Prior to the initiation of in vivo experimentation, study protocols were reviewed and approved by the IIT Research Institute 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).
2.1. Subchronic oral toxicity study in rats
Male and female CD rats (Crl:CD®[SD]IGS) were received at approximately four weeks of age from virus-free colonies maintained at Charles River Laboratories (Raleigh, NC). Rats were housed individually in suspended stainless steel cages in a temperature-controlled room maintained on a 12 h light/dark cycle, and were held in quarantine for two weeks prior to the initiation of dosing. With the exception of scheduled fasting periods, rats were allowed free access to Certified Rodent Diet 5002 (PMI Nutrition International, Inc., 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 20 per sex using a computer-based randomization procedure that blocks for body weights. Rats received daily oral (gavage) administration of resveratrol (Pharmascience, Inc. [formerly Royalmount Pharma], Montreal, Quebec) at doses of 200, 400, or 1000 mg/kg/day (1200, 2400, or 6000 mg/m2
/day) for a minimum of 90 days; resveratrol doses were selected on the basis of the results of a previous 28-day toxicity study in rats (Crowell et al., 2004
). Resveratrol was administered in a vehicle of 0.5% (w/v) aqueous methycellulose containing 0.2% (w/v) Tween 80 (Sigma Chemical Co., St. Louis, MO); a dosing volume of 10 ml/kg/day was used. Rats in the vehicle control group received daily oral (gavage) administration of vehicle only (10 ml/kg/day) for the same period.
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 5 rats/sex/group at pretest and during week 13 of dosing; the week 13 FOB was performed at 2 to 4 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, hindlimb extension, hearing (click response), vision, catalepsy, righting reflex, grip strength (forelimb and hind limb), and foot splay. Blood samples for clinical chemistry and hematology evaluations were collected from fasted rats (10/sex/group) during weeks 4 and 12 of dosing; coagulation parameters were evaluated in blood samples obtained from the same animals at study termination. 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]; MLA Electra 900 Automatic Coagulation Timer [Hemoliance, Raritan, NJ]).
Levels of free (unconjugated) resveratrol and total (unconjugated + conjugated) resveratrol were quantitated in plasma samples collected at 1 h post-dosing on day 1 and during week 13. Plasma levels of free resveratrol were measured by HPLC, using an analytical method developed in our laboratories (Muzzio et al., submitted for publication). Total resveratrol values were quantitated by HPLC after enzymatic hydrolysis of plasma samples with β-glucuronidase/sulfatase (from Helix pomatia; Type H-5, lot number 084K3795, Sigma-Aldrich, St. Louis, MO).
After 13 weeks of resveratrol exposure, all rats were euthanized with sodium pentobarbital followed by exsanguination, and received a complete gross necropsy with tissue collection. At necropsy, weights of the adrenals, brain, heart, kidneys, liver, ovaries/testes, spleen, thyroids and uterus were collected. 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 underwent histologic processing and were evaluated microscopically. Histologic processing and microscopic evaluation of tissues from rats in the middle and low dose groups were limited to gross lesions and identified target tissues.
2.2. Subchronic oral toxicity study in dogs
Male and female purebred beagle dogs were received at approximately five months of age from Ridglan Farms, Inc. (Mount Horeb, WI), and were held in quarantine for approximately four weeks prior to randomization into experimental groups. Dogs were housed individually in floor-level pens in a temperature-controlled room maintained on a 12 h light/dark cycle. Dogs were provided with 400 g of Certified Canine Diet 5007 (PMI Nutrition International, Inc.) for a minimum of 2 h 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 experimental groups of four dogs per sex using a computerized randomization procedure that blocks for body weight. Dogs received daily oral (capsule) exposure to resveratrol at doses of 200, 600, or 1200 mg/kg/day (4000, 12,000 or 24,000 mg/m2/day) for 91 consecutive days. Dogs in the control group received empty capsules only. Dose levels used in the study were selected on the basis of the results of a preliminary 28-day oral toxicity study in dogs (unpublished).
Throughout the study, dogs were observed a minimum of twice daily to monitor their general health status. Detailed clinical examinations and body weight measurements were performed once weekly, and food consumption was quantitated daily. Indirect funduscopic ophthalmic examinations and electrocardiographic (ECG) evaluations were performed on all dogs during quarantine (pre-test) and during the final week of the treatment period. ECGs were reviewed for heart rate and rhythm, amplitude of the P wave and QRS complex, and duration of the P wave, PR, QRS, and QT intervals. To identify possible neurotoxic effects of resveratrol, all dogs underwent FOB evaluations at pretest and during the final week of resveratrol administration. FOB parameters included body weight, body temperature, cage-side observation, gait and posture, wheelbarrow test, hopping tests (forelimb and hindlimb), placing response, patellar reflex, perineal reflex, hindlimb and forelimb flexor reflexes, menace reflex, papillary reflex, and righting reflex.
Blood samples for determination of levels of free resveratrol, resveratrol glucuronide, and resveratrol sulfate were collected from all resveratrol-treated animals at one hour post-dosing on day 1 and during week 13. Plasma levels of free resveratrol and resveratrol conjugates were quantitated using a tandem mass spectrometer (API 3000; Applied Biosystems/MDS Sciex, Foster City, CA) equipped with a high performance liquid chromatograph (Agilent 1100; Agilent Technologies, Wilmington, DE) and methods established in our laboratories. Pharmacokinetic analyses were performed using WinNonlin Professional Edition software, version 5.0.1 (Pharsight Corporation, Mountain View, CA).
Blood samples for clinical chemistry, hematology and coagulation evaluations were collected from fasted dogs during pre-test and weeks 4 and 13 of dosing. Clinical pathology assays were performed as described for the 90-day toxicity study in rats, except that clinical pathology parameters were evaluated using a Synchron LX20 and coagulation parameters were measured using the STA Compact CT automatic coagulation instrument (Diagnostica Stago, Parsippany, NJ). Urine samples were collected from fasted dogs pre-test and during weeks 4 and 13 of dosing, and were analyzed by dipstick and microscopy.
One day after the final dose of resveratrol or vehicle, dogs were euthanized by barbiturate overdose and subjected to a complete necropsy with tissue collection. Weights of the adrenals, brain, heart, kidneys, liver, spleen, testes, thymus and thyroids were collected, 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 vehicle control groups were processed by routine histologic methods and evaluated microscopically. Histologic processing and microscopic evaluation of tissues from dogs in the middle and low dose groups were limited to gross lesions and identified target tissues.
2.3 Cardiovascular safety pharmacology study in dogs
A separate cardiovascular safety pharmacology (telemetry) study was performed using a balanced Latin square design. In this study, eight naïve beagle dogs (4 per sex; Ridglan Farms) were implanted with telemetry transmitters (Data Sciences International, St. Paul, MN [DSI]) for remote monitoring of cardiovascular function. Telemetry data acquisition was performed using a signal receiver, ambient pressure monitor, DSI Data Exchange Network, and the DSI data acquisition system based on DataquestART™ software.
After recovery from implantation surgery, groups of two dogs (one per sex) received a single oral (capsule) dose of resveratrol at one of four dose levels (0 mg/kg [empty capsules; control], 200 mg/kg, 400 mg/kg, and 1000 mg/kg). After a one-week washout period, animals were rotated to a different dose level; weekly dosing was continued in this fashion until each animal had received a single capsule dose of resveratrol at each dose level.
General toxicology evaluations performed during the safety pharmacology study included twice daily mortality/moribundity observations, daily clinical observation, and weekly body weights. Body temperature and cardiovascular function were monitored continuously via telemetry for at least 2 hours prior to and 24 hours after each dose of resveratrol; cardiovascular parameters monitored included heart rate; diastolic, systolic and mean blood pressure; and electrocardiography. ECG waveforms were evaluated using DSI ECG Analysis 4.0 software, and were reviewed by a veterinary cardiologist. The ECG Analysis software recognizes ECG waveforms and measures the distance between waveform peaks to determine PR, RR, QRS and QT interval times within each 30-second interval. The QT interval was normalized for changes in heart rate by conversion to the corrected QT (QTc) interval using Fridericia’s formula [QTcF=QT/(RR)1/3].
2.4. Statistical analyses
Statistical evaluations of continuous data from toxicology studies 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.
Statistical analyses of plasma drug level data (Tmax, Cmax, AUC) were performed using Systat software, version 10.2 (Systat Software Inc., Chicago, IL); data were analyzed either by t-test or by analysis of variance (ANOVA) followed, as necessary, by the post hoc Tukey’s test. Data for Cmax and AUC were normalized to the dose prior to log-transformation for statistical evaluation of dose-proportionality responses. A minimum significance level of p < 0.05 was used in all comparisons.