DESC:FIELD OF THE INVENTION
[0001] The present disclosure relates generally to formulations of anti-inflammatory therapeutic proteins. More specifically, the disclosure is directed to an oral formulation of anti-inflammatory proteins. Further, the disclosure also relates to a method of preparing the oral formulation.

BACKGROUND OF THE INVENTION
[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 present invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Biotechnology has seen a rise in the past few decades. This has influenced medicinal biochemistry as well. Therapeutic proteins are a class of proteins synthesized in the laboratory for pharmaceutical purposes- such as treatment of cancer, arthritis or diabetes. They have evolved as an alternative field of medical research owing to their biological compatibility, specificity, efficacy and high toleration levels compared to commonly administered drugs. However, therapeutic proteins are difficult to formulate especially, for oral consumption. They are usually anti-bodies however owing to stability issues their suitability for administration has been limited. Physical and chemical degradation of proteins is inevitable if not kept under proper conditions and temperature. Upon degradation, the proteins lose their pharmaceutical activity and instead may cause negative consequences in a subject’s body. Thus, stabilizing proteins is an essential requirement to ensure suitability for administration.
[0004] Inflammation is a natural immune response of the body for defense against pathogens, toxins, injury, etc. Non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids are widely used to reduce inflammation. Therapeutic proteins as anti-inflammatory agents with a potential to modulate protein functioning provide an alternative to conventional drugs. But when it comes to anti-inflammatory therapeutic proteins, the problem of making their formulations is even more difficult as a majority of these proteins are hydrophilic. Existing formulations are mostly intravenous formulations that are sensitive to initial break down by the enzymes. Oral formulations are not associated with stability for long durations.
[0005] Thus, there is a need in the art to develop formulations of anti-inflammatory proteins that are stable and orally deliverable.

OBJECTS OF THE INVENTION
[0006] An object of the present disclosure is to provide an oral formulation of anti-inflammatory proteins.
[0007] An object of the present disclosure is to provide an oral formulation of anti-inflammatory proteins that is stable and easy to prepare.
[0008] Another object of the present disclosure is to provide a method of preparation of an oral formulation of anti-inflammatory proteins.

SUMMARY OF THE INVENTION
[0009] 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 subject matter, nor is it intended to be used as an aid in determining the scope of the subject matter.
[0010] Aspects of the present disclosure provide an oral formulation of anti-inflammatory proteins for therapeutics.
[0011] In an aspect, the present disclosure provides an oral formulation for inflammation comprising (i) one or a plurality of anti-inflammatory protein(s); (ii) lactitol; and (iii) mannitol.
[0012] In an embodiment, the anti-inflammatory proteins may be selected from IL-4, IL-6, IL-10, TGF-ß, combinations thereof and the like. In an embodiment, the anti-inflammatory proteins maybe IL-4, IL-6, IL-10, and TGF-ß. In a preferred embodiment, the anti-inflammatory proteins are IL-4, IL-6, IL-10, and TGF-ß wherein the proteins IL-4, IL-6, IL-10, and TGF-ß may be present in the ratio of about 2:4:2:6.
[0013] In an embodiment, the anti-inflammatory proteins may be present in a range of about 10mg/ml to about 100mg/ml of the formulation.
[0014] In an embodiment, lactitol may be present in about 15% w/v to about 40% w/v aqueous solution of lactitol. Preferably, lactitol may be a 40% w/v aqueous solution.
[0015] In an embodiment, mannitol may be present in about 1% w/v to about 10% w/v aqueous solution of mannitol. Preferably, mannitol may be a 5% aqueous solution.
[0016] In an embodiment, the present disclosure provides an oral formulation for inflammation comprising: (i) one or a plurality of anti-inflammatory protein(s) in a range of about 10mg/ml to about 100mg/ml of the formulation; (ii) lactitol in a range of about 15% w/v to about 40% w/v aqueous solution of lactitol; and (iii) mannitol in a range of about 1% w/v to about 10% w/v aqueous solution of mannitol.
[0017] In an aspect, the present disclosure provides a method of stabilization of one or a plurality of anti-inflammatory protein(s), wherein the protein is stabilized by lactitol and mannitol.
[0018] In an aspect, the present disclosure provides a method for preparation of an oral formulation for inflammation comprising (i) one or a plurality of anti-inflammatory protein(s); (ii) lactitol; and (iii) mannitol.
[0019] In an embodiment, the method comprises the steps of: (a) dissolving each of the anti-inflammatory proteins in lactitol independently to give a solution; (b) mixing all the solutions and centrifuging; (c) replacing the supernatant with fresh lactitol; (d) slowly adding mannitol and freezing in liquid nitrogen to give a frozen formulation; and (e) thawing the frozen formulation to give the formulation.
[0020] Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0022] Figure 1 provides the high performance liquid chromatogram of the formulation as per an embodiment of the present disclosure.
[0023] Figure 2 is Western blot analysis of an embodiment of the oral formulation showing the anti-inflammatory proteins in the intact form after keeping at room temperature in the oral formulation developed.

DETAILED DESCRIPTION OF THE INVENTION
[0024] 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 within the spirit and scope of the present disclosure.
[0025] 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 definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0026] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0027] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe 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 are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0028] Various terms as used herein are shown below. To the extent a term used is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0029] As used in the description herein, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0030] 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.”
[0031] 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.
[0032] 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. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0033] 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 individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0034] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0035] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0036] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0037] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0038] The term ‘room temperature’ herein refers to a temperature in the range of about 20- 22ºC.
[0039] Aspects of the present disclosure provide an oral formulation of anti-inflammatory proteins for therapeutics.
[0040] In an embodiment, the present disclosure provides an oral formulation for inflammation comprising (i) one or a plurality of anti-inflammatory protein(s); (ii) lactitol; and (iii) mannitol.
[0041] In an embodiment, the anti-inflammatory proteins may be selected from IL-4, IL-6, IL-10, TGF-ß, combinations thereof and the like. In an embodiment, the anti-inflammatory proteins may be IL-4, IL-6, IL-10, and TGF-ß. In a preferred embodiment, the anti-inflammatory proteins maybe IL-4, IL-6, IL-10, and TGF-ß wherein the proteins IL-4, IL-6, IL-10, and TGF-ß may be present in the ratio of about 2:4:2:6.
[0042] In an embodiment, the anti-inflammatory proteins may be present in high concentrations in the formulation. The formulation effectively reduces swelling, pain, and infection related to inflammation.
[0043] In an embodiment, the anti-inflammatory proteins may be present in a range of about 10mg/ml to about 100mg/ml of the formulation.
[0044] In an embodiment, lactitol may be present in about 15% w/v to about 40% w/v aqueous solution of lactitol. Preferably, lactitol may be a 40% w/v aqueous solution.
[0045] In an embodiment, mannitol may be present in about 1% w/v to about 10% w/v aqueous solution of mannitol. Preferably, mannitol may be a 5% aqueous solution.
[0046] In an embodiment, the present disclosure provides an oral formulation for inflammation comprising: (i) one or a plurality of anti-inflammatory protein(s) in a range of about 10mg/ml to about 100mg/ml of the formulation; (ii) lactitol in a range of about 15% w/v to about 40% w/v aqueous solution of lactitol; and (iii) mannitol in a range of about 1% w/v to about 10% w/v aqueous solution of mannitol.
[0047] In an embodiment, the ratio of lactitol to mannitol may range from 2 to 5.
[0048] In an embodiment, the formulation may be a solid, liquid or semi-solid. Preferably the oral formulation is a liquid formulation.
[0049] In an embodiment, the formulation has storage stability.
[0050] In an embodiment, the formulation may further comprise pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers are those approved for use in oral formulations. The pharmaceutically acceptable carriers may be selected from buffer, solvent, emulsifier, surfactants and the like. The carriers do not affect the stability of the formulation.
[0051] In an embodiment, the oral formulation for inflammation comprises one or a plurality of anti-inflammatory protein(s); lactitol; mannitol; Labrafil M (15%), tween 80 - ethanol (35%), and water (50%).
[0052] In an embodiment, the formulation may be stable for extended periods of time upto a temperature of 37ºC. Unlike formulations known in the art, the presently disclosed formulation is suitable for oral consumption. The anti-inflammatory protein(s) are stabilized in the present formulation such that they retain their activity in the physiological environment. In an embodiment, the formulation is surprisingly synergistic. The present disclosure provides an oral formulation of anti-inflammatory proteins that is less viscous ensuring ease of manufacture, processing, finishing and delivery.
[0053] In an embodiment, the present disclosure provides an oral formulation for inflammation for use in treatment of inflammation caused by arthritis, inflammatory bowel disease, obesity, cardiovascular diseases, cancer, autoimmune diseases, neurological disorders and the like, wherein the formulation comprises (i) one or a plurality of anti-inflammatory protein(s); (ii) lactitol; and (iii) mannitol. However, a person of skill in the art would understand that the use of the formulation may be extended to associated diseases.
[0054] In an embodiment, the present disclosure relates to a method of treating inflammation in a subject by administering a therapeutically effective amount of the oral formulation for inflammation.
[0055] In an embodiment, the present disclosure provides a method of stabilization of one or a plurality of anti-inflammatory protein(s), wherein the protein is stabilized by lactitol and mannitol.
[0056] In an embodiment, the present disclosure provides a method for preparation of an oral formulation for inflammation comprising (i) one or a plurality of anti-inflammatory protein(s); (ii) lactitol; and (iii) mannitol.
[0057] In an embodiment, the method comprises the steps of: (a) dissolving each of the anti-inflammatory proteins in lactitol independently to give a solution; (b) mixing all the solutions and centrifuging; (c) replacing the supernatant with fresh lactitol; (d) adding mannitol and freezing in liquid nitrogen to give a frozen formulation; and (e) thawing the frozen formulation to give the formulation.
[0058] In an embodiment, the mixing of solutions of different anti-inflammatory proteins may be performed at room temperature. In an embodiment, mannitol may be added slowly, preferably drop-wise.
[0059] In an embodiment, thawing may be performed at room temperature for about 4 to 5 hours. In an embodiment, rate of thawing would depend on the ambient temperature, volume of formulation and the like.
[0060] Figure 2 is Western blot analysis of an embodiment of the oral formulation showing the anti-inflammatory proteins in the intact form after keeping at room temperature in the oral formulation developed.
[0061] Referring now to figure 2, it can be seen that the protein is stabilized by lactitol and mannitol.
EXAMPLES
[0062] 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 similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Materials and Methods:
[0063] All chemicals and proteins were procured from Sigma in pure form.
EXAMPLE 1: Preparation of oral formulation
[0064] An oral formulation was prepared comprising IL-4, IL-6, IL-10, and TGF-ß in the ratio 2:4:2:6. Individual anti-inflammatory molecules were taken and aqueous solution of 40% lactitol (100 ul) was added to each. The solutions were kept at room temperature thereafter they were mixed and centrifuged. The lactitol solution was discarded and 500 ul of 40% lactitol was added. 5% of mannitol solution was added drop-wise till up to 250 mL of the solution was prepared. The solution was then frozen in liquid nitrogen and kept once again at room temperature for 4 hours to give the oral formulation. The solution was subjected to high performance liquid chromatography (HPLC) after 24 hours and the proteins were detected via western blotting. Figure 1 provides the HPLC data showing three peaks. The peaks correspond to IL-4, IL-6, IL-10, and TGF-ß with overlapping peak of IL-6 & IL-10. This indicates the protein were intact in the formulation.
[0065] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The 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.

ADVANTAGES OF THE PRESENT INVENTION
[0066] The present disclosure provides an oral formulation of anti-inflammatory proteins that is stable and easy to prepare.
[0067] The present disclosure provides an oral formulation of anti-inflammatory proteins that is less viscous ensuring ease of manufacture, processing, finishing and delivery.
[0068] The present disclosure provides an oral formulation comprising high concentration of anti-inflammatory proteins for effective reduction in inflammation.

WE CLAIMS:

1. An oral formulation for inflammation comprising
(i) one or a plurality of anti-inflammatory protein(s);
(ii) lactitol; and
(iii) mannitol.
2. The oral formulation as claimed in Claim 1, wherein the anti-inflammatory proteins being selected from any or a combination of IL-4, IL-6, IL-10, TGF-ß.
3. The oral formulation as claimed in Claim 1, wherein the anti-inflammatory proteins are in a range of 10mg/ml to 100mg/ml of the formulation.
4. The oral formulation as claimed in Claim 1, wherein lactitol is present 15% w/v to 40% w/v aqueous solution of lactitol.
5. The oral formulation as claimed in Claim 1, wherein Mannitol is present 1% w/v to 10% w/v aqueous solution of mannitol.
6. The oral formulation as claimed in Claim 1, wherein the oral formulation for inflammation comprises:
(i) one or a plurality of anti-inflammatory protein(s) in a range of 10mg/ml to 100mg/ml of the formulation;
(ii) Lactitol in a range of 15% w/v to 40% w/v aqueous solution of Lactitol;
(iii) Mannitol in a range of 1% w/v to 10% w/v aqueous solution of Mannitol.
7. The oral formulation as claimed in Claim 1, wherein the anti-inflammatory proteins are IL-4, IL-6, IL-10, and TGF-ß; the proteins IL-4, IL-6, IL-10, and TGF-ß being present in a ratio of 2:4:2:6.
8. A method for preparation of an oral formulation for inflammation comprising
(i) one or a plurality of anti-inflammatory protein(s);
(ii) lactitol; and
(iii) mannitol.
9. The method as claimed in claim 8, wherein the method comprises the steps of:
(a) dissolving each of the anti-inflammatory proteins in lactitol independently to obtain a solution;
(b) mixing the solution and centrifuging;
(c) replacing a supernatant with lactitol;
(d) slowly adding mannitol to the solution of lactitol and the and the anti-inflammatory protein;
(e) freezing in liquid nitrogen to obtain a frozen formulation; and
(f) thawing the frozen formulation to obtain the formulation for usage.