Formulation and Characterization of Glucosamine Sulphate Loaded Carbopol Based Hydrogels for the Management of Osteoarthritis

Osteoarthritis is emerging as themost ordinary form of arthritis, affecting 2239% of the Indian population. A wide range of medications and therapies are available for the treatment of osteoarthritis. With a desire to develop a therapeutically effective dosage form, the present study was carried out to formulate glucosamine sulfate loaded carbopol based hydrogel. Hydrogels H1 to H6 were formulated without permeation enhancers while formulations H7 to H12 were developed with a different class of permeation enhancers such as PEG400, oleic acid, Tween 40, DMSO and PG. Based on viscosity, it was detected that formulation H4 containing polymer 1% was ideal for incorporating drug. Considering H4 as a placebo, H6 was used for further evaluation. Drug content was found to be 99.2±0.64, with in vitro drug release of 15±0.86, 22±1.59, 28±0.72, 35±0.68, 40±0.31, 47±0.83, 58±1.59, and 70±0.9% at a duration of 1, 2, 3, 4, 5, 6, 7, 8 hours respectively. Skin irritation tests carried out on Wistar rats revealed that skin was intact with no in lammation or erythema detected, compared to untreated site. By diffusion disc method, it was evident that the levels of microbial load were relatively low, and no harmful microorganisms were identi ied. There were no signi icant changes in physicochemical properties on stability studies. Due to a simple method of preparation and effective drug delivery, glucosamine sulfate loaded hydrogels could be contemplated as a prominent formulation in the bene iciary treatment of osteoarthritis.


INTRODUCTION
Osteoarthritis, the most common form of arthritis, is a degenerative joint disease characterized by cellular stress and deterioration of the extracellular matrix. These, in turn, activate in lammatory pathways. The anatomic modi ications include degradation of cartilage and bone remodelling accompanied by in lammation of joints and formation of osteophytes. And thus, affect the normal routine activities. Osteoarthritis was believed to be caused only due to mechanical degradation of cartilage. But it was later discovered to be a complex condition affecting the joint. According to the Global Burden of Disease study conducted in 2010, osteoarthritis was the 11 th highest contributor to global disability (for Disease Control and Prevention, 2018;Sharma, 2016;Glyn-Jones et al., 2015). It is also the most cited reason for locomotor impairment in elders (Thamvasupong and Viravaidya-Pasuwat, 2016). Various strategies are being employed for the treatment of osteoarthritis, which includes lifestyle modi ication, surgical pro-cedures and drug treatment. It is usually treated with nonsteroidal anti-in lammatory drugs. Chondroitin and glucosamine are being widely studied as they exhibit anti-in lammatory and anticatabolic properties. They tend to provide symptomatic relief and delay anatomic degradation (Glyn-Jones et al., 2015). Glucosamine, an amino saccharide, decreases the production of prostaglandin E2 which is responsible for in lammatory response. Shreds of evidence have demonstrated that administration of glucosamine sulfate orally tends to reduce the symptoms of osteoarthritis (Reginster et al., 2012). Hydrogels are 3-D, cross-linked networks of watersoluble polymers that are highly preferred for topical delivery. These biocompatible formulations are hydrophilic, which holds potential in controlled drug release (Chellaswamy and Natrajan, 2020). Once the drug is incorporated into the hydrogel, it can be injected in a liquid state. On achieving the body temperature, it converts into a gel to release the drug content at the site of administration (Thamvasupong and Viravaidya-Pasuwat, 2016). Glucosamine sulphate being a potential candidate in the treatment of osteoarthritis, the present study was designed to formulate glucosamine sulphate into hydrogels, which are considered as promising drug carriers. The hydrogel was constructed using carbopol (Ultrez 20) due to the ability to increase elasticity and bio adhesion. The optimized formulation was further added with permeation enhancers, and the various evaluation results were compared.

Formulation Of Glucosamine Loaded Hydrogel
The gels (0.2, 0.5 and 1% w/w, respectively) were prepared by the following procedure (Škalko et al., 1998). Carbopol (Ultrez 20) is found to have outstanding dispersion ability and forms gels rapidly. Carbopol resin (w/v) was dispersed in distilled water. The dispersion was stirred using mechanical stirrer at 500 rpm until achieving uniform dispersion and then neutralized by using triethanolamine (drop-wise addition) to form gel consistency. Once the polymer concentration was optimized, drug glucosamine sulphate 1 % w/v was incorporated. Hydrogel formulation was developed with a different class of permeation enhancers such as PEG400, oleic acid, Tween 40, DMSO and PG. Benzalkonium chloride was used as a preservative, which is added to all the batches. The prepared hydrogel formulations were stored at room temperature for 24 hours to stabilize.

Physical appearance
Initial characterization involves analyzing physical appearance. The formulations were tested for their homogeneity by visual appearance after the hydrogels have been set in a suitable container. Also, a small quantity of each formulation was pressed between the thumb and the index inger, then the consistency of the hydrogel was noticed homogeneity (Vasudevan and Rajan, 2012).

pH analysis
The pH of hydrogel formulations was determined using pH meter. pH meter was calibrated before each use with standard buffer solutions. A quantity of 1 g of hydrogel was dissolved in 100 mL freshly prepared distilled water and stored for 2 hours. The electrode was inserted into the sample solution 10 min before recording the reading at 25 • C temperature. Each analysis was carried out in triplicate (Zakaria et al., 2016).

Viscosity
The viscosity of the hydrogel formulations was determined using Brook ield viscometer with spindle no. 7 at 100 rpm at the temperature of 25 • C (Monica and Gautami, 2014).

Spreadability
Spreadability (g.cm/sec) is expressed in terms of time consumed in seconds by two slides to slip off from the hydrogel placed between them, under a speci ic load. The standardized load tied on the upper plate was 20g, and the length of the glass slide was 7.5 cm. Spreadability was calculated by using the following formulaSpreadability = (W eight × Length) /T ime

Drug content analysis
A speci ic quantity of each drug-loaded hydrogel (Code: H6-H11) was weighed and transferred into a volumetric lask containing 100ml of methanol. The hydrogel was stirred for 1 hour on a magnetic stirrer, at 250 rpm, to get complete solubility of the drug. The solution was iltered to remove the undissolved particles and analyzed the drug content using UV spectroscopical method (Aly, 2012).

In vitro drug release analysis
A sample of 1g hydrogel was accurately weighed and placed on a semipermeable standard cellophane membrane (previously immersed in phosphate buffer, pH 7.4, for 24 hours). The loaded membrane was stretched over the lower open end of a glass tube of 3 cm diameter and sealed with a rubber band. The glass cylinder was then immersed in a 250 ml beaker containing 200 ml of the phosphate buffer solution (pH 7.4) in such a manner that the membrane was located just below the surface of the sink solution. The whole dialysis assembly was placed in a thermostatically controlled shaker water-bath adjusted at 37±1 • C with constant stirring at 50 rpm. At predetermined time intervals aliquots of 2ml, was collected and immediately replaced by an equal volume of the fresh phosphate buffer solution at the same temperature to keep the volume of the sink solution constantly during the experimentation. Samples were then assayed UV spectrophotometrically at 240 nm (Aly, 2012;Muthukumar et al., 2019).

Accelerated stability studies
Stability studies were carried out on optimized hydrogel (Code: H8) according to International Conference on Harmonization (ICH) guidelines. The hydrogel tightly packed in an aluminium tube was subjected to accelerated stability testing for three months duration as per ICH norms at a temperature (40 ± 2 • C) and relative humidity 75 ± 5%. Samples were taken at every one month for three months and investigated for the change in physical appearance, pH, viscosity and drug content.

Skin Irritation test
Wistar rats (200-250 g) of either sex were used for skin irritation test for hydrogel formulation. The animals were maintained on standard animal feed and had free access to water. The animals were kept under standard conditions, and hair was gen- tly removed at the back area (∼ 4cm 2 ). The hydrogel formulation was applied and observed for any sensitivity and reaction if any. It was graded as 0, 1, 2, 3 for no reaction, slight patchy erythema, slight but con luent or moderate but patchy erythema and severe erythema with or without oedema, respectively (C et al., 2020).

Microbial load test by disc diffusion method
The hydrogel formulation was dissolved in methanol and iltrated through 0.45 µm Millipore ilters. The microbial load was then carried out by disc diffusion method using 100 µL of suspension containing 110 CFU/mL of bacteria, 115 CFU/mL of yeast and 128 spores/mL of fungi spread on NA, SDA and PDA medium, respectively. The blank discs were impregnated with 20 µL of dissolved compounds and placed on the inoculated agar. Negative controls were prepared using the same volume of methanol employed to dissolve the synthetic compounds. O loxacin (5µg/disc) and nystatin (100 µl/ disc) were used as positive reference standards for bacteria and fungi, respectively, to determine the sensitivity of one strain/isolate in each microbial species tested. The inoculated plates were incubated at 36±1 • C for 24 h for bacterial strains and 48 h for yeast, at 27±1 • C for 72 h for fungi isolates. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms.

Formulation Of Glucosamine Loaded Hydrogel
Around 11 formulations were prepared and coded as H1 to H11. Formulations H1 to H6 were prepared using different concentrations of carbomer and without permeation enhancers. After determining the optimum concentration of carbomer, glucosamine sulphate was incorporated with the addition of permeation enhancers (Table 1).

Physical appearance
Each formulation is expected to have a uniform appearance and elegant. Formulations H1 to H6 were formulated without incorporation of the drug. On evaluation, they appeared clear and transparent. Formulations H7 to H11 were observed to be white and opaque (Figure 1). This was due to the presence of glucosamine. All the formulations exhibited good homogeneity without any clumps. This denotes complete dispersion of polymer and added drug.

pH analysis
As hydrogel is intended for topical application, pH plays a vital role. The pH of all formulations ranged within 5.1-6.1. It was found that the observed pH range is compatible with skin and will not produce any redness or irritation.

Viscosity
Viscosity re lects the consistency of the formulation. The viscosity of formulation depicts the penetration of the drug into skin and ease administrationconcentration and type of polymer in luence the viscosity of the product. Increase in viscosity was noticed with an increase in the concentration of polymer. When the polymer was used at 0.25% concentration, viscosity was found to be 3712 cps. On 0.5% and 0.75%, viscosity was 7204 cps and 9827cps respectively. And at 1 % and 1.25 %, it increased to 12529 cps and 13454 cps. It was detected that formulation H4 containing polymer 1% was ideal for incorporating drug. And formulation H5 seemed to be very thick, and it was not feasible in terms of administration.

Spreadability
Spreadability is considered as a critical parameter for topical formulations. It is predicted that the lesser the time consumed for separation of the two slides, the better its spreadability. H7 formulation showed higher spreadability with 35±1.14 g.cm/Sec (Table 2).   Based on the viscosity analysis, we considered a placebo formulation (H4) and hydrogel (H6) developed with the same variables and added glucosamine (1%w/v). Formulations H7-11 used different permeation enhancer with other variables constant. The drug content was in the limit. It ranged from 97 to 99% (Table 3). The formulations also exhibited content uniformity.

In vitro drug release
For the treatment of osteoarthritis, we aimed to develop glucosamine hydrogel with better pene-tration through various skin membranes and the simultaneously prolong therapeutic effectiveness. Since glucosamine sulphate is highly soluble in an aqueous medium, there was a necessity of addition to permeation enhancer. Various permeation enhancers like PEG 400, Oleic acid, Tween 40, DMSO and PG were chosen for the formulation of glucosamine hydrogel. The results compared with a reference coded as R. It was observed that 15-20% of drug released at a irst one-hour time. At 6 th hour, 50% drug was released. And at the end of    Figure 2). As a result, the performance of permeation enhancers was comparable. Formulation H8 containing oleic acid permeation enhancer was selected, as oleic acid is acknowledged for topical delivery and well tolerated by skin compared to DMSO and Tween 40. Therefore, H8 was considered for stability studies.

Skin irritation studies
Animals were treated with H6 formulation. The treated skin was intact; no in lammation and erythema compared to untreated site. There was no signi icant in lammation at the time of application, and after 4 hours (Figures 3, 4 and 5). All the animals were tolerated with applied hydrogel, and there were no signs of irritations/ redness noticed during the whole period of study.

The microbial load of the hydrogel by disc diffusion method
It was observed that levels of microbial load were relatively low, and no harmful microorganisms were identi ied. The total microbial count was found to lie within the speci ied limit (Figures 6, 7 and 8). Hence the formulation passed the microbial quality test.

Stability analysis
Stability analysis showed no signi icant changes in physicochemical properties of optimized formulation even after its exposure to accelerated conditions of temperature (40 • C and 75 ±5%RH).
The optimized formulation was found to be stable after subjecting to accelerated stability conditions (Table 5).

CONCLUSIONS
A versatile, biodegradable hydrogel has been successfully prepared using Carbomer Ultrez 20 loaded with Glucosamine sulphate for topical delivery for the treatment of osteoarthritis. Selection of Suitable polymers and their concentration is a prerequisite for formulating an effective transdermal drug delivery system. Various concentrations of polymer and different permeation enhancers have been tested successfully to optimize the formulation. The optimized glucosamine loaded hydrogels exhibited agreeable formulation characteristics such as physical appearance, pH, drug uniformity, rheology, microbial load, skin irritation, stability, and in vivo drug release. Further preclinical and clinical studies can be performed to support the use of this hydrogel for the patients suffering from osteoarthritis.