The absorption, distribution, and excretion of radioactivity were investigated following a single oral administration of ¹⁴C-cabergoline to rats. The protein binding of the drug was also investigated in vivo and in vitro.
1. After oral administration of ¹⁴C-cabergoline of a dose of 0.25-1.0 mg/kg to male rats, the plasma level of radioactivity increased and reached the C_max within 1 hour, which was followed by a biexponential decreased with a t_{1/2(1-8 hr)} of 4.0-6.2 hours and t_{1/2(24-168 hr)} of 33.3-38.4 hours, respectively. Both C_max and AUC_(0-∞) increased approximately in proportion to the dose over the 0.25-1.0 mg/kg dose range. The t_{1/2(1-8 hr)} was increased by food taking.
2. More than 50% of the rad ioactivity was absorbed within 4 hours after injection of ¹⁴C-cabergoline into the jejunum or ileum. On the other hand, absorption from the stomach was low.
3. The radioactivity in most tissues after oral dosing of male and female rats with ¹⁴C-cabergoline at 0.5 mg/kg reached C_max at 4 to 8 hours, being higher in liver, lung, and spleen, and lower in the central nervous system of both sexes. And at 168 hours after oral dosing, the radioactivity in testis, hypophysis, harderian gland, brown fat, and trachea decreased to below 30% of each maximum concentration.
4. Within 168 hours after oral dosing with ¹⁴C-cabergoline (0.5 mg/kg), the radioactivity excreted in the urine and feces of fasting male rats were 5.6 and 93.1% of dose, respectively; i.e., 98.7% of the radioactivity was excreted. No significant difference was observed between non-fasting, and fasting male rats.
5. Within 48 hours after oral dosing of bile duct-cannulated male, and female rats with ¹⁴C-cabergoline (0.5 mg/kg) 33.0 and 25.4%, respectively, of the radioactivity were excreted in the bile.
6. Within 24 hours after intraduodenal injection of the radioactive bile obtained from other rats which had been administered ¹⁴C-cabergoline (0.5 mg/kg) orally, 12.3 and 5.0% of the injected radioactivity were excreted in the bile and urine, respectively.
7. The percentage of radioactivity bound to rat serum protein after oral dosing of male and female rats with ¹⁴C-cabergoline (0.5 mg/kg) was 62.6-69.2 and 62.4-68.4%, respectively. The percentage of ¹⁴C-cabergoline at concentrations of ca. 1.0-15.0 ng /ml bound to rat and human serum protein in vitro was 59.5-70.3 and more than 59.3-65.5%, respectively.

View full abstract

The absorption, distribution, and excretion of radioactivity were investigated following a 21-day period of daily oral administration of ¹⁴C-cabergoline (0.5 mg/kg/day) to male rats. Furthermore, the transfer of radioactivity into the fetus and milk was also investigated on the 19th day pregnant rats and in lactating female rats, respectively.
1. When measured 24 hours after each of 21 repeated dosings of male rats with ¹⁴C-cabergoline (0.5 mg/kg), the plasma radioactivity concentration reached the steady-state by the 14th dosing. After repeated dosing for 21 days the C_max and t_1/2 were increased to 1.8 times and 3 times, respectively, over those of the single oral dosing.
2. The radioactivity in blood, plasma, eyes, harderian glands, heart, lung, spleen, and skin reached the steady-state condition probably before the 14th dosing. However the radioactivity concentrations in most tissues except the above did not reach the steady-state before the 21st dosing. The highest increase was observed in the hypophysis. At 24 hours after the 21st dosing, radioactivity in most tissues was 4-6 times higher than that after the first dosing. At 1344 hours after the 21st dosing, the radioactivity decreased to below the detection limit in about 1/3 of the tissues, and remained more than 10% of maximum level in several tissues.
3. Within the 7th dosing of ¹⁴C-cabergoline, the cumulative urinary and fecal excretions reached the steady-state condition. Within 168 hours after the 21st dosing, the cumulative excretion of radioactivity was 5.4% in urine and 92.8% in feces, for a total of 98.1% of the cumulative dose. The ratio of excretion between urine and feces was approximately the same as after single oral dosing of non-fasting rats.
4. On the 19th day of gestation, the concentration of radioactivity in most tissues of the dams reached C_max at 4-8 hours after a single dosing with ¹⁴C-cabergoline. The maximum radioactivity in most fetal tissues was less than half of the C_max in the dam's plasma
5. After oral dosing of lactating female rats with ¹⁴C-cabergoline, the concentration of radioactivity in the milk was much higher than that in plasma at all times, with the C_max in milk being about 6 times higher than that in plasma.

View full abstract

The metabolites of cabergoline in liver were investigated in rats after a single oral administration. The identification was performed by HPLC and mass spectrometry equipped with an APCI interface. In addition to cabergoline, the identified metabolites are FCE-27395 which is the demethylated derivative of cabergoline and, possibly, one more demethylated of FCE-27395.
Analysis of the metabolites in liver after a single oral administration of [¹⁴C]-cabergoline (0.5 mg/kg) showed a difference in the content of the metabolites between male and female rats. This sexual difference was due to the much higher content in FCE-27395 in male rat liver compared to female rat liver. For both sexes the major metabolite was FCE-27395, and other metabolites were found in minor quantities.
The consecutive administration of cabergoline did not cause any induction of hepatic drug metabolizing enzymes, such as cytochrome P450, cytochrome b5, aniline hydroxylase, and aminopyrine demethylase.
The distribution in the brain after a single intravenous administration of [³H]-cabergoline (0.5 mg/kg) was also investigated. The collected tissues were cerebral cortex, striatum, and hypophysis. The highest radioactivity was in the hypophysis, in which tissue the radioactivity tended to remain until 24 hour after dosing. Most of the radioactivity in these tissues could be attributed to cabergoline, as there were only traces of its metabolites.

View full abstract

The metabolism of human granulocyte colony-stimulating factor (G-CSF) derivative, nartograstim (NTG), was investigated using SDS-PAGE/Western blotting technique.
When NTG was incubated with rat liver, kidney and bone marrow homogenates at 37°C, the several metabolites of NTG were detected. In the liver and kidney homogenates, NTG was metabolized to six immunoreacive matabolites at pH 4.5 in the presence of the surfactant, but no metabolites was detected at pH 7.5. The molecular weights of the metabolites were ranged from 12.7 to 18.5 kDa. The similar metabolites of NTG were detected in the homogenate of bone marrow, which is one of target tissue of G-CSF. However, the composition of the metabolites in the bone marrow homogenate was slightly different from those in liver or kidney homogenates. The lysosomal enzymes would be responsible for these metabolism/degradation of NTG. In addition, NTG was metabolized to 9 and 11 kDa metabolites at pH 7.5 only in the bone marrow homogenates. This suggests that the neutral peptidases may play some important roles in the metabolism of NTG in the target tissue.
The apparent me tabolic pathway of G-CSF has been suggested by Western blotting technique.

View full abstract

After a single subcutaneous administration of ¹⁴C-clonidine at a dose of 1 mg/kg (principally) to rats, dogs and a monkey, the absorption, distribution and excretion of radioactivity were investigated.
1. In the male rats, the radioactivity level in plasma reached a maximum (C_max) at 2 hr and then declined with half-lives of 7.1 hr up to 24 hr and 37 hr thereafter. The plasma levels were dose-proportional at the dosing range of 0.25 to 1 mg/kg. No marked difference was observed between the males and females in the plasma level.
Most tissues showed higher radioactivity levels than that observed in the plasma, especially high levels were seen in the mandibular gland, kidney, liver and stomach. The elimination from tissues was more rapid than that in the plasma except the aorta and thyroid gland until 24 hr. High radioactivity was also found in the gastro-intestinal contents, some exocrine glands and nasal cavity as revealed by the whole body autoradiograms.
Urinary and fecal excretions within 168 hr were 69.5 and 28.4% of the dose, respectively. Biliary excretion accounted for 33.2% of the dose within 48 hr. Entero-hepatic circulation was partly observed. In the females, urinary excretion was slightly higher than that in the males.
2. In the male dogs, the Cmax was 6 times higher and the elimination of second phase was slower than those in the male rats. Within 480 hr, 87.0 and 10.2% of the dose were excreted in urine and feces, respectively.
3. In the male monkey, the radioactivity level in plasma and urinary and fecal excretions were similar to those in dogs.

View full abstract

After application of a transdermal delivery system for ¹⁴C-clonidine, ¹⁴C-M-5041T, to male rats and a dog for 24 hr, the absorption, distribution and excretion of radioactivity were investigated.
1. In the male rats, applied with a dose of 2.5, 5, 10 and 20 mg/kg, the radioactivity levels in plasma reached respective maxima at 6 or 8 hr and then declined in a biphasic manner. Within 168 hr, 4.3 to 11.1% and 1.2 to 4.3% of the dose were excreted in the urine and feces, respectively. The percutaneous absorption ratio was estimated to be about 11%.
Most tissues showed higher radioactivity levels than that in the plasma, especially high levels were observed in the mandibular gland and kidney at a dose of 5 mg/kg. After removal of the system, the elimination was rapid except the aorta, thyroid gland and liver. High radioactivity was also found in the gastro-intestinal contents, some exocrine glands and nasal cavity as evidenced by the whole body autoradiograms.
2. The microautoradiograms taken at the application site of a male rat revealed tha t the radioactivity was found in the stratum corneum and then distributed uniformly in the dermis. At 120 hr (96 hr after removal), the radioactivity remained in the stratum corneum.
3. When the stratum corneum of skin was removed in a male rat at a dose of 1 mg/kg, the absorption ratio calculated from AUC was about 60%.
4. In the male dog, applied with a dose of 5 mg/kg, 4.3% of the dose was excreted in the urine and feces within 480 hr. The radioactivity level in plasma reached a maximum at 8 hr after application and then declined with a biphasic fashion.

View full abstract

After repeated subcutaneous administration of ¹⁴C-clonidine to male rats once a day for 21 days at a dose of 1 mg/kg/day, the absorption, distribution and excretion of radioactivity were investigated. 1. No marked variation was observed in the radioactivity level of the plasma at every 24 hr after daily administration. After the final dosing, the elimination of radioactivity from the plasma after 24 hr was slower than that after single dosing.
2. The radioactivity levels in most tissues at 24 hr after daily administration achieved nearly a steady state by the 21st dosing, but the level in aorta increased cumulatively and the value was 10 times higher than that after the first dosing. After the final dosing, the elimination of radioactivity from the tissues after 24 hr was slower than that after single dosing. On the whole body autoradiograms, high radioactivity was also found in the gastro-intestinal content, some exocrine glands and nasal cavity.
3. The total excretion of radioactivity in the urine and feces during each 24 hr after daily dosing was almost constant after the 2nd dosing and 71.6 and 26.4% of the cumulative dose were excreted in the urine and feces, respectively, within 168 hr after the final dosing.

View full abstract

The plasma protein binding of ¹⁴C-clonidine in vitro and in vivo and its transfer into the fetus and milk in rats were investigated.
1. The binding ratios of radioactivity in vitro to male rat, dog, monkey and human plasma proteins did not change with drug concentration in any species and no marked species difference was observed, however, in vivo binding to male rat, dog and monkey plasma proteins tended to increase with the elapse of time.
2. After subcutaneous administration to pregnant rats at a dose of 1 mg/kg, the radioactivity levels in the fetal kidney and liver were higher than that in the maternal plasma. The radioactivity was rapidly eliminated from the fetal tissues as that from the maternal plasma. On the whole body autoradiograms, comparable radioactivity was found in the fetus with the maternal plasma.
3. The radioactivity level in the milk reached a maximum at 30 min after subcutaneous administration at a dose of 1 mg/kg, being 5.5 times higher than that in the plasma, but was comparable with that in the plasma after 8 hr and decreased to undetectable level at 72 hr after administration.

View full abstract

The metabolism of clonidine in rats, dogs and monkeys was investigated after subcutaneous administration of ¹⁴C-clonidine or after application of a transdermal delivery system containing ¹⁴C-clonidine, ¹⁴C-M-5041T.
1. In male rats after subcutaneous dosing, unchanged clonidine was mainly found in the urine, followed by the conjugate of CM-4, a hydroxylated metabolite of the phenyl ring. CM-4 was also found in the feces and bile.
2. The metabolic profile in male rats after 21 times repeated subcutaneous dosing was similar to that after single dosing. Certain metabolites were bound tightly to the constituent proteins of aorta.
3. Unchanged clonidine was mainly found in the skin at the application site of male rats after application of ¹⁴C-M-5041T. The metabolic profile was similar to that after subcutaneous dosing.
4. In male dogs after subcutaneous dosing, CM-1, a metabolite formed by cle avage of the imidazolidine ring, was mainly found, whereas the unchanged clonidine was found in minor quantities in the urine and feces. The metabolic profile in a male monkey was similar to that in dogs.
5. From the above results, main metabolic pathways of clonidine were proposed to be the hydroxylation and conjugation of the phenyl ring in rats, and the oxidation and cleavage of the imidazolidine ring in dogs and monkeys.

View full abstract

The present article reviews our recent efforts for physiologically-based modeling of oral drug absorption. We demonstrated that the intestinal absorption rate constant (k_a) in vivo can be estimated by analyzing remaining fraction versus time profiles for stomach and small intestine after oral administration, using a model incorporated with first-order gastric emptying followed by first-order intestinal absorption. The intestinal membrane permeability clearance (CL_app) was obtained as the product of k_a and the average luminal volume (V_av). We also demonstrated that k_a can be alternatively estimated from the fraction absorbed (F_a) and small intestinal transit time (T_si), assuming first-order intestinal absorption, F_a=1−exp(−k_a·T_si). For L-glucose and D-xylose as model drugs, k_a was shown to be affected by dosing conditions due to the changes in V_av, but CL_app was unaltered, consistent with information in situ and supporting the model assumptions. The proposed model should be useful in establishing in vitro, _{in situ} and _{in vivo} correlations in intestinal transport and developing a method to predict oral drug absorption.

View full abstract

Cytochrome P450 including in CYP3A subfamily is a major form and is induced by the treatment with several drugs in livers of humans and experimantal animals. Human CYP3A forms were recently reported to relate to a half of clinical available drugs in the metabolism. To better understand the molecular mechanism of the tissue specific expression and the induction, we have purified four CYP3A forms from rat livers, and isolated their cDNAs and genes. Enzymatic properties of recombinant CYP3A forms expressed in COS-1 cells were similar to those of purified forms. Expression profile of these forms in a rat liver differed in sexes and changed during the development. In addition, extent of induction level showed different manners in individual form. Isolated genes (P450/6βA and P450/6βB corresponding to CYP3A2 and CYP3A1 genes, respectively) were analyzed nucleotide sequences of the exon-intron boundary and compared. Both genes spread of 25-35 Kb and consisted of 13 exons. Nucleotide sequences of these proximal promoter regions conserved a high similarity with each other and included three banding sites similar to a consensus sequence of hepatocyte nuclear factor 4 (HNF-4). By further study of CAT and gel mobility shift asseys, the site (6βA-A or 6βB-A site) binding with HNF-4 related to the liver specific expression of CYP3A2 or CYP3A1 gene. Other site (6βA-B or 6βB-B) interacting with unknown nuclear transfactors, however, related to suppressive regulation of these genes.

View full abstract

Various endogenous macromolecular substances have become a new class of therapeutic agents with recent progress in biotechnology. For a successful use of the macromolecular drugs, optimal delivery systems should be developed. In our laboratory, a series of pharmacokinetic studies have been carried out aiming at the development of delivery systems for macromolecular drugs. Aa the first step towards the development of delivery systems, we studied pharmacokinetic characteristics of macromolecules in relation to their physicochemical properties such as molecular weight and electric charge. Based on the findings, we first developed macromolecular prodrugs of an antitumor antibiotic, mitomycin C (MMC). MMC was covalently conjugated with dextran and various types of macromolecular prodrug of MMC were developed for tumor targeting. Similar strategy was applied to the development of delivery systems for various protein drugs. For example, successful targeting of recombinant superoxide dismutase (SOD) to the liver, kidney and blood circulation was achieved by chemical modification of the protein drug. Very recently, we have been trying to develop delivery systems for nucleic acid drugs such as antisense oligonucleotides and plasmid DNA. Based on the same concept, delivery systems have been tested to control the in vivo behavior of the nucleic acid drugs. The strategy for rational design of delivery systems for various types of macromolecular drugs has thus been establised through the pharmacokinetic studies.

View full abstract