The purposes of the review are to: a) Provide a comprehensible introduction of the-state-of-the-art sciences of solubility and dissolution, b) introduce typical technologies to assess solubility and dissolution, and c) propose the best practice strategy. The theories of solubility and dissolution required in drug discovery were reviewed especially from the view point of oral absorption. The physiological conditions in the gastrointestinal fluid in humans and animals were then briefly summarized. Technologies to assess solubility and dissolution in drug discovery were then introduced. Recently, these technologies have been improved by the laboratory automation and computational technologies. Finally, the strategies to apply these technologies for a drug discovery project were discussed.
The basement membrane at the blood-brain barrier (BBB) plays important roles in maintaining the structure and function of capillary vessels. The BBB is constructed from endothelial cells, astrocytes and pericytes, but their interactions in the formation or maintenance of basement membrane have not been established. Transforming growth factor-β1 (TGF-β1) is known to increase fibronectin in brain capillary basement membrane with deposition of β-amyloid. We previously reported that the mRNA level of α-smooth muscle actin in a brain capillary pericyte cell line TR-PCT1 was increased by treatment with TGF-β1. In this study, expression of mRNAs encoding basement membrane-related molecules in TR-PCT1, a rat endothelial cell line TR-BBB13, and a type 2 astrocyte cell line TR-AST4 was evaluated by RT-PCR. The effects of TGF-β1 on expression of basement membrane-related genes in these cell lines were also examined. Fibronectin, MMP-9, tPA, TIMP-1, and PAI-l in TR-PCT1 were higher than in TR-BBB13 and TR-AST4. In TR-PCT1 treated with TGF-β1, collagen type IV, PAI-1, and MMP-9 were increased, and TIMP-2 was reduced. The change in PAI-1 mRNA was faster than those in MMP-9, TIMP-2, collagen type IV mRNAs. These results suggest that pericytes may be key cells in the maintenance of the basement membrane at the BBB.
We investigated the inhibitory effects of the angiotensin receptor blockers (ARBs), candesartan, irbesartan, losartan, losartan active metabolite (EXP-3174), olmesartan, telmisartan and valsartan (0.3-300 μM), on the CYP2C9 activity in human liver microsomes using (S)-(−)-warfarin as a typical CYP2C9 substrate. Except for olmesartan and valsartan, these ARBs inhibited the activity of 7-hydroxylation of (S)-(−)-warfarin with IC50 values of 39.5-116 μM. Of six synthetic derivatives of olmesartan, five compounds which possess either alkyl groups or a chloro group at the same position as that of the hydroxyisopropyl group in olmesartan inhibited CYP2C9 activity with IC50 values of 21.7-161 μM. Olmesartan and the olmesartan analogue, RNH-6272, both having a hydroxyisopropyl group, showed no inhibition, indicating that the hydrophilicity of this group greatly contributes to the lack of CYP2C9 inhibition by these two compounds. A three-dimensional model for docking between EXP-3174 and CYP2C9 indicated that the chloro group of EXP-3174 is oriented to a hydrophobic pocket in the CYP2C9 active site, indicating that the lipophilicity of the group present in ARBs at the position corresponding to that of the hydroxyisopropyl group in olmesartan is important in inhibiting CYP2C9 activity.
In the present study, we have utilized a target selective human pregnane X receptor-siRNA (hPXR-siRNA)-adenovirus expression system to examine the contribution of hPXR on the gene regulation of drug-metabolizing P450s in human hepatocytes. Introduction of the hPXR-siRNA adenoviral vector reduced the level of PXR mRNA. After infection with Ad hPXR-siRNA, the basal and ligand-activated CYP2A6, CYP2C8, CYP3A4 and CYP3A5 mRNA levels were decreased significantly in dose-dependent manners, whereas CYP2B6, CYP2C9 and CYP2C19 mRNA levels were moderately influenced after infection with Ad hPXR-siRNA. These data suggest the distinct PXR influences on the regulation of these genes. The expression of CYP1A2 and CYP2D6 mRNA were not affected by the introduction of hPXR-siRNA, suggesting that PXR plays no functional role in the expression of either of these genes. This is the first report to compare simultaneously the relative contribution of hPXR on the expression of nine forms of P450 in primary cultured human hepatocytes. Mutual sharing among nuclear receptors of their binding cis-elements becomes clear now. Thus, the present method using the combination of adenovirus-mediated hPXR-siRNA expression and human hepatocytes may offer clear information on the relative role of nuclear receptors such as hPXR on the expression of drug metabolizing genes.
Hepatocyte nuclear factor 4α (HNF4α) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4α in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4α-small interfering RNA (hHNF4α-siRNA). The hHNF4α-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4α in a concentration-dependent manner. The hHNF4α-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4α-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4α-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4α-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4α-suppressed cells fell significantly compared to the control. These results suggest that HNF4α plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4α expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.
In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-Mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition sudies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalized solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.
Amiodarone (AMD) is a benzofurane derivative with class III antiarrhythmic activity that is effective in controlling intractable cardiac arrhythmias. One of the most common and serious drug interactions in clinical practice is the interaction between digoxin and an antiarrhythmic agent. It has been reported that AMD and N-monodesethylamiodarone (DEA), the active metabolite of AMD, inhibit the P-glycoprotein (P-gp/MDR1)-mediated digoxin transport. However, the intestinal transport processes of AMD and DEA have not been fully revealed. In this study, we focused on the intestinal transport mechanism of DEA and characterized the intestinal transport of DEA using Caco-2 cells. Basal-to-apical transport of DEA by Caco-2 cells was greater than apical-to-basal transport. The relationship between concentration and basal-to-apical flux rate appeared to approach saturation. The uptake of DEA by Caco-2 cells was increased in the presence of typical ATP-depletion compounds and thyroid hormones. On the other hand, substrates for P-gp, multidrug resistance-associated proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2) had no effect on the efflux of DEA. These results suggest that an ATP-binding cassette (ABC) transporter, which is different from P-gp, MRPs and BCRP, mediates the efflux of DEA across the apical membrane in Caco-2 cells and that thyroid hormone inhibits this transporter.
Adjuvant-induced arthritis (AA) rats have been used as an animal model for rheumatoid arthritis. Several studies have shown that the pharmacokinetics of a number of drugs are altered in AA rats. We investigated the effects of AA on the barrier functions of the intestine using a rat model. Intestinal CYP3A activities (midazolam 1′-hydroxylation and 7-benzyloxy-4-(trifluoromethyl)-coumarin 7-hydroxylation) in AA rats were significantly decreased compared with those in normal rats, with marked decrease observed in the upper segment of intestine. Intestinal P-glycoprotein (P-gp) activitiy at upper segment was also significantly decreased in AA rats to 60% of that in normal rats, and the other segments (middle and lower) of intestine also exhibited tendencies toward decrease in P-gp activity. This decrease was supported by the finding that levels of mdr1a mRNA and P-gp protein were decreased in AA rats. No significant differences were observed in intestinal paracellular and transcellular permeability between AA and normal rats. These results suggest that intestinal CYP3A and P-gp activities are decreased in AA rats, and that the pharmacokinetics and bioavailabilities of drugs whose membrane permeation is limited by intestinal CYP3A and/or P-gp may be altered in rheumatic diseases.
Cytochrome P450 oxidoreductase (POR) is the single flavoprotein which donates electrons to the microsomal cytochrome P450 enzymes for oxidation of their substrates. In this study, we sequenced all 15 exons and the surrounding intronic sequences of POR in 100 human liver samples to identify novel and confirm known genetic polymorphisms in POR. Thirty-four single nucleotide polymorphisms (SNPs) were identified including 9 in the coding exons (5 synonymous and 4 nonsynonymous), 20 in the intronic regions, and 5 in the 3′-UTR. Of these, 9 were novel SNPs, including three nonsynonymous SNPs, SNH313003 (817733G>C; K49N), SNH313020 (848661C>A; L420M), and SNH313029 (849577T>C; L577P) with minor allele frequencies of 0.005, 0.045, and 0.020, respectively. We also confirmed a previously reported non-synonymous SNP rs1057868 (A503V) as well as five synonymous SNPs (G5G, T29T, P129P, S485S, and S572S) all with allele frequencies similar to those previously reported. Structurally, these polymorphisms occur in different regions: SNH313003 (K49N) in the amino-terminal tail, SNH313020 (L420M) in the connecting domain, SNH313029 (L577P) in the NADPH-binding domain, and rs1057868 (A503V) in the FAD binding domain.