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FORMULATION AND EVALUATION OF POLYMYXIN B AND BACITRACIN OINTMENT M. Pharm Dissertation Protocol Submitted to Rajiv Gandhi University of Health Sciences, Karnataka Bangalore– 560 041 By Mr. S.SHADAB AZHAR B.Pharm Under the Guidance of Mr. ANUP KUMAR ROY M.Pharm,(Ph.D) Asst. Professor & HOD Department of Industrial Pharmacy, Acharya & B.M. Reddy College of Pharmacy, Soldevanahalli, Chikkabanavara (Post) Hesaraghatta, Main Road, Bangalore – 560 090. 2010-2011 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE. ANNEXURE - II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. Name of the Candidate and Address 2. Name of the Institution 3. Course of Study and Subject 4. Date of Admission 5. Mr. S.SHADAB AZHAR . H-NO-HIG-1/2,ABBAS NAGAR, KURNOOL-518002 ANDHRA PRADESH. ACHARYA & B.M. REDDY COLLEGE OF PHARMACY, Hesaraghatta Main Road, Soldevanahalli, Chikkabanavara Post. Bangalore-560090 M. Pharm (Industrial pharmacy) 30-May-2010 TITLE OF THE PROJECT: FORMULATION AND EVALUATION OF POLYMYXIN B AND BACITRACIN OINTMENT 6. BRIEF RESUME OF THE INTENDED WORK: 6.1 NEED FOR THE STUDY: The objective of the work is to formulate and evaluate ointment of combination of the drugs Polymyxin B and Bacitracin which are antibiotics with an aim to improve its bioavailability and better management of diseases . The ointment will be useful to treat wounds (e.g., cuts, scrapes, burns) and to help prevent or treat skin infections. Minor skin infections and wounds usually heal without treatment, but some minor skin wounds may heal faster when an antibiotic is applied to the affected area. This will contain antibiotics that work by slowing or stopping the growth of bacteria. Polymyxin B is an antibiotic primarily used for resistant gram-negative infections. It is derived from the bacterium Bacillus polymyxa. Polymyxin B sulfate has a bactericidal action against almost all gram-negative bacilli except the Proteus group. Polymyxins bind to the cell membrane and alter its structure making it more permeable. The resulting water uptake leads to cell death. MECHANISM OF ACTION Polymyxin alters bacterial outer membrane permeability by binding to a negatively charged site in the lipopolysaccharide layer, which has an electrostatic attraction for the positively charged amino groups in the cyclic peptide portion (this site normally is a binding site for calcium and magnesium counter ions); the result is a destabilized outer membrane Fatty acid portion dissolves in hydrophobic region of cytoplasmic membrane and disrupts membrane integrity Leakage of cellular molecules, inhibition of cellular respiration Binds and inactivates endotoxin Relative absence of selective toxicity: nonspecific for cell membranes of any type, highly toxic Bacitracin is a mixture of related cyclic polypeptides produced by organisms of the licheniformis group of Bacillus subtilis , isolation of which was first reported in 1945. As a toxic and difficult-to-use antibiotic, bacitracin does not work well orally. However, it is very effective topically, and is a common ingredient of eye and skin antibiotic preparations. Its action is on gram-positive cell walls. It can cause contact dermititis and cross-reacts with allergic sensitivity to sulfa-drugs. MECHANISM OF ACTION Bacitracin interferes with the dephosphorylation of the C55-isoprenyl pyrophosphate, a molecule that carries the building-blocks of the peptidoglycan bacterial cell wall outside of the inner membrane. Bacitracin has been claimed to be a protein disulfide isomerase inhibitor in cells, but this is disputed by in vitro studies 6.2 REVIEW OF LITERATURE: Jeffrey S.Robin et al., studied that the use of ointments as an ocular drug vehicle adds an important dimension to topical therapy. Ointments are well tolerated, fairly safe, and provide an excellent means for enhanced ocular contact time. In the case of certain antibiotics, this improved contact time yields increased ocular drug levels. However, experimental data indicate that corticosteroid ointments do not penetrate into the eye as well as do suspension preparations. This may related to the binding of the drug to the ointment base and also to the particular steroid compound. Like other ophthalmic preparations, ointments may become contaminated. Ophthalmic ointments should not be instilled into eyes with open wounds. Instillation of ointments into postoperative eyes where wound closure is secure appears to be safe and effective1 . H. Yoshida et al., studied that the in vitro release profiles and the bleeding phenomenon of Tacrolimus and propylene carbonate (PC) as a dispersing solvent for Tacrolimus drug substance in Tacrolimus ointment were investigated when changing concentrations of Tacrolimus and PC in the ointment were used, respectively. The bleeding test result indicated that Tacrolimus was in equilibrium between inside and outside of PC droplets in intact ointment base. A cumulative release amount of Tacrolimus from ointment, plotted against the square root of time, showed a straight line initially with a slope of q1 followed to change a slope to be q2 at a certain time, where the relation of these slopes being q1<q2. The q1 values increased with the concentration of Tacrolimus but decreased with PC concentration in Tacrolimus ointment. And the q2 values increased with Tacrolimus concentration but were independent of PC concentration. These profiles indicated that there were two phases for Tacrolimus release from ointment, namely, first phase was related with the period during PC release and the second phase was related with the state of ointment after PC release. When the PC release was applied to the Higuchi’s release equation, the above slope q1 was found to be correlated to the parameter of A/φ0, where A was a parameter of release rate of PC and φ0 was an initial volume fraction of PC droplets. It should be indicated that more rapid release rate of PC rather than that of Tacrolimus resulted in the generation of amorphous phase of Tacrolimus outside of remaining PC droplets. During PC release, the slope q1 could be influenced by the thermodynamic activity of Tacrolimus dissolved in PC droplets. After PC release, it would be reasonable to speculate that the amorphous cluster of Tacrolimus with a constant thermodynamic activity would give constant q2 values regardless of PC contents in Tacrolimus ointment2. MDRaymond J Roberge et al., studied that thirty-four patients with diminutive peripheral veins requiring peripheral IV access (PIVA) in the emergency department setting were randomly assigned to one of two treatment groups. One group of patients (Group A) received application of a bland ointment to the dorsum of the hand prior to attempts at cannulation, while the other group (Group B) received application of 2% nitroglycerin ointment to the dorsum of the hand prior to cannulation attempts. Cannulation was achieved in all 34 patients in the study, but required significantly fewer attempts in Group B patients (P = .04). No side effects of the drug were observed in any patient or any staff member applying the ointments. They concluded that application of 2% nitroglycerin ointment to the dorsum of the hand is a safe, effective method of inducing local venodilation that will ensure PIVA with significantly fewer attempts at cannulation3. F.W. Ezzedeen et al., assessed the rate and extent of release of sorbic acid from 6 ointment bases . The amount of drug released depended on the composition of the base and the concentration of sorbic acid employed. The rate of release decreased in the following order: water soluble > o/w emulsion > oleaginous > hydrophobic > w/o emulsion base. The rate of diffusion from the ointment bases was pH dependent. The data obtained may be useful in the formation of topical sorbic acid formulations4. Tacey X. Viegas et al., conducted In-vitro studies to study the efficacy of mixed and self-emulsifying creams and hydrophobic ointment formulations in delivering peldesine (BCX-34) into and across cryopreserved human cadaver skin (HCS). Oil-in-water cream formulations, containing 1% w/w of radiolabeled C14 BCX-34 and propylene glycol (PG), glycerin(GLY), isopropyl myristate (IPM), oleic acid (OA) and capric-caprylic esters (CE) were prepared. Petrolatum and lanolin based ointments were also prepared with PG. Sections of the HCS, 250 μm thick, were fitted to vertical Franz diffusion chambers containing a receptor medium of pH 7.4 phosphate buffer solution maintained at 37°C. Using the finite dose technique, 4–6 mg of a formulation sample was applied to the epidermal surface of each section and drug diffusion was permitted for 12 and 24 h periods. The distribution of drug into the HCS epidermis, dermis and into the receptor medium was measured by scintillation spectroscopy. The results show good correlation of the calculated in-vitro values for flux and skin-vehicle partition coefficients against the observed amounts of drug detected in the HCS. The mixed emulsion cream formulation containing PG delivered higher amounts of drug into the skin when compared to the same formulation containing GLY cream.The self-emulsifying cream formulation containing IPM had a higher skin-vehicle partition coefficient and delivered more drug into the dermis when compared to those formulations that contained OA and CE. The petrolatum ointment delivered six times more drug into the epidermis than the lanolin ointment, and had higher skin-vehicle partition values. In conclusion, creams containing PG and petrolatum-base formulations would be suitable for BCX-34 dermal delivery5. A.S. Velissaratou et al., studied the in vitro release of chlorpheniramine maleate from ointments using the Sartorius Absorption Simulator and its artificial epidermis barrier. The amount of drug released depended on the composition of the vehicle and the concentration of drug incorporated in it. The rate of release increased in the following order: ointment < o/w emulsion < w/o emulsion6. Kozo Takayama et al., applied a computer optimization technique for the designing of a formulation for a gel ointment of indomethacin (IMC) containing d-limonene as an absorption enhancer. The combined use of an adaptive least-squares (ALS) method with general optimization techniques was investigated for dealing with response variables which were given as non-quantitative data. Plasma concentrations of IMC absorbed from model formulations were determined in rats as prime response variables. Physicochemical characteristics such as the spreadability of the ointments and chemical stability of IMC in the formulations were also determined as response variables. These variables were predicted by multiple regression equations composed of a combination of formulation factors. On the other hand, irritation of the skin by each formulation and the appearance of the ointments were obtained as non-quantitative data. For these variables, the ALS method was applied to define discriminant equations composed of the combination of formulation factors. The regression and discriminant equations for response variables were assembled as the constrained optimization problem. This problem was solved on the basis of external transformation. Experimental results obtained for the optimum formulation agreed well with the predictions, indicating the usefulness of the optimization techniques in which the ALS method was incorporated7. D. Love et al., described a simple and reproducible method to assess the effect of formulation on the bactericidal and fungicidal activity of topical antimicrobial agents. The technique involves direct inoculation of test and control articles followed by aliquot sampling, neutralization of active antimicrobial ingredients and enumeration of viable microorganisms by standard methods. Two silver sulphadiazine cream formulations, differing in their aliphatic alcohol content, were evaluated as manufactured and in diluted forms in this manner using three bacterial and one fungal burn wound isolates. Results were analysed by a multivariate analysis of variance to test formulation effects, concentration effects and their interaction over time. The results demonstrate that the antimicrobial activity of both creams is equivalent8. Milan Petelin et al., studied that local drug delivery to the oral cavity tissues has been used for treatment of periodontal disease, aphthosus stomatitis, lichen planus, bacterial and fungal infections. The liposome stability in different mucoadhesive ointments, their transport into dog’s oral mucosa and gingiva and washing out of ointments from tissue surface was investigated by electron paramagnetic resonance (EPR). Liposomes were composed of soya lecithin, cholesterol and lipoaminosalt (55:35:10 w:w) and encapsulated with spin labeled water soluble spin probe ASL (N-1-oxyl-2,2,6,6-tetramethyl-4piperidinyl- N-dimethyl-N-hydroxyethylammonium iodide). Polymethyl methacrylate, Carbopol 934P and Orabase as the bioadhesive ointments were used. The stability of liposomes did not change significantly after mixing with polymethyl methacrylate, but decreases in Carbopol and even more in Orabase. Washing out experiment shows that all three ointments adhere well to oral mucosa and gingiva. After 10 min of washing, approximately 30% of hydrogels were washed out. The transport experiment showed that liposomes limit the transport of hydrophilic substance to the superficial layer of epithelium. In oral mucosa, as opposed to gingiva, hydrogels enhance the transport as compared to solution of ASL or liposomes. Among the examined ointments, polymethyl methacrylate proved to be the most appropriate for local application of liposome entrapped drug to oral mucosa or gingiva9. . Paul Nathan et al., described an in vitro method which predicts the antimicrobial effects of the drugs which are used for topical application in the treatment of infected burn wounds. Standard in vitro disc sensitivities are not available for this panel of drugs. The test incorporates an evaluation of the drug carriers, creams and ointments, which are not factors in standard disc or dilution sensitivity methods. Quantitative bacteriological data following treatment of infected burn wounds in children support the conclusion that the in vitro test correctly selects effective antimicrobial agents10. Stefan J. Sveinsson et al., formulated a mucoadhesive ointment by using a co-polymer of methacrylic acid and methacrylic acid methyl ester (Eudispert) . Liposomes containing triamcinolone acetonide were incorporated into (a) the Eudispert ointment, which contains 11% (w/w) of the neutralized polymet and 0.5% (w/w) gelatin, and (b) Orabase. The in vitro drug release and dissolution behaviour of these formulations were investigated. A clinical trial is currently being carried out and the initial findings indicate that the liposomal formulations are well tolerated and no local irritation has been observed.11 6.3 OBJECTIVE OF THE STUDY: The main objective of the present study is to formulate polymyxin B and Bacitracin ointment with an aim to improve patient compliance and to improve bioavailability by using base such as liquid paraffin and preservatives such as methyl paraben and propyl paraben. The intended work will be as follows 1. To carryout preformulation studies for possible drug/polymer/excipient interaction IR/DSC analysis. 2. To formulate ointment of Polymyxin B and Bacitracin.. 3. Evaluate of prepared ointment by A) Physical studies such as Test of non-irritancy Test of rate of penetration Test of rate of drug release Test of rheological properties Test of content uniformity B) And Microbiological methods such as Test of microbial content Test of preservative efficacy 4. To carry out short term stability studies on the most satisfactory formulation as per ICH guidelines. 7. MATERIALS AND METHODS: 7.1 SOURCE OF DATA: Review of literature from: a. Journals such as:1. Indian Journal of Pharmaceutical Science 2. International Journal of Pharmaceutics 3. Survey of opthalmology 4. International society for Burns and Injuries 5. Annals of emergency medicine b. World Wide Web. c. J-Gate@Helinet. d. Science direct. 7.2 METHODS : 1. To carryout preformulation studies for possible drug/polymer/excipient interaction by FTIR. 2. To formulate ointment of Polymyxin B and Bacitracin by using base such as liquid paraffin and preservatives such as methyl paraben and propyl paraben. 3. To evaluate of prepared ointment by A) Physical studies such as Test of non-irritancy Test of rate of penetration Test of rate of drug release Test of rheological properties Test of content uniformity B) And Microbiological methods such as Test of microbial content Test of preservative efficacy 4. To carry out short term stability studies on the most satisfactory formulation as per ICH guidelines. 7.3 DOSE THE STUDY REQUIRES ANY INVESTIGATION TO BE CONDUCTED ON PATIENT OR OTHER HUMANS OR ANIMALS? “NO” 7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3? “NOT APPLICABLE” 8. REFERENCE:- 1. J.S Robin, P.P. Ellis. .Ophthalmic ointments. Surv Ophthalmol (22); 5; Mar- Apr 1978: 335-40 2. H. Yoshida, S. Tamura, T. Toyoda, K. Kado, N. Ohnishi, R. Ibuki. In vitro release of Tacrolimus from Tacrolimus ointment and its speculated mechanism. Int J Pharm (270); 1-2; 11 Feb 2004: 55-64 3. MDRaymond J Roberge, MDMichael Kelly, MDTC Evans, MD Elgin Hobbs,MDMichael Sayre PhD Eric Cottington.Facilitated intravenous access through local application of nitroglycerin ointmet . Ann Emerg Med (16); 5; May 1987: 546-49. 4. F.W. Ezzedeen, F.A. Shihab, S.J. Stohs. Release of sorbic acid from ointment bases. Int J Pharm (28); 2-3; Feb 1986: 113-17. 5. T.X. Viegas, L. L.Van Winkle, P.A. Lehman, S.F. Franz, T.J. Franz. Evaluation of creams and ointments as suitable formulations for peldesine. Int J Pharm (219); 2001:73–80 6. A.S. Velissaratou, G. Papaioannou. In vitro release of chlorpheniramine maleate from ointment bases. Int J Pharm (52); 1; 15 May 1989: 83-86 7. K.Takayama, H.Okabe, Y.Obata, Tsuneji. Formulation design of indomethacin gel ointment containing d limonene using computer optimization methodology. Int J Pharm (61); 3; 30 Jun 1990: 225-34 8. D. Love, W. Kubey and C. J. Holmes. A laboratory method to evaluate formulation effects on the In vitro antimicrobial activity of topical creams and ointments. International Society for Burns and Injuries. (13); 3; Jun 1987: 204-07 9. M.Petelin.a,b, Marjeta.S, Entjurc c, Zorka Stolic c, Uros Skaleric. EPR study of mucoadhesive ointments for delivery of liposomes into the oral mucosa. Int J Pharm (173); 1998: 193–202 10. P.Nathan, E.J. Law, D.F. Murphy, B.G. MacMillan.A laboratory method for selection of topical antimicrobial agents to treat infected burn wounds. International Society for Burns and Injuries. (4); 3; Mar 1978; 177-87 11. S.J.Sveinsson, W.P.Holbrook Oral mucosal adhesive ointment containing liposomal corticosteroid Int. J. Pharm. (95); 1-3;30 Jun 1993 :105-09 9. Signature of the candidate 10. Remark of the Guide 11. Name and designation of: Mr. ANUP KUMAR ROY M.Pharm, ( Ph.D) 11.1 Institutional Guide: Asst. Professor and HOD Dept. Of Industrial Pharmacy 11.2 Signature Mr.ANUP KUMAR ROY M.Pharm, (Ph.D) 11.5 Head of the Department: 11.6 Signature 12.1 Remarks of Principal Professor & HOD Dept. of Industrial Pharmacy Dr. DIVAKAR GOLI M.Pharm, Ph.D 12 Principal 12.2 Signature Acharya & B.M.Reddy College Of Pharmacy.