Simple determination of o-phenylphenol in skin lotion by high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization with 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole.

o-Phenylphenol (OPP) in skin lotion was quantitated by high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization with 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) in borate buffer (pH 8.5) at room temperature for 2 min. The column [150 mm x 3.0 mm internal diameter (i.d.)], which contained 5 µm particles of C18 packing material, was eluted at room temperature (flow rate: 0.5 ml/min) with mobile phase prepared by addition of acetonitrile (550 ml) to 450 ml of Milli-Q water containing trifluoroacetic acid (0.1 v/v%). 2-Hydroxyfluorene was used as an internal standard. The retention times of NBD-CO-OPP and NBD-CO-IS derivatives were 16.2 and 22.2 min, respectively. The calibration plot was linear in the range of 0.01-0.2 µg/ml with an r2 value of 0.9960, and the lower limit of detection was 0.003 µg/ml (at a signal-to-noise ratio of 3:1; absolute amount of 12 pg/20 µl injection). The coefficient of variation was less than 8.8%. Contents of OPP in three skin lotions were determined with the present system, and the recovery from spiked samples was satisfactory.

Effects of eugenol-reduced clove extract on glycogen phosphorylase b and the development of diabetes in db/db mice.

We found that the 50% aqueous EtOH extract of clove (Syzygium aromaticum) had potent dose-dependent inhibitory activity toward glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes. Among the components, eugeniin inhibited glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes, with IC50 values of 0.14 and 4.7 µM, respectively. In sharp contrast, eugenol showed no significant inhibition toward glycogen phosphorylase b, even at a concentration of 400 µM. Eugenol-reduced clove extracts (erCE) were prepared and when fed to a db/db mouse they clearly suppressed the blood glucose and HbA1c levels. Furthermore, plasma triglyceride and non-esterified fatty acid levels in 5% and 10% erCE-fed db/db mice were significantly lowered, compared with control db/db mice without erCE supplementation. These results suggested that dietary supplementation with the erCE could beneficially modify glucose and lipid metabolism and contribute to the prevention of the progress of hyperglycemia and metabolic syndrome.

Determination of hinokitiol in skin lotion by high-performance liquid chromatography-ultraviolet detection after precolumn derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

Hinokitiol, a potent, broad-spectrum antibacterial agent, is a component of various personal care products. In this study, the concentration of hinokitiol in skin lotion was analyzed by means of high-performance liquid chromatography-ultraviolet detection (380 nm) after precolumn derivatization with 4-fluoro-7-nitro-2,1, 3-benzoxadiazole (NBD-F). A standard curve was obtained after derivatization of the authentic compound with NBD-F in borate buffer (pH 9.0) at 60°C for 10 min. The retention time of NBD-hinokitiol was 7.2 min. The calibration plot was linear in the range of 0.2 to 4 mg/ml with an r2 value of 0.9985, and the lower limit of detection was 0.05 µg/ml (at a signal-to-noise ratio of 3, absolute amount of 0.33 ng/20 µl injection). The coefficient of variation was less than 9.4%. It was found that the amount of hinokitiol in the tested skin lotion was 194 ± 14 µg/ml (range: 180-212 µg/ml). Recovery in addition-recovery tests was within the range of 84.5% to 98.0%. This system is simple, sensitive, and convenient, and should be suitable for routine quality assessment of personal care products containing hinokitiol.

Determination of kojic acid in a skin-whitening cosmetic by high-performance liquid chromatography coupled with ultraviolet detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

Various methods for the determination of kojic acid (KA), a skin-whitening agent, have been reported by high-performance liquid chromatography (HPLC). In this study, the concentration of KA in a skin-whitening cosmetic was analyzed by HPLC with ultraviolet detection (380 nm) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in order to improve the sensitivity. The HPLC column was 150 mm x 3.0 mm i.d., containing 5 microm particles of C18 packing material. The mobile phase was prepared by the addition of acetonitrile (550 ml) to 450 ml of Milli-Q water containing trifluoroacetic acid (0.1 v/v%). The samples were eluted from the column at room temperature at a flow rate of 0.35 ml/min. The retention time of NBD-KA was 7.8 min. A standard curve was obtained after derivatization with NBD-F in borate buffer (pH 9.0) at 40 degrees C for 7 min. The calibration plot was linear, in the range of 0.25-5 microg/ml with an r2 value of 0.9982, and the lower limit of detection was 0.06 microg/ml (at a signal-to-noise ratio of 3:1; absolute amount of 0.4 ng/20 microl injection). The coefficient of variation was less than 9.6%. It was found that the amount of KA in a skin-whitening cosmetic was 237 +/- 14 microg/ml (range: 219-255 microg/ml). Recovery in addition-recovery tests was within the range of 83.4% to 98.1%.

High-performance liquid chromatography with dual-wavelength ultraviolet detection for measurement of hinokitiol in personal care products.

Hinokitiol is found in the heartwood of several cupressaceous plants and is frequently added to cosmetic products such as hair restorers, skin lotions, and body soaps because of its potent and broad-spectrum antibacterial activity. In this study, we established a simple method of hinokitiol determination by high-performance liquid chromatography (HPLC) with dual-wavelength ultraviolet detection at 240 and 345 nm, using a reversed-phase C4 column (RP-4). The retention time of hinokitiol was 7.1 min at both wavelengths. The value of the symmetry coefficient of the hinokitiol peak was close to 1 when the RP-4 column, not an RP-8 or RP-18 column, was used. With the RP-4 column, the regression equation for hinokitiol showed good linearity in the range of 0.05-5 microg/ml, with a detection limit (signal-to-noise ratio of 3) of 0.005 microg/ml at 240 nm and 0.01 microg/ml at 345 nm. The coefficients of variation at 240 and 345 nm were less than 8.2% and 8.7%, respectively, and the recovery was good. The proposed method was used for the determination of hinokitiol in commercial hair restorers, skin lotions, and body soaps.

Sensitive determination of 4-(4-bromophenyl)-4-hydroxypiperidine, a metabolite of bromperidol, in rat plasma by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

The purpose of this study was to determine the level of 4-(4-bromophenyl)-4-hydroxypiperidine (BPHP), a bromperidol (BRO) metabolite, in rat plasma by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was utilized through the procedure as an internal standard (IS). Retention times of the BPHP and IS derivatives were 7.7 and 11.5 min, respectively. The regression equation for BPHP showed good linearity in the range of 0.01-1 mg/ml with the detection limit of 0.003 microg/ml. The coefficient of variation was less than 12.0%. The recovery was satisfactory. This method was applied for a pharmacokinetic study of BPHP in comparison with 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), the corresponding haloperidol (HAL) metabolite, in rats. The ratio of the area under the plasma concentration curve (AUC) after p.o. administration of BPHP to the AUC after i.p. administration of BPHP (46%) was lower than that of CPHP (56%), indicating that intestinal absorption of BPHP is lower than that of CPHP. The ratio of BRO metabolism to BPHP (48%) was 1.8-fold higher than that of HAL metabolism to CPHP (27%); the ratio was estimated as (AUCp.o.,A-->B/AUCp.o.,B)x100, where AUCp.o.,A-->B is the AUC value of BPHP or CPHP after p.o. administration of BRO or HAL, and AUCp.o.,B is the AUC of BPHP or CPHP after administration of BPHP or CPHP, respectively. Our method provides a sensitive procedure for determination of BPHP in rat plasma and is suitable for pharmacokinetic studies of BPHP after BRO administration.

Effect of imatinib mesilate on the disposition kinetics of ciclosporin in rats.

The purpose of this study was to investigate the effect of imatinib mesilate on the disposition kinetics of ciclosporin in rats. The blood concentration-time course and pharmacokinetic parameters of ciclosporin did not significantly change after intravenous injection of ciclosporin (10 mg kg(-1)) in rats treated with imatinib mesilate (50 mg kg(-1)) as compared with a control. When ciclosporin (10 mg kg(-1)) was orally administered, the time course, area under the curve, bioavailability and peak blood concentration of ciclosporin were significantly increased in rats that had been treated with imatinib mesilate 2 h before ciclosporin administration as compared with the control. Because both drugs are transported via P-glycoprotein and breast cancer resistance protein and metabolized by cytochrome P450 3A2, the interaction of imatinib mesilate with these proteins may be responsible for the increased intestinal absorption of ciclosporin in rats. These results indicate that imatinib mesilate enhanced the intestinal absorption of ciclosporin in rats with only the oral administration of ciclosporin, suggesting that our results support clinical data. In addition, imatinib mesilate may increase the pharmacological effects and possibly toxicity of ciclosporin.

Sensitive determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, in a rat biological sample by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

4-(4-Chlorophenyl)-4-hydroxypiperidine (CPHP), one of the metabolites of haloperidol, is considered to exhibit brain toxicity. CPHP concentrations in plasma and tissue homogenates (each 200 microL) from rats were analyzed by HPLC fluorescence detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, the derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was carried through the procedure as an internal standard. The regression equation for CPHP showed a good linearity in the range of 0.03-1 microg/mL with a detection limit of 0.008 microg/mL. The coefficient of variation was less than 11.6%. Plasma concentration-time courses of CPHP after intraperitoneal or per oral administration of CPHP, haloperidol or reduced haloperidol were examined, and the pharmacokinetic parameters were estimated. Additionally, CPHP levels in various tissues at 8 h after intraperitoneal administration of these compounds were compared. The method was simple and sensitive, useful for determination of CPHP in rat biological samples using as little as 200 microL of sample volume and could be applied for pharmacokinetic study.

Simultaneous analysis of haloperidol, its three metabolites and two other butyrophenone-type neuroleptics by high performance liquid chromatography with dual ultraviolet detection.

We investigated simultaneous determination of haloperidol (HAL), its three metabolites [reduced HAL (R-HAL), 3-(4-fluorobenzoyl)propionic acid (FBPA) and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP)] and two related compounds [spiperone (SPI) and droperidol (DRO)] in phosphate-buffered saline using high-performance liquid chromatography (HPLC) coupled with dual ultraviolet detection (220 and 250 nm). Retention times of HAL, R-HAL, FBPA, CPHP, SPI and DRO were 16.8, 11.8, 10.2, 4.1, 12.6 and 8.3 min, respectively. Their lower limits of detection were 7.5, 14, 4.5, 12, 10 and 20 ng/mL in the same order. The coefficients of variation for their intra- and inter-day assays were less than 7.8 and 9.4%, respectively. Of the other centrally acting drugs, only amoxapine interfered with the peak of DRO. Using our procedure, the binding study of tested compounds to synthetic melanin, human serum albumin and alpha1-acid glycoprotein was performed by determining the unbound concentration to total concentration ratio. These results indicated that simultaneous assay of HAL, R-HAL, FBPA, CPHP, SPI and DRO in phosphate-buffered saline by HPLC equipped with dual ultraviolet detection is simple, sensitive and reproducible. Also, our assay system can be applied to the binding study of these compounds to synthetic melanin, human serum albumin and alpha1-acid glycoprotein.

Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagent.

Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60 degrees C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human alpha(1)-acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein.

Simultaneous determination of the binding of amantadine and its analogues to synthetic melanin by liquid chromatography after precolumn derivatization with dansyl chloride.

For the determination of amantadine (1-ADA), 2-adamantanamine (2-ADA), memantine (MEM), and rimantadine (RIM) in melanin binding studies, the simultaneous determination of 1-ADA or 2-ADA, MEM, and RIM is investigated by high-performance liquid chromatographic assay with dansyl chloride as a fluorescent derivative reagent. Dansyl derivatives with fluorescent intensity are detected at an excitation wavelength of 370 nm and an emission wavelength of 506 nm. Retention times of 1-ADA, 2-ADA, MEM, and RIM derivatives are 12.2, 12.2, 15.2, and 16.6 min, respectively. The peak of 1-ADA derivative coelutes with the 2-ADA derivative. The limits of detection for 1-ADA, 2-ADA, MEM, and RIM are 0.014, 0.007, 0.012, and 0.020microM, respectively (signal-to-noise ratio of 3:1). In the intra- and interday assay, the range of standard deviation to the average of 1-ADA, 2-ADA, MEM, and RIM is 4.6-12.7%. Their recovery is also good. The ranking order for synthetic melanin binding among these compounds is RIM > MEM > 2-ADA = 1-ADA. The method is simple, sensitive, and reproducible for simultaneously measuring 1-ADA or 2-ADA, MEM, and RIM. Also, it is useful to investigate their binding kinetics to melanin.

Pharmacokinetic interaction with digoxin and glucocorticoids in rats detected by radio-immunoassay using a novel specific antiserum.

We previously prepared a more specific antiserum (Antiserum-I) to digoxin (Dx) compared with commercially available anti-Dx antiserum (Antiserum-II), clinically used in the therapeutic drug monitoring of Dx. The aims of this study are to compare Dx disposition kinetics by radio-immunoassay (RIA) using Antiserum-I and Antiserum-II, and evaluate the drug-drug interaction with Dx and glucocorticoids in rats. When Dx metabolites were added to rat serum containing Dx, the recovery ratios using Antiserum-I showed 100 to 110% and were remarkably lower than those using Antiserum-II. In rats, serum concentration-time courses of Dx after a single i.v. or p.o. administration of Dx (0.017 mg/kg) by RIA using Antiserum-I were much lower than those using Antiserum-II. The area under the concentration-time course of Dx was significantly lower than that using Antiserum-II and the total body clearance values were significantly higher, while an obvious change of bioavailability was not observed. When using Antiserum-I, rats twice and six times pretreated with dexamethasone (75 mg/kg/day, i.p.) and prednisolone (69 mg/kg/day, i.p.), respectively, showed significant change of the pharmacokinetic parameters of Dx compared with the control rats. In contrast, using Antiserum-II, it took three and nine times of pretreatment with dexamethasone and prednisolone, respectively, to significantly change the parameters of Dx. In conclusion, these results demonstrate that Antiserum-I is very useful not only to more precisely monitor serum Dx levels, but also to determine earlier the drug-drug interaction with glucocorticoids than Antiserum-II.

Simultaneous determination of amantadine and rimantadine by HPLC in rat plasma with pre-column derivatization and fluorescence detection for pharmacokinetic studies.

We investigated simultaneous high-performance liquid chromatographic (HPLC) determination of amantadine hydrochloride (AMA) and rimantadine hydrochloride (RIM) levels in rat plasma after fluorescent derivatization with o-phthalaldehyde and 2-mercaptoethanol. Afterwards, the method was applied to determine their pharmacokinetics. The retention times of AMA and RIM derivatives were 12.6 and 22.2 min and the lower limits of detection were 0.025 and 0.016 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay of AMA and RIM were less than 5.1 and 7.6%, respectively. After i.v. administration of AMA or RIM to rats, the total body clearance and distribution volume at the steady-state of RIM were higher than those of AMA. Bioavailability of AMA and RIM was 34.9 and 37.2%, respectively. When AMA and RIM were p.o. co-administered, the area under the plasma concentration--time curve of RIM was significantly lower than that after RIM alone. On the other hand, pharmacokinetic parameters of AMA did not significantly change. These results indicate that our HPLC assay is simple, rapid, sensitive and reproducible for simultaneously determining AMA and RIM concentrations in rat plasma and is applicable to their pharmacokinetic studies. Also, co-administration of AMA and RIM may result in the lack of pharmacological effects of RIM.

Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.

Liquid chromatographic determination of 1-adamantanamine and 2-adamantanamine in human plasma after pre-column derivatization with o-phthalaldehyde and 1-thio-beta-D-glucose.

We investigated high-performance liquid chromatographic (HPLC) determination of 1-adamantanamine hydrochloride (1-ADA) and 2-adamantanamine hydrochloride (2-ADA) in human plasma after the derivatization with o-phthalaldehyde (OPA) and 1-thio-beta-D-glucose (TG). Extracted human plasma samples were mixed with OPA and TG at room temperature for 6 min and injected onto HPLC. Retention times of 1-ADA and 2-ADA derivatives were 12.6 and 14.1 min, respectively. The lower limits of detection of 1-ADA and 2-ADA were 0.02 and 0.008 microg/ml, and the lower limits of quantitation of 1-ADA and 2-ADA were 0.025 and 0.01 microg/ml, respectively. The coefficients of variation for intra-day and inter-day assay of 1-ADA and 2-ADA were less than 4.4 and 6.0%, respectively. L-Dopa and dopamine were not found to interfere with the peaks of 1-ADA and 2-ADA derivatives. Human plasma unbound fraction (f(p)) values of 1-ADA varied between 0.32 and 0.48, while those of 2-ADA varied between 0.38 and 0.68. These results indicate that HPLC assay of 1-ADA and 2-ADA by derivatization with OPA and TG is simple, rapid, sensitive and reproducible for determining 1-ADA and 2-ADA in human plasma.

Measurement of beta-methyldigoxin level in serum from patients by enzyme immunoassay using novel specific antiserum with a phenyl boric acid column.

The authors compared serum beta-methyldigoxin (MDx) levels in digitalized patients by enzyme immunoassay (EIA) using anti-MDx 3'-hemisuccinate BSA antiserum (antiserum-I) with commercial antidigoxin antiserum (antiserum-II). The usefulness of a phenyl boric acid (PBA) column for pretreatment of the serum samples was also investigated. The assay using antiserum-I demonstrated good accuracy and precision in the concentration range of 0.5 to 5 ng/mL. When the specificities of antiserum-I and antiserum-II were assessed by cross-reactivity studies with various related compounds, antiserum-I was much more specific for MDx antiserum-II. Using a phenyl boric acid (PBA) column, MDx, and digoxigenin, which exhibits a negligible cross-reactivity, were separated from serum, including MDx and its metabolites. The recovery tests of MDx using antiserum-I with a PBA column in human serum were satisfactory and no interference of metabolites of MDx was observed. Mean MDx concentrations in serum samples (n = 30) from digitalized patients by EIA using antiserum-I with PBA column, antiserum-I, and antiserum-II were 1.06, 1.30, and 1.74 ng/mL, respectively. These results indicate that our EIA system using antiserum-I with a PBA column for pretreatment of serum samples is useful to more precisely measure the unchanged type of MDx in patients.

Pharmacokinetic study of beta-methyldigoxin by enzyme immunoassay using a novel specific antiserum in rats.

We previously showed that enzyme immunoassay (EIA) of beta-methyldigoxin (MDx3) using anti-MDx3 3'-hemisuccinate-bovine serum albumin antiserum (Antiserum-I) was superior to that using commercial anti-digoxin antiserum (Antiserum-II) in terms of specificity and that pretreatment of human serum with phenyl boric acid (PBA) column was effective. In the present study, we examined the precision of EIA using Antiserum-I and the recovery of MDx3 after PBA column treatment in rat serum, and also investigated pharmacokinetic changes of MDx3 in rats. The intra- and inter-assay variations and recovery tests using Antiserum-I were good. The PBA column was effective in selectively separating MDx3 from rat serum containing MDx3 and its metabolites. The recovery tests using Antiserum-I with PBA column showed about 110% and the interference of metabolites of MDx3 was negligible. Serum concentration-time courses of MDx3 by EIA using Antiserum-I with PBA column and Antiserum-I were lower than that using Antiserum-II. The distribution volume at steady state and total body clearance values of MDx3 in these conditions were significantly higher than those using Antiserum-II. The usefulness of PBA column was ascertained, while effects of PBA column on these parameters were not significant. In addition, rapid absorption of MDx3 was observed by EIA using Antiserum-I with PBA column. These results suggest that EIA using Antiserum-I with PBA column for the pretreatment of serum samples should be a more useful and valuable system in therapeutic drug monitoring and pharmacokinetic studies of the unchanged type of MDx3 than Antiserum-II.

Establishment of enzyme immunoassay for measuring beta-methyldigoxin levels in human serum by specific antiserum.

We investigated the specificity of obtained antisera to beta-methyldigoxin by the enzyme immunoassay. Three types of hapten-bovine serum albumin (BSA) conjugates were synthesized to obtain high specific antisera to beta-methyldigoxin. The haptens were linked to the carrier protein through hemisuccinate at C-3' and C-3'' positions in the digitoxose chain and at C-12 position in the aglycone. Anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum showed a low detection limit (0.2 ng/ml) and possessed high specificity for beta-methyldigoxin, exhibiting low cross-reactions with digoxigenin bisdigitoxoside (8.3%), dihydrodigoxin (4.8%), digitoxin (1.5%), and digoxigenin monodigitoxoside (0.95%), except for cross-reaction with digoxin (43%). Compared with commercial antidigoxin antiserum, clinically used to monitor beta-methyldigoxin concentration in human serum, cross-reaction data of anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum showed higher specificity for beta-methyldigoxin. The intra-assay and inter-assay variations using this antiserum were less than 6.9% and 8.1%, respectively. The recovery tests were good, within the range of 96.2-104.3%. Phenyl boric acid (PBA) column treatment was effective to rapidly and selectively separate beta-methyldigoxin from the mixture of beta-methyldigoxin and its metabolites in human serum. The recovery tests of beta-methyldigoxin with PBA column in human serum were about 110% and interference of metabolites of beta-methyldigoxin was negligible. These results suggest that anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum and PBA column treatment are useful to more precisely monitor the unchanged type of beta-methyldigoxin concentration in human serum.

Modulation of mdr1a and CYP3A gene expression in the intestine and liver as possible cause of changes in the cyclosporin A disposition kinetics by dexamethasone.

We investigated the effect of dexamethasone (DEX) on the disposition kinetics of cyclosporin A (CyA) and the mechanism of this drug interaction. Rats were treated with DEX (1 or 75mg/kg per day, i.p.) once a day for 1-7 days, and the blood concentration of CyA was measured after an i.v. or p.o. dose of CyA (10mg/kg) at 1.5hr after the last DEX treatment. In rats treated with a low dose of DEX (1mg/kg), the blood concentration of CyA after i.v. administration was unchanged compared with that of untreated rats, whereas the blood concentration after oral administration was significantly decreased, and this decrease was dependent on the duration of DEX administration. The total clearance (CL(tot)) of CyA was unchanged, but the bioavailability was significantly decreased to about one-third of that in DEX-untreated rats after 7 days of DEX treatment. At this time, the expression of mdr1a mRNA and P-gp in the liver and intestine was increased, whereas CYP3A2 was unaffected at both the mRNA and protein levels. In rats treated with a high dose of DEX (75mg/kg), the blood concentration of CyA was significantly decreased after both i.v. and p.o. administrations compared with those of untreated rats. The bioavailability of CyA was decreased, and the CL(tot) was significantly increased. The P-gp and CYP3A2 in the liver and intestine were increased at both the mRNA and protein levels. Our results indicate that the drug interaction between CyA and DEX is a consequence of modulation of P-gp and CYP3A2 gene expression by DEX, with differential dose-dependence.