An iminium ion metabolite hampers the production of the pharmacologically active metabolite of a multikinase inhibitor KW-2449 in primates: irreversible inhibition of aldehyde oxidase and covalent binding with endogenous proteins.

We previously reported that KW-2449, (E)-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}piperazine, a novel multikinase inhibitor developed for the treatment of leukemia patients, was oxidized to an iminium ion intermediate by monoamine oxidase B (MAO-B) and then converted to its oxo-piperazine form (M1) by aldehyde oxidase (AO). However, it was found that the significant decrease in the pharmacologically active metabolite M1 following repeated administration of KW-2449 in primates might hamper the effectiveness of the drug. The mechanism underlying this phenomenon was investigated and it was found that the AO activity was inhibited in a time-dependent manner in vitro under the co-incubation of KW-2449 and MAO-B, while neither KW-2449 nor M1 strongly inhibited MAO-B or AO activity. These results clearly suggest that MAO-B catalysed iminium ion metabolite inhibited AO, prompting us to investigate whether or not the iminium ion metabolite covalently binds to endogenous proteins, as has been reported with other reactive metabolites as a cause for idiosyncratic toxicity. The association of the radioactivity derived from 14 C-KW-2449 with endogenous proteins both in vivo and in vitro was confirmed and it was verified that this covalent binding was inhibited by the addition of sodium cyanide, an iminium ion-trapping reagent, and pargyline, a MAO-B inhibitor. These findings strongly suggest that the iminium ion metabolite of KW-2449 is highly reactive in inhibiting AO irreversibly and binding to endogenous macromolecules covalently.

Quantitative investigation of the brain-to-cerebrospinal fluid unbound drug concentration ratio under steady-state conditions in rats using a pharmacokinetic model and scaling factors for active efflux transporters.

A pharmacokinetic model was constructed to explain the difference in brain- and cerebrospinal fluid (CSF)-to-plasma and brain-to-CSF unbound drug concentration ratios (Kp,uu,brain, Kp,uu,CSF, and Kp,uu,CSF/brain, respectively) of drugs under steady-state conditions in rats. The passive permeability across the blood-brain barrier (BBB), PS1, was predicted by two methods using log(D/molecular weight(0.5)) for PS1(1) or the partition coefficient in octanol/water at pH 7.4 (LogD), topologic van der Waals polar surface area, and van der Waals surface area of the basic atoms for PS1(2). The coefficients of each parameter were determined using previously reported in situ rat BBB permeability. Active transport of drugs by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) measured in P-gp- and Bcrp-overexpressing cells was extrapolated to in vivo by introducing scaling factors. Brain- and CSF-to-plasma unbound concentration ratios (Kp,uu,brain and Kp,uu,CSF, respectively) of 19 compounds, including P-gp and Bcrp substrates (daidzein, dantrolene, flavopiridol, genistein, loperamide, quinidine, and verapamil), were simultaneously fitted to the equations in a three-compartment model comprising blood, brain, and CSF compartments. The calculated Kp,uu,brain and Kp,uu,CSF of 17 compounds were within a factor of three of experimental values. Kp,uu,CSF values of genistein and loperamide were outliers of the prediction, and Kp,uu,brain of dantrolene also became an outlier when PS1(2) was used. Kp,uu,CSF/brain of the 19 compounds was within a factor of three of experimental values. In conclusion, the Kp,uu,CSF/brain of drugs, including P-gp and Bcrp substrates, could be successfully explained by a kinetic model using scaling factors combined with in vitro evaluation of P-gp and Bcrp activities.

[Motivation for change and recidivism among prison inmates for drug-related offences].

Programs for improving motivation to recover drug dependence were conducted in penal institutes in Japan. This study examined the effects of these programs, in order to increase their efficacy. Furthermore, relationship between increased motivation and prevention of recidivism was examined. The following programs had been conducted in penal institutions: (1) speed learning program using audio-visual aids, (2) meeting program (meet a experienced person in recover), in cooperation with DARC (Drug Addiction Rehabilitation Center), and (3) relapse prevention program. Participants were female inmates (N = 94) that had taken part in one of the programs. In order to examine the relationship between motivation for change and recidivism, the motivation for change score was assessed before and after the treatment program. Furthermore, a followed-up was conducted to assess recidivism after release. Results indicated that all the programs resulted in improvements in motivation for change. However, motivation for change resulting from none of the programs had any significant effect on recidivism. The objectives of the treatment programs were reached as expected, since motivation for change improved after the treatment However, it is suggested that to prevent recidivism, more effective treatment methods for improving motivation to change are required.

[Relationship between recidivism and self-efficacy among inmates of drug-related offences].

UNLABELLED: Aims. Treatment programme for drug-related offenders in Japanese prisons is aimed at acquiring coping skills for preventing recidivism and improving self-efficacy for preventing recidivism. In this study, we examined if improvement of self-efficacy actually contributes to the prevention of recidivism. Methods. In order to measure the relationship between recidivism and self-efficacy, we examined the degree of self-efficacy at the beginning and end of the programme, and we monitored 144 females upon their release from prison. The women were all incarcerated for drug-related offences and had been participated in the self-efficacy programme. Results. In this study, we obtained the following results: 1) Change in self-efficacy through the programme was not statistically significant; 2) The recidivism rate of inmates with high self-efficacy at the end of the program was low; 3) The amount of change in self-efficacy through the programme did not affect the recidivism rate. CONCLUSION: Enhancing self-efficacy through the programme is effective in preventing recidivism. It is necessary to improve the programme.

Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.

Species differences in the pharmacokinetics of KW-7158 [(2S)-(+)-3,3,3-Trifluoro-2-hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydrothieno[3,2-c][1]benzothiepin-9-yl)propanamide]: formation of hydrolyzed metabolite in human and animals.

Species differences in the pharmacokinetics of KW-7158 [(2S)-(+)-3,3,3-Trifluoro-2-hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydrothieno[3,2-c][1]benzothiepin-9-yl)propanamide] were studied in in vivo and in vitro experiments. The exposure ratio of hydrolyzed metabolite (M2, primary metabolite in human plasma)/KW-7158 was higher than the ratio of thiophen-to-furan converted metabolite (M1)/KW-7158 in human subjects after oral administration, but the mouse, rat and dog studies gave opposite results. M2 was produced in the highest amount by the 9000g supernatant of small intestine, followed by that of liver and kidney in human subjects. After correction for protein contents, the results obtained suggested that the small intestine plays a major role in the metabolism to M2 for the first pass effect after oral administration of KW-7158. The formation of M2 was independent of the presence of NADPH and was inhibited by various esterase inhibitors. These observations suggested that the predominant enzymes or isozymes involved in the formation of M2 are esterases, which differ between humans and animals. Such differences may be one of the reasons for the species differences in the pharmacokinetics of KW-7158 between humans and animals.

Quantitative evaluation of the impact of active efflux by p-glycoprotein and breast cancer resistance protein at the blood-brain barrier on the predictability of the unbound concentrations of drugs in the brain using cerebrospinal fluid concentration as a surrogate.

This study investigated the impact of the active efflux mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) at the blood-brain barrier (BBB) on the predictability of the unbound brain concentration (C(u,brain)) by the concentration in the cerebrospinal fluid (CSF) (C(u,CSF)) in rats. C(u,brain) is obtained as the product of the total brain concentration and unbound fraction in the brain (f(u,brain)) determined in vitro in brain slices. Twenty-five compounds, including P-gp and/or Bcrp substrates, were given a constant intravenous infusion, and their plasma, brain, and CSF concentrations were determined. P-gp and/or Bcrp substrates, such as verapamil, loperamide, flavopiridol, genistein, quinidine, dantrolene, daidzein, cimetidine, and pefloxacin, showed a higher CSF-to-brain unbound concentration ratio (K(p,uu,CSF/brain)) compared with non-P-gp and non-Bcrp substrates. K(p,uu,CSF/brain) values of P-gp-specific (quinidine and verapamil) and Bcrp-specific (daidzein and genistein) substrates were significantly decreased in Mdr1a/1b(-/-) and Bcrp(-/-) mice, respectively. Furthermore, consistent with the contribution of P-gp and Bcrp to the net efflux at the BBB, K(p,uu,CSF/brain) values of the common substrates (flavopiridol and erlotinib) were markedly decreased in Mdr1a/1b(-/-)/Bcrp(-/-) mice, but only moderately or weakly in Mdr1a/1b(-/-) mice and negligibly in Bcrp(-/-) mice. In conclusion, predictability of C(u,brain) by C(u,CSF) decreases along with the net transport activities by P-gp and Bcrp at the BBB. C(u,CSF) of non-P-gp and non-Bcrp substrates can be a reliable surrogate of C(u,brain) for lipophilic compounds.

Kinetic analysis of the cooperation of P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp/Abcg2) in limiting the brain and testis penetration of erlotinib, flavopiridol, and mitoxantrone.

A synergistic effect of P-glycoprotein (P-gp)/Abcb1a and breast cancer resistance protein (Bcrp)/Abcg2 was reported to limit the brain penetration of their common substrates. This study investigated this based on pharmacokinetics using Mdr1a/1b(-/-), Bcrp(-/-), and Mdr1a/1b(-/-)/Bcrp(-/-) mice. Comparison of the brain- and testis-to-plasma ratios (C(brain)/C(plasma) and C(testis)/C(plasma), respectively) of the reference compounds quinidine and dantrolene for P-gp and Bcrp, respectively, indicates that impairment of either P-gp and Bcrp did not cause any change in the efflux activities of Bcrp or P-gp, respectively, at both the blood-brain barrier (BBB) and blood-testis barrier (BTB). The C(brain)/C(plasma) and C(testis)/C(plasma) of the common substrates erlotinib, flavopiridol, and mitoxantrone were markedly increased in Mdr1a/1b(-/-)/Bcrp(-/-) mice even compared with Mdr1a/1b(-/-) and Bcrp(-/-) mice. Efflux activities by P-gp and Bcrp relative to passive diffusion at the BBB and BTB were separately evaluated based on the C(brain)/C(plasma) and C(testis)/C(plasma) in the knockout strains to the wild-type strain. P-gp made a larger contribution than Bcrp to the net efflux of the common substrates, but Bcrp activities were also significantly larger than passive diffusion. These parameters could reasonably account for the marked increase in C(brain)/C(plasma) and C(testis)/C(plasma) in the Mdr1a/1b(-/-)/Bcrp(-/-) mice. In conclusion, the synergistic effect of P-gp and Bcrp on C(brain)/C(plasma) and C(testis)/C(plasma) can be explained by their contribution to the net efflux at the BBB and BTB without any interaction between P-gp and Bcrp.

Effect of benidipine on simvastatin metabolism in human liver microsomes.

Benidipine, which is a calcium channel blocker that has clinical advantages in the treatment of hypertension, is metabolized by CYP3A4 in humans. The effect of benidipine on the metabolism of simvastatin by human liver microsomes was investigated in order to predict the potential of in vivo drug-drug interactions between benidipine and other substrates of CYP3A4. The results were compared with data generated with azelnidipine, which is also metabolized by CYP3A4. Both benidipine and azelnidipine inhibited simvastatin metabolism in vitro in a concentration-dependent manner. Assuming competitive inhibition, the K(i) values based on the unbound concentrations, were calculated to be 0.846 and 0.0181 microM for benidipine and azelnidipine, respectively. If simvastatin (10 mg) and benidipine (8 mg, the clinically recommended highest dose) were to be administered concomitantly, the ratio of the areas under the concentration-time curves of simvastatin with and without benidipine (AUC((+I))/AUC) was predicted to be 1.01. On the other hand, if simvastatin (10 mg) and azelnidipine (8 mg) were co-administered, the AUC((+I))/AUC for simvastatin was predicted to be 1.72, which is close to the observed value (1.9) in healthy volunteers. These data suggest that benidipine is unlikely to cause a drug interaction by inhibiting CYP3A4 activity in the liver.

Potent and selective inhibitors of PDGF receptor phosphorylation. 2. Synthesis, structure activity relationship, improvement of aqueous solubility, and biological effects of 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives.

4-[4-(N-Substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives such as KN1022 are potent inhibitors of the phosphorylation of platelet derived growth factor receptor (PDGFR). Structure activity relationships in the (thio)urea moiety, the phenyl ring itself, the linker between these two moieties, and the piperazine moiety were investigated. The role of the linker was found to be quite different, where ureas yielded decreasing activity, while thioureas provided increasing activity. Cyanoguanidine as a bioisostere of thiourea and related dicyanovinyl or nitrovinyl groups were not suitable for potent activity. A hydrogen atom on the (thio)urea moiety was essential for activity. Stereochemistry was also important for inhibition of PDGFR phosphorylation. Through the modification of these moieties, benzylthiourea analogues with a small substituent on the 4-position and the 3,4-methylenedioxy group (KN734/CT52923) were found to be optimal for selective and potent activity. Replacement of the phenyl ring by heterocycles improved aqueous solubility without loss of activity and kinase selectivity. Introduction of a methyl group on 5-position of the piperazine ring and replacement by homopiperazine reduced inhibitory activity. An efficient synthetic method was also developed for 2-pyridylurea-containing analogues, via carbonylation of 2-aminopyridine with N,N'-carbonyldiimidazole. A potent analogue, KN734, inhibited smooth muscle cell proliferation and migration induced by platelet derived growth factor-BB (PDGF-BB) and suppressed neointima formation following balloon injury in rat carotid artery by oral administration. Therefore, 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives may be expected to have potential as therapeutic agents for the treatment of restenosis.

Potent and selective inhibitors of platelet-derived growth factor receptor phosphorylation. 3. Replacement of quinazoline moiety and improvement of metabolic polymorphism of 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives.

We have previously reported that a series of 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives were potent and selective inhibitors of platelet-derived growth factor receptor (PDGFR) phosphorylation and demonstrated several biological effects such as suppression of neointima formation following balloon injury in rat carotid artery by oral administration. Here, we investigated structure-activity relationships of the 6,7-dimethoxyquinazolinyl moiety. In regard to 6,7-dimethoxy groups, ethoxy analogues showed potent activity (IC(50) of 16b is 0.04 microM; IC(50) of 17a is 0.01 microM) and further extension of the alkyl group reduced activity. Interestingly, methoxyethoxy (IC(50) of 16j is 0.02 microM; IC(50) of 17h is 0.01 microM) and ethoxyethoxy (IC(50) of 17j is 0.02 micro M) analogues showed the most potent activity, suggesting that the inserted oxygen atom significantly interacts with beta-PDGFR. Among tricyclic quinazoline derivatives, the 2-oxoimidazo[4,5-e]quinazoline derivative 21a showed potent activity (IC(50) = 0.10 microM). Regarding replacements of quinazoline by other heterocyclic rings, pyrazolo[3,4-d]pyrimidine (39a, IC(50) = 0.17 microM) and quinoline (IC(50) of 40a is 0.18 microM; IC(50) of 40b is 0.09 microM) derivatives showed potent activity. Isoquinoline and some pyridopyrimidine derivatives were completely inactive; therefore, 1-aza has an important role. Also 7-aza and 8-aza substitution on the parent quinazoline ring has a detrimental effect on the interaction with beta-PDGFR. We also demonstrated that the substituents on the quinazoline ring possess major consequences for metabolic polymorphism. Although there existed extensive metabolizers and poor metabolizers in Sprague-Dawley rats administrated 6,7-dimethoxyquinazoline derivatives (1b and 1c), 6-(2-methoxy)ethoxy-7-methoxyquinazoline analogue 16k showed no metabolic polymorphism.

Potent and selective inhibitors of platelet-derived growth factor receptor phosphorylation. 1. Synthesis, structure-activity relationship, and biological effects of a new class of quinazoline derivatives.

A new series of 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives were found to show potent and selective inhibition of platelet-dervied growth factor (PDGF) receptor phosphorylation. In this exploration of the structure-activity relationships (SARs) of the prototype inhibitor KN1022, the 4-nitrophenylurea moiety was probed. We found that 4-substitution on the phenyl ring was optimal and the introduction of more than two substituents on the phenyl ring decreased activities. Bulky substituents on the phenyl ring enhanced activities. Thiourea analogues were also prepared, and the SARs were found to be slightly different from those of the urea derivatives. Through this research, we obtained some potent KN1022 derivatives such as 4-(4-methylphenoxy)phenyl (36, IC(50) 0.02 micromol/L), 4-tert-butylphenyl (16, IC(50) 0.03 micromol/L), and 4-phenoxyphenyl (21, IC(50) 0.08 micromol/L) analogues, which had almost a 10-fold increase in activity against KN1022. These potent compounds retained their high selectivity against the PDGF receptor family similar to KN1022. We also observed that these compounds could inhibit the PDGF-BB-induced proliferation of porcine vascular smooth muscle cells without cell toxicity almost at the same IC(50) values observed for PDGF receptor phosphorylation. To evaluate the biological effects in vivo, we selected some analogues on the basis of the measurement of the plasma drug concentration after oral administration to rats. Oral administration of the 4-chlorophenyl (6), 4-bromophenyl (9), or 4-isopropoxyphenyl (20) analogue to Sprague-Dawley rats (30 mg/kg, twice daily) resulted in significant inhibition (24-38%) of neointima formation in the carotid artery of the balloon catheter deendothelialized vessel in the rats. Therefore, 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives, which are potent inhibitors of PDGFR phosphorylation, may be expected to represent a new therapeutic approach for the treatment of various aspects of atherosclerosis and other cellular proliferative disorders.

Involvement of the active metabolites in the inhibitory activity of K579 on rat plasma dipeptidyl peptidase IV.

K579 ((S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile), which is a long-acting and a slow binding dipeptidyl peptidase IV inhibitor, preserved the endogenously secreted active forms of glucagon-like peptide-1, augmented the insulin response and ameliorated the glucose excursion during oral glucose tolerance test in rats. In this study, we measured plasma concentrations of K579 after oral administration to rats. However, K579 was eliminated rapidly from plasma after oral administration to rats. Therefore, we postulated that there are active metabolites of K579 in rat plasma. We investigated the effect of K579 on plasma dipeptidyl peptidase IV activity using bile duct-cannulated rats. The duration of inhibitory action of plasma dipeptidyl peptidase IV after the administration of K579 in bile duct-cannulated rats was shorter than that in sham-operated rats. Moreover, we investigated the effect of bile obtained from K579-treated rat on plasma dipeptidyl peptidase IV activity in normal rats. The bile collected from K579-treated rats exhibited tardive and potent inhibitory activity of normal rat plasma. These results suggest that K579 sustained the duration of inhibitory action of plasma dipeptidyl peptidase IV by the character as a slow-binding inhibitor and, as well, by the presence of metabolites of K579, which exhibit the inhibitory activity of dipeptidyl peptidase IV.

L-ascorbic acid stimulates expression of smooth muscle-specific markers in smooth muscle cells both in vitro and in vivo.

The dedifferentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the progression of atherosclerosis and restenosis after angioplasty. Thus, factors that stimulate smooth muscle cell differentiation should be useful for therapy for these diseases. Previously, we found that l-ascorbic acid (L-Asc) induces the expression of smooth muscle-specific genes in a pluripotent bone marrow stromal cell line, TBR-B. This finding suggests that l-Asc stimulates the differentiation of smooth muscle cells. In the present study, we investigated the effects of l-Asc and its derivatives on the differentiation state of VSMCs in vitro and in vivo. l-Asc and its long-lasting derivatives stimulated the production of smooth muscle-specific myosin heavy chain-1 (SM1) and calponin 1 in a dose-dependent manner in rat cultured VSMCs, and the elevated production of SM1 and calponin 1 was maintained for at least 2 weeks. Moreover, oral administration of 3 g/kg of l-Asc to the balloon-injured rats induced a higher expression of SM1 and calponin 1 in the injured arteries compared with that from administration of the delivery vehicle alone. These data demonstrated new biologic activity, such as the stimulation of VSMC differentiation, of l-Asc and its long-lasting derivatives. In addition, these compounds may serve as useful tools for analysis of the differentiation of VSMCs and for therapy for vascular diseases.