FIELD OF THE INVENTION
[0001] The present disclosure relates generally to pharmaceutical compositions. More specifically, the disclosure is directed to a composition for hirsutism comprising solid lipid microparticles comprising Eflornithine or 2,5-diamino-2-(difluoromethyl)pentanoic acid, cetyl palmitate, glycerol stearate, and a surfactant. The present disclosure also provides a process of preparing the composition.

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 presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Hirsutism is characterized as excess terminal hair which is commonly found in females in a male pattern in the feminine body's androgen sensitive areas (upper lip, throat, chin, back, belly, thighs and arms). Hirsutism is a typical reproductive-age women's medical complaint that affects most females with polycystic ovary syndrome. Hirsutism exists in about 7 percent of women and has an estimated economic burden of over $600 million annually in the United States. Among women, hirsutism leads to major social and psychological challenges. It has a detrimental impact on women's standards of living, and is source of anxiety and depressive symptoms. Hirsutism can make women extremely self-conscious, threatening both their sense of femininity and self-esteem. All the sufferers want to get rid of this disease at any cost. They end up using the existing treatments without even considering their side-effects; therefore, new techniques need to be explored.
[0004] Available alternatives for hirsutism treatment include androgen suppression, peripheral androgen blockade, mechanical or cosmetic removal of unwanted hair and topical application of eflornithine cream. Androgen suppression could be achieved using oral contraceptive but produces certain side effects like suppression of luteinizing and follicle stimulating hormones which leads to decrease in ovarian as well as adrenal androgen production. Another alternative is receptor blocker but they have teratogenic potential. Mechanical or cosmetic removal of unwanted hair through bleaching, shaving or chemical depilation possibly can be helpful for temporary elimination of unwanted hair. Nevertheless, plucking and waxing can cause skin irritation and scarring while shaving may be psychologically unacceptable and lead to blunt hair. Chemical depilatories might cause skin irritation and electrolysis may be painful while short-wave diathermy can cause scarring.
[0005] Looking at these drawbacks, topical application of eflornithine seems to be an appropriate preference to get rid of hirsutism. Eflornithine hydrochloride (EFH) is the only non-hormonal and non-systemic prescription option available for women who suffer from hirsutism. Application of EFH topical cream is approved by the food and drug administration (FDA) for treatment of unwanted facial hair growth. EFH inhibits the activity of L-ornithine decarboxylase enzyme which decreases the production of spermidine protein which regulates hair growth and differentiation. However, EFH has poor permeability characteristics (log P is approximately -2.9); therefore, frequent application of topical cream containing high percentage of EFH is required for successful treatment of hirsutism. Continuous use of highly concentrated EFH topical cream may cause local side effects such as irritation, acne, skin itching, stinging, burning and rashes. Other challenges with conventional topical formulations includes need of very high dosage, high frequency of applications, short remission period of eight weeks and post application skin scaling.
[0006] There is a need in the art to look for novel compositions of Eflornithine that provide improved drug permeability, decreased dosage and frequency of applications.

OBJECTS OF THE INVENTION
[0007] An object of the present disclosure is to provide a composition comprising Eflornithine that gives targeted delivery, and high drug permeability.
[0008] Another object of the present disclosure is to provide a composition comprising microparticles of Eflornithine that reduces dosage amounts, frequencies of application and adverse side effects.
[0009] Yet another object of the present disclosure is to provide a formulation comprising Eflornithine that is suitable for topical application.
[0010] Still another embodiment of the present disclosure is to provide a process of preparation of the composition comprising Eflornithine.

SUMMARY OF THE INVENTION
[0011] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0012] Aspects of the present disclosure provide compositions of Eflornithine that comprise Eflornithine in the form of microparticles for increased permeation through the skin barriers.
[0013] In an aspect, the present disclosure provides a composition for hirsutism comprising solid lipid microparticles comprising Eflornithine or 2,5-diamino-2-(difluoromethyl)pentanoic acid, an isomer, a solvate, or a pharmaceutically acceptable salt thereof; cetyl palmitate, glycerol stearate, and a surfactant.
[0014] In an embodiment, Eflornithine may be present in the form of a pharmaceutically acceptable salt, including Eflornithine hydrochloride.
[0015] In an embodiment, Eflornithine may be present in the composition in a range of about 15% w/w to about 25% w/w of the composition.
[0016] In an embodiment, cetyl palmitate may be present in the composition in a range of about 35% w/w to about 40% w/w of the composition.
[0017] In an embodiment, glycerol stearate may be present in the composition in a range of about 35% w/w to about 40% w/w of the composition.
[0018] In an embodiment, the ratio of weight of Eflornithine to weight of cetyl palmitate and glycerol stearate may range from about 1:3 w/w to about 1:5 w/w.
[0019] In an embodiment, the surfactant may be selected from polyethylene glycol sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sorbitan monooleate, sorbitan monolaurate, or combinations thereof. In an embodiment, surfactant may be present in the composition in a range of about 1% w/w to about 4% w/w of the composition.
[0020] In an embodiment, the composition comprises solid lipid microparticles comprising Eflornithine hydrochloride in a range of 15% w/w to 25% w/w of the composition; cetyl palmitate in a range of 35% w/w to 40% w/w of the composition; glycerol stearate in a range of 35% w/w to 40% w/w of the composition, and polyethylene glycol sorbitan monooleate in a range of 1% w/w to 2% w/w of the composition.
[0021] In an aspect, the present disclosure provides a pharmaceutical formulation for topical application for management of hirsutism comprising: (a) solid lipid microparticles comprising Eflornithine or 2,5-diamino-2-(difluoromethyl)pentanoic acid, an isomer, a solvate, or a pharmaceutically acceptable salt thereof; cetyl palmitate, glycerol stearate, and a surfactant; and (b) one or more pharmaceutically acceptable excipient(s).
[0022] In an embodiment, the microparticles may be present in a range of about 30% w/w to about 40% w/w of the formulation.
[0023] In an embodiment, the excipient may be selected from one or more of paraffin, beeswax, liquid paraffin, cetyl alcohol, stearyl alcohol, isopropyl myristate, propyl paraben, sorbitan monostearate, polyoxyethylene sorbitan monooleate, or combinations thereof. In an embodiment, the excipient(s) may be present in a range of about 60% w/w to about 70% w/w of the formulation.
[0024] In an embodiment, the formulation comprises microparticles in a range of 30% w/w to 35% w/w of the formulation comprising Eflornithine hydrochloride, cetyl palmitate, glycerol stearate, and polyethylene glycol sorbitan monooleate; and excipient(s) in a range of 65% w/w to 70% w/w of the formulation comprising paraffin, beeswax, liquid paraffin, stearyl alcohol, isopropyl myristate, and propyl paraben.
[0025] In an aspect, the present disclosure provides a process of preparation of a composition for hirsutism comprising solid lipid microparticles, wherein the process comprises the steps of : (a) weighing and melting cetyl palmitate and glycerol stearate at 60 oC; (b) dissolving eflornithine in an acetone-ethanol mixture and transferring to the molten mixture of step (a) to give an organic phase; (c) transferring with stirring the organic phase to an aqueous phase comprising a surfactant; (d) ultrasonicating to fabricate an oil-in-water microemulsion and cooling with continuous stirring till complete evaporation to give a solid lipid microparticles dispersion; and (e) lyophilizing the dispersion to give the composition.
[0026] 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
[0027] 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.
[0028] Figures 1(a) provide the contour plot and response surface plot of effect of surfactant (%v/v) (X2) and drug:lipid (w/w) (X1) on the entrapment efficiency (% w/w)(Y1). Figures 1(b) provide the contour plot and response surface plot of effect of stirring time (h) (X3) and drug:lipid (w/w) (X1) on the entrapment efficiency (% w/w)(Y1).
[0029] Figures 2(a) provide the contour plot and response surface plot of effect of surfactant (%v/v)(X2) and drug:lipid (w/w) (X1) on the drug loading (% w/w) (Y2). Figures 2(b) provide the contour plot and response surface plot of effect of stirring time (h) (X3) and drug:lipid (w/w) (X1) on the drug loading (% w/w) (Y2). Figures 2(c) provide the contour plot and response surface plot of effect of stirring time (h) (X3) and surfactant (% v/v) (X2) on the drug loading (% w/w) (Y2).
[0030] Figures 3(a) provide the contour plot and response surface plot of effect of surfactant (%v/v) (X2) and drug:lipid (w/w) (X1) on the mean diameter (µm) (Y3). Figures 3(b) provides the contour plot and response surface plot of effect of stirring time (h) (X3) and surfactant (% v/v) (X2) on the mean diameter (µm) (Y3).
[0031] Figures 4(a) provide the contour plot and response surface plot of effect of surfactant (%v/v) (X2) and drug:lipid (w/w) (X1) on the process yield (% w/w) (Y4). Figures 4(b) provide the contour plot and response surface plot of effect of stirring time (h) (X3) and drug:lipid (w/w) (X1) on the process yield (% w/w) (Y4).
[0032] Figure 5 provides a comparative cumulative drug permeation profile(µg/cm2) over time(h) for (a) cream loaded with plain drug (eflornithine hydrochloride) (EFH-C); (b) cream loaded with physical mixture of drug and other constituents (EFH-PM-C); and (c) cream loaded with composition comprising solid lipid microparticles of eflornithine hydrochloride (EFH-SLM-C) as per an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION
[0033] 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 as defined by the appended claims.
[0034] 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.
[0035] 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.
[0036] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired 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.
[0037] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0038] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0039] 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.”
[0040] 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.
[0041] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0042] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other 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.
[0043] 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.
[0044] 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.
[0045] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0046] 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.
[0047] As described herein, the term ‘effective amount’ refers to the amount of the composition required to bring about a change or improvement in a subject without side effects or overdosing.
[0048] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia' , including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.
[0049] The term, ‘management’, or ‘treatment’ as used herein refers to alleviate, slow the progression, attenuation, prophylaxis or as such treat the existing disease or condition. Treatment also includes treating, preventing development of, or alleviating to some extent, one or more of the symptoms of the diseases or condition.
[0050] Entrapment efficiency, as used herein, may be defined as percentage of drug successfully entrapped into the solid lipid microparticles.
[0051] Percentage drug loading, as used herein, may be defined as percentage of drug loaded per unit weight of the solid lipid microparticles.
[0052] Aspects of the present disclosure provide a composition of eflornithine that overcomes the drawbacks of the known compositions of eflornithine such as low permeability.
[0053] In an embodiment, the present disclosure provides a composition comprising eflornithine hydrochloride, wherein eflornithine hydrochloride is in the form of microparticles that enhance its permeation through layers of the skin.
[0054] In an embodiment, the present disclosure provides a composition for hirsutism comprising solid lipid microparticles comprising Eflornithine or 2,5-diamino-2-(difluoromethyl)pentanoic acid, an isomer, a solvate, or a pharmaceutically acceptable salt thereof; cetyl palmitate, glycerol stearate, and a surfactant.
[0055] In an embodiment, Eflornithine may be present in the form of a pharmaceutically acceptable salt, including Eflornithine hydrochloride. In an embodiment, the solvate may include hydrates such as, monohydrate, dehydrate or sesquihydrate of Eflornithine.
[0056] In an embodiment, Eflornithine may be present in the composition in a range of about 15% w/w to about 25% w/w of the composition. In a preferred embodiment, Eflornithine may be present in the composition in a range of about 20% w/w to about 22% w/w of the composition.
[0057] In an embodiment, cetyl palmitate may be present in the composition in a range of about 35% w/w to about 40% w/w of the composition. In a preferred embodiment, cetyl palmitate may be present in the composition in a range of about 38% w/w to about 40% w/w of the composition.
[0058] In an embodiment, glycerol stearate may be present in the composition in a range of about 35%w/w to about 40% w/w of the composition. In a preferred embodiment, glycerol stearate may be present in the composition in a range of about 38% w/w to about 40% w/w of the composition.
[0059] In an embodiment, weight of Eflornithine to weight of cetyl palmitate and glycerol stearate ratio may range from about 1:3 w/w to about 1:5 w/w.
[0060] In an embodiment, the surfactant may be selected from polyethylene glycol sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sorbitan monooleate, sorbitan monolaurate, or combinations thereof.
[0061] In an embodiment, surfactant may be present in the composition in a range of about 1% w/w to about 4% w/w of the composition. In a preferred embodiment, surfactant may be present in the composition in a range of about 1% w/w to about 2% w/w of the composition.
[0062] In an embodiment, the composition comprises solid lipid microparticles comprising Eflornithine hydrochloride in a range of 15% w/w to 25% w/w of the composition; cetyl palmitate in a range of 35% w/w to 40% w/w of the composition; glycerol stearate in a range of 35% w/w to 40% w/w of the composition, and polyethylene glycol sorbitan monooleate in a range of 1% w/w to 2% w/w of the composition.
[0063] The composition of the present disclosure comprising lipid based carriers like solid lipid microparticles (SLM) are capable of targeted and prolonged drug release to hair follicles due to enhancement in drug penetration via skin layers. Therefore, formulations loaded with eflornithine solid lipid microparticles composition may enhance permeation along with minimization of dose, frequency of application and therefore, will reduce local adverse effects such as irritation, acne, skin itching, skin scaling, stinging, burning and rashes caused by plain drug usage.
[0064] The composition of the present disclosure has a synergistic effect, in that the components of the composition when present in the form of microparticles, as in the present disclosure, provide higher permeation compared to a mere physical mixture of the components and the pure drug.
[0065] In an embodiment, the composition may have entrapment efficiency in the range of about 46% w/w to about 75% w/w. In an embodiment, the composition may have an entrapment efficiency of about 73.21% w/w. Thus, the composition provides high entrapment efficiency.
[0066] In an embodiment, the composition may provide drug loading in the range of about 13% to about 19%. In an embodiment, the composition may have a drug loading of about 15.3%. Thus, the composition provides high drug loading efficacy.
[0067] In an embodiment, the microparticles may have a mean diameter in a range of about 62µm to about 155µm. In a preferred embodiment, the microparticles may have a mean diameter of about 100.7 µm. Thus, the composition effectively reduces the size of the microparticles.
[0068] In an embodiment, the present disclosure provides a pharmaceutical formulation for topical application for management of hirsutism comprising: (a) solid lipid microparticles comprising Eflornithine or 2,5-diamino-2-(difluoromethyl)pentanoic acid, an isomer, a solvate, or a pharmaceutically acceptable salt thereof; cetyl palmitate, glycerol stearate, and a surfactant; and (b) one or more pharmaceutically acceptable excipient(s).
[0069] In an embodiment, the formulation may be in the form of a liquid or semi-solid. In an embodiment, the formulation may be in the form of a gel, cream, lotion, oil, paste, or suspension for topical application.
[0070] In a preferred embodiment, the formulation is in the form of a cream formulation.
[0071] In an embodiment, the microparticles may be present in a range of about 30% w/w to about 40% w/w of the formulation. In a preferred embodiment, the microparticles may be present in a range of about 30% w/w to about 35% w/w of the formulation.
[0072] In an embodiment, the excipient may be selected from one or more of paraffin, cetyl alcohol, beeswax, liquid paraffin, stearyl alcohol, isopropyl myristate, propyl paraben, polyoxyethylene sorbitan monooleate, sorbitan monostearate, or combinations thereof.
[0073] In an embodiment, the excipient(s) may be present in a range of about 60% w/w to about 70% w/w of the formulation. In a preferred embodiment, the excipient(s) may be present in a range of about 65% w/w to about 70% w/w of the formulation.
[0074] In an embodiment, the formulation comprises microparticles in a range of 30% w/w to 35% w/w of the formulation comprising Eflornithine hydrochloride, cetyl palmitate, glycerol stearate, and polyethylene glycol sorbitan monooleate; and excipient(s) in a range of 65% w/w to 70% w/w of the formulation comprising paraffin, beeswax, liquid paraffin, stearyl alcohol, isopropyl myristate, propyl paraben, and sorbitan monostearate.
[0075] The formulation has a pH in the range of 6-7 suitable for application on skin.
[0076] The formulations may be prepared by methods well-known in the art.
[0077] In an embodiment, the present disclosure provides use of a composition or formulation as recited above for treatment and management of hirsutism.
[0078] In an embodiment, the present disclosure provides a process of preparation of a composition for hirsutism comprising solid lipid microparticles that comprises the steps of emulsion-solvent evaporation technique and ultrasonication.
[0079] In an embodiment, the present disclosure provides a process of preparation of a composition for hirsutism comprising solid lipid microparticles, wherein the process comprises the steps of : (a) weighing and melting cetyl palmitate and glycerol stearate at 60 oC; (b) dissolving eflornithine in an acetone-ethanol mixture and transferring to the molten mixture of step (a) to give an organic phase; (c) transferring with stirring the organic phase to an aqueous phase comprising a surfactant; (d) ultrasonicating to fabricate an oil-in-water microemulsion and cooling with continuous stirring till complete evaporation to give a solid lipid microparticles dispersion; and (e) lyophilizing the dispersion to give the composition.
[0080] In an embodiment, the acetone-ethanol mixture may be acetone to ethanol mixture in a ratio of 1:1. In an embodiment, the stirring time may range from 1 hour to 5 hours. Preferably the stirring is performed at 37±0.5oC at 2000 rpm.
[0081] In an embodiment, the process of the present disclosure may provide a yield in the range of about 47.36% to about 78.7%. In a preferred embodiment, the process of the present disclosure may provide a yield of about 76.84%.
[0082] The drug: total lipid (cetyl palmitate and glycerol stearate), concentration of the surfactant and stirring time influence the process yield.
[0083] In another embodiment, the present disclosure provides a method of treatment, amelioration or prophylaxis of hirsutism by administering an effective amount of the composition or formulation as recited above.
[0084] The method may be effective in reducing the growth of hair, removing existing hair, slowing the growth of hair, decreasing the density of hair growth, decreasing the thickness of hair, or combinations thereof.
[0085] 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 scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0086] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
[0087] MATERIALS: The chemicals Monecol® PC, Softemul® 165, polyethylene glycol sorbitan monooleate and Eflornithine hydrochloride were obtained from Mohini Organics Private Limited (Mumbai, India). The chemicals hard paraffin, beeswax, liquid paraffin, cetyl alcohol, stearyl alcohol, propyl paraben, polyoxyethylene sorbitan monooleate, sorbitan monostearate and isopropyl myristate were obtained from Loba Chemicals Private Limited, Mumbai, India.
[0088] Example 1: Preparation of the solid lipid microparticles composition
[0089] Central composite design was used to systemically investigate the influence of independent variables over dependent parameters specified in Table 1. Twenty batches of solid lipid microparticles (SLM) were synthesized according to design outline generated by Stat-Ease Design-Expert software version 11.1.2.0 (Table 2). Second-order polynomial models were developed for analyzing main, interaction and quadratic effects of factors over dependent variables:
Eq. (1)

[0090] Where, Y denotes observed response variable, ß0 is constant coefficient and ? is residual correlated to experiments. ßi, ßii and ßij represents coefficients of linear, quadratic parameter and interaction parameters, respectively. Xi represents average outcome of changing one variable at a time from low to high, polynomial terms Xi2 was employed to assess non-linearity effect of variable Xi, interaction terms XiXj illustrate how the response transforms when two variables Xi and Xj were altered concurrently.
Table 1.Independent and dependent variables used in central composite design
Independent variables Coded levels of variables
-1.68 (?a) -1 0 +1 +1.68 (+a)
X1= Drug: lipid (w/w) 1:1.32 1:2 1:3 1:4 1:4.68
X2= Surfactant concentration (% v/v) 0.16 0.5 1 1.5 1.84
X3= Stirring time (h) 1.32 2 3 4 4.68
Dependent variables Constraints
Y1 = Entrapment efficiency (% w/w) Maximize
Y2 = Drug loading (% w/w) Maximize
Y2 = Mean diameter (µm) Minimize
Y4 = Process yield (% w/w) Maximize

Table 2.Central composite experimental design matrix with coded value

Run X1 (Drug: lipid) (w/w) X2 (Surfactant concentration) (% v/v) X3 (Stirring time) (h) Design
1 -1 -1 -1 Factorial Design
2 1 -1 -1
3 -1 1 -1
4 1 1 -1
5 -1 -1 1
6 1 -1 1
7 -1 1 1
8 1 1 1
9 -1.68 0 0 Axial Points
10 1.68 0 0
11 0 -1.68 0
12 0 1.68 0
13 0 0 -1.68
14 0 0 1.68
15 0 0 0 Center points
16 0 0 0
17 0 0 0
18 0 0 0
19 0 0 0
20 0 0 0

[0091] Solid lipid microparticle composition of eflornithine hydrochloride (EFH-SLM) was manufactured via emulsion-solvent evaporation technique in conjunction with ultra-sonication. Briefly, accurately weighed amount of Monecol® PC and Softemul® 165 were melted at 60oC and 5 mL solution of eflornithine hydrochloride (EFH) in acetone-ethanol (1:1) mixture was transferred to molten lipids to produce organic phase. Subsequently, organic phase was transferred slowly through syringe into aqueous phase containing PEG-20 sorbitan monolaurate as surfactant with continuous magnetic stirring at 37±0.5oC at 2000 rpm (Remi, India). The resulting pre-emulsion was ultra-sonicated for ten minutes using an ultrasonic probe sonicator (PCI analytical, India) to fabricate an oil-water microemulsion which was cooled with continuous magnetic stirring at room temperature till complete evaporation of organic solvent to generate Eflornithine Hydrochloride- solid lipid microparticles (EFH-SLM) dispersion. Lyophilization was accomplished under vacuum (0.5 kPa) at -65oC for 24 h using D-mannitol (lyoprotectant) in freeze dryer (Allied Frost, India) to give the microparticles composition (EFH-SLM).
1.1 Determination of EFH-SLM response variables (Y1-Y4):
[0092] Entrapment efficiency (EE) (Y1) and drug loading (Y2) of EFH-SLM was determined using the ultra-filtration method. A lipid drug delivery system must be capable to entrap utmost possible quantity of drug with minimum amount of lipid, therefore, optimum drug: lipid for highest % EE should be acquired. Effect of design variables on % EE can be described by second-order polynomial Eq. (2).
Y1 = 70.20 + 9.26X1 + 0.56X2 + 1.64X3 + 1.21X1X2 + 0.51X1X3 – 1.07X2X3 – 3.53X12– 0.746X22 – 2.41X32 (r2= 0.9807) Eq. (2)
[0093] Figures 1(a) provide the contour plot and response surface plot of effect of surfactant (%v/v)(X2) and drug:lipid(w/w) (X1) on the entrapment efficiency (w/w%) (Y1). Figures 1(b) provide the contour plot and response surface plot of effect of stirring time (h) (X3) and drug:lipid(w/w) (X1) on the entrapment efficiency (w/w%)(Y1).
[0094] Model proposed quadratic polynomial equation for estimating the effect of selected variables on % drug loading (DL) (Y2). Synergistic effects X2, X3 and antagonistic effect of X1 was observed for % DL as illustrated by following equation:
Y2 = 17.55 – 1.77X1 + 0.093X2 + 0.408X3 + 0.3231X1X2 + 0.0062X1X3 – 0.2588X2X3 – 0.4056X12– 0.2059X22 – 0.6213X32 (r2 = 0.9574) Eq. (3)
[0095] Figures 2(a) provide the contour plot and response surface plot of effect of surfactant (%v/v)(X2) and drug:lipid(w/w) (X1) on the drug loading (% w/w) (Y2). Figures 2(b) provide the contour plot and response surface plot of effect of stirring time (h) (X3) and drug:lipid(w/w) (X1) on the drug loading (% w/w) (Y2). Figures 2(c) provide the contour plot and response surface plot of effect of stirring time (h) (X3) and surfactant (% v/v) (X2) on the drug loading (% w/w) (Y2).
[0096] Mean diameter (Y3) of EFH-SLM were determined by optical microscopy employing optical microscope. A second-order polynomial quadratic developed by multiple regression analysis of mean diameter values could satisfactorily illustrate outcome of independent variables on mean diameter as indicated in following equation:
Y3 = 115.38 + 27.97X1 – 2.24X2 – 5.09X3 + 0.0837X1X2 – 0.1738X1X3 – 0.4413X2X3 – 3.58X12– 8.08X22 – 7.74X32 (r2 = 0.9772) Eq. (4)
[0097] Figures 3(a) provide the contour plot and response surface plot of effect of surfactant (%v/v) (X2) and drug : lipid (w/w) (X1) on the mean diameter (µm) (Y3).Figures 3(b) provides the contour plot and response surface plot of effect of stirring time (h) (X3) and surfactant (% v/v) (X2) on the mean diameter (µm) (Y3).
[0098] The completely dried EFH-SLM recovered from each experimental run was accurately weighed and the percentage process yield (Y4) was calculated. Independent variables significantly influencing % process yield of EFH-SLM were drug: lipid (X1), surfactant concentration (X2) and stirring time (X3) (p< 0.05). The effect can be elucidated through mathematical relationship depicted in following equation:
Y4 = 72.8 + 9.09X1 + 1.22X2 + 1.6X3 + 1.47X1X2 + 0.5863X1X3 – 0.3462X2X3 – 4.23X12– 1.11X22 – 2.4X32 (r2 = 0.9791) Eq. (5)
[0099] Figures 4(a) provide the contour plot and response surface plot of effect of surfactant (%v/v) (X2) and drug:lipid(w/w) (X1) on the process yield (%) (Y4).Figures 4(b) provides the contour plot and response surface plot of effect of stirring time (h) (X3) and drug:lipid(w/w) (X1) on the process yield (%) (Y4).
1.2 Determination of optimized EFH-SLM batch by numerical optimization method
[00100] A mathematical relationship among independent and response parameters was generated by response surface regression analysis and optimum values of variables were estimated using Design Expert software. The composition of the software-suggested optimized EFH-SLM was 1:3.74 drug: lipid (w/w) (X1), 1.84% surfactant concentration (X2) and 3.5 h stirring time (X3). The values of response variables Y1, Y2, Y3, and Y4 of optimized EFH-SLM were 73.21%, 15.3%, 100.7µm, and 76.84%, respectively.
Example 2: Manufacturing of Optimized EFH-SLM Loaded Topical Cream
[00101] An oil-phase was produced through melting the hard paraffin and beeswax at 75°C followed by addition and gentle mixing of liquid paraffin, cetyl alcohol, stearyl alcohol, and isopropyl myristate. The aqueous phase was prepared by dissolving the Tween® 80, Span® 60 and propyl paraben in distilled water and heating to 75-80°C for complete dissolution. Aqueous phase was mixed with oil phase for emulsification which was cooled to room temperature under constant stirring. An optimized EFH-SLM of Example 1 was incorporated in a pre-prepared cream base with moderate stirring for 10 min to produce microparticles loaded cream (EFH-SLM-C). Likewise, creams incorporating plain drug eflornithine hydrochloride (EFH-C) and cream with physical mixture (PM) (EFH-PM-C) of drug, Softemul® 165 and Monecol® PC were manufactured for comparative study.
2.1 Evaluation of Physical appearance, spreadability, pH value and viscosity of optimized EFH-SLM loaded topical cream (EFH-SLM-C)
[00102] The EFH-SLM-C appeared aesthetically acceptable, pleasant to feel, non-gritty as well as homogenous in texture without any signs of phase separation. The spreadability time was 14±2 seconds with merely small shear stress. The pH of the EFH-SLM-C was found to be 6.3±0.2 which ensures that it could be indicated to treat hirsutism with lower risk of skin irritation since topical dosage form should have a pH range of 6-7 to prevent skin pH alteration. The viscosity of EFH-SLM-C was found to be 12500, 8100, 4600 and 3100 cP at 10, 20, 50 and 100 rpm shear rate, respectively.
Example 3: In-vitro drug permeation study
[00103] In-vitro drug permeation study from the cream loaded with plain drug (EFH-C), cream loaded with physical mixture (PM) of drug and other constituents (EFH-PM-C) and cream loaded with composition comprising solid lipid microparticles of drug (EFH-SLM-C) was performed in phosphate buffer, pH 6.8 using Franz diffusion cell over 24 h. It was observed that the quantities of EFH permeated across membrane by EFH-SLM-C were considerably higher (*p< 0.05) compared to EFH-C and EFH-PM-C (refer Figure 5). The in-vitro steady state flux (Jss) (µgcm-2h-1) through artificial membrane from EFH-C, EFH-PM-C and EFH-SLM-C were 17.27, 17.41 and 36.65 while apparent permeability coefficient (Papp) (cmh-1) were 0.046, 0.0464 and 0.0977, respectively. These statistics revealed significant differences in flux and Papp of EFH-SLM-C and EFH-C (*p< 0.05). The significantly enhanced permeation of EFH from EFH-SLM-C over EFH-C is accredited to existence of drugs and other constituents at micro scale (as microparticles) which synergistically improve the permeation of eflornithine hydrochloride.
Example 4: Ex-vivo hair re-growth study
[00104] Healthy Swiss albino mice (female mice, 15-20 g weight) were divided into three groups (two treatment groups and one control group). Hair on the dorsal sides of the mice was removed using marketed hair remover (Veet, Health-Hygiene-Home) to ensure complete denual of a 6 cm2 area.After being depilated, the animal skin was treated by application of thin layer (50 mg) of EFH-SLM-C cream of the present disclosure (once a day) while other creams (EFH-PM-C and EFH-C) (twice a day) for three weeks. The animal skin was observed at specific time intervals of Day 1, 7, 14, and 21, to record pattern of hair re-growth, and percentage of hair re-growth demonstrated by the formulation of the present disclosure compared to controls. Usually, the dorsal part of the animals is used for the testing.
[00105] Hair growth study/efficacy of the treatment was evaluated by scoring on the basis of density of hair and visualization of skin area:
Type IV (high hair density, full, thick fur)
Type III (moderate hair density with no visible skin area)
Type II (low hair density, with the visualization of the skin)
Type I (uneven hair growth on the test area, skin easily seen)
[00106] The results of the tests are provided in Table 3. On day 21, modest hair-density without visible skin area were observed for animals treated with pure drug - EFH-Cream while small hair density was observed for animals treated with EFH-SLM-Cream. This indicated the greater efficiency of cream loaded with composition comprising microparticles of the present invnetion, in comparison to plain EFH-Cream.
Table 3. Hair re-growth potential scores in different groups of animals
Group Name Administration Hair re-growth potential scores
Day 1 Day 7 Day 14 Day 21
Control Group Cream base (50 mg) I II III IV
Treatment Group-1 EFH cream (50 mg) I I II III
Treatment Group-2 EFH-SLM cream (50 mg) I I I II

[00107] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

Claims:

1. A composition for hirsutism comprising solid lipid microparticles comprising Eflornithine or 2,5-diamino-2-(difluoromethyl)pentanoic acid, an isomer, a solvate, or a pharmaceutically acceptable salt thereof; cetyl palmitate, glycerol stearate, and a surfactant.

2. The composition as claimed in claim 1, wherein Eflornithine is present in the form of Eflornithine hydrochloride.

3. The composition as claimed in claim 1, wherein Eflornithine is present in the composition in a range of 15% w/w to 25% w/w of the composition.

4. The composition as claimed in claim 1, wherein cetyl palmitate is present in the composition in a range of 35% w/w to 40% w/w of the composition.

5. The composition as claimed in claim 1, wherein glycerol stearate is present in the composition in a range of 35%w/w to 40% w/w of the composition.

6. The composition as claimed in claim 1, wherein weight of Eflornithine to weight of cetyl palmitate and glycerol stearate ratio is in a range from 1:3 w/w to 1:5 w/w.

7. The composition as claimed in claim 1, wherein surfactant is selected from polyethylene glycol sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sorbitan monolaurate, sorbitan monooleate or combinations thereof.

8. The composition as claimed in claim 1, wherein surfactant is present in the composition in a range of 1% w/w to 4% w/w of the composition.

9. The composition as claimed in claim 1, wherein the composition comprises Eflornithine hydrochloride in a range of 15% w/w to 25% w/w of the composition; cetyl palmitate in a range of 35% w/w to 40% w/w of the composition; glycerol stearate in a range of 35% w/w to 40% w/w of the composition, and polyethylene glycol sorbitan monooleate in a range of 1% w/w to 2% w/w of the composition.

10. A pharmaceutical formulation for topical application for management of hirsutism comprising: (a) solid lipid microparticles comprising Eflornithine or 2,5-diamino-2-(difluoromethyl)pentanoic acid, an isomer, a solvate, or a pharmaceutically acceptable salt thereof; cetyl palmitate, glycerol stearate, and a surfactant; and (b) one or more pharmaceutically acceptable excipient(s).

11. The formulation as claimed in claim 10, wherein the formulation is in the form of a gel, cream, lotion, oil, paste, or suspension for topical application.

12. The formulation as claimed in claim 10, wherein the microparticles are present in a range of 30% w/w to 40% w/w of the formulation.

13. The formulation as claimed in claim 10, wherein the excipient(s) are present in a range of 60% w/w to 70% w/w of the formulation.

14. The formulation as claimed in claim 10, wherein the excipient may be selected from one or more of paraffin, beeswax, liquid paraffin, cetyl alcohol, stearyl alcohol, isopropyl myristate, propyl paraben, polyoxyethylene sorbitan monooleate, sorbitan monostearate, or combinations thereof.

15. The formulation as claimed in claim 10, wherein the formulation comprises microparticles in a range of 30% w/w to 35% w/w of the formulation comprising Eflornithine hydrochloride, cetyl palmitate, glycerol stearate, and polyethylene glycol sorbitan monooleate; and excipient(s) in a range of 65% w/w to 70% w/w of the formulation comprising paraffin, beeswax, liquid paraffin, cetyl alcohol, stearyl alcohol, isopropyl myristate, propyl paraben, polyoxyethylene sorbitan monooleate, and sorbitan monostearate.

16. A process of preparation of a composition for hirsutism comprising solid lipid microparticles, wherein the process comprises the steps of : (a) weighing and melting cetyl palmitate and glycerol stearate at 60 oC; (b) dissolving eflornithine in an acetone-ethanol mixture and transferring to the molten mixture of step (a) to give an organic phase; (c) transferring with stirring the organic phase to an aqueous phase comprising a surfactant; (d) ultrasonicating to fabricate an oil-in-water microemulsion and cooling with continuous stirring till complete evaporation to give a solid lipid microparticles dispersion; and (e) lyophilizing the dispersion to give the composition.