The binding of drugs to plasma proteins, such as albumin and α₁-acid glycoprotein (AGP) is a major determinant in the disposition of drugs. A topology analysis of drug binding sites on HSA and AGP was determined using various methods, including spectroscopy, QSAR, photoaffinity labeling and site directed mutagenesis. Recombinant albumin was found to be useful for rapidly identifying drug binding sites. The binding sites on AGP are not completely separated but are partially overlapped, and Trp, Tyr, Lys and His residues in the drug binding pockets play important roles in this process. Drug displacement is somewhat complex, due to the involvement of multiple effects. The reduced binding in uremic patients may be explained by a mechanism that involves a combination of direct displacement by free fatty acids as well as cascade effects of free fatty acids and unbound uremic toxins for significant inhibition in serum binding. Albumin-containing dialysate is useful for the extracorporeal removal of endogenous toxins and in the treatment of drug overdoses. Oxidized albumin is a useful biomarker for the quantitative and qualitative evaluation of oxidative stress. Interestingly, AGP undergoes a structural transition to a unique structure that differs from the native and denatured states, when it interacts with membranes.

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   We have noticed that the trough level of blood concentration of cyclosporin A (CyA) tends to be lower in patients receiving long-term oral levothyroxine (LTX) than in patients not receiving LTX. We confirmed this clinical observation in experiments using Wistar rats orally given LTX (8 μg/kg) or saline (control) for 3 weeks, followed by CyA (10 mg/kg). The LTX treatment had little effect on the blood concentrations of CyA after i.v. administration, whereas they were decreased significantly after p.o. administration. After p.o. administration, the value of the area under the blood concentration-time curve from 0 to 24 hr and the bioavailability of CyA in the LTX group were decreased to only about one-fifth and a quarter of those in the control group, respectively. After treatment with LTX, the expression levels of mdr1a, mdr1b and CYP3A2 mRNAs in the duodenum were markedly increased to about twice the control, but in jejunum, ileum and liver the expression levels were little changed. These findings suggest that the absorption of CyA, which occurs mainly from the upper intestine, is reduced as a result of efflux transport via P-glycoprotein induced by LTX. In conclusion, careful monitoring of CyA levels is required in the event of LTX administration to patients receiving immunotherapy with CyA.

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  In vivo percutaneous absorption of emedastine difumarate was investigated in rats and compared with rat skin in vitro. Since emedastine entering the systemic circulation is mostly excreted in bile, we first came up with the method of collecting bile with a minimal skin incision. In vivo skin permeation of the drug was estimated from biliary excretion data by deconvolution analysis. Prior to applying deconvolution analysis, it was confirmed that biliary excretion of emedastine was linear against its dose. When the in vivo permeation profile estimated by deconvolution was compared with the in vitro profile, the lag time for permeation was significantly shorter in vivo than in vitro, whereas the skin permeability coefficient was almost the same. If we presume a two-layer diffusion model, then this finding may primarily be due to the shorter diffusion length of the dermis.

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  Objectives: We conducted a randomized, double-blind, placebo-controlled study to evaluate the pharmacokinetics and pharmacodynamics of landiolol hydrochloride in a dose escalation regimen in healthy male volunteers.
   Methods: We set two-dose escalation regimen (LM and MH groups) using three different doses [L (low): 0.03 mg/kg/min (1 min) loading→0.01 mg/kg/min (10 min) continuous, M (medium): 0.06 mg/kg/min (1 min) loading→0.02 mg/kg/min (10 min) continuous, H (high): 0.125 mg/kg/min (1 min) loading→0.04 mg/kg/min (10 min) continuous]. Sixteen subjects were allocated randomly to the LM, MH, and placebo groups (n=6, 6, and 4, respectively).
   Results: In both the LM and MH groups, the blood concentration of landiolol hydrochloride changed within a constant range from 2 minutes after initiation of administration to just before the higher dose escalation. By 2 minutes following the higher dose escalation, the concentration of landiolol hydrochloride reached C_max, and reached almost steady state levels until 6 minutes following administration of the higher dose. The t_1/2 of landiolol hydrochloride was 3.5 minutes. The heart rates and blood pressures of subjects administered landiolol hydrochloride decreased, but there were no adverse events in any subject.
   Conclusions: The concentration of landiolol hydrochloride rapidly reached steady state levels, and rapidly dissipated after completion of administration.

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   We encountered DNA samples which showed a positive product using a long PCR-based method for the detection of CYP2D6^*5, indicating deletion of the entire CYP2D6 gene, but the samples did not show a band related to CYP2D6^*5 in either XbaI- or EcoRI-RFLP analysis. To achieve genotyping with accuracy, we performed a further genetic analysis to clarify the discrepancy. An unknown 1.6-kb insert was identified in a region downstream from the CYP2D6 stop codon where a specific primer was designed for long-PCR analysis for CYP2D6^*5 genotyping. This finding suggested that the CYP2D6 gene might not be deleted in the samples even if a positive product was detected by the long-PCR method. Furthermore, the allelic frequency of this type was found to be approximately 0.3% (4 heterozygous/771 samples) in a Japanese population. In conclusion, we found a novel structure of the CYP2D6 gene, which might lead to incorrect genotyping for CYP2D6^*5. Although the long PCR-based strategy for the detection of CYP2D6^*5 has been widely used due to its usefulness and convenience, we recommend caution when adopting this method and propose re-evaluating the method for detecting CYP2D6^*5.

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  1-Aminobenzotriazole (ABT) is extensively used as a non-specific cytochrome P450 (CYP) inhibitor. In this study, the inhibitory effect of ABT on CYP-dependent drug oxidations was investigated in human liver microsomes (HLM) and compared with that of SKF-525A, another non-specific inhibitor. The following probe activities for human CYP isoforms were determined using pooled HLM: phenacetin O-deethylation (CYP1A2); diclofenac 4′-hydroxylation (CYP2C9); S-mephenytoin 4′-hydroxylation, (CYP2C19); bufuralol 1′-hydroxylation (CYP2D6); chlorzoxazone 6-hydroxylation (CYP2E1); midazolam 1′-hydroxylation, nifedipine oxidation, and testosterone 6β-hydroxylation (CYP3A). ABT had the strongest inhibitory effect on the CYP3A-dependent drug oxidations and the weakest effect on the diclofenac 4′-hydroxylation. SKF-525A potently inhibited the bufuralol 1′-hydroxylation, but weakly inhibited chlorzoxazone 6-hydroxylation. The inhibitory effects of ABT and SKF-525A were increased by preincubation in some probe reactions, and this preincubation effect was greater in ABT than in SKF-525A. The remarkable IC₅₀ shift (> 10 times) by preincubation with ABT was observed on the phenacetin O-deethylation, chlorzoxazone 6-hydroxylation, and midazolam 1′-hydroxylation. In conclusion, ABT and SKF-525A had a wide range of IC₅₀ values in inhibiting the drug oxidations by HLM with and without preincubation.

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  Microsomal alcohol oxygenase catalyzes the stereoselective oxidation of 7α- and 7β-hydroxy-Δ⁸-tetrahydrocannabinol (7α- and 7β-hydroxy-Δ⁸-THC) to 7-oxo-Δ⁸-THC in monkey liver, and the activity for 7β-hydroxy-Δ⁸-THC is relatively higher than that for 7α-hydroxy-Δ⁸-THC. We previously reported that purified P450JM-E, assumed to be CYP3A8, is a major enzyme responsible for the oxidation of 7-hydroxy-Δ⁸-THC to 7-oxo-Δ⁸-THC in monkey liver and is capable of catalyzing the oxidative reaction by NADH as well as NADPH. In the present study, we demonstrated that some steroids such as testosterone and progesterone stimulated both the NADH- and NADPH-dependent conversions of 7β-hydroxy-Δ⁸-THC to 7-oxo-Δ⁸-THC in monkey liver microsomes. Kinetic analyses revealed that both the NADH- and NADPH-dependent 7-oxo-Δ⁸-THC formation showed sigmoid kinetics. Testosterone caused a decrease in S₅₀ and an increase in V_max for the NADH-dependent activity, and resulted in a decrease in S₅₀ without changing the V_max for the NADPH-dependent activity. On the other hand, NADH-dependent testosterone 6β-hydroxylation activity showed Michaelis-Menten kinetics and was also inhibited by 7β-hydroxy-Δ⁸-THC, resulting in a decrease in V_max with no effect on the K_m. NADPH-dependent testosterone 6β-hydrozylation activity was also inhibited by 7β-hydroxy-Δ⁸-THC, resulting in a decrease in both S₅₀ and V_max. In order to explain the metabolic interaction between 7β-hydroxy-Δ⁸-THC and testosterone, we propose a kinetic model involving at least three binding sites, for the mechanism of activation by testosterone.

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  Objective: Among various adverse reactions of atypical antipsychotics, weight gain and impaired glucose tolerance are clinically significant. The aim of this study is to analyze quantitatively the contributions of various receptors to these antipsychotics-induced adverse reactions based on the receptor occupancy theory.
   Methods: Two indices of antipsychotics-induced weight gain (the values estimated by a meta-analysis and the observed values in clinical trials) and the morbidity rate of type 2 diabetes mellitus during treatment with antipsychotics were taken from the literature. We calculated the estimated mean receptor occupancies of α₁ adrenergic, α₂ adrenergic, dopamine D₂, histamine H₁, muscarinic acetylcholine (mACh), serotonin 5-HT_1A, 5-HT_2A and 5-HT_2C receptors by antipsychotics by using the pharmacokinetic parameters and receptor dissociation constants, and analyzed the correlation between the occupancies and the extent of adverse reactions as assessed using the aforementioned indices.
   Results: There were statistically significant correlations between the estimated occupancies of H₁ and mACh receptors and antipsychotics-induced weight gain estimated by meta-analysis (r_s=0.81 and r_s=0.83, respectively, p<0.01). There were also statistically significant correlations between these receptor occupancies and observed weight gain in clinical trials (r_s=0.66 in each case, p<0.01). The morbidity rate of type 2 diabetes mellitus was highly correlated with H₁, mACh, and 5-HT_2C receptor occupancies (r_s=0.90 in each case, p<0.05). However, H₁ receptor occupancy was also highly correlated with mACh receptor occupancy among antipsychotics, so that only one of them may be critically associated with the adverse reactions. Considering that these adverse reactions have not been reported for drugs with mACh receptor antagonistic action, other than antipsychotics, the H₁ receptor may contribute predominantly to the antipsychotics-induced weight gain and diabetes mellitus.
   Discussion/Conclusion: Model analysis based on receptor occupancy indicates that H₁ receptor blockade is the primary cause of antipsychotics-induced weight gain and diabetes mellitus.

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  Although metformin, a cationic agent for type II diabetes, shows its pharmacological effect in the liver, the drug is mainly eliminated into urine. The tissue selectivity based on the function of drug transporters is unclear. In the present study, the transport of metformin was examined using HEK293 cells transiently transfected with five human renal organic ion transporter cDNAs. Human OCT1 and OCT2, but not OAT1, OAT3 or OCT2-A, stimulated the uptake. A kinetic analysis of metformin transport demonstrated that the amount of plasmid cDNA for transfection was also important parameter to the quantitative elucidation of functional characteristics of transporters, and both human and rat OCT2 had about a 10- and 100-fold greater capacity to transport metformin than did OCT1, respectively. In male rats, the mRNA expression level of rOCT2 in the whole kidneys was 8-fold greater than that of rOCT1 in the whole liver. The in vivo distribution of metformin in rats revealed that the expression level of renal OCT2 was a key factor in the control of the concentrative accumulation of metformin in the kidney. These findings suggest that metformin is a superior substrate for renal OCT2 rather than hepatic OCT1, and renal OCT2 plays a dominant role for metformin pharmacokinetics.

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  The genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) have been associated with increased toxicity of methotrexate (MTX), a folic acid antagonist that is widely used to treat cancer and immunosuppressive disorders such as rheumatoid arthritis. In this study, we analyzed all the exons and exon/intron junctions of the MTHFR gene from 200 Japanese individuals. We detected a novel single nucleotide polymorphism (SNP) 148C>T (Arg46Trp) in exon 1. The allele frequency of this polymorphism in the Japanese population appears to be extremely low (0.25%).

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