[0001] The present disclosure relates generally to the field of pharmaceuticals.
Specifically, the present disclosure is directed to a compound diminazene aceturate for
attenuation of diazepam dependence induced 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] Addiction or drug dependence is a psychiatric disorder that exhibits itself as
15 compulsive drug seeking and taking despite knowing detrimental negative consequences such
as uncontrolled drug intake, craving, anxiety, and relapse resulting in major health hazards
and socioeconomic impacts worldwide (Balfour, 2004; Adinoff et al., 2011).
[0004] Benzodiazepines (BZD) is a group of positive allosteric modulators of
GABAA receptors, having familiar names including Velum and Xanax which are widely
20 prescribed for the management of anxiety, insomnia, and seizure disorders (Solomon et al.,
2019). However, widespread BZDs prescription upsurges the amount of patients who develop
dependence. The abrupt discontinuation of BZD may upshot withdrawal symptoms, such as
anxiety, sleep disturbance, irritability, increased tension and anxiety, panic attacks, hand
tremor, sweating, nausea, weight loss, palpitations, muscular pain and stiffness, agitation, and
25 seizures (Griffiths & Johnson, 2005). The induction of BZD dependence can ensue even at
low dose with 23% becoming habituated within 3 months of use (Barthelmé et al., 2008).
BZD long term use is around 6–76% globally (Kurko et al., 2015) and while attempting BZD
discontinuation, withdrawal syndrome was reported among 10% of patients which can persist
for many months or year (Faccini et al., 2016). Overall, it ranges from approximately 2.0% to
30 7.4% in the general population (Kurko et al., 2015). In India, BZDs were the most commonly
prescribed i.e. around 64% (Sahana et al., 2010). Moreover, long-term users are more
expected to be elder, female, with more significant chronic health and/or emotional state
(Jones et al., 2010).
3
[0005] BZDs does not upsurge its dopamine (DA) neurotransmission so its addictive
effects seem to be facilitated through circuits other than the mesocorticolimbic dopamine
system (DiChiara et al., 1993; Robinson and Berridge, 1993). The cause of withdrawal is due
to down-regulation of GABAA complex (BZD Binding sites) in specific brain region such as
5 cortex, hippocampus and amygdala. Moreover, there is significant increase in calcium flux
and serotonin (5-HT) level in brain during withdrawal (Begg et al., 2005).
[0006] Acid sensing ion channels (ASICs) are amiloride-sensitive, cation-selective
channels that belong to DEG/ENaC family activated at the pH between 5-6.9 which cause a
small increase in calcium permeability and an inward flow of calcium facilitates the
activation of the voltage gated Ca2+ 10 and NMDA receptor due to initial depolarization leading
to increase in intracellular calcium in neuronal cell (Cushman et al., 2007; Chu et al., 2011),
which is abundant in the nucleus accumbens (NAc), a region known for its role in addiction.
ASIC1a plays prominent role in synaptic plasticity, learning/memory, fear conditioning, and
retinal physiology (Chu et al., 2012) as abundantly expressed in the nucleus accumbens core
15 (NAcore), a region of the mesolimbocortical system that has an established role in regulating
drug-seeking behaviour (Gutman et al., 2018). ASIC has not been explored much as a target
for remedying the withdrawal syndrome.
[0007] Hence, the inventors of the present invention have arrived at the role of
diminazene aceturate, an ASIC modulator, for attenuation of diazepam dependence induced
20 withdrawal syndrome that is precipitated by flumazenil.
OBJECTS OF THE INVENTION
[0008] An object of the present disclosure is to provide a compound, diminazene
aceturate, for the attenuation of diazepam dependence induced withdrawal syndrome.
25 [0009] An object of the present disclosure is to provide a compound, diminazene
aceturate, for the treatment, amelioration or prevention of diazepam induced withdrawal
syndrome that is precipitated using flumazenil.
[0010] An object of the present disclosure is to provide a compound, diminazene
aceturate, for the treatment, amelioration or prevention of diazepam induced withdrawal
30 syndrome that is affected via the ASIC1a channel.
[0011] An object of the present disclosure is to provide a compound, diminazene
aceturate, for the improvement in severity of anxiety, tremor frequency, sniffing, grooming
behaviour, impairment in locomotion induced by diazepam dependence related withdrawal
syndrome.
4
SUMMARY OF THE INVENTION
[0012] 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
5 intended to be used as an aid in determining the scope of the claimed subject matter.
[0013] The present disclosure is directed to a compound, diminazene aceturate, for
attenuation of diazepam dependence induced withdrawal syndrome.
[0014] In an aspect, the present disclosure provides a compound, diminazene
aceturate, for the treatment, prevention or amelioration of diazepam dependence induced
10 withdrawal syndrome.
[0015] In an aspect, the present disclosure provides a compound, diminazene
aceturate, for the treatment, prevention or amelioration of diazepam dependence induced
withdrawal syndrome precipitated by flumazenil.
[0016] In an embodiment, the present disclosure provides a compound, diminazene
15 aceturate, which affects the attenuation of the diazepam dependence induced withdrawal
syndrome via modulation of the ASIC1a channel.
[0017] In an embodiment, the compound is administered in a therapeutically effective
amount to a subject.
[0018] In another aspect, the present disclosure relates to a composition comprising
20 the compound, diminazene aceturate, for the prevention or amelioration of diazepam
dependence induced withdrawal syndrome.
[0019] In an embodiment, the composition further comprises pharmaceutically
acceptable excipients.
[0020] In yet another aspect, the present disclosure provides the use of a compound,
25 diminazene aceturate, for the prevention or amelioration of diazepam dependence induced
withdrawal syndrome.
[0021] In still another aspect, the present disclosure provides a method of treatment of
diazepam dependence induced withdrawal syndrome by administering to a subject in need
thereof, a therapeutically effective amount of the compound diminazene aceturate.
30 [0022] 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
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.
5
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0023] 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
5 by reference to the drawings in combination with the detailed description of the specific
embodiments presented herein.
[0024] Figure 1: Effect of treatment(s) on Flumazenil induced increase in withdrawal
severity score in Diazepam dependent/ vehicle treated mice. Doses employed in the study
were as follows: vehicle (Saline 10 ml/kg), Diazepam (15 mg/ kg, i.p.) was administered once
10 daily, Flumazenil (25 mg/kg, i.p.), Diminazene aceturate (10 & 30 mg/kg, i.p.) [Values are
mean ± S.E.M.] a =P<0.05 vs. Vehicle control; b =P<0.05 vs. Diazepam–Flumazenil
Control
[0025] Figure 2: Effect of treatment(s) on Flumazenil induced anxiety like behaviour
in Diazepam dependent/ vehicle treated mice in terms of time spent in open arm in elevated
15 plus maze test.
[0026] Figure 3: Effect of treatment(s) on Flumazenil induced anxiety like behaviour
in Diazepam dependent/ vehicle treated mice in terms of time spent in closed arms in
elevated plus maze test.
[0027] Figure 4: Effect of treatment(s) on Flumazenil induced anxiety like behaviour
20 in diazepam dependent/vehicle treated mice in terms of head dipping frequency in elevated
plus maze test.
[0028] Figure 5: Effect of treatment(s) on Flumazenil induced Body tremor in
Diazepam dependent/ vehicle treated mice.
25 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
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
30 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
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
6
definition of that term provided herein applies and the definition of that term in the reference
does not apply.
[0031] Reference throughout this specification to ―one embodiment‖ or ―an
embodiment‖ means that a particular feature, structure or characteristic described in
5 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.
Furthermore, the particular features, structures, or characteristics may be combined in any
suitable manner in one or more embodiments.
10 [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.‖
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
15 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
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
20 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.
[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
25 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
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‖
30 unless the context clearly dictates otherwise.
[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.‖
7
[0036] 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.
Unless otherwise indicated herein, each individual value is incorporated into the specification
as if it were individually recited herein.
5 [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
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
10 should be construed as indicating any non-claimed element essential to the practice of the
invention.
[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
15 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
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
20 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.
[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
25 processes. In general, the order of the steps of the disclosed processes may be altered within
the scope of the invention.
[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
30 inventive subject matter. Although each embodiment represents a single combination of
inventive elements, the inventive subject matter is considered to include all possible
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
8
matter is also considered to include other remaining combinations of A, B, C, or D, even if
not explicitly disclosed.
[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.
5 [0044] The present disclosure relates to a compound, diminazene aceturate, for
attenuation of diazepam dependence induced withdrawal syndrome.
[0045] Benzodiazpines (BZDs) sometimes called ―benzos‖ are a class of psychoactive
drugs whose core chemical structure is the fusion of a benzene ring and a diazepine ring and
are one of the most widely prescribed tranquilizers in the United States under the name of
10 Velum and Xanax. BZDs are one of the largest classes of abused drugs; they are classed as
schedule IV controlled drugs because of their recognized medical uses (Garnier et al., 2010).
The main actions of BZDs includes hypnotics, insomnia, alcohol withdrawal, anxiety
disorders, panic disorder, phobias, post-traumatic stress disorder, obsessive-compulsive
disorder, cardiovascular, gastrointestinal, acute status epilepticus, neonatal seizures or febrile
15 convulsions, preeclampsia, multiple sclerosis, paraplegia that confer a therapeutic value in a
wide range of conditions (Liu et al., 2010; Chen et al., 2011).
[0046] Discontinuation of BZDs or abrupt reduction of the dose, even after a
relatively short course of treatment (three to four weeks), may result in two groups of
symptoms—rebound and withdrawal. Withdrawal symptoms typically consist of a mirror
20 image of the drug's effects: sedative effects and suppression of REM and SWS stages of sleep
can be replaced by insomnia, nightmares, and hypnogogic hallucinations; its anti-anxiety
effects are replaced with anxiety and panic; muscle-relaxant effects are replaced with
muscular spasms or cramps; and anticonvulsant effects are replaced with seizures, especially
in cold turkey or overly-rapid withdrawal (Wilkinson et al.).
25 [0047] In an embodiment, the present disclosure provides a compound, diminazene
aceturate, for the treatment, prevention or amelioration of diazepam dependence induced
withdrawal syndrome. The disclosure also covers pharmaceutically acceptable salts or
derivatives of diminazene aceturate.
[0048] Diminazene is a solid, yellow coloured aromatic diamidine of synthethic origin
30 connected by a triazene bridge, chemically known as 4,4'-(1-Triazene-1,3-diyl)
bis(benzenecarboximidamide) having chemical formula C14H15N7 that is derived from Surfen
C. Moreover it is stable for 2-3 days in water (Fairclough, 1962; Vial et al., 2006) having
Molar mass: 281.316 g/mol and molecular weight 515.531g/mol. According to the study
conducted by Chen et al. (chen et al., 2010) diminazene aceturate presented high capacity to
9
inhibit the acid sensitive ion channels (ASICs) through in vitro study using a primary culture
of hippocampal neurons having IC50 value of 0.30 ± 0.11 M. Diminazene aceturate showed
no activity on the epithelial sodium channel (ENaC), which indicates that this aromatic
diamidine presents a greater selectivity for ASIC.
5 [0049] In an embodiment, the present disclosure provides a compound, diminazene
aceturate, for the treatment, prevention or amelioration of diazepam dependence induced
withdrawal syndrome affected via ASIC 1a modulation.
[0050] Flumazenil- Flumazenil (Ro 15-1788) is a 1,4- imidazo BZD that possesses
specific antagonism at the level of BZD receptors. In an embodiment, the present disclosure
10 provides a compound, diminazene aceturate, for the treatment, prevention or amelioration of
diazepam dependence induced withdrawal syndrome precipitated by flumazenil.
[0051] In an embodiment, the compound maybe administered in a therapeutically
effective amount to a subject. This is determined based on the patient history, body weight
and severity of the condition.
15 [0052] In an embodiment, the compound may preferably be administered in a dose
range of about 1 mg/kg of the body weight to about 100 mg/kg of the body weight each day,
more preferably in the range of about 10mg/kg of the body weight to about 40mg/kg of the
body weight.
[0053] In an embodiment, the compound is administered to a subject. The subject
20 may be a mammal, including humans and non-humans, for examples, humans, cats, dogs,
rats, bats, pigs, cattle and the like.
[0054] In an embodiment, the compound may be administered orally, intracerebrally,
transdermally, subcutaneously, intravenously, intraperitoneally, or intramuscularly. The
compound may be administered in any form known in the art and suitable for patient
25 compliance including pills, tablets, capsules, lozenges, powders, granules, patches,
suspensions, solutions, microparticles, nanoparticles, aerosols, and sustained release particles.
[0055] In an embodiment, the compound may be delivered via injection or infusion.
[0056] In an embodiment, the compound may be in the form of a medicament. The
compound or the medicament may be administered as a supplementary treatment.
30 [0057] In an embodiment, the compound may also be supplemented with an
alternative therapeutically effective drug for diazepam dependence induced withdrawal
syndrome for treatment of further symptoms or side effects. The compound may also be
administered suitably along with drugs for other ailments like hypertension, nicotine
addiction, blood sugar, depression, insomnia and the like.
10
[0058] In another embodiment, the present disclosure relates to a composition
comprising the compound, diminazene aceturate, for the prevention or amelioration of
diazepam dependence induced withdrawal syndrome.
[0059] In an embodiment, the disclosure relates to a composition comprising the
5 compound diminazene aceturate for the prevention or amelioration of diazepam dependence
induced withdrawal syndrome along with a pharmaceutically acceptable excipient.
[0060] In an embodiment, the pharmaceutically acceptable excipient includes those
generally known in the art and that do not affect the therapeutic activity of diminazene
aceturate. These excipients may be selected from dispersants, binders, buffering agents,
10 preservatives, stabilizers, colouring agents, flavouring agents, sugars, diluents and the like
[0061] In another embodiment, the present disclosure provides the use of a
compound, diminazene aceturate, for the prevention or amelioration of diazepam dependence
induced withdrawal syndrome. It also relates to the use of a compound, diminazene aceturate,
for the prevention or amelioration of diazepam dependence induced withdrawal syndrome
15 precipitated with flumazenil.
[0062] In an embodiment, the present disclosure provides the use of a compound,
diminazene aceturate, in the form of a medicament for the prevention or amelioration of
diazepam dependence induced withdrawal syndrome.
[0063] In still another embodiment, the present disclosure provides a method of
20 treatment of diazepam dependence induced withdrawal syndrome by administering to a
subject in need thereof, a therapeutically effective amount of the compound diminazene
aceturate.
[0064] While the foregoing describes various embodiments of the disclosure, other
and further embodiments of the disclosure may be devised without departing from the basic
25 scope 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.
30 [0065] 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
11
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.
5 MATERIAL AND METHODS:
Animals:
[0066] Swiss albino male mice weighing 22±5g obtained from ISF, College of
Pharmacy, Moga, 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
10 housed in the departmental animal house and were exposed to a regular 12 hr cycle of light
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
15 (CPCSEA), Ministry of Environment and Forests, Government of India (Reg. No.
1181/PO/ReBi/S/08/CPCSEA).
Drugs and chemicals:
[0067] Diazepam, Flumazenil, and diminazene aceturate (Sigma-Aldrich Chemicals
Pvt. Ltd., St. Louis, USA), were dissolved / diluted in normal saline and DMSO. The
20 chemicals used were of analytical grade and flumazenil and diminazene aceturate were
freshly prepared before use.
EXAMPLE 1:
Induction of diazepam withdrawal syndrome in mice:
[0068] Acute administration of diazepam followed by a single injection of flumazenil
25 was used to induce diazepam induced withdrawal syndrome in mice (Chan et al., 1989).
Diazepam dependence was induced by one dose of diazepam (15 mg/kg, i.p.) daily for seven
days. The control groups were treated with saline following the same schedule. On the test
day (day 7), to precipitate diazepam abstinence mice were given flumazenil (25 mg kg−1,
i.p.), 1 h after the last diazepam injection. The somatic signs of withdrawal were evaluated
30 for 30 min, immediately after flumazenil administration. The observations were made in a
transparent perspex observation chamber with dimensions of 30 cm X 30 cm X 30 cm.
EXAMPLE 2:
Experimental protocol
12
[0069] Five groups were employed in the present study, with each group comprising
of 08 male animals.
[0070] Group I (Vehicle-vehicle control): Vehicle (Saline, 10 ml/kg, i.p.) for
diazepam was administered once daily for a period of seven days. Vehicle (10% DMSO in
5 water, 10 ml/kg, i.p.) for diminazene aceturate was simultaneously injected once daily for the
same period of seven days. Vehicle (10% DMSO in water 10 ml/kg, i.p.) for flumazenil was
then injected on the day 7, 1 hr after administering vehicle (Saline, 10 ml/kg, i.p.) for
diazepam.
[0071] Group II (Vehicle-flumazenil control): Vehicle (Saline, 10 ml/kg, i.p.) for
10 diazepam was administered once daily for a period of seven days. Vehicle (10% DMSO in
water, 10 ml/kg, i.p.) for diminazene aceturate was simultaneously injected once daily for the
same period of seven days. Flumazenil (25 mg/kg, i.p.) was administered on the day 7, 1 hr
after last dose of diazepam vehicle.
[0072] Group III (Diazepam–Flumazenil control): diazepam (15 mg/ kg, i.p.) was
15 administered once daily for a period of seven days. Vehicle (10% DMSO in water, 10 ml/kg,
i.p.) for diminazene aceturate was simultaneously injected once daily for the same period of
seven days. Flumazenil (25 mg/kg, i.p.) was then injected 1 hr after the last dose of diazepam
to precipitate the withdrawal syndrome in mice.
[0073] Group IV-V (Diminazene aceturate treatment + Diazepam- flumazenil):
20 diazepam (15 mg/ kg, i.p) was administered once daily for a period of seven days.
Diminazene aceturate (at a dose level of 10 and 30 mg/kg/d, i.p. for groups number IV-V
respectively) was simultaneously injected once daily for the same period of seven days.
Flumazenil (25 mg/kg, i.p.) was then injected 1 hr after the last dose of diazepam to
precipitate the withdrawal syndrome in mice.
25 EXAMPLE 3
Statistical analysis
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
30 was compared in saline- and diazepam-treated mice. A value of P<0.05 was considered to be
statistically significant. The statistical analysis was done using Sigma Stat 6.0 software.
3.1 Effect of various treatments on flumazenil induced alteration in withdrawal severity score
in Diazepam dependent mice
13
[0074] A set of modified withdrawal severity score was employed to quantitate the
magnitude of withdrawal syndrome in mice in terms of the earlier reported characteristic
behavioural patterns seen in mice suffering from experimental diazepam dependence induced
withdrawal syndrome viz., piloerection, sniffing, grooming, tail lift, paw licking, head
5 nodding, impairement in locomotion, cage scratching all in a composite manner (Table 1)
(Chan et al., 1989). The test was performed immediately after flumazenil administration and
the results were based on observations spanning first 30 minutes (refer Table 1 below).
Table 1: Modified Diazepam dependence induced Withdrawal Severity Score
14
[0075] Administration of one dose of Diazepam (15 mg/ kg, i.p.) for a period of 7
days, followed by a single injection of Flumazenil (25 mg/kg, i.p.), precipitated withdrawal
syndrome in mice as reflected by a significant increase (p<0.01) in the composite withdrawal
5 severity score particularly in the Diazepam–Flumazenil group, when compared to that of the
vehicle treated control groups. Administration of Diminazene aceturate (10 & 30 mg/kg, i.p.)
significantly (p<0.01 each) and dose dependently attenuated Diazepam–Flumazenil induced
withdrawal syndrome in mice, when measured in the terms of the total withdrawal severity
score (refer Figure 1).
10 3.2 Effect of various treatments on Flumazenil induced anxiety like behaviour in Diazepam
dependent mice:
[0076] Anxiety like behaviour was monitored in mice using an elevated plus maze
test (Walf et al., 2007). The mouse is placed in the centre of the maze, facing one of the
15
enclosed arms. During a 5 min test period the following measures were taken: time spent in
the open and enclosed. Likewise, different ethological measures were also quantified:
Headdipping (HD): movement of the head over the side of the maze and down towards the
floor. The elevated plus maze test was performed 30 minutes after the administration of
5 flumazenil on day 7 of the Diazepam dependence procedure to assess the level of anxiety like
behaviour in mice after the treatment schedule is over.
[0077] Administration of one dose of Diazepam (15 mg/ kg, i.p.) for a period of 7
days, followed by a single injection of Flumazenil (25 mg/kg, i.p.), precipitated withdrawal
syndrome in mice as reflected by a significant increase (p<0.01) in anxiety like behaviour as
10 measured in terms of average time spent in the open arm and head dipping frequency
(P<0.01) particularly in the Diazepam–Flumazenil group, when compared to that of the
vehicle treated control groups. Administration of Diminazene aceturate (10 & 30 mg/kg, i.p.)
significantly (p<0.01 each) and dose dependently attenuated Diazepam–Flumazenil induced
anxiety like behaviour as measured in terms of the average time spent in the open arm, Close
15 arm and head dipping frequency ( refer Figures 2, 3 & 4).
3.3 Effect of various treatments on Flumazenil induced body tremor in Diazepam dependent
mice:
[0078] Body tremor frequency observations were made for a period of 30 min to
quantitate the severity of the experimental withdrawal phenomenon immediately after
20 flumazenil administration. These parameters have been noted to be indicative of the intensity
of withdrawal syndrome as also reported earlier.
[0079] Diazepam (15 mg/ kg, i.p.) was administered daily for a period of seven days
to induce dependence in mice. On the test day (day 7), to precipitate diazepam abstinence
mice were given selective GABAA antagonist Flumazenil (25 mg/kg, i.p.), 1 hr after last
25 dose of diazepam precipitated withdrawal syndrome in mice as reflected by a significant
increase (p<0.01) in frequency of tremors in Diazepam–Flumazenil group, when compared to
that of the vehicle treated control group. Administration of Diminazene aceturate (10 & 30
mg/kg, i.p.) significantly (p<0.01 each) and dose dependently attenuated Flumazenil induced
withdrawal syndrome in Diazepam dependent mice, when measured in terms of stereotyped
30 tremor behaviour (refer Figure 5).
[0080] Overall, the study evaluated the withdrawal severity score (WSS) along with
effect on anxiety after administration of diminazene aceturate as the treatment for attenuation
of diazepam induced withdrawal syndrome. The administration of diminazene aceturate
showed improvement in WSS scale such as reduction in erection of hair, sniffing, grooming
16
behavior, tail lift, paw licking head nodding and impairment in locomotion. Moreover, the
severity of anxiety and tremor frequency also diminished by administration of diminazene
aceturate.
[0081] On the basis of the above discussion, it may be concluded that diminazene
5 aceturate, a slow pore blocker of ASIC 1a, attenuates the development of diazepam
dependence as observed in the flumazenil induced precipitation of withdrawal symptoms in
diazepam dependent mice.
[0082] While the foregoing describes various embodiments of the disclosure, other
and further embodiments of the disclosure may be devised without departing from the basic
10 scope 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.
15
ADVANTAGES OF THE PRESENT INVETION
[0083] The present disclosure provides a compound for the attenuation of diazepam
dependence induced withdrawal syndrome.
[0084] The present disclosure provides a compound for the attenuation of diazepam
20 dependence induced withdrawal syndrome precipitated with flumazenil.
[0085] The present disclosure provides a compound that shows improvement in WSS
scale such as reduction in erection of hair, sniffing, grooming behaviour, tail lift, paw licking
head nodding and impairment in locomotion.
[0086] The present disclosure provides a compound that decreases the severity of
25 anxiety and tremor frequency.
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We Claim:

1. A compound, diminazene aceturate, for the treatment, prevention or amelioration of
diazepam dependence induced withdrawal syndrome.
5 2. The compound as claimed in claim 1, wherein the diazepam dependence induced
withdrawal syndrome is precipitated with flumazenil.
3. The compound as claimed in claim 1, wherein the diminazene aceturate is an Acid-sensing
ion channel (ASIC) modulator.
4. The compound as claimed in claim 1, wherein the compound is administered in a
10 therapeutically effective amount to a subject.
5. The compound as claimed in claim 1, wherein the compound is administered in a dose
range of 1 mg/kg of the body weight to 100mg/kg of the body weight each day.
6. The compound as claimed in claim 1, wherein the compound is administered in a dose
range of 10 mg/kg of the body weight to 40mg/kg of the body weight each day.
15 7. The compound as claimed in claim 4, wherein the subject is a mammal.
8. The compound as claimed in claim 1, wherein the compound is administered orally,
intracerebrally, transdermally, subcutaneously, intravenously, intraperitoneally, or
intramuscularly.
9. The compound as claimed in claim 1, wherein the compound is formulated in the form of a
20 medicament.
10. A composition comprising the compound as claimed in claim 1, along with a
pharmaceutically acceptable excipient.