Masamichi Inami

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.

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.

Effects of FK317, a novel anti-cancer agent, on survival of mice bearing B16BL6 melanoma and Lewis lung carcinoma

The effects of FK317 (11-acetyl-8-carbamoyloxymethyl-4-formyl-6-methoxy-14- oxa-1,11-diazatraacylo[7.4.1.0(2.7).0(10.2)]-tetradeca-2,4,6-trien-9-yl acetate), a novel anti-cancer agent, and mitomycin C (MMC) on survival time of mice bearing B16BL6 melanoma and Lewis lung carcinoma (LLC), induced by intravenous inoculation of the tumor, were investigated. Treatment with FK317 resulted in a significant prolongation of survival time in both tumor models. Four of ten mice bearing B16BL6 were disease-free following FK317 treatment. In contrast, MMC was not effective in prolonging survival time. Overall, this study demonstrated that FK317 shows more potent survival extension in mice bearing B16BL6 and LLC than MMC, suggesting that FK317 may have therapeutic utility for cancer chemotherapy.

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.

Discovery of 3,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2(1H)-one derivatives as novel JAK inhibitors.

Because Janus kinases (JAKs) play a crucial role in cytokine-mediated signal transduction, JAKs are an attractive target for the treatment of organ transplant rejection and autoimmune diseases such as rheumatoid arthritis (RA). To identify JAK inhibitors, we focused on the 1H-pyrrolo[2,3-b]pyridine derivative 3, which exhibited moderate JAK3 and JAK1 inhibitory activities. Optimization of 3 identified the tricyclic imidazo-pyrrolopyridinone derivative 19, which exhibited potent JAK3 and JAK1 inhibitory activities (IC50=1.1 nM, 1.5 nM, respectively) with favorable metabolic stability.

FR177391, a new anti-hyperlipidemic agent from Serratia. II. Pharmacological activity of FR177391.

The pharmacological effect of FR177391, isolated from Serratia liquefaciens No. 1821, was studied in normal animals and various types of animal models of hypertriglyceridemia. Treatment of normal mice with FR177391 resulted in an increase in heparin-releasable lipoprotein lipase (LPL) activity in the blood and epididymal fat tissue. FR177391 treatment decreased triglyceride (TG) and increased high-density lipoprotein cholesterol in the blood in normal rats following 7 days treatment, suggesting potent LPL activating properties of FR177391. Both Triton WR1339-induced severe and fructose-induced mild hypertriglyceridemia in rats were attenuated by FR177391 treatment. Severely elevated levels of TG in db/db mice, an insulin resistant diabetic animal model, also significantly decreased from 14 days of treatment with FR177391. FR177391 treatment for 9 days caused a decrease in the elevated levels of TG in mice induced by intraperitoneal inoculation of murine lymphoma EL-4. Overall, this study demonstrated that FR177391 can be possibly a LPL activating agent and that FR177391 treatment improved hypertriglyceridemia in various rat and mouse animal models. These results suggest that FR177391 is a promising candidate compound for the management of hypertriglyceridemia.

AS2553627, a novel JAK inhibitor, prevents chronic rejection in rat cardiac allografts

ABSTRACT Janus family kinases (JAKs) are essential molecules for cytokine responses and attractive targets for the treatment of transplant rejection and autoimmune diseases. Several JAK inhibitors have shown demonstrable effects on acute rejection in experimental cardiac transplant models. However, little is known about the potential benefits of JAK inhibitors on chronic rejection outcomes such as vasculopathy and fibrosis. Here, we examined the pharmacological profile of a novel JAK inhibitor, AS2553627, and explored its therapeutic potential in chronic rejection as well as acute rejection in a rat cardiac transplant model. AS2553627 potently inhibited JAK kinases but showed no inhibition of other kinases, including TCR‐associated molecules. The compound also suppressed proliferation of IL‐2 stimulated human and rat T cells. In a rat cardiac transplant model, oral administration of AS2553627 alone or co‐administration with a sub‐therapeutic dose of tacrolimus effectively prolonged cardiac allograft survival, suggesting the efficacy in treating acute rejection. To evaluate the effect on chronic rejection, recipient rats were administered a therapeutic dose of tacrolimus for 90 days. In combination with tacrolimus, AS2553627 significantly reduced cardiac allograft vasculopathy and fibrosis that tacrolimus alone did not inhibit. AS2553627 at the effective dose in rat transplantation models did not significantly reduce reticulocyte counts in peripheral whole blood after in vivo erythropoietin administration, indicating a low risk for anemia. These results suggest that AS2553627 may be a therapeutic candidate for the prevention of not only acute but also chronic rejection in cardiac transplantation.

Synthesis and evaluation of novel 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives as potent and orally efficacious immunomodulators targeting JAK3.

Janus kinases (JAKs) regulate various inflammatory and immune responses and are targets for the treatment of inflammatory and immune diseases. As a novel class of immunomodulators targeting JAK3, 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives are promising candidates for treating such diseases. In chemical modification of lead compound 2, the substitution of a cycloalkyl ring for an N-cyanopyridylpiperidine in C4-position was effective for increasing JAK3 inhibitory activity. In addition, modulation of physical properties such as molecular lipophilicity and basicity was important for reducing human ether-a-go-go-related gene (hERG) inhibitory activity. Our optimization study gave compound 31, which exhibited potent JAK3 inhibitory activity as well as weak hERG inhibitory activity. In cellular assay, 31 exhibited potent immunomodulating effect on IL-2-stimulated T cell proliferation. In a pharmacokinetic study, good metabolic stability and oral bioavailability of 31 were achieved in rats, dogs, and monkeys. Further, 31 prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.