Download polymyxin b and bacitracin ointment

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.