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(Ϫ/Ϫ), Bcrp(Ϫ/Ϫ), and Mdr1a/1b(Ϫ/Ϫ)/ Bcrp(Ϫ/Ϫ) mice. Comparison of the brain-and testis-toplasma 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 bloodbrain 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(Ϫ/Ϫ)/Bcrp(Ϫ/Ϫ) mice even compared with Mdr1a/1b(Ϫ/Ϫ) and Bcrp(Ϫ/Ϫ) 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(Ϫ/Ϫ)/ Bcrp(Ϫ/Ϫ) 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.It is well accepted that the penetration of xenobiotic compounds into the brain and testis is restricted by the blood-brain barrier (BBB) and blood-testis barrier (BTB), respectively. The BBB is formed by brain capillary endothelial cells, whereas, in addition to endothelial cells, myoid and Sertoli cells form the BTB (Bart et al., 2002;Kusuhara and Sugiyama, 2005). Tight junctions between adjacent cells in the BBB and BTB are highly developed and limit the penetration of substances via the paracellular route. Moreover, drug transporters act as active barriers to limit the tissue penetration of substrates from the blood by extruding them back into the blood in the BBB and BTB and, thereby, modulating pharmacological or adverse reactions. It has been shown that ATP binding cassette (ABC) transporters, which are known to mediate resistance to anticancer drugs and antiviral drugs, are expressed in the BBB and BTB. These include P-glycoprotein (P-gp/MDR1/ABCB1), breast cancer resistance protein (BCRP/ABCG2), multidrug resistance-associated protein (MRP)-1/ABCC1, MRP2/ABCC2, MRP4/ABCC4, and MRP5/ABCC5 (Leggas et al., 2004;Zhang et al., 2004;Lee et al., 2005). In particular, P-gp is a well known transporter that plays a pivotal role in barrier function, and disruption of the Mdr1a gene, a predominant isoform expressed in the barriers, causes accumulation of a number of its substrates (S...

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(Ϫ/Ϫ) and Bcrp(Ϫ/Ϫ) 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(Ϫ/Ϫ)/Bcrp(Ϫ/Ϫ) mice, but only moderately or weakly in Mdr1a/1b(Ϫ/Ϫ) mice and negligibly in Bcrp(Ϫ/Ϫ) 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.

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.

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.

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.