In-Vitro Study on Permeation of different Semi-solid dosage forms of Timolol Maleate using Franz cell

 

Tarang R Bhatt¹*, Dharmesh Golwala²

¹Research Scholar, Faculty of Pharmaceutical Science, C. U. Shah University,

Wadhwan, Surendranagar, Gujarat, India.

²Shankersinh Vaghela Bapu, Institute of Pharmacy, Vasan, Gandhinagar, Gujarat, India.

 

ABSTRACT:

The aim of the present study was to formulate different semi-solids dosage form for Timolol Maleate (0.5%) in oleaginous ointments, hydrocarbon gel and hydrogels and to study in-vitro comparison of flux and permeability into cornea of these semi-solids dosage form in comparison to ophthalmic solution using Franz diffusion cell. Objective of the study compare rate and extent of steady state flux in between semi-solids and solution of timolol maleate and obtain permeation co-efficient of all formulations from steady state flux using Fick’s first law of diffusion. An modified Franz diffusion cell consisting of 20 ml glass receptor along with a glass donor was for used for study and analysis of permeation  was carried out using high performance liquid chromatography at time points 0,0.5,1,2,4,8,16 and 24 h. Results concluded that hydrogel formulation containing hydroxy propyl methyl cellulose as an gelling agent  was found to have better flux and permeability than the reference solution formulation, while other two formulation containing paraffin base had less flux and permeability than reference solution formulation.

 

 

 

INTRODUCTION:

Recently, ophthalmic drug delivery has achieved great standards in the modern pharmaceutical design and intensive research are being carried out for achieving better drug product effectiveness, reliability, and safety. Topical medication to eye continues to account for the largest share  of drug delivery systems for ophthalmological disorders¹. As up to 95 percent  of drug administered topical fails to permeate various pre-corneal and corneal barriers which leads to substantial loss of drug adding to unnecessary cost to patient^2,3. And continuous rise in annual cost of medication therapy in last decade for chronic ophthalmological disorder like glaucoma, dry eye, and age-Related macular degeneration (AMD) has raised serious concern to patients in India.

 

To overcome the challenge of rising cost of medication there is a pressing need for research into novel formulation which can provide both increased permeation through cornea barriers and decreased loss of product in pre-corneal barriers. Semisolids ophthalmological dosage form like Oleaginous ointments, hydrocarbon gel and hydrogels provide better precorneal retention of drug compared to other conventional dosage forms like solution, suspension and gel forming solutions⁴. However, there has been a lack of understanding of formulation design and processing, as well as in vitro, ex vivo and in vivo performance of ocular ointments, with only a few recent publications covering these aspects⁵.

 

Apart from being first line choice of drug for glaucoma, different studies around the globe has suggested beta blockers to be the most economic  drug therapy to patients^6–11. US-FDA considers timolol as their standard drug for glaucoma therapy, thus it is against timolol maleate that all the new medications must be compared prior to approval. It lowers intra-ocular pressure by decreasing aqueous humour formation. Average  reduction in of timolol is  range of  20-35 percent¹²¹³. It is very effective during waking hours and causes less reduction in intra-ocular pressure in night¹⁴. Trials demonstrated that it is more effective in lowering intra-ocular pressure, as compared to epinephrine and pilocarpine¹⁵. Formulations of timolol  and other anti-glaucoma drugs containing bio adhesive polymers  presented a significantly higher in vitro tolerance than the same compound in traditional formulations^16–23, but almost no research has been carried out to compare efficacy  of  timolol in semi-solid form containing bio adhesive polymers and  different paraffin  bases with respect to traditional formulations.

 

Although there are US-FDA guidance documents and USP monographs covering some aspects of semisolid formulations, there are no FDA guidance documents nor any USP monographs for dissolution study for ophthalmic ointments²⁴. For these tests to be valuable, the correlation between in-vitro and in-vivo data is imperative in order to drive out human and animal testing²⁵. Following the EU ban on animal testing on many categories of formulations  , the value of accurate in vitro data has increased²⁶.

 

MATERIAL AND METHODS:

Chemicals and reagents:

Timolol maleate, white soft paraffin (Savita Polymers Ltd), liquid paraffin (Savita Polymers Ltd), low density polyethylene polymer (Acron Finevest Pvt. Ltd.), hypromellose (The Dow Chemicals Company), benzalkonium chloride (Sigma Aldrich) were kindly supplied by Indiana Ophthalmics. Sodium chloride, potassium chloride, calcium chloride, magnesium chloride, citric acid, benzalkonium chloride and sodium citrate were procured of analytical grade (Merck Ltd). While dihydrogen orthophosphate anhydrous and sodium hydroxide pellets were procured of Laboratory grade (Loba Chemie Pvt. Ltd.). 0.45micron nylon and 0.45 micron cellulose acetate (Advance Microdevices Ltd.) were used as filtration membrane and diffusion cell membrane respectively.

 

Preparation of Timolol Maleate Ointment in Oleaginous base (TR001).

Stainless steel container with 199gms of white soft paraffin was heated up to 160ºC in an oven for two hours.  Thereafter it was allowed to cool to 40ºC at room temperature. As soon as the desired temperature was achieved 1gm timolol maleate was added and mixed vigorously using stirrer until uniform distribution of timolol maleate was achieved into white soft paraffin.

 

Preparation of Timolol Maleate Hydrocarbon gel (TR002):

Stainless steel container with 189grams of heavy liquid paraffin and 10gm of low density polyethylene polymer heated in oven at 150ºC for 120minutes. Preparation was than cooled upto 30ºC with occasional stirring using chiller to achieve thick hydrocarbon gel base. 1gm timolol maleate was added to hydrocarbon gel base with continuous stirring until uniform distribution of timolol maleate was achieved.

 

Preparation of Timolol Maleate Hydrogel (TR003):

Beaker containing 0.980grams of sodium chloride and 150ml of distilled water was heated on a water bath at 90 ºC for 5 minutes. Thereafter 4.0grams Hypromellose powder was slowly dispersed in the pre-heated solution using stirrer. Resultant solution was autoclaved at 121ºC for 30 minutes and cooled at room temperature to form hydrogel. 1.0gram of timolol maleate, 0.150gm of potassium chloride, 0.0960gm of calcium chloride, 0.060gm of magnesium chloride were dissolved in hydrogel  and distilled water was added to make up weight of 200gm. Preparation was then set aside for 12 h until a transparent gel of was formed.

 

Preparation of Timolol Maleate Ophthalmic Solution as reference formulation(TR004):

1.0 gram of timolol maleate and 1.8gram of sodium chloride were solubilised in 100mL of distilled water in a 200ml volumetric flask.  There after 0.1gm of benzalkonium chloride, 0.8 gm of sodium citrate, 0.6gm of citric acid, 0.1gm of edetate disodium and 0.2gm of sodium hydroxide pellets were added one by one into the flask until they dissolved completely and 200ml volume was made up distilled water.

 

Preparation of phosphate buffer (For Franz cell receptor compartment):

11.1g of Sodium dihydrogen orthophosphate anhydrous was dissolved using 900mL of distilled water in 1000ml volumetric flask. pH of solution was adjusted to 7.4  using sodium hydroxide. Solution was then made up to volume of 1000ml using distilled water. Buffer was then filtered using 0.45 micron nlyon filter and degassed for 30 mins before use. 

 

Preparation of Franz diffusion cell:

A modified Franz diffusion cell consisting of 20ml glass receptor along with a glass donor cell, which was connected to outlet assembly was used to study permeability of different timolol maleate formulation. The donor compartment represented the conjunctival sac whereas the receiver compartments represented the anterior segment of the eye.  An 0.45 microns cellulose  acetate membrane used as a mimic of cornea ²⁷. Membrane was cut into appropriate size to fit appropriately in between donor and receptor compartments. Membrane was allowed to be pre-soaked in receptor media for 30 minutes prior to use. Receptor compartment was filled with phosphate buffer solution up to mark on side. A side arm of receptor compartment was allowed for sampling of the receptor cell and the receptor chamber. A Teflon coated magnetic bead was placed at the bottom of the receptor compartment  to ensure homogeneity of the receptor solution. The entire cell was clamped over a magnetic stirrer. All the cells were equilibrated for a minimum of 15 minutes before sample introduction.

 

Parameters of Franz diffusion cell:

All the four formulations where subjected with exactly same experimental parameter throughout the experiment. 2.5mg timolol maleate equivalent samples of all the formulations were placed onto donor compartment. Temperature was maintained at 37ºC throughout the tenure of experiment. Stirring speed of magnetic bar placed at bottom of receptor compartment was kept constant at 100 rotation per minute throughout the experiment²⁸. 0.5ml of sample was withdrawn at eight time points of 0, 0.5, 1, 2, 4, 8, 16, and 24 hours. Immediately after withdrawal of sample at each time point, the receptor compartment was replenished with 0.5ml of PBS of 37ºC.

 

Chromatographic analysis:

In order to obtain results of samples and standards a High-Performance liquid chromatography (HPLC) analysis were conducted at Sci prec Lab (Surendranagar, India) using a Shimadzu HPLC 2030 PLUS system comprising of LC 2030 plus UV-vis detector, series 2030 plus quaternary pump and series 2030 plus autosampler, Shimadzu C18 column, 150 mm × 4.6 mm, 5μm (part number: 227-30017-07) and lab solution software. Analysis of timolol maleate was achieved with a run time of 12 min using the method adapted from (USP-41). The mobile phase consisted of a mixture of methanol and phosphate buffer (35:65) under isocratic conditions, a flow rate was used at 1.2ml/min at 40ºC and detected with a UV detector (295nm). The retention time of timolol maleate was about 4.6min.

 

Standard preparation:

25mg of Timolol Maleate was accurately weighed and transferred to a 200mL volumetric flask which was then diluted to volume to 200ml with mobile phase. Solution was degassed for 30min and filtered using 0.45micron Nylon filter. 0.5ml of filtered solution was transferred into amber coloured HPLC vial with pre-punctured septa, of which 10µL was subjected to HPLC analysis.

 

Sample preparation:

0.5ml of sample drawn from diffusion cell was filtered using 0.45micron Nylon filter was placed directly into amber coloured HPLC vial with pre-punctured septa, of which 10µL was subjected to HPLC analysis.

 

Table 1 Equation used in experiment

 

Table 2: Cumulative drug release in µg and Percentage of TR001

 

Table 3: Average Cumulative Drug Release in Percentage and µg – ALL

 

Table 4: Cumulative amount of drug permeated per CM² in TR001 and TR002

 

Table 5: Cumulative amount of drug permeated per CM² in TR003 and TR004

 

RESULTS:

Determination of standard area:

Average standard area was obtained to be 2650915 determined using equation 1. While Table 2 gives insight of calculation from area to its conversion of average cumulative drug release in µg and per and percentage of all 3 sets of TR001 formulation. Similar calculations were used to determine cumulative drug releases in µg and percentage of TR002, TR003 and TR004. And summarised cumulative results of all formulations are described in Table 3. Cumulative drug permeated per CM² obtained by applying equation 6 for formulations TR001 and TR002 were summarised in Table 4, while for formulations TR003 and TR004 were summarised in Table 5.

 

Based on results of cumulative amount of drug permeated per CM² Table 4 and Table 5 slope at steady state were determined to be 0.0005, 0.0007, 0.0016 and 0.0012 for formulations TR001, TR002, TR003 and TR004 respectively. And by applying equation 7 to steady state slopes of each formulation permeation co-efficient was determined to be 0.0002, 0.00028, 0.00064 and 0.00048 for formulations TR001, TR002, TR003 and TR004 respectively.

DISCUSSION:

In vitro permeation testing with synthetic or non-viable membrane is mainly diffusion based and relies on random walk – so called Brownian motion²⁹. Diffusion of a substance is dependent on temperature, higher temperature increases the internal energy of the system, yielding more effective Brownian motion. An concentration gradient between two systems yields a driving force towards equilibrium, which increases diffusion rates²⁹. Fick's first law can be used to describe diffusion in the Franz cells. The more conventional form of Fick's law is the following³⁰:

 

𝐽𝑠𝑠 = 𝐾𝑝 ∙ 𝐶0                                 

 

Jss corresponds to the steady state,  Kp is the permeability constant for a given solute in a given vehicle and C0 is the concentration of the solute in the donor compartment³⁰.

 

The present research work was planned to provide the data about the selection of suitable vehicle for semi-solid dosage forms for timolol maleate to achieve better flux and permeation through cornea in comparison to traditional solution formulation. Higher flux and increased permeability could be suggestive indication for lowering dose or frequency of administration to patient.

 

From results of cumulative amount of drugs released for all the formulation it was reported that highest cumulative release of timolol was reported in hydrogel formulation while least cumulative release was reported in oleaginous base. Though at sampling interval of time 0 hour no formulation showed release at all the other time point the release of timolol was reported to be in increasing and linear manner. Although comparative release of timolol in oleaginous base was reported to be less in comparison to that of hydrocarbon gel base, its initial flux was higher during first 30 mins compared to later formulation. And overall cumulative release percentage of timolol formulation in hydrocarbon gel and solution were reported to be very similar to reference solution, while release percentage of hydrogel and oleaginous base were significantly different to reference solution. Timolol maleate in hydrogel was released almost twice compared to that of in hydrocarbon gel and reference solution formulation.

 

Considering Timolol in oleaginous base provides less but constant release, while timolol in hydrogel provides quick and prominent initially release. Release patterns for timolol in hydrocarbon gel and timolol in solution formulation was found to be very similar. All the formulation of timolol reached its steady state release after 8 hours. And permeation coefficient of timolol maleate in hydrogel was found to be about 33 percent higher than the in-reference solution formulation, but permeation co-efficient of reference solution formulation was found to be almost 2.5 times higher than both the ointment-based formulations indicating that permeation of timolol malate into eye can be increased in presence of hydro propyl methyl cellulose, and decreased in presence of white soft paraffin and liquid paraffin.

 

CONCLUSION:

Study for development of semi-solid formulations of Timolol Maleate for ophthalmic use was conducted in which 3 formulations were developed with the aim of enhancing the current available formulation of ophthalmic solution by prolonged duration and improved bioavailability for glaucoma treatment. In the in-vitro study, the hydrogel formulation containing hydroxy propyl methyl cellulose as a gelling agent was found to have released the maximum amount of drug which was significantly better than the reference solution formulation. While formulations containing paraffin bases did not perform better then reference solution formulation in terms of the drug release and the permeability co-efficient of timolol maleate.

 

FUNDING:

No competing financial interests exist

 

CONFLICT OF INTERESTS:

The authors declare no conflict of interest.

 

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Received on 20.04.2021           Modified on 11.06.2021

Accepted on 08.07.2021         © RJPT All right reserved