Lloyd A. Dethloff

Apoptosis and Nitrative Stress Associated with Phosphodiesterase Inhibitor-Induced Mesenteric Vasculitis in Rats

Nitric oxide may play a role in phosphodiesterase (PDE) inhibitor-induced rat mesenteric vasculitis. The present study was conducted to identify cellular sources of iNOS, determine the distribution of nitrotyrosine (NT) residues as a footprint of peroxynitrite (ONOO-) production, and evaluate their association with vascular apoptosis. To dissociate primary events from secondary changes associated with the inflammatory response, rats were given the PDE IV inhibitor CI-1018 orally at 750 mg/kg alone or concurrently with dexamethasone (DEX) intraperitoneally at 1 mg/kg for 4—5 days. Neutrophil (PMN) involvement in apoptosis was investigated in CI-1018 treated rats dosed with rabbit anti-rat PMN serum (APS). iNOS expression, NT residues, and caspase-3 were detected by immuno-histochemistry. Apoptosis was evaluated by TUNEL assay. CI-1018 induced vascular lesions were associated with iNOS expression in endothelial cells and inflammatory infiltrates; NT was evident only in the latter. Caspase-3 and TUNEL-positive staining were prominent only in medial smooth muscle cells (SMC) from CI-1018-treated rats and only when associated with active inflammation. iNOS- and NT-positive inflammatory cells were present in close proximity to SMC with caspase-3 staining. Inflammatory infiltrates were absent in rats given DEX with minimal SMC necrosis and hemorrhage remained. DEX eliminated apoptosis and immunoreactivity associated with caspase-3, iNOS, and NT. APS depletion of PMNs decreased the incidence and severity of vasculitis but failed to abolish completely caspase-3 immunoreactivity. Expression patterns for caspase-3, iNOS, and NT demonstrated that nitrative stress is a prominent feature of PDE inhibitor-induced vasculitis, with a possible role in medial SMC apoptosis. Further, medial SMC apoptosis may not be a primary event, but instead may be secondary to the inflammatory response.

Inhibition of gastrin-stimulated cell proliferation by the CCK-B/gastrin receptor ligand CI-988

The gastrointestinal hormone gastrin functions as a trophic factor for oxyntic mucosa as well as a secretagogue for gastric acid. In preclinical toxicology studies CI-988, a peptoid cholecystokinin (CCK) ligand with nanomolar affinity for the CCK-B/gastrin receptor, caused gastric gland degeneration and mucosal atrophy in cynomolgus monkeys, perhaps consistent with an expected pharmacological outcome of inhibition of the trophic effect of gastrin on stomach mucosa. Because of the expense and difficulty associated with experimental use of non-human primates, we investigated the effects of CI-988 on signal transduction pathways associated with gastrin-stimulated cell proliferation using the AR42J rat pancreatic tumour cell line as a model. The AR42J cell line was selected because it is known to express the CCK-B/gastrin receptor and because it is responsive to the growth promoting effects of gastrin in vitro. Gastrin-17 at 1 nM stimulated proliferation of AR42J cells 26% and 104% above control after 24 and 96 hours, respectively. CI-988 at 1 nM had no apparent effect on basal cell proliferation rates, but decreased gastrin-17 stimulated cell proliferation 13% and 47%, respectively, after 24 and 96 hours of treatment, consistent with competitive antagonism at the gastrin receptor. Because the trophic effect of gastrin towards AR42J cells has been linked to intracellular calcium ([Ca2+]i) mobilization and/or cyclic AMP, the effect of CI-988 on these second messengers were also investigated. Gastrin-17 at 10 nM stimulated both ([Ca2+]i) and cAMP, while CI-988 alone at 100 nM had no effect, but blocked the gastrin-stimulated increases in both mediators. Therefore, using the AR42J pancreatic tumour cell line as a model, the dipeptoid CCK-B/gastrin receptor ligand CI-988 behaves as an antagonist towards gastrin receptor-stimulated signal transduction pathways and cell proliferation in vitro.

Gastric gland degeneration induced in monkeys by the CCK-B/gastrin receptor antagonist CI-988.

Gastric effects of subchronic treatment with the cholecystokinin-B (CCK-B)/gastrin receptor antagonist CI-988 were investigated in cynomolgus monkeys. In preliminary range-finding studies, CI-988 was given orally to 1 monkey per sex for 14 days at doses of 50, 100, 200, and 500 mg/kg/day. Subchronic studies of CI-988 were subsequently conducted using 5 monkeys per sex at doses of 0, 5, 25, and 75 mg/kg for 4 or 13 wk. High-dose monkeys were dosed initially at 100 mg/kg, but the dose was not well tolerated and was decreased to 75 mg/kg after 8 days of treatment. One male monkey at 75 mg/kg was euthanatized in extremis on day 23. In the range-finding study, minimal to moderate, multifocal to diffuse degeneration of gastric glands, primarily in the fundic region, was observed at 100 mg/kg and above, with frank gastric mucosal atrophy occurring at 200 and 500 mg/kg. Minimal to mild gastric gland degeneration was also observed in the subchronic study after 4 wk at 25 and 75 mg/kg, but histopathologic gastric changes were remarkably absent after 13 wk. Mucosal height in the stomach fundus was decreased 19.8% in 75-mg/kg males at week 4, and although gastric mucosa appeared histologically normal after 13 wk, mucosal height remained 28.6% less than that of controls. In females at 75 mg/kg, fundic mucosal height was decreased 7% and 5% at weeks 4 and 13, respectively, but decreases were not statistically significant. Mean serum gastrin concentrations were increased 10-fold in males only after 4 wk at 75 mg/kg, but were comparable to controls during week 13. CI-988-induced gastric gland degeneration is consistent with antagonism of gastrins trophic activity toward gastric mucosa. Notwithstanding decrements in gastric mucosal height, disappearance of mild histopathologic findings despite continued treatment with the ligand suggests some degree of adaptation to subchronic CCK-B/gastrin inhibition, although the mechanism of accomodation has yet to be delineated.

Divergent proliferative responses to a gastrin receptor ligand in synchronized and unsynchronized rat pancreatic AR42J tumour cells

Depending upon experimental model, the CCK-B/gastrin receptor ligand CI-988 exhibits either agonist or antagonist activity. To confirm that CI-988 behaves as an antagonist toward gastrin-stimulated growth, its effects on cell proliferation were investigated in unsynchronized and synchronized AR42J rat pancreatic tumour cells. In unsynchronized cultures CI-988 alone had no effect, but inhibited gastrin-stimulated cell proliferation. In contrast, in synchronized cultures, CI-988 stimulated cell proliferation. Similarly, CI-988 inhibited gastrin-stimulated cAMP production in unsynchronized cells, but stimulated cAMP formation in synchronized cultures. Therefore, CI-988 stimulation of cAMP production and proliferation in AR42J cell cultures appears to be cell cycle-dependent. CI-988 inhibited gastrin-stimulated intracellular calcium ([Ca2+]i) mobilization in both populations and thus acted as an antagonist toward this pathway. Because CCK receptor densities and affinities were similar in both cell populations, the data suggest that CI-988s divergent effects on cell proliferation are governed by postreceptor signalling events which vary with cell cycle.

Gastric effects of the CCK-B/gastrin receptor ligand CI-988 in cynomolgus monkeys

We have previously demonstrated that the CCK-B/gastrin receptor ligand CI-988 induces gastric gland degeneration and atrophy in cynomolgus monkeys, an effect consistent with gastrin receptor antagonism and inhibition of gastrins trophic effects on oxyntic mucosa. However, gastrin receptor ligands of the dipeptoid chemical series to which CI-988 belongs have been reported to act as agonists or antagonists towards gastrin-related events, depending on the animal model and the functional endpoint examined. To investigate further these apparently conflicting data, basal gastric acid secretion was monitored acutely in conscious monkeys given CI-988 orally at 10 mg/kg or intravenously at 0.01 mumol/kg/hr and histological changes in gastric mucosa were evaluated in monkeys given CI-988 orally at 5, 25 or 75 mg/kg/day for 4 weeks. Degeneration and atrophy of gastric glands occurred at 25 and 75 mg/kg with statistically significant decrements in gastric mucosal height at 75 mg/kg. In addition, CI-988 stimulated gastric acid secretion when given either orally or intravenously. Co-administration of the structurally unrelated CCK-B/gastrin antagonist L-365,260 completely blocked CI-988-stimulated acid secretion, confirming that CI-988s agonist effect on acid secretion is mediated by the gastrin receptor. Assuming that gastric mucosal degeneration is the result of inhibition of gastrins trophic activity, CI-988 appears to induce paradoxical agonist and antagonist gastrin-receptor mediated effects.

Subacute and subchronic toxicology studies of CI-986, a novel anti-inflammatory compound

CI-986 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)- thione-2-hydroxy-N,N,N-trimethylethanaminium salt) is a novel anti-inflammatory compound classified as a dual inhibitor of cyclooxygenase and 5-lipoxygenase. Studies were undertaken to characterize the preclinical toxicology of the compound. CI-986 was administered to rats for 2 weeks (0, 50, 250, 750, and 1500 mg/kg) or 13 weeks (0, 20, 250, 500, and 1000 mg/kg), dogs for 2 weeks (0, 50, 150, and 500 mg/kg) or 13 weeks (0, 20, 100, and 200 mg/kg), and to monkeys for 2 weeks (0, 50, 250, and 1000 mg/kg). No drug-related deaths resulted. Mild clinical signs of toxicity were noted in rats given doses of 250 mg/kg and above. Drug-related emesis and diarrhea were absent at the low dose in the dog and monkey but increased in incidence and severity at higher doses. Severe clinical signs in monkeys (emesis and diarrhea) necessitated the lowering of the top dose to 500 mg/kg/day (administered b.i.d.) during the second week of the monkey study. Slight decreases (< 23%) in serum protein and/or albumin were noted in all studies at the higher doses. A dose-related increase in alkaline phosphatase was noted in both dog studies, with no other drug-related effect on clinical pathology parameters. A gastric ulcer occurred in one rat administered 500 mg/kg CI-986 for 13 weeks. Gastrointestinal ulcers were not noted at any other dose in rats or at any dose in dogs or monkeys. A dose-related eosinophilia of glandular stomach submucosa was noted in rats after 2 and 13 weeks of drug administration but not in dogs or monkeys. In the 2-week rat study, mean combined sex plasma drug concentrations monitored 2 hr after dose on Day 14 were 0.59, 1.10, 2.64, and 3.43 micrograms/ml for the 50, 250, 750, and 1,500 mg/kg dose groups, respectively. In the 2-week dog studies, maximum plasma drug concentrations on Day 10 or Day 11 were achieved within 2 hr of dose with mean combined sex Cmax values of 0.73, 2.05, and 2.62 micrograms/ml for the 50, 250, and 750 mg/kg groups, respectively. Hepatic microsomal induction characterized by increased microsomal protein, increased microsomal cytochrome P450 content, and increased p-nitroanisole O-demethylation activity was noted in dogs and monkeys but not rats. CI-986 was well tolerated in rats and dogs at the doses employed and in monkeys at doses up to 500 mg/kg (b.i.d.).(ABSTRACT TRUNCATED AT 250 WORDS)

Perspective on the carcinogenic potential of phenytoin based on rodent tumor bioassays and human epidemiological data

1 Phenytoin is a hydantoin-type anticonvulsive agent used extensively for nearly sixty years in the prophylactic treatment of grand mal and psychomotor seizures. 2 Based upon somewhat contentious evidence of pheny toin-induced lymphoma in mice and upon epidemiologic evidence of an association between anticonvulsive ther apy and lymphoma in epilepsy patients, the International Agency for Research on Cancer (IARC) has collectively regarded these data as limited evidence of carcinogeni city. 3 Two year carcinogenicity studies of standard bioassay design conducted in mice and rats yielded statistically significant increased incidence of hepatocellular adeno mas in mice at phenytoin plasma concentrations approx imating the therapeutic anticonvulsive range. Tumor incidence in rats was not affected. Previous carcinogeni city studies have found similar increases in hepatic tumor incidence in mice. 4 Phenytoin is a known enzyme inducer and shows tumor promoting activity in chemically initiated mouse liver. Evidence for genotoxicity is weak or equivocal, consequently phenytoin-induced liver tumors appear to occur through nongenotoxic mechanisms. 5 Finally, despite six decades of extensive therapeutic use and thorough epidemiologic evaluation, there is no evidence for an association between liver cancer and phenytoin therapy in epilepsy patients. Thus, hepatocel lular neoplasia in phenytoin-treated rodents appears to be of little significance to man.

Chronic Toxicity of the Anticancer Agent Trimetrexate in Rats

Trimetrexate was administered to rats in an interrupted treatment regimen comparable to proposed human clinical treatment. Forty-five rats of each sex were dosed intravenously with trimetrexate at 0, 1, 10, or 30 mg/kg (0, 6, 60, or 180 mg/m2), once daily for 5 consecutive days, followed by a 23-day recovery period. This cycle of dosing and recovery was repeated for a total of six cycles. Hematology, urinalysis, clinical chemistry, and gross and microscopic pathology examinations were conducted for animals euthanatized 3 and 21 days after dosing cycles 1, 3, and 6. Additional rats in each group were maintained without dosing for an additional 56 days (77 days after the last trimetrexate dose) to assess the long-term reversibility of pathologic changes. Target organs were typical for an antifolate and included gastrointestinal tract, lymphoid tissues, and the hematopoietic and male reproductive systems. No toxicity was observed at 1 mg/kg. Treatment-related changes in hematology parameters following 10 and 30 mg/kg were fully reversible within 3 weeks of each dosing cycle. Except for testis and cecum, histopathological changes were also reversible within 21 days of dosing. Trimetrexate-induced testicular changes persisting during the course of multiple cycles of dosing were not reversible within 21 days, but required an additional 56 days for essentially complete recovery.

Toxicokinetic comparison of a CCK‐B/gastrin receptor antagonist given orally and intravenously

CI‐988 is a dipeptoid CCK‐B/gastrin receptor ligand with potential clinical indications in anxiety and panic disorder. The toxicity and toxicokinetics of CI‐988 given orally (PO) or intravenously (IV) were evaluated to delineate the relationship between administered dose, plasma CI‐988 concentrations, and target organ changes in order to establish appropriate safety margins for human clinical studies. CI‐988 was given to monkeys PO at doses of 5, 25, and 75 mg/kg/day or IV at doses of 0.2, 2, and 5 mg/kg/day for up to 4 weeks. When given PO, CI‐988 caused dose‐related, mild to moderate gastric gland degeneration at 25 and 75 mg/kg. There were no adverse effects at 5 mg/kg. Associated with these doses were mean (± standard deviation) maximum plasma concentrations (Cmax) of 5.6 ± 4.9, 12.9 ± 6.2, and 10.4 ± 5.1 ng/mL and areas under the plasma concentration vs. time curve (AUC) of 24.1 ± 14.2, 63.8 ± 27.1 and 100 ± 43 nghmL–1, respectively. Given IV, CI‐988 had no effect on the gastric mucosa despite achieving plasma concentrations as high as 239 ± 116, 2,250 ± 1,230, and 5,490 ± 1,890 ng/mL approximately 5 min postdose at 0.2, 2, and 5 mg/kg, respectively. The AUCs associated with IV CI‐988 at 5 mg/kg were at least 10‐fold greater than those associated with PO doses shown to induce gastric lesions. These data indicate that plasma CI‐988 concentrations are not predictive for, or directly associated with, gastric gland degeneration and suggest that these mucosal changes may be related to local, rather than systemic, exposure to CI‐988. Accordingly, safety margins for CI‐988 appear to be more appropriately based upon administered dose than upon plasma concentrations. Drug Dev. Res. 40:292–298, 1997.