Development of PH Triggered in-Situ Gel for Ophthalmic Delivery of Pilocarpine.

Ophthalmic delivery of drug has always considered among the foremost demanding aspects for researchers worldwide as the organization of eye bears some of the physiological barriers that have to be targeted sequentially to accomplish a good rate of absorption and bioavailability. [1] Eye is considered to be a fascinating organ due to its uniqueness. In the advancement of an ocular drug delivery system, parameters of absorption, distribution, metabolism and elimination (ADME) have to be given emphasis by a pharmaceutical scientist so that a better ocular system could be developed and less problems are encountered. [2, 3]

 

Ocular delivery of drug has number hurdles such as ocular barriers, drainage of the drug due to tear flow which ultimately leads to poor bioavailability of drug, thus reducing the desired therapeutic effect of the drug. [4, 5] But ocular delivery has one of the advantage that drug enters the systemic circulation by passing the first-pass metabolism. [6, 7] Ocular formulations such as Ointments, suspensions and aqueous gels have certain drawbacks such as poor patient compliance, blurred vision, cumbersome for self-administration, ocular drainage of drug, instability of the formulation, poor retention and bioavailability of drug in ocular cavity etc. [8, 9]

 

To overcome the problem of poor retention and bioavailability of drug in ocular cavity pH triggered in situ gel can be a one of the good alternative approach. [10] This in-situ gelling system consists of pH-sensitive polymers which are polyelectrolytes contain an acidic (carboxylic or sulfonic) or a basic group (ammonium salts) that either accept or release protons in response to alteration in pH in the surrounding environment. [11, 12] At lower pH (pH 4.4), the formulation exists as a regular solution, however, it undergoes gel formation at pH 7.4, that is the pH of tear fluid. [13] The most commonly used pH-responsive polymers in ophthalmic preparation are Polyacrylic acid (PAA, Carbopol 940), polycarbophil, and cellulose acetate phthalate (CAP). [14]

 

pH triggered in situ gel offers several advantages such as ease of administration, ease of manufacturing, patient compliance, good sol to gel transformation by the tears, prolong ocular retention time of drug, increase bioavailability, sustained release of drug due to the gel network formation etc. [15]

 

 

III. Formulation of in situ ocular gel using IHM:

In situ gel formulations containing different concentrations of Isabgol Husk Mucilage and Carbopol-940 in combination with HPMC-K4M were prepared by dispersion method. Briefly, about 20 mL distilled water was preheated to 70 °C to dissolve Benzalkonium chloride and then sodium chloride (NaCl), HPMC and Carbopol-940 were incorporated into the solution. The mixture was left at room temperature overnight to allow the polymer to hydrate. Pilocarpine was dissolved in 5 mL distilled water separately. It was added into above polymeric solution and stirred until a uniform solution was obtained. The final product was filled into sterile amber colour bottles and sterilized in autoclave at 121°C for 15 min. The prepared formulations were stored in refrigerator at 4 °C until further use. [20]

 

Table 1: Formula for in situ ocular gel using IHM

*All values in the table are in % W/V.

 

IV. Evaluation of in situ ocular gel containing IHM

A. Organoleptic examination: A general visualization of prepared formulations was done to check out the changes in color, odor as well as the appearance and clarity on the first third, seventh, fourteenth, twenty first and twenty eighth day of the preparation. [20]

 

B. In situ gelation: The determination of in vitro gelling capacity was done by visual method. Colored solutions were prepared by adding 1% amaranth dye solution in water and mixed with in situ ocular gel. The in vitro gelling capacities of prepared formulations were measured by placing 5 mL of the STF pH 7.4 in glass tube. To this 20 µl-50 µl of colored formulation solution was added with the help of pipette. As the solution comes in contact with STF fluid 7.4, it gets converted into stiff gel. The gelling capacity of solution was evaluated on the basis time required to form the gel. Colored dye was added in the formulation in order to visualize the appearance of in situ gel formation. The in vitro gelling capacity was graded in two categories on the basis of gelation time and time period for which the formed gel remains as such i.e, gel intact time. [21]

 

C. Rheological study: Viscosity of the formulation was checked by using Brookfield viscometer (Cole Parmer), before and after gelation using spindle number. L3. The angular velocity of the spindle was increased 10 to 100, and the viscosity of the formulation was measured before and after gelation. [21]

 

D. Determination of pH: The developed formulations were evaluated for pH by using digital pH meter. [22]

E. Drug content: The specific amount of gel was taken and diluted with STF pH 7.4. It was then stirred in order to dissolve the pilocarpine as well as formulation uniformly with the STF pH 7.4. The solution was further diluted with STF pH 7.4 in such a way that the resultant solution should have the concentration of pilocarpine 10 µg/mL. This solution was then filtered and filtrate was subjected to UV visible spectrophotometric analysis at 213.3 nm and finally the absorbance was measured. [22]

 

It is possible to assess drug content by using the formula:

 

F. In vitro drug release: In vitro release studies were carried out using franz diffusion cell. For this study the dialysis membrane was soaked overnight in STF fluid. The sample was applied on to the membrane and the membrane was placed in between donor and receptor compartment of the cell consisting of STF fluid pH 7.4. Samples were withdrawn at periodic intervals and replaced with fresh buffer to maintain sink condition. The drug release was analyzed using UV visible spectrophotometer at 213.3 nm using STF fluid as blank. [23, 24]

 

G. Ex vivo drug permeation studies: Ex-vivo trans corneal permeation study was carried out on freshly excised goat cornea. The fresh, whole eye balls of the goat were obtained from local slaughter shop and transported to the laboratory in cold condition at 4º C in normal saline. The cornea was then carefully excised along with 2- 4 mm of surrounding scleral tissue and was washed with normal saline until the washings were free from proteins. The excised cornea was fixed between the clamped donor and receptor compartments of glass modified franz diffusion cell in such a way that its epithelial surface faced the donor compartment. The corneal area available for diffusion was 0.50 cm2. The receptor compartment was filled with 20 ml of freshly prepared simulated tear fluid pH 7.4 and all air bubbles were expelled from the compartment. Aliquot 1 ml of the prepared in situ gel was placed on the cornea and the opening of the donor cell was sealed in a glass cover slip. The receptor fluid was kept at 37 ºC with constant stirring using a teflon coated magnetic stirrer beads. The permeation study was carried out and the samples were withdrawn from the receptor and analyzed for drug concentration by measuring absorbance at 213.3 nm in a UV-Visible spectrophotometer. [25, 26]

 

H. Test of sterility: The formulations were kept in Fluid Thioglycolate medium (20 ml) to check the growth of bacteria and in Soyabean Casein digest medium (20 ml) to detect the growth of any fungi in the preparation. The method of direct inoculation was incorporated and 2 ml of the optimized formulation was transferred in to the respective mediums with the help of a sterile pipette or a syringe. The incubation of the inoculated media was done for not less than fourteen days at a temperature of 30-35 ◦C in Fluid Thioglycolate medium and at a temperature between 20-25 ◦C in Soyabean Casein digest medium. At the end, a visual inspection was done to check the growth of any undesirable microbial growth. [27, 28]

 

I. Osmolality or Isotonicity studies: The isotonicity was determined by using Digital Osmometer. The value in mOsm/kg is determined by the osmometer's measurement of freezing point depression, which is then converted into the osmolality reading. To obtain a reading, a sample is loaded into the instrument, which then cools it to its freezing point, measures the temperature at which freezing occurs, and displays the corresponding osmolality. [29]

 

J. Stability study: To assess the stability of the formulation, accelerated stability study was performed. The different evaluation parameters such as the clarity, gel intact time, viscosity after gelation, pH and drug content were assessed at the initial time, after 3 months and after 6 months which can be referred to as the accelerated stability studies. For this study, the guidelines laid by the ICH were strictly adhered and the prepared optimized formulation was kept in a glass vial and the stability chamber was set at 40 ˚C ± 2 ˚C temperatures and 75 % ± 5 % relative humidity (RH) was also constantly maintained for the conduction of this study. [30]

I. Preformulation studies on pilocarpine

A. Melting point determination of pilocarpine: The melting point of Pilocarpine by using melting point apparatus was noted to be 204-205 °C.

 

B. Determination of maximum wavelength (λmax): λmax of PLC of concentration 20 µg/mL in simulated tear fluid (STF) pH 7.4 was noted to be 213.3 nm.

 

Figure 1: λmax of pilocarpine in STF pH 7.4

 

C. Standard calibration curve: The standard calibration curve of prepared solutions of known concentration of PLC is mentioned in figure 2.

 

Figure 2: Calibration curve of pilocarpine in STF pH 7.4

 

D. Compatibility between drug and excipients

a. IR (Infra-red) study: Various prominent peaks with their corresponding functional groups are given in table 2.

 

Table 2: Interpretation of pilocarpine by IR

 

Figure 3: IR spectra of PLC

 

Figure 4: IR spectra of IHM

 

Figure 5: IR spectra of physical mixture of PLC and IHM

 

By cross checking the prominent peaks of some characteristic functional groups in PCL, Isabgol  Husk Mucilage (IHM) and physical mixture of PLC and IHM, it can be revealed that there is no significant shifting of peaks observed. As a result, there appears to be no chemical interaction among the PLC and IHM.

 

b. DSC (Differential Scanning Calorimetry) study: DSC spectra of PLC, IHM and physical mixtures of PLC and IHM as shown in fig. 6, 7 and 8 respectively were recorded to see the thermal behavior of drug and also to check drug-excipients compatibility.

 

Figure 6: DSC spectrum of PLC

 

Figure 7: DSC spectrum of IHM

 

Figure 8: DSC spectrum of physical mixture of PLC and IHM

 

Table 3: Interpretation of Pilocarpine by DSC

 

Figure 6 illustrates the DSC thermogram of pilocarpine indicated a characteristic endothermic peak at 205.77 °C which suggests the melting point of pilocarpine. DSC thermogram of IHM does not show sharp endothermic peak as these are natural compounds. When DSC spectrum of PLC alone was compared with physical mixture of PLC and IHM, no shifting of the endothermic peak of drug was found. Thus it can be revealed that, there is no interaction among the PLC and IHM.

 

II. Formulation of in situ ocular gel using IHM: The in situ ocular gel was formulated using IHM powder in the batch of 30 mL. Here the ability of IHM to transform sol to gel by pH triggered approach was highlighted for the development of in situ ocular gel.

 

III. Evaluation of in situ ocular gel containing IHM

Organoleptic examination: After the general visualization of the all the prepared seven formulations, it was observed that all the prepared batches of in situ gel formulations were found to be clear, transparent and free from particulate matter. No changes in color, odor, appearance and clarity was noted on the third, seventh, fourteenth, twenty first and twenty eighth day of the preparation.

 

In situ gelation: The determination of in vitro gelling capacity was done by visual method. The gelling capacity of solution was evaluated on the basis time required to form the gel. Actually the in vitro gelling capacity was performed by two ways on the basis of gelation time and time period for which the formed gel remains as such. The results of the in situ gelation time and time of gel remain as such i.e, gel intact time are mentioned in table 4.

 

Table 4: Gelation behavior of in situ ocular gel of IHM powder

*n=3; values are expressed as mean ± SD

 

From table 4 it could be said that the in situ gelation time of all batches lies within 36 to 156 sec. An ideal time of in situ gelation for an ocular gel is up to 2 min. It is very acceptable range for the in situ ocular gel. Because the formulation should be transformed from sol to gel immediately when instilled in to ocular cavity in order to avoid the loss of formulation through ocular drainage.  On the other hand all batches showed the gel intact time in the range 3 to 8.5 h. Here formulations I1, I2 and I3 contain only IHM powder in the concentration 1, 3 and 5 %W/V respectively. In situ gel containing IHM showed the increase in in situ gelation time as well as gel intact time with increase in the concentration of IHM. On the other hand the formulations I4, I5 and I6 contain only Carbopol-940 in the concentration 0.3, 0.5 and 0.8 %W/V respectively. In situ gel containing Carbopol-940 also showed the increase in in situ gelation time as well as gel intact time with increase in the concentration of Carbopol-940. The last batch, I7 contains the combination of both IHM and Carbopol-940 in the concentration 3 and 0.5 %W/V respectively. Here batch I7 showed better results of the in situ gelation time as well as gel intact time i.e, 36 sec. and 8.5 h. This indicate that when IHM and Carbopol-940 are used in combination shows better results than the formulations containing IHM alone or Carbopol-940 alone.

 

Rheological study: In rheological study the viscosity of the formulation was measured before and after gelation. The results of the rheological study are mentioned in table 5.

 

Table 5: Rheological study of in situ ocular gel of IHM powder

*n=3; values are expressed as mean ± SD

Figure 9:  Comparison of viscosity before gelation of I1 to I7

 

Figure 10:  Comparison of viscosity after gelation of I1 to I7

 

An ideal viscosity of the in situ ocular gel before gelation and after gelation should be in the range 50 to 160 cps and 500 to 6000 cps respectively. From figure 9 and 10 it could be said that the viscosity of in situ ocular gel before as well as after the gelation increases for both IHM powder and Carbopol-940 as their corresponding concentration increases. But the better viscosity was obtained when both IHM and Carbopol-940 were used in the combination.

 

Determination of pH: pH values of the in situ ocular gel from batch I1 to I7 are mentioned in table 6. pH values of all the formulations were found in the range 5.7 to 6.5 which could be considered as ideal and non-irritating for the ocular cavity and stable under storage conditions.

 

Table 6: pH of in situ ocular gel of IHM powder

Batch	I1	I2	I3	I4	I5	I6	I7
pH	6±0.5	5.7±0.4	6.5±0.3	6.3±0.2	6.3±0.7	6.5±0.4	6.3±0.4

*n=3; values are expressed as mean ± SD

 

Drug content: Drug content values of all the batches of in situ ocular gel were found within acceptable range i.e, 96.25 to 103.45 %.

 

In vitro drug release: Drug release pattern of in situ ocular gel containing IHM is shown in table 7 and figure 11. In situ gel containing IHM such as I1, I2 and I3 showed the decrease in drug release with increase in the concentration of IHM. It showed the drug release up to 5 h. On the other hand the in situ gel containing Carbopol-940 also showed the decrease in drug release with increase in the concentration of Carbopol-940. It showed the drug release up to 7 h. The last batch, I7 contains the combination of both IHM and Carbopol-940 in the concentration 3 and 0.5 %W/V respectively. Here batch I7 showed better sustained release profile up to 9 h. This indicate that when IHM and Carbopol-940 are used in combination shows better sustained release profile than the formulations containing IHM alone or Carbopol-940 alone.

 

So it can be said that when in situ gel remain in the ocular cavity for prolong period of time i.e, 9 h, it will provide sustained release of pilocarpine for the same time period. So ultimately the in situ ocular gel would increase the bioavailability of pilocarpine in the ocular cavity.

 

Table 7: Drug release of in situ ocular gel of IHM powder

*n=3; values are expressed as mean ± SD

 

Figure 11: Drug release of in situ ocular gel of IHM powder

 

Ex vivo drug permeation studies: The prepared in situ gel formulations were subjected to ex vivo drug permeation studies to understand the diffusion pattern of the drug through the goat cornea. The rate of drug permeation of all the formulation was observed to be slow because the pilocarpine is highly hydrophilic in nature. On the other hand the corneal epithelium is highly lipophilic. Hence the corneal epithelium is expected to be the rate-limiting barrier for ocular absorption of pilocarpine. It results into a slower diffusion rate of drug through cornea. The results of the ex vivo drug permeation studies are mentioned in table 8 and figure 12.

 

Table 8: Ex vivo drug diffusion of in situ ocular gel of IHM powder

*n=3; values are expressed as mean ± SD

 

Figure 12: Ex vivo drug diffusion of in situ ocular gel of IHM powder

 

Test of sterility: After the incubation time period of 14 days at a temperature of 30-35 °C in Fluid Thioglycolate medium and 20-25 °C in Soyabean Casein digest medium, no turbidity or growth of microorganisms observed in any of the 7 in situ ocular gel formulations. So it could be said that the prepared in situ ocular gel passed the test of sterility.

 

Osmolality or Isotonicity studies: An ideal osmolality for ophthalmic formulations is generally considered to be isotonic with human tears, falling within the range of 290 to 310 mOsm/kg (or mOsm/L). If it is altered drastically it may cause cell swelling or cell shrinkage. Ophthalmic gels should be isotonic with the human tear to minimize irritation and promote a longer ocular residence time.

 

The results of the osmolarity are mentioned in table 9 which suggest that the osmolarity of all the formulation batches of in situ ocular gel are in the acceptable range.

 

Table 9: Osmolality of in situ ocular gel of IHM powder

*n=3; values are expressed as mean ± SD

 

Stability study: The results of the stability study of optimized formulation I7 are mentioned in table 10.

 

Table 10: Accelerated stability study

* n=3; values are expressed as mean ± SD

 

The accelerated stability study was carried out for the optimized in situ ocular gel formulation i.e, batch I7. The stability study results suggested that clarity, gel intact time, viscosity after gelation, pH and drug content are within acceptable limits. No considerable changes in the results observed after 3rd and 6th month of stability study. Thus, the formulation I7 can be said to be stable.

On the basis of result and discussion in this research, formulation I7 of in situ ocular gel containing IHM powder and Carbopol-940 at 3 % and 0.5 % concentration respectively showed pH triggered in situ gelation behavior i.e, in situ gelation time 40 sec. and gel intact time 8.5 h. On the other hand, in situ gel showed expected sustained release profile for 9 h with the use of HPMC-K4M. The results suggested that as the concentration of IHM powder increases the gel intact time as well as viscosity after gelation increases. The novel polymers IHM has very less sustained release effect. The sustained release effect of in situ ocular gel is mainly attributed to HPMC-K4M a release retardant polymer used in the formulation. The research says that IHM could be used as effective pH triggered in situ gelling novel natural polymer with good gelation behavior and viscosity after gelation to prepare in situ ocular gel of pilocarpine. An accelerated stability studies showed that, no considerable changes in the results observed after 3rd and 6th month of stability study. Thus, the formulation I7 can be said to be stable.

 

ACKNOWLEDGEMENT:

The authors gratefully acknowledge Madhyanchal Professional University, Bhopal, Madhya Pradesh (India) for providing the essential facilities which were required for the successful completion of this research project.

1.                Brambilla E, Locarno S, Gallo S, Orsini F, Pini C, Farronato M, et al. Poloxamer-Based Hydrogel as Drug Delivery System: How Polymeric Excipients Influence the Chemical-Physical Properties. Polymers (Basel). 2022 Sep 1;14(17).

2.                Dhyani A, Kumar G. Ocular Delivery of Atenolol Loaded Microsponge in-situ Gel: Development, Characterization and in-vitro Evaluation. Indian Journal of Pharmaceutical Education and Research. 2022 Jan 1;56(1):S75–80.

3.                Szalai B, Jójárt-Laczkovich O, Kovács A, Berkó S, Balogh GT, Katona G, et al. Design and Optimization of In Situ Gelling Mucoadhesive Eye Drops Containing Dexamethasone. Gels. 2022 Sep 1;8(9).

4.                Jacob S, Nair AB, Shah J. Emerging role of nanosuspensions in drug delivery systems. Vol. 24, Biomaterials Research. BioMed Central Ltd.; 2020.

5.                Destruel PL, Zeng N, Brignole-Baudouin F, Douat S, Seguin J, Olivier E, et al. In situ gelling ophthalmic drug delivery system for the optimization of diagnostic and preoperative mydriasis: In vitro drug release, cytotoxicity and mydriasis pharmacodynamics. Pharmaceutics. 2020 Apr 1;12(4).

6.                Huang H, Qi X, Chen Y, Wu Z. Thermo-sensitive hydrogels for delivering biotherapeutic molecules: A review. Vol. 27, Saudi Pharmaceutical Journal. Elsevier B.V.; 2019. p. 990–9.

7.                Sasaki H, Yamamura K, Nishida K, Nakamurat J, Ichikawa M. Delivery of Drugs to the Eye by Topical Application. 1996.

8.                Gaudana R, Ananthula HK, Parenky A, Mitra AK. Ocular drug delivery. Vol. 12, The AAPS journal. 2010. p. 348–60.

9.                Weinreb RN, Aung T, Medeiros FA. The pathophysiology and treatment of glaucoma: A review.Vol. 311, JAMA. American Medical Association; 2014. p. 1901–11.

10.             Brambilla E, Locarno S, Gallo S, Orsini F, Pini C, Farronato M, et al. Poloxamer-Based Hydrogel as Drug Delivery System: How Polymeric Excipients Influence the Chemical-Physical Properties. Polymers (Basel). 2022 Sep 1;14(17).

11.             Macovei L, Gheorghe A, Schmitzer S, Burcea M, Morosanu M. Ocular drug delivery systems: A review. Vol. 69, Farmacia. Romanian Society for Pharmaceutical Sciences; 2021. p. 1018–31.

12.             Hegde RR, Verma A, Ghosh A. Microemulsion: New Insights into the Ocular Drug Delivery. ISRN Pharm. 2013 Jun 27;2013:1–11.

13.             Janagam DR, Wu L, Lowe TL. Nanoparticles for drug delivery to the anterior segment of the eye. Vol. 122, Advanced Drug Delivery Reviews. Elsevier B.V.; 2017. p. 31–64.

14.             Subrizi A, del Amo EM, Korzhikov-Vlakh V, Tennikova T, Ruponen M, Urtti A. Design principles of ocular drug delivery systems: importance of drug payload, release rate, and material properties. Vol. 24, Drug Discovery Today. Elsevier Ltd; 2019. p. 1446–57.

15.             Hoare TR, Kohane DS. Hydrogels in drug delivery: Progress and challenges. Vol. 49, Polymer. Elsevier BV; 2008. p. 1993–2007.

16.             Alton M.E. Eds. Pharmaceutics: The Science of Dosage Form Design, Churchill Livingstone: Edinburgh, 2005:398-416.

17.             Ahmed NR. Indirect spectrophotometric determination of captopril in pharmaceutical tablets and spiked environmental samples. Iraqi National Journal of Chemistry. 2013;49:1-11.

18.             Naskar S, Sharma S, Kuotsu K. A smart gelatin nanoparticle for delivery of metoprolol succinate: A strategy for enhancing the therapeutic efficacy by improving bioavailability. J Drug Deliv Sci Technol. 2019;53:1-7.

19.             Pawar S.S., Malpure P.S., et al. Formulation and evaluation of sustained release matrix tablets of Captopril. Journal of drug delivery & therapeutics. 2019;9(4-A):260-268.

20.             Acharya A, Goudanavar P, Chitti R, Dinnimath BM. Preparation of Gellan Gum and Chitosan based In-Situ Gel of Timolol Maleate for Ophthalmic Drug Delivery and Evaluation of Physicochemical Properties and Drug Release Profile. Acta Scientific Pharmaceutical Sciences. 2019; Vol. 3. 1-10.

21.             Jethava J, Jethava G. Design, formulation, and evaluation of novel sustain release bioadhesive in-situ gelling ocular inserts of ketorolac tromethamine. Int J Pharm Investig. 2014;4(4):226.

22.             Askarkar S. S., Gupta K. R. Design and evaluation of ophthalmic delivery of bepotastine besilate from eye drop. Pharm Methods. 2016;7:104‑15.

23.             Patil J., Rajput R., Nemade R., et al. Preparation and characterization of artemether loaded solid lipid nanoparticles: a 32 factorial design approach. Mater Technol. 2020;35:719-726.

24.             Shirsath N. R., Goswami A. K. Vildagliptin loaded gellan gum mucoadhesive beads for sustained drug delivery: design, optimization and evaluation. Mater Technol. 2020:1-13.

25.             Parakh D. R., Patil M. P. Comparison of in vitro dissolution profiles of marketed Dicyclomine hydrochloride tablets. PHARMANEST An International Journal of Advances in Pharmaceutical Sciences. 2014;5(3):2109-2119.

26.             Yadav G., Bansal M., et al. Multilayer tablets and their drug release kinetic models for oral controlled drug delivery system. Middle-East J. Sci. Res. 2013;16(6):782-795.

27.             Kumar U, Islam S, et al. Assessment of once daily sustained release hydrophilic matrix tablet of carvedilol. Dhaka Univ. J. Pharm. Sci. 2017;16(1):43-53.

28.             Shirsand S. B., Sarasija S., Swamy P. V. Formulation design and optimization of fast dissolving clonazepam tablets. Indian J Pharm Sci. 2009;71:567-572.

29.             Gozcu S., Polat H. K., Gultekin Y., et al. Formulation of hesperidin-loaded in situ gel forocular drug delivery: a comprehensive study. J Sci Food Agric. 2024; 104: 5846–5859.

30.             Oliveira PR, Mendes C, et al. Formulation development and stability studies of Norfloxacin extended-release matrix tablets. BioMed Research International. 2013:1-9.