9-Amino-8-fluoro-1, 2, 3, 4-tetrahydro-2, 4-methanoacridine citrate (SM-10888) was given to rats orally or intravenously, and its absorption, distribution and excretion were investigated.
1. After a single oral administration of ¹⁴C-SM-10888 (5mg/kg), the serum ¹⁴C level reached a maximum at 30min (1.09μg eq./g), with an AUC value corresponding to about 90% of that observed after intravenous administration, clearly revealing rapid and efficient absorption from the gastrointestinal tract.
Under fasted conditions, C_max was 2-fold higher than that of non-fasted animals, without significant change in AUC, suggesting that diet causes delayed absorption. Both C_max and AUC increased as the dose was raised from 1 to 20mg/kg. Repeated dosing (5mg/kg/day for 14 days) exerted little effect on serum ¹⁴C levels.
2. After a single oral administration of ¹⁴C-SM-10888 (5mg/kg), the radioactivity was distributed throughout the body, with relatively higher activities being observed in the digestive tract, the liver and the salivary gland. Radioactivity in each tissue had drastically decreased by 24hr post administration.
3. After a single oral administration of ¹⁴C-SM-10888 (5mg/kg), cumulative excretion into the urine and feces after 168hr was 52.2% and 44.2%, respectively. When administered to bile-duct cannulated rats, cumulative excretion after 72hr into the bile and urine was 65.1% and 29.1%, respectively, providing further support for a high absorption rate.
Repeated dosing had little effect on the patterns of excretion into the urine and feces.

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rG·CSF is a recombinant human granulo cyte colony-stimulating factor (rhG-CSF) produced in CHO cells. rG·CSF is a glycoprotein which has one O-linked sugar chain. We have studied the role of this sugar chain on the disposition of rG·CSF using enzymatically deglycosylated rG·CSF (degly-rG·CSF) and rhG-CSF produced in E. coli (Eco-rG-CSF).
1. rG·CSF or degly-rG·CSF was administered to rats at the dose of 10μg/kg intravenously and subcutaneously. After intravenous administration, the pharmacokinetic parameters (T_1/2 (α), T_1/2 (β), MRT, Vc and Vss) of two G-CSFs were not significantly different between each other except that the AUC in rG·CSF was greater and the CLtotal in rG·CSF was smaller than those in degly-rG·CSF. After subcutaneous administration, T_1/2 and MRT in degly-rG·CSF were significantly smaller than those parameters in rG·CSF. Mean absorption time (MAT) and absolute bioavailability (F) in degly-rG·CSF were smaller than those in rG·CSF.
2. rG·CSF or Eco-rG-CSF was administered to rats at the dose of 10μg/kg intravenously and subcutaneously. After intravenous administration, the pharmacokinetic parameters of two G-CSFs were not significantly different between each other except that the AUC in rG·CSF was greater and the CLtotal in rG·CSF was smaller than those in Eco-rG-CSF. After subcutaneous administration, T_1/2, MRT and AUC in Eco-rG-CSF were significantly smaller than those parameters in rG·CSF, respectively. MAT and F in Eco-rG-CSF were smaller than those in rG·CSF.
3. These results showed that after intravenous administration, there were little changes related to sugar chain. These results also suggested that sugar chain contributed to stabilization of rG·CSF at the administration site and delayed absorption of rG·CSF from the administration site after subcutaneous administration. Thus, the sugar chain of rG·CSF seemed to play a role in the elongation of plasma concentration after subcutaneous administration.

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We have studied the effect of sialic acid in rG·CSF on the disposition of rG·CSF using monosialo-rG·CSF and disialo-rG·CSF.
1. Monosialo-rG·CSF and disialo-rG·CSF were administered to male rats at the dose of 50μg/kg intravenously and subcutaneously ; plasma and urinary concentrations were measured by EIA.
2. After intravenous administration, the half-lives of α and β phases, Vc, Vss, MRT, CLtotal and CLrenal of monosialo-rG·CSF were 0.24h, 1.07h, 55.25ml/kg, 90.78ml/kg, 1.07 h, 93.30ml/h/kg and 0.004ml/h/kg, respectively. Half-lives of α and β phases, Vc, Vss, MRT, CLtotal and CLrenal of disialo-rG·CSF were 0.27h, 1.17h, 51.84ml/kg, 80.46ml/kg, 1.24h, 61.56ml/h/kg and 0.026ml/h/kg, respectively. The CLtotal of monosialo-rG·CSF was significantly greater than that of disialo-rG·CSF.
3. After subcutaneous administration, the Tmax of monosialo-rG·CSF was 1.75h and was shorter than 3.5h of disialo-rG·CSF. C_max, apparent half-life, MRT, AUC, CLrenal, MAT and F for monosialo-rG·CSF were 54.01ng/ml, 2.06h, 3.99h, 302.18ng·h/ml, 0.003ml/h/kg, 3.08h and 0.56, respectively. The C_max, apparent half-life, MRT, AUC, CLrenal, MAT and F for disialo-rG·CSF were 57.84ng/ml, 2.37h, 5.31h, 396.92ng·h/ml, 0.030ml/h/kg, 4.07h and 0.49, respectively. The AUC and MRT of disialo-rG·CSF were significantly greater than those of monosialo-rG·CSF.
4. There were some differences in dispositions between monosialo-rG·CSF and disialo-rG ·CSF. These results have suggested that sialic acids in rG·CSF molecule modify the elimination and absorption of rG·CSF.

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The metabolites of rG·CSF present in the serum were investigated in rats after intravenous administration of ¹²⁵I-rG·CSF at the dose of 10μg/kg, or nonlabeled-rG·CSF at the dose of 100-400μg/kg.
1. Using gel filtration on the Superose 12 column, most of the radioactivity in rat serum at 30min after administration of ¹²⁵I-rG·CSF was eluted at a point corresponding to molecular weight of 19kd, this point was the same as that of intact-rG·CSF. The radioactivity was immunoreactive with anti-rG·CSF antibody and showed biological activity against NFS-60 cell lines. At 2 and 6h after administration, the radioactivity present in the serum was eluted from the column at a point corresponding to molecular weight of 46kd and 19kd. The radioactivity corresponding to 46kd showed immunoreactivity, but was biologically inactive.
2. The SDS-polyacrylamide gel electrophoresis revealed that most of the radioactivity in the serum present at 30min after administration of ¹²⁵I-rG·CSF was detected at a point corresponding to intact-rG·CSF (molecular weight of 19kd). The radioactivity was also detected at a point corresponding to molecular weight of 19kd and 5kd at 2h, 19kd, 14kd and 5kd at 6h after administration.
3. The chromatofocusing using Mono-P column showed that most of the radioactivity in the serum was focused at a point corresponding to isoelectric points of 5.8 and 5.5 at 30min after administration of ¹²⁵I-rG·CSF, these points were related to monosialo-rG·CSF and disialo-rG· CSF, respectively. At 2 and 6h after administration, the radioactivity showing immunoreactivity was newly found at a point corresponding to isoelectric points of 5.2 and 4.5.
4. During immunoblotting of serum samples after administration of nonlabeled-rG·CSF, the presence of a band corresponding to molecular weight of 19kd was found in all sampling times after administration. Beside the band of 19kd, only bands at a point corresponding to molecular weight of 5kd and 14kd were detected at 6h after administration.
5. Based of the fact that only an intact form of rG·CSF exists at early stage after intrave nous administration of rG·CSF, and since 2h the metabolites which have not biological activity appeared in the serum, we have concluded that an intact form mainly participate in hematologic activity of rG·CSF in vivo.

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To evaluate the role of the kidney in the elimination of rG·CSF, the pharmackinetics of rG·CSF was studied in rats upon removal of one (1/2-nephrectomized rat) or of two kidneys (nephrectomized rat). Plasma concentrations of rG·CSF were measured by enzyme immunoassay method.
1. The elimination half lives of rG·CSF in nephrectomized and 1/2-nephrectomized rats, which were 4.85h and 1.llh respectively, were longer than those in sham operated rats given rG·CSF 10μg/kg intravenously. Total nephrectomy and 1/2-nephrectomy resulted in 70% and 30% reduction of total body clearance of rG·CSF in rats, respectively.
2. The result suggested that the kidney is the major elimination site of rG·CSF.

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The change of the oxidative m etabolism of buprenorphine (BN) during repeated administration of BN was investigated in rats. The animals were pretreated intramuscularly with 4 or 8 doses of BN (0.6mg/kg) every 12hr. When BN (0.6mg/kg dose) was given intravenously to rats at 12hr after the 8 doses, it was estimated that about 95% of administered dose was recovered within 24hr as the sum of BN and norbuprenophine (NBN) conjugates in the bile. This value was not significantly different as compared with 90% in rats given intravenously at a dose of BN (0.6mg/kg) without prior intramuscular administration. However, the excretion ratio of NBN conjugate (12.8%) in the pretreated rats was significantly small as compared with 20% in the unpretreated rats (p <0.05). The pharmacokinetic profiles of NBN after intravenous administration of NBN (0.6mg/kg dose) did not change by the repeated administration of BN. The results of in vitro metabolism experiments using the tissue homogenates showed that the oxidation rate of BN in the liver pretreated with BN was decreased to one-ninth of the controls, which were given saline. These findings suggested that the decrease of the biliary excretion ratio of NBN conjugate during repeated administration of BN may be due to the decrease in metabolizing rate from BN to NBN. In exploratory studies, BN (0.003, 0.03, 0.3 and 0.6mg/kg dose) were administrated in four different doses repeatedly. The contents of cytochrome P-450 in the hepatic microsome fraction after repeated administration of 0.03, 0.3 and 0.6mg/kg doses were decreased significantly as compared with those in the control rats given saline, and were decreased 91, 84 and 71% of the contents in the pretreated rats, respectively. Both glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities in the plasma did not change during repeated administration. These results suggested that the decrease in the metabolizing clearance from BN to NBN may be due to the decrease of cytochrome P-450 contents in the hepatic microsome.

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Absorption, distribution, metabolism and excretion of [¹⁴C] 4-amino-1-β-D-arabinofuranosyl-2 (1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate (YNK01) were studied in rats after single oral administration of 25mg/kg.
1. Blood concentration reached the highest concentration at 4 hours after dosing, and declined up to 12 hours with half life of 3.7 hours.
2. Radioactivity secreted to urine, feces and expired air amounted to 40.6, 43.8 and 15.6% of administered dose, respectively up to 120 hours after dosing. Radioactivity remaining in the body 120 hours after administration accounted for 1.4%.
3. Radioactivity in bile up to 48 hours after dosing amounted to 2.4% of the dose.
4. Radioactivity levels in tissues other than the eyes and the central nerve system were similar to or higher than that in blood. Radioactivity was distributed at particularly high levels in the small intestinal mucosa, liver, mesenteric lymph nodes, kidneys and preputial glands.
5. YNK01 was not detected in urine up to 24 hours after dosing. As a major metabolite, 1-β-D-arabinofuranosylcytosine (ara-C) (26.7%) was found. 1-β-D-arabinofuranosyluracil (ara-U) (4.9%) and 1-β-D-arabinofuranosylcytosine 5'-(3-carboxypropyl) phosphate (C-C₃ PCA) (1.3%) were found as minor metabolites.

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Absorption, distribution, and excretion of [¹⁴C] 4-amino-1-β-D-arabinofuranosyl-2(1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate (YNK01) were studied during and after repeated administrations to rats once a day at 25mg/kg for the maximum of 21 days.
1. Blood concentration of [¹⁴C] YNK01 at 24 hours after dosing showed a tendency to increase with repetition.
2. Excretion of [¹⁴C] YNK01 in urine and feces at 24 hours after dosing did not show changes by increasing dosing times after the 7 th dosing, and no marked difference was observed compared with the single dose group.
3. After the 21st dosing, an in crease tendency in tissue concentration was observed in all tissues, except thymus and digestive tract. Tissue which showed the highest increase in concentration was the adipose tissue. Metabolite in the adipose tissue was assumed to be structure of more lipophilic properties that of YNK01.
4. The effect of YNK01 on the hepatic drug-metabolizing enzymes was examined after repeated administration of YNK01 for 10 days. A slight increase was observed in the P-450 content in the 75 mg/kg group. Significant, but dose-independent, increase in the activities of aminopyrine-N-demethylase and aniline hydroxylase was observed.

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Fetoplacental transfer and excretion into the milk were studied in rats after oral administration of [¹⁴C] 4-amino-1-β-D-arabinofuranosyl-2 (1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate (YNK01) at 25mg/kg.
1. After oral administration of [¹⁴C]YNK01 to fasting pregnant rats at 25mg/kg, the radioactivity transferred to fetuses was comparable to that in maternal plasma on 18th day of gestation. Similarly as in the case of mothers, radioactivity disappeared slowly from the fetal brain, eyeball, thymus, skin, etc. Whole body autoradiograms revealed that transfer of radioactivity to fetuses on the 12th day of gestation was of a low level compared to that on 18th day.
2. After oral administrati on of [¹⁴C]YNK01 to non-fasting lactating rats at 25mg/kg on 10th day after delivery, the concentration in milk increased with time, reaching maximum at 8 hours after dosing, and decreased to the levels below the detection limit at 48 hours. Although the plasma concentration was higher in the early stage after dosing, milk-to-plasma concentration ratio rose with time, reaching 1.48 at 24 hours after dosing. Subsequently, radioactivity in milk decreased to the levels below the detection limit.

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A new radioactivity detector, a bioimage analyzer, was coupled to a commercially available image analyzer to establish an image analyzing system for radioactive thin-layer chromatography. An image showing radioactivity distribution was obtained by the bioimage analyzer and transferred to the image analyzer. At this step, the transfer program could detect most of the locations of radioactive spots automatically. The operation required was, therefore, greatly simplified. The relative amount of radioactivity in each spot was determined by using the image analyzing system. The results were compared with those obtained by the conventional scraping method followed by the liquid scintillation counting. Both methods gave identical results. The image analyzing method was superior to the conventional method in that i) quick determination was possible, ii) it showed greater sensitivity and iii) the amount of radioactive waste could be reduced. Furthermore, the image data and the data showing the spot area selection could be stored in a magnetic-optical disc. Hence the determination using the image analyzing system was readily checked and reexamined when required.

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A simple and sensitive analytical method for the determination of a drug and its metabolites in plasma samples has been developed. The method is based on image analyses of radioactive thin layer chromatograms obtained by using a bioimage analyzer and an imaging plate. To simplify the determination using thin layer chromatography (TLC), a small volume of plasma sample was directly applied on the reverse phase layer of the dual phase TLC plate and developed toward the silica layer. The radioactive material could be sufficiently separated from degraded plasma proteins. Time course studies of plasma concentrations of indomethacin and its metabolites were performed after i.v. and p.o. administration of [¹⁴C]indomethacin to rats. Specific radioactivity of [¹⁴C]indomethacin used in the present studies was relatively low (about 1.5 MBq / mg). However, the volume of plasma required for each time point was only 20μl due to high sensitivity of the image analyzing system. A complete concentration-time curve including nine time points could be obtained from each rat in the present studies. Pharmacokinetic parameters of unchanged indomethacin in the plasma could, therefore, be calculated for each rat. Hence the present method allowed to reduce the number of animals required for the preclinical pharmacokinetic studies using small laboratory animals.

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