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Corning Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 Deliveries: 3301 Kinsman Blvd. Madison, WI 53704 608.241.4471 608.241.7227 Fax /lR te -o % 0 Sponsor: 3M St. Paul, Minnesota C O R N IN G Hazleton FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study o f T-6564 in Rabbits Author: F. Bud W. McDonald Study Completion Date: April 18, 1997 Performing Laboratory: Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: CHW 6329-186 Page 1 o f 34 Corning Pharmaceutical Services 002942 QUALITY ASSURANCE STATEMENT CHW 6329-186 This report has been reviewed by the Quality Assurance Unit o f Corning Hazleton Inc., in accordance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations, 21 CFR 58. The following inspections were conducted and findings reported to the Study Director and management. Inspection Dates From To 06/27/96 06/27/96 09/03/96 09/03/96 10/15/96 10/15/96 12/03/96 12/05/96 Date Reported to Study Date to Phase_________ Director Management Protocol Review Sample Collection Protocol Amendment Data/Report Review 06/27/96 09/03/96 10/15/96 12/05/96 06/27/96 09/03/96 10/15/96 12/05/96 Date 2 C02943 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study o f T-6564 in Rabbits CHW 6329-186 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental Termination Date Study Completion Date T-6564 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Roger G. Perkins, PhD, DABT 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612)733-3222 F. Bud W. McDonald Coming Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 242-7901 Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, WI 53704 August 2, 1996 August 6,1996 S eptem ber3,1996 April 18,1997 April 18, 1997 3 002944 Acute Studies Steven M. Glaza Manager F. Bud W. McDonald Study Director Jeffrey B. Hicks In-life Supervisor Rose M. Bridge Administrative Supervisor Toxicology Support Kathy Myers Manager Calvin L. Horton Supervisor KEY PERSONNEL Quality Assurance Sherry R. W. Petsel Manager CHW 6329-186 Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data 4 C02945 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Table 1 Individual Body Weights (g) 2 Individual Clinical Signs 3 Individual Animal Tissue Weights and Bile Volumes Appendix A Protocol Deviations Protocol TP6350 Amendment No. 1 to the Protocol CHW 6329-186 Page 2 3 4 6 8 8 8 9 10 12 13 13 14 16 18 19 20 21 33 5 002946 SUMMARY CHW 6329-186 This study was done to assess the level o f systemic exposure o f T-6564 when administered by a single intravenous injection to rabbits. The study was conducted using four male and four female acclimated rabbits o f the Hra:(NZW)SPF strain for each treatment group as follows: Group 1 (Control) 2 J 4 5 Control/Test Material Sterile Water T-6564 T-6564 T-6564 T-6564 Dose Level (mg/kg)* 0.0 3.0 10.0 100.0 300.0 Number of Animals6 Male Female 44 44 44 44 4C 4 a Administered at a dose volume of 0.5 mL/kg. b Two animals/sex/dose level were sacrificed on Day 14. The remaining animals (two animals/sex/dose level) were sacrificed on Day 29. c One male died approximately 20 minutes after treatment (possibly due to the volume and rate of injection). A replacement animal was then selected and treated in the same manner. The animals received a single intravenous injection o f the control or test material at the indicated dose level into the marginal ear vein o f the right ear. The test material was mixed with Sterile Water for Injection (sterile water) to a specific concentration for each dose level and administered at a dose volume of 0.5 mL/kg o f body weight. One o f the initial males treated at 300 mg/kg (Group 5) died approximately 20 minutes after treatment. This animal, discarded without an abbreviated necropsy examination or tissue collection, was replaced with another animal and treated in the same manner. Two animals/sex/dose level were sacrificed on Day 14 and the remaining animals (two animals/sex/dose level) were sacrificed on Day 29. Clinical observations were conducted predose and at approximately 0.5, 2, and 4 hours after intravenous injection. Additional clinical observations and twice a day mortality checks were conducted daily thereafter until the scheduled sacrifice interval (a.m. mortality check only on days o f scheduled sacrifice). Body weights were determined on Day -6 for randomization 6 C02947 CHW 6329-186 purposes, before test or control material administration (Day 1), and at the scheduled sacrifice interval (Day 14 or Day 29). A blood sample (approximately 4 mL) was collected from each animal four days before control or test material administration with the exception o f the one Group 5 replacement male which had a blood sample collected on Day 1 (before test material administration). Blood samples were also collected from the animals at approximately 4-, 8-, 12-, 24-, and 48-hours post-injection, and on Day 8. An exception to these intervals was the replacement animal (Group 5 male), from which blood samples were collected at approximately 3.5-, 6.5-, and 10.5-hours postdose at the 4-, 8-, and 12-hour postdose intervals, respectively. An approximate 4-mL blood sample was also collected on Days 14 and 22 from each animal scheduled for sacrifice on Day 29. In addition, at the time o f the scheduled sacrifice (Day 14 or Day 29), approximately 20 to 40 mL o f blood was obtained from each control animal and approximately 20 mL o f blood was obtained from each Group 2-5 animal with the exception o f one Group 4 female on Day 14 from which only approximately 12 mL o f blood was collected. All samples were centrifuged and separate samples of serum and cellular fractions were obtained and sent frozen on dry ice to the Sponsor. On Day 14 or 29, the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected, weighed (volume only determined for bile), and sent frozen to the Sponsor. After being intravenously injected with T-6564 or the sterile water control, all animals appeared clinically normal throughout the study with the exception o f the one Group 5 male (300 mg/kg) that died within 20 minutes of dose administration. In addition, one Group 4 female (100 mg/kg) and one Group 5 female (300 mg/kg) exhibited a slight loss in weight at their respective termination interval (Day 14). 7 C02948 OBJECTIVE CHW 6329-186 The objective o f this study was to assess the level o f systemic exposure to the test material, T-6564, when administered as a single intravenous injection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis o f the test mixtures for concentration, homogeneity/solubility, and stability was not conducted. All procedures used in this study were in compliance with the Animal Welfare Act Regulations. In the opinion o f the Sponsor and study director, the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification The test material was identified as T-6564 and described as a clear colorless liquid. The control material was Sterile Water for Injection, USP (Abbott Laboratories, Lot No. 10-382-JT; Expiration November 1997), and was described as a clear, colorless liquid. Purity and Stability The Sponsor assumes responsibility for test material purity and stability determinations (including under test conditions). Analysis of the test material mixtures for concentration, homogeneity/solubility, and stability was not conducted or requested by the Sponsor. The purity and stability o f the control material were considered to be adequate for the purposes o f this study. Storage and Retention The test material was stored at room temperature. The control material was stored refrigerated. Reserve samples o f the test and control materials were taken and stored in a freezer set to maintain a temperature of -20C 10C. The control material reserve sample will be retained at CHW for 1 year in accordance with the Wisconsin facility o f Coming Hazleton (CHW) Standard Operating Procedure (SOP) and then discarded. The test material reserve sample and unused test material were returned to the Sponsor. Any remaining control material may be used for other testing and will not be discarded after issuance o f the final report. 8 002949 BEST COPY AVAI1 ABLE CHW 6329-186 Safety Precautions The test and control material handling procedures were according to CHW SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits o f the Hra:(NZW)SPF strain were received from HRP, Inc., Kalamazoo, Michigan on June 26, 1996 and maintained at the CHW facility at 3301 Kinsman Boulevard, Madison, Wisconsin. Housing After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in suspended stainless steel cages in temperature- and humidity- controlled quarters. Environmental controls for the animal room were set to maintain a temperature o f 19 to 23 C, a relative humidity o f 50% 20%, and a 12-hour light/12-hour dark lighting cycle. The dark cycle was interrupted to conduct in-life procedures. In cases where variations from these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad libitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples o f the water are periodically analyzed. There were no known contaminants in the feed or water at levels that would have interfered with or affected the results o f the study. Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were placed into study groups based on a stratified body weight randomization program. The randomization body weights were determined on Day -6. A Group 5 replacement male (Animal No. F60093) was arbitrarily selected from the extra animals acclimated for the study. 9 C02950 CHW 6329-186 Study Design Animals weighing from 2,613 to 2,998 g at initiation o f treatment were placed into the following study groups: Group 1 (Control) 2 3 4 5 Control/Test Material Sterile Water T-6564 T-6564 T-6564 T-6564 Dose Level (mg/kg)* 0.0 3.0 10.0 100.0 300.0 Number o f Animals1* Male Female 44 44 44 44 4C 4 a Administered at a dose volume o f 0.5 mL/kg. b Two animals/sex/dose level were sacrificed on Day 14. The remaining animals (two animals/sex/dose level) were sacrificed on Day 29. c One male died approximately 20 minutes after treatment (possibly due to the volume and rate o f injection). A replacement animal was then selected and treated in the same manner. Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal o f choice because o f the large amount o f background information on this species. PROCEDURES Dose P reparation and Administration The test material was diluted with sterile water to achieve a specific concentration for each dose level in Groups 2 to 5. An individual dose of each respective control material or test solution was calculated for each animal based on its body weight on the day o f treatment. The control material or respective test solution was administered by intravenous injection into the marginal ear vein o f the right ear over a period of 34 to 52 seconds. The prepared test solutions were stored at room temperature until administered. After administration, any remaining test solutions were discarded. 10 002951 Reason for Route of Administration Intravenous injection is an acceptable route to assess systemic exposure. CHW 6329-186 Observations of Animals Clinical observations were conducted predose and at approximately 0.5, 2, and 4 hours after intravenous injection. Additional clinical observations and twice a day mortality checks were conducted daily thereafter until the scheduled sacrifice interval (a.m. mortality check only for animals sacrificed on Days 14 or 29). Body weights were determined on Day -6 for randomization purposes, before test or control material administration (Day 1), and at the scheduled sacrifice interval (Day 14 or Day 29). Blood Sample Collections/Shipment A blood sample (approximately 4 mL) was collected from a marginal ear vein (left ear) of each animal on Day -4, with the exception o f Animal No. F60093 (Group 5 replacement male) which had a blood sample collected from the left ear on Day 1 (before test material administration). Blood samples (approximately 4 mL) were then collected from a marginal ear vein (left ear) of each animal at approximately 4-, 8-, 12-, 24-, and 48-hours post-injection, and on Day 8. An exception to these intervals was the replacement animal (Group 5 male), from which blood samples were collected at approximately 3.5-, 6.5-, and 10.5-hours postdose at the 4-, 8-, and 12-hour postdose intervals, respectively. An approximate 4-mL blood sample was also collected on Days 14 and 22 from each animal scheduled for sacrifice on Day 29. In addition, at the time of the scheduled sacrifice (Day 14 or Day 29) via the posterior vena cava, approximately 20 to 40 mL o f blood was obtained from each control animal and approximately 20 mL o f blood was obtained from each animal in Groups 2-5, with the exception o f Animal No. F60085 (Group 4 female) from which only approximately 12 mL o f blood was obtained. All samples were stored at room temperature until centrifuged. After centrifugation, separate samples of serum and cellular fractions were obtained. The serum and cellular fraction samples obtained through Day 14 were stored in a freezer set to maintain a temperature o f -20C 10C until shipped frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.T. & S, Bldg. 2-3E-09, 935 Bush Avenue. St. Paul, MN, 55106), on Day 21. The serum and cellular fraction samples obtained on Days 22 and 29 were stored at CHW and shipped to the Sponsor six days after experimental (in life) termination in the same manner as the samples obtained prior to Day 22. Pathology The Group 5 animal that died shortly after dose administration was discarded without an abbreviated necropsy examination or tissue collection. On Day 14, the first two animals/sex assigned to each dose level (based on the group assignment randomization) were anesthetized 11 GG2952 CHW 6329-186 with sodium pentobarbital (via injection in the marginal ear vein), bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected, weighed (volume only determined for bile), and immediately placed in a freezer set to maintain a temperature o f -20C 10C. After tissue/bile collection, the animals were discarded. The remaining two animals/sex/dose level were anesthetized, bled, and exsanguinated on Day 29 in the same manner as the animals sacrificed on Day 14. Shipment of Tissues The tissues (whole livers and kidneys) and bile collected on Days 14 and 29, along with documentation o f their corresponding weights or volumes, were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.T. & S, Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106) one week after collection and six days after in-life termination, respectively. The Sponsor is responsible for the retention and disposition o f the samples. CHW does not accept any responsibility for the analysis o f the samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data, Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives o f CHW in accordance with CHW SOP. RESULTS Body Weights Individual body weights are in Table 1. All animals gained weight during the study with the exception of one Group 4 female (100 mg/kg) and one Group 5 female (300 mg/kg), sacrificed at Day 14. which exhibited weight losses o f 125 g and 13 g, respectively. Clinical Observations Individual clinical signs are in Table 2. All animals appeared normal throughout the study with the exception o f the Group 5 male (Animal No. F60071) that died approximately twenty minutes postdose. The death o f this animal is believed due to the rate and volume o f the administered dose. 12 002953 CHW 6329-186 Pathology Individual animal tissue weights and bile volumes are in Table 3. The animals were not examined grossly, although tissues were saved (with the exception o f the Group 5 male that died shortly after dosing - no tissues were saved on this animal). DISCUSSION The level of systemic exposure o f T-6564 was evaluated in male and female albino rabbits when administered as a single intravenous injection at levels o f 3.0,10.0, 100.0, and 300.0 mg/kg. All animals appeared clinically normal throughout the study with the exception o f the one Group 5 male (300.0 mg/kg) that died within 20 minutes o f dose administration (believed due to the rate and volume o f the dose). All surviving animals gained weight during the study with the exception o f one Group 4 female (100 mg/kg) and one Group 5 female (300 mg/kg) that were sacrificed at Day 14. SIGNATURE F. Bud W. McDonald Study Director Acute Studies Date 13 C02954 Table 1 Individual Body Weights (g) CHW 6329-186 Animal Randomization Initial Sex Number (Day -6) (Day I) Terminal (Day 14) (Day 29) Male Female F60052 F60053 F60054 F60055 F60072 F60073 F60074 F60075 Group 1 (Control) - Sterile Water (0.0 mg/kg) 2,643 2,752 2,653 2,591 2,828 2,774 2,797 2,777 2,724 2,788 2,686 2,685 2,904 2,881 2,805 2,880 2,919 2,884 - 2,914 3,008 - - * 0 2,833 2,907 * 0 3,207 3,229 Male Female F60056 F60057 F60058 F60059 F60076 F60077 F60078 F60079 Group 2 - T-6564 (3.0 mg/kg) 2,898 2,798 2,716 2,821 2,823 2,698 2,699 2,882 2,962 2,851 2,790 2,940 2,924 2,873 2,799 2,950 3,012 2,937 3,122 3,141 - * 3,024 3,149 * 3,082 3,242 Male Female F60060 F6006I F60062 F60063 F60080 F60081 F60082 F60083 Group 3 - T-6564 (10.0 mg/kg) 2,738 2,634 2,716 2,832 2,732 2,520 2,769 2,786 2,822 2,667 2,912 2,749 2,797 2,613 2,838 2,795 2,924 2,822 2,923 2,818 - * * 3,293 2,984 0 * 3,147 3,108 Not required. * Animal sacrificed on Day 14. 14 002955 Table 1 (Continued) Individual Body Weights (g) CHW 6329-186 Animal Randomization Initial Sex Number (Day -6) (Day 1) Group 4 -T-6564 (100.0 mg/kg) Male Female F60064 F60065 F60066 F60067 F60084 F60085 F60086 F60087 2,686 2,726 2,740 2,745 2,538 2,632 2,728 2,892 2,697 2,792 2,734 2,750 2,653 2,696 2,825 2,998 Group 5 -T-6564 (300.0 mg/kg) Male F60068 F60069 F60070 F6007I F60093b 2,774 2,590 2,917 2,633 2,989 2,865 2,722 2,936 2,707 2,951 Female F60088 F60089 F60090 F60091 2,837 2,661 2,913 2,711 2,872 2,737 2,953 2,781 Not required. * Animal sacrificed on Day 14. a Animal died approximately 20 minutes postdose, b Replacement animal for Animal No. F6007I. Terminal (Day 14) (Day 29) 2,836 2,874 - - 2,789 2,571 - * 3,071 2,962 * * 3,137 3,289 3,128 2,784 - - - 3,106 2,724 * * 3,217 a 3,174 * * 3,269 3,070 15 02956 Table 2 Individual Clinical Signs CHW 6329-186 Animal Sex Number Observation Hour (Day 1)* 0.5 2.0 4.0 2-8 Group 1 (Control) - Sterile Water (0.0 mg/kg) Male F60052 F60053 F60054 F60055 Appeared Normal Appeared Normal Appeared Normal Appeared Normal / S / Female F60072 F60073 F60074 F60075 Appeared Normal Appeared Normal Appeared Normal Appeared Normal S/ // // / / / / Group 2 - T-6564 (3.0 mg/kg) Male F60056 F60057 F60058 F60059 Appeared Normal Appeared Normal Appeared Normal Appeared Normal s/ // // / / / S Female F60076 F60077 F60078 F60079 Appeared Normal Appeared Normal Appeared Normal Appeared Normal // s/ s/ / / / / Group 3 - T-6564 (10.0 mg/kg) Male F60060 F6006I F60062 F60063 Appeared Normal Appeared Normal Appeared Normal Appeared Normal / S / / / Female F60080 F6008I F60082 F60083 Appeared Normal Appeared Normal Appeared Normal Appeared Normal / // // / / / / / a Each animal appeared normal prior to control or test material administration. * Animal sacrificed on Day 14. Condition existed. Days 9-14 15-29 /* /* / / * * // * * * * / * /* / / 16 C02957 Table 2 (Continued) Individual Clinical Signs CHW 6329-186 Animal Sex Number Observation Hour (Day 1)* 0.5 2.0 4.0 Group 4 - T-6564 (100.0 mg/kg) Male F60064 F60065 F60066 F60067 Appeared Normal Appeared Normal Appeared Normal Appeared Normal / Female F60084 F60085 F60086 F60087 Appeared Normal Appeared Normal Appeared Normal Appeared Normal / Group 5 T-6564 (300.0 mg/kg) Male F60068 Appeared Normal F60069 Appeared Normal F60070 Appeared Normal F60093 Appeared Normal / / Female F60088 F60089 F60090 F60091 Appeared Normal Appeared Normal Appeared Normal Appeared Normal / / / / Days 2-8 9-14 15-29 / // * * / / * / / / / 0 0 / / * * a Each animal appeared normal prior to control or test material administration. Animal sacrificed on Day 14. Condition existed. 17 002958 CHW 6329-186 C 02959 Corning Hazleton Inc. Madison, Wisconsin USA TABLE 3 Individual Animal Tissue Weights and Bile Volumes TABLE INCLUDES: SEX-ALL;GROUP-ALL;WEEKS-ALL DEATH-ALL;SUBSET-ALL ORGAN ABBREVIATION: LI - LIVER, KD - KIDNEY, SEX DOSE ANIMAL GROUP NUMBER LI (g) KD (g) BI (mL) : DOSE ANIMAL GROUP NUMBER BI - BILE (ML) LI (g) KD (g) M 1 F60052* M 1 F600S3* H 1 F600S4b M 1 F6005Sb M 2 F600S6* M 2 F60057* M 2 F60058b M 2 F60059b M3 M3 M3 M3 F60060* F60061* F60062b F60063b M 4 F60064* M 4 F60065* M 4 F60066b M 4 F60067b M 5 F6006B* M 5 F60069* M 5 F60070b M 5 F60093b 90.3302 73.7863 48.1630 76.1902 81.8360 80.8815 77.3457 90.2474 74.6403 75.0707 93.3989 68.3306 63.9420 81.3660 74.4153 66.7900 99.4912 81.8480 75.3822 80.5678 14.6620 16.7332 15.2486 14.6761 15.1615 15.8406 17.9012 16.7928 15.0898 16.0551 16.2391 15.0642 15.2450 15.6753 15.4621 14.1424 15.2020 17.1618 16.5091 16.9704 Dose Level 0.0 mq/kq 1.1000 F 1 F60072* 0.7000 F 1 F60073* 1.1000 F 1 F60074b 1.0000 F 1 F60075b Dose Level ,3.0 nw/Kq 0.3000 F 2 F60076* 0.7000 F 2 F60077* 0.2000 F 2 F60078b 1.0000 F 2 F60079b Dose Level 10.0 mg/kg 0.7000 F 3 F60080* 0.1000 F 3 F600B1* 1.6000 F 3 F60082b 0.6000 F 3 F60083b Dose Level 100.0 mq/kq 1.5000 F 4 F60084* 0.6000 F 4 F60085* 0.9000 F 4 F60086b 0.3000 F 4 F60087b Dose Level 300.0 mq/kq 1.1000 F 5 F60088* 0.9000 F 5 F60089* 0.4000 F 5 F60090b 0.9000 F 5 F60091b 79.9848 82.8800 71.9963 92.6407 91.0291 101.0659 80.7474 92.7357 86.8055 69.6640 99.7506 89.2276 69.7363 80.6815 88.9171 87.2201 91.9828 73.9520 91.9623 100.2084 15.7815 14.6450 15.1548 18.5087 15.8879 15.9660 13.9903 19.1657 16.3950 16.0235 18.3869 15.7710 14.4089 13.6620 13.2784 16.3357 15.3894 12.3374 18.2069 17.9672 BI (mL) 0.8000 0.8000 0.7000 1.5000 0.4000 1.2000 1.1000 1.4000 1.6000 0.7000 0.8000 0.1000 o.eooo 0.9000 1.0000 0.9000 1.5000 0.6000 2.0000 2.5000 a Sacrificed on Day 14. b Sacrificed on Day 29. CHW 6 3 2 9 -1 8 6 PAGE: 1 APPENDIX A Protocol Deviations Protocol TP6350 Amendment No. 1 To The Protocol CHW 6329-186 19 002960 Protocol Deviations CHW 6329-186 ________PlfltOC.Ql________ Page 8, 7. Experim ental Design, D. O bservation of Animals, (3) Blood Sample Collections, (b) M ethod of Collection/Number of Animals, Second Paragraph, Last Two Lines, "approximately 20 mL o f blood will be obtained from each Group 2-5 animal sacrificed on Days 14 or 29." ______ A ctual P rocedure Only approximately 12 mL o f blood was collected from one Group 4 female (Animal No. F60085) at the Day 14 sacrifice interval. Page 8, 7. Experim ental Design, D. O bservation of Animals, (3) Blood Sample Collections, (a) Frequency, Second Line, "administration to Day 1), 4-, 8-, 12-, 24-, and 48-hours post-" . The blood samples collected from the replacement animal (Animal No. F60093) at the 4-, 8-, and 12-hour postdose intervals were collected 3-hours/21 minutes, 6-hours/41 minutes, and 10-hours/28 minutes postdose, respectively. These deviations are not considered to have 1 an adverse effect on the outcome o f the study. 20 C 029S! Coming Hazleton Inc. P.O. Box 7J45 Medium. SH SJ7U7.74 Delhvries: JJOI K ittfu n Bird. V I SJT04 60S.2C1.C47I 60S.241.7227 Fix Sponsor: 3M St. Paul, Minnesota CHW 6329-186 C O R N IN G Hazleton PROTOCOL TP6350 Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6564 in Rabbits Date: August 2, 1996 Performing Laboratory: Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: CHW 6329-186 Corning Pharmaceutical Service* 21 C029S2 CHW 6329-186 STUDY ID E N T inC A T IO N CHW 6329-186 TP6350 Page 2 Single-Dose Intravenous Pharmacokinetic Study of T-6564 in Rabbits CHW No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-186 T-6564 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Roger G. Perkins, PhD, DABT 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-3222 F. Bud W. McDonald Coming Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 242-7901 Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, WI 53704 August 6, 1996 September 3, 1996 October 15, 1996 22 002963 CHW 6329*186 1. Study Single-Dose Intravenous Pharmacokinetic Study in Rabbits CHW 6329-186 TP6350 Page 3 2. Purpose To assess the level o f systemic exposure when the test material is administered as a single intravenous injection to rabbits 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines with the exception that analysis o f the test material mixtures for concentration, solubility, homogeneity, and stability will not be conducted. [ ] Conduct as a Nonregulated Study [X] 21 CFR58 (FDA) [ ] 40 CFR 160 (EPA-FIFRA) [ ] 40 CFR 792 (EPA-TSCA) [ ] C(81)30 (Final) (OECD) ( ] 59 NohSan No. 3850 (Japanese MAFF) [ j Notification Nos. 313 and 870 (Japanese MOHW) All procedures in this protocol are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. 4. Quality Assurance The protocol, study conduct, and the final report will be audited by the Quality Assurance Unit in accordance with the Wisconsin facility o f Coming Hazleton Inc. (CHW) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. Identification T-6564 B. Physical Description (To be documented in the raw data) 23 002964 CHW 6329-186 CHW 6329-186 TP6350 Page 4 C. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). Samples of test material/vehicle mixture(s) for concentration, homogeneity/solubility, and stability analyses will not be taken before administration unless requested otherwise by the Sponsor. These samples (if taken) will be sent to the Sponsor after experimental termination. D. Storage Room temperature E. Reserve Samples Reserve sample(s) of each batch/lot of test material will be taken for this study. The test material reserve sample(s) will be stored at CHW in a freezer set to maintain a temperature of -20C I0C and then returned to the Sponsor after completion of the in-life phase of the study. F. Retention Any unused test material will be returned to the Sponsor after completion of all related in-life testing. G. Safety Precautions As required by CHW SOPs and policies Control Material A. Identification Sterile Water for Injection (sterile water) B. Physical Description Clear colorless liquid C. Purity and Stability The purity and stability of this USP grade material is considered adequate for the purposes of this study. D. Storage Refrigerated 24 002965 CHW 6329-186 CHW 6329-186 TP6350 Page 5 E. Reserve Samples Reserve $ampie(s) of each batcb/lot o f control material will be taken for this study. The control material reserve sample(s) m il be stored at CHW in a freezer set to maintain a temperature of -20C 10#C. F. Retention Any remaining control material may be used for other testing and will not be discarded after issuance of the final report G. Safety Precautions As required by CHW SOPs and policies Experimental Design A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. (3) Age at Initiation Adult (4) Weight at Initiation 2.5 to 3.5 kg (5) Number and Sex 20 males and 20 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually, in suspended screen-bottom stainless steel cages 25 002966 CHW 6329-186 CHW 6329-186 TP6350 Page 6 (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinely analyzed by the manufacturer for nutritional components and environmental contaminants (c) W ater A d libitum from an automatic system. Samples o f the water are analyzed for total dissolved solids, specified microbiological content, selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interfere with this study. (e) Environment Environmental controls for the animal room will be set to maintain a temperature of 19C to 23C, a relative humidity of 50% 20%, and a 12-hour light/12-hour dark cycle. The dark cycle may be interrupted due to in-life procedures. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to CHW SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. The animals will be placed into study groups using a stratified body weight randomization program within nine days of study initiation. (9) Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. 26 002967 CHW 6329-186 B. Dose Administration (1) Test Groups CHW 6329-186 TP6350 Page 7 Contro1/Test Dose Level Group Material (mg/kg)* 1 (Control) Sterile Water 0.0 2 T-6564 3.0 3 T-6564 10.0 4 T-6564 100.0 5 T-6564 300.0 Number o f Animals* Male Female 4 4| 4 41 44 44 44 a Administered at a dose volume of O.S mL/kg. b Two animals/sex/dose level will be sacrificed on Day 14. The remaining animals (two animals/sex/dose level) will be sacrificed on Day 29. C. Dosing Procedures (1) Dosing Route Intravenous injection into the marginal ear vein of the right ear over approximately 30 to 60 seconds. (2) Reason for Dosing Route Intravenous injection is an acceptable route to assess systemic exposure. (3) Dosing Duration Single dose (4) Dose Preparation The day of treatment will be designated as Day 1. Individual doses will be calculated based on the animal's body weight taken on Day 1. The Group 1 animals will be treated with sterile water at a dose volume of O.S mL/kg. The test material will be diluted with sterile water to achieve a specific concentration for each dose level in Groups 2-5 and administered at a dose volume of 0.5 mL/kg. The prepared test mixtures will be stored at room temperature until administered. 27 002968 CHW 6329-186 D. Observation of Animals CHW 6329-186 TP6350 Page 8 (1) Clinical Observations Before test or control material administration, at approximately 0.5, 2.0, and 4.0 hours post-injection (Day 1) for clinical signs, daily thereafter for clinical signs, and twice daily (a.m. and p.m.) for mortality until the scheduled sacrifice interval (Day 14 or Day 29). The animals sacrificed on Days 14 and 29 will be observed only once for mortality on those respective days. Observations may be extended when directed by the study director. (2) Body Weights For randomization, before test or control material injection (Day 1), at the scheduled sacrifice interval (Day 14 or Day 29), or at unscheduled death and sacrifices (when survival exceeds 1 day) (3) Blood Sample Collections (a) Frequency Pre-injection (anytime from up to four days before test material administration to Day 1), 4-, 8-, 12-, 24-, and 48-hours post injection, on Days 8, 14, 22, and at the scheduled sacrifice interval (Day 14 or Day 29) (b) Method of Collection/Number of Animals Blood samples (approximately 4 mL) will be collected from the marginal ear vein (either ear) of all animals on the day before test or control material injection, and from the marginal ear vein (left ear) at 4-hours postdose through Day 8. On Days 14 and 22, additional blood samples (approximately 4 mL) will be collected from the marginal ear vein (left, ear) of the animals scheduled for sacrifice on Day 29. From the posterior vena cava, approximately 20 mL of blood will be obtained from each animal sacrificed in a moribund condition (if possible), approximately 20 to 40 mL of blood will be obtained from each control animal sacrificed on Days 14 or 29 (the maximum volume possible will be obtained), and approximately 20 mL of blood will be obtained from each Group 2-5 animal sacrificed on Days 14 or 29. 28 002969 CHW 6329-186 CHW 6329-186 TP6350 Page 9 The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractions stored in a freezer set to maintain a temperature o f -20C 10C. The serum and cellular fractions obtained through Day 14 will be sent frozen on dry ice to the Sponsor one to two weeks prior to in-life termination. The serum and cellular fractions obtained after Day 14 will be sent frozen on dry ice to the Sponsor within one week after in-life termination. The Sponsor is responsible for the retention and disposition of the samples. The serum and cellular fraction samples will be shipped to: James D. Johnson 3M E.T. & S Bldg. 2-3E-09 933 Bush Avenue St. Paul, MN 55106 James D. Johnson or his alternate will be notified regarding the shipment of the samples. E. Termination (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificed in a moribund condition wilt be subjected to an abbreviated gross necropsy examination and all abnormalities will be recorded. Animals in a moribund condition will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section 7.E.(3) Sample Collection, will be collected from any animal dying during the study or sacrificed in a moribund condition. After necropsy, the animals will be discarded. (2) Scheduled Sacrifices On Day 14, the first two animals/sex assigned to each dose level (based on the group assignment randomization) will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. The remaining two animals/sex/dose level will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated on Day 29. An abbreviated gross necropsy examination 29 002970 CHW 6329-186 CHW 6329-186 TP6350 Page 10 will not be done, however, tissues (as described in section 7.E.(3) Sample Collection) will be collected. (3) Sample Collection The whole liver, bile, and both kidneys (collected as one sample) from each animal will be collected, weighed (volume only determined for bile), and immediately placed into a freezer set to maintain a temperature of -20SC 10C. After sample collection, the animals will be discarded. The samples (liver, bile, and kidneys) will be sent frozen on dry ice to the Sponsor within one week after collection. The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.D.(3)(b). The Sponsor is responsible for the retention and disposition of the samples. F. Statistical Analyses No statistical analyses are required. 8. Report A final report including those items listed below will be submitted. Description of the test and control materials Description of the test system Procedures Dates o f experimental initiation and termination Description of any toxic effects Gross pathology findings (if applicable) Gross pathology report (if applicable and requested by the study director) Individual animal tissue weights and bile volumes 30 002971 CHW 6329-186 CHW 6329-186 TP6350 Page 11 9. Location of Raw Data, Reserve Sample(s), Records, and Final Report Original data, or copies thereof, will be available at CHW to facilitate auditing the study during its progress and before acceptance o f the final report When the final report is completed, control material reserve sample(s), all original paper data, including those items listed below will be retained in the archives of CHW for a period o f one year following signing of the final report One year after signing of the final report all of the aforementioned materials will be sent to the Sponsor and a return fee will be charged. The Sponsor may elect to have the materials retained in the CHW Archives for an additional period of time and CHW will charge a storage fee. If the Sponsor chooses to have CHW dispose of the materials, a disposal fee will be charged. Protocol and protocol amendments Dose preparation records In-life records Body weights Dose administration Observations Sample collection records Shipping records Pathology Records Study correspondence Final report (original signed copy) The following supporting records will be retained at CHW but will not be archived with the study data. Animal receipt/acclimation records Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records 31 =02972 BEST COPY AYAH ABLE CHW 6329-186 PROTOCOL APPROVAL CHW 6329-186 TP6350 Page 12 Sponsor's Representative 3M F. Bud W. McDonald Study Director Acute Studies Coming Hazleton Inc. Representative Quality Assurance Unit Coming Hazleton Inc. (6329-186)HD S'* Date Date Date 32 002973 BEST copy M M Corning Hazleton Inc. 1*0. Bo* T .U S M jdm m . VH 5j* 0 7 .7 J4 5 D W w t t i r t : J JO I Ktnsmjm BlvJ. xujiiu*. vrr S2704 >*.24|.4471 w.241.7227Fj* CHW 6329-186 C O R N IN G Hazleton AMENDMENT NO. 1TO THE PROTOCOL PROTOCOL TP6350 Single-Dose Intravenous Pharmacokinetic Study of T-6564 in Rabbits CHW 6329-186 Sponsor Testing Facility 3M Toxicology Service Medical Department 3M Center. Bldg. 220-2E-02 P.O. Box 33220 St. Paul. MN 55133-3220 Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Study Director Roger G. Perkins. PhD F. Bud W. McDonald This amendment modifies the following portion of the protocol: Effective August 6,1996 1. Page 7,7. Experimental Design, C. Dosing Procedures. Add the following section to the protocol: (5) Died on Test/Replacement Animals Animal No. F6007I (Group 5 male) died approximately twenty minutes after treatment. Discard the animal without an abbreviated gross necropsy examination or tissue collection. Arbitrarily select a replacement animal (male), weighing from 2.5 to 3.5 kg. from the extra animals and obtain a predose (Day I) 4-mL blood sample. Process the sample according to the procedure used for Day -4 samples. Treat the replacement animal on Day 1 in the same manner used for the initial animal and follow all subsequent study procedures as indicated in the protocol. C o rn in g P h jm u c e u u e jJ Services 33 002974 09 BESTCOPY AVAUA CHW6329-186 Amendment No. 1 CHW 6329-186 Page 2 Reason for above change: To address the procedures associated with the death and replacement of the male treated at 300.0 mg/kg o f body weight (Group 5). PROTOCOL AMENDMENT APPROVAL Sponsor's Representative 3M F. Bud W. McDonald Study Director Acute Studies Coming Hazleton Inc. RcDresantative Quality Assurance Unit Coming Hazleton Inc. (6329-I86.Aml/HD) Date Date 34 002975