 microM), except for the S-oxidase catalyzing the reaction from FS to R-FSO. Enzymes induced by clofibrate and ethanol were not involved in the oxidations at a low substrate concentration. The activities of S-oxidases were correlated with the contents of cytochrome P450 (CYP)3A enzymes. Anti-CYP3A2 antisera inhibited the activities of the S-oxidases catalyzing the reactions from FS to R-FSO (40%) and to S-FSO (60%) at the high substrate concentration and inhibited the activities of the S-oxidases, thus catalyzing reactions from R-FSO and S-FSO to FSO2 (70%) at both high and low substrate concentrations. These results suggest that CYP3A is the major enzyme involved in all S-oxidation pathways in flosequinan metabolism in rats. On the other hand, except for the S-oxidation of FS to R-FSO, the rates of the other three S-oxidations by liver microsomes from 30 individual humans correlated highly with each other, suggesting that the same enzyme would be involved in the three S-oxidations. Anti-CYP3A2 antisera inhibited the activities of all the S-oxidases in human liver microsomes ranging from 40 to 80%, suggesting that CYP3A is also involved in all of the S-oxidations of flosequinan in humans.

To elucidate the penetrability of carteolol, a beta-adrenoceptor antagonist (beta-blocker) into the brain of rats, intracerebral and serum concentrations of the compound were determined in male rats receiving single or repetitive oral administration of carteolol hydrochloride at 30 mg/kg. The time-course of the intracerebral concentration of carteolol following single IV administration of the compound at 10 and 30 mg/kg was also studied in male rats. A high-performance liquid chromatography method was used to determine the intracerebral and serum concentrations. Following single oral dosing, the intracerebral concentration of carteolol reached a maximum of 0.074 microgram/g at 2 h postdosing and declined with a half-life of 3.7 h, and the Cmax and AUC of carteolol in the brain were 12.5% and 19.8% of those in serum. The intracerebral and serum concentrations of carteolol were determined in male rats receiving repetitive oral dosing of the compound once daily for 7 days. The concentration of carteolol in the brain and serum at 1 h postdosing varied within a range of 0.059-0.091 microgram/g and 0.321-0.443 microgram/ml, respectively, throughout the dosing period, showing no changes in the penetrability of the compound into the brain due to repeated dosing. The concentration of carteolol in the brain and serum increased in a dose-dependent manner in rats receiving a single IV administration of the compound. The elimination half-life of carteolol in the serum and brain was 0.6-0.8 h and 1.3-1.7 h, respectively, in rats following single IV dosing of the compound. The half-life in the brain was about twice as long as that in the serum. The brain to serum concentration ratio was 0.306:0.499. From the above results, it was concluded that carteolol is distributed from the circulation to the brain with low penetrability.

A simple and rapid quantitative method for 13C-labelled urea ([13C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [13C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [13C]urea from intrinsic urea present at high concentration in serum. In addition, a 13C nuclear magnetic resonance spectroscopic quantitative method for [13C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [13C]urea in human serum and urine is also presented.

BOF-A2 (emitefur: 3-(3-[6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl]benzoyl)-1-ethoxy- methyl-5- fluorouracil), a novel 5-FU (5-fluorouracil)-derived drug, was co-administered with other conventional 5-FU-derived drugs or BV-araU [sorivudine: 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyluracil)] for 8 consecutive days to rats. BOF-A2 (6 or 8 mg/kg, p.o.) co-administered with other 5-FU-derived drugs elevated the plasma 5-FU concentration 3- to 23.3-fold and decreased the peripheral white blood cell (WBC). The percentage decreases of WBC by 5-FU (4 mg/kg, i.p.), UFT (16 mg/kg, p.o.), tegafur (FT; 16 mg/kg, p.o.), carmofur (HCFU; 15 mg/kg, p.o.), doxifluridine (5'-DFUR; 16 mg/kg, p.o.) and flucytosine (200 mg/kg, p.o.) were 25.7%, 31.9%, 70.3%, 32.0%, 58.6% and 30.0%, respectively, compared with each drug alone. On the other hand, these phenomena did not occur with BV-araU. These findings can be attributed to the fact that the inhibitory activity of CNDP (3-cyano-2,6-dihydroxypyridine) for 5-FU degradation (IC50: 6.3 x 10(-9) M) is potent and 6000 times greater than that of BVU [(E)-5-(2-bromovinyl) uracil], another inhibitor of 5-FU degradation.

To investigate the distribution and elimination of carteolol in pigmented rabbits, 14C-carteolol eye drops were instilled singly and repeatedly. After single ocular instillation, the radioactivity in the iris and ciliary body reached maximum levels at 24 h. The elimination rate of pigmented tissues decreased at a half-life of approximately 15 days. The concentration of radioactivity in pigmented tissues increased markedly by repeating the ocular instillation and reached a maximum after the 80th repeated instillation. The concentration of radioactivity at 1 h after 80th instillation was 63.7 times that in the iris, 61.1 times that in the ciliary body and 17.2 times that in the retina & choroid after single instillation. No accumulation of radioactivity was found in other ocular tissues.

Rat hepatic flavin-containing monooxygenase (FMO) 1A1 expressed in yeast catalysed the S-oxidation of flosequinan sulfide (7-fluoro-1-methyl-3-methylthio-4-quinolone) to R(+)-flosequinan (sulfoxide form, R(+)-7-fluoro-1-methyl-3-methylsulfinyl-4-quinolone) but not to S(-)-flosequinan, and did not catalyse the oxidation of R(+)- and S(-)-flosequinan to flosequinan sulfone. The Km and Vmax for the stereoselective S-oxidation were 33 microM and 6.2 nmol per min per mg of microsomal protein, respectively. The S-oxidation was inhibited by 1-(1-naphthyl)-2-thiourea and thiobenzamide. n-Octylamine activated the S-oxidation with little change in stereoselectivity. The ability of the recombinant yeast to produce R(+)-flosequinan from flosequinan sulfide could be maintained for at least 2 days and exemplifies the value of a recombinant yeast expressing FMO as a stereoselective bioreactor.

A high-performance liquid chromatographic assay method for the quantification of OPC-18790 enantiomers in human plasma and urine is described. A human plasma or urine was extracted with organic solvent under alkaline conditions following the addition of internal standard. The enantiomers and internal standard were then derivatized by reaction with the chiral reagent GITC (2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate), followed by octadecylsilica chromatographic separation of the diastereomeric products. The mobile phase consisted of acetonitrile-water (41:59). The fluorescence of the eluate was monitored at 355/405 nm. The lowest quantification limit of each enantiomer was 10 ng/ml in plasma and 0.1 micrograms/ml in urine. Both intra- and inter-day coefficients of variation were below 10%. The assay is sensitive, specific and applicable for stereoselective pharmacokinetic studies in human.

The pharmacokinetics, safety, and pharmacologic effects of OPC-21268, a nonpeptide orally active vasopressin V1 receptor antagonist, have been investigated in 33 healthy subjects. First, 24 subjects were randomly divided into 3 groups of 8, 6 of whom were given 2 ascending single oral doses out of 6 (10, 50, 150, 300, 450, and 600 mg) of OPC-21268 after an overnight fast. The remaining two subjects in each group received placebo as control at each dosing. Additionally, after this procedure, the 6 subjects who received 50-mg single doses were given the same dose in a nonfasting condition. After the single-dose study was completed and the safety and tolerability were ascertained, the remaining 9 subjects, including 3 controls, were given 300 mg of the drug 3 times daily for 7 days (days 3-9) and were given single 100-mg oral doses before (day 1) and after (day 10) this repeated-dose study. OPC-21268 plasma concentrations declined in a monoexponential or biexponential pattern after reaching the maximum plasma concentrations (Cmax). The mean (+/- standard error of the mean) plasma half-life (t1/2) of the alpha phase ranged from 1.31 +/- 0.11 to 1.78 +/- 0.15 hours, and the mean t1/2 of the beta phase ranged from 4.31 +/- 0.28 to 6.28 +/- 0.59 hours. The area under the concentration (AUC0-infinity) and Cmax were proportional to the dose (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)