Marograstim (KW-2228) is a derivative of human granulocyte colony-stimulating factor(rhG-CSF). In order to determine biological activity of KW-2228 in the plasma, a bioassay method of G-CSF was developed. Determination limit of the bioassay was 2ng/ml for plasma samples. A comparison of plasma concentrations, determined by the bioassay and the enzyme-linked imm unosorbent assay using specific anti KW-2228 antibody (ELISA), was performed on samples after intravenous or subcutaneous administration of KW-2228 to human volunteers (2μg/kg), monkeys (10μg/kg), rats (50μg/kg) and mice (50μg/kg). In all species, there was no difference between the plasma concentrations of KW-2228 determined by the bioassay and ELISA. There was a significant correlation between plasma concentrations of KW-2228 determined by the bioassay and ELISA (p<0.01). This result suggests that ELISA is a specific method to measure only the drug retaining biological activity of GCSF. Therefore the ELISA is thought to be a very useful method for pharmacokinetic and pharmacodynamic studies. Plasma concentration of KW-2228 measured by the bioassay showed slower elimination after subcutaneous administration than after intravenous administration. The route of subcutaneous administration is the better method for maintenance of plasma concentration. Based on the pharmacokinetic parameters of KW-2228 after intravenous and subcutaneous administration there was no species difference in drug disposition.

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Pharmacokinetics of Marograstim (KW-2228) was studied in rats with cyclophosphamide (CY)-induced leukopenia. On 5 days after CY-treatment, the rats were administered 5 or 50μg/kg of KW-2228 intravenously or 50μg/kg subcutaneously.
1. After intraperitoneal administration of 100mg/kg of CY to rats, the white blood cell (WBC) and platelet (PLT) counts in the peripheral blood were decreased to about 1/10 and 1/2 of the control rats, respectively. Red blood cell (RBC) count, GOT, GPT and BUN did not differ from those in the control rats, suggesting that neither hepatic nor renal failure occ urred. KW-2228 administration increased WBC count in the control rats, but not in the CYtreated rats.
2. After intravenous administration of 5 or 50μg/kg of KW-2228, the KW-2228 disappeared biexponentially from the plasma both in the control and CY-treated rats. During the elimination phase, the plasma levels of KW-2228 in CY-treated rats were significantly higher than those in the control rats. The elimination half-lives and mean residence time (MRT) in CY-treated rats were prolonged by 1.6 to 1.9 times over the control group. The results of the compartment model analysis including a Michaelis-Menten type elimination were in good agreement with actual measurements in both groups. In this model, V_max in CY-treated rats was 9.678ng/ml/hr, as low as about 1/2 of the control rats, however, there was no difference in the Km of both groups, 5.012 and 5.645ng/ml, respectively. This suggests that WBC will play an important role in the saturable elimination of KW-2228.
3. After subcutaneous administration of 50μg/kg of KW-2228, its plasma levels reached the C_max 2.5hr after administration and then showed monoexponential elimination in both groups. The elimination half-life in the CY-group was about 1.7-fold and the MRT about 1.4-fold longer than in the control. In contrast the C_max in CY group was significantly reduced to about 70% of the control. Bioavailability hardly differed between the control (49.9%) and the CY group (45.8%).

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The tissue levels of KW-2228 in rats were determined by the ELISA after intravenous or subcutaneous administration.
1. At 5 min after intravenous administration of 50μ g/kg of KW-2228, the plasma level of KW-2228 was 846.0±49.4ng/ml (mean±S.D.), and then biexponentially disappeared, reaching 2.976±0.62ng/ml at 8hr. The elimination half life of KW-2228 in plasma was 1.23hr. The levels of KW-2228 in tissues were lower than those in plasma at all time points. The kidney showed higher level of KW-2228 than any other tissues, but C_max in kidney was 214.9±26.7 ng/g, about 1/4 of that in plasma. The C_max in other tissues were lower than in kidney, ranging 1/10 to 1/40 of that in plasma. The elimination half-lives of KW-2228 in spleen and bone marrow were 2.48 and 3.08hr, respectively, which were 2 ?? 3 fold longer than those in plasma or other tissues.
2. After subcutaneous administration, the level of KW-2228 in plasma reached the C_max of 118.3±22.8ng/ml at 2hr then monoexponentially disappeared. The half-life of KW-2228 in plasma was 1.72hr. The levels of KW-2228 in tissues also reached the C_max at 2hr, and then monoexponentially disappeared as in plasma. C_max in tissues were in the following order, kidney, lung, heart, adrenal gland, liver, spleen and bone marrow. The Cmax in kidney was 7/10 of that in plasma, and in other tissues, below 1/7. The half-lives of KW-2228 in kidney, lung, heart, liver and adrenal gland were 1.39 ?? 2.33hr, almost same as in plasma. However, the half-lives in spleen and bone marrow were 2.70 and 4.28hr, longer than those in other tissues. In all tissues, the half-lives of KW-2228 after subcutaneous administration were longer than those after intravenous administration. The mean residence time of KW-2228 in the bone marrow, its target tissue, after subcutaneous administration, was longer than that after intravenous administration. The difference in effectiveness of KW-2228 resulting from the administration route might be due to the difference of the mean residence time of KW-2228 in the bone marrow.

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The absorption, distribution and excretion of PERGOLIDE MESYLATE have been investigated in rats and monkeys after administration of a single oral dose of ¹⁴C-PERGOLIDE MESYLATE labeled on the thiomethylene carbon.
1. Male rats, receiving orally 2 mg/kg of ¹⁴C-PERGOLIDE MESYLATE, had maximum blood concentrations of 28 ng-euuivalents/ml of PERGOLIDE 2hr after dosing. Blood levels of radioactivity decreased with a half-life of 45 hours. The concentration of radioactivity in the blood increased in a dose-dependent manner at doses ranging from 1 ?? 5 mg/kg. Female rats dosed orally with 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE had blood concentrations similar to those observed in male rats. However, female rats exhibited two peaks of radioactivity at 2 and 24 hours after dosing. Peak blood concentrations of 57ng equivalents/ml of PERGOLIDE were achieved in male rats 30min after intravenous dose of 1mg/kg of ¹⁴C-PERGOLIDE MESYLATE. The half-life of radioactivity in the blood, as measured between 0.5 and 4 hours, was 4.0 hours, and 29 hours when measured from 6 ?? 48 hours. In male monkeys, the peak plasma concentrations of 326ng equivalents/ml were observed 1 hour after an oral dose of 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE. The half-life of radioactivity was 5.4 hours.
2. Male rats dosed orally with 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE excreted 18.2%of the dose in the urine and 75.7% in the feces during 168 hours after dosing. Similar excretion patterns were observed in fasting male rats, female rats, and in male rats receiving either 1 or 5mg/kg of the drug. After intravenous administration of 1mg/kg of ¹⁴C-PERGOLIDE MESYLATE, male rats during 168 hours after administration excreted 23.3% and 69.9% of the dose in the urine and feces, respectively. In female rats dosed orally with 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE, corresponding excretion was 21.4% and 72.2% of administered dose. Male monkeys, dosed p.o. with 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE, excreted 54.3% and 39.4% of the dose in the urine and feces, respectively, during 240 hours after dosing.
3. Bile duct cannulated male and female rats, dosed orally with 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE, excreted 17.4% and 16.0% of the dose, respectively, in the bile during 48 hours after dosing. Entero-hepatic circulation was observed after intraduodenal injection of radioactive bile.
4. After oral administration of 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE to male rats, high concentrations of radioactivity were noted in the gastro-intestinal tract, liver, kidney and submaxillary gland, while low levels were found in the central nervous system and eyeball. Radioactivity was eliminated slowly from the thyroid gland, spleen, kidney and brown fat.

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The absorption, distribution and excretion of ¹⁴C-PERGOLIDE MESYLATE were investigated after daily oral administration to male rats.
1. The radioactivity in the blood of rats receiving daily oral doses of ¹⁴C-PERGOLIDE MESYLATE showed a continuous increase at 24 hours after each dose for up to 18 doses. The amount of radioactivity in the blood 24 hours after the 18th dose was 4.2 times higher than that observed at 24 hours after the first dose. The rate of elimination of radioactivity from the blood after the 21st dose was slower than that observed after a single dose.
2. The excretion of radioactivity in the urine and feces reached near steady state after 4 doses. The excretion of radioactivity in the urine and feces was 24.4% and 72.1% of the cumulative dose, respectively, during 168 hours after the 21st dose.
3. The distribution of radioactivity in the tissues was measured 24 hours after the 1st, 7th, 14th and 21st dose, and in most tissues the radioactivity accumulated throughout the dosing period. The radioactivity accumulated greatly in the spleen, submaxillary gland, thyroid gland, bone marrow and kidney with continuous dosing. The radioactive content in the rest of the tissues after the 21st dose was less than 6 times as high as that after the 1st dose. Also, the elimination of radioactivity from all tissues was slower after the 21st dose than that after the 1st dose.

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The plasma protein binding, site of absorption, and placental and mammary transfer of ¹⁴C-PERGOLIDE MESYLATE were investigated after oral administration to rats and monkeys.
1. In pregnant rats receiving orally 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE on the 12th day of gestation, the peak plasma concentrations of radioactivity were found 2 hours after dosing and were two times higher than the levels found in the fetus, indicating a low placental transfer. The radioactivity in the fetal liver, lung and kidney 48 hours after dosing on the 18th day of gestation was 2.0 ?? 5.9 times higher than that found in maternal plasma.
2. The concentration of radioactivity in the milk peaked at 2 and 24 hours after dosing and declined with a half-life of 9.0 hours during the observation period of 72 hours. The milk contained 14 times higher radioactivity than that found in the plasma 24 hours after administration.
3. In vivo binding to plasma proteins was 70.3 ?? 72.4% in male rats and 62.3 ?? 69.1% in male monkeys. Plasma protein binding in vitro as determined at a concentration of 1000ng/ml of PERGOLIDE to male rats, monkeys and human plasma, ranged from 97.1 ?? 97.8%. L-DOPA had no effect on these in vitro binding ratios.
4. Six hours after injection of radioactivity into various loops of prepared from the gastrointestinal tract of male rats, the order of residual radioactivity was stomach >ileum, colon, rectum >duodenum >jejunum.

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1. After repeated oral administration of 0.06 or 2 mg/kg of PERGOLIDE MESYLATE to male rats, liver microsomal protein content, cytochrome p-450 content, aniline hydroxylase activity and p-nitrophenyl UDP-glucuronyltransferase activity were elevated compared to controls. Aminopyrine N-demethylase activity was higher than controls only in the group receiving 0.06mg/kg dose.
2. After oral administration of 2 mg/kg of ¹⁴C-PERGOLIDE MESYLATE, the plasma samples collected at 0.5 ?? 24 hours after dosing contained PERGOLIDE SULFOXIDE and an unknown metabolite, M7, as major metabolites and PERGOLIDE as a minor component. Unknown metabolites, M6 (conjugate of PERGOLIDE or unknown metabolite M10) and M8, as well as PERGOLIDE SULFOXIDE were major metabolites excreted in the urine. PERGOLIDE was a major component excreted in the feces. The bile contained two major unknown metabolites, M6 and M8. Major radioactive spots, corresponding to PERGOLIDE and PERGOLIDE SULFONE, were found in the liver, PERGOLIDE, PERGOLIDE SULFOXIDE and PERGOLIDE SULFONE were found to be major metabolites in the lung. However, in the kidney, PERGOLIDE was the major component at 2 hours after dosing while at 24 hours the major component in the kidney was the unknown metabolite, M5. The relative amounts of metabolites after repeated oral dosing were similar to those observed in the group receiving a single oral dose.
3. PERGOLIDE was a minor component and the unknown metabolites, M8 and M2, were major components in the plasma and urine of male monkeys dosed orally with 2mg/kg of ¹⁴C-PERGOLIDE MESYLATE. None of the metabolites found in monkey plasma was present in the form of a conjugate.

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The distribution of sodium prasterone sulfate (PS, a common name is sodium dehydroepiandrosterone sulfate) was studied in pregnant rats after single and multiple vaginal administration of ¹⁴C-PS.
The radioactivity was high in the vagina, liver and kidney, and low in the central nervous system and fat tissues after single vaginal administration.
During multiple vaginal administration, plasma levels of radioactivity and the rate of urinary and fecal excretion of radioactivity were almost constant. Tissue distribution of radioactivity after multiple vaginal administration was similar to that after single vaginal administration. There were no tissues showing a pronounced retaining of the radioactivity after the multiple vaginal administration.
Unchanged PS and its metabolites were transported to the cervix of uterus being an action site for PS and its some metabolites, to much higher extent after vaginal administration than after intravenous administration.

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The metabolism of sodium prasterone sulfate (PS) was investigated in pregnant rats after vaginal administration. Major metabolites in rat urine and bile were identified by means of thin layer chromatography/secondary ionization mass spectrometry and capillary gas chromatography/mass spectrometry. The urinary and biliary excretion of identified metabolites according to above menntioned methods were determined after vaginal administration of ¹⁴C-labelled PS. Main metabolites in the urine and bile were androst-5-ene-3β, 17β-diol 3-sulfate (3β, 17β-diol-MS) and androst-5-ene-3β, 17β-diol 3, 17-disulfate, respectively. Androst-5-ene-3β, 7α-diol 3-sulfate, androst-5-ene-3β, 7β-diol 3-sulfate and androst-5-ene-3β, 7α, 17β-triol 3-sulfate were also identified as minor metabolites in the urine and/or bile. Furthermore, the excretion ratio of unchanged PS in the urine and bile after vaginal administration was lower than that after intravenous one. PS was also converted to 3β, 17β-diol-MS during the incubation with the 9000 × g supernatant of the vaginal membrane including uterine cervix. These results suggest that a part of PS was metabolized to 3β, 17β-diol-MS during the process of the absorption after vaginal administration.

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Glutathione(GSH)has various important roles in maintaining cell functions. It is a major non-protein tissue thiol, supporting the redox potential of cells, regulating enzyme activities, processing endo-or exogenous substances and protecting cells from deleterious effects of active oxygen species and free radicals. GSH has been shown to be a regulator of gluconeogenesis, glycolysis or cholesterol biosynthesis, and, more recently, a component of several autacoids. Many reactive xenobiotics bind to SH-group of GSH and are transported out of the cells. Elevation of circular or tissue GSH levels is often effective for preventing the development of toxic and pathological processes. Various attempts have been made to explore the defense action of GSH, but our studies have led to an interesting finding that prostaglandin A₂ (PGA ₂) promotes biosynthesis of GSH and protects cells from oxidative stress. The finding suggests that PGA₂ may be involved in regulating the physiological functions of cells by affecting the GSH status. The extension of work on the GSH status should reveal answers to the elaborate mysteries of bioorganisms.

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