2.1. Animal welfare considerations
Prior to the initiation of in vivo experimentation, study protocols were reviewed and approved by the IIT Research Institute Institutional Animal Care and Use Committee. All aspects of the program involving animal care, use, and welfare were performed in compliance with United States Department of Agriculture regulations and the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Both studies were conducted in full compliance with the Good Laboratory Practice regulations of the United States Food and Drug Administration (21 CFR Part 58).
2.2. Test article
CP-31398 (> 99% purity; Indofine Chemical Co., Hillsborough, NJ) was obtained through the Chemopreventive Agent Repository maintained by the Division of Cancer Prevention, National Cancer Institute. Bulk CP-31398 was protected from light and stored desiccated at 2–8°C under nitrogen. Dosing formulations were prepared by dissolving CP-31398 in ASTM Type 1 water; after preparation, dosing formulations were stored under nitrogen at 2–8°C until used. CP-31398 was protected from light during all dose preparation, storage, and dose administration operations.
2.3. Twenty-eight day oral toxicity study (with recovery) in rats
Male and female CD rats (Crl:CD® [SD]IGS) were received at approximately five to six weeks of age from virus-free colonies maintained at Charles River Laboratories (Portage, MI). Rats were housed individually in suspended stainless steel cages in a temperature-controlled room maintained on a 12-hour light/dark cycle, and were held in quarantine for approximately two weeks prior to the initiation of dosing. With the exception of overnight fasts prior to scheduled necropsies, rats were allowed free access to Certified Rodent Diet 5002 (PMI Nutrition International, Brentwood, MO) throughout the study. City of Chicago drinking water was supplied to rats ad libitum using an automatic watering system.
After release from quarantine, rats were assigned to groups of 15/sex using a computer-based procedure that blocks for body weights. Within each dose group, 10 rats/sex were assigned to the Main Study cohort (scheduled for necropsy on Study Day 29), and five rats/sex were assigned to the Recovery cohort (scheduled for necropsy on Study Day 43). After group assignment, rats received daily oral (gavage) administration of CP-31398 (in ASTM water) at doses of 0 (control), 40, 80, or 160 mg/kg/day (0, 240, 480, or 960 mg/m2/day) for 28 days; a constant dosing volume of 10 ml/kg/day was used.
Doses were selected for the 28-day definitive toxicity study in rats on the basis of the results of a preliminary 14-day range-finding study in which groups of 5 rats/sex received oral exposure to CP-31398 at doses of up to 500 mg/kg/day. In this study, administration of CP-31398 at 500 mg/kg/day induced 100% mortality, and drug administration at 250 mg/kg/day induced mortality in 0/5 male rats and 1/5 female rats. At both dose levels, mortality was associated with severe coagulation necrosis in the liver (hepatic infarcts); hepatic infarcts were also a common microscopic finding in surviving rats receiving drug at 250 mg/kg/day. By contrast, toxicity in rats receiving CP-31398 at 100 mg/kg/day was limited to minimal to mild microscopic alterations in the liver (hypertrophy and vacuolation of Kupffer cells) and heart (cytoplasmic alterations); no clear evidence of drug-related toxicity was seen in rats receiving CP-31398 at 50 mg/kg/day. On this basis, doses for the definitive study were selected with the expectation that significant toxicity would be seen in rats exposed to the high dose (160 mg/kg/day) of CP-31398, and that little or no toxicity would be seen at the low dose (40 mg/kg/day).
Throughout the study, rats were observed a minimum of twice daily to monitor their general health status; detailed clinical examinations and measurements of body weight and food consumption were performed weekly. Indirect funduscopic ophthalmic examinations were performed on all animals during the quarantine period (pre-test) and during the final week of the treatment period. A Functional Observational Battery (FOB) was performed on five rats/sex/group at pretest, during Weeks 1 and 4 of the treatment period, and during the final week of the recovery period; during Weeks 1 and 4, FOBs were performed at two to four hours post-dosing. Evaluations in the FOB included home cage observation, hand-held observation, open field observation (mobility/gait), body weight, body temperature, eye blink, pupil response, tail pinch, hind limb extension, hearing (click response), vision, catalepsy, righting reflex, grip strength (forelimb and hind limb), and foot splay.
Blood samples for clinical chemistry, hematology, and coagulation evaluations were collected from fasted rats prior to their scheduled necropsy. Clinical pathology assays were performed using automated instruments (Synchron CX5 Clinical Chemistry Analyzer [Beckman Instruments, Brea, CA]; Advia System 120 Hematology Analyzer [Bayer Corp., Tarrytown, NY]; STA Compact CT automatic coagulation analyzer [Diagnostica Stago, Parsippany, NJ]).
On Study Day 29, surviving Main Study rats were humanely euthanized with sodium pentobarbital followed by exsanguinations, and were subjected to a complete necropsy with tissue collection. Surviving Recovery rats in each dose group were euthanized and submitted for necropsy on Study Day 43 (following a 14-day recovery period). At both scheduled necropsies, weights of the adrenals, brain, heart, kidneys, liver, ovaries/testes, spleen, thyroid, and uterus were collected from each animal. All gross lesions plus approximately 45 tissues per rat were collected and fixed in 10% neutral buffered formalin. All tissues from all rats in the high dose and vehicle control groups were processed by routine histologic methods, cut at 5 μm, stained with hematoxylin and eosin, and evaluated microscopically. Microscopic evaluation of tissues from rats in the mid and low dose groups in the Main Study and from all Recovery animals was limited to gross lesions and identified target tissues.
2.4. Metabolite profiling of CP-31398 in rats
Blood samples for plasma drug level analysis and metabolite profiling were collected from cohorts of four rats/sex/group at 1, 4, 8, 12, and 24 hours post-dosing on Study Day 1 and during Week 4 of CP-31398 administration; samples analyzed for metabolite profiles reflect a subset of samples that were collected to characterize CP-31398 pharmacokinetics (data reported elsewhere).
Levels of CP-31398 and metabolites in blood samples were quantitated by high performance liquid chromatography/quantitative time-of-flight mass spectrometry (LC/QTOF), using analytical methods developed in our laboratory (Muzzio et al., 2011
). Separations were performed using an Agilent 1200 LC (Agilent Technologies, Wilmington, DE) and a Luna 3 μ PFP(2) 100 Å, 100 × 2.0 mm column (Phenomenex, Torrance, CA). The mobile phases consisted of Solvent A (50 mM ammonium acetate in water/methanol/acetonnitrile [v:v:v::90:6:4]) and Solvent B (50 mM ammonium acetate in water/methanol/acetonnitrile [v:v:v::10:54:36)). The separation program was A:B::80:20 for 16 minutes and A:B::5:95 for 6 minutes, followed by 6 minutes of post-run time at A:B::80:20. Parent compound and metabolites were identified and quantitated using an Agilent 6250 QTOF mass spectrometer with electrospray ionization; data were analyzed using Agilent Mass Hunter software (revision B.01.03).
CP-31398 has a formula weight of 362.46804 daltons and a mono-isotopic mass of 362.21066 daltons. Under the analytical conditions used, CP-31398 was detected in the LC/QTOF system as the protonated molecular ion, with an exact mass of 363.21794 and a retention time of approximately 18 minutes. Metabolites would be expected to produce ions with molecular weight shifts characteristic of the biotransformation of the parent molecule; ions resulting from common biotransformation reactions (e.g., hydroxylation, demethylation, demethylation + hydroxylation, etc.) were detected by inspecting the total ion chromatogram file for the suspected metabolite protonated molecular ions. When such ions were found, the extracted ion chromatogram was integrated and the concentration of the metabolite was estimated using the calibration curve established for CP-31398.
2.5. Twenty-eight day oral toxicity study (with recovery) in dogs
Male and female purebred beagle dogs were received at approximately six months of age from Ridglan Farms, Inc. (Mount Horeb, WI), and were held in quarantine for three weeks prior to assignment to experimental groups. Throughout the study, dogs were housed individually in floor-level pens in a temperature-controlled room maintained on a 12-hour light/dark cycle. Dogs were provided with 400 g of Certified Canine Diet 5007 (PMI Nutrition International, Inc.) for a minimum of two hours each day, and were permitted free access to City of Chicago drinking water supplied via an automatic watering system.
After release from quarantine, dogs were assigned to groups of five/sex (three Main Study dogs/sex [scheduled for necropsy on Day 29] + two Recovery dogs/sex [scheduled for necropsy on Day 43]) using a computerized procedure that blocks for body weight. Dogs received daily oral (gavage) exposure to CP-31398 at doses of 0 (control), 10, 20, or 40 mg/kg/day (0, 200, 400, or 800 mg/m2/day) for 28 consecutive days, using a dosing volume of 2 ml/kg.
Doses of CP-31398 for the 28-day definitive toxicity study in dogs were selected on the basis of the results of a preliminary 14-day range-finding study in which groups of 1 dog/sex received oral administration of CP-31398 at doses of up to 50 mg/kg/day. In this range-finding study, administration of CP-31398 at 50 mg/kg/day induced mortality in one male dog; the probable cause of death was hepatic necrosis. Drug-related toxicity in dogs exposed to CP-31398 at 25 mg/kg/day was limited to minimal cytoplasmic alterations in the liver (basophilic clumping, cytoplasmic clearing); no evidence of toxicity was seen in dogs receiving CP-31398 at 5 or 10 mg/kg/day for 14 days. On the basis of these results, doses for the definitive toxicity study were selected with the expectation that clear evidence of CP-31398 toxicity would be seen in dogs receiving the high dose (40 mg/kg/day) of CP-31398, and that little or no toxicity would be seen in dogs receiving the low dose (10 mg/kg/day).
Dogs were observed a minimum of twice daily to monitor their general health status. Detailed clinical examinations and body weight measurements were performed weekly, and food consumption was measured daily. Blood samples for clinical chemistry, hematology, and coagulation evaluations were collected from all surviving dogs during pre-test and on Study Days 4, 8, 15, 22, 29, and 43. Clinical chemistry, hematology, and coagulation assays were performed as described for the 28-day toxicity study in rats. Urine samples were collected on the same schedule and analyzed by dipstick and microscopy.
Surviving Main Study dogs were humanely euthanized by barbiturate overdose on Study Day 29, and were subjected to a complete necropsy with tissue collection; this process was repeated on Study Day 43 for dogs in the Recovery groups. Weights of the adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, and thyroids were collected at necropsy, and all gross lesions and approximately 45 tissues were collected from each animal and fixed in 10% neutral buffered formalin. All tissues collected from dogs in the high dose and control groups in the Main Study were processed by routine histologic methods, cut at 5 μm, stained with hematoxylin and eosin, and evaluated microscopically. Microscopic evaluation of tissues from Main Study dogs in the mid and low dose groups and from Recovery dogs in all dose groups were limited to gross lesions and identified target tissues.
2.6. Metabolite profiling of CP-31398 in dogs
Blood samples for plasma drug level analysis and metabolite profiling were collected from all CP-31398-treated dogs at 0.5, 4, and 12 hours post-dosing on Study Days 1 and 25. Levels of CP-31398 and metabolites in blood samples were quantitated by LC/QTOF, as described for rats in Section 2.4.
2.7. Statistical analyses
Statistical evaluations of continuous data were performed by analysis of variance (ANOVA), with post-hoc analyses performed using Dunnett’s test. Incidence data were compared by χ2 analysis or Fischer’s Exact Test. A minimum significance level of p < 0.05 was used in all comparisons.