We have recently reported that cepharanthine (CEP) incorporated in liposomes increased IgM antibody production to a greater extent than CEP solution [Shinoda, et al.; Drug Delivery System, 10: 43-48 (1995)]. In order to provide some insights into the relationship between the immuno-modulating activity of liposomal CEP and its biodistribution, this study examined the disposition of liposomal CEP after intraperitoneal (i.p.) administration to mice in comparison with CEP solution. The liposomes were composed of egg phosphatidylcholine and cholesterol in a molar ratio of 7 : 2. Multilamellar liposomes (MLV) were prepared by vortexing dried lipid films, and small unilamellar liposomes (SUV) by ultrasonication of MLV. CEP-containing liposomes and CEP solution (as a control) were injected intraperitoneally to mice. The half-life and mean residence time of CEP were longer after administration of liposomal CEP compared with CEP solution. After CEP injection in liposomes or solution, the mean transit time of CEP in the spleen was higher than those in the liver and lung. However, the liposomes containing CEP reduced the tissue distribution of CEP into the liver, lung, and spleen, compared with CEP solution. In conclusion, it was suggested that immuno-modulating action of CEP may not be directly related to the in vivo disposition of CEP.

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Plasma concentration-time profile, distribution and excretion of ONO-1101 after a single intravenous administration were investigated in male and female rats.
1. AUC_0-∞ obtained from plasma levels of radioactivity following intravenous administration of ¹⁴C-ONO-1101 at the doses of 0.3, 1, 3, 10 mg/kg to male and female rats, increased approximately in proportion to the dose, and linearity to the dose was observed. The half-life of terminal phase was 27-33 min. There was no difference in the plasma concentration-time profile between male and female rats.
2. Following intravenous administration of ¹⁴C-ONO-1101 to male and female rats at the dose of 1 mg/kg, the kidney, liver, lung, trachea, skin and carcass showed high levels or high distribution of radioactivity within 15 min and declined to about or below detection limit in almost all tissues at 24 hr. Radioactivity levels in the brain and fetus were much lower than in the plasma.
3. Following intravenous administration of ¹⁴C-ONO-1101 at the dose of 1 mg/kg to lactating rats, the radioactivity in milk reached the highest levels (278.8 ng eq/ml) at 1 hr and then began to decline. At 24 hr the radioactivity in milk was 2.4 ng eq/ml.
4. Within 72 hr after intravenous administration of ¹⁴C-ONO-1101 at the dose of 1 mg/kg, 85.6% and 9.9% of administered radioactivity were excreted into urine and feces in male, 84.2% and 12.0% in female rats, respectively.
5. Within 24 hr after intravenous administration of ¹⁴C-ONO-1101 at the dose of 1 mg/kg, 6.1% of administered radioactivity were excreted into bile in male, 9.5% in female rats. Sex-difference was observed.

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Plasma concentration-time profile, distribution and excretion of ONO-1101 after repeated intravenous administration were investigated in rats. Also the influence on the hepatic drug metabolizing enzyme activities was examined.
1. The radioactivity in plasma at 24 hr after each intravenous administration of ¹⁴C-ONO-1101 at the dose of 1 mg/kg/day for 7 days was not detected or was around the detection limit. Repeated administration had no effect on plasma concentration-time profile.
2. Following repeated intravenous admini stration of ¹⁴C-ONO-1101 to male rats at the dose of 1 mg/kg, the kidney, liver, lung, trachea contained high levels of radioactivity that declined to about or below detection limit in almost all tissues at 24 hr. Also in skin and carcass, a high degree of radioactivity were observed within 15 min then declined. Compared with a single administration, the elimination of radioactivity in kidney and liver was slightly delayed. But repeated intravenous administration was not associated with any accumulation of radioactivity in organ or tissue.
3. The daily excretion of radioactivity in urine and feces was constant since 4-th administration. After repeated intravenous administration, the excretion of radioactivity into urine and feces was respectively 89.7, 14.9%, for male, 86.5, 17.0% for female rats.
4. Repeated intravenous administration of ONO-1101 at the doses of 1, 10, 50 mg/kg/day for 7 days had no effect on microsomal protein content, liver weight and hepatic drug metabolizing enzyme activities.

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The metabolism of ONO-1101 after intravenous administration was examined in rats and dogs. Beside species-related differences in metabolic rate, the effects of drugs with ester group on the metabolism of ONO-1101 in human plasma and the binding to serum protein were also investigated.
1. After a single intravenous administration of ¹⁴C-ONO-1101 in male rats, 81.8% of blood radioactivity at 5 min consisted of M-1, and 2.1% was ONO-1101. Main metabolite in urine and bile was M-1, and ONO-1101 was less than 7.0%.
2. After a single intravenous administration of ONO-1101 in male dogs, the concentrations of ONO-1101 and M-1 in plasma at 5 min were 1.87 μg/ml and 1.35 μg/ml, respectively. At 30 min, M-2, the β-oxidized of M-1, was detected besides ONO-1101 and M-1. Within 24 hr after intravenous administration of ONO-1101, 38.1%, 40.5% and 2.7% of administered dose were excreted to urine as M-1, M-2 and ONO-1101, respectively. Within 8 hr after intravenous infusion, 0.10% and 2.83% of administered dose were excreted to bile as M-1 and ONO-1101, respectively.
3. Enzymatic activity for the hydrolysis of ONO-1101 was higher in rat, human and dog plasma in that order. The major enzyme that hydolyzed ONO-1101 was supposed to be pseudocholinesterase in human plasma and carboxylesterase in rat plasma and dog liver.
4. Acetylcholine and enalapril did not affect t he hydolysis of ONO-1101 inhuman plasma. Procaine and succinylcholine, known as the substrate of pseudocholinesterase, competitively inhibit the hydrolysis of ONO-1101.
5. The serum protein bindings of ¹⁴C-ONO-1101 (0.1 μg/ml) in rat, dog and human were 2.8, 21.3 and 1.6%, respectively.

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Plasma concentration-time profiles of ONO-1101 and its metabolites after intravenous administration were investigated in male beagle dogs. Additionally the influence of anesthesia on the plasma concentration-time profile was investigated after intravenous infusion in male beagle dogs.
1. Following intravenous administration of ONO-1101 at the doses of 0.3 and 3.0 mg/kg, the AUC_0-∞ obtained from plasma levels of the unchanged form increased approximately in proportion to the dose. The half-life of terminal phase was 6 min and its metabolites, M-1 and M-2, were slowly eliminated at the dose of 3.0 mg/kg.
2. Compared with anesthetized dogs, plasma levels of ONO-1101 and its metabolites during and after intravenous infusion were higher in conscious dogs though significant difference was not observed.

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[¹⁴C]menaquinone-4, a therapeutic agent for osteoporosis, was administered orally to male dogs at a dose of 4 mg/kg, and the absorption, distribution, metabolism and excretion were investigated.
1. The plasma concentration of radioactivity reached the maximum level (C_max) of 4540.4 ng eq./ml at 1 to 2 hours after administration and thereafter decreased slowly. At T_max, the unchanged menaquinone-4 in the plasma corresponded to 79.6% of the total radioactivity.
2. Within 168 hours, 2.3 and 78.1% of the radioa ctivity was excreted into the urine and feces, respectively. Unchanged menaquinone-4 corresponded to 28.5% of the radioactivity in the feces excreted within 24 hours.
3. The liver (33164.0 ng eq./g) and spleen (12542.2 ng eq./g) showed much higher concentrations of radioactivity than the other tissues and plasma (2994.5 ng eq./ml) at 1.5 hours after administration. The concentration in bile (109392.1 ng eq./ml) was also high, suggesting that the menaquinone-4 was immediately excreted into the bile. The elimination of radioactivity from tissues was relatively slow compared to that from plasma. In bone tissues, the target organ of the drug, the marrow (1424.9 ng eq./g) and cancellous tissue (2465.7 ng eq./g) of thighbone and the marrow (3533.4 ng eq./g) of ribs showed levels as high as the plasma concentrations at 1.5 hours and were 2 to 3 times higher than that of the plasma at 24 hours after dosing.
4. The unchanged menaquinone-4 was the major form present in the plasma, liver, kidney, spleen, adrenal, adipose and cancellous tissue of thighbone at 1.5 hours after administration. The known metabolites, ω-COOH, K acid I and K acid II, were found in these tissues at 1.5 hours. In the adipose and cancellous tissue of thighbone, the unchanged menaquinone-4 was the major form up to 168 hours, while the relative amount of metabolites increased by 24 hours in the other tissues. Polar metabolites were found in the urine excreted within 24 hours and bile at 1.5 and 24 hours after administration. The unchanged menaquinone-4 was mainly found in the feces excreted within 24 hours.
5. The levels of radioactivity in the marrow and cancellous tissue of thighbone and the marrow of ribs at 1.5 and 24 hours after dosing (1.5 hours: 3.3−8.0×10^-6 M, 24 hours: 1.9−3.1×10^-6 M) were comparable to the pharmacologically effective concentrations of menaquinone-4 (10^-6−10^-5 M) in in vitro studies on bone formation. This finding suggests that menaquinone-4 distributes sufficiently into bone tissues after a single oral administration in dogs, and can be expected to maintain pharmacologically effective concentrations by repeated administration as a therapeutic agent for osteoporosis.

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[¹⁴C]menaquinone-4, a therapeutic agent for osteoporosis, was administered orally to male dogs at a dose of 4 mg/kg once a day for seven days, and the absorption, distribution, metabolism and excretion were investigated.
1. The mean plasma concentration of radioactivity reached a maximum level at 1.5 hours after the 1st administration and thereafter decreased slowly. The concentrations at 1.5, 4 and 24 hours after each dose increased daily and approached a steady state by the 7th (final) dose, whereas constant plasma concentrations of unchanged menaquinone-4 were observed during repeated administration. The concentrations of radioactivity in erythrocytes increased during repeated administration and had not reached a steady state by the 7th dose.
2. The concentrations of radioactivity in the bile, liver, adipose, gall bladder and spleen at 1.5 hours after the final dose were 1.0, 0.9, 10.1, 0.6 and 1.2 fold, respectively, and were greater than those after a single dose. Radioactivity was highly accumulated in adipose tissues. In bone tissues, the target organ of the drug, the marrow and cancellous tissue of thighbone and the marrow of ribs showed higher concentrations than plasma levels at 1.5 hours, and were 3.7, 2.6 and 2.7 fold higher, respectively, than those after a single dose.
3. In urine and feces, 2.4 and 84.0% of the dosed radioactivity was excreted within 168 hours after the 7th dose, respectively. Unrecovered radioactivity was present in the adipose and other tissues at 168 hours.
4. The metabolic profile of [¹⁴C]menaquinone-4 after repeated administration was similar to that after single administration. The unchanged menaquinone-4 was the major form present in the plasma, liver, kidney, spleen, adrenal, adipose and cancellous tissue of thighbone at 1.5 hours and in feces excreted within 24 hours after the 7th dose. In the adipose and cancellous tissue of thighbone, the unchanged menaquinone-4 was the major form up to 168 hours, while the relative amounts of metabolites increased by 24 hours in the other tissues.
5. The levels of radioactivity in the marrow and cancellous tissue of thighbone and the marrow of ribs increased with repeated administration, and in the end were comparable to the pharmacologically effective concentrations of menaquinone-4 (10^-6−10^-5 M) observed in n vitroi studies on bone formation. It is, therefore, expected that repeated administration of menaquinone-4 may contribute to the pharmacological action of the drug as a treatment for osteoporosis.

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Sex difference in the elimination of drugs can be due to either a difference in excretion or in metabolism. Sex differences in drug metabolism are well known and occur in both phase I and II metabolism, while the situation with excretion, especially renal excretion, is unclear.
Zenarestat, [3-(4-bromo-2-fluorobenzyl)-7-chloro-2, 4-dioxo-1, 2, 3, 4-tetrahydroquinazolin-1-yl] acetic acid is an aldose reductase inhibitor. Rat shows a marked sex difference in the excretion of ¹⁴C-zenarestat: only 1 % of the dose was excreted in the urine of males, about 45% of the dose was excreted in the urine of females. ¹⁴C in the urine of female rats was almost entirely unchanged drug. Renal clearance experiments, and inhibition of urinary excretion by probenecid, indicated that female rats may possess an active secretory mechanism which is lacking or relatively inactive in male rats.
Castration of male rats at 22 days of age abolished th e adult sex difference in urinary excretion of zenarestat, while castration at 5 weeks of age produced urinary excretion of the drug about half that in females. Ovariectomy of females at 22 days or 5 weeks of age had no effect on the urinary excretion of zenarestat. Treatment of male and female gonadectomized rats with testosterone resulted in the urinary excretion of zenarestat characteristic of intact adult male rats.
Hypophysectomized male rats and normal female rats has similar urinary excretion of zenarestat which was much greater than that of normal male rats. Treatment of male and female hypophysectomized rats with testosterone resulted in the urinary excretion of zenarestat characteristic of intact adult male rats. Urinary secretion of zenarestat in rats is independent of pituitary hormone and is suppressed only by androgen.
Urinary excretion of zenarestat scarcely differed between 3-week-old male and female rats; it decreased in males from 4 weeks of age, but in females urinary excretion of the drug was essentially constant during ageing.
Mice show a sex difference in the excretion of zenarestat similar to that seen in rats, but dogs and humans show no significant sex difference. Female rats and mice, and both sexes of dogs and humans, appear to possess an active secretory mechanism in the renal excretion of zenarestat, which is lacking or relatively inactive in male rats and mice.
The effect of acute ( ?? 1 week) and chronic ( ?? 3 weeks) streptozocin induced diabetes on the pharmacokinetics of zenarestat was studied in male and female rats. In male acute and chronic diabetic rats, the urinary excretion increased compared with the control after the dose with ¹⁴C-zenarestat. It did not change in female acute diabetic rats and decreased in female chronic diabetic rats compared with the control. Renal clearance experiments suggested that the decrease in urinary excretion in female chronic diabetic rats is due to a decrease in active secretion. The increase in the urinary excretion in male rats was probably due to a decrease in testostereone levels in the diabetic state.

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