FORM 2

THE PATENTS ACT  1970
(39 of 1970)
&
THE PATENTS RULES  2003

PROVISIONAL SPECIFICATION
(See section 10  rule 13)

“TOPICAL OIL COMPOSITION FOR THE TREATMENT OF FUNGAL INFECTIONS”

VYOME BIOSCIENCES PVT LTD  a company registered under the companies act of India at Plot No.459 F.I.E.  First floor  Patparganj Industrial Area  Delhi 110 092  India

The following specification particularly describes the invention and the manner in which it is to be performed.
TOPICAL OIL COMPOSITION FOR THE TREATMENT OF FUNGAL INFECTIONS

FIELD OF THE INVENTION
The present invention provides topical oil composition comprising an anti-fungal agent  base-oil and excipients or additives  for the treatment of fungal infections. More particularly  the invention provides the composition free from fatty acids (from C-10 to C-24) or their esters for the treatment of topical fungal infections.

BACKGROUND OF THE INVENTION
Fungal infections of the skin are also known as ‘mycoses’. They are common and generally mild. However  in very sick or otherwise immune suppressed people  fungi can sometimes cause serious disease. Fungal infections in humans range from superficial  i.e.  skin surface to deeply invasive type or disseminated infection.

In general superficial fungal infections (also known as cutaneous mycosis) can affect the outer layers of the skin  the nails and hair. The main groups of fungi causing superficial fungal infections are dermatophytes (tinea)  yeasts e.g. candida  malassezia  piedra  etc. and moulds. These infections include dandruff/seborrheic dermatitis (D/SD)  ringworm  onychomysis  intertrigo  and those in psoriasis amongst others.

Seborrheic dermatitis is a common  chronic  superficial skin disorder causing scaly  itchy  red skin on the scalp  eyebrows  nasolabial creases  lips  ears  sternal area  axillae  submammary folds  umbilicus  groins  and gluteul crease. The disease is characterized by many shapes  sizes  and surface textures and is often crust-like  yellowish  and accompanied by itching. Seborrheic dermatitis is one of the leading causes of stubborn dandruff and occurs in all age groups. This condition primarily affects the sebaceous cysts present in the skin.

Currently  fungi of the genus Malassezia  are believed to be the most likely responsible agents for causing dandruff (Dawson TL  J. Investig. Dermatol. Symp. Proc. (2007)  12:1519).Most cases of seborrhoeic dermatitis likely involve an inflammatory reaction to the proliferation of the yeast Malassezia  These fungi are highly dependent on external lipids for in vitro growth (Chen TA and Hill PV  Vet Dermatol  (2005)  16:4). Further  the inability to synthesize fatty acids may be complimented by the presence of multiple secreted lipases to aid in utilizing host lipids. Consequently  these fungi metabolize triglycerides present in sebum through these lipases resulting in lipid byproducts. Penetration of the top layer of the epidermis  the stratum corneum  by these lipid byproducts results in an inflammatory response in susceptible persons  which disturbs homeostasis causing erratic cleavage of stratum corneum cells which further leads to dandruff and seborrheic dermatitis.

Most common treatment of fungal infections is the topical application of antifungal agents that reduce the level of Malassezia on the scalp. Maintaining the scalp clean is mandatory for sufferers of seborrheic dermatitis and therefore using anti-dandruff shampoos which are effective may be one way of preventing getting this condition.

Typically  the antifungal agent is applied to the scalp as a component of a shampoo or other hair care composition. The disadvantage of such shampoo formulations is that during normal usage  the formulation does not remain on the scalp for a period of time sufficient to allow the antifungal agent to achieve its maximal therapeutic effect ((Ralph M.Trüeb  JDDG  (2007)  5:356). These are designed to be applied  for example in the shower or bath  and shortly after  rinsed off with water. Typically  the application instructions for such shampoos suggest that the formulation be removed after 3-5 minutes.
One of the anti-fungal agents  ketoconazole is among the most potent and widely used in anti dandruff shampoo. However  the exposure time of shampoo is less  due to which the efficacy is poor and relapse rates are higher.
US application 2002/0172648 describes anti-dandruff composition comprising a combination of three known anti-dandruff agents  preferably in the form of shampoo to prevent or reduce the occurrence of dandruff. Said compositions contain fatty acids as an additive or auxiliary substance which directly serves as a nutrient for the fungus.
Another  the US patent application No. 2010/0016271 discloses a hair conditioning composition comprising of cationic surfactant  triglyceride oil and an anti-dandruff agent. Said composition contains triglyceride oil  as an essential ingredient. These triglycerides are fatty acid esters of glycerol  and hence act as a nutrient or aid in the growth of the fungus. Said compositions contain fatty material up to 10% having carbon chains from 8 to 30 carbon atoms.

U.S. Patent No. 5 624 666 exemplifies and claims shampoo compositions  containing anionic surfactants  cationic polymers  and zinc pyridinethione as an anti-dandruff agent. It describes that conditioning agents such as silicone fluids can optionally be incorporated into the compositions therein. Head & Shoulders® Dandruff Shampoo Plus Conditioner is an example of a marketed product which provides both anti-dandruff and conditioning benefits upon application of the shampoo to hair. Patent describes about shampoo formulation  the exposure time of shampoos are less and hence relapse rate are higher.
The other alternative commercially available formulations for treatment of dandruff are leave-on hair formulations such as hair oil  styling gel etc having antidandruff or antifungal agents. Conventional leave-on formulations especially oil or cream has fatty acid or their esters as an essential ingredient. These fatty acid esters help in the growth of dandruff-causing fungus (Malassezia) and works as a source of nutrient.
Nevertheless  consumers desire a personal care composition that provides improved cleansing and anti-dandruff efficacy versus currently marketed products. The present invention addresses this need by providing topical oil compositions having antifungal agents which are free from fatty acid(s) or their ester(s) in order to achieve maximal therapeutic effect.

OBJECTS OF THE INVENTION:
The primary objective of the present invention is to provide a topical oil composition for the treatment of fungal infection.
Another objective of the invention is to provide a topical oil composition which is free from fatty acid(s) or their ester(s).
Yet another objective of the invention is to provide better retention/penetration of anti-fungal agent onto the hair  skin  scalp  and/or nail to reduce relapse of fungal infection

SUMMARY OF THE INVENTION
The present invention provides anti-fungal topical compositions which is free from fatty acid(s) or their ester(s). Fungi of the genus Tinea  Malassezia and Candida lack fatty acid synthase. As these fungi cannot produce their own fatty acids required for biosynthesis of its cell wall  they metabolize mono-  di- and triglycerides present in sebum  through lipase secretion to release Free Fatty Acids (FFAs) for the synthesis of ergosterol (an important constituent of the fungal cell wall). Thus  the invention provides anti-fungal topical composition which is devoid of fatty acid(s) and their ester(s). Since these serve as nutrients for the growth of the fungus.
Furthermore  said topical oil composition prevent or reduce relapse of dandruff formation or elimination/alleviation of already existing dandruff on the scalp.

DETAILED DESCRIPTION
Accordingly  the present invention relates to a topical oil composition for the treatment of fungal infections  comprising:
(A) atleast an anti-fungal agent;
(B) a base oil and;
(C) solvents  excipients or additives.

The anti-fungal agent as used herein is selected from piroctone olamine  ciclopirox olamine  ketoconazole  climbazole  miconazole nitrate  itraconazole  fluconazole  econazole  terconazole  saperconazole  amorolfine  oxiconazole  clotrimazole  luliconazole  terbinafine  butenafine  naftifine  selenium disulfide  salicylic acid  sulfur  tar preparations  undecanoic acid derivatives  zinc pyrithione  hinokitol etc. and chemical compound from natural sources  such as extract of arnica  walnut shells  tea tree oil  rosemary oil   birch etc.

More preferably  the ant-fungal agent used in the composition is piroctone olamine or ketoconazole or combination thereof.

The amount of anti-fungal agent used in the composition is in the range of 0.01 % or 10 % by weight of the total composition more preferably from 0.01 % to 5 % by weight of the total composition or more preferably from 0.01 % to 2 % by weight of the total composition

The base oil used herein is paraffin oil  silicone oils  oleyl alcohols (fatty alcohols which are liquid at room temperature) and mixture(s) thereof. Particularly preferred is paraffin oil.

The amount of base oil used in the composition is in the range of 50 % to 99 % by weight of the total composition.

The amounts of used solvents  excipients or additives are in the range of 0.5 % to 20 % by weight of the total composition.

The solvents as used herein are selected from C-1 to C-6 lower aliphatic alcohols comprising of ethanol  isopropyl alcohol etc.  lower alkyl acetate  lower carbon carboxylic acid (caprylic acid  capric acid etc.) or mixture/s thereof  fatty alcohols such as oleyl alcohol etc.

The composition also comprises of additive/s used in the cosmetic field  such as thickener  antioxidants  perfumes/fragrances  essential oils  preserving agents  hair conditioning substances  hair care ingredients  emollient  dyestuffs  moisturizers  vitamins  sphingoceryls  sunscreens  surfactants  oil-soluble polymers which are compatible with the base oil and/or skin care agents including skin-nutrient agents  anti-wrinkle agents  light and dust protector etc.

For example  the above-described additive substances  which can be contained in the compositions include thickener (for example bentonite  cellulose etc.)  antioxidants (for example  butylated hydroxytoluene (BHT)  butylated hydroxyanisole (BHA)  tert-butylhydroquinone (TBHQ)  ferulic acid  Vit A  Vit E (Tocopherol) etc.)  preservatives (for example  methyl p-hydroxybenzoate or propyl p-hydroxybenzoate  sorbic acid etc.)  hair care ingredients (for examples  fatty alcohols  peptides  proteins  vitamins etc. and mixture thereof)  light protective agents or sunscreens (for examples  p-methoxycinamic acid isoamyl ester etc.)

The surfactants as used herein are selected from cetearths  ceteth  isoceteths  laureths  oleths  steareths  lauramide DEA  linoleamide DEA and other surfactants which are suitable for topical application.

The essential oils as used herein are selected natural/synthetic such as eucalyptus oil  rosemary oil  pine needle oil  tea tree oil  sage oil  cinnamon oil  lemon oil  lime oil  orange oil  peppermint oil  spearmint oil  wintergreen oil  sweet birch oil  clove leaf oil  camphor oil  cardamon oil  cedar leaf oil sweet birch oil and others.

The pH adjuster as used herein is selected from inorganic or organic acids (e.g. citric acid  lactic acid  succinic acid  acetic acid  fumaric acid  glycolic acid  benzoic acid)  bases  salts and/or buffers thereof.

The oil soluble herbal extract as used herein are selected from Amla fruit extract  Arnica Extract  Brahmi extract etc.

The hair care adjuncts as used herein are selected from ones beneficial in the treatment of hair loss or the promotion of hair growth such as taurine  caffeine  minoxidil  azelaic acid  marine cartilage  hydrolysed keratin  biotin  niacin  panthenol  vitamin B6  zinc  copper  peptides  horsetail silica  beta sitosterols  pycnogenol  PABA  green tea extract  folic acid  iron  L-cysteine  magnesium and ginseng.

Skin care adjuncts as used herein are selected from ones beneficial for the treatment of various skin conditions (like dry skin  oily skin  fine lines  pigmentation  etc.) such as proteins  vitamins (e.g. A  B  C  D  E  and K)  trace metals (e.g. zinc  calcium and selenium)  moisturizers (including  e.g.  emollients  humectants  film formers  occlusive agents  and agents that affect the natural moisturization mechanisms of the skin)  UV absorbers (physical and chemical absorbers such as paraminobenzoic acid (PABA)  titanium dioxide  zinc oxide  etc.)  anti-irritants (e.g. steroids and non-steroidal anti-inflammatories)  botanical extracts (e.g. aloe vera  chamomile  cucumber extract  ginkgo biloba  ginseng  and rosemary)  absorbents (e.g.  aluminum starch octenylsuccinate  kaolin  corn starch  oat starch  cyclodextrin  talc  and zeolite)  skin bleaching and lightening agents (e.g.  hydroquinone and niacinamide lactate)  humectants (e.g.  sorbitol  urea  and manitol)  exfoliants  skin conditioning agents (e.g.  aloe extracts  allantoin  bisabolol  ceramides  dimethicone  hyaluronic acid  and dipotassium glycyrrhizate) and other natural components (e.g. oatmeal).

The topical oil composition potent in antifungal activity of the present invention is applied topically in the form of oil.

The topical oil composition of the present invention is used in the treatment of diseases associated with Malassezia but are not limited to tinea pedis  tinea capitis  tinea cruris  tinea glabrosa  tinea corporis  onychomycosis  pityriasis capitis  pityriasis vesicolor  pityrosporum folliculitis  seborrheic dermatitis etc.

Further  the term "treatment" covers any topical fungal treatment in a mammal  particularly a human.

Further  these formulations could be of veterinary use in the topical treatment of dermatological fungal infections.

Further  the said composition gives better retention/penetration of anti fungal agent onto the hair  skin  scalp and/or nail.

Further aspect of the present invention is to provide a method of treating fungal infections of a skin  scalp  hair or nail  using said composition containing anti-fungal agent/s dissolved in minimum quantity of solvent and solubilized  suspended or emulsified in base oil.
A preferred embodiment the topical antifungal compositions are provided as anti-dandruff oil.

EXAMPLES
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1: Preparation of various oil compositions containing Piroctone Olamine
The compositions were prepared by dissolving the active agent in ethanol or isopropyl alcohol (IPA). The oleyl alcohol was then added and stirred until a homogenous solution was obtained. Other excipients or additives were added and stirred to get clear solution except liquid paraffin. Weight was finally made up with liquid paraffin and stirred until homogenous solution was obtained. Final formulations were clear transparent oil solutions. Table 1 describes anti-fungal clear oil compositions containing piroctone olamine as anti-fungal agent using various excipients or additives.

Observations:
Table 1: Piroctone olamine – oil compositions
Formulation. Code PO
(mg) Ethanol
(mL) IPA
(mL) Oleyl Alco(mL) Triacetin
(mL) Caprylic Acid
(mL) Tocopherol acetate
(mg) Tea Tree Oil
(mL)

(mL) Cyclomethicone
(mL) Liq. Paraffin
(mLupto) upto) pH Appearance

VPO-001 50 0.4 -- 0.4 -- 0.1 -- 0.5 -- 10 6-7 C
VPO-002 50 0.4 -- 0.4 -- 0.1 -- -- -- 10 6-7 C
VPO-018 10 -- 0.2 0.2 -- 0.02 -- -- -- 10 6-7 C
VPO-019 10 -- 0.2 0.2 0.02 0.02 -- -- -- 10 6-7 C
VPO-020 10 -- 0.2 0.3 0.04 0.02 -- -- -- 10 6-7 C
VPO-021 10 -- 0.2 0.5 0.06 0.02 -- -- -- 10 6-7 C
VPO-022 10 -- -- 0.4 -- 0.02 -- -- -- 10 6-7 C
VPO-023 10 -- -- 0.4 0.02 0.02 -- -- -- 10 6-7 C
VPO-024 10 -- -- 0.5 0.04 0.02 -- -- -- 10 6-7 C
VPO-025 10 0.05 -- 0.05 --- 0.02 -- -- -- 10 6-7 ST
VPO-026 10 0.05 -- 0.3 0.02 0.02 -- -- -- 10 6-7 C
VPO-027 10 0.05 -- 0.3 0.02 0.02 -- 0.1 -- 10 6-7 C
VPO-028 10 0.05 -- 0.1 -- 0.02 -- -- -- 10 6-7 C
VPO-030 10 -- 0.2 0.2 -- 0.02 -- -- 0.1 10 6-7 C
VPO-031 10 0.05 -- 0.1 -- 0.02 -- -- 0.1 10 6-7 C
VPO-032 10 -- 0.2 0.2 -- 0.02 -- -- 1.0 10 6-7 C
VPO-033 10 0.05 -- 0.1 -- 0.02 -- -- 1.0 10 6-7 C
VPO-034 10 -- 0.2 0.2 -- 0.02 50 -- -- 10 6-7 C
VPO-035 10 0.05 -- 0.1 -- 0.02 100 -- -- 10 6-7 C
VPO-036 10 -- 0.2 0.2 -- 0.02 25 -- -- 10 6-7 C
VPO-037 10 -- 0.2 0.2 -- 0.02 50 -- -- 10 6-7 C
VPO-038 10 -- 0.2 0.2 -- 0.02 75 -- -- 10 6-7 C
VPO-039 10 -- 0.2 0.2 -- 0.02 100 -- -- 10 6-7 C
VPO-040 10 -- 0.2 0.2 -- 0.02 100 -- 1.0 10 6-7 C
VPO-041 10 -- 0.2 0.2 -- 0.02 100 0.2 1.0 10 6-7 C
VPO-042 10 0.05 -- 0.1 -- 0.02 25 -- -- 10 6-7 C
VPO-043 10 0.05 -- 0.1 -- 0.02 50 -- -- 10 6-7 C
VPO-044 10 0.05 -- 0.1 -- 0.02 75 -- -- 10 6-7 C
VPO-045 10 0.05 -- 0.1 -- 0.02 100 -- -- 10 6-7 C
VPO-046 10 0.05 -- 0.1 -- 0.02 100 -- 1.0 10 6-7 C
VPO-047 10 0.05 -- 0.1 -- 0.02 100 0.2 1.0 10 6-7 C
VPO-049 10 -- -- -- -- 0.3 -- -- -- 10 6-7 C
VPO-050 10 -- -- 0.2 -- 0.2 -- -- -- 10 6-7 C
VPO-051 10 -- -- -- -- 0.3 50 0.1 0.5 10 6-7 C
VPO-052 10 -- -- 0.1 -- 0.3 50 0.2 0.5 10 6-7 C
C- Clear  ST- Slight turbid

Result:
1. Compositions using liquid paraffin as base oil containing piroctone olamine  were clear oil solutions.
2. Addition of substances such as tea tree oil  cyclomethicone (D4)  tocopherol acetate etc. did not affect the physical stability of formulations as compositions appeared as clear oil solution.

Example: 2 Preparation of various oil compositions containing ketoconazole
The compositions were prepared by dissolving the active agent in ethanol. The oleyl alcohol was then added and stirred until homogenous solution was obtained. Other excipients or additives were added and stirred to get clear solution except liquid paraffin. Weight was finally made up with liquid paraffin and stirred until homogenous solution was obtained. Final formulations were clear transparent oil solutions. Table 2 describes anti-fungal clear oil compositions containing ketoconazole as anti-fungal agent using various excipients or additives.

Observations:

Table 2: Ketoconazole – oil compositions

Formula-
Tion Code Keto
(mg) Ethanol
(mL) Oleyl Alco
(mL) Caprylic acid
(mL) Tea Tree Oil (mL) Terpene-4-0l (mL) Toco-pherol acetate
(mg) Cyclomethicone
(mL) Liq. Paraffin
(mL  upto) pH Appear-
ance

VK-001 10 0.5 0.45 -- 0.5 -- -- -- 10 6-7 C
VK-002 10 0.5 0.45 -- -- -- -- -- 10 6-7 C
VK-012 5 0.3 0.3 -- -- -- -- -- 10 6-7 C
VK-013 5 0.3 0.3 -- 0.1 -- -- -- 10 6-7 C
VK-014 5 0.3 0.3 -- 0.2 -- -- -- 10 6-7 C
VK-015 5 0.3 0.3 -- 0.3 -- -- -- 10 6-7 C
VK-016 5 0.3 0.3 -- 0.4 -- -- -- 10 6-7 C
VK-017 5 0.3 0.3 -- 0.1 -- -- 0.2 10 10 C
VK-018 5 0.3 0.3 -- 0.1 -- -- 0.5 10 10 C
VK-019 5 0.3 0.3 -- 0.1 -- -- 1.0 10 10 C
VK-020 5 0.3 0.3 -- -- -- -- 1.0 10 6-7 C
VK-030 5 -- 0.1 0.3 -- 0.1 20 -- 10 10 ST
VK-031 5 -- 0.1 0.3 -- 0.2 20 -- 10 10 C
VK-032 5 -- 0.1 0.3 -- 0.3 20 -- 10 10 C
VK-036 5 -- 0.1 0.4 -- 0.1 20 -- 10 10 C
VK-037 5 -- 0.1 0.4 -- 0.2 20 -- 10 10 C
VK-038 5 -- 0.1 0.4 -- 0.3 20 -- 10 10 ST
VK-040 5 -- 0.4 0.4 -- 0.4 20 -- 10 10 C
C-Clear transparent  ST-Slight turbid

Result:
1. Compositions using liquid paraffin as base oil containing ketoconazole  were clear oil solutions.
2. Addition of substances such as tea tree oil  terpene-4-ol  caprylic acid  cyclomethicone (D4) etc. did not affect the physical stability of formulations and compositions appeared as clear oil solution.

Example 3: Study of oil containing fatty acid/esters and fatty acids/esters as source of nutrient for the growth of M.furfur under in vitro condition.
Malassezia species are lipophilic unipolar yeasts recognized as commensals of skin that may be pathogenic under certain conditions (Indian Journal of Medical Microbiology  (2004) 22 (3):179-181). To compare lipid requirements of the fungus most closely associated with dandruff/seborrheic dermatitis  the best studied Malassezia species is M. furfur. Lipid assimilation in vitro assay was designed to investigate lipid effect on growth of M. furfur (MTCC 1374).

Method: Briefly  Sabouraud Dextrose containing low-melt agar was melted  cooled to 38oC. Fatty acids/esters constituents eg  capric acid  caprylic acid  linoleic acid  oleic acid  lauric acid  palmitic acid ethyl oleate  isopropyl myristate and oils containing fatty acid/esters eg  coconut oil  mustard oil etc.  were added to study the growth of the fungus (Kaw Bing CHUA  et al Malaysian J Pathol (2005) 27(2): 99). After solidification  agar plates were streaked with M.furfur innoculum adjusted to appropriate cfu/ml  aseptically. Positive control with 2% olive oil and negative control without fatty substance were also maintained.

Observations:

Fig. 1: Picture showing growth/no growth of M.furfur depending on the nutrients provided in culture media

Results:
1. Results showed that there was no growth of M. furfur in absence of fatty acids/esters or oils in in vitro condition (Fig. 1) up to 6 days.
2. Culture media which contained fatty acids or esters eg  capric acid  caprylic acid  linoleic acid  sefsol  oleic acid  lauric acid  palmitic acid ethyl oleate  isopropyl myristate and oils containing fatty acid/esters eg  coconut oil  mustard oil etc. were showed confluent growth of fungus up to 6 days.
3. Culture media with lower carbon fatty acids (C≤10) caprylic acid(C8) and capric (C10) were failed to provide nutrient for the growth of fungus and no growth was observed up to 6 days.

Example 4: Bioactivity of oil compositions described in example 1 against M. furfur
The Minimum Inhibitory Concentration (MIC) is considered as an index for indicating Anti-fungal efficacy. Therefore lower the value of MIC of the composition  the better is its anti-fungal efficacy.

Method: The in vitro activities of some of the oil compositions containing piroctone olamine against Malassezia furfur (MTCC 1374) were determined by agar dilution methods (Jan Faergemann  et al Acta Derm Venereol  (2006)  86:312; Irith Wiegand  et al Nature Protocols (2008)  3:163) Appropriate dilutions of solubilized antifungal compositions were added to molten Leeming Notman Medium. Once the plates were set  M.furfur innoculum adjusted to appropriate cfu/ml was streaked on the agar plates and incubated for 6 days. After incubation  the plates were observed at day 3 and day 6 for visible M.furfur growth. The MIC is defined as as the lowest concentration of antifungal agents that inhibits visible growth of fungus.

Observations:
Table 3: Results of MIC for oil compositions of piroctone olamine
Day 3 and Day 6 Piroctone Olamine concentration Final concentration of piroctone olamine in culture medium of fungus.
Formulation Codes 8 16 32 64
VPO-001 5 mg/mL + + -- --
VPO-018 1 mg/mL + + -- --
VPO-022 1 mg/mL + + -- --
VPO-028 1 mg/mL + + -- --
VPO-030 1 mg/mL + + -- --
VPO-031 1 mg/mL + + -- --
VPO-032 1 mg/mL + + -- --
VPO-033 1 mg/mL + + -- --
VPO-034 1 mg/mL + + -- --
VPO-035 1 mg/mL + + -- --
VPO (Negative control) --- + + + +
PO in DMSO (Positive control) 1 mg/mL + + -- --
“+” indicates growth of fungus and “ –“ indicates no growth of fungus
Result:
1. Piroctone olamine containing oil compositions VPO-018  VPO-022  and VPO-028 with different solvents isopropyl alcohol  oleyl alcohol  and ethanol  respectively  showed MIC at 32µg/ml which is similar to the MIC of positive control where drug is dissolved in DMSO at the same concentration.
2. Addition of substances such as caprylic acid  cyclomethicone (D4)  tocopherol acetate etc. did not affect the MIC of oil compositions.

Example 5: Bioactivity of oil compositions described in example 2 against M. furfur

Method: The in vitro activities of some of the oil compositions containing ketoconazole against Malassezia furfur (MTCC 1374) were determined by agar dilution methods. Appropriate dilutions of solubilized antifungal compositions were added to molten Leeming Notman Medium. Once the plates were set  M.furfur innoculum adjusted to appropriate cfu/ml was streaked on the agar plates and incubated for 6 days. After incubation  the plates were observed at day 3 and day 6 for visible M.furfur growth. The MIC is defined as the lowest tested dilution of antifungal active that yields no growth.

Observations:

Table 4: Results of MIC for oil compositions of ketoconazole
Day 6 Ketoconazole concentration Final concentration of ketoconazole(µg/ml) in fungus culture medium of fungus
Formulation Codes 0.0625 0.125 0.25 0.5 1 2
VK-001 1 mg/mL + + -- -- -- --
VK-002 1 mg/mL + + -- -- -- --
VK-013 0.5 mg/mL + + -- -- -- --
VK-019 0.5 mg/mL + + -- -- -- --
Negative control --- + + + + + +
Keto in DMSO (Positive control) 1 mg/mL + + -- -- -- --
“+” indicates growth of fungus and “ –“ indicates no growth of fungus

Result:
1. Ketoconazole containing oil compositions showed MIC at 0.25 µg/ml  which is similar to the MIC of positive control where drug is dissolved in DMSO at the same concentration.