U.S. patent application number 15/432198 was filed with the patent office on 2017-06-01 for treatment of schizophrenia using beta-caryophyllene and cb2 receptor agonists.
This patent application is currently assigned to ARIEL SCIENTIFIC INNOVATIONS LTD. The applicant listed for this patent is ARIEL SCIENTIFIC INNOVATIONS LTD. Invention is credited to Sharon Anavi-Goffer, Juerg Gertsch.
Application Number | 20170151182 15/432198 |
Document ID | / |
Family ID | 49221922 |
Filed Date | 2017-06-01 |
United States Patent
Application |
20170151182 |
Kind Code |
A1 |
Anavi-Goffer; Sharon ; et
al. |
June 1, 2017 |
TREATMENT OF SCHIZOPHRENIA USING BETA-CARYOPHYLLENE AND CB2
RECEPTOR AGONISTS
Abstract
Disclosed are compositions comprising beta-caryophyllene (BCP)
for use in the treatment of schizophrenia, methods of making such
compositions and methods of treating schizophrenia using BCP.
Disclosed are also compositions comprising CB2 receptor agonists
for use in the treatment of schizophrenia, methods of making such
compositions and methods of treating schizophrenia using CB2
receptor agonists.
Inventors: |
Anavi-Goffer; Sharon;
(Ashdod, IL) ; Gertsch; Juerg; (Schaffhausen,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARIEL SCIENTIFIC INNOVATIONS LTD |
Ariel |
|
IL |
|
|
Assignee: |
ARIEL SCIENTIFIC INNOVATIONS
LTD
ARIEL
IL
|
Family ID: |
49221922 |
Appl. No.: |
15/432198 |
Filed: |
February 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14385739 |
Sep 16, 2014 |
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PCT/IB2013/052182 |
Mar 19, 2013 |
|
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15432198 |
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61612411 |
Mar 19, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/5415 20130101;
A61K 31/4515 20130101; A61K 47/20 20130101; A61K 31/5513 20130101;
A61K 9/0019 20130101; A61K 31/519 20130101; A61K 9/48 20130101;
A61P 43/00 20180101; A61K 31/015 20130101; A61P 25/18 20180101;
A61K 9/0053 20130101; A61K 45/06 20130101; A61K 31/015 20130101;
A61K 2300/00 20130101; A61K 31/5513 20130101; A61K 2300/00
20130101; A61K 31/5415 20130101; A61K 2300/00 20130101; A61K
31/4515 20130101; A61K 2300/00 20130101; A61K 31/519 20130101; A61K
2300/00 20130101 |
International
Class: |
A61K 31/015 20060101
A61K031/015; A61K 45/06 20060101 A61K045/06; A61K 47/20 20060101
A61K047/20; A61K 9/00 20060101 A61K009/00 |
Claims
1. A composition comprising beta-caryophyllene (BCP) and a
pharmaceutically effective carrier for use in treating
schizophrenia.
2. The composition of claim 1, wherein said schizophrenia is
selected from the group consisting of paranoid schizophrenia;
disorganized schizophrenia; undifferentiated schizophrenia;
catatonic schizophrenia; and residual schizophrenia.
3. The composition of any of claims 1 to 2, wherein said treating
comprises treating at least one symptom of schizophrenia selected
from the group consisting of a negative symptom of schizophrenia
and a positive symptom of schizophrenia.
4. The composition of any of claims 1 to 3, wherein said
pharmaceutically effective carrier comprises dimethyl sulfoxide
(DMSO).
5. The composition of any of claims 1 to 4, a single discrete unit
of the composition comprises BCP at a weight in the range of from
about 25 to about 100 mg.
6. The composition of any of claims 1 to 5, formulated as an
injectable solution dosage form.
7. The composition of any one of claims 1 to 5, wherein the
composition is formulated as an orally-administrable dosage
form.
8. The composition of any of claims 1 to 7, further comprising at
least one additional antipsychotic agent.
9. The composition of claim 8, wherein said at least one additional
antipsychotic agent is selected from the group consisting of
chlorpromazine, haloperidol, perphenazine, fluphenazine, clozapine,
risperidone, olanzapine, quetiapine, ziprasidone, aripiprazole, and
paliperidone, or combinations thereof.
10. Use of beta-caryophyllene (BCP) and a pharmaceutically
effective carrier in the manufacture of a composition for treating
schizophrenia in a subject in need thereof.
11. The use according to claim 10, wherein said schizophrenia is
selected from the group consisting of paranoid schizophrenia;
disorganized schizophrenia; undifferentiated schizophrenia;
catatonic schizophrenia; and residual schizophrenia.
12. The use according to any of claims 10 to 11, wherein said
treating comprises treating at least one symptom of schizophrenia
selected from the group consisting of a negative symptom of
schizophrenia and a positive symptom of schizophrenia.
13. The use of any of claims 10 to 12, wherein said
pharmaceutically effective carrier comprises dimethyl sulfoxide
(DMSO).
14. The use according to any of claims 10 to 13, wherein a single
discrete unit of said composition comprises BCP at a weight in the
range of from about 25 to about 100 mg.
15. The use according to any of claims 10 to 14, wherein said
composition is made as an injectable solution dosage form.
16. The use according to any of claims 10 to 14, wherein said
composition is made as an orally administrable dosage form.
17. The use according to any of claims 10 to 16, wherein said
composition further comprises at least one additional antipsychotic
agent.
18. The use according to claim 17, wherein said at least one
additional antipsychotic agent is selected from the group
consisting of chlorpromazine, haloperidol, perphenazine,
fluphenazine, clozapine, risperidone, olanzapine, quetiapine,
ziprasidone, aripiprazole, and paliperidone, or combinations
thereof.
19. A method for treating schizophrenia in a subject in need
thereof, the method comprising administering a therapeutic
composition comprising beta-caryophyllene (BCP) and a
pharmaceutically effective carrier.
20. The method of claim 19, wherein said schizophrenia is selected
from the group consisting of paranoid schizophrenia; disorganized
schizophrenia; undifferentiated schizophrenia; catatonic
schizophrenia; and residual schizophrenia.
21. The method of any of claims 19 to 20, wherein said treating
comprises treating at least one symptom of schizophrenia selected
from the group consisting of a negative symptom of schizophrenia
and a positive symptom of schizophrenia.
22. The method of any of claims 19 to 21, wherein an average daily
amount of said BCP administered is from about 0.4 mg/kg to about 2
mg/kg.
23. The method of any of claims 19 to 22, wherein said
pharmaceutically effective carrier comprises dimethyl sulfoxide
(DMSO).
24. The method of any of claims 19 to 23, wherein said
administering comprises injecting said composition to said
subject.
25. The method of any of claims 19 to 23, wherein said
administering comprises orally administering said composition to
said subject.
26. The method of any of claims 19 to 25, further comprising
co-administering at least one additional antipsychotic agent.
27. The method of claim 26, wherein said at least one additional
antipsychotic agent is selected from the group consisting of
chlorpromazine, haloperidol, perphenazine, fluphenazine, clozapine,
risperidone, olanzapine, quetiapine, ziprasidone, aripiprazole, and
paliperidone, or combinations thereof.
28. The method of any of claims 26 to 27, wherein said at least one
additional antipsychotic agent is co-administered in a single
dosage form together with said BCP.
29. The method of any of claims 26 to 27, wherein said at least one
additional antipsychotic agent is co-administered in a dosage form
separate from said BCP.
30. A composition comprising a CB2 receptor agonist and a
pharmaceutically effective carrier for use in treating
schizophrenia.
31. Use of a composition comprising a CB2 receptor agonist and a
pharmaceutically effective carrier in the manufacture of a
composition for treating schizophrenia in a subject in need
thereof.
32. A method for the treating schizophrenia in a subject in need
thereof, the method comprising administering a therapeutic
composition comprising a CB2 receptor agonist and a
pharmaceutically effective carrier.
Description
RELATED APPLICATION
[0001] The present application gains priority from U.S. Provisional
Patent Application No. 61/612,411 filed 19 Mar. 2012, which is
included by reference as if fully set-forth herein.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The invention, in some embodiments, relates to the field of
therapy and more particularly, but not exclusively, to compositions
comprising beta-caryophyllene (BCP), methods of making the
compositions and methods using BCP, for the treatment of
schizophrenia. The invention, in some embodiments, relates to the
field of therapy and more particularly, but not exclusively, to
compositions comprising Cannabinoid Receptor Type 2 (CB2) receptor
agonists, methods of making the compositions and methods using CB2
receptor agonists for the treatment of schizophrenia.
[0003] Schizophrenia affects about 1% of the population (Lewis
& Lieberman, 2000), and genetic and environmental factors
underlie the eventual eruption of the disease (Ross, 2006).
Schizophrenia is often chronic, characterized by deterioration of
social contact, cognitive deficits, anxiety and depression,
resulting in suicide in about 10% of the schizophrenic population
(Lewis & Lieberman, 2000).
[0004] Different subtypes of schizophrenia are defined according to
the most significant and predominant characteristics present, as
follows: Paranoid schizophrenia; Disorganized schizophrenia;
Undifferentiated schizophrenia; Catatonic schizophrenia; and
Residual schizophrenia.
SUMMARY OF THE INVENTION
[0005] Some embodiments of the invention relate to compositions
comprising beta-caryophyllene (BCP), methods of making the
compositions and methods using BCP for the treatment of
schizophrenia. Some embodiments of the invention relate to
compositions comprising Cannabinoid Receptor Type 2 (CB2) receptor
agonists, methods of making the compositions and methods using CB2
receptor agonists for the treatment of schizophrenia.
[0006] According to an aspect of some embodiments of the invention,
there is provided a therapeutic composition comprising
beta-caryophyllene (BCP) and a pharmaceutically effective carrier
for use in treating schizophrenia. In some embodiments, the
composition is for use in the treatment of a human subject. In some
embodiments, the composition is for use in the treatment of a
non-human subject.
[0007] In some embodiments, the schizophrenia is selected from the
group consisting of paranoid schizophrenia; disorganized
schizophrenia; undifferentiated schizophrenia; catatonic
schizophrenia; and residual schizophrenia.
[0008] In some embodiments, the treating comprises treating at
least one symptom of schizophrenia selected from the group
consisting of a negative symptom of schizophrenia and a positive
symptom of schizophrenia.
[0009] In some embodiments, the pharmaceutically effective carrier
comprises dimethyl sulfoxide (DMSO). In some such embodiments, the
pharmaceutically effective carrier comprises DMSO, saline and
Cremophor EL. In some such embodiments, the pharmaceutically
effective carrier comprises DMSO, saline and Cremophor EL at a
ratio of 1:0.6:18 Cremophor EL:DMSO:saline.
[0010] In some embodiments, a single discrete unit (e.g., a single
tablet, capsule, metered liquid) of the composition comprises BCP
at a weight in the range of from about 25 to about 100 mg.
[0011] In some embodiments, the composition is formulated as an
injectable solution dosage form. In some embodiments, the
injectable solution is configured to be administered by a route
selected from the group consisting of intravenous injection,
intramuscular injection, intradermal injection, intraperitoneal
injection, intrathecal injection and subcutaneous injection.
[0012] In some embodiments, the composition is formulated as an
orally-administrable dosage form. In some such embodiments, the
composition is formulated in a dosage form selected from the group
consisting of a tablet, a capsule, a dragee, a powder, granules,
and an ingestible solution, especially a tablet or capsule.
[0013] In some embodiments, the composition further comprises at
least one additional antipsychotic agent. In some such embodiments,
at least one additional antipsychotic agent is selected from the
group consisting of chlorpromazine, haloperidol, perphenazine,
fluphenazine, clozapine, risperidone, olanzapine, quetiapine,
ziprasidone, aripiprazole, and paliperidone, or combinations
thereof.
[0014] According to an aspect of some embodiments of the invention,
there is also provided the use of beta-caryophyllene (BCP) and a
pharmaceutically effective carrier in the manufacture of a
composition (also known as a medicament) for treating schizophrenia
in a subject in need thereof. In some embodiments, the composition
is configured for use in the treatment of a human subject. In some
embodiments, the composition is configured for use in the treatment
of a non-human subject.
[0015] In some embodiments, the schizophrenia is selected from the
group consisting of paranoid schizophrenia; disorganized
schizophrenia; undifferentiated schizophrenia; catatonic
schizophrenia; and residual schizophrenia.
[0016] In some embodiments, the treating comprises treating at
least one symptom of schizophrenia selected from the group
consisting of a negative symptom of schizophrenia and a positive
symptom of schizophrenia.
[0017] In some embodiments, the pharmaceutically effective carrier
comprises dimethyl sulfoxide (DMSO). In some such embodiments, the
pharmaceutically effective carrier comprises DMSO, saline and
Cremophor EL. In some such embodiments, the pharmaceutically
effective carrier comprises DMSO, saline and Cremophor EL at a
ratio of 1:0.6:18 Cremophor EL:DMSO:saline.
[0018] In some embodiments, a single discrete unit (e.g., a single
tablet, capsule, metered liquid) of the composition that is
manufactured comprises BCP at a weight in the range of from about
25 to about 100 mg.
[0019] In some embodiments, the composition is made as an
injectable solution dosage form. In some embodiments, the
injectable solution is configured to be administered by a route
selected from the group consisting of intravenous injection,
intramuscular injection, intradermal injection, intraperitoneal
injection, intrathecal injection and subcutaneous injection.
[0020] In some embodiments, the composition is made as an
orally-administrable dosage form. In some such embodiments, the
dosage form selected from the group consisting of a tablet, a
capsule, a dragee, a powder, granules, and an ingestible solution,
especially a tablet or capsule.
[0021] In some embodiments, the composition further comprises at
least one additional antipsychotic agent. In some such embodiments,
at least one additional antipsychotic agent is selected from the
group consisting of chlorpromazine, haloperidol, perphenazine,
fluphenazine, clozapine, risperidone, olanzapine, quetiapine,
ziprasidone, aripiprazole, and paliperidone, or combinations
thereof.
[0022] According to an aspect of some embodiments of the present
invention, there is also provided a method for treating
schizophrenia in a subject in need thereof, the method comprising
administering a therapeutic composition comprising
beta-caryophyllene (BCP) and a pharmaceutically effective carrier.
In some embodiments, the subject is a human subject. In some
embodiments, the subject is a non-human subject.
[0023] In some embodiments, the schizophrenia is selected from the
group consisting of paranoid schizophrenia; disorganized
schizophrenia; undifferentiated schizophrenia; catatonic
schizophrenia; and residual schizophrenia.
[0024] In some embodiments, the treating comprises treating at
least one symptom of schizophrenia selected from the group
consisting of a negative symptom of schizophrenia and a positive
symptom of schizophrenia.
[0025] In some embodiments, the average daily amount of the BCP
administered to the subject is from about 0.4 mg/kg to about 2
mg/kg.
[0026] In some embodiments, the pharmaceutically effective carrier
comprises dimethyl sulfoxide (DMSO). In some such embodiments, the
pharmaceutically effective carrier comprises DMSO, saline and
Cremophor EL. In some such embodiments, the pharmaceutically
effective carrier comprises DMSO, saline and Cremophor EL at a
ratio of 1:0.6:18 Cremophor EL:DMSO:saline.
[0027] In some embodiments, the administering comprises injecting
the composition to the subject. In some embodiments, the injecting
comprises injecting by a route selected from the group consisting
of intravenous injection, intramuscular injection, intradermal
injection, intraperitoneal injection, intrathecal injection and
subcutaneous injection.
[0028] In some embodiments, the administering comprises orally
administering the composition to the subject.
[0029] In some embodiments, the method further comprises
co-administering at least one additional antipsychotic agent. In
some such embodiments, the at least one additional antipsychotic
agent is selected from the group consisting of chlorpromazine,
haloperidol, perphenazine, fluphenazine, clozapine, risperidone,
olanzapine, quetiapine, ziprasidone, aripiprazole, and
paliperidone, or combinations thereof.
[0030] In some such embodiments, the at least one additional
antipsychotic agent is co-administered in a single dosage form
together with the BCP. In some such embodiments, the at least one
additional antipsychotic agent is co-administered in a dosage form
separate from the BCP. In some such embodiments, the
co-administration comprises sequential or simultaneous
administration. In some such embodiments, the sequential
administration comprises administration of the at least one
additional antipsychotic agent prior to administration of the BCP.
In some such embodiments, the sequential administration comprises
administration of the at least one additional antipsychotic agent
subsequent to administration of the BCP.
[0031] When found in nature, BCP (beta-caryophyllene) typically
appears as a mixture of two pharmaceutically-active isomers E-BCP
and Z-BCP, together with substantially inactive sesquiterpenes such
as alpha-humulene and derivatives such as BCP oxide. Typically,
natural sources include a greater proportion of E-BCP than
Z-BCP.
##STR00001##
[0032] For implementing the teachings herein, the BCP includes both
E-BCP and Z-BCP, alone or in combination.
[0033] In some embodiments, the BCP used for implementing the
teachings herein is at least 65%, at least 75%, at least 85% and
even at least 95% by weight E-BCP. In some embodiments, the BCP is
substantially pure (at least 99% by weight) E-BCP.
[0034] In some embodiments, the BCP used for implementing the
teachings herein is at least 65%, at least 75%, at least 85% and
even at least 95% by weight Z-BCP. In some embodiments, the BCP is
substantially pure (at least 99% by weight) Z-BCP.
[0035] In some embodiments, the BCP used for implementing the
teachings herein is at least 65%, at least 75%, at least 85% and
even at least 95% by weight E-BCP and/or Z-BCP. In some
embodiments, the BCP is substantially pure (at least 99% by weight)
E-BCP and/or Z-BCP.
[0036] For example, in some embodiments the BCP used for
implementing the teachings herein comprises 45-49% E-BCP, 45-49%
Z-BCP, 1-5% BCP oxide and 1-5% alpha humulene.
[0037] For example, in some embodiments BCP used for implementing
the teachings herein comprises 45-90% E-BCP, 5-30% Z-BCP, 1-5% BCP
oxide and traces alpha humulene.
[0038] According to an aspect of some embodiments of the invention,
there is also provided a composition comprising a CB2 receptor
agonist and a pharmaceutically effective carrier for use in
treating schizophrenia.
[0039] According to an aspect of some embodiments of the invention,
there is also provided a use of a composition comprising a CB2
receptor agonist and a pharmaceutically effective carrier in the
manufacture of a composition for treating schizophrenia in a
subject in need thereof.
[0040] According to an aspect of some embodiments of the invention,
there is also provided a method for the treating schizophrenia in a
subject in need thereof, the method comprising administering a
therapeutic composition comprising a CB2 receptor agonist and a
pharmaceutically effective carrier.
[0041] Any suitable CB2 receptor agonist may be used in
implementing the composition, the use or the method of treating, in
some embodiments BCP and/or HU308. In some embodiments, the various
features, options and embodiments are as explicitly discussed with
reference to BCP.
[0042] In some embodiments, the teachings herein are applied to the
treatment of human subjects, for example, humans suffering from
schizophrenia.
[0043] In some embodiments, the teachings herein are applied to the
treatment of non-human animal subjects suffering from
schizophrenia.
[0044] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. In case
of conflict, the specification, including definitions, takes
precedence.
[0045] As used herein, the terms "comprising", "including",
"having" and grammatical variants thereof are to be taken as
specifying the stated features, integers, steps or components but
do not preclude the addition of one or more additional features,
integers, steps, components or groups thereof.
[0046] As used herein, the indefinite articles "a" and "an" mean
"at least one" or "one or more" unless the context clearly dictates
otherwise.
[0047] As used herein, when a numerical value is preceded by the
term "about", the term "about" is intended to indicate +/-10%.
[0048] As used herein, the term "treating" includes curing a
condition, treating a condition, preventing a condition, treating
symptoms of a condition, curing symptoms of a condition,
ameliorating symptoms of a condition, treating effects of a
condition, ameliorating effects of a condition, and preventing
results of a condition
[0049] As used herein a "therapeutic composition" refers to a
preparation of one or more of the active ingredients with other
components such as pharmaceutically-acceptable carriers and
excipients. The purpose of a therapeutic composition is to
facilitate administration of an active ingredient to a subject.
[0050] The term "pharmaceutically acceptable carrier" refers to a
carrier or a diluent that does not cause significant irritation to
a subject and does not substantially abrogate the activity and
properties of the administered active ingredients. An adjuvant is
included under these phrases. The term "excipient" refers to an
inert substance added to a therapeutic composition to further
facilitate administration of an active ingredient.
[0051] Therapeutic compositions used in implementing the teachings
herein may be formulated using techniques with which one of average
skill in the art is familiar in a conventional manner using one or
more pharmaceutically-acceptable carriers comprising excipients and
adjuvants, which facilitate processing of the active ingredients
into a pharmaceutical composition and generally includes mixing an
amount of the active ingredients with the other components.
Suitable techniques are described in "Remington's Pharmaceutical
Sciences," Mack Publishing Co., Easton, Pa., latest edition, which
is incorporated herein by reference. For example, pharmaceutical
compositions useful in implementing the teachings herein may be
manufactured by one or more processes that are well known in the
art, e.g., mixing, blending, homogenizing, dissolving, granulating,
emulsifying, encapsulating, entrapping and lyophilizing
processes.
[0052] Pharmaceutical compositions suitable for implementing the
teachings herein include compositions comprising active ingredients
in an amount effective to achieve the intended purpose (a
therapeutically effective amount). Determination of a
therapeutically effective amount is well within the capability of
those skilled in the art, for example, is initially estimated from
animal models such as monkey or pigs.
BRIEF DESCRIPTION OF THE FIGURES
[0053] Some embodiments of the invention are described herein with
reference to the accompanying figures. The description, together
with the figures, makes apparent to a person having ordinary skill
in the art how some embodiments of the invention may be practiced.
The figures are for the purpose of illustrative discussion and no
attempt is made to show structural details of an embodiment in more
detail than is necessary for a fundamental understanding of the
invention. For the sake of clarity, some objects depicted in the
figures are not to scale.
[0054] In the Figures:
[0055] FIGS. 1A and 1B relate to mouse body weight at PND 16-17:
FIG. 1A is a line graph showing changes in body weight at postnatal
days 3 to 17 in mice treated with phencyclidine (PCP), PCP+BCP or
control (vehicle) and FIG. 1B is a bar graph showing body weight
for the 3 groups at postnatal day 17;
[0056] FIGS. 2A-2C relate to open field test at PND 16-17: FIGS. 2A
and 2B are line graphs showing ambulation (2A) and rearing (2B) at
PND 16-17 and FIG. 2C is a bar graph showing body weight at PND
17;
[0057] FIGS. 3A-3F relate to open field test at PND 16-17: FIGS. 3A
and 3D arc bar graphs showing body weight for males (3A) and
females (3D), FIGS. 3B and 3E are line graphs showing ambulation in
males (3E) and females (3F) and FIGS. 3C and 3F are line graphs
showing rearing in males (3C) and females (3F);
[0058] FIGS. 4A-4F relate to open field test at PND 35-37: FIGS. 4A
and 4D are bar graphs showing body weight for males (4A) and
females (4D), FIGS. 4B and 4E are line graphs showing rearing in
males (4B) and females (4E) and FIGS. 4C and 4F are line graphs
showing ambulation in males (4C) and females (4F);
[0059] FIGS. 5A-5D relate to pre-pulse inhibition at age 8 weeks:
FIGS. 5A and 5C are bar graphs showing response to startle for 8
week old males (5A) and females (5C); FIGS. 5B and 5D are line
graphs showing percentage inhibition of prepulse inhibition for
males (5B) and females (5D);
[0060] FIGS. 6A-6H relate to elevated plus maze test at age 13
weeks: female duration closed (6A), male duration closed (6B),
female duration open (6C), male duration open (6D), female duration
distal open (6E), male duration distal opem (6F), female open/close
duration (6G) and male open/close duration (6H);
[0061] FIGS. 7A-7I are bar graphs showing mRNA expression of
cannabinoid receptors in 9 day old mice for glyceraldehyde
3-phosphate dehydrogenase (GAPDH) in the left cortex (7A), right
cortex (7B) and brain stem (7G); for Cannabinoid Receptor Type 1
(CB1) in the left cortex (7C), right cortex (7D) and brain stem
(7H); and for CB2 in the left cortex (7E), right cortex (7F), and
brain stem (7I) for control mice and mice treated with PCP;
[0062] FIG. 8 is a Table detailing the percentage change (%)
relative to control animals of protein expression of cannabinoid
receptors in 2 week old mice;
[0063] FIGS. 9A-9C are bar graphs showing protein expression of 67
kDa glutamic acid decarboxylase (GAD67)/actin in the left cortex
(9A), right cortex (9B) and brain stem (9C) of 2-week old mice
treated with saline or PCP;
[0064] FIGS. 9D-9F are bar graphs showing protein expression of 67
kDa glutamic acid decarboxylase (GAD67)/actin in the left cortex
(9D), right cortex (9E) and brain stem (9F) of 9-day old mice
treated with saline or PCP;
[0065] FIGS. 10A-10C relate to monoacylglycerol lipase (MGL)
expression in 2 week old mice treated with saline or PCP: bar graph
relating to the left cortex (10A), bar graph relating to the right
cortex (10B) and Western blot (10C);
[0066] FIGS. 11A and 11B are schematic representations of the
endocannabinoid synthesizing and degrading pathways as described in
Anavi-Goffcr, ChemBioChem 2009;
[0067] FIGS. 12A-12C relate to PND17 using a DMSO-based vehicle:
line-graph showing male ambulation (12A), line-graph showing male
rearing (12B) and line graph showing male body weight (12C);
[0068] FIGS. 13A-13C relate to PND16: line-graph showing body
weight over PND 3-17 (13A), line-graph showing male and female
ambulation (13B) and line-graph showing male and female rearing
(13C);
[0069] FIGS. 14A-14E show results demonstrating that BCP treatment
at adolescence reversed the effect of PCP on ambulation but did not
affect body weight: line graph of body weight at PND 40-68 (14A),
bar graph of female and male body weight at PND63 (14B), line graph
of male ambulation at PND 63 (14D), line graph of female ambulation
at PND 63 and line graph of male and female ambulation at PND
63;
[0070] FIGS. 15A-15C show results demonstrating that BCP treatment
at adolescence reversed the effect of PCP on rearing: line graph of
male and female rearing at PND63 (15A), line graph of male rearing
at PND63 (15B) and line graph of female rearing at PND63 (15C);
[0071] FIGS. 16A-16C show results demonstrating that BCP treatment
at adolescence reversed the effect of PCP on PPI: line graph of %
PPI at PND68 (16A); bar graph of female startle response at PND68
(16B) and bar graph of male startle response at PND68 (16C);
[0072] FIGS. 17A-17C show results demonstrating that BCP treatment
at adolescence reversed the effect of PCP on the response to tone
(PPI test): line graph of response to tone at PND68 (17A); line
graph of female response to tone at PND68 (17B) and line graph of
male response to tone at PND68 (17C);
[0073] FIGS. 18A-18C show results demonstrating that BCP treatment
at adolescence did not affect the startle response at the end of
the PPI test: female startle response at PND 68 (18A), male startle
response at PND 68 (18B) and all-mice startle response at PND68
(18C);
[0074] FIGS. 19A-19F show results demonstrating that BCP treatment
at adolescence did not reverse the effects of PCP in plus maze
test: open/close duration at PND 64 (19A), open/(close+open)
duration at PND 64 (19B), distal open/(close+open) duration at PND
64 (19C), open/close frequency at PND 64 (19D), open/(open+close)
frequency at PND 64 (19E) and distal open/(open+close) frequency at
PND 64 (19F);
[0075] FIGS. 20A-20E show results demonstrating that BCP treatment
at adolescence reversed the effects of PCP on the time spent in the
hidden zone (behavior in the Phenotyper cage): bar graph of female
hidden zone duration at PND 91 (20A), bar graph of male hidden zone
duration at PND 91 (20B), bar graph of male and female hidden zone
duration at PND 91 (20C), bar graph of male hidden zone frequency
at PND 91 (20D) and bar graph of female hidden zone frequency at
PND 91 (20E);
[0076] FIGS. 21A-21C show results demonstrating that BCP treatment
at adolescence reversed the effects of PCP on frequency of entries
to the wheel (motor behavior in the Phenotyper cage: bar graph of
female wheel zone frequency at PND 91 (21A), bar graph of male
wheel zone frequency at PND 91 (21B) and bar graph of male and
female wheel zone frequency at PND 91 (21C);
[0077] FIGS. 22A-22F show results demonstrating that BCP treatment
at adolescence on the time spent at drinking and food zones
(Phenotyper cage): bar graph showing male food zone duration at
PND91 (22A), bar graph showing female food zone duration at PND91
(22B), bar graph showing male and female food zone duration at
PND91 (22C), bar graph showing male drink zone duration at PND91
(22D), bar graph showing female drink zone duration at PND91 (22E)
and bar graph showing male and female drink zone duration at PND91
(22F);
[0078] FIGS. 23A-23E show results demonstrating that BCP treatment
at adolescence improved exploration and rearing behaviors of male
PCP treated mice at PND 104: line graph of female ambulation at PND
104 (23A), line graph of male ambulation at PND 104 (23B), line
graph of female rearing at PND 104 (23C), line graph of male
rearing at PND 104 (23D) and bar graph of male and female body
weight at PND 104 (23E);
[0079] FIGS. 24A-24C show results demonstrating that BCP treatment
at adolescence did not reverse the effect of PCT on grooming at
PND104: bar graph of female grooming at PND 104 (24A), bar graph of
male grooming at PND 104 (24B) and bar graph of male and female
grooming at PND 104 (24C);
[0080] FIGS. 25A-25I show results demonstrating that BCP treatment
at adolescence reversed the effect of PCP on attention at PND106
(PPI test): bar graph of female startle at PND 106 (25A), bar graph
of male startle at PND 106 (25B), bar graph of male and female
startle at PND 106 (25C), line graph of female response to tone of
varying intensity (25D), line graph of female response to tone of
varying intensity (25E), line graph of female response to tone of
varying intensity (25F), line graph of % prepulse inhibition for
females at PND 106 (25G), line graph of % prepulse inhibition for
males at PND 106 (25H) and line graph of % prepulse inhibition for
males and females at PND 106 (25I);
[0081] FIGS. 26A-26F show results demonstrating that BCP treatment
at adolescence reversed the effect of PCP on frequency of entries
to hidden zone (Phenotyper cage) at PND 105: bar graph of male
hidden zone frequency at PND 105 (26A), bar graph of female hidden
zone frequency at PND 105 (26B), bar graph of male and female
hidden zone frequency at PND 105 (26C), bar graph of male wheel
zone frequency at PND 105 (26D), bar graph of female wheel zone
frequency at PND 105 (26E) and bar graph of male and female wheel
zone frequency at PND 105 (26F);
[0082] FIGS. 27A-27E show results demonstrating that BCP treatment
at adolescence reversed the effect of PCP on time spent at the
hidden zone but not the time spent in the wheel zone (Phenotyper
cage) at PND 105: bar graph of male hidden zone duration at PND 105
(27A), bar graph of female hidden zone duration at PND 105 (27B),
bar graph of male wheel zone duration at PND 105 (27C), bar graph
of female wheel zone duration at PND 105 (27D) and bar graph of
male and female wheel zone duration at PND 105 (27E); and
[0083] FIGS. 28A-28B show results demonstrating that AM630 reversed
the effect of BCP on PCP-induced inhibition of ambulation and
rearing: line graph of male ambulation at 17 days (28A) and line
graph of male rearing at 17 days (28B).
DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION
[0084] Some embodiments of the invention relate to compositions
comprising beta-caryophyllene (BCP), methods of making the
compositions and methods using BCP for the treatment of
schizophrenia. Some embodiments of the invention relate to
compositions comprising Cannabinoid Receptor Type 2 (CB2) receptor
agonists, methods of making the compositions and methods using CB2
receptor agonists for the treatment of schizophrenia.
[0085] The principles, uses and implementations of the teachings
herein may be better understood with reference to the accompanying
description and figures. Upon perusal of the description and
figures present herein, one skilled in the art is able to implement
the invention without undue effort or experimentation.
[0086] The Cannabinoid Receptor Type 2 (CB2) is a guanine
nucleotide-binding protein (G protein)-coupled receptor that in
humans is encoded by the CNR2 gene.
[0087] Recent studies have identified the cannabinoid CB2 receptor
in the brain. Up-regulation of CB2 receptor expression in the brain
during central nervous system pathologies has been demonstrated for
certain neurological diseases.
[0088] Beta-caryophyllene (BCP, CAS 87-44-5) is a CB2-receptor
agonist (Gertsch et al. 2008, Anavi-Goffer et al., 2012). The fact
that orally-administered BCP is absorbed by the digestive tract and
becomes systemically available and apparent substantial
non-toxicity makes BCP attractive as a potential active
pharmaceutical ingredient.
[0089] The role of CB2 receptor agonists in general, and BCP in
particular, in the treatment of schizophrenia, has not previously
been studied.
[0090] The Inventors have studied the effect of BCP in a murine
model of schizophrenia, produced by administration of the
N-methyl-D-aspartic acid (NMDA) antagonist, phenylcyclidine (PCP).
Administration of phencyclidine to rats (e.g. Josselyn and
Vaccarino, 1998; Wang & Johnson, 2005; Ballmaier, 2007;
Takahashi, 2006) or mice (e.g. Long, 2006; Hashimoto 2005) has been
used as an animal model for schizophrenia. Phencyclidine may be
administered acutely or chronically, during adulthood or during
postnatal development, using different dose ranges (2.5 mg/kg to 20
mg/kg). In order to induce chronic, long lasting schizophrenic-like
behaviors and neurochemical changes in the endocannabinoid system,
the Inventors have now developed a neonatal mouse model, based on a
neonatal phencyclidine model previously described for rats
(Takahashi, 2006). As the Inventors have extensively studied the
endocannabinoid system in Sabra strain mice (Harlan, Israel), see
for example Fride 2005 and Fride 2007, these mice have been used in
the present study.
[0091] The Inventors have found that following administration of
PCP, the CB2 receptor expression level is selectively
down-regulated in different brain areas. Furthermore, up-regulation
of putative CB2 receptor expression has been detected in the right
cortex and basal ganglia/diencephalon of mice which were neonatally
treated with phencyclidine. These results support some aspects of
the teachings herein, where putative CB2 receptors are up-regulated
in specific brain areas in schizophrenia.
[0092] Thus, according to an aspect of some embodiments of the
teachings herein, there is provided a composition comprising
beta-caryophyllene (BCP) and a pharmaceutically effective carrier
for use in treating schizophrenia.
[0093] According to an aspect of some embodiments of the teachings
herein, there is also provided the use of beta-caryophyllene (BCP)
and a pharmaceutically effective carrier in the manufacture of a
medicament for treating schizophrenia in a subject in need
thereof.
[0094] In some embodiments, such a composition is configured for
administration to a human subject. In some embodiments, such a
composition is configured for administration to a non-human animal
subject.
[0095] According to an aspect of some embodiments disclosed herein,
there is also provided a method for treating schizophrenia in a
subject in need thereof, the method comprising administering a
pharmaceutically-effective amount of beta-caryophyllene (BCP) to
the subject. In some embodiments, the subject is a human subject.
In some embodiments, the subject is a non-human animal.
[0096] The efficacy of the methods and compositions according to
the teachings herein are demonstrated in the experimental section
hereinbelow.
[0097] According to some embodiments, the compositions and methods
of treatments disclosed herein are useful for treating one or more
of paranoid schizophrenia; disorganized schizophrenia;
undifferentiated schizophrenia; catatonic schizophrenia; and
residual schizophrenia.
[0098] In some embodiments, the compositions and methods of
treatments disclosed herein are useful in the treatment of a
negative symptom of schizophrenia.
[0099] In some embodiments, the compositions and methods of
treatments disclosed herein are useful in the treatment of a
positive symptom of schizophrenia.
[0100] The duration of treatment according to the method of
treating schizophrenia according to the teachings is any suitable
duration as determined by a treating health-care professional,
typically a psychiatric doctor.
[0101] In some embodiments of the method of treating schizophrenia
according to the teachings herein, the average daily dose of BCP
administered to a human subject is from about 0.4 mg/kg to about 2
mg/kg, such as, for example, from about 0.4 mg/kg to about 1.5
mg/kg, from about 0.4 mg/kg to about 1.8 mg/kg, from about 0.4
mg/kg to about 1.6 mg/kg, from about 0.4 mg/kg to about 1.4 mg/kg,
from about 0.4 mg/kg to about 1.2 mg/kg, from about 0.4 mg/kg to
about 1 mg/kg, from about 0.4 mg/kg to about 0.8 mg/kg, from about
0.4 mg/kg to about 0.6 mg/kg or from about 0.4 mg/kg to about 0.5
mg/kg.
[0102] In some embodiments of the method of treating schizophrenia
according to the teachings herein, the average daily dose for a
human subject (especially an adult human, weighing between about 40
kg and about 120 kg) is in the range of from about 25 mg to about
100 mg, such as about 25 mg, about 30 mg, about 35 mg, about 40 mg,
about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg,
about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg,
about 95 mg, or about 100 mg.
[0103] In some embodiments of the method of treating schizophrenia
according to the teachings herein, the average daily dose is
administered with a frequency of between about once a week to about
3 times per day, for example once per week, twice per week, 3 times
per week, 4 times per week, 5 times per week, 6 times per week,
once per day, twice per day or 3 times per day.
[0104] In some embodiments, a composition according to the
teachings herein is provided as or made as a dosage form including
a plurality of discrete units (e.g., discrete solids or metered
liquids), especially discrete solid units such as pills (including
tablets and caplets) and capsules (including gelcaps), where each
unit includes BCP in the range of from about 25 mg to about 100 mg,
such as about 25 mg, about 30 mg, about 35 mg, about 40 mg, about
45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70
mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95
mg, or about 100 mg. In some such embodiments, such a dosage form
is exceptionally useful for the once-daily administration of the
desired average daily dosage.
[0105] According to some embodiments, the composition disclosed
herein may be administered by any suitable route of administration,
including but not limited to oral administration, parenteral
administration (including intravenous injection, intramuscular
injection, intradermal injection, intraperitoneal injection,
intrathecal injection and subcutaneous injection), and rectal
administration. That said, in some embodiments, oral administration
is preferred due to the proven oral availability and
substantial-non toxicity of BCP.
[0106] For oral administration, the composition disclosed herein
may comprise a pill, a capsule, a dragee, a powder, granules, an
ingestible solution (such as a liquid, a gel, a syrup, or a
suspension) and the like, for oral ingestion by a subject. In a
preferred embodiment, a composition for oral administration
comprises a pill or a capsule.
[0107] In a preferred embodiment, the composition is a
gastroresistant orally-administrable dosage form, that is to say,
an orally-administrable dosage form configured to carry the BCP
through the stomach to be released into contact with the digestive
tract only after passage through the duodenum. For example, in some
such embodiments, the composition is in the form of a
gastroresitant soft gel capsule, comprising between 25 mg and about
100 mg BCP in a carrier comprising vegetable oil. Some embodiments
of the method, when implemented with an adult human subject,
comprise orally ingesting a single such capsule twice a day for at
least one month, so that the average daily dose is between about 50
mg and about 200 mg BCP.
[0108] In some embodiments, the composition described herein
further comprises at least one additional antipsychotic agent, such
as, for example, a typical antipsychotic agent (including, but not
limited to, one or more of chlorpromazine, haloperidol,
perphenazine, or fluphenazine), and/or an atypical antipsychotic
agent (including, but not limited to, one or more of clozapine,
risperidone, olanzapine, quetiapine, ziprasidone, aripiprazole, and
paliperidone), or combinations thereof.
[0109] In some embodiments of the method of treatment, the BCP is
administered together with at least one additional antipsychotic
agent, such as, for example, a typical antipsychotic agent
(including, but not limited to, one or more of chlorpromazine,
haloperidol, perphenazine, or fluphenazine), and/or an atypical
antipsychotic agent (including, but not limited to, one or more of
clozapine, risperidone, olanzapine, quetiapine, ziprasidone,
aripiprazole, and paliperidone), or combinations thereof.
[0110] In some embodiments where the BCP and an antipsychotic agent
are administered together, BCP and the additional antipsychotic
agent are co-administered in a single dosage form.
[0111] In some embodiments where the BCP and an antipsychotic agent
are administered together, BCP and the additional antipsychotic
agent are co-administered in separate dosage forms, either
sequentially or simultaneously. For example, the additional
antipsychotic agent may be administered prior to administration of
BCP, or the additional antipsychotic agent may be administered
subsequent to administration of BCP.
[0112] Although not wishing to be bound to any one theory, the
Inventors consider that it is likely that at least part, if not
all, of the herein demonstrated efficacy of BCP in treating
schizophrenia relates to the CB2 receptor agonist properties of
BCP.
[0113] Thus, according to an aspect of some embodiments of the
teachings herein, there is provided a composition comprising a CB2
receptor agonist and a pharmaceutically effective carrier for use
in treating schizophrenia.
[0114] According to an aspect of some embodiments of the teachings
herein, there is also provided the use of a CB2 receptor agonist
and a pharmaceutically effective carrier in the manufacture of a
medicament for treating schizophrenia in a subject in need
thereof.
[0115] According to an aspect of some embodiments of the teachings
herein, there is also provided a method for treating schizophrenia
in a subject in need thereof, the method comprising administering a
pharmaceutically-effective amount of a CB2 receptor agonist to the
subject.
[0116] Exemplary embodiments of the teachings herein are discussed
hereinbelow with reference to specific materials, methods and
examples. The material, methods and examples discussed herein are
illustrative and not intended to be limiting. In some embodiments,
methods and materials similar or equivalent to those described
herein are used in the practice or testing of embodiments of the
invention. It is to be understood that the invention is not
necessarily limited in its application to the details of
construction and the arrangement of the components and/or methods
set forth in the following description and/or illustrated in the
drawings. The invention is capable of other embodiments or of being
practiced or carried out in various ways.
Examples
Materials and Methods
[0117] BCP was obtained from Sigma-Aldrich (St. Louis, Mo., USA),
catalogue Nr. W225207 and further purified using preparative HPLC
(HP1090 series; column, PEGASIL ODS (Senshu Sci. i.d. 10.times.250
mm); solvent, 70% CH3OH; flow rate, 2.0 mL/min; detection, UV 220
nm] to remove other sesquiterpenes. GC-MS analysis showed that the
BCP used in the below included 95% E-BCP, 3% Z-BCP, 1% BCP Oxide
and traces of alpha humulene. AM630 was obtained from Cayman
Chemical Company (Ann Arbor, Mich., USA). PCP, Cremophor EL and
DMSO were obtained from Sigma-Aldrich (St. Louis, Mo., USA).
Animal Model of Schizophrenia:
[0118] The mouse model of schizophrenia was established.
Phencyclidine (PCP), an NMDA antagonist which induces schizophrenia
and psychotic effects in humans, was administered to murine pups
(injection of 5 mg/kg in saline) on postnatal days 3, 5, 7, 9, 11,
13, and 16. This treatment induces long-lasting schizophrenic-like
effects in mice that lasted into adulthood. The therapeutic effects
of beta-caryophyllene, a dietary cannabinoid and CB2 receptor
agonist, in accordance with the teachings herein were
evaluated.
I. Treatment of Mice with BCP at Postnatal Days 3-16
[0119] BCP (final dose 10 mg/kg in 1:0.6:18 Cremophor
EL:ethanol:saline) was administered by injection 1 hour after PCP.
Results were obtained from two different litters, each of which was
divided into 3 groups: [0120] Group 1: vehicle (n=6 and 4 pups,
respectively); [0121] Group 2: PCP (n=6 and 5 pups, respectively);
[0122] Group 3: PCP+BCP (n=5 and 5 pups, respectively).
[0123] In FIG. 17 BCP (final dose 10 mg/kg in 1:0.6:18 Cremophor
EL:DMSO:saline) was administered by injection 1 hour after PCP.
Results were obtained from one litter which was divided into the
three groups.
Assessment of Positive/Negative Schizophrenic-Like Behavior:
Open-Field Test (Crossing and Rearing)
[0124] Mice were assessed for hyperactivity behavior on postnatal
day 16 (FIG. 2). Mice were placed in the center of a transparent
glass cube cage 30.times.40.times.31 cm divided into squares of
7.5.times.7.5 cm. The number of squares and rearing activity were
counted for 8 min. Positive Symptoms. Prepulse Inhibition (PPI) of
the Startle Reflex
[0125] In this experimental model, a weak stimulus (74-90 dB tone)
inhibited the subsequent response to a strong stimulus (120 dB
tone). Reduced prepulse inhibition of the startle reflex (PPI) was
taken as an index of the positive symptoms of schizophrenia
(Josselyn and Vaccarino, 1998).
[0126] PPI was assessed similarly to the method described by Varty
et al. In the employed model, mice were placed in a startle chamber
and allowed to acclimate for 5 min. A loudspeaker produced a 65 dB
background white noise or the various acoustic pre-pulse stimuli
(dB): 74, 78, 82, 86, and 90 (20 ms). A 120 dB (40 ms) stimulus was
given first to induce a response to startle. The response of the
mouse was transduced and stored by a computer. Each test session
lasted for 11 min and consisted of 5 presentations of each of the
trial types presented in random order and separated by 15 second
intervals. The amount of prepulse inhibition was calculated as %
PPI=[1-(startle response for prepulse+pulse)/(startle response for
pulse alone)].times.100.
Negative Symptoms
[0127] Anxiety as measured by the paucity of time spent on the two,
anxiety-provoking, open arms (as opposed to the two enclosed arms)
of an "Elevated Plus Maze" was used as a parameter of negative
symptoms of schizophrenia (Josselyn and Vaccarino, 1998). The plus
maze was elevated 50 cm above the table top. Behavior of each mouse
was recorded for 5 min by a video camera and scored using the
"EthoVision" software (Noldus Information Technology, Wageningen,
The Netherlands), measuring the number of entries as well as the
amount of time spent in each arm, open or closed. Increased time
spent in the closed arms indicated increased anxiety. Increased
time spent in the opened arms indicated anxiolytic behavior
(reduced stress).
Results
Body Weight
[0128] Body weight was recorded at each day of injection (FIG. 1A).
PCP was shown to significantly reduce the body weight. The results
show that BCP significantly reversed the inhibitory effect of PCP
on body weight (p<0.0001 for PCP+BCP vs. PCP).
[0129] It was shown that at PND 7, PCP induced a significant
reduction in body weight. BCP reversed the reduction in body weight
during the first week of life (FIG. 1B) but, under these conditions
(injections in ethanol-based vehicle as described above) did not
restore the weight completely. However, when vehicle mixture was
changed to DMSO-based, BCP completely reversed the effect of PCP on
body weight (FIG. 12C).
[0130] At postnatal day (PND) 17, the difference between vehicle-
and PCP-treated groups in body weight was still significant
(p<0.0001) (FIG. 2C). There was no significant difference
between groups treated with PCP with or without BCP.
Rearing and Exploration
[0131] At PND 17, at the end of treatment with BCP, locomotor
activity, hyperactivity, and exploratory behaviors were tested with
the open-field test (FIGS. 2A, 2B). PCP significantly inhibited
both ambulation and rearing behaviors. Treatment with BCP reversed
the effects of PCP on rearing and exploration. In addition, it is
seen that the effect of BCP is not dependent on ethanol as its
solvent. It is seen that a DMSO-based carrier completely reversed
the effects of PCP on ambulation (FIG. 12A) and rearing behavior
(FIG. 12B).
Results According to Sex
[0132] Changes in body weight and results from the open-field test
at PND 17 were separated according to the sex of mice (FIGS.
3A-F).
[0133] Females appeared to be more sensitive to the effect of PCP
(ambulation p<0.006; p<0.03 rearing). Treatment with BCP
reversed a proportion of the effects of PCP on ambulation and
rearing behavior of female mice (FIGS. 3E, 3F, respectively) but
not the effect of PCP on body weight (FIG. 3D). Locomotor activity
of male mice appeared to be less sensitive to the treatment of PCP
or PCP+BCP (FIGS. 3A-3C). However, the effect of BCP on PCP-induced
reduction of body weight in male mice appeared to be more prominent
(FIG. 3A).
[0134] These results suggest that in some embodiments the
administration of BCP for the treatment of schizophrenia has a
different effect on male and female mice, being more effective in
the treatment of female mice.
[0135] Mice were re-evaluated in the open-field test at PND 35-37.
Results of body weight, rearing and ambulation were separated
according to the sex of mice (FIGS. 4A-F). Treatment with BCP
significantly reversed the effect of PCP on female body weight
(FIG. 4D). At this age there was no significant difference in male
body weight between the groups (FIG. 4A).
[0136] In males, treatment with BCP significantly reduced rearing
behavior as compared to vehicle and PCP-treated groups (FIG.
4B).
[0137] In males, treatment with PCP significantly increased the
locomotor activity compared with vehicle treatment, while
administration of BCP reduced locomotor activity (FIG. 4C).
[0138] In females, no differences in rearing and exploration
behaviors were seen between the different groups (FIGS. 4E, 4F,
respectively).
[0139] It was concluded that treatment with BCP significantly
reversed the effect of PCP in males.
Prepulse Inhibition Testing
[0140] Mice were tested at age 8 weeks in the Pre-Pulse Inhibition
test (FIGS. 5A-D).
Response to Startle
[0141] The response to startle stimuli was not significantly
different between groups (in males (FIG. 5A) or in females (FIG.
5C). It was concluded that mice in all groups had no problem in
hearing and were not apathetic.
% Inhibition
[0142] Males treated with PCP showed a significant reduction in
their ability to adjust to sound stimulus as compared to vehicle
treated mice. BCP reversed this effect (FIG. 5B).
[0143] No significant difference in in the ability to adjust to
sound stimulus was seen in females for the PCP-treated group or
PCP+BCP treated group (FIG. 5D).
[0144] It was concluded that treatment with BCP significantly
reversed the effect of PCP on the sensorimotor-gate in males.
Elevated-Plus Maze Test
[0145] At age 13 weeks old, mice were tested in the Elevated-Plus
Maze test which indicates the level of anxiety (FIGS. 6A-H).
[0146] Phencyclidine alters the level of anxiety. However its
effect is dependent on the strain of mice, sex and possibly age
(Turgeon, 2011; Wily, 1995).
[0147] There was no significant change in the time spent in the
closed arm or open arm (all length of arm), for either females or
males (FIGS. 6A, 6B, 6C, 6D, respectively).
PCP reduced the time spent in the distal end of the opened arm (the
very far end of the arm from the center) for females, and BCP
reversed this effect (FIGS. 6E, 6G). In FIGS. 6F, 6H, PCP increased
the time spent in the distal end of the opened arm (the very far
end of the arm from the center) for males, and BCP reversed this
effect.
[0148] It was concluded that PCP induced anxiety in female mice and
anxiolytic effect in male mice. BCP reversed both effects.
Expression Level of CB1 and CB2 Receptor in Mice at 9 Days Old
(mRNA) or 2 Weeks Old (Protein)
[0149] Brain tissue of control mice (saline treated) and mice
treated with PCP were analyzed in the left and right cortex and
brain stem (FIGS. 7A-I; FIG. 8. Results from the cortex and brain
stem were reported by the Inventors in 2011 (Anavi-Goffer et
al).
[0150] No difference in GAPDH was seen in the left cortex, right
cortex or brain stem (FIGS. 7A-7C, respectively).
[0151] No significant increase in mRNA expression of CB1 receptor
was found in the left or right cortex of the PCP-treated mice
(FIGS. 7D, 7E, respectively). A significant increase in mRNA
expression of CB1 receptor was found in the brain stem of the
PCP-treated mice (FIG. 7F).
[0152] A significant increase in mRNA expression of CB2 receptor
was found in the left cortex of the PCP-treated mice (FIG. 7G) but
in the right cortex there was no difference between control and
PCP-treated mice (FIG. 7H). A significant increase in mRNA
expression of CB2 receptor was found in the brain stem of the
PCP-treated mice (FIG. 7I).
[0153] It is noteworthy that these results are the opposite of the
observed in Western blotting, (FIG. 8) being about 50 kDa (the
predicted molecular weight of the CB2 receptor) but in line with
these at 64 kDa, suggesting that the CB2 receptor may form a
complex with another protein X. This suggests that the change in
cannabinoid receptor expression is specific (as no change was found
for GAPDH or actin). This also suggests a malfunction in the
regulation of cannabinoid receptor synthesis, leading to the
accumulation of cannabinoid receptor mRNA in the left cortex and
brain stem. On the other hand, synthesis in the right cortex
appeared to lead to the formation of CB2 receptor-protein X
complexes.
Expression Level of GAD67 in Mice at 9 Days Old (mRNA) or 2 Weeks
Old (Protein)
[0154] Brain tissue of control mice and mice treated with PCP were
analyzed for GAD67, a neurochemical marker for schizophrenia (FIG.
9A-F). In the left cortex, GAD67 protein level was significantly
decreased (FIG. 9A) but no change was found at the mRNA level FIG.
9D). In the right cortex, the reduction in protein level of GAD67
did not reach a significant level and no change was seen in the
mRNA level (FIG. 9B, FIG. 9E). In the brain stem, GAD67 mRNA level
was increased in the PCP-treated group (FIG. 9F), although the
results of Western blotting showed a non-significant reduction in
GAD67 protein level (FIG. 9C). This suggests that there might be a
common mechanism which links the changes in GAD67 to those of CB1
and CB2, and this mechanism may be related to the function of
GABAergic neurons.
Expression of MGL in 2 Week Old Mice
[0155] Brain tissues were analyzed for MGL, an enzyme which
degrades 2-AG an endocannabinoid (FIGS. 10A-C, FIG. 11B) in control
mice and mice treated with PCP. mRNA levels of MGL decreased in the
left cortex of the PCP-treated group (FIG. 10A), but not in the
right cortex (FIG. 10B). The direction of these results was
correlated with the reduction in protein level of MGL as analyzed
with Western blotting (FIG. 10C).
[0156] A scheme of synthesis and degradative enzymes of the
endocannabinoid system is shown in FIG. 11A (FIGS. 11A and 11B were
published by Anavi-Goffer & Mulder, Chembiochem. 2009
10:1591-8).
II. Postnatal Induction of Schizophrenia (Days 3-15) Followed by
Treatment of Adolescent Mice with BCP (Postnatal Days 43-61)
Methods
[0157] PCP 5 mg/kg was administered by injection on PND 3, 5, 7,
10, 12, 13, 15 and 17. Body weight was measured at every injection
between PND 3-17. The open field test was conducted on PND 16.
[0158] When the mice were adolescent (PND 43-61), BCP (5 mg/kg in a
mixture of DMSO:Cremophor EL:saline 0.6:1:18.4) was injected twice
a week (on Sunday and Wednesday) for 3 weeks, a total of 6
injections. After the final BCP injection, mice were tested in the
open field test (PND 63), Elevated Plus Maze test (PND 64), PPI
test (PND 68) and behavior at the Phenotyper cage (PND 91). Mice
were re-tested at adulthood on PND 104 (open field), PND 105
(Phenotyper) PND 106 (PPI).
Results
Body Weight
[0159] As seen in FIG. 13A, PCP significantly reduced body weight
in male and female mice as measured on days 3, 5, 7, 10, 12, 15 and
17.
[0160] Injections of BCP (5 mg/kg) on PND 43-61 did not affect body
weight (FIG. 14A). At age PND 63 there was no significant
difference in the body weight between vehicle-treated mice and
PCP-treated mice (FIG. 14B).
Ambulation, Rearing
[0161] At PND 16, PCP significantly inhibited ambulation, rearing
behaviors (FIGS. 13B, 13C).
[0162] BCP treatment during adolescence significantly reversed the
effect of PCP on ambulation in male and female mice at PND 63
(FIGS. 14C-14E). BCP treatment in adolescence significantly
inhibited the exploration of mice compared with saline-treated
mice. BCP reversed the effect of PCP on rearing in both females and
males (FIGS. 15A-15C).
[0163] On PND 104 a relapse in exploratory behavior was evident in
the schizophrenic female mice that had been treated with BCP (last
BCP treatment was on PND 61), as seen in FIGS. 23A, 23C. Rearing
behavior of male PCP-treated mice that had been treated with BCP
was still significantly higher than that of PCP-treated mice with
no BCP treatment (FIGS. 24B, 24C). There was no difference in body
weight between groups (FIG. 24E). BCP treatment did not increase
body weight in females and males, rather reduced body weight of
PCP+BCP treated group (vehicle vs. PCP+BCP, P=0.06).
[0164] BCP treatment in adolescence did not reverse the effect of
PCP on self-grooming (total, without stimuli) in females or males
at PND 104 (FIGS. 24A, 24B, 24C).
Prepulse Inhibition and Startle Response
[0165] BCP treatment in adolescence significantly reversed the
effect of PCP on pre-pulse inhibition (FIG. 16A). BCP had no
substantial effect on saline-treated mice. BCP treatment at
adolescent significantly reversed the effect of PCP on startle
response in females (FIG. 16B). In males, there was no difference
in the response to the startle stimuli between groups (FIG.
16C).
[0166] BCP treatment at adolescence significantly reversed the
effect of PCP on response to pre-pulse tones (FIGS. 17A-17C). BCP
had no effect on saline-treated mice.
[0167] There was no difference between groups in the response to
the startle stimuli at the end of the PPI test (FIGS. 18A-18C). BCP
treatment at adolescence did not affect the response to startle at
the end of the PPI test (FIGS. 18A-18C).
[0168] At PND 106, there was no difference in the response to the
startle stimuli (120 dB) between groups (FIGS. 25A-25C). BCP
reversed the effect of PCP on the response to tones (FIGS.
25D-25F). BCP treatment at adolescence reversed the effect of PCP
on the % pre-pulse inhibition (PPI) (FIGS. 25G-25I).
Plus-Maze Test
[0169] BCP treatment at adolescence did not reverse the effects of
PCP on behavior in the Plus Maze test at age 64 days (6 weeks)
(FIGS. 19A-19F).
PhenoTyper Test
[0170] At PND 91, PCP increased the time spend at the Hidden Zone,
indicating the PCP-treated mice had higher level of anxiety of mice
at the PhenoTyper cage compared with vehicle-treated mice. BCP
treatment at adolescence reversed the effects of PCP on the time
spend in the Hidden Zone of PhenoTyper cage in females and males,
respectively (FIGS. 20A, 20B). FIG. 20C shows combined results,
suggesting that BCP reversed the effect of PCP on anxiety level.
PCP appeared to reduce the frequency of entries to the Hidden Zone
in males (FIG. 20D) but not in females (FIG. 20E). BCP reversed the
effect of PCP in males (FIG. 20D).
[0171] PCP reduced the frequency of entries to the wheel. BCP
treatment at adolescence reversed the effects of PCP on the
frequency of entries to the wheel in the Phenotyper cage both in
females and males (FIGS. 21A, 21B). FIG. 21C, shows combined
results of both sex. These results indicate that BCP reversed the
action of PCP on loco/motor behavior.
BCP treatment at adolescence did not affect the time spend in the
food zone (FIGS. 22A, 22B, 22C).
[0172] Compared with PCP-treated group, BCP treatment at
adolescence appeared to reduce the time spend at the drinking zone
(FIGS. 22D, 22E, 22F).
[0173] At PND 105, PCP increased the frequency of entries to the
Hidden Zone, indicating an increased level of anxiety.
[0174] BCP treatment at adolescence reversed the effects of PCP on
the frequency of entries to the hidden zone (FIGS. 26A, 26B, 26C).
These results suggest that treatment with BCP reduced the level of
stress and anxiety. BCP treatment appeared to reduce the effect of
PCP on the frequency of entries to the Wheel Zone FIG. 26. This
effect was prominent in males than in females (FIGS. 26D, 26E).
[0175] At PND 105, BCP treatment at adolescence reversed the
effects of PCP on the time spend at the hidden zone (FIGS. 27A,
27B). These results suggest that treatment with BCP reduced the
level of stress and anxiety. BCP treatment at adolescence showed a
trend to reverse the effect of PCP on the time spend in the wheel
(FIGS. 27C, 27D, 27E).
III. Effects of AM630 (6-Iodopravadoline)
[0176] AM630 (6-Iodopravadoline, CAS 164178-33-0) is a molecule
that acts as a potent and selective inverse agonist for the
cannabinoid receptor CB2, with a Ki of 32.1 nM at CB2 and
165.times. selectivity over CB1, at which it acted as a weak
partial agonist. It is used in the study of CB2 mediated
responses.
Materials and Methods
Murine Model of Schizophrenia:
[0177] Mice were injected with PCP (5 mg/kg in saline) at postnatal
days PND 4, 6, 8, 11, 13, 15, and 18 to provide a murine model of
schizophrenia. A control group was injected with vehicle
(0.6:1:18.4 DMSO:Cremophor EL:saline) alone. Each experiment was
repeated twice. In each experiment, male mice were divided into 4
groups:
[0178] Group 1: vehicle (n=4 pups, 1+3 pups, respectively);
[0179] Group 2: PCP (n=5 pups, 2+3 pups, respectively);
[0180] Group 3: PCP+BCP (n=6 pups, 2+4, respectively); and
[0181] Group 4:PCP+BCP+AM630 (n=3 pups, 1+2 respectively).
Administration of BCP or BCP+AM630
[0182] The effect of co-administering AM630 with BCP was
studied.
[0183] One hour after each injection with PCP, mice were injected
with vehicle or BCP (final dose 10 mg/kg in 1:0.6:18 Cremophor
EL:DMSO:saline) or BCP+AM630 (equal parts of 20 mg/kg BCP in DMSO
and 20 mg/kg AM630 in DMSO, providing a final concentration of 10
mg/kg each of BCP and AM630, mixed together).
Results
Rearing and Exploration
[0184] At PND 17, locomotor activity, hyperactivity, exploratory
and grooming behaviors were tested with the open-field test (FIGS.
29A and 29B). PCP significantly inhibited both ambulation and
rearing behaviors. Treatment with BCP reversed the effects of PCP
on rearing and exploration. AM630 reversed the effects of BCP on
ambulation and rearing behaviors. The behaviors of mice that had
been treated with AM630 were not significantly different from these
of PCP-treated mice.
[0185] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0186] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the scope of the appended claims.
[0187] Citation or identification of any reference in this
application shall not be construed as an admission that such
reference is available as prior art to the invention.
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