Tamarind seed polysaccharide (TSP) uses in ophthalmic drug delivery

author: matheuscastro123@hotmail.com Abstract At present, ophthalmic drug delivery remains a major challenge, given the eye's protective structure and susceptibility to irritation, resulting in poor patient adherence. In order to overcome these constraints, new formulations are continually being developed. The inclusion of Galactoxyloglucan (Tamarind seed polysaccharide (TSP) in such formulations, a natural substance extracted from the seeds of Tamarindus indica , has shown great potential due to its physicochemical properties, high biocompatibility and safety profile. Such properties, have led to its use in formulations for the treatment of dry eye disease, glaucoma, and bacterial keratitis, as well as in dilating eye drops used in eye examinations. In this article, we highlight the most recent TSP-based ophthalmologic formulations, which indicate that this polymer is a strong candidate to reduce adverse effects, improve patient tolerability and rufloxacin administration resulted in slightly higher intraocular drug levels than rufloxacin alone, as shown by the values observed in uninfected (4.62 g/ml; P 0.01), P. aeruginosa -infected (5.63 g/ml; P 0.01), and S. aureus-infected (6.91 g/ml; P 0.001) eyes. In the cornea, TSP-rufloxacin was slightly more effective than rufloxacin alone in regulating P. aeruginosa bacterial proliferation. With regard to S. aureus, TSP-rufloxacin administration resulted in a significant drop in corneal bacteria relative to both rufloxacin-treated and ofloxacin-treated eyes (P 0.05). A further study was conducted evaluating different formulations of rufloxacin (RUF) with TSP, namely two pH 7.2 suspensions of non-salified rufloxacin base, one of which was thickened with TSP; two pH 7.2 solutions of RUF obtained using hydroxypropyl- β cyclodextrin (CD), one with TSP; and two pH 5.0 solutions of rufloxacin hydrochloride (RUF-HCl), one containing TSP 33 . In this study, the suspension containing TSP showed twice the concentration in aqueous humor compared to S-RUF (187.88 vs 67.63 min µg/mL), given the viscosity potential of the polysaccharide. Additionally, pH levels were also shown to influence drug concentration, since the concentration was higher at pH 7.2 when compared to the same formulation at pH 5.0. Rufloxacin solubilization in HP- β -CD (pH 7.2) irrespective of whether or not it contained TSP, showed, poor performance in the final concentration. This could be explained by the fact that the drug interacted strongly with the cyclodextrins and that


INTRODUCTION
Tamarind (Tamarindus indica) is a medium-sized evergreen tree native to Africa with a history of use in traditional medicine across tropical Asia. It is cultivated as a commercial crop on a large scale throughout a number of different countries in tropical regions including India, Thailand, Brazil, Mexico and the United States ( Figure 1), with annual fruit production reaching up to 275 tonnes in sub-tropical countries alone 1 . According to research, each part of the tree contains different components that are used in a number of applications 2 including the food, clothing, and pharmaceutical industries, and also aid in environmental matters 3 . Pharmacological interest in tamarind seed and its components (TSP) first arose with the discovery of their use in traditional medicine for the treatment of diarrhea, dysentery and bleeding, and intensified following the identification of many active constituents 2,4 . TSP, also known as galactoxyloglucan, is composed of the monomers glucose, galactose and xylose in a 3:1:2 ratio, with a molecular weight of roughly 52 kDa ( Figure 2). According to literature, TSP is hydrophilic, stable over a wide pH range, has no ionic charge, and is neither toxic nor carcinogenic, making it an excellent candidate for use in pharmaceutical preparations 5 . In this article we will demonstrate TSP's potential for use in ophthalmic formulations, especially for those diseases/conditions in which TSP's properties prove especially useful such as: dry eye disease (DED), glaucoma, bacterial keratitis, in addition to improving the performance of mydriasis stimulants used in clinical examinations.

OPHTHALMIC POTENTIAL OF TSP
Several findings have verified TSP's beneficial properties. Historically, TSP has been used throughout Asia in healing skin wounds, and in intestinal and ocular drug delivery [6][7][8] . TSP has ideal properties for ocular administration, such as a tendency to swell, favouring ocular bioadhesion, non-Newtonian rheological behaviour and a ferning pattern similar to natural tear film 9 . Furthermore, TSP is a "mucin-like" compound, conferring properties similar to the MUC1 glycoprotein, which also provides mucoadhesive properties 10 . Studies evaluating TSP's interaction with hyaluronic acid, an excipient commonly used in ophthalmic formulations, have shown a positive synergistic relationship between the two 11,12 . A summary of the ophthalmic formulations to which this study refers is provided in Tables 1 and 2 below.

TSP in Formulations for Dry Eye Disease (DED)
As part of a study, an animal model for DED was developed and commercially available eye drops (Lacrisif ® , Hyalistil ® and Viscotears ® ) containing commonly used polysaccharides were compared to 1.0% TSP in relation to their power to reduce the signs and symptoms of DED 13 . The study found that Hyalistil ® and the eye drops developed with TSP produced type I fern structures, which are directly linked to the formulation's muco-mimetic properties. The capabilities of commercially available eye drops containing TSP (Xiloial ® and 0.5%TSP ® ) were measured in an in-vitro model for dry eye disease, significantly improving inflammatory indicators, increasing the lipid layer, and enhancing mucin production within 24 hours 14 .
A study was undertaken comparing the effectiveness of 0.5% and 1.0% TSP eye drops to a hyaluronate-based eye lubricant (Hyalistil ™ ) 15,16 . In this study, in which 30 patients were monitored for 90 days, improvements in tear breakup time (TBUT) were detected in comparison to the commercial formulation, as well as reduced clinical symptoms. In addition, 1.0% TSP achieved even better results when taking into consideration patientscore levels and the absence of any side effects, suggesting that the formulations of both concentrations of TSP are interesting candidates for further development. A further randomized clinical study involving 28 TFOS DEWS severity level 2 patients compared VISINE INTENSIV ® 1% (commercially available eye drops containing 1.0% TSP), and SYSTANE UD ® (composed of HPC-guar) 17 . The use of VISINE INSENTIV ® 1% resulted in substantial Ocular Surface Disease Index (OSDI) improvement (from 50.0 to 34.1). Schrimer's test results also improved in comparison to the baseline for both eye drops (15.3 SYSTANE UD -11.9 baseline; 15.1 VISINE -10.75 baseline).
In a comparison of Xiloial Monodose ® (0.2% TSP and 0.2% hyaluronic acid (Farmigea, Italy)) and Optive ® (Allergan, USA), comprising 49 patients with moderate DED, OSDI values demonstrated that the response to the former was twice that observed in the Optive ® group (-14,8 and -7,9 respectively) 18 . More recently, the synergistic relationship between hyaluronic acid and TSP was further evaluated in a study involving 25 patients. The study compared two formulations: one containing 0.4% uncross-linked hyaluronic acid and 0.2% TSP (Tear A), the other containing 15% cross-linked hyaluronic acid, crocin, and liposomes (Tear B) 19 . The study found that the results of all evaluated parameters (TBUT, OSDI) showed significant statistical improvement for both formulations (p <0.0001).
In a further study aimed at assessing discomfort related to disposable soft contact lenses, participants were submitted to treatment with Xiloial ® for 60 days 20 . Significant reduction in subjective symptoms was observed after one month of treatment, according to OSDI, with 80% of patients reaching normal scoring (8.5 ± 3 versus 20.2 ± 1.6). Schirmer's test results showed a significant increase at the endpoint evident after two months of treatment (14.6 ± 1.1 versus 12 ± 2.1). Overall, TSP use in formulations demonstrates several benefits for DED (Table 3).

TSP in Formulations for Glaucoma
TSP has been used in the development of new formulations containing timolol and pilocarpine, two drugs widely used to treat glaucoma. The first of these consisted of an experimental formulation containing 5 mg/ml timolol which was developed and tested on rabbits, comparing it to the commercially available formulations Droptimol ® and Timoptic ® (based on in-situ gelling system). The TSP-based formulation saw higher concentrations of timolol in tears, aqueous humour and cornea, alongside lower concentrations in plasma, indicating lower systemic absorption and greater local absorption. The formulation containing TSP reduced intraocular pressure to 3.1 mm Hg, lasting 19 hours and returning to basal values after 24 hours, suggesting a long-lasting hypotensive effect 21 . A novel ocular film timolol-based formulation containing TSP has also been subject to comparative testing against a regular commercial eye drop formulation (Glaucomol) 22 . According to Draize testing, this formulation was found to be non-irritant. In addition, the reduction in the animals' IOP was found to be rapid upon the eye drop formulation reaching maximum reduction (AUC 88 mmHg) four hours after instillation, whereas the hydrogel reduction peaked (AUC 148.425 mmHg) after six hours and was maintained for a total of 12 hours.
Pilocarpine was investigated through the development of an in-situ gelling system 23 . It released approximately 25% of the drug in the first hour and 80% during the next 12 hours, and was considered the most sustained release. In comparison to a standard eye drop solution, the longer drug activity and higher pharmacodynamic activity strongly suggest that pilocarpine bioavailability had improved.
Benzalkonium chloride (BAK) is an antibacterial substance widely used in eye dropformulations in varying concentrations. However, despite its usefulness as an excipient, it causes conjunctival and corneal damage, representing a problem for long-term therapies in patients with glaucoma associated with cellular damage and inflammation 24 . TSP has shown potential as an antioxidant in the food industry (decreasing visual changes in naturally sensitive products), though not as a preservative 25,26 . TSP's efficacy in reducing toxicity has also been subject to evaluation in a formulation containing 0.5% TSP (Oftagen ® ) as part of a prospective clinical study involving 20 patients 27 . Corneal epithelial defects assessed by fluorescein staining completely disappeared after three months. Improvements in the results of clinical parameters, such as TBUT and Schirmer's test, were also detected. In addition, a further study involving 42 patients from different ophthalmic centers evaluated the benefits of using 0.5% TSP-containing eye drops in patients undergoing treatment with BAKcontaining prostaglandins (PGAs) 28 . After three months of daily instillation, a decline in the incidence of conjunctival hyperaemia was observed, as well as a reduction in subjective symptoms such as discomfort, swelling, and itching resulting in ocular irritation. However, an increase in Schirmer's and TBUT test results was also observed.
Recently it has been reported that the use of nerve growth factor (BDNF) can assist in retinal recovery in an animal model for glaucoma 29 . Evaluations of how different excipients for ocular formulations increased BDNF bioavailability in animals with lightinduced retinal damage (LE) have also been conducted 30 . A pre-LE BDNF/TSP topical treatment maintained fERG responses while avoiding dramatic a and b-wave shifts. Compared to vehicle-treated retinas, LE eyes treated with a single drop of BDNF/TSP revealed a substantially higher number of rows of photoreceptor nuclei. Pharmacokinetic analysis of BDNF/TSP topical eye application revealed that, six hours after application, retinal BDNF levels were higher than in the control eye and remained elevated for 12 hours. Protection against damage induced by UV radiation in cornealderived cells (SIRC) was obtained when using a formulation containing TSP. This formulation led to a decrease in the number of reactive species, significantly reducing DNA damage to corneal cells 31 . In conclusion, TSP would appear to offer several benefits when used in glaucoma formulations (Table 3).

TSP in Formulations for Bacterial Keratitis
In order to investigate TSP's ability to increase the concentration of antibiotics in the cornea, formulations of rufloxacin either containing or not containing TSP were compared in an animal model for keratitis, in addition to a traditional formulation of oflocaxin 32 . TSP-rufloxacin administration resulted in slightly higher intraocular drug levels than rufloxacin alone, as shown by the values observed in uninfected (4.62 g/ml; P 0.01), P. aeruginosa-infected (5.63 g/ml; P 0.01), and S. aureus-infected (6.91 g/ml; P 0.001) eyes. In the cornea, TSPrufloxacin was slightly more effective than rufloxacin alone in regulating P. aeruginosa bacterial proliferation. With regard to S. aureus, TSP-rufloxacin administration resulted in a significant drop in corneal bacteria relative to both rufloxacin-treated and ofloxacin-treated eyes (P 0.05). A further study was conducted evaluating different formulations of rufloxacin (RUF) with TSP, namely two pH 7.2 suspensions of non-salified rufloxacin base, one of which was thickened with TSP; two pH 7.2 solutions of RUF obtained using hydroxypropyl-βcyclodextrin (CD), one with TSP; and two pH 5.0 solutions of rufloxacin hydrochloride (RUF-HCl), one containing TSP 33 . In this study, the suspension containing TSP showed twice the concentration in aqueous humor compared to S-RUF (187.88 vs 67.63 min µg/mL), given the viscosity potential of the polysaccharide. Additionally, pH levels were also shown to influence drug concentration, since the concentration was higher at pH 7.2 when compared to the same formulation at pH 5.0. Rufloxacin solubilization in HP-β-CD (pH 7.2) irrespective of whether or not it contained TSP, showed, poor performance in the final concentration. This could be explained by the fact that the drug interacted strongly with the cyclodextrins and that combination with the viscous agent proved a greater hinderance to drug delivery. Accordingly, the viscosity of the final formulation required further evaluation.
Recently, an emulgel formulation containing different concentrations of rice bran oil (RBO) and TSP has been developed for controlled release of ciprofloxacin 34 . To evaluate the formulation's effectiveness in inhibiting the growth of microorganisms, an ex-vivo corneal permeation test was performed in goats. This revealed that the drug's ability to penetrate tissue increased relative to RBO concentration. The formulation was tested against B. subtilis and E. coli, inhibiting microorganism development based on an increase in RBO concentration and the stabilization of TSP levels, indicating an increase in permeability and drug release into the medium.
An additional study using ciprofloxacin was conducted with the objective of developing a TSP (2%) based ocular film formulation 35 . When tested for the efficacy of drug release against S. aureus, the formulation succeeded in inhibiting microbiological growth throughout the test period (48 hours) with no signs of irritation found (Draize test).
Results suggest that TSP provides for a more effective antibacterial reaction, potentially raising the antibiotic's precorneal residence period and increasing drug concentration in the cornea (Table 3).

TSP in Formulations for Mydriasis
Tropicamide -TSP (0.1%) suspension was developed as a safe and effective alternative method of drug delivery. In comparison to the commercial formulation, the suspension scored much lower (between 0 and 1 point) in ex-vivo biocompatibility testing (HET-CAM) (between 3 and 4 points). In bioadhesion testing, the formulation adhered to 88% of the mucin 36 , indicating mucoadhesive characteristics. Mydriasis improvement was addressed with a TSP/poloxamer-based nanoaggregates formulation 37 . The tropicamide nanoaggregates permeated the cornea more intensely in an ex-vivo study (0.661%) than the commercial solution Tropicacyl (0.573%), explained by its capacity to stimulate organism endocytic system and the absence of preservative in the developed formulation. However, the premise of increasing corneal permeation in future in-vivo studies remains. In bioadhesion testing, the formulation succeeded in binding to 87.35% of the mucin, a finding that is closely associated with the formulation's TSP. In addition, there was no significant score in the HET-CAM test for the developed formulation and Tropicacyl ® , nor any relevant cytotoxic activity (Table 3).

CONCLUSION
After careful analysis of recent articles regarding ophthalmic formulations, TSP undoubtedly shows potential to increase drug corneal permeation by prolonging precorneal retention time in eye drop formulations, therefore safely increasing patient adherence by improving ocular comfort. Further research of TSP-based ophthalmic formulations should therefore be encouraged. This paper paves the way for new research involving this natural polymer.