DESC:PREAMBLE TO THE DESCRIPTION
COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.
FIELD OF THE INVENTION

The present invention relates to a pharmaceutical formulation comprising of Apremilast in the form of topical composition. Another aspect of the invention relates to a stable topical formulation comprising of Apremilast for prevention and/or treatment of Psoriasis and other skin disorders and also manufacturing processes thereof. Yet another aspect of the invention relates to method of prevention and/or treatment of Psoriasis and other skin disorders using topical Apremilast.

BACKGROUND

Psoriasis is a chronic autoimmune skin disease that speeds up the growth cycle of skin cells. It causes patches of thick red skin and silvery scales. Patches are typically found on the elbows, knees, scalp, lower back, face, palms, and soles of feet, but can affect other places (fingernails, toenails, and mouth).

There are various types of psoriasis viz. plaque, guttate, psoriatic arthritis, pustular, inverse or flexural, erythrodermic, scalp psoriasis, Nail Psoriasis, Palmoplanter but most common type of psoriasis is a plaque psoriasis.

Plaque psoriasis is the most common type of psoriasis. Approximately, 9 out of 10 people with psoriasis have plaques. The disease begins with a small scaling red bump that coalesces with other similar bumps to form an elevated plaque of red skin that is covered with silvery scales. Circular- to oval-shaped red plaques that sometimes itch or burn are typical of plaque psoriasis. The plaques usually are found on the elbows, knees, trunk, or scalp, but they may be found on any part of the skin. Most plaques of psoriasis are persistent (they stay for years and do not tend to come and go).

Guttate psoriasis are small salmon-pink (or red) bumps on the skin. Guttate psoriasis usually occurs on the trunk, arms, or legs. However, it may cover a large portion of the body. This type of psoriasis often "runs its course" and may even go away without treatment in a few weeks. Sometimes guttate psoriasis can be more persistent, and it may evolve into plaque psoriasis. The guttate form of psoriasis is the second most common form of psoriasis. About 2% of those with psoriasis have the guttate type. This type of psoriasis is more common in children and adults younger than 30 years of age.

The trigger to the disease is often a streptococcal bacterial throat infection. The eruption of the lesions on the skin usually occurs two to three weeks after a streptococcal sore throat. Outbreaks may resolve only to return with the next strep throat.

Psoriatic arthritis is an inflammatory type of arthritis that eventually occurs in 10% to 20% of people with psoriasis. It is different from more common types of arthritis (such as osteoarthritis or rheumatoid arthritis) and is thought to be related to the underlying problem of psoriasis. Psoriasis and psoriatic arthritis are sometimes considered together as psoriatic disease.

Pustular psoriasis is an uncommon form of psoriasis. People with pustular psoriasis have clearly defined, raised bumps on the skin that are filled with pus (pustules). The skin under and around these bumps is reddish. Pustular psoriasis may cause large portions of the skin to redden. Pustular psoriasis can occur alone or with plaque-type psoriasis.

Inverse psoriasis or Flexural often appears in skinfolds, such as under the breasts or in the armpits or groin area. This type of psoriasis is red and often shiny and smooth. The sweat and moisture from skinfolds keeps this form of psoriasis from shedding skin scales. Sometimes it’s misdiagnosed as a fungal or bacterial infection. The skin-on-skin contact can make inverse psoriasis very uncomfortable. Most people with inverse psoriasis also have a different form of psoriasis in other places on the body.

Erythrodermic is an uncommon but very serious form of psoriasis. Only 3 percent of people with psoriasis will develop erythrodermic psoriasis in their lifetime. This type usually occurs in people who have uncontrolled plaque psoriasis. Erythrodermic psoriasis causes fiery-looking skin that affects most of the body.

Scalp psoriasis shows up as red, itchy areas with silvery-white scales on the scalp. These scaly patches may bleed if person remove them. At least half of people who have psoriasis have it on their scalp. At the onset, scalp psoriasis often is confused with seborrheic dermatitis (dandruff). Dandruff is a flaky, itchy scalp without signs of inflammation (such as redness or swelling). Scalp psoriasis can spread beyond hairline and affect the forehead, back of the neck, and areas around the ears. Often psoriasis in kids is first found on the scalp.

Nail Psoriasis which affects the nails by causing them to grow abnormally, dent (pitting) become discoloured and/or separate from the nail bed. Nails can crumble away from the finger in severe cases.

Palmoplanter Psorias is which affects the palms of the hands and soles of the feet. Patches of skin may become red and expand to form scales and thickening of the skin. In these cases skin can itch and crack; this may make walking or performing certain task with the hands difficult. In some cases deep yellow pustules can appear.

Common treatments available for psoriasis are as follows: (https://www.mayoclinic.org/diseases-conditions/psoriasis/diagnosis-treatment/drc-20355845)

Topical treatments: Used alone, creams and ointments that may apply to the skin can effectively treat mild to moderate psoriasis. When the disease is more severe, creams are likely to be combined with oral medications or light therapy. Topical psoriasis treatments include topical corticosteroids, vitamin D analogues, anthralin, topical retinoids, calcineurin inhibitors, salicylic acid, coal tar and moisturizers.

Light therapy (phototherapy): treatment uses natural or artificial ultraviolet light. The simplest and easiest form of phototherapy involves exposing your skin to controlled amounts of natural sunlight. Other forms of light therapy include the use of artificial ultraviolet A (UVA) or ultraviolet B (UVB) light, either alone or in combination with medications. Light therapy include exposure to sunlight, UVB phototherapy, Narrow band UVB phototherapy, Goeckerman therapy, Psoralen plus ultraviolet A (PUVA) and Excimer laser.

Oral or injected medications: If psoriasis becomes severe or it's resistant to other types of treatment, oral or injected drugs are prescribed. This is known as systemic treatment. Because of severe side effects, some of these medications are used for only brief periods and may be alternated with other forms of treatment. Oral or injected medications include Retinoids, Methotrexate, Cyclosporine, Drugs that alter the immune system (biologics) viz. Apremilast (Otezla), Etanercept (Enbrel), Infliximab (Remicade) and Adalimumab (Humira) etc.

Alternative medicines: number of alternative therapies can be used to ease the symptoms of psoriasis, including special diets, creams like aloe vera extract cream, dietary supplements like Omega-3 fatty acids and herbs like topical applications of Oregon grape.

Other skin disorder includes but not limited to ichthyosis, ulcer, synovitis, acne, pustulosis, hyperostosis and osteitis, ankylosing spondylitis, prostatitis, vulvodynia, Behcet disease, cutaneous lupus, lichenplanus, cutaneous sarcoidosis and rosacea etc.

PCT application no. 2017216738 discloses Apremilast can be used in treatment of Psoriatic arthritis, Plaque psoriasis and atopic dermatitis and inflammatory skin disease condition. Also, it can be efficacious for the treatment of dermatomycosis, scleroderma, epidermolysis bullosa, eczema and systemic lupus erythematous affecting skin.

Apremilast nail lacquer formulation was prepared to improve the ugual and trans ungual delivery for the treatment of nail psoriasis and nail lacquer formulation was found to be capable of delivering sufficient amount of apremilast into and across the nail plate. (A Novel Apremilast Nail Lacquer Formulation for the Treatment of Nail Psoriasis; Kushwaha A et al; AAPS PharmSciTech, 18(8), 2949-2956, 2017).

Apremilast has recently completed the phase 2 clinical trial (NCT02087943) for oral treatment of Atopic Dermatitis in adults. Apremilast as a treatment for Atopic Dermatitis in children is disclosed (Apremilast Use for Moderate-to-Severe Atopic Dermatitis in Pediatric Patients; Case Rep Dermatol 8(2), 179-184, 2016).

NCT01045551, Open Label Pilot Study of Apremilast in Treatment of Rosacea discloses Phase 2 clinical trial of oral treatment Apremilast for erythematotelangiectatic rosacea and papulopustular rosacea.

All the treatments have some or the other disadvantage like light therapy, systemic side effects due to oral or injected medications, and other alternatives which do not completely treat psoriasis and other conventional topical treatments which are less effective. Thus, there is a need to develop new and improved pharmaceutical composition for treatment of Psoriasis and other skin diseases.

The nanostructured lipid carriers (NLCs), the particles are produced by using a blend of a solid lipid with a liquid lipid, this blend also being solid at body temperature. NLCs are the new generation of lipid nanoparticles. They were developed to resolve issues associated with first generation lipid nanoparticles i.e. solid lipid nanoparticles (SLNs). NLCs have been looked upon as promising carriers for presenting several attractive features for transdermal drug delivery. NLC possess numerous features that are advantageous for topical route of application. NLC are colloidal carrier systems providing controlled release profiles for many substances. These carriers are composed of physiological and biodegradable lipids exhibiting low systemic toxicity and low cytotoxicity. The small size of the lipid particles ensures close contact to stratum corneum and can increase the amount of drug penetrating into mucosa or skin. Due to their solid lipid matrix, a controlled release from these carriers is possible. This becomes an important tool when it is necessary to supply the drug over prolonged period of time, to reduce systemic absorption, and when drug produces irritation in high concentrations. (Nanostructured lipid carrier (NLC) based gel of celecoxib; Vandana Patravale et al, International Journal of Pharmaceutics, 346,124-132, 2008).

NLCs are prepared by hot homogenization, cold homogenization, and microemulsion method. The essential ingredients for NLCs include lipids such as solid and liquid lipids, water, and emulsifiers. Both solid and liquid lipids are included in NLCs for constructing the inner cores. The emulsifiers (lipophilic, hydrophilic and amphiphilic) are used to stabilize the lipid dispersions. Also, the combination of emulsifiers can prevent particle aggregation more efficiently. NLCs are composed of oily droplets embedded in a solid lipid matrix. The morphology of particles of NLCs is not necessary spherical and their particulate structure is solid platelets with oil present between the solid platelets and the emulsifier layer. (Nanostructured Lipid Carriers (NLCs) for Drug Delivery and Targeting; Chia Lang Fang et al, Recent Patents on Nanotechnology, 7, 41-55, 2013).

PCT application no. 2017216738 discloses a topical pharmaceutical composition comprising Apremilast in an amount of about 0.1 to 5 % w/w and it further discloses wherein composition is in the form of cream, ointment, gel, transdermal formulations, foam, spray, lotion, solution, emulsion or suspension.

An US application no. 2019/0060221 discloses a topical pharmaceutical composition of apremilast wherein, composition of apremilast in gel form comprising apremilast and its pharmaceutical acceptable salts in range of 0.01 to 25% w/w, isopropyl myristate and optionally with other pharmaceutically acceptable excipients.

The nanostructured lipid carriers based topical gel of Valdecoxib was prepared using the lipid phase consisted of 1:1 mixture of Caproyl 90 and Glyceryl Dilaurate. The surfactant phase consisted of a mixture of Cremophor RH 40, Solutol HS 15, and Transcutol (ratio 1:2:0.17) while the aqueous phase was double-distilled water and Carbopol was selected as gelling agent (Formulation and Evaluation of Nanostructured Lipid Carrier (NLC)–based Gel of Valdecoxib; Vandana Patravale et al; Drug Development and Industrial Pharmacy, 32 (8), 911-918, Sep 2006).

Nanostructured lipid carriers (NLC) based topical gel of celecoxib was prepared using the lipid phase consisted of a 1:1 mixture of Capmul MCM and Glyceryl dilaurate. The surfactant phase consisted of a mixture of Cremophor RH 40 and Transcutol (ratio 1:1.66), while the aqueous phase was double distilled water and Carbopol was selected as gelling agent (Nanostructured lipid carrier (NLC) based gel of celecoxib; Vandana Patravale et al, International Journal of Pharmaceutics, 346,124-132, 2008).

The Tacrolimus loaded nanostructured lipid carrier were prepared using Glyceryl monocaprylate (Capmul MCMC8®), Macrogol 15 hydroxy stearate (Solutol HS15), glycerol dibehenate (Compritol 888ATO®), L-a-phosphatidyl choline 30% (soy lecithin 30%), Taurocholic acid, pancreatin, tris maleate, cycloheximide, zinc sulphate hepatahydrate, ammonium acetate and calcium chloride (Tacrolimus-loaded nanostructured lipid carriers for oral delivery-in vivo bioavailability enhancement; Saba Khan et al, European Journal of Pharmaceutics and Biopharmaceutics; 109, 149-157, 2016).

The Tacrolimus loaded nanostructured lipid carrier were prepared using Stearic acid, oleic acid, glyceryl monostearate, glyceryl monocaprylate (Capmul MCMC8®), glycerol polyoxyl 35 castor oil (Cremophore EL), glyceryl monocaprylate/monocaprate (Capmul MCMC 8/10®), propylene glycol monocaprylate (Capryol 90), macrogol 15 hydroxy stearate (Solutol HS15), polyoxypropylene polyoxyethylene triblock copolymer (Poloxamer 108), glyceryl triacetate or triacetin (Captex 500®), fatty acid ester (Cutina P®), groundnut oil, glycerol distearate (Precirol ATO 5®), glycerol dibehenate (Compritol 888ATO®), olive oil, soyabean oil, polyoxypropylene polyoxyethylene triblock copolymer (Poloxamer 407), sunflower oil, peanut oil, sesame oil, Span 80, corn oil, sunflower oil, ethyl oleate, oleoyl macrogol-6 glycerides (Labrafil® M1944CS), oleic acid, plurol oleic, omega-3-fatty acid, linseed oil, propylene glycol dicaprylocaprate (LabrafacTM WL1349), caprylo caproyl macrogol-8-glyceride (Labrasol®) and polyethylene glycol (PEG), L-a-phosphatidyl choline 30% (soy lecithin 30%), Tween 20, PEG-8- beewax (Apifil®), Tween 80, Span 20, miglyol812, castor oil, glyceryl stearate and PEG-75 stearate (Gelot 64®), lauroglycol 90, glycerol mono-oleates (Peceol®), macrogol glycerol (Gelucire 44/14®) and methanol, Acetone, Ethanol, acetonitrile, water, n-hexane and sodium chloride (Tacrolimus-loaded nanostructured lipid carriers for oral delivery - Optimization of production and characterization; Saba Khan et al, European Journal of Pharmaceutics and Biopharmaceutics; 108, 277-288, 2016).

The present invention relates to a topical composition. More particularly, the present invention relates to a topical composition in the form of Nanostructured Lipid Carriers (NLCs), which achieves desired efficacy with considerably enhanced drug penetration into the skin or mucosa, in comparison with a conventional oral treatment and/ or composition and can considerably avoids systemic side effects and helps in improving the physical condition of the skin in comparison with other available conventional treatment and/ or composition. Topical compositions include cream, ointment, gel, transdermal formulations, foam, spray, lotion, solution, emulsion, or suspension. Topical pharmaceutical compositions can be used for the treatment psoriasis and other skin diseases.

The prior art refers to some of the topical composition of Apremilast, but do not provide any teaching for topical composition of Apremilast loaded in NLCs.

OBJECTIVE OF THE INVENTION

It is an object of the present invention is to provide topical composition.

It is an object of the invention is to manufacture Nanostructured Lipid Carriers.

It is an object of the present invention is to provide topical composition comprising Nanostructured Lipid Carriers.

Yet another object of the invention is to manufacture phosphodiesterase 4 (PDE4) inhibitor loaded Nanostructured Lipid Carriers.

Yet another object of the invention is to manufacture Apremilast loaded Nanostructured Lipid Carriers.

It is another object of the invention is to provide a safe and effective means for the topical administration of phosphodiesterase 4 (PDE4) inhibitor.

Yet another object of the invention is to provide a safe and effective means for the topical administration of Apremilast.

It is another object of the present invention is to provide for compositions and methods for the prevention of psoriasis.

Yet another object of the present invention is to provide for compositions and methods for the prevention of other skin diseases

It is another object of the present invention to provide for compositions and methods for the treatment of psoriasis.

Yet another object of the present invention to provide for compositions and methods for the treatment of other skin diseases.

It is an object of the invention to provide topical composition for prevention of psoriasis.

Yet another object of the invention to provide topical composition for prevention of other skin diseases.

It is an object of the invention to provide topical composition for treatment of psoriasis.

Yet another object of the invention to provide topical composition for treatment of other skin diseases.

It is another object of the invention is to provide a topical gel to prevent psoriasis.

Yet another object of the invention is to provide a topical gel to prevent other skin diseases.

It is another object of the invention is to provide a topical gel to treat psoriasis.

Yet another object of the invention is to provide a topical gel to treat other skin diseases.

It is an object of the invention is to provide a method of preventing psoriasis.

Yet another object of the invention is to provide a method of preventing other skin diseases.

It is an object of the invention is to provide a method of treating psoriasis.

Yet another object of the invention is to provide a method of treating other skin diseases.

It is an object of the invention is to provide a method of making a topical gel to prevent psoriasis.

Yet another object of the invention is to provide a method of making a topical gel to prevent other skin diseases.

It is an object of the invention is to provide a method of making a topical gel to treat psoriasis.

Yet another object of the invention is to provide a method of making a topical gel to treat other skin diseases.

It is an object of the invention is to provide a method of application of a topical gel to prevent other skin diseases.

Yet another object of the invention is to provide a method of application of a topical gel to treat other skin diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Solubility of Apremilast in Solid Lipids.

Figure 2: Solubility of Apremilast in Liquid Lipids.

Figure 3: Solubility of Apremilast in Surfactants.

Figure 4: Solubility of Apremilast in Co-Surfactants

Figure 5: Lipid Content: Surfactant Evaluation Pseudo ternary phase diagram

Figure 6: Lipid Content: Surfactant System Optimization Pseudo ternary phase diagram

Figure 7: SEM image of NLC

Figure 8: Physical Appearance of developed Gel Composition

Figure 9: Rabbit Patch Study for Safety Profile

Figure 10: MTT assay result

Figure 11: Efficacy result of oxazolone induced dermatitis on rat ear

Figure 12: HET-CAM Analysis for Irritation Potential Study

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific pharmacologically active carriers, formulation types, treatments, so forth, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The present invention is of topical pharmaceutical composition comprising PDE-4 Inhibitors or pharmaceutically acceptable salt, ester, prodrug thereof for the treatment of psoriasis and other skin diseases. It also relates to the topical pharmaceutical composition comprising Apremilast or pharmaceutically acceptable salt, ester, prodrug thereof for the treatment of psoriasis and other skin diseases.

The present invention relates to a topical pharmaceutical composition comprising PDE-4 Inhibitors loaded in Nanostructured Lipid Carriers for the treatment of psoriasis and other skin diseases. The present invention also relates to a topical pharmaceutical composition comprising Apremilast loaded in Nanostructured Lipid Carriers for the treatment of psoriasis and other skin diseases.

The composition of present invention further comprises combination of Apremilast with at least one additional active agent. The at least one additional active agent comprises, a cosmetic active agent or a pharmaceutical active agent having a cosmetic or pharmaceutical effect or combination thereof.

The pharmaceutically active agent of present invention that can be used in combination is selected from the group consisting of active agents including but not limited to, herbal extracts, acaricides, corticosteroids, age spot and keratose removing agents, allergen, analgesics, local anesthetics, antiacne agents, antiallergic agents, antiaging agents, antibacterials, antibiotics, antiburn agents, anticancer agents, antidandruff agents, antidepressants, antidermatitis agents, antiedemics, antihistamines, antihelminths, antihyperkeratolyte agents, antiinflammatory agents, antiirritants, antilipemics, antimicrobials, antimycotics, antiproliferative agents, antioxidants, anti-wrinkle agents, antipruritics, antipsoriatic agents, antirosacea agents antiseborrheic agents, antiseptic, antiswelling agents, antiviral agents, anti-yeast agents, astringents, topical cardiovascular agents, chemotherapeutic agents, dicarboxylic acids, disinfectants, fungicides, hair growth regulators, hormones, hydroxy acids, immunosuppressants, immunoregulating agents, insecticides, insect repellents, keratolytic agents, lactams, metals, metal oxides, mitocides, neuropeptides, non-steroidal anti-inflammatory agents, oxidizing agents, pediculicides, photodynamic therapy agents, retinoids, sanatives, scabicides, self-tanning agents, skin whitening agents, vasoconstrictors, vasodilators, vitamins, vitamin derivatives, vitamin A and derivatives, vitamin D and derivatives, wound healing agents and wart removers.

The composition of present invention further comprises combination of Apremilast with therapeutically effective amount of corticosteroids such as steroidal anti-inflammatory agents, including but not limited to, corticosteroids such as mometasone, fluticasone, clobetasone, hydrocortisone, hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone phosphate, beclomethasone dipropionate, alclometasone, clobetasol valerate, deoxymethasone, diflorasone, fluocinolone, fluocinonide, halobetasol, desonide, deoxycorticosterone acetate, dexamethasone, dichlorisone, deflorasonediacetate, diflucortolone valerate, fluadronolone, fluclarolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocionide, flucortine butylester, fluocortolone, flupredylidene acetate, flurandronolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenalone acetonide, medrysone, amciafel, amcinafide, betamethasone, and esters of said compounds chlorprednisone acetate, Clocortolone, clescinolone, difluprednate difluprednate, flucloronide, flunisolide, Fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, (hydrocortamate), meprednisone, paramethasone, prednisolone, prednisone, beclomethasone, triamcinolone and mixtures thereof.

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific pharmacologically active carriers, formulation types, treatments, so forth, and as such may vary. The invention is capable of other embodiments or of being practiced or carried out in various ways. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Before describing the present invention in detail, it is to be understood that this invention is not limited to Solid lipid nanoparticles (SLNs), Nanostructured Lipid carriers (NLCs), Liposomes, Niosomes, Ethosomes, Transferosomes, etc. having meaning as understood by general knowledge with person of ordinary skilled in art so forth, and as such may vary.

SLNs, the first generation of lipid nanocarriers and also known as solid lipid nanospheres at room temperature. The solid lipid is used as a matrix material for drug encapsulation and can be selected from a variety of lipids, including monoglycerides to triglycerides; glyceride mixtures; and lipid acids.

NLCs are the second generation of lipid nanoparticles. NLC nanosystems are lipid nanoparticles composed of a solid lipid matrix incorporated with liquid lipid or oil. Liposomes are mainly composed of phospholipid bilayer vesicles containing phosphatidylcholine and phosphatidylethanolamine, the most common phospholipids found in nature, with other membrane bilayer constituents, such as cholesterol and hydrophilic polymers around each liposomal vesicle. Niosomes are formed by the hydration of nonionic surfactant with cholesterol incorporation as an excipient. Ethosomes are a modification of classical liposomes and are composed of multiple, concentric layers of flexible phospholipid bylayers, with a relative high concentration of ethanol (20-45%), glycols and water. Transfersomes are elastic liposomes composed of phosphatidylcholine and an edge activator.

The NLCs and formulations present in the invention it is to be evaluated that this invention is not limited to specific evaluation such as Particle Size, Polydispersity Index (PDI), Zeta Potential (ZP), pH and Scanning Electron Microscopy (SEM) etc.
Particle Size and PDI evaluation is performed on Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK). The Particle size & PDI is determined by diluting 0.1µl of micro-emulsion samples (n=3), with 0.9µl of deionized water and then loading the sample in a glass cell/cuvettes. The particle size and its distribution is determined at 25°C based on the principle of dynamic light scattering. Zeta Potential evaluation is performed on Zetasizer (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). The zeta potential is determined by diluting 0.1µl of micro-emulsion samples (n=3), with 0.9µl of deionized water and the sample is placed in an electrophoretic cell. The mean zeta potential is calculated from the electrophoretic mobility using the Smoluchowski equation. Electrophoretic mobility is measured by a combination of laser doppler velocimetry and phase analysis light scattering technique at 25°C. pH value evaluation is performed on pH Tutor (Eutech, Singapore). The 1 gm of the developed gel sample is taken, dispersed in 10ml of distilled water and analysed for its pH (n=3). pH meter is calibrated using standard buffer solution of pH 4.0, 7.0 and 10.0 prior to use. Scanning Electron Microscopy evaluation is performed on SEM (JEOL, JSM 50A, Tokyo, Japan). An appropriate amount of sample (NLC’s) is dropped on silicon wafer fractured with a razor blade and mounted onto aluminum specimen stubs using double-sided adhesive tape. The samples are sputter-coated with gold/palladium for 120 sec at 14 mA under argon atmosphere for secondary electron emissive SEM and observed for morphology, at an acceleration voltage of 20 kV.

DEFINITIONS:
The term "topical composition" or "topical formulation" means a composition in which the drug may be placed for direct application to a skin surface and from which an effective amount of the drug is released. Such formulations may include but are not limited to plasters, ointment, paste, cream, solution, suspension, emulsion, lotion, liniment, jelly, gel, poultice, foam, collodion, paints, powder, transdermal patch, and any combination thereof or any other dosage form suitable for topical application and the like. In some aspects, such formulations may be applied to the skin in an un-occluded form with/without additional backing, structures or devices.

The term "skin" or "skin surface" is meant to include the outer skin of a subject comprising one or more of epidermal layers to which a drug composition may be administered.

The term "skin disease”, is any condition of the diseases or disorders that affect the human/animal skin.

The term "treatment" or "treating" of a state, disorder or condition as used herein means treating psoriasis or other skin diseases.

The term “nanostructured lipid carriers” as used herein means nanoparticles composed of solid lipid and liquid lipid, and an aqueous phase containing a surfactant or a mixture of surfactants.

The term “lipid” as used herein, refers to any lipophilic compound. Non-limiting examples of lipid compounds include fatty acids and their derivatives, including straight chain, branched chain, saturated and unsaturated fatty acids, carotenoids, terpenes, bile acids, and steroids, including cholesterol and derivatives or analogues thereof.

The term "pharmaceutically acceptable carrier" or a "pharmaceutically acceptable salt/solvate or derivative" is meant a compound that is not biologically or otherwise undesirable, i.e., the compound may be incorporated into a topical formulation of the invention and administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.

The term "carriers" or "vehicles" as used herein refer to pharmaceutically acceptable carrier materials suitable for topical drug administration. Carriers and vehicles useful herein include any such materials known in the art that are non-toxic and do not interact with other components of the composition in a deleterious manner.

The term “pharmaceutically acceptable salt” as used herein is meant to include salts of the compounds of the invention which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the alts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compounds in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents. In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds or complexes described herein readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment.

The term "Apremilast" as used herein means Apremilast and its pharmaceutically acceptable salts in various solid state forms such as polymorphs, solvates and hydrates. Also, it refers to various isomers and enantiomers of Apremilast, specifically S-form and its pharmaceutically acceptable salts. Apremilast remains in substantially solubilized form in the composition.

Apremilast or pharmaceutically acceptable salt, ester, pro drug thereof, as per present invention can be used in the concentration of about 0.001% - about 30 %, preferably about 0.01% – about 20%, more preferably about 0.1% - about 15%. “Apremilast” is a novel, orally available small molecule inhibitor of type-4 cyclic nucleotide phosphodiesterase (PDE-4). Apremilast is chemically N-[2-[(1S)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)-ethyl]-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl]acetamide. The molecular formula and weight of Apremilast are C22H24N2O7S and 460.5 g/mole, respectively.

In March 2014, US FDA approved oral tablet of Apremilast for adults with active psoriatic arthritis. Subsequently, in September 2014 US FDA approved oral tablet of Apremilast for the treatment of moderate-to-severe plaque psoriasis in patients for whom phototherapy or systemic therapy is appropriate.

NLCs are a nanoparticulate carrier system derived from O/W nanoemulsions. Like nano- and micro-emulsions, the major ingredients of NLCs are lipids like solid lipids and liquid lipids, surface active agent and water.

The formulations of the present invention include those suitable for topical, transdermal, rectal and buccal (e.g., sub-lingual), vaginal, nasal, rectal, ophthalmic, injectable, administration etc. having meaning as understood by general knowledge with person of ordinary skilled in art. Preferably the formulations of the present invention are administered topically and are provided in the form of semisolid dosage forms.

The topical pharmaceutical composition of the present invention may include suitable pharmaceutically acceptable carriers. A wide range of ingredients including, lipids such as solid lipids, liquid lipid; surfactant and co-surfactant; emulsion stabilizers; and viscosity builders; emollients; humectants; odorants; preservatives, antioxidants, and chemical stabilizers; solvents; and thickening, stiffening, suspending agents; buffers, neutralizing agents and agents to adjust pH; opacifiers and colouring agents, pigments; and antifoaming agents, skin feel modifiers and the like.

PDE-4 inhibitors is selected, but not limited to, the roflumilast, apremilast, Cilomilast, crisaborole.

The PDE-4 inhibitors can be used in the concentration of about 0.001% - about 30 %, preferably about 0.01% – about 20%, more preferably about 0.1% - about 15%.

Exemplary solid lipid is selected, but not limited to, the group consisting of solid triglycerides such as trilaurin, tricapriloína, tripalmitin and tristearin, glyceryl trilaurate, glyceryl trimyristate or trimyristin, glyceryl tripalmitate, glyceryl Stearate, glyceryl tristearate, glycerol distearate, glyceryl hydroxystearate, glyceryl monostearate, glyceryl palmitostearate, glyceryl behenate, glyceryl dibehenate, or tribehenin; diglycerides solids such as dipalmitin and distearin; solid monoglycerides such as glyceryl monostearate; combinations of glycerides such as glyceryl palmitostearate, glyceryl stearate citrate and fats Witepsol series; aliphatic long chain alcohols such as cetyl alcohol and stearyl alcohol; fatty acid medium and long chain (C10-C22) such as stearic acid, palmitic acid, behenic acid and capric acid, and their esters with polyols such as polyethylene glycol, propylene glycol, polyoxylethylene stearates (like Tefose 63, Tefose 1500); esters of fatty alcohols with fatty acids long, such as cetyl palmitate, cetyl alcohol, cetearyl olivato and hydroxypropyl stearate, 2-hydroxypropyl stearate hydroxystearate hydroxyoctacosanol chain; sterols, cholesterol and cholesterol esters such as cholesteryl hemisuccinate, cholesteryl butyrate, Cholesterol butyrate, and cholesteryl palmitate; fatty amines such as stearylamine; waxes such as Emulsifying wax, beeswax, shea butter, cocoa butter, carnauba wax, ozokerite wax and paraffin wax; ceramides; 1-Pentadecanecarboxylic acid, , 2-Hydroxypropyl stearate, octadecyl ether, polyethylene glycol octadecyl ether, Triglycerides, Behenoyl polyoxyl-8 glycerides; hydrogenated vegetable oils like hydrogenated castor oil; quaternary derivatives, such as behenyl trimethyl ammonium chloride (INCI: Alkyltrimethyl ammonium chloride) ammonium, and / or mixtures thereof. Also certain lipophilic active can act as solid lipid matrix at room temperature, for example and without limiting sense, Lipochroman-6 (INCI: Dimethylmethoxy chromanol), Chromabright (INCI: Dimethylmethoxy chromanyl palmitate), coenzyme Q10 and / or mixtures thereof. Exemplary liquid lipid is selected, but not limited to, the group consisting of vegetable oils such as soybean oil, sunflower oil, corn oil, olive oil, palm oil, seed oil, cottonseed oil, rapeseed oil peanut, coconut oil, castor oil, linseed oil, borage oil, Oleic acid, evening primrose oil; marine oils, such as fish oils and algal oils; petroleum oils, such as mineral oil, liquid paraffin and petrolatum; short chain fatty alcohols; aliphatic fatty alcohols mid-chain branched; fatty acid esters with short chain alcohols, such as isopropyl myristate, isopropyl palmitate and isopropyl stearate and dibutyl adipate; medium chain triglycerides (MCT, Medium Chain Triglycerides) as triglycerides of capric and caprylic acids (INCI: Capric / caprilyc triglycerides) and other oils Miglyol® series (such as Miglyol 812); octanoates C12-C16; fatty alcohol ethers such as dioctyl ether, Glyceryl caprylate, Glyceryl monocaprylate (Capmul MCM), propylene glycol monocaprylate (Capmul PG 8, Capryol 90), Propylene glycol monolaurate (Capmul PG 12) etc. and / or mixtures thereof. Also certain lipophilic active can act as lipid matrix liquid at room temperature, for example and not limited to, beta-carotene, vitamin E and retinol, and / or mixtures thereof.

The percentage ratio of the liquid lipid to solid lipid in NLCs ranges from about 0.5 to about 3: about 1 to about 5, preferably about 1 to about 2.5: about 1 to about 4 and more preferably about 1.5 to about 2: about 1 to about 3.

The formation of micro- or nanoemulsions requires the addition of surfactants. In turn, the aqueous dispersions of lipid nanoparticles are stabilized by the addition of surfactants, cosurfactants, antiflocculants and / or viscosifying agents, favoring nanoparticle formation while minimizing the formation of aggregates of these. Exemplary surfactant and co-surfactant is selected from, but not limited to, the group consisting of nonionic surfactants, amphoteric surfactants, anionic surfactants, cationic surfactants and / or mixtures thereof. Macrogol 15 hydroxystearate, Polyoxyl 15 Hydroxystearate, Diethylene glycol monoethyl ether, Caprylocaproyl macrogol-8, Caprylocaproyl Polyoxyl-8 glycerides, Polyglycerol oleate, Polyglyceryl-3 dioleate, Polyoxylglycerides, Oleoyl polyoxyl-6 glycerides, Propylene glycol stearate, Polyethylene glycol hexadecyl ether, aluminium starch octenylsuccinate, ammonium hydroxide, amphoteric-9, beeswax, white beeswax, synthetic beeswax, carrageenan, carbomer 934, carbomer 934P, carbomer 940, ceteareth-20, ceteareth-30, cetearyl alcohol, ceteth 20, cetyl alcohol, cholesterol, cyclomethicone, diglycerides, dimethicone (e.g., dimethicone 350), disodium monooleamido sulfosuccinate, NF emulsifying wax, fatty acid pentaerythritol ester, glycerides, glyceryl monooleate, glyceryl monostearate, glyceryl stearate, lanolin, lanolin alcohol, hydrogenated lanolin, magnesium stearate, mineral oil, monoglycerides, polyethylene glycol, PEG stearate, polyethylene glycol 6000 distearate, polyethylene glycol 1000 monocetyl ether, polyethylene glycol monostearate, polyethylene glycol 400 monostearate, polyoxyethylene glycol fatty alcohol ethers, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbates, PPG-26 oleate, propylene glycol stearate, quatemium-15, simethicone, sodium laureth sulfate, sodium lauryl sulfate, sorbitan esters, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan palmitate, sorbitan sesquioleate, steareth-2, alcohols and low molecular glycols, such as propanol, isopropanol, butanol and hexanol weight; long fatty acids such as octanoic acid and butyric acid chain; monoesters of phosphoric acid; benzyl alcohol; bile acid salts such as sodium cholate, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate and / or mixtures thereof.
The percentage of the surfactant and/or co-surfactant of the NLCs in a ratio ranging from about 5 to about 1: about 0.5 to about 3, preferably from about 4 to about 1.5: about 0.7 to about 2.5, and more preferably about 3 to about 2: about 1 to about 2.
The percentage of the lipid and surfactant and/or co-surfactant of the NLCs in a ratio ranging from about 0.5 to about 5: about 1 to about 5. Preferably about 0.7 to about 4: about 1.5 to about 4 and more preferably about 1 to about 3: about 2 to about 3.

Exemplary antiflocculant is selected, but not limited to, the group consisting of sodium citrate, sodium pyrophosphate, sodium sorbate, amphoteric surfactants, cationic surfactants and / or mixtures thereof. Exemplary emulsion stabilizers and viscosity builders or gelling agent is selected, but not limited to, the group consisting of cellulose ethers and esters, such as methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and sodium carboxymethylcellulose; polyvinyl derivatives such as polyvinyl alcohol, polyvinylpyrrolidone and polyvinylacetate; alginates; polyacrylates, xanthanes; pectins; polymers such as carbomer 934, carbomer 934P, carbomer 940, cetearyl alcohol, cetostearyl alcohol, cetyl alcohol, cetyl stearyl alcohol, dextrin, diglycerides, disodium edetate, edetate disodium, glycerides, glyceryl monostearate, glyceryl stearate, monoglycerides, plasticized hydrocarbon gel, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 1450, polyethylene glycol 8000, polyethylene glycols, propylene glycol stearate, stearyl alcohol and the like. Exemplary emollients is selected, but not limited to, octyldodecanol, caprylic/capric triglycerides, castor oil, ceteareth-20, ceteareth-30, cetostearyl alcohol, ceteth 20, cetostearyl alcohol, cetyl alcohol, cetyl stearyl alcohol, cocoa butter, diisopropyl adipate, glycerine, gyceryl 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, propylene glycol mono stearate squalane, steareth-2 or -100, stearic acid, stearyl alcohol, urea and the like. Exemplary humectants is selected, but not limited to, glycerine, propylene glycol, sorbitol, urea and the like. Exemplary odorants is selected, but not limited to hypoallergenic perfume, menthol, eucalyptus oil, rose, jasmine, sandalwood, vanilla and the like. Exemplary preservatives, antioxidants, and chemical stabilizers include alcohol, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, calcium acetate, castor oil, chlorocresol, Potassium sorbate, 4-chloro-m-cresol, citric acid, disodium edetate, Dowicil 200 (Dow), edetate disodium, ethoxylated alcohol, ethyl alcohol, glycerine, Glydant Plus (Lonza), 1 ,2,6-hexanetriol, Kathon CG (Rohm & Haas), Liquid Germall Plus (ISP Sutton Labs), Liquipar (ISP Sutton Labs), methyl paraben, parabens, potassium sorbate, propyl gallate, propylene glycol, propyl paraben, sodium bisulfite, sodium citrate, sodium metabisulfite, sorbic acid, tannic acid, triglycerides of saturated fatty acids, Ucarcide (Union Carbide), Vitamin E, zinc stearate and the like. Exemplary solvents is selected, but not limited to, alcohol, castor oil, diisopropyl adipate, ethoxylated alcohol, ethyl alcohol, fatty alcohol citrate, glycerine, 1,2,6-hexanetriol, hexylene glycol, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, mineral oil, phosphoric acid, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 1450, polyethylene glycol 8000, polyethylene glycol 1000 monocetyl ether, polyethylene glycol monostearate, polyethylene glycol 400 monostearate, polyethylene glycols, polyoxyl 20 cetostearyl ether, polyoxypropylene 15-stearyl ether, polysorbates, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, octyldodecanol, propylene carbonate, propylene glycol, purified water, and SD alcohol 40, triglycerides of saturated fatty acids and the like. Exemplary thickening, stiffening and suspending agents is selected, but not limited to, aluminium stearate, beeswax, white beeswax, synthetic beeswax, carbomer 934, carbomer 934P, carbor?er 940, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, dextrin, glyceryl monostearate, hydroxypropyl cellulose, kaolin, paraffin, white soft paraffin, petrolatum, white petrolatum polyethylene, propylene glycol stearate, starch, stearyl alcohol, wax, white wax, xanthan gum, bentonite and the like. Exemplary buffers, neutralizing agents and agents to adjust pH is selected, but not limited to, phosphoric add, ammonium hydroxide, citric acid, diisopropanolamine, hydrochloric acid, lactic acid, monobasic sodium phosphate, sodium citrate, sodium hydroxide, sodium acid phosphate, triethanoiamine, trolamine and the like. Exemplary opacifiers/colorants is selected, but not limited to, for use may be organic and/or inorganic and the like. Suitable examples include titanium dioxide, and pre-dispersed titanium dioxide. Exemplary antifoaming agents is selected, but not limited to, cyclomethicone, dimethicone (e.g., dimethicone 350), simethicone and the like. Exemplary skin feel modifiers is selected, but not limited to Aluminium starch octenylsuccinate (gamma irradiated) and the like.

The temperature used in the process for synthesis of NLCs can be used in the temperature of about 10 oC - about 90 oC, preferably about 30 oC – about 70 oC, more preferably about 50 oC - about 80 oC.

The speed of cyclomixer/overhead stirrer used in the process for synthesis of NLCs can be used in the RPM of about 10 RPM - about 5000 RPM, preferably about 100 RPM – about 1000 RPM, more preferably about 100 RPM - about 3000 RPM.

The topical formulations of the present invention with Apremilast loaded nanostructured lipid carriers supposed to be found effective in the treatment of psoriasis and other skin diseases.

The process according to the invention is illustrated in details by the following but not limiting examples.

EXAMPLE 1: Solubility Study
1a: Solubility of Apremilast in Solid Lipids: The solubility of Apremilast was performed in various Solid Lipids such as stearic acid, glyceryl stearate (Softisan 601), Polyoxylethylene stearates (Tefose 63, Tefose 1500), polyethylene glycol octadecyl ether (Brij S20), 2-hydroxypropyl stearate (Monosteol) etc. FIG. 1 Shows solubility of Apremilast in Solid Lipids.

Procedure: A fixed amount of drug (5 mg) was taken in a microemulsion tube. Solid lipids were melted on a water bath maintained at temperature 50 C above the melting point of each solid lipid. Melted-solid-lipid/s were added to tube drop by drop in increasing amounts till the drug was completely solubilised in the lipid. The solubility of drug in solid lipids were calculated using total solid lipid quantity required to solubilize drug completely.

Observation: The amount of solid lipid quantity required to solubilize the drug completely is tabulated in Table 1 & figure 1.
Table 1: Solid Lipid Quantity to solubilize drug
Solid Lipid Concentration (mg/gm)
Stearic Acid 2.78
Softisan 601 8.33
Tefose 63 6.25
Tefose 1500 4.17
Brij S20 7.14
Monosteol 2.50

Results: The solubility of drug was found to be in the following order of solid lipids:
Softisan 601 > BrijS20 > Tefose 63 > Tefose 1500 > Stearic Acid > Monosteol.

1b: Solubility of Apremilast in Liquid Lipids: The solubility of Apremilast was performed in various Liquid Lipids such as Triglycerides (Miglyol 812), Propylene glycol monolaurate (Capmul PG 12, Lauroglycol 90), Propylene glycol monocaprylate (Capryol 90, Capmul PG 8), Glyceryl monocaprylate (Capmul MCM), etc. FIG. 2 Shows solubility of Apremilast in liquid Lipids.

Procedure: A fixed amount of drug (20 mg) was taken in an eppendorf tube and individual oil/s (500 mg) were added. Sealed eppendorf tubes were kept in water-bath-shaker at 37 0C for 24 hrs. After 24 hrs tubes were removed and centrifuged at 10,000 RPM for 15 min. Supernatant phase was diluted with suitable solvent and analysed using HPLC.

Observation: The amount of liquid lipid quantity required to solubilize the drug completely is tabulated in Table 2 & figure 2.
Table 2: Liquid Lipid Quantity to solubilize drug
Liquid Lipid Concentration (mg/gm)
Miglyol 812 3.74
Capmul PG 12 1.78
Lauroglycol 90 3.12
Capryol 90 4.95
Capmul MCM 3.81
Capmul PG 8 5.94

Results: The solubility of drug was found to be in following order of liquid lipid:
Campul PG 8 > Caproyl 90 > Campul MCM > Miglyol 812 > Lauroglyrol 90 > Campul PG 12

1c: Solubility of Apremilast in Surfactant: The solubility of Apremilast was performed in various surfactants like Tween 20, Tween 80 and Kolliphore HS 15.
Procedure: A fixed amount of drug (20 mg) was taken in an eppendorf tube and 1% solution of individual surfactant (500 mg) was added. Sealed eppendorf tubes were kept in water-bath-shaker at 37 0C for 24 hrs. After 24 hrs tubes were removed and centrifuged at 10,000 RPM for 15 min. Supernatant phase was diluted with a suitable solvent and analysed using HPLC.

Observation: The amount of surfactant quantity required to solubilize the drug completely is tabulated in Table 3 & figure 3.
Table 3: Surfactant Quantity to solubilize drug
Surfactant Concentration (mg/gm)
Tween 20 0.27
Tween 80 0.39
Kolliphor HS 15 0.25

Results: The solubility of drug was found to be in following order of Surfactants:
Tween 80 > Tween 20 > Kolliphore HS 15

1d: Solubility of Apremilast in Co-surfactant: The solubility of Apremilast was performed in various co-surfactants like Transcutol P, Labrasol, Plurol Oleique and Labrafil MS2125.

Procedure: A fixed amount of drug (20 mg) was taken in an eppendorf tube and 1% solution of individual co-surfactant (500 mg) was added. Sealed eppendorf tubes were kept in water-bath-shaker at 37 0C for 24 hrs. After 24 hrs tubes were removed and centrifuged at 10,000 RPM for 15 min. Supernatant phase was diluted with a suitable solvent and analysed using HPLC.

Observation: The amount of co-surfactant quantity required to solubilize the drug completely is tabulated in Table 4 & figure 4.
Table 4: Co-Surfactant Quantity to solubilize drug
Co-Surfactant Concentration (mg/gm)
Transcutol P 0.25
Plurol Oleique 0.08
Labrasol 0.15
Labrafil M2125 0.15

Results: The solubility of drug was found to be in following order of Co-surfactant:
Transcutol P > Labrasol = Labrafil MS2125 > Plurol Oleique

EXAMPLE 2: Nanostructured Lipid Carrier component compatibility Study
The compatibility/miscibility of liquid lipids and solid lipids were tested in various ratios to evaluate lipid phase formation.

Procedure: Blend of Solid Lipids and Liquid Lipids were taken in a micro-emulsion tube in a fixed ratio and heated on a water bath at a temperature 5 0C above the melting point of the solid lipids, and mixed well using cyclomixer. Table 5 gives the proportion ratios at which studies were carried out along with observation for each system. Systems were then allowed to stand for around 10 min, to observe for any layer/ phase separation at 250C ± 20C.

Observation: The systems that were clear and miscible at lowest concentrations of solid as well as liquid lipid will be most preferred, as our purpose is deposition in skin and not permeation to systemic circulation for the maximum efficacy.
Table 5: Miscibility of Solid Lipid and Liquid lipid at various ratios.
Sr. No. Mixture Solid Lipid: Liquid Lipid Observation
A Softisan 601
+
Capryol 90
1:1 System solidify slowly, Miscible
1:2 Clear System, Miscible
1:3 Clear System, Miscible
1:4 Clear System, Miscible
2:1 System solidify fast, Miscible
B Softisan 601
+
Capmul PG 8
1:1 System solidify, Miscible
1:2 System solidify, Miscible
2:1 System solidify fast, Miscible
C Softisan 601
+
Capmul MCM
1:1 System solidify, Miscible
2:1 System solidify, Miscible
1:2 System solidify fast, Miscible
D Softisan 601
+
Miglyol 812
1:1 System solidify, Miscible
1:2 System solidify, Miscible
2:1 System solidify fast, Miscible
E Softisan 601
+
Lauroglycol 90 1:1 System solidify slowly, Miscible
2:1 System solidify slowly, Miscible
1:2 System solidify fast, Miscible

Results: The system of Softisan 601 + Capryol 90 at various tested ratios provided the clear & miscible system & thus was most preferred Liquid lipid & Solid Lipid combination. The ratio of 1:2 of Softisan 601 + Capryol 90 provided desired system effect & was selected for further evaluations & used for preparation of NLCs.

EXAMPLE 3: Lipid: Surfactant System Optimization
A) Selection of Surfactant(s): The lipid phase of example 2 was evaluated with various surfactants such as Tween 20, Tween 80, Solutol HS 15 and Transcutol P. The aim of the study was evaluating the water uptake capacity of the micro-emulsion formed. Higher was the water uptake, greater was the micro-emulsion area & better was stability of system.

Procedure: The lipid phase of example 2 was introduced in a micro-emulsion tube in ascending order from 100 micrograms to 900 micrograms and heated on a water bath at a point till mixture just melts (i.e. to melting point of the solid lipid), and mixed well using cyclomixer, this forms the Lipid content of system. Surfactants were added in descending order from 900 micrograms to 100 micrograms to make final concentration of mixture [Solid Lipid: Liquid lipid (1:2) + surfactant/co-surfactant)] in each tube 1 gram and heated on water bath at melting temperature of solid lipid. The system was titrated using milli-Q water till the system turns from clear to translucent/cloudy. The table 6 provides the Lipid Content: Surfactant Titrations that were evaluated.

Table 6: Lipid: Surfactant Titrations
Sr. No Lipid Content: Surfactant
1 Softisan 601:Caproyl 90 Tween 80
2 Softisan 601:Caproyl 90 Tween 20
3 Softisan 601:Caproyl 90 Solutol HS 15
4 Softisan 601:Caproyl 90 Transcutol P

Observation: The pseudo ternary phase diagrams indicating water uptake capacity of micro-emulsions formed are depicted in figure 5.

Result: The figure 5 provides that system of lipid content with Tween 20 and Transcutol P were found to be satisfactory. The results also suggest that system using combination of Tween 20 as surfactant & Transcutol P as co-surfactant shall provide more stable system

B) Optimization of Surfactant(s): The surfactant & co-surfactant system of example (A) was further optimised using varied concentrations of surfactant & co-surfactant system.

Procedure: Blend of Solid Lipid and Liquid Lipid in a fixed ratio of 1:2 taken in a microemulsion tube in ascending order from 100 micrograms to 900 micrograms and heated on a water bath at a point till mixture just melts (melting point of the solid lipid), and mixed well using cyclomixer. Surfactants were added in descending order from 900 micrograms to 100 micrograms to make final concentration of mixture [Solid Lipid: Liquid lipid (1:2) + Tween 20: Transcutol P (in various concentration)] in each tube to 1 gram and heated on water bath at melting temperature of solid lipid. The system was titrated using milli-Q water and the water uptake capacity till the end point i.e. system turns clear to translucent/cloudy was observed. The table 7 provides the various Lipid Content: Surfactant System titrations that were evaluated.
Table 7: Lipid Content: Surfactant System titrations
Sr. No [Lipid(1:2): Surfactant Mixture (1:1 to 3:1)]
1 [Softisan 601:Capryol 90 (1:2)] : [Surfactant Mixture (1:1)]
2 [Softisan 601:Capryol 90(1:2)] : [Surfactant Mixture (2:1)]
3 [Softisan 601:Capryol 90(1:2)] : [Surfactant Mixture (3:1)]

Observation: The various ratios of surfactant/co-surfactant were evaluated for stability of emulsion. Higher the water uptake greater the micro-emulsion area, better is stability of micro-emulsion. The water uptake plots are depicted in figure 6.

Results: The figure 6 indicates that system with 2:1 ratio of surfactant: co- surfactant were showing more stable micro-emulsions. Thus system comprising [Softisan 601: Capryol 90(1:2)]: [Surfactant Mixture (2:1)] was taken for further development.

EXAMPLE 4: Drug Loading NLCs
The formulations were prepared using the lipid phase and surfactant mixture as depicted in Table 8.
Table 8: NLCs Composition
Sr. No. API (mg) % Lipid Phase % Surfactant mixture % water
A 10 15 30 55
B 10 20 50 30
C 10 15 40 45
D 10 10 40 50
E 10 20 45 35
F 10 10 30 60
G 10 10 50 40
H 10 22 38 40
I 10 15 35 50
J 10 20 45 35
K 10 20 45 35

Manufacturing Process:
i) Preparation of Drug in co-surfactant system: Drug was solubilised in optimised-ratio concentration of Transcutol P.
ii) Preparation of Lipid phase: The solid lipid and liquid lipid were added in drug test-tube to form lipid mixture and melted in a water bath at around 60 0C.
iii) Preparation of aqueous surfactant phase: In another test tube surfactant/s in preheated water around 50 °C to 80 °C was/were added.
iv) Emulsification Step: The lipid phase and aqueous surfactant phase were mixed using cyclomixer/overhead stirrer at 100 – 1000 RPM speed to form microemulsion; which was then dispersed in water under stirring at 100 – 3000 RPM.

Observation: The NLCs Dispersion formed was observed visually for clarity (translucency), instability, precipitation of particles or separation of oily phase at various time points and was evaluated for particle size, Scanning Electron Microscopy (SEM) and polydispersity index, zeta potential, pH and appearance.

Results: Particle size, PDI, and zeta potential values for the optimized formulation was found in the range of 100nm-600nm, 0.1-0.7%, and -3.81 ± 10 mV, respectively. The prepared formulation was found in the expected submicron range i.e. less than 600 nm, which was further confirmed by SEM analysis is depicted in figure 7. PDI is a measure of particle homogeneity and PDI result suggested that optimized ratio form nanoparticles with more homogenous size distributions due to the size reduction. Zeta potential result exhibiting a very good physical stability during the shelf-life. The results of tests performed reported in table no. 9.
Table 9: Physicochemical Evaluation
Particle Size 100nm to 600nm
Polydispersity Index (PDI) 0.1 to 0.7
Zeta Potential -3.81 ± 10
pH 3 to 7
Appearance Translucent

EXAMPLE 5: NLC’s Kinetic Stability
Apremilast NLC’s were screened for its kinetic stability. At room temperature samples were diluted 10 times with milli-q water, visual inspection of diluted samples were carried out for its stability at room temperature until 24hrs.

Observation: Glass vials were inspected for any change in colour, precipitation and phase separation.

Result: Samples of formulations (n=3) were analyzed visually and found stable for 24hrs after comparing it with initial 0hrs sample.

EXAMPLE 6: Gelling Agent optimization
The various commercial grades of carbomer polymers were selected as gelling agent and they were screened for parameters like gelling strength, dispersion ease, aesthetic appeal, feel, clarity and viscosity. The Table 10 depicts various commercial grades of carbomer along with their evaluated parameters.
Table 10: Screening of Gelling Agent
Gelling Agent Parameters
Gelling Strength Dispersion Ease Aesthetic Appeal Desired Viscosity Feel Clarity
Carbopol Ultrez 10 ? ? ? ? ? ?
Carbopol ETD 2020 ? ? ? ? ? ?
Carbopol 974-NF X ? ? X ? ?
Carbopol 71G X ? X X X X
Carbopol 940 ? ? X ? X X
Carbopol 980-NF ? ? X ? X ?

Results: The carbomer polymer “Carbopol Ultrez 10” and “Carbopol ETD 2020” showed the most desired effects in terms of parameters tested.
EXAMPLE 7: Topical Compositions
7a: Cream Composition: The NLCs of example 4 were formulated in cream composition using category of excipients as depicted in Table 11.
Table 11: Cream composition
Sr. No. Ingredients Concentration (% w/w)
1 Drug 0.001 – 30
2 Lipid base 0 – 70
3 Solubilizer & Penetration enhancer 0 – 50
4 Cosolvent 0 – 90
5 Emulsifier 0 – 30
6 Preservatives 0 – 5
7 Colourant 0 – 10
8 Perfume 0 – 5
9 Purified water q.s to 100

7b: Ointment Composition: The NLCs of example 4 were formulated in ointment composition using category of excipients as depicted in Table 12
Table 12: Ointment composition
Sr. No. Ingredients Concentration (% w/w)
1 Drug 0.001 – 30
2 Solubilizer & Penetration enhancer 0 – 50
3 Emollient 0 – 99
4 Liquid Paraffin 0 – 50
5 Hard Paraffin 0 – 50
6 Petroleum White 0 – 80
7 Colourant 0 – 10
8 Purified water qs.to. 100

7c: Gel Composition: The NLCs of example 4 were formulated in gel composition using category of excipients as depicted in Table 13
Table 13: Gel Composition
Sr. No. Ingredients Concentration (% w/w)
1 Drug 0.001 – 30
2 Gelling agent 0 – 15
3 Water for M.E. 0 – 10
4 Liquid Lipid 0 – 70
5 Solid Lipid 0 – 70
6 Surfactant 0 – 30
7 Co-surfactant 0 – 30
8 pH modifier 0 – 10
9 Water (NLC Dispersion) qs.to. 100

7d: Gel Composition: The NLCs of example 4 were formulated in gel composition using carbomer polymer viz Carbopol Ultrez 10 in varied concentration as depicted
in Table 14 & was evaluated for various physicochemical properties like viscosity, spreadability, particle size, appearance, pH etc.
Table 14: Gel composition
Sr. No. Ingredients A
Concentration (% w/w) B
Concentration (% w/w)
1 Drug 0.2 0.2
2 Carbopol Ultrez 10 0.7 0.9
3 Water 3.5 3.5
4 Capryol 90 1.33 1.33
5 Softisan 601 0.66 0.66
6 Tween 20 3 3
7 Transcutol P 1.5 1.5
8 Triethanolamine 0.01-1.2 0.01-1.2
9 Water (NLC Dispersion) qs.to. 100 qs.to. 100

Procedure: A drug was solubilized in a co-surfactant at a temperature of 60 ºC, in a beaker. Molten lipid mix was added to the said solubilized drug in beaker. The weighted quantity of surfactant and water was taken and mixed well in a separate container at a temperature of 60 ºC. Surfactant-water mixture was then poured into beaker under mild mixing conditions to get homogenous-clear micro-emulsion (M.E) system. The system was diluted using purified water under mild mixing conditions to form NLC dispersion. Carbopol was dispersed in said NLC dispersion formed above, followed by addition of Triethanolamine as a pH adjuster to form nano-gel under constant mixing.

Observation: The gel compositions developed as per table no. 14 were evaluated physicochemical properties as noted in table no 15.

Table 15: Physiochemical Evaluation of Gel.
Sr. No. Parameter Result
A B
1 Viscosity (cPs) 126000 ± 3000 154800 ± 3000
2 Torque 79.4 97.5
2 Particle Size (nm) 100-800 100-800
3 Firmness (g) 221.809 ± 50 300.993± 50
4 Spreadability (Work of Shear) (g.sec) 186.512 ± 30 232.686 ± 30
5 Appearance Translucent Translucent
6 Texture Smooth Smooth
7 Colour Faint Milky White Faint Milky White
8 pH 5-7 5-7

Results: The gel developed with 0.9 % of Carbopol Ultrez 10 was found to have better gelling property. The visual appearance of gel developed using 0.7 % Carbopol Ultrez 10 vs 0.9 % Carbopol Ultrez 10 is depicted in figure 8.

EXAMPLE 8: In-Vitro Release Testing (IVRT)
The cellulose nitrate membranes would be soaked in iso-propyl-myristate for 1hr and individually placed in between donor and receptor compartment of Franz diffusion cells set at 32 oC with thermostated water bath. 10 ml of ethanol: water (1:1 v/v) hydro-alcoholic media would be used as the receptor phase. Assurance would be made that the skin is fully in contact with the receptor phase, leaving out any air bubbles. The stopper would be placed on the withdrawal port for removing the receptor phase. Next, about 300 to 350 mg/cm2 of formulations (0.5 gm) would be uniformly placed in the donor phase, in contact with the cellulose nitrate membrane. Receptor phase would be stirred constantly throughout the experiment and the temperature kept constant. At set intervals of 0, 1, 2, 4, 6, 12 and 24 hours, 0.5 ml of the receptor phase would be removed and immediately replaced by the same volume of hydro-alcoholic media. The amount of drug released into the receptor phase from the gel formulations would be determined by injecting 100µl sample to HPLC system.
EXAMPLE 9: In vitro Skin permeation study
In vitro Skin permeation study would be conducted on skin (Rat skin/Abdominal skin of guinea/Human cadaver skin) using Franz diffusion cells to determine the amount of drug released. The skin specimen would be cut carefully as short as possible using scissors, without damaging or scratching the skin surface. Then, the skin would be freed from muscle, fat or vasculature, the same would be kept in deep freezer for a period of 24 hours before experimentation. The prepared skin specimens would be individually placed in Franz diffusion cells set at 32 0C with thermostated water bath. The diffusion medium (10 ml) would be used as the receptor phase. The excised sections of skin would be carefully placed in between the donor and receptor compartments of the diffusion cells and firmly stuck in place assuring that the skin would be fully in contact with the receptor phase, leaving out any air bubbles. The stopper would be placed on the withdrawal port for removing the receptor phase. Further, around 300 to 350 mg/cm2 of formulation (0.5 g) would be placed uniformly in the donor phase, in contact with the excised section of skin specimens. The receptor phase would be stirred constantly throughout the experiment while keeping the temperature constant. At set of predetermined intervals, 1 ml of the receptor phase would be removed and immediately replaced with the same volume of diffusion media-solution. The amount of drug released into the receptor phase would be determined. After determining the amount of drug released at pre-determined time intervals, the plot would be drawn of cumulative drug permeated (percent) versus time (hours). The amount of drug remaining on the skin (i.e. in the donor compartment) would be calculated by using HPLC after diluting suitably with the mobile phase/acetonitrile/methanol/ethanol (5-20 ml). To calculate the amount of drug deposited in the skin, the formulation would be gently wiped off from the skin. The skin would be minced, transferred to a test tube containing mobile phase/acetonitrile/methanol/ethanol (5-20 ml), and then would be vortexed using a cyclomixer followed by sonication for 10 minutes in bath sonicator. The resulting solution would be filtered through 0.45 µm membrane and injected into the HPLC system to determine the drug concentration.

EXAMPLE 10: Skin Irritation Study
Skin Irritation Study was performed on rabbits. One day prior to the experimentation, three rabbits weighing 2.5 - 3 kg was prepared by removing the hair from the back and sides using an animal clipper and razor. Formulation of 0.5 g was applied on the hair free skin of the rabbits by uniformly spreading within an area of 4 cm2 and the skin was observed for any visible changes such as erythema (redness) or oedema (swelling) at predetermined time interval. The ‘Primary irritation index’ of the formulation was evaluated using the scale; Draize’s scale. The Draize test is used to measure irritation or corrosion caused to the skin. The animals are observed for up to 14 days for signs of erythema and edema in the skin test and is subsequently measured on a numerical scale. The Draize’s scale is noted in table 16. Further the microscopy study of Rabbit Patch Study would be conducted.
Table 16: Scale for evaluation of skin irritation reaction
Skin Reaction Observation Score
Erythema No erythema 0
Very slight erythema 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema 4
Oedema No oedema 0
Very slight oedema 1
Slight oedema 2
Moderate oedema 3
Severe oedema 4

Observation: The ‘Primary irritation index’ of the formulation was evaluated using the Draize’s scale as noted in table no 16 and rabbit patch study for safety profile is depicted in figure 9.

Result: The tested gel on the basis of scoring of Draize scale was found to be non-irritant, showed no erythema and oedema. Thus, the prepared gel formulation is non-irritant and safe for topical application.

EXAMPLE 11: In-Vitro cytotoxicity studies on murine fibroblasts
The In-Vitro cytotoxicity study was performed on 3T3 murine fibroblasts cell line using MTT assay. The cytotoxicity of the developed gel formulation was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. Cells were seeded into 96-well plate and incubated it for 24 hr. After 24 hr, investigational sample (gel formulation) was added into wells in triplicate and incubated it for 24 hr. After an exposure of 24 hr to the investigational samples, MTT reagent was added in each well. Again plates will be incubated at 37 0C for 4 hr in dark. After 4 hr sodium dodecylsulfate reagent (DMSO) was added to dissolve formazan crystals and plate was kept at room temperature for 15 minutes. Readings were taken and compared the investigational sample with the positive control was evaluated by using 10% solution of Triton X-100 and negative control was evaluated by using Cells culture media - Dulbecco's Modified Eagle Medium (DMEM).

Observation: The readings were taken and compared the investigational samples with the control as noted in table no 17 and depicted in figure 10.
Table 17: The in-vitro cytotoxicity studies on murine fibroblasts
Sr. No. Parameter IC50 value
1 In-Vitro cytotoxicity
(conc in µg/mL) 55 ± 0.5

Result: MTT assay of cytotoxicity studies on murine fibroblast confirmed IC50 values of gel exhibits very high potency against 3T3 cells of fibroblast.

EXAMPLE 12: Anti-elastase activity for skin elasticity
The study of Anti-elastase activity for skin elasticity was performed on Porcine Pancreatic Elastase (PPE, Sigma, and Type IV) sample by using Enzyme inhibition assay. The test sample was added to Tris- HCL buffer initially then enzyme was added and incubated for 15 minutes. The substrate was added to initiate the reaction. Each test reaction was incubated for 30 minutes at 25 0C and readings was taken. An appropriate blank was ran and conducted the experiment in triplicate to check its mean percentage inhibition.

Observation: The experiment of Anti-elastase activity for skin elasticity was conducted and the percentage inhibition is noted in table no 18.
Table 18: Anti-elastase activity for skin elasticity
Sr. No. Parameter % Inhibition
1 Anti-elastase 84 ± 5

Result: The prepared gel formulation exhibited very good enzyme inhibitory activity. The elastase enzyme inhibitory activity suggests that it can help in restoration of skin elasticity, thereby slowing down aging.

EXAMPLE 13: Anti-hyaluronidase activity for skin elasticity
The study of Anti-hyaluronidase activity for skin elasticity was performed on Bovine hyaluronidase using Enzyme inhibition assay technique. The bovine hyaluronidase was mixed with test formulation of various concentration which was dissolved in DMSO. It was incubated for 20 min at 37 0C. The control group was treated with DMSO instead of test formulation. Hyaluronidase was activated by calcium chloride in reaction mixture and incubated for 20 min at 37 0C. The Ca+2 activated hyaluronidase was subjected to sodium hyaluronate and was incubated in water batch exactly for 3 minutes at 100 0C. The reaction mixture was cooled to room temperature, p-dimethyl amino benzaldehyde was added and incubated in water bath for 20 minutes at 37 0C. The sample was ran and its absorbance was checked and its percentage anti-hyaluronidase activity was calculated.

Observation: The experiment of Anti-hyaluronidase activity for skin elasticity was conducted and percentage inhibition is noted in table no 19.
Table 19: Anti-hyaluronidase activity for skin elasticity
Sr. No. Parameter % inhibition
1 Anti-hyaluronidase 21 ± 1

Result: The prepared gel formulation exhibited very good enzyme inhibitory activity. The hyaluronidase enzyme inhibitory activity suggests that it can help in diminishing skin drying and wrinkling.

EXAMPLE 14: Efficacy testing using ‘Oxazolone induced dermatitis’ or other relevant pharmacodynamic models.
Study of Efficacy testing using ‘Oxazolone induced dermatitis’ or other relevant pharmacodynamic models was performed on Female Sprague Dawley Rats using Skin lesions in rats. For sensitization, 300µl of 2% oxazolone (dissolved in ethanol) was applied to abdomen region of Sprague Dawley rats. On 7th day total of 60µl of 60% [dissolved in acetone: olive oil (4:1)] oxazolone was applied to both sides of every 3 days starting from after sensitization. Application of oxazolone was repeated for five times. The thickness will be measured using digital caliper for 72 hr after each application of oxazolone. The 100mg of test, standard and control formulation was applied to both sides of ear before and after each application of oxazolone. On the last day of experiment (day 22), ear thickness was measured and animals was sacrificed after 1 hr of final topical administration of formulations (2 hr after the final oxazolone challenge), and ear for histopathological examination for hematoxylin and eosin staining would be excised. The stastaical analysis was performed using student’s ‘t’ test and one-way analysis of variance (ANOVA) followed by Tukey-Kramer Multiple Comparison test.

Observation: The efficacy testing using ‘Oxazolone induced dermatitis’ was evaluated and is noted in table no 20 and depicted in figure 11.
Table 20: Efficacy of prepared gel on rat skin tissue
Sr. No. Parameter Evaluation
1 Oxazolone induced dermatitis
(Visual Inspection)
(Using Vernier Caliper) Moderate to Fast
Reduction in Inflammation after Oxazolone Sensitization

Result: Visual inspection reflects better efficacy of gel than standard marketed formulation. Measured ear thickness concludes very fast recovery of inflamed tissue.

EXAMPLE 15: Dermal Penetration Study using confocal microscopy
Oxazolone (60µl of 60% [dissolved in acetone: olive oil (4:1)] would be applied to both sides of ears of Female Sprague Dawley Rats for sensitization. After sensitization, 100mg of coumarin-6 (radioactive dye - ? max = 525nm) loaded investigational samples, standard marketed formulation, placebo gel and control formulation (oxazolone) would be applied to both sides of ear before and after each application of oxazolone. At 0hrs, 4hrs, 12hrs, 24hrs, 48hrs and 72hrs animals would be sacrificed after, and ears would be excised. Tissue sectioning would be done on cryotome and slides would be observed on confocal microscope on DAPI emission band range. Dermal penetration would be calculated after confocal analysis.

EXAMPLE 16: Immunohistochemistry study of CD-4 antibody
For sensitization, 300µl of 2% oxazolone (dissolved in ethanol) would be applied to abdomen region of Sprague Dawley rats. On 7th day total of 60µl of 60% [dissolved in acetone: olive oil (4:1)] oxazolone would be applied to both sides of every 3 days starting from after sensitization. Application of oxazolone would be repeated for five times. The 100mg of investigational samples, standard (marketed), placebo gel and control formulation (oxazolone) would be applied to both sides of ear before and after each application of oxazolone. On the last day of experiment (day 22), animals would be sacrificed after 1 hr of final topical administration of formulations (2 hrs after the final oxazolone challenge), and ears would be excised for microtome sectioning of hematoxylin and eosin staining. Further, CD-4 inflammatory marker would be studied using its immunohistochemistry protocol.

EXAMPLE 17: HET-CAM (hen's egg-chorioallantoic membrane test) study for irritation-toxicity analysis
The purpose of this study was to determine corrosive and severe irritation of test substance. Fresh, clear fertile (not older than 7 days around 50-70 gm) chicken eggs were collected and assembled vertically in incubator at 38.3±0.20C and 58 ± 2% RH for a day. On 9th day eggs were candled and marked on its air cell. Dentist saw blade was used to cut the marked portion of egg shell without disturbing inner white membrane linings of shell. 0.9% NaCl solution was used to moisten inner membrane; after NaCl treatment eggs were incubated for 30min. Before application of test substance NaCl solution was decanted from eggs. Inner white membrane linings were removed carefully with the help of forceps. 0.9% NaCl solution and 10% KOH solution was used as negative and positive control respectively. Applied 0.3ml equivalent test substance, negative and positive control on fertile egg sac separately (n=6). Changes were observed for hemorrhage, vascular lysis and coagulation of blood vessels form 0 seconds to 300 secs. Time dependent scoring was given to hemorrhage, vascular lysis and coagulation parameters for calculating index of irritation potential by IS(B) analysis method.

Observation: There were no signs of hemorrhage, vascular lysis and/or coagulation observed from 0 to 300 secs after the treatment/application of gel formulation.

Result: On the basis of time dependent visual inspection is depicted in figure 12 value of gel HET-CAM scoring was found to be less than 270, prepared gel formulations were confirmed as safe and non-irritant for its topical application as per ICCVAM Hen’s Egg Test, Recommended Protocols, 2006
,CLAIMS:WE CLAIM:

1. A nanocarrier pharmaceutical composition comprising:
a) a nanoparticle loaded with PDE-4 inhibitor or its pharmaceutically acceptable salt, ester, prodrug; and
b) one or more pharmaceutically acceptable excipients.

2. The pharmaceutical composition as claimed in claim 1, wherein the PDE-4 inhibitor is selected from roflumilast, apremilast, Cilomilast and crisaborole, or combination thereof.

3. The pharmaceutical composition as claimed in claim 1, wherein the composition is Nanostructured Lipid carriers (NLCs), Solid lipid nanoparticles (SLNs), Liposomes, Niosomes, Ethosomes, Transferosomes, etc.

4. The pharmaceutical composition as claimed in claim 1-3, wherein the composition is a topical dosage form.

5. The pharmaceutical composition as claimed in claim 4, wherein the topical dosage form is in the form of gel, solution, ointment, suspension, cream, paste, powder and tincture.

6. The pharmaceutical composition as claimed in claim 4, wherein Apremilast is present in an amount of from 0.001 to 30.00 wt. %.

7. The pharmaceutical composition as claimed in any of claims 1-5, wherein the composition further comprises a solid lipid, a liquid lipid, a polymer, a co-polymer, a gelling agent, a surfactant, a co-surfactant, a preservative, a pH adjusting agent, an occlusive agent, an emollient, an anti-oxidant, a humectant and moisturizer, a chelating agent, colouring agent and opacifying agent, perfume, penetration enhancer and a solvent.
8. A composition for treating psoriasis and other skin disorders comprising Apremilast compound or its pharmaceutically acceptable salt, ester, prodrug and a solid lipid, a liquid lipid, a polymer, a co-polymer, a gelling agent, a surfactant, a co-surfactant, a preservative, a pH adjusting agent, an occlusive agent, an emollient, an anti-oxidant, a humectant and moisturizer, a chelating agent, colouring agent and opacifying agent, perfume penetration enhancer and a solvent.

9. The pharmaceutical composition as claimed in any of claims 1-8, wherein the composition optionally comprises an additional active.

10. Process for synthesis of NLC comprises solid lipid, liquid lipid, surfactant, co-surfactant and water and this method is feature with the following step:
A) Preparation of Drug in co-surfactant system: Drug is solubilised in optimised co-surfactant system.
B) Preparation of Lipid phase: The solid lipid and liquid lipid is added in drug test-tube to form lipid mixture and melted in a water bath at around 60 0C.
C) Preparation of aqueous surfactant phase: In another test tube surfactant/s in preheated water around 50 °C to 80 °C is added.
D) Emulsification Step: The lipid phase and aqueous surfactant phase is mixed using cyclomixer/overhead stirrer at around 100 – 1000 RPM speed to form microemulsion; which is then dispersed in water under stirring at around 100 – 3000 RPM.

Dated this 18th day of May, 2020

Mehul Shah
For Applicant Encube Ethicals Pvt Ltd