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Research Article | DOI: https://doi.org/10.31579/2690-1919/055
1 College of Pharmacy, National University of Science and Technology, Muscat, Sultanate of Oman.
*Corresponding Author: Alka Ahuja, College of Pharmacy, National University of Science and Technology, Muscat, Sultanate of Oman
Citation: Harith Salam Mohammed, Ahmad Makame Mwadini, Nida’a Mohammed Ali Wadi and Alka Ahuja. Formulation and Evaluation of Ophthalmic Antibacterial Gels and Comparison of Different Polymers Using Factorial Design. J Clinical Research and Reports, 3(3); DOI:10.31579/2690-1919/055
Copyright: © 2020 Alka Ahuja. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: 19 February 2020 | Accepted: 03 March 2020 | Published: 09 March 2020
Keywords: ophthalmic antibacterial gels; polymers; HPMC, HPC, HEC; HP-P-CD
Different formulations for the treatment of eye infections are usually administered in the form of conventional ocular drug delivery systems which are topical eye drops or ointments. Typically ophthalmic bio-availabilities of only 1–10% are achieved due to the short precorneal residence time of ophthalmic solutions. The preparation and evaluation of gel containing antibiotic azithromycin combined with different polymers like Carbopol, sodium alginate and Hydroxypropyl cellulose (HPC) was done and assessed to find out which polymer could best be used in preparing ophthalmic gels for this antibiotic using factorial design. Since the efficacy of these gels is dependent on factors like viscosity and pH, the polymers in these gels were also examined for different parameters such as pH, in vitro release, permeation and microbiological evaluation.
Several ophthalmic problems have been investigated including bacterial conjunctivitis, blepharitis and keratitis, and topical antibiotics have been used in the treatment of these problems. Different formulations for treatment of eye infections are usually administered in the form of conventional ocular drug delivery systems which are topical eye drops or ointments [1]. Eye drops and suspensions are often used for topical administration of ophthalmic active drug to tissue around ocular cavity.
The limitation of the conventional liquid ophthalmic formulations is that they demonstrate low bioavailability because of constant lacrimal drainage in the eye. The normal drainage of an instilled drug dose commences immediately upon instillation and is essentially completed within 5 min. Typically ophthalmic bio-availabilities of only 1–10% are achieved due to the short precorneal residence time of ophthalmic solutions [2].
Consequently, there is a need for frequent instillation of concentrated solutions to achieve the desired therapeutic effect, which may lead to some undesirable side effects. Many techniques are applied to prolong the drug stay at site of action and attain optimal concentration. The use of gels for ocular administration of drug offers many advantages compared to conventional ocular drug delivery systems like eye drops which leads to prolonged corneal contact time [3].
Jain SP et al. (2008) prepared and evaluated ophthalmic delivery system for ciprofloxacin hydrochloride based on the concept of pH-triggered in situ gelation. The in situ gelling system involved the use of Polyacrylic acid (Carbopol 980) as a phase transition polymer, hydroxypropyl methylcellulose as a release retardant, and ion exchange resin as a complexing agent. The developed formulation was stable and in vitro drug release was found to be around 98% over a period of 24 hours [4].
Ma WD et al. investigated the in vitro drug release of Pluronic acid copolymer gels, as well as the in vivo resident properties of in situ gel ophthalmic formulations. These in vivo experimental results demonstrated that in situ gels containing Pluronic acid copolymer may significantly prolong the drug residence time and thus improve bioavailability [5].
Al-Kassas RS et al. also performed a study to design controlled release ophthalmic delivery systems for ciprofloxacin based on polymeric carriers in an attempt to prolong the effect of ciprofloxacin and improve its ocular bioavailability. Carbopol and alginates polymers were used to confer gelation properties to the formulations. Controlled release formulated gels consisting of carbopol and cellulose derivatives showed an increase in viscosity, gelling capacity and adhesiveness as the concentration of each polymeric component was increased [6].
Vyas SP et al. described ophthalmic delivery system of an antiglaucoma drug, timolol maleate (TM) based on the use of polymers. The results clearly demonstrated that developed carbopol-chitosan based formulation was therapeutically efficacious and showed a diffusion controlled release behaviour over 24 h periods [7].
Padma Preetha J et al. formulated and evaluated in-situ gels of Diclofenac sodium. These formulations were prepared by combination of HPMC, HPC, HEC and Carbopol derivatives. They performed the studies such as drug content, pH of the Ophthalmic gels, in-vitro diffusion study, sterility testing, anti-microbial studies, anti-bacterial and anti-fungal studies. The percentage cumulative drug release of the best formulation was 87.23% compared to other formulations for the period of 8 hours. The selected formulations showed the antimicrobial, antibacterial and antifungal efficacy [8].
Kesavan K et al. studied HP-p-CD based mucoadhesive hydrogel for ophthalmic delivery of Dexamethasone (DXN) to treat uveitis and improve ocular retention and sustained action. The developed HP-P-CD-based mucoadhesive system was alternative to conventional eye drops of DXN due to its ability to enhance bioavailability through its longer precorneal residence time and ability to sustain the release of the drug [9].
Wu H et al. investigated the correlation between the stability of baicalin and drug release. Carbopol 974P (0.3%, w/v) was used as the gelling agent combined with Hydroxypropyl methylcellulose E4M (0.6%, w/v) which acted as a viscosity enhancing agent. The results demonstrated that ophthalmic gels have better ability to keep baicalin stable and retain drug release than marketed baicalin eye drops. Gels had enhanced the ocular bioavailability [10].
Piyush B. Patel designed a study to optimize and characterize an ophthalmic gel of an antibacterial agent, ofloxacin using Carbopol as the gelling agent along with Polycarbophil as viscosity enhancer. A 32 factorial design was applied for the optimization of the final formulation. The optimized formulation was found to be stable, therapeutically efficacious and provided sustained release of the drug over a 8 h period [11].
Lokhande Umesh Ramachandra et al. designed and developed a pH-triggered in-situ gelling system of Ciprofloxacin using Carbopol and HPMC. The poor bioavailability and therapeutic response exhibited by conventional ophthalmic solutions due to rapid pre-corneal elimination of the drug was overcome by the use of in-situ gel forming systems that are instilled as drops into eye and undergo sol-gel transition in the cul-de-sac. The in-vitro release studies showed the release as 97.33% for the period of 8 hours [12].
Nisha Shetty G et al. formulated Naphazoline and antazoline in-situ gelling systems for ocular delivery and carried out stability studies. The polymers used for preparation of in situ gel were Carbopol 940 and HPMC K4M. The formulations were evaluated by using their pH, Isotonicity, gelling capacity, rheological characteristics, in-vitro release, sterility and in-vivo studies. Their formulations were proved to be very stable at room temperature and at higher temperature (40ºC) with satisfactory drug release [13].
Nnamani PO designed the study for the formulation of ocular gels based on polymer Carbopol to evaluate suitability for enhanced topical delivery of aminoglycoside antibiotic gentamicin. The formulated gels were evaluated in terms of drug content, pH, physical assessment, viscosity, stability as well as drug release. The study demonstrated that gels of gentamicin made with polymer may offer a promising approach for topical delivery of gentamicin for the treatment of infections caused by gentamicin-susceptible bacteria [14].
Prachi Saxena et al. (2014) developed the pH sensitive hydrogels of Levofloxacin hemihydrates for ophthalmic drug delivery using Carbopol 940 and HPMC. Levofloxacin is widely used for the treatment of acute conjunctivitis. The drug suffers the drawbacks of poor bioavailability due to its pH dependent solubility. Evaluation parameters were clarity, drug content, gelling capacity, viscosity studies and in vitro release studies. In-vitro release revealed that best formulation showed the release of 84.31% [15].
Prolongation of residence time due to these ophthalmic gels helps to deliver a drug continuously in a controlled manner to the anterior chamber of the eye and eliminates frequent administration of the drug, thus leading to better patient compliance and extended action. This results in a dose reduction and helps to minimize local and systemic side effects. The preparation and evaluation of gel containing antibiotic azithromycin combined with different polymers like Carbopol, sodium alginate and Hydroxypropyl cellulose (HPC) was done and assessed to find which polymer could best be used in preparing ophthalmic gels for this antibiotic using factorial design. Since the efficacy of these gels is dependant on factors like viscosity and pH, the polymers in these gels were also examined for different parameters such as pH, in vitro release, permeation and microbiological evaluation.
In the present study ophthalmic gels were prepared for Azithromycin using various polymers and evaluated. Azithromycin, 1-oxa-6-azacyclopentadecan-15-one, 13-[(2,6-dideoxy-3-C-methyl-3-O -methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,0,12,14-heptamethyl-11-[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl)oxy], dihydrate, an azalide is , a subclass of macrolide antibiotics. The macrocyclical lactone expansion and the presence of nitrogen atom in the lactone ring are responsible for the best pharmacological and microbiological characteristics. Azithromycin has similar antimicrobial spectrum as erythromycin, but is more effective against certain gram-negative bacteria, particularly Haemophilus influenzae.
Azithromycin prevents bacteria from growing by interfering with their protein synthesis. Azithromycin binds to the 50 S subunit of the bacterial ribosome, and thus inhibits translation of mRNA but nucleic acid synthesis is not affected. It can be used to treat ophthalmic bacterial infections like bacterial conjunctivitis.
2.1 Aim
The aim of the study was to formulate and evaluate the ophthalmic gels of azithromycin using different polymers and find out which polymer is best suited in preparing ophthalmic gels for the antibiotic gel.
2.2 Objectives
The objectives of the present study were:
To prepare ocular gels using different polymers like Carbopol 940 and Hydroxypropyl Cellulose (HPC)
To evaluate the prepared formulations for parameters like pH, in vitro release, antimicrobial activity and drug permeation.
3.1 Materials
3.1.1 Chemicals
Carbopol 940P was gifted by Hangzhou Lingeba technology Co. Ltd. Hydroxypropyl Cellulose (HPC) and Azithromycin were gifted by National Pharmaceutical Industries (NPI) Oman. All other chemicals including sodium chloride, hydrated calcium chloride, sodium carbonate, sodium hydroxide were taken from College of Pharmacy Laboratory.
3.1.2 Equipments
Beakers, measuring cylinders, conical flasks, reagent bottles, glass rods, watch glasses
3.1.3 Apparatuses
Dissolution apparatus (veego), UV-spectrophotometer (6705 UV/Vis. Spectrophotometer by Jenway), pH meter (Martini by Milwaukee Instrument Company) and Franz apparatus (fabricated).
3.2 Methods
3.2.1 Factorial design
Optimization was done using 32 full factorial design in which two factors were evaluated and experimental trials were performed on all 9 possible combinations. In this design there were two independent variables and 3 levels which were low, medium, and high of each variable applied.
The advantages of a factorial design include greater precision. Using a factorial design allows examination of the effect of one variable when other factors are changed, something which is not possible using traditional methods of investigation.
3.2.2 Preparation of ophthalmic gels
3.2.2.1 Composition of Carbopol (940) gel
Formulations were prepared using a 32 factorial design (Table 1). The weighed amount of Carbopol was added in the beaker with a little amount of water and then stirred using glass rod to dissolve the added Carbopol. Hydroxypropyl Cellulose (HPC) was then added to previous formulation and water was added and stirred. Azithromycin was finally added with remaining amount of water to make volume up to 50ml. To provide alkaline medium for the swelling of Carbopol to form gel, 0.5N NaOH was added drops-wise until gel was formed.
3.2.3 Evaluation of gels
3.2.3.1 Physical Appearance
The formulated gels were observed for clarity, homogeneity and presence of bubbles in them.
3.2.3.2 pH evaluation
1ml of gel was added in beaker and 10ml of water was added and stirred completely. pH was measured by digital pH meter in triplicate to get mean and standard deviation.
3.2.3.3 In vitro release
The drug release from different Azithromycin containing polymer gels was studied using dissolution testing apparatus type 1. 1ml of the gel was filled inside cellulose bag using 2.5 ml syringe and kept inside the basket of the dissolution apparatus. The container was immersed in 700 ml freshly prepared simulated tear fluid (NaCl 6.7g, NaHCO3 2g, CaCl2 0.08g and distilled water up to 1000ml) with adjusted pH at 6.85, which was used as dissolution medium. Temperature of 37±0.50 °C and rotations of 100 rpm were maintained throughout the test. Aliquots of 5ml were withdrawn from release medium at each sampling time and replaced with an equal volume of the prepared buffer. The drug content in the samples was determined at 240nm using UV-Visible spectrophotometer.
3.2.3.4 Preparation of standard curve for the release study
The stock solution of 20µg/ml was prepared using the drug and methanol. 2mg of azithromycin was weighed and dissolved in methanol and made up to 100ml using volumetric flask. The solution was stirred well using magnetic stirrer to get uniform solution. Then six dilutions of concentrations 2,4,8,10,16 and 20µg/ml were made from stock solution and made up to 10 ml. These dilutions were finally used for the plotting of standard curve. Calibration of UV-spectrophotometer was done by the use of blank of buffer and methanol.
3.2.3.5 Drug permeation
A modified device (modified Franz diffusion cell using epithelial tissues facial part of the sheep was taken from the slaughter house in Muscat and washed with 0.9% normal saline and used as a diffusion membrane) was used for evaluation of drug permeation. The membrane was tied to a specifically designed glass cylinder (open at both ends). 13 ml Simulated tear fluid pH 6.8 (NaCl 8g, Na2HPO4 2.38g, K2HPO4 0.19g and 1000ml distilled water and the pH adjusted using H3PO4) was used as the diffusion medium in the receptor chamber. 1ml of the formulation which was to be tested was added to the donor chamber with the help of a syringe. The donor surface of the membrane was constantly in contact with simulated tear fluid. Temperature of 37 ± 0.5 °C was maintained throughout the test. A magnetic stirrer provided continuous agitation. 1 mL of sample was withdrawn every 1 hour up to 6 hours and replaced with same volume of fresh simulated tear fluid to maintain constant volume. The withdrawn samples were diluted to 10mL in a test-tube with the prepared buffer and analysed by UV spectrophotometer at 240nm.
3.2.3.6 Preparation of standard curve for the permeation study
The stock solution of 20µg/ml was prepared using the drug and methanol. 2mg of azithromycin was weighed and dissolved in methanol and made up to 100ml in a volumetric flask. The solution was stirred well using magnetic stir to get uniform solution. Then concentrations; 2,4,8,10,16 and 20µg/ml were made from stock solution. These dilutions were finally used for plotting of the standard curve. Calibration of UV-spectrophotometer was done by the use of blank (buffer and methanol).
3.2.3.7 Antimicrobial activity
Among prepared formulations, the gel which showed immediate release from the in-vitro drug release study was taken as the optimized gel for microbiological study to determine the antibacterial properties of Azithromycin against gram positive bacteria namely Staphylococcus aureus, streptococcus, Escherichia coli, and gram negative bacteria Klebsiella and Proteus using cup-plate method. Fresh cultures of different strains of bacteria were prepared and bacteria were sub-cultured in the media at room temperature. Then with the help of sterile cork borer, cups of around 4mm diameter were made on media and the developed formulation (1ml) was poured into the cups made and incubated for 24 hours at 37ºC. The zone of inhibition was measured after incubation.
4.1 Physical Appearance
The gels formulated were clear, viscous enough and homogenous with few bubbles present in them.
4.2 pH readings
The pH of the formulated gels was satisfactory and in acceptable ranges for the ophthalmic drug delivery systems as shown by (Table 2)
Key: SD=Standard Deviation
In vitro drug release
4.2.1 Standard curve from release study
The curve was obtained by using different concentrations made from the stock solution of 20 µg/ml. The absorbance was measured and results are shown in (figure 1).
4.2.2 Release Characteristics
The nine gel formulations of azithromycin were subjected to in vitro release studies for 6 hours and samples were taken over one hour interval. These in-vitro release studies were carried out using simulated tear fluid (STF) of pH 6.8 as the dissolution medium using basket type dissolution apparatus. The cumulative percentages of drug released from each formulation are shown in (Table 3).
Key: % DR indicates percentage of cumulative drug release
The drug release from formulated gels followed a linear pattern where the amount increased as the time increased from 1 to 6 hours. The drug was released more from the formulation containing 0.25 (% w/w) of Carbopol 940. The release was 95, 98 and 90 from the Gel 2, Gel 1 and Gel 3 respectively. From these three gels nearly the entire drug was released after 6 hours. The same was demonstrated by Syed et al. (16). The drug release by the gels containing 0.375(%w/w) of Carbopol 940 were 76, 79 and 78 for the Gel 6, Gel 4 and Gel 5 respectively. The gels containing 0.5(%w/w) of Carbopol 940 showed a percentage of release of 72, 67 and 77 for the Gel 8, Gel 9 and Gel 7 respectively.
Hence the percentage of drug release from the formulated gels followed the pattern, 0.25(%w/w) ˃ 0.375(%w/w) ˃ 0.5(%w/w) for the Carbopol concentrations. Gel 1 showed highest drug release whereas gel 9 showed delay in release of drug after 6 hours.
The higher amount of drug release was attributed to low concentration of the polymer which gave the drug chance to be easily released from the gel formulations. These results indicate that structure of the gel functioned as a resistant barrier to drug release as the concentration of polymers increased.
4.2.3 Factorial design of percent release
Gel dissolution provides an indication of the release from the gel formulation. A decrease in release of the drug from gel formulation was seen as the concentration of the polymers increased (Figure 2). This may be due to formation of new bond between the Azithromycin and Carbopol 940 polymer which explained the reduction in release. The coefficient of correlation was good. The use of mucoadhesive polymers such as HPC and Carbopol 940 directly affect the gel release. Carbopol played a role in drug release while HPC was not having an effect in drug release which was proved by the study.
4.3 Standard curve for permeation study
The curve (Figure 3) was obtained by preparing six concentrations made from the stock solution of 20µg/ml and absorbance from UV spectrophotometer was measured at 240nm.
4.3.1 Diffusion study
Using UV-Spectrophotometer, samples from permeation were analysed and absorbance data was used to calculate concentration and amount of drug permeated across diffusion membrane. The results are shown in (Table 4).
Key :
The purpose of present research work was to develop the ocular pharmaceutical formulations which can overcome the problem related to the conventional dosage form by formulating gels using different polymers. The prepared formulations were shown to give required release profile from in vitro release study as the drug release decreased as the concentration of polymers increased with respect to time. Diffusion study of optimized formulation indicated that most of drug in the gel permeated across the epithelial cell. Antimicrobial studies indicated the effectiveness of the formulation to different strains of bacteria.
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Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, I hope this message finds you well. I want to express my utmost gratitude for your excellent work and for the dedication and speed in the publication process of my article titled "Navigating Innovation: Qualitative Insights on Using Technology for Health Education in Acute Coronary Syndrome Patients." I am very satisfied with the peer review process, the support from the editorial office, and the quality of the journal. I hope we can maintain our scientific relationship in the long term.
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Dear Dr. Jessica Magne, Editorial Coordinator 0f Clinical Cardiology and Cardiovascular Interventions, The review process for the article “The Handling of Anti-aggregants and Anticoagulants in the Oncologic Heart Patient Submitted to Surgery” was extremely rigorous and detailed. From the initial submission to the final acceptance, the editorial team at the “Journal of Clinical Cardiology and Cardiovascular Interventions” demonstrated a high level of professionalism and dedication. The reviewers provided constructive and detailed feedback, which was essential for improving the quality of our work. Communication was always clear and efficient, ensuring that all our questions were promptly addressed. The quality of the “Journal of Clinical Cardiology and Cardiovascular Interventions” is undeniable. It is a peer-reviewed, open-access publication dedicated exclusively to disseminating high-quality research in the field of clinical cardiology and cardiovascular interventions. The journal's impact factor is currently under evaluation, and it is indexed in reputable databases, which further reinforces its credibility and relevance in the scientific field. I highly recommend this journal to researchers looking for a reputable platform to publish their studies.
Dear Editorial Coordinator of the Journal of Nutrition and Food Processing! "I would like to thank the Journal of Nutrition and Food Processing for including and publishing my article. The peer review process was very quick, movement and precise. The Editorial Board has done an extremely conscientious job with much help, valuable comments and advices. I find the journal very valuable from a professional point of view, thank you very much for allowing me to be part of it and I would like to participate in the future!”
Dealing with The Journal of Neurology and Neurological Surgery was very smooth and comprehensive. The office staff took time to address my needs and the response from editors and the office was prompt and fair. I certainly hope to publish with this journal again.Their professionalism is apparent and more than satisfactory. Susan Weiner
My Testimonial Covering as fellowing: Lin-Show Chin. The peer reviewers process is quick and effective, the supports from editorial office is excellent, the quality of journal is high. I would like to collabroate with Internatioanl journal of Clinical Case Reports and Reviews.
