Formulation for treating acne

Abstract

The present invention pertains to a formulation designed for the treatment of acne, possessing potent antibacterial activity The invention additionally outlines a method for preparing this formulation tailored for acne treatment. The formulation, presented as a topical gel, incorporates a synergistic blend of doxycycline and riboflavin, along with key components such as carbopol, emollient, penetration enhancer, parabens, distilled water, and triethanolamine. Utilizing a 32 factorial design approach, the formulation is statistically optimized to achieve optimal characteristics. Notably, the formulation demonstrates a harmonious interaction between doxycycline and riboflavin, resulting in enhanced antibacterial efficacy. Furthermore, the formulation exhibits proven nonirritant properties and maintains stability during storage.

Description

[0001] FORMULATION FOR TREATING ACNE FIELD OF THE INVENTION: The present invention relates to a formulation for treating acne and more specifically to a topical formulation of doxycycline and riboflavin for treating acne. BACKGROUND OF THE INVENTION: Acne, also known as acne vulgaris is chronic skin condition that primarily affects the pilo-sebaceous unit of hair follicles. The underlying pathology involves the obstruction of hair follicles due to the accumulation of oil and dead cells, resulting in various manifestations such as blackheads, whiteheads (comedones), papules, pustules, nodules, pimples, oily skin, and the potential for scarring. The complexity of acne is heightened by the emergence of inflammatory lesions, notably on facial, neck, shoulder, and chest regions. The interplay between human hormones, sebum production, and bacterial activity further influences the progression and severity of the disorder. Onset typically occurring in early adolescence, acne is a skin condition that spans across various age groups. The formation of acne is influenced by a multitude of factors. These encompass genetic predisposition, smoking habits, stress levels, hormonal imbalances, high-calorie diets, lifestyle disorders, insufficient physical activity, excessive cosmetic usage, poor hygiene practices, heightened androgen and PCOD levels, as well as underlying medical conditions like diabetes and kidney diseases. During puberty, usually the hormonal changes contribute to formation of acne. Increased androgen production leads to an increase in sebum production, as a result leading to acne formation. Further, a major contributing factor for acne is the gram-positive anaerobic bacterium Propionibacterium acnes (P. acnes). P. acnes forms a part of the resident flora of the pilosebaceous follicles and contributes to the inflammatory process due to the production of follicular lipases, proteases, hyaluronidases, and several other enzymes. Inflammation is a key component in the pathogenesis of acne. There is development of papules, pustules and nodules after rupture of comedones. Their content is extruded into dermis and there is initiation of inflammation by a non- immune foreign body reaction. Significant research has been dedicated to both the treatment and prevention of acne. Approaches to address acne encompass lifestyle modifications, pharmaceutical interventions, and various medical procedures. Medications can be administered through different routes, including topical and oral applications. Additionally, therapeutic modalities such as light therapy, chemical peeling, drainage, extraction, and the use of steroids contribute to the spectrum of available treatments for acne. Various antibiotics like minocycline, tetracycline, erythromycin etc. and retinoids are available in topical and oral formulations. The medications available for acne usually work by reducing oil production and swelling or by treating bacterial infection. However, resistance to antibiotics may develop as a result of long-term antibiotic therapy. Tetracyclines are widely used in the treatment of acne due to their antimicrobial effect on P. acnes and their anti‐inflammatory mechanisms. However, there is development of resistance to tetracycline‐class of antibiotics due to the mechanism of efflux of antibiotics and conjugal transfer of plasmids and transposons. The U.S. Patent Application US2004228908A1, authored by Lin Tongho and team, details a topical composition designed for transdermal administration. The primary constituents of this composition comprise multivitamins, a thickening agent, and a surfactant. The disclosed composition demonstrates utility in the treatment of acne, comedones, and zits. The U.S. Patent Application US2011217249A1, to Dreher Frank, pertains to compositions and methods for the topical application of compounds that sequester antimicrobial peptides, targeting the treatment of skin disorders in individuals. Furthermore, the U.S. Patent US8513304B2, to Kisak Edward T and others, outlines a topical formulation comprising a combination of an antibacterial agent and an active agent, providing insights into potential therapeutic applications. The formulations found in prior art demonstrate utility in treating skin conditions broadly. However, formulations incorporating antibiotics, such as tetracyclines, exhibit inconsistent results attributed to the emergence of antibiotic resistance in microorganisms. There is a need for an improved anti-acne formulation having consistent antimicrobial effect and leading to reduction in inflammation. SUMMARY OF THE INVENTION: The present invention discloses a formulation for treating acne. The formulation contains 8-10% doxycycline 8-10%, 1-2% riboflavin, 1-2% carbopol, 1-2% emollient, 3-7% penetration enhancer, and 0.1-0.5% parabens as preservatives. The formulation further contains 100ml distilled water for adjusting the volume of the formulation, and triethanolamine for adjusting the pH of the formulation in the range 6 to 6.5. The emollient in the said formulation is glycerin and the penetration enhancer is clove oil. The parabens are methyl paraben and propyl paraben. The said formulation is preferably a topical formulation in gel form. The invention further describes a method 100 of preparing the formulation for treating acne. The method includes a first step 105 of dispersing 1-2% of carbopol in 50 ml of distilled water containing 1-2% of glycerin with vigorous stirring and keeping it overnight at 4°C to attain complete hydration. In a second step 110, 8-10% of doxycycline and 1-2% of riboflavin is dispersed in water and this is added to the gel base of first step 105. In a third step 115, 3-7% of clove oil, 0.10% methyl paraben and 0.02% propyl paraben preservatives are added to the gel base. A fourth step 120 includes adjusting pH of the formulation to 6-6.5 with triethanolamine. In a fifth step 125, the final weight of the gel product is adjusted to 100 g with distilled water. The formulation of the present invention has a gel viscosity of 2200 Cps. The gel formulation has a drug content in the range of 85% to 100%. The maximum amount of the doxycycline and riboflavin is dispersed uniformly in the gel. The enhancement ratio is 2.25 fold for doxycycline and 3.91 fold for riboflavin in the said formulation. The formulation for treating acne effectively acts against the bacterium P. acnes and suppresses the inflammatory responses of acne caused due to the activation of inflammatory cells. The formulation shows enhanced ROS generation ensuring augmentation of pathogen clearance. Further the gel exhibits demonstrated nonirritant properties and is stable on storage. BRIEF DESCRIPTION OF DRAWINGS: The objectives and advantages of the present invention will become apparent from the following description read in accordance with the accompanying drawings wherein, Figure 1 demonstrates the peak intensities observed in the infrared spectrum in the IR analysis studies of pure form of doxycycline drug in accordance with the present invention; Figure 2 demonstrates the peak intensities observed in the infrared spectrum in the IR analysis studies of pure form of riboflavin drug in accordance with the present invention; Figure 3 demonstrates the peak intensities observed in the infrared spectrum in the IR analysis studies of combination of pure form of Doxycycline and Riboflavin in accordance with the present invention; Figure 4 depicts the calibration curve of doxycycline in accordance with the present invention; Figure 5 depicts the calibration curve of riboflavin in accordance with the present invention; Figure 6A demonstrates the 2D contour and Figure 6B demonstrates the 3D response surface plots showing effect of Carbopol concentration and clove oil concentration on the viscosity of gel formulation in accordance with the present invention; Figure 7A depicts a line graph showing effect of different concentration of (A) Carbopol 940 on viscosity of gel formulation and Figure 7B depicts a line graph showing effect of different concentration of(B) clove oil on viscosity of gel formulation in accordance with the present invention; Figure 8 depicts the graphical representation of desirability and optimized conditions of Carbopol concentration and clove oil concentration for viscosity of gel formulation in accordance with the present invention; and Figure 9 demonstrates the permeation flux values of both the active compounds and their formulations in accordance with the present invention. DESCRIPTION OF THE INVENTION: The present invention describes a formulation for treating acne and a method of preparing the formulation. The antiacne formulation of the present invention is a topical gel formulation containing Doxycycline and Riboflavin. References in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, characteristic, or function described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment. References in the specification to “preferred embodiment” means that a particular feature, structure, characteristic, or function described in detail thereby omitting known constructions and functions for clear description of the present invention. The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed and obviously many modifications and variations are possible in light of the above teaching. In one aspect, the present invention relates to formulation for treatment of acne. The formulation of the present invention incorporates a blend of Doxycycline and Riboflavin (Vitamin B2) in safe and therapeutically effective amounts, along with other compatible ingredients. In another aspect, the present invention describes a method of preparation of the formulation for treating acne containing a blend of Doxycycline and Riboflavin. In yet another aspect, the present invention discloses a process of optimizing the formulation for treating acne. In accordance with an embodiment, the formulation for treating acne includes: 1) Doxycycline 8 - 10%; 2) Riboflavin 1 - 2%; 3) Carbopol 1 - 2%; 4) Emollient 1 - 2%; 5) Penetration enhancer 3 - 7%; 6) Paraben 0.1 - 0.5% as preservatives; 7) Distilled water 100 ml; and 8) Triethanolamine in the range 0.1% for adjusting the pH. In accordance with a preferred embodiment, the pH of the formulation is adjusted to 6 to 6.5 by triethanolamine; the emollient is glycerin; the penetration enhancer is clove oil; and the parabens are methyl paraben and propyl paraben. In accordance with the preferred embodiment, the formulation is a topical formulation in gel form. In accordance with the present invention a method 100 of preparation of the formulation for treating acne includes a series of steps. 1. In a first step 105, 1 - 2% of Carbopol is dispersed in 50 ml of distilled water containing 1 - 2% of glycerin with vigorous stirring. The solution is then kept overnight at 4°C to attain complete hydration. 2. A second step 110 includes dispersing 8 - 10% of doxycycline and 1 - 2% of riboflavin in water and adding it to the gel base of first step 105. 3. In a third step 115, 3 - 7% of clove oil, 0.10% methyl paraben and 0.02% propyl paraben preservatives are added to the gel base. 4. In a fourth step 120, the pH of the formulation is adjusted to 6 - 6.5 with triethanolamine. 5. A fifth step 125 includes adjusting the final weight of the gel product to 100g with distilled water. In accordance with the present invention, a process 200 of optimization of the antiacne topical gel formulation by a 32factorial design approach with a Design Expert software is described. The process includes a first step 205 of investigating the effect of two independent variables X1 (Concentration of Carbopol 940) and X2 (Concentration of clove oil) on the viscosity (Y1) of gel formulation. In a second step 210, the effect of independent variables on the responses is studied using ANOVA (F Value). Further, a third step 215 includes calculating the sum of square, degree of freedom, F-value, and p-value of each factor for studied response. In a fourth step 220, the effect of individual as well as combined independent variables over viscosity is studied by 2D contour and 3D response surface plots. In a fifth step 225, an analysis of the closeness between the actual and predicted value of each response of all formulations is performed. A sixth step 230 includes deciding the concentration of independent variables to optimize to achieve desired gel viscosity, by considering the extrudability and spreadability of all the prepared gels. In a seventh step 235, the concentration of clove oil required to get desired permeation of gel formulation across the skin is obtained. The final eighth step 240 includes choosing the most desirable formulation, preparing the optimized formulation, and contrasting the findings with values projected by the software. EXAMPLES: Only a few examples and implementations are disclosed. Variations, modifications, and enhancements to the described examples and implementations and other implementations can be made based on what is disclosed. Examples are set forth herein below and are illustrative of different amounts and types of reactants and reaction conditions that can be utilized in practicing the disclosure. It will be apparent, however, that the disclosure can be practiced with other amounts and types of reactants and reaction conditions than those used in the examples, and the resulting devices various different properties and uses in accordance with the disclosure above and as pointed out hereinafter. EXAMPLE 1: INFRARED (IR) SPECTROSCOPY ANALYSIS Study: IR analysis of pure drug Doxycycline; pure drug Riboflavin; and a combination of Doxycycline and Riboflavin was performed to obtain functional groups and to study the interaction between groups in combination. Objectives: IR spectroscopy was performed i. to determine functional groups in the molecules, as IR Spectroscopy measures the vibrations of atoms; ii. to obtain an infrared spectrum that is a plot of measured infrared intensity versus wavelength (or frequency) of light; and iii. to determine the peak intensities in infrared spectra for deducing the concentration of molecules in the sample. Methodology: IR analysis of pure form of Doxycycline; pure form of Riboflavin; and physical mixture of Doxycycline and Riboflavin in 1:1 ratio was performed. Observation: As illustrated in Table 1, the IR analysis of pure form of Doxycycline demonstrated values of Primary Aliphatic Amine, C-F(halide) and Conjugated Ketone C=O Stretch functional groups comparable with the reference range. Figure 1 demonstrates the peak intensities in the infrared spectrum of pure form of Doxycycline drug. Sr. Functional Group Observed Value Reference range No. (cm-1) (cm-1) 1 Primary Aliphatic Amine 3423 3300 - 3400 2 C-F (halide) 2975.62 2700 - 3200 Conjugated Ketone C=O 3 1612.2 1666 - 1685 Stretch Table 1: IR analysis studies of pure form of Doxycycline drug As illustrated in Table 2, the IR analysis of pure form of Riboflavin demonstrated values of Alcoholic O-H Stretch and Ketone C=O Stretch functional groups comparable with the reference range. Figure 2 demonstrates the peak intensities in the infrared spectrum of pure form of Riboflavin. Sr. No Functional Group Observed Value Reference range (cm-1)(cm-1) Alcoholic O-H 1 2935.13 2700 - 3200 Stretch Ketone C=O 2 1731.76 1705- 1728 Stretch Table 2: IR analysis studies of pure form of Riboflavin The IR analysis of physical mixture of Doxycycline and Riboflavin in 1:1 ratio demonstrated no specific drug interaction and intact functional group peaks. Figure 3 demonstrates the peak intensities in the infrared spectrum of combination of pure form of Doxycycline and Riboflavin. Result: The physical mixture of Doxycycline and Riboflavin in 1:1 ratio did not show any specific chemical interaction between the two components under study. This implies that the individual properties of the drugs are retained when in combination, maintaining their individual activity. EXAMPLE 2: ANTIMICROBIAL POTENTIAL OF DOXYCYCLINE, RIBOFLAVIN (VITAMIN B2) AND THEIR COMBINATION Study: Evaluation of antimicrobial potential of Doxycycline, Riboflavin (Vitamin B2) and their combination in relation to Propionibacterium acnes (P acnes) in vitro. Objectives: Antimicrobial potential of doxycycline, riboflavin (vitamin b2) and their combination were evaluated i. to study MIC (Minimum inhibitory concentration); ii. to study MBC (Minimum bacterial concentration); and iii. to study synergism. In vitro interactions are calculated algebraically and interpreted as synergistic, indifferent or antagonistic depending on whether the antibacterial activity of the combination is greater than, equivalent to or less than the activities of the individual agents respectively. Study Drugs and Chemicals: a) Riboflavin (Vitamin B2) - Purchased from Yarrow Chem Products. b) Doxycycline- Purchased from Yarrow Chem Products. c) Propionibacterium acnes MTCC 1951 -purchased from MTCC (Microbial Type Culture Collection and Gene Bank), Chandigarh. Strain and culture specification - Genus Name: Propionibacterium Species Name: acnes Organism: Bacteria Risk Group: N / A Authority: (Gilchrist) Douglas & Gunter Collection Acc. No 1951TAHIFPC / Other culture collections NCTC737, ATCC6919, DSM1897, Received as genus: Propionibacterium Preservation suspending medium: 10% Glycerol, 10% Skim milk Received as species: acnes Isolated or derived from: Facial acne Received from NCTC, London Growth Condition: Anaerobic Strain designation *737(Type Strain) Incubation: 48 Hours Subculturing period: 30 Days Temperature: 37℃ Special features: Type strain Anatomical: N / A Reference: N / A Growth medium: 50 d) Sterilized syringe - needles e) Agar and broth - HIMEDIA M210-500G Subculture of organisms: Subculture of the P. acnes acquired from MTCC was performed according to guidelines from MTCC at Maratha Mandal’s Central Research Laboratory Belgaum, Karnataka. Methodology: The antibacterial activity of Riboflavin (vitamin B2 and Doxycycline against P. acnes was studied by antibiotic assay and culture sensitivity technique recommended by National Committee for Clinical Laboratory Standards (NCCLS) and performed at Maratha Mandal’s Central Research Laboratory Belgaum, Karnataka. The methods included: A. (Minimum inhibitory concentration) MIC determination; B. Disc diffusion method (to study MBC); and C. Checkerboard titration (to study synergism). A. (Minimum inhibitory concentration) MIC determination Media Used: Brain Heart Infusion broth - HIMEDIA M210-500 gm Ingredients gm / litre Calf brain, infusion 200.00 Beef heart, infusion 250.00 Proteose peptone 10.00 Dextrose 2.00 Sodium chloride 5.00 Disodium phosphate 2.50 Final pH (at 25ºC) 7.4+ / -0.2 Process: 1) Inoculum preparation: P. acnes were harvested from a single colony on the agar. It was washed three times using sterile tryptic soy broth (TSB) without bovine serum and then suspended in the same solvent (sterilized TSB without bovine serum) with adjusting the concentration to 1 × 107 CFU (Colony-Forming Units) per ml using the plate count method. 2) Preparation of McFarland Standards a) 1% solution of anhydrous barium chloride (BaCl2) and 1% solution of sulfuric acid (H2SO4) was prepared. b) The solutions were combined and completely mixed to form a turbid suspension. This mixture was placed in a foil-covered screw-cap tube. It was Stored at McFarland standard at a room temperature (25°C) when not in use. (McFarland standard density solution will precipitate and clump over time, and it needs vigorous agitation (vortexing) or shaking before each use. The tubes are marked to indicate the level of liquid, and checked before use to be sure that no evaporation has occurred.) c) The turbidity was visually compared in the presence of good lighting, by holding the bacterial sample and McFarland Standard tubes up against the black and white bars printed on the enclosed card. d) In case of heavy turbidity, the turbidity of the log growth of the bacterial suspension was adjusted with the addition of broth or saline via sterile pipette to match the turbidity to that of known McFarland Equivalence Standard. (If the test suspension is too light, the tubes are inoculated with additional organisms or incubated until turbidity matches that of the standard.) e) The turbidity standard of the broth that has been vortexed was visually adjusted equal to that of a 0.5 McFarland turbidity standard. Alternatively, the suspension is standardized with a photometric device. ) Stock solution preparation: 10 mg each of Doxycycline and Riboflavin was weighed and dissolved in 1ml of broth separately to prepare stock solutions of the active compounds Doxycycline and Riboflavin respectively. 4) MIC test (Aerobic) a) 9 dilutions of each compound were prepared with BHI (Brain Heart Infusion) broth. b) In the first tube, 20µl of prepared stock solution of the compound was added into 380µl of BHI (Brain Heart Infusion) broth. c) For preparing the dilutions, 200µl of BHI (Brain Heart Infusion) broth was added into the next 9 tubes separately. d) From the first tube, 200µl of the solution was transferred to the second tube containing 200 µl of BHI (Brain Heart Infusion) broth. This was considered as 10-1dilution. e) From the second tube containing 10-1solution, 200µl of the solution was transferred to the third tube to prepare 10-2dilution. f) The serial dilution was repeated up to 10-9dilution for each active compound. g) From the maintained stock cultures of P. acnes, 5µl was taken and added into 2ml of BHI (Brain Heart Infusion) broth. h) In each of the serially diluted tube, 200µl of above prepared culture suspension was added. i) The tubes were incubated for 24 hours and observed for turbidity. j) Thus, the serial dilutions generated P. acnes cultures incubated in solution of active compound having concentrations of 100µg / ml, 50µg / ml, 25µg / ml, 12.5µg / ml, 6.25µg / ml, 3.12µg / ml, 1.6µg / ml, 0.8µg / ml, 0.4µg / ml and 0.2µg / ml. k) MIC tests for both active compounds were carried out in triplicates. Observations: Table 3 shows the (Minimum inhibitory concentration) MIC test for both active compounds Doxycycline and Riboflavin carried out in triplicates. Sr.Samples10050 25 12.5 6.25 3.12 1.6 0.8 0.4 0.2 No µg / µg / µg / µg / µg / µg / µg / µg / µg / µg / . ml ml ml ml ml ml ml ml ml ml 1 Doxycyclin S S S S S S S S S S e 10mg / ml 2 Riboflavi S S S S S S R R R R n1 3 Riboflavi S S S S S S S R R R n2 4 Riboflavi S S S S S S S R R R n3 5 Doxycyclin S S S S S S S S S S e 1mg / ml 6 Doxycyclin S S S S S S S S S S e 1mg / ml 7 Doxycyclin S S S S S S S S S S e 1mg / ml S –Sensitive, R–Resistant Table 3: (Minimum inhibitory concentration) MIC determination For Doxycycline, sensitivity was observed at 1mg / ml concentration. For Riboflavin, sensitivity was observed at 10mg / ml concentration of Riboflavin. Standard Error of Mean (SEM) was calculated for each active compound separately. S.E.M. of MIC of Riboflavin at 10mg / ml concentration S.E.M = 3.12 + 1.6 + 1.6 / 3 = 1.6 µg / ml. MIC of Riboflavin = 1.6µg / ml. S.E.M. of MIC of Doxycycline at 1mg / ml and 10mg / ml concentration S.E.M = 0.2 = 0.2 µg / ml. B. Disc diffusion method Media Used: Brain Heart Infusion broth, Brain heart infusion agar. Temperature: The agar plates were brought to room temperature before use. Process: 1) Inoculation of Agar plate: a) The inoculum of P. acnes was adjusted to a McFarland 0.5 turbidity standard, and within 15 mins, a sterile cotton swab was dipped into the inoculum and rotated against the wall of the tube above the liquid to remove excess inoculum. b) The entire surface of agar plate was swabbed three times, while rotating the plate approximately 60º between streaking to ensure even distribution. During this process, creation of aerosols by hitting the sides of the Petri plate was avoided. c) The inoculated plate was allowed to stand for at least 3 minutes but not longer than 15 min before making wells in the agar. d) A hollow tube of 5mm diameter was heated and pressed on the inoculated agar plate. The tube was removed immediately to make a well in the plate. Likewise, five wells were made on each plate. 2) Preparation of stock solutions: 10 mg each of Doxycycline and Riboflavin was weighed and dissolved in 1ml of broth separately to prepare stock solutions of the active compounds Doxycycline and Riboflavin respectively. 3) Addition of compound into plate: With the help of micropipette, 50µl of the stock solutions were added in each well for the two active compounds. Incubation: a) The plates were incubated within 15 min of the active compound application. b) The plates were inverted and stacked for not more than five minutes. c) The plates were then incubated for 18-24 hrs. at 37 ºC in an incubator. Reading the plates: a) The plates were read only when the lawn of growth was confluent or nearly confluent. b) The diameter of the inhibition zone was measured to the nearest whole millimeter by holding the measuring device. Observation: Table 4 shows the zone of inhibitions for determining the Minimum Bacterial Concentration (MBC) of Doxycycline and Riboflavin. Sr. No. Samples Riboflavin Doxycycline10mg / ml1 P. acneR >50mmSr. No. SamplesRiboflavin Doxycycline1mg / ml 2 P. acne1 R 40mm 3P. acne2R 40mm4P. acne3R 40mmS - Sensitive, R – Resistant Table 4: Minimum Bacterial Concentration (MBC) of Doxycycline and Riboflavin Results: MBC of Doxycycline: Zone of inhibition was observed at i.10mg / 1ml concentration: > 50mm ii.1mg / ml concentration: 40mm Inference: Doxycycline is bactericidal at 10mg / ml as well as 1mg / ml concentration. MBC of Riboflavin: No Zone of inhibition was observed at 10mg / ml concentration. Inference: Riboflavin is bacteriostatic. C. Checkerboard Titration Materials: i. Supplemented blood agar (with hemin 5ug / ml and menadione 1ug / ml) ii. Supplemented thioglycolate broth (with hemin 5ug / ml, menadione 1ug / ml and horse serum 1%) iii.96-well polystyrene U-bottom microdilution plates iv. Eppendorf tubes 1.8ml, sterile v. Micropipettes variable volume 1-10ul, 10-100ul vi. Multichannel pipette 10-100ul vii. Sterile, serological pipettes 2ml and 5ml viii. Sterile micropipette tips ix. Diluents for test compounds x. McFarland standards Culture: Bacterial Strain: P. acnes Process: 1) Revival of frozen bacterial strains a) Frozen cultures of two strains of P. acnes used for the study were thawed by exposing the culture vials to a temperature of 37℃ in water bath till the cultures were completely liquefied. After gentle centrifugation, the storage medium was removed, the cultures were resuspended in supplemented thioglycolate broth and incubated anaerobically at 37℃ for 72hrs. b) The cultures were then plated on to a supplemented blood agar plate and incubated under anaerobic conditions further for 72 hrs. c) The colonies of each isolate were inspected, checked for purity and the morphology was confirmed by Gram staining. d) At least 5-10 colonies of each isolate were suspended in 1ml of supplemented thioglycolate broth, properly emulsified and the turbidity was adjusted to 0.5 McFarland standard. This suspension was used as an inoculum for checker-board titration. 2) Checkerboard Titration a) Single agent MICs for both strains of P. acnes against both active compounds were determined, and these results were reconfirmed by repeat testing (for the fresh batch of test compounds). b) Concentrations ranging from eight times the expected MIC levels for each test compound were included in the test panel. c) Number of panels needed for each test isolate was determined (for preparation of adequate amount of reagents). d) Sufficient quantity of stock solutions of each test compound was prepared in respective solvent. e) Doxycycline i.e., compound A was serially diluted in well numbers 2 to 12 in each row (A to H). f) Riboflavin i.e., compound B was serially diluted in separate sets of tubes and then different concentrations were added to columns 1-12 from A to H. This implies that one compound was serially diluted along X axis and other one was serially diluted along Y axis. Now each well had combination of different concentrations of each compound. With each plate both active compound controls and sterility controls were set up. g) 10ul of the suspension of revived culture was added to all the wells except the sterility control well. h) The microtiter plates were incubated anaerobically at 37℃ for 48 hrs. i) Each combination well was examined, and results were recorded as growth or no growth for each well. Observations: Single Agent MIC test: Active Compound P. acnes A. Doxycycline 0.2µg / ml B. Riboflavin 1.6µg / ml Table 5 below shows the checkerboard titration results of Doxycycline and Riboflavin. 1 2 3 4 5 6 7 8 9 10 11 12 Broth A0. A0. A0. A0. A0. A0. A0. A0. A0. A A+ B + 000 000 000 001 003 006 012 025 05 0.1 S Organ 2 4 8 6 R R 5 S S S ism R R R R R R B A0. A0. A0. A0. A0. A A0. A0. A0. A A0.2 0.025 000 000 000 001 001 0.00 012 025 05 0.1 B R 2 4 8 6 6 6 5 B B B0.0 0.025 B B B B B0.0 B B 0.02 0.02 25 S 0.02 0.02 0.02 0.02 25 0.02 0.02 5 5 S 5 5 5 5 R 5 5 S S R R R R R R B A0. A0. A0. A0. A0. A A0. A0. A0. A A0.2 0.05 000 000 000 001 003 0.00 012 025 05 B 0.1 B0.05 R 2 4 8 B 6 B B0.0 6 5 B B 0.05 B0.0 S B B0.0 0.05 0.05 5 B 0.05 0.05 S 5 S 0.05 5 R R R 0.05 R S R R R B A0. A0. A0. A0. A0. A0. A0. A0. A0. A A0.2 0.1 000 000 000 001 003 006 012 025 05 B 0.1 B 0.1 R 2 B 4 B 8 B 6 B B0.1 B 5 B 0.1 B0.1 S 0.1 0.1 0.1 0.1 R 0.1 B 0.1 S S R R R R R 0.1 S S B A0. A0. A0. A0. A0. A0. A0. A A0. A A0.2 0.2 000 000 000 001 003 006 012 0.01 05 0.1 B0.2 R 2 4 8 6 B0.2 B0.2 5 2 B0.2 B0.2 S B0.2 B0.2 B0.2 B0.2 R R B0.2 B0.2 S S R R R R S S B A0. A0. A0. A0. A0. A0. A0. A0. A0. A A0.2 0.4 000 000 000 001 003 006 012 025 05 0.1 B0.4 R 2 4 8 6 B0.4 B0.4 5 B0.4 B0.4 B0.4 S B0.4 B0.4 B0.4 B0.4 R R B0.4 S S S R R R R S B A A0. A0. A0. A0. A0. A0. A0. A0. A A0.2 0.8 0.00 000 000 001 003 006 012 025 05 0.1 B0.8 R 02 4 8B0. 6B0. B0.8 B0.8 5 B0.8 B0.8 B0.8 S B0.8 B0.8 8 R 8 R R S B0.8 S S S R R S B A A0. A0. A0. A0. A0. A0. A0. A0. A BRO 1.6 0.00 000 000 001 003 006 012 025 05 0.1 TH R 02 4 8 6B1. B1.6 B1.6 5 B1.6 B1.6 B1.6 B1.6 B1.6 B1.6 6 R S B1.6 S S S S R R R R S A - Doxycycline, B - Riboflavin, Against - P. acnes. Table 5: Checkerboard titration results of Doxycycline and Riboflavin Calculations: Fractional Inhibitory Concentration (FIC) FIC of agent A = [MIC of agent A in combination / MIC of agent A alone] = 0.006 / 0.2 = 0.03 FIC of agent B = [MIC of agent B in combination / MIC of agent B alone] = 0.8 / 1.6 = 0.5 The summation of FIC for each combination was calculated. ∑FIC = FIC of agent A + FIC of agent B ∑FIC = (FIC of A + FIC of B) ∑FIC = 0.03 + 0.5 ∑FIC = 0.53 Results: Fractional Inhibitory Concentration of Doxycycline is 0.03. The fractional Inhibitory Concentration of Riboflavin is 0.5. Fractional Inhibitory Concentration of combination of Doxycycline and Riboflavin is 0.53. Interpretation: With reference to the results of the broth microdilution method, FIC of combination of Doxycycline and Riboflavin is 0.53. Hence, the combination shows synergism (Synergism, x ≤ 0.5) against P. acnes. (Interpretation standards: Synergism x ≤ 0.5, Indifference 0.5 < x ≤ 4, Antagonism x > 4). EXAMPLE 3: CALIBRATION CURVE STUDIES Objective: Calibration curve studied were performed to determine the concentration of active compound in an unknown sample by comparing the unknown to a set of standard samples of known concentration of active compound. Methodology: 1) Calibration curve of doxycycline Aliquots of standard solution of active compound (100µg / ml) in phosphate buffer (pH 5.5) were transferred in a series of 10ml volumetric flask and diluted with phosphate buffer to give several dilutions in concentration range of 5-40µg / ml of doxycycline.3 ml of the samples were analyzed spectrophotometrically at 272nm against buffer having pH 5.5 to draw a calibration curve of absorbance vs. concentration. 2) Calibration curve of riboflavin The standard solution (100μg / ml) of riboflavin (vitamin B2) was prepared by dissolving 10mg of riboflavin in a 100ml volumetric flask in HPLC grade water. The standard working solutions of riboflavin (1,5, 10, 20, 30, 40, and 50μg / ml) were prepared from primary standard solution by dilution with HPLC grade water. Absorbance of these solutions was measured using a double beam UV spectrophotometer against deionized water as reference, at 440 nm wavelength to draw a calibration curve of absorbance vs. concentration. Observations: 1) Doxycycline The standard curve extended from 5µg / ml to 40µg / ml. It followed the Beer- Lamberts law over the concentration range of 5µg / ml to 40µg / ml. The equation of the line was y = 27.82x + 1.364 (intercept at origin) with coefficient of determination (R²) of 0.9984. This corroborated a strong correlation between concentration of doxycycline and their absorbances. Figure 4 depicts the plot of concentration vs absorbance for doxycycline. 2) Riboflavin The standard curve extended from 1µg / ml to 50µg / ml. It followed the Beer- Lamberts law over the concentration range of 1µg / ml to 50µg / ml. The equation of the line was y = 32.45x -0.212 (intercept at origin) with coefficient of determination (R²) of 0.9996. This corroborated the strong correlation between the concentration of riboflavin and their absorbances. Figure 5 depicts the plot of concentration vs absorbance for riboflavin. EXAMPLE 4: FORMULATION OF DOXYCYCLINE AND RIBOFLAVIN The composition of the antiacne gel formulation consists of: 1) Doxycycline 10gm 2) Riboflavin 1gm 3) Carbopol 1-2gm; 4) Glycerin 2gm; 5) Clove Oil 3-7ml; 6) Methyl Paraben 0.10gm; 7) Propyl Paraben 0.02gm; 8) Distilled water 100ml; and 9) Triethanolamine to adjust the pH to 6-6.5. EXAMPLE 5: PREPARATION OF THE GEL FORMULATION 1.5gm of Carbopol was dispersed in 50ml of distilled water containing 2gm of glycerin with vigorous stirring and kept overnight at 4°C to attain complete hydration. Subsequently, 10gm of doxycycline and 1gm of riboflavin were dispersed in water separately and were added to the gel base. Next, appropriate 5ml of clove oil and 10mg methyl paraben and 20mg propyl paraben were added to the gel base. pH of the formulation was adjusted to 6.2 with triethanolamine. The final weight of the gel product was adjusted to 100g with distilled water. EXAMPLE 6: OPTIMIZATION OF GEL FORMULATION BY FACTORIAL DESIGN APPROACH Objective: The gel formulation containing Doxycycline, Riboflavin and clove oil as a permeation enhancer was prepared and further statistically optimized by 32factorial design approach. Methodology: 32factorial design was employed using Design Expert® software (Version 13.0, Stat-Ease Inc., USA). The effect of two independent variables namely X1: Concentration of Carbopol 940 and X2: Concentration of clove oil was investigated on viscosity (Y1) of gel formulation. The effect of independent variables on the responses was studied using ANOVA (F Value). 3D Response surface plots and Contour plots were used to visualize the relationship and interaction between the variables and responses studied. Optimization of the gel formulation was carried out using desirability function. Process: a) 32factorial design of design expert software was used to optimize the best experimental condition that was meant to produce the batches with anticipated viscosity. The values of viscosity of experimental condition were recorded. At the recorded experimental conditions, an extra checkpoint formulation (B10) was prepared in triplicate to validate the design. Furthermore, the values for the dependent variables i.e., viscosity of gel formulation (Y1) was measured experimentally. Later, this value (Y1) was compared with the recorded values (obtained from the software) and further their statistical significance of difference was observed using a t-test at a 95% confidence interval (p < 0.05). b) The gel formulation was prepared using 32factorial design approach. The effect of concentration of Carbopol 940 in %w / w (gelling agent) and concentration of clove oil in %w / w (permeation enhancer) was systematically studied using the design of expert. c) The linear model of response Y1 was further verified by ANOVA and the sum of squares, degree of freedom, F-value, and p-value of each factor for studied response was calculated. d) The effect of individual as well as combined independent variables over viscosity was studied by the 2D contour and 3D response surface plots e) The closeness between the actual and predicted value of each response of all formulations (F1 - F9) was analyzed. f) Considering the extrudability and spreadability of all the prepared gels, concentration of independent variables was decided to optimize such that to achieve desired gel viscosity. Concentration of clove oil to get desired permeation of gel formulation across skin was obtained. The most desirable formulation was chosen, and the optimized formulation was prepared. The findings were contrasted with values that the software had projected. Observations: Table 6 depicts the formulation runs for optimization of the formulation and their results. A: Conc. of carbopol B: Conc. of clove Batch 940 oil Viscosity Code % w / w % w / w Cps F1 1 3 1200 F2 1 5 1167 F3 1 7 847 F4 1.5 5 2380 F5 1.5 7 1648 F6 1.5 3 1468 F7 2 7 1689 F8 2 5 2645 F9 2 3 2420 Table 6: Formulations with their results used for optimization The viscosity of developed gel formulation was found between 847cps (F3) and 2645 Cps (F8). The difference in the viscosity was found due to the variation in the formulation composition. The formulation (F3) having Carbopol 940 concentration (1% w / w) and clove oil concentration (3% w / w) showed the lowest value of viscosity. The formulation (F8) with Carbopol 940 concentration (2% w / w) and clove oil concentration (5% w / w) showed highest viscosity. It was observed that increasing the concentration of gelling agent (Carbopol 940), increased the viscosity of gel. On the contrary, increasing the concentration of clove oil, reduced the viscosity of gel formulation. Carbopol showed significant positive effect on the viscosity of gel while clove oil exhibited non-significant negative effect on the viscosity of gel formulation. Figure 6A demonstrates the 2D contour and Figure 6B demonstrates the 3D response surface plots showing effect of Carbopol concentration and clove oil concentration on the viscosity of gel formulation. Further, it was observed that on increasing the concentration of Carbopol from 1% to 1.5%, the viscosity increased drastically relative to the increase in Carbopol from 1.5% to 2%. Also, on increasing the clove oil concentration from 3% to 5%, the viscosity of gel formulation increased initially, but further increase in the concentration of clove oil to 7% led to reduction in the viscosity of gel formulation. Figure 7A and 7B demonstrate the line graphs showing effect of different concentrations of Carbopol 940 (A) and clove oil (B) on viscosity of gel formulation, respectively. Result: The concentrations of independent variables were decided for optimization to achieve gel viscosity of 2200 Cps. Concentration of clove oil was decided to be 5% to get desired permeation of gel formulation across skin. The most desirable formulation, with a desirability rating of 1.00, was chosen. Optimized formulation was prepared using 1.91% of Carbopol 940 and 5% of clove oil. Figure 8 depicts the graphical representation of desirability and optimized conditions of Carbopol concentration and clove oil concentration for viscosity of gel formulation. EXAMPLE 7: CHARACTERIZATION OF THE GEL FORMULATION 1) Visual Appearance: The prepared gels were visually evaluated for clarity, color, and particle's existence. All the gel formulations containing doxycycline and riboflavin were found to be yellow colored and uniform in consistency. 2) Microscopic study: The gel formulations were checked in terms of uniformity, gel texture, and air bubble by optical microscope with a magnification of 10 and 40 within 48hrs. No crystals and no phase separation were observed in the gel systems. 3) pH: 1% aqueous solution of the gel formulation was prepared and stored for 2h. The pH of the formulation was determined using a digital pH meter. The pH analysis of each gel formulation was performed in triplicate, and the average pH value and standard deviation was calculated. All the gel formulations had pH in the range of 6.40 to 7.11. This pH range was found to be suitable for skin care and caused no irritancy effect. Table 6 below depicts the pH recorded for all formulations. 4) Viscosity: The viscosity of the developed gel formulations was determined using Brookfield digital viscometer. The spindle no.6 was dipped in gel sample rotated at 10rpm and 20 ± 1°C temperature for 15min. The reading in triplicate was noted. Viscosity was measured in centipoise (cp). The measured viscosity is demonstrated in table 6. 5) Spreadability by Arvouet-Grand Method: The spreadability of gels was determined by pressing 1g of a gel sample between two 20 X 20 cm horizontal plates, the upper plate weighing 125g. The spread diameter was measured after 1min. Under the said experimental conditions, semi stiff gel should show spreadability diameter ϕ ≤ 50 mm and semifluid gel should show spreadability diameter ϕ ˃ 50 mm but ˂ 70 mm. The spreadability of formulations was measured in triplicates and average value was calculated. The values obtained by Arvouet-Grand Method indicated that prepared gels were semi stiff gels. Table 7 below depicts the spreadability recorded for all formulations. 6) Drug content:100 mg of gel sample was dissolved in 100 ml phosphate buffer having pH 5.5 and the gel solution was shaken for 2hrs. on a mechanical shaker to dissolve the active compound completely. Then the solution of the prepared gel formulation was filtered, and the drug content was determined spectrophotometrically at 270 nm for doxycycline and 440 nm for riboflavin against phosphate buffer having pH 5.5 as blank solution. The drug content of the gel formulations was measured in the triplicate and the average value ± standard deviation was calculated. Table 7 shows that all gel formulations demonstrated drug content in the range of 85% to 100%. These results suggest that doxycycline and riboflavin were uniformly distributed in the Carbopol gels and the loss of drug during formulation processes was insignificant. This indicates that maximum amount of the drug was dispersed uniformly in the gel matrix, and it was undesirable character of the prepared gel formulations. The high drug content supports the authenticity of the used method to prepare the gel formulations. Batch pH Spreadability Homogeneity Drug content (%) (mm) D R F1 6.48 40 homogeneous 97.45 96.47 F2 6.57 41 homogeneous 96.23 96.40 F3 6.95 48 homogeneous 95.32 97.11 F4 7.04 39 homogeneous 96.93 94.16 F5 6.40 45 homogeneous 96.28 95.20 F6 7.08 46 homogeneous 97.32 95.37 F7 6.57 44 homogeneous 95.20 96.56 F8 7.11 36 homogeneous 94.33 96.78 F9 6.62 38 homogeneous 95.19 97.22 D: Doxycycline and R: Riboflavin Table 7: Evaluation results of the topical gel formulations for pH, Viscosity, Spreadability and Homogeneity EXAMPLE 8: PERMEATION STUDY 1) Objective: The in vitro permeation study was performed to evaluate the permeation of doxycycline and riboflavin from developed gel formulation using an eggshell. 2) Methodology: The in-vitro permeation study of developed gel was performed using eggshell membrane as egg membrane have similar properties to the stratum corneum of human skin. The eggs used for the study were incubated at 37℃ to prepare the Chorioallantoic Membrane (CAM). CAM was extracted from the egg by making a hole at the blunt end of the egg. The CAM adhered to the eggshell was washed with the normal saline and then the CAM was carefully removed. The CAM was collected and further used for the permeation study. The membrane was placed between donor and acceptor compartments of a Franz diffusion cell having an effective area (2cm2) and volume (20ml). Phosphate buffer having pH 5.5 was used as the permeation media. The permeation media was filled into the acceptor compartment and the temperature was fixed at 37 ± 0.5℃ with continuous stirring. The gel samples were added to the donor compartment. The permeated sample (2ml) was taken from the acceptor compartment at a fixed time interval of 1 hr upto 6 hrs fixed time and replaced with the same volume of fresh buffer. The samples were filtered, diluted, and analyzed at 270 nm for doxycycline and 440 nm for riboflavin. 3) Observations: The study results revealed a significant difference in the permeation flux values between both the active compounds. Figure 8 demonstrates the permeation flux values of both the active compounds and their formulations. The amount of doxycycline permeated from pure doxycycline and prepared gel formulation was found to be 27.79 µg / cm2 / h and 62.56 µg / cm2 / h, respectively. The amount of riboflavin permeated from pure riboflavin and prepared gel formulation was found to be 23.15 µg / cm2 / h and 90.64 µg / cm2 / h. 4) Results: The enhancement ratio was found to be 2.25 fold for doxycycline and 3.91 fold for riboflavin. EXAMPLE 9: IN-VITRO IRRITATION STUDIES 1) Objective: HET-CAM test (hen’s egg-chorioallantoic membrane test) was used to evaluate the irritation study of the prepared gel formulation using egg membrane and the irritation score was calculated. 2) Methodology: The irritation study of the gel formulation was assessed utilizing hen egg chick chorioallantoic membrane. The hen’s eggs obtained from the poultry farm were incubated for ten days at 37 ± 0.5℃ at 55 ± 2% RH in a humidity chamber. The eggs were manually rotated on a regular basis. The eggs were retrieved from the air chamber side on the tenth day, and the outer shell of the eggs was carefully removed. The eggs were checked for CAM development. 0.9% w / v regular saline was employed as negative control. The assessment was performed by pouring the gel formulation, a 0.1M NaOH solution and the negative control on top of the CAM. A visual assessment of the irritation potential was performed, and a score based on the reference chart was assigned. The irritation potential was scored based on the hemorrhage, lysis, and coagulation scales. The scoring was applied as 0 (no reaction) to 3 (strong reaction). Accordingly, the irritation score was classified as ≤0.8 (slightly irritating); >0.8 to <1.2 (moderately irritating); ≥1.2 to <2.0 (irritating); and ≥2.0 severely irritating. Observation: The samples were tested for lysis, hemorrhage, and coagulation for 5 min. The negative control (0.9% w / v, sodium chloride) exhibited no hemorrhage and no damage to blood capillary. It was therefore scored as zero score. The positive control (0.1M NaOH) exhibited a cumulative score of 6.67 (>2). The CAM treated with the gel formulation also showed a cumulative score of 0.67. The CAM showed no signs of lysis, hemorrhage, or coagulation. Table 8 demonstrates the observation score of in-vitro irritation study by HET - CAM test.

[0002] Test Time (min) Egg Overall Sample 0 0.5 2 5 score Gel Sample 1 0 0 0 0 Sample 2 0 0 0 1 Sample 3 0 0 0 1 0.67 Mean score 0.00 0.00 0.00 0.67 0.1 M Sample 1 0 1 2 3 NaoH Sample 2 0 1 3 3 Sample 3 0 1 3 3 6.67 Mean score 0 1 2.67 3 Normal Sample 1 0 0 0 0 saline Sample 2 0 0 0 0 Sample 3 0 0 0 0 0 Mean score 0 0 0 0 Table 8: HET - CAM test for in-vitro irritation study Result: As the negative control exhibited no hemorrhage and no damage to blood capillary, it is considered as non-irritant. The high score of the positive control indicated severe irritation to the tissue. The gel formulation showed no signs of lysis, hemorrhage, or coagulation. The gel formulation showed a cumulative score of 0.67. The overall study confirmed the nonirritant property of the developed gel formulation. EXAMPLE 10: STABILITY STUDIES Objective: The stability study of optimized gel formulation was performed as per ICH guidelines. Methodology: The stability studies were carried out at different temperatures of 4℃, 25℃ at 60%RH, and 40℃ at 75%RH. Each sample was packed into a borosilicate glass vial and kept in a stability chamber (REMI, India) for 3 months. The gel was evaluated for physical appearance, viscosity, spreadability, and drug content as per a pre-determined time interval. Stability samples are tested for a pre- determined time interval of 0 time reading and after 1, 2, 3 months subsequently. Observations: The observed values of the stability study of the optimized gel formulation were as shown in Table 9. It was observed that there were no significant changes (p>0.05) in the physical appearance (color, consistency, homogeneity) at the tested condition. Time Spreadability Viscosity (Cps) Drug content (%) (month (mm) s) 4± 4±1 ℃ 25±1 ℃ 40±1 ℃ 4±1 25±1 40±1 25±1 40±1 1 D R D R D R ℃ ℃ ℃ ℃ ℃ ℃ 0 2259 2253 2212 40 40 41 96. 97. 96. 96. 96. 97. 27 18 13 82 23 14 1 2243 2238 2234 41 40 42 96. 96. 95. 96. 95. 96. 01 57 93 39 65 96 2 2245 2245 2220 41 41 42 95. 96. 95. 96. 95. 96. 94 23 33 10 31 73 3 2238 2233 2226 41 41 42 95. 95. 95. 95. 94. 96. 45 88 14 82 80 21 Table 9: Stability study results of the optimized gel at per ICH guidelines Result: The drug content, viscosity, and spreadability of the optimized gel formulation on storage in the stability chamber at different temperatures and relative humidity, were found to be within the standard limit. It was concluded that the prepared formulation is stable in all storage conditions. The anti-acne topical formulation of the present invention effectively acts against the bacterium P. acnes and suppresses the inflammatory responses of acne caused due to the activation of inflammatory cells. The formulation further shows enhanced ROS generation ensuring augmentation of pathogen clearance. The formulation has demonstrated synergistic activity between doxycycline and riboflavin, further providing antibacterial efficacy. The formulation is statistically optimized by a 32factorial design approach to obtain the most desirable formulation based on permeation and viscosity. Further, characterization of the gel by standardized protocols demonstrates uniform characteristics. Further the gel exhibits demonstrated nonirritant properties and is stable on storage. The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the scope of the present invention.

Claims

CLAIMS:

1. A formulation for treating acne comprising: a) Doxycycline 8-10%; b) Riboflavin 1-2%; c) Carbopol 1-2%; d) Emollient 1-2%; e) Penetration enhancer 3-7%; f) Paraben 0.1-0.5% as preservatives; g) Distilled water 100 ml; and h) Triethanolamine for adjusting the pH of the formulation.

2. The formulation for treating acne as claimed in Claim 1, wherein the pH of the formulation being in the range 6 to 6.5, and the pH being adjusted by triethanolamine.

3. The formulation for treating acne as claimed in Claim 1, wherein the emollient being glycerin; the penetration enhancer being clove oil; and the parabens being methyl paraben and propyl paraben.

4. The formulation for treating acne as claimed in Claim 1, wherein the formulation being a topical gel formulation.

5. A method 100 of preparing the formulation for treating acne as claimed in Claim 1 including a) a first step 105 of dispersing 1-2% of Carbopol in 50 ml of distilled water containing 1-2% of glycerin with vigorous stirring and keeping it overnight at 4°C to attain complete hydration;b) a second step 110 of dispersing 8-10% of doxycycline and 1-2% of riboflavin in water and adding this to the gel base of first step 105; c) a third step 115 of adding 3-7% of clove oil, 0.10% methyl paraben and 0.02% propyl paraben preservatives to the gel base; d) a fourth step 120 of adjusting pH of the formulation to 6-6.5 with triethanolamine; and e) a fifth step 125 of adjusting the final weight of the gel product to 100 g with distilled water.

6. The formulation for treating acne as claimed in Claim 1, wherein the gel viscosity of the formulation being 2200 Cps.

7. The formulation for treating acne as claimed in Claim 1, wherein the formulation having a drug content in the range of 85% to 100%, and wherein the maximum amount of the doxycycline and riboflavin being dispersed uniformly in the gel.

8. The formulation for treating acne as claimed in Claim 1, wherein the enhancement ratio being 2.25 fold for doxycycline and 3.91 fold for riboflavin.

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