The absorption and excretion of radioactivity were investigated in Sprague-Dawly rats following a single oral administration of ¹⁴C-labeled OPB-2045, and the following results were obtained:
1. Serum levels of radioactivity reached the maximum of 0.09-0.11μg eq. /ml at 30 min in male and female rats after oral administration of ¹⁴C-OPB-2045 at 10 mg/kg. The half-life of radioactivity in serum was 5.9-8.6 hr as determined at 2 hr to 24 hr after administration. AUC_0-infinite was 0.46-0.59 μg eq.·hr/ml. Radioactivity in blood cells accounted for 7.5-53.1% of total radioactivity in the blood.
2. Urinary and fecal excretion of radioactivity was 1.2-4.6% and 92.1-94.5% of the administered dose, respectively, in rats receiving oral doses of 10 mg/kg. Biliary excretion of radioactivity was 2.9% of the dose within 24 hr after oral administration.
3. No sex difference in absorption or excretion of ¹⁴C-OPB-2045 was observed in rats after oral administration of the compound.

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1. The pharmacokinetics of OPB-2045, a new antiseptic antimicrobial agent possessing a biguanide moiety in its molecule, was investigated in Sprague-Dawley rats after a single oral, subcutaneous, and intravenous administrations. A high-performance liquid chromatographic method with detection limit of 8 ng/ml was developed and then used to determine serum concentration of the compound.
2. Following single oral doses at 10, 100, and 1, 000 mg/kg, serum OPB-2045 could be detected only at 1, 000 mg/kg with the concentration below 15 ng/ml.
3. The time-courses of serum OPB-2045 were almost flat in rats after subcutaneous administrations of the compound at 1, 3, 10, and 30 mg/kg, and no dose-dependent increase of serum concentration was observed.
4. Adequate OPB-2045 serum profiles following intravenous administrations at 1, 3, and 6 mg/kg could be obtained using a two-compartment open model with AIC of -3.0 ?? -58.1. Serum OPB-2045 after intravenous administration exhibited a biphasic decline with half-lives of 0.25 ?? 0.32 hr in the α-phase and 2.16 ?? 4.07 hrs in the β-phase. The volume of distribution in the β-phase was 9.8 ?? 19.8 l/kg, suggesting considerable diffusion of the compound. Total serum clearance of OPB-2045 was 2.0 ?? 3.9 l/hr/kg.
5. The bioavailability of OPB-2045 after oral administration thus appeared to be lower than that subsequent to subcutaneous administration.

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Plasma concentration-time profile, distribution, metabolism and excretion of [¹⁴C]NB-506 were investigated in male and female rats after single and repeated intravenous administration.
1. After a single intravenous administration of [¹⁴C]NB-506 to male rats, plasma levels of radioactivity decreased triexponentially with the t_1/2 of 22 min, 2.4 hr and 2.1 day. The pharmacokinetic parameters obtained from female rats, were similar to those in male rats.
2. After a single intravenous administration of [¹⁴C]NB-506 to male rats, the maximum concentration in almost all tissues was observed at 10 min. The radioactivity was highly distributed in liver followed by kidney, lung, mandibular gland, skin and pancreas. At 24 hr post dose, the radioactivities in tissues were almost same as that of plasma except liver and kidney, and then decreased and were lower than 10% of maximum radioactivity at 72 hr.
3. Within 120 hr after a single intravenous administration of [¹⁴C]NB-506 to male rats, 9.8 and 88.3% of dose were excreted into urine and feces, respectively. Biliary excretion was 82.9% in male rats. The enterohepatic circulation of [¹⁴C]NB-506 was not observed in male rats. The excretion of radioactivity in female rats was similar to that in male rats.
4. After a single intravenous administration of [¹⁴C]NB-506 to male rats, 40.0, 23.8, 6.3 and 1.5% of dose were excreted to bile (0-6 hr) as intact NB-506, NB-506 glucuronide (ED-594), NB-506 deformyl form (ED-501) and ED-501 glucuronide (ED-595), respectively. The 6.2% of administered dose in urine (0-4 hr) corresponded to intact NB-506 and 0.7% of dose was excreted to urine as ED-594 and ED-501.
5. The plasma levels of radioactivity at 5 min, 4, 24 hr after the 5th dosing were higher than those after the first dosing. Moreover, the levels of AUC_(0-∞) and t_1/2γ of the 5th dosing were 2.1 and 2.3 times higher than those of the first dosing, respectively. These results indicate that [¹⁴C]NB-506 tend to accumulate in plasma after the multiple dosing. The accumulation ratio was calculated based on the plasma levels of radioactivity at 24 hr after the first and the 5th dosing; this value was 3.85. On the other hand, the accumulation factor, which was estimated from the elimination rate constant, was calculated to be 3.56. Therefore, the accumulation ratio was almost of the same value as the predicted accumulation factor. These results suggest that it is possible to predict the disposition of [¹⁴C]NB-506 after the multiple dosing and the possibility that the crucial accumulation of [¹⁴C]NB-506 may arise from the multiple dosing is low.
6. No change in the daily excretion of radioactivity in urine and feces was observed during 5-day repeated administration. Within 168 hr after the last dosing, urinary and fecal excretion of radioactivity were 7.5 and 90.1% of dose, respectively, indicating that the main elimination route is fecal excretion.

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To investigate the metabolism and disposition of OPB-2045, 1-(3, 4-dichlorobenzyl)-5-octylbiguanide, 14C-labeled OPB-2045 was subcutaneously injected to male Sprague-Dawley rats at a dose of 1 mg/kg and the resulting radioactivity in the urine and bile was analyzed using a high-performance liquid radiochromatography. The radioactivity retained at the injection site was also analyzed.
1. The metabolites excreted in the urine included 6-[5-(3, 4-dichlorobenzyl)-1-biguanidino] hexanoic acid (DM-210), 4-[5-(3, 4-dichlorobenzyl)-1-biguanidino] butanoic acid (DM-212), 5-[5-(3, 4-dichlorobenzyl)-1-biguanidino] pentanoic acid (DM-213) and 3, 4-dichlorobenzoic acid. Within 72 hr after the dosing, the metabolites DM-210, DM-212, DM-213, and 3, 4-dichlorobenzoic acid excreted in the urine accounted for 4.6, 1.6, 1.5 and 1.4% of the administered dose, respectively. No excretion of the unchanged compound was detected in the urine.
2. The metabolites excreted in the bile included DM-210, DM-212 and DM-213. The combined excretion rate of these metabolites accounted for 15.6% of the dose within 24 hr postdosing. No excretion of 3, 4-dichlorobenzoic acid or the unchanged OPB-2045 was detected in the bile.
3. The radioactivity retained at the injection site at 1, 8 and 24 hr postdosing accounted for 51.5%, 34.6% and 13.0% of the administered dose, respectively. The unchanged compound was found to account for at least 80.8% of the radioactivity, and no metabolites including DM-210, DM-212, DM-213 and 3, 4-dichlorobenzoic acid, were detected. These results indicated that OPB-2045 was not metabolized by the skin/tissues at the injection site, but instead by other organ(s).

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RS-8359 is a selective and reversible inhibitor of A type monoamine oxidase. In order to elucidate the metabolism of RS-8359, the metabolites were isolated from the urine after oral administration to rats and dogs. The chemical structures of the seven isolated metabolites, including the unchanged form, were determined by NMR and mass spectrometry. Hydroxylation on the cyclopentanol moiety and oxidation on the pyrimidine moiety of the molecule were preferentially responsible for the phase I metabolism. The tentative main metabolic pathways of RS-8359 in the rat and dog are proposed and discussed.

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Sulpho-conjugation and glucuronic acid conjugation of 4 stereoisomers of troglitazone, a new anti-diabetic agent, were comparatively investigated in vitro using microsomal and cytosolic fractions of livers from male KK and male RFVL mice, male and female F344 rats, male cynomolgus monkeys and male and female humans. Isomerization at the 5-position of the thiazolidinedione ring was relatively rapid in the incubation medium, while there was no marked difference in the conjugation reactions between the stereoisomers at this position. Therefore, the comparison was made between the stereoisomers at the 2-position, i.e., 2S(5S or 5R) isomer and 2R(5S or 5R) isomer. Following results were obtained:
1. There was no marked species difference in the sulpho-conjugation reaction. The 2S isomers were more rapidly sulpho-conjugated than the 2R isomers. Rats exhibited a sex-related difference in this conjugation reaction, and the activity in male rats was about two times greater than that in female rats.
2. There was a species difference in the glucuronic acid conjugation reaction, as revealed by much higher activities in mice and monkeys than in other animal species. KK mice showed a higher glucuronidation activity for the 2R isomers than the 2S isomers. In contrast, other animal species, including the human, showed a higher activity for the 2S isomers than the 2R isomers.

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Uricase from Candida utilis, an enzyme containing essential thiol, was examined for its sensitivity to myeloperoxidase (MPO), an oxidant present in neutrophils. Upon exposure to a system composed of MPO, hydrogen peroxide and bromide at moderately acidic pH, but not at neutral to alkaline pH, the uricase lost almost all of its enzyme activity. Thus the MPO-H₂O₂-bromide system significantly inactivated uricase only at moderately acidic pH. This inactivation by the MPO system was prevented by the presence of N-acetylmethionine, a thioether compound, or glutathione, a thiol compound analogous to amino acid, the remaining uricase activity being respectively 86.0±2.0 and 81.0±2.0% (mean±SEM, n=5). These findings suggest that the inactivation was due to oxidation and damage of thio-group present in the catalytic site of uricase by the MPO system. They also suggest that methionine and glutathione modulate the strength of an oxidative stress by this MPO system. Therefore, it appears that these compounds are clinically applicable as a therapeutic antioxidant in leukocytoses such as inflammatory diseases and granulocytic leukemia.

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The pharmacokinetic study of nicardipine in the same human after intravenous and oral adminstration was examined. The profile of nicardipine after bolus 1.05 mg injection fit well to a bi-exponential curve. Plasma nicardipine concentrations for constant rate infusion at 1.27 mg/hr for 3 hr declined from 3 to 7 hr with the same pattern as the bolus injection, but exhibited another slope after 7 hr that declined more gently. The γvalue was 0.0463 hr^-1, 1/5 the β-values. On the other hand, absolute bioavailability values for Perdipine LA® sustained-release capsule containing 40 mg nicardipine as hydrochloride salt after single and multiple doses were calculated by dividing AUCPO by AUCIV, correcting for administered dose. Bioavailability values calculated based on the bolus injection (about 20%) represented a 2-fold difference when compared to values derived from constant rate infusion data. Consequently, differences in bioavailability values must be considered carefully to calculate bioavailability accurately. When the γ-phase is detected as in the case of nicardipine disposition, we need to select the intravenous administration for detecting γ-phase.

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With the increasing need for reliable and comprehensive pharmacokinetic data from diverse nonclinical as well as clinical studies, a sensitive and specific bioanalytical technique is essential. Such a technique is liquid chromatography-mass spectrometry (LC-MS). This article provides a brief overview of the key developments in LC-MS and focuses on the more recent LC-MS ionisation/interfacing technique, that of atmospheric pressure ionisation (API). The advantages of LC-API-MS/MS for routine bioanalysis, in terms of sensitivity, specificity and reproducibility are discussed. Recent scientific literature is cited that exemplifies the capabilities, applications and usefulness of this powerful analytical technique in pharmacokinetics/toxicokinetics studies.

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To date, it is very difficult to extrapolate the data obtained from experimental animals (for example rodents) to humans, since there are species differences in the characteristics of drug metabolising enzymes. The monkeys is often used for the preclinical study. However, the similarities for properties and capacities of drug metabolizing enzymes between monkeys and humans were not well recognized. Therefore, we started to characterize the drug metabolizing enzymes, especially cytochrome P450s, in non-human primates.
Four forms of cytochrome P450 enzymes (termed P450 CMLa-d) were purified from hepatic microsomes of cynomolgus monkeys. P450 CMLa was classified into the CYP2B subfamily, in its N-terminal amino acid sequence, catalytic activities, and immunochemical properties and act as a testosterone 16, 8-hydroxylase in cynomolgus monkseys. P450 CMLa is expressed constitutively as a minor form of P450 and is one of the forms inducible by phenobarbital. The nucleotide sequence of a cDNA coding for the monkey CYP2B was also determined and is now called CYP2B17. P450 CMLb and P450 CMLc purified from hepatic microsomes of cynomolgus monkeys were classified into CYP2A and CYP3A subfamilies, respectively. The content of the protein immunoreactive with anti-P450 CMLc in cynomolgus monkeys was decreased by the administration of aztreonam. P450 CMLd was one of CYP2C enzymes and was the S-mephenytoin 4'-hydroxylase in cynomolgus monkeys. The cDNA coding for monkey CYP2C (reffered to as CYP2C37) was isolated and was expressed in yeast. The expressed protein act as S-mephenytoin 4'-hydroxylase.

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Liver microsomal carboxylesterases (E. C. 3.1.1.1) function in the hydrolysis of a wide variety of endogenous and xenobiotic compounds, and play an important role in drug and lipid metabolism in many mammalian species. Multiple isozymes of liver microsomal carboxylesterases exist in various animal species, and the enzyme is involved in the metabolic activation of ester and amide-type prodrugs. In the present study, twenty-seven carboxylesterase isozymes were purified to electrophoretic homogeneity from liver microsomes of ten mammalian species and humans, and their physical, enzymological and immunological properties were compared with each other. And also we tried to compare the primary structure of carboxylesterase isozymes in various animal species and humans using cDNA cloning and analysis. The carboxylesterase isozymes from various species examined here showed considerable similarities in physicochemical and immunochemical properties, but not similar in substrate specificity. The deduced amino acid sequence of the clone possessed many structural characteristics that are highly conserved among rat RLl, RH1, RHlec, RS1, RS2, mouse ML1, MH1, MS1, guinea pig GP, beagle dog D1 and human HU1, including active site sequence (GESAGG, NKQEXG, GDHXD), and four cysteines that may be involved in the specific disulfide bond. It is well known that proteins that are retained in the endoplasmic reticulum (ER) lumen contain the retention signal at their carboxy terminal of the tetrapeptide (KDEL-COOH). The seven carboxylesterase clones (RL1, RH1, RHlec, MH1, GP, Dl and HUI) also contained an ER-retention signal of carboxylesterase (HXEL), and three clones (RS1, RS2, MS1) did not contain this peptide. When clone was expressed in COST and V79 cells, the plasmid-coded protein was retained. The cells expressing carboxylesterase is very high activity towards xenobiotic ester and amide. Site-specific mutation of the three amino acid residues of catalytic triad (Ser203 to Thr203, Glu335 to Ala 335, or His 448 to A1a448) greatly reduced the carboxylesterase activity. In conclusion, liver microsomal carboxylesterase in mammals and humans are closely involved in drug and lipid metabolism in the endoplasmic reticulum, and it is noteworthy that the isozymes from various species examined here showed considerable similarities in amino acid sequences, but not similar in substrate specificity. These reasons may be due to the variances of substrate binding site.

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