[0001] The present disclosure relates generally to the field of pharmaceuticals.
Specifically, the present disclosure is directed to a compound ML-141 for attenuation of
mecamylamine-induced precipitation of nicotine dependence withdrawal syndrome.
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] Nicotine addiction means maladaptive drug-seeking behavior generally
15 performed despite having knowledge of negative health consequences. Nicotine meets the
established criteria for a drug that produces addiction, specifically, dependence and
withdrawal (Chen et al., 2012). Previous studies depict that smoking cessation after nicotine
dependence develops nicotine dependence (ND) induced withdrawal symptoms such as
anxiety, cravings, depression, difficulty in concentrating, impaired performance, increased
20 appetite and weight gain, irritability, frustration and anger, restlessness and impatience, sleep
disturbances such as insomnia or sleeping too much (Kirsten et al., 2012). Approved
Pharmacotherapy of nicotine dependence includes nicotine gums, transdermal nicotine
patches, nicotine nasal spray as nicotine replacement therapy (NRT) (Carpenter et al., 2013),
along with this antidepressant drugs like nortriptyline and bupropion have been approved by
25 the FDA to help people quit smoking (Marc et al, 2008; Hugo et al, 2009). Varenicline
tartrate is another medication that recently received FDA approval for smoking cessation,
which acts at the sites in the brain affected by nicotine and may help people quit by easing
withdrawal symptoms and blocking the effects of nicotine if people resume smoking. Side
effects of pharmacotherapy include sleep disturbance and dry mouth, nausea, headache,
30 insomnia and vivid dreams. Rarely, varenicline has been associated with serious psychiatric
symptoms, such as depressed mood, agitation and suicidal thoughts (Uma et al, 2008).
[0004] Nicotine from tobacco smoke is absorbed rapidly into the pulmonary venous
circulation. It then enters the arterial circulation and moves quickly to the brain. Nicotine
diffuses readily into brain tissue, where it binds to nicotinic acetylcholine receptors
3
(nAChRs), which are ligand-gated ion channels. Whenever a cholinergic agonist binds to the
outside of the channel, the channel opens, allowing the entry of cations, including sodium and
calcium. These cations further activate voltage-dependent calcium channels, leading to
further calcium entry and releasing Dopamine (DA) in dopaminergic neurons in the Ventral
5 Tegmental Area (VTA). Dopamine is involved in information processing, memory, and
emotions in regions such as the nucleus accumbens (NAc), hippocampus, amygdala, and
prefrontal cortex (PFC). Increase in dopamine levels due to Nicotine, within the mesolimbic
system gives rise to rewarding effects. Nicotine also modulates dopamine release indirectly
by binding to nAChRs located on excitatory glutamatergic and inhibitory gamma
10 aminobutyric acid (GABAergic) neurons in the VTA. These glutamatergic and GABAergic
neurons originate from a number of brain areas, such as the NAc, hippocampus, PFC,
amygdala, ventral pallidum, and pedunculopontine tegmental nucleus, and regulate the
activity of dopaminergic neurons resulting in further enhancement of the rewarding effects
(Benowitz, 2008; Souza and Markou, 2011).
15 [0005] Microarray studies on the time–course of acute response to nicotine in mouse
brain identified 95 genes regulated in VTA. Out of these, 30 genes were part of a gene
network, with functions relevant to neural plasticity. Among these the genes RhoA and
Ywhag were selected for human association studies. A synteny search has identified their role
in tobacco smoking and nicotine dependence (ND) in a human case–control association
20 study. Haplotype analyses were negative for the Ywhag gene, whereas for RhoA gene
showed highly significant genotypic association with both smoking initiation (SI) and
nicotine dependence (Chen et al., 2006). It has been reported that the Rho family of small
GTPases comprises some 21 genes in humans, encoding at least 23 signalling proteins. These
proteins have been found to control various neuronal and cellular functions (Wherlock and
25 Mellor, 2002). Regular use of nicotine modulates Rho GTPase signalling that might
contribute to physiological alterations in brain functioning observed in nicotine withdrawal
syndrome. Nicotine activates Rho small GTPase family and its downstream effectors,
including Rho-associated kinase (ROCK) and p21-activated kinase (PAK), are responsible
for forming and organizing the actin cytoskeleton (Clark and Paluc, 2011) which binds with
30 spinophilin and protein phosphatase-1. This association activates a signalling cascade for
dopaminergic neurotransmission through DA receptors into VTA and nucleus accumbens
(Milgram et al., 1999). This activation of dopaminergic tracts further modulates the release of
various neurotransmitters like serotonin, epinephrine and GABA, that plays a prominent role
in nicotine dependence induced withdrawal symptoms. Laminin-induced AChR clustering is
4
also mediated by the activation of the Rho GTPases (Christi et al., 2007). It was studied that
nicotine and psychological stress act upon the hypothalamo-pituitary-adrenal axis, thus leads
release of corticotrophin releasing factor (Cam and Bassett, 1984). This nicotine mediated
activation of CRF-CRF1 is modulated through Rho GTPase and mitogen-activated protein
5 kinase (Jerome et al., 2006), which is directly associated with nicotine dependence. Based
upon these studies it can be hypothesized that Rho GTPase is involved in nicotine
dependence. However, there is a deficiency in the art wherein this association is used for
improving the withdrawal symptoms associated with nicotine dependence.
[0006] Therefore, the inventors of the present invention have, hitherto unexplored,
10 investigated the effect of ML141 on the development of mecamylamine-induced precipitation
of nicotine dependence withdrawal syndrome.
OBJECTS OF THE INVENTION
[0007] An object of the present disclosure is to provide a compound, ML-141, for the
15 treatment, prevention or amelioration of mecamylamine-induced nicotine dependence
withdrawal syndrome.
[0008] An object of the present disclosure is to provide a compound, ML-141, for the
treatment, prevention or amelioration of mecamylamine-induced nicotine dependence
withdrawal syndrome which is affected via the Rho-GTPase protein.
20 [0009] An object of the present disclosure is to provide a compound, ML-141, for the
treatment, prevention or amelioration of mecamylamine-induced nicotine dependence
withdrawal syndrome which leads to a reduction in jumping frequency, withdrawal
symptoms related anxiety, nicotine-induced hyperalgesia, piloerection frequency and body
tremor frequency.
25
SUMMARY OF THE INVENTION
[0010] 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
30 intended to be used as an aid in determining the scope of the claimed subject matter.
[0011] The present disclosure relates generally to nicotine dependence withdrawal
syndrome. Specifically the present disclosure relates to the attenuation of the mecamylamineinduced precipitation of nicotine dependence withdrawal syndrome.
5
[0012] In an aspect, the present disclosure is directed to a compound ML-141 for
attenuation of mecamylamine-induced precipitation of nicotine dependence withdrawal
syndrome.
[0013] In an embodiment, the present disclosure is directed to a compound ML-141
5 for treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome.
[0014] In an embodiment, the present disclosure is directed to a compound ML-141
for treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome wherein the ML-141 affects the therapeutic effect via the
10 Rho-GTPase protein.
[0015] In an embodiment, the compound ML-141 for treatment, amelioration or
prevention of mecamylamine-induced precipitation of nicotine dependence withdrawal
syndrome wherein ML-141 is 4-[4,5-dihydro-5-(4-methoxyphenyl)-3-phenyl-1H-pyrazol-1-
yl]-benzenesulfonamide.
15 [0016] In an embodiment, the compound ML-141 for treatment, amelioration or
prevention of mecamylamine-induced precipitation of nicotine dependence withdrawal
syndrome may be administered in a therapeutically effective amount to a subject.
[0017] In an embodiment, the subject may be a mammal, including but not limited to,
humans, rats, dogs, cats, pigs or cows.
20 [0018] In an embodiment, the present disclosure provides a compound ML-141 for
treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome wherein the compound may be administered
intraperitoneally, orally, intracerebrally, intramuscularly, intravenously, subcutaneously or
transdermally .
25 [0019] In an aspect, the present disclosure relates to a composition comprising the
compound ML-141 for treatment, amelioration or prevention of mecamylamine-induced
precipitation of nicotine dependence withdrawal syndrome along with a pharmaceutically
acceptable excipient.
[0020] In another aspect, the present disclosure relates to the use of a compound ML30 141 for treatment, amelioration or prevention of mecamylamine-induced precipitation of
nicotine dependence withdrawal syndrome.
[0021] 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
6
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
5 [0022] The following drawings form part of the present specification and are
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.
[0023] Figure 1: Effect of treatment(s) on mecamylamine induced increase in
10 withdrawal severity score in nicotine dependent/ vehicle treated mice. Doses employed in the
study were as follows: vehicle (10% DMSO in water, 10 ml/kg, i.p.), nicotine (2.5 mg/kg,
s.c.) was administered four times daily, mecamylamine (3mg/kg, i.p.), ML-141 (1, 5, 10
mg/kg, i.p.) [Values are mean ± S.E.M.] a =P<0.05 vs. VEH-VEH control; b =P<0.05 vs.
NIC-VEH-MEC] NIC: Nicotine; SAL: Saline; VEH: Vehicle; MEC: Mecamylamine.
15 [0024] Figure 2: Effect of treatment(s) on mecamylamine induced increase in
jumping frequency in nicotine dependent/ vehicle treated mice.
[0025] Figure 3: Effect of treatment(s) on mecamylamine induced hyperalgesia in
nicotine dependent/ vehicle treated mice.
[0026] Figure 4: Effect of treatment(s) on mecamylamine induced piloerection in
20 nicotine dependent/ vehicle treated mice.
[0027] Figure 5: Effect of treatment(s) on mecamylamine induced anxiety like
behaviour in nicotine dependent/ vehicle treated mice in terms of time spent in open arm in
elevated plus maze test.
[0028] Figure 6: Effect of treatment(s) on mecamylamine induced body tremor in
25 nicotine dependent/ vehicle treated mice.
DETAILED DESCRIPTION OF THE INVENTION
[0029] 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
30 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.
[0030] All publications herein are incorporated by reference to the same extent as if
each individual publication or patent application were specifically and individually indicated
7
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.
5 [0031] 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
throughout this specification are not necessarily all referring to the same embodiment.
10 Furthermore, the particular features, structures, or characteristics may be combined in any
suitable manner in one or more embodiments.
[0032] In some embodiments, numbers have been used for quantifying weights,
percentages, dosages, and so forth, to describe and claim certain embodiments of the
invention and are to be understood as being modified in some instances by the term “about.”
15 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
and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges
20 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
in their respective testing measurements.
25 [0033] 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.
[0034] As used in the description herein and throughout the claims that follow, the
30 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.
8
[0035] Unless the context requires otherwise, throughout the specification which
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.”
[0036] The recitation of ranges of values herein is merely intended to serve as a
5 shorthand method of referring individually to each separate value falling within the range.
Unless otherwise indicated herein, each individual value is incorporated into the specification
as if it were individually recited herein.
[0037] 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
10 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.
15 [0038] 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
20 occurs, the specification is herein deemed to contain the group as modified.
[0039] 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.
25 [0040] 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.
30 [0041] The headings and abstract of the invention provided herein are for convenience
only and do not interpret the scope or meaning of the embodiments.
[0042] 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
9
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.
5 [0043] 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.
[0044] The present disclosure relates generally to nicotine dependence withdrawal
syndrome. Specifically the present disclosure relates to the attenuation of the mecamylamineinduced precipitation of nicotine dependence withdrawal syndrome.
10 [0045] In an embodiment, the present disclosure is directed to a compound ML-141
for attenuation of mecamylamine-induced precipitation of nicotine dependence withdrawal
syndrome.
[0046] In an embodiment, the present disclosure is directed to a compound ML-141
for treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
15 dependence withdrawal syndrome.
[0047] In an embodiment, the present disclosure is directed to a compound ML-141
for treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome wherein the therapeutic effect of ML-141 is via the RhoGTPase protein. ML-141 is a GTPase inhibitor.
20 [0048] In an embodiment, the present disclosure provides a compound ML-141 for
treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome wherein ML-141 chemically is 4-[4,5-dihydro-5-(4-
methoxyphenyl)-3-phenyl-1H-pyrazol-1-yl]-benzenesulfonamide. Said reference to the
compound also includes pharmaceutically acceptable salts or derivatives of the compound
25 ML-141. This would include salts of the compound that are non-toxic and possess the
pharmaceutical activity of the parent compound.
[0049] In an embodiment, the compound ML-141 may be synthesised by any process
or technique known in the art.
[0050] In an embodiment, the source for nicotine dependence may be those well
30 known in the art, primarily including cigarettes or tobacco.
[0051] In an embodiment, the compound ML-141 for treatment, amelioration or
prevention of mecamylamine-induced precipitation of nicotine dependence withdrawal
syndrome may be administered in a therapeutically effective amount to a subject. This
10
therapeutically effective amount may be determined based on the subject‟s condition, body
weight and other factors.
[0052] In an embodiment, the compound ML-141 for treatment, amelioration or
prevention of mecamylamine-induced precipitation of nicotine dependence withdrawal
5 syndrome may preferably be administered in a dose range of about 1 mg/kg to about 100
mg/kg of the body weight, more preferably the dose range is about 1 mg/kg to about 10
mg/kg of the body weight. ML-141 shows dose dependent attenuation within this range of 1
to 10 mg/kg.
[0053] In an embodiment, the subject may be a mammal, including humans and non10 humans, for example, humans, rats, dogs, cats, pigs and cows.
[0054] In an embodiment, the present disclosure provides a compound ML-141 for
treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome wherein the compound may be administered
intraperitoneally, orally, intracerebrally, intramuscularly, intravenously, subcutaneously or
15 transdermally.
[0055] In an embodiment, the compound may be administered in the form of a
capsule, tablet, gel, powder, solution, aerosol, suspension, microparticle, sustained release
particles or nanoparticles.
[0056] In an embodiment, the present invention provides a compound ML-141 for
20 treatment, amelioration or prevention of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome wherein the compound is used for the preparation of a
medicament.
[0057] In an embodiment, the administration of ML-141 for treatment, amelioration
or prevention of mecamylamine-induced precipitation of nicotine dependence withdrawal
25 syndrome maybe done as a supplementary treatment along with other treatments provided to
a subject. ML-141 may be supplemented with a nicotine patch, nicotine nasal sprays or
nicotine gum for gradual cessation of nicotine dependence.
[0058] Further, it may also be supplemented with an alternative therapeutically
effective drug for mecamylamine-induced precipitation of nicotine dependence withdrawal
30 syndrome. ML-141 may be administered along with other pharmaceutically active drugs that
do not affect the activity of ML-141; this may include for example, drugs for depression,
hypertension, blood dilution, blood coagulation, insomnia and the like.
11
[0059] In an embodiment, the present disclosure relates to a composition comprising
the compound ML-141 for treatment, amelioration or prevention of mecamylamine-induced
precipitation of nicotine dependence withdrawal syndrome.
[0060] In an embodiment, the present disclosure relates to a composition comprising
5 the compound ML-141 for treatment, amelioration or prevention of mecamylamine-induced
precipitation of nicotine dependence withdrawal syndrome along with a pharmaceutically
acceptable excipient.
[0061] In an embodiment, the pharmaceutically acceptable excipient may be selected
from those well known in the art, including dispersants, binders, preservatives, stabilizers,
10 colouring agents, flavouring agents, sugars, diluents and the like. The excipients are
chemically unreactive with ML-141.
[0062] In another embodiment, the present disclosure relates to the use of a compound
ML-141 for treatment, amelioration or prevention of mecamylamine-induced precipitation of
nicotine dependence withdrawal syndrome.
15 [0063] In an embodiment, the present disclosure relates to the use of a compound
ML-141 in the form of a medicament for the attenuation of mecamylamine-induced
precipitation of nicotine dependence withdrawal syndrome.
[0064] In yet another embodiment, the present disclosure relates to the method of
treatment of mecamylamine-induced precipitation of nicotine dependence withdrawal
20 syndrome by administering to a subject in need thereof, a therapeutically effective amount of
the compound, ML-141.
[0065] 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
25 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.
[0066] The disclosure will now be illustrated with working examples, which is intended
30 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
similar or equivalent to those described herein can be used in the practice of the disclosed
12
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.
5 MATERIALS AND METHODS:
[0067] Swiss albino male mice weighing 25±2g obtained from Central Research
Institute, Kasauli, India, maintained on standard laboratory diet (Kisan Feeds Ltd., Mumbai,
India) and having free access to tap water were employed in the present study. They were
housed in the departmental animal house and were exposed to a regular 12 hr cycle of light
10 and dark. The experiments were conducted in a semi-sound proof laboratory. The observer
was blind to the treatment group assignment. The experimental protocol was approved by the
institutional animal ethical committee and care of the animals was done as per the guidelines
of Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA), Ministry of Environment and Forests, Government of India (Reg. No.
15 1181/ab/08/CPCSEA).
DRUGS AND CHEMICALS
[0068] Mecamylamine hydrochloride, ML-141 and nicotine tartrate (Sigma-Aldrich
Chemicals Pvt. Ltd., St. Louis, USA), were dissolved / diluted in normal saline & DMSO.
The chemicals used were of analytical grade and all drug solutions were freshly prepared
20 before use. The doses of nicotine employed in the present study of nicotine were calculated
in terms of weight of tartrate salt. Mecamylamine hydrochloride was dissolved in normal
saline prior to dosing.
EXAMPLE 1: INDUCTION OF NICOTINE WITHDRAWAL SYNDROME IN MICE
[0069] Sub-acute administration of nicotine followed by a single injection of
25 mecamylamine was used to induce nicotine withdrawal in mice (Rehni et al., 2012; Damaj et
al., 2003; Biala and Weglinska, 2005; Singh et al., 2013 a, b, Singh et al., 2016). Nicotine
dependence was induced by repeated s.c. injections, four times daily, at an interval of 4 h
starting at 06:00 a.m, for 7 days (2.5 mg/kg, s.c). The control groups were treated with saline
following the same schedule. On the test day (day 8), mice received one nicotine injection in
30 the morning. To precipitate nicotine abstinence mice were given the nicotine receptor
antagonist mecamylamine (3 mg kg−1, i.p.), 1 h after the last nicotine injection. The somatic
signs of withdrawal were evaluated for 30 min, immediately after mecamylamine
administration. The observations were made in a transparent perspex observation chamber
with dimensions of 30 cm X 30 cm X 30 cm.
13
Experimental protocol
[0070] Seven groups were employed in the present study, with each group comprising
of 08 animals out of which half were males and half females.
Group I (Vehicle-vehicle control): Vehicle (Saline, 10 ml/kg, i.p.) for Nicotine was
5 administered four times daily for a period of seven days. Vehicle (10% DMSO in water, 10
ml/kg, i.p.) for ML 141 was simultaneously injected once daily for the same period of seven
days. Vehicle (10 ml/kg, i.p.) for Mecamylamine was then injected on the morning of day 8,
1 hr after administering vehicle (Saline, 10 ml/kg, i.p.) for nicotine.
Group II (Vehicle-mecamylamine control): Vehicle (Saline, 10 ml/kg, i.p.) for Nicotine was
10 administered four times daily for a period of seven days. Vehicle (10% DMSO in water, 10
ml/kg, i.p.) for ML 141 was simultaneously injected once daily for the same period of seven
days. Mecamylamine (3 mg/kg, i.p.) was then injected on the morning of day 8, 1 hr after
administering vehicle (Saline, 10 ml/kg, i.p.) for nicotine.
Group III (Nicotine–Mecamylamine control): Nicotine (2.5 mg/ kg, s.c.) was administered
15 four times daily for a period of seven days. Vehicle (10% DMSO in water, 10 ml/kg, i.p.) for
ML 141 was simultaneously injected once daily for the same period of seven days.
Mecamylamine (3 mg/kg, i.p.) was then injected on the morning of day 8, 1 hr. after
administering Nicotine (2.5 mg/ kg, s.c.)
Group IV (ML 141 treatment + Vehicle- Mecamylamine control): Vehicle (Saline, 10 ml/kg,
20 i.p.) for nicotine was administered four times daily for a period of 7 days. ML 141 (10 mg/kg,
i.p.) was simultaneously injected once daily for the same period of 7 days. Mecamylamine (8
mg/kg, i.p.) was then injected on the morning of day 8, 1 hr. after administering vehicle
(Saline, 10 ml/kg, i.p.) for Nicotine.
Group V-VII (ML 141 treatment + nicotine-mecamylamine): nicotine (2.5 mg/ kg, s.c.) was
25 administered four times daily for a period of seven days. ML 141 (at a dose level of 1,5 and
10 mg/kg/d, i.p. for groups number V, VI, and VII, respectively) was simultaneously injected
once daily for the same period of seven days. Mecamylamine (3 mg/kg, i.p.) was then
injected on the morning of day 8, 1 hr. after administering nicotine (2.5 mg/ kg, s.c.).
EXAMPLE 2: ASSESSMENT
30 Statistical analysis
[0071] All the results were expressed as mean ± standard error of mean (S.E.M.).
Data of the results was analyzed using ANOVA followed by post-hoc comparison using
Sheffe‟s multiple range test. For elevated plus maze, the numbers of entries and time (in
seconds) spent in both arms was compared in saline- and nicotine-treated mice. A value of
14
P<0.05 was considered to be statistically significant. The statistical analysis was done using
Sigma Stat 6.0 software.
2.1 Effect of various treatments of ML-141 on mecamylamine induced alteration in
withdrawal severity score in nicotine dependent mice.
5 [0072] A set of withdrawal severity score was employed to quantitate the magnitude
of withdrawal syndrome in mice in terms of the earlier reported characteristic behavioral
patterns seen in mice suffering from experimental nicotine withdrawal syndrome viz.,
grooming, scratching, chewing, fore paw tremor, wet dog shake, cage scratching, head
nodding, paw licking, all in a composite manner (Rehni et al., 2012; Damaj et al., 2003; Biala
10 and Weglinska, 2005; Singh et al., 2013 a, b; Singh et al., 2016). The severity of the
withdrawal phenomenon was graded on a scale of 0–24 (normal score, 0; maximal
withdrawal severity score, 24). In each of the individual behavioural components of severity
scores of withdrawal, 0 score point is awarded for no change in the normal behaviour of mice
with respect to each observation criteria, 1 score point is awarded for a mild increase in the
15 respective observation criteria in mice, 2 score point is awarded for a moderate increase in the
respective observation criteria in mice, 3 score point is awarded for a severe increase in the
respective observation criteria in mice. Thus, the higher the score, the more severe is the
withdrawal syndrome. The test was performed immediately after mecamylamine
administration and the results were based on observations spanning first 30 minutes.
20 [0073] Administration of four doses of nicotine (2.5 mg/kg, s.c) for a period of 7
days, followed by a single injection of mecamylamine (3 mg/kg, i.p.) precipitated withdrawal
syndrome in mice as reflected by a significant increase (p<0.01) in the composite withdrawal
severity score (Rehni et al., 2012; Singh et al., 2013 a, b; Singh et al., 2016) measured in
terms of a concomitant rise in the severity of grooming, scratching, chewing, fore paw
25 tremor, wet dog shake, cage scratching, head nodding, paw licking, particularly in the
nicotine-mecamylamine group, when compared to that of the vehicle treated control groups.
Administration of ML 141 (1, 5 & 10 mg/kg, i.p.) significantly (p<0.01 each) and dose
dependently attenuated nicotine-mecamylamine induced withdrawal syndrome in mice, when
measured in the terms of the total withdrawal severity score (refer Figure 1).
30 2.2 Effect of various treatments of ML 141 on mecamylamine induced alteration in jumping
behaviour in nicotine dependent mice
[0074] Stereotyped jumps precipitated by nicotine receptor antagonist mecamylamine
(3 mg kg−1, i.p.), 1 h after the last nicotine injection, has been considered as a predominant
sign for quantification of Nicotine withdrawal syndrome in mice (Biala and Weglinska, 2005;
15
Singh et al., 2013 a, b; Singh et al., 2016). Jumping frequency observed in a period of 30 min
was used as a quantitative symptom of Nicotine withdrawal immediately after mecamylamine
administration.
[0075] Nicotine (2.5 mg/ kg, s.c.) was administered four times daily for a period of
5 seven days. On the test day (day 8), mice received one nicotine injection in the morning. To
precipitate nicotine abstinence mice were given the nicotine receptor antagonist
mecamylamine (3 mg kg−1, i.p.), 1h after the last nicotine injection precipitated withdrawal
syndrome in mice as reflected by a significant increase (p<0.01) in stereotyped jumping
behaviour in nicotine/ mecamylamine group, when compared to that of the vehicle treated
10 control group. Administration of ML-141 (1, 5 & 10 mg/kg, i.p.) significantly (p<0.01 each)
and dose dependently attenuated mecamylamine induced withdrawal syndrome in nicotine
dependent mice, when measured in terms of stereotyped jumping behavior (Figure 2).
2.3 Measurement of the effect of drug treatment(s) on nicotine-induced hyperalgesia using
tail flick test
15 [0076] Subcutaneous (s.c.) administration of nicotine produced an antinociceptive
effect (i.e. an increase in pain thresholds) (Kiguchi et al. 2008). However, sub-chronic
administration of nicotine is documented to cause the induction of hyperalgesia which has
been quantified in terms of tail flick test (Jackson et al. 2010). Therefore, nociceptive
threshold was measured by the tail flick test in mice (D'Amour and Smith 1941; Singh et al.,
20 2013 a, b; Singh et al., 2016). The tail flick latency was considered as the time between tail
exposure to radiant heat and tail withdrawal. Electrically heated nichrome wire was used as a
source of radiant heat in the analgesiometer. The intensity of radiant heat was regulated in
order to obtain pretreatment latency between 2 and 3 sec in the animals. A cut off latency
time was fixed at 10 sec. Thus, tail flick latency was observed five minutes later, after
25 mecamylamine administration.
[0077] After chronic intermittent administration of nicotine (2.5 mg kg−1, 7 days,
four injections/day) produced significant hyperalgesia, as indicated by decreased tail-flick
latencies (P <0.05), when compared to vehicle control group. Moreover, pair-wise
comparisons confirmed that administration of ML 141 (1, 5 & 10 mg/kg, i.p.) significantly
30 (p<0.05 each) and dose dependently attenuated nicotine-mecamylamine induced hyperalgesia
measured in terms of reversal of withdrawal induced decrease in the tail flick latency (refer
Figure 3).
2.4 Effect of ML 141 treatment(s) on piloerection behaviour in nicotine dependent mice:
16
[0078] Piloerection frequency observations were made for a period of 30 min to
quantitate the severity of the experimental withdrawal phenomenon immediately after
mecamylamine administration. This parameter has been noted to be indicative of the intensity
of withdrawal syndrome as also reported earlier (Damaj et al. 2003; Biala and Weglinska
5 2005; Singh et al., 2016).
[0079] Statistical analysis showed that there was a main effect of group in ANOVA
when applied to piloerection frequency data. Nicotine (2.5 mg/ kg, s.c.) was administered
four times daily for a period of seven days. On the test day (day 8), mice received one
nicotine injection in the morning. To precipitate nicotine abstinence mice were given the
10 nicotine receptor antagonist mecamylamine (3 mg/kg, i.p.), 1 h after the last nicotine
injection precipitated withdrawal syndrome in mice as reflected by a significant increase
(p<0.05) in stereotyped piloerection behaviour in nicotine/ mecamylamine group, when
compared to that of the vehicle treated control group. Further, pair-wise comparisons
confirmed that administration of ML 141 (1, 5 & 10 mg/kg, i.p.) significantly (p<0.01 each)
15 and dose dependently attenuated mecamylamine induced withdrawal syndrome in nicotine
dependent mice, when measured in terms of stereotyped piloerection behavior (refer Figure
4).
2.5 Effect of various treatments of ML 141 on mecamylamine induced anxiety like behaviour
in nicotine dependent mice:
20 [0080] Anxiety like behaviour was monitored in mice using an elevated plus maze
test (Navarro et al., 2006; Singh et al., 2013 a, b; Singh et al., 2016). The plus maze consists
of two open arms, 16 X 5 X 12 cm and two enclosed arms, 16 X 5 X 16 cm with an open
roof, arranged so that the two open arms are opposite to each other. The maze was elevated to
a height of 25 cm. The mouse is placed in the center of the maze, facing one of the enclosed
25 arms. During a 5 min test period the following measures were taken: the number of entries
into and time spent in the open and enclosed. Likewise, different ethological measures were
also quantified: (a) Stretched attend posture (SAP): a body posture in which the mouse
stretches forward and then retracts to its original position without moving the feet, and (b)
Head-dipping (HD): movement of the head over the side of the maze and down towards the
30 floor. The elevated plus maze test was performed 30 minutes after the administration of
mecamylamine on day 8 of the nicotine dependence procedure to assess the level of anxiety
like behavior in mice before and after the treatment schedule is over.
[0081] Administration of four doses of nicotine (2.5 mg/kg, s.c) for a period of 7
days, followed by a single injection of mecamylamine (3mg/kg, i.p.) precipitated withdrawal
17
syndrome in mice as reflected by a significant increase (p<0.01) in anxiety like behaviour as
measured in terms of average time spent in the open arm and head dipping frequency
(P<0.01) particularly in the nicotine-mecamylamine group, when compared to that of the
vehicle treated control groups. Administration of ML 141 (1, 5 & 10 mg/kg, i.p.) significantly
5 (p<0.01 each) and dose dependently attenuated nicotine-mecamylamine induced anxiety like
behaviour as measured in terms of the average time spent in the open arm and head dipping
frequency (refer Table 1 below and Figure 5).
Table 1: Effect of ML 141 treatment(s) on mecamylamine induced increase in anxiety like
behaviour in Nicotine dependent/ saline treated mice.
10
Doses employed in the study were as follows: Vehicle (10% DMSO in water, 10 ml/kg, i.p.),
nicotine (2.5 mg/ kg, s.c.) was administered four times daily), mecamylamine (3 mg/kg, i.p.),
ML 141 (Low; 1, Med: 5 & High dose: 10 mg/kg, i.p.) [Values are mean ± S.E.M.] *=P<0.05
vs. SAL-VEH-VEH; **=P<0.05 vs. NIC-VEH-MEC. HD: head-dipping; SAP: stretched
15 attend posture; NIC: Nicotine; SAL: Saline; VEH: Vehicle; MEC: Mecamylamine.
2.6 Effect of ML 141 treatment(s) on body tremor in nicotine dependent mice:
[0082] Body tremor frequency observations were made for a period of 30 min to
quantitate the severity of the experimental withdrawal phenomenon immediately after
mecamylamine administration. These parameters have been noted to be indicative of the
20 intensity of withdrawal syndrome as also reported earlier (Damaj et al. 2003; Biala and
Weglinska 2005; Singh et al., 2016).
[0083] Statistical analysis showed that there was a main effect of group in ANOVA
when applied to body tremor frequency data. Nicotine (2.5 mg/ kg, s.c.) was administered
18
four times daily for a period of seven days. On the test day (day 8), mice received one
nicotine injection in the morning. To precipitate nicotine abstinence mice were given the
nicotine receptor antagonist mecamylamine (3 mg/kg, i.p.), 1 h after the last nicotine
injection precipitated withdrawal syndrome in mice as reflected by a significant increase
5 (p<0.05) in stereotyped tremor behaviour in nicotine/ mecamylamine group, when compared
to that of the vehicle treated control group. Further, pair-wise comparisons confirmed that
administration of ML-141 (1, 5 & 10 mg/kg, i.p.) significantly (p<0.01 each) and dose
dependently attenuated mecamylamine induced withdrawal syndrome in nicotine dependent
mice, when measured in terms of stereotyped body tremor behavior (refer Figure 6).
10 [0084] From the above data it can be interpreted that ML-141 is suitable for
treatment, prevention or amelioration of mecamylamine-induced precipitation of nicotine
dependence withdrawal syndrome.
[0085] 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
15 thereof. The scope of the invention is determined by the claims that follow. The 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.
20 ADVANTAGES OF THE PRESENT INVENTION
[0086] The present invention provides a compound, ML-141, for treatment,
prevention or amelioration of nicotine dependence and its associated withdrawal syndrome.
[0087] The present invention provides a compound, ML-141, for treatment,
prevention or amelioration of mecamylamine-induced precipitation of nicotine dependence
25 withdrawal syndrome which is affected via the Rho-GTPase protein.
[0088] The present invention provides a compound, ML-141, for treatment,
prevention or amelioration of mecamylamine-induced precipitation of nicotine dependence
withdrawal syndrome that results in reduction in jumping frequency, withdrawal symptoms
related anxiety, nicotine-induced hyperalgesia, piloerection frequency and body tremor
30 frequency.
[0089] The present invention provides a compound, ML-141, for treatment,
prevention or amelioration of mecamylamine-induced precipitation of nicotine dependence
withdrawal syndrome that aids the patient in alcohol abstinence.
19
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We Claim:

1. A compound, ML-141, for treatment, prevention or amelioration of mecamylamineinduced precipitation of nicotine dependence withdrawal syndrome.
5 2. The compound as claimed in claim 1, wherein ML-141 is a Rho-GTPase inhibitor.
3. The compound as claimed in claim 1, wherein ML-141 is 4-[4,5-dihydro-5-(4-
methoxyphenyl)-3-phenyl-1H-pyrazol-1-yl]-benzenesulfonamide.
4. The compound as claimed in claim 1, wherein the compound is administered in a
therapeutically effective amount to a subject.
10 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 100 mg/kg of body weight.
6. 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.
7. The compound as claimed in claim 4, wherein the subject is a mammal.
15 8. The compound as claimed 1, wherein the compound is administered intraperitoneally,
orally, intracerebrally, intramuscularly, intravenously, subcutaneously or transdermally.
9. The compound as claimed in claim 1, wherein the compound is formulated in the form of a
medicament.
10. A composition comprising the compound as claimed in claim 1, along with a
20 pharmaceutically acceptable excipient.