Fusao Komada

CYP2C19 genotype–related efficacy of omeprazole for the treatment of infection caused by Helicobacter pylori

Omeprazole is used for the treatment of infection caused by Helicobacter pylori, and it is metabolized by the polymorphic cytochrome P4502C19 (CYP2C19). We have found that the anti–H pylori efficacy by the combination of omeprazole and antibiotics is related to the CYP2C19 genotype.

Intratracheal delivery of insulin : absorption from solution and aerosol by rat lung

Abstract In this study, we investigated the dynamics of insulin absorption from the lung with reference to bioavailability. Human insulin in 10 μl of aqueous solution and an insulin aerosol were administered into the exposed trachea of anesthetized rats. Blood samples were collected from the jugular vein at specified intervals and the plasma concentration of insulin was determined by an EIA. The relative bioavailability of insulin after intratracheal administration in 10 μl of pH 7.0 isotonic phosphate buffer and pH 3.0 isotonic citrate buffer was 13 and 42%, respectively, of that obtained after subcutaneous administration. Although insulin absorption in the presence of surfactants such as glycocholate, surfactin, and Span 85 was 3–4-times greater than that without surfactants, the co-administration of EDTA and salicytate, which previously enhanced the absorption of rectally administered insulin, did not increase the intratracheal absorption of insulin. The concomitant administration of nafamostat, a protease inhibitor, produced modest effects. The relative bioavailability of insulin given as an intratracheal aerosol was similar to that after subcutaneous administration. These observations indicate that the intratracheal route may be useful for the delivery of insulin.

Genotyping of N‐acetylation polymorphism and correlation with procainamide metabolism

We studied the genotypes of polymorphic N‐acetyltransferase (NAT2) in 145 Japanese subjects by the polymerase chain reaction‐restriction fragment length polymorphism method. The rapid‐type NAT2*4 was expressed at a higher frequency (68.6%) than the slow‐type genes with specific point mutations (NAT2*6A, 19.3%; NAT2*7B, 9.7%; NAT2*5B, 2.4%). The frequency of NAT2* genotypes consisted of 44% of a homozygote of NAT2*4, 49% of a heterozygote of NAT2*4 and mutant genes, and 7% of a combination of mutant genes. The metabolic activity for procainamide to N‐acetylprocainamide was measured in 11 healthy subjects whose genotype had been determined. Although the acetylation activity substantially varied interindividually, the variability was considerably reduced after classification according to the genotype. The N‐acetylprocainamide/procainamide ratio in urinary excretion was 0.60 ± 0.17 (mean ± SD) for those with NAT2*4/ *4, 0.37 ± 0.06 for NAT2*4/ *6A, 0.40 ± 0.03 for NAT2*4/ *7B, and 0.17 for NAT2*6A/ *7B. The results indicated that the NAT2* genotype correlates with acetylation of procainamide.

Interaction of docetaxel ('Taxotere') with human P-glycoprotein

The interaction of docetaxel (“Taxotere”) with P‐glycoprotein (P‐gp) was examined using porcine kidney epithelial LLC‐PK1 and LLC‐GA5‐COL150 cells, overexpressing human P‐gp selectively on the apical plasma membrane by transfection of human MDR1cDNA into the LLC‐PK1 cells. The basal‐to‐apical transport of [14C]docetaxel in LLC‐GA5‐COL150 cells significantly exceeded that in LLC‐PK1 cells, but the apical‐to‐basal transport was decreased in LLC‐GA5‐COL150 cells. The intracellular accumulation after its basal or apical application to LLC‐GA5‐COL150 cells was 4‐ to 20‐fold lower than that of LLC‐PK1 cells. Multidrug resistance (MDR) modulators, i.e., cyclosporin A and SDZ PSC 833, inhibited the basal‐to‐apical transport and increased the apical‐to‐basal transport of [14C] docetaxel in LLC‐GA5‐COL150 cells, but verapamil affected only apical‐to‐basal transport. The intracellular accumulation after basal or apical application to LLC‐GA5‐COL150 cells was also increased by these three MDR modulators. These observations demonstrated that docetaxel is a substrate for human P‐gp, suggesting that docetaxel‐drug interactions occur via P‐gp. The inhibition of [14C]docetaxel transport by the MDR modulators, as well as daunorubicin and vinblastine, was also found in LLC‐PK1 cells, which endogenously express P‐gp at lower levels, and concentrations showing similar levels of inhibition were lower than those in the case of LLC‐GA5‐COL150 cells. These observations indicate that it is necessary to consider the Pharmacokinetic and pharmacodynamic interactions of docetaxel via P‐gp.

Effects of 12 Ca2+ antagonists on multidrug resistance, MDR1-mediated transport and MDR1 mRNA expression

The effects of 12 Ca(2+) antagonists on MDR1 were examined by two independent models: the inhibitory effect on MDR1-mediated transport of [(3)H]digoxin using MDR1-overexpressing LLC-GA5-COL150 cell monolayers and the reversal effect on cytotoxicity of vinblastine or paclitaxel using MDR1-overexpressing Hvr100-6 cells. The inhibitory effects on [(3)H]digoxin transport were assessed as the 50% inhibitory concentration during 4 h exposure, and the values were the lowest for nicardipine (4.54 microM), manidipine (4.65 microM) and benidipine (4.96 microM), followed by bepridil (10.6 microM), barnidipine (12.6 microM), efonidipine (13.0 microM), verapamil (13.2 microM) and nilvadipine (18.0 microM). The reversal effect on cytotoxicity was assessed by the 50% growth inhibitory concentration after 3 days exposure, and the resistance to vinblastine or paclitaxel in Hvr100-6 cells was reversed by manidipine, verapamil, benidipine, barnidipine, and nicardipine, in that order. Bepridil, barnidipine, efonidipine, verapamil and nilvadipine showed similar inhibitory effects on [(3)H]digoxin transport, but barnidipine and verapamil showed a stronger effect in reversal of cytotoxicity. Real-time quantitative RT-PCR assay indicated a decrease in MDR1 mRNA expression by barnidipine and verapamil. It is concluded that Ca(2+) antagonists cannot only be direct inhibitors of MDR1 but that some may at the same time act as inhibitors of expression of MDR1 via down-regulation of MDR1 mRNA.

Improvement in Wound Healing by Epidermal Growth Factor (EGF) Ointment. I. Effect of Nafamostat, Gabexate, or Gelatin on Stabilization and Efficacy of EGF

The healing effect of human epidermal growth factor (hEGF) on open wounds was studied in rats. No improvement in wound healing was found by topical application of EGF alone to open wound sites. We found an ointment containing EGF and a protease inhibitor, nafamostat mesilate or gabexate mesilate, or gelatin accelerated the healing rate of open wounds. Significant increases in the dry weight of the wound site granulation tissue, uronic acid (as an index of acid mucopolysaccharide) and hydroxyproline (as an index of collagen) were observed by treatment with EGF ointment containing nafamostat compared with the controls. The effects of the protease inhibitor on wound healing were dose dependent. Nafamostat was more efficient than gabexate or gelatin on wound healing. The degradation of 125I-EGF in wound tissue homogenate was significantly decreased in the presence of a protease inhibitor, such as nafamostat or gabexate, or gelatin. These findings indicate that the stabilization of EGF at the wound site is an important factor in permitting the expression of its healing effects and suggest that the ointment containing EGF and a stabilizing agent would be a suitable dosage form for acceleration of wound repair.

CYP2C19 genotype related effect of omeprazole on intragastric pH and antimicrobial stability.

AbstractPurpose. A combination of proton pump inhibitors and antimicrobials has been applied as an anti–Helicobacter pylori (H. pylori) therapy. Omeprazole, one of the proton pump inhibitors, is metabolized by CYP2C19, which exhibits genetic polymorphism. It was reported previously that the overall anti–H. pylori efficacy can be related to the CYP2C19 genotype. The main aim of the present study was to obtain a rational explanation for the relationship between the overall anti–H. pylori efficacy and the CYP2C19 genotype. Methods. Six healthy volunteers were classified as extensive metabolizers and poor metabolizers, according to their CYP2C19 genotypes. Plasma concentrations and intragastric pH were monitored prior to and until 24 h after the administration of 20 mg omeprazole. The stability of amoxicillin, clarithromycin, and metronidazole was examined using buffer solutions with monitored intragastric pH, and their remaining percentage in the intragastric space was simulated. Results. The poor metabolizers, classified by the CYP2C19 genotypes, showed the higher effectiveness in anti–H. pylori therapy, via the higher plasma concentration of omeprazole and the higher intragastric pH, and possibly the higher stability of antimicrobials in the higher intragastric pH. Conclusions. CYP2C19 genotyping is a very useful method to determine the effective and safe dosage regimen including the selection of the dual and triple therapy in anti–H. pylori therapy.

Inhibitory effects of a cyclosporin derivative, SDZ PSC 833, on transport of doxorubicin and vinblastine via human P-glycoprotein

The inhibitory effects of SDZ PSC 833 (PSC833), a non‐immunosuppressive cyclosporin derivative, on the P‐glycoprotein (P‐gp)‐mediated transport of doxorubicin and vinblastine were compared with those of cyclosporin A (Cs‐A). The transcellular transport of the anticancer drugs and PSC833 across a monolayer of LLC‐GA5‐COL150 cells, which overexpress human P‐gp, was measured. Both PSC833 and Cs‐A inhibited P‐gp‐mediated transport of doxorubicin and vinblastine in a concentration‐dependent manner and increased the intracellular accumulation of doxorubicin and vinblastine in LLC‐GA5‐COL150 cells. The values of the 50%‐inhibitory concentration (IC50) of PSC833 and Cs‐A for doxorubicin transport were 0.29 and 3.66 μM, respectively, and those for vinblastine transport were 1.06 and 5.10 μM, respectively. The IC50 of PSC833 for doxorubicin transport was about 4‐fold less than that for vinblastine transport, suggesting that the combination of PSC833 and doxorubicin might be effective. PSC833 itself was not transported by P‐gp and had higher lipophilicity than Cs‐A. These results indicated that the inhibitory effect of PSC833 on P‐gp‐mediated transport was 5‐ to 10‐fold more potent than that of Cs‐A, and this higher inhibitory effect of PSC833 may be related to the absence of PSC833 transport by P‐gp and to the higher lipophilicity of PSC833.

Enhanced bioavailability of subcutaneously injected insulin by pretreatment with ointment containing protease inhibitors

The present study was undertaken to develop an ointment preparation containing a protease inhibitor for stabilizing subcutaneously injected insulin. The ointment containing the protease inhibitor, gabexate mesilate or nafamostat mesilate, was applied to the skin around the insulin injection site. Three results were obtained. First, gabexate and nafamostat inhibited insulin degradation in subcutaneous tissue homogenates in vitro. Second, after application of gabexate or nafamostat ointment, an appreciable amount of gabexate or nafamostat appeared in the subcutaneous tissue of rats or hairless mice and their concentrations were comparable to those seen in the in vitro experiment. Third, insulin degradation at the subcutaneous injection site in the rat was depressed after pretreatment with gabexate or nafamostat ointment. Pretreatment with gabexate or nafamostat ointment increased the plasma immunoreactive insulin (IRI) levels and the hypoglycemic effect of insulin in healthy volunteers. These results indicate that gabexate or nafamostat ointments stabilize subcutaneously injected insulin.

Identification of N-acetyltransferase 2 and CYP2C19 genotypes for hair, buccal cell swabs, or fingernails compared with blood

Genotyping of polymorphic drug metabolizing enzymes may be useful to estimate the blood concentration, efficacy, and toxicity of drugs before administration. Blood samples are most generally used for genotyping; however, sampling is invasive and complicated by handling and transport. Therefore, the authors developed genotyping methods using nonblood specimens, and then each genotype was compared with that from blood. Healthy Japanese volunteers provided hairs (n = 50), buccal cell swabs (n = 50), and fingernails (n = 30) for N-acetyltransferase 2 and CYP2C19 genotyping. Recovery of genomic DNA from each nonblood specimen was lower than that from 0.5 mL blood. Using a modification of the DNA extraction and polymerase chain reaction amplification method, genotypes were diagnosed without failure, even for those with very low levels of DNA. Both genotypes from these specimens completely matched the genotypes from the blood of the same subject. These nonblood specimens can be convenient, accessible, and economical alternatives to blood as a source of DNA for genotyping.