WO2008119710A1

WO2008119710A1 - Two-phase test method for characterizing the effect of a chemical compound on a test animal and maze for carrying out the method

Info

Publication number: WO2008119710A1
Application number: PCT/EP2008/053588
Authority: WO
Inventors: John Paul Redrobe; Dan Peters; Gunnar M. Olsen
Original assignee: Neurosearch AS
Current assignee: NTG Nordic Transport Group AS
Priority date: 2007-03-30
Filing date: 2008-03-27
Publication date: 2008-10-09
Anticipated expiration: 2009-09-30

Abstract

The present invention relates to a two-phase method for characterizing the effect of a chemical compound on a test animal. The invention also relates to a maze for carrying out the test method and to a drug development method.

Description

TWO-PHASE TEST METHOD FOR CHARACTERIZING THE EFFECT

OF A CHEMICAL COMPOUND ON A TEST ANIMAL AND

MAZE FOR CARRYING OUT THE METHOD

TECHNICAL FIELD

BACKGROUND ART

An important stage in the development of a new drug is the animal trial stage. In this stage, the drug is administered to one or more test animals, such as laboratory mice, in order to determine possible drug benefits and side effects. Information concerning the behaviour of an animal in response to a drug, including cognitive function, can provide valuable information concerning the efficacy or safety of the drug.

One device used in the art to obtain behavioural information of a test animal subject during a trial is a maze. A test animal is placed in a physical construction of obstacles and passageways. By evaluating the ability of the test animal to correctly navigate the maze, such as the time it takes for the animal to traverse the maze, investigators can gain insight into the effect of a drug on the behaviour of the animal.

One drawback of many existing methods is that they are cumbersome and time consuming. Another is that many require extensive pre-training sessions and involve the use of reinforcers and aversive stimuli that may interfere with the interpretation of drug effects.

Thus there is a great need for animal test methods that are simpler and yet as reliable when compared to the existing methods.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a two-phase test method for characterizing the effect of a chemical compound on a test animal . In a second aspect, the invention provides a maze for performing the test method.

In a third aspect, the invention provides a drug development method.

Details and other objects of the invention will be apparent to the person skilled in the art from the following detailed description and examples. DETAILED DISCLOSURE OF THE INVENTION

The two-phase test method In a first aspect, the invention provides a two-phase test method for characterizing the effect of a chemical compound on a test animal, which method comprises the following steps:

1 ) a first phase in which the test animal is placed in a first constrained environment B for a first period of time; and 2) a second phase in which the test animal is placed in a second constrained environment consisting of the first familiar environment B and a novel environment C, the two environments being connected by a constrained passage D; and

3) calculation of the effect of the chemical compound based on the observance of the animal behaviour during the second phase.

In one embodiment of the test method, prior to step (1 ), the method comprises step (1 ') including the sub-steps:

1 'a) the test animal is placed in a constrained runway A connected to the constrained environment B by the constrained passage D; 1 'b) whereafter the test animal is left at its own will to move to the constrained environment B; 1 'c) whereafter the access between the constrained environment B and the constrained passage D is blocked by a partition.

In a second embodiment, prior to step (2), the method comprises step (2') including the sub-steps:

2'a) the test animal is placed in a constrained runway A connected to the constrained environments B and C by the constrained passage D; 2'b) whereafter the test animal is left at its own will to move away from the constrained runway A; 2'c) whereafter the access between the constrained runway A and the constrained passage D is blocked by a partition.

In a further embodiment, the novel environment C - in size and geometric form - is a mirror image of the familiar environment B.

In a still further embodiment, the visible areas as seen from environment B are optically different from the visible areas as seen from environment C.

In a further embodiment, at least parts of the barriers constraining environments B and C are transparent. In a still further embodiment, the partition - if present - between runway A and passage D, environment B and passage D or environment C and passage D is opaque.

In a further embodiment the test animal is rodent, such as a mouse. In a still further embodiment, the chemical compound is administered to the test animal prior to step 1.

In a further embodiment, the chemical compound is administered to the test animal prior to step 2.

In a special embodiment, the route of administration of the compound is intraperitoneal (i.p.), intraveneous (i.v.), peroral (p.o.) or subcutaneous (s.α).

In a still further embodiment, observance of the animal behaviour during the second phase is measured by recording the following parametres:

• the cumulative time spent in environment B, environment C novel and passage D; • the number of entries into environment B, environment C and passage D;

• the total distanced travelled.

In a special embodiment, the parameters are recorded by an automated video tracking system.

In a still further embodiment, the effect of the compound to be tested is the effect on cognition. In a special embodiment, the effect on cognition is calculated based on the time spent in environment B as opposed to the time spent in environment C during the second phase.

The maze In a second aspect, the invention provides a maze for performing the above test method characterized by having two constrained environments B and C connected by a constrained passage D.

In one embodiment of the maze, the maze has a further constrained runway A connected to the constrained passage D. In a second embodiment, access between passage D and each of runway A, environment B and environment C may be blocked by a partition.

In a further embodiment, environment C - in size and geometric form - is a mirror image of the familiar environment B.

In a further embodiment, the maze is equipped with an automated video tracking system for measuring of the animal behaviour during the second phase.

The drug development method

In a third aspect, the invention provides a drug development method, which comprises the identification of a compound as a potential development compound by the above method.

BRIEF DESCRIPTION OF THE DRAWING

The present invention is further illustrated by reference to the accompanying drawing, in which:

Figs. 1a-1d show the various compartments and steps of the test method: Fig. 1a shows the various compartments: Runway A, environments B and C and passage D; Fig. 1 b shows step (1 '); Fig. 1c shows step (1 ); and Fig. 1d shows step (2). In the following drawings environment B is labelled "familiar", environment C is labelled "novel" and passage D is labelled "conflict".

Figs. 2a-2c show the results when mice were treated with scopolamine. Figs. 3a-3c show the results when mice were treated with chlordiazepoxide. Figs. 4a-4c show the results when mice were treated with caffeine. Figs. 5a-5c show the results when mice were treated with Zolpidem.

Figs. 6a-6c show the results when mice were treated with SSR-180711 and scopolamine.

Figs. 7a-7c show the results when mice were treated with PNU-282987 and scopolamine. Figs. 2a, 3a, 4a, 5a, 6a and 7a show time in the zones; Fig. 2b, 3b, 4b, 5b, 6b and 7b show number of entries in the zones; and Fig. 2c, 3c, 4c, 5c, 6c and 7c show the total distance run.

The following example will illustrate the invention further; however, it is not to be construed as limiting. EXAMPLES

Animals:

Male C57BL/6J mice (Harlan, Netherlands), 22-25 g, with free access to food and water before and after testing are used. The mice are allowed to habituate to the laboratory in a pre-designated Scantainer for 1 week.

Test Procedure:

Test compounds are administered p.o., i.p., or s.c, at a determined pre-treatment time. Testing is carried out in a clear plexiglass maze composed of 2 perpendicular arms connected to a vertical runway. The 2 arms and runway are 50 cm long and 8 cm wide, surrounded by clear plexiglass walls 30 cm high. Two perpendicular arms are available for exploration, hence the name V-maze. Each arm meets at a central platform equipped with 3 blackened doors enabling arms to be opened and closed as desired. The whole maze is enclosed in a triangular black plexiglass box (1 m x 1 m x 1 m). The walls of the outer box surrounding each exploration arm are covered with distinct optical cues, e.g., horizontal, vertical, diagonal lines. The area surrounding the runway does not contain optical cues and is black in colour. In phase 1 , each mouse is placed at the end of the runway arm and is allowed access to one of the perpendicular arms by forced choice (by occluding one arm with an opaque partition). The forced choice arm is alternated for each mouse to avoid any place preference. The mouse is then confined in this arm and is allowed to explore for a period of 5 min. In phase 2, the door is opened enabling the mouse to explore the familiar perpendicular arm or the unfamiliar perpendicular arm, but not the runway arm for a period of 2 min. The cumulative time spent in each arm, the number of entries and the total distance travelled are recorded during this test session by an automated video tracking system. Data are compared using Students t-test (time in each arm) or one-way ANOVA (entries, distance travelled). A skilled observer, blinded to the treatment groups, may also perform scoring. In this case, an individual entry into an arm is defined as the animal placing all four paws in that arm.

Experiment 1

In a first set of experiments, mice were treated with: vehicle, scopolamine (0.25-1 mg/kg), chlordiazepoxide (10 & 20 mg/kg), caffeine (5 & 10 mg/kg) or Zolpidem (1 & 3 mg/kg), 30 min before phase 1 testing. Results:

Vehicle-treated mice displayed a clear and robust preference to explore the unfamiliar arm, without changes in the number of entries to either arm. This preference was abolished when mice were treated with scopolamine (1 mg/kg, Figure 2) or chlordiazepoxide (20 mg/kg, Figure 3), without changes in number of entries or distance travelled. Caffeine (10 mg/kg, Figure 4) increased the number of arm entries and total distance travelled, but did not affect preference for novel arm exploration. Zolpidem (1 & 3 mg/kg, Figure 5) decreased entries and distance travelled, but only attenuated novel arm exploration at the higher dose. Based on these data, scopolamine was chosen for use in combination studies with two alpha7 nicotinic receptor agonists.

Experiment 2

In a second set of experiments, mice were pretreated with: SSR-180711 (0.3-3 mg/kg) or PNU-282987 (1 -10 mg/kg), 15 min before scopolamine (1 mg/kg), administered 30 min prior to phase 1 testing.

Results:

Results from these experiments showed that both alpha7 agonists restored preference for novel arm exploration, when administered prior to scopolamine: SSR-180711 (MED: 3 mg/kg) and PNU-282987 (MED: 3 mg/kg), at doses largely corresponding to their respective ex-vivo [3H]-bungarotoxin ED₅₀ values.

Claims

CLAIMS:

1. A two-phase test method for characterizing the effect of a chemical compound on a test animal, which method comprises the following steps: 1 ) a first phase in which the test animal is placed in a first constrained environment B for a first period of time; and

2) a second phase in which the test animal is placed in a second constrained environment consisting of the first familiar environment B and a novel environment C, the two environments being connected by a constrained passage D; and

2. The method according to claim 1 , wherein prior to step (1 ), the method comprises step (1 ') including the sub-steps:

1 'a) the test animal is placed in a constrained runway A connected to the constrained environment B by the constrained passage D;

1 'b) whereafter the test animal is left at its own will to move to the constrained environment B; 1 'c) whereafter the access between the constrained environment B and the constrained passage D is blocked by a partition.

3. The method according to claims 1 or 2, wherein prior to step (2), the method comprises step (2') including the sub-steps: 2'a) the test animal is placed in a constrained runway A connected to the constrained environments B and C by the constrained passage D; 2'b) whereafter the test animal is left at its own will to move away from the constrained runway A;

2'c) whereafter the access between the constrained runway A and the constrained passage D is blocked by a partition.

4. The method according to any one of claims 1 -3, wherein the novel environment C - in size and geometric form - is a mirror image of the familiar environment B.

5. The method according to any one of claims 1 -4, wherein the visible areas as seen from environment B are optically different from the visible areas as seen from environment C.

6. The method according to any one of claims 1 -5, wherein at least parts of the barriers constraining environments B and C are transparent.

7. The method according to any one of claims 1 -6, wherein the partition - if present 5 - between runway A and passage D, environment B and passage D or environment C and passage D is opaque.

8. The method according to any one of claims 1 -7, wherein the test animal is rodent, such as a mouse.

10

9. The method according to any one of claims 1 -8, wherein the chemical compound is administered to the test animal prior to step 1.

10. The method according to any one of claims 1 -8, wherein the chemical 15 compound is administered to the test animal prior to step 2.

11. The method according to any one of claims 1 -10, wherein observance of the animal behaviour during the second phase is measured by recording the following para metres:

20 • the cumulative time spent in environment B, environment C novel and passage D;

• the number of entries into environment B, environment C and passage D;

• the total distanced travelled.

25 12. The method according to claim 11 , wherein the parameters are recorded by an automated video tracking system.

13. The method according to any one of claims 1 -12, wherein the effect of the compound to be tested is the effect on cognition.

30

14. The method according to claim 13, wherein the effect on cognition is calculated based on the time spent in environment B as opposed to the time spent in environment C during the second phase.

35 15. A maze for performing the test method according to any one of claims 1 -14 characterized by having to constrained environments B and C connected by a constrained passage D.

16. The maze according to claim 15 having a further constrained runway A connected to the constrained passage D.

17. The maze according to claims 15 or 16 wherein access between passage D and 5 each of runway A, environment B and environment C may be blocked by a partition.

18. The maze according to any one of claims 15-17 wherein environment C - in size and geometric form - is a mirror image of the familiar environment B.

10 19. The maze according to any one of claims 15-18 wherein the visible areas as seen from environment B are optically different from the visible areas as seen from environment C.

20. The maze according to any one of claims 15-19 wherein at least parts of the 15 barriers constraining environment B and C are transparent.

21. The maze according to any one of claims 15-20 wherein the partition - if present - between runway A and passage D, environment B and passage D or environment C and passage D is opaque.

20

22. The maze according to any one of claims 15-21 equipped with an automated video tracking system for measuring of the animal behaviour during the second phase.

23. A drug development method, which comprises the identification of a compound 25 as a potential development compound by the method according to any one of the claims 1 -14.