U.S. patent application number 16/124549 was filed with the patent office on 2019-03-07 for use of cannabidivarin in the treatment of autism spectrum disorder, associated disorders and schizophrenia.
The applicant listed for this patent is GW Research Limited. Invention is credited to James BRODIE, Geoffrey GUY, Livio LUONGO, Rafael MALDONADO, Joanna NEILL, Daniela PAROLARO, Marie WOOLLEY-ROBERTS, Stephen WRIGHT.
Application Number | 20190070128 16/124549 |
Document ID | / |
Family ID | 58579203 |
Filed Date | 2019-03-07 |
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United States Patent
Application |
20190070128 |
Kind Code |
A1 |
GUY; Geoffrey ; et
al. |
March 7, 2019 |
USE OF CANNABIDIVARIN IN THE TREATMENT OF AUTISM SPECTRUM DISORDER,
ASSOCIATED DISORDERS AND SCHIZOPHRENIA
Abstract
The present invention relates to the use of cannabidivarin
(CBDV) in the treatment of autism spectrum disorder (ASD) and
ASD-associated disorders such as Fragile X syndrome (FXS); Rett
syndrome (RS); or Angelman syndrome (AS). In a further embodiment
the invention relates to the use of CBDV in the treatment of
schizophrenia. CBDV has been shown to be particularly effective in
improving cognitive dysfunction in rodent models of ASD, FXS, RS,
AS and schizophrenia. The CBDV is preferably substantially pure. It
may take the form of a highly purified extract of cannabis such
that the CBDV is present at greater than 95% of the total extract
(w/w) and the other components of the extract are characterised.
Alternatively, the CBDV is synthetically produced.
Inventors: |
GUY; Geoffrey; (Cambridge,
GB) ; WRIGHT; Stephen; (Cambridge, GB) ;
BRODIE; James; (Cambridge, GB) ; WOOLLEY-ROBERTS;
Marie; (Cambridge, GB) ; MALDONADO; Rafael;
(Barcelona, ES) ; PAROLARO; Daniela; (Varese,
IT) ; LUONGO; Livio; (Naples, IT) ; NEILL;
Joanna; (Manchester, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GW Research Limited |
Cambridge |
|
GB |
|
|
Family ID: |
58579203 |
Appl. No.: |
16/124549 |
Filed: |
September 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/GB2017/051007 |
Apr 11, 2017 |
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16124549 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 31/05 20130101; A61K 31/352 20130101; A61K 45/06 20130101;
A61P 25/18 20180101; A61K 36/185 20130101 |
International
Class: |
A61K 31/05 20060101
A61K031/05; A61P 25/18 20060101 A61P025/18; A61P 25/28 20060101
A61P025/28 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 2016 |
GB |
1606098.0 |
Claims
1. A method of treating one or more symptoms or disease
characteristics associated with autistic spectrum disorder (ASD) or
ASD-associated disorders, as defined by DSM-IV, in a subject in
need thereof, the method comprising administering cannabidivarin to
the subject, wherein the one or more symptoms or disease
characteristics are one or more of: (i) qualitative impairment in
social interaction; (ii) qualitative impairment in communication;
and (iii) restricted repetitive and stereotyped patterns of
behaviour interest and activities.
2. The method according to claim 1, wherein the symptoms or disease
characteristics of (i) qualitative impairment in social interaction
include one or more of: (a) marked impairment in the use of
multiple nonverbal behaviours; (b) failure to develop peer
relationships appropriate to developmental level; (c) a lack of
spontaneous seeking to share enjoyment, interests, or achievements
with other people; and (d) lack of social or emotional
reciprocity.
3. The method according to claim 1, wherein the symptoms or disease
characteristics of (ii) qualitative impairment in communication
include one or more of: (a) delay in, or total lack of, the
development of spoken language (not accompanied by an attempt to
compensate through alternative modes of communication such as
gesture or mime); (b) in individuals with adequate speech, marked
impairment in the ability to initiate or sustain a conversation
with others; (c) stereotyped and repetitive use of language or
idiosyncratic language; and (d) lack of varied, spontaneous
make-believe play or social imitative play appropriate to
developmental level.
4. The method according to claim 1, wherein the symptoms or disease
characteristics of (iii) restricted repetitive and stereotyped
patterns of behaviour interest and activities include one or more
of: (a) encompassing preoccupation with one or more stereotyped and
restricted patterns of interest that is abnormal either in
intensity or focus; (b) apparently inflexible adherence to
specific, non-functional routines or rituals; (c) stereotyped and
repetitive motor mannerisms; and (d) persistent preoccupation with
parts of objects.
5. The method according to claim 1, wherein the symptoms or disease
characteristics associated with autistic spectrum disorder comprise
two or more symptoms associated with (i) qualitative impairment in
social interaction; one or more symptoms associated with (ii)
qualitative impairment in communication, and one or more symptoms
associated with (iii) restricted repetitive and stereotyped
patterns of behavior interest and activities.
6. A method of treating one or more symptoms or disease
characteristics associated with autistic spectrum disorder (ASD) or
ASD-associated disorders, as defined by DSM-V, in a subject in need
thereof, the method comprising administering cannabidivarin to the
subject, wherein the one or more symptoms or disease
characteristics are one or more of: (a) persistent deficits in
social communication and social interaction across contexts, not
accounted for by general developmental delays, and (b) restricted,
repetitive patterns of behaviour, interests, or activities.
7. The method according to claim 6, wherein the symptoms or disease
characteristics of (a) persistent deficits in social communication
and social interaction across contexts, not accounted for by
general developmental delays include one or more of: (i) deficits
in social-emotional reciprocity; (ii) deficits in nonverbal
communicative behaviours used for social interaction; and (iii)
deficits in developing and maintaining relationships.
8. The method according to claim 6, wherein the symptoms or disease
characteristics of (b) restricted, repetitive patterns of
behaviour, interests, or activities include one or more of: (i)
stereotyped or repetitive speech, motor movements, or use of
objects; excessive adherence to routines, (ii) ritualized patterns
of verbal or nonverbal behaviour, or excessive resistance to
change; (iii) highly restricted, fixated interests that are
abnormal in intensity or focus; and (iv) hyper-or hypo-reactivity
to sensory input or unusual interest in sensory aspects of
environment.
9. The method according to claim 6 wherein the symptoms or disease
characteristics associated with autistic spectrum disorder comprise
all three of (i) deficits in social-emotional reciprocity; (ii)
deficits in nonverbal communicative behaviours used for social
interaction; and (iii) deficits in developing and maintaining
relationships together with two or more of (i) stereotyped or
repetitive speech, motor movements, or use of objects; excessive
adherence to routines, (ii) ritualized patterns of verbal or
nonverbal behaviour, or excessive resistance to change; (iii)
highly restricted, fixated interests that are abnormal in intensity
or focus; and (iv) hyper-or hypo-reactivity to sensory input or
unusual interest in sensory aspects of environment.
10. The method according to claim 1, wherein the ASD-associated
disorder is Fragile X syndrome.
11. The method according to claim 1, wherein the ASD-associated
disorder is Rett syndrome.
12. The method according to claim 1, wherein the ASD-associated
disorder is Angelman syndrome.
13.-32. (canceled)
33. A method of treating schizophrenia in a subject in need
thereof, comprising administering an effective amount of
cannabidivarin (CBDV) to the subject.
34. A method of treating cognitive dysfunction in a subject in need
thereof, comprising administering an effective amount of
cannabidivarin (CBDV) to the subject.
35. The method according to claim 6, wherein the ASD-associated
disorder is Fragile X syndrome.
36. The method according to claim 6, wherein the ASD-associated
disorder is Rett syndrome.
37. The method according to claim 6, wherein the ASD-associated
disorder is Angelman syndrome.
38. The method according to claim 2, wherein the (a) marked
impairment in the use of multiple nonverbal behaviours is one or
more of eye-to-eye gaze, facial expression, body postures, and
gestures to regulate social interaction.
39. The method according to claim 2, wherein the (c) a lack of
spontaneous seeking to share enjoyment, interests, or achievements
with other people is a lack of showing, bringing, or pointing out
objects of interest.
40. The method according to claim 4, wherein the (c) stereotyped
and repetitive motor mannerisms are one or more of hand or finger
flapping or twisting or complex whole-body movements.
Description
[0001] This application is a continuation application of
International Application No. PCT/GB2017/051007, filed on Apr. 11,
2017, which claims the benefit of priority of British Patent
Application No. GB 1606098.0, filed on Apr. 11, 2016. The contents
of these applications are each incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the use of cannabidivarin
(CBDV) in the treatment of autism spectrum disorder (ASD) and
ASD-associated disorders such as Fragile X syndrome (FXS); Rett
syndrome (RS); or Angelman syndrome (AS). In a further embodiment
the invention relates to the use of CBDV in the treatment of
schizophrenia.
[0003] CBDV has been shown to be particularly effective in
improving cognitive dysfunction in rodent models of ASD, FXS, RS,
AS and schizophrenia.
[0004] The CBDV is preferably substantially pure. It may take the
form of a highly purified extract of cannabis such that the CBDV is
present at greater than 95% of the total extract (w/w) and the
other components of the extract are characterised. Alternatively,
the CBDV is synthetically produced.
[0005] Alternatively the CBDV may be used as a botanical drug
substance (BDS) from a cannabis plant in which CBDV is the
predominant cannabinoid. The CBDV may also be present in
combination with other cannabinoids and non-cannabinoid components
such as terpenes.
[0006] In yet a further embodiment the CBDV may be present with one
or more other cannabinoids such as CBD and/or CBDA in defined
ratios in which the CBDV is the predominant cannabinoid.
[0007] In use the CBDV may be used concomitantly with one or more
other medicaments. The CBDV may be formulated for administration
separately, sequentially or simultaneously with one or more
medicaments or the combination may be provided in a single dosage
form. Where the CBDV is formulated for administration separately,
sequentially or simultaneously it may be provided as a kit or
together with instructions to administer the one or more components
in the manner indicated. It may also be used as the sole
medication, i.e. as a monotherapy.
BACKGROUND TO THE INVENTION
[0008] Autism spectrum disorder (ASD) is a condition that presents
in children usually before three years of age and is characterized
by a lack of social interaction, communication, interests and
problems with behaviour.
[0009] The condition is relatively common as it is estimated that 1
in 100 children have some form of ASD. The condition is more
prevalent in boys than girls.
[0010] In children under four the signs and symptoms of ASD include
those in the area of spoken language such as delayed speech
development (for example, speaking less than 50 different words by
the age of two), or not speaking at all; frequent repetition of set
words and phrases; speech that sounds very monotonous or flat and
preferring to communicate using single words, despite being able to
speak in sentences.
[0011] When responding to others these younger children will often
not respond to their name being called, despite having normal
hearing; reject cuddles initiated by a parent; react unusually
negatively when asked to do something by someone else.
[0012] When interacting with others younger children show signs of
not being aware of other people's personal space, or being
unusually intolerant of people entering their own personal space;
little interest in interacting with other people, including
children of a similar age; not enjoying situations that most
children of their age like, such as birthday parties; preferring to
play alone, rather than asking others to play with them; rarely
using gestures or facial expressions when communicating; and
avoiding eye contact.
[0013] Symptoms involving behaviour in pre-school children include:
having repetitive movements, such as flapping their hands, rocking
back and forth, or flicking their fingers; playing with toys in a
repetitive and unimaginative way, such as lining blocks up in order
of size or colour, rather than using them to build something;
preferring to have a familiar routine and getting very upset if
there are changes to this routine; having a strong like or dislike
of certain foods based on the texture or colour of the food as much
as the taste; and unusual sensory interests for example, children
with ASD may sniff toys, objects or people inappropriately.
[0014] In older school age children some of the signs and symptoms
of ASD are similar to those experienced by younger children and
also include other symptoms. With spoken language an older child
with ASD often prefers to avoid using spoken language; has speech
that sounds very monotonous or flat; may speak in pre-learned
phrases, rather than putting together individual words to form new
sentences; may seem to talk "at" people, rather than sharing a
two-way conversation.
[0015] When responding to others, school age children with ASD
often take people's speech literally and are unable to understand
sarcasm, metaphors or figures of speech they may also react
unusually negatively when asked to do something by someone
else.
[0016] When interacting with others school age children with ASD
may not be aware of other people's personal space, or are unusually
intolerant of people entering their own personal space; show little
interest in interacting with other people, including children of a
similar age, or have few close friends, despite attempts to form
friendships; not understand how people normally interact socially,
such as greeting people or wishing them farewell; are unable to
adapt the tone and content of their speech to different social
situations for example, speaking very formally at a party and then
speaking to total strangers in a familiar way; not enjoy situations
and activities that most children of their age enjoy; rarely use
gestures or facial expressions when communicating; and avoid eye
contact.
[0017] With respect to the problems with behaviour that older
children with ASD experience these include repetitive movements,
such as flapping their hands, rocking back and forth, or flicking
their fingers; playing in a repetitive and unimaginative way, often
preferring to play with objects rather than people; developing a
highly specific interest in a particular subject or activity;
preferring to have a familiar routine and getting very upset if
there are changes to their normal routine; having a strong like or
dislike of certain foods based on the texture or colour of the food
as much as the taste; unusual sensory interests for example,
children with ASD may sniff toys, objects or people
inappropriately.
[0018] According to DSM-IV autism is diagnosed with three core
characteristics using the following criteria: A total of six (or
more) items from lists (1), (2), and (3), with at least two items
from list (1), and one item from each of lists (2) and (3).
[0019] List (1) qualitative impairment in social interaction, as
manifested by at least two of the following: [0020] a. marked
impairment in the use of multiple nonverbal behaviours, such as
eye-to-eye gaze, facial expression, body postures, and gestures to
regulate social interaction; [0021] b. failure to develop peer
relationships appropriate to developmental level; [0022] c. a lack
of spontaneous seeking to share enjoyment, interests, or
achievements with other people (e.g., by a lack of showing,
bringing, or pointing out objects of interest); and [0023] d. lack
of social or emotional reciprocity.
[0024] List (2) qualitative impairments in communication, as
manifested by at least one of the following: [0025] a. delay in, or
total lack of, the development of spoken language (not accompanied
by an attempt to compensate through alternative modes of
communication such as gesture or mime); [0026] b. in individuals
with adequate speech, marked impairment in the ability to initiate
or sustain a conversation with others; [0027] c. stereotyped and
repetitive use of language or idiosyncratic language; and [0028] d.
lack of varied, spontaneous make-believe play or social imitative
play appropriate to developmental level.
[0029] List (3) restricted, repetitive, and stereotyped patterns of
behaviour, interests, and activities as manifested by at least one
of the following: [0030] a. encompassing preoccupation with one or
more stereotyped and restricted patterns of interest that is
abnormal either in intensity or focus; [0031] b. apparently
inflexible adherence to specific, non-functional routines or
rituals; [0032] c. stereotyped and repetitive motor mannerisms
(e.g., hand or finger flapping or twisting or complex whole-body
movements); and [0033] d. persistent preoccupation with parts of
objects.
[0034] According to DSM-V which was published in May 2013, the new
diagnostic criteria for autism spectrum disorder is that the
patient must either currently, or by history, meet criteria from A,
B, C, and D as set out below. [0035] A. Persistent deficits in
social communication and social interaction across contexts, not
accounted for by general developmental delays, and manifest by all
3 of the following: [0036] 1. Deficits in social-emotional
reciprocity; which may range for example from abnormal social
approach and failure of normal back and forth conversation, to
reduced sharing of interest, emotions, or affect, to failure to
initiate or respond; [0037] 2. Deficits in communicative behaviours
used for social interaction; ranging for example, from poorly
integrated verbal and non-verbal communication, to abnormalities in
eye contact and body language or deficits in understanding and use
of gestures, to a total lack of facial expressions and
non-verbalisation; and [0038] 3. Deficits in developing,
maintaining and understanding relationships, ranging for example
from difficulties adjusting behaviour to suit various social
contexts, difficulties in sharing imaginative play or in making
friends, to absence of interest in peers. [0039] B. Restricted,
repetitive patterns of behaviour, interests, or activities as
manifested by at least two of the following: [0040] 1. Stereotyped
or repetitive motor movements, use of objects, or speech, (such as
simple motor stereotypies, lining up toys or flipping plates,
echolalia, idiosyncratic phrases); [0041] 2. Insistence on
sameness, inflexible adherence to routines, or ritualized patterns
of verbal or non-verbal behaviour (such as extreme distress at
small changes, difficulties with transitions, rigid thinking
patterns, greeting rituals, need to take same route or eat same
food every day); [0042] 3. Highly restricted, fixated interests
that are abnormal in intensity or focus (such as strong attachment
to or preoccupation with unusual objects, excessively circumscribed
or perseverative interests); and [0043] 4. Hyper- or
hypo-reactivity to sensory input or unusual interest in sensory
aspects of the environment (such as apparent indifference to
pain/temperature, adverse response to specific sounds or textures,
excessive smelling or touching of objects, visual fascination with
lights or movement). [0044] C. Symptoms must be present in early
childhood but may not become fully manifest until social demands
exceed limited capacities. [0045] D. Symptoms together limit and
impair everyday functioning.
[0046] In certain genetic syndromes there is a strong prevalence of
ASD or characteristics of ASD. Such syndromes can be said to be
ASD-associated disorders. Such genetic syndromes include: Tuberous
Sclerosis Complex, Fragile X syndrome, Cornelia de Lange syndrome,
Down syndrome, Angelman syndrome, Coffin-Lowry syndrome, Cohen
Laurence-Moon-Biedel syndrome, Marinesco-Sjogren syndrome, Moebius
syndrome, Phelan-McDermid syndrome, CDKL5, Dup15q, Potocki-Lupski
syndrome, Smith Lemli Optiz syndrome, Timothy syndrome,
Prader-Willi syndrome, Rett syndrome and Williams syndrome.
[0047] It has been suggested that the genes underlying those
syndromes in which ASD characteristics are very common, lead to
common differences at the biological and neurological level, which
in turn give rise to the presentation of ASD characteristics.
[0048] Fragile X syndrome (FXS) co-occurs with autism in many cases
and is the most common cause of inherited learning disability,
occurring in 1 in 3,600 males and 1 in 8,000 females. FXS is caused
by the presence of an apparently unstable or `fragile` site located
on the FMR1 gene on the X chromosome. The instability is caused by
an excess of genetic code in this region. Males with FXS typically
show mild to severe learning disability while females with FXS
usually have a mild learning disability.
[0049] Recent studies of individuals with FXS show a fairly
consistent pattern of association with ASD. The percentage of
individuals with FXS showing ASD characteristics or meeting ASD
criteria is up to 50%. Severe ASD is relatively rare in FXS and a
milder presentation of ASD-like features is more
characteristic.
[0050] Impairments in social interaction in FXS are characterised
by social anxiety, extreme shyness and eye gaze avoidance. These
characteristics are also observed in individuals with ASD. The
social impairments associated with FXS often increase as the
patient gets older.
[0051] The major symptom of FXS is intellectual disability with an
average IQ of 40 in males who have complete silencing of the FMR1
gene. The main difficulties in individuals with FXS are with
working and short-term memory, executive function, visual memory,
visual-spatial relationships, and mathematics, with verbal
abilities being relatively spared.
[0052] FXS sufferers also present with prominent characteristics
which may include an elongated face, large or protruding ears, flat
feet, larger testes (macro-orchidism), and low muscle tone.
[0053] FXS patients also suffer from recurrent middle ear infection
and sinusitis. Speech may be cluttered or nervous. Behavioural
characteristics may include stereotypic movements such as
hand-flapping and atypical social development, particularly
shyness, limited eye contact, memory problems, and difficulty with
face encoding. These features mean that individuals with FXS also
meet the diagnostic criteria for autism. Genetic mouse models of
FXS have also been shown to have autistic-like behaviours.
[0054] Attention deficit hyperactivity disorder (ADHD) is found in
the majority of males with FXS and 30% of females, making it the
most common psychiatric diagnosis in those with FXS. Hyperactivity
and disruptive behaviour peak in the preschool years and then
gradually decline with age, although inattentive symptoms are
generally lifelong.
[0055] From their 40s onward, males with FXS begin developing
progressively more severe problems in performing tasks that require
the central executive of working memory.
[0056] There is currently no drug treatment that has shown benefit
specifically for FXS. However, medications are commonly used to
treat symptoms of attention deficit and hyperactivity, anxiety, and
aggression.
[0057] Rett syndrome (RS) is a neurological disorder that is caused
by a mutation on the X chromosome. RS predominantly affects females
and occurs in 1 in 15,000 to 22,800 live female births. Typically,
development appears to be normal in the first six to eighteen
months but this is followed by a period of regression resulting in
a loss of language and motor skills, leading to severe or profound
learning and physical disabilities.
[0058] Autistic-like behaviours were noted in the very first
description of RS in 1966. Studies have since estimated that 25% to
40% of individuals with RS show ASD-like characteristics. ASD is
the most common misdiagnosis in children with RS, with many
individuals being diagnosed with ASD prior to receiving a diagnosis
of RS.
[0059] RS is caused by a mutation in the MECP2 gene which is found
on the X chromosome. The MECP2 gene codes for the MeCP2 protein
which is essential for brain development. Without this protein
nerve cells in the brain are prevented from developing
properly.
[0060] The symptoms associated with RS usually go unnoticed for the
first few months of a child's life. The symptoms then tend to
progress over several stages as outlined below.
[0061] Stage one consists of early signs and slow development,
these usually appear in the first six to twelve months of the
child's life. The symptoms include: a general slowness in
development; hypertonia; difficulty feeding; abnormal hand
movements; lack of interest in toys; and poor coordination of trunk
and limbs.
[0062] Stage two is known as the regression or rapid destruction
stage. This stage usually begins between the age of one and four
and may last for weeks or months. The child will develop severe
cognitive impairment. Problems arise with communication, language,
learning, co-ordination and brain functions. Signs at this stage
include: repetitive and uncontrollable hand movements; periods of
distress, irritability and screaming; social withdrawal;
unsteadiness when walking; rapid or slow breathing; problems
sleeping; small head size; difficulty eating and gastrointestinal
problems.
[0063] Many children with RS also start to suffer from epileptic
seizures at this stage; up to 80% of children with the syndrome
suffer from epilepsy at some stage of their illness.
[0064] Stage three is known as the plateau stage and usually begins
between the ages of two and ten. This stage can last for years
indeed many RS sufferers will remain in this stage for the majority
of their life. The prominent symptoms include: floppiness of limbs
and inability to move around; inability to use hands to hold, carry
or manipulate objects; repetitive hand movements; teeth grinding;
abnormal tongue movements; and lack of gain in bodyweight.
[0065] The final stage is characterized by deterioration in
movement. This stage can again last for years or even decades. The
main problems are caused by scoliosis of the spine; spasticity and
loss of the ability to walk.
[0066] The lifespan of a child born with RS is generally shortened
often due to life threatening seizures or arrhythmias.
[0067] There is no cure for RS however anti-epileptic medications
are often prescribed to control the seizures along with a high
calorie diet and physiotherapy to help control the symptoms.
[0068] Angelman syndrome (AS) occurs in approximately 1 in 12,000
to 15,000 individuals and is caused by abnormalities on chromosome
15. Individuals with AS typically show severe to profound learning
disability, significant difficulties with mobility and
communication in addition to seizures.
[0069] It has been suggested that between 50% and 80% of
individuals with AS meet the criteria for ASD.
[0070] Typical characteristics of Angelman syndrome include:
delayed development which is usually noticeable from 6-12 months of
age; severe language impairment with little or no speech; movement
and balance problems (ataxia); frequent seizures (epilepsy) in
around 85% of cases; a small head size (microcephaly); sociable
behaviour with frequent smiling.
[0071] A genetic anomaly responsible for AS which occurs by chance
around the time of conception. The UBE3A gene is either absent or
malfunctions. A child usually inherits one copy of the UBE3A gene
from each parent. Both copies are switched on (active) in most of
the body's tissues. However, in certain areas of the brain, only
the gene inherited from the mother is active. In most cases of AS
(about 70%), the child's maternal copy of the UBE3A gene is
missing, which means there's no active copy of the UBE3A gene in
the child's brain.
[0072] Schizophrenia is a psychiatric diagnosis that describes a
mental illness characterised by impairments in the perception or
expression of reality, most commonly manifesting as auditory
hallucinations, paranoid or bizarre delusions or disorganised
speech and thinking in the context of significant social,
occupational or cognitive dysfunction.
[0073] Schizophrenia is often described in terms of positive and
negative symptoms. Positive symptoms include delusions, auditory
hallucinations, and thought disorder, and are typically regarded as
manifestations of psychosis. Negative symptoms are so-named because
they are considered to be the loss or absence of normal traits or
abilities, and include features such as flat or blunted affect and
emotion, poverty of speech (alogia), anhedonia, and lack of
motivation (avolition). A third symptom grouping, the
disorganisation syndrome, includes chaotic speech, thought, and
behaviour. The disorder is also thought to affect cognition, which
also usually contributes to chronic problems with behaviour and
emotion.
[0074] Cognitive symptoms are often detected when
neuropsychological tests are performed on schizophrenia patients.
They include the following: poor "executive functioning" (the
ability to absorb and interpret information and make decisions
based on that information); inability to sustain attention; and
problems with "working memory" (the ability to keep recently
learned information in mind and use it right away). Such cognitive
dysfunction often interferes with the patient's ability to lead a
normal life and earn a living. They can cause great emotional
distress.
[0075] Treatment for the positive symptoms of schizophrenia is
usually with antipsychotic medications; however the negative and
cognitive symptoms remain largely untreated.
[0076] Testing compounds for their effectiveness on signs and
symptoms of ASD, ASD-associated disorders and schizophrenia is
challenging given that these disorders have so many different
affected symptom domains.
[0077] The rodent valproic acid model is a widely accepted model of
ASD. Rat foetuses are exposed to valproic acid on the 12.5th day of
gestation to produce VPA rats. The VPA rats present behavioural
aberrations observed in autism such as delayed maturation, lower
body weight, delayed motor development, and attenuated integration
of a coordinated series of reflexes, delayed nest-seeking response
mediated by olfactory system, and normal negative geotaxis.
[0078] Additionally there are particular animal models which can be
used to test particular syndromes which present with ASD like
characteristics such as FXS, RS and AS or ASD-associated
disorders.
[0079] Male patients with FXS lack the FMR1 protein due to
silencing of the FMR1 gene by amplification of a CGG repeat and
subsequent methylation of the promoter region. A knockout model for
FXS in mice is a well-known model used to test compounds for their
effectiveness in the treatment of FXS. Mice lack normal FMR1
protein and show macro-orchidism, learning deficits, and
hyperactivity.
[0080] The MeCP2 knockout mouse model is able to evaluate the
effectiveness of a treatment the symptoms that present in RS. Mice
lacking the MeCP2 gene show severe neurological symptoms at
approximately six weeks of age. After several months, heterozygous
female mice also show autism like behavioural symptoms.
[0081] The UBE3A mouse model is used to evaluate a compounds
effectiveness in the treatment of AS. This model has been shown to
recapitulate many of the phenotypic features of AS, including motor
dysfunction, increased seizure susceptibility, and
hippocampal-dependent learning and memory deficits in mice with the
knockout gene.
[0082] The phencyclidine (PCP) model is used to evaluate the
effectiveness of a compound on cognitive dysfunction in
schizophrenia. PCP is a non-competitive N-methyl-D-aspartate (NMDA)
receptor antagonist, and reproduces a schizophrenia-like psychosis
including positive symptoms, negative symptoms and cognitive
dysfunction. PCP-treated animals exhibit hyper-locomotion as an
index of positive symptoms, and a social behavioural deficit in a
social interaction test and enhanced immobility in a forced
swimming test as indices of negative symptoms. They also show a
sensorimotor gating deficit and cognitive dysfunctions in several
learning and memory tests such as the Novel Object Recognition
(NOR) test.
[0083] The NOR test is used to evaluate cognition, particularly
recognition memory, in rodent models of CNS disorders, such as ASD,
ASD-associated disorders and schizophrenia. The test is based on
the tendency of rodents to spend more time exploring a novel object
than a familiar one. The choice to explore the novel object
reflects the use of learning and recognition memory.
[0084] The NOR test is conducted in an open field arena with two
different kinds of objects. Both objects are generally consistent
in height and volume, but are different in shape and appearance.
During habituation, the animals are allowed to explore an empty
arena. Twenty-four hours after habituation, the animals are exposed
to the familiar arena with two identical objects placed at an equal
distance. The next day, the mice are allowed to explore the open
field in the presence of the familiar object and a novel object to
test short-term and long-term recognition memory. The time spent
exploring each object and the discrimination index percentage is
recorded. This test is useful for assessing cognitive dysfunction
in rodent models of ASD, FXS, RS, AS and schizophrenia.
[0085] The Food and Drug Administration (FDA) has approved two
drugs for treating irritability associated with the autism
(risperidone and aripiprazole) which are both antipsychotic
medications. However, there are currently no approved medications
for treating autism's core characteristics. Antipsychotics can ease
core symptoms to some extent, for example relieving irritability
often improves sociability, reduces tantrums, aggressive outbursts
and self-injurious behaviour. The disadvantages associated with
antipsychotics are that this class of medicaments is known to have
side effects including severe weight gain, stiffness and shakiness.
Accordingly it would be desirable to provide a more effective
medication able to treat the core characteristics of ASD and offer
an improved side effect profile.
[0086] The endocannabinoid system has been linked to physiological
progression of autism spectrum disorders, possibly implicating CB1
and CB2 receptors.
[0087] The phytocannabinoids are known to interact with the
endocannabinoid system.
[0088] The phytocannabinoid tetrahydrocannabinol (THC) in the form
of dronabinol, a CB1 agonist, has been used to treat an autistic
child (Kurz and Blass, 2010). Problems associated with the use of
CB1 agonists are psychoactivity, anxiety and hallucinations.
[0089] Patent applications GB 2,492,487 and GB 2,434,312 describe
the use of cannabinoids in the treatment of neurodegenerative
diseases and disorders. Furthermore patent application WO
2006/017892 describes the use of CBD in the treatment of
schizophrenia.
[0090] Patent application WO 2014/146699 describes the use of CB1
receptor antagonists in the treatment of diseases associated with
dendritic abnormalities. Such diseases include AS and RS. The
application is exemplified by the use of rimonabant in the FMR1
knockout mouse model which is a model of FXS.
[0091] The CB1 antagonist, rimonabant, has been shown to have
serious side effects such as suicide ideation which limit its
use.
[0092] The present application relates to the use of a
phytocannabinoid which is neither a CB1 agonist nor antagonist.
Cannabidivarin (CBDV) is known to have a low affinity for the CB1
receptor. Hill et al. (2013) demonstrated that a plant extract
comprising CBDV showed greater affinity for CB1 cannabinoid
receptors than purified CBDV in both MF1 mouse brain and hCB1-CHO
cell membranes; however neither bound with high enough affinity to
be described as an agonist or antagonist.
[0093] CBDV has been shown to be effective in animal models of
seizure (Hill et al., 2012) and WO 2011/121351.
[0094] To date there are no studies of the use of CBDV in the
treatment of ASD or ASD-associated disorders such as FXS, RS and AS
or schizophrenia. Such symptoms as described above are difficult to
treat, therefore many patients with ASD or ASD-associated disorders
such as FXS, RS and AS and schizophrenia have unmet needs with
respect to the treatment of their disease.
BRIEF SUMMARY OF THE DISCLOSURE
[0095] In accordance with a first aspect of the present invention
there is provided Cannabidivarin (CBDV) for use in the treatment of
one or more symptoms or disease characteristics associated with
autistic spectrum disorder (ASD) or ASD-associated disorders, as
defined by DSM-IV, wherein the symptoms or disease characteristic
is one or more selected from the group consisting of: (i)
qualitative impairment in social interaction; (ii) qualitative
impairment in communication; and (iii) restricted repetitive and
stereotyped patterns of behaviour interest and activities.
[0096] Preferably the symptoms or disease characteristics of
qualitative impairment in social interaction include one or more
of: (a) marked impairment in the use of multiple nonverbal
behaviours, such as eye-to-eye gaze, facial expression, body
postures, and gestures to regulate social interaction; (b) failure
to develop peer relationships appropriate to developmental level;
(c) a lack of spontaneous seeking to share enjoyment, interests, or
achievements with other people (e.g., by a lack of showing,
bringing, or pointing out objects of interest); and (d) lack of
social or emotional reciprocity.
[0097] Preferably the symptoms or disease characteristics of
qualitative impairment in communication include one or more of: (a)
delay in, or total lack of, the development of spoken language (not
accompanied by an attempt to compensate through alternative modes
of communication such as gesture or mime); (b) in individuals with
adequate speech, marked impairment in the ability to initiate or
sustain a conversation with others; (c) stereotyped and repetitive
use of language or idiosyncratic language; and (d) lack of varied,
spontaneous make-believe play or social imitative play appropriate
to developmental level.
[0098] Preferably the symptoms or disease characteristics of
restricted repetitive and stereotyped patterns of behaviour
interest and activities include one or more of: (a) encompassing
preoccupation with one or more stereotyped and restricted patterns
of interest that is abnormal either in intensity or focus; (b)
apparently inflexible adherence to specific, non-functional
routines or rituals; (c) stereotyped and repetitive motor
mannerisms (e.g., hand or finger flapping or twisting or complex
whole-body movements); and (d) persistent preoccupation with parts
of objects.
[0099] Preferably the symptoms or characteristics associated with
of autistic spectrum disorder comprise at least two symptoms
associated with qualitative impairment in social interaction; at
least one symptom associated with qualitative impairment in
communication and at least one symptom associated with restricted
repetitive and stereotyped patterns of behaviour interest and
activities.
[0100] In accordance with a second aspect of the present invention
there is provided Cannabidivarin (CBDV) for use in the treatment of
autistic spectrum disorder (ASD) or ASD-associated disorders as
defined by DSM-V, wherein the symptoms or disease characteristic is
one or more selected from the group consisting of: (a) persistent
deficits in social communication and social interaction across
contexts, not accounted for by general developmental delays, and
(b) restricted, repetitive patterns of behaviour, interests, or
activities.
[0101] Preferably the symptoms or disease characteristics of (a)
persistent deficits in social communication and social interaction
across contexts, not accounted for by general developmental delays
include one or more of: (i) deficits in social-emotional
reciprocity; (ii) deficits in nonverbal communicative behaviours
used for social interaction; and (iii) deficits in developing and
maintaining relationships.
[0102] Preferably the symptoms or disease characteristics of (b)
restricted, repetitive patterns of behaviour, interests, or
activities include one or more of: (i) stereotyped or repetitive
speech, motor movements, or use of objects; excessive adherence to
routines, (ii) ritualized patterns of verbal or nonverbal
behaviour, or excessive resistance to change; (iii) highly
restricted, fixated interests that are abnormal in intensity or
focus; and (iv) hyper-or hypo-reactivity to sensory input or
unusual interest in sensory aspects of environment.
[0103] Preferably the symptoms or disease characteristics
associated with of autistic spectrum disorder comprise all three
symptoms associated with (a) persistent deficits in social
communication and social interaction across contexts, not accounted
for by general developmental delays together with at least two of
(b) restricted, repetitive patterns of behaviour, interests, or
activities.
[0104] In this aspect, treatment of ASD and ASD-associated
disorders encompass the treatment of the condition as a whole as
opposed to the individual symptoms. Accordingly the present
invention does not encompass CBDV for use in the treatment of
seizures.
[0105] Preferably the ASD-associated disorder is taken from the
group: Fragile X syndrome; Rett syndrome; or Angelman syndrome.
[0106] In accordance with a third aspect of the present invention
there is provided Cannabidivarin (CBDV) for use in the treatment of
schizophrenia.
[0107] Preferably the CBDV is for use in the treatment of positive
or negative symptoms associated with schizophrenia. In particular
the CBDV is particularly useful in the treatment of the negative
symptoms associated with social withdrawal.
[0108] In accordance with a fourth aspect of the present invention
there is provided Cannabidivarin (CBDV) for use in the treatment of
cognitive dysfunction.
[0109] Preferably the cognitive dysfunction occurs in patients with
ASD, ASD-associated disorders or schizophrenia.
[0110] In one embodiment treatment of the cognitive dysfunction is
associated with the treatment of memory. Preferably the treatment
is of short term memory and/or long term memory.
[0111] In a further embodiment the CBDV is for use in combination
with one or more concomitant medicaments which may be taken by the
patient to treat the condition and/or one or more symptoms
associated therewith. Such as, for example, melatonin for sleeping
problems, SSRI for depression, anticonvulsants for epilepsy,
methylphenidate for ADHD or antipsychotics for aggression or
self-harming behaviour. In this respect the CBDV of the present
invention is not used to treat seizures.
[0112] Preferably the one or more concomitant medicament is an
anti-epileptic drug (AED). The AED may be the cannabinoid CBD and
as such a combination of CBDV and CBD may be used.
[0113] In a further embodiment the CBDV is substantially pure. The
CBDV may be present as a highly purified extract of cannabis which
comprises at least 95% (w/w) CBDV. Preferably the extract comprises
less than 0.15% THC.
[0114] In an alternative embodiment the CBDV is present as a
synthetic compound.
[0115] Alternatively the CBDV may be used as a botanical drug
substance (BDS) from a cannabis plant in which CBDV is the
predominant cannabinoid. The CBDV may also be present in
combination with other cannabinoids and non-cannabinoid components
such as terpenes.
[0116] In yet a further embodiment the CBDV may be present with one
or more other cannabinoids such as CBD and/or CBDA in defined
ratios in which the CBDV is the predominant cannabinoid.
Determining an effective dose in humans will depend on, for example
the mode of delivery (i.v. or oral), the formulation and the
bioavailability of the CBDV when delivered and might range between
0.01 and 100 mg/kg/day. Furthermore the fact that cannabinoids
often show bell-shaped dose response curves makes determining a
dose of CBDV more difficult.
[0117] Preferably the dose of CBDV is greater than 0.01 mg/kg/day.
Thus for a 15 kg patient a dose of greater than 0.15 mg of CBDV per
day would be provided. Doses greater than 0.1 mg/kg/day, such as
greater than 1 mg/kg/day, such as greater than 5 mg/kg/day, greater
than 10 mg/kg/day, greater than 15 mg/kg/day and greater than 20
mg/kg/day are also envisaged to be effective.
[0118] In use the CBDV may be effective in a therapeutic amount of
between 1 to 30 mg/kg/day and it may also be administered as an
oral formulation.
[0119] Preferably the CBDV is provided over an extended period;
more preferably this period is at least seven days.
[0120] In a further embodiment the CBDV may be used as a dietary
supplement or food additive in order to improve symptoms in ASD,
ASD-associated conditions or schizophrenia.
[0121] In accordance with a fifth aspect of the present invention
there is provided a method of treating one or more symptoms or
disease characteristics associated with autistic spectrum disorder
(ASD) or ASD-associated disorders in a subject, as defined by
DSM-IV, wherein the symptoms or disease characteristic is one or
more selected from the group consisting of: qualitative impairment
in social interaction; qualitative impairment in communication; and
restricted repetitive and stereotyped patterns of behaviour
interest and activities, comprising administering an effective
amount of cannabidivarin (CBDV) to the subject in need thereof.
Preferably the subject is a human.
[0122] In accordance with a sixth aspect of the present invention
there is provided a method of treating one or more symptoms or
disease characteristics associated with autistic spectrum disorder
(ASD) or ASD-associated disorders in a subject, as defined by
DSM-V, wherein the symptoms or disease characteristic is one or
more selected from the group consisting of: (a) persistent deficits
in social communication and social interaction across contexts, not
accounted for by general developmental delays, and (b) restricted,
repetitive patterns of behaviour, interests, or activities,
comprising administering an effective amount of cannabidivarin
(CBDV) to the subject in need thereof.
[0123] Preferably the ASD-associated disorder is taken from the
group: Fragile X syndrome; Rett syndrome; or Angelman syndrome.
[0124] In accordance with a seventh aspect of the present invention
there is provided a method of treating schizophrenia in a subject
comprising administering an effective amount of cannabidivarin
(CBDV) to a subject in need thereof. Preferably the subject is a
human.
[0125] In accordance with an eighth aspect of the present invention
there is provided a method of treating cognitive dysfunction
comprising administering cannabidivarin (CBDV) to a subject in need
thereof. Preferably the subject is a human.
[0126] The human dose equivalent (HED) can be estimated using the
following formula:
HED = Animal dose ( mg / kg ) multiplied by Animal K m Human K m
##EQU00001##
The K.sub.m for a mouse is 3, for a rat the K.sub.m is 6 and the
K.sub.m for a human is 37.
DEFINITIONS
[0127] Definitions of some of the terms used to describe the
invention are detailed below:
[0128] The cannabinoids described in the present application are
listed below along with their standard abbreviations.
TABLE-US-00001 TABLE 1 Cannabinoids and their abbreviations CBD
Cannabidiol ##STR00001## CBDV Cannabidivarin ##STR00002## THC
Tetrahydrocannabinol ##STR00003##
[0129] The table above is not exhaustive and merely details the
cannabinoids which are identified in the present application for
reference. So far over 60 different cannabinoids have been
identified and these cannabinoids can be split into different
groups as follows: Phytocannabinoids; Endocannabinoids and
Synthetic cannabinoids (which may be novel cannabinoids or
synthetically produced phytocannabinoids or endocannabinoids).
[0130] "Phytocannabinoids" are cannabinoids that originate from
nature and can be found in the cannabis plant. The
phytocannabinoids can be isolated from plants to produce a highly
purified extract or can be reproduced synthetically.
[0131] "Substantially pure CBDV" is defined as CBDV that is 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.
[0132] "Highly purified cannabinoid extracts" are defined as
cannabinoids that have been extracted from the cannabis plant and
purified to the extent that other cannabinoids and non-cannabinoid
components that are co-extracted with the cannabinoids have been
substantially removed, such that the highly purified cannabinoid is
greater than or equal to 95% (w/w) pure.
[0133] "Botanical drug substance" or "(BDS)" is defined in the
Guidance for Industry Botanical Drug Products Draft Guidance,
August 2000, US Department of Health and Human Services, Food and
Drug Administration Centre for Drug Evaluation and Research as: "A
drug derived from one or more plants, algae, or microscopic fungi.
It is prepared from botanical raw materials by one or more of the
following processes: pulverisation, decoction, expression, aqueous
extraction, ethanolic extraction or other similar processes." A
botanical drug substance does not include a highly purified or
chemically modified substance derived from natural sources. Thus,
in the case of cannabis, BDS derived from cannabis plants do not
include highly purified Pharmacopoeial grade cannabinoids. In a BDS
comprising cannabinoids the cannabinoid will be present in an
amount of less than 95% (w/w).
[0134] "Synthetic cannabinoids" are compounds that have a
cannabinoid or cannabinoid-like structure and are manufactured
using chemical means rather than by the plant.
[0135] Phytocannabinoids can be obtained as either the neutral
(decarboxylated form) or the carboxylic acid form depending on the
method used to extract the cannabinoids. For example it is known
that heating the carboxylic acid form will cause most of the
carboxylic acid form to decarboxylate into the neutral form.
[0136] "Cognitive dysfunction" is defined as the loss of
intellectual functions such as thinking, remembering, and reasoning
with sufficient severity to interfere with daily functioning.
Patients with cognitive dysfunction have trouble with verbal
recall, basic arithmetic, and concentration.
[0137] An ASD-associated disorder is defined as genetic syndromes
where there is a strong prevalence of ASD or characteristics of
ASD. Such genetic syndromes include: Tuberous Sclerosis Complex,
Fragile X syndrome, Cornelia de Lange syndrome, Down syndrome,
Angelman syndrome, Coffin-Lowry syndrome, Cohen
Laurence-Moon-Biedel syndrome, Marinesco-Sjogren syndrome, Moebius
syndrome, Phelan-McDermid syndrome, CDKL5, Dup15q, Potocki-Lupski
syndrome, Smith Lemli Optiz syndrome, Timothy syndrome,
Prader-Willi syndrome, Rett syndrome and Williams syndrome.
[0138] A symptom or disease characteristic associated with ASD or
ASD-associated disorders are defined as the diagnostic criteria as
defined by either DSM-IV or DSM-V as described above (under the
section "Background to the Invention").
BRIEF DESCRIPTION OF THE DRAWINGS
[0139] Embodiments of the invention are further described
hereinafter with reference to the accompanying drawings, in
which
[0140] FIG. 1A shows the effect of CBDV on sociability and social
recognition deficits in the rat VPA model of general autism;
[0141] FIG. 1B shows the effect of CBDV on sociability and social
recognition deficits in the rat VPA model of general autism;
[0142] FIG. 10 shows the effect of CBDV treatment on social novelty
preference in offspring of VPA- and vehicle-exposed rats, as
measured through the three chamber apparatus.
[0143] FIG. 2A shows the effect of CBDV on repetitive behaviours,
hyperactivity, cognitive deficits and biomarkers in the rat VPA
model of general autism;
[0144] FIG. 2B shows the effect of CBDV on repetitive behaviours,
hyperactivity, cognitive deficits and biomarkers in the rat VPA
model of general autism;
[0145] FIG. 2C shows the effect of CBDV on repetitive behaviours,
hyperactivity, cognitive deficits and biomarkers in the rat VPA
model of general autism;
[0146] FIG. 2D shows the effect of CBDV on repetitive behaviours,
hyperactivity, cognitive deficits and biomarkers in the rat VPA
model of general autism;
[0147] FIG. 3A shows the effect of CBDV on discrimination index
after acute and chronic treatment in the mouse model of fragile X
syndrome;
[0148] FIG. 3B shows the effect of CBDV on discrimination index
after acute and chronic treatment in the mouse model of fragile X
syndrome;
[0149] FIG. 4A shows the effect of CBDV on bodyweight in a mouse
model of Rett syndrome;
[0150] FIG. 4B shows the effect of CBDV on bodyweight in a mouse
model of Rett syndrome;
[0151] FIG. 5 shows the effect of CBDV on survival in a mouse model
of Rett syndrome;
[0152] FIG. 6A shows the effect of CBDV on symptoms in a mouse
model of Rett syndrome;
[0153] FIG. 6B shows the effect of CBDV on symptoms in a mouse
model of Rett syndrome;
[0154] FIG. 6C shows the effect of CBDV on symptoms in a mouse
model of Rett syndrome;
[0155] FIG. 6D shows the effect of CBDV on symptoms in a mouse
model of Rett syndrome;
[0156] FIG. 7A shows the effect of CBDV on symptoms in a mouse
model of Rett syndrome;
[0157] FIG. 7B shows the effect of CBDV on symptoms in a mouse
model of Rett syndrome;
[0158] FIG. 8 shows the effect of CBDV on total symptom score in in
a mouse model of Rett syndrome;
[0159] FIG. 9 shows the effect of CBDV on short-term memory in a
mouse model of Rett syndrome;
[0160] FIG. 10 shows the effect of CBDV on long-term memory in a
mouse model of Rett syndrome;
[0161] FIG. 11 shows the effect of CBDV on clasping duration in a
mouse model of Angelman syndrome;
[0162] FIG. 12 shows the effect of CBDV in the rotarod test in a
mouse model of Angelman syndrome;
[0163] FIG. 13 shows the effect of CBDV in the novel object
recognition test in a mouse model of Angelman syndrome;
[0164] FIG. 14 shows the effect of CBDV in the tail suspension test
in a mouse model of Angelman syndrome;
[0165] FIG. 15 shows the effect of CBDV on audiogenic seizures in a
mouse model of Angelman syndrome;
[0166] FIG. 16 shows the effect of CBDV in the novel object
recognition test in a rat model of schizophrenia;
[0167] FIG. 17 shows the effect of CBDV on the discrimination index
in the novel object recognition test in a rat model of
schizophrenia, data is expressed as **P<0.01; Significant
reduction in DI compared to vehicle, # P<0.05-## P<0.01;
Significant reversal of the reduction in DI compared to PCP;
and
[0168] FIG. 18 shows the effect of CBDV on social interaction in a
rat model of schizophrenia.
[0169] For all figures, except FIG. 17, data are expressed as mean
.+-.S.E.M. *p<0.05, ***p<0.001 vs WT-vehicle;
.sup..smallcircle..smallcircle.p<0.01,
.sup..smallcircle..smallcircle..smallcircle.p<0.001 vs
KO-vehicle. Two-way ANOVA followed by Bonferroni post hoc test.
DETAILED DESCRIPTION
Example 1: Use of Cannabidivarin (CBDV) in a Mouse Model of Autism
Spectrum Disorder
[0170] The phytocannabinoid cannabidivarin (CBDV) was evaluated in
a rodent model of autism spectrum disorder (ASD).
[0171] In utero exposure of rodents to valproic acid (VPA) has been
shown to induce a phenotype with behavioural characteristics
similar to those observed in ASD and provides a robust animal model
for social cognitive impairment understanding and a potential
screen for the development of novel therapeutics for this condition
(Foley et al. 2012).
[0172] Thus, in utero exposure to VPA has been used as a reliable
model to increase the understanding of behavioural effects
evaluated by specific tests as sociability, social preference and
stereotypic behaviour, also observed in human patients (Schneider
and Przewlocki, 2005).
[0173] Example 1 describes the use of prenatal VPA exposure in rats
to evaluate the efficacy of chronic CBDV administration in
reversing the autism-like behaviours present in this model.
Materials and Methods
Prenatal VPA Administration
[0174] Pregnant Sprague-Dawley rats (Charles River, Calco, Italy),
received a single intraperitoneal injection of 500 mg/kg sodium
valproate on the 12.5 day after conception, and control females
were injected with physiological saline at the same time. Sodium
valproate (Sigma Aldrich, Milan, IT) was dissolved in saline at a
concentration of 250 mg/ml.
[0175] Females were housed individually and were allowed to raise
their own litters. The offspring was weaned on postnatal day (PND)
21, separated by sex and the animals were kept four to a cage, with
controlled temperature and light conditions. Rats had free access
to food (standard laboratory pellets) and water. All the
experiments were performed in the light phase between 09:00 and
15:00.
CBDV Treatment
[0176] CBDV was dissolved in ethanol, cremophor and saline
(1:1:18). Symptomatic treatment with CBDV at doses of 0.2, 2, 20,
and 100 mg/kg/day i.p. was performed starting from PND 34 (early
adolescence) till 56 (early adulthood), both in male and female
offspring of dams injected with VPA 500 mg/kg (or vehicle) on day
12.5 after conception. At PND 56, a series of behavioural tests was
performed in order to assess the effect of chronic CBDV on
sociability, social novelty, short-term memory, locomotion and
stereotyped/repetitive behaviours.
Behavioural Studies
[0177] Locomotor activity and repetitive behaviours: Locomotor
activity was recorded in an activity cage for 20 minutes with the
aid of Anymaze program (Ugo Basile, Italy). In this period,
repetitive behaviours (self-grooming and digging) were measured by
an observer blind to the treatment group.
[0178] Sociability and preference for social novelty: These
behaviours were investigated in a 3-chamber apparatus which allows
for the measurement of social approach and social preference. In
brief, animals were placed into a novel arena (80 cm.times.31.5 cm)
composed of three communicating chambers separated by Perspex walls
with central openings allowing access to all chambers for 5
min.
[0179] Distance moved (m) and time spent (s) in the various
compartments was assessed during this time to evaluate general
locomotor activity and ensure that animals did not have a
preference for a particular side of the arena.
[0180] Following this acclimatisation period, animals were briefly
confined to the central chamber while an unfamiliar rat confined in
a small wire cage was placed in one of the outer chambers. An
identical empty wire cage was placed in the other chamber. The
unfamiliar rat was randomly assigned to either the right or left
chamber of the arena. The test animal was then allowed to explore
the arena/chambers for a further 5 min. Time spent engaging in
investigatory behaviour with the rat was evaluated with the aid of
Anymaze program (Ugo Basile, Italy) in order to examine social
approach.
[0181] To investigate the preference for social novelty, a novel
unfamiliar rat was then placed in the empty cage and the test
animal was then allowed to explore the arena/chambers for a further
5 min. Time spent engaging in investigatory behaviour with the
novel unfamiliar rat was evaluated with the aid of Anymaze program
(Ugo Basile, Italy) in order to examine preference for social
novelty.
[0182] Short-term memory: The experimental apparatus used for the
object recognition test was an open-field box (43.times.43.times.32
cm) made of Plexiglas, placed in a dimly illuminated room. The
experiment was performed and analysed as previously described
(Zamberletti et al, 2014). Animals performed each test
individually.
[0183] Each animal was placed in the arena and allowed to explore
two identical previously unseen objects for 5 minutes
(familiarization phase). After an inter-trial interval of 3 minutes
one of the two familiar objects was replaced by a novel, previously
unseen object and rats were returned to the arena for the 5-minute
test phase. During the test phase the time spent exploring the
familiar object (Ef) and the new object (En) was videotaped and
recorded separately by two observers blind to the treatment groups
and the discrimination index was calculated as follows:
[(En-Ef)/(En+Ef)].times.100.
Neuroinflammation Studies
[0184] At the end of the study, on PND 59, male rats treated with
CBDV 20 mg/kg i.p. were sacrificed and the hippocampus was
collected 24 hours after the last CBDV injection and stored at
-80.degree. C. for evaluation of changes in protein expression of
components of the endocannabinoid system (CB1 and CB2 receptors,
the endocannabinoid degrading enzymes, FAAH and MAGL and the
synthetic enzymes, DAGL.alpha. and NAPE-PLD); neuroinflammatory
markers (GFAP (astrocytic marker), Iba1 (microglial marker), CD11 b
(activated microglial marker), TNF-.alpha. (proinflammatory
cytokine) and iNOS (inflammatory mediator)); synaptic markers
(Synaptophysin (synaptic vesicle protein), PSD-95 (post synaptic
density protein) and the neurotrophins, BDNF and IGF by Western
blotting.
[0185] Statistical analysis: Data were expressed as mean .+-..
Results were analysed by unpaired Student's t test or two-way
ANOVA, followed up by Bonferroni's post hoc test. The level of
statistical significance was set at p<0.05.
Results
[0186] FIG. 1A shows the effect of the different doses of CBDV
treatment on social novelty preference in offspring of VPA- and
vehicle-exposed rats, as measured through the three chamber
apparatus. CBDV at 20 mg/kg was demonstrated to have a
statistically significant effect (p<0.001 in males and p<0.01
in females) on restoration of social novelty preference.
[0187] During the habituation phase, no differences in the time
spent in each compartment of the maze were observed, suggesting
that animals did not show a preference for a particular side of the
arena (data not shown).
[0188] During the sociability test, two-way ANOVA revealed
significant main effects of VPA and CBDV on sociability.
VPA-exposed rats spent significantly less time in the chamber
containing the unfamiliar rat with respect to the time spent in the
empty compartment when compared to controls. The deficit in
sociability present in VPA-treated rats was confirmed by the
preference index that showed a significant reduction by about 48%
in the percentage of time spent exploring the unfamiliar rat with
respect to the empty compartment.
[0189] FIG. 1B shows that CBDV treatment at all doses significantly
reduced the impairment in sociability observed in VPA rats, the
preference index being only reduced by about 13% in VPA-CBDV
animals compared to controls.
[0190] FIG. 1C shows the effect of CBDV treatment on social novelty
preference in offspring of VPA- and vehicle-exposed rats, as
measured through the three chamber apparatus.
[0191] Control mice spent significantly more time exploring the
novel rat than the known rat (p<0.01). In contrast, VPA animals
spent similar time exploring the two stimuli.
[0192] Treatment with CBDV at doses of 20 mg/kg and 100 mg/kg
reversed the deficit in social preference in VPA rats, as
demonstrated by the fact that VPA-CBDV rats spent significantly
more time exploring the novel rats with respect to the familiar
one.
[0193] FIG. 2A shows the effect of CBDV treatment on repetitive
behaviours (compulsive self-grooming), in VPA-exposed offspring.
Prenatal VPA exposure significantly increased the time spent in
compulsive self-grooming. CBDV administration at 20 mg/kg in males
and 2 and 100 mg/kg in females was able to significantly normalize
this behaviour.
[0194] FIG. 2B shows the effect of CBDV treatment on locomotor
activity in VPA offspring. VPA administration significantly
increased locomotion by about 70% compared to control and CBDV
administration at 2, 20 and 100 mg/kg in males was able to
normalize this.
[0195] FIG. 2C shows the effect of CBDV treatment on short-term
memory, evaluated through the novel object recognition test.
Prenatal VPA administration significantly impaired short-term
memory, as demonstrated by a significant reduction of the
discrimination index by about 59% with respect to controls. CBDV at
doses of 2, 20 and 100 mg/kg completely and significantly reversed
the short-term memory deficit in both male and female VPA rats,
without affecting per se recognition memory when administered to
vehicle-treated (non VPA) rats.
[0196] FIG. 2D shows there were significant increases in GFAP and
CD11b expression in the hippocampus, paralleled by a concomitant
enhancement of TNF.alpha. levels. In contrast, no changes were seen
in Iba1 and iNOS levels after prenatal VPA exposure. Chronic CBDV
administration completely normalised the expression of those
inflammatory markers that were increased when administered to
VPA-exposed rats and had no effect in control rats.
Conclusions
[0197] These data demonstrate that CBDV provided an effective
treatment on the alterations in sociability, social novelty
preference, short-term memory, repetitive behaviours and locomotion
induced by prenatal VPA exposure offspring.
[0198] CBDV administration does not affect per se any of the
behaviours that were under investigation.
[0199] These data indicate that CBDV at doses of 2, 20 and 100
mg/kg was able to reverse the autism-like phenotype in VPA-exposed
rats and is therefore a potential novel treatment option for
ASD.
[0200] Furthermore CBDV was able to normalise the overexpression of
particular inflammatory biomarkers which were found to be increased
in rats treated with VPA. These data suggest that the CBDV
treatment was able to modulate the deficits that occur in autism
such as cognitive and behavioural deficits at a cellular level.
Example 2: Use of Cannabidivarin (CBDV) in a Mouse Model of Fragile
X Syndrome
[0201] The phytocannabinoid cannabidivarin (CBDV) was evaluated in
a mouse model of Fragile X syndrome (FXS). Such model evaluates the
treatment on cognitive deficits and seizures present in Fmr1
knockout (KO) mice.
Materials and Methods
Animals
[0202] Fmr1 KO mice and wild type mice were obtained and housed
four per cage in a temperature of 21.degree. C..+-.1.degree. C. and
humidity of 55%.+-.10% controlled environment. Food and water were
available ad libitum. All experiments were performed during the
light phase of a 12 hour light/dark cycle (08:00 to 20:00). Animals
were handled for one week before the start of experiments. All
behavioural tests were performed by researchers blind to the
different experimental groups.
Novel Object Recognition Task
[0203] On day one mice were habituated for 10 minutes to the empty
V-maze in which the task was to be performed. On the second day the
mice went back to the maze for 10 minutes which contained two
identical objects at the end of each corridor in the V-maze.
[0204] The following day the mice were placed again in the same
maze for a further 10 minutes but one of the familiar objects was
replaced for a novel object. The total time spent exploring the
novel and familiar object was recorded. A discrimination index was
calculated as the difference between the time spent exploring the
novel and familiar object divided by the total time exploring the
two objects. A high discrimination index (0.3-0.5) is considered to
reflect memory retention for the familiar object.
Seizure Susceptibility
[0205] This trait was evaluated in PND21 mice after acute
administration of the compounds or its vehicle, 30 min before
starting the procedure. To measure audiogenic seizure sensitivity
mice were placed individually into a novel environment, a glass
cylinder (40 cm high, 16 cm diameter) and allowed to explore for 1
min. Next, a bell (100 dB) was rung for 30 sec and seizure activity
scored according to the following scale: no response, 0; wild
running, 1; clonic seizure, 2; tonic seizure, 3; status
epilepticus/respiratory arrest/death, 4.
Experimental Protocol
[0206] Adult mice (10-15 weeks old) were treated either acutely
with one dose of CBDV on PND 21 or were treated once daily with
CBDV for 7 consecutive days. Mice were evaluated in the novel
object recognition task after the first and sixth day of CBDV
administration. Doses of CBDV used were 2, 10, 20, 100 and 200
mg/kg.
Results
[0207] FIG. 3A shows the acute treatment had no effect on the
discrimination index for knockout Fmr1 mice at any of the doses
administered. At the lowest dose of 2 mg/kg CBDV there was an
increase of the discrimination index in the knockout mice over that
of the vehicle treated mice however this was not significant, nor
did it reach the values considered to reflect memory retention.
[0208] FIG. 3B demonstrates that after chronic treatment (once
daily for 7 days) with CBDV at doses of 20 and 100 mg/kg there was
a statistically significant increase in the discrimination index in
the knockout mice over the vehicle treated knockout mice.
[0209] The discrimination index level are both over 0.3 and as such
are considered to represent that the mice treated with these doses
of CBDV were able to retain memory for familiar objects.
[0210] On postnatal day 21 (PND21) WT and Fmr1 KO mice were
injected with CBDV (2, 10, 20 or 100 mg/kg) or vehicle, 30 min
before exposure to a bell ring (100 dB). Mice exposed to this noise
for 30 sec ceased from their normal exploratory behaviour and
reacted with gradually to wild-running, clonic seizure, tonic
seizure or death. The maximum response was annotated for each mouse
as detailed in Table 2 below. A score was calculated according to
the severity of the symptoms as explained in the Methods
section.
[0211] As can be seen the FXS mice provided with the highest dose
of 100 mg/kg CBDV remained seizure free whereas those on the lower
doses experienced seizures of varying severity.
TABLE-US-00002 TABLE 2 Susceptibility to audiogenic seizures in
Fmr1 KO and WT mice (PND21) after acute (30 min) administration of
CBDV or vehicle WT CBDV CBDV CBDV CBDV (2 (10 (20 (100 Vehicle
mg/kg) mg/kg) mg/kg) mg/kg) n = 6 n = 6 n = 5 n = 6 n = 6 No 6 6 5
5 6 response Wild 0 0 0 1 0 running Clonic 0 0 0 0 0 seizure Tonic
0 0 0 0 0 seizure Death 0 0 0 0 0 FXS CBDV CBDV CBDV CBDV (2 (10
(20 (100 Vehicle mg/kg) mg/kg) mg/kg) mg/kg) n = 7 n = 5 n = 5 n =
5 n = 5 No 2 2 0 2 5 response Wild 3 1 3 1 0 running Clonic 0 0 0 0
0 seizure Tonic 2 2 2 2 0 seizure Death 0 0 0 0 0
Conclusions
[0212] These data demonstrate that the treatment of 20 and 100
mg/kg CBDV once daily for 7 days to mice which were deficient in
the Fmr1 gene and subsequently suffered similar cognitive deficits
to individuals with FXS, were able to reverse these cognitive
deficits.
[0213] Furthermore at the higher dose of CBDV (100 mg/kg) the
compound was able to completely eliminate the seizures that occur
in the FXS type mice.
[0214] As such CBDV is considered to be a viable treatment option
for FXS.
Example 3: Use of Cannabidivarin (CBDV) in a Mouse Model of Rett
Syndrome
[0215] The phytocannabinoid cannabidivarin (CBDV) was evaluated in
a mouse model of Rett syndrome (RS). Such model evaluates the
treatment on motor alterations and cognitive deficits present in
MeCP2 KO mice.
[0216] CBDV was administered daily at the dose of 2, 20 and 200
mg/kg i.p. starting from PND 28 and the following signs were scored
every other day: hindlimb clasping (indication of motor imbalance),
tremor, gait (measure of coordination), breathing, mobility and
general condition.
[0217] Furthermore, the efficacy of CBDV in reverting/attenuating
the short- and long-term memory deficits present in these mice was
evaluated. The Novel Object Recognition (NOR) test was performed
before the starting of the treatment Post Natal Day 28 (PND 28), at
PND 41 when the first motor symptoms appear and at PND 56 and 66
when the disease is fully manifested.
Materials and Methods
[0218] The CBDV was dissolved in ethanol, cremophor and saline
(1:1:18).
[0219] Starting from PND 28, mice received a daily intraperitoneal
injection of CBDV (or vehicle) at the dose of 2, 20 and 200 mg/kg.
Animals were then scored every other day to evaluate the effect of
CBDV treatment on motor symptoms (hindlimb clasping, gait,
mobility) as well as neurological signs and general conditions
(breathing abnormalities, tremors and general condition) present in
MeCP2 knockout (KO) mice.
Symptom Scoring
[0220] Each of the six symptoms was scored from 0 to 2 (0
corresponds to the symptom being absent or the same as in the wild
type (WT) animal; 1 when the symptom was present; 2 when the
symptom was severe).
[0221] Mobility: the mouse is observed when placed on bench, then
when handled gently. 0 =as WT. 1=reduced movement when compared
with WT: extended freezing period when first placed on bench and
longer periods spent immobile. 2=no spontaneous movement when
placed on the bench; mouse can move in response to a gentle prod or
a food pellet placed nearby.
[0222] Gait: 0=as WT. 1=hind legs are spread wider than WT when
walking or running with reduced pelvic elevation, resulting in a
`waddling` gait. 2=more severe abnormalities: tremor when feet are
lifted, walks backward or `bunny hops` by lifting both rear feet at
once.
[0223] Hindlimb clasping: mouse observed when suspend by holding
base of the tail. 0=legs splayed outward. 1=hindlimbs are drawn
toward each other (without touching) or one leg is drawn into the
body. 2=both legs are pulled in tightly, either touching each other
or touching the body.
[0224] Tremor: mouse observed while standing on the flat palm of
the hand. 0=no tremor. 1=intermittent mild tremor. 2=continuous
tremor or intermittent violent tremor.
[0225] Breathing: movement of flanks observed while animal is
standing still. 0=normal breathing. 1=periods of regular breathing
interspersed with short periods of more rapid breathing or with
pauses in breathing. 2=very irregular breathing-gasping or
panting.
[0226] General condition: mouse observed for indicators of general
well-being such as coat condition, eyes and body stance. 0=clean
shiny coat, clear eyes, and normal stance. 1=eyes dull, coat
dull/un-groomed, and somewhat hunched stance. 2=eyes crusted or
narrowed, piloerection, and hunched posture.
[0227] At PND 28, 41, 56 and 66 the effect of CBDV on short- and
long-term memory deficits was investigated through the Novel Object
Recognition (NOR) test.
Novel Object Recognition (NOR) test
[0228] The experiment was performed as previously described
(Zamberletti E. et al. 2014 and Kruk-S omka M. et al. 2014).
[0229] The experimental apparatus used for the Novel Object
Recognition test was an open-field box (43.times.43.times.32 cm)
made of Plexiglas, placed in a dimly illuminated room. Animals
performed each test individually.
[0230] Briefly, each animal was placed in the arena and allowed to
explore two identical previously unseen objects for 10 minutes
(familiarization phase). After an inter-trial interval of 30
minutes and 24 hours one of the two familiar objects was replaced
by a novel, previously unseen object and mice were returned to the
arena for the 10-minute test phase.
[0231] During the test phase the time spent exploring the familiar
object (Ef) and the new object (En) was recorded separately by two
observers blind to the groups and the discrimination index was
calculated as follows: [(En-Ef)/(En+Ef)].times.100.
Results
[0232] FIG. 4A shows the effect of CBDV (2, 20 and 200 mg/kg) on
body weight gain in KO and wild type animals. Analysis of body
weight gain as measured during the entire treatment period (PND
28-66) revealed that KO mice treated with vehicle were leaner than
controls.
[0233] Treatment with CBDV 2, 20 and 200 mg/kg was able to
partially prevent the reduction in body weight present in MeCP2 KO
mice.
[0234] This was evident when the area under the curve (AUC) was
calculated (FIG. 4B), clearly showing that body weight of KO mice
treated with vehicle was significantly lower than WT mice and
treatment with CBDV significantly rescued body weight in KO mice
without affecting body weight in WT controls.
[0235] FIG. 5 shows the percentage of survival of MeCP2 KO and WT
mice treated with three different doses of CBDV (2, 20 and 200
mg/kg). The results are displayed as percent survival with respect
to the time (PND).
[0236] No lethality was observed for WT mice treated with vehicle
or CBDV. In contrast, analysis of survival measured at PND 67
revealed that CBDV at all three doses tested was able to increase
survival in KO mice.
[0237] In particular, survival rates were 62.5% in KO mice treated
with CBDV 2 and 20 mg/kg and 75% in those treated with 200 mg/kg,
compared with 25% of KO mice treated with vehicle.
[0238] FIGS. 6 and 7 describes the effect of chronic CBDV (2, 20
and 200 mg/kg) on six different signs of the phenotype (FIG. 6A
tremor; 6B breathing; 6C hindlimb clasping; 6D gait, FIG. 7A
general condition and 7B mobility) in KO mice at different stages
of the disease.
[0239] All three doses of CBDV were effective in significantly
delaying and reducing tremors in KO mice in the first stages of the
disease (PND 45-49), whereas only 200 mg/kg dose was still able to
reduce tremors at later stages of the disease (PND 55 and 63). The
lower dose, 2 mg/kg, was still protective at the later time point
(PND 63).
[0240] Similarly, CBDV administration was able to improve breathing
in KO mice. This improvement was more pronounced in KO mice treated
with CBDV 2 mg/kg. CBDV 2 mg/kg significantly improve breathing
from PND 56 to 60, whereas CBDV 20 mg/kg showed a significant
effect at PND 57 and CBDV 200 mg/kg was effective at PND 57 and
60.
[0241] The lower doses of CBDV were also able to significantly
attenuate hindlimb clasping at PND 47 and 49, whereas CBDV 200
mg/kg did not significantly affect this parameter. Conversely, CBDV
200 mg/kg significantly improved gait from PND 53 to 57.
[0242] General conditions of KO mice (coat and eyes conditions)
were significantly improved after treatment with CBDV 2 and 200
mg/kg (PND 52-64); whereas the dose of 20 mg/kg was significantly
effective only at the earlier time point (PND 52). CBDV effect on
mobility was less intense, as it reached statistical significance
only at PND 64.
[0243] To better analyse the effect of chronic CBDV (2, 20 and 200
mg/kg) on progression of the signs in KO animals the single scores
were grouped in a total symptom score that is represented in FIG.
8.
[0244] The total symptom score confirms the observation that CBDV
treatment at all three doses tested is able to delay and attenuate
the appearance of the phenotype in KO mice as compared with
KO-vehicle animals.
[0245] The dose of 2 mg/kg significantly improved total symptom
score at every time point (PND 45, 47, 49, 55, 56, 57, 58, 60 and
64). The 20 mg/kg dose had a similar effect, but was less effective
at the later time point (PND 64). The 200 mg/kg dose exerted
beneficial effect at a later time point (PND 47) and its protective
effect was evident until PND 58.
[0246] FIG. 9 shows the effect of CBDV (2, 20 and 200 mg/kg) on
short-term memory in the NOR test at different ages.
[0247] In KO animals, a significant cognitive impairment in
short-term memory was found at PND 28, a time point when the motor
symptoms are not present. This impairment was still present and
significant at PND 41, 56 and 66, when the disease is fully
manifested.
[0248] At each considered time point, administration of the three
different doses of CBDV significantly reverted the cognitive
impairment in short-term memory present in KO mice without
affecting recognition memory when administered to WT
littermates.
[0249] FIG. 10 shows the effect of CBDV (2, 20 and 200 mg/kg) on
long-term memory in the NOR test at different ages.
[0250] Similar to what has been observed for short-term memory; all
three doses of CBDV administered were also able to significantly
counteract the cognitive impairment in long-term memory present in
KO mice without affecting recognition memory when administered to
WT littermates.
Conclusions
[0251] These data demonstrate that CBDV treatment was effective in
delaying and attenuating the phenotype of MeCP2 KO mice. The CBDV
treatment was able to significantly recover the deficits in short-
and long-term memory present in those animals.
[0252] Moreover all doses tested had similar beneficial effects in
attenuating the phenotype and reversing the cognitive deficits.
[0253] Besides the beneficial effect in terms of total symptoms
appearance, it must be underlined the striking recovery of the
cognitive deficits induced by CBDV in MeCP2 KO mice, that is still
present and significant also at very late stages of the disease
(PND 66).
[0254] Importantly the survival rates in KO mice treated with CBDV
is in a range between 62.5 and 75% with respect to the value of 25%
in KO mice treated with vehicle.
[0255] These data indicate that CBDV offers a significant treatment
option in the treatment of RS.
Example 4: Use of Cannabidivarin (CBDV) in a Mouse Model of
Angelman Syndrome
[0256] The effect of CBDV was tested in the treatment of
neurological, behavioural and motor disorders and seizures in a
transgenic mouse model of Angelman Syndrome.
Materials and Methods
Animals
[0257] Heterozygous mice with maternal deficiency of Ube3A
(Ube3am-/p+) and wild type (Ube3am+/p+) were purchased from The
Jackson Laboratory (Jackson code: B6.12957-Ube3a tm1Alb/J) and
maintained in a C57BL/6 background.
[0258] Animals were housed under controlled illumination
(12:12-hour light/dark cycle; light on 6 hours) and environmental
conditions (room temperature: 20.degree. C.-22.degree. C.,
humidity: 55%-60%) with food and tap water were available ad
libitum.
Drugs and Treatment
[0259] Drugs were dissolved in 1:1:18 ethanol:cremophor:0.9%
saline, for intraperitoneal (i.p.) administration. Drug treatment
was performed daily for 35 days. CBDV was administered at 20
mg/kg.
[0260] For the audiogenic seizure tests CBDV was administered at
20, 200 and 400 mg/kg.
Behavioural Tests
[0261] Rotarod: The rotarod test assesses balance and motor
coordination of mice. Mice have been measured for the time (in
seconds) of equilibrium before falling on a rotary cylinder by a
magnet that, activated from the fall of the mouse on the plate,
allows to record the time of permanence on the cylinder. After a
period of adaptation of 30 s, the spin speed gradually increased
from 3 to 30 rpm for a maximum time of 5 min. The animals were
analysed by 2 separate tests at 1-h interval in the same day.
[0262] Clasping: The clasping test assesses ataxia in mice. Mice
were suspended by the base of the tail and their behaviours were
recorded for 30 seconds. The time for which the mice clasped their
hind limbs was recorded. The time was then scored as follows: 4,
15-30 s, 3, 10-15 s, 2, 5-10 s, 1, 0-5 s and 0.0 s
[0263] Tail suspension: The tail suspension test assesses
depressive-like behaviour in mice. Mice were individually suspended
by the tail on a horizontal bar (50 cm from floor) using adhesive
tape placed approximately 4 cm from the tip of the tail. The
duration of immobility was recorded in seconds over a period of 6
minutes by a time recorder. Immobility time was defined as the
absence of escape-oriented behaviour.
[0264] Novel Object Recognition: The novel object recognition
assesses recognition memory in mice. The experiment started with
the habituation period, during which mice were allowed to freely
explore for 1 hour the apparatus which consists of a rectangular
open box (40.times.30.times.30 cm width.times.length.times.height)
made of grey polyvinyl chloride (PVC) illuminated by a dim light.
The day after each mouse was allowed to explore two identical
objects positioned in the back left and right corners for 5 min
(acquisition). A video camera recorded the time spent on
exploration of each object. In the test trial, which was carried
out for 2 hrs after the acquisition, one of the two objects was
replaced with a new different object. The time spent exploring the
object was the time that the mouse spent with its nose directed and
within 1 cm from the object. The behaviour of mice was analyzed by
an observer blind to the treatment. Data are expressed as
percentage of recognition index (RI %), which was calculated as the
percentage of the time spent exploring the novel object/time spent
exploring the novel object+time spent exploring the familiar
object.times.100.
[0265] Seizure susceptibility: Audiogenic seizures were induced by
vigorously scraping scissors across the metal grating of the cage
lid which produces a 100 DB high frequency loud noise. This was
done for 20 seconds. This test consists of 3 phases. In phase 1 all
mice were tested for epilepsy. In phase 2 all mice were injected
daily with either vehicle or a drug for 7 or 10 days. In phase 3
mice were tested for epilepsy (one hour after last injection).
[0266] Statistical Analysis: Behavioural data are represented as
means .+-.SEM and statistical analysis of these data was performed
by two way analysis of variance (ANOVA) for repeated measured
followed by the Student Newman-Keuls for multiple comparisons to
determine statistical significance between different treated groups
of mouse. p<0.05 was considered statistically significant.
Results
[0267] FIG. 11 shows that AS mice treated with vehicle showed
significantly longer clasping duration at 10 weeks of age compared
to WT mice treated with vehicle. In AS mice chronic treatment (30
days) with CBDV significantly reduced clasping duration at 10 weeks
of age compared to AS mice treated with vehicle.
[0268] FIG. 12 demonstrates that AS mice treated with vehicle
showed a significant motor impairment at 10 weeks of compared to WT
mice treated with vehicle. In AS mice, chronic treatment (30 days)
with CBDV reduced latency to fall compared to AS mice treated with
vehicle.
[0269] Mice were assessed at the age of 7-8 weeks in the novel
object recognition test. AS mice treated with vehicle showed a
significant decrease in the discrimination index compared with WT
mice that received the same treatment. FIG. 13 shows that AS mice
treated with CBDV significantly increased the discrimination index
compared to AS mice treated with vehicle.
[0270] FIG. 14 shows that in the tail suspension test the time of
immobility were significantly higher in AS mice that received
vehicle compared to WT mice that received the same treatment. In AS
mice treatment with CBDV significantly reduced the duration of
immobility time compared to AS mice treated with vehicle.
[0271] FIG. 15 demonstrates that the wildtype mice were not
susceptible to audiogenic seizures. CBDV administered at 20 mg/kg
or 200 mg/kg had no effect on the seizure susceptibility as there
was very little change in the number of mice with seizures compared
to baseline after either 7 or 10 days treatment. However in the
mice treated with 400 mg/kg CBDV there was a statistically
significant decrease in the number of mice with seizures, whereby
after 7 days treatment the number of mice experiencing seizures
dropped from 100% down to 60% and after 10 days treatment this
number had decreased further to 30%.
Conclusion
[0272] These data demonstrate that the treatment of 20 mg/kg CBDV
to mice which were deficient in the Ube3A gene and subsequently
suffered similar cognitive deficits to individuals with AS, were
able to reverse these cognitive deficits.
[0273] Furthermore at a higher dose of 400 mg/kg CBDV was able to
reduce the seizure susceptibility of Angelman mice by 70% after 10
days treatment.
[0274] As such CBDV is considered to be a viable treatment option
for AS.
Example 5: Use of Cannabidivarin (CBDV) in a Rat Model of
Schizophrenia
[0275] The phytocannabinoid cannabidivarin (CBDV) was evaluated in
a rat model of schizophrenia.
[0276] These data demonstrate the ability of CBDV (2, 10 and 20
mg/kg), in comparison with the positive control risperidone, to
attenuate the disruption of a cognitive task induced by sub-chronic
treatment with phencyclidine (PCP) in rats.
[0277] The effect of PCP in the novel object recognition (NOR) test
is thought to model visual recognition memory deficits similar to
those observed in schizophrenia. The atypical antipsychotics,
clozapine and risperidone can attenuate the deficit.
[0278] A further animal model for assessing the avolition domain of
negative symptoms of schizophrenia, lack of social behaviour has
also been tested with CBDV using the sub-chronic administration of
PCP to rats. PCP induces social behaviour deficit in rats in their
interaction with a vehicle treated weight matched control rat.
Materials and Methods
Animals
[0279] Female hooded-Lister rats were used for this experiment.
Rats were housed in groups of 5 under standard laboratory
conditions under a 12 hr light: dark cycle, lights on at 0700 hr.
Testing was carried out in the light phase.
Treatment
[0280] Rats were randomly assigned to two treatment groups and
treated with vehicle, n=20 (distilled water, i.p.) or Phencyclidine
hydrochloride (PCP), n=100 (2 mg/kg, i.p. twice daily for 7-days).
PCP was dissolved in distilled water. This was followed by a 7-day
wash out period before the rats were tested following acute
treatment with CBDV, risperidone or vehicle.
[0281] Risperidone (0.1 mg/kg) was dissolved in a minimum volume of
acetic acid, made up to volume with distilled water and pH adjusted
to 6 with 0.1M NaOH and administered via the i.p. route in a volume
of 1 ml/kg, 60 min prior to testing.
[0282] CBDV at 2, 10 or 20 mg/kg was dissolved in 2:1:17
(Ethanol:Cremofor:Saline 0.9%) and administered via the i.p. route
in a volume of 5 ml/kg, 60 min prior to testing.
Novel Object Recognition Test
Habituation:
[0283] Rats were allowed to habituate to the empty test box and the
behavioural test room environment for 1 h on day 1. Prior to
behavioural testing on day 2 rats are given a further 3 min
habituation.
Behavioural Testing:
[0284] Following the 3 min habituation period, the rats are given
two 3 min trials (T1 and T2) which are separated by a 1 min
inter-trial interval in the home cage during which the objects are
changed. Behaviour in all trials was recorded on video for
subsequent blind scoring.
T1=Trial 1, the Acquisition Trial:
[0285] In this trial, the animals are allowed to explore two
identical objects (A1 and A2) for 3 min.
T2=Trial 2, the Retention Trial:
[0286] In this trial, the animals explore a familiar object (A)
from T1 and a novel object (B) for 3 min. The familiar object
presented during T2 is a duplicate of the object presented in T1 in
order to avoid any olfactory trails.
Object Exploration:
[0287] The object exploration is defined by animals licking,
sniffing or touching the object with the forepaws whilst sniffing,
but not leaning against, turning around, standing or sitting on the
object. The exploration time (s) of each object (A, B, familiar and
novel) in each trial are recorded using two stopwatches and the
following factors are calculated: [0288] Total exploration time of
both objects in the acquisition trial (s). [0289] Total exploration
time of both objects in the retention trial (s). [0290] Habituation
of exploratory activity includes the exploration time, as measured
by the number of lines crossed, for both the trials. [0291]
Discrimination index (DI), which is calculated as shown below:
[0291] ( Time spent exploring novel object - time spent exploring
familiar object ) Total time spent in exploring the objects
##EQU00002##
Social Interaction Test
[0292] The social interaction test is performed in an open-field
comprising a square box made of Plexiglas (52.times.52.times.31 cm)
placed 27 cm above the floor on a moveable trolley. The floor of
the box is white with black gridlines forming 9 identical squares
on it. All other walls are black. A video camera connected to a
video recorder and monitor is positioned above the box. The object
used for the test consists of a heavy structure made of metal that
cannot be displaced by the animals. Care is taken to ensure that
these objects do not have natural significance for the rats.
[0293] The rats were habituated to the test environment and arena
prior to the test day. Habituation consists of placing all rats
from one cage together in the empty test arena for one hour for
three days including the day before the test day.
[0294] Pairs of rats, weight matched (15-20 g) and unfamiliar to
each other, receiving either no treatment (n=70 "conspecific" rats)
or different treatments (PCP and Vehicle; n=10 "tested" rats or
PCP+Drug; n=50 "tested" rats) were placed in the test arena
together for 10 min and behaviour assessed as described below.
[0295] An inanimate object such as an unopened drinks can is also
placed in the centre of the arena to measure any differences in
interaction of the test animal with an unfamiliar animal as opposed
to an unfamiliar object. After each 10 minute trial, the object and
arena are cleaned with 10% alcohol in an attempt to remove traces
of any olfactory cues. All testing is carried out under standard
room illumination levels (70 cd/m2).
[0296] Behaviour in both trials was recorded on video for
subsequent blind scoring. A behavioural scoring software program
(Hindsight, Scientific programming services) is used to score the
following parameters: [0297] Investigative sniffing behaviour:
sniffing the conspecific's snout or parts of the body including the
anogenital region; [0298] Following: rat moves after the
conspecific i.e. a vehicle treated rat of the same species, around
the arena; [0299] Avoidances: actively turning away when approached
by the conspecific animal; [0300] Investigation of object:
exploration of object placed in centre of the arena; and [0301]
Locomotor activity is recorded by counting the total number of
sectors (i.e. lines) crossed by the test rat.
Results
[0302] FIG. 16A shows the time spent investigating two identical
objects in Trial 1, as can be seen the rats show no real preference
for the two identical objects whether they were treated with CBDV,
risperidone or vehicle.
[0303] FIG. 16B shows the time spent investigating the two objects
when one object is familiar and another is novel. In the PCP
treated rats that were not given either CBDV or the positive
control risperidone there is no difference between the time spent
investigating both objects. In the vehicle treated rats who were
not treated with PCP and as such suffered no cognitive dysfunction
there was a statistically significant difference between the time
spent investigating the objects with a preference for the novel
object. This therefore shows that the animals without cognitive
dysfunction were able to discriminate between the object that was
familiar to them and that which was novel. The PCP treated mice in
comparison showed that because of cognitive dysfunction they were
unable to remember the object that they had been familiarised with
in Trial 1.
[0304] FIG. 16B also shows that at a dose of 10 and 20 mg/kg CBDV
was able to reverse the cognitive dysfunction brought about in the
PCP treated rats such that they were able to discriminate between
familiar and novel objects. This effect was also shown in the
positive control risperidone.
[0305] FIG. 17 shows the discrimination index (DI) for the
treatments. In agreement with the data shown in FIG. 15B the rats
treated with 10 and 20 mg/kg produced a statistically significant
increase in the DI in PCP rats.
[0306] FIG. 18A to C describe the data produced in the social
interaction test. As can be seen rats that were treated with PCP
showed a decrease in sniffing and following the other rat. In
addition treatment with PCP produced an increase in the time spent
avoiding the other rat. Taken together these data demonstrate that
the PCP treatment produced symptoms of social withdrawal in the
rats.
[0307] CBDV was able to reverse these effects in a statistically
significant manner as was the positive control risperidone. FIG.
18A shows that CBDV at doses of 10 and 20 mg/kg significantly
increased the time spent sniffing the other rat. FIG. 18B shows
that CBDV at a dose of 20 mg/kg also increased the amount of time
spent following the other rat. This effect was not seen to be
significant in the positive control risperidone; however the amount
of time was increased over that of the PCP alone group.
[0308] FIG. 18C demonstrates that CBDV at 20 mg/kg was able to
reduce the amount of time the test rat spent avoiding the other
rat.
Conclusion
[0309] The data described in this example demonstrate that the
administration of CBDV to
[0310] PCP-treated rats was able to treat both the cognitive
dysfunction and negative effects which occur in these animals.
[0311] Such data demonstrates that CBDV is a suitable treatment
option for schizophrenia, in particular symptoms of schizophrenia
associated with cognitive dysfunction and negative symptoms such as
social withdrawal.
Overall Conclusion
[0312] Examples 1 to 5 above describe the use of CBDV in a model of
ASD, three models of ASD-associated disorders and a model of
schizophrenia. Unexpectedly CBDV has been shown to produce
statistically significant reversal of the symptoms associated with
these disorders.
[0313] In particular CBDV has been shown to produce positive
results in the Novel Object Recognition (NOR) test in all models
and as such demonstrates unequivocally that this phytocannabinoid
could reverse cognitive dysfunction in these disorders.
[0314] In addition, other tests in these models provide support
that CBDV could be used to treat additional symptoms associated
with these disorders. CBDV was able to reduce repetitive
behaviours, hyperactivity and sociability in the model of ASD. CBDV
was also able to reduce the decrease in bodyweight and survival in
a model of Rett syndrome in addition to improvement of overall
general condition and symptoms in these animals such as mobility
and breathing. Furthermore CBDV was able to reduce ataxia and
anxiety symptoms in a model of Angelman syndrome. Lastly CBDV
treatment was able to reduce negative symptoms of social withdrawal
in a model of schizophrenia.
[0315] Taken together CBDV is able to provide an effective
treatment option to individuals suffering from ASD, ASD-associated
disorders and schizophrenia.
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