A new severe acute necrotizing pancreatitis model induced by L-ornithine in rats.

OBJECTIVE: Intraperitoneal administration of large doses of L-arginine is known to induce severe acute pancreatitis in rats. We therefore set out to determine whether metabolites of L-arginine (L-ornithine, L-citrulline, and nitric oxide) cause pancreatitis. DESIGN: The authors conducted an in vivo animal study. SETTING: This study was conducted at a university research laboratory. SUBJECTS: Study subjects were male Wistar rats. INTERVENTIONS: Dose-response and time course changes of laboratory and histologic parameters of pancreatitis were determined after L-arginine, L-ornithine, L-citrulline, or sodium nitroprusside (nitric oxide donor) injection. MEASUREMENTS AND MAIN RESULTS: Intraperitoneal injection of 3 g/kg L-ornithine but not L-citrulline or nitroprusside caused severe acute pancreatitis; 4 to 6 g/kg L-ornithine killed the animals within hours. Serum and ascitic amylase activities were significantly increased, whereas pancreatic amylase activity was decreased after intraperitoneal injection of 3 g/kg L-ornithine. The increase in pancreatic trypsin activity (9-48 hrs) correlated with the degradation of IkappaB proteins and elevated interleukin-1beta levels. Oxidative stress in the pancreas was evident from 6 hrs; HSP72 synthesis was increased from 4 hrs after L-ornithine administration. Morphologic examination of the pancreas showed massive interstitial edema, apoptosis, and necrosis of acinar cells and infiltration of neutrophil granulocytes and monocytes 18 to 36 hrs after 3 g/kg L-ornithine injection. One month after L-ornithine injection, the pancreas appeared almost normal; the destructed parenchyma was partly replaced by fat. Equimolar administration of L-arginine resulted in lower pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, and histologic damage compared with the L-ornithine-treated group. L-ornithine levels in the blood were increased 54-fold after intraperitoneal administration of L-arginine. CONCLUSIONS: We have developed a simple, noninvasive model of acute necrotizing pancreatitis in rats by intraperitoneal injection of 3 g/kg L-ornithine. Interestingly, we found that, compared with L-arginine, L-ornithine was even more effective at inducing pancreatitis. Large doses of L-arginine produce a toxic effect on the pancreas, at least in part, through L-ornithine.

Polyamines and regulation of ornithine biosynthesis in Escherichia coli.

The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.

Antitumor properties of (2R,5R)-6-heptyne-2,5-diamine, a new potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, in rodents.

(2R,5R)-6-Heptyne-2,5-diamine hydrochloride (MDL 72175) is a new, potent, and selective inhibitor of mammalian ornithine decarboxylase. MDL 72175 given p.o. in drinking fluid reduced by 80% the growth of EMT6 sarcoma in mice and of HTC hepatoma in rats. It prolonged the survival of mice bearing L1210 or P388 leukemias and inhibited the development of Lewis lung carcinoma in mice at doses 10- to 20-fold lower than those of alpha-difluoromethylornithine, the most widely used irreversible inhibitor of ornithine decarboxylase. MDL 72175 depleted putrescine and spermidine levels in the tumors to the same extent as did alpha-difluoromethylornithine. In the EMT6 sarcoma, MDL 72175 achieved at low doses a greater maximal antitumor effect than did alpha-difluoromethylornithine. In combination therapy, MDL 72175 plus Adriamycin gave at least additive antitumor effects on solid tumors and experimental leukemias in animals. The combination MDL 72175 plus methylglyoxal bis(guanylhydrazone) also gave additive antitumor effects on P388 leukemia, associated with an increased uptake of methylglyoxal bis(guanylhydrazone); in contrast, antagonistic effects were observed with this combination on EMT6 tumors in mice. Since MDL 72175 did not present toxicity at effective antitumor doses, this new ornithine decarboxylase inhibitor can be considered as a promising antitumor drug.

Inhibition of growth of human or hamster pancreatic cancer cell lines by alpha-difluoromethylornithine alone and combined with cis-diamminedichloroplatinum(II).

A major problem in the therapy of pancreatic adenocarcinoma is its inherent resistance to most chemotherapeutic agents. Previously, we have reported that the four pancreatic cancer cell lines studied here have elevated levels of ornithine decarboxylase, a growth-regulating enzyme, and further that the degree of elevation tends to parallel the degree of chemoresistance. On the basis of these prior findings, we investigated the effects of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, alone and in combination with cis-diamminedichloroplatinum(II) (cisplatin), to which two of the four cell lines display relative resistance. The cell lines studied were: two of human origin, PANC-1 and COLO-357; and two of hamster origin, WD PaCa and PD PaCa. Colony formation (clonogenic) assays were used to evaluate drug effects. Cells were exposed continuously to DFMO in medium. For the combined treatments, cells were exposed to cisplatin for 1 hr, washed, and then plated in DFMO-containing medium. The inhibitory effects of DFMO were predominantly cytostatic, were reversible by putrescine, and were roughly additive when combined with cisplatin. Our panel of cell lines responded heterogeneously to DFMO, with PANC-1 and WD PaCa showing the most sensitivity. The combination of DFMO and cisplatin appears to be a promising experimental approach to overcoming drug resistance in pancreatic cancer.

Effects of alpha-difluoromethylornithine on the growth of experimental Wilms' tumor and renal adenocarcinoma.

Because polyamines are essential for cellular growth and differentiation, and because human renal carcinomas have spermidine levels that are higher than those in normal renal tissue, effects of 2-difluoromethylornithine (DFMO) on the growth of experimental renal tumors were investigated. DFMO is a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme controlling polyamine biosynthesis. DFMO (2%) in drinking water was administered to BALB/c mice with intrarenal transplants of a renal adenocarcinoma cell suspension and to Wistar/Furth rats with s.c. transplants of a Wilms' tumor. At 28 days, renal carcinomas in DFMO-fed mice weighed 72% less than those in control animals (p less than 0.001). Wilms' tumor weight was not affected by DFMO feeding. DFMO caused 72 to 75% inactivation of ornithine decarboxylase activity and reduced putrescine levels in renal carcinoma and Wilms' tumor, reduced spermidine levels in Wilms' tumor, and apparently raised spermine levels in the latter as a consequence. DNA content was not affected by DFMO feeding. The mean number of lung metastases in DFMO-fed, renal carcinoma-bearing mice was 0.1 and in controls was 1.4 (p less than 0.001). DFMO feeding increased survival of mice bearing renal carcinomas by 3.0 +/- 0.8 (S.E.) days (p less than 0.05), i.e., from 30.5 +/- 0.8 days to 33.5 +/- 1.2 days. DFMO did not affect the growth of Wilms' tumor; however, in renal adenocarcinoma, it reduced growth, prevented lung metastases, and increased survival.

Successful treatment with DL-alpha-difluoromethylornithine in established human small cell variant lung carcinoma implants in athymic mice.

We report that p.o. administration of DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, markedly inhibits the growth of established implants of cultured human small cell lung carcinoma (SCC) in athymic (nude) mice. Human SCC tumor cells, from a cell line which exhibited cell death in culture in the presence of DFMO, were inoculated s.c. into athymic mice. The tumors were permitted to grow until they became palpable (0.05 cu cm, 3- to 5-mm-diameter nodules). The animals were then randomized into control, and early (low tumor burden) and late (high tumor burden) treatment groups which received 3% DFMO in the drinking water (5.0 g/kg/day). The tumors in the untreated control group grew to a size of 29 cu cm by 9 weeks, and these animals had a median survival of 9 weeks. The late treatment group began DFMO treatment 3 weeks after clinical tumor engraftment, when mean tumor size was 1.5 cu cm (1.2- to 1.5-cm-diameter nodules). Tumor growth was inhibited by 60% (11.4 cu cm) by Week 9 and survival was prolonged, with 83% survival at 10 weeks and a 56% increase in median survival to 14 weeks (p less than 0.05). The early treatment group received the same dose of DFMO beginning 1 week after tumor engraftment, when their mean tumor size was 0.1 cu cm (4- to 6-mm-diameter nodules). The early DFMO group had a 99% inhibition in tumor growth (0.3 cu cm) (p less than 0.05). Survival was also prolonged compared to the untreated controls, with 83% survival at 10 weeks and a median survival of 15 weeks (p less than 0.05). In both the early- and late-DFMO-treatment groups, no significant clinical toxicities were observed in the first 10 weeks, during which antitumor therapeutic effects were seen. DFMO may have a potential role in the treatment of sensitive human tumors such as SCC. The data suggest that DFMO may be most useful clinically in patients with SCC who have a low tumor burden. Thus, DFMO might be an important tool to produce long-term maintenance of initial clinical remissions induced by combination chemotherapy.