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-18005-IN-NP
2
PHARMACEUTICAL COMPOSITIONS AND METHODS
RELATED PATENT APPLICATIONS
This application claims the priority to and benefit of Indian Provisional
Patent Application No. 201821028333 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
O
N
F
O
COOH
R
wherein R is H or COC(CH3)NH2.
WK-18005-IN-NP
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
O
N
F
O
COOH
R
wherein R is H or COC(CH3)NH2.
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.
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
WK-18005-IN-NP
4
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 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,
WK-18005-IN-NP
5
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
O
N
F
O
COOH
R
wherein R is H or COC(CH3)NH2.
In some embodiments, the compound of Formula (I) is:
N
O
N
F
HO
COOH
In some other embodiments, the compound of Formula (I) is:
WK-18005-IN-NP
6
N
O
N
F
HO
COOH
H N 2 NH
NH
NH2
. COOH . 4H2O
In some other embodiments, the compound of Formula (I) is:
N
O
N
F
O
COOH
H C 3
O
NH2
.CH3SO3H
.
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).
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.
WK-18005-IN-NP
7
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 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.
WK-18005-IN-NP
8
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
O
N
F
O
COOH
R
wherein R is H or COC(CH3)NH2.
In some embodiments, the compound used in the methods according to the
invention is selected from the following:
N
O
N
F
HO
COOH
;
N
O
N
F
HO
COOH
H N 2 NH
NH
NH2
. COOH . 4H2O
; or
N
O
N
F
O
COOH
H C 3
O
NH2
.CH3SO3H
.
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
WK-18005-IN-NP
9
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 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
LPS induced Ex-Vivo cytokine secretion in whole blood:
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
WK-18005-IN-NP
10
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 (LPS) (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 known literature procedures.
LPS in concentration of 10 ng/mL was used to study the release of IL-6, TNF-α, and IL-1β, whereas, 50 ng/mL was used for the GM-CSF. Heparinized whole blood samples (received from the human donor) were kept at room temperature and processed within 2 hrs of collection. Blood was diluted in 1:3 ratio with sterile RPMI 1640 and L-glutamine, and 25 mM HEPES (Gibco, Life Technologies; USA). A final volume of 1 mL was added to each well of 24-well tissue culture treated flat-bottom polystyrene plates (Eppendorf, Germany). The experimental plates were then incubated for 6 h except for GM-CSF release assay (incubated for 48 h) at 37 °C in a humidified incubator at 5% CO2. Samples were collected at 6 h and 48 h time point from respective wells, centrifuged at 8000 rpm for 8 minutes at 4°C, and the supernatant was stored at -80°C until analysis.
The supernatants were analysed for IL-6, TNF-, IL-1and GM-CSF by ELISA assay. 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.
Supernatant cytokine concentrations of samples treated with test compounds were expressed as absolute levels and percentage compared to cytokine concentrations induced by LPS alone, which were defined as 100%.
LPS induced In-Vivo cytokine production in rat:
Male Wistar rats, 10-12 weeks of age were received from the Animal House Facility, Wockhardt Research Centre, Aurangabad, India. The animals were housed in individually ventilated cages at a room temperature of 22 ± 2 °C, humidity 60 ± 10% with a 12:12 h light-dark cycle and have access to food and water ad-libitum. Animals were acclimatised at least for a week before initiation of the study. They were fasted overnight before the start of an experiment. During fasting and throughout the study, animals were kept on fasting grills not more than four animals/cages with free access to water throughout the study period. Following fasting, the blood was withdrawn from a retro-orbital puncture in micro-centrifuge tubes containing K2EDTA as anticoagulants at the concentration of 1.75 mg/mL of blood. LPS was administered at 5 (for TNF-α, IL-6, IL-1β) and 12.5 (for GM-CSF) mg/kg as a single intraperitoneal dose for sepsis induction. Test (Drug) compound and placebo were administered subcutaneously 30 min before the LPS administration. Blood samples were collected at 0 h (before LPS),
WK-18005-IN-NP
11
2 and 6 h after LPS administration, centrifuged and plasma stored at -80°C until
analysis.
The samples were analysed for IL-6, TNF-, IL-1and GM-CSF by
ELISA assay. 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
Data Analysis:
Statistical analysis was performed using One-Way ANOVA post-hoc
Tukey‘s test for the comparison of ex-vivo data (% inhibition of cytokines) in
more than two groups. Two-Way ANOVA post-hoc Bonferroni’s multiple
comparison test was applied for the comparison of in-vivo study data. Paired ttest
was used to compare the mean differences in ex-vivo absolute cytokine levels
between two groups. The data obtained was expressed at a significance level of
p<0.05. All data were presented as mean ± SEM. All statistical analyses were
carried out using GraphPad Prism version 5.00 for Windows (GraphPad Software,
San Diego, CA, USA).
Example 1
Activity of test Compound A, Levofloxacin (LVFX) and Linezolid (LNZ)
in modulating Lipopolysaccharide (LPS)-simulated cytokine release in human
whole blood (HWB) assay and in-vivo in rat was evaluated using the above
experimental procedure. In general, the activity was evaluated with respect to IL-
6, TNF-, IL-1and GM-CSF. The results are presented in Table 1-5, below.
N
O
N
F
HO
COOH
H N 2 NH
NH
NH2
. COOH . 4H2O
Compound A
WK-18005-IN-NP
12
Table 1. Effect of Compound A, Levofloxacin (LVFX) and Linezolid (LNZ) on pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, GM-CSF) release in LPS-stimulated HWB
Treatment
IL-6 (pg/mL)
TNF - α (pg/mL)
IL-1β (pg/mL)
GM-CSF (pg/mL)
Absolute levels
% inhibition
Absolute levels
% inhibition
Absolute levels
% inhibition
Absolute levels
% inhibition
LPS + Dextrose
11170.5±1773.1
NA
4371.6 ± 743.0
NA
4414.8 ± 892.6
NA
15.4 ± 4.0
NA
Levofloxacin
(25 μg/mL)
9093.7± 1684.6**
19.4± 3.6
3393.7 ± 659.7***
23.4 ± 1.9
1964.5 ± 506.1**
57.2 ± 3.6##
11.7 ± 3.7
25.4 ± 9.2
Levofloxacin
(100 μg/mL)
5209.5± 1236.7***
55.0± 3.2+++ ###, $$$
1986.9 ± 456.0**
56.0 ± 2.6+++, $$$
214.3 ± 90.9**
95.6 ± 0.9+++, ###, $$$, @@@
4.5 ± 1.3*
68.6 ± 7.0+, ##, $$
LPS + DMSO
11784.9± 1809.2
NA
4917.3 ± 1164.8
NA
6098.3 ± 1439.6
NA
19.4 ± 4.8
NA
Linezolid
(200 μg/mL)
9757.3± 1856.0***
18.6± 2.6
2509.0 ± 668.3**
50.0 ± 1.8+++, $$$
3546.0 ± 804.3*
40.8 ± 2.8
17.5 ± 5.0
10.6 ± 8.8
Lipopolysaccharide (LPS)
10446.9± 1657.8
NA
4402.6 ± 743.7
NA
4182.0 ± 815.9
NA
14.4 ± 3.8
NA
Compound A (15 μg/mL)
8430.3± 1746.7***
21.2± 3.1
3146.9±686.2**
30.0 ± 4.6
2615.2±567.3**
38.0 ± 2.1
10.8± 2.5
18.6 ± 6.6
Compound A (30 μg/mL)
5714.7± 1415.4***
47.6± 3.8+++, ###, $$$
2183.8± 465.4**
51.6 ± 2.3+++ $$$
1438.9 ± 392.7**
67.0 ± 2.8 $$$, ###
9.2 ± 2.3*
33.6 ± 5.9
+p<0.05, +++p<0.001 vs. Levofloxacin (25 μg/mL), ##p<0.01, ### p<0.001 vs. Linezolid, $$ p<0.01, $$$ p<0.001 vs. Compound A (15 μg/mL), @@@ vs. Compound A (30 μg/mL) by One Way ANOVA post-hoc Tukey’s Multiple Comparison test; *p< 0.05, **p<0.01, ***p<0.001 vs. respective control group calculated by paired t-test. LPS (lipopolysaccharide), IL-6 (interleukin-6), TNF-α (tumour necrosis factor - α), IL-1β (interleukin-1β), GM-CSF (granulocyte-macrophage-colony-stimulating factor)
Effect on IL-6:
Compound A 15 and 30 μg/mL concentration showed significant reduction in IL-6 levels 8430.3±1746.7 pg/mL (p<0.001) and 5714.7 ± 1415.4 pg/mL (p<0.001) as compared to LPS control (10446.9 ± 1657.8 pg/mL) in HWB assay, respectively. Similarly, Levofloxacin (25,100 μg/mL) and Linezolid (200 μg/mL) demonstrated significant reduction in IL-6 levels (p<0.001) as compared to respective control, respectively.
Compound A caused 21.2±3.1% and 47.6±3.8% inhibition of IL-6 release at 15 and 30 μg/mL, respectively, while Levofloxacin showed 19.4±3.6% and
WK-18005-IN-NP
13
55±3.2% inhibition of IL-6 release at 25 and 100 μg/ml concentrations, respectively. Thus, Compound A was found to be almost three times more effective compared to Levofloxacin. Linezolid at 200 μg/mL concentration demonstrated 18.6±2.6% inhibition of IL-6 release. These results indicate that Compound A is 10 times more effective compared to Linezolid in inhibiting IL-6 release (p<0.001, Table 1). Compound A and Levofloxacin inhibited the production of IL-6 in a dose-dependent fashion.
Effect on TNF-α :
Compound A at doses 15 and 30 μg/mL caused significant reduction in TNF-α levels 3146.9 ± 686.2 pg/mL (p<0.01) and 2183.8 ± 465.4 pg/mL (p<0.01) as compared to LPS control (4402.6 ± 743.7 pg/mL) in HWB assay, respectively. Similarly, Levofloxacin (25 and 100 μg/mL) and Linezolid (200 μg/mL) showed significant reduction in TNF-α levels as compared to control, respectively.
Compound A at 15 and 30 μg/mL demonstrated 30±4.6% and 51.6±2.3% inhibition, whereas, Levofloxacin at 25 and 100 μg/ml concentrations showed 23.4±1.9% and 56±3% inhibition, respectively. Linezolid at 200 μg/mL concentration demonstrated 50±2% inhibition of TNF-α release. Overall, Compound A was found to be three times more effective than Levofloxacin and six times more effective than Linezolid in inhibiting TNF-α release (Figure 1). Compound A and Levofloxacin inhibited the production of TNF-α in a dose-dependent fashion.
Effect on IL-1β:
Compound A (15 and 30 μg/mL) demonstrated significant reduction in IL-1β levels (p<0.01) compared to LPS control. Similarly, Levofloxacin and Linezolid showed significant reduction in IL-1β levels (p<0.01) as compared to respective control. Compound A showed 38±2.1% and 67±2.8% inhibition of IL-1β release at 15 and 30 μg/mL concentrations, respectively. Levofloxacin exhibited 57.2±3.6% and 95.6±0.9% inhibition of IL-1β release at 25 and 100 μg/ml concentrations, respectively. Linezolid at 200 μg/mL concentration demonstrated 40.8±2.8% inhibition of IL-1β release. Thus, Compound A was 10 times more effective compared to Linezolid (Table 1).
Effect on GM-CSF:
Compound A (30 μg/mL) demonstrated significant reduction in GM-CSF levels compared to LPS control (9.2 vs. 14.4 pg/mL, p<0.05). Similarly, Levofloxacin 100 μg/mL showed significant reduction in GM-CSF levels compared to LPS+Dextrose control (4.5 vs. 15.4 pg/mL, p<0.05). Compound A at 15 and 30 μg/mL concentrations demonstrated 18.6±6.6% and 33.6±5.9% inhibition, whereas, Levofloxacin at 25 and 100 μg/ml concentrations showed 25.4±9.2% and 68.6±7% inhibition, respectively. Linezolid at 200 μg/mL concentration demonstrated 10.6±8.8% inhibition of GM-CSF release. Overall,
WK-18005-IN-NP
14
Compound A was found to be equally effective as Levofloxacin and twenty times more effective than Linezolid in inhibiting GM-CSF release (Table 1).
Table 2. Effect of Compound A and Levofloxacin on TNF-α release in LPS-induced sepsis rat model
Treatment
Tumour necrosis factor-α (TNF-α)
Absolute levels (pg/mL)
% Inhibition
0 h
1 h
2 h
0 h
1 h
2 h
Placebo control
0.7 ± 0.3
0.0 ± 0.0
0.0 ± 0.0
-
-
-
LPS Control
(5mg/kg)
1.3 ± 0.4
7827.0 ± 1906.7###
11114.1 ± 4413.4###
-
-
-
Levofloxacin 50 mg/kg
0.7 ± 0.1
5295.2 ± 1064.3
3641.9 ± 364.6***
-
32.4 ± 13.6
67.2 ± 3.3
Compound A 50 mg/kg
0.5 ± 0.2
4668.1 ± 599.8
3765.4 ± 776.7***
-
40.4 ± 7.7
66.1 ± 7.0
###p<0.001 vs. placebo control, ***p<0.001 vs. LPS control by Two Way ANOVA post-hoc Bonferroni’s Multiple comparison test
Table 3. Effect of Compound A and Levofloxacin on IL-6 release in LPS-induced sepsis rat model
Treatment
Interleukin 6 (IL-6)
Absolute levels (pg/mL)
% Inhibition
0 h
2 h
6 h
0 h
2 h
6 h
Placebo control
18.3 ± 3.2
288.2 ± 57.5
303.9 ± 111.1
-
-
-
LPS Control
(5mg/kg)
21.6 ± 4.4
46990.2 ± 3096.1###
73649.9 ± 12428.8###
-
-
-
Levofloxacin 50 mg/kg
16.3 ± 2.1
42578.3 ± 4267.2
47330.9 ± 7501.7**
-
9.4 ± 9.1
35.7 ± 10.2
Compound A 50 mg/kg
16.6 ± 3.0
36949.4 ± 3787.2
54128.6 ± 15871.1*
-
21.4 ± 8.1
31.7 ± 18.5
###p<0.001 vs placebo control, *p<0.05, **p<0.01, vs LPS control by Two Way ANOVA post hoc Bonferroni’s Multiple comparison test
Table 4. Effect of Compound A and Levofloxacin on IL-1β release in LPS-induced sepsis rat model
Treatment
Interleukin-1β (IL-1β)
Absolute levels (pg/mL)
% Inhibition
0 h
2 h
6 h
0 h
2 h
6 h
Placebo control
14.4 ± 2.2
172.2 ± 49.8
214.4 ± 47.6
-
-
-
LPS Control
(5mg/kg)
18.7 ± 6.8
1450.4 ± 85.3***
1565.4 ± 232.1***
-
-
-
Levofloxacin 50
12.0 ±
1241.1 ±
1288.2 ±
-
14.4 ±
17.7 ±
WK-18005-IN-NP
15
mg/kg
3.8
150.3
215.2
10.4
13.7
Compound A 50 mg/kg
21.4 ± 5.8
1388.8 ± 181.5
1402.6 ± 278.8
-
4.2 ± 12.5
10.4 ± 17.8
***p<0.001 vs placebo control by Two Way ANOVA post hoc Bonferroni’s Multiple comparison test
Table 5. Effect of Compound A and Levofloxacin on GM-CSF release in LPS-induced sepsis rat model.
Treatment
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
Absolute levels (pg/mL)
% Inhibition
0 h
2 h
0 h
2 h
Placebo control
0.0 ± 0.0
0.0 ± 0.0***
-
-
LPS Control (12.5 mg/kg))
0.0 ± 0.0
3.4 ± 0.9###
-
-
Levofloxacin 50 mg/kg
0.0 ± 0.0
1.2 ± 0.1***
-
65.8 ± 2.3
Compound A 50 mg/kg
0.0 ± 0.0
1.1 ± 0.3**
-
68.2 ± 7.2
###p<0.001 vs. placebo control, ***p<0.001 vs LPS control by Two Way ANOVA post hoc Bonferroni’s Multiple comparison test.
In line with our ex-vivo observations, we found a selective down-modulation of TNF-α, IL-6 and GM-CSF levels in the course of endotoxin triggered sepsis, whereas, IL-1β cytokine remained unaffected by the treatment (Table 2-5). Compound A and Levofloxacin showed significant reduction in TNF-α levels compared to LPS control at 2 h. Overall, Compound A was equally effective as Levofloxacin at 50 mg/kg in LPS-induced sepsis rat model (Table 2). Compound A and Levofloxacin demonstrated significant reduction in IL-6 levels 54128.6 pg/mL (p<0.05) and 47330.9 pg/mL (p<0.01) compared to LPS control (73649.9 pg/mL) at 6 h, respectively. The observed reductions in IL-6 levels were 31.7% and 35.7% in Compound A and Levofloxacin group, respectively. Thus, no significant difference between Compound A and Levofloxacin was observed on IL-6 production (Table 3). Compound A and Levofloxacin did not show significant reduction in IL-1β levels compared to LPS control (Table 4). Significant reduction in GM-CSF levels were observed with Compound A and Levofloxacin (1.1 and 1.2 pg/mL, p<0.001) compared to LPS control (3.4 pg/mL) at 6h, respectively.
Example 2
Activity of test Compound B and Linezolid 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 6 and 7, below.
WK-18005-IN-NP
16
N
O
N
F
O
COOH
H C 3
O
NH2
.CH3SO3H
Compound B
Table 6. Effect of Compound B and Linezolid on Interleukin-6 (IL-6) release in
whole blood assay after 6 hours of incubation
Treatment
Conc.
(μg/mL)
IL-6 (n=1)
Absolute levels
(pg/mL)
% Inhibition vs.
positive control
LPS 0.01 5909 -
Compound B 25 2452 42
LPS + Dimethyl sulfoxide 0.01 7158 -
Linezolid 200 6721 6
As can be seen, Compound B showed significant inhibition of LPSstimulated
IL-6 release. The data also indicates that Compound B is more potent
compared to Linezolid.
Table 7. Effect of Compound B and Linezolid on Tumor necrosis factor α (TNF-
α) release in whole blood assay after 6 hours of incubation
Treatment
Conc.
(μg/mL)
TNF-α (n=1)
Absolute
levels (pg/mL)
% Inhibition vs.
positive control
LPS 0.01 2579 -
Compound B 25 1267 51
LPS + Dimethyl sulfoxide 0.01 2996 -
Linezolid 200 2886 3.6
As can be seen, Compound B showed significant inhibition of LPSstimulated
TNF-α release. The data also indicates that Compound B is potent
compared to Linezolid.
WK-18005-IN-NP
17
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
O
N
F
O
COOH
R
wherein R is H or COC(CH3)NH2.
2. The pharmaceutical composition according to Claim 1, wherein the
compound of formula (I) is selected from:
N
O
N
F
HO
COOH
;
N
O
N
F
HO
COOH
H N 2 NH
NH
NH2
. COOH . 4H2O
; or
N
O
N
F
O
COOH
H C 3
O
NH2
.CH3SO3H
.
3. 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
WK-18005-IN-NP
18
(IL-1), Interferon-g (IFN-g), and Granulocyte-macrophage colony stimulating
factor (GM-CSF).
4. A method for modulating one or more cytokines in a subject, said
method comprising administering to the subject a pharmaceutical composition
according to any one of the Claims 1-2.
5. The method according to Claim 4, 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).
6. 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
O
N
F
O
COOH
R
wherein R is H or COC(CH3)NH2.
7. The method according to Claim 6, wherein the compound of
formula (I) is selected from:
N
O
N
F
HO
COOH
;
N
O
N
F
HO
COOH
H N 2 NH
NH
NH2
. COOH . 4H2O
; or
WK-18005-IN-NP
19
N
O
N
F
O
COOH
H C 3
O
NH2
.CH3SO3H
.
8. The method according to Claim 6 or 7, 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).
9. The pharmaceutical composition according to any one of the
claims 1-3, 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).
10. The pharmaceutical composition for modulating one or more
cytokines, comprising a compound of Formula (I) or a pharmaceutically
acceptable salt thereof according to any one of claims 1 to 2 and one or more
pharmaceutically acceptable excipients.
WK-18005-IN-NP