A METHOD FOR EVALUATING THE PRO-OR ANTI CONVULSIVE PROPERTIES OF TEST COMPOUNDS

Abstract

The present invention relates to a method of evaluating the pro- or anti convulsive properties of test compounds that is both streamlined and is capable of providing a clear indication for the selection of candidate compounds during preclinical assessment.

Claims

1. A method of assessing the pro- or anti-convulsant properties of a test compound comprising the following steps: a. dosing a number of animals with one of either a vehicle, the test compound or a positive control compound; b. determining if the animals produce a tonic hind limb extensor convulsion at a defined period of time post-dose from a single electroshock at a defined current; c. decreasing or increasing the defined current if the preceding animal did or did not produce tonic hind limb extensor convulsion, respectively and d. collecting CC.sub.50 values for the treated animals; characterized in that the number of animals used is at least 6.

2. The method according to claim 1, wherein the number of animals used is no more than 11.

3. The method according to claim 1, wherein the current of the electroshock is decreased or increased in a logarithmic scale.

4. The method according to claim 1, wherein the defined period of time post-dose is at least 15 minutes.

5. The method according to claim 1, wherein the defined period of time post-dose is about 30 minutes.

6. The method according to claim 1, wherein the defined period of time post-dose is 120 minutes.

7. The method according to claim 1, wherein the positive control compound is diazepam.

8. The method according to claim 1, wherein the positive control compound is sodium valproate.

9. The method according to claim 1, wherein the animal used is a mouse.

10. The method according to claim 1, wherein the animal used is a rat.

11. The method according to claim 1, wherein the animal used is a pig.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0025] FIG. 1 shows the effect of the Compound 1, as shown as Formula I, in the mini-MEST test in the mouse as described in Example 1.

[0026] FIG. 2 shows the effect of Compound 2, as shown as Formula II, in the mini-MEST test in the mouse as described in Example 2.

[0027] FIG. 3 shows the effect of Compound 3, as shown as Formula III, in the mini-MEST test in the mouse as described in Example 3.

[0028] FIG. 4 shows the effect of Compound 3, as shown as Formula III, in the MEST test in the mouse as described in Example 4.

DEFINITIONS

[0029] Cannabinoids are a group of compounds including the endocannabinoids, the phytocannabinoids and those which are neither endocannabinoids or phytocannabinoids, hereinafter syntho-cannabinoids.

[0030] Endocannabinoids are endogenous cannabinoids, which are high affinity ligands of CB1 and CB2 receptors.

[0031] Phytocannabinoids are cannabinoids that originate in nature and can be found in the cannabis plant. The phytocannabinoids can be present in an extract including a botanical drug substance, isolated, or reproduced synthetically.

[0032] Syntho-cannabinoids are those compounds that are not found endogenously or in the cannabis plant. Examples include WIN 55212 and rimonabant.

[0033] An isolated phytocannabinoid is one which has been extracted from the cannabis plant and purified to such an extent that all the additional components such as secondary and minor cannabinoids and the non-cannabinoid fraction have been removed.

[0034] A synthetic cannabinoid is one which has been produced by chemical synthesis. This term includes modifying an isolated phytocannabinoid, by, for example, forming a pharmaceutically acceptable salt thereof.

[0035] A substantially pure cannabinoid is defined as a cannabinoid which is present at greater than 95% (w/w) pure. More preferably greater than 96% (w/w) through 97% (w/w) thorough 98% (w/w) to 99% % (w/w) and greater.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The following Examples describe for the first time how the mini-MEST test was used to assess the anti-convulsant activity of the following CBD analogues, Compound 1 as shown as Formula I, Compound 2 as shown as Formula II, and Compound 3 as shown as Formula III.

##STR00001##

Example 1: Evaluation of Cannabinoid Derivative for Anticonvulsant Activity Using the Maximal Electroshock Seizure Threshold (MEST) Test in the Mouse Using Minimal Sample Sizes (Mini MEST)

Methods

Study Details

[0037] Na?ve mice were acclimatised to the procedure room in their home cages up to 7 days following arrival to the test facility, with food and water available ad libitum (see Table 1 for details).

[0038] All animals were weighed at the beginning of the study and assigned to treatment groups (n=6/group) based on a mean distribution of body weight across groups. All animals were dosed at 10 mL/kg via intraperitoneal (i.p.) injection, with either vehicle, test compound (50 mg/kg) or diazepam (2.5 mg/kg) (Tables 2 and 3 for details).

[0039] Animals were individually assessed for the production of a tonic hind limb extensor convulsion at 30 min post-dose for vehicle, test compound (50 mg/kg) or diazepam, from a single electroshock (see Table 4 for details). The first animal within a treatment group was given a shock at the expected or estimated CC.sub.50 current. For subsequent animals, the current was lowered or raised depending on the convulsion outcome from the preceding animal in log scale intervals. Data generated from each treatment group were used to calculate the CC.sub.50?SEM values for the treatment group (see Table 5 for details).

Euthanasia and Sample Collection

[0040] Each animal was humanely killed immediately after production of a convulsion by destruction of the brain from striking the cranium, followed by confirmation of permanent cessation of the circulation from decapitation under The Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986. Terminal blood and brain collection were performed following decapitation.

[0041] Blood was collected in Lithium-heparin tubes and centrifuged at 4? C. for 10 min, at 1500?g. The resulting plasma was removed (>100 ?L) and split into 2 aliquots stored in 0.5 mL Eppendorf tubes, containing 100 ?L of ascorbic acid (100 mg/mL) for stabilisation. Brains were removed, washed in saline and halved. Each half was placed into separate 2 mL screw cap cryovials, weighed and frozen on cardice. Samples were stored at ?80? C. until shipment.

Animal Details

[0042]

TABLE-US-00001 TABLE 1 Details of animal species, strain, sex, order details and environmental conditions Species Mouse Strain C57BL/6J Sex Male No. of animals/group n = 6/group Weight range at study start 20.8-24.8 g Estimated age range at study 8-9 weeks start Environmental conditions Housed in groups of 4-5, with standard conditions. Lighting conditions 12 h/2 h light cycle; 7 am lights on, 7 pm lights off; light intensity: 25-75 lux at bench level Food and water Food: Certified Rodent CR 14% Protein Rodent Diet, LabDiet? 5CR4 Water: pathogen-free water from test facility

Compounds Details

[0043]

TABLE-US-00002 TABLE 2 Details of Compound 1, batch, appearance, supplier, vehicle used for formulation Storage conditions Room temperature Appearance Cream powder Vehicle used for formulation 1:2:17 Ethanol: Kolliphor HS (Solutol): saline

TABLE-US-00003 TABLE 3 Details of Diazepam, batch, appearance, supplier, vehicle used for formulation Appearance White powder Vehicle used for formulation 1:1:18 Ethanol: Kolliphor EL (Cremaphor): saline

Vehicle Preparation:

[0044] 5% ethanol, 10% Kolliphor HS (Solutol) in 85% Saline solution

[0045] 1 mL of Ethanol, 2 mL of Kolliphor HS (Solutol)warmed to 60? C., in 17 mL of saline (1:2:17).

Data Recorded and Analysis

[0046]

TABLE-US-00004 TABLE 4 Details of data recorded in visual observations, mini MEST test, mini MEST data analysis and statistical analysis. Visual observations Animals were observed throughout the study from the start of dosing. Any abnormal signs were recorded and reported. Mini MEST test Mini MEST was run between 8 am to 4 pm under normal light conditions. Electroshock was delivered using a Hugo Sachs Electronik stimulator, with an adjustable constant current (1-300 mA). Electroshock duration is 0.1 seconds delivered via corneal electrodes on both eyes. Induction of seizure from the electroshock was measured as an all-or-nothing effect scored as either present (+) or absent (0) of tonic hind limb extensor convulsions for each animal. Up-and-down method based on The current was lowered or raised in log 0.06:10.sup.? Kimball AW et al., 1957 (1 + x*0.06) mA intervals (see raw results in Appendix) if the preceding animal did or did not show tonic hind limb extension, respectively. If tonic hind limb extension was absent in the animal, the subsequent animal will receive a raised current level. If tonic hind limb extension was present in the animal, the subsequent animal will receive a lowered current level. This procedure was continued for all mice within a treatment group. Mini MEST data analysis The data for each treatment group were recorded as the number of +'s and 0's at each current level employed and this information was then used to calculate the CC.sub.50 value (current required for 50% of the animals to show seizure behaviour) ? standard error of mean (SEM) based on Kimball et al. (1957). Test compound effects were also calculated as percentage change in CC.sub.50 from the vehicle control group. Statistical analysis Significant difference between drug-treated animals and controls were assessed according to Litchfield and Wilcoxon (1949), using Microsoft Excel macro.

Results

[0047] FIG. 1 and Table 5 describe the data produced in this experiment, and raw results are shown in the Appendix.

[0048] In the vehicle group, the CC.sub.50 value was calculated to be 24.5 mA.

[0049] In the diazepam (2.5 mg/kg) treated group, administered i.p. 30 minutes before the test, the CC.sub.50 value was 75.0 mA. This result was statistically significant (p<0.001) compared to the vehicle control.

[0050] In the test compound treatment group, administered i.p. 30 minutes before the test, the compound produced a statistically significant CC.sub.50 value compared to vehicle, 119.5 mA.

[0051] Such data are indicative that this compound will be of therapeutic benefit.

TABLE-US-00005 TABLE 5 mini-MEST results table and statistical analysis. Test time % change Dose post CC.sub.50 ? from Signi- Treatment (mg/kg) dose (min) N SEM vehicle ficance Vehicle 0 30 6 24.5 ? 0.9 Diazepam 2.5 30 6 75.0 ? 206% P < 0.001 3.4 Compound 50 30 6 119.5 ? 388% P < 0.001 1 1.9

Conclusion

[0052] The positive control, diazepam (2.5 mg/kg) administered at 30 min post-dose (i.p.) produced a significant increase in seizure threshold. This result clearly demonstrates the robustness of the presently claimed method and validates the method used.

[0053] Compound 1 (50 mg/kg) administered at 30 min post-dose (i.p.) produced a produced a significant increase in seizure threshold, which suggests this compound exhibits anticonvulsive properties.

[0054] Thus, the mini-MEST method used was capable of providing a clear indication of the test compound's anticonvulsant properties.

Example 2: Evaluation of Cannabinoid Derivative for Anticonvulsant Activity Using the Maximal Electroshock Seizure Threshold (MEST) Test in the Mouse Using Minimal Sample Sizes (Mini MEST)

[0055] The Example below was carried out similar to Example 1 outlined above using Compound 2 as according to Formula II.

Methods

Study Details

[0056] Na?ve mice were acclimatised to the procedure room in their home cages up to 7 days following arrival to the test facility, with food and water available ad libitum (see Table 6 for details).

[0057] All animals were weighed at the beginning of the study and assigned to treatment groups (n=6/group) based on a mean distribution of body weight across groups. All animals were dosed at 10 mL/kg via intraperitoneal (i.p.) injection, with either vehicle, test compound (5 or 50 mg/kg) or diazepam (2.5 mg/kg) (Tables 7 and 8 for details).

[0058] Animals were individually assessed for the production of a tonic hind limb extensor convulsion at 30 min post-dose for vehicle, 15 and 30 minutes for test compound at 5 and 50 mg/kg respectively or diazepam, from a single electroshock (see Table 9 for details). The first animal within a treatment group was given a shock at the expected or estimated CC.sub.50 current. For subsequent animals, the current was lowered or raised depending on the convulsion outcome from the preceding animal in log scale intervals. Data generated from each treatment group were used to calculate the CC.sub.50?SEM values for the treatment group (see Table 10 for details).

Euthanasia and Sample Collection

[0059] Each animal was humanely killed immediately after production of a convulsion by destruction of the brain from striking the cranium, followed by confirmation of permanent cessation of the circulation from decapitation under The Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986. Terminal blood and brain collection were performed following decapitation.

[0060] Blood was collected in Lithium-heparin tubes and centrifuged at 4? C. for 10 min, at 1500?g. The resulting plasma was removed (>100 ?L) and split into 2 aliquots stored in 0.5 mL Eppendorf tubes, containing 100 ?L of ascorbic acid (100 mg/mL) for stabilisation. Brains were removed, washed in saline and halved. Each half was placed into separate 2 mL screw cap cryovials, weighed and frozen on cardice. Samples were stored at ?80? C. until shipment.

Animal Details

[0061]

TABLE-US-00006 TABLE 6 Details of animal species, strain, sex, order details and environmental conditions Species Mouse Strain C57BL/6J Sex Male No. of animals /group n = 6/group Weight range at study start 21.5-25.9 g Estimated age range at study start 8-9 weeks Environmental conditions Housed in groups of 4-5, with standard conditions. Lighting conditions 12 h/12 h light cycle; 7 am lights on, 7 pm lights off; light intensity: 25-75 lux at bench level Food and water Food: Certified Rodent CR 14% Protein Rodent Diet, LabDiet? 5CR4 Water: pathogen-free water from test facility

Compounds Details

[0062]

TABLE-US-00007 TABLE 7 Details of Compound 2, batch, appearance, supplier, vehicle used for formulation Storage conditions Room temperature Appearance White powder Vehicle used for formulation 1:2:17 Ethanol: Kolliphor HS (Solutol): saline

TABLE-US-00008 TABLE 8 Details of Diazepam, batch, appearance, supplier, vehicle used for formulation Appearance White powder Vehicle used for formulation 1:1:18 Ethanol: Kolliphor EL (Cremaphor): saline Vehicle preparation: 5% ethanol, 10% Kolliphor HS (Solutiol) in 85% Saline solution 1 mL of Ethanol, 2 mL of Kolliphor HS (Solutol)-warmed to 60? C., in 17 mL of saline (1:2:17).

Data Recorded and Analysis

[0063]

TABLE-US-00009 TABLE 9 Details of data recorded in visual observations, mini MEST test, mini MEST data analysis and statistical analysis. Visual observations Animals were observed throughout the study from the start of dosing. Any abnormal signs were recorded and reported. Mini MEST test Mini MEST was run between 8 am to 4 pm under normal light conditions. Electroshock was delivered using a Hugo Sachs Electronik stimulator, with an adjustable constant current (1-300 mA). Electroshock duration is 0.1 seconds delivered via corneal electrodes on both eyes. Induction of seizure from the electroshock was measured as an all-or-nothing effect scored as either present (+) or absent (0) of tonic hind limb extensor convulsions for each animal. Up-and-down method based on The current was lowered or raised in log 0.06:10.sup.? Kimball AW et al., 1957 (1 + x*0.06) mA intervals (see raw results in Appendix) if the preceding mouse did or did not show tonic hind limb extension, respectively. If tonic hind limb extension was absent in the animal, the subsequent animal will receive a raised current level. If tonic hind limb extension was present in the animal, the subsequent animal will receive a lowered current level. This procedure was continued for all rats within a treatment group. Mini MEST data analysis The data for each treatment group were recorded as the number of +'s and 0's at each current level employed and this information was then used to calculate the CC.sub.50 value (current required for 50% of the animals to show seizure behaviour) ? standard error of mean (SEM) based on Kimball et al. (1957). Test compound effects were also calculated as percentage change in CC.sub.50 from the vehicle control group. Statistical analysis Significant difference between drug-treated animals and controls were assessed according to Litchfield and Wilcoxon (1949), using Microsoft Excel macro.

Results

[0064] FIG. 2 and Table 10 describe the data produced in this experiment, and raw results are shown in the Appendix.

[0065] In the vehicle group, the CC.sub.50 value was calculated to be 22.5 mA.

[0066] In the diazepam (2.5 mg/kg) treated group, administered i.p. 30 minutes before the test, the CC.sub.50 value was 89.0 mA. This result was statistically significant (p<0.001) compared to the vehicle control.

[0067] In the test compound treatment groups, administered i.p. 15 and 30 minutes before the test, the compound at both doses produced statistically significant CC.sub.50 values compared to vehicle.

[0068] Such data are indicative that this compound will be of therapeutic benefit.

TABLE-US-00010 TABLE 10 mini-MEST results table and statistical analysis. Test time % change Dose post CC.sub.50 ? from Signi- Treatment (mg/kg) dose (min) N SEM vehicle ficance Vehicle 0 30 6 22.5 +/? 0.9 Diazepam 2.5 30 6 89.0 +/? 3.4 296% P < 0.001 Compound 5 15 6 28.3 +/? 1.1 26% P < 0.001 2 Compound 50 30 6 70.5 +/? 5.8 213% P <0 .001 2

Conclusion

[0069] The positive control, diazepam (2.5 mg/kg) administered at 30 min post-dose (i.p.) produced a significant increase in seizure threshold. This result clearly demonstrates the robustness of the presently claimed method and validates the method used.

[0070] Compound 2 tested at 5 & 50 mg/kg administered 15 and 30 mins respectively before testing (i.p.) produced a significant increase in seizure threshold as compared to vehicle, which suggests this compound exhibits anticonvulsive properties.

[0071] The data generated provides clear evidence of a dose-related increase in mini-MEST, further confirming the consistency of the method used.

[0072] The following example demonstrates the anti-convulsant activity for the CBD analogue, Compound 3 as shown as Formula III in the mini-MEST model. Additionally, data is provided from the same compound in the standard MEST model in example 4. Such data demonstrate the efficacy of the mini-MEST model in predicting the anti-convulsant effects of a test compound.

Example 3: Evaluation of Cannabinoid Derivative for Anticonvulsant Activity Using the Maximal Electroshock Seizure Threshold (MEST) Test in the Mouse Using Minimal Sample Sizes (Mini MEST)

[0073] The Example below was carried out similarly to Examples 1 and 2 outlined above using Compound 3 as according to Formula III.

Methods

Study Details

[0074] Na?ve mice were acclimatised to the procedure room in their home cages up to 7 days following arrival to the test facility, with food and water available ad libitum (see Table 11 for details).

[0075] All animals were weighed at the beginning of the study and assigned to treatment groups (n=6/group) based on a mean distribution of body weight across groups. All animals were dosed at 10 mL/kg via intraperitoneal (i.p.) injection, with either vehicle, test compound (200 mg/kg) or diazepam (2.5 mg/kg) (Tables 12 and 13 for details).

[0076] Animals were individually assessed for the production of a tonic hind limb extensor convulsion at 120 minutes post-dose for vehicle, 120 minutes for test compound and 30 minutes for diazepam, from a single electroshock (see Table 14 for details). The first animal within a treatment group was given a shock at the expected or estimated CC.sub.50 current. For subsequent animals, the current was lowered or raised depending on the convulsion outcome from the preceding animal in log scale intervals. Data generated from each treatment group were used to calculate the CC.sub.50?SEM values for the treatment group (see Table 15 for details).

Euthanasia and Sample Collection

[0077] Each animal was humanely killed immediately after production of a convulsion by destruction of the brain from striking the cranium, followed by confirmation of permanent cessation of the circulation from decapitation under The Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986. Terminal blood and brain collection were performed following decapitation.

[0078] Blood was collected in Lithium-heparin tubes and centrifuged at 4? C. for 10 min, at 1500?g. The resulting plasma was removed (>100 ?L) and split into 2 aliquots stored in 0.5 mL Eppendorf tubes, containing 100 ?L of ascorbic acid (100 mg/mL) for stabilisation. Brains were removed, washed in saline and halved. Each half was placed into separate 2 mL screw cap cryovials, weighed and frozen on cardice. Samples were stored at ?80? C. until shipment.

Animal Details

[0079]

TABLE-US-00011 TABLE 11 Details of animal species, strain, sex, order details and environmental conditions Species Mouse Strain C57BL/6J Sex Male No. of animals/group n = 6/group Weight range at study start 19.2-24.7 g Estimated age range at study start 8-9 weeks Environmental conditions Housed in groups of 4-5, with standard conditions. Lighting conditions 12 h/12h light cycle; 7 am lights on, 7pm lights off; light intensity: 25-75 lux at bench level Food and water Food: Certified Rodent CR 14% Protein Rodent Diet, LabDiet? 5CR4 Water: pathogen-free water from test facility

Compounds Details

[0080]

TABLE-US-00012 TABLE 12 Details of Compound 3, batch, appearance, supplier, vehicle used for formulation Storage conditions Room temperature Appearance White powder Vehicle used for formulation 1:2:17 Ethanol: Kolliphor HS (Solutol): saline

TABLE-US-00013 TABLE 13 Details of Diazepam, batch, appearance, supplier, vehicle used for formulation Appearance White powder Vehicle used for formulation 1:1:18 Ethanol: Kolliphor EL (Cremaphor): saline

Vehicle Preparation:

[0081] 5% ethanol, 10% Kolliphor HS (Solutiol) in 85% Saline solution

[0082] 1 mL of Ethanol, 2 mL of Kolliphor HS (Solutol)warmed to 60? C., in 17 mL of saline (1:2:17).

Data Recorded and Analysis

[0083]

TABLE-US-00014 TABLE 14 Details of data recorded in visual observations, mini MEST test, mini MEST data analysis and statistical analysis. Visual observations Animals were observed throughout the study from the start of dosing. Any abnormal signs were recorded and reported. Mini MEST test Mini MEST was run between 8 am to 4 pm under normal light conditions. Electroshock was delivered using a Hugo Sachs Electronik stimulator, with an adjustable constant current (1-300 mA). Electroshock duration is 0.1 seconds delivered via corneal electrodes on both eyes. Induction of seizure from the electroshock was measured as an all-or-nothing effect scored as either present (+) or absent (0) of tonic hind limb extensor convulsions for each animal. Up-and-down method based on The current was lowered or raised in log 0.06:10.sup.? Kimball AW et al., 1957 (1 + x*0.06) mA intervals (see raw results in Appendix) if the preceding mouse did or did not show tonic hind limb extension, respectively. If tonic hind limb extension was absent in the animal, the subsequent animal will receive a raised current level. If tonic hind limb extension was present in the animal, the subsequent animal will receive a lowered current level. This procedure was continued for all rats within a treatment group. Mini MEST data analysis The data for each treatment group were recorded as the number of +'s and 0's at each current level employed and this information was then used to calculate the CC.sub.50 value (current required for 50% of the animals to show seizure behaviour) ? standard error of mean (SEM) based on Kimball et al. (1957). Test compound effects were also calculated as percentage change in CC.sub.50 from the vehicle control group. Statistical analysis Significant difference between drug-treated animals and controls were assessed according to Litchfield and Wilcoxon (1949), using Microsoft Excel macro.

Results

[0084] FIG. 3 and Table 15 describe the data produced in this experiment, and raw results are shown in the Appendix.

[0085] In the vehicle group, the CC.sub.50 value was calculated to be 23.5 mA.

[0086] In the diazepam (2.5 mg/kg) treated group, administered i.p. 30 minutes before the test, the CC.sub.50 value was 46.5 mA. This result was statistically significant (p<0.001) compared to the vehicle control.

[0087] In the test compound treatment group, administered i.p. 120 minutes before the test, the compound tested at 200 mg/kg produced a CC.sub.50>173 mA; an exact value was not calculated as a + was not seen within the 6 animals tested. Although CC.sub.50 was not determined and statistical significance was not achieved, the drug showed a clear increase in seizure threshold in the mini-MEST as compared to vehicle.

[0088] Such data are indicative that this compound will be of therapeutic benefit.

TABLE-US-00015 TABLE 15 mini-MEST results table and statistical analysis. Test time post % change Dose dose CC.sub.50 ? from Signi- Treatment (mg/kg) (min) N SEM vehicle ficance Vehicle 0 120 6 23.5 +/? 0.3 Diazepam 2.5 30 6 46.5 +/? 1.0 98% P < 0.001 Compound 200 120 6 >173 >636% # 3 # Statistical significance not calculated due to CC.sub.50 not reached

Conclusion

[0089] The positive control, diazepam (2.5 mg/kg) administered at 30 min post-dose (i.p.) produced a significant increase in seizure threshold. This result clearly demonstrates the robustness of the presently claimed method and validates the method used.

[0090] Compound 3 tested at 200 mg/kg administered 120 mins before testing (i.p.) showed a clear increase in seizure threshold as compared to vehicle, which suggests this compound exhibits anticonvulsive properties.

[0091] The data generated using the mini-MEST method presents clear evidence of the potential of this compound as an anticonvulsant.

Example 4: Evaluation of Cannabinoid Derivative for Anticonvulsant Activity Using the Maximal Electroshock Seizure Threshold (MEST) Test in the Mouse

[0092] The efficacy of Compound 3 was tested in a mouse model of generalised seizure, the maximal electroshock seizure threshold (MEST) test.

Methods

Study Details

[0093] Na?ve mice were acclimatised to the procedure room in their home cages for up to 7 days, with food and water available ad libitum.

[0094] All animals were weighed at the beginning of the study and randomly assigned to treatment groups (n=12/group) based on a mean distribution of body weight across groups. All animals were dosed at 10 ml/kg via intraperitoneal (i.p) injection, with either vehicle, test compound at 2, 20 or 200 mg/kg or diazepam at 2.5 mg/kg.

[0095] Animals were individually assessed for the production of a tonic hind limb extensor convulsion at 30 min post-dose for vehicle, 30 min post-dose for test compound and 30 min post-dose for diazepam, from a single electroshock.

[0096] The first animal within a treatment group was given a shock at the expected or

[0097] estimated CC.sub.50 current. For subsequent animals, the current was lowered or raised depending on the convulsions outcome from the preceding animal in 5 mA intervals.

[0098] Data generated from each treatment group were used to calculate the CC.sub.50?SEM values for the treatment group.

Test Compounds:

[0099] Vehicle: (5% ethanol, 10% solutol, 85% Saline) was prepared as follows: 1 mL of ethanol, 2 mL of solutol were warmed to 60? ? C., in 17 mL of saline (1:2:17).

[0100] Positive control: diazepam was used at 2.5 mg/kg.

[0101] The test compound used was Compound 3. Test compound was administered at 2, 20 and 200 mg/kg (i.p.) in a 1:2:17 ethanol:solutol:0.9% saline formulation.

Sample Collection:

[0102] Each animal was humanely killed immediately after production of a convulsion by destruction of the brain from striking the cranium, followed by the confirmation of permanent cessation of the circulation from decapitation under The Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986. Terminal blood and brain collection were performed following decapitation.

[0103] Blood was collected in Lithium-heparin tubes and centrifuged at 4? C. for 10 minutes at 1500?g. The resulting plasma was removed (>100 ?L) and split into 2 aliquots of 0.5 mL Eppendorf tubes containing 100 ?L of ascorbic acid (100 mg/mL) for stabilisation. Brains were removed, washed in saline and halved. Each half was placed into separate 2 mL screw cap cryovials, weighed and frozen on cardice.

Statistical Analysis

[0104] The data for each treatment group were recorded as the number of +'s and 0's at each current level employed and this information is then used to calculate the CC.sub.50 value (current required for 50% of the animals to show seizure behaviour)?standard error.

[0105] Test compound effects were also calculated as percentage change in CC.sub.50 from the vehicle control group.

[0106] Significant difference between drug-treated animals and controls were assessed according to Litchfield and Wilcoxon (1949).

Results

[0107] FIG. 4 and Table 16 describe the data produced in this experiment, and raw results are shown in the Appendix.

[0108] In the vehicle group, the CC.sub.50 value was calculated to be 24.3 mA.

[0109] In the diazepam (2.5 mg/kg) treated group, administered i.p. 30 minutes before the test, the CC.sub.50 value was 78.5 mA. This result was statistically significant (p<0.001) compared to the vehicle control. One animal in the diazepam group, was not dosed due to welfare issues from fighting.

[0110] In the test compound treatment groups, administered i.p. 30 minutes before the test, the compound produced a statistically significant CC.sub.50 value compared to vehicle at all three doses of the compound.

TABLE-US-00016 TABLE 16 Evaluation of effect of Compound 3 in the MEST test Test time % change Dose post dose CC.sub.50 +/? Signi- from Treatment (mg/kg) (min) N SEM ficance vehicle Vehicle 30 12 24.3 ? 0.4 Diazepam 2.5 30 11 78.5 ? 1.0 P < 0.001 223% Compound 2 30 12 30.8 ? 1.0 P < 0.001 27% 3 Compound 20 30 12 52.5 ? 1.3 P < 0.001 116% 3 Compound 200 30 12 197.5 ? 20.4 P < 0.001 712% 3

Conclusions

[0111] These data demonstrate a therapeutic effect for Compound 3 with a dose-related increase in MEST, which suggests that this compound exhibits anticonvulsive properties.

[0112] Thus, the data produced using the standard MEST model is consistent with the results of the mini-MEST model from Example 3 and reaffirms its conclusion. This consistency proves how the novel method of this application is able to generate robust results in an effective manner to be a useful predictor for the full MEST model.

[0113] Further, it has been shown that through the use of a logarithmic scale to increase or decrease the current, a smaller group of animals could be used in the mini-MEST method, thus achieving the overall aim of lowering number of animals and quantity of test compounds used.

APPENDIX

[0114]

TABLE-US-00017 Raw data for Example 1 Current Animal no. Treatment: Vehicle (mA) 1 2 3 4 5 6 +'s 0's Dose: 22 0 0 0 2 Route: i.p. 25 + + 0 2 1 Predose: 30 mins 29 + 1 0 Current Animal no. Treatment: Diazepam (mA) 7 8 9 10 11 12 +'s 0's Dose: 2.5 mg/kg 66 0 0 0 2 Route: i.p. 75 + 0 0 1 2 Predose: 30 mins 87 + 1 0 Current Animal no. Treatment: Compound 1 (mA) 13 14 15 16 17 18 +'s 0's Dose: 50 mg/kg 87 0 0 1 Route: i.p. 100 0 0 1 Predose: 30 mins 114 0 0 0 2 131 + 0 1 1

TABLE-US-00018 Raw data for Example 2 Treatment: Current Animal no. Vehicle (mA) 1 2 3 4 5 6 +'s 0's Dose: 19 0 0 1 Route: i.p. 22 + 0 0 1 2 Predose: 30 mins 25 + + 2 0 Treatment: Current Animal no. Diazepam (mA) 7 8 9 10 11 12 +'s 0's Dose: 2.5 mg/kg 75 0 0 1 Route: i.p. 87 + 0 0 1 2 Predose: 30 mins 100 + + 2 0 Treatment: Current Animal no. Compound 2 (mA) 13 14 15 16 17 18 +'s 0's Dose: 5 mg/kg 25 0 0 0 2 Route: i.p. 29 + + 0 2 1 Predose: 15 mins 33 + 1 0 Treatment: Current Animal no. Compound 2 (mA) 19 20 21 22 23 24 +'s 0's Dose: 50 mg/kg 57 0 0 1 Route: i.p. 66 0 + 1 1 Predose: 30 mins 75 0 + 1 1 87 + 1 0

TABLE-US-00019 Raw data for Example 3 Current Animal no. Treatment: Vehicle (mA) 1 2 3 4 5 6 +'s 0's Dose: 22 0 0 0 0 3 Route: i.p. 25 + + + 3 0 Predose: 120 mins Current Animal no. Treatment: Diazepam (mA) 7 8 9 10 11 12 +'s 0's Dose: 2.5 mg/kg 43 0 0 1 Route: i.p. 50 + 0 1 1 Predose: 30 mins 57 0 0 1 66 0 0 1 75 0 0 1 Current Animal no. +'s 0's Treatment: Compound 3 (mA) 13 14 15 16 17 18 Dose: 200 mg/kg 87 0 0 1 Route: i.p. 100 0 0 1 Predose: 120 mins 114 0 0 1 131 0 0 1 151 0 0 1 173 0 0 1

TABLE-US-00020 Raw data for Example 4 Current Animal no. Treatment: Vehicle (mA) 1 2 3 4 5 6 7 8 9 10 11 12 +'s 0's Dose: 22 0 0 0 2 Route: i.p. 24 + + 0 0 0 0 2 4 Predose: 30 mins 26 + + + + 4 0 Current Animal no. Treatment: Diazepam (mA) 13 14 15 16 17 18 19 20 21 22 23 24 +'s 0's Dose: 2.5 mg/kg 75 0 0 0 0 0 0 5 Route: i.p. 80 0 + + + + 4 1 Predose: 30 mins 85 + 1 0 Current Animal no. Treatment: Compound 3 (mA) 25 26 27 28 29 30 31 32 33 34 35 36 +'s 0's Dose: 2 mg/kg 25 0 0 0 2 Route: i.p. 30 0 + 0 + 0 0 2 4 Predose: 30 mins 35 + + + + 4 0 Current Animal no. Treatment: Compound 3 (mA) 37 38 39 40 41 42 43 44 45 46 47 48 +'s 0's Dose: 20 mg/kg 45 0 0 1 Route: i.p. 50 0 0 0 + 0 1 4 Predose: 30 mins 55 + + + + 0 4 1 60 + 1 0 Current Animal no. Treatment: Compound 3 (mA) 49 50 51 52 53 54 55 56 57 58 59 60 +'s 0's Dose: 200 mg/kg 180 0 0 0 2 Route: i.p. 185 + 0 1 1 Predose: 30 mins 190 0 0 1 195 0 0 1 200 0 0 0 2 205 + 0 0 1 2 210 + 1 0