Prior to initiation of in vivo experimentation, study protocols were reviewed and approved by the institutional Animal Care and Use Committee. All aspects of 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). All studies were conducted in full compliance with the Good Laboratory Practice Regulations of the United States Food and Drug Administration (21 CFR Part 58).
16 male and 16 female Beagle dogs, 4 animals per gender per group (Marshall Farms USA, Inc., North Rose, NY), approximately 5.7-7.2 kg and 5-6 months at the start of the study, were used in the chronic study (scheduled 9-month study) performed in a different laboratory than the earlier 13-week study (Johnson et al. 1999
). They were housed individually in stainless steel cages and 300-400 g/day of Certified Diet # 5007 (PMI Feeds, Inc., St. Louis, MO) was available for 2 hours starting 2 hours after dosing.
Fifteen male Beagle dogs, 3 animals per group (Covance Laboratories, Inc., Kalamazoo, MI), approximately 6.4-8.5 kg and 5-6 months at the start of the study, were used in a follow-up subchronic 13-week study performed in a third laboratory. They were singly housed in runs and approximately 350 g of Harlan Teklad Certified Diet No. 2025C (Harlan Teklad, Madison, WI) was provided daily from arrival until termination.
In all studies, the animals were housed in an AALAC Intl.-accredited facility in a temperature (64-84°F) and humidity (50 ± 20%) controlled room with a 12 hour light/12 hour dark cycle. Local tap water from an automatic watering system was provided ad libitum from arrival until termination. Animals were quarantined for approximately 21 days prior to randomization into experimental groups and start of the study. During the quarantine period, animals underwent a complete physical examination, including body temperature, a fecal examination for parasites, clinical pathology, and body weights. They were also observed for general health and acceptability for use in these studies.
2.2. Test article
PPE (lot A, PE-041014 and lot B, PE-050808) was obtained from Mitsui Norin Co., LTD., Shizuoka, Japan and its clinical formulation (lot C, 21110905) from ThermoFisher, Rockville, MD. Lot A was used in both the chronic 9-month study and the follow-up 13-week study. In addition to lot A, two additional lots (newer lot B and a clinical lot C) were used in the follow-up 13-week study for comparison. Lot C was a formulated clinical lot and contained microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxide, and magnesium stearate excipients in size 0 gelatin capsules. Different lots contained approximately 63.3-64.8% (–)-epigallocatechin gallate (EGCG; CAS 989-51-5), 3.0-3.7 % (–)-epigallocatechin (EGC; CAS 970-74-1), 6.0-8.0 % (–)-epicatechin gallate (ECG; CAS 1257-08-5), and 7.6-12.3% (–)-epicatechin (EC; CAS 490-46-0) (). PPE was stored at approximately 2-8°C at ambient humidity and protected from light. All test article lots were shown to be stable under the storage conditions. Except for lot C, PPE was administered as neat, unformulated substance in gelatin capsules. Lot C was formulated and already in gelatin capsules and was administered as such. Control group animals received the same number of the same size empty gelatin capsules each day as test animals.
It was re-confirmed that the levels of heavy metals, pesticides, microbial contamination, decomposition products, caffeine and methylxanthines in the PPE lots used in these studies were within acceptable ranges and comparable to other lots.
In the chronic study, animals were dosed with 0, 200, 500, and 1000/800 mg/kg/day (approximately 0, 4000, 10,000, and 20,000/16,000 mg/m2/day) of neat (unformulated) PPE in gelatin capsules (1-2 capsules/dog/day) on empty stomach. Food was available 2 hours after dosing for 2 hours.
In the 13-week follow-up study, animals were grouped according to test article lot. PPE (200 mg/kg/day ~ 4000 mg/m2/day) was orally administered neat (unformulated) in gelatin capsules, with the exception of lot C. This lot was administered as 6 clinical capsules each containing approximately 300 mg of PPE (176 mg/kg/day ~ 3416 mg/m2/day). PPE was administered on an empty stomach in 3 groups and to fed animals (lot A) in one group. Starting from day 1, the “fed” group had food available approximately two hours prior to dosing. All other groups had food available for 2 hours starting 2 hours after dosing.
2.4. Toxicological evaluation
Throughout the study, animals were observed a minimum of twice daily at least 4 hours apart to monitor their general health status and for signs of mortality/morbidity. Morning observation was within an hour following dosing to look for the onset and duration of any clinical signs of toxicity.
Body weight measurements were performed weekly and food consumption was quantified daily. Each dog was removed from its cage once each week for a physical exam that involved close examination for detailed signs of toxicity. This detailed physical examination including the eyes and all orifices. All dogs underwent ophthalmic examination by indirect ophthalmoscopy at least once during the quarantine period and during the study. The eyelids, conjunctiva, cornea, sclera, iris, lens, fundus, anterior chamber lens, anterior vitreous and posterior chambers were examined. A tear production test (Schirmer Test) was also performed as deemed warranted.
All dogs also underwent electrocardiographic examinations during the quarantine period and during the study. Electrocardiograms were evaluated for heart rate and rhythm, amplitude of the P wave and QRS complex, and duration of the P wave, PR, QRS, and QT intervals.
Blood samples for clinical chemistry, hematology and coagulation evaluations were collected from fasted dogs at pre-test, at the terminal necropsy and when significant toxicity was observed. Clinical pathology assays were performed using standard automated instruments. Hematology parameters were measured using an Advia 120 Hematology Analyzer by using standardized methods. Tests included Hemoglobin (cyanomethemoglobin method); Hematocrit, Erythrocyte Count, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Leukocyte Count, Platelet Count, Reticulocyte Count, Leukocyte Differential Count, Nucleated RBCs, and RBC Morphology. In addition to this routine battery of tests, spherocytes were hand counted (10 fields at 100x magnification). The following coagulation parameters were measured on an MLA 900 coagulation machine: Activated Partial Thromboplastin Time (APTT); Prothrombin Time (PT); Fibrinogen. The following clinical chemistry parameters were measured using a Hitachi 912 Clinical Chemistry Analyzer using standardized methods: Glucose, Urea Nitrogen (BUN), Inorganic Phosphorus, Creatinine, Total Protein, Albumin, Calcium, Aspartate Aminotransferase (AST/GOT), Alanine Aminotransferase (ALT/GPT), Alkaline Phosphatase (AKP), Total Bilirubin, Creatine Kinase (CK), sodium, Potassium, and Chloride. The following chemistry parameters were measured using specific assay kits: Folic Acid (Dualcount Reagent Kit, DPC); Insulin (Diagnostics Products Coat-A Insulin Kit); Histamine (Histamine RIA kit, Beckman Coulter); IgE (Dog IgE ELISA Quantitation Kit, Bethyl Laboratories); IgG, IgA, IgM (Radial Immune Diffusion Reagent Kits, Bethyl Laboratories); Glucagon (Glucagon RIA kit, Linco Research); Lipase (Randox method of Colorimetric Hitachi 717, Randox); Pancreatic Amylase (Pancreatic α-Amylase test, Randox); Glutathione peroxidase (Glutathione Peroxidase Assay Kit, Cayman Chemical Company); Lipid Peroxidation (Lipid Peroxidation Assay Kit, Cayman Chemical Company); Total antioxidant activity (Antioxidant Activity Kit, Cayman Chemical Company); Total Glutathione (Measured by fluorescence HPLC); Reduced Glutathione (Measured by fluorescence HPLC); Oxidized Glutathione (calculated from difference between total and reduced glutathione measurements).
Urine samples were collected from fasted dogs at pre-test and during the final week of dosing, and were analyzed for color, appearance, specific gravity, urinalysis parameters by dipstick and sediment microscopically.
At necropsy, all gross lesions and approximately 45 tissues were collected from each animal and fixed in 10% neutral buffered formalin. All tissues collected from all study dogs were processed by routine histological methods and evaluated histopathologically.
2.5. Plasma analysis
The concentration of EGCG, EGC, ECG and EC in plasma (EDTA-tubes) was determined using high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Chromatographic separation was achieved using Waters Symmetry Shield™ RP8 (50 × 2.1mm, 3.5 μm) (Waters, Milford, MA) analytical and Thermo Hypersil Gold (10 × 2.1 mm, 3 μm) (Thermo Fisher Scientific, Inc., Waltham, MA) guard columns at ambient temperature and a flow rate of 400 μl/min. Linear gradient elution was from 95% A (5 mM ammonium acetate with 0.4% formic acid): 5% B (50:50 methanol:acetonitrile with 0.4% formic acid) to 10% A:90% B over 6.5 min followed by a 1.5 min re-equilibration. Detection was performed using multiple reaction monitoring mode with negative polarity on Micromass Quattro Micro™ API (Waters, Milford, MA) unit. The following parent-daughter transitions were monitored 457.10 to 169.2, 305.20 to 125.20, 441.20 to 169.30, 289.20 to 125.35, and 301.24 to 151.24 for EGCG, EGC, ECG, EC and quercetin (internal standard), respectively. Quantification was based on peak areas. Free catechins (unconjugated) and total (unconjugated plus conjugated) were determined without and with hydrolysis by β-glucuronidase/arylsulfatase (Sigma-Aldrich, St. Louis, MO) for 2 hr at 37°C. After combining plasma samples (250 μl) with 25 μl of 10% ascorbic acid, 500 μl of acetonitrile and 50 μl of the internal standard were added to each tube. Tubes were vortexed, centrifuged and supernatants were transferred to clean tubes and evaporated to dryness under nitrogen. The dried extracts were reconstituted in 100 μl of mobile phase (95% A: 5% B), centrifuged, and supernatants were transferred to autosampler vials for analysis.
2.6. Pharmacokinetic analysis
Due to low levels of some catechins in a number of samples, data were not adequate for reliable determination of the elimination half-lives so full pharmacokinetic modeling could not be performed. Area-under-plasma concentration-time curve was determined using the trapezoidal rule from time equals zero to the last time point, 24 hr. Maximal plasma concentration (Cmax) was based on the highest measured plasma test article concentration.
2.7. Ancillary tests
Fasting serum levels of amylase, lipase, insulin, and glucagon were measured once in the quarantine period (day -11) and in weeks 4 (day 23), 8 (day 51) and 13 (day 86) as indices of pancreatic function. Immunoglobulins (IgE, IgG, IgM and IgA) and folic acid levels in serum were tested once from serum collected during quarantine period and from serum collected on the day of necropsy from fasted animals. Histamine levels were tested from EDTA-plasma collected on the day of necropsy from fasted animals. Tests for oxidative stress (levels of reduced and oxidized glutathione, glutathione peroxidase, lipid peroxidation and total antioxidant activity) were performed by AniLytics (Gaithersburg, MD) in plasma processed from the heparinized blood samples collected from all animals at necropsy. Additional liver samples (approximately 1.0 g) were obtained from all scheduled sacrifice animals at necropsy, rinsed three times in separate aliquots of ice-cold phosphate buffered saline until free of blood and stored frozen at approximately -80°C until shipped on dry ice to AniLytics, Inc. (Gaithersburg, MD), for the five tests for oxidative stress (reduced and oxidized glutathione, glutathione peroxidase, lipid peroxidation and total antioxidant activity).
2.8. Statistical analyses
Where appropriate, group comparisons were made by analysis of variance. If a significant F ratio (P < 0.05) was obtained, Dunnett’s Test was used for comparisons with a control. In all cases, the lower limit for statistical significance was defined as p < 0.05.