The pharmacokinetics, distribution and excretion of NKH477, a novel water-soluble forskolin derivative, improving acute cardiac failure, were investigated in rats and dogs after a single intravenous administration of ¹⁴C-labeled NKH477 and unlabeled NKH477.
1. After intravenous bolus administration of ¹⁴C-NKH477 (0.011 ?? 0.3 mg/kg) to male rats, the elimination half life of radioactivity in the blood ranged from 70.67 to 118.23 hr. The elimination half life of radioactivity in the blood was prolonged compared with that in the plasma, which should be attributed to binding of the radioactivity to blood cells.
2. After intravenous bolus dosing of NKH477 to male rats, plasma concentrations of unchanged NKH477 and active metabolite, M-1, declined rapidly with half lives of 0.23 and 0.25 hr, respectively. The AUC of M-1 was markedly lower than that of unchanged NKH477.
3. In dogs, the radioactivity levels in the blood and plasma declined two-exponentially following administration, and the slow elimination of the blood radioactivity, but less compared to rats, was also observed.
4. Following intravenous infusion of NKH477 for 2 hours to male dogs under anesthesia at doses ranging from 0.15 to 0.60 mg/kg/min, plasma concentrations of unchanged NKH477 and M-1 decreased with the elimination half lives of 1.57 ?? 2.36 hr. The AUC of M-1 was half as much as that of unchanged NKH477.
5. After administration to male rats, the radioactivity was rapidly and extensively distributed to the tissues except those belonging to the central nervous system. The liver showed much higher concentration of radioactivity than the other tissues. The radioactivity declined rapidly from most tissues, except the heart, blood, spleen and adrenal gland.
6. After dosing to male rats, excretion of the radioactivity in urine and feces within 144 hr was 6.4 and 90.2% of dose, respectively. In bile duct-cannulated rats, biliary and urinary excretion of radioactivity was 77.1 and 7.8% of the dose, respectively within 72 hr after administration, suggesting that the main excretory route of ¹⁴C-NKH477 is feces through bile.
7. In dogs, 78.2% of the dose was excreted into feces during 144 hr after dosing.

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The blood concentration, distribution and excretion of radioactivity were investigated in male rats after daily intravenous administration of ¹⁴C-NKH477 for 21 days. The effects of NKH477 on hepatic drug metabolizing enzyme system were also investigated in male rats.
1. The radioactivity in the blood at 24 hr after daily administration rose as number of dose increased, the elimination of radioactivity from the blood after the last dosing was slower than that after a single dosing.
2. In most tissues, the radioactivity accumulated during the period of repeated administration, especially in the spleen, blood, kidney and mesenteric lymph node that rose markedly. Disappearance of radioactivity from most tissues after the last dosing was slower compared with that after a single dosing.
3. Within 168 hr after a repeated administration for 21 days, urinary and fecal excretion amounted to 7.8% and 88.9% of cumulative dose, respectively. The ratio of excretion between urine and feces was approximately the same as after a single dosing.
4. Repeated administration of NKH477 at doses of 0.3 and 1.0 mg/kg/day for 7 days had no effect on liver weight, microsomal cytochrome P-450 contents and hepatic drug-metabolizing enzyme system.

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Feto-placental transfer and excretion into milk of ¹⁴C-NKH477 were investigated in pregnant and lactating rats after a single intravenous administration.
1. From the results of whole body autoradiography in pregnant rats performed on the 17th of gestation, the levels of radioactivities in placenta and amnion were similar as that in the maternal blood within 6 hours after a single intravenous administration of ¹⁴C-NKH477, and the radioactivities in these tissues decreased to tracer levels at 24 hours after administration. There were no radioactivities present in fetus and amniotic fluid to 24 hours after administration.
2. The radioactivity levels in amniotic fluid, fetus and fetal liver were lower than those of amnion and placenta after a single intravenous administration of ¹⁴C-NKH477 to rats on the 14th or 18th day of pregnancy. The levels of radioactivity in fetus at 5 min and 1 hour after administration were 1/3 ?? 1/9 of the concentrations in maternal blood. The amounts of the radioactivity transferred to fetus were low both on the 14th and 18th day of pregnancy, and the radioactivity slightly distributed in fetus was eliminated rapidly.
3. After a single intravenous administration of ¹⁴C-NKH477 to lactating rats on the 11th day after delivery, the radioactivity was detected in the milk, and the ratio of milk to plasma within 24 hours after administration was 1.0 ?? 2.2.

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The metabolism of NKH477 by the rat liver 9000×g supernatant was investigated. Also the metabolites of NKH477 in plasma, tissue, urine, feces and bile were analyzed after intravenous administration of ¹⁴C-NKH477 to rats and dogs or NKH477 to human.
1. The structure of NKH477 and its metabolites in the rat liver 9000×g supernatant was confirmed by comparing with authentic compound using LC/FRIT-FAB-MS. The metabolic pathways of NKH477 were N-demethylation, hydroxylation and intramolecular cyclization.
2. The metabolites of NKH477 in plasma and various tissues were analyzed after a single intravenous administration of ¹⁴C-NKH477 to rats. At 15 and 30 min after administration, NKH477 and M-1 (N-demethyl form) were major compounds found in plasma, heart and kidney. At 5 min after administration, various metabolites in the liver were found, suggesting that the liver was main metabolizing organ.
3. The metabolites of NKH477 in urine, feces and bile were analyzed after a single intravenous administration of ¹⁴C-NKH477 to rats. Within 24 hr after administration, in urine 0.8% of dose was excreted as NKH477, 2.2% as M-1, 1.7% fraction D containing M-3 (hydroxy form) and M-4 (N-demethyl hydroxy form) and 1.7% as fraction C containing M-5 (intramolecular cyclic form) and M-6 (N-demethyl intramolecular cyclic form). Main metabolite in rat urine was M-1. Within 48 hr after administration, in feces 20.9% of dose was excreted as fraction D, 15.8% as fraction C, and 14.6% as fraction B containing M7 (hydroxy M-5) and M-8 (hydroxy M-6), while the NKH477 and urinary main metabolite M-1 were found in minimal quantities. Within 6 hr after administration, in bile 13.4% of dose was excreted as fraction D, 14.4% as fraction C, and 11.8% as fraction B, and the profile in bile was the same as that in feces.
4. The metabolites of NKH477 in urine and feces were analyzed after a single intravenous administration of ¹⁴C-NKH477 to dogs. Within 24 hr after administration, in urine 2.8% of dose was excreted as NKH477, 5.9% as M-1 and 1.3% as fraction D. Main metabolite in dog urine was M-1. Within 72 hr after administration, in feces 20.9% of dose was excreted as fraction D, 9.9% as fraction C, and 6.0% as fraction B, and metabolic profile in dog urine was similar to that in rat urine and feces.
5. The metabolites of NKH477 in urine were analyzed after a single intravenous infusion of NKH477 to healthy volunteers. Within 24 hr after administration, the major compounds in human urine were NKH477 and M-1, and other metabolites were found in trace amount.

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Plasma concentrations, distribution, and excretion of [¹⁴C]-alendronate (MK-217/GTH-42) were investigated after oral administration to male and female rats.
1. The urinary excretion of radioactivity within 168 hr was 0.46% after oral administration at the dose of 5 mg/kg to male rats. The remaining drug was recovered in the feces quantitatively.
2. The radioactivity levels in the bone, trachea, and kidney were 3 to 6 times higher than that in the plasma at 30 minutes after a single oral administration at 5 mg/kg to male and female rats. The levels in the bone and trachea showed a plateau at 4 hr and decreased very slowly thereafter. High radioactivity levels were detected in the gastro-intestinal tract, but this was probably due to unabsorbed drug. In the other organs and tissues, the radioactivity was similar to or less than that in the plasma and decreased to almost undetectable levels at 24 hr after dosing. Sex differences were not observed in the tissue distribution of [¹⁴C]-alendronate in the rats.
3. After multiple oral administration at 5 mg/kg once daily for 7 days to female rats, the radioactivity levels in the trachea and bone were markedly higher than those after a single dosing. In the other organs and tissues, the overall profile of drug disposition was similar to that after a single dose.
These data indicated that the oral absorption of alendronate was very low (approximately 1 %), and that the drug distributed and accumulated selectively in the target site, the bone tissue.

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The absorption, distribution, metabolism and excretion of tacrolimus (FK506) were studied in the rat after intravenous (i.v.) and oral administration of ¹⁴C-labeled FK506 (¹⁴C-FK506).
1. After i.v. injection at a dose level of 1.0 mg/kg, the pharmacokinetic parameters of FK506 in the whole blood were as follows: elimination half life, 6.4 hours; total body clearance, 1.59 l/h ·kg; and volume of distribution at steady state, 11.8 l/kg. After oral administration at a dose level of 3.2 mg/kg, the parameters in the whole blood were as follows: maximal blood concentration (C_max), 41 ng/ml; time to reach C_max, 0.25 hour; and area under the concentration-time curve (AUC_0-24h), 255 ng·h/ml. Absorption of radioactivity and FK506 was 37 and 14%, respectively, calculated from the values of AUC0-24 h in the whole blood. Distribution of FK506 in the blood changed depending on its levels. The ratio of whole blood to plasma levels was more than 2.5 at less than the whole blood levels of 50 ng/ml but decreased above these levels.
2. Radioactivity was distributed throughout the body after i.v. injection of ¹⁴C-FK506 at a dose of 0.32 mg/kg. Radioactivity was highest in most of tissues at 5 minutes after injection, the first sampling point after injection, and the levels were higher than those in the plasma except that in the white fat, testis, cerebellum and cerebrum. At 72 hours after injection, radioactivity in most tissues decreased to less than 10% of the maximal concentrations except that in the cerebrum, testis and urinary bladder, in which about half of the concentration was detected. After oral administration at a dose of 1.0 mg/kg, radioactivity was distributed mainly in the gut and liver and was hardly detected in the other tissues.
3. During 72 hours after administration of ¹⁴C_ FK506 at a dose of 1.0 mg/kg, 8 and 95%of the dosed radioactivity were respectively excreted to the urine and feces after i.v. injection and 4 and 96% after oral administration. During 48 hours after i.v. injection to the bile duct-cannulated rats, 3, 8 and 82% of the dosed radioactivity were recovered in the urine, feces and bile, respectively, and 3, 6 and 35% during the same period after oral administration.
4. Less than 0.4% of the dosed radioactivity was excreted as unchanged FK506 in the urine, feces and bile, and the elution pattern of excreted radioactivity was very complex on high performance liquid chromatography and no major but many small radioactive peaks were observed. The major in vitro metabolite, 13-O-mono-demethylated metabolite, was detected in the urine, feces and bile but its portion to total radioactivity was very small.

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Molecular genetic studies of familial adenomatous polyposis (FAP) kindreds led to the discovery of the APC (adenomatous polyposis coli) gene on human chromosome 5q21. Mutations in APC appear to be responsible for not only FAP but also many sporadic cancers of the colorectal axis, stomach, and esophagus. The APC protein contains regions that may form an α-helical coiled-coil structure, and a subdomain of the first 55 as form a stable, parallel helical dimer. Antibody studies showed that the wild-type, but not mutant, APC protein is associated with the microtubule cytoskeleton. The predicted structure of APC, its localization, and its interaction with β-catenin suggested its involvement in cell adhesion. In fact, recent studies demonstrated that APC is localized to plasma membrane sites involved in active cell migration. At the same time, β-catenin interacts with hTcf-4 and Lef transcription factors. hTcf-4 transactivates transcription only when associated with β-catenin. We recently constructed a gene knockout mouse strain in which the mouse homolog of the human APC was inactivated by homologous recombination. Using this mouse strain, we elucidated the mechanism how the polyp adenomas are formed in both morphological and genetic aspects. At the same time, we investigated the effects of carcinogens and anticancer agents on the polyposis. Accumulating evidence indicates that nonsteroidal antiinflammatory drugs (NSAIDs) reduce the incidence of colorectal cancers in human and experimental animals, and reduce the polyp number and size in FAP patients. Recently, evidence has been presented that COX-2 is induced in human colorectal cancers, and in the polyps of mouse FAP models. Accordingly, we inactivated the COX-2 gene in our FAP model mice, and demonstrated that both the number and size of polyps are reduced dramatically. In addition, a COX-2 selective inhibitor caused similar results to COX-2 gene knockout mutations. These genetic and pharmacological data open the possibility of effectively treating human FAP and various cancers with COX-2 selective inhibitors, a new class of NSAIDs.

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Discussions on the hepatic uptake mechanisms for Pravastatin and Temocaprilat were made. The uptake of Pravastatin by primary cultured rat hepatocytes was temperature-dependent, saturated at high substrate concentrations, and inhibited by metabolic inhibitors such as rotenone, indicating an involvement of carrier-mediated active transport system. The hepatic uptake of Pravastatin was also inhibited competitively by various organic anions such as dibromosulfophthalein and bile acids, and was dependent neither on Na⁺ ion nor on Cl^- ion. Pravastatin competitively inhibited the uptake of Na⁺-independent bile acids, indicating that Pravastatin serves as the substrate of the Na⁺-independent bile acids transporter. Therefore, the organic anion transporting polypeptide (oatp) was strongly suggested to be involved in the hepatic uptake of Pravastatin, while the expression system of oatpl in COS-7 cells failed to transport Pravastatin, suggesting that the uptake of Pravastatin is mediated either by other isoforms of oatp, or by bilitranslocase or by organic anion binding protein. The hepatic uptake of Temocaprilat, the active metabolite of Temocapril, was also found to be the carrier-mediated active transport according to the observations obtained in the similar uptake experiments as described above. Oatpl accounted for most part of the hepatic uptake of Temocaprilat.

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The rates and routes of the metabolism of a compound, as well as its potential to interfere with the metabolism of other, co-administered compounds, can often be the major factors that determine the clinical usefulness of a drug. There are, therefore, obvious economic advantages in being able to make predictions of these factors in drug discovery, thus enabling informed choices to be made between candidate molecules in a discovery series. Many of the major drug-drug interactions recognised to date occur at the level of liver cytochromes P450. All of the major human cytochromes P450 involved in drug metabolism have been cloned and expressed in transformed cell lines. These are commercially available, either as the original cell lines, or as microsomes or purified enzyme prepared from them. In these formats, the P450s can be used to determine the enzymology of a compound's metabolism and its potential for inhibition-based interactions. Current adaptations of the methods used in these types of study are enabling expressed P450s to be used in high throughput metabolism screens. It is possible that, in the future, they will also be incorporated into novel techniques, such as hyphenated screening systems. Thus, the expressed P450s will continue to be an important tool for drug discovery in the pharmaceutical industry.

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