Note: Descriptions are shown in the official language in which they were submitted.
CA 02929286 2016-04-29
WO 2014/085480 PCT/US2013/072049
TREATMENT OF AUTISM SPECTRUM DISORDERS USING GLYCYL-L-2-
METHYLPROLYL-L-GLUTAMIC ACID
Claim of Priority
This application claims priority to United States Provisional Patent
Application No.
61/730,829, filed 28 November 2012, entitled "Treatment of Autism Spectrum
Disorders Using
Glycy1-2-Methylproly1 Glutamic Acid," Larry Glass, Michael John Bickerdike,
Michael Fredrick
Snape, and Patricia Perez DeCogram, inventors. This application is
incorporated herein fully by
reference.
Field of the Invention
This invention relates generally to therapy of Autism Spectrum Disorders
(ASD),
including autism, Fragile X Syndrome, Rea Syndrome (RTT), Autistic Disorder,
Asperger
Syndrome, Childhood Disintegrative Disorder and Pervasive Developmental
Disorder Not
Otherwise Specified (PDD-NOS), and Pathological Demand Avoidance (PDA). In
particular, this
invention relates to treatment of ASD using Glycy1-2-methyl-Prolyl-Glutamate
(G-2-MePE).
BACKGROUND
Autism Spectrum Disorders and neurodevelopment disorders (NDDs) are becoming
increasingly diagnosed. According to the fourth edition of the American
Psychiatric Association's
(APA) Diagnostic and Statistical Manual of Mental Disorders (DSM-4), Autism
spectrum
disorders (ASD) are a collection of linked developmental disorders,
characterized by
abnormalities in social interaction and communication, restricted interests
and repetitive
behaviours. Current classification of ASD according to the DSM-4 recognises
five distinct forms:
classical autism or Autistic Disorder, Asperger syndrome, Rett syndrome,
childhood
disintegrative disorder and pervasive developmental disorder not otherwise
specified (PDD-
NOS). A sixth syndrome, pathological demand avoidance (PDA), is a further
specific pervasive
developmental disorder.
More recently, the fifth edition of the American Psychiatric Association's
(APA) Diagnostic
and Statistical Manual of Mental Disorders (DSM-5) recognizes recognises
Asperger syndrome,
childhood disintegrative disorder, and pervasive developmental disorder not
otherwise specified
(PDD-NOS) as ASDs.
This invention applies to treatment of disorders, regardless of their
classification as either
DSM-4 or DSM-5.
1
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Neurodevelopment Disorders (NDDs) include Fragile X Syndrome (FXS), Angelman
Syndrome, Tuberous Sclerosis Complex, Phelan McDermid Syndrome, Rett Syndrome,
CDKL5
mutations (which also are associated with Rett Syndrome and X-Linked Infantile
Spasm Disorder)
and others. Many but not all NDDs are caused by genetic mutations and, as
such, are sometimes
referred to as monogenic disorders. Some patients with NDDs exhibit behaviors
and symptoms of
autism.
As an example of a NDD, Fragile X Syndrome is an X-linked genetic disorder in
which
affected individuals are intellectually handicapped to varying degrees and
display a variety of
associated psychiatric symptoms. Clinically, Fragile X Syndrome is
characterized by intellectual
handicap, hyperactivity and attentional problems, autism spectrum symptoms,
emotional lability and
epilepsy (Hagerman, 1997a). The epilepsy seen in Fragile X Syndrome is most
commonly present
in childhood, but then gradually remits towards adulthood.
Hyperactivity is present in
approximately 80 percent of affected males (Hagerman, 1997b). Physical
features such as prominent
ears and jaw and hyper-extensibility of joints are frequently present but are
not diagnostic.
Intellectual handicap is the most common feature defining the phenotype.
Generally, males are more
severely affected than females. Early impressions that females are unaffected
have been replaced by
an understanding of the presence of specific learning difficulties and other
neuropsychiatric features
in females. The learning disability present in males becomes more defined with
age, although this
longitudinal effect is more likely a reflection of a flattening of
developmental trajectories rather than
an explicit neurodegenerative process.
The compromise of brain function seen in Fragile X Syndrome is paralleled by
changes in
brain structure in humans. MRI scanning studies reveal that Fragile X Syndrome
is associated with
larger brain volumes than would be expected in matched controls and that this
change correlates with
trinucleotide expansion in the FMRP promoter region (Jakala et al., 1997). At
the microscopic level,
humans with Fragile X Syndrome show abnormalities of neuronal dendritic
structure, in particular,
an abnormally high number of immature dendritic spines (Irwin et al, 2000).
Currently available treatments for NDDs are symptomatic ¨ focusing on the
management of symptoms ¨ and supportive, requiring a multidisciplinary
approach.
Educational and social skills training and therapies are implemented early to
address core
issues of learning delay and social impairments. Special academic, social,
vocational, and
support services are often required. Medication, psychotherapy or behavioral
therapy may
be used for management of co-occurring anxiety, ADHD, depression, maladaptive
behaviors (such as aggression) and sleep issues. Antiepileptic drugs may be
used to
control seizures.
2
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Description of Related Art
EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-
Pro-Glu) and
its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that
GPE is effective as a
neuromodulator and is able to affect the electrical properties of neurons.
W095/172904 discloses that GPE has neuroprotective properties and that
administration
of GPE can reduce damage to the central nervous system (CNS) by the prevention
or inhibition of
neuronal and glial cell death.
WO 98/14202 discloses that administration of GPE can increase the effective
amount of
choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and
nitric oxide synthase
(NOS) in the central nervous system (CNS).
W099/65509 discloses that increasing the effective amount of GPE in the CNS,
such as
by administration of GPE, can increase the effective amount of tyrosine
hydroxylase (TH) in the
CNS to increase TH-mediated dopamine production in the treatment of diseases
such as
Parkinson's disease.
W002/16408 discloses certain GPE analogs having amino acid substitutions and
certain
other modification that are capable of inducing a physiological effect
equivalent to GPE within a
patient. The applications of the GPE analogs include the treatment of acute
brain injury and
neurodegenerative diseases, including injury or disease in the CNS.
SUMMARY
There is no current, effective, treatment of ASDs or NDDs, and patient care is
limited to
management of the symptoms.
This invention relates to synthetic analogs and peptidomimetics of glycyl¨L-
prolyl-L-
glutamic acid (GPE). In particular, this invention relates to GPE analogs and
peptidomimetics
that are anti-apoptotic and anti-necrotic, to methods of making them, to
pharmaceutical
compositions containing them, and to their use to enhance cognitive function
and/or treat memory
disorders and to improve neuronal connectivity in animals. More specifically,
this application
relates to the methods of use of the GPE analog, L-Glycy1-2-methyl-L-Prolyl-L-
Glutamate
2MePE) in the treatment of ASD.
The U.S. Patent No. 7,041,314 discloses compositions of matter and methods of
use of G-
2-MePE.
3
CA 02929286 2016-04-29
WO 2014/085480 PCT/US2013/072049
In one aspect, this invention provides compounds of Formula 1 and Formula 2:
, R3
2 R3
R4
0
y
y
1 0
0 R
0 (CH2)n (CH2)0
1
(CH2)m (CH2)n,
X
Foula 1 Formula 2
rm
where m is 0 or 1;
n is 0 or 1;
X is H or ¨NR6R7;
Y is H, alkyl, --0O2R5, or ---CONR6R7;
Z is I-1, alkyl, ¨CO2R5 or CONR6R7;
R1 is H, alkyl, or aralkyl;
R2, R3, and R4 are independently H or alkyl;
each R5 is independently H, alkyl, or a fatty alcohol residue;
each R6 and R7 is independently H, alkyl, or aralkyl, or -NR6R7 is
pyrrolidino, piperidino, or
morpholino;
or a lactone formed when a compound where Y is ¨0O2(alkyl) and Z is ¨CO2H or
where Y is
¨CO2H and Z is ¨0O2(allcyl) is lactonized;
and the pharmaceutically acceptable salts thereof,
provided that the compound is not GPE, N-Me-GPE, GPE amid; APE, GPQ or a salt
thereof.
Another aspect the invention provides methods for treatment of an animal
having a
Autism Spectrum Disorder comprising administration of an effective amount of
Glycyl-L-2-
Methylprolyl-L-Glutamic Acid (G-2-MePE) to the animal.
BRIEF DESCRIPTION OF THE DRAWINGS
This invention is described with reference to specific embodiments thereof.
Other aspects
and features of this invention can be understood with reference to the
Figures, in which:
FIG. 1 is a general scheme for preparation of synthetic analogs of GPE of the
invention.
FIGs. 2 and 3 depict schemes for modifying glycine residues on GPE.
4
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
FIGs. 4 through 9 depict schemes for modifying glutamic acid residues of GPE.
FIGs. 10 and II depict schemes for modifying peptide linkages of GPE.
FIGs. 12 - 15 depict graphs summarizing results of testing neurons in vitro
with GPE or
G-2-MePE and okadaic acid.
FIG. 12 depicts a graph showing effects of GPE on cortical neurons injured
with okadaic
acid.
FIG. 13 depicts a graph showing effects of G-2-MePE on cortical neurons
injured with
okadaic acid.
FIG. 14 depicts a graph showing effects of G-2-MePE, GPE on cerebellar
microexplants
injured with okadaic acid.
FIG. 15 depicts a graph showing effects of G-2-MePE or GPE on striatal cells
injured
with okadaic acid.
FIG. 16 shows the effects of subcutaneous injection of G-2-MePE (at doses of
0.012,
0.12, 1.2 and 12 mg/kg) on the number of ChAT-positive neurons in the striatum
of 18-month old
rats.
FIG. 17 shows effects of G-2-MePE treatment on spatial memory retention in
middle-
aged 12-month old rats.
FIGs. 18A and 18B show effects of G-2-MePE on spatial working memory of aged
(17-
month old) rats in an 8-arm radial maze following 3-weeks of treatment and a
nine day washout.
FIG. 18A shows the maze acquisition profiles across days for the different
groups. FIG. 18B
shows the proportion of correct maze choices averaged across days for the
groups.
FIG. 19A shows effects of a single intraperitoneal administration of 4 doses
of G-2-
MePE on neuroblast proliferation as assessed by the number of PCNA positive
cells in the
subventricular zone (SVZ) of aged rats.
FIG. 19B shows effects of a single intraperitoneal administration of 4 doses
of G-2-
MePE on co-localisation of PCNA and doublecortin staining in a rat treated
with the highest dose
of G-2-MePE (right panel) compared to the vehicle treated rat (left panel).
FIG. 19C shows effects of G-2-MePE on neuroblast proliferation as assessed by
PCNA
immunohistochemical staining in middle-aged rats.
FIG. 20A shows a significant increase in the number of reactive astrocytes as
assessed by
GFAP staining in the hippocampus in aged rats compared to young rats (*p<0.01)
and middle
aged rats (*p<0.01).
FIG. 20B shows a photograph of a section of cerebral cortex of an aged rat,
showing
astrocytes as assessed with GFAP staining, some of which are associated with
formation of
capillaries (arrows).
5
CA 02929286 2016-04-29
WO 2014/085480 PCT/US2013/072049
FIG. 20C shows dose-dependent effects of G-2-MePE treatment (at doses of 0.12,
0.12,
1.2 and 12 mg/kg/day) on reduction of the number of astrocytes as assayed
using GFAP staining
in the CA4 sub-region of the hippocampus in aged rats.
FIG. 20D shows dose-dependent effects of G-2-MePE treatment (at doses of 0.12,
0.12,
1.2 and 12 mg/kg/day) on reduction of the number of astrocytes as assayed
using GFAP staining
in the cerebellar cortex.
FIG. 21 shows pharrnacokinetic properties of GPE and G-2-MePE in the
circulation of
rats after intravenous injection.
FIG. 22 shows the effects of G-2-MePE on increased survival duration in MeCP2
deficient mice compared to saline-treated MeCP2 deficient mice.
FIG. 23 shows the effects of G-2-MePE on the hippocampal long-term
potentiation as
measured by the fEPSP slope in MeCP2 deficient mice, compared to saline-
treated MeCP2
deficient mice.
FIG. 24 depicts a graph showing effects of G-2-MePE on dendrite length as a
function of
distance from the cell soma.
FIGs. 25 and 26 depict graphs illustrating the open field behaviour of wild-
type
and finrl knockout mice, treated with either saline vehicle or G-2-MePE (100
mg/kg, i.p.
in saline; 28 days). Movement (FIG. 25) and rearing activity (FIG. 26) are
both elevated
in fmrl knockout mice. These effects are attenuated by treatment with G-2-MePE
on
trials 2 and 3 regarding locomotion (most likely indicative of enhanced
cognition), and on
all three trials regarding rearing (indicative of ablation of the
hyperactivity seen in the
vehicle-treated fmrl knockout mice). V: vehicle, NNZ: G-2-MePE; FX: fmrl KO
and
WT=wild-type. Filled circles: vehicle treated wild-type; open circles: vehicle
treated
fmrl KO; filled triangles: G-2-MePE treated wild-type; open triangles: G-2-
MePE treated
finrl KO.
FIG. 26 is a graph showing effects of G-2-MePE (100 mg/kg, i.p.; 28 days) on
behavior of wild-type andfinr/ knockout mice in the Successive Alley Test as
compared
to vehicle (saline) treated fmrl KO and wild-type animals. Fmrl KO mice showed
no
alley preference, attributed to the hyperactive phenotype of the model. G-2-
MePE
treatment for 28 days reversed this behavioural pattern. Filled circles:
vehicle treated
wild-type; open circles: vehicle treated fmrl KO; filled triangles: G-2-MePE
treated wild-
type; open triangles: G-2-MePE treated fmrl KO.
FIG. 27A-D show behavior of wild-type and fmrl knockout mice given either
saline vehicle or G-2-MePE (100 mg/kg, i.p.; 28 days) on the elevated plus
maze. FIG.
27A is a graph showing the effects of G-2-MePE on the total number of arm
entries.
6
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Open arm entries, expressed as a percentage of total arm entries are depicted
in FIG. 27B.
FIG. 27C is a graph showing the effects of G-2-MePE on the time spent in the
center of
the apparatus. Open bars: vehicle-treated wild type; shaded bars: G-2-MePE
treated wild-
type; hatched bars: vehicle treated fmr 1 KO; filled bars: G-2-MePE treated
fmr 1 KO.
FIG. 28A ¨ B depict graphs of the effects of G-2-MePE on numbers of arm
entries in wild type and fmr 1 knock-out mice. Open bars: vehicle-treated wild
type;
shaded bars: G-2-MePE treated wild-type; hatched bars: vehicle treated fmr I
KO; filled
bars: G-2-MePE treatedfinr/ KO.
FIG. 28C depicts a graph of the time spent in the center of a maze in G-2-MePE
treated and vehicle-treated wild type and fmr1 knockout mice. Open bars:
vehicle-treated
wild type; shaded bars: G-2-MePE treated wild-type; hatched bars: vehicle
treated fmr1
KO; filled bars: G-2-MePE treated finr 1 KO.
FIG. 29 shows a graph depicting the effects of G-2-MePE (100 mg/kg, i.p.; 28
days) on contextual-fear conditioning in wild-type and filar/ knockout mice.
Filled bar:
G-2-MePE treated wild-type; open bar: G-2-MePE treated fmr 1 KO; shaded bar:
vehicle
treated wild-type; hatched bar: vehicle treated frm 1 KO.
FIG. 30A depicts a graph showing the effects of G-2-MePE treatment on social
behavior: sniffing of a conspecific odor. FIG. 3013 shows the count of marbles
buried by
wild-type and fmr1 knockout mice given either saline vehicle or G-2-MePE (100
mg/kg,
i.p.; 28 days). FIG. 30C shows the nest building score of wild-type and finr 1
knockout
mice given either saline vehicle or G-2-MePE. Open bars: vehicle-treated wild
type;
shaded bars: G-2-MePE treated wild-type; hatched bars: vehicle treated fmr1
KO; filled
bars: G-2-MePE treated fmr1 KO.
FIG. MA shows an illustration depicting the microfluidic chamber used to
culture
the hippocampal cells. FIG. 31B shows a confocal micrograph showing GFP-
positive
untreated WT axons. FIG. 31C shows fmrl KO axons showing spine supernumeracy.
FIG. 31D shows a clear reduction in spine number when hippocampalfmrl KO
neurons
were cultured with G-2-MePE at a concentration of 50nM, compared to untreated
fmrl
KO neurons (FIG. 31E). FIG. 31F shows the effects of 0.5 nM G-2-MePE on finr 1
KO
hippocampal neurons.. FIG. 31G shows a substantial reduction in spine number
observed
in fmr 1 KO neurons cultured with G-2ePE at a concentration of 5nM, indicating
a mild,
treatment-positive effect. FIG 31H shows a vehicle treated neuron indicating
no toxicity,
and no effect in the number of neuronal spines. FIG. 311 shows finr 1 KO
hippocampal
7
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
culture treated with MPEP at 20uM. The mean (+SD) spine density was measured
as
number of spines per micrometer.
FIG. 32 is a graph showing the testis weight of each animal group ('" p <0.01
versus vehicle-treated wild-type mice).
FIG. 33A is a photograph of a Western blot of pERK expression in lymphocytes
obtained from the wild-type and finr 1 KO animals treated either with vehicle
or G-2-
MePE (100 mg/kg, i.p.; 28 days). FIG. 33B is a graph representing levels of
pERK in the
animal groups. FIG. 33C is a Western blot analysis of total ERK and FIG. 33D
is a graph
showing total levels of ERK in each animal group of the study.
FIG. 34A is a photograph of a Western blot of pAkt and Akt levels in
lymphocytes obtained from wild-type and finr1 knockout mice administered
either
vehicle or G-2-MePE (100 mg/kg, i.p.; 28 days). FIG. 34B is a graph showing
levels of
pAkt in the animal groups of the study.
DETAILED DESCRIPTION
Definitions
The term "about" with reference to a dosage or time refers to a particular
variable and a
range around that variable that is within normal measurement error is within
20% of the value of
the variable. The term "about" with reference to a result observed means the
variation is within
20% of the value of the observed variable.
The term "alkyl" means a linear saturated hydrocarbyl group having from one to
six
carbon atoms, or a branched or cyclic saturated hydrocarbyl group having from
three to six carbon
atoms. Exemplary alkyl groups include straight and branched chain, or cyclic
alkyl groups,
methyl, ethyl, isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, and
hexyl.
The term "animal" includes humans and non-human animals, such as domestic
animals
(eats, dogs, and the like) and farm animals (cattle, horses, sheep, goats,
swine, and the like).
The term "araIkyl" means a group of the formula ¨(CH2)1.2Ar, where Ar is a 5-
or
6-membered carbocyclic or heterocyclic aromatic ring, optionally substituted
with 1 to 3
substituents selected from Cl, 13r, ¨OH, CO2R8
(where R8 is H or alkyl), or ¨
NR8R9, where R8 is as described previously and R9 is H or alkyl. Exemplary
aralkyl groups
include benzyl, 2-chlorobenzyl, 4-(dimethylamino)benzyl, phenethyl, 1-
pyrrolylmethyl, 2-
thienylmethyl, and 3-pyridylmethyl.
The term "disease" includes any unhealthy condition of an animal including
particularly
Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple
sclerosis, diabetes,
motor disorders, seizures, cognitive dysfunctions due to aging and Autism
Spectrum Disorders
8
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
including autism, Fragile X Syndrome, Rett Syndrome (RTT), Autistic Disorder,
Asperger
Syndrome, Childhood Disintegrative Disorder and Pervasive Developmental
Disorder Not
Otherwise Specified (PDD-NOS), and Pathological Demand Avoidance (FDA). .
The term "fatty alcohol residue" is a linear hydrocarbyl group having from
seven to
twenty carbon atoms, optionally containing up to three carbon-carbon double
bonds. Exemplary
fatty alcohol residues include decyl, pentadecyl, hexadecyl (eetyl), octadecyl
(stearyl), oleyl,
linoleyl, and eicosyl.
The term "growth factor" means an extracellular polypeptide-signaling molecule
that
stimulates a cell to grow or proliferate.
The term "injury" includes any acute damage of an animal including non-
hemorrhagic
stroke, traumatic brain injury, perinatal asphyxia associated with fetal
distress such as that
following abruption, cord occlusion or associated with intrauterine growth
retardation, perinatal
asphyxia associated with failure of adequate resuscitation or respiration,
severe CNS insults
associated with near miss drowning, near miss cot death, carbon monoxide
inhalation, ammonia
or other gaseous intoxication, cardiac arrest, coma, meningitis, hypoglycemia
and status
epilepticus, episodes of cerebral asphyxia associated with coronary bypass
surgery, hypotensive
episodes and hypertensive crises, cerebral trauma and toxic injury.
"Memory disorders" or "cognitive disorders" are disorders characterized by
permanent or
temporary impairment or loss of ability to learn, memorize or recall
information. Memory
disorder can result from normal aging, injury to the brain, tumors,
neurodegenerative disease,
vascular conditions, genetic conditions (Huntington's disease), hydrocephalus,
other diseases
(Pick's disease, Creutzfeld-Jakob disease, AIDS, meningitis), toxic
substances, nutritional
deficiency, biochemical disorders, psychological or psychiatric dysfunctions.
The presence of
memory disorder in a human can be established thorough examination of patient
history, physical
examination, laboratory tests, imagining tests and neuropsychological tests.
Standard
neuropsychological tests include but are not limited to Brief Visual Memory
Test-Revised
(BVMT-R), Cambridge Neuropsychological Test Automated Battery (CANTAB),
Children's
Memory Scale (CMS), Contextual Memory Test, Continuous Recognition Memory Test
(CMRT),
Controlled Oral Word Association Test and Memory Functioning Questionnaire,
Denman
Neuropsychology Memory Scale, Digit Span and Letter Number Sequence sub-test
of the
Wechsler Adult Intelligence Scale-III, Fuld Object Memory Evaluation (FOME),
Graham-
Kendall Memory for Designs Test, Guild Memory Test, Hopkins Verbal Learning
Test, Learning
and Memory Battery (LAMB), Memory Assessment Clinic Self-Rating Scale (MAC-S),
Memory
Assessment Scales (MAS), Randt Memory Test, Recognition memory Test (RMT), Rey
Auditory
and Verbal Learning Test (RAVLT), Rivermead Behavioral Memory Test, Russell's
Version of
the Wechsler Memory Scale (RWMS), Spatial Working Memory, Test of Memory and
Learning
9
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
(TOMAL), Vermont Memory Scale (VMS), Wechsler Memory Scale, Wide Range
Assessment of
Memory and Learning (WRAML).
The term "pharmaceutically acceptable excipient" means an excipient that is
useful in
preparing a pharmaceutical composition that is generally safe, non-toxic, and
desirable, and
includes excipients that are acceptable for veterinary use as well as for
human pharmaceutical use.
Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol
composition,
gaseous.
The term "pharmaceutically acceptable salt" means a salt that is
pharmaceutically
acceptable and has the desired pharmacological properties. Such salts include
salts that can be
formed where acidic protons present in the compounds react with inorganic or
organic bases.
Suitable inorganic salts include those formed with the alkali metals, e.g.
sodium and potassium,
magnesium, calcium, and aluminum. Suitable organic salts include those formed
with organic
bases such as amines e.g. ethanolamine, diethanolamine, triethanolamine,
tromethamine, N-
methylglucamine, and the like. Salts also include acid addition salts formed
by reaction of an
amine group or groups present in the compound with an acid. Suitable acids
include inorganic
acids (e.g. hydrochloric and hydrobromic acids) and organic acids (e.g. acetic
acid, citric acid,
maleic acid, and alkane- and arene-sulfonic acids such as methanesulfonic acid
and
benzenesulfonic acid). When there are two acidic groups present in a compound,
a
pharmaceutically acceptable salt may be a mono-acid mono-salt or a di-salt;
and similarly where
there are more than two acidic groups present, some or all of such groups can
be salified. The
same reasoning can be applied when two or more amine groups are present in a
compound.
The term "protecting group" is a group that selectively blocks one or more
reactive sites
in a multifunctional compound such that a chemical reaction can be carried out
selectively on
another unprotected reactive site and such that the group can =readily be
removed after the
selective reaction is complete.
The term "therapeutically effective amount" means the amount of an agent that,
when
administered to an animal for treating a disease, is sufficient to effect
treatment for that disease as
measured using a test system recognized in the art.
The term "treating" or "treatment" of a disease may include preventing the
disease from
occurring in an animal that may be predisposed to the disease but does not yet
experience or
exhibit symptoms of the disease (prophylactic treatment), inhibiting the
disease (slowing or
arresting its development), providing relief from the symptoms or side-effects
of the disease
(including palliative treatment), and relieving the disease (causing
regression of the disease).
The term "functional deficit" means a behavioral deficit associated with
neurological
damage. Such deficits include deficits of gait, as observed in patients with
Parkinson's disease,
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
motor abnormalities as observed in patients with Huntington's disease.
Functional deficit also
includes abnormal foot placement and memory disorders described herein.
The terms "cG-2A11y1P" and "NNZ-2591" and "NNZ" means cyclic Glycy1-2-
Ally1Proline.
The term "seizure" means an abnormal pattern of neural activity in the brain
that results
in a motor deficit or lack of motor control resulting in abnormal motion,
including spasmodic
motion. "Seizure" includes electroencephalographic abnormalities, whether or
not accompanied
by abnormal motor activity.
Implicit hydrogen atoms (such as hydrogen atoms on a pyrrolidine ring, etc.)
are omitted
from the formulae for clarity, but should be understood to be present.
Autism Spectrum Disorders
Autism spectrum disorders (ASDs) are a collection of linked developmental
disorders,
characterized by abnormalities in social interaction and communication,
restricted interests and
repetitive behaviours. Current classification of ASDs recognises five distinct
forms: classical
autism or Autistic Disorder, Asperger syndrome, Rett syndrome, childhood
disintegrative disorder
and pervasive developmental disorder not otherwise specified (PDD-NOS). A
sixth syndrome,
pathological demand avoidance (PDA), is a further specific pervasive
developmental disorder.
However, while PDA is increasingly recognised as an ASD, it is not yet part of
the Diagnostic
and Statistical Manual of Mental Disorders (DSM-IV), published by the American
Psychiatric
Association, nor is it part of the proposed revision, the DSM-V.
Autism
Classical autism is a highly variable neurodevelopmental disorder. It is
typically
diagnosed during infancy or early childhood, with overt symptoms often
apparent from the age of
6 months, and becoming established by 2-3 years. According to the criteria set
out in the DSM-
IV, diagnosis of autism requires a triad of symptoms to be present, including
(a) impairments in
social interaction, (b) impairments in communication and (c) restricted and
repetitive interests and
behaviours. Other dysfunctions, such as atypical eating, are also common but
are not essential for
diagnosis. Of these impairments, social interaction impairments are
particularly important for
diagnosis, and two of the following impairments must be present for a
diagnosis of autism:
(i) impairments in the use of multiple nonverbal behaviors (e.g. eye
contact) to regulate
social interaction;
(ii) failure to develop peer relationships appropriate to developmental level;
(iii) lack of spontaneous seeking to share enjoyment, interests, or
achievements;
(iv) lack of social or emotional reciprocity.
11
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Communication impairments in autism may be manifest in one or more of the
following
ways: delay in (or total lack of) the development of spoken language; marked
impairment in the
ability to initiate or sustain a conversation; stereotyped and repetitive use
of language; and/or a
lack of spontaneous make-believe play. Restricted, repetitive and stereotyped
patterns of behavior
is also required for diagnosis, such as preoccupation with one or more
interest considered
abnormal in intensity, inflexible adherence to routines or rituals, repetitive
motor mannerisms
and/or persistent focus on parts of objects.
Lastly, for a diagnosis of autism, it is necessary that the impairment in the
functioning of
at least one area (i.e. social interaction, language, or imaginative play)
should have an onset at less
than 3 years of age.
Autism is commonly associated with epilepsy or epileptiform activity in the
electroencephalogram (EEG). As many as 60 percent of patients with autism have
epileptiform
activity in their EEGs (Spence and Schneider, 2009 Ped Res 65: 599-606).
Autism is also associated with disturbances in function of IGF-1, which is
depleted in the
Central Nervous System (CNS) in patients with autism (Riikonen et al., 2006
Devel Med Child
Neural 48: 751-755). IGF-1 levels in the CNS increase in patients with autism
after treatment
with agents that reduce symptoms such as fluoxetine (Makkonen et al., 2011
Neuropediatrics
42:207-209).
Importantly, autism shares features of Rett Syndrome and Fragile X Syndrome in
relation
to neuronal connectivity. All three disorders are characterised by defects in
synaptic function and
neuronal connectivity. This is reflected in studies of post mortem human brain
in these patient
groups, which all show failure to form normal synaptic connections. This is
reflected in altered
morphological characteristics, being either a reduction in neuron dendritic
spine density, or
enhanced dendritic spine density but associated with immature synapses. This
is reflected in
animal models of autism, Rett Syndrome and Fragile X Syndrome, which are based
on genetic
changes known to be pathological in these disorders. In these animal models,
neuronal
connectivity defects are revealed morphologically, and also as a failure of
Long Term Potentiation
(LTP). This is important since IGF-1, IGF-111-31 and G-2-MePE increase synapse
formation.
Asperger Syndrome
Asperger syndrome or Asperger Disorder is similar to autism, and shares
certain features.
Like autism, Asperger syndrome is also characterized by impairment in social
interaction and this
is accompanied by restricted and repetitive interests and behavior. Thus,
diagnosis of Asperger
syndrome is characterized by the same triad of impairments as autism. However,
it differs from
the other ASDs by having no general delay in language or cognitive development
and no deficit in
interest in the subject's environment. Moreover, Asperger syndrome is
typically less severe in
12
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
symptomology than classical autism and Asperger's patients may function with
self-sufficiency
and lead relatively normal lives.
Childhood Disintegrative Disorder
Childhood disintegrative disorder (CDD), also known as Heller syndrome, is a
condition
in which children develop normally until age 2-4 years (i.e. later than in
Autism and Rett
syndrome), but then demonstrate a severe loss of social, communication and
other skills.
Childhood disintegrative disorder is very much like autism and both involve
normal development
followed by significant loss of language, social play and motor skills.
However, childhood
disintegrative disorder typically occurs later than autism, involves a more
dramatic loss of skills
and is far less common.
Diagnosis of CDD is dependent on dramatic loss of previously acquired skills
in two or
more of the following areas: language, social skills, play, motor skills (such
as a dramatic decline
in the ability to walk, climb, grasp, etc), bowel or bladder control (despite
previously being toilet-
trained). The loss of developmental skills may be abrupt and take place over
the course of days to
weeks or may be more gradual.
Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS)
Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS) is an ASD
that
describes patients exhibiting some, but not all, of the symptoms associated
with other well defined
ASDs. The key criteria for diagnosis of an ASD include difficulty socializing
with others,
repetitive behaviors, and heightened sensitivities to certain stimuli. These
are all found in the
ASDs described above. However, autism, Asperger syndrome, Rett syndrome and
childhood
disintegrative disorder all have other features that enable their specific
diagnosis. When specific
diagnosis of one of these four disorders cannot be made, but ASD is apparent,
a diagnosis of
PDD-NOS is made. Such a diagnosis may result from symptoms starting at a later
age than is
applicable for other conditions in the spectrum.
Rett Syndrome
Rett Syndrome (RTT) is a neurodevelopmental disorder that almost exclusively
affects
females (1 in 10:000 live births). RTT is classified as an autism spectrum
disorder (Diagnostic
and Statistical Manual of Mental Disorders, Fourth Edition ¨ Revised (DSM-1V-
R).
Approximately 16,000 patients are currently affected by it in the U.S.A. (Rett
Syndrome Research
Trust data). For a diagnosis of Rett syndrome, the following symptoms are
characteristic:
impaired development from age 6-18 months; slowing of the rate of head growth
starting from
between age 3 months and 4 years; severely impaired language; repetitive and
stereotypic hand
13
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
movements; and gait abnormalities, e.g. toe-walking or unsteady stiff-legged
walk. There are in
addition, a number of supportive criteria that may help diagnosis of Rett
Syndrome, but are not
essential for a diagnosis. These include breathing difficulties, EEG
abnormalities, seizures,
muscle rigidity and spasticity, scoliosis (curving of the spine), teeth-
grinding, small hands and
feet in relation to height, growth retardation, decreased body fat and muscle
mass, abnormal sleep
patterns, irritability or agitation, chewing and/or swallowing difficulties,
poor circulation and
constipation.
The onset of RTT usually begins between 6-18 months of age with a slowing of
development and growth rates. This is followed by a regression phase
(typically in children aged
1-4 years of age), pseudo-stationary phase (2-10 years of age) and a
subsequent progressive late
motor deterioration state. RTT symptoms include sudden deceleration of growth
and regression
in language and motor skills including purposeful hand movements being
replaced by
stereotypical movements, autistic features, panic-like attacks, sleep cycle
disturbances, tremors,
seizures, respiratory dysfunctions (episodic apnea, hyperpnea), apraxia,
dystonia, dyskinesia,
hypotonia, progressive kyphosis or scoliosis and severe cognitive impairment.
Most RTT patients
survive into adulthood with severe disabilities and require 24-hour-a-day
care.
Between 85% and 95% cases of RTT are reported to be caused by a mutation of
the
Mecp2 gene (Amir et al. 1999. Nat Genet 23:185-188; Rett Syndrome Research
Trust) - a gene
encoding methyl-CpG-binding protein 2 (MeCP2). Mecp2 maps to the X-chromosome
(location
Xq28) and for this reason, mutations to the gene in males are usually lethal.
While RTT is a
genetic disorder, less than I% of recorded cases are inherited; almost all
mutations of Mecp2
occur de novo, with two thirds caused by mutations at 8 CpG dinucleotides
(R106, R133, T158,
R168, R255, R270, R294 and R306) located on the third and fourth exons.
MeCP2 is a protein that binds methylated CpG dinucleotides to exert
transcriptional
silencing of DNA in the CNS. The key effect of a reduction or absence of MeCP2
appears to be
an impairment of dendritic spine development and the formation of synapses.
MeCP2 expression
appears to temporally correlate with brain maturation, explaining why symptoms
typically appear
around 18 months of age.
Presenting Features Common to ASDs
Taking the ASDs together, it is clear that there are commonalities in
presenting symptoms
among all 5 forms. These common features are impairments in normal social
competences, and
repetitive behaviours. In all but Asperger syndrome there is also a consistent
presentation of
delayed intellectual development most commonly manifest as a shortfall in
language skills.
Cognitive loss relative to normal parameters for the age is often quite marked
in autism, Rett
Syndrome, CDD and PDD-NOS. The presence of epilepsy or abnormal activity in
the EEG is
14
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
also common to autism, Fragile X Syndrome and Rett Syndrome. Epilepsy arises
in situations of
abnormal neuronal connectivity. Impaired neuronal connectivity and deranged
synaptic function
is a common feature of autism, Fragile X Syndrome and Rett Syndrome and of
animal models of
these conditions.
Genetic Models of ASD
To offer validity, animal models of ASDs and neurodevelopment disorders, must
demonstrate similar symptoms to the clinical conditions and have a reasonable
degree of face
validity regarding the etiology of those symptoms. It is known that classical
autism may be
caused by many different genetic impairments and no single genetic defect is
thought to account
for more than a few percent of autism cases. Indeed, recent studies have
revealed numerous de
novo structural variations of chromosome locations thought to underlie ASD, in
addition to rare
inherited genetic defects (Marshall et al, 2008; Sebat et al, 2007). Thus,
copy number variation
(CNV), translocation and inversion of gene sequences at 20 key sites or more,
including lp, 5q,
7q, 15q, 16p, 17p and Xq, have been mapped as ASD loci.
However, despite the polygenetic background underlying ASD, and the complexity
of the
etiology, it is known that certain genetic defects can produce ASD. Some of
the best
characterized defects arise from chromosomal aberrations of genes that code
for a cluster of
postsynaptic density proteins, including neuroligin-3 (NLGN3), neuroligin-4
(NLGN4), neurexin-
I a (NRXN1) and shank3 (Sebat et al, 2007). Importantly, these defects point
to altered synaptic
function and therefore disturbed neuronal connectivity as a final common
pathway in autism and
related disorders (Minshew and Williams 2007 Arch Neurol. 64:945-950; Gilman
et al., 2011
Neuron. 70:898-907). Such connectivity deficits are reflected in morphological
findings in post
mortem examination, which reveal increased dendritic spine density in autism
(Hutsler and Zhang
2010 Brain Res. 1309:83-94).
NLGN3 and NLGN4 are postsynaptic cell-adhesion molecules present in
glutamatergic
synapses. They play a role in coordinating presynaptic contact to the
postsynaptic site and also
interact with the postsynaptic scaffolding protein shank3. Mutations to NLGN3
and NLGN4 have
been observed in the ASD population and account for perhaps 1% of all ASD
cases (Lintas &
Persico, 2008). Jamain and colleagues first reported a missense to NLGN3 and a
frameshift to
NLGN4 in two unrelated subjects, resulting in Asperger syndrome and classical
autism
respectively (Jamain et al, 2003). While the incidence of NLGN3 or NLGN4
mutations in the
ASD population is low (indeed, no much mutations were observed in a study of
96 ASD patients
in a Canadian study; Gauthier et al, 2005), it has been confirmed in
preclinical studies that
neuroligin mutations can indeed produce of model of autistic symptoms. Thus,
introduction to
mice of the same R451C missense to NLGN3 that has been reported clinically
results in a mutant
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
mouse strain showing reduced social interaction and enhanced inhibitory
synaptic transmission
(Tabuchi et al, 2007).
The R451C mutant therefore mouse represents a model for ASD based upon NLGN3
mutation. In this case, mutation at the R451 position of NLGN3 results in a
'gain-of-function'
mutation.
In contrast, modeling the clinical mutation of NLGN4 in mice is achieved by a
'loss-of-
function' mutation of NLGN4 (a classical knockout model). In this model,
mutant mice display a
social interaction deficit and reduced ultrasonic vocalization (Jamain et al,
2008).
Communication deficits are central to clinical ASDs and in the NLGN4 knockout
mice a
reduction in ultrasonic vocalizations from male mice exposed to wild-type
female counterparts
supports the face validity of the strain as a model of ASD.
Presynaptic neurexin proteins induce postsynaptic differentiation in opposing
dendrites
through interactions with postsynaptic neuroligin counterparts. Mutations of
the neurexin-1 a
(NRXN1) gene have been reported in numerous studies (Sebat et al, 2007;
Marshall et at, 2008;
Kim et al, 2008; Yuri et al, 2008) and these have been observed in the form of
copy-number
variants. As with NLGN mutations, when a mutation of the NRXN1 gene is
introduced to mice
(in the form of gene knockout), a mutant strain with certain ASD-like features
is produced
(Etherton et at, 2009). These NRXN1 knockout mice show a decrease in
hippocampal miniature
excitatory postsynaptic current (mEPSC) frequency and a decreased input-output
relationship of
evoked currents. These electrophysiological effects relate to decreased
excitatory transmission in
the hippocampus. In addition to decreased excitatory neurotransmission, NRXN1
knockout mice
exhibit a decrease in pre-pulse inhibition, though social behaviour appears to
be unaffected
(Etherton et al, 2009).
Sharing certain features with the neurexin-NLGN trans-synaptic construct, cell
adhesion
molecule 1 (CADM1) is an imrnunogolbulin family protein present both pre- and
post-
synaptically that is also involved in synaptic trans-cell adhesion activity
(Biederer et at, 2002).
Mutations to the CADM1 gene have been detected in ASD patients and appear to
represent a
further possible cause of these conditions (Zhiling et at, 2008).
Analysis of CADM1 knockout mice reveals that these animals show increased
anxiety-
related behavior, impaired social interaction and impaired social memory and
recognition. In
addition CADM1 knockout mice demonstrate poorer motor skills (Takayanagi et
al, 2010). These
dysfunctions are again consistent with ASD syrnptomatology.
22q13 deletion syndrome (also known as Phelan-McDermid Syndrome), is a rare
genetic
disorder caused by a micro-deletion at the q13.3 terminal end of chromosome
22. This micro-
deletion is rarely uncovered by typical genetic screening and a fluorescence
in situ hybridization
test is recommended to confirm the diagnosis. Recent work indicates the
syndrome is caused by
16
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
errors in the gene shank3, a postsynaptic density protein critical for normal
neuronal functioning.
Interestingly, errors in this gene have also been associated with ASD and
22q13 deletion
syndrome can commonly lead to an ASD diagnosis (Durand et al, 2007; Moessner
et al, 2007;
Sykes et al, 2009). Given
the close association of 22q13 deletion syndrome and the
consequential diagnosis of ASD, a mutant mouse model of this mutation has been
developed.
The shank3 knockout mouse exhibits several deficits that mirror ASD symptoms,
including reduced ultrasonic vocalizations (i.e. diminished social
communication) as well as
impaired social interaction time between mice. In addition, these mice have
impaired
hippocampal CA1 excitatory transmission, measured by input-output relationship
of evoked
currents and impaired long-term potentiation (LTP). LTP is believed to be a
physiological process
underlying memory formation and consolidation. Thus, the model exhibits a
similar phenotype to
the NLGN4 knockout, consistent with ASD.
As has been noted, 22q13 deletion syndrome itself is very rare. However, it
provides
important information that involvement specific genes may have in the etiology
of ASDs. In
addition to shank3, this disorder reveals a further possible gene defect in
ASD. Of the 50 or so
cases of 22q13 deletion syndrome described, all but one have a gene deletion
that extends beyond
shank3 to include a further gene, known as the Islet Brain-2 gene (IB2) (Sebat
et al, 2007). The
IB2 protein interacts with many other proteins including MAP kinases and
amyloid precursor
protein, appears to influence protein trafficking in neurites, and is enriched
at postsynaptic
densities (Giza et al, 2010). Mice lacking the protein (1B2-/- knockout mice)
exhibit impaired
social interaction (reduced social sniffing and interaction time), reduced
exploration and cognitive
and motoric deficits (Giza et al, 2010). This behavioral phenotype was
associated with reduced
excitatory transmission in cerebellar cells. As with shank3 knockout, the
phenotype of IB2
mutation is therefore also consistent with ASD.
In addition to the animal models of postsynaptic density protein defects
described above,
other monogenetic syndromes that share various features with ASDs can lead to
autism offer
another avenue for drug targeting of ASD or neurodevelopment disorders. An
excellent example
of this is Fragile X Syndrome.
Fragile X Syndrome (FXS) is caused by the expansion of a single trinucleotide
gene
sequence (CGG) on the X-chromosome that results in failure to express the
protein coded by the
fmr I gene. FMRI (fragile X mental retardation 1) is a protein required for
normal neural
development. FXS can cause a child to have autism (Hagerman et al, 2010); in 2-
6% of all
children diagnosed with autism the cause is FXS gene mutation. Moreover,
approximately 30% of
FXS children have some degree of autism and a further 30% are diagnosed with
PDD-NOS
(Hagerman et al, 2010). Indeed, Fragile X Syndrome is the most common known
single gene
cause of autism. FMR1 knockout mice have been developed as a model of FXS and,
therefore, as
17
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
a further model of ASD. Knockout mutation of the FIVIR1 gene has been shown to
result in
neuronal connectivity deficits such as abnormal dendritic spine development
and pruning
(Comery et al, 1997), along with an associated dysregulation of dendritic
scaffold proteins
(including shankl) and glutamate receptor subunits in postsynaptic densities
(Schlitt et at, 2009).
These effects on dendrite morphology results deficits in functional measures
of connectivity such
as impaired LTP in the cortex and amygdala (Zhao et al, 2005) and hippocampus
(Lauterborn et
al, 2007), as well as impaired cognition (Kreuger et al, 2011) and an
enhancement in social
anxiety (Spencer et at, 2005). These connectivity deficits are mirrored in FXS
patients, who show
enhanced dendritic spine density in post mortem analyses (Irwin et al., 2000
Cereb Cortex
10:1034-1048). This enhanced dendritic spine density is accompanied by
immature synapses
(Klemmer et al., 2011 J Biol Chem. 286:25495-25504), i.e. may represent a
functionally
immature state.
In contrast to the ASDs of autism, Asperger, CDD and PDD-NOS, Rat syndrome
appears
to have an almost monogenetic basis and may be modelled in mice with good face
validity. Rett
syndrome is thought be caused, in up to 96% of cases, by a defect in the Meep2
gene (Zoghbi,
2005). As a result, MeCP2 knockout mutant mice provide an animal model with
all the hallmarks
of clinical Rett syndrome, with a phenotype showing some overlap with the
NLGN4, shank3 and
IB2 knockout models of ASD. Thus, MeCP2 knockout mice display a clear
impairment in LTP in
the hippocampus along with a corresponding decrease in social and spatial
memory (Moretti et al,
2006) and impaired object recognition (Schaevitz et al, 2010). This impairment
in LTP is
accompanied by a decrease in dendritic spine density. Patients with Rett
Syndrome show reduced
dendritic spine density (Bel ichenko et al., 1994 Neuroreport 5:1509-1513).
Thus, ASDs in human beings share many features of cognitive or developmental
disorders in animals, including rodents. Therefore, studies of therapies of
ASDs in rodents such
as mice and rats are reasonably predictive of results obtained in human
beings. A common
feature seen in autism, Fragile X Syndrome and Rett Syndrome is the presence
of neuronal
connectivity deficits, reflected in either decreased dendritic spine density
or enhanced dendritic
spine density with immature synapses. The functional consequences of these
morphological
changes are similar in animal models of these disorders, reflected as deficits
in LTP, for example.
Treatment of Clinical ASD and ASD Animal Models With G-2MePE
As described above, a conserved pathology is observed in ASDs that comprises
impaired
neurite development, impaired synaptic connectivity and a corresponding
impairment in social
and cognitive functioning as a result. Such synaptic dysfunctions result from
genetically altered
functions of postsynaptic density proteins. Normal neurite growth and
postsynaptic development
may be regulated and augmented by growth factors such as brain derived
neurotrophic factor
18
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
(BDNF; Chapleau et al, 2009) and insulin-like growth factor-I (IGF-1; Riikonen
et al, 2006;
Tropea et al, 2009). Indeed, IGF-1 is essential for normal dendritic spine
growth and synapse
formation (Cheng et al., 2003 J Neurosci Res. 73:1-9). Drugs that promote
growth factor function
are therefore of use in the treatment of progressive developmental disorders
such as ASDs. cG-
2A1ly1P is a small molecule analog of the terminal tripeptide of IGF-1, IGF1(I-
3). As an IGF-1
mimetic analog, cG-2A11y1P exerts trophic and neuroprotective effects in
various animal models.
cG-2AllyIP is therefore effective at treating ASD and FXS symptoms such as
those relating to
synaptic dysfunctions resulting from the gene mutations described above.
In clinical terms, ASD patients, presenting with autism, Asperger syndrome,
Rett
syndrome, childhood disintegrative disorder and PDD-NOS, as well as patients
with 22q13
deletion syndrome, Fragile X Syndrome and pathological demand avoidance are
treated with cG-
2Ally1P. Patients exhibit social and communication impairments as well as
cognitive deficit.
Treatment with cG-2Al1y1P, for example, on a daily basis and in another
example, by the oral
route, is observed to induce an improvement in stereotypic repetitive
movements, improved social
functioning and improved cognitive performance following drug treatment.
In animal models of ASDs, daily G-2MePE treatment by oral gavage or
intraperitoneal
injection to knockout mice will improve ASD-like symptoms. G-2MePE is
effective in the
following ASD mutant mouse models: NLGN3 (R451C) mutant, NLGN4 knockout, NRXN1
knockout, CADM1 knockout, shank3 knockout, IB2 knockout, FMR1 knockout and
MeCP2
knockout. When administered sub-chronically (1-10 weeks) on a daily basis, G-
2MePE is
effective at improving LTP in the hippocampus following burst stimulation or
high frequency
stimulation. Similarly, G-2MePE increases excitatory neurotransmission as
measured by field
extracellular postsynaptic potential electrophysiological recordings in
cortex, hippocampus and
cerebellum. As a result of improved excitatory neurotransmission (reversal of
observed ASD-like
neurotransmission deficit), G-2MePE is observed to improve cognitive and
motoric outcome
tests of cognitive performance. Thus, G-2-MePE improves performance in the
Morris water maze
and radial arm maze tests. In models of social interaction, G-2MePE,
administered to ASD
mutant mice, increases time spent by knockout males in social interaction with
wild-type females.
In addition, ultrasonic vocalizations to female wild type mice is increased.
Because G-2MePE is a member of the compounds of GPE analogs disclosed
herein, any of the disclosed compounds also can be effective in treating
symptoms of
ASDs. Further, because compounds and methods of this invention address
underlying
neurological mechanisms (e.g., decrease neural inflammation by inhibiting
release of
inflammatory cytokines), this invention can provide more than short-term
management of
symptoms. Rather, compounds and methods of this invention can improve neural
19
CA 02929286 2016-04-29
WO 2014/085480 PCT/US2013/072049
function, promote neuronal cell migration, promote neurogenesis, promote
neuronal stem
cell differentiation, promote axonal and dendritic outgrowth, and promote
synaptic
transmission, thereby relieving adverse symptoms of ASDs.
Compounds of the Invention
While the broadest definition of the invention is set out in the Summary,
certain
compounds of this invention are presently described.
In one aspect, this invention provides compounds of Formula 1 and Formula 2:
2 R3 R3
2
\R I
R I
R4
/R4
O)NN N
Y _ Y
1 0
0 R
0 (0112)n (CHOn
(01-12), Z (0F126
Formula I Formula 2
where m is 0 or 1;
n is 0 or 1;
X is H or ----NR6R7;
Y is H, alkyl, ¨0O2R5, or ¨CONR6R7;
Z is H, alkyl, ¨0O2R5 or ¨CONR6R7;
R. is H, alkyl, or aralkyl;
R2, R3, and R4 are independently H or alkyl;
each R5 is independently H, alkyl, or a fatty alcohol residue;
each R6 and R7 is independently H, alkyl, or arallcyl, or -NR6R7 is
pyrrolidino, piperidino, or
morpholino;
or a lactone formed when a compound where Y is ¨0O2(alkyl) and Z is ¨CO2H or
where Y is
¨CO2H and Z is ¨0O2(alkyl) is lactonized;
and the pharmaceutically acceptable salts thereof,
provided that the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ or a salt
thereof.
In some aspects, this invention includes:
(a) the compounds are compounds of Formula 1;
(b) m is 0;
(c) nisi;
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
(d) at least one of X, Y, R', RL, R, R`4, and R5 is not hydrogen;
(e) X is --NR6R7; and
(f) Y is ¨0O2R5 or ¨CO2NR6R7; and
(g) Z is ¨0O2R5 or ¨CO2NR6R7.
Other compounds of the invention are compounds of Formu-l-a-l-wherein X is -
NR6R7 and
R6 and R7 are independently alkyl or aralkyl. The more preferred embodiment is
a compound of
Formula I wherein X is -NR6R7 and both R6 and R7 are alkyl.
Yet another compound of the invention is G-2-MePE, a compound of Formula 1
wherein
m is 0, n is I, RI -43-R4-H, R2 is methyl, X is NR6R7 where R6=R7=H, Y is
CO2R5 where R5
=H, Z is CO2R5where R5 =11.
Pharmacology and Utility
Compounds of this invention can have anti-inflammatory, anti-apoptotic, anti-
necrotic
and neuroproteetive effects. Their activity in vivo can be measured by cell
counts, specific
staining of desired markers, or by methods such as those discussed in Klempt
ND et al: Hypoxia-
ischemia induces transforming growth factor 111 mRNA in the infant rat brain.
Molecular Brain
Research: 13: 93-101. Their activity can also be measured in vitro using
methods known in the
art or described herein.
Conditions affecting brain function become prevalent in aging populations.
Memory loss
and memory impairment are distressing to patients affected and their families.
Memory loss or
impairment can result from normal aging, injury to the brain,
neurodegenerative disease and
psychological or psychiatric dysfunctions. It is therefore of great benefit to
patients, their families
and to society that novel compounds are identified and characterized that
enhance memory and/or
cognitive function, and treat or prevent memory loss or impairment.
It is desirable to study effects of potential therapeutic agents in animal
systems. One such
useful system is the rat. It is known that with aging, rats and other animals
(including human
beings) can exhibit symptoms of memory loss, memory impairment and other
cognitive
dysfunctions. Further, it is known that studies in rats of therapeutic agents
are predictive of
therapeutic effects in humans. Thus, studies of effects of GPE and G-2-MePE
and cognitive
function in aging rats are reasonably predictive of therapeutic effects of
those agents in aging
human beings that have or are prone to acquiring memory deficits or other
cognitive dysfunction.
Compounds of this invention can enhance cognitive function and/or treat memory
disorders. The
cognitive enhancing activity and therapeutic activity in vivo can be measured
by standard
neuropsychological or behavioral tests known to individuals skilled in the
art. Such tests can be
chosen from a wide range of available tests described above, and will vary
depending on the
cognitive function to be tested and the condition of the animal.
21
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Standard behavioral tests useful for testing cognitive function in
experimental animals
include but are not limited to the Morris Water Maze test, passive avoidance
response test, novel
object recognition test, olfactory discrimination test, the 8-arm radial maze
test and the T-maze
test. These tests are directly applicable to studies of effects of GPE and G-2-
MePE on cognitive
function in aging rats.
The compounds of this invention are also expected to have pharmacological and
therapeutic activities similar to those of GPE, and these activities may be
measured by the
methods known in the art, and discussed in the documents cited herein, and by
methods used for
measuring the activity of GPE.
The therapeutic ratio of a compound can be determined, for example, by
comparing the
dose that gives effective anti-inflammatory, anti-apoptotic and anti-necrotic
activity in a suitable
in vivo model such as a hypoxic-ischemic injury (Sirimanne ES, Guan J,
Williams CE and
Gluckman PD: Two models for determining the mechanisms of damage and repair
after hypoxie-
ischemic injury in the developing rat brain (Journal of Neuroscience Methods:
55: 7-14, 1994) in a
suitable animal species such as the rat, with the dose that gives significant
observable side-effects in
the test animal species.
The therapeutic ratio of a compound can also be determined, for example by
comparing the
dose that gives effective cognitive function enhancement or treats a memory
disorder in a suitable in
vivo model (Examples 4, 5 and 6 below) in a suitable animal species such as
the rat, with the dose
that gives significant weight loss (or other observable side-effects) in the
test animal species.
Compounds of this invention can be useful in treatment of a variety of
neurodegenerative
disorders, including hypoxia/ischemia and neuronal degeneration (U.S. Pat. No.
7,041,314),
traumatic brain injury, motor disorders and seizures, stroke, and cardiac
artery bypass graft
surgery (U.S. Pat. No. 7,605,177), non-convulsive seizures (U.S. Pat. No.
7,714,020), and
disorders of cognitive function (U.S. Appl. No. 12/903,844). Additionally, as
described more
fully herein below, compounds of this invention can be useful for treating
Rett Syndrome,
including prolonging life, increasing neuronal activity and treating seizures
associated with Rett
Syndrome.
In one study of Rett Syndrome in mice (using the MeCP2 knock-out model), GPE
was
found to have effects to prolong life and increase neuronal function (U.S,
Publication No.
2009/0099077). However, as disclosed further herein, GPE, being a naturally
occurring peptide,
is rapidly degraded in vivo and in vitro, and its utility in chronic therapy
of patients with Rett
Syndrome is therefore unclear.
22
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Pharmaceutical Compositions and Administration
In general, compounds of this invention can be administered in therapeutically
effective
amounts by any of the usual modes known in the art, either singly or in
combination with at least
one other compound of this invention and/or at least one other conventional
therapeutic agent for
the disease being treated. A therapeutically effective amount may vary widely
depending on the
disease or injury, the severity of the disease, the age and relative health of
the animal being
treated, the potency of the compound(s), and other factors. As anti-
inflammatory, anti-apoptotic,
anti-necrotic, anti-neurodegenerative, therapeutically effective amounts of
compounds of this
invention can range from about 0.001 milligrams per kilogram (mg/kg) to about
100 (mg/kg)
mass of the animal, for example, about 0.1 to about 10 mg/kg, with lower doses
such as about
0.001 to about 0.1 mg/Kg, e.g. about 0.01 mg/Kg, being appropriate for
administration through the
cerebrospinal fluid, such as by intracerebroventricular administration, and
higher doses such as
about 1 to about 100 mg/Kg, e.g. about 10 mg/Kg, being appropriate for
administration by methods
such as oral, systemic (e.g. transdermal), or parenteral (e.g. intravenous)
administration. A person
of ordinary skill in the art will be able without undue experimentation,
having regard to that skill
and this disclosure, to determine a therapeutically effective amount of a
compound of this
invention for a given disease or injury.
In general, compounds of this invention can be administered as pharmaceutical
compositions by one of the following routes: oral, topical, systemic (e.g.
transdermal, intranasal,
or by suppository), or parenteral (e.g. intramuscular, subcutaneous, or
intravenous injection), by
administration to the CNS (e.g. by intraspinal or intercisternal injection);
by implantation, and by
infusion through such devices as osmotic pumps, implantable pumps, transdermal
patches, and
the like. Compositions can take the form of tablets, pills, capsules,
semisolids, powders, sustained
release formulation, solutions, suspensions, elixirs, aerosols, soluble gels
or any other appropriate
compositions; and comprise at least one compound of this invention in
combination with at least
one pharmaceutically acceptable or physiological acceptable excipient.
Suitable excipients are
well known to persons of ordinary skill in the art, and they, and the methods
of formulating the
compositions, may be found in such standard references as Gennaro AR:
Remington: The Science
and Practice of Pharmacy, 20' ed., Lippincott, Williams & Wilkins, 2000.
Suitable liquid carriers,
especially for injectable solutions, include water, aqueous saline solution,
aqueous dextrose
solution, glycols, and the like, with isotonic solutions being preferred for
intravenous, intraspinal,
and intracisternal administration and vehicles such as artificial
cerebrospinal fluid being also
especially suitable for administration of the compound to the CNS. The above
text is expressly
incorporated herein fully by reference.
Compounds of this invention can be administered orally, in tablets or
capsules. In some
embodiments, compounds of this invention can be prepared in water-in-oil
emulsions in the form
23
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
or microemuisions, coarse emulsions, liquid crystals, or nanocapsules (U.S.
App!. No.
12/283,684, now U.S. Pat. No. 7,887,839 issued February 15, 2011). Because
compounds of this
invention can have substantial oral bioavailability, they can be
advantageously used for
convenient and chronic administration. Additionally, orally available
compositions include
soluble hydrogels containing active compounds, thus permitting oral
administration of
neuroprotective compounds without the need for a patient to swallow a tablet
or capsule. Such
slow-release materials and gels are known in the art.
Compounds of this invention can be administered after or before onset of a
condition that
is likely to result in neurodegeneration or a symptom thereof. For example, it
is known that
hypoxia/ischemia can occur during coronary artery bypass graft (CABG) surgery.
Thus, a patient
can be pre-treated with a compound of this invention before being placed on an
extracorporeal
oxygenation system. In some embodiments, it can be desirable to administer a
compound of this
invention beginning about 4 hours before surgery or before an event that is
likely to lead to
traumatic or other neurological injury. In other embodiments, it can be
desirable to infuse a
compound of this invention during the surgery or during a surgical procedure
to repair a
neurological injury. Compounds of this invention can also be used in emergency
situations, for
example in a patient that has just experienced a stroke, hypoxic event,
traumatic brain injury or
other acute insult. In such situations, a compound of this invention can be
administered
immediately after a diagnosis of neural injury is made.
In some situations, kits containing compound of this invention can be prepared
in advance
of use in the field. A kit can contain a vial containing a compound of the
invention in a
pharmaceutically acceptable formulation (e.g., for injection or oral
administration), along with a
syringe or other delivery device, and instructions for use. In situations in
which a seizure is
diagnosed, a compound of this invention can be administered along with an
anticonvulsant. Many
anticonvulsants are known in the art and need not be described in detail
herein.
Additionally, "secondary" neurological injuries can occur after a primary
insult such as a
traumatic injury, stroke or surgical procedure. For example, after a stroke,
penetrating brain
injury or a CABG procedure, inflammation of neural tissue can lead to
neurodegeneration.
Secondary injuries can be reflected by increased activation of inflammatory
cells (e.g., astrocytes
and/or microglia), and actions of inflammatory mediators can cause
neurological damage. Thus,
in some embodiments, it can be desirable to administer a compound of this
invention for periods
beginning before the insult, to up to about 100 hours after the insult. In
other embodiments, it can
be desirable to administer a compound of this invention beginning before the
insult, during the
insult and after the insult, either continuously, as an infusion, or in
discrete doses separated by a
desired time interval.
24
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Compounds of this invention can also be suitably administered by a sustained-
release
system or gel material with G-2-MePE incorporated therein. Suitable examples
of sustained-
release compositions include semi-permeable polymer matrices in the form of
shaped articles,
e.g., films, or mierocapsuIes. Sustained-release matrices include polylactides
(U.S. Pat. No.
3,773,919; EP 58,481), copolymers of L-glutamie acid and gamma-ethyl-L-
glutamate (Sidman et
al., 1983), poly(2-hydroxyethyl methacrylate) (Langer et al., 1981), ethylene
vinyl acetate
(Langer et al., supra), or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
Additionally, gel
compositions based on polysaccharides (e.g., carboxymethyl cellulose,
carboxyethyl cellulose,
chitosan or other cellulose derivatives) and polyethylene oxide derivatives
(e.g., polyethylene
glycols) can be used used. These gel compositions are soluble in aqueous
solutions, are
biocompatible, non-toxic and therefore can be used for administering compounds
of this invention
to any mucosa] surface, including the oral cavity, nasopharynx, urogenital
tract, intestine or
rectum.
Sustained-release compositions also include a liposomally entrapped compound.
Liposomes containing the compound are prepared by methods known per se: DE
3,218,121;
Epstein et al., 1985; Hwang et al., 1980; EP 52,322; EP 36,676; EP 88,046; EP
143,949; EP
142,641; Japanese Pat. Appin. 83-118008; U.S. Pat. Nos. 4,485,045 and
4,544,545; and EP 102,
324. Ordinarily, liposomes are of the small (from or about 200 to 800
Angstroms) unilamellar
type in which the lipid content is greater than about 30 mole percent
cholesterol, the selected
proportion being adjusted for the most efficacious therapy. Each and every of
the above-
identified publications is expressly herein incorporated fully by reference,
as if each had been
separately so incorporated.
Compounds of this invention can also be attached to polyethylene glycol
("PEGylated")
to increase their lifetime in vivo, based on, e.g., the conjugate technology
described in WO
95/32003.
Desirably, if possible, when administered as an anti-inflammatory, an anti-
apoptotic
agent, an anti-necrotic agent, or an anti-neurodegenerative agent, compounds
of this invention can
be administered orally. The amount of a compound of this invention in the
composition can vary
widely depending on the type of composition, size of a unit dosage, kind of
excipients, and other
factors well known to those of ordinary skill in the art. In general, the
final composition can
comprise from about 0.0001 percent by weight (% w) to about 10% w of the
compound of this
invention, preferably about 0.001% w to about 1% w, with the remainder being
an excipient or
excipients.
A composition may optionally contain, in addition to a compound of this
invention, at
least one agent selected from, for example, growth factors and associated
derivatives (insulin-like
growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), transforming
growth factor-131,
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
activin, growth hormone, nerve growth factor, brain-derived neurotrophic
factor (BDNF), growth
hormone binding protein, IGF-binding proteins (especially IGFBP-3), basic
fibroblast growth
factor, acidic fibroblast growth factor, the hst/K.fgk gene product, FGF-3,
FGF-4, FGF-6,
keratinocyte growth factor, androgen-induced growth factor. Additional members
of the FGF
family include, for example, int-2, fibroblast growth factor homologous factor-
1 (FHF-1), FHF-2,
FHF-3 and FHF-4, karatinocyte growth factor 2, glial-activating factor, FGF-10
and FGF-16,
ciliary neurotrophic factor, brain derived growth factor, neurotrophin 3,
neurotrophin 4, bone
morphogenetic protein 2 (BMP-2), glial-cell line derived neurotrophic factor,
activity-dependant
neurotrophic factor, cytokine leukaemia inhibiting factor, oncostatin M,
interleukin), a-, y-, or
consensus interferon, and TNF-a. Other forms of neuroprotective therapeutic
agents include, for
example, clomethiazole; kynurenic acid, Semax, tacroIimus, L-threo-l-pheny1-2-
decanoylamino-
3-morpholino-1-propanol, andrenocorticotropin-(4-9) analog [ORG 2766] and
dizolcipine (MK-
801), selegiline; glutamate antagonists such as mematine (Namenda) NF'S1506,
0V1505260,
MK-801, GV150526; AMPA antagonists such
as 2,3 -dihydroxy-6-nitro-
7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070 and LY300164; anti-
inflammatory agents
directed against the addressin MAdCAM-1 and/or its integrin a4 receptors
(a4131 and a407),
such as anti-MAdCAM- 1 rnAb MECA-367 (ATCC accession no. 11B-9478).
Combination
therapy with metabotropic glutamate receptor antagonists such as fenobam may
also be useful.
Also, in addition to a compound of this invention, a composition may include a
selective
serotonin reuptake inhibitor such as fluoxetine, a selective norepinephine
reuptake inhibitor such
as viloxazine, or an atypical anti-psychotic such as risperidone. Most of
these agents, especially
the peptides such as the growth factors, etc., are not orally active, and will
require administration
by injection or infusion.
Preparation of Compositions
The starting materials and reagents used in preparing these compounds are
either
available from commercial suppliers such as Aldrich Chemical Company
(Milwaukee, Wis.),
Bachem (Torrance, Calif.), Sigma (St.Louis, Mo.), or are prepared by methods
well known to the
person of ordinary skill in the art following procedures described in such
references as Fieser and
= 30 Fieser's Reagents for Organic Synthesis, vols 1-17, John
Wiley and Sons, New York, N.Y., 1991;
= Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supplements, Elsevier
Science
Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New
York, N.Y., 1991;
March 1; Advanced Organic Chemistry, 4th ed. John Wiley and Sons, New York,
N.Y., 1992; and
Larock: Comprehensive Organic Transformations, VCH Publishers, 1989. In most
instances,
amino acids and their esters or amides, and protected amino acids, are widely
commercially
available; and the preparation of modified amino acids and their amides or
esters are extensively
26
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
described in the chemical and biochemical literature and thus well-known to
persons of ordinary
skill in the art. For example, N-pyrrolidineacetic acid is described in Dega-
Szafran Z and
Pryzbylak R. Synthesis, IR, and NMR studies of zwitterionic a-(1-
pyrrolidine)alkanocarboxylic
acids and their N-methyl derivatives. J. Mol. Struct.: 436-7, 107-121, 1997;
and N-
piperidineacetic acid is described in Matsuda 0, Ito S, and Sekiya M. Reaction
of N-
(alkoxymethyDdialkylamines and N,Nr-methylenebisdialkylamines with
isocyanides. Chem.
Pharm. Bull.: 23(1), 219-221, 1975. Each of the above-identified publications
is herein expressly
incorporated fully by reference as though individually so incorporated.
Starting materials, intermediates, and compounds of this invention may be
isolated and
purified using conventional techniques, including filtration, distillation,
crystallization,
chromatography, and the like. They may be characterized using conventional
methods, including
physical constants and spectral data.
Compounds of this invention may be prepared by the methods described below and
as
given in the Examples.
Compounds of Formula 1 are analogs of GPE, or modifications thereof, such as
esters or
amides. In general, they may be prepared by methods such as are already well-
known to persons
of ordinary skill in the art of peptide and modified peptide synthesis,
following the reaction
schemes set forth in the FIGs 1-11 accompanying this specification, or by
following other
methods well-known to those of ordinary skill in the art of the synthesis of
peptides and analogs.
Conveniently, synthetic production of the polypeptides of the invention may be
according
to the solid-phase synthetic method described by Merrifield et al. Solid phase
peptide synthesis. I.
The synthesis of a tetrapeptide: J. Amer. Chem. Soc.: 85, 2149-2156, 1963.
This technique is well
understood and is a common method for preparation of peptides. The general
concept of this
method depends on attachment of the first amino acid of the chain to a solid
polymer by a
covalent bond. Succeeding protected amino acids are added, one at a time
(stepwise strategy), or
in blocks (segment strategy), until the desired sequence is assembled.
Finally, the protected
peptide is removed from the solid resin support and the protecting groups are
cleaved off. By this
procedure, reagents and by-products are removed by filtration, thus
eliminating the necessity of
purifying intermediaries.
Amino acids may be attached to any suitable polymer as a resin. The resin must
contain a
functional group to which the first protected amino acid can be firmly linked
by a covalent bond.
Various polymers are suitable for this purpose, such as cellulose, polyvinyl
alcohol,
polymethylmethacryIate and polystyrene. Suitable resins are commercially
available and well
known to those of skill in the art. Appropriate protective groups usable in
such synthesis include
tert-butyloxycarbonyl (BOC), benzyl (Bzl), t-amyloxycarbonyi (Aoc), tosyl
(Tos), o-bromo-
phenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (Bz1C12), and
phenylmethoxycarbonyl (Z or
27
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
CBZ). Additional protective groups are identified in Merrifield, cited above,
as well as in
McOmie WW: Protective Groups in Organic Chemistry, Plenum Press, New York,
1973, both
references expressly incorporated fully herein.
General procedures for preparing peptides of this invention involve initially
attaching a
carboxyl-terminal protected amino acid to the resin. After attachment the
resin is filtered, washed
and the protecting group (desirably 130C) on the 1-amino group of the carboxyl-
terminal amino
acid is removed. The removal of this protecting group must take place, of
course, without
breaking the bond between that amino acid and the resin. The next amino, and
if necessary, side
chain protected amino acid, is then coupled to the free 1-amino group of the
amino acid on the
resin. This coupling takes place by the formation of an amide bond between the
free carboxyl
group of the second amino acid and the amino group of the first amino acid
attached to the resin.
This sequence of events is repeated with successive amino acids until all
amino acids are attached
to the resin. Finally, the protected peptide is cleaved from the resin and the
protecting groups
removed to reveal the desired peptide. The cleavage techniques used to
separate the peptide from
the resin and to remove the protecting groups depend upon the selection of
resin and protecting
groups and are known to those familiar with the art of peptide synthesis.
Alternative techniques for peptide synthesis are described in Bodanszky et al,
Peptide
Synthesis, 2nd ed, John Wiley and Sons, New York, 1976. For example, the
peptides of the
invention may also be synthesized using standard solution peptide synthesis
methodologies,
involving either stepwise or block coupling of amino acids or peptide
fragments using chemical or
enzymatic methods of amide bond formation. (See, e.g., H. D. Jakubke in The
Peptides, Analysis,
Synthesis, Biology, Academic Press, New York, 1987, p. 103-165; J. D. Glass,
ibid., pp. 167-184;
and European Patent 0324659 A2, describing enzymatic peptide synthesis
methods.) These
solution synthesis methods are well known in the art. Each of the above-
identified publications is
expressly incorporated herein fully by reference as though individually so
incorporated.
Commercial peptide synthesizers, such as the Applied Biosystems Model 430A,
are
available for the practice of these methods.
A person of ordinary skill in the art will not have to undertake undue
experimentation,
taking account of that skill and the knowledge available, and of this
disclosure, in developing one
or more suitable synthetic methods for compounds of this invention.
For example, analogs in which the glycine residue of GPE is replaced by an
alternative
amino acid, or by a non-amino acid, may conveniently be prepared by the
preparation of a C-
protected proline-glutamic acid dipeptide (such as the dibenzyl ester), and
coupling that dipeptide
with an N-protected glycine analog, such as BOC-N-methylglycine, BOC-L-valine,
N-
pyrrolidineacetic acid, and the like, followed by deprotection, as illustrated
in FIGs. 2 and 3.
Analogs in which the glutamic acid residue of GPE is replaced by an
alternative amino acid or an
28
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
amino acid amide or ester may conveniently be prepared by the preparation of
an N-protected
glycine-L-proline dipeptide (such as BOC-glycyl-L-proline), and coupling that
dipeptide with a
C-protected glutamic acid or analog thereof, such as tert-butyl -y-
aminobutyrate, methyl 4-amino-
4-dimethylcarbamoylbutyrate, L-glutamine methyl ester, dimethyl I-
methylglutamate, etc.
Lactones may be prepared by the preparation of an appropriate mono-acid-mono-
ester derivative
and reduction Analogs in which R2 is alkyl may conveniently be prepared simply
by use of the
appropriate 2-alkylproline in the synthesis, and similarly analogs in which R3
is alkyl may
conveniently be prepared by the use of the appropriate N-alkylglutamic acid or
analog in the
synthesis. Where modifications are to be made to two or more amino acids, the
coupling
techniques will still be the same, with just more than one modified amino acid
or analog being
used in the synthesis. The choice of appropriate protecting groups for the
method chosen (solid-
phase or solution-phase), and of appropriate substrates if solid-phase
synthesis is used, will be
within the skill of a person of ordinary skill in the art.
Compounds of Formula 2 may be prepared from suitably protected 5-oxo-L-proline
or
analogs or derivatives thereof, following methods such as the coupling of the
proline carboxyl
group with a protected glutamic acid or analog or derivative to give an analog
of intermediate A
of FIG. 2, comparable to the coupling reaction shown in FIG. 2, and then
alkylating the
pyrrolidine nitrogen with a group of the formula A----(CH2),õ--CH(R1)---CH2R,
protected at A if
necessary, where R is a leaving group under alkylation conditions.
Alternatively, the suitably
protected 5-oxo-L-proline may first by alkyIated at the pyffolidine nitrogen
to give an analog of
intermediate B of FIG. 4, and then coupling this with a suitably protected
glutamic acid or analog
or derivative in the manner shown in FIGs. 4 though 9.
EXAMPLES
The following examples are intended to illustrate embodiments of this
invention, and are
not intended to limit the scope to these specific examples. Persons of
ordinary skill in the art can
apply the disclosures and teachings presented herein to develop other
embodiments without undue
experimentation and with a likelihood of success. All such embodiments are
considered part of
this invention.
Example 1: Synthesis of N,N-Dimethylglycyl-L-prolyl)-L-glutamic acid
The following non-limiting example illustrates the synthesis of a compound of
the
invention, N,N-Dimethylglycyl-L-prolyl-L-glutamic acid
29
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
N)i3OH 112 NN7 CO2Bn EtOCOCI
INNCO2Bfl
=-
Et3N
1
I a
B OC 0
CO2 B n BOC 0
BOC = u0C 0 C 02B n
TFA
H020õ,
CO2 Bn 1
N DC C 19.44tr, CO2B n
/ \
0/), \ 0 14 ________________________________________ 0 "NI
CO2Bn Intermediate I C 02B n
11, H2, Pd
c,CO2E1
150
0;
CO21-1
All starting materials and other reagents were purchased from Aldrich;
BOC=tert-butoxycarbonyl;
Bn¨benzyl.
BOC-L-proline-(i-benzyl)-L-glutamic acid benzyl ester
To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem.
Soc.:
79, 6810, 1994] (10 mmol) in dichloromethane (50 ml), cooled to 0 C, was added
triethylamine
(1.39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant
mixture was
stirred at 0 C for 30 minutes. A solution of dibenzyl-L-glutamate (10 mmol)
was then added and
the mixture stirred at 0 C for 2 hours then warmed to room temperature and
stirred overnight.
The reaction mixture was washed with aqueous sodium bicarbonate and citric
acid (2 mol 1-1) then
dried (MgSO4) and concentrated at reduced pressure to give BOC-L-proline-L-
gIutamic acid
dibenzyl ester (5.0 g, 95%).
L-proline-L-glutamic acid dibenzyl ester
A solution of BOC-L-glutamyl-L-proline dibenzyl ester (3.4 g, 10 mmol), cooled
to 0 C,
was treated with trifluoroacetic acid (25 ml) for 2 h. at room temperature.
After removal of the
volatiles at reduced pressure the residue was triturated with ether to give L-
proline-L-glutamic
acid dibenzyl ester.
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
N,N-Dimethylglycyl-L-prolyl-L-glutainic acid
A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml)
was
added to a stirred and cooled (0 C) solution of L-proline-L-glutamic acid
dibenzyl ester (10
mmol), NN-dimethylglyeine (10 mmol) and triethylamine (10.3 mmol) in
dichloromethane (30
m1). The mixture was stirred at 0 C overnight and then at room temperature for
3 h. After
filtration, the filtrate was evaporated at reduced pressure. The resulting
crude dibenzyl ester was
dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml)
containing 10% palladium on
charcoal (0.5 g) then hydrogenated at room temperature and pressure until the
uptake of hydrogen
ceased. The filtered solution was evaporated and the residue recrystallised
from ethyl acetate to
yield the tripeptide derivative.
It can be appreciated that following the method of the Examples, and using
alternative
amino acids or their amides or esters, will yield other compounds of Formula
1.
31
CA 02929286 2016-04-29
WO 2014/085480 PCT/US2013/072049
Eample 2: Synthesis of Glycyl-L-2-Methyl-L-Prolyl-L-Glutamate
Glycyl-L-2-Methylprolyi-L-Glutamic Acid (G-2MePE)
(i) .,\\me Me
OH 0 OH
B 02C ¨N
&N)r\hle
0 = H¨Cl
(L)-2-methylproline I 2 3
Me OM e OH
(ii) 0 (iii)
0 ''s)
C 02Bn
NHCO2Bn NHCO2Bn
=pTs0H
4 5 6
&N . \µµIvi%
C 02H
0 2Bn
(iv) (v) a
0 0 0N 0
CO2Bn CO2H
NHCO2Bn NHR
7 G-2M
ePE: R = H (73:27 trans:cis)
8: R = CH3
Scheme 1 Reagents, conditions and yields: (i) SOC12, Me0H, 79 C, N2, 24 h
(104%); (ii) Et3N,
DCC, CH2C12, 0 C to RI, N2, 20 h; (iii) 1M aq. NaOH, 1,4-dioxane, 19 h (60%, 2
steps); (iv) Et3N,
B oPC1, CI-12C12, RI, N2, 17 h (19%); (v) H2. 10% Pd/C, 91;9 Me0H-H20, RI, 23
h (86%).
L-2-Methylproline and L-glutamic acid dibenzyl ester p-toluenesulphonate were
purchased from Bachem, N-benzyloxycarbonyl-glycine from Acros Organics and
bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (B0PC1, 97%) from Aldrich Chem. Co.
Methyl L-2-methylprolinate hydrochloride 2
Thionyl chloride (5.84 cm3, 80.1 num was cautiously added dropwise to a
stirred
solution of (L)-2-methylproline 1 (0.43 g, 3.33 rnmoI) in anhydrous methanol
(30 cm3) at -5 C
under an atmosphere of nitrogen. The reaction mixture was heated under reflux
for 24 h, and the
32
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
resultant pale yellow-coloured solution was concentrated to dryness in vacuo.
The residue was
dissolved in a 1:1 mixture of methanol and toluene (30 cm3) then concentrated
to dryness to
remove residual thionyl chloride. This procedure was repeated twice more,
yielding
hydrochloride 2 (0.62 g, 104%) as an hygroscopic, spectroscopically pure, off-
white solid: mp
127-131 C; [a]0 -59.8 (c 0.24 in CH2C12); v.,õ (film)/cm-1 3579, 3398 br,
2885, 2717, 2681,
2623, 2507, 1743, 1584, 1447, 1432, 1374, 1317, 1294, 1237, 1212, 1172, 1123,
981, 894, 861
and 764; (300 MHz; CDCI3; Me4Si) 1.88 (3H, s, Proa-CH3), 1.70-2.30 (3H, br
m, Pr0J3-1441-1B
and Pro1'-H2), 2.30-2.60 (1H, br m, Pro13-HAHB), 3.40-3.84 (2H, br m, Pro-H2),
3.87 (3H, s,
CO2CH3), 9.43 (1H, br s, NH) and 10.49 (1H, br s, HC1); gc (75 MHz; CDC13)
21.1 (CH3, Proa-
CH3), 22.4 (CH2, Proy-C), 35.6 (CH2, Prop-C), 45.2 (CH2, Pros-C), 53.7 (CH3,
CO2CH3), 68.4
(quat., Proa-C) and 170.7 (quat., CO); m/z (FAB+) 323.1745 [M2.H35C1.H+:
(C7H13NO2)2.
H35C1.H requires 323.1738] and 325.1718 [M2.1-137C1.H+: (C71113NO2)2. H37C1.H
requires
325.17081.
N-Benzyloxycarbonyl-glycyl-L-2-methylproline 5
Anhydrous triethylamine (0.45 cm3, 3.23 mmol) was added dropwise to a mixture
of
methyl L-2-methylprolinate hydrochloride 2 (0.42 g, 2.34 mmol) and N-
benzyloxyearbonyl-
glycine (98.5%) 3 (0.52 g, 2.45 mmol) in methylene chloride (16 cm), at 0 C,
under an
atmosphere of nitrogen. The resultant solution was stirred for 20 min and a
solution of 1,3-
dicyclohexylcarbodiimide (0.56 g, 2.71 mmol) in methylene chloride (8 cm3) at
0 C was added
- dropwise and the reaction mixture was warmed to room temperature and
stirred for a further 20 h.
The resultant white mixture was filtered through a Celiti4 pad to partially
remove 1,3-
dicyclohexylurea, and the pad was washed with methylene chloride (50 cm3). The
filtrate was
washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated
aqueous sodium
hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and concentrated to
dryness in vacuo.
Further purification of the residue by flash column chromatography (35 g Si02;
30-70% ethyl
acetate ¨ hexane; gradient elution) afforded tentatively methyl N-
benzyloxycarbonyl-glycyl-L-2-
inethylprolinate 4 (0.56 g), containing 1,3-dicyclohexylurea, as a white semi-
solid: Rf 0.65
(Et0Ac); m/z (EI+) 334.1534 (Mt. C17H22N205 requires 334.1529) and 224 (1,3-
dicycIohexylurea).
To a solution of impure prolinate 4 (0.56 g, ca. 1.67 mmol) in 1,4-dioxane (33
cm3) was
added dropwise 1M aqueous sodium hydroxide (10 cm3, 10 mmol) and the mixture
was stirred for
19 h at room temperature. Methylene chloride (100 cm3) was then added and the
organic layer
extracted with saturated aqueous sodium hydrogen carbonate (2 x 100 cm). The
combined
aqueous layers were carefully acidified with hydrochloric acid (32%),
extracted with methylene
chloride (2 x 100 cm3), and the combined organic layers dried (MgSO4),
filtered, and
33
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
concentrated to dryness in vacuo. Purification of the ensuing residue (0.47 g)
by flash column
chromatography (17 g SiO2; 50% ethyl acetate ¨ hexane to 30% methanol ¨
dichloromethane;
gradient elution) gave N-protected dipeptide 5 (0.45 g, 60%) as a white foam
in two steps from
hydrochloride 2. Dipeptide 5 was shown to be exclusively the trans-orientated
conformer by
NMR analysis: Rf 0.50 (20% Me0H ¨ CH2C12); [a],, -62.3 (c 0.20 in CH2C12); v..
(film)/cm'
3583, 3324 br, 2980, 2942, 1722, 1649, 1529, 1454, 1432, 1373, 1337, 1251,
1219, 1179, 1053,
1027, 965, 912, 735 and 698; SH (300 MHz; CDC13; Me48i) 1.59 (3H, s, Proa-
CH3), 1.89 (1H, 6
lines, J18.8, 6.2 and 6.2, Prop-HAHB), 2.01 (2H, dtt, J18.7, 6.2 and 6.2, Proy-
H2), 2.25-2.40 (11-1,
m, Prop-HAHB), 3.54 (2H, t, J6.6, Proo-H2), 3.89 (111, dd, J17.1 and 3.9, Glya-
HAHB), 4.04 (1H,
dd, J17.2 and 5.3, Glyoc-HAHB), 5.11 (2H, s, OCH2Ph), 5.84 (1H, br t, J4.2, N-
H), 7.22-7.43 (5H,
m, Ph) and 7.89 (1H, br s, -COOH); 5,c (75 MHz; CDC13) 21.3 (CH3, Proa-CH3),
23.8 (CH2, Proy-
C), 38.2 (CH2, Prop-C), 43.6 (CH2, Glya-C), 47.2 (CH2, Proo-C), 66.7 (quat,
Proa-C), 66.8 (CH2,
OCH2Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4, (CH, Ph), 136.4 (quat., Ph),
156.4 (quat.,
NCO2), 167.5 (gnat., Gly-CON) and 176.7 (quat., CO); m/z (EP-) 320.1368 (Mt
C16H20N205
requires 320.1372).
Dibenzyl N-benzy1oxycarbony1-g1ycyl-L-2-methylprolyl-L-glutannne 7
Triethylamine (0.50 cm3, 3.59 mmol) was added dropwise to a solution of
dipeptide 5
(0.36 g, 1.12 mmol) and L-glutamic acid dibenzyl ester p-toluenesulphonate 6
(0.73 g, 1.46
mmol) in methylene chloride (60 cm') under nitrogen at room temperature, and
the reaction
mixture stirred for 10 min. Bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(BoPC1, 97%) (0.37 g,
1.41 mmol) was added and the colourless solution stirred for 17 h. The
methylene chloride
solution was washed successively with 10% aqueous hydrochloric acid (50 cm')
and saturated
aqueous sodium hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and
evaporated to dryness
in vacua. Purification of the resultant residue by repeated (2x) flash column
chromatography (24
g Si02; 30-70% ethyl acetate ¨ hexane; gradient elution) yielded fully
protected tripeptide 7 (0.63
g, 89%) as a colourless oil. Tripeptide 7 was shown to be exclusively the
trans-orientated
conformer by NMR analysis: Rf 0.55 (Et0Ac); [c]p -41.9 (c 0.29 in CH2C12);
Vrnax (filnl)/CM
3583, 3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188,
1166, 1051, 911,
737 and 697; 81-1 (400 MHz; CDC13; Me4Si) 1.64 (3.11, s, Proa-CH3), 1.72 (1H,
dt, J 12.8, 7.6 and
7.6, Pro[3-HAHB), 1.92 (2H, 5 lines, J 6.7, Proy-H2), 2.04 (111, 6 lines, J
7.3 G1ui3-HAHB), 2.17-
2.27 (1H, m, Glul3-HAHB), 2.35-2.51 (3H, m, Pro13-HAHB and Gluy-H2), 3.37-3.57
(2H, m, Pro&
H2), 3.90 (1H, dd, J 17.0 and 3.6, Glya-HAHB), 4.00 (1H, dd, J 17.1 and 5.1,
Glya-HAHB), 4.56
(1H, td, J7.7 and 4.9, Glua-H), 5.05-5.20 (6H, m, 3 x OCH2Ph), 5.66-5.72 (1H,
br m, Gly-N11),
7.26-737 (15H, m, 3 x Ph) and 7.44(114, d, J7.2, Glu-NH); gc (100 MHz; CDC13)
21.9 (CH3,
Proa-CH3), 23.4 (CH2, Pray-C), 26.6 (CH2, Gluf3-C), 30.1 (CH2, Gluy-C), 38.3
(CH2, Prof3-C),
34
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
43.9 (CH2, Glya-C), 47.6 (CH2, Pro5-C), 52.2 (CH, Glua-C), 66.4 (CH2, OCH2Ph),
66.8 (CH2,
OCH2Ph), 67.1 (CH2, OCH2Ph), 68.2 (quat, Proa-C), 127.9 (CH, Ph), 128.0 (CH,
Ph), 128.1,
(CH, Ph), 128.2, (CH, Ph), 128.2, (CH, Ph), 128.3, (CH, Ph), 128.4, (CH, Ph),
128.5, (CH, Ph),
128.5, (CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat., Ph),
156.1 (quat., NCO2), 167.3
(quat., Gly-CO), 171.4 (gnat., CO), 172.9 (quat., CO) and 173.4 (quat., CO);
m/z (FAB+)
630.2809 (MH+. C35H40N308 requires 630.2815).
Glycyl-L-2-methylprolyl-L-glutarnic acid (G-2-MePE)
A mixture of the protected tripeptide 7 (0.63 g, 1.00 mmol) and 10 wt. %
palladium on
activated carbon (0.32 g, 0.30 mmol) in 91:9 methanol ¨ water (22 cm') was
stirred under an
atmosphere of hydrogen at room temperature, protected from light, for 23 h.
The reaction mixture
was filtered through a Celite- pad and the pad washed with 75:25 methanol ¨
water (200 cm).
The filtrate was concentrated to dryness under reduced pressure and the
residue triturated with
anhydrous diethyl ether to afford a 38:1 mixture of G-2-MePE and tentatively
methylamine
(0.27 g, 86%) as an extremely hygroscopic white solid. Analytical reverse-
phase HPLC studies on
the mixture [Altech Econosphere C18 Si column, 150 x 4.6 mm, 5 Elm; 5 min
flush with H20
(0.05% TFA) then steady gradient over 25 min to MeCN as eluent at flow rate of
1 ml/min;
detection using diode array] indicated it was a 38:1 mixture of two eluting
peaks with retention
times of 13.64 and 14.44 min at 207 and 197 nm, respectively. G-2-MePE was
shown to be a
73:27 trans:cis mixture of conformers by 111 NMR analysis (the ratio was
estimated from the
relative intensities of the double doublet and triplet at 5 4.18 and 3.71,
assigned to the Glua-H
protons of the major and minor conformers, respectively): mp 144 d; [a]p -
52.4 (c 0.19 in
H20); gn (300 MHz; D20; internal Me0H) 1.52 (3H, s, Proa-CH3), 1.81-2.21 (6H,
m, Pro-H2,
Proy-H2 and Gluf3-H2), 2.34 (1.46H, t, J7.2, Gluy-H2), 2.42* (0.54H, t, J7.3,
Gluy-H2), 3.50-3.66
(2H, m, Proo-H2), 3.71* (0.27H, t, J6.2, Glua-H), 3.85 (1H, d, J16.6, Glya-
HAHB), 3.92 (1H, d, J
16.6, Glya-HAHB) and 4.18 (0.73H, dd, J 8.4 and 4.7, Glucc-H); ac (75 MHz;
D20; internal
Me0H) 21.8 (CH3, Proa-CH3), 25.0 (CH2, Proy-C), 27.8* (CH2, Glu13-C), 28.8
(CH2, Glu13-C),
32.9 (CH2, Gluy-C), 40.8 (CH2, Prop-C), 42.7 (CH2, Glycc-C), 49.5 (CH2, Pro6-
C), 56.0* (CH,
Glua-C), 56.4 (CH, Glua-C), 69.8 (quat, Prooc-C), 166.5 (quat., Gly-CO), 177.3
(quat., Pro-
CON), 179.2 (quat., Glua-00), 180.2* (quat., Gluy-CO) and 180.6 (quat., Gluy-
00); m/z (FAB+)
316.1508 (MHf. C13H22N306 requires 316.1509).
Example 3: In Vitro Neuroprotection
Therapeutic effects of GPE analogs were examined in a series of experiments in
vitro to
determine their effects on neurodegeneration of neural cells of different
origin. The in vitro
systems described herein are well-established in the art and are known to be
predictive of
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
neuroprotective effects observed in vivo, including effects in humans
suffering from
neurodegenerative disorders.
Material and Methods
The following experimental protocol followed guidelines approved by the
University of
Auckland Animal Ethics Committee.
Preparation of cortical astrocyte cultures for harvest of metabolised cell
culture
supernatant
One cortical hemisphere from a postnatal day 1 rat was used and collected into
4m1 of
DMEM. Trituration was performed using a 5m1 glass pipette and an 18-gauge
needle. The cell
suspension was sieved through a 100[Irri cell strainer and washed in 50 ml
DMEM (centrifitgation
for 5 min at 250g). The sediment was resuspended in 20m1 DMEM+10% fetal calf
serum. The
suspension was added into two 25cm3 flasks (10 ml per flask) and cultivated at
37 C in the
presence of 10% CO2 followed by a change of the medium twice a week. When
cells reached
confluence, they were washed three times with PBS, adjusted to Neurobasal/B27
and incubated
for another 3 days. This supernatant was frozen for transient storage at -80
C.
Preparation of Stratial and Cortical Tissue from Rat E18/E19 Embryos
A dam was sacrificed by CO2-treatment, and then was prepared for caesarean
section.
After surgery, the embryos were removed from their amniotic sacs and
decapitated. The heads
were placed on ice in DMEM/F12 medium for striatum and PBS + 0.65% D(+)-
glucose for
cortex.
Striatal Tissue Extraction Procedure and Preparation of Cells
A whole brain was removed from the skull with the ventral side facing upwards
in
DMEM/F12 medium. The striatum was dissected out from both hemispheres under a
stereomicroscope and the striatal tissue was placed into a Falcon tube on ice.
Striatal tissue was
then triturated using a P1000 pipettor in 1 ml of volume. The tissue was
triturated by gently
pipetting the solution up and down into the pipette tip about 15 times, using
shearing force on
alternate outflows. The tissue pieces settled to the bottom of the Falcon tube
within 30 seconds.
The supernatant containing a suspension of dissociated single cells was then
transferred to a new
sterile Falcon tube on ice. The tissue pieces were triturated again to avoid
excessively damaging
already dissociated cells, by over triturating them. 1 milliliter of ice-cold
DMEM/F12 medium
was added to the tissue pieces in the first tube and triturated as before. The
tissue pieces were
36
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
allowed to settle and the supernatant was removed to a new sterile Falcon tube
on ice. The cells
were centrifuged at 250g for 5 minutes at 4 C.
Plating and Cultivation of Striatal Cells
Striatal cells were plated into Poly-L-Lysine (0.1mg/m1) coated 96-well plates
(the inner
60 wells only) at a density of 200,000 cells/cm2 in Neurobasal/B27 medium
(Invitrogen). The
cells were cultivated in the presence of 5% CO2 at 37 C under 100% humidity.
Medium was
changed on days 1, 3 and 6.
Cortical Tissue Extraction Procedure and Preparation of Cells
The two cortical hemispheres were carefully removed by spatula from the whole
brain
with the ventral side facing upside into a PBS +0.65%D(+)- glucose containing
petri dish.
Forceps were put into the rostra! part (near B. olfactorius) of the cortex in
order to fix the tissue
and two lateral-sagittal oriented cuts were made to remove the paraform and
entorhinal cortices.
A frontal oriented cut at the posterior end was made to remove the hippocampal
formation. A
final frontal cut was done a few millimetres away from the last cut in order
to get hold of area
17/18 of the visual cortex.
Cortices were placed on ice in PBS+0.65%(+)- glucose and centrifuged at 350g
for 5
minutes. The supernatant was removed and trypsin/EDTA (0.05%/0.53mM) was added
for 8min
at 37 C. The reaction was stopped by adding an equal amount of DMEM and 10%
fetal calf
serum. The supernatant was removed by centrifugation followed by two
subsequent washes in
Neurobasal/B27 medium.
The cells were triturated once with a glass Pasteur pipette in 1 ml of
Neurobasal/B27
medium and subsequently twice by using a 1 ml insulin syringe with a 22 gauge
needle. The cell
suspension was passed through a 100um cell strainer and rinsed by 1 ml of
Neurobasal/B27
medium. Cells were counted and adjusted to 50,000 cells per 60111.
Plating and Cultivation of Cortical Cells
96-well plates were coated with 0.2mg/m1 Poly-L-Lysine and subsequently coated
with
2ug/mI laminin in PBS, after which 60u1 of cortical astrocyte-conditioned
medium was added to
each well. Subsequently, 60i.11 of cortical cell suspension was added. The
cells were cultivated in
the presence of 10% CO2 at 37 C under 100% humidity. At day 1, there was a
complete medium
change (1:1 ¨ Neurobasal/B27 and astrocyte-conditioned medium) with addition
of 1 M
cytosine-I3-D-arabino-fiiranoside (mitosis inhibitor). On days 2 and 5, 2/3 of
the medium was
changed.
37
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Cerebellar Mieroexplants from P8 Animals; Preparation, Cultivation and
Fixation
Laminated cerebellar cortices of the two hemispheres were explanted from a P8
rat, cut
into small pieces in PBS +0.65% D(+) glucose solution and triturated with a
23gauge needle and
subsequently pressed through a 1251.tm pore size sieve. The obtained
microexplants were
centrifuged (60g) twice (media change) into serum-free BSA-supplemented STARTV-
medium
(Biochrom). For cultivation, 40 1 of cell suspension was adhered for 3 hours
on a 0.1mg/m1 Poly-
L-Lysine coated cover slip placed in 35 mm sized 6 well plates in the presence
of 5% CO2 under
100% humidity at 34 C. Subsequently, 1 ml of STARTV-medium was added together
with the
toxins and drugs. The cultures were monitored (evaluated) after 2-3 days of
cultivation in the
presence of 5% CO2 under 100% humidity. For cell counting analysis, the
cultures were fixed in
rising concentrations of paraformaldehyde (0.4%, 1.2%, 3% and 4% for 3 mm
each) followed by
awash in PBS.
Toxin and Drug Administration to Neural Dells In Vitro and Analysis of Data
To study neuroprotective effects of GPE analogs, we carried out a series of
experiments
in vitro using okadaic acid to cause toxic injury to neural cells. Okadaic
acid is an art-recognized
toxin that is known to cause injury to neurons. Further, recovery of neural
cells or neural cell
function after injury by okadaic acid is recognized to be predictive of
recoveries from injuries
caused by other toxins.
To cause toxic injury to neurons, we exposed neurons to 1:100 parts of okadaic
acid at
concentrations of 30nM or 100nM and 0.5mM 3-nitropropionic acid (for
cerebellar microexplants
only). GPE (I nM-1mM) or G-2-MePE (lriM-1mM) was used at 8 days in vitro (DIV)
for cortical
cultures and 9D1V for striatal cultures. The incubation time was 24 hours. The
survival rate was
determined by a colorimetric end-point MTT-as say at 595nm in a multi-well
plate reader. For the
cerebellar microexplants four windows (field of 0.65 mm2) with highest cell
density were chosen
and cells displaying neurite outgrowth were counted.
Results
The GPE analog G-2-MePE exhibited comparable neuroprotective effects within
all three
tested in vitro systems (FIGs 12-15).
Cortical cultures responded to 10p.M concentrations of GPE (FIG. 12) or G-2-
MePE
(10 M, FIG. 13) with 64% and 59% neuroprotection, respectively.
The other 2 types of cultures demonstrated neuroprotection at lower doses of G-
2-MePE
(cerebellar microexplants: FIG. 14 and striatal cells: FIG. 15). Striatal
cells demonstrated
neuroprotection within the range of lM to 1mM of G-2-MePE (FIG. 15), while the
postnatal
cerebellar microexplants demonstrated neuroprotection with G-2-MePE in the
dose range between
38
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
about inivt and about' uuntvt 14).
ihus, we conclude that G-2-MePE is a neuroprotective
agent and can have therapeutic effects in humans suffering from
neurodegenerative disorders.
Because G-2-MePE can be neuroprotective when directly administered to neurons
in culture, that
G-2-MePE can be effective in vivo when directly administered to the brains of
affected animals.
Example 4: Effects of G-2-MePE on Striatal Cholinergic Neurons in Aging Rats
To determine whether G-2-MePE can affect cholinergic neurons, we studied aging
rats.
Choline acetyltransferase (ChAT) is an enzyme that is involved in the
biosynthesis of the
neurotransmitter for cholinergic nerves, acetylcholine. It is well known that
immunodetection of
ChAT can be used to determine the numbers of cholinergic nerves present in a
tissue. It is also
known that the numbers of cholinergic nerves present is associated with the
physiological
function of cholinergic neural pathways in the brain.
In this experiment, we tested the effects of G-2-MePE on the number of ChAT-
positive
neurons in brains of 18-month old rats.
Methods
Eighteen-month old male rats received one of five treatments. A control group
was
treated with vehicle (saline alone (n=4) and four groups were treated with a
single dose of G-2-
MePE. Doses of 0.012 (n-4), 0.12 (n=5), 1.2 (In=5) and 12 mg/kg (n--3),
respectively, were
given sub-cutaneously. Rats were sacrificed with an overdose of pentobarbital
3 days after drug
treatment. Brains were perfused with normal saline and 4% paraformaldehyde and
fixed in
perfusion fixative overnight. Brains were stored in 25% sucrose in 0.1M PBS
(pH7.4) until the
tissue sank. Frozen coronal sections of striatum were cut with a microtome and
stored in 0.1%
sodium azide in 0.1M PBS at 4 C. Immunoreactivity for choline
acetyltransferase (ChAT) was
established by staining using a free floating section method. Briefly,
antibodies were diluted in
1% goat serum. The sections were incubated in 0.2% triton in 0.1 M
PBS/TritonTm at 4 C.
overnight before Immunohistochemical staining. The sections were pre-treated
with 1% H202 in
50% methanol for 20 min. The sections were then incubated with rabbit (Rb)
anti-ChAT (1:5000)
antibodies (the primary antibodies) in 4D on a shaker for two days. The
sections were washed
using PBS/TritonTm (15 minutes x 3d) and then incubated with goat anti-rabbit
biotinylated
secondary antibodies (1:1000) at room temperature overnight. The sections were
washed and
incubated in ExtrAvidinTM (Sigma) (1:1000) for 3 hours and followed by H202
(0.01%) in 3,3-
diaminobenzine tetrahydrochloride (DAB, 0.05%) to produce a coloured reaction
product. These
sections were mounted on chrome alum-coated slides, dried, dehydrated and
covered.
The striatal neurons in both hemispheres exhibiting specific
immunoreactivities
corresponding to ChAT were counted using a light microscope and a 1 mm 2xI000
grid. The size
39
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
of the striatal region used for the count was measured using an image
analyser. The total counts of
neurons/mm2 were compared between the groups.
Data were analysed using a paired t-test and presented as mean +/- SEM.
Results are
presented in FIG. 16.
Results
FIG. 16 shows that the number of ChAT-immunopositive neurons increased in the
brains
of animals treated with G-2-MePE. This clearly indicates that administration
of 0-2-MePE is
effective in increasing the level of ChAT in the brains of aged rats. Because
ChAT is an enzyme
involved in the synthesis of the cholinergic neurotransmitter acetylcholine,
we conclude that G-2-
MePE can increase the amount of cholinergic transmitter in the brains of
middle-aged rats.
Example 5: Effects of G-2-MePE on Spatial Reference Memory in Rats
Having demonstrated that G-2-MePE can increase ChAT and therefore has the
potential
to *prove cholinergic neural function, we then examined whether 0-2-MePE can
be useful in
treating age-related changes in cognition and/or memory. Therefore, we carried
out a series of
studies in rats using well-established tests for memory.
Experiment 1: The Morris Water Maze Test
The Morris water maze test is a well-recognized test to assess spatial
reference memory in
rats.
Subjects
We used male Wistar rats 12, 8 or 4 months of age.
Methods
Testing Environment and Apparatus
The Morris water maze test was conducted using a black plastic pool filled to
a depth of
25 cm with water colored black with a non-toxic dye. The pool had a circular
black insert so that
the walls also appeared uniform black The pool was divided into four quadrants
(north, south, east
and west) by two imaginary perpendicular lines crossing at the pool's center A
metal platform
was placed in the geographical centre of the SE quadrant 50cm from the edge of
the pool, so that
it was 2cm below the water surface and invisible. The platform remained in
that position though
the training.
The experiment used extra-maze cues (i.e. objects in the room surrounding the
pool) that
the rats could use to navigate to the platform. Distinctive posters or
paintings were hung on the
walls. Furniture in the room was not moved during the testing period. The
placement of the pool
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
allowed the experimenter an easy access to it from all sides. The pool was
emptied and refilled
daily during testing, with water at 25 C +1- 2 C.
The furthermost point in the pool (relative to the position of the
experimenter) was
designated as "north", and the other compass points "east", "south" and "west"
were the right-
most, bottom and left-most points of the pool respectively. These points were
marked with tape
on the outside of the pool.
Acquisition Phase
Rats in each group were trained to swim to the submerged platform. The rats
received six
60-second trials per day for four consecutive days. A trial began by placing
the rat into the water
facing the wall of the pool, at one of four start locations (north, south,
east, west). The sequence
of start locations was chosen pseudorandomly, so that the start location of
any given trial was
different from that of the previous trial, and no start location was used more
than twice during
daily training. The same sequence of locations was used for all the rats on a
given day but varied
between days. The trial ended when the rat had found the platform, or in 60
seconds, which ever
occurred first. The trials were timed with a stop watch. If the rat found the
platform, it was
allowed to remain there for 15 seconds before being removed to a holding
container. If the
platform was not found, the rat was guided there manually and placed on the
platform for 15-
seconds. The inter-trial interval was 60 seconds. The holding container was
covered in order to
minimize any inter-trial interference. At the completion of daily testing for
a rat, the animal was
towel-dried and placed under the heat lamp in the holding bucket until his
coat was dry. The time
needed to locate the platform (latency, secs) was obtained for each rat in
each training trial. If the
rat did not find the platform in a given trial their latency score was the
maximum length of that
trial (60 seconds).
Drug Treatment
Three days after the completion of the acquisition phase, mini-osmotic pumps
(Alzet)
were implanted subcutaneously under halothane anesthesia) to dispense drug or
vehicle
continuously for 1 or 3 weeks. At the completion of the infusion the pumps
were removed and the
wounds re-sutured.
The 5 treatment groups were:
I. saline 1 week (n was originally 7, but one rat that lost weight rapidly was
excluded
and later found to have had a pituitary tumor);
2. saline 3 weeks (n=8);
3. G-2-MePE low dose (0.96 mg/day) 1 week (n=8);
41
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
4. 0-2-MePE low dose (0.96 mg/day) 3 weeks (n-8);
5. G-2-MePE high dose (4.8 mg/day) 3 weeks (n.--7).
The four (n=3) and eight month old (n=9) control rats received no drug
treatment. The 12-
month old rats were assigned to one of five groups on the basis of their swim
times over
acquisition, such that the groups were approximately equivalent in their mean
performance prior
to receiving any drug.
Retention (Reference Memory) Phase
The ability of the rats to remember or to relearn the original platform
location was tested
four weeks after original training. This means that residual drug would have
been washed out for
a minimum of 7 days in the case of the 3-week pumps, and 21 days in the case
of the 1-week
pumps. The retention testing procedure was identical to that of acquisition.
Pharmacokinetic
studies indicate that the plasma concentration of subcutaneously administered
G-2-MePE rose to a
peak and then declined with an approximately first order kinetic pattern, with
a plasma half-life (t
1/2) of between about 30 and 60 minutes. Thus, by the time the retention study
was performed, at
least 7 days after removal of the G-2-MePE containing minipumps, nearly all of
the G-2-MePE
had been cleared from the animals' circulation.
Data Analysis
The swim latency for each rat was recorded for each trial for each day of the
acquisition
and retention phases and changes between phases were examined using Analysis
of Variance.
The 3-week vehicle and 3-week high dose 0-2-MePE were compared in acquisition
and
retention. The high dose of G-2-MePE, given over 3 weeks improved the
retention of the original
water maze task after a 4-week delay.
Results
FIG. 17 shows the comparison between high-dose (4.8 mg/day) G-2-MePE-treated
and
low-dose-treated (0.96 mg/day) aged rats and saline treated aged rats, with
the young controls (4
months) used as controls. Prior to treatment with G-2-MePE, there were no
differences between
the aged (12 month old) groups. In contrast, the 4 month old animals required
less time to reach
the platform than older animals. After a 3-week period of no testing, during
which time either
saline or G-2-MePE were administered, animals that received saline only did
not show improved
ability to reach the platform, as indicated by the similar times required at
test day 4 of the
acquisition phase and test day 1 of the retention phase. In contrast, animals
that received
treatment with G-2-MePE at either the high or low doses, had improved memory
as reflected in a
decrease in the time needed to reach the platform compared to saline-treated
controls. Further,
42
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
the 0-2-MePE-treated animals had similar performance to the 4 month old young
animals (FIG.
17) and 8 month old animals (data not shown). Thus, we conclude that G-2-MePE
can improve
memory in middle-aged rats animals that had previously shown memory deficits
in relation to
young rats. Further, because by the time of retesting, the G-2-MePE had washed
out from the
circulation, we conclude that the memory-enhancing effects of G-2-MePE were
likely due to the
improvement in function of cholinergic neurons.
Experiment 2: 8-Arm Radial Maze Test
Five months after the original experiment the now 17 month old rats were
retested for
spatial working memory in a radial arm maze.
Methods
Apparatus
The apparatus consists of a central platform communicating with 8 identical
arms, each
with a food cup at the end of the arm
Testing Procedure
Rats were partially food-deprived for at least 10 days prior to, and
throughout the radial
maze procedure.
The maze was assembled and positioned so that the experimenter could clearly
observe
the rats' behavior from a predetermined location. The experimenter numbered
the arms of the
maze according to their orientation from one to eight in a clock-wise
direction.
Pre-Training (Pre-Drug)
On day one the doors were inserted into the arms and each rat was confined in
the central
platform with 20 food pellets for 5 minutes. This continued once a day for
four days, and all of
the rats were observed to consume some of the pellets. The following day the
rats were allowed
five minutes to explore the whole maze. All arms were baited with two food
pellets in the food
cup located at the end of each arm, and one pellet at both the entrance and
middle of each arm.
This was repeated for at least five, but up to eight days for rats that
explored fewer than eight
arms in two consecutive sessions. All rats had a final session on the ninth
day of pre-training. At
this point it was decided that one of the old rats that had made only one arm
entry on eight of the
nine days should be excluded from future testing in this procedure. Otherwise
all rats were
included regardless of the amount of exploring they performed in pre-training.
There was no
statistically significant difference between the old groups in the number of
arms entered on the
final pre-training session (Drug: F(2,31)=0.44, p=0.65).
43
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Drug Treatment
30 days before the test (five days after pre-training) the 17 Male Wistar
month old rats
were implanted (under halothane anesthesia) with sub-cutaneous mini-osmotic
pumps (Alzet) to
dispense drug continuously for 3 weeks. At the completion of the infusion the
pumps were
removed and the wounds re-sutured (9-day washout allowed).
The treatment groups were:
I. young controls (4 months old), n =6;
2. saline n=10;
3. G-2-MePE low dose (2.4mg/kg/day) n=13
4. G-2-MePE high dose (12.4mg/kg/day) n=5
Saline and the low dose groups are comprised of all the rats that received
those treatments
in phase 1 of this experiment (when the rats were 12 months old) regardless of
whether they had
the one or three week treatment One rat in each of the saline and high dose
groups have been
dropped because of skin tumors. One of the low dose rats did not participate
in this experiment
due to the fact that it could not be pre-trained (see below).
Testing (Post-Drug)
Working memory testing commenced on the ninth day of washout. Rats received 10
daily
training sessions over 12 days. The procedure was the same as for pre-training
but only the food
cups were baited. Rats had 6 minutes to make up to 16 choices by visiting any
of the eight arms.
A choice was defined as occurring when all four paws were inside an arm. The
experimenter
recorded the sequence of arm entries with pen and paper. Sessions were
terminated after all eight
arms had been entered, 16 choices made, or 6 minutess had elapsed. The time
taken to enter all
eight arms, when this occurred, was recorded.
Data Analysis
An arm choice was considered correct when the rat entered an arm not
previously visited.
Performance was classified daily according to the following parameters:
1) Correct Choice (CC) 8-12 is the number of correct choices made divided by
the total
number of choices made. For animals that failed to visit all 8 arms in a test,
the denominator of
this ratio is considered to be 12.
2) Working Correct Choice (WCC) 8-12 is the measure from which the working
memory
data are derived. Data were collected as described for CC 8-12 above, but for
this parameter, only
the rats that entered all 8 arms in a session were included.
44
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Rats that made fewer than 8 arm entries were not used to ascertain working
memory
because they couldn't remember which arms they had previously visited and
therefore had
memory so impaired that they could not complete the test, as opposed to the
animals that, for
whatever reason, did not explore the maze.
Results
CC8-12: There was a general improvement by all of the groups across the 10
days
(F(9,324)=4.01, p<0.0001), but no significant group effect (F(3,36)=1.19, ns)
or Group X Days
interaction (F(27,324)=1.05, ns) (data not shown)
WCC8-12: FIG. 18A shows the acquisition profile according to WCC8-12 score
across
the 10 days of testing. There was a significant effect of Group (F(3,12)=4.27,
p=0.029) and Days
(F(9,108)=2.09, p=0.036) but the interaction between these factors was not
significant
(F(27,108)=1.06, ns). The high dose G-2Me-PE group showed the greatest
improvement across
days, followed by the young controls. There was very little difference between
the low dose G-
2Me-PE and saline.
FIG. 18B shows results indicating that rats exposed to the higher dose of G-2-
MePE
(n=5) had made more correct entries for getting food pellets compared to the
vehicle treated rats
(*p<0.05, n=10). We conclude from this study that G-2-MePE improves spatial
memory in aged
rats.
Example 6: G-2-MePE Increases Neuroblast Proliferation and Decreases
Astrocytosis in
Brains of Aged Rats
Because neuronal degeneration can result in decreased numbers of neurons, one
desirable
therapeutic aim is increasing the numbers of neurons in the brain. Neurons are
derived from
neuroblasts, a less differentiated cell than a neuron, but within the neural
lineage. Typically, a
neuroblast is exposed to conditions that cause it to mature into a mature
phenotype, having a
defined soma, neural processes (axons and dendrites) and ultimately, making
connections with
other neurons (e.g., synapses). Thus, measuring neuroblast proliferation has
become a well-
known early marker for nerve cell proliferation. Thus, detecting an increase
in neuroblast
proliferation induced by a pharmaceutical agent is an accepted method for
predicting growth of
neural cells in animals. Because rats and humans share similar mechanisms in
neural cell
proliferation, detection of changes in neuroblast proliferation in rats in
vivo is predictive of similar
effects in human beings.
It is also known that one histological correlate of impaired cognitive
function is an
increase in the numbers of astrocytic cells in the brain of affected animals.
Thus, to determine
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
whether G-2-MePE might be useful in stimulating neuroblast proliferation and
in treating
astrocytosis, we carried out a series of studies in aging rats.
Methods and Materials
Immunohistoehemistry
To carry out these studies, tissues were fixed and embedded in paraffin and
sections
obtained using standard methods. Coronal sections (6 lam) containing the level
of the
hippocampus were cut and mounted on chrome-alum-coated slides for staining.
The sections
were deparaffinized in xylene, dehydrated in a series of ethanol and incubated
in 0.1 M phosphate
buffered saline (PBS).
Primary antibodies against glial fibrillary acidic protein (GFAP) and
proliferating cell
nuclear antigen (PCNA) were used to mark reactive glial cells and cells
undergoing apoptosis and
proliferation, respectively. For antigen unmasking (caspase-3 and PCNA
staining), sections were
heated in 10 mM sodium citrate buffer (pH 6.0) for 1 min at high power. All
sections were
pretreated with 1% H202 in 50% methanol for 30 min to quench the endogenous
peroxidase
activity. Then either 1.5% normal horse serum or 2.5% normal sheep serum in
PBS was applied
for 1 h at room temperature to block= nonspecific background staining. The
sections were then
incubated with following primary antibodies: monoclonal mouse anti-GFAP
antibody (Sigma, St.
Louis, MO, U.S.A. diluted 1:500); mouse anti-PCNA antibody (DAKA, A/S,
Denmark, diluted 1:
100). After incubation with primary antibodies at 4 C for 2 d (except for PCNA
staining which
was incubated overnight) the sections were incubated with biotinylated horse
anti-mouse or goat
anti-rabbit secondary antibody (1:200, Sigma) at 4 C overnight. The
ExtrAvidinIm (Sigma,
1:200), which had been prepared 1 h before use, was applied for 3 h at room
temperature, and
then reacted in 0.05% 3,3-diaminobenzidine (DAB) and PBS to produce a brown
reaction
product. Sections were dehydrated in a series of alcohols to xylene and
coverslipped with
mounting medium.
Immunohistochemical staining was performed on brain samples taken from both
control
and G-2-MePE treated groups of young (4 months old), middle-aged (9 months
old) and aged rats
(18 months old).
Control sections were processed in the same way except the primary antibody
was
omitted from the incubation solution. The number of PCNA positive cells was
counted in the
subventricular zone and the GFAP positive cells was scored in the cerebral
cortex.
Experiment 1: G-2-MePE Stimulates Neuroblast Proliferation in Brains of Aged
Rats
The subventricular zone (SVZ) and the dentate gyms (DG) are two brain regions
hosting
adult neurogenesis. The reduction of neurogenesis in both SVZ and the DG has
been well
46
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
reported to be co-related to the memory decline with aging and effects of
Nerve Growth Factor
and Epidermal Growth Factor on memory improvement are reported to be due to
increase in
progenitors proliferation of the SVZ. Using PCNA as a marker of cell
proliferation, cellular
proliferation in the SVZ was examined by counting the numbers of cells that
are positive for
PCNA. In selected animals, at least some of the proliferating cells were
identified as neuroblasts,
as stained with the neural-cell specific agent, doublecortin.
Eighteen month old male rats were treated intraperitoneally with single does
of G2-MePE
(doses of either 0, 0.012, 0.12. 1.2, 12mg/kg). Brains were collected 3 days
after the treatments
and the immunohistochemical staining of PCNA and GFAP were performed. The
number of
PCNA positive cells was counted in the SVZ and the number of cells was then
averaged as
cells/mm depending on the length of ventricle wall used for counting (FIG.
19A). The group
treated with the highest dose (12 mg/kg, n=5) showed a significant increase in
the number of
PCNA positive cells compared to the group treated with vehicle (*p<0.05, 11-
7). The data
indicated a dose-dependent effect of G-2PE on improving neurogenesis.
Fluorescence double labelling indicated co-localisation of PCNA with
doublecortin, a
marker for neuroblasts. FIG. 19B is a photograph of a portion of a rat's brain
showing an increase
in both PCNA (green, x20) and doublecortin (red, x20) in the rat treated with
the highest dose of
G-2-MePE (right panel) compared to the vehicle treated rat (left panel). The
two markers clearly
co-localised (Figure 19B, photo, x100). We conclude that G-2-MePE can
stimulate proliferation
of brain cells, including neuroblasts. Because neuroblasts are precursor cells
for neurons, we
further conclude that G-2-MePE can increase the population of neurons in the
brains of animals
treated with the compound of this invention.
Experiment 2: G-2-MePE Stimulates Neuroblast Proliferation in the SVZ of
Brains of
Middle-Aged Rats
Effects of G-2-MePE (1.2mg/kg) were studied in a group of middle-aged, 9 month
old
rats. G-2-MePE (1.2 mg/kg) or vehicle was administered intraperitoneally
(i.p.). The proliferation
of cells in the SVZ was examined 3 days after the treatment using PCNA
immunohistochemical
staining. FIG. 19C shows a significant increase in number of PCNA positive
cells after the
treatment of G-2-MePE ("p<0.005, n=4). Because some of the proliferating cells
stained with
PCNA were identified as neuroblasts (see Experiment 1 above), we conclude that
G-2-MePE can
stimulate neuroblast proliferation in middle-aged rat brains.
Experiment 3: Astrocytosis in Aging Brains
Growing evidence suggests that dysfunction of astrocytes in advanced age can
trigger
inflammation, leading to further neuronal degeneration. Up-regulation of
activated astrocytes has
47
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
been well reported and is closely associated with memory decline with aging,
perhaps through
depressed endogenous neurogenesis.
Using GFAP as a marker for reactive astrocytes, the number of GFAP-positive
cells was
counted in the CA4 sub-region of the hippocampus of aged rats treated with G-
2MeP or vehicle.
We found a significant increase in reactive astrocytes in the hippocampus of
aged animals (Figure
20A), and in the cerebral cortex. Some of the astrocytes were associated with
capillaries (Figure
20B photo, arrows) in aged rats compared to both young (*p<0.01) and middle
aged rats
(*#p<0.01).
As part of the vascular component, GFAP positive astrocytes also play a role
in
angiogenesis (FIG. 20B, arrows), which also contribute to inflammatory
response in brains.
Therefore the elevated GFAP astrocytes seen in aged brains may indicate a
chronic stage of brain
degeneration.
Experiment 4: G-2-MePE Reduces Astrocytosis in Aged Brains
We also evaluated effects of G-2-MePE on astrocytosis in the CA4 sub-region of
the
hippocampus in aged rats. 18-month old male Wistar rats were assigned to 5
treatment groups as
follows: vehicle, 0.12 mg/kg/day, 0.12, 1.2 and 12 mg/kg/day (each n=6).
GFAP-positive cells were counted using a computerised program (Discovery 1).
Results
are shown in FIGs 20C and 200. G-2-MePE was administered intra-peritoneally
and the numbers
of GFAP-positive cells were assessed 3d after the injection. Using a visual
scoring system (0 = no
astrocytes, I = few astrocytes, 2 <50%, 3>50%) we estimated the number of
astrocytes in 5
different cortical regions.
Treatment with G-2-MePE reduced number of reactive astrocytes in the CA4
region of
the hippocampus compared to the vehicle treated group (FIG. 20C; *p<0.05),
particularly the
groups treated with doses of 0.12 and 12 mg/kg. A similar effect was observed
for G-2-MePE in
the cerebral cortex (FIG. 200).
Normally there are few GFAP-positive astrocytes located in the deep layer of
cortex of rat
brains and those that are present are usually in close association with white
matter tracks.
However, we have found there were GFAP-positive cells in the middle layer of
the cortex, closely
associated with blood vessels.
Results of the studies presented herein indicate that aging is associated with
several
changes in the brain. First, there is an age-dependent loss of memory and
cognitive function.
Second, there is an age-depended increase in astrocytes. All of these findings
in the rat are
consistent with each other and the known roles of cholinergic nerves in
maintaining cognitive
function and memory in experimental animals and in humans.
48
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
We unexpectedly found that a GPE analog, G-2-MePE, delivered to aged animals
at least
partially reverses all of the above age-associated changes. First, 0-2-MePE
increases the amount
of ChAT present in the brain cells of animals exposed to the neurotoxins
okadaic acid or 3-NP.
This effect of G-2-MePE mimicked that of a well-known neuroprotective agent,
GPE. These
effects were seen in cortical cells, cerebellar cells and in striatal cells,
indicating that the effects
were widespread in different portions of the brain. Second, G-2-MePE increased
ChAT in the
striatum, indicating that cholinergic neurons are sensitive to G-2-MePE. These
observed
chemical and histological changes were paralleled by behavioral changes. Aged
animals treated
with G-2-MePE exhibited improved memory in two well-known test systems
compared to
vehicle-treated controls. Next, G-2-MePE induced neuroblast proliferation in
aging brains.
Finally, treatment with G-2-MePE reversed the increase in astrocytosis
observed in the
hippocampus and cortex of aging brains. The effects of G-2-MePE were not due
to acute effects
of the agent; because in many of the studies cited herein, sufficient time had
elapsed from
cessation of drug delivery to the test, that there was likely little or no
drug present.
Example 7: Comparison of the Pharmaeokinetics of GPE and G-2-MePE
The purpose of these studies was to compare pharmacokinetic profiles of GPE
and G-2-
MePE in animals in vivo using standard pharmacokinetic methods.
Methods
Adult male Wistar rats weighing between 180 and 240g were used to determine
the
pharmacokinetics of GPE and G2MePE. To facilitate intravenous bolus injections
and blood
sampling, all rats were surgically implanted with an indwelling jugular venous
cannula under
halothane anesthesia three days before the experiment. Groups of six rats were
given a single
intravenous bolus injection of either 30 mg/kg GPE or 10 mg/kg G2MePE
dissolved in 0.1M
succinate buffer (pH 6.5). Blood samples (about 220 ul each) were collected
into heparinized
tubes containing Sigma protease inhibitor cocktail for mammalian tissues at 10
and 0 min before
injection of either GPE or G2MePE, and 1, 2, 4, 8, 16, 32, 64 and 128 min
after injection of either
GPE or G2MePE. The samples were centrifuged at 3000g for 15 min at 4 C and the
plasma
removed and stored at -80 C until extraction and assay by either
radioimmunoassay ("RIA") or
reverse phase HPLC. The RIA and HPLC methods used were conventional.
Drug elimination after a single intravenous bolus injection was found to be a
first-order
process following the equation C= Coe-kt, where C represents drug
concentration in any time
point, Co is the concentration when time (t) equals zero and k is the first-
order rate constant
expressed in units of concentration per hour. The k and half-life (t112) were
calculated from the
slope of the linear regression line in the elimination phase of the semi-
logarithmic plot of plasma
49
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
concentration versus time as: Log C = -kt/2.3 + log Co Results were expressed
as mean
standard error.
Results
FIG. 21 shows a graph of plasma concentrations in vivo of GPE and G-2-MePE
after
intravenous (iv.) injection. Filled squares represent concentrations of GPE at
each time point,
and filled triangles represent concentrations of G-2-MePE at each time point.
Plasma concentrations of GPE and 0-2-MePE were markedly increased within 1 min
after injection. After injection of 30 mg/kg GPE, a peak concentration of 40.0
10.8 mg/ml was
observed. Plasma concentrations of GPE then rapidly declined according to a
first-order kinetic
process. The first order rate constant for GPE was found to be 0.15 + 0.014
ng/ml/min, the t112
was found to be 4.95 + 0.43 min and the estimated clearance of GPE from plasma
was found to be
137.5 + 12.3 ml/hr.
After injection of 10 mg/kg G-2-MePE, the peak concentration was found to be
191
16.1 mg/ml. Plasma concentrations of 0-2-MePE then declined according to a
first-order kinetic
process. The first order rate constant for G-2-MePE was found to be 0.033 +
0.001 ng/ml/min,
the t112 was found to be 20.7 + 0.35 min and the estimated clearance was found
to be 30.1 + 0.5
ml/hr.
After injection, the maximal plasma concentration of G-2-MePE was about 4.8
times
greater than the maximal plasma concentration of GPE, in spite of the larger
dose of GPE
delivered (30 mg/kg) compared to the dose of G-2-MePE delivered (10 mg/kg).
The finding of greater plasma concentrations of G-2-MePE than for GPE at all
time
points less than 125 minutes, in spite of a lower delivered dose of G-2-MePE,
was totally
unexpected based on known plasma concentrations of GPE. The tia, for G-2-MePE
was over 4
times longer than the t1,2 for GPE.
The finding of increased half-life of G-2-MePE compared to that of GPE was
completely
unexpected based on the t1,2 of GPE. The increased t112 of G-2-MePE means that
G-2-MePE is
cleared more slowly from the circulation. This finding is totally unexpected
based on the
clearance rate of GPE.
We conclude from these studies that G-2-MePE is a potent agent capable of
reversing
many of the adverse effects of aging in the brains of animals, including
humans. GPE analogs,
including G-2-MePE therefore, can produce desirable therapeutic effects,
including
neuroprotection, improved memory, increased neuroblast proliferation and
reduction in
astrocytosis, and can be valuable in reversing or mitigating adverse effects
of aging in humans.
While this invention has been described in terms of certain preferred
embodiments, it will
be apparent to a person of ordinary skill in the art having regard to that
knowledge and this
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
disclosure that equivalents of the compound of this invention may be prepared
and administered
for the conditions described in this application, and all such equivalents are
intended to be
included within the claims of this application.
Example 8: Treatment of Rett Syndrome I
Effects of G-2-MePE on Lifespan and Long-Term Potentiation in Rett
Syndrome (RTT) Model
To determine whether G-2-MePE treatment can impact the development and
progression
of Rett Syndrome in a murine model of the disorder, we used hemizygous
MeCP2(lIox) male
mice. The MeCP2 knock-out (MeCP2-KO) mouse system is widely accepted in the
art as closely
mimicking the range and the severity of physiological and neurological
abnormalities
characteristic of the human disorder, Rett Syndrome.
All experiments were performed at the University of Texas Southwestern Medical
Center
and approved by the University of Texas Southwestern Medical Center Animal
Care and Use
Committee G-2-MePE was synthesised Albany Molecular Research Inc. (Albany, NY)
and
supplied by Neuren Pharmaceuticals Limited.
Methods
Treatment
We treated hemizygous MeCP2(1Iox) male mice with 20 mg/kg/day of G-2-MePE or
(0.01%BSA, n=15 per group in survival experiment and n=20 in the LIP
experiment).
The treatments were administered intraperitoneally from 4 weeks after birth.
For the survival
experiments the treatment was maintained through the course of the experiment.
For the LTP
experiment the mice were treated until week 9 when they were used for slice
preparation.
Survival
MeCP2 deficient mutant mice develop RTT symptoms at about 4-6 weeks of age and
die
between 10-12 weeks (Chen et al., 2001. Nat Genet 27: 327-331). We compared
the survival of
the wild type controls and the MeCP2 deficient animals in vehicle- and G-2-
MePE-treated groups.
Survival was measured weekly from start of treatment (4 weeks) and used to
produce Kaplan-
Meier survival curves to show the proportion of mice that survived (y axis) at
each weekly
interval (x axis) (see FIG. 22).
Long-Term Potentiation (Electrophysiology)
MeCP2 deficient mice have been previously reported to suffer from functional
and
ultrastructural synaptic dysfunction, significant impairment of hippocampus-
dependent memory
and hippocampal long-term potentiation (LIP) (Moretti et al. The Journal of
Neuroscience. 2006.
51
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
26(I):319-327). To test the effects of the G-2-MePE treatment on synaptic
function in the RTT
model we compared hippocampal LTP in both vehicle and G-2-MePE treated animals
at 9 weeks
of age. To do so, we measured the slope of the fEPSP as a % of baseline
potential in neurons in
slices of hippocampus from MeCP2 deficient mice treated with either saline or
G-2-MePE (FIG.
23).
Results
FIG. 22 shows that G-2-MePE treatment increased survival of MeCP2 deficient
mice.
Wild-type mice (top line) are control animals, and therefore their survival
was 100% at each time
point. MeCP2 deficient mice treated with saline only died much more rapidly
(dotted line) than
wild-type mice, such that by about 11 weeks, only 50% of the MeCP deficient
mice survived. In
striking contrast, however, we unexpectedly found that MeCP2 deficient mice
treated with G-2-
MePE survived substantially longer than saline-treated mice. At about 15
weeks, 50% of the
animals survived. Data initially presented showed that MeCP2 mice were
impacted in terms of
survival such that 50 percent of animals had died by llweeks in the untreated
case. G-2-MePE
treated animals showed improved survival, with 50 percent having died at 16
weeks. In this
study, the longevity data were compromised by inconsistent veterinary
procedures, such that mice
did not have their teeth clipped consistently ¨ a requirement in mecp2 mice
unrecognized at the
start of the experiment. A consequence was the observation of early animal
deaths unrelated to
Rett Syndrome (particularly in the control group). Re-examination of the data
showed that the
effect of G-2-MePE persisted when the control group was re-run, albeit the
difference in groups
being smaller (time to 50 percent death 13.5 weeks in controls, 16 weeks in G-
2-MePE treated
animals). No safety concerns were raised by G-2-MePE treatment of mecp2 mice.
These results demonstrated that G-2-MePE can substantially increase survival
of MeCP2
deficient mice. Because MeCP2 deficient mice are predictive of the pathology
and therapeutic
efficacy in human beings with Rett Syndrome, we conclude that G-2-MePE can
increase life span
of human beings with Rett Syndrome.
FIG. 23 shows results of our studies to determine if G-2-MePE treatment
increased
hippocampal long-term potentiation (LTP) as measured by the fEPSP slope in
MeCP2 deficient
animals compared to saline-treated mutant mice. As shown in FIG. 23, we
unexpectedly found
that G-2-MePE increased the slope of fESPS in MeCP2 deficient mice compared to
animals
treated with saline only.
These results demonstrated that 0-2-MePE can be effective in treating MeCP2
deficient
mice in vivo. Because MeCP2 deficient mice are predictive of the pathology and
therapeutic
efficacy in human beings with Rett Syndrome, we conclude that 0-2-MePE can be
an effective
therapy for people with Rett Syndrome.
52
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Example 9: G-2-MePE
Improves Dendritic Arborization and Increases Dendritic Spine
Length
We assess the effects of G-2-MePE treatment on dendrites. Transgenic mecp2
knockout mice (n = 15 to 20) were administered G-2-MePE intraperitoneally at a
dose of
20 mg/kg once daily. Following sacrifice dendritic spine density, spine length
and aborization
were examined after Golgi staining after nine weeks, as per the Table 1 below:
Table 1: Sample size for all neuron morphologic and spine analysis
MALE
Analysis AGE Sample size (no. of mice)
Sample size (average
(Weeks) number of neurons or
dendrites per animal)
KO-vehicle KO-NNZ- KO-vehicle KO-NNZ-
G-2MePE G-2MePE
Neuron 9 3 3 4 4
morphology
Spine Analysis 9 3 3 10 10
Dendritic length was assessed by distance from the soma of representative
hippocampal CAI
neurons from 9 week old male mecp2 null mutant mice treated with either saline
(3 neurons
analysed from 3 separate mice, tr=9) or G-2-MePE (20 mg/kg i.p. 1/day, from
week 4; 3 neurons
analysed from 3 separate mice, n=9).
We observed that G-2-MePE improved dendritic arborization and increased
dendritic spine length. FIG. 24 depicts results of this study. Dendritic
length in um (vertical
axis) is plotted against the distance (in um; horizontal axis) from the soma
of the cells. For cells
with dendrites close to the somas, the dendrites were short. However, as the
distance from the
somas increased saline-treatment (open squares) produced dendritic lengths
that increased to a
maximum at a distance of 70 um from the soma and declined at distances further
away from the
somas. In contrast, treatment with G-2MePE (filled squares) produced longer
dendrites over
much of the range of distances from the somas.
Example 10: Treatment of Rett Syndrome in Mice II
Mice Mating and Genotyping
The MeCP2 germline null allele mice are used (Chen et al., 2001). Genotyping
is
performed as in Chen et al. (Chen et al., 2001).
G-2-MePE Treatment
For the survival measurements, the nocturnal activity analysis and the
immunoblot
analysis, G-2-MePE (synthesised Albany Molecular Research Inc. (Albany, NY)
and supplied by
53
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Neuren Pharmaceuticals Limited) is administered daily via intra-peritoneal
injections (20 mg/kg,
vehicle=saline, 0.01% BSA). The treatment starts at P15 and is maintained
throughout the course
of the experiments. For intracellular physiology experiments, the mice are
injected daily with G-
2-MePE (20 mg/kg body weight, vehicle¨saline, 0.01% BSA) for 2 weeks, from P15
to P28-P32
when they are used for acute slice preparation. For optical imaging
experiments, mice are injected
with G-2-MePE (20 mg/kg body weight, vehicle=saline, 0.01% BSA) daily from the
day of the lid
suture to the day of imaging.
Slice Physiology Preparation
Coronal sections (300 fim thick) at or near sensorimotor cortex are cut in <4
C ACSF
using a Vibratorne. Slices are incubated at 37 C for 20 minutes after slicing,
and at room
temperature for the remainder of the experiment. Slices are transferred to a
Warner chamber and
recordings are taken from visually identified pyramidal neurons located in
layer 5. Artificial
cerebral spinal fluid (ACSF) containing 126 mM NaCI, 25 mM NaHCO3, 1 mM
NaHPO4, 3 mM
KC1, 2 mM MgSO4, 2 mM CaC12, and 14 mM dextrose, is adjusted to 315-320 mOsm
and 7.4
pH, and bubbled with 95% 02/5% CO2. The intracellular pipette solution
contained 100 rriM
potassium gluconate, 20 mM KC1, 10 mM HEMS, 4 mM MgATP, 0.3 mM NaGTP, and 10
mM
Na-phosphocreatine.
Intracellular Whole-Cell Recordings
Borosilicate pipettes (3-5 MR WPI) are pulled using a Sutter P-80 puller
(Sutter
Instruments). Cells are visualized with an Achroplan 40x water-immersion lens
with infrared-D1C
optics (Zeiss) and detected with an infrared camera (Hamamatsu) projecting to
a video monitor.
Experiments are driven by custom acquisition and real-time analysis software
written in Matlab
(Mathworks, Natick, Mass.) using a Multiclamp 70013 amplifier (Axon
Instruments) connected to
a BNC-2110 connector block and M-Series dual-channel acquisition card
(National Instruments).
Gigaseal and rupture is achieved and whole-cell recordings are continuously
verified for low
levels of leak and series resistance. For each recording, a 5 mV test pulse is
applied in voltage
clamp ¨10 times to measure input and series resistance. Then in current clamp
¨10 pulses (500
ms, 40-140 pA at 10 pA increments), are applied to quantify evoked firing
rates and cellular
excitability. Access resistance, leak, and cellular intrinsic excitability are
verified to be consistent
across groups. Finally, spontaneous EPSCs under voltage clamp at -60 mV are
sampled at 10 kHz
and low-pass filtered at 1 kHz. Analysis is performed using a custom software
package written in
Matlab, with all events detected according to automated thresholds and blindly
verified for each
event individually by the experimenter.
54
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Golgi Staining
Samples (<1 cm) from P28 mice are fixed in 10% formalin and 3% potassium
bichromate
for 24 hours. Tissue is then transferred into 2% silver nitrate for 2 days in
the dark at room
temperature. Sections from these samples are then cut at 50 m thickness into
distilled water.
Sections corresponding to motor cortex are mounted onto slides, air dried for
10 minutes, and
then dehydrated through sequential rinses of 95% alcohol, 100% alcohol, and
xylene, and then
sealed with a coverslip. Images re acquired at 10x (whole cell) and 100x
(spine imaging) using a
Zeiss Pascal 5 Exciter confocal microscope.
Optical Imaging of Intrinsic Signals
Adult (>P60) wild type (SVEV or MA) and MeCP2 (+/-) mutant females (BIL6) are
used
for this experiment. The wild type control group is composed of both wild type
littermates of
MeCP2+/- females or wild type age matched SVEV females. For monocular
deprivation, animals
are anesthetized with Avertin (0.016 ml/g) and the eyelids of one eye is
sutured for 4 days. Prior
to imaging, the suture is removed and the deprived eye re-opened. Only animals
in which the
deprivation sutures are intact and the condition of the deprived eye appears
healthy are used for
the imaging session. For G-2-MePE signaling activation, a solution containing
G-2-MePE is
injected intra-peritoneally (IP) daily for the entire period of deprivation.
For the imaging sessions
mice are anesthetized with urethane (1.5 g/kg; 20% of the full dosage is
administered IP each 20-
30 minutes up to the final dosage, 0.02 ml of cloroprothixene 1% is also
injected together with the
first administration). The skull is exposed and a custom-made plate is glued
on the head to
minimize movement. The skull is thinned over V1 with a dremel drill and
covered with an
agarose solution in saline (1.5%) and a glass coverslip. During the imaging
session, the animal is
constantly oxygenated, its temperature maintained with a heating blanket and
the eyes
periodically treated with silicone oil; physiological conditions are
constantly monitored. The
anesthetized mouse is placed in front of a monitor displaying a periodic
stimulus presented to
either eye, monocularly; the stimulus consisted of a drifting vertical or
horizontal white bar of
dimensions 9 x72 , drifting at 9 sec/cycle, over a uniformly gray background.
The skull surface is
illuminated with a red light (630 nm) and the change of luminance is captured
by a CCD camera
(Cascade 512B, Roper Scientific) at the rate of 15 frames/sec during each
stimulus session of 25
minutes. A temporal high pass filter (135 frames) is employed to remove the
slow signal noise,
after which the signal is computer processed in order to extract, at each
pixel, the temporal Fast
Fourier Transform (FFT) component corresponding to the stimulus frequency. The
FFT amplitude
is used to measure the strength of the visual evoked response to each eye. The
ocular dominance
index is derived from each eye's response (R) at each pixel as ODI=(Rcontra-
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Ripsi)/(Rcontra+Ripsi). The binocular zone is defined as the region activated
by the stimulation of
the eye ipsilateral to the imaged hemisphere.
Heart Rate Measurements
Real time cardiac pulse rate is measured using a tail clip sensor (Mouse OX
Oximeter--
Oakmont, PA). Mice are not anesthetized but physically restrained in a fitted
open plastic tube.
Prior to the recording session the tube is placed overnight in the cages
housing the experimental
animals to allow habituation. Body temperature is maintained at ¨82-84 F
throughout the
recording time. We record 3 trials of 15 minutes for each mouse, mice are 8
weeks old and treated
with vehicle or G-2-MePE from P15.
Nocturnal Activity Measurements
Spontaneous motor activity is measured by using an infrared beam-activated
movement-
monitoring chamber (Opto-Varimax-MiniA; Columbus Instruments, Columbus, Ohio).
For each
experiment, a mouse is placed in the chamber at least 3 h before recordings
started. Movement is
monitored during the normal 12-h dark cycle (7 p.m. to 7 a.m.). One dark cycle
per animal per
time point is collected.
Results
To test whether G-2-MePE treatment will impact the development of cardinal
features of
the RTT disease, 2 week old mutant animals are given daily intra-peritoneal
injections for the
course of their lifespan. Measurements of synaptic physiology, synaptic
molecular composition,
and cortical plasticity are then acquired as detailed below, along with health-
related
measurements such as heart rate, locomotor activity levels, and lifespan.
Effects of G-2-MePE on the Synaptic Physiology of MeCP2 Mutant Mice
Recent studies have reported that neurons across multiple brain regions of
MeCP2-/y
mice display a profound reduction in spontaneous activity (Chang et al., 2006;
Chao et al., 2007;
Dani et al., 2005; Nelson et al., 2006) a phenotype that is rescued by over-
expression of BDNF
(Chang et al., 2006). Similarly, acute application of an IGF1 derivative has
been shown to elevate
evoked excitatory postsynaptic current (EPSC) amplitudes by 40% in rat
hippocarnpal cultures
(Ramsey et al., 2005; Xing et al., 2007). To test the efficacy of G-2-MePE in
rescuing the
MeCP2-/y physiological phenotype, we acquire intracellular whole cell
recordings in acute brain
slices, measuring excitatory synaptic drive (spontaneous EPSC amplitude and
frequency) in layer
5 cortical neurons. Here, EPSCs recorded from -/y animals are significantly
reduced in amplitude
compared to EPSCs measured in wild-type animals. The trend is partially
reversed in EPSCs
56
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
recorded from MeCP2-/y animals treated with G-2-MePE, which are significantly
larger in
amplitude than EPSCs from MeCP2-/y mice treated with vehicle. These
differences are also seen
when averaging across cells. Throughout these measurements, access resistance,
leak, and cellular
intrinsic excitability are also verified to be consistent across groups.
Quantifying EPSC intervals
also shows a slight increase in the interval between EPSC events (reduced EPSC
frequency)
between wild-type and MeCP2-/y animals (P-0,04, Kolmogorov-Smirnov test). Our
findings thus
indicate that the reduction of excitatory synaptic drive in cortical cells of
MeCP2-/y mice, and its
partial rescue following G-2-MePE treatment, are due in part to a change in
EPSC amplitude as a
consequence of a change in the strength of the synapses mediating excitatory
transmission in this
region.
G-2-MePE Treatment Stimulates Cortical Spine Maturation
We use Golgi staining to label neurons sparsely and distinctly, and applied
high-
resolution confocal imaging to measure dendritic spine density and morphology
in the labelled
cells, restricting analysis to layer 5 pyramidal neurons in sections of motor
cortex from critical
period mice (P28).
While low-magnification imaging clearly delineates the extent of the dendrites
of the
pyramidal cells we use higher magnifications to count synaptic contacts and
determine the
morphological class of each spine. We classify spines as either large and
bulbous ("mushroom",
M), short and stubby ("stubby", S), short and thin ("thin", T) or filopodia
(F). Comparing the
density of spines per unit branch exhibits a trend of decreased spine density
in knockout neurons
that is largely ameliorated in the knockout with treatment.
Together these results indicate the potential for deficits in the number and
maturational
status of dendritic contacts in the knockout to underpin functional defects in
excitatory
transmission, in a manner that can be treated following administration of G-2-
MePE.
Ocular Dominance (OD) Plasticity in Adult MeCP2+/- Mice Is Reduced By
G-2-MePE
Developmental changes in OD plasticity are controlled in part by the
activation of the
IGF-1 pathway, and administration of (1-3)IGF-1 can reduce OD plasticity in
wild type young
mice (Tropea et al., 2006). We therefore test if G-2-MePE treatment could
stabilize the prolonged
OD plasticity observed in adult MeCP2 mutants. Female MeCP2+/- mice, aged P60
or more, are
monocularly deprived for 4 days and treated concurrently with G-2-MePE. G-2-
MePE treatment
reduces the OD plasticity in the adult Mecp2+/- mice, indicating that indeed G-
2-MePE can
rapidly induce synapse stabilization or maturation.
Bradycardia in MeCP2-/y. Mice Is Treated By G-2-MePE
57
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
in addition to examining the efficacy of G-2-MePE in ameliorating
neurophysiological
symptoms, we seek to characterize its effects on the general health of the
organism. Clinical and
experimental evidence shows autonomic system dysfunctions such as labile
breathing rhythms
and reduced baseline cardiac vagal tone in Rett Syndrome patients (lulu et
al., 2001). A poor
control of the feedback mechanisms that regulate blood pressure homeostasis
through the
sympathetic system, for example hyperventilation-induced decrease in heart
rate, is common in
Rett Syndrome patients and can cause life threatening cardiac arrhythmias
(Acampa and Guideri,
2006; Julu et al., 2001).
The pathogenesis of the cardiac dysautonomia, although not well understood,
suggests
that immature neuronal connections in the brainstern could be the cause. To
examine heart rate
abnormalities in MeCP2-/y mice and the effect of G-2-MePE treatment, we
monitor real time
cardiac pulse rate in non-anesthetized wild type and MeCP2-/y animals treated
with vehicle or G-
2-MePE, Wild type mice exhibit a regular distribution of heart rate
measurements centred near
750 beats per minute. In contrast, MeCP2-/y mice exhibit a more irregular
heart rate with a lower
average rate, the occurrence of which is significantly reduced following
treatment with G-2-
MePE.
G-2-MePE Administration Improves Locomotor Activity and Life Span
MeCP2-/y mice develop Rett-like symptoms beginning at 4-6 weeks of age when
they
progressively become lethargic, develop gait ataxia and die between 10 and 12
weeks of age
(Chen et al., 2001). Baseline locomotor activity is also recorded in mice
after 6 weeks by counting
nocturnal infrared beam crossing events within a caged area. MeCP2 knockout
mice (KO)
exhibits markedly reduced locomotor activity levels compared to wild-type mice
(WT), but
treatment with G-2-MePE (KO-T) elevates these levels.
Finally, compared to MeCP2 KO littermates, MeCP2-/y mice treated with G-2-MePE
also show a ¨50% increase in life expectancy (an increase in the 0.5
probability survival rate).
We also measure the effect of 0-2-MePE treatment on neuron soma size in the
hippocampus. Mice are treated with G-2-MePE as described above for locomotor
activity. Soma
size in neurons in the CA3 region of the hippocampus is significantly impaired
in MeCP2 KO
animals relative to wild-type animals. G-2-MePE treatment increases average
soma size in KO
animals, but has little or no effect on soma size in wild type animals.
Example 11: Effect of Oral G-2-MePE on Survival in Rett Syndrome in Mice
Because Rett Syndrome is a chronic, debilitating disorder involving loss of
motor skills, it
is desirable to treat Rett Syndrome using easily administered preparations. To
this end, we can
take advantage of unexpectedly beneficial therapeutic and pharmacokinetic
properties of G-2-
58
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
MePE and related compounds (U.S. Pat. Nos. 7,041,314, 7,605,177, 7,714, 070,
7,863,309 and
U.S. Appl. Nos. 11/315,784 and 12/903,844).
Therefore, we administer G-2-MePE orally to MeCP2 deficient mice as described
in US
2009/0074865. Briefly, an aqueous solution, a water-in-oil emulsion (micro-
emulsion, coarse
emulsion or liquid crystal), or a gel composition containing a
pharmaceutically effective amount
of G-2-MePE (20 or 80 mg/kg per animal) is administered daily. In control
MeCP2 deficient
animals, we administer saline only, and wild-type animals are used to obtain
baseline data similar
to the design of studies described in Example 8 above.
In wild-type animals, survival is defined to be 100% at each time point. In
MeCP2
deficient animals, survival is decreased substantially. However, after oral
administration of G-2-
MePE to MeCP2 deficient mice, survival is increased substantially.
Example 12: Effect of G-2MePE on Seizure Activity in Rett Syndrome in Mice
Because seizures are a prominent, hazardous and a difficult to treat aspect of
Rett
Syndrome, we determine the effects of G-2MePE on seizure activity in MeCP2
deficient animals.
0-2-MePE can be effective in treating seizure activity in animals with
neurodegenerative disease
(U.S. Pat. No. 7,714,020). Therefore, we carry out experiments to determine
whether G-2-MePE
can also treat seizure activity in MeCP2 deficient mice.
Electroencephalograpic recordings of wild-type mice and MeCP2 deficient mice
treated
with either saline or G-2-MePE are obtained using methods described in U.S.
Pat. No. 7,714,020.
We find that G-2MePE can be effective in decreasing both motor seizures and
non-
convulsive seizures.
Conclusions
Based on our in vivo and in vitro studies in MeCP2 deficient animals, we
conclude that
G-2-MePE can be an effective therapy for treating human beings with Rett
Syndrome. Moreover,
because 0-2-MePE has unexpectedly longer half life than a naturally occurring
compound ((1-3)
IGF-1; Glycyl-Prolyl-Glutamate or GPE) (PIG. 21), we conclude that use of 0-2-
MePE has
distinct and substantial advantages over other pharmacological agents,
including GPE.
For example, G-2-MePE is not degraded by gastrointestinal cells, is taken up
by
gastrointestinal cells, and is active in the central nervous system after oral
administration (Wen et
al., U.S. Appl. No. 12/283,684; U.S. 2009/0074865, U.S. Pat. No. 7,887,839,
incorporated herein
fully by reference), Therefore, G-2MePE need not be delivered intravenously,
subcutaneously,
intraventricularly, or parenterally. In fact, oral formulations comprising
micro-emulsions, coarse
emulsions, liquid crystal preparations, nanocapsules and hydrogels can be used
in manufacture of
orally administered preparations such as tablets, capsules and gels that can
improve neurological
59
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
function and treat neurodegenerative conditions (U.S. Pat. No. 7,887,839).
Compounds of this
invention can be used in situations in which a patient's motor functioning is
below that needed to
swallow a table or capsule. There are several types of soluble gels for oral
administration of
compounds, and these can be used to deliver a compound or composition of this
invention to a
patient. Because G-2-MePE can be easily administered orally and is orally
effective in treating
neurodegenerative disorders, including Rett Syndrome, we conclude that G-2-
MePE can be
convenient and beneficial for long-term therapy of patients with Rett
Syndrome.
Further, because Rett Syndrome shares key features with other autism spectrum
disorders,
compounds of this invention can be useful in providing therapeutic benefit
from animals having
other ASD, and in humans with autism, Asperger Syndrome, Childhood
Disintegrative Disorder,
and Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS).
Example 13: Treatment of ASD
Shank3-Deficient Mouse Model
Shank3- deficient mice are used in the study as a model of 22q13 deletion
syndrome
associated with ASD,
22q13 deletion syndrome has been linked with deletions or mutations in Shank3
gene
(Bonaglia et al, 2006). The Shank3 gene codes for a master scaffolding protein
which forms the
framework in glutamatergic synapses (Boeckers et al, 2006). Shank3 is a
crucial part of the core
of the postsynaptie density (PSD) and recruits many key functional elements to
the PSD and to
the synapse, including components of the a-amino-3-hydroxyl-5-methyl-4-
isoxazole-propionic
acid (AMPA), metabotropie glutamate (mGIu), and N-methyl-D-aspartic acid
(NMDA) glutamate
receptors, as well as cytoskeletal elements. Recent studies exploring the rate
of 22q13 deletions/
Shank3 mutations suggest that haploinsufficiency of Shank3 can cause a
monogenic form of ASD
with a frequency of 0.5% to 1% of ASD cases (Durand et al, 2007; Moessner et
al, 2007; Gauthier
et al, 2008).
The generation of the mouse model with disrupted expression of full-length
Shank3 has
been previously described in the art (Bozdagi et al., Molecular Autism 2010,
1:15, p4). Briefly,
Bruce4 C57B116 embryonic stem cells were used to generate a mouse line that
had loxP sites
inserted before exon 4 and exon 9. The foxed allele was excised and a line was
maintained with a
deletion of exons 4 to 9, i.e. a complete deletion of the ankyrin repeat
domains of Shank3. Wild-
type (+1+), heterozygous (+1-) and knockout (-/-) mice were produced, with
Mendelian
frequencies from heterozygote-heterozygote crosses. A 50% reduction of full
length Shank3
mRNA was confirmed in heterozygotes (qPCR) as well as a reduced expression of
Shank3 protein
(by immunoblotting with Shank3 antibody N69/46).
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Heterozygous mice generated by crossing wild-type mice with heterozygotes are
used in
this example to best model the haploinsufficiency of Shank3, responsible for
22q13 deletion
syndrome.
Methods
Drug Treatment
1 to 3 month old wild type and heterozygous Shank3-deficient mice are divided
into 4
treatment groups: placebo treated wild-type, placebo treated Shank3-deficient
group and two
Shank3-deficient G-2-MePE treated groups. The animals are given placebo
(water) or G-2-MePE
formulated in water administered orally, b.i.d for 14 days. G-2-MePE is
administered at two
doses: 15 or 60 mg/kg.
Methodology
A detailed description of the methodology can be found in Bozdagi et al.
(Molecular
Autism 2010, 1:15).
Behavioral Analyses
Behavioral assessments are made at several time points, and include analysis
of social
interactions and ultrasonic social communication, in line with the methodology
described by
Bozdagi et al. Briefly, male-female social interactions in each treatment
group are evaluated. The
subject males are group-housed and individually tested in clean cages with
clean litter. Each
testing session lasts 5 min. Each of the subject mice is paired with a
different unfamiliar estrus
C57BL/6J female. A digital closed circuit television camera (Panasonic,
Secaucus, NJ, USA) is
positioned horizontally 30 cm from the cage. An ultrasonic microphone (Avisoft
UltraSoundGate
condenser microphone capsule CM15; Avisoft Bioacoustics, Berlin, Germany) is
mounted 20 cm
above the cage. Sampling frequency for the microphone is 250 kHz, and the
resolution is 16 bits.
While the equipment used cannot distinguish between calls emitted by the male
subject and
female partner, the preponderance of calls during male-female interactions in
mice is usually
emitted by the male. The entire apparatus is contained in a sound-attenuating
environmental
chamber (ENV-018V; Med Associates, St Albans, VT, USA) illuminated by a single
25-Watt red
light. Videos from the male subjects are subsequently scored by an
investigator uninformed of the
subject's genotype and treatment group on measures of nose-to-nose sniffing,
nose-to-anogenital
sniffing and sniffing of other body regions, using Noldus Observer software
(Noldus Information
Technology, Leesburg, VA, USA). Ultrasonic vocalizations are identified
manually by two highly
trained investigators blinded to genotype/treatment group information, and
summary statistics are
61
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
calculated using the Avisoft package. IntelTater reliability is 95%. Data are
analysed using an
unpaired Student's t-test.
Olfactory habituation/dishabituation testing is conducted in male and female
mice for
each group. The methodology is as previously described (Silverman et al 2010,
Yang et al 2009
and Silverman et al 2010). Non-social and social odors are presented on a
series of cotton swabs
inserted into the home cage sequentially, each for 2 min, in the following
order: water, water,
water (distilled water); almond, almond, almond (1:100 dilution almond
extract); banana, banana,
banana (1:100 dilution artificial banana flavouring); social 1, social 1,
social 1 (swiped from the
bottom of a cage housing unfamiliar sex-matched B6 mice); and social 2, social
2, social 2
(swiped from the bottom of a second cage housing a different group of
unfamiliar sex-matched
129/SvImJ mice). One-way repeated measures ANOVA is performed within each
treatment group
for each set of habituation events and each dishabituation event, followed by
a Tukey post hoc
test.
Hippocampal Slice Electrophysiology
Post-mortem, acute hippocampal slices (350 gm) are prepared from mice using a
tissue
chopper. Slices are maintained and experiments are conducted at 32 C. Slices
are perfused with
Ringer's solution containing (in inM): NaC1, 125.0; KC1, 2.5; MgSO4, 1.3;
NaH2PO4, 1.0;
NaHCO3, 26.2; CaC12, 2.5; glucose, 11Ø The Ringer's solution is bubbled with
95% 02/5% CO2,
at 32 C, during extracellular recordings (electrode solution: 3 M NaC1).
Slices are maintained for
1 hr prior to establishment of a baseline of field excitatory postsynaptic
potentials (fEPSPs)
recorded from stratum radiatum in area CAI, evoked by stimulation of the
Schaffer collateral-
commissural afferents (100 Rs pulses every 30 s) with bipolar tungsten
electrodes placed into area
CA3. Test stimulus intensity is adjusted to obtain fEPSPs with amplitudes that
are one-half of the
maximal response. The EPSP initial slope (mV/ms) is determined from the
average waveform of
four consecutive responses. Input-output (I/O) curves are generated by
plotting the fEPSP slope
versus fiber volley amplitude in low-Mg2+ (0.1 MIVI) solution. AMPA receptor-
mediated and
NMDA receptor-mediated I/O relationships are measured in the presence of ion
otropic glutamate
receptor antagonists: 2-amino-2-phosphonopentanoic acid APV (50 M) and 6-cyano-
7-
nitroquinoxaline-2,3-dione CNQX (100 M). Paired-pulse responses are measured
with
interstimulus intervals of 10 to 200 ms, and are expressed as the ratio of the
average responses to
the second stimulation pulse to the first stimulation pulse.
LTP is induced either by a high-frequency stimulus (four trains of 100 Hz, 1 s
stimulation
separated by 5 min), or by theta-burst stimulation (T13S) (10 bursts of four
pulses at 100 Hz
separated by 200 ms), or by a single 100 Hz stimulation, for control and
genetically-modified
mice. To induce long-term depression (LTD), Schaffer collaterals are
stimulated by a low
62
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
frequency or paired-pulse low trequency stimulus (900 pulses at I Hz for 15
mm) to induce mGlu
receptor-dependent LTD. Data are expressed as means SD, and statistical
analyses are
performed using analysis of variance (ANOVA) or student's t-test, with
significance set at an a
level of 0.05.
Results
Behavioral
Cumulative duration of total social sniffing by the male test subjects is
lower in placebo
treated Shank-3-deficient group than in placebo treated wild-type group. In
addition, fewer
ultrasonic vocalizations are emitted by the placebo treated Shank3-deficient
group than by the
wild-type controls during the male-female social interactions.
G-2-MePE treatment in the two Shank3-deficient groups results in a significant
increase
in the cumulative duration of total social sniffing in comparison to the
placebo treated Shank3-
deficient group. Moreover, the G-2-MePE treated groups display an increased
number of
ultrasonic vocalizations than the placebo treated mutant group.
In the olfactory habituation/dishabituation study, intended to confirm that
the mice are
able to detect social pheromones, all 4 groups display normal levels of
habituation (indicated by
decreased time spent in sniffing the sequence of three same odors), and the
expected
dishabituation (indicated by increased spent in sniffing the different odor).
Eleetrophysiology
Plotting field excitatory postsynaptic potential (fEPSP) slope versus stimulus
intensity
demonstrates a reduction in the I/O curves in the placebo treated Shank-3-
deficient group versus
the control group. In the heterozygous placebo treated group we also observe a
decrease in AMPA
receptor-mediated field potentials, reflected in a 50% decrease in the average
slope of I/O
function compared to the wild-type control group. In contrast, when the I/O
relationship is
analysed in the presence of the competitive AMPA/kainate receptor antagonist
CNQX to measure
synaptic NMDA receptor function, there is no difference between the wild-type
and placebo
treated heterozygous groups. These results indicate that there is a specific
reduction in AMPA
receptor-mediated basal transmission in the Shank3 heterozygous mice.
G-2-MePE treatment in both heterozygous groups normalizes the AMPA receptor-
mediated field potentials and causes an increase in the average slope of 1/0
function compared to
the placebo treated Shank3-deficient group.
The maintenance of LTP in the placebo treated Shank-3-deficient group is
clearly impaired
in comparison to the wild-type control. TBS LTP tests (10 bursts of four
pulses at 100 Hz
separated by 200 ms) also show a significant decrease in the potentiation at
60 min after TBS in
63
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
the placebo treated Shank3-deficient group. In contrast to the altered
synaptic plasticity observed
with LTP, long-term depression (LTD) was not significantly changed in the
mutant group.
G-2-MePE treatment increased hippocampal long-term potentiation (LTP) and its
maintenance in
both Shank3-deficient group in comparison to the placebo treated Shank3-
deficient group.
Discussion
Poor social competencies and repetitive behaviors are the common features and
key
diagnostic measures of all forms of ASD. Delayed intellectual development and
underdeveloped
language skills are also a common feature present in all ASD, excluding
Asperger syndrome.
The animal models described above have been accepted in the art as
demonstrating
similar symptoms to the clinical human conditions. All mutant models discussed
above (NLGN3,
NLGN4, CADM1, NRXN1, FMR1, shank3) exhibit impaired social skills or increased
social
anxiety. Decreased excitatory transmission into the hippocampus has been
identified in NRXN1,
shank3, MeCP2 and FMR1 mutant animal models. At present no polygenetic or
multifactorial
models of ASD have been described. The animal models described above, based on
genetic
defects that are known to produce ASD in human population, provide the best
opportunity to test
the efficacy of ASD therapies.
Therefore the efficacy of G-2-MePE in animal models of ASD is reasonably
predictive of
its efficacy in a human subject suffering from ASD.
Example 14: G-2-MePE Treatment Changes the Morphology of Neurons in an in
vitro Human Model of Rett Syndrome
To test the effects of G-2-MePE on neuronal morphology, we used an in vitro
model of
RTT described in Marchetto et al., A model for neural development and
treatment of Rett
syndrome using human induced plunpotent stem cells, Cell 143:527-539 (2010)
(including
supplemental information). The model uses induced pluripotent stem cells
(iPSCs) generated
from fibroblasts of human RTT patients carrying different MeCP2 mutations.
Methods
Cell Culture and Retrovirus Infection
RTT fibroblasts (carrying 4 distinct MeCP2 mutations) and control fibroblasts
are
generated from explants of dermal biopsies. The shRNA against target MeCP2
gene is cloned
into the LentiLox3.7 lentivirus vector (as described in Marchetto et al.). The
fibroblasts are
infected with retroviral reprogramming vectors (Sox2, Oct4, c-Myc and K1f4).
Two days after
infection, fibroblasts are plated on mitofically inactivated mouse embryonic
fibroblasts with hESC
medium. After 2 weeks, iPSC colonies that emerge from the background of
fibroblasts are
manually picked and transferred to feeder-free conditions on matrigel-coated
dishes (BD) using
64
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
embryonic stem cell culture media mTeSRTm (Stem Cell Technologies) and
passaged manually.
Gene expression profiles of the generated clones are measured using human
genorne Affymetrix
Gene ChipTM arrays to confirm that reprogramming is successful.
Neural Differentiation: NPCs and Mature Neurons
To obtain neural progenitor cells (NPCs), embryoid bodies (EBs) are formed by
mechanical dissociation of cell clusters and plating onto low-adherence dishes
in hESC medium
without FGF2 for 5-7 days. After that, EBs are plated onto poly-
ornithine/laminin-coated dishes
in DMEM/F 12 plus N2 medium (serum-free supplement for growth and expression
of post-
mitotic cells). Resulting rosettes are collected after 7 days and dissociated
with accutase and
plated onto coated dishes with NPC media (DMEM/F12; 0.5X N2; 0.5X B27 and
FGF2).
Homogeneous populations of NPCs are achieved after 1-2 passages with accutase
in the same
condition. To obtain mature neurons, floating EBs are treated with luM or
retinoic acid for 3
weeks (giving the total time of differentiation of 4 weeks). Mature EBs are
dissociated with
papain and DNAse for 1 h at 37 C and plated in poly-ornithine/laminin-coated
dishes in NPC
media without FGF2.
Treatment with G-2-MePE
RTT neuronal cultures are treated with G-2-MePE (1nM-I0uM) for I week.
Immunocytochemistry and Quantification of Neuronal Morphology
Cells are fixed in 4% paraformaIdehyde and permeabilized with 0.5% Triton-X100
in
PBS. Cells are then blocked in PBS containing 0.5% Triton-X100 and 5% donkey
serum for I h at
room temperature. Fluorescent signals are detected using a Zeiss inverted
microscope and images
are processed with Photoshop CS3. The following primary antibodies are used:
TRA-1-60,
TRA-1-81 (1:100), Nanog and Lin28 (1:500), human Nestin (1:100), Tuj-1
(1:500), Map2
(1:100); meCP2 (1:1000; VGLUTI (1:200), Psd95 (1:500), GFP (1:200), Soxl
(1:250), Mushasil
(1:200) and me3H3K27 (1:500). Cell soma size is measured using suitable
software (e.g. ImageJ)
after identification of neurons using the Syn::EGFPTM. The morphologies of
neuronal dendrites
and spines are studied from an individual projection of z-stacks optical
sections and scanned at
0.5um increments that correlate with the resolution valued at z-plane. Each
optical section is the
result of 3 scans at 500 lps followed by Kalman filtering. For synapse
quantification, images are
taken by a z-step of lum using Biorad radiance 2100TM confocal microscope.
Synapse
quantification is done blinded to genotype. Only VGLUT1 puncta along Map2-
positive processes
are counted. Statistical significance is tested using 2-way ANOVA test and
Bonferroni post-test.
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Calcium Imaging
Neuronal networks derived from human iPSCs are infected with the lentiviral
vector
carrying the Syn:DsRed reporter construct. Cell cultures are washed twice with
sterile Krebs
HEPES Buffer (KHB) and incubated with 2-5 !AM Fluo-4AMTm (Molecular
Probes/Invitrogen,
Carlsbad, CA) in KHB for 40 minutes at room temperature. Excess dye is removed
by washing
twice with KHB, and an additional 20 minutes incubation is done to equilibrate
intracellular dye
concentration and allow de-esterification. Time-lapse image sequences (100X
magnification) of
5000 frames are acquired at 28 Hz with a region of 336 x 256 pixels, using a
Hamamatsu ORCA-
ERTM digital camera (Hamamatsu Photonics K.K., Japan) with a 488 nm (FITC)
filter on an
Olympus IX81 inverted fluorescence confocal microscope (Olympus Optical,
Japan). Images are
acquired with MetaMorph 77TM (MDS Analytical Technologies, Sunnyvale, CA).
Images are
subsequently processed using ImageJTM and custom written routines in Matlab
72TM (Mathworks,
Natick, MA).
Electrophysio logy
Whole-cell patch clamp recordings are performed from cells co-cultured with
astrocytes
after 6 weeks of differentiation. The bath is constantly perfused with fresh
HEPES-buffered saline
(see supplemental methods for recipe). The recording micropipettes (tip
resistance 3-6 Mil) are
filled with internal solution described in the Supplemental materials.
Recordings are made using
Axopatch 200BTM amplifier (Axon Instruments). Signals are filtered at 2 kHz
and sampled at 5
kHz. The whole-cell capacitance is fully compensated. The series resistance is
uncompensated but
monitored during the experiment by the amplitude of the capacitive current in
response to a 10-
mV pulse. All recordings are performed at room temperature and chemicals are
purchased from
Sigma. Frequency and amplitude of spontaneous postsynaptic currents are
measured with the
Mini Analysis ProgramTm software (Synaptosoft, Leonia, NJ). Statistical
comparisons of WT and
RTT groups are made using the non-parametric Kolmogorov-Smirnov two-tailed
test, with a
significance criterion of p = 0.05. EPSCs are blocked by CNQX or DNQX (10-20
uM) and IPSPs
are inhibited by bicuculine (20 ItM).
Results
RTT iPSC-derived neurons are characterized by decreased number of
glutamatergic
synapses, reduced spine density and smaller soma size. RTT neurons also show
certain
electophysio logical defects, i.e. a significant decrease in frequency and
amplitude of spontaneous
synaptic currents when compared to controls. The RTT neurons show a decreased
frequency of
intracellular calcium transients.
66
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
We test G-2-MePE in the above model to test whether any of the pathologies of
the RTT
phenotype can be attenuated.
Treatment of the cell cultures with each drug concentration improves all of
the
morphological and physiological parameters of the treated RTT cell cultures in
comparison to the
non-treated RTT controls. Specifically, we observe a significant increase in
glutamatergic
synapse numbers in the G-2-MePE treated RTT cells. All concentrations of G-2-
MePE treatment
increase VGLUT1 puncta number in the RTT-derived neurons. G-2-MePE treatment
normalizes
the frequency and amplitude of spontaneous post-synaptic currents as well as
the frequency of
calcium transients generated by synaptic activity of the G-2-MePE treated RTT
neurons.
In the present in vitro model of human RTT, the iPSCs derived from RTT
patients and
neurons differentiated from them are characterized by abnormalities in the
MeCP2 expression.
As discussed in the detailed description of the invention above, the vast
majority of RTT cases are
associated with mutations of the MeCP2 gene. Therefore the efficacy of 0-2-
MePE in the present
in vitro model of human RTT is reasonably predictive of its efficacy in a
human subject suffering
from ATT.
Example 15: Effects of G-2-MePE in Human Beings with Rett Syndrome
Methods
Thirty subjects with Rett Syndrome are recruited. Subjects are female and aged
between
16 and 29 years (Mean = 12.1 SD = 4.4). All subjects have an IQ <60 and
mutations of the
MECP2 gene. Subjects also show ether spike activity in the EEG or an increase
in lower
frequency bands of the EEG as detected by Fast Fourier Transform (FFT).
Subjects are instructed
that concomitant medications are to be stable for at least six weeks prior to
study. Subjects
receiving medication to treat signs of inattention are tested in the morning
and instructed to take
their medication in the afternoon. Subjects with QTc interval >451 msec are
excluded.
The study is a randomized double blind placebo controlled parallel study with
three doses
of either placebo, 10 mg/kg T.I.D oral G-2-MePE for five days, or 30 mg/kg
T.I.D. oral G-2-
MePE.
Subjects are tested at baseline using the following instruments: The Rett
Syndrome
Natural History / Clinical Severity Scale, Aberrant Behavior Checklist
Community Edition
(ABC), Vinelands, Clinical Global Impression of Severity (CGI-S) and their
carers completed the
Caregiver Strain Questionnaire (CSQ).
Subjects are brought into clinic on an inpatient basis to enable initial
baseline recordings
of EEG, ECG and respiratory rate continuously for 24 hours using
polysornnography technology.
Hand movements are also recorded using the Q-SensorTM. Derived EEG measures
include:
67
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
spikes per unit time in the EEG, overall power of frequency bands of the EEG,
QTc and heart rate
variability (HRV), and respiratory irregularities.
Adverse events are also recorded using standard safety measures and the SMURF
elicitation of adverse events
Statistically, the effect of treatment with G-2-MePE is analysed by conducting
a repeated
analysis of covariance (ANCOVA) on the effect of treatment on change from
baseline scores.
Results
Treatment with G-2-MePE produces no more adverse events than are present
during
treatment with placebo, with all adverse events being of short duration and
mild severity. No
Serious Adverse Events are reported. No instances of increases in QTC are
reported.
No effects are seen on respiratory rate or heart rate variability.
Treatment with 0-2-MePE produces a significant overall reduction of spikes per
unit time
in the EEG. Treatment with 30 mg/kg T.I.D. oral G-2-MePE decreases spike
activity compared
to placebo. This dose of G-2-MePE also decreases the power of the delta band
of the EEG
compared to placebo.
Treatment with G-2-MePE also reduces total hand movements per twenty-four hour
period as counted using the Q-Sensor'' device. This effect is significant for
the 30 mg/kg T.I.D.
dose compared to placebo.
Treatment with G-2-MePE has no significant effect overall on the Rett Syndrome
Natural
History / Clinical Severity Score. However, 30 mg/kg T.I.D. oral G-2-MePE,
compared to
placebo, produces significant effects on the following subscales: "Nonverbal
Communication at
this visit by exam"; "Epilepsy/Seizures at this visit: and "Hand use".
Conclusions
Treatment with G-2-MePE produces significant improvements in Central Nervous
System function in the present study. Despite relatively short term treatment,
abnormalities in the
electrical activity of the brain is reduced, a clear signal of efficacy. This
effect is dose dependent,
seen after treatment 30 mg/kg T.I.D. oral G-2-MePE. These effects mirror the
improvements in
CNS function seen in the mecp2 knockout transgenic mouse model of Rett
Syndrome after
administration of G-2-MePE.
Dose dependent effects are also seen on hand use, as assessed by an objective
counting
device and subjective rating. This is of interest because purposeless hand
wringing is both
characteristic to the Rett Syndrome clinical phenotype and is unique to this
disorder.
The Non-verbal communication rating of the Rett Syndrome Natural History /
Clinical
Severity Scale is improved by treatment. This measure primarily assesses eye
contact. This
68
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
raises the prospect that longer term treatment with G-2-MePE may improve
social relatedness in
the population.
0-2-MePE is well tolerated in this population. No effects are seen in either
standard
measures or areas of specific concern in the patient population, such as QTc
interval prolongation
or apnea.
Example 16: Effects of G-2MePE on Human Beings with Autism Spectrum
Disorders
Methods
To determine whether G-2-MePE can treat symptoms of ASD, we carry out a study
in
human beings with ASD. Twenty subjects with an Autism Spectrum Disorder are
recruited.
Subjects are male and aged between 16 and 65 years (Mean = 18.1 SD = 3.4). All
subjects have
an IQ >60 and strict DSM-IV-TR diagnosis of Autistic Disorder or Asperger
Disorder. Subjects
also meet criteria for an Autism Spectrum Disorder according the ADI-R and
ADOS-G
instruments, and fulfill the proposed DSM-V criteria for and Autism Spectrum
Disorder. Subjects
are instructed that concomitant medications are to be stable for at least six
weeks prior to study.
Subjects receiving medication to treat signs of inattention are tested in the
morning and instructed
to take their medication in the afternoon. Subjects better treated with
atypical anti-psychotic
medications indicated for autism are excluded. Subjects are screened for known
genetic disorders
including and those with Fragile X Syndrome or tuberous sclerosis excluded.
Subjects with
uncontrolled epilepsy are excluded.
The study is a double blind placebo-controlled crossover study with three
phases.
Subjects enter each phase of the crossover in a randomized order. In the test
phases, subjects
receive either placebo, 10 mg/kg T.I.D oral G-2-MePE for five days, or 30
mg/kg T.I.D. oral G-2-
MePE. Each phase of the crossover is separated by a washout period of fourteen
days.
Subjects are tested at baseline using the following instruments: Wechsler IQ,
Abberant
Behavior Checklist Community Edition (ABC), Vinelands, Yale-Brown Obsessive
Compulsive
Scale (YBOCS) compulsion subscale, Social Responsiveness Scale (SRS), Clinical
Global
Impression of Severity (CGI-S) and their carers complete the Caregiver Strain
Questionnaire
(CSQ).
Subjects are administered two tasks ¨ the Reading the Mind in the Eyes Test-
Revised
(RMET) and an Eye Tracking (ET) task, as well as Clinical Global Impression of
Improvement
(CGI-I). Tasks commence two hours following administration of placebo or
either dose of G-2-
MePE. The RMET is a computer based task that assesses one's ability to read
emotions from the
eyes of subtle affective facial expressions and is a widely used test of
emotion recognition in
patients with autism (2001). Importantly, the RMET is capable of detecting
improvement with
69
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
even a single dose of a pharmacological agent (Guastella et al., 2010). Eye
tracking issues are
characteristic of patients with autism who spend less time looking at the eyes
of photographs of
human faces. Again, a single administration of a pharmacologic intervention
can ameliorate eye
tracking deficits in autism (Andari et al, 2010).
Adverse events are also recorded using standard safety measures.
Statistically, the effect of treatment with G-2-MePE is analysed by conducting
a repeated
analysis of covariance (ANCOVA) on the effect of treatment on change from
baseline scores.
Results
Treatment with G-2-MePE produces no more adverse events than were present
during
treatment with placebo, with all adverse events being of short duration and
mild severity. No
Serious Adverse Events are reported.
Treatment with G-2-MePE produces a significant overall improvement in
performance of
the RMET test. Treatment with 30 mg/kg T.I.D. oral G-2-MePE increases the
percent correct
responses on the RMET.
Treatment with G-2-Men produces a significant overall improvement in time
spent
looking at the eye region in the ET test. CG1-I scores at the end of treatment
periods show a
significant difference. Positive treatment effects are correlated with
baseline CSQ scores.
Conclusions
Treatment with G-2-MePE produces significant improvements in performance in
the
Reading the Mind in the Eyes Test ¨ Revised, and in performance of an Eye
Tracking task. This
effect is dose dependent, seen after treatment 30 mg/kg TAD. oral G-2-MePE.
Improvement in these measures is reflective of an improvement in processing of
social
information processing. Social interaction deficits are a core symptom
diagnostic for autism
spectrum disorders, and this is therefore a key finding.
G-2-MePE also produces an overall improvement in function as indexed by the
Clinical
Global Impression of Improvement. Free text annotation of the Case Report
Forms from the
study indicate this effect related to an improvement in social relatedness.
This implies that the
changes seen in the RMET and ET task may have relevance to social activity in
daily life.
G-2-MePE is well tolerated in this population.
70
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Example 17: Animal Models for Determining Effects of G-2-MePE on Autism
Spectrum
Disorders
Effects of G-2-MePE are further tested in the following genetic models of ASD:
the Tbxl
heterozygous mouse, the Cntnap2 knockout mouse and the S1c9a6 knockout mouse.
G-2-MePE is
also tested in the fmrl knockout mouse model of Fragile X Syndrome.
Tbxl . Mutations of the TBX1 gene are associated with Autism Spectrum
Disorders
(Paylor et al., 2006). Transgenic Tbxl mice are selectively impaired in social
interaction,
ultrasonic vocalization, repetitive behaviors and working memory (Hiramoto et
al., 2011).
Cntnap2. Two-thirds of patients with mutations of the contactin associated
protein-like 2
(CNTNAP2) gene are diagnosed with an Autism Spectrum Disorder (Alarcon et al.,
2008; Arking
et al., 2008; Bakkaloglu et al., 2008; Strauss et al., 2006; Vernes et al.,
2008). Cntnap2 knockout
(KO) mice exhibit ASD-related phenotypes in social behavior, ultrasonic
vocalization and
repetitive behaviors (Penagarikano et al., 2011).
81c9a6. This gene has been implicated in syndromic ASD and encodes the sodim-
hydrigen exchanger 6 (NHE6). Mutations in SLC9A6 are associated with
intellectual disability
(Gilfillan et al., 2008) and autistic behavior (Garbern et al., 2010). On
S1c9a6 KO mice exhibit
motor hyper-activity and cerebellar dysfunction (Stromme et al., 2011).
Fmrl. Silencing of the FMR1 gene produces Fragile X Syndrome, the phenotype
of
which includes autism; two thirds of patients with Fragile X Syndrome meet
screening criteria for
an Autism Spectrum Disorder (Harris et al., 2008). Pediatric patients with
Fragile X Syndrome
also show lowered seizure threshold. The fmr/ knockout mouse replicates much
of the phenotype
of Fragile X Syndrome, including juvenile seizure susceptibility (Yan et al.,
2004).
Methods
Animals in each of the above models are generated in accordance with the
methodology
described in the cited literature. Wild type equivalents are also obtained for
each genetic model.
Animals in each model are divided into three groups (n=10 to n=20): placebo
treated wild type
mice, mutant G-2-MePE-treated group and mutant placebo-treated control group.
The treatments are administered intraperitoneally: placebo (saline) or 20
mg/kg/day of G-2-
MePE.
Measures of key features of ASD as displayed in each model are taken in
accordance with
the cited literature.
Results
G-2-MePE treatment significantly improves all measures associated with the ASD
phenotype.
71
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Example 18: The Effects of G-2-MePE in Fragile-X Syndrome
Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, or Marker X
Syndrome is the most common monogenetic cause of autism and the most common
inherited
cause of mental retardation. It is characterised by a range of intellectual
disabilities, physical
characteristics such as elongated face, large ears and enlarged testes (macro-
orchidism), and a
neurobehavioral phenotype that includes stereotypic movements, social anxiety
and attention-
deficit hyperactivity disorder (ADHD). Symptoms and signs of FXS may include
delays in
crawling, walking or twisting, hand clapping or hand biting, hyperactive or
impulsive behavior,
mental retardation, delays in development of speech and language, tendency to
avoid eye contact,
stereotypic movements, social anxiety, and attention-deficit hyperactivity
disorder (ADHD).
The genetic basis underlying FXS is expansion of the CGG-repeat of the fragile
X mental
retardation 1 gene (FMR1) on the X chromosome. The FMR1 gene produces a
protein called
fragile X mental retardation protein, or FMRP. The FMR1 gene in normal people
contains a
trinucleotide segment (CGG) that is repeated from fewer than 10 times to about
40 times. These
repeated CGG segments are interspersed with other trinucleotide segments
(AGG), which may
stabilize the oligonucleotide encoding FMRP. In affected males, the CGG
trinucleotide may be
repeated from about 200 times to over 1000 times, which makes the
oligonucleotide encoding
FMRP (i.e., FMR1 RNA) unstable. This results in failure to express sufficient
amounts of the
fragile X mental retardation protein (FMRP), which is required for normal
neural development
There is currently no drug treatment that has shown benefit specifically for
Fragile X Syndrome.
FMR/-Knockout Mouse Model of Fragile X Syndrome
Because the genetic basis of FXS is known, it is possible to create animal
systems that are
useful in studying causes and characteristics of FXS. Fmrl gene-knockout
mutant mice are
available, and show many of the features of clinical FXS, including macro-
orchidism, learning
deficits and hyperactivity. Similarly to clinical FXS, the/int-1 knockout
(finr1 KO) model displays
failure in neuronal pruning, showing dendritic supemumeracy and ERK and Akt
hyperphosphorylation. Consequently, finr1 knockout mice represent a valuable
tool for testing
potential drug treatments of FXS in human subjects. We demonstrated that in
fmrl KO mice,
treatment with G-2-MePE is effective in ameliorating many of the signs and
symptoms of FXS.
We investigated the effects of G-2-MePE in the fmr1 knockout mouse model. The
following measures were analysed:
a) Anxiety state,
locornotor behavior and social activity, learning and species-
typical behaviors were assessed using (i) the open field test, (ii) the
successive alley test, (iii) the
elevated plus maze test, (iv) contextual fear-conditioning test and (v) social
behavior test.
72
CA 02929286 2016-04-29
WO 2014/085480 PCT/US2013/072049
Exposure to novel environments typically induces anxiety in rodents. Alongside
punishment and frustrative non-reward, novelty is capable of inducing
behavioral inhibition,
reflective of an internal anxiety state (Gray (1987) Br J Psycho] 69:417-434).
The behavioral
inhibition inherent to anxiety is manifest in rodents as freezing behavior
i.e. a reduction in
locomotor activity or a reduction in ongoing behaviors such as social
interaction. This freezing
behavior parallels the increase in attention and interruption seen during the
experience of anxiety
in human subjects (Gray (1987) Br J Psychol 69:417-434), including Fragile X
Syndrome
patients who are susceptible to anxiety and avoidance (Tranfaglia (2011) Dev
Neurosci. 33:337-
348). Anxiety in rodents is also manifest as a reluctance to remain in exposed
environments such
as the center of an open space or elevated situations (Pellow et al, (1985) .1
Neurosci Methods
14:149-167. Therefore, measurement of locomotor activity or preference for
enclosed spaces can
index anxiety in the open field, successive alleys and elevated plus maze
apparatus.
Typically, this anxiety reduces upon repeated exposure. This reduction in
anxiety is
reflected as increased ongoing behavior and therefore either decreased
freezing or increased
locomotor activity. This decrease in anxiety can be described as a habituation
to the experimental
situation. This habituation reflects a decrease in perception of novelty and
relies upon memory of
prior experience. That is, locomotor activity will change during repeated
exposure to a test
environment as a rodent becomes familiar with that environment. This change
will not occur if
the rodent does not form memory of prior exposure to the environment.
Therefore, habituation to
the open field may be altered if cognitive ability is impaired in rodents.
Rodents will also learn to
associate an environment with presentation of punishment, and upon exposure to
an environment
associated with punishment will show freezing behavior. This response is known
as contextual
fear conditioning, which also relies on the ability to code environmental
information into memory
(Garcia et al (1997) J Neurophysiol. 78:76-81).
b) Anatomical aspects
of the phenotype were also measured, including (i) dendritic
spine density in the hippocampus, and (ii) testis weight.
c) Brain levels of total and phosphorylated ERK and Akt were assayed.
Activation of ERK is reflected in phosphorylation of this intraneuronal
signaling protein. ERK is
not normally revealed as activated by immunohistochemistry in human brain
(Perry et al (1999)
Neuroreport. 10:2411-2415). However, in disorders affecting CNS function, ERK
may be
aberrantly activated. This occurs in Alzheimer disease (Perry et al (1999) and
in autism (Zou et al
(2011) Genes Brain Behav. 10:615-624). Phosphorylation of ERK also occurs in
the brain in
Fragile X Syndrome and in the frnrI knockout mouse model of this disorder
(Wang et al (2012) J
Neurochem. 121:672-679).
Activation of Akt is also seen in pathological states in the brain, such as
Alzheimer
disease (Griffin et al (2005) J Neurochem. 93:105-117) and suicide (Karege et
al (2007) Biol
73
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Psychiatry. 61:240-245). In Fragile X Syndrome, abnormal phosphorylation of
the Akt ¨ mToR
pathway is also seen in the brain (Hoeffer eta! (2012) Genes Brain Behav.
11:332-341).
These findings of abnormal activation of ERK and Alct in human brain in autism
spectrum disorders such as Fragile X Syndrome are reflected in the finri
knockout mouse model
of Fragile X Syndrome, which can therefore be considered to be a validated
model.
Animals
Fmrl knockout (finr 1 KO mice and wild-type litterrnates (Jackson Laboratory)
were
generated on a C57B116.1 background and repeatedly backcrossed onto a C57B116J
background
for more than eight generations. Mice were group housed (4-6 per cage) and all
animals were
provided with ad libitum food and water unless otherwise stated. Mice were
maintained on a 12 h
light/dark cycle (lights off 19:00 to 7:00) in a temperature-controlled
environment (21 1 C).
Tasks were performed in the order described with no more than one task
performed per
day.
Animals were allowed a minimum acclimatization period of one week prior to
commencing the tests. No prophylactic or therapeutic treatment was
administered during the
acclimatization period. Only healthy animals were placed in a study.
During the experiments all mice were tested once in the same apparatus and non-
experimental mice were placed in the apparatus for some minutes before the
experiment. The
apparatus was then cleaned with moist and dry tissues before testing each
mouse. The aim was to
create a low but constant background mouse odor for all experimental subjects.
Experimenters were blind to the genotype and treatment during all testing and
data
analysis.
Experiments were conducted in line with the requirements of the UK Animals
(Scientific
Procedures) Act, 1986.
The animals were divided into the following treatment groups:
1. fmr1 KO + Vehicle (saline)
2. lVt + Vehicle (saline)
3. fmrl KO + G-2-MePE (100 mg/kg in saline)
4. Wt G-2-MePE (100 mg/kg in saline).
G-2-MePE was administered at the dose of 100 mg/kg i.p. in 0.1 ml saline, once
daily for
28 days. Vehicle-treated animals were injected with 0.1 ml saline daily for 28
days. Thereafter,
drug or vehicle treatments were stopped and animals were tested on successive
days until all tests
were completed. Finally, the animals were sacrificed for determining
expression of pAkt and
pERK
74
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
As used in this Example, the terms "0-2-MePE," "NNZ," "NNZ 2566," "NNZ-2566"
and
"2566" have the same meaning and are interchangeable. 0-2-MePE was shown to
have an
unexpectedly long half-life in vivo compared to IGF(1-3) ("GPE") and thus was
considered to be
a good candidate for therapy of animals including human beings with Fragile X
Syndrome.
1. Anatomical Analysis
Hippocampal cell cultures were prepared from wild-type and film/ KO foetal
mice (14 ¨
16 d of gestation). After 3d in vitro, green fluorescent protein (GFP) was
used to monitor
dendritic spine density during time-course of culture (Ethel! and Yamaguchi,
1999; Ethell et al.,
2001, Henkemeyer et al., 2003). Dendritic spines are usually formed between 7
and 14 days in
vitro (DIV). By 14 DIV most dendritic protrusions are spines. We evaluated the
effect of G-2-
MePE, a positive control (the mGluR5 antagonist 2-methyl-6-(phenylethyny1)-
pyridine or MPEP)
and vehicle controls on dendritic spine density in fmrl KO and WT hippocampal
primary cell
cultures. Fmr1 KO and wild-type cultures were treated at 17 DIV.
Treatment groups:
1. G-2-MePE at 0.5 nM
2. G-2-MePE at 5 nM
3. G-2-MePE at 50 nM
4. MPEP at a concentration of 20 gM
5. MPEP-Vehicle and G-2-MePE ¨vehicle (saline)
We used the compartmentalized culture system, a microfluidic chamber (see FIG.
31A)
which opens the possibility for fast drug testing with the capacity to detect
in vitro drug effects
such as spine morphology, neurite outgrowth and synapse formation. The treated
and untreated
neurons in the compartmentalized chambers can be also being use for western
blot analysis. The
compartmentalised culture is a new approach to evaluate the effect of drugs on
anterograde and
retrograde transport of messengers and proteins relevant to FXS. Other new
possibility that offers
this approach its access into exclusive axon compartment, in this area
irnmunodetection and
treatments are also possible.
Statistical Analysis
Parametric data were analysed using two-way ANOVAs (genotype and sex as
between-
subject factors). For all studies, the data was normally distributed. A p-
value < 0.05 was
considered statistically significant throughout.
75
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
2. Western Blot Analysis of Expression of Phosphorylated ERK and Akt
Phosphorylated Extracellular Signal-Regulated Protein Kinase (ERK) and Protein
Kinase
B (Akt) expression was measured by Western blot analysis in lymphocytes from
fmr1 KO and
wild-type animals administered ether vehicle or G-2MePE (100 mg/kg, i.p.; 28
days) treated in
the behavior experiments as previously described (Lopez Verrilli et al. 2009).
ERK is a classical
MAPK signal transduction protein, responsible for growth factor transduction,
proliferation,
cytokine response to stress and apoptosis. Akt is a key component in the
PI3KJAkt/mTOR
signalling pathway and regulates cellular survival and metabolism by binding
and regulating
many downstream effectors, such as Nuclear Factor-KB (NficB) and Bc1-2 family
proteins.
To conduct the western blots, antibodies were used against Akt (1/1000) (Cell
Signaling),
ERK1/2 (1/2000) (Cell Signaling), antiphospho-Akt (1/1000) and antiphospho-ERK
(1/2000)
(Cell Signaling). Total Ala and ERK 1/2 protein content (phosphorylated) were
evaluated by
blotting membranes with antiphospho-Akt (1/1000) and antiphospho-ERK
antibodies(1/2000)
(Cell Signaling). Akt or ERK phosphorylation was normalized to protein content
in the same
sample and expressed as % of change with respect to basal conditions,
considering basal levels as
100%. Protein loading was evaluated by stripping and re-blotting membranes
with b-actin
antibody (1/1000) (Sigma Chemical Co.).
3. Behavioral Tests
a. Open Field Test
The open field is a grey PVC enclosed arena 50 x 30 cm divided into 10 cm
squares. Mice
were brought to the experimental room 5-20 min before testing. A mouse was
placed into a corner
square facing the corner and observed for 3 min. The number of squares entered
(whole body) and
rears (both front paws off the ground, but not as part of grooming) were
counted. The latency to the
first rearing (rear) was also noted. The movement of the mouse around the
field was recorded with
a video tracking device for 300s (vNT4.0, Viewpoint). The latency for the
mouse to enter the
brightest, central part of the field total time spent in this central region,
and total activity (in terms
of path length in centimeters), were recorded in each trial, the initial
exposure, 10 mm after the
initial exposure and 24h after the initial exposure.
During exposure to the open field chamber mice habituated to the environment
and thus
explored less, decreasing the amount movement shown over time. Movement and
rearing were
recorded during the 3 exposures. Failures to reduce locomotion or rearing at
10 minutes and 24
hours indicated deficits in short and long term memory, respectively.
76
CA 02929286 2016-04-29
WO 2014/085480 PCT/US2013/072049
Results
Time travelled
Vehicle-treated fmrl KO mice moved a greater distance in the open field in
comparison
to the vehicle-treated wild-type group, demonstrating hyperactivity in the
fmrl KO animals. The
hyperactivity was reversed in the fmrl KO animals by administration of G-2-
MePE. G-2-MePE
was observed to significantly reduce movement in the 2"d and 3rd trials in
fmrl knockout mice,
suggesting improved memory retention of the test environment, as well as
reducing rearing
overall in these animals (i.e. attenuating hyperactivity) (FIG. 25).
These effects of G-2-MePE treatment were substantial and statistically
significant, and
were completely unexpected based on the prior art.
Rearing Activity
Vehicle-treated Awl KO mice showed increased rearing activity in comparison to
the
vehicle-treated wild-type controls. G-2-MePE had no effect in wild-type mice,
but significantly
reduced rearing activity in fmrl KO mice. The fmrl KO mice did not show
habituation at 10
minutes, which is an indication of a short term memory deficit. During both
short term and long
term memory tests, G-2-MePE treated fmrl KO mice did not differ from wild-type
+ vehicle
controls. (FIG. 26).
These effects of G-2-MePE treatment were substantial and statistically
significant, and
were completely unexpected based on the prior art.
b. Successive Alleys Test
The successive alleys test was used to assess the effects of G-2-MePE on
anxiety in the
fmrl KO mice. The apparatus consists of four successive, increasingly
anxiogenic, linearly
connected alleys (each succeeding alley is painted a lighter colour, has lower
walls and/or is
narrower than the previous alley). Animals were placed at the closed end of
alley 1, facing the
end wall. The latency to first enter each alley, the amount of time spent in
each alley, and the
number of entries into each alley was recorded during a total test time of 300
s.
Results
Wild-type mice spent most time in Alley 1, and progressively less time in
subsequent
alleys. Fmrl KO mice were hyperactive and spent time in all alleys. In this
case the lack of choice
of alley is likely to relate to the hyperactive nature of the FXS model mice.
Treatment with 0-2-
MePE reinstated the normal pattern of behavior in film/ knockout animals,
consistent with
reversing the behavioral phenotype of FXS (FIG. 27).
77
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
These effects of G-2-MePE treatment were substantial and statistically
significant, and
were completely unexpected based on the prior art.
c. Elevated Plus Maze
The Elevated Plus Maze (EPM) is a further test system for examining anxiety
and
locomotor activity. The EPM was built according to the description of Lister
(Lister 1987).
Briefly, the maze consisted of two open and two closed arms opposite each
other in a plus shape,
raised above floor height. The open arms were more exposed and therefore more
anxiogenie.
Wild-type mice therefore spent more time in the closed arms and visited them
more. Mice were
tested for 5 min and their behaviour was recorded. Measures taken included
time spent in the
arms and the center of the maze, and number of arm entries.
Results
Compared to wild type mice, fmr1 knockout mice showed a pattern of behavior
that
combined hyperactivity and cognitive impairment. Surprisingly, fmr1 knockout
mice showed a
slight reduction in the total number of arm entries (indicative of lower
activity) (FIG. 28A), and a
clear increase in the ratio of entries made into the open arms as a percentage
of the total arm
entries (FIG. 28B). This behavioral phenotype was not observed in fmr1
knockout animals given
G-2-MePE (FIGs. 28A and 28B). Indeed, the 'percent open arm entries' measure
was completely
normalised by drug treatment. Time spent in the center (where open or closed
arm entry choice
is made), is known to be reduced by treatment with anxiolytic compounds, such
as
benzodiazepines. Vehicle-treated fmr./ knockout mice showed a very marked
increase in the time
spent in the center (FIG. 28C). This may therefore reflect indecision over
which arm to enter,
and be indicative of cognitive deficits in the mutant animals. The extra time
spent in the centre
space (FIG. 28C) presumably underlies the reduced number of total arm entries.
Time spent in
the centre was normalised by 28-days G-2-MePE treatment.
These effects of G-2-MePE treatment were substantial and statistically
significant and
were completely unexpected based on the prior art.
d. Contextual Fear Conditioning
Contextual fear conditioning is the most basic of the conditioning (learning)
procedures.
It involves placing the animal in a novel environment (dark chamber),
providing an aversive
stimulus (a 1-sec electric shock at 0.2 mA to the paw), and then removing it.
When the animal is
returned to the same environment, it generally will demonstrate a freezing
response if it
remembers and associates that environment with the aversive stimulus. Freezing
is a species-
specific response to fear and is defined as absence of movement except for
respiration.
78
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
The clinical manifestation of Fragile X Syndrome is mental retardation in
which learning
and memory may be profoundly impaired. We tested G-2-MePE and vehicle treated
fmrl KO and
wild-type animals in a fear-conditioning task in which we measured freezing
behavior in response
to context. In these tests the mice had to learn and remember an association
between an aversive
experience (a foot shock) and environmental cues (a dark chamber). Before the
test, we observed
the mice for their reactions to a I-sec foot shock at 0.2 mA, and we noted
vocalizations, jumping
behavior, and excessive running in both genotypes as evidence that they had
detected the
stimulus.
Results
Vehicle-treated flair] KO mice showed a significantly lower percentage of
freezing
behavior on exposure to conditioned context compared to wild-type mice. When
treated with G-2-
MePE, the fmr1 KO group did not show significant differences in freezing
behavior when
compared to G-2-MePE treated wild-type group. We conclude that G-2-MePE
treatment
normalizes the deficit seen in contextual fear conditioning in find KO mice
model. We also
concluded that G-2-MePE decreased the percent of freezing behavior in fmrl KO
animals
compared to vehicle, in= a statistically significant fashion (**), reflecting
a reversal of a cognitive
deficit seen in fmrl knockout mice. This effect of G-2-MePE treatment was
completely
unexpected (FIG. 29).
These effects of G-2-MePE treatment were substantial and completely unexpected
based
on the prior art.
e. Social Behavior
Mice are a social species, which engage in easily scored social behaviors
including
approaching, following, sniffing, allogrooming, aggressive encounters, sexual
interactions,
parental behaviors, nesting and sleeping in a group huddle. In the present
studies, social
recognition and social memory in mice were evaluated by assessing the amount
of time spent
sniffing a novel mouse upon repeated exposures, to induce familiarity, and
reinstatement of high
levels of sniffing when a novel stimulus animal is introduced.
Results
Bouts of Sniffing
Fmrl knockout mice displayed heightened sniffing of the presented mouse (*** p
<0.001
versus vehicle-treated wild-type controls) (FIG. 30A), suggesting a
dysfunction in social
behavior. This effect was nullified by 28-day treatment with G-2-MePE (100
mg/kg, i.p.). G-2-=
MePE had no significant effect in the wild-type group. Sniffing behavior of G-
2-MePE treated
79
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
fmrl KO animals was not significantly different to G-2-MePE-treated wild-type
group. We
conclude that G-2MePE normalized the abnormalities in social behavior seen in
fmrl KO mice.
We also found that G-2-MePE treated fmr1 KO animals showed substantially and
statistically
significantly less sniffing than vehicle treated fmrl KO animals. These
effects of G-2-MePE were
substantial, statistically significant, and completely unexpected based on the
prior art (FIG. 30A).
Species Typical Behaviors
Mice spontaneously dig in many substrates in the laboratory. This behavior
comes from
their ancestry in the wild, where they would forage for seeds, grain, insects,
and other food to be
found buried in the soil or leaf litter in their natural habitat. It exploits
a common natural rodent
behavior, provides quantitative data under controlled laboratory conditions,
and has proved
extremely sensitive to prion disease, Fragile X, strain differences, and brain
lesions. We tested the
effects of G-2-MePE on both wild-type and the fmrl KO group's nesting and
marble burying
behavior.
Results
FIG. 30B and 30C show that fmrl KO mice engaged in significantly less marble
burying
(FIG. 30A) and nest building (FIG. 30B) than wild-type mice. The strong
impairment produced
by hippocampal lesions on nesting and marble burrowing, all of which are
species-typical
behaviors, complements the impairments in spatial learning and memory which
are well-
established symptoms in FXS patients. G-2-MePE had no significant effect in
wild-type mice.
After G-2-MePE administration to finrl KO mice, this group was not
significantly different to the
G-2-MePE ¨ treated wild-type group. We conclude that G-2-MePE treatment
normalized marble
burying behavior in fmrl KO animals, The same profile of effect was seen in
nest building
behavior. We also found that in fmrI KO animals G-2-MePE increased both marble
burying and
nest building behaviors.
These effects of G-2-MePE treatment were substantial and were completely
unexpected
based on the prior art.
4. Dendritic Spines Increase in fmrl KO Mouse Neurons in Vitro
Dendritic spine numbers are increased in fmrl KO mice compared to normal, wild-
type
animals. To determine whether G-2-MePE could be useful in reversing this
abnormality, Fmrl
KO mouse neurons were cultured in microfluidic chambers and GFP was used to
visualize
morphology. We observed that neurons from fmr 1 KO animals, when treated with
G-2-MePE at
0.5 nM, did not show a significant improvement (mean SD, of n = 3
independent
experiments: 0.33 0.07), and nor at 5 nM (0.29 0.05) when compared to
control cultures (0.26
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
0.08). Interestingly, we did observe significant dendritic spine reduction
when G-2-MePE was
added into culture at 50 nM (0.26 0.10) when compare to WT controls (0.28
0.09). These
values are significantly different (p<0.005) by pairwise post hoc analyses,
and were completely
unexpected based on the prior art.
5. Macro-Orehidism
Fmrl knockout mice showed an increase in testis size and weight, as do human
Fragile X
Syndrome patients (macro-orchidism). We measured testis weight in WT-vehicle
and 0-2-MePE
injected and fmrl KO-vehicle and G-2-MePE-treated animals. Macro-orchidism was
evident in
fmrl KO mice compared to WT liftermate animals showing a 12-28% increased
testis weight.
Average weights of testes in wild-type mice vehicle treated was 18 mg, in fmrl
knockout mice-
vehicle treated it was 22.47 mg (p< 0.001). In G-2-MePE-treated WT mice, the
average testis
weight was 18.36 mg and thefmrI KO G-2-MePE-treated mice the testis weight was
significantly
reduced to 18.7. Supporting our observation of testis reduction in the FXS
mice after 0-2-MePE
treatment, it was previously published by Storto (2001) that GpI mGluR RNAs
are abundantly
expressed in the testicles, with high levels of both mGluR5 and mGluR1
expression in the
seminiferous tubuli and germ cells, although their function there is not
known. However, it is
interesting to note that there are contradictory reports of reduction in macro-
orchidism reduction
in FXS mice after treatment with mGluR antagonist, (even coming from a same
laboratory)
indicating that at present the role of mGluR in the testis it is not well
understood. Regardless of
the mechanism of action, we found that G-2-MePE was effective in reversing
marco-orchidism in
fi-mi KO animals.
G-2-MePE had little effect on testis weight in wild-type mice. However, G-2-
MePE
administration significantly reversed the increase in testis weight seen in
fmrl knockout mice
(FIG. 32) This effect of G-2-MePE was substantial, statistically significant,
and completely
unexpected based on the prior art.
5. Ras-Mek-ERK and Akt Expression
Neurons are critically influenced by Fragile X Mental Retardation Protein,
which
regulates local dendritie translation through phosphatidylinositol 3-kinase-
Akt-mammalian target
of rapamycin (mTOR) and Ras¨ERK signalling cascades and implicated in the
mGluR5
signalling cascade. To determine if these pathways are affected by G-2-MePE
treatment, after the
behavioural tests were completed (12 days after the last injection of G-2-
MePE), the animals were
sacrificed, and expression of pERK and pAlct were measured. Here we
demonstrated that after
four weeks treatment of fmrl KO mice by 0-2-MePE (100 mg/kg) affected the
levels of
phosphorylated ERK1/2 pathway proteins as assayed by semi quantitative
phosphospecific
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
western bloting. Quite unexpectedly, this effect was observed in animals 12
days after the last
treatment with G-2-MePE was administered.
Total ERK expression was not significantly affected by either frnr./ knockout
or drug
treatment (FIGs. 33C and 33D), although an increase in ERK expression levels
was observed in
find KO G-2-MePE treated mice (p< 0.05).
ERK. activation, as indexed by levels of phosphorylated ERK (pERK), was
increased in
vehicle-treated find knockout mice compared to wild-type controls (FIGs. 33A
and 33B). Four
weeks treatment with G-2-MePE significantly reduced the elevated pERK in the
brains of the
find KO mice when compared to the find KO vehicle treated mice. Western blots
were
normalized to the amounts of GAPDH protein present. Elevated ERK I /2
phosphorylation
(Thr202/Tyr204) in find KO-vehicle treated mice was significantly reduced by
treatment with G-
2-MePE (p<0.05).
As previously reported on individuals with Fragile X Syndrome, we observed
that the
levels of phosphorylated Akt, a protein that activates mTOR signalling, and
eIF4F, another
translation initiator that is activated by mTOR, were significantly elevated
in the brains of the
vehicle treated fmr./ KO mice compared with vehicle treated wild-type mice.
Overall we observed
a significant reduction in Akt phosphorylation when compared to total Akt in
fmrl KO mice
treated with G-2-MePE (p<0.05) (FIGs. 34A and 34B). This result of 0-2-MePE
treatment was
substantial, statistically significant, and completely unexpected based on the
prior art.
Conclusions
In conclusion, the described study in find knockout mice ¨ a model of Fragile
X
Syndrome with excellent face validity ¨ has clearly demonstrated numerous
phenotypic changes
compared to wild-type mice that are all reversed with treatment with G-2-MePE,
as summarized
below.
= Hyperactivity:
¨ find KO mice showed hyperactivity in the open field, successive alleys,
and
elevated plus maze tests. This hyperactivity was reversed by 0-2-MePE.
Overall,
these data are consistent with the other behavioral testing data obtained and
with
key deficits shown in Fragile X Syndrome patients. These abnormalities were
reversed by treatment with G-2-MePE.
= Learning:
¨ find KO mice showed a deficit in habituation (short term memory) which
was
reversed by G-2-MePE treatment.
82
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
¨ fmrl KO mice showed a learning deficit in contextual fear conditioning,
which
was reversed by G-2-MePE.
= Social behaviors and complex species typical behaviors:
- fmrl KO mice showed abnormal social interactions, which were reversed by
G-2-
MePE;
¨ fmrl KO mice showed abnormalities in marble burying and next building,
which
were reversed by G-2-MePE.
= CNS and peripheral morphology and molecular pathways:
- Hippocampal neurons from fmr1 KO mice showed increased numbers of
dendritic spines in vitro, an effect that was reversed by G-2-MePE; and
¨ G-2-MePE reversed the abnormal activation of ERK and Akt in brains of
finrl
KO mice.
= Macro-orchidism:
¨ fmr1 KO control mice showed macro-orchidism, which was reversed by G-2-
MePE.
Because the signs and symptoms of mice with fmr1 deficiency mirror effects
seen in
human beings with Fragile X Syndrome (FXS), and because the underlying genetic
defect in finrl
KO mice is the same as in human beings with FXS, we conclude that these
results are highly
predictive of effects in human beings with Fragile X Syndrome.
We conclude that the effects of 0-2-MePE observed in the finrI knockout
animals were
likely due to the drug's ability to induce normal neuronal and synaptic state
in the animals. This
conclusion is supported by the findings that: (1) 0-2-MePE had a plasma half-
life in vivo of about
minutes, (2) that the normalized behavioral tests of the finrI knockout
animals was observed
long after the G-2-MePE had presumably been completely washed out of the
animals bodies by
25 the time the behavioral tests were carried out, and (3) the measurements
of expression of pAlct
and pERK were performed long after the G-2-MePE had presumably been washed
out.
Therefore, we conclude that G-2-MePE can be an effective treatment for human
beings
suffering from Fragile X Syndrome and other autism spectrum disorders.
30 References
The following references and all patents, patent applications and other
publications cited
herein are incorporated fully by reference.
Alarcon,M., Abrahams,B.S., Stone,J.L., Duvall,J.A., Perederiy,J.V.,
Bomar,J.M., Sebat,J.,
Wigler,M., Martin,C.L., Ledbetter,D.H., Nelson,S.F., Cantor,R.M., and
Geschwind,D.H. (2008).
83
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Linkage, association, and gene-expression analyses identify CNTNAP2 as an
autism-
susceptibility gene. Am. J. Hum. Genet. 82, 150-159.
Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett
syndrome is
caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.
Nat Genet.
1999 23:185-188
Andari E, Duhamel JR, Zalla T, Herbrecht E, Leboyer M, Sirigu A. (2010)
Promoting social behavior with oxytocin in high-functioning autism spectrum
disorders.
PNAS 107:4389-4394
Arking,D.E., Brune,C.W., Teslovich,T.M., West,K., Ikeda,M., Rea,A.,
Guy,M.,
Lin,S., Cook,E.H., and Chakravarti,A. (2008). A common genetic variant in the
neurexin
superfamily member CNTNAP2 increases familial risk of autism. Am. J. Hum.
Genet. 82, 160-
164.
Balckaloglu,B., O'Roak,B.J., Louvi,A., Gupta,A.R., Abelson,J.F., Morgan,T.M.,
Chawarska,K.,
Klin,A., Ercan-Sencicek,A.G., Stillman,A.A., Tanriover,G., Abrahams,B.S.,
Duvall,J.A.,
Robbins,E.M., Geschwind,D.H., Biederer,T., Gunel,M., Lifton,R.P., and State MW
(2008).
Molecular cytogenetic analysis and resequencing of contactin associated
protein-like 2 in autism
spectrum disorders.
Bakkaloglu,B., O'Roak,B.J., Louvi,A., Gupta,A.R., Abelson,J.F., Morgan,T.M.,
Chawarska,K.,
Klin,A., Ercan-Sencicek,A.G., Stillman,A.A., Tanriover,G., Abrahams,B.S.,
Duvall,J.A.,
Robbins,E.M., Geschwind,D.H., Biederer,T., Gunel,M., Liftori,R.P., and State
MW (2008).
Molecular cytogenetic analysis and resequencing of contactin associated
protein-like 2 in autism
spectrum disorders. Am. J. Hum. Genet. 82, 165-173.
Baron-Cohen S. Wheelwright S, Hill J, Raste Y, Plumb 1(2001) The "Reading the
Mind in the
Eyes" test, revised version: A study with normal adults, and adults with
Asperger's syndrome or
high-functioning autism. J Child Psycho! Psychiatry 42:241-251.
Belichenko PV, Oldfors A, Hagberg B, Dahlstrom A. Rett syndrome: 3-D confocal
microscopy
of cortical pyramidal dendrites and afferents. Neuroreport. 1994 5:1509-1513
84
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Biederer T, Sara Y, Mozhayeva M, Atasoy D, Liu X, Kavalali ET, Stidhof IC.
(2002) SynCAM,
a synaptic adhesion molecule that drives synapse assembly. Science 297(5586):
1525-1531.
Chapleau CA, Larimore JL, Theibett A, Pozzo-Miller L. (2009) Modulation of
dendritic spine
development and plasticity by BDNF and vesicular trafficking: fundamental
roles in
neurodevelopmental disorders associated with mental retardation and autism. J.
Neurodev.
Disord. 1: 185-196.
Cheng CM, Mervis RF, Niu SL, Salem N Jr, Witters LA, Tseng V, Reinhardt R,
Bondy CA.
Insulin-like growth factor I is essential for normal dendritic growth. J
Neurosci Res. 2003 73:1-9
Comery TA, Harris 113, Willems PJ, Oostra BA, Irwin SA, Weiler IJ, Greenough
WT. (1997)
Abnormal dendritic spines in fragile X knockout mice: maturation and pruning
deficits.
Proc. Nati Acad. Sci. USA 94: 5401-5404.
Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, Nygren
G, Rastam
M, Gillberg IC, Anckarsater H, Sponheim E, Goubran-Botros H, Delorme R,
Chabane N,
Mouren-Simeoni MC, de Mas P, Bieth E, Rage B, Heron D, Burglen L, Gillberg C,
Leboyer M,
Bourgeron T. (2007) Mutations in the gene encoding the synaptic scaffolding
protein SHANK3
are associated with autism spectrum disorders. Nat Genet. 39: 25-27.
Etherton MR, Blaiss CA, Powell CM, Siidhof TC. (2009) Mouse neurexin- la
deletion causes
correlated electrophysiological and behavioural changes consistent with
cognitive impairments.
Proc. Nat. Acad. Sci. 106: 17998-18003.
Garbern,J.Y., Neumann,M., Trojanowski,J.Q., Lee,V.M., Feldman,G.,
Friez,M.J.,
Schwartz,C.E., Stevenson,R., and Sima,A.A. (2010). A mutation affecting the
sodium/proton
exchanger, SLC9A6, causes mental retardation with tau deposition. Brain 133,
1391-1402.
Gauthier J, Bonnel A, St-Onge J, Karemera L, Laurent S. Mottron L, Fombonne E,
Joober R,
Rouleau GA. (2005) NLGN3/NLGN4 gene mutations are not responsible for autism
in the
Quebec population. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 132B(1): 74-
75.
Gilfillan,G.D., Selmer,K.K., Roxrud,I., Smith,R., Kyllerman,M., Eiklid,K.,
Kroken,M.,
Mattingsdal,M., Egeland,T., Stenmark,H., Sjoholm,H., Server,A., Samuelsson,L.,
Christianson,A., Tarpey,P., Whibley,A., Stratton,M.R., Futreal,P.A.,
Teague,J., Edkins,S.,
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Geez,J., Turner,G., Raymond,F.L., Schwartz,C., Stevenson,R.E., Undlien,D.E.,
and Stromme,P.
(2008). SLC9A6 mutations cause X-linked mental retardation, microcephaly,
epilepsy, and ataxia,
a phenotype mimicking Angelman syndrome, Am. J. Hum. Genet. 82, 1003-1010.
Gilman SR, Iossifov I, Levy D, Ronemus M, Wigler M, Vitkup D. Rare de novo
variants
associated with autism implicate a large functional network of genes involved
in formation and
function of synapses. Neuron. 2011 70:898-907
Giza J, Urbanski MJ, Preston i F, Bandyopadhyay B, Yam A, Friedrich V, Kelley
K, D'Angelo E,
Goldfarb M. (2010) Behavioural and cerebellar transmission deficits in mice
lacking autism-
linked gene Islet Brain-2. J. Neurosci. 30: 14805-14816.
Guastella AJ, EinfeId SL, Gray KM, Rinehart NJ, Tonge BJ, Lambert TJ, Hickie
IB. (2010)
Intranasal oxytocin improves emotion recognition for youth with autism
spectrum disorders.
Biol Psychiatry. 67:692-694
Hagerman R, Hoem G, Hagerman P. (2010) Fragile X and autism: Intertwined at
the molecular
level leading to targeted treatments. Mol. Autism 1: 12-24.
Harris SW, Hess! D, Goodlin-Jones B, Ferranti J, Bacalman S, Barbato I,
Tassone F, Hagerman
PJ, Herman H, Hagerman RJ. (2008) Autism profiles of males with fragile X
syndrome. Am J
Merit Retard. 113:427-438.
Hiramoto T, Kang G, Suzuki G, Satoh Y, Kucherlapati R, Watanabe Y, Hiroi N.
(2011) Tbxl:
identification of a 22q11.2 gene as a risk factor for autism spectrum disorder
in a mouse model.
Hum Mol Genet. 2011 20:4775-4785.
Hutsler JJ, Zhang H. Increased dendritic spine densities on cortical
projection neurons in autism
spectrum disorders. Brain Res. 2010 1309:83-94
Irwin SA, Galvez R, Greenough WT. Dendritic spine structural anomalies in
fragile-X mental
retardation syndrome. Cereb Cortex. 2000 10:1038-1044
Jarnain S, Quach H, Betancur C, Rastam M, Colineaux C, Gillberg IC, Soderstrom
H, Giros B,
Leboyer M, Gillberg C, Bourgeron T; Paris Autism Research International
Sibpair Study. (2003)
86
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are
associated with
autism. Nat. Genet. 34: 27-29.
Jamain S, Radyushkin K, Hammerschmidt K, Granon S, Boretius S, Varoqueaux F,
Ramanantsoa
N, Gallogo J, Ronnenberg A, Winter D, Frahm J, Fischer J, Bourgeron T,
Ehrenreich H, Brose N.
(2008) Reduced social interaction and ultrasonic communication in a mouse
model of monogenic
heritable autism. Proc. Nat. Acad. Sci. 105: 1710-1715.
Kim HG, Kishikawa S, Higgins AW, Seong IS, Donovan DJ, Shen Y, Lally E, Weiss
LA, Najm J,
Kutsche K, Descartes M, Holt L, Braddock S, Troxell R, Kaplan L, VoIkmar F,
Klin A, Tsatsanis
K, Harris DJ, Noens I, PauIs DL, Daly MI, MacDonald ME, Morton CC, Quade BJ,
Gusella JF.
(2008) Disruption of neurexin 1 associated with autism spectrum disorder. Am.
J. Hum. Genet.
82: 199-207.
Klemmer P, Meredith RM, Holmgren CD, Klychnikov OI, Stahl-Zeng I, Loos M, van
der Schors
RC, Wortel J, de Wit H, Spijker S, Rotaru DC, Mansvelder HD, Smit AB, Li KW.
Proteomics,
ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome
mouse model
reveal presynaptic phenotype. J Biol Chem. 2011 286:25495-25504
Krueger DD, Osterweil EK, Chen SP, Tye LD, Bear MF. (2011) Cognitive
dysfunction and
prefrontal synaptic abnormalities in a mouse model of fragile X syndrome.
Proc. Natl Acad. Sci.
USA 108: 2587-2592.
Lauterborn JC, Rex CS, Kmmar E, Chen LY, Pandyarajan V. Lynch G, Gall CM.
(2007) Brain-
derived neurotrophic factor rescues synaptic plasticity in a mouse model of
fragile X syndrome. J.
Neurosci. 27: 10685-10694.
Lintas C, Persico AM. (2009) Autistic phenotypes and genetic testing: state-of-
the-art for the
clinical geneticist. J. Med. Genet. 46: 1-8.
Makkonen I, Kokki H, Kuikka J, Turpeinen U, Riikonen R. Effects of fluoxetine
treatment on
striatal dopamine transporter binding and cerebrospinal fluid insulin-like
growth factor-I in
children with autism. Neuropediatrics. 2011 42:207-209
87
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Marchetto et al. (2010) A model for neural development and treatment of Rett
syndrome
using human induced pluripotent stem cells. Cell 143:527-539 (incl.
supplemental
information)
Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, Skaug J, Shago M, Moessner
R, Pinto D,
Ren Y, Thiruvahindrapduram B, Fiebig A, Schreiber S, Friedman J, Ketelaars CE,
Vos YJ,
Ficicioglu C, Kirkpatrick S, Nicolson R, Sloman L, Summers A, Gibbons CA,
Teebi A, Chitayat
D, Weksberg R, Thompson A, Vardy C, Crosbie V. Luscombe S, Baatjes R,
Zwaigenbaum L,
Roberts W, Fernandez B, Szatmari P, Scherer SW. (2008) Structural variation of
chromosomes in
autism spectrum disorder. Am J Hum Genet. 82: 477-488.
Minshew NJ, Williams DL. The new neurobiology of autism: cortex, connectivity,
and neuronal
organization. Arch Neurol. 2007 64:945-950
Moessner R, Marshall CR, Sutcliffe JS, Skaug J, Pinto D, Vincent J,
Zwaigenbaum L, Fenandez
B, Roberts W, Szatmari P, Scherer SW. (2007) Contribution of SHANK3 mutations
to autism
spectrum disorder. Am. J. Hum. Genetics 81: 1289-1297.
Moretti P, Levenson JM, Battaglia F, Atkinson R, Teague R, Antalffy B,
Armstrong D, Arancio
0, Sweatt JD, Zoghbi HY. (2006) Learning and memory and synaptic plasticity
are impaired in a
mouse model of Rett syndrome. J. Neurosci. 26: 319-327.
Paylor,R., Glaser,B., Mupo,A., Ataliotis,P., Spencer,C., Sobotka,A.,
Sparks,C., Choi,C.H.,
Oghalai,J., Curran,S., Murphy,K.C., Monks, S., Williams,N., O'Donovan,M.C.,
Owen,M.J.,
Scambler,P.J., and Lindsay,E. (2006). PNAS 103, 7729-7734.
Penagarikano,O., Abrahams,B.S., Herrnan,E.I., Winden,K.D., Gdalyahu,A.,
Dong,H.,
Sonnenblick,L.I., Gruver,R., Almajano,J., Bragin,A., Golshani,P.,
Trachteriberg,J.T., Peles,E., and
Geschwind,D.H. (2011). Absence of CNTNAP2 Leads to Epilepsy, Neuronal
Migration
Abnormalities, and Core Autism-Related Deficits. Cell 147, 235-246.
Riikonen R, Makkonen I, Vanhala R, Turpeinen U, Kuikka J, Kokki H. (2006)
Cerebrospinal
fluid insulin-like growth factors 1GF-1 and IGF-2 in infantile autism. Dev.
Med. Child Neurol. 48:
751-755.
Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, Yamrom B,
Yoon S, Krasnitz
A, Kendall 3, Leotta A, Pai D, Zhang R, Lee YH, Hicks 3, Spence SJ, Lee AT,
Puura K,
88
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Lehtimdki T, Ledbetter D, Gregersen PK, Bregman J, Sutcliffe JS, Jobanputra V,
Chung W,
Warburton D, King MC, Skuse D, Geschwind DH, Gilliam IC, Ye K, Wigler M.
(2007) Strong
association of de novo copy number variation mutations with autism. Science
316(5823): 445-
449.
Schaevitz LR, Moriuchi JIM, Nag N, Mellot TJ, Berger-Sweeney J. (2010)
Cognitive and social
functions and growth factors in a mouse model of Rett syndrome. Physiol.
Behav. 100: 255-263.
Schtitt J, Falley K, Richter D, Kreienkamp HJ, Kindler S. (2009) Fragile X
mental retardation
protein regulates the levels of scaffold proteins and glutamate receptors in
postsynaptic densities.
J. Biol. Chem. 284: 25479-25487.
Silverman JL, Turner SM, Barkan CL, Tolu SS, Saxena R, Hung AY, Sheng M,
Crawley TN:
Sociability and motor functions in Shank! mutant mice. Brain Res 2010.
Silverman JL, Yang M, Lord C, Crawley JN: Behavioural phenotyping assays for
mouse models
of autism. Nat Rev Neurosci 2010, 11:490-501
Spence Si, Schneider MT. The role of epilepsy and epileptiform EEGs in autism
spectrum
disorders. Pediatr Res. 2009 65:599-606.
Spencer CM, Alekseyenko 0, Serysheva E, Yuva-Paylor LA, Paylor R. (2005)
Altered anxiety-
related and social behaviors in the Fmrl knockout mouse model of fragile X
syndrome. Genes
Brain Behav. 4: 420-430.
Strauss,K.A., Puffenberger,E.G., Huentelman,M.J., Gottlieb,S., Dobrin,S.E.,
Parod,J.M.,
Stephan,D.A., and Morton,D.H. (2006). Recessive symptomatic focal epilepsy and
mutant
contactin-associated protein-like 2. N. Engl. J. Med. 354, 1370-1377.
Stromme,P., Dobrenis,K., Sillitoe,R.V., Gulinello,M., Ali,N.F., Davidson,C.,
Micsenyi,M.C.,
Stephney,G., ElIevog,L., Klungland,A., and Walkley,S.U. (2011). X-linked
Angelman-like
syndrome caused by S1c9a6 knockout in mice exhibits evidence of endosomal-
lysosomal
dysfunction. Brain. 134:3369-3383.
Sykes NH, Toma C, Wilson N, Volpi EV, Sousa I, Pagnamenta AT, Tancredi R,
Battaglia A,
Maestrini E, Bailey AJ, Monaco AP; International Molecular Genetic Study of
Autism
89
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Consortium (IMGSAC). (2009) Copy number variation and association analysis of
SHANK3 as a
candidate gene for autism in the IMGSAC collection. Eur. J. Hum. Genet. 17:
1347-1353.
Tabuchi K, Blundell J, Etherton MR, Hammer RE, Liu X, Powell CM, Stidhof TC.
(2007) A
neuroligin-3 mutation implicated in autism increases inhibitory synaptic
transmission in mice.
Science 318(5847): 71-76.
Takayanagi Y, Fujita E, Yu Z, Yamagata T, Momoi MY, Momoi T, Onaka T. (2010)
Impairment
of social and emotional behaviors in Cadm I -knockout mice. Biochem. Biophys.
Res. Commun.
396: 703-708.
Tropea D, Giacometti E, Wilson NR, Beard C, McCurry C, Fu DD, Flannery R,
Jaenisch R, Sur
M. (2009) Partial reversal of Rett Syndrome-like symptoms in MeCP2 mutant
mice. Proc. Nati
Acad. Sci. USA 106: 2029-2034.
Vemes,S.C., Newbury,D.F., Abrahams,B.S., Winchester,L., Nicod,J., Groszer,M.,
Alarcon,M.,
Oliver,P.L., Davies,K.E., Geschwind,D.H., Monaco,A.P., and Fisher,S.E. (2008).
A functional
genetic link between distinct developmental language disorders. N. Engl. J.
Med. 359, 2337-2345.
Yan J, Noltner K, Feng J, Li W, Schroer R, Skinner C, Zeng W, Schwartz CE,
Sommer SS.
(2008) Neurexin lalpha structural variants associated with autism. Neurosci
Lett. 438: 368-370.
Yan QJ, Asafo-Adjei PK, Arnold HM, Brown RE, Bauchwitz RP. (2004) A phenotypic
and
molecular characterization of the fmrl-tmlCgr fragile X mouse. Genes Brain
Behav. 3:337-359.
Yang M, Crawley IN: Simple behavioural assessment of mouse olfaction. CUIT
Protoc Neurosci
2009, Chapter 8(Unit 8):24.
Zhiling Y, Fujita E, Tanabe Y, Yamagata T, Momoi T, Momoi MY. (2008) Mutations
in the gene
encoding CADM1 are associated with autism spectrum disorder. Biochem. Biophys.
Res.
Commun. 377: 926-929.
Zhao MG, Toyoda H, Ko SW, Ding HK, Wu LJ, Zhuo M. (2005) Deficits in trace
fear memory
and long-term potentiation in a mouse model for fragile X syndrome. J.
Neurosci. 25: 7385-7392.
(Erratum in: J Neurosci. 2005, 25: 8112)
CA 02929286 2016-04-29
WO 2014/085480
PCT/US2013/072049
Zoghbi HY. (2005) MeCP2 dysfunction in humans and mice. J Child Neural. 20:
736-740.
91
