Kyoichi Shimomura

Transcriptional inhibition of insulin by FK506 and possible involvement of FK506 binding protein-12 in pancreatic β-cell

FK506 (tacrolimus) is a strong immunosuppressant: it has been approved as a drug for liver transplantation in Japan, the United States, and the United Kingdom. One of its main adverse effects is hyperglycemia. Thus, in this study, we investigated the mechanism and the reversibility of the hyperglycemia caused by FK506. FK506 did not affect the glucose uptake by insulin into rat strio-muscle cell line, but suppressed insulin production in rat insulinoma cells. Two-week oral administration of FK506 at 10 mg/kg/day suppressed insulin production time-dependently at the transcriptional step in pancreatic β-cells, while glucagon content in pancreatic α-cells was not affected. When FK506 administration was stopped in these rats, insulin mRNA transcription and insulin production returned to normal. This recovery indicates that the adverse effect of FK506 on the pancreas is reversible. A high content of FK506 binding protein-12 (FKBP-12) in the pancreatic β-cells was confirmed by immunostaining with anti-human FKBP-12 mAb, but the content was less in the pancreatic α-cells and almost negligible in the acinar cells. In contrast, a high content of calcineurin in the pancreatic α-cells was confirmed by using anti-calcineurin polyclonal antibody, but this content was less in the pancreatic β-cells and not found in the acinar cells. Thus, as in the case with NF-AT in T cells, these findings point to the reduction of unidentified nuclear factors for insulin mRNA transcription caused by the binding of FK506 to FKBP-12 and a subsequent inhibition of calcineurin in the β-cells.

Chemical synthesis and analgesic effect of morphine ethereal sulfates

Abstract Chemical synthesis of morphine-3- and -6-ethereal sulfate was accomplished, utilizing chlorosulfonic acid as sulfonating reagent. The analgesic effect of morphine-6-sulfate in mice was about same order of magnitude as that of morphine-6-glucuronide, being much higher in potency and longer in duration than that of morphine. On the other hand, no effect was observed with morphine-3-sulfate (20 mg/kg, s. c.). Morphine-6-sulfate, an active conjugate, could not be detected in the urine of cats injected with morphine by careful examination with thin-layer chromatography.

Effect of FR145237, a novel ACAT inhibitor, on atherogenesis in cholesterol-fed and WHHL rabbits. Evidence for a direct effect on the arterial wall

The hypocholesterolemic and antiatherosclerotic activities of FR145237, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, were evaluated in cholesterol-fed and Watanabe heritable hyperlipidemic (WHHL) rabbits. In the first experiment, rabbits were fed a high cholesterol (1% cholesterol) diet for 2 weeks and further fed a high cholesterol diet containing FR145237 for 8 weeks. FR145237 (0.1, 0.32 and 1.0 mg/kg) dose-dependently lowered the plasma total cholestrol levels by 80%, 96% and 97%, respectively. and reduced aortic atherosclerosis by 44%, 90% and 90%, respectively. To clarify a direct effect of FR145237 at the aortic wall, a second experiment was performed. Rabbits were fed a high-cholesterol diet for 8 weeks to establish aortic atherosclerosis and then fed a normal diet with or without FR145237 for 8 weeks. Cholesterol content in the aorta and the liver was significantly reduced in the FR145237 group (10 mg/kg) by 50% and 43%, respectively, though plasma total cholesterol level did not differ from that in the control group. In the WHHL rabbits, FR145237 (10 mg/kg) did not affect plasma cholesterol level but significantly reduced the atherosclerotic lesion in the coronary arteries by 61%. These results suggest that FR145237 potently lowers the plasma cholesterol level in hypercholesterolemia induced by dietary cholesterol but not that by LDL receptor deficiency, and that FR145237 has a direct antiatherosclerotic activity on the arterial wall independent of its hypocholesterolemic activity.

FK317: a novel substituted dihydrobenzoxazine with potent antitumor activity which does not induce vascular leak syndrome.

Purpose: FK973, a substituted dihydrobenzoxazine, is an antitumor antibiotic which has shown high therapeutic efficacy in a phase I study, but its development has been abandoned because of the side effect of vascular leak syndrome (VLS) in the clinical study. This study was performed to investigate whether or not FK317, a new benzmethoxy derivative of FK973, retains the antitumor activity of FK973 without the side effect of VLS. Methods: VLS was evaluated by the volume of pleural effusion in rats. Cytotoxic activities were determined by a tetrazolium-based colorimetric assay (MTT assay) against murine (B16, P388) and human (HeLa S3, KB) tumor cell lines. Antitumor activities against murine ascitic leukemia (P388, L1210), murine solid tumors (reticulum cell sarcoma M5076, Colon 38 carcinoma) and human xenografts (mammary carcinoma MX-1, lung carcinoma LX-1) were examined. Results: FK973 (1.8 mg/kg) given i.v. to rats induced pleural effusion, one of the elements of VLS, 36 days after the first dosing, but did not 28 days after dosing. This model reflects clinical VLS delayed-type effusion with high protein concentrations. In contrast, FK317 (1.0–3.2 mg/kg) did not induce pleural effusion at all. FK317 had stronger cytotoxic effects against in vitro cultured B16, P388, HeLa S3 and KB tumor cell lines, and in in vivo experiments, FK317 showed equivalent antitumor activity against P388, M5076 and MX-1, and more potent antitumor activity against L1210, Colon 38 and LX-1 compared with FK973. Conclusion: These results suggest that FK317 retains the antitumor activity of FK973 and does not induce VLS, and FK317 is a drug with high clinical potential for treating tumors in humans.

The efficacy of combined treatment with recombinant human tumor necrosis factor-α and 5-fluorouracil is dependent on the development of capillaries in tumor

The antitumor effects of recombinant human tumor necrosis factor-alpha (rTNF-alpha) and 5-fluorouracil (5-FU) in combination treatment were examined on Meth A fibrosarcoma implanted intradermally in mice. Growth of the tumor was inhibited when rTNF-alpha was given i.v. on day 7 or 11 after implantation, but the effect was countered when 5-FU was additionally given i.p. once a day on days 1-4 after implantation. Conversely, 5-FU given on days 5-8 after implantation augmented the antitumor effects of rTNF-alpha. Injection of carbon particles showed that fine capillaries did not develop in the tumors of mice treated with 5-FU on days 1-4 after implantation, but that a delicate network of capillaries developed in the tumors of both the mice treated with 5-FU on days 5-8 after implantation and the controls given saline. The results show that the timing of 5-FU treatment is important when attempting to enhance the antitumor effects of rTNF-alpha, and suggest that these effects are directly associated with newly formed fine capillaries in the tumor.

Anti‐cachectic Effect of FK317, a Novel Anti‐cancer Agent, in Colon26 and LX‐1 Models in Mice

The effects of FK317 (11‐acetyl‐8‐carbamoyloxymethyl‐4‐formyl‐6‐methoxy‐14‐oxa‐1,11‐diazatetra‐cyclo[7.4.1.02,7.010,2]tetradeca‐2,4,6‐trien‐9‐yl acetate), a novel anti‐cancer agent, on murine adenocarcinoma colon26‐ and human lung carcinoma LX‐1‐induced cachexia were investigated in mice. Mice bearing colon26 or LX‐1 s.c. lost weight and became cachectic, associated with tumor growth. FK317 and mitomycin C (MMC) inhibited the growth of both tumors. FK317 ameliorated the weight loss induced by the presence of colon26 or LX‐1, while MMC enhanced it. An attenuation of the reduction in the weights of epididymal fat, gastrocnemius muscle and carcass was observed in FK317‐treated tumor‐bearing mice in both cachexia models, but not in MMC‐treated mice. The decreases in the circulating levels of triglyceride, glucose and non‐esterified fatty acid, which were induced by the presence of colon26, was partially inhibited by treatment with FK317. Overall, this study revealed that FK317 is a potent anti‐cancer drug with anti‐cachectic activity, suggesting that FK317 has potential utility for the treatment of cancer.

FK317, a novel substituted dihydrobenzoxazine, exhibits potent antitumor activity against human tumor xenografts in nude mice.

The antitumor effects of FK317, a novel substituted dihydrobenzoxazine, were evaluated using human tumor xenografts (small cell lung cancer, non‐small cell lung cancer, stomach cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer and ovarian cancer). Tumor growth‐inhibitory effects and the effective dose‐range of FK317 were much stronger and broader, respectively, than those of reference drugs such as mitomycin C, adriamycin, cisplatin, taxol and irinotecan. Furthermore, the body weight decrease and myelosuppression in FK317‐treated mice were less than in the animals given any of the reference drugs. To explain this tumor selectivity, the distribution of FK317 was investigated after dosing tumor‐bearing mice with the 14C‐labelled compound. The concentration of FK317 in tumor tissues was relatively low, and long tumor retention was not observed. However, thin‐layer chromatographic separation revealed that the radioactivity in the tumor resided mainly in strongly cytotoxic metabolites, while that in other tissues resided mainly in non‐cytotoxic metabolites. These results suggest that FK317 shows strong antitumor activity without side effects, and one reason for this is its specific metabolite pattern. FK317 is now undergoing phase I clinical trials.

Cytotoxic Mechanisms of FK317, a New Class of Bioreductive Agent with Potent Antitumor Activity

FK317 is a member of a new class of bioreductive agents that exhibit strong cytotoxicity against various human cancer cells. The effect of FK317 was found to be stronger than that of mitomycin C (MMC), adriamycin (ADR) or cisplatin (CDDP). Alkaline elution analysis indicated that FK317 formed interstrand DNA‐DNA and DNA‐protein cross‐links in cells. On the other hand, no DNA single‐strand breaks were observed in the cells treated with FK317. In a cell‐free system the deacetylated metabolites produced cross‐linked DNA under reductive conditions, though FK317 itself did not form DNA‐DNA cross‐links. In order to elucidate the metabolic activation mechanisms, we established an FK317‐resistant subline from human non‐small cell lung cancer cells (Lu99) by stepwise and brief exposure (1 h) to FK317. The resistant subline (Lu99/317) showed cross‐resistance to MMC and carboquone (CQ), but not to ADR or CDDP. DT‐diaphorase, which is one of the activation enzymes of MMC and CQ, was deficient in Lu99/317 cells as determined by enzyme activity assay. However, the levels of NADPH:cytochrome P450 reductase, which is another activation enzyme for MMC and CQ, were comparable in resistant and parent cell lines. Treatment of the cells with dicumarol, an inhibitor of DT‐diaphorase, reduced the cytotoxicity of FK317 to Lu99 cells, but not to Lu99/317 cells. These results indicate that deacetylation of FK317 is necessary for its reductive activation, and deacetylated FK317 is reduced by DT‐diaphorase to form an active metabolite, which produces DNA‐DNA interstrand and DNA‐protein cross‐links that lead to cell death.

Different Effects of FK317 on Multidrug‐resistant Tumor in vivo and in vitro

FK317, a novel substituted dihydrobenzoxazine, was examined for antitumor effects on multidrug‐resistant (MDR) tumor cells in vitro and in vivo. In nude mice, FK317 markedly inhibited the growth of s.c. implanted KB‐V1 vinblastine (VLB)‐resistant human epidermal carcinoma KB cells, as well as the parent cells (KB‐3‐1). However, KB‐V1 showed much greater resistance to FK317 than to VLB and adriamycin (ADM) in the in vitro study. This resistance was reversed by the addition of verapamil, whereby intracellular accumulation of FK317 in the KB‐V1 cells was also decreased. After incubation of FK317 in human and mouse blood, it was shown to be rapidly metabolized to a monodeacetylated form, and slowly metabolized further to a dideacetylated form. With the removal of the acetyl groups from FK317, resistance indexes in KB‐V1 and SBC‐3/ADM, ADM‐resistant human lung carcinoma, decreased. In addition, photolabeling of P‐glycoprotein with [3H]azidopine in KB‐V1 plasma membrane was completely inhibited by FK317, but not by the deacetylated metabolites. These results indicate that FK317 is metabolized to deacetylated forms, which do not bind to P‐glycoprotein and are incorporated into MDR cells, causing cytotoxic effects.

An approach to high-level production of a mecasermin (somatomedin C) fused protein in Escherichia coli HB101

Abstract A method to produce a mecasermin fused protein on an industrial scale was investigated. We constructed a new expression vector (designated pLSD1) derived from pLHSdMmtrp (Saito, Y. et al.: J. Biochem., 101, 123–134, 1987) by introduction of a synthetic fd phage terminator and deletion of the rop region originating from pBR322. The plasmid, pLSD1, exhibited high stability and was present at high copy number in Escherichia coli HB101. Although the growth of E. coli HB101 pLSD1 was not sufficient for high-level production of the fused protein in a Trp-deficient medium such as M9CA medium, it was improved by growing the strain in a medium containing 0.7% yeast extract which was constantly supplied with glucose. E. coli HB101 requires Pro and Leu for its growth; however, excess Leu tended to inhibit the cell growth. From the results of investigation of the mechanism of inhibition by Leu, addition of Ile was found to prevent the inhibition. Consequently, high-level production of the fused protein was attained by (i) using pLSD1 as the expression vector, (ii) cultivation of E. coli HB101 pLSD1 in a medium containing 0.7% yeast extract, (iii) fed-batch cultivation with periodic addition of glucose, and (iv) addition of Pro, Leu and Ile during cultivation. The cell density reached an OD at 600 nm of 50.4 and production of the fused protein was 1.24 g/l broth in a 150 l fermentor.