[0001] The present disclosure relates generally to the field of pharmaceuticals.
Specifically, the present disclosure is directed to a compound SMND-309 for neuroprotective effect against MPTP-induced Parkinson‟s disease via PI3-kinase/Akt pathway.
BACKGROUND OF THE INVENTION
10 [0002] Background description includes information that may be useful in
understanding the present invention. It is not an admission that any of the information
provided herein is prior art or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0003] Parkinson‟s disease (PD) is the most prevalent neurodegenerative disease
15 manifested by the degeneration of dopaminergic neurons in the substantia nigra pars
compacta (SNpc) and a remarkable reduction of striatal dopamine primarily among the
geriatric people around the world (Thomas et al., 2007). It is a progressive neurodegenerative
disorder characterized by neuronal cell death in the specific brain region like basal ganglia,
cerebral cortex and hippocampus. The genesis of this neuronal loss is unclear, but there is
20 increasing evidence suggesting that oxidative stress, via free radical production and apoptosis
plays a crucial role in the process (Owen et al., 1996). Other potential reasons of neuronal
damage are protein aggregation, protein misfolding, excitotoxicity and the loss of trophic
support associated with rigidity, akathesia, tremor, postural imbalance, cognitive and memory
dysfunctions like symptoms (Yacoubian et al., 2009). Pathological hallmarks are
25 dopaminergic neuronal degeneration, neuro-inflammation, oxidative stress and free radical
generation. Current PD treatment is symptomatic and primarily involves pharmacological
dopamine agonist therapies such as levodopa and with drugs that increase the dopamine
supply by inhibiting catechol-O-methyl transferase (COMT) and monoamine oxidase B
(MAO-B).
30 [0004] Protein phosphorylation is a specifically regulated post translational
modification that influences nearly all neuronal processes. A balance is normally regulated
through the actions of multiple kinases and phosphatases. The PI3K- PKB/Akt pathway is
highly conserved, tightly controlled and a multistep signalling cascade termed
phosphoinositide 3-kinases (PI3Ks) has been proven time and time again to facilitate crucial
3
cellular dynamics viz. survival, proliferation and differentiation. PI3Ks critically operate
downstream of receptor tyrosine kinases (RTKs) and G protein coupled receptors (GPCRs)
and are responsible for propagating a wide array of signals arising out from numerous growth
factors and cytokines into intracellular communications by generating phospholipids, which
5 in turn activate the serine/threonine kinase AKT and several other effector pathways (Zang et
al., 2012). PI3Ks can be divided into three classes based on their structural physiognomies
and substrate specificity; of these, the most commonly investigated are the class I enzymes
that are activated directly by cell surface receptors. Class I PI3Ks can further be segmented
into class IAs that are activated by RTKs, GPCRs and oncogenes like the small G protein
10 Ras, and class IBs, that are entirely moderated by GPCRs. Activated receptors can directly
trigger class 1A PI3Ks bound via their regulatory subunit or adapter molecules like the
insulin receptor substrate (IRS) proteins. Once it is activated, class I PI3Ks generates three
phospholipids PI (3, 4, 5) which serves as a secondary messenger, driving multiple effector
pathways influencing key cellular processes. For the full activation of AKT, the
15 phosphorylation within the Carboxyl-terminal regulatory domain (Ser 473 in AKT1) of AKT
by PDK2 is required. Once activated, AKT moves to the cytoplasm and nucleus, where it
phosphorylates, activates, or inhibits many downstream targets to regulate various cellular
functions (Griffin et al., 2005). AKT inhibits the GTPase activating protein (GAP) activity of
the tuberous sclerosis complex 1 (TSC1) and TSC2 complex by phosphorylating TSC2
20 tuberin protein, leading to the accumulation and activation of the mTOR complex (kitagishi
et al., 2014). The mTOR mediates the phosphorylation of the ribosomal protein S6 kinases
and eukaryotic translation initiation factor 4E-binding protein 1 leading to the release of the
translation initiation factor eIF4E. Suppression of the PI3K/AKT/mTOR signaling modulates
PARKIN expression (Klinkeberg et al., 2012), which is an ubiquitin ligase involved in PD.
25 [0005] Glycogen synthase kinase 3 (GSK-3) is also a serine/threonine kinase that was
initially identified as playing a role in the regulation of glycogen synthesis in response to
insulin receptor stimulation (Jolivalt et al., 2008). This molecule has also been shown to be
involved in cellular proliferation, programmed cell death, embryogenesis and circadian
entrainment, in addition to the regulation of glycogenesis. Phosphatase and tensin homolog
30 (PTEN) is a dual-specificity phosphatase which has protein phosphatase activity and lipid
phosphatase activity that antagonizes PI3K activity (Wang et al., 2012). Dysregulation in the
PI3-kinase/Akt transcriptional cascade play a promising role in neurobiology of Parkinson‟s
disease (Wang et al., 2012). The PI3-kinase pathway may be explored for the treatment of
Parkinson‟s induced by multiple causes.
4
[0006] The inventors of the present disclosure have studied, experimented and arrived
at the neuro-protective effects of the compound, SMND-309 against MPTP-induced
Parkinson‟s disease.
5 OBJECTS OF THE INVENTION
[0007] An object of the present disclosure is to provide a therapeutic or prophylactic
treatment of MPTP-induced Parkinson‟s disease.
[0008] An object of the present disclosure is to provide a compound, SMND-309, for
neuro-protective effects against MPTP-induced neurotoxicity.
10 [0009] Another object of the present disclosure is to provide a compound to alleviate
the degeneration of dopamine caused by MPTP.
[0010] An object of the present disclosure is to provide a compound that significantly
alleviates MPTP-induced lewy bodies and impairment in motor functions such as
bradykinesia, stiffness and hind limb paralysis.
15
SUMMARY OF THE INVENTION
[0011] This summary is provided to introduce a selection of concepts in a simplified
form that are further described below in Detailed Description section. This summary is not
intended to identify key features or essential features of the claimed subject matter, nor is it
20 intended to be used as an aid in determining the scope of the claimed subject matter.
[0012] The present disclosure is directed to a compound SMND-309 for neuroprotective effect against MPTP-induced Parkinson‟s disease via PI3-kinase/Akt pathway.
[0013] In an aspect of the present disclosure, there is provided a compound, SMND309, for treatment, prevention or amelioration of MPTP-induced Parkinson‟s disease via the
25 PI3k-Akt pathway.
[0014] In an embodiment, the compound SMND-309 is (2E)-2-{6-[(E)-2-
carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid.
[0015] In an embodiment, wherein the compound is administered in the dose range of
about 1 mg/kg to about 100 mg/kg of the body weight per day, preferably from about 1
30 mg/kg to about 10 mg/kg of the body weight per day.
[0016] In an embodiment, the present disclosure relates to a pharmaceutical
composition comprising SMND-309 for the treatment, prevention or amelioration of MPTPinduced Parkinson‟s disease via the PI3k-Akt pathway.
5
[0017] In another embodiment, the present disclosure relates to a pharmaceutical
composition comprising SMND-309 along with a pharmaceutically acceptable excipient for
the treatment, prevention or amelioration of MPTP-induced Parkinson‟s disease.
[0018] In still another embodiment, the present disclosure relates to the use of
5 SMND-309 for the preparation of a medicament for the treatment, prevention or amelioration
of MPTP-induced Parkinson‟s disease.
[0019] These and other features, aspects, and advantages of the present subject matter
will be better understood with reference to the following description and appended claims.
This summary is provided to introduce a selection of concepts in a simplified form. This
10 summary is not intended to identify key features or essential features of the claimed subject
matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0020] The following drawings form part of the present specification and are
15 included to further illustrate aspects of the present disclosure. The disclosure may be better
understood by reference to the drawings in combination with the detailed description of the
specific embodiments presented herein.
[0021] Figure 1: Effect of various treatments on Catalepsy Score. All values are
represented as mean ± standard error of the mean; n = 6, a
represents significant difference (P
< 0.05) compared to vehicle control and b
20 represents significant difference (P < 0.05)
compared to MPTP control. Results were analysed using analysis of variance Tukey‟s posthoc test. a =P<0.05 vs. VEH-VEH control; b =P<0.05 vs. Control; c =P<0.05 vs. MPTP+
SMND-309 Control (10 mg/kg, i.p.); VEH: Vehicle; MPTP: 1-methyl-4-phenyl-1, 2, 3, 6-
tetrahydropyridine hydrochloride.
25 [0022] Figure 2: Effect of various treatments on fall time in seconds in Accelerated
Rotarod Test.
[0023] Figure 3: Effect of various treatments on fall time in seconds in 0.5 mm Bar
Test.
[0024] Figure 4: Effect of various treatments on fall time in seconds in 2 mm Bar
30 Test.
[0025] Figure 5: Effect of various treatments on fall time in seconds in 4 mm Bar
Test.
[0026] Figure 6: Effect of various treatments on locomotor activity in actophotometer
test. All values are represented as mean ± standard error of the mean; n = 6, a
represents
6
significant difference (P < 0.05) compared to vehicle control and b
represents significant
difference (P < 0.05) compared to MPTP control. Results were analysed using analysis of
variance Tukey‟s post-hoc test. a =P<0.05 vs. VEH-VEH control; b =P<0.05 vs. MPTP
Control; VEH: Vehicle; Nec 1: SMND-309.
5 [0027] Figure 7: Effect of various treatments on gripping time in seconds in Hang
Test. All values are represented as mean ± standard error of the mean; n = 6, a
represents
significant difference (P < 0.05) compared to vehicle control and b
represents significant
difference (P < 0.05) compared to MPTP control. Results were analysed using analysis of
variance Tukey‟s post-hoc test. a =P<0.05 vs. VEH-VEH control; b =P<0.05 vs. Control; c
10 =P<0.05 vs. MPTP+ SMND-309 Control (10 mg/kg, i.p.); VEH: Vehicle; MPTP: 1-methyl4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride.
[0028] Figure 8: Effect of various treatments on Catalase Test.
[0029] Figure 9: Effect of various treatments on TBARS Test.
[0030] Figure 10: Effect of various treatments on GSH Test.
15 [0031] Figure 11: Effect of various treatments on Myeloperoxidase Test.
[0032] Figure 12: Effect of various treatments on Dopamine test.
[0033] Figure 13: Histopathological observation of the mouse brain (HE stain).
Vehicle Control (a), MPTP Control (b), Syndopa (100 mg/kg, p.o) (c) MPTP + SMND-309
(5 mg/kg, i.p.) (d) MPTP + SMND-309 (10 mg/kg, i.p.) (e) MPTP + LY294002 (3mg/kg, i.p)
20 + SMND-309 (10 mg/kg, i.p.).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The following is a detailed description of embodiments of the disclosure. The
embodiments are in such detail as to clearly communicate the disclosure. However, the
25 amount of detail offered is not intended to limit the anticipated variations of embodiments; on
the contrary, the intention is to cover all modifications, equivalents, and alternatives falling
within the spirit and scope of the present disclosure as defined by the appended claims.
[0035] All publications herein are incorporated by reference to the same extent as if
each individual publication or patent application were specifically and individually indicated
30 to be incorporated by reference. Where a definition or use of a term in an incorporated
reference is inconsistent or contrary to the definition of that term provided herein, the
definition of that term provided herein applies and the definition of that term in the reference
does not apply.
7
[0036] Reference throughout this specification to “one embodiment” or “an
embodiment” means that a particular feature, structure or characteristic described in
connection with the embodiment is included in at least one embodiment. Thus, the
appearances of the phrases “in one embodiment” or “in an embodiment” in various places
5 throughout this specification are not necessarily all referring to the same embodiment.
Furthermore, the particular features, structures, or characteristics may be combined in any
suitable manner in one or more embodiments.
[0037] In some embodiments, numbers have been used for quantifying weights,
percentages, dosages, and so forth, to describe and claim certain embodiments of the
10 invention and are to be understood as being modified in some instances by the term “about.”
Accordingly, in some embodiments, the numerical parameters set forth in the written
description and attached claims are approximations that can vary depending upon the desired
properties sought to be obtained by a particular embodiment. In some embodiments, the
numerical parameters should be construed in light of the number of reported significant digits
15 and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges
and parameters setting forth the broad scope of some embodiments of the invention are
approximations, the numerical values set forth in the specific examples are reported as
precisely as practicable. The numerical values presented in some embodiments of the
invention may contain certain errors necessarily resulting from the standard deviation found
20 in their respective testing measurements.
[0038] Various terms as used herein are shown below. To the extent a term used in a
claim is not defined below, it should be given the broadest definition persons in the pertinent
art have given that term as reflected in printed publications and issued patents at the time of
filing.
25 [0039] As used in the description herein and throughout the claims that follow, the
meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates
otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on”
unless the context clearly dictates otherwise.
[0040] Unless the context requires otherwise, throughout the specification which
30 follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising”
are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0041] The recitation of ranges of values herein is merely intended to serve as a
shorthand method of referring individually to each separate value falling within the range.
8
Unless otherwise indicated herein, each individual value is incorporated into the specification
as if it were individually recited herein.
[0042] All methods described herein can be performed in any suitable order unless
otherwise indicated herein or otherwise clearly contradicted by context. The use of any and
5 all examples, or exemplary language (e.g. “such as”) provided with respect to certain
embodiments herein is intended merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No language in the specification
should be construed as indicating any non-claimed element essential to the practice of the
invention.
10 [0043] Groupings of alternative elements or embodiments of the invention disclosed
herein are not to be construed as limitations. Each group member can be referred to and
claimed individually or in any combination with other members of the group or other
elements found herein. One or more members of a group can be included in, or deleted from,
a group for reasons of convenience and/or patentability. When any such inclusion or deletion
15 occurs, the specification is herein deemed to contain the group as modified.
[0044] The description that follows, and the embodiments described therein, is
provided by way of illustration of an example, or examples, of particular embodiments of the
principles and aspects of the present disclosure. These examples are provided for the
purposes of explanation, and not of limitation, of those principles and of the disclosure.
20 [0045] It should also be appreciated that the present disclosure can be implemented in
numerous ways, including as a system, a method or a device. In this specification, these
implementations, or any other form that the invention may take, may be referred to as
processes. In general, the order of the steps of the disclosed processes may be altered within
the scope of the invention.
25 [0046] The headings and abstract of the invention provided herein are for convenience
only and do not interpret the scope or meaning of the embodiments.
[0047] The following discussion provides many example embodiments of the
inventive subject matter. Although each embodiment represents a single combination of
inventive elements, the inventive subject matter is considered to include all possible
30 combinations of the disclosed elements. Thus if one embodiment comprises elements A, B,
and C, and a second embodiment comprises elements B and D, then the inventive subject
matter is also considered to include other remaining combinations of A, B, C, or D, even if
not explicitly disclosed.
9
[0048] The term „therapeutically effective amount‟ generally refers to the amount that
when administered to a subject is sufficient to affect the treatment intended by the drug.
[0049] The present disclosure is directed to a compound SMND-309 for neuroprotective effect against MPTP-induced Parkinson‟s disease via PI3-kinase/Akt pathway.
5 [0050] In an embodiment of the present disclosure, there is provided a compound,
SMND-309, for treatment, prevention or amelioration of MPTP-induced Parkinson‟s disease
via the PI3k-Akt pathway.
[0051] In an embodiment, the compound SMND-309 is (2E)-2-{6-[(E)-2-
carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid. SMND-309
10 may be prepared by conventional methods of preparation, isolation and purification known in
the art. SMND-309, a derivative of salvianolic acid B, has been reported to exert a great
number of neuroprotective effects.
[0052] In an embodiment, the present disclosure provides the compound SMND-309
or its derivatives for treatment, prevention or amelioration of MPTP-induced Parkinson‟s
15 disease via the PI3k-Akt pathway.
[0053] MPTP is 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) known as a
neurotoxicant inducer of Parkinsonism in rodents. MPTP crosses the blood–brain barrier and
is subsequently converted to the neurotoxic metabolite 1-methyl-4-phenylpyridinium
(MPP+), a substrate of the dopamine transporter. MPTP potentiates the lipid peroxidation in
20 substantia nigra and striatum, which may be the result of diminished activities of antioxidant
enzymes, low level of glutathione, dopamine, cytokines and partial loss of tyrosine
hydroxylase (Sauer and Oertel, 1994).
[0054] In an embodiment of the present disclosure, the compound is administered
based on the patient history, body weight and the like. Preferably it may be administered to a
25 subject in the dose range of about 1 mg/kg to about 100 mg/kg of the body weight per day,
more preferably from about 1 mg/kg to about 10 mg/kg of the body weight per day.
[0055] In an embodiment, the compound maybe formulated for administration via
injection or infusion orally, intravenously, intraperitoneally, subcutaneously, topically,
transdermally, intracerebrally or intramuscularly to the subject.
30 [0056] In an embodiment, the subject administered a therapeutically effective amount
may be a mammal, including human and non-human mammals such as dogs, pigs, cats,
horses, rats, monkeys and the like.
[0057] In an embodiment, the compound having neuro-protective activity is used as a
medicament for the treatment, prevention or amelioration of MPTP-induced Parkinson‟s
10
disease. This includes the alleviation of symptoms and side-effects of the disease and relief
from the diseases.
[0058] In an embodiment, the compound improves the neurobehaviour of MPTPinduced Parkinson‟s disease by alleviating the degeneration of dopamine, MPTP-induced
5 lewy bodies and motor function in-coordination.
[0059] In an embodiment, the neuro-protective effect of the compound is attributed to
the suppressed levels of Myeloperoxidase (MPO), thiobarbituric acid reactive substances
(TBARS) and elevated levels of Glutathione (GSH) and catalase in MPTP-induced PD
patients due to downregulation of PI3K/Akt pathway. In an embodiment, the neuro-protective
10 effect is dose dependent.
[0060] In an embodiment, the compound is formulated in the form of a medicament
for the therapeutic or prophylactic treatment of MPTP-induced Parkinson‟s disease.
[0061] In an embodiment, the medicament may be administered as a suspension,
granules, syrups, tablets, powders, sustained release powders, oils, solution, aerosols,
15 nanoparticles or microparticles.
[0062] In an embodiment, the present disclosure relates to a pharmaceutical
composition comprising SMND-309 for the treatment, prevention or amelioration of MPTPinduced Parkinson‟s disease via the PI3k-Akt pathway.
[0063] In another embodiment, the present disclosure relates to a pharmaceutical
20 composition comprising SMND-309 along with a pharmaceutically acceptable excipient for
the treatment, prevention or amelioration of MPTP-induced Parkinson‟s disease.
[0064] In an embodiment, the pharmaceutically acceptable excipient includes but is
not limited to adjuvants, preservatives, pharmaceutically acceptable carriers, wetting agents,
dispersants, sweetening agents, flavouring agents, diluents and the like well known to a
25 person of skill in the art. The pharmaceutically acceptable excipient is safe, non-toxic and
does not interfere with the therapeutic activities of the pharmaceutical active.
[0065] In still another embodiment, the present disclosure relates to the use of
SMND-309 for the preparation of a medicament for the treatment, prevention or amelioration
of MPTP-induced Parkinson‟s disease.
30 [0066] In yet another embodiment, the present disclosure relates to a method of
treatment of a subject with MPTP induced Parkinson‟s disease using SMND-309.
[0067] While the foregoing describes various embodiments of the disclosure, other
and further embodiments of the disclosure may be devised without departing from the basic
scope thereof. The scope of the invention is determined by the claims that follow. The
11
invention is not limited to the described embodiments, versions or examples, which are
included to enable a person having ordinary skill in the art to make and use the invention
when combined with information and knowledge available to the person having ordinary skill
in the art.
5 [0068] The disclosure will now be illustrated with working examples, which is
intended to illustrate the working of disclosure and not intended to take restrictively to imply
any limitations on the scope of the present disclosure. Unless defined otherwise, all technical
and scientific terms used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs. Although methods and materials
10 similar or equivalent to those described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and materials are described
herein. It is to be understood that this disclosure is not limited to particular methods, and
experimental conditions described, as such methods and conditions may vary.
MATERIALS AND METHODS:
15 [0069] All animal experiments were conducted in accordance as per the guidelines of
the Committee for the purpose of control and supervision of experimentation on animals
(CPCSEA). Male Swiss albino mice (25±2g) were housed in animal house exposed to regular
12 hr cycle of light and dark (lights on at 7:00 h and off at 19:00 h), obtained from ISF
College of Pharmacy, Moga, India. Animals were maintained on standard laboratory diet and
20 experiments were conducted in semi– sound proof laboratory. For all behavioral estimations,
mice were brought in experimental room 20 minutes before testing. Each animal was kept
back to the home cage after each motor test so as to allow proper recovery of muscular
strength.
DRUGS AND CHEMICALS:
25 [0070] MPTP, LY294002 (PI3K inhibitor) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA) and SMND-309 from Chem scene, China. The chemicals used were of
analytical grade and all drug solutions were freshly prepared before use.
EXPERIMENTAL DESIGN
[0071] A total of 36 male mice were divided in 6 groups, each group contained 6
30 mice.
Group 1: Vehicle Control Group: To examine the motor co–ordination, neuromuscular
strength on 21st day, Vehicle (Saline-10 ml/kg, i.p.) was administered to mice. The mice were
sacrificed by cervical dislocation on 21st day after the last injection of saline for biochemical
and histopathological analysis.
12
Group 2: MPTP Group (Disease Control Group): Four doses of MPTP (17 mg/kg, i.p.) at
every 2 hour intervals were injected to mice. On the 21st day, after the last injection of MPTP,
motor coordination, neuromuscular strength, biochemical estimations and histopathological
5 analysis were conducted by cervical dislocation of mice. The entire tests were carried out
between 9.00 AM and 18.00 PM.
Group 3: MPTP + Syndopa (Standard drug): After MPTP intoxication mice were
received Levodopa/Carbidopa (100/10 mg/kg/twice/day, p.o.) from day 8 to day 21. Motor
coordination test, neuromuscular strength and biochemical test estimation were performed on
21st 10 day.
Group 4: MPTP + SMND-309 (Low dose): After MPTP intoxication mice were
administered SMND-309 (5 mg/kg, i.p.) from day 8 to day 21. Motor coordination test,
neuromuscular strength and biochemical test estimation were performed on 21st day.
Group 5: MPTP + SMND-309 (High dose): After MPTP intoxication mice were
15 administered SMND-309 (10 mg/kg, i.p.) from day 8 to day 21. Motor coordination test,
neuromuscular strength and biochemical test estimation were performed on 21st day.
Group 6: MPTP + LY294002 + SMND-309 (High dose): After MPTP intoxication mice
were administered LY294002 (3 mg/kg, i.p.) and 1 hour later administered SMND-309 (10
mg/kg, i.p.) was administered from day 8 to day 21. Motor coordination test, neuromuscular
strength and biochemical test estimation were performed on 21st 20 day.
EXAMPLE 1:
INDUCTION OF PARKINSONISM:
[0072] To prepare the mouse model of Parkinson‟s disease with neurotoxic effect on
the nigrostriatal system, MPTP (17 mg/kg, i.p.) total of four doses were injected every 2
25 hours over an 8h period in day with modification. Safety measurements for the use of MPTP
during preparation of chemicals and animal injections were taken as per previously published
procedures (Jackson-Lewis, 2007).
EXAMPLE 2:
BEHAVIORAL ASSESSMENT:
30 2.1 MPTP induced catatonia
[0073] This test was conducted according to the protocol described by (Kulkarni et
al., 1980) with slight modifications. In this test, front paw of the animals was placed on a
horizontal bar located 3 cm above and parallel to the base. The time at which the animal
13
removes its paw from the bar was noted. The maximum cut off time of bar test was fixed at
300 sec.
Stage I: Mouse moves normally when placed on the table, score=0.
Stage II: Mouse moves only when touched or pushed, score=0.5.
5 Stage III: Mouse has marked in-coordination and gait abnormalities, score=1.
Stage IV: Mouse placed on the table with front paws set alternatively on 3 cm high
block fails to correct the posture in 10 sec, score=1 for each paw with a
total of 2 for the stage.
[0074] Thus, for a single mice, the maximum possible score would be (0.5+1+2) 3.5
10 revealing total catatonia.
[0075] Catatonia is one of the primary symptoms in rodents suffering from
Parkinsonism, which has been quantified by catalepsy test. MPTP induces catatonia
significantly (P<0.05), when compared to vehicle control group. Administration of Syndopa
(100 mg/kg, p.o) and SMND-309 (5 & 10 mg/kg, i.p.) significantly and dose dependently
15 (p<0.05) decreased catatonia when compared to MPTP control group. Pre-treatment with
LY294002 (3 mg/kg. i.p.), (Selective inhibitor of PI3-kinase), significantly (p<0.05)
abolished the protective effect of SMND-309 by showing marked increase in catatonia score
(Figure 1).
2.2 Assessment of Motor coordination
20 2.2.1 Assessment of motor coordination by using Rota Rod test
[0076] The Rota rod system (Imcorp Ltd., Ambala) for assessing locomotor skills
measures the time that an animal maintains balance on a moving rod (2.5cm diameter).
General training is employed in different treatment and control groups, animals are trained to
stay on the rods at speed higher than normal walking. When the animal falls, pressure plate is
25 activated yielding a digital read out to mark the maximal duration the rodent remained on the
rod. Speed of the Rota rod was set at 4 rpm, acceleration rate at 20 rpm/min. At 300 seconds,
after placing the mouse on the rod, acceleration was initiated.
[0077] Administration of four doses of MPTP (17 mg/kg, i.p) on day one was
documented to cause motor incoordination in mice which was quantified by falling time in
30 rota rod test. MPTP reduces fall time significantly (P<0.05) when compared to vehicle
control group. Moreover pair wise comparison via ANOVA confirmed that administration of
Syndopa (100 mg/kg, p.o) and SMND-309 (5 & 10 mg/kg, i.p.) significantly and dose
dependently increased (P<0.05) fall time when compared to MPTP control group. Pretreatment with LY294002 (3 mg/kg. i.p.), (Selective inhibitor of PI3-kinase), significantly
14
(p<0.05) abolished the protective effect of SMND-309 by showing marked decrease in fall
time (Figure 2).
2.2.2 Assessment of motor coordination by using Horizontal Bars Test
[0078] To evaluate the fore and hind limb motor co–ordination, horizontal bars test
5 was performed. The mouse was lifted by tail, and was placed on the bench in front of the
apparatus; mouse was slid in reverse rapidly about 20 cm (this adjusts it opposite to the bar),
quickly raised and allowed to hold flat bar with its forepaws just, tail was discharged, at the
same time stopwatch was begun. A few mice may fail to hold better if the tail was discharged
all of a sudden. On the off chance that the mouse failed to hold a handle on the bar
10 legitimately first time, the test was performed again after brief rest and rehashed maximum 3
attempts. Best score was taken. The scoring framework for 2 mm bar was the same all
through the protocol, and last score was recorded. Bar test was performed for add up to 30
seconds (Deacon et al., 2013).
[0079] Administration of MPTP leads to cause motor incoordination in mice which
15 has been quantified by 0.5 MM, 2 MM & 4 MM bar test. MPTP reduces griping time
significantly (P<0.05) when compared to vehicle control group. Moreover pair wise
comparison confirmed that administration of Syndopa (100 mg/kg, p.o) and SMND-309 (5 &
10 mg/kg, i.p.) significantly (P<0.05) and dose dependently increased griping time when
compared to MPTP control group (Figures 3, 4 and 5). Pretreatment with LY294002 (3
20 mg/kg. i.p.), (Selective inhibitor of PI3-kinase), significantly (p<0.05) abolished the
protective effect of SMND-309 by showing marked decrease in griping time on 0.5 MM, 2
MM & 4 MM bars.
2.2.3 Assessment of activity by using Open Field Activity test
[0080] Horizontal and vertical activity as well as the pattern of the activity was
25 measured by animal activity monitor equipment. Locomotor activity can be indicated by the
infrared beam and digital counter (Anstrom et al., 2003). General training was performed for
five minutes. Individually animals were placed and observed in the chamber for five minutes.
Activity was expressed as count per five minutes, the maximum scores on the actometer were
recorded (Anstrom et al., 2003).
30 [0081] Administration of four doses of MPTP (17 mg/kg, i.p.) on day one reduced
locomotor activity significantly (P<0.05) as compared to vehicle control group.
Administration of Syndopa (100 mg/kg, p.o) and SMND-309 (5 & 10 mg/kg, i.p.)
significantly (P<0.05) and dose dependently increased locomotor activity, when compared to
MPTP control group. Pre-treatment with LY294002 (3 mg/kg. i.p.), (Selective inhibitor of
15
PI3-kinase), significantly (p<0.05) abolished the protective effect of SMND-309 by showing
marked decrease in locomotor activity (Figure 6).
2.2.4 Assessment of motor coordination by using Hang Test
[0082] Neuromuscular strength was evaluated by Hang test. Apparatus (Rolex Pvt
Ltd., Ambala) consisted of a horizontal grid mesh (total size: 12 cm2
, openings 0.5 cm2
5 )
mounted 20 cm above the hard surface, thus deterring falling, but not leading to injury in case
of falling. Opaque black plexi glass was used for 3 – inch wall in the apparatus. At 90 degree
angle wall was mounted onto the grid and did not restrict the movement on the grid. To
prevent the injury edges of the grid were blunted. Mice were raised by their tails, slowly
10 placed on a flat grid and supported until they hold the grid with their fore and hind paws later
on inverted the grid so that the mice were allowed to hang upside down. Maximum hanging
time was recorded and animals were required to stay on the grid for 300 seconds.
[0083] Administration of MPTP reduced hang time significantly (P<0.05) as
compared to vehicle control group. Administration of Syndopa (100 mg/kg, p.o) and SMND15 309 (5 & 10 mg/kg, i.p.) significantly (P<0.05) and dose dependently increased hanging time,
when compared to MPTP control group. LY294002 (3 mg/kg. i.p.), (Selective inhibitor of
PI3-kinase) pretreatment significantly (p<0.05) attenuated the protective effect of SMND-309
by showing marked decrease in hanging time (Figure 7).
EXAMPLE 3:
20 BIOCHEMICAL ASSESSMENT
[0084] Animals were sacrificed by cervical dislocation for the biochemical assays.
After removal, the brains were homogenized in a phosphate buffer (pH 7.4, 10% w/v) using a
homogenizer. After centrifugation at 3000 rpm for the time interval of 15 minutes, clear
supernatant was obtained to estimate TBARS, GSH, AChE activity, Catalase and Total
25 protein.
3.1 Estimation of thiobarbituric acid reactive substances (TBARS)
[0085] Addition of 0.2 ml of 8.1 % sodium dodecyl sulphate, 1.5 ml of 30% acetic
acid (pH 3.5), 1.5 ml of 0.8 % thiobarbituric acid to 0.2 ml of pipette supernatant of
homogenate in a test tube and then volume was made up to 4ml with distilled water. Test
30 tubes were incubated for 1 hour at 95 degree Celsius, followed by cooling for half an hour in
chilled water then added 5 ml of distilled water followed by addition of n–butanol–pyridine
mixture (5ml, 15: 1 v/v). At the temperature of 40 – 100 C, tubes were centrifuged at 4000 g
for 15 minutes. Dark pink colour was developed later on; absorbance was measured
spectrophotometrically at 532 nm. Standard calibration was prepared using 1,1,3,3 –
16
tetramethoxy propane (1 – 10 nM) (y = 0.03 x – 0.003; r2
= 0.999). TBARS value was
expressed as nanomoles per mg of protein.
3.2 Estimation of Reduced Glutathione
[0086] Homogenate supernatant was mixed with 10% w/v trichloroacetic acid in 1:1
5 ratio. The tubes were centrifuged at 1000 g for 10 minutes at 4 degree Celsius. 0.5 ml of
obtained supernatant was mixed with 0.3 M, 2ml of disodium hydrogen phosphate. 0.001 M
freshly prepared DNTB [5, 5‟-dithiobis (2-nitro benzoic acid) dissolved in 1 % w/v sodium
citrate] was added and with the help of spectrophotometer absorbance was noted at 412 nm.
Standard curve of reduced glutathione was plotted using 10 - 100µM of reduced form of
glutathione. Results were calculated using equation (y = 0.001 x + 0.002; r2
10 = 0.999). The
values were expressed as micromoles of reduced glutathione per mg of protein (Bhatia et al.,
2003).
3.3 Estimation of Catalase activity
[0087] 50 µl of supernatant was added to 3.0 ml cuvette which contained 1.95 ml of
15 50 mM phosphate buffer (pH 7.0). 30 mM hydrogen peroxide (H2O2) was added and changes
in absorbance were noted at 240 nm for 30 seconds at 15 seconds interval. The activity of
catalase was calculated using milli molar extinction coefficient of H2O2 (0.071 mM cm – 1
)
and was expressed as micromoles of H2O2 oxidized per minute per milligram protein. The
activity of catalase was calculated using formula
20
CAT activity = δ OD
E x Volume of Sample (ml) x total protein (mg)
Where: δ OD is change in absorbance/minutes, E is extinction coefficient of H2O2 (0.071 m
mol cm -1
25 )
3.4 Estimation of Brain Myeloperoxidase (MPO) activity
[0088] At the wavelength of 460 nm MPO activity was measured using
spectrophotometer. Pellet obtained during sample collection was used for MPO activity. 50
mM phosphate buffer (10 ml, pH 6.0) which was ice cold, containing 0.5% hexadecyl
30 trimethyl ammonium bromide (HETAB) and 10 mM EDTA was added to the pellet, followed
by one cycle of freezing and thawing, brief period (15 seconds) of sonication. After that the
solution was centrifuged at 19000 g for 15 minutes. Supernatant (0.1 ml) was combined with
2.9 ml of phosphate buffer containing 0.167 mg/ml o – dianisidine hydrochloride and 0.0005
% H2O2, after that change in absorbance was measured.
17
MPO activity = X
Weight of the piece of tissue taken
5 X = 10 x change in absorbance per minute
Volume of supernatant taken in final reaction (ml)
[0089] The brain homogenate of MPTP treated groups showed increased lipid
peroxidation indicated by significantly (p<0.05) increased levels of TBARS and
10 Myeloperoxidase compared with the vehicle control group. The brain homogenate of animals
of these groups also showed significantly decreased levels of reduced glutathione, catalase
activity when compared with vehicle treated groups. The Syndopa (100 mg/kg, p.o) and
SMND-309 (5 & 10 mg/kg, i.p.) treated groups showed significantly and dose dependently
(p<0.05) amelioration of oxidative stress and by decreasing TBARS and Myeloperoxidase
15 levels and increasing the levels of reduced glutathione, catalase compared to MPTP control
group. Pre-treatment with LY294002 (3 mg/kg, i.p.), (Selective inhibitor of PI3-kinase),
significantly (p<0.05) abolished the protective effect of SMND-309 by showing increased
levels of TBARS, Myeloperoxidase and decreased levels of reduced glutathione, catalase
(Figures 8, 9, 10 and 11).
20 EXAMPLE 4
NEUROCHEMICAL ESTIMATION
4.1 Estimation of dopamine levels in the brain.
[0090] Dopamine was estimated by HPLC method as described by Patel et al.
Dopamine levels were estimated by high-performance liquid chromatography (HPLC) using
25 electrochemical detector. Waters standard system consisting of a high-pressure isocratic
pump, a 20-l manual injector valve, C18 reverse phase column, and electrochemical detector
was used in the study. Mobile phase consisted of sodium citrate buffer (pH4.5)–acetonitrile
(87:13), v/v). Sodium citrate buffer consisted of 10 mM citric acid, 25 mM NaH2PO4, 25 mM
ethylenediaminetetraacetic acid (EDTA), and 2 mM of 1-heptane sulfonic acid.
30 Electrochemical conditions for the experiment were +0.75 V, and sensitivity ranged from 5 to
50 nA. Separation was carried out at a flow rate of 0.8 ml/min. Samples (20 μl) were injected
manually. On the day of experiment, frozen brain hippocampus samples were thawed and
they were homogenized in homogenizing solution containing 0.2 M perchloric acid. After
that, samples were centrifuged at 12,000×g for 5 min. The supernatant was filtered through
18
0.22-m nylon filters before injecting in the HPLC sample injector. Concentrations of
neurotransmitter and their metabolites were calculated from the standard curve generated by
using standard in a concentration range of 10–100 ng/ml.
[0091] The MPTP administered mice showed a significant decrease (p<0.05) in
5 dopamine level in the mice brain homogenate as compared with that of the control group.
Treatment with Syndopa, SMND-309 (10 mg/kg, i.p.) imparted a significant increase
(p<0.05) in dopamine levels dose dependently, when compared to that of MPTP group.
However, LY294002 (3 mg/kg. i.p.) attenuated the protective effect of SMND-309 in terms
of dopamine level in brain (Figure 12).
10 EXAMPLE 5
HISTOPATHOLOGICAL EXAMINATION
[0092] After completion of the above behavior tests, 3 mice in every group were
anesthetized with pentobarbital sodium (50mg/kg, i.p.) and the brains were transcardially
perfused with phosphate buffer solution (PBS, pH 7.4), followed by 4% paraformaldehyde in
15 PBS. The brains were removed and kept overnight in PBS containing 4% paraformaldehyde
at 4oC, and embedded in paraffin. Sections of 5-μm thickness were stained with hematoxylin
and eosin. Hematoxylin is used to stain nuclei blue, while eosin stains cytoplasm and the
extracellular connective tissue matrix pink (Senior et al., 2008). The lesions of brain tissues
were observed with light microscope (NIKON E600, Japan) and the images were collected by
20 image analysis system (Image Pro-Plus 7200, America, SONY 3CCD, Japan).
[0093] Histopathological examinations of brain parts were carried out, which
contained a large number of dopaminergic neuronal tracts and were involved in pathogenesis
and neurobiology of Parkinsonism. Administration of MPTP specifically damaged the
dopamine producing neurons in Substantia Nigra and pars compacta. In the present study
25 MPTP induced Parkinsonism in mice brain clearly demonstrated by the presence of Lewy
bodies viewed under light microscope in comparison to vehicle control group. Syndopa (100
mg/kg, p.o) and SMND-309 (5 & 10 mg/kg, i.p.) significantly and dose dependently (p<0.05)
completely ameliorated MPTP induced defect as seen in the figure 13. Pretreatment with
LY294002 (3 mg/kg. i.p.), (Selective inhibitor of PI3-kinase), significantly (p<0.05)
30 abolished the protective effect of SMND-309 by showing marked increase in Lewis bodies
and degeneration in neurons (Figure 13). Said figure is of Hematoxylin/Eosin (H/E) stained
paraffin sections (100 X H&E) of Vehicle Control (a), MPTP Control (b), Syndopa (100
mg/kg, p.o) (c) SMND-309 (10 mg/kg, i.p.) (d) LY294002 (3 mg/kg. i.p.) + SMND-309 (10
mg/kg, i.p.) (e).
19
STATISTICAL ANALYSIS
[0094] The results were expressed as mean ± standard deviation (S.E.M.). All the
statistical analysis was performed by one-way ANOVA followed by tukey‟s test as post hoc
analysis using Sigma Stat software, USA. A value of P<0.05 was considered to be
5 statistically significant.
[0095] In the present disclosure MPTP was used to induce Parkinson‟s disease(PD) in
mice and the effect of SMND-309 on the induced PD was investigated. Data indicates that
SMND-309 caused neuroprotection in MPTP-induced PD, through PI3K/Akt signaling
pathway. Syndopa, the most widely used drug for PD therapy, contains two active
10 ingredients, levodopa and carbidopa, which contributes to the supplementation of dopamine
in the brain and was used as a positive control drug in the study.
[0096] The results showed significant decrease in the GSH, dopamine, catalase and
increase in the TBARS, MPO level; impairment in the motor functions such as bradykinesia,
stiffness & hind limb paralysis and formation of lewy bodies within substantia nigra pars
15 compacta (SNpc) in MPTP control group. Results from actophotometer test and traction test
showed that SMND-309 improved the neurobehavior of MPTP-treated mice, indicating that
SMND-309 treatment prevented bradykinesia and alleviated the degeneration of dopamine
caused by MPTP. Moreover, SMND-309 treatment significantly alleviated the MPTPinduced lewy bodies (hippocampal damage) as observed in H&E staining, impairment in the
20 motor functions such as bradykinesia, stiffness & hind limb paralysis, suppressed the levels
of MPO, TBARS and elevated the levels of GSH, catalase and dopamine which might be due
to downregulation of PI3K/Akt pathway. This hypothesis has been proved by administering
LY294002, a PI3K inhibitor, which attenuated the effect of SMND-309 on behavioral,
biochemical, neurochemical and histological parameters. Therefore, the data from experiment
25 indicates that SMND-309 improved the neurobehavior and attenuated PD in MPTP-treated
mice through PI3K/Akt signaling pathway.
ADVANTAGES OF THE PRESENT INVENTION
[0097] The present disclosure provides a compound SMND-309 for neuro-protective
30 effect in MPTP-induced Parkinson‟s disease via PI3-kinase/Akt pathway.
[0098] The present disclosure provides a compound that suppresses the levels of
Myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS) and elevated
levels of Glutathione (GSH) and catalase.
20
[0099] The present disclosure provides a compound capable of improving
neurobehaviour of MPTP-induced Parkinson‟s disease by alleviating the degeneration of
dopamine, MPTP-induced lewy bodies and motor function in-coordination.
REFERENCES:
5 1. Thannickal, T.C., Lai, Y.Y. and Siegel, J.M., 2007. Hypocretin (orexin) cell loss in
Parkinson's disease. Brain, 130(6), pp.1586-1595.
2. Owen AD, Schapira AH, Jenner P, Marsden CD. Indices of oxidative stress in
Parkinson‟s disease, Alzheimer‟s disease and dementia with Lewy bodies. InDementia in
Parkinsonism 1997 (pp. 167-173). Springer, Vienna.
10 3. Yacoubian TA, Standaert DG. Targets for neuroprotection in Parkinson's disease.
Biochimica et biophysica acta (BBA)-Molecular basis of disease. 2009 Jul 1;1792(7):676-87.
4. Zhang, L., Ding, W., Sun, H., Zhou, Q., Huang, J., Li, X., Xie, Y. and Chen, J., 2012.
Salidroside protects PC12 cells from MPP+-induced apoptosis via activation of the PI3K/Akt
pathway. Food and chemical toxicology, 50(8), pp.2591-2597.
15 5. Alquézar, C., Barrio, E., Esteras, N., De la Encarnación, A., Bartolomé, F., Molina, J.A.
and Martín‐Requero, Á., 2015. Targeting cyclin D3/CDK 6 activity for treatment of
Parkinson's disease. Journal of neurochemistry, 133(6), pp.886-897.
6. Griffin, R.J., Moloney, A., Kelliher, M., Johnston, J.A., Ravid, R., Dockery, P.,
O'Connor, R. and O'Neill, C., 2005. Activation of Akt/PKB, increased phosphorylation of
20 Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's
disease pathology. Journal of neurochemistry, 93(1), pp.105-117.
7. Kitagishi, Y., Wada, Y. and Matsuda, S., 2014. Roles of PI3K/AKT/PTEN pathway in
the pathogenesis of parkinson‟s disease and the neuropsychiatric symptoms. International
Neuropsychiatric Disease Journal, pp.1-12.
25 8. Klinkenberg, M., Gispert, S., Dominguez-Bautista, J.A., Braun, I., Auburger, G. and
Jendrach, M., 2012. Restriction of trophic factors and nutrients induces PARKIN expression.
Neurogenetics, 13(1), pp.9-21.
9. Jolivalt, C.G., Lee, C.A., Beiswenger, K.K., Smith, J.L., Orlov, M., Torrance, M.A. and
Masliah, E., 2008. Defective insulin signaling pathway and increased glycogen synthase
30 kinase‐3 activity in the brain of diabetic mice: parallels with Alzheimer's disease and
correction by insulin. Journal of neuroscience research, 86(15), pp.3265-3274.
10. Wang, G., Pan, J. and Chen, S.D., 2012. Kinases and kinase signaling pathways:
potential therapeutic targets in Parkinson's disease. Progress in neurobiology, 98(2), pp.207-
221.
21
11. Sauer, H. and Oertel, W.H., 1994. Progressive degeneration of nigrostriatal dopamine
neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined
retrograde tracing and immunocytochemical study in the rat. Neuroscience, 59(2), pp.401-
415.
5 12. Jackson-Lewis, V. and Przedborski, S., 2007. Protocol for the MPTP mouse model of
Parkinson's disease. Nature protocols, 2(1), p.141.
13. Kulkarni, S.K., Arzi, A. and Kaul, P.N., 1980. Modification of drug-induced catatonia
and tremors by quipazine in rats and mice. The Japanese Journal of Pharmacology, 30(2),
pp.129-135.
10 14. Deacon, B.J., 2013. The biomedical model of mental disorder: A critical analysis of its
validity, utility, and effects on psychotherapy research. Clinical psychology review, 33(7),
pp.846-861.
15. Anstrom, K.K., Schallert, T., Woodlee, M.T., Shattuck, A. and Roberts, D.C., 2007.
Repetitive vibrissae-elicited forelimb placing before and immediately after unilateral 6-
15 hydroxydopamine improves outcome in a model of Parkinson's disease. Behavioural brain
research, 179(2), pp.183-191.
16. Senior, S.L., Ninkina, N., Deacon, R., Bannerman, D., Buchman, V.L., Cragg, S.J. and
Wade‐Martins, R., 2008. Increased striatal dopamine release and hyperdopaminergic‐like
behaviour in mice lacking both alpha‐synuclein and gamma‐synuclein. European Journal of
20 Neuroscience, 27(4), pp.947-957.

We Claim:

1. A compound, SMND-309, for treatment, prevention or amelioration of MPTP-induced
Parkinson‟s disease via the PI3k-Akt pathway.
5 2. The compound as claimed in claim 1, wherein SMND-309 is (2E)-2-{6-[(E)-2-
carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid.
3. The compound as claimed in claim 1, wherein MPTP is 1-methyl-4-phenyl-1, 2, 3, 6-
tetrahydropyridine.
4. The compound as claimed in claim 1, wherein the compound is administered to a subject in
10 a dose range of 1 mg/kg to 100 mg/kg of body weight per day.
5. The compound as claimed in claim 1, wherein the compound is administered to a subject in
a dose range of 1 mg/kg to 10 mg/kg of body weight per day.
6. The compound as claimed in claim 1, wherein the compound is formulated for
administration orally, intravenously, intraperitoneally, subcutaneously, topically,
15 transdermally, intracerebrally or intramuscularly.
7. The compound as claimed in claim 1, wherein the compound is administered via injection
or infusion.
8. The compound as claimed in claim 1, wherein the compound is formulated in the form of a
medicament for the therapeutic or prophylactic treatment of MPTP-induced Parkinson‟s
20 disease.
9. The compound as claimed in claim 1, can be administered in a therapeutically effective
amount to a mammal.
10. A pharmaceutical composition comprising the compound as claimed in claim 1, along
with a pharmaceutically acceptable excipient.