INTRODUCTION:

Drug delivery through ocular route in the form of eye drops is associated with plenty of issues like limited drug retention time, rapid drug removal due to washout effect resulting into poor bio-availability etc. In order to increase drug retention in eye, dosage forms like ointments or ocular inserts are used but these often face patient non-compliance due to blurred vision or difficulty in usage. To overcome these limitations, in-situ gelling systems has emerged. These formulations upon ocular instillation, show alteration in their physico-chemical properties such as cross-linking, viscosity which results in sol-gel transition. This phenomenon results in good ocular residence of drug along with improved bioavailability.¹

Because of its unique 'Sol to Gel' transition, the in-situ gelling technology aids in the continuous and regulated release of medication, as well as increased patient compliance and comfort.²

Glaucoma is the second most common cause of visual damage worldwide and is characterized by optic neuropathy and vision loss. It affects more than 70 million people worldwide^.³ Glaucoma is a chronic ophthalmic disorder caused by increased intraocular pressure (IOP) due to retention and backup of aqueous humor, which is excreted from a ciliary gland of the eye. The elevated IOP may lead to stringent complications such as optic neuropathies resulting in a decrease in neurons and axons of the retina. The eye consists of trabecular mesh-work spongy tissues which act as a drain for the eye. The problem arises when this trabecular mesh-work become clogged so prevents aqueous humor from leaving the eye. As a result, the accumulated aqueous humor inside the eye increases the IOP which affects the nerve cells of eye make the nerve cells more compressed and then finally destroy them. Nerve cells are responsible for the occurrence of visual mechanism as they deliver visual information to the brain, so the death of these cells results in loss of vision. Additionally, high levels of endothelin-1 (ET-1), a vasoactive peptide released by the vascular endothelial cells, have been reported to be a part of the glaucoma pathogenesis process. High levels of ET-1 may reduce the blood flow in the optic nerves by constricting the extra-ocular vessels. Also, it may increase the retinal venous pressure by constricting the retinal vein.⁴

Ocular drug delivery is very challenging and at the same time effective for the treatment of eye disorders. Many physiological barriers represent a problem for effective ocular drug delivery such as small volume of the conjunctival sac, rapid wash off outside the eye, nasolacrimal drainage and dilution by the tears. Based on these facts, topical ocular application of the drugs using conventional dosage forms such as solutions and suspensions shows low ocular bioavailability and very short duration of action. Furthermore, the cornea serves as a barrier to drug molecules entering the eye due to the tight epithelial junctions that reduce the Para cellular drug permeation mechanism.⁴ bypassing the complex defence mechanisms of the eye and thus achieving effective ocular drug delivery poses challenges for pharmaceutical technologists.⁵

The principles of the bioadhesion and stimuli sensitive drug delivery systems can be combined together. Bio adhesion can increase the residence time and the contact area of drug at the site of action. This will be advantageous over the conventionally used gels and semisolid preparations which are readily washed-off by the body fluids.⁶

In situ gel formation offers an attractive alternative to instillation of solutions because the in-situ gel increases pre-corneal drug residence time. The gelling process involves a phase transition in which the instilled solution forms a gel in the cul-de-sac of the eye as a result of response to some stimuli by the polymer. Therefore, these systems offer the dual advantage of an easy to administer liquid formulation along with the increased residence time of a gel. Parameters that can change and trigger this sol-gel phase transition include pH, temperature or ionic strength of the tear fluid. Among all in situ gel-forming systems, activation by change in ionic strength is most effective.⁷

Brimonidine Tartrate is a highly selective α₂-adrenoceptor agonist which reduces intra-ocular pressure (IOP) by reducing aqueous humour production and increasing aqueous humour outflow via the uveoscleral pathway.⁸ Chemically, brimonidine tartrate is 5-bromo-6-(2- imidazolidinylideneamino) quinoxaline L-tartrate (Fig. 1). It is a water soluble α₂ adrenergic agonist which works by reducing aqueous humor production, and by increasing aqueous humor outflow through the trabecular meshwork. Brimonidine binds extensively and reversibly to melanin in ocular tissues without any untoward effects.⁹ It is a promising drug for the treatment of open-angle glaucoma and high intraocular pressure (IOP). Studies using fluorophotometry in animals and humans showed that it has a dual mechanism of IOP-lowering. It can not only reduce the production of aqueous humor, but also increase the scleral-pigmentary outflow of aqueous humor. At the same time, it has an optic neuroprotective effect. Compared to thermal and pH-sensitive in-situ gel delivery systems, ion-sensitive in situ gels have the advantages of low polymer concentration, suitable pH and no eye irritation.⁹

Figure 1: Structure of Brimonidine Tartrate

Thus, objective of this study was to prepare and optimize ion responsive in situ gel for ophthalmic delivery, which is used for the treatment of glaucoma of the eye, to reduce raised intraocular pressure, to get better patient compliance by increasing residence time and bioavailability and to investigate BRT release in vitro and in vivo¹⁰

MATERIALS AND METHODS:

Brimonidine Tartrate reference material was received as gift sample from Micro Labs, Bangalore. Sodium alginate, Carbopol 971P and HPMC K4M were procured from Noveon Ltd, Mumbai. All other chemicals and solvents used were of highest analytical quality.

The Newzeland White Rabbits weighing 2±0.5Kg, free of any signs of ocular inflammation or gross abnormalities were procured from Animal house, Shardachandra Pawar college of Pharmacy, Otur, Pune. (CPSCEA No.1197/PO/Re/S/08/CCSEA)

METHODS:

Compatibility Studies Fourier Transform Infrared (FTIR):

The FTIR spectra of pure drug Brimonidine tartrate and physical mixture of Brimonidine tartrate, Sodium alginate, Carbopol 971P and HPMC K4M were recorded using an FTIR Spectrophotometer (Agilent). FTIR analysis was carried out to assess the physicochemical interaction of drug with excipients.

Differential Scanning Colorimetry (DSC):

Thermal DSC analyzer was used to obtain DSC thermograms of pure drug Brimonidine tartrate and Physical mixture of Brimonidine tartrate, Carbopol 971P, HPMC K4M, and Sodium alginate.¹¹

Preparation of in situ gel:¹²

To prepare Boric acid buffer, 8.61gm of Boric acid was dissolved in 500ml distilled water. Sodium alginate was overnight soaked in 50ml Boric acid buffer. HPMC K4M or Carbopol 971P were soaked overnight in 25ml Boric acid buffer. Brimonidine tartrate and Benzalkonium chloride were dissolved in 25ml of Boric acid buffer. Polymers sodium alginate-HPMC K4M and Sodium alginate-Carbopol 971P were mixed together to get clear solution. Drug solution was added to the polymeric mixture slowly with constant stirring and the formulations were equilibrated for 24hours. All the batches were filled in glass containers under aseptic conditions. (Table-1).

Evaluation of prepared in situ gel:

Visual appearance, homogeneity, and clarity:

The prepared in-situ gel formulations were evaluated visually. For the colour and homogeneity study, was performed with the help of a white and dark background.¹³

Determination of gelation time and gelling capacity:

A drop of the formulation was put into a watch glass holding 2ml of newly manufactured simulated tear fluid at a temperature of 37°C (680mg NaCl, 200mg NaHCO₃, 8mg sodium chloride, and volume made with water to 1000ml); the gelation time and gelling capacity of the prepared in-situ forming gel were determined. The time it took to produce the gel and the time it took to dissolve, was observed to estimate the gelling capacity.¹⁴

Drug Content:

0.5ml of the formulation was diluted to 50ml simulated tear fluid (STF). From 50ml, 1ml was pipetted out and diluted to 10ml with STF and absorption of this solution was measured at 254nm using UV spectrophotometer (UV-1900, Shimadzu) to calculate the percentage of drug content.¹⁵

Gel Strength Study:

Drug dissolution profile is affected by Gel strength of the formulation. A lab designed apparatus was used for the determination of gel strength. The apparatus consists of a syringe with sealed opening and the piston with arrangements to keep varying weights to apply pressure on the contents of the syringe from above. Pressure results in the penetration of piston into the contents of the syringe.¹⁶

6 ml of the gel was added to a beaker containing 25ml simulated tear fluid. Gelation was allowed to take place. After 15 minutes supernatant STF was decanted without disturbing the gel. After completely removing the supernatant STF, the formed gel was transferred to a syringe. 4gm constant weight was put on a plunger and it was allowed to penetrate the gel in syringe. The time required for the plunger to travel down the syringe was noted in seconds.¹⁷

Pourability Test:

Pourability of all the batches was checked by pouring the formulation from its container and pourability and flow ability was checked visually.¹⁷

Table 1: Formulation of in situ gelling ophthalmic solution

Batch	Sodium alginate (gm)	HPMC K4M (gm)	Carbopol 971P (gm)	Brimonidine Tartrate (gm)	Benzalkonium chloride (gm)	Buffer qs 100 ml
M1	1	0.25	--	0.2	0.05	100
M2	1	0.5	--	0.2	0.05	100
M3	1	0.75	--	0.2	0.05	100
M4	1.25	0.25	--	0.2	0.05	100
M5	1.25	0.5	--	0.2	0.05	100
M6	1.25	0.75	--	0.2	0.05	100
M7	1.5	0.25	--	0.2	0.05	100
M8	1.5	0.5	--	0.2	0.05	100
M9	1.5	0.75	--	0.2	0.05	100
F1	1	--	0.15	0.2	0.05	100
F2	1	--	0.30	0.2	0.05	100
F3	1	--	0.45	0.2	0.05	100
F4	1.25	--	0.15	0.2	0.05	100
F5	1.25	--	0.30	0.2	0.05	100
F6	1.25	--	0.45	0.2	0.05	100
F7	1.5	--	0.15	0.2	0.05	100
F8	1.5	--	0.30	0.2	0.05	100
F9	1.5	--	0.45	0.2	0.05	100

Measurement of the pH:

pH plays a very important role in ophthalmic formulations as the eye can tolerate formulations within a specific pH range (6.5–8.5)¹⁷. The pH for each of the formulations was measured using a calibrated pH meter, which was previously calibrated using standard buffers of pH 4 and pH 7 as per the established procedure. The readings were recorded three times for each of the formulation and the averages of the readings were considered.¹⁸

Viscosity Determination:

The viscosity values of prepared formulations were measured by using Brookfield viscometer at room temperature.¹⁹The viscosity of all the formulations either in solution or in gel made with simulated tear fluid, was determined with a Brookfield’s viscometer using a 50mL aliquot of the sample. Measurements were performed using suitable spindle number at 50rpm, and the temperature was maintained at 37°C. The viscosity was read directly from the viscometer display. All measurements were made in triplicate.²⁰

In-vitro Drug Release Study:²¹

An aliquot (1ml) of the formulation was transferred to the Franz diffusion cell donor chamber. The receptor chamber was filled with 18 ml Simulated Tear Fluid (STF) (pH 7.4) and constantly stirred. A cellophane membrane was pre-soaked in 0.1 N HCL overnight before the experiment and used to separate the donor and receptor chambers. The temperature was maintained at 37^◦C±0.5^◦C. At scheduled time intervals for up to 24 h, 5 ml samples were removed from the receptor solutions and replaced with an equivalent fresh volume of simulated tear fluid. The experiments were done in triplicate, and the drug concentrations were measured spectrophotometrically at λmax 254nm against a standard calibration curve.²¹

Mucoadhesion Study:

Mucoadhesion testing was done in order to determine mucoadhesion of various concentrations of polymers.²² Mucoadhesive potential of each formulation was determined by measuring a force required to detach the formulation from membrane. It was measured by modified balance. Goat intestinal mucosa was used for the study. Intestinal mucosa of Goat was obtained from slaughterhouse. Gel equivalent was placed on membrane surface. Empty beaker was attached to another side of the balance. Membrane surface with gel formulation and upper membrane surface were held in contact with each other for 2 min to ensure intimate contact. Water was added to the beaker until detachment takes place.²³

Ocular Irritation Studies (Hen’s egg test–chorioallantoic membrane HET‑CAM)²⁴

Ex-vivo ocular irritation and tolerance studies were performed using the HET-CAM; 8-day-old, incubated hen’s eggs were used for the same. Initially, one egg was cut open using a surgical sterile blade and checked for the development of CAM. The maturation of visible veins on the surface confirmed the development of CAM. Furthermore, the eggs were divided into 4 groups: positive control (10% w/v KOH; which is a known ocular irritant), negative control (0.9% saline; which does not cause irritation), Brimonidine tartrate in situ gel, and a placebo group. After removing the egg surface, 0.3ml of the test sample was applied to the CAM surface with the help of a micropipette ensuring that at least 50% of the CAM surface area was covered. Reactions on the CAM surface were observed for a period of 300 s. The time for the appearance of each of the endpoints (haemorrhage, vascular lysis, and coagulation) was monitored and recorded, in seconds. Based on the time required for the endpoints to develop, a potential irritancy (PI) score was calculated based on the following equation

PI = {[(301 − h) ∗ 5] 300} + {[(301 − v) ∗ 7] 300} + {[(301 − c) ∗ 9] 300}

Where, h appearance time in seconds of haemorrhage

v appearance time in seconds of vasoconstriction

c appearance time in seconds of coagulation.

In vivo Ocular Irritation Studies:

Ocular irritancy study was carried out using Modified Draize technique. The Newzeland White Rabbit were used for the study. The standard and test substance placed in the conjunctival sac of left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were gently held together for about one second in order to prevent loss of the material. The other eye, which remains untreated, serves as a control. Ocular response was examined and scored visually. Animals were observed for 0, 1, 24, 48, and 72 h post-dose.^25,26

Stability Study:

The optimized formulation was stored at room temperature for three months. After the first, second, and third months, the appearance, pH, gelling capacity, in vitro drug release and drug content of the formulations were evaluated. Optimized formulation M5 was subjected to stability study as per ICH guidelines. Parameters like visual appearance, gelling capacity, and in vitro drug release were evaluated. There is no much variation in visual appearance, gelling capacity, and in vitro drug release indicating the stability of formulation based on 3 months study.²⁷

RESULTS:

Compatibility Study:

From Fig 2, it was observed that there was no significant change in the peak values of drug when compared with the standard values. This indicated absence of any chemical reaction between drug and polymer.

DSC Study:

From figure 3, it was observed that DSC graphs are having sharp peaks that means drug Brimonidine tartrate is crystalline and pure in form as amorphous and impure drugs show broad peaks. Melting point of drug Brimonidine tartrate is seen at 217°C in individual drug graph and in drug and polymer mixture. The characteristic peak of drug Brimonidine tartrate is observed unaltered in its physical mixture with polymers. Above 300°C, some peaks are observed showing interaction but the interaction is above 300°C that is above the melting point of drug Brimonidine Tartrate hence there is no compatibility issue. From this study it can be concluded that the peak of drug remains unchanged in combination with the polymer indicating absence of any chemical interaction between drug and polymer.

Visual appearance, homogeneity, and clarity:

The appearance of the formulation was found to be clear and homogenous.

Pourability Test:

Pourability of all the batches was checked by pouring the formulation from its container and pourability and flow ability was checked visually. Batch M6, batch M8 batch M9, batch F8 and batch F9 showed poor pourability. The remaining batches were easily pourable.

Drug Content:

The percentage drug content in Brimonidine tartrate in situ gel ranged from 92.91 % to 98.86 %, as mentioned in Table 3, indicating uniform distribution of drug Brimonidine Tartrate in the prepared in situ gels.

Measurement of pH:

It is known that normal physiological pH of eye is 7.4. pH of all the formulations was within the acceptable range i.e., between 6.70-7.33 and hence would not cause any irritation upon administration which is desirable for the ophthalmic formulations.

Table 2: Evaluation parameters for in situ gel

Batch	Appearance	pH	Gelling Capacity	Viscosity (Cps)		Drug Content
Batch	Appearance	pH	Gelling Capacity	Before Gelation	After Gelation	Drug Content
M1	Clear	6.81	+	32.2	80.40	95.12 %
M2	Clear	6.92	+ +	35.6	90.92	94.38 %
M3	Clear	7.2	+ +	54.3	85.22	95.86 %
M4	Clear	7.12	+ +	59.8	102.13	94.38 %
M5	Clear	7.31	+ + +	71.3	115.33	97.80 %
M6	Clear	7.21	+ + +	74.8	120.18	92.91 %
M7	Clear	6.89	+ + +	92.1	118.92	95.12 %
M8	Clear	6.76	+ +	98.7	135.11	98.07 %
M9	Clear	6.65	+ + +	112.4	148.92	97.24 %
F1	Clear	6.26	+	15.5	69.21	91.32 %
F2	Clear	6.93	+ +	20.2	67.19	91.38 %
F3	Clear	6.87	+ + +	19.2	62.34	93.67 %
F4	Clear	7.02	+ +	21.6	92.23	95.83 %
F5	Clear	6.95	+ + +	36.6	115.21	98.86 %
F6	Clear	6.49	+ + +	54.8	119.62	97.12%
F7	Clear	6.38	+ + +	43.6	125.69	94.35%
F8	Clear	7.06	+ + +	59.5	152.39	91.37%
F9	Clear	7.11	+ + +	96.1	163.54	95.91%

Determination of gelation time and gelling capacity:

Immediate gelation of all the formulations was observed in simulated tear fluid. Batch M1 and F1 showed poor gelling. Batch M5, batch M6, batch M7, M9 were best in gelling ability.

Viscosity Determination:

An increase in viscosity was observed upon mixing the formulation with simulated tear fluid. Increased viscosity assures better retention at the site of application and enhanced bioavailability.

Gel strength Study:

Gel strength gives an indication about the tensile strength of the gelled mass. It demonstrates the ability of the gelled mass to withstand the peristaltic movement in in vivo. The batches M5, M6, M8, M9 and F9 exhibited satisfactory gel strength. Figure 4 gives the gel strength of all the formulations.²⁶

Figure 4: Gel Strength Study

Mucoadhesion study:

It was observed that polymeric combination of HPMC K4M and sodium alginate exhibit good mucoadhesion. All the batches M1 to M9 and F1 to F9 showed excellent mucoadhesion.

Figure 5: Mucoadhesion Study

In vitro Drug release study:

Figure 6 : In-vitro drug rease profile

All the batches showed extended drug release upto 6 hours which show a greater residence time of the formulation in ophthalmic cavity. Batch F1 and M5 were best in retarded drug release from the formulation. The dissolution profiles of the formulations are depicted in Fig. 6

Ocular Irritation Studies (hen’s egg test–chorioallantoic membrane HET‑CAM):

M5 formulation was applied on the choroiallantoic membrane on the 9th day. Effects are assessed in the near surroundings of the test item within 5 minutes. The time point is noted when one of the following effects occurs: hemorrhage, lysis, and coagulation. An irritation score (IS) is calculated, and the test item is classified with this score.

After the treatment, the main reaction was scored within 5 minutes of time (either hemorrhage or lysis, or coagulation) according to the following scheme:

0 = no reaction

1 = slight reaction

2 = moderate reaction

3 = severe reaction, and mean irritation score was determined.

Table 3: Irritation score, severity and classification of effect in the in vitro HET-CAM Assay

Compound	Irritation score (mean)	Irritation severity (mean)	Classification of the effect
0.9% NaCI Negative control	0.07	0	No reaction
0.1 N NaOH SDS Positive control	12.14	3	Severe reaction
Test (Formulation M6)	0.10	0	No reaction

Figure 7 HET-CAM assay

Viscosity Determination:

Viscosity of each batch was found to be increased after mixing with simulated tear fluid. Increase in viscosity indicates that the formulation in sol form is converted to gel form. Increase in viscosity enhances retention time of formulation in eyes this ultimately results in increased bioavailability. Table 3 shows viscosity values of each batch, before and after gelation.

Stability Studies:

Stability study was carried out for the duration of three months. After the first, second, and third months, the appearance, pH, gelling capacity and drug content of the optimized formulation was evaluated. The physical stability of in situ gel formulation was found to be maintained throughout the testing period, as only negligible changes were observed. There was no significant difference in the appearance as well as pH of the formulation. Also, the drug content of the formulation was found almost similar, which suggested the absence of any drug degradation at these conditions.

Figure 8 In vivo ocular irritancy test of in situ gel in rabbit

Table 4: Stability Study

Interval	pH	Gelling capacity	Drug content (%)	Viscosity (Cps)
After 1 month	7.35	+ + +	97.56	115.23
After 2 months	7.29	+ + +	97.23	120.12
After 3 months	7.41	+ + +	97.89	116.78

Ocular Irritation Studies:

The results obtained from study reveals that after ocular administration of the standard marketed formulation and test drug showed no Degree of Opacity, iris (Reaction to Light), and conjunctiva (Redness, Chemosis, Discharge) during the study as per the Draize scale for scoring ocular irritancy. As compared to the standard marketed formulation there is no any change was observed with the parameters like, redness, Chemosis, discharg.The second parameter IRIS is normal in both standard and test sample. In the third parameter in which degree of opacity of Cornea (most dense area used) no opacity was observed. All standard treatment and test treatment group of animals were observed upto 72Hr. As per the observation and images showed there is no any sign of irritation with the test formulation.

DISCUSSION:

For effective therapy for eye diseases, such as Glaucoma, it is important to prolong the pre-corneal contact time of ocular drugs. In situ gel systems have been developed well and showed beneficial effect over other traditional dosage forms. These profits contain sustained and prolonged release of the drug, biocompatibility, easy instillation, minimum chances of irritation, etc. Formulation M5 containing 1.25% Sodium alginate and 0.5% HPMC K4M exhibited good therapeutic effect.

CONCLUSION:

In this study, we formulated the ion activated in situ gels containing Brimonidine tartrate successfully. Formulation M5 was optimized batch as it exhibited good results for pH, Viscosity, drug content and in vitro drug release study and in vivo ocular irritancy study. The release study showed that the release of Brimonidine tartrate from in situ gels had good sustained release ability. No ocular damage or abnormal clinical signs to the cornea, iris, or conjunctivae were visible. Consistent with the in vitro studies, Brimonidine tartrate in situ gels were highly efficient in treating glaucoma and improving the ocular bioavailability. Ophthalmic in situ gels containing Brimonidine tartrate are safe and promising therapeutic alternatives to existing medications for Glaucoma.

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Received on 28.05.2024 Modified on 02.07.2024

Research J. Pharm. and Tech 2024; 17(10):4981-4990.

DOI: 10.52711/0974-360X.2024.00766


Figure 3a Brimonidine Tartrate	Figure 3b Sodium alginate

Figure 3c HPMC K4M	Figure 3d Brimonidine Tartrate + Sodium alginate + HPMC K4M

Figure 3e Carbopol971P	Figure 3f Brimonidine Tartrate + Sodium alginate + Carbopol 971P