FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule13)
1.
TITLE OF THE INVENTION
PHARMACEUTICAL COMPOSITIONS AND METHODS
2.
APPLICANT
(a).
NAME:
WOCKHARDT LIMITED
(b).
NATIONALITY:
INDIAN
(c).
ADDRESS:
D-4, M. I. D. C INDUSTRIAL AREA,
CHIKALTHANA, AURANGABAD – 431006, MAHARASHTRA, INDIA.
The following specification particularly describes the invention and the manner in which it is to be performed.
WK-18006-IN-NP
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PHARMACEUTICAL COMPOSITIONS AND METHODS
RELATED PATENT APPLICATIONS
This application claims the priority to and benefit of Indian Provisional
Patent Application No. 201821028332 filed on July 27, 2018; the disclosures of
which are incorporated herein by reference in its entirety as if fully rewritten
herein.
FIELD OF THE INVENTION
The invention relates to pharmaceutical compositions and their use in
modulating one or more cytokines.
BACKGROUND OF THE INVENTION
Cytokines are important biological molecules that are involved in several
biological functions. Cytokines are small proteins, and particularly play important
role in the immune system. Cytokines may include chemokines, interferons,
interleukins, lymphokines, and tumour necrosis factors. The modulation of
cytokines, for example by increasing or decreasing their levels, can help in
achieving outcome of many intended treatments (e.g. infections, allergies,
inflammation).
SUMMARY OF THE INVENTION
Accordingly, there are provided pharmaceutical compositions for
modulating one or more cytokines, said compositions comprising a compound of
Formula (I) or a pharmaceutically acceptable salt thereof:
N
S
N
N
O
O
O
O
O
N
HO
O
O
N
O
NH2
OMe
O
H
(I)
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3
In another aspect, there is provided a method for modulating one or more
cytokines in a subject, said method comprising administering to the subject a
pharmaceutical composition comprising a compound of Formula (I) or a
pharmaceutically acceptable salt thereof.
In another general aspect, there is provided a method for modulating one
or more cytokines in a subject, said method comprising administering to the
subject a compound of Formula (I) or a pharmaceutically acceptable salt thereof:
N
S
N
N
O
O
O
O
O
N
HO
O
O
N
O
NH2
OMe
O
H
(I)
The details of one or more embodiments of the invention are set forth in
the description below. Other features, objects and advantages of the invention will
be apparent from the following description including claims.
DETAILED DESCRIPTION OF THE INVENTION
Reference will now be made to the exemplary embodiments, and specific
language will be used herein to describe the same. It should nevertheless be
understood that no limitation of the scope of the invention is thereby intended.
Alterations and further modifications of the inventive features illustrated herein,
and additional applications of the principles of the invention as illustrated herein,
which would occur to one of ordinary skills in the relevant art and having
possession of this disclosure, are to be considered within the scope of the
invention. It must be noted that, as used in this specification and the appended
claims, the singular forms “a”, “an” and “the” include plural referents unless the
content clearly dictates otherwise. All references including patents, patent
applications, and literature cited in the specification are expressly incorporated
herein by reference in their entirety.
The inventors have surprisingly discovered that certain compounds exhibit
ability to modulate cytokines.
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The term “pharmaceutically acceptable salt” as used herein refers to one or more salts of a given compound which possesses the desired pharmacological activity of the free compound and which are neither biologically nor otherwise undesirable. In general, the “pharmaceutically acceptable salts” refer to and include those salts that are suitable for use in contact with the tissues of human and animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. (J. Pharmaceutical Sciences, 66: 1-19 (1977)), incorporated herein by reference in its entirety, describes various pharmaceutically acceptable salts in details.
The term “treat”, “treating” or “treatment” as used herein refers to administering a medicament, including a pharmaceutical composition, or one or more pharmaceutically active ingredients, for prophylactic and/or therapeutic purposes.
The term “pharmaceutically effective amount” or “therapeutically effective amount” or “effective amount” as used herein refers to an amount, which has a therapeutic effect or is the amount required to produce a therapeutic effect in a subject. The compounds and/or pharmaceutical compositions according to the invention are used in amounts that are effective in providing the desired therapeutic effect or result.
The term “administration” or “administering” includes delivery of a composition or one or more pharmaceutically active ingredients to a subject, including for example, by any appropriate methods, which serves to deliver the composition or its active ingredients or other pharmaceutically active ingredients to the desired site of action. The method of administration may vary depending on various factors, such as for example, the components of the pharmaceutical composition, nature of the pharmaceutically active or inert ingredients, the desired site of action, age and physical condition of the subject and a like. Some non-limiting examples of ways to administer a composition or a pharmaceutically active ingredient to a subject according to this invention includes oral, intravenous, topical, intra-respiratory, intra-peritoneal, intra-muscular, parenteral, sublingual, transdermal, intranasal, aerosol, intra-ocular, intra-tracheal, intra-rectal, vaginal, gene gun, dermal patch, eye drop, ear drop or mouthwash.
The term “synergistic” or “synergy” as used herein refers to the interaction of two or more agents so that their combined effect is greater than their individual effects.
The term “pharmaceutically inert ingredient” or “carrier" or "excipient" refers to a compound or material used to facilitate administration of a compound, including for example, to increase the solubility of the compound. Typical, non-limiting examples of solid carriers include, starch, lactose, dicalcium phosphate, sucrose, and kaolin and so on. Typical, non-limiting examples of liquid carriers
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include sterile water, saline, buffers, non-ionic surfactants, and edible oils such as
oil, peanut and sesame oils and so on. In addition, various adjuvants commonly
used in the art may be included. These and other such compounds are described in
the literature, for example, in the Merck Index (Merck & Company, Rahway,
N.J.). Considerations for inclusion of various components in pharmaceutical
compositions are described, for example, in Gilman et al. (Eds.) (1990); Goodman
and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon
Press., which is incorporated herein by reference in its entirety.
The term “subject” as used herein refers to a vertebrate or invertebrate,
including a mammal. The term “subject” includes human, animal, a bird, a fish, or
an amphibian. Typical, non-limiting examples of a “subject” includes humans,
cats, dogs, horses, sheep, bovine cows, pigs, lambs, rats, mice and guinea pigs.
The term “modulating” or “modulation” of cytokines refers to and
includes increasing or decreasing level of cytokines. The term “modulating” or
“modulation” also refers to and includes stimulating cytokines.
In one general aspect, there are provided pharmaceutical compositions for
modulating one or more cytokines, said composition comprising a compound of
Formula (I) or a pharmaceutically acceptable salt thereof:
N
S
N
N
O
O
O
O
O
N
HO
O
O
N
O
NH2
OMe
O
H
(I)
In some embodiments, the pharmaceutical composition comprising a
compound of Formula (I) or a pharmaceutically acceptable salt thereof, for use in
modulating one or more cytokines in a subject.
In some embodiments, the cytokine is one or more of Tumor necrosis
factor-(TNF-), Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8),
Interleukin-10 (IL-10), Interleukin-1(IL-1), Interferon-g (IFN-g), and
Granulocyte-macrophage colony stimulating factor (GM-CSF).
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The pharmaceutical compositions according to the invention may include one or more pharmaceutically acceptable carriers or excipients or a like. Typical, non-limiting examples of such carriers or excipients include mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatine, sucrose, magnesium carbonate, wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, lubricants, stabilizing agents, binding agents and a like.
The pharmaceutical compositions according to this invention can exist in various forms. In some embodiments, the pharmaceutical composition is in the form of a powder or a solution. In some other embodiments, the pharmaceutical compositions according to the invention are in the form of a powder that can be reconstituted by addition of a compatible reconstitution diluent prior to parenteral administration. Non-limiting example of such a compatible reconstitution diluent includes water.
In some other embodiments, the pharmaceutical compositions according to the invention are in the form of a frozen composition that can be diluted with a compatible diluent prior to parenteral administration.
In some other embodiments, the pharmaceutical compositions according to the invention are in the form ready to use for parenteral administration.
The pharmaceutical composition or the active ingredients according to the present invention may be formulated into a variety of dosage forms. Typical, non-limiting examples of dosage forms include solid, semi-solid, liquid and aerosol dosage forms; such as tablets, capsules, powders, solutions, suspensions, suppositories, aerosols, granules, emulsions, syrups, elixirs and a like.
In the methods according to the invention, the pharmaceutical composition and/or other pharmaceutically active ingredients disclosed herein may be administered by any appropriate method, which serves to deliver the composition or its constituents or the active ingredients to the desired site. The method of administration can vary depending on various factors, such as for example, the components of the pharmaceutical composition, nature of the active ingredients, age and physical condition of the subject. Some non-limiting examples of administering the composition to a subject according to this invention include oral, intravenous, topical, intra-respiratory, intra-peritoneal, intra-muscular, parenteral, sublingual, transdermal, intranasal, aerosol, intraocular, intra-tracheal, intra-rectal, vaginal, gene gun, dermal patch, eye drop, ear drop or mouthwash.
The compositions according to the invention can be formulated into various dosage forms wherein the active ingredients and/or excipients may be present either together (e.g. as an admixture) or as separate components. When the various ingredients in the composition are formulated as a mixture, such composition can be delivered by administering such a mixture. The composition
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or dosage form wherein the ingredients do not come as a mixture, but come as
separate components, such composition/dosage form may be administered in
several ways. In one possible way, the ingredients may be mixed in the desired
proportions and the mixture is then administered as required. Alternatively, the
components or the ingredients (active or inert) may be separately administered
(simultaneously or one after the other) in appropriate proportion so as to achieve
the same or equivalent therapeutic level or effect as would have been achieved by
administration of the equivalent mixture.
In another general aspect, there are provided methods for modulating one
or more cytokines in a subject, said method comprising administering to the
subject a pharmaceutical composition disclosed herein.
In another general aspect, there are provided methods for modulating one
or more cytokines in a subject, said method comprising administering to the
subject a compound of Formula (I) or a pharmaceutically acceptable salt thereof:
N
S
N
N
O
O
O
O
O
N
HO
O
O
N
O
NH2
OMe
O
H
(I)
In the methods according to the invention, the active ingredients disclosed
herein may be administered to a subject in several ways depending on the
requirements. In some embodiments, the active ingredients are admixed in
appropriate amounts and then the admixture is administered to a subject. In some
other embodiments, the active ingredients are administered separately. Since the
invention contemplates that the active ingredients agents may be administered
separately, the invention further provides for combining separate pharmaceutical
compositions in kit form. The kit may comprise one or more separate
pharmaceutical compositions, each comprising one or more active ingredients.
Each of such separate compositions may be present in a separate container such as
a bottle, vial, syringes, boxes, bags, and the like. Typically, the kit comprises
directions for the administration of the separate components. The kit form is
particularly advantageous when the separate components are preferably
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administered in different dosage forms (e.g., oral and parenteral) ore are administered at different dosage intervals. When the active ingredients are administered separately, they may be administered simultaneously or sequentially.
It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. For example, those skilled in the art will recognize that the invention may be practiced using a variety of different compounds within the described generic descriptions.
EXAMPLES
The following examples illustrate the embodiments of the invention that are presently best known. However, it is to be understood that the following are only exemplary or illustrative of the application of the principles of the present invention. Numerous modifications and alternative compositions, methods, and systems may be devised by those skilled in the art without departing from the spirit and scope of the present invention. The appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been described above with particularity, the following examples provide further detail in connection with what are presently deemed to be the most practical and preferred embodiments of the invention.
Experimental Procedure
The activity of test compounds in modulating one or more cytokines was evaluated using a whole blood cytokine secretion assay, using the following general procedure. Peripheral venous blood was collected with consent from healthy adult volunteers between 25 to 40 years of age and stored in tubes containing heparin. All volunteers confirmed that they did not take any concomitant medication within the last 2 weeks prior to the start of study and also that they had no known hypersensitivity to any antibacterial drugs. All blood collections were in accordance to protocols approved by the Institutional Review Board of Wockhardt Ltd, India. Written informed consent was obtained from study participants. Lipopolysaccharide (Sigma, USA) was used as stimulating agent for immune cells. Quantikine ELISA human cytokine kits were purchased from, R & D Systems, Inc, USA. Test compounds were prepared using literature procedures.
Heparinised whole blood samples were kept at room temperature and processed within 2 hours of collection. Blood was diluted 1:3 with sterile RPMI 1640 with L-glutamine and 25 mM HEPES (Gibco, Life Technologies; USA) and a final volume of 1 mL was added to each well of 24-well tissue culture treated flat-bottom polystyrene plates (eppendorf, Germany). Samples were stimulated
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with LPS (Lipopolysaccharide) reagent (10 ng/mL) with or without test
compounds or their respective vehicle control, and cultured for the 6 hours under
each experiment at 37°C in a humidified incubator at 5% CO2. After incubation,
the blood was centrifuged at 8000 rpm for 8 minutes at 4°C and supernatants were
collected at 6 hour intervals. The supernatants were stored at -80°C prior to the
detection of cytokines.
The supernatants were analysed for IL-6 and TNF-sandwich
immunoassay using a BioTek 96 well Plate Reader. The assay was performed
according to the manufacturer’s instructions (R & D Systems, Inc, USA) and
analysed with the BioTek 96 well Plate Reader software. The sensitivity of TNF-
method was 1.6 pg/mL and the assay could accurately detect cytokines in the
range of 15.6 – 1,000 pg/ml. The sensitivity of IL-6 method was 0.7 pg/mL and
the assay could accurately detect cytokines in the range of 3.13 – 200 pg/ml.
Supernatant cytokine concentrations of samples treated with test
compounds were expressed as a percentage compared to cytokine concentrations
induced by LPS alone, which were defined as 100%. Linear mixed models, which
take into account the possible dependence among measurements from the same
sample under different drug concentrations, were employed to analyse drug
concentration response data for each cytokine, stimulation condition and
antibacterial agent independently. Data were expressed as means ± standard error
of mean (SEM).The results were analysed via a one-way ANOVA with Graph pad
Prism 5 software, and Tukey’s post hoc test was used to compare the differences
between groups.
Example 1
Activity of Compound of formula (I), Azithromycin, Clarithromycin and
Telithromycin in modulating LPS-simulated cytokine release in whole blood
assay was evaluated using the above experimental procedure. In general, the
activity was evaluated with respect to Interleukin-6 (IL-6) and Tumor necrosis
factor α (TNF-α). The results are presented in Table 1 and 2, below.
N
S
N
N
O
O
O
O
O
N
HO
O
O
N
O
NH2
OMe
O
H
(I)
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Table 1. Effect of Compound of Formula (I), Azithromycin, Clarithromycin and Telithromycin on interleukin-6 (IL-6) release in whole blood assay after 6 hours of incubation
Treatment
Conc. (μg/mL)
IL-6 (n=5)
Absolute levels (pg/mL)
% Inhibition vs. positive control
Lipopolysaccharide (LPS)
0.01
6599 ± 1236
-
Compound of Formula (I)
2.5
6030 ± 1221
10 ± 3
5
5628 ± 1103
15 ± 4
10
5284 ± 1026
20 ± 4
20
4283 ± 866
35 ± 4
40
3734 ± 810
44 ± 4
Telithromycin
2.5
6240 ± 1066
4 ± 6
5
5949 ± 996
8 ± 3
10
5498 ± 892
15 ± 3
20
5174 ± 787
19 ± 4
40
4236 ± 737
33 ± 8
Azithromycin
2.5
6228 ± 1321
7 ± 6
5
6721 ± 1353
-1 ± 6
10
6489 ± 1253
2 ± 5
20
6453 ± 1198
0 ± 9
40
5372 ± 1051
17 ± 7
Clarithromycin
2.5
6404 ± 1057
2 ± 5
5
6742 ± 1267
-3 ± 7
10
6713 ± 1097
-4 ± 6
20
5670 ± 907
7 ± 13
40
6229 ± 1019
3 ± 6
As can be seen, Compound of Formula (I) and Telithromycin showed concentration-dependent inhibition of LPS-stimulated IL-6 release with 44% and 33% inhibition at highest concentration, respectively. Compound of Formula (I) showed statistically non-significant higher inhibition of IL-6 release compared to Telithromycin at all the tested concentrations. Azithromycin and Clarithromycin did not show any effect on LPS-stimulated IL-6 release. Thus, Compound of Formula (I) was found to be more potent modulator when compared to Telithromycin, Azithromycin and Clarithromycin.
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Table 2. Effect of Compound of Formula (I), Azithromycin, Clarithromycin and Telithromycin on Tumor necrosis factor α (TNF-α) release in whole blood assay after 6 hours of incubation
Treatment
Conc.
(μg/mL)
TNF-α (n=5)
Absolute levels (pg/mL)
% Inhibition vs. positive control
LPS
0.01
3622 ± 733
-
Compound of Formula (I)
2.5
2866 ± 673
23 ± 4
5
2638 ± 568
28 ± 4
10
2326 ± 549
38 ± 4
20
1869 ± 426
50 ± 3
40
1367 ± 348
64 ± 3
Telithromycin
2.5
3228 ± 699
12 ± 5
5
2915 ± 596
20 ± 4
10
2765 ± 534
23 ± 4
20
2312 ± 403
35 ± 4
40
1648 ± 254
52 ± 3
Azithromycin
2.5
3400 ± 767
8 ± 5
5
3462 ± 750
6 ± 4
10
3681 ± 727
-2 ± 4
20
4030 ± 662
-15 ± 8
40
3452 ± 602
2 ± 7
Clarithromycin
2.5
3422 ± 706
6 ± 5
5
3686 ± 648
-4 ± 5
10
4270 ± 815
-19 ± 6
20
4367 ± 611
-27 ± 10
40
3959 ± 567
-15 ± 10
As can be seen, Compound of Formula (I) and Telithromycin showed concentration-dependent inhibition of LPS- stimulated TNF–α release with 64 and 52% inhibition at highest concentration, respectively. Compound of Formula (I) showed statistically non-significant higher inhibition of TNF–α release compared to Telithromycin at all the tested concentrations. Azithromycin and Clarithromycin did not show any effect on LPS-stimulated TNF–α release. Thus, Compound of Formula (I) was found to be more potent modulator when compared to Telithromycin, Azithromycin and Clarithromycin.
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Claims
1. A pharmaceutical composition for modulating one or more
cytokines, said composition comprising a compound of Formula (I) or a
pharmaceutically acceptable salt thereof:
N
S
N
N
O
O
O
O
O
N
HO
O
O
N
O
NH2
OMe
O
H
(I)
2. The pharmaceutical composition according to Claim 1, wherein the
cytokine is one or more of Tumor necrosis factor-(TNF-), Interleukin-1 (IL-1),
Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-1
(IL-1), Interferon-g (IFN-g), and Granulocyte-macrophage colony stimulating
factor (GM-CSF).
3. A method for modulating one or more cytokines in a subject, said
method comprising administering to the subject a pharmaceutical composition
according to Claim 1.
4. The method according to Claim 3, wherein the cytokine is one or
more of Tumor necrosis factor-(TNF-), Interleukin-1 (IL-1), Interleukin-6 (IL-
6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-1(IL-1), Interferong
(IFN-g), and Granulocyte-macrophage colony stimulating factor (GM-CSF).
5. A method for modulating one or more cytokines in a subject, said
method comprising administering to the subject a compound of Formula (I) or a
pharmaceutically acceptable salt thereof:
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N
S
N
N
O
O
O
O
O
N
HO
O
O
N
O
NH2
OMe
O
H
(I)
6. The method according to Claim 5, wherein the cytokine is one or
more of Tumor necrosis factor-(TNF-), Interleukin-1 (IL-1), Interleukin-6 (IL-
6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-1(IL-1), Interferong
(IFN-g), and Granulocyte-macrophage colony stimulating factor (GM-CSF).
7. The pharmaceutical composition according to any one of the
claims 1-2, for use in modulating one or more cytokines in a subject, wherein the
cytokine is one or more of Tumor necrosis factor-(TNF-), Interleukin-1 (IL-1),
Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-1
(IL-1), Interferon-g (IFN-g), and Granulocyte-macrophage colony stimulating
factor (GM-CSF).
8. The pharmaceutical composition for modulating one or more
cytokines, comprising a compound of Formula (I) or a pharmaceutically
acceptable salt thereof according to Claim 1 and one or more pharmaceutically
acceptable excipients.