FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
Title: TOPICAL COMPOSITION COMPRISING TACROLIMUS
GLENMARK PHARMACEUTICALS LIMITED,
An Indian Company registered under
The Companies Act, 1956, India
and having its office at
Glenmark House, HDO - Corporate Bldg,
Wing A, B. D. Sawant Marg,
Chakala, Andheri (East),
MUMBAI- 400 099
The following specification particularly describes the invention and the manner in which it is to be performed.

TOPICAL COMPOSITION COMPRISING TACROLIMUS
PRIORITY
This patent application claims priority to Indian Provisional Patent Application No. 2890/MUM/2009 (filed on December 15, 2009), the contents of which are incorporated by reference herein.
TECHNICAL FIELD The present patent application relates to a topical composition comprising tacrolimus. Particularly, the present patent application relates to a stable topical composition comprising tacrolimus and a pharmaceutically acceptable solvent; process of preparing such composition; and its use for treating an inflammatory or immunological disorder in a subject.
BACKGROUND
Tacrolimus (Formula 1) is a macrolide immunosuppressant and is represented by the chemical formula, [3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*, 9E, 12R*,14R*,15S*,16R*,18SM9S*l26aR*]I-5,6,8,11,12,13,14,15,16,17,18, 19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1 -methylethenyl]-14,16-dimethoxy-4,10, 12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1 -c][1,4] oxaazacyclothcosine-1,7,20,21 (4H,23H)-tetrone, monohydrate.


Tacrolimus for topical application is commercially available as ointment in the United States as PROTOPIC® by Astellas Pharma. Tacrolimus ointment is indicated as second-line therapy for the short-term and non-continuous chronic treatment of moderate to severe atopic dermatitis in non-immunocompromised adults and children who have failed to respond adequately to other topical prescription treatments for atopic dermatitis, or when those treatments are not advisable.
U.S. Patent No. 5385907 discloses ointment compositions of tacrolimus. U.S. Patent Nos. 6352998 and 5939427 also disclose topical compositions of tacrolimus.
However, there still exists a need for stable topical compositions of tacrolimus.
SUMMARY OF THE INVENTION
The present invention provides a topical composition comprising tacrolimus and a pharmaceutically acceptable solvent.
The inventors of the present invention have surprisingly found that a topical composition comprising tacrolimus dispersed in dimethyl isosorbide shows increased stability as compared to the compositions that are devoid of dimethyl isosorbide.
Thus, in an embodiment, the present invention refates to a stable topical composition comprising tacrolimus and dimethyl isosorbide.
In the context of present invention, the topical composition can be a semi-solid formulation or a liquid formulation. Thus, the topical composition of the present invention can be an ointment, gel, cream, paste, suspension, emulsion, solution, lotion, dispersion, shampoo, and the like. Preferably, the topical composition of the present invention is an ointment.
In a further embodiment, the present invention relates to a stable topical ointment composition comprising therapeutically effective amount of tacrolimus and dimethyl isosorbide.
The therapeutically effective amount of tacrolimus, in the context of present invention ranges from about 0.001 % to about 2 % w/w, or preferably

from about 0.01 % to about 1 % w/w (based on the total weight of the composition).
In an embodiment, the present invention relates to a stable topical composition comprising from about 0.01 % to about 1 % w/w tacrolimus, and dimethyl isosorbide.
In the topical composition of the present invention, the weight ratio of tacrolimus to dimethyl isosorbide ranges from about 1: 1 to about 1:50, or from about 1:5 to about 1 ;40. Preferably, the weight ratio of tacrolimus to dimethyl isosorbide ranges from about 1: 10 to about 1:30, or from about 1:15 to about 1:25.
The content of dimethyl isosorbide in the topical composition of the present invention ranges from about 0.2 to about 25 % w/w, or from about 0.5 to about 20% w/w.
In a specific embodiment, the present invention relates to a stable topical ointment composition comprising from about 0.01 % to about 0.5 % w/w tacrolimus, and from about 0.5 to about 10 % w/w dimethyl isosorbide.
The present invention also relates to use of therapeutically effective amount of tacrolimus and dimethyl isosorbide in preparation of a topical composition for the treatment of an inflammatory or immunological disorder in a subject in need thereof.
In another embodiment, the present invention relates to a stable topical composition comprising therapeutically effective amount of tacrolimus and dimethyl isosorbide for the treatment of an inflammatory or immunological disorder in a subject in need thereof.
The present invention also provides a process for the preparation of a topical composition that comprises tacrolimus and dimethyl isosorbide. Preferably, the process comprises dispersing tacrolimus in dimethyl isosorbide, and further formulating this dispersion to get the topical composition.

DETAILED DESCRIPTION
The terms used herein are defined as follows. If a definition set forth in the present application and a definition set forth earlier in a provisional application from which priority is claimed are in conflict, the definition in this non-provisional application shall control the meaning of the terms.
The term "topical composition" (synonymously, "topical formulation") is used herein to refer to a pharmaceutical composition that is intended for topical (or local) application to the affected skin or mucosa regions of a subject. Such a topical composition can be in the form of an ointment, gel, cream, paste, suspension, emulsion, solution, lotion, dispersion, shampoo, and the like.
The term "topical" as employed herein relates to the use of the active ingredient incorporated in a suitable pharmaceutical carrier, and applied at the site of the disease for exertion of local action. Accordingly, such topical compositions include those pharmaceutical forms in which the active ingredient is applied externally by direct contact with the skin surface (including hair and scalp) of the subject to be treated.
Generally, the terms "topical application" or "local application" refer to external application of an active agent or its composition to the hair, scalp or skin of the subject.
As used herein, the term "subject" as used herein refers to a mammal, and preferably a human.
As used herein, the terms "treating" or "treatment" of a state, disorder or condition mean: (1) inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or (2) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the subject or to the physician.
The term "pharmaceutical acceptable" as used in connection with components includes those components approved by a governmental

regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, such as humans.
As used herein, the terms "effective amount" or a "therapeutically effective amount" of a drug refers to a non-toxic but sufficient amount of the drug to provide the desired effect. The effective amount will vary depending on the disease and its severity and the age, weight, physical condition and responsiveness of the mammal to be treated.
The present invention provides a topical composition comprising tacrolimus and a pharmaceutically acceptable solvent. Generally, it has been observed that the topical composition of tacrolimus has inherent problems related to its stability, particularly in the ointment dosage form.
The inventors of the present invention have surprisingly discovered that a topical composition comprising tacrolimus dispersed in dimethyl isosorbide shows increased stability over compositions that are devoid of dimethyl isosorbide. Particularly, the inventors of the present invention surprisingly found that a topical composition comprising tacrolimus dispersed in dimethyl isosorbide shows improved stability as compared to the topical compositions that contain tacrolimus and few other commonly used solvents such as propylene carbonate, propylene glycol, benzyl alcohol, and diethylene glycol monoethyl ether (Transcutol® P).
Thus, in an embodiment, the present invention relates to a stable topical composition comprising tacrolimus and dimethyl isosorbide.
For example, the tacrolimus can be dispersed or dissolved in dimethyl isosorbide.
In the context of present invention, the topical Composition can be a semi-solid formulation or a liquid formulation. Thus, the topical composition of the present invention can be an ointment, gel, cream, paste, suspension, emulsion, solution, lotion, dispersion, shampoo, and the like. Preferably, the topical composition of the present invention is an ointment.
Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide

for optimum drug delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency. As with other carriers or vehicles, an ointment base should be inert, stab)e, nonirhtating and nonsens'itizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable-bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (0/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight; again, see Remington: The Science and Practice of Pharmacy for further information.
Creams, as also well known in the art, are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic, or amphoteric surfactant.
Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from a single-phase aqueous gel. The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethyl cellulose or the like as a base.

In a further embodiment, the present invention provides a topical composition in the form of an ointment, wherein the composition comprises tacrolimus and dimethyl isosorbide.
The therapeutically effective amount of tacrolimus, in the context of present invention ranges from about 0.001 % to about 2 % w/w, or preferably from about 0.01 % to about 1 % w/w (based on the total weight of the composition).
in an embodiment, the present invention relates to a stable topical composition comprising from about 0.01 % to about 1 % w/w tacrolimus, and dimethyl isosorbide.
In the topical composition of the present invention, the weight ratio of tacrolimus to dimethyl isosorbide ranges from about 1:1 to about 1:50, or from about 1:5 to about 1:40. Preferably, the weight ratio of tacrolimus to dimethyl isosorbide ranges from about 1: 10 to about 1:30, or from about 1:15 to about 1:25.
The content of dimethyl isosorbide in the topical composition of the present invention ranges from about 0.2 to about 25 % w/w, or from about 0.5 to about 20 % w/w.
In a specific embodiment, the present invention relates to a stable topical ointment composition comprising from about 0.01 % to about 0.5 % w/w tacrolimus, and from about 0.5 to about 10 % w/w dimethyl isosorbide.
In the context of the present invention, a topical composition of tacrolimus is said to be stable if the composition should contain not more than 4.0 % w/w of total impurities at initial sampling (i.e., at to) within 1 month after the preparation of the topical composition. Preferably, the topical composition should contain not more than 3.0 % w/w of total impurities at to-
Apart from dimethyl isosorbide, the topical composition of the present invention can contain pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients include, but are not limited to, chelating agents, preservatives, surfactants, co-solvents, absorbents, antioxidants, emollients, and mixtures thereof. Examples of these excipients are described in, for example, Howard C. Ansel et. a/., Pharmaceutical

Dosage Forms and Drug Delivery Systems, (7th Ed. 1999); Alfonso R. Gennaro et a/., Remington: The Science and Practice of Pharmacy, (20th Ed. 2000); and A. Kibbe, Handbook of Pharmaceutical Excipients, (3rd Ed. 2000), the contents of which are incorporated by reference herein.
Examples of emollients include, but are not limited to, caprylic/capric triglyerides, castor oil, ceteareth-20, ceteareth-30, cetearyl alcohol, ceteth 20, cetostearyl alcohol, cetyl alcohol, cetyl stearyl alcohol, cocoa butter, diisopropyl adipate, glycerin, glyceryl monooleate, glyceryl monostearate, glyceryl stearate, isopropyl myristate, isopropyl palmitate, lanolin, lanolin alcohol, hydrogenated lanolin, liquid paraffins, linoleic acid, mineral oil, oleic acid, white petrolatum, polyethylene glycol, polyoxyethylene glycol fatty alcohol ethers, polyoxypropylene 15-stearyl ether, propylene glycol stearate, squalane, steareth-2 or -100, stearic acid, stearyl alcohol, urea and mixtures thereof.
Examples of conditioners include, but not limited to silicone compound (e.g., amodimethicone, dimethicone, trimethylsilyl amodimethicone, cyclomethicone or dimethiconol) isododecane and mixtures thereof.
Suitable preservatives include, by way of example and without limitation, phenoxyethanol, butylated hydroxytoluene, parabens such as methylparaben and propylparaben, propylene glycols , sorbates, urea derivatives such as diazolindinyl urea, and the like and mixtures thereof.
Examples of co-solvents include, but are not limited to, tetrahydrofuran, isopropyl alcohol, propylene glycol, liquid petrolatum, ether, petroleum ether, alcohols (e.g., methanol, ethanol and higher alcohols), aromatics (e.g., benzene and toluene), alkanes (e.g., pentane, hexane and heptane), ketones (e.g., acetone and methyl ethyl ketone), chlorinated hydrocarbons (e.g., chloroform, carbon tetrachloride, methylene chloride and ethylene dichloride), alkyl pyrolidones (e.g., caprylyl pyrrolidone and lauryl pyrrolidone), acetates (e.g., ethyl acetate), oils (e.g., isopropyl myristate, diisopropyl adipate and mineral oil) its salts and mixtures thereof.
In another embodiment, the present invention relates to a stable topical composition comprising therapeutically effective amount of tacrolimus and

dimethyl isosorbide for the treatment of an inflammatory or immunological disorder in a subject in need thereof.
The present invention also provides a process for the preparation of a topical composition that comprises tacrolimus and dimethyl isosorbide. Preferably, the process comprises dispersing tacrolimus in dimethyl isosorbide, and further formulating this dispersion to get the topical composition.
The following examples are merely illustrative of the invention. The examples should not be read as limiting the scope of the invention.
EXAMPLES
EXAMPLE 1: Ointment composition of tacrolimus.

Sr. No. Ingredients Composition (% w/w)
1. Tacrolimus 0.105
2. White Soft Paraffin 74.39
3. Liquid Paraffin 17.00
4. White Beeswax 3.50
5. Hard Paraffin 3.00
6. Dimethyl Isosorbide (Arlasolve®) 2.00
Manufacturing process:
1. White soft paraffin, liquid, paraffin, beeswax and hard paraffin were melted together in a steam-jacketed stainless steel vessel heated at 70-72 °C.
2. Dimethyl isosorbide was placed in a steam-jacketed stainless steel vessel and heated at 55-60 °C.
3. Tacrolimus was added to the dimethyl isosorbide of Step 2 and dispersed under stirring to obtain a dispersion.

4. The dispersion of Step 3 was gradually added to the mixture of Step 1 under stirring to obtain dispersion.
5. Under homogenization, the dispersion of Step 4 was allowed to cool to about 40-45 0C and allowed to congeal into ointment.
6. The ointment of Step 5 was filled into aluminum tubes and crimped.
COMPARATIVE EXAMPLES A-D: Topical compositions of tacrolimus (not part of the invention)

Sr. No. Ingredients Composition (% w/w)

Comparative Example A Comparative Example B Comparative Example C Comparative Example D
1. Tacrolimus 0.105 0.105 0.105 0.105
2. White Soft Paraffin 74.39 74.39 74.39 74.39
3. Liquid Paraffin 17.00 17.00 17.00 17.00
4. White Beeswax 3.50 3.50 3.50 3.50
5. Hard Paraffin 3.00 3.00 3.00 3.00
6. Propylene Carbonate 2.00 ~ ~ —
7. Propylene Glycol — 2.00 — —
8. Diethylene
Glycol
Monoethyl
Ether
(Transcutol
P) — — 2.00 —
9. Benzyl Alcohol - -- 2.00
Manufacturing process:
1. White soft paraffin, liquid, paraffin, beeswax and hard paraffin were melted together in a steam-jacketed stainless steel vessel heated at 70-72 °C.

2. Propylene Carbonate/Propylene Glycol/ Diethylene Glycol Monoethyl Ether/Benzyl Alcohol was placed in a steam-jacketed stainless steel vessel and heated at 55-60 °C.
3. Tacrolimus was added to Propylene Carbonate/Propylene Glycol/ Diethylene Glycol Monoethyl Ether/Benzyl Alcohol of Step 2 and dispersed under stirring to obtain dispersion.
4. The dispersion of Step 3 was gradually added to the mixture of Step 1 under stirring to obtain dispersion.
5. Under homogenization, the dispersion of Step 4 was allowed to cool to about 40-45 °C and allowed to congeal into ointment.
6. The ointment of Step 5 was filled into aluminum tubes and crimped.
EXAMPLE 2: Stability data of the Example 1 and Comparative Examples A-D Stability Pack: Laminated tube
Storage condition: Temperature of about 40 °C and about 75 % relative humidity for 3 months (3 M).

Composition Example Stability data by HPLC

Total Impurities
(%w/w) Assay
(%w/w)

Initial 3M Initial 3M
Example 1 1.80 5.69 100.74 100.2
Comparative Example A 5.40 6.39 105.70 93.8
Comparative Example B 6.25 6.01 98.08 77.1
Comparative Example C 4.81 7.39 99.96 74.9
Comparative Example D 6.04 6.63 91.71 71.9
Brief analytical method:

A, For related substances (RS):
Sample equivalent to 5 mg of tacrolimus was dispersed in 100 ml n-heptane and extracted with the mobile phase to obtain test sample.
A reversed phase HPLC method was developed. The analysis was performed using C18 HPLC column (Inertsil ODS 3V, 150 mm x 4.6 mm, 5 u). The mobile phase was a mixture of buffer (6.89 gm of Potassium dihydrogen orthophosphate in 800 ml water and 0.57ml of orthophosphoric acid, made up the volume with water to 1000 ml) and a solvent mixture (Acetonitrile and Methanol in the ratio of 5:1) in the ratio 43:57 v/v. The flow rate was 1.5 ml/min; injection volume of 100 μl; and column temperature of 40 °C. Suitable dilutions of the placebo, standard and test samples were injected in the column. Areas for RS in the chromatogram were integrated, and accordingly the total RS was determined.
B. For Assay:
Sample equivalent to 5 mg of tacrolimus was dispersed in 100 ml n-heptane and extracted with the mobile phase to obtain test sample. A reversed phase HPLC method was developed. The analysis was performed using C18 HPLC column (Discovery RP-18, 150 mm x 4.6 mm, 5u). The mobile phase was a mixture of buffer (6.89 gm of Potassium dihydrogen orthophosphate in 800 ml water and 0.57ml of orthophosphoric acid, made up the volume with water to 1000 ml), acetonitrile and methanol in the ratio 40:50:10 v/v. The flow rate was 1.5 ml/min; injection volume of 100 pi; and column temperature of 60 °C. Suitable dilutions of the placebo, standard and test samples were injected in the column. The assay values were determined by comparing with the internal standard (of betamethasone dipropionate of concentration 24 ug/ml).
All publications, patents, and patent applications cited in this application are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.

CLAIMS
We claim:
1. A stable topical composition comprising tacrolimus and dimethyl isosorbide.
2. The topical composition according to claim 1, wherein the composition is an ointment.
3. The topical composition according to claim 1 or 2, wherein the composition comprises from 0.001 % to 2 % w/w tacrolimus.
4. The topical composition according to claim 1 or 2, wherein the composition comprises from 0.01 % to about 1 % w/w tacrolimus.
5. The topical composition according to claim 1 or 2, wherein the weight ratio of tacrolimus to dimethyl isosorbide ranges from 1: 1 to 1:50.
6. The topical composition according to claim 1 or 2, wherein the weight ratio of tacrolimus to dimethyl isosorbide ranges from 1: 10 to 1:30.
7. The topical composition according to claim 1 or 2, wherein the weight ratio of tacrolimus to dimethyl isosorbide ranges from 1: 15 to 1:25
8. The topical composition according to claim 1 or 2, wherein the content of dimethyl isosorbide ranges from 0.2 to 25 % w/w.
9. The topical composition according to claim 1 or 2, wherein the content of dimethyl isosorbide ranges from 0.5 to 20 % w/w.
10. A stable topical ointment composition comprising from 0.01 % to 0.5 % w/w tacrolimus, and from 0.5 to 10 % w/w dimethyl isosorbide.