C The Effect of CHDI on Behavior, Survival and Magnetic Resonance Imaging in R6/2 Huntington s Mice. Cerebricon Ltd.

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1 Cerebricon Ltd. C521-1 The Effect of on Behavior, Survival and Magnetic Resonance Imaging in R6/2 Huntington s Mice Prepared for: CHDI Inc. c/o MRSSI Inc. 35 Seventh Avenue, Suite 61 New York, NY 11 USA

2 C521-1 R6/2 Mice Final Report 6 Purpose of Study The objective of this study was to investigate the effect of treatment on body weight, survival, motor deficits, cognitive performance and volumetry of whole brain, striatum and cortex in R6/2 Huntington s mice. 6 male and female R6/2 mice (TG) (strain R62 (CHDI-1)) and 12 male and female wild-type littermate control mice (WT) were used in the study. The mice were genotyped and TG mice were randomly divided into vehicle and treated groups (female/male balance equal) whereas the WT mice received the corresponding vehicle. The twice-a-day p.o. treatment with CHDI (1 or 3 mg/kg) or vehicle started at age week 4. Body weights were measured twice a week starting at age week 4 and continued until 12 weeks of age. Motor function testing including open field and rear climbing tests were commenced at 4 weeks of age and continued at weeks 6, 8 and 12, for grip strength test at age weeks 4 and 12 and for rotarod at age weeks 4, 6 and 8. Cued two-choice swim test (Cued 2-CST) was performed at 9 weeks of age. At 13 weeks of age all the mice were subjected to in vivo T2-MRI analysis of brain, striatum and cortical volume. The mice were followed until death (=spontaneous death or fulfillment of euthanasia criteria). 7 Materials and Methods 7.1 Animals All animal experiments were carried out according to the National Institute of Health (NIH) guidelines for the care and use of laboratory animals, and approved by the Ethical Committee of the National Laboratory Animal Center, Kuopio, Finland. 6 male and female R6/2 TG mice (R6/2J CAG 12 (strain R62 (CHDI-1)) and 12 male and female wild-type littermate control mice were bred at National Animal Facility Center Kuopio. The experimental groups were: 12 WT littermate control mice treated with vehicle (1 ml/kg) (p.o., BID) starting at 4 weeks of age and continuing until 23 weeks of age 2 TG mice treated with vehicle (1 ml/kg) (p.o., BID) starting at 4 weeks of age and continuing until 23 weeks of age 2 TG mice treated with (1 mg/kg, 1 ml/kg) (p.o., BID) starting at 4 weeks of age and continuing until 23 weeks of age 2 TG mice treated with (3 mg/kg, 1 ml/kg) (p.o., BID) starting at 4 weeks of age and continuing until 23 weeks of age 11(39)

3 C521-1 R6/2 Mice Final Report 7.2 Husbandry All the mice were housed in groups of up to 4 per cage (mixed genotypes, single sex), in a temperature (22±1 C) and humidity (3-7%) controlled environment with a normal light-dark cycle (7:-2:). All mice were housed in shoe-box cages with clean bedding covering the ground that is changed as frequently as needed to provide the animals with dry bedding. This basic environment was enriched with the addition of play tunnels, wooden nesting material, and plastic bones for all mice; i.e. an environmentally-enriched cage contains a Mouse Tunnel, (amber color, certified, transparent, BioServ Product# K3323) and a Petite Green Gumabone (BioServ Product # K3214) and wooden nesting material. Food (Purina Lab Diet 51) and water were available ad libitum to the mice in their home cages. R6/2 and corresponding WT mice also receive wet powdered food (Purina Lab Diet 51 mixed with water to form a paste) placed inside a cup on the floor of the cage; this additional food was replaced fresh daily and started from the time of weaning. In addition, water spouts were fitted with extensions to allow mice to easily access from floor level. 7.3 Breeding and Weaning 6 male and female R6/2 mice and 12 male and female wild-type littermate control mice (F 1 generation) were bred at FELASA compliant National Animal Facility Center Kuopio by mating (F generation) WT males (C57BL/6J, JAX) with ovarian transferred (OT) TG females (JAX). The F mice arrived at 1-12 weeks of age to allow 1 week quarantine and breeding in two consecutive weeks. Breeder animals received igloos instead of play tunnels, nylabone and cotton nestlets. Upon weaning, pups received Purina diet 51. Pups were weaned from their mothers and segregated to new cages for male and females, not exceeding 4-5 mice per cage. Ear snips were taken during the weaning process at 2-3 weeks for genotype analysis as described below. 7.4 Genotyping Mice were ear marked at the age of days and ear samples were collected at the same time for genotyping with PCR. Genotypes were determined between 15 and 21 days (weaning age) of age by PCR of ear samples. In mutant mice, the genotype was a simple PCR assay and CAG repeat length was analyzed by GeneAmp 272 Thermal Cycler sequencer. 7.5 Experimental Set Up of Mice In setting up groups for study (i.e. vehicle or compound treated), transgenic and wildtype mice were randomized into groups so that whole litters of mice did not end up in a single testing group. Mice were housed in groups of 4 and separated by sexes. In each cage, one wild-type mouse of the same gender, but different litter, was included in an attempt to provide normal social stimulation. Mice were allowed to acclimate to the experimental room for at least one hour prior to the beginning of any experiment. Mice were transported from the colony room to experimental rooms in their home cages. Experimentation was conducted in a 12(39)

4 C521-1 R6/2 Mice Final Report blinded manner. Tail samples were taken at the end of the study for possible verification of genotypes and CAG sizes of individual mice. 7.6 General Health Status and Humane End-Points Animals were monitored twice-a-day by laboratory personnel (8 am and 16 pm). In case general health status of an animal was significantly worsened, the mouse was sacrificed by an overdose of CO 2 and decapitated. Definitions of acceptable endpoints include: weight loss of more than 25 % of the maximum, no spontaneous movements and inability to drink or eat in 24-h observation period, massive bleeding, spontaneous inflammation, missing anatomy, swelling or tumors. 7.7 Compound Delivery Compound was dissolved in 3% Propylene Glycol, 2% Solutol HS15, 5% 5mM Citrate, and administered p.o. twice-a-day starting at age week 4 and continuing until endpoint. On the days of behavioral testing the dosing was done at least 1.5 h before the test. Genotype N (F/M) Compound Dose (mg/kg) Regimen Route Dose volume (ml/kg) Wild-type 6/6 R6/2 1/1 3% Propylene Glycol, 2% Solutoll HS15, 5% 5mM Citrate BID p.o. 1 3% Propylene Glycol, 2% Solutoll HS15, 5% 5mM Citrate BID p.o. 1 R6/2 1/1 1 BID p.o. 1 R6/2 1/1 3 BID p.o. 1 Table 4. Details of the genotype, number and gender of animals (F: female, M: male) included in each treatment group, the compound, dose, dosage regime and route and volume of administration. 7.8 Body Weight and Survival Body weight was measured starting at the age of 4 weeks and two times per week on the same day (i.e. Monday and Friday) until the end of the study. This was done just prior to animals receiving doses for those days. In the case that the general health status of an animal had significantly worsened, the mouse was sacrificed by an overdose of CO 2, and decapitated. Definitions of acceptable endpoints include: no spontaneous movements and inability to drink or eat in 24-h observation period, massive bleeding, spontaneous inflammation, missing anatomy, swelling or tumors larger than 2 mm, and inability to right itself in 3 sec period. The mice were monitored until death or the fulfillment of the euthanasia criteria. No tissues were collected at the end point. 13(39)

5 C521-1 R6/2 Mice Final Report 7.9 Neurological Index The mice were observed for 1-2 minutes per mouse at 13 weeks of age. The following 3 behaviors were evaluated: Head Tremor, Head Twitch, Head Bobbing, Head Searching, Body Tremor, Body Twitch, Tail Tremor, Tail Twitch, Straub Tail, Piloerection, Shallow Respiration, Flattened Body Posture, Swollen Face, Ptosis, Irritability, Seizure, Urine Staining, Lacrimation, Salivation, Limb Splay, Catalepsy, Abnormal Gait, Tip Toe Walking, Slow Careful Movements, Excessive Grooming, Circling, Sniffing, and Chewing. In addition to the above occurrences the chronic observational phase includes excessive locomotor acitivty, loss of startle response, loss of righting reflex, dehydration and tail pinch. The assessment was performed as follows: A score of was assigned for normal features (such as locomotor activity) or for the absence of abnormal features (such as absence of piloerection); a score of 1 was given when mild abnormalities were observed; and a score of 2-3 was given when severe abnormalities were observed. All of these features were scored from simple observation of the mice in their cage, except for the startle, tail pinch and righting reflexes which are direct manipulations. To test the startle reflex, a small hand clicker was used to generate a loud popping noise and the following behaviors were identified during this process: jumping, freezing, and rapid eye blinks. For the righting reflex, each mouse was then removed from its home cage and placed on its back allowing the mouse to correct itself. Tail pinch was tested by gently squeezing the end of the tail with forceps. 7.1 Motor Function Motor function testing including open field and rear climbing tests were commenced at 4 weeks of age and continued at weeks 6, 8 and 12, for grip strength test at age weeks 4 and 12 and for rotarod at age weeks 4, 6 and 8. Compound treatment was started at 4 weeks of age after the baseline behavioral tests Rotarod Mice were tested at age weeks 4, 6 and 8 over 3 consecutive days (Wed, Thu, Fri). Each daily session included a training trial of 5 min at 4 RPM on the rotarod apparatus (AccuScan Instruments, Columbus, USA). One hour later, the animals were tested for 3 consecutive accelerating trials of 6 min with the speed changing from to 4 RPM over 36 seconds and an inter-trial interval at least 3 min. The latency to fall from the rod was recorded. Mice remaining on the rod for more than 36 s were removed and their time scored as 36 sec Grip Strength Grip strength measurement was performed according to PGI SOP at age weeks 4 and 12. The mice were tested on Monday afternoon. Mice were taken to the experimental room and, one at a time, were placed on the grip-strength apparatus (San Diego Instruments, San Diego, USA) in such a way that the animal grabs a small mesh grip with its forepaws. The entire apparatus is placed on a table top for testing. Animals are lowered to the platform and then slowly pulled away from the handle by the tail until the animal releases the handle. The equipment automatically measures the strength of the animal s grip in grams. Five scores were recorded per 14(39)

6 C521-1 R6/2 Mice Final Report animal in consecutive sequence. testing. Mice were returned to their home cage after Open Field Test Open field test was performed according to PGI SOP at age weeks 4, 6, 8 and 12. The mice were tested on Monday at approximately 1-3 lux of red light. The mice were brought to the experimental room for at least 1 h acclimation to the experimental room conditions prior to testing. Activity chambers (Med Associates Inc, St Albans, VT; 27 x 27 x 2.3 cm) are equipped with IR beams. Mice were placed in the center of the chamber and their behavior was recorded for 3 min in 5-minute bins. Quantitative analysis was performed on the following five dependent measures: total locomotion, locomotion in the center of the open field, rearing rate in the center, total rearing frequency and velocity Rear Climbing Test Rear climbing test was performed according to PGI SOP at age weeks 4, 6, 8 and 12. The mice were tested on Tuesday. Each animal was placed into custom made pencil holder, and was scored for 5 minutes. During this period the following events were noted: 1) number of times the mouse rears without touching the mesh walls, 2) latency to climb (lifting 4 paws from the floor is labeled climbing), 3) climbing time, and 4) climbing instances Cognitive Function Cued Two-Choice Swim Test The cued two-choice swim test was performed at 9 weeks of age. Testing was conducted under low light, white light conditions. The mice were taught to swim to (acquisition), or away from (reversal), a light positioned over the escape platform. Animals were placed within a rectangular tank (76 cm x 3 cm x 3 cm) filled with water maintained at 25 C ± 1 C. The water was rendered opaque by the addition of non-toxic white paint. The escape platform was located.5 cm below the surface of the water at one end of the tank. A light source with a 25W bulb (white light), oriented toward the water, was clipped to one side of the tank above the escape platform (7 cm in diameter with the borders slanted and roughened to help the rodents climb to it). Under these conditions, the light intensity is measured as 2 lux on the illuminated side of the tank, and 3 lux on the dark side of the tank. The platform was located on the lit side of the arena across acquisition trials, although the location of the lit side within the room relative to the mouse s entry point was varied. The location of the bulb and platform was different on different trials for the same mouse. On each trial, mice were placed into the middle of the tank and allowed to swim for up to 6 sec, or until they found the platform. If an animal did not reach the platform within 6 sec, the mouse was placed on it by the experimenter and allowed to remain there for 3 sec and then removed to a pre-warmed holding cage placed on a warming pad. During all acquisition days, mice were given 8 trials per day (4 blocks of 2 trials, separated by an interblock interval of 2-3 minutes). Training continued in this way until each mouse performed at 75% correct choices for 2 consecutive days; upon reaching this criterion for successful acquisition of the task, mice were placed into the reversal phase on an individual basis. During reversal training, (4 blocks of 2 15(39)

7 C521-1 R6/2 Mice Final Report trials per day), the platform was placed on the dark side of the tank. On each trial during testing days, the latency to reach the platform was recorded (6 seconds if the platform was not reached) and the trial was scored as either correct, incorrect or no choice. The maximum number of days of acquisition was 12 days. All mice went from acquisition into the reversal phase and the experiment was stopped when 2 consecutive days of 75% criterion was reached or after a maximum of 7 days of reversal testing. A correct choice was scored if the animal turned in the direction of the platform and successfully mounted the platform. An incorrect choice was scored if the animal swam in the direction opposite the platform. No choice was scored if the animal either did not make a choice, by swimming in the middle of the tank for the 6 sec, or turned initially toward the platform but did not mount the platform. To make a choice, a mouse had to swim within 2 cm of either end of the tank MRI MRI analysis was performed for all mice at the age of 13 weeks in a horizontal 7T magnet with bore size 16 mm (Magnex Scientific Ltd., Oxford, UK) equipped with Magnex gradient set (max. gradient strength 4 mt/m, bore 1 mm) interfaced to a Varian DirectDrive console (Varian, Inc., Palo Alto, CA) using a volume coil for transmission and surface phased array coil for receiving (Rapid Biomedical GmbH, Rimpar, Germany). Isoflurane -anesthetized mice were fixed to a head holder and positioned in the magnet bore in a standard orientation relative to gradient coils. Rectal temperature (TC-1 Temperature Controller, CWE Inc., Ardmore, PA, USA) and respiration (ECG Trigger Unit, Rapid Biomedical GmbH) were monitored throughout the study and the body temperature was kept at C. For determination of volume of brain, striatum, cortex and lateral ventricles T2-weighted multi-slice (17 continuous slices) images were acquired using fast spin-echo sequence with TR/TE eff = 3/36 ms, matrix size of 256x128, FOV of 2x2 mm 2, slice thickness of.7 mm and 8 averages. 8 Statistical Analysis All values are presented as mean ± Standard Error of Mean (SEM), and differences are considered to be statistically significant at the p <.5 level. Statistical analysis was performed using StatsDirect statistical software. Differences between group means were analyzed by 1-way-ANOVA followed by Dunnett s test (comparison to the control (=vehicle treated TG mice) group). The acquisition of the Cued two-choice swim test was analyzed using Fisher s exact test. 16(39)

8 Weight (g) C521-1 R6/2 Mice Final Report 9 Results 9.1 Body Weight The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on body weight of the R6/2 mice from 4-23 weeks of age are presented in Figure 1. No significant differences with were found at any time point in body weight (p >.5) (Fig. 1). C521-1 Body Weight Age Week Figure 1. The effects of chronic administration of on body weight from 4-23 weeks of age. Data are presented as mean ± SEM. 17(39)

9 Latency (sec) C521-1 R6/2 Mice Final Report 9.2 Rotarod The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on rotarod performance of the R6/2 mice are presented in Figure 2. No significant differences with were found at any time point in rotarod latency (p >.5) (Fig. 2). C521-1 Rotarod Age Week / Test Day Figure 2. The effects of chronic administration of on rotarod performance from 4-8 weeks of age. Data are presented as mean ± SEM. 18(39)

10 Force (g) C521-1 R6/2 Mice Final Report 9.3 Grip Strength The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on grip strength of the R6/2 mice are presented in Figure 3. The mice treated with 3 mg/kg of had increased grip strength at 12 weeks of age compared to vehicle-treated R6/2 mice (p <.5) (Fig. 3). C521-1 Grip Strength 1 8 * Weeks 12 Weeks Figure 3. The effects of chronic administration of on grip strength from 4-12 weeks of age. Data are presented as mean ± SEM 19(39)

11 Distance (cm) C521-1 R6/2 Mice Final Report 9.4 Open Field Total Distance Traveled The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on total distance traveled in the open field by the R6/2 mice are presented in Figure 4. The mice treated with 3 mg/kg of traveled a shorter total distance at 12 weeks of age compared to vehicle-treated R6/2 mice (p <.5) (Fig. 4). C521-1 Open Field - Distance Traveled Age Week / Session Time (min) * Figure 4. The effects of chronic administration of on total distance traveled from 4-12 weeks of age. Data are presented as mean ± SEM. 2(39)

12 Distance (cm) C521-1 R6/2 Mice Final Report Distance Traveled in Center The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on distance traveled in the center area of the open field by the R6/2 mice are presented in Figure 5. The mice treated with 3 mg/kg of traveled a shorter distance in the center area at 12 weeks of age compared to vehicle-treated R6/2 mice (p <.5) (Fig. 5). C521-1 Open Field - Distance Traveled in Center Area Age Week / Session Time (min) * Figure 5. The effects of chronic administration of on distance traveled in the center of the open field from 4-12 weeks of age. Data are presented as mean ± SEM. 21(39)

13 Rearing Rate (count / min) C521-1 R6/2 Mice Final Report Rate of Rears in Center The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on rearing rate in the center area of the open field of the R6/2 mice from are presented in Figure 6. No significant differences with were found at any time point on rearing rate in the center area (p >.5) (Fig. 6). C521-1 Open Field - Rearing Rate in Center Area Age Week Figure 6. The effects of chronic administration of on rearing rate in the center area of the open field from 4-12 weeks of age. Data are presented as mean ± SEM. 22(39)

14 Vertical Count C521-1 R6/2 Mice Final Report Total Rearing Frequency The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on total rearing frequency in the open field of the R6/2 mice are presented in Figure 7. No significant differences with were found at any time point on total rearing frequency (p >.5) (Fig. 7). C521-1 Open Field - Total Rearing Frequency Age Week Figure 7. The effects of chronic administration of on total rearing frequency in the open field from 4-12 weeks of age. Data are presented as mean ± SEM. 23(39)

15 Average Velocity (cm / sec) C521-1 R6/2 Mice Final Report Average Velocity The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on average velocity in the open field of the R6/2 mice from are presented in Figure 8. No significant differences with were found at any time point on average velocity (p >.5) (Fig. 8). C521-1 Open Field - Average Velocity Age Week Figure 8. The effects of chronic administration of on average velocity in the open field from 4-12 weeks of age. Data are presented as mean ± SEM. 24(39)

16 Vertical Count C521-1 R6/2 Mice Final Report 9.5 Rear Climbing Rearing Frequency The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on rearing frequency of the R6/2 mice in the rear climbing test are presented in Figure 9. No significant differences with were found at any time point on rearing frequency (p >.5) (Fig. 9). C521-1 Rear Climbing - Rearing Frequency Age Week Figure 9. The effects of chronic administration of on rearing frequency in the rear climbing test from 4-12 weeks of age. Data are presented as mean ± SEM. 25(39)

17 Latency (sec) C521-1 R6/2 Mice Final Report Latency to Climb The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on latency to climb of the R6/2 mice in the rear climbing test are presented in Figure 1. No significant differences with were found at any time point on latency to climb (p >.5) (Fig. 1). C521-1 Rear Climbing - Latency to Climb Age Week Figure 1. The effects of chronic administration of on latency to climb in the rear climbing test from 4-12 weeks of age. Data are presented as mean ± SEM. 26(39)

18 Time Spent Climbing (sec) C521-1 R6/2 Mice Final Report Time Spent Climbing The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on time spent climbing by the R6/2 mice in the rear climbing test are presented in Figure 11. No significant differences with were found at any time point on time spent climbing (p >.5) (Fig. 11). C521-1 Rear Climbing - Time Spent Climbing Age Week Figure 11. The effects of chronic administration of on time spent climbing in the rear climbing test from 4-12 weeks of age. Data are presented as mean ± SEM. 27(39)

19 Number of Climbing Instances C521-1 R6/2 Mice Final Report Climbing Instances The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on climbing instances of the R6/2 mice in the rear climbing test are presented in Figure 12. No significant differences with were found at any time point on climbing instances (p >.5) (Fig. 12). C521-1 Rear Climbing - Climbing Instances Age Week Figure 12. The effects of chronic administration of on climbing instances in the rear climbing test from 4-12 weeks of age. Data are presented as mean ± SEM. 28(39)

20 % of Mice Reaching Criterion 9.6 Cued Two-Choice Swim Test Acquisition Percent of Mice Reaching the Criterion C521-1 R6/2 Mice Final Report The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on the acquisition learning of the R6/2 mice in the cued two-choice swim test are presented in Figure 13. The acquisition learning is presented as the percent of mice in each group reaching the criterion. No significant differences with were found in the acquisition learning (p >.5) (Fig. 13). C521-1 Cued Two-Choice Swim Test - Acquisition Test Day Figure 13. The effects of chronic administration of on the acquisition learning in the cued two-choice swim test at 9 weeks of age. Data are presented as mean ± SEM. 29(39)

21 Daily Average Correct (%) C521-1 R6/2 Mice Final Report Reversal - Correct Choices The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on the number of correct choices of the R6/2 mice in the reversal phase of the cued two-choice swim test are presented in Figure 14. No significant differences with were found in the number of incorrect choices (p >.5) (Fig. 14). C521-1 Cued Two-Choice Swim Test - Reversal - Correct Choice Test Day Figure 14. The effects of chronic administration of on the number of correct choices in the reversal phase of the cued two-choice swim test at 9 weeks of age. Data are presented as mean ± SEM. 3(39)

22 Daily Average Incorrect (%) Reversal - Incorrect Choices C521-1 R6/2 Mice Final Report The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on the number of incorrect choices of the R6/2 mice in the reversal phase of the cued two-choice swim test are presented in Figure 15. No significant differences with were found in the number of incorrect choices (p >.5) (Fig. 15). C521-1 Cued Two-Choice Swim Test - Reversal - Incorrect Choice Test Day Figure 15. The effects of chronic administration of on the number of incorrect choices in the reversal phase of the two-choice swim test at 9 weeks of age. Data are presented as mean ± SEM. 31(39)

23 Daily Average No Choice (%) C521-1 R6/2 Mice Final Report Reversal - No Choice The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on no choice of the R6/2 mice in the reversal phase of the cued two-choice swim test are presented in Figure 16. No significant differences with were found in no choice (p >.5) (Fig. 16). C521-1 Cued Two-Choice Swim Test - Reversal - No Choice Test Day Figure 16. The effects of chronic administration of on no choice in the reversal phase of the two-choice swim test at 9 weeks of age. Data are presented as mean ± SEM. 32(39)

24 Daily Average Latency (%) C521-1 R6/2 Mice Final Report Reversal - Latency The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on latency of the R6/2 mice in the reversal phase of the cued two-choice swim test are presented in Figure 17. No significant differences with were found in latency (p >.5) (Fig. 17). C521-1 Cued Two-Choice Swim Test - Reversal - Latency Test Day Figure 17. The effects of chronic administration of on latency in the reversal phase of the two-choice swim test at 9 weeks of age. Data are presented as mean ± SEM. 33(39)

25 Score C521-1 R6/2 Mice Final Report 9.7 Neurological Index The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on Neurological Index of the R6/2 mice at 13 weeks of age are presented in Figure 18. No significant differences with were found in Neurological Index (p >.5) (Fig. 18). C521-1 Neurological Index Figure 18. The effects of chronic administration of on neurological index at 13 weeks of age. Data are presented as mean ± SEM. 34(39)

26 Volume (mm 3 ) C521-1 R6/2 Mice Final Report 9.8 MRI Volumetry Whole Brain Volume The whole brain volume was measured at the age of 13 weeks using MRI techniques. Initially the purpose was to perform the measurements at 12 weeks of age but due to technical problems with the MRI machinery the measurements had to be postponed by one week. Due to the same reason the MR spectroscopy measurements and analysis could not be performed in this study. Total n MRI Volumetry Table 5. Number of mice used for the MRI volumetry measurements in study C The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on whole brain volume are presented in Figure 19. No significant differences with treatment were found in whole brain volumes (p >.5) (Fig. 19). C521-1 MRI Volumety - Whole Brain Figure 19. The effects of chronic administration of on whole brain volume at 13 weeks of age. Data are presented as mean ± SEM. 35(39)

27 Volume (mm 3 ) C521-1 R6/2 Mice Final Report Striatum Volume The striatum volume was measured at the age of 13 weeks using MRI techniques. The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on striatum volume are presented in Figure 2. No significant differences with CHDI treatment were found in striatum volumes (p >.5) (Fig. 2). C521-1 MRI Volumetry - Striatum Figure 2. The effects of chronic administration of on striatum volume at 13 weeks of age. Data are presented as mean ± SEM. 36(39)

28 Volume (mm 3 ) C521-1 R6/2 Mice Final Report Cortical Volume The cortical volumes were measured at the age of 13 weeks using MRI techniques. The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on cortical volumes are presented in Figure 21. No significant differences with CHDI treatment were found on cortical volumes (p >.5) (Fig. 21). C521-1 MRI Volumetry - Cortex Figure 21. The effects of chronic administration of on cortical volume at 13 weeks of age. Data are presented as mean ± SEM. 37(39)

29 C521-1 R6/2 Mice Final Report 9.9 Survival The survival of the mice was monitored until 25 weeks of age. The effects of chronic administration of (1 and 3 mg/kg, p.o., BID) on survival of the R6/2 mice is displayed as a Kaplan-Meier plot in Figure 22. The mean/median survival times of the R6/2 mice treated with vehicle was 115/16 days, with 1 mg/kg of 115/11 days, and with 3 mg/kg of 122/121 days. There were no differences in survival between the treatment groups (Logrank and Wilcoxon tests, p >.5). C521-1 Survival Plot (PL estimates) Survivor 1,,75,5,25, Times Figure 22. The effects of chronic administration of on survival until 25 weeks of age in R6/2 mice. Data are presented as Kaplan-Meier plot 38(39)

30 1 Discussion and Conclusions C521-1 R6/2 Mice Final Report As a summary, the twice-a-day oral treatment with (1 or 3 mg/kg) had no major beneficial effects on body weight, survival, motor deficits, cognitive performance, or the volume of whole brain, striatum or cortex in the R6/2 mice of Huntington s disease. However, the mice treated with 3 mg/kg of had increased grip strength at 12 weeks of age and had a decreased number of no choice on the test day two of the reversal phase of cued two-choice swim test compared to vehicle-treated R6/2 mice. Also, the mice treated with 3 mg/kg of showed decreased horizontal activity in the open field at 12 weeks of age compared to vehicle-treated R6/2 mice. 11 Storage of the Raw Data, Specimens, and the Final Report The raw data and the final report are stored in the premises of Cerebricon Ltd., Microkatu 1, FIN-7211 Kuopio, Finland until delivered to the client. ELECTRONIC ANNEXES Annex 1 C521-1 Final Report, Statistical Analyses and Excel Data Sheets (electronic files) 39(39)