Formulation, Development and Evaluation of In-Situ Gel of Anti-Inflammatory Drug for Ocular Drug Delivery Systems

OCULAR DRUG DELIVERY SYSTEM:

The eye is considered one of the most complex organs due to its unique capacity to eliminate administered drugs.  Ocular chemotherapy is predominantly administered via the topical route owing to its simplicity of application and inherent protective characteristics (Bassi et al., 2015). A significant problem for the formulator is to circumvent the eye's natural defences without inflicting irreparable tissue damage (Dasankoppa et al., 2017). Drugs are often administered to the eye, either on the surface or within the eye, for a localized effect.  The establishment of an optimal medication concentration at the site of action is a critical challenge in ocular therapies.  The low bioavailability of ocular drug formulations is primarily attributed to precorneal factors, including tear turnover, inefficient absorption, short residence time in the conjunctival sac, and the limited permeability of the corneal epithelium  Due to ocular anatomical and physiological limitations, the bioavailability of administered drugs is extremely low, with absorption typically restricted to about 1% or less (Patel et al., 2013). To date, attempts have been undertaken to increase the bioavailability of ocular medications by increasing the length of drug residency in the conjunctival sac and improving drug penetration through the cornea, the major route of drug entrance into the inner eye (Draize, 1944). The development of new, more sensitive diagnostic procedures and innovative therapy molecules has resulted in great therapeutic effectiveness for ocular delivery systems. Conventional ophthalmic dosage forms, including solutions, suspensions, and ointments, exhibit several limitations within the ocular cavity that lead to poor drug absorption (Tangri & Khurana, 2011a). The primary objective of therapeutic device design is to maintain an optimal drug concentration at the active site for a reasonable amount of time.  The physicochemical qualities, as well as the associated ocular anatomy and physiology, are determined by the ocular disposition and removal of a therapeutic substance. An integrated understanding of the drug molecule and the constraints of the ocular administration path is necessary for an efficient drug delivery system design (Gadad et al., 2016). Apart from anti-glaucoma medications, antibiotics and antibacterial medicines that need to be used often in normal vehicles to treat conditions like trachoma and acanthamoeba keratitis may also be distributed via log-acting inserts. In addition to these, drugs chosen for topical delivery via ocular inserts include antivirals, antifungals, anti-filarial, antiallergic agents, anti-inflammatory drugs (both NSAIDs and steroids), fibrinolytic agents, immunosuppressants, and growth factors (Goel et al., 2010).

Obstacles in Ocular Drug Delivery System

Developing an ocular drug delivery system that can accurately deliver the drug at the optimal concentration to the target site while ensuring effective therapy remains a significant challenge.  Because of the corneal structure, physiology, and barrier functions that cause rapid drug absorption, it is necessary to apply eye drops quickly in order to balance the therapeutic level in tear film or at specific places.  Frequent usage of medication solution might result in cellular damage and toxicity at the ocular surface (Gupta et al., 2015). Ocular drug absorption is significantly reduced by precorneal losses, which involve tear dynamics, nasolacrimal drainage, dilution effects, conjunctival and non-productive absorption, and limited residence time within the cul-de-sac.  Transporting medications to the anterior segment is hindered by the corneal epithelial barrier, which exhibits low permeability to topically applied drugs.  Owing to various anatomical and physiological barriers, only approximately 1% or less of a topically applied drug reaches intraocular tissues. Enhancing corneal residence time and optimizing the balance between lipophilicity and hydrophilicity are essential for improving therapeutic efficacy (Goel et al., 2010).

Ocular Drug Administration Pathways

Below is a description of the several potential pathways for ocular medication administration.:

Intravitreal route: This method involves injecting the drug into the vitreous fluid of the eye.  Figure 1 illustrates the delivery via this ocular route, which is used to treat a variety of eye conditions.

Intracameral route: This delivery pathway allows direct action on the anterior and posterior ocular compartments, illustrated by the use of anaesthetics injected into the anterior chamber during surgery (Ghate & Edelhauser, 2006).

Periocular route: This approach entails delivering the drug in the tissues surrounding the eye. An example is periocular steroid injection, where steroids are administered around the eye to reduce intraocular inflammation or oedema.

Suprachoroidal route: This administration method targets the supra choroid area of the eye.  Suprachoroidal space is the area that exists between the choroid & the sclera.

Subconjunctival route: This delivery technique targets the mucous membrane of the eye, involving both the inner eyelid lining and the open conjunctival surface.

Topical route: Among ophthalmic dosage forms used for topical drug delivery, eye drops are the most common, particularly for treating anterior segment disorders. Their affordability and ease of administration make them the most practical method for delivering medication to the eye (Gaudana et al., 2010).

Systemic route: The systemic delivery of ophthalmic drugs is often limited by the blood-aqueous barrier in the anterior segment and the blood-retinal barrier (BRB) in the posterior segment of the eye.

Figure 1: Routes of Ocular Drug Delivery

Limitations and Barriers in Ocular Drug Delivery

The development of ocular drug delivery systems capable of delivering medication at the desired therapeutic concentration faces challenges due to the cornea’s structural and functional barriers. Rapid drug absorption necessitates frequent eye drop application to maintain adequate therapeutic levels at the site of action. Frequent usage of medication solution might result in cellular damage and toxicity at the ocular surface.  Precorneal factors, including tear turnover, lacrimation, drainage, dilution, conjunctival absorption, non-productive uptake, and short residence time in the cul-de-sac, contribute to the poor ocular bioavailability of most administered drugs (Ramesh et al., 2017). Another challenge is the limited permeability of the corneal epithelium, which hinders the delivery of drugs to the anterior segment following topical application. Various ocular anatomical and physiological factors significantly reduce drug absorption, such that only a minimal portion of the administered dose, approximately 1% or less, reaches the intraocular tissues. Higher contact duration and a balance between lipophilicity and hydrophilicity are necessary for topical dose forms to produce better therapeutic outcomes (Kaur et al., 2004a) Difficulties encountered in ocular drug delivery are generally divided into the following categories:

Difficulties in Delivering Drugs to the Anterior Segment of the Eye

Figure 2: Precorneal variables affecting topically applied ophthalmic medications' bioavailability

The preference for topical over systemic ocular drug delivery arises from the need to overcome precorneal barriers, such as the tear film and conjunctiva, which slow the entry of the drug into and across the cornea. The majority of ocular formulations have low drug bioavailability due to precorneal loss factors, as seen in figure. 2.  Additionally, in order to maintain a therapeutic drug level in the tear film or at the site of action, eye drops must be instilled often. However, using highly concentrated medication solutions frequently may result in harmful side effects and cellular damage at the ocular surface (Patel et al., 2010).

In-Situ Gel

Owing to its distinctive sol-to-gel transition, the in-situ gel system has emerged as a highly innovative approach in drug delivery. It aids in the prolonged and regulated release of the medications and enhances patient compliance and comfort.  An in-situ gelling system is a formulation that, under certain physiological conditions, transforms from a solution to a gel before entering the body.  Factors such as temperature, pH variations, solvent exchange, ultraviolet light, and the presence of specific molecules or ions can influence the sol-to-gel transition.  Drug delivery systems with the aforementioned "sol to gel transition" characteristics can be widely utilized for the synthesis of bioactive compounds in sustained delivery vehicles. Drug delivery systems with the aforementioned "sol to gel transition" characteristics can be widely utilized for the synthesis of bioactive compounds in sustained delivery vehicles (Gupta et al., 2007).

Importance of in situ gelling system

• Its distinctive sol–gel transition facilitates controlled and sustained drug release.
• It contributes to the body administering drugs less often.
• A low dosage of the medication is needed, and there won't be any side effects or drug build-up.
• The drug is expected to exhibit enhanced bioavailability.
• Because of gel formation, the medicine will have a longer residence period.
• The in-situ gel system decreases wastage of the drug.
• The best dose form is a liquid that can maintain medication release and stay in touch with the cornea of the eye for a long time.
• Some unfavourable side effects may arise from decreased systemic absorption of medication discharged through the nasolacrimal duct (Patil et al., 2015).

Advantages of in situ gel system

• controlled and prolonged medication release.
• The medication's ease of administration.
• Patients who are unconscious can get it.
• Greater comfort and compliance from patients Reducing medicine toxicity and dosage frequency.
• Enhanced bioavailability
• Natural polymers offer biodegradation and biocompatibility.
• Natural polymers inherently possess properties such as biocompatibility, biodegradability, and the presence of bioactive moieties that support cellular functions.
• The structures of synthetic polymers are often well-defined and may be altered to produce acceptable functionality and degradability.
• For non-invasive drug delivery, in situ gels can also be designed to be bio adhesive to enable drug targeting, particularly across mucous membranes.
• Because of their hydrophilicity, which prolongs the delivery device's in vivo circulation duration by avoiding the host immunological reaction and reducing phagocytic activities, in situ gels provide a crucial "stealth" feature (Paul et al., 2023).

Disadvantages of in situ gel system

• The system requires the presence of a sufficient amount of fluid for proper functioning.
• The sol form of the drug is more susceptible to degradation.
• The possibility of stability issues brought on by chemical deterioration.
• Food and fluid intake may need to be limited for a certain period after administration of the drug.
• Limitations may exist regarding the drug loading capacity and the uniform distribution of drugs within hydrogels, particularly in the case of hydrophobic compounds.
• Only medications with minimal dosage requirements may be administered.
• A lower mechanical strength might cause the hydrogel to dissolve too quickly or to flow away from the intended local location (Rathore, 2010).
• Ideal characteristics of polymers for preparation of in situ gel
• The polymer should possess adequate mucoadhesive properties to effectively adhere to the mucosal membrane.
• It should not have any harmful effects and be very compatible.
• It ought to exhibit pseudoplastic behaviour.
• As the shear rate increases, the polymer should be able to reduce its viscosity.
• The polymer's preferred pseudo-plastic characteristic.
• Optical clarity and good tolerance are desirable.
• It ought to affect how tears behave (Pahuja et al., 2012).

Drug Profile

Bromfenac

For usage in the eyes, bromfenac is a nonsteroidal anti-inflammatory medication (NSAID).  Ocular discomfort and inflammation are increasingly being treated with ophthalmic NSAIDs.  NSAIDs are a crucial tool for improving surgical outcomes because to their well-established safety record, analgesic properties, and well-characterized anti-inflammatory action.  In 1998, reports of severe liver damage led to the withdrawal of non-ophthalmic versions of bromfenac in the United States.

Chemical Identity

• IUPAC Name: 2-amino-3-(4-bromobenzoyl) benzene acetic acid
• Molecular Formula: C15H12BrNO3
• Molecular Weight: 334.17 g/mol

Structure

Physicochemical Properties

• Appearance        Yellow to orange crystalline powder
• Solubility (Bromfenac base) Practically insoluble in water
• Solubility (Sodium salt)  Soluble in water
• LogP (octanol/water) ~2.99–3.50 (lipophilic)
• pKa (Carboxylic acid group) ~4.2–4.4
• Melting Point ~198–200 °C

Pharmacodynamics

A sterile topical nonsteroidal anti-inflammatory medication (NSAID) for use in the eyes is called bromfenac ophthalmic solution (Kalgutkar & Soglia, 2005).

Mechanism of action

It’s believed to work by preventing prostaglandin formation by blocking cyclooxygenase 1 and 2.  Numerous animal models have demonstrated prostaglandins as mediators of specific types of ophthalmic inflammation.  Prostaglandins have been demonstrated to cause leucocytosis, increased vascular permeability, vasodilation, disruption of the blood-aqueous humour barrier, & elevated intraocular pressure an experiments conducted in animal eyes (Sheppard et al., 2018a).

Pharmacokinetics

i) Absorption

Route: Topical (ophthalmic)

Onset of Action: Within 24 hours of administration

Systemic Absorption: Minimal, after ocular administration, bromfenac is absorbed into the aqueous humour and ocular tissues. Systemic plasma levels are very low or undetectable.

Time to Peak Concentration (Tmax): Approximately 2 to 3 hours in aqueous humor after a single topical dose.

ii) Distribution

Ocular Distribution: Well-distributed within ocular tissues such as: Cornea Conjunctiva, Aqueous humour, Iris/ciliary body, Retina/choroid.

Plasma Protein Binding: ~99.8% (very high)

iii) Metabolism

Primary Site: Liver (if systemically absorbed)

Pathway: Oxidation followed by conjugation (primarily glucuronidation)

Enzymes: Likely CYP450-mediated oxidation, though minimal systemic exposure means hepatic metabolism is typically insignificant in clinical settings.

iv) Elimination

Elimination Half-life (ocular tissues): Estimated at ~1–2 hours in aqueous humor

Systemic Half-life (if absorbed): ~1.4 hours

Excretion: Primarily renal if systemically absorbed (as metabolites) (Walters et al., 2007).

Dosing Considerations

Typical Dosing: Once daily (0.07%, 0.09%, or 0.075% solutions depending on formulation) (Henderson et al., 2011).

MATERIALS AND METHODS

Pre-formulation Study

Identification of Selected Drugs

The identification of drug carried out by UV spectroscopy, IR spectroscopy and thermal study (melting point determination solubility, partition co-efficient determination and DSC thermogram & DSC thermogram).

Determination of absorption maxima (λ max)

Preparation of simulated tear fluid (STF) of pH 7.4: The previously published formula was utilized to produce STF1.  To put it briefly, a tiny amount of distilled water was used to dissolve sodium chloride (0.670 g), sodium bicarbonate (0.200 g), and dehydrated calcium chloride (0.008 g).  Ten minutes of sonication were applied to the resultant solution, lastly the weight was changed (Sheppard et al., 2018b).

Preparation of stock solution: 50 mg of the medication was dissolved in 50 ml of deionized water and STF pH 7.4 to create a standard stock solution with 1 mg/ml.  Ten minutes of sonication were applied to the resultant solution.  A stock solution containing 100 μg/ml of medication was obtained by diluting 5 ml of this solution with 50 ml of deionized water and STF pH 7.4.  Working standards were further prepared using this stock solution.

Identification of absorption maxima: STF 7.4 for PM was used to dilute the stock solution to a concentration of 18 μg/ml.  A UV spectrophotometer was used to perform further scanning between 200 and 700 nm.  STF was utilized as a blank in order to determine the absorption maxima, and all measurements were obtained in triplicate.  For the ciprofloxacin solution (10μg/ml), a similar process was used with deionized water.  After being gathered, the spectra were compared to a standard.

Infrared spectrum of drug: The Agilent FT-IR spectrometer was utilized to perform IR spectroscopy on the drug sample, and the KBr pellet technique was employed for the analysis.  The spectra were scanned between 4000 and 400 cm-1 in wave number (Ramarao & Preethi, 2022).

Determination of melting point: The Veego digital melting point device was used to determine the melting point.  The temperature at which the medication began to melt and finished melting was used to determine the melting point range.  For every medication, the melting point was determined in triplicate (Gabr et al., 2023).

DSC analysis: The Netezch DSC 204 F1 Phoenix Differential scanning calorimeter was used for the DSC investigation. Samples were weighed, put in aluminum pans, and heated at a rate of 20 K/min using a calorimeter to get the necessary temperature range. As a coolant, liquid N2 was flushed at flow rates of 40 and 50 milliliters per minute. Temperature and change in enthalpy (mW/mg) were displayed on the graphs (Shelley, Grant, et al., 2018).

Determination of Solubility: The shaking flask method was used to assess the drug's solubility in water and STF pH 7.4. Twenty milliliters of solvents (deionized water and STF pH 7.4) were used to dissolve the extra medication. For the following 48 hours, the supersaturated solution was maintained at room temperature on a revolving shaker. After that, it was examined once every five hours until three consecutive readings were similar. Before sampling, the supernatant solutions were filtered to remove any particulate matter, and the residual solution was diluted for UV spectroscopic analysis.

Determination of Partition Coefficient: The study was performed in n-octanol/water system using shake flask technique4, 5. The amount of the drug substance was fixed in such a manner that the concentration remained 0.01 mol. L-1. A pre-saturated solution of water and octanol was used for three different ratios 1:1, 1:5 and 5:1 in the study. Following a 24-hour period on a flask shaker, the samples were separated using a separating funnel. Once the aqueous phase reached a steady state, it was centrifuged at 5000 rpm.  UV spectroscopy was used to measure the drug's concentration in the aqueous phase.  Next, the apparent partition coefficient and the drug's concentration in n-octanol were computed.  An average of three parallel measurements is used to determine each value.

Compatibility Study of Selected Drugs and Polymers: The suitability and compatibility of the polymers with the drugs used in the study was evaluated by performing compatibility study. The drug and polymers alone and their 1:1 mixture were kept in stability chamber for two weeks at 50 °C & ambient RH6. Further the samples were tested for any physical and chemical changes by using DSC and IR spectroscopy.

Preparation of In-Situ Gelling Ocular Delivery System

Literature suggested that thermosensitive polymers and pH sensitive polymers show prominence role in in-situ gelling delivery. We have selected poloxamer 407 is approved (USFDA) ophthalmic polymers, and belongs to thermosensitive and pH sensitive category, respectively. Firstly, Poloxamers is a thermosensitive tri-block co-polymer which consist of hydrophilic polyoxyethylene (PEO) and hydrophobic polyoxypropylene (PPO) units. It has reversible thermal gelation property at specific CMC and CMT. At higher concentration in an aqueous solution, it exhibits transformation from a low- viscosity solution to non-cross-linked hydro-gel at ambient temperature. It is also reported that the lachrymal fluid dilution significantly increases the gelation temperature so, a relatively high concentration is required for the poloxamer solution, to become gel under physiological conditions. In that case, there might be a risk of gelling of formulation at room temperature, which is ultimately unfavourable for instillation15. The poloxamer as alone, also has a serious drawback that due to non-cross-linking nature of its gel it shows weak mechanical strength, which ultimately leads to rapid erosion. The problem can be overcome by incorporation of other polymers like low molecular weight chitosan (muco-adhesive polymer), HPMC (viscosity imparting), which might increase the residence time and mechanical strength of the gel. Besides that, the Pluronic F68 can also be used with a combination of the above polymers, as it has a tendency to reduce the viscosity and delaying gelation temperature at low concentration. The potential of the poloxamer 407 in combination with low molecular weight chitosan, HPMC E4M, Pluronic F68, for sustaining drug release and for overcoming cornea impermeability, still needed to be evaluated. Hence in this study, we proposed to design formulations containing different proportion of the above polymers, for evaluating their possible effects on gelation temperature, release, residence time, viscosity and stability (Shelley, Rodriguez-Galarza, et al., 2018).

Preparation of Formulations

Poloxamer solution preparation: According to Schmolka, the "cold method" was used to prepare the poloxamer solution.  Using a magnetic stirrer, the necessary amount (% w/w) of polymer, determined on a mass basis, was dissolved in half of the deionized cold water while being continuously stirred.  Ice was used to keep the temperature close to freezing.  The partly dissolved poloxamer solutions were kept in a refrigerator between 2 and 8 °C for the whole night, with periodic stirring to produce clear, homogeneous solutions.

Preparation of low molecular weight chitosan solution: A modest volume of 0.5% acetic acid solution was used to dissolve the necessary amount of chitosan.  The resultant mixture was kept in storage at room temperature.

Preparation of Methocel E4M: The HPMC solution was equipped, simply by dissolving the required quantity in water with constant stirring.

Drug solution: The required amount of drug (BM) was dissolved in previously prepared solution of edetate disodium and benzalkonium chloride. The required amount of mannitol, was added to this solution to maintain, the isotonicity of the formulation.

Preparation of formulations BG1-BG12: The chitosan solution was added to poloxamer 407 solution using ice bath, and restored at refrigerator for further 24 hours. Drug solution was added to Methocel E4M with continuous stirring, and finally it was mixed with chitosan-poloxamer solution. The pH was adjusted to 6 using with the use of 0.5 M NaOH & 0.5 M HCl.

Preparation of formulations BG1a-BG12a: The pluronic F68 was initially mixed with poloxamer 407, and then the mixture was dissolved in the same manner as described for poloxamer solution, further steps will be the same as discussed above (Table 1).

Table 1: Preparation of in-situ gel without Pluronic F68

BM= Bromfenac; DW= Deionized Water; DSE- Di-Sodium Edetate; BC= Benzalkonium Chloride, before being characterized, each formulation was given a full day to equilibrate in the refrigerator.

In-Vitro Characterization of In-Situ Gelling Ocular Drug Delivery System

Following their successful preparation, each formulation was evaluated using the following criteria:

Visual Appearance and Clarity: Every formulation's look and clarity were assessed visually.

Determination of pH: The pH of the formulations was determined using calibrated micro pH meter. The observation was repeated in triplicate manner.

Determination of Gelation Temperature Visual Method: The "Visual Tube Inversion Method" was used to calculate the gelation temperature (GT) of the developed formulations.  A glass vial (13 mm) was filled with one gram of the material.  A thermometer with a 0.1°C accuracy rating was submerged in a water bath close to the vial.  The vial was heated in a water bath at a regulated rate of 2 °C per minute.  When the sample solution stopped flowing upon tilting, the gelation temperature (t1) was recorded.  The temperature at which the gel began to flow was recorded as the gel melting temperature (t2) after the water bath was gradually cooled.  The practical was conducted in triplicate, and the results were presented as a mean ± SD of the measured t1 and t2 values.

Viscosity Analysis: The viscosity was determined as a function of temperature, using Brookfield LVDV IIP viscometer. The formulations were kept on temperature-controlled circulating water bath, and temperature probe of viscometer was inserted in to the bottom of the container. Temperature was slowly increased, with rate of 1 K/min from 18-37 °C to locate the sol-gel transition point. The viscosity was measured, using S95 spindle at 20 rpm, at respective temperature. The inflection point on the apparent viscosity (cp) vs. temperature (°C) curve was used to graphically quantify the gelling temperature.  The results were presented as mean± SD after all readings were repeated three times (Ramarao & Preethi, 2022)

Effect of Dilution on Gelation Temperature: STF pH 7.4 was used to dilute each mixture., in the ratio of 30:7, for assessing the effect of lachrymal fluid, on gelation phenomenon in the eye. The gelation temperature was recorded as discussed above.

Rheology Study

Viscosity measurement: The viscosities of the formulations were measured using a Brookfield LVDV II P viscometer at three different temperatures: 10, 25, and 35 °C. Due to large viscosity range, it was not possible to measure the viscosities using single spindle in rotational viscometer. The analysis was done using S62 spindle (at 10 °C) and S95 spindle (25 & 35 °C) at 20 rpm for accurate measurement. Each point is the mean of three readings.

Visco-elastic analysis: A study was also performed, to ensure the behavior of the formulation at, non-physiological condition [25 °C, pH-5.8 or 6.0 (all other)] and physiological condition (35 °C, pH 7.4) with change in shear rate (rpm). The study was performed using spindle S63 at non physiological condition, and viscosity was measure as a function of shear rate (1-20 rpm). While in case of physiological condition the viscosity was recorded as a function of shear rate (0.4-4 rpm) using S64 spindle of Brookfield LVDV II P viscometer. All the measurements were recorded as mean ± SD of three measurements.

Estimation of Drug Content: A one-gram sample was combined with a little amount of deionized water and vortexed for five minutes.  A 0.45 μ membrane filter was used to filter the final volume, which was increased to 100 ml using deionized water.  One millilitre of the mixture was taken out and diluted with five milliliters of distilled water.  The drug content of the resultant solution was measured at 272 nm using the Labindia UV 3000+ UV visible spectrophotometer.

In vitro Release Study: The investigation was conducted utilizing a modified diffusion cell to assess each formulation's diffusivity and release pattern.

Donor Compartment: It consists of a cylindrical glass tube fabricated diffusion cell, open at one end and covered with, cellulose acetate sheet (soaked overnight in simulated tear fluid) at the other open end on which 1 gram of the gel was placed.

Receptor Compartment: 100 ml of simulated tear fluid was taken in the receptor compartment and temperature was maintained at 35 °C. The receptor compartment was continuously stirred at 100 rpm. After a certain amount of time, a 1 ml aliquot was removed from the trial, which lasted for six hours.  The concentration was measured using the UV visible spectrophotometer Labindia UV 3000+ at 272 nm30 after the aliquot was further diluted to 3 ml using STF pH 7.4.  To determine the precise release pattern, all of the data from release studies was subjected to different kinetic models: zero order as cumulative percentage amount of drug released vs. time, first order as log cumulative percentage of drug remaining vs. time, and Higuchi's model as cumulative percentage of drug released vs. square root of time. The precise release mechanism was ascertained by fitting all of the kinetic data to the Korsmeyer-Peppas equation:

Mt/Mα= ktn as log cumulative percentage of drug released vs. log time, where k was a constant associated with the formulation's structural and geometrical properties as release rate, n was the release exponent that indicated the drug release mechanism, and Mt/Mα was the proportion of drug released at time t (Si et al., 2011).

Osmolarity Measurement: It was determined by measuring freezing point depression of the solution, the method was adopted from European Pharmacopoeia.

Microbiological Study: Microbial study was performed to compare antimicrobial efficacy of optimized formulations, with marketed formulations. The turbidity method (broth assay), using nutrient broth (Luria) was performed for two bacterial strain Staphylococcus aureus (MTCC-4322 strain) and Pseudomonas aeruginosa (MTCC-3222). Suspensions of S. aureus and P. aeruginosa, were prepared to give 0.5 McFarland standards. The luria broth was prepared and sterilized, using autoclave at 121 °C for 20 minutes. The 10 ml of sterilized broth was filled aseptically in all glass inoculum tubes and 100μl bacterial suspension was seeded aseptically to the tubes. Finally, 25 μl of formulations and marketed drops were mixed aseptically with respective tubes. After incubation at 37 °C for 4h and 24 h, the growth of each sample was determined by measuring OD at 420 nm using spectrophotometer. The non-seeded nutrient broth was considered as blank and, seeded broth without treatment was considered as positive control, for the analysis. Each reading was taken in triplicate manner (Cho et al., 2009).

Effect of Sterilization: All of the formulations underwent autoclave sterilization in accordance with US Pharmacopeia XXXI guidelines in order to examine the impact of moist heat sterilization (autoclave) on the physicochemical characteristics of improved ocular formulations. All the optimized formulations were exposed to moist heat sterilization using autoclave, at 121 °C, 15 psi pressure for 20 minutes. Then, the samples were evaluated for the pH, drug content, & viscosity and, comparison was made between the obtained values before and after autoclaved (Ramarao & Preethi, 2022)

RESULTS AND DISCUSSION

Pre-formulation Study

Identification of Bromfenac (BM): The identification of BM carried out by UV spectroscopy, IR spectroscopy, melting point, solubility, partition co-efficient determination and DSC thermogram.

Determination of absorption maxima (λmax): As shown in figure 3, the λmax of BM was determined to be 582 nm in simulated tear fluid at pH 7.4.

Figure 3: UV. Spectra of Bromfenac

Figure 4: Calibration curve of Bromfenac

Infrared spectrum of Bromfenac (BM): The identity of the BM was confirmed by verification of presence of functional group. For this, the pure sample of BM was subjected to IR spectroscopy using Bruker FT-IR spectrometer & KBr pellet method. FT-IR spectrum of BM exhibited characteristics peaks for carboxylic group with stretching (3200-3600 cm-1), carbonyl group and unsaturated >C=O (1714, 1629 cm-1) and bands for aromatic ring (1483, 895 & 780 cm-1.

Figure 5: FTIR Spectra of procured drug Bromfenac

Determination of melting point: The Veego digital melting point device was used to determine the melting point. The BM's identification and purity were confirmed by the melting point range of 283 to 285 °C, with a mean value of 282.11 ± 0.87 °C.

Determination of Solubility: The shake flask technique was used to evaluate the solubility of BM in water and STF at pH 7.4. They were found to be well soluble in both solvents. At room temperature, BM's solubility was determined to be 0.13422±0.0031 in distilled water and 0.15322±0.0042 mole L-1 in STF pH 7.4. Therefore, it can be said that both solutions may be used to formulate and analyze BM.

DSC analysis: The BM sample was subjected to DSC analysis using Netzsch DSC instrument. DSC pattern of BM exhibited three characteristic endothermic peaks (figure 6): first peak further splitted into two peaks which confirmed the presence of dihydrate molecule in the BM; the second one appeared at 200 °C and third melting point peak was at 202 °C.

Figure 6: DSC Spectra of Bromfenac

Determination of Partition Coefficient: The partition coefficient of BM was determined using n-octanol/water system at room temperature by shake flask method. The Log P was found to be 0.2655± 0.0054 at room temperature which was in line with the previously reported results. The results suggested that BM interact more strongly with an organic solvent than with an aqueous solvent. The observed Log P value indicate that BM has good corneal permeability.

Compatibility Study of Drug and Polymers: The compatibility of drug with the polymers used in the study was confirmed by IR spectroscopy. The presence of characteristic peaks of the drug in the IR spectra of drug with different polymer mixture confirmed that no interaction was found in between them. Similarly, the characteristic endothermic peaks of BM were retained in physical mixture of drug with polymers. These studies confirmed that no physical and chemical interaction was present in the selected polymers with BM so, they could be used as a formulation candidate with the drug. The ocular preparations were successfully prepared and were evaluated for their performances and stability.

Characterization and Evaluation

Visual appearance and clarity: It are prerequisites for the ocular in-situ gelling system that they should be clear and transparent before and after gelation to avoid irritation and blurring of vision. All the formulation were visually assessed for the appearance and clarity in dark and white background.

pH: As USP guidelines “The pH and buffering capacity of an ophthalmic preparation are probably of equal importance to proper preservation of ophthalmic preparations”. Ideally the pH of the formulation should match the pH of tear fluid (7.4) but there is a flexibility in selecting pH, depending on their best performance and stability. The reported iso electric point of PM is at pH 5.11–7.23 so the pH of prepared formulations containing BM was selected 6.12. For confirming the uniformity and buffering capacity of prepared formulations pH determination were made performed. The results of the study suggested that the pH was in the range of 5.11-7.23 for all the formulations (BG1-BG12) containing a combination of poloxamer 407, chitosan, Methocel E4M, and pluronic F68.

Table 2: Visual appearance, clarity, and pH of various formulations

Drug Content: Drug content of all the formulations was found to be 0.89 to 1.18 mg/gm, which suggested that drug was uniformly distributed in the ocular formulations.

Table 3: Drug Content of all formulation

Gelation Temperature (GT): The main prerequisites for the ocular in situ gelling system are gelation temperature and viscosity. To identify the optimum formulations which would form gel at surface temperature of cul-di-sac of eye (35 °C) we measured the GT of all formulations by two methods namely “visual tube inversion method” and “viscosity analysis”. In addition to this, effect of dilution by lachrymal fluid was also observed. Results of effect of dilution by lachrymal fluid on geletion temperature is tabulated in 4. It was observed that in all formulations (BG1-BG12) which contain poloxamer 407 (14- 18% w/w) gelation temperature was significantly reduced, with the increase in concentration of poloxamer 407. The pattern of decrease of GT, was in line with the previous findings, but the values of GT was slightly different from the previously reported method, this might be due to the difference in selected drug. The dilution of formulation with lachrymal fluid, significantly increase the GT, this might be due to low ionic dilution with STF. The results are in line with the previous findings. Overall, the GT observed from viscosity method was slightly less than the GT observed using visual method.

Table 4: GT and effect of lachrymal dilution on various formulations

All the values of table represented as Mean±SD (n=3); * no gelation

The poloxamer 407, a non-ionic triblock copolymer (PPO-PEO), aggregate into micelles at above critical micellar temperature which generally decrease with increase in concentration. The dehydration of polymer blocks with temperature is the cause of this micellization. Micellar expansion and packing have been found to cause gel formation, and at greater poloxamer 407 concentrations, the gel is more entangled. Due to the process of micelle entanglements, it cannot split easily from each other, and responsible for gelation and high viscosity at higher concentration of poloxamer 407.

Effect of chitosan and methocel E4M (HPMC): The formulations (BF1, BG12) containing poloxamer 407(14%w/w) alone or in combination methocel E4M and chitosan shown no gelling up to 40 °C. In the formulations (BG4-BG8), the presence of chitosan and Methocel E4M at low level (0.1, 0.5 % w/w) with poloxamer 407 (at 16% w/w) could not affect GT much more in comparison to BG1, but in BG9 where they were at high level (0.2,1 % respectively), significantly decreased the GT. The BG12 slightly decreased the gelation. The overall result of this study suggested that combination of polymers might be reduces total polymer content and increases the gelling process hence, GT can be achieved around physiological temperature by the combination of poloxamer 407 with other polymers like pluronic F68, chitosan and HPMC E4M.

Rheology Study: Rheological behaviour and viscosity under both physiological and non-physiological conditions are important criteria for the ocular in-situ gelling system. The appropriate viscosity for an ocular in-situ formulation is one that facilitates facile instillation into the eye as a drop that quickly undergoes a sol-gel transition. Additionally, it should maintain its integrity for a longer amount of time without deteriorating or disintegrating.

Viscosity study: The rheological property of formulation as a function of temperature was studied. The viscosity of formulations, at three different temperatures (10, 25 and 35 ?C) which could represent the behaviour of formulation at storage, non-physiological and physiological conditions, respectively.

Table 5: Viscosity of formulations at different temperature

All the values of table represented as Mean ± SD (n=3)

All the formulations were exhibited temperature dependent increase of viscosity, but it was observed that by the addition of chitosan and HPMC E4M the viscosity of formulations was not increased that much high as they might have individually. The viscosity of formulation was significantly increased at 35°C. All the formulations were solution at 10°C but at 25 °C some formulations (BG1-BG12) shown higher index of viscosity.

Visco-elastic analysis: This study was performed to assess the flow behavior and nature of the formulation under various shear rate and force condition, which might be exerted by the formulation while non physiological condition (instillation) and during physiological condition (blinking and inter blink period of eyes). In this study, the formulations BG1-BG12 were excluded from the study on the basis of results obtained from GT study. We only studied those formulations which showed some promising results. The dynamic viscosity of ocular formulations was measured as a function of shear rate (rpm) under non-physiological (at 25 °C, pH-6) and physiological conditions (at 35 °C, pH- 7.4).

In-vitro Release Study: The in-vitro drug release was performed using modified diffusion cell apparatus for a period of 6 hours. The cumulative percentage drug release as a function of time was plotted for all the formulations and shown in figures. The release of drug at 2 hour and 6 hour was considered as the comparison criteria of release. The release at 2 hour provides burst release information (required for instant action) and 6 hours provided information regarding sustainability. All the release data were fitted to the various kinetic models like zero order, first order, Higuchi kinetics and, the results obtained from kinetic studies. At last, all the kinetic data were fitted to korsmeyer-peppas equation to know the exact release mechanism of the formulations. The results of different formulations in group are as follows: The percentage cumulative drug release at the end of 2 hours was found to be 41.83- 83.84% for the formulations BG1-BG4. For the formulations BG1-BG3, almost all the drug was released before 6 hours while, for the formulations BG5-BG8 it was found to be 87.81- 96.70 % at 6 hours. The resistance towards release was observed as the concentration of poloxamer 407 increased. The formulations BG9-BG12 were not studied for the release study because they did not give promising results in GT study.

Table 6: In-vitro drug release study

All the values of table represented as Mean ± SD (n=3).

Figure 7: Cumulative drug release profile of the formulations (BG1-BG4)

The formulations, BG5-BG8 (containing poloxamer 16%, Chitosan (0.1-0.2%), and HPMC (0.5-1%) shown 45.97 -70.00 % drug release at the end of 2 hours, while, 94.17- 100 % at 6 hours. All the formulations clearly shown, decrease in drug release at all time point in comparison to formulation PF2. This might be due to increase in viscosity and mechanical strength of the formulations by the addition of mucoadhesive polymers, which ultimately decrease the erosion rate and diffusion process.

Table 7: In-vitro drug release study

All the values of table represented as Mean ± SD (n=3).

Figure 8: Cumulative drug release profile of the formulations (BG5-BG8)

Table 8: In-vitro drug release study

All the values of table represented as Mean ± SD (n=3)

Figure 9: Cumulative drug release profile of the formulations (BG9-BG12)

The results can be explained by the fact that these formulations produced a prehydrated gel matrix with water penetration and hydration acting as the rate-limiting phase of drug release when they came into contact with the simulated tear fluid at 35 °C and gelation took place. The presence of chitosan and HPMC increases the resistance for water penetration in to the gel matrix hence decrease in release, was observed at single level of poloxamer 407. From the kinetic study, it was observed that all of the formulations exhibited highest linearity (0.966-0.996) for the cumulative percentage release vs square root of time plots, hence, it was proposed that in vitro release of all the formulations could be best explained by Higuchi kinetics. The R2 value of all the formulations was found to be highest in Higuchi, followed by first order and zero order.

Table 9: In vitro release kinetic parameters of BM in-situ gelling formulations

The remaining three formulations BG6, & BG10 might be follow fickian release mechanism that might be due to, no proper gelation occurred in these formulations at 35°C in STF.

Selection of Optimized Drug Formulations: The main prerequisites for the ocular in-situ gelling system are gelation temperature and rheological property. In addition to this, we also focused the release pattern (sustainability) of the formulations as the criteria of optimization process. Total two formulations namely, BG6, & BG10 were best outcome from GT study. Finally, the formulations BG6, & BG10 considered as optimized formulations and processed to further important in vitro, in vivo and stability studies for further confirmation of their suitability as ocular delivery system.

Osmolarity: All the optimized formulation were subjected to osmolarity study, using osmometer based on freezing point depression. All the five formulations were found to be isotonic with 0.9 % NaCl. The osmolarity was found to be 254±2.87 - 287±3.65 mMol.L-1 for all the optimized formulations.

Microbiological Study: As per USP guidelines, any new formulations containing antimicrobial agent, must be checked for their efficacy and potency in terms of their antimicrobial activity. This study was performed to assess the efficacy of optimized formulations and compared with conventional BM solution based on the formula of marketed eye drops. The result of the study against Staphylococcus aureus (MTCC-87) and Pseudomonas aeruginosa (MTCC-424) proved remarkable antimicrobial activity of all the prepared formulations.

Effect of Sterilization: Effect of sterilization was assessed on all four optimized formulations. It was found that no significant changes occurred in the viscosity of the formulations before and after sterilization by autoclave (Labtech). The result of the study was in line with the previous finding which stated that autoclaving could not significantly alter the rheological behaviours of the formulations containing poloxamer 407. In addition to this, autoclaving could not significantly affect the pH, and drug content of the formulations (table 10). This suggested that autoclaving sterilization might be suitable for sterilization purpose of the developed formulations, as it could not change the physicochemical properties of developed ocular formulations.

Table 10: Effect of sterilization on optimized ocular formulations