INTRODUCTION:

All controlled release products share the common goal of improving drug therapy over that achieved with their non- controlled counter parts. The term ‘’sustained release’’ has been constantly used to describe a pharmaceutical dosage form formulated to retard the release of the therapeutic agents such that its appearance in the systemic circulation is delayed and or prolonged and its plasma profile is sustained in duration¹. The onset of pharmacological action is often delayed, and the duration of its therapeutic effect is sustained. Methotrexate is an antimetabolite which competitively inhibits dihydrofolate reductase (DHFRase)-blocking the conversion of dihydrofolic acid (DHFA) to tetrahydrofolic acid (THFA) which is an essential coenzyme required for one carbon transfer reactions in denovo purine synthesis and amino acid interconversions. Methotrexate has a cell cycle specific action-kills cells in S phase; primarily inhibits DNA Synthesis, but also affects RNA and protein synthesis. It is an anti-metabolite and antifolate drug used in treatment of neoplastic diseases (cancers), Rheumatoid arthritis, Psoriasis.

Development of sustained release oral dosage forms is beneficial for optimal therapy regarding efficacy, safety and patient compliance. In case of sustained release (SR) dosage forms the release of the active agent, although, is lower than in the conventional formulations^2,3, however, it is still substantially affected by the external environments into which it is going to be released. The advantages of sustained-release tablets or capsules are that they can often be taken less frequently than instant formulations of the same drug, and that they keep steadier levels of the drug in the bloodstream. Therefore, the need for the formulation and evaluation of sustained release dosage form is obvious and essential for optimal therapy.

MATERIALS AND METHODS:

Methotrexate was received as a gift sample from Mac Mohan Pharma Ltd, Hyderabad, Eudragit S 100 and Ethyl cellulose are purchased from Loba chemicals Pvt. Ltd. Dichloro methane was obtained from Nice chemicals Pvt. Ltd, Cochin.Tween80 was purchased from Merck Pvt. Ltd, Mumbai. Distilled water was obtained from Leo scientific Erode.

PREPARATION OF METHOTREXATE MICROCAPSULES:

The microcapsules were prepared by emulsion solvent evaporation technique. In first stage, solid dispersion of methotrexate with different polymer ratio of ethyl cellulose, eudragit S100 such as1:1:2(F1), 1:1:1(F2),1:0.5:1.5(F3) were prepared ,which is shown in the following table .All ingredients were accurately weighed and transferred to a china dish .Then added 14ml of dichloromethane to the china dish containing drug and different polymer ratio. The china dish was heated on water bath to evaporate the solvent. Now 1gm of ethyl cellulose was accurately weighed and dissolved in 25ml of acetone to form a homogenous polymer solution .To this solid dispersion containing methotrexate was poured the polymer solution and dispersed thoroughly. The resulting mixture was added to light liquid paraffin (100ml) containing 1gm tween 80 in a 250ml beaker. The system was stirred at 600rpm for 1hr to form fine droplets. The solvent gets evaporated at room temperature and small uniform microcapsules were formed. The microcapsules were separated from oil phase by filtration and washed with petroleum ether to remove the adhering liquid paraffin. The microcapsules were kept in a desiccator for 24 hrs. The ethyl cellulose was used as coating polymer and took 1g constant in each formulation.

Fig. 1: STANDARD GRAPH OF METHOTREXATE

TABLE-1 STANDARD GRAPH OF METHOTREXATE:

S. No	Concentration In(µgm/ml)	Absorbance Mean ± S.D*
1	3	0.1919 ± 0.0007
2	6	0.3332 ± 0.0014
3	9	0.5266 ± 0.0010
4	12	0.6868 ± 0.0018
5	15	0.8556 ± 0.0020

PHYSICOCHEMICAL CHARACTERIZATION OF THE MICROCAPSULES:

PERCENTAGE YIELD:

The dried microcapsules were weighed and percentage yield of the prepared microcapsules was calculated by using the following formula,

Percentage yield = {The weight of microcapsules / The weight of polymer +drug} ×100

DRUG CONTENT:

The various batches of the microcapsules were subjected for drug content analysis. Accurately weighed microcapsules were mechanically powdered. The powdered microcapsules were dissolved inadequate quantity of phosphate buffer PH 7.2then filter. The UV absorbance of the filtrate was measured using a UV spectrometer at 303nm.

DRUG LOADING AND ENCAPSULATION EFFICIENCY:

Drug loading and encapsulation efficiency was determined for all batches using the following formulas. Values are expressed as percentage.

Drug loading = {Weight of drug in microspheres / Microspheres sample weight}×100

Encapsulation efficiency = {Actual weight of drug in sample/ Theoretical weight of drug} ×100

PARTICLE SIZE ANALYSIS:

Size distribution plays a very important role in determining the release characteristics of the microsphere. Particle size distribution analysis was done by optical microscopy method, using calibrated eye piece micrometer, nearly 200 particles were measured and the results were determined.

IN VITRO DISSOLUTION STUDY:

The drug release studies of the microspheres were carried out for up to 24 hours using the dissolution test apparatus (paddle method). Five hundred milliliters of phosphate buffer P^H7.2 was taken as the dissolution medium. Microspheres containing 50mg –equivalent of methotrexate were accurately weighed and tied in the muslin cloth .The paddle were rotated at 50 rpm. The dissolution medium was thermally controlled at 37^οC. Samples of 1ml were withdrawn from the dissolution media at suitable time intervals. The same volume was replaced by an equal volume of fresh medium. The drawn 1ml samples are made up to 10ml and analyzed using a UV spectrophotometer at 303 nm.(Table-2)

Table-2: in vitro Cumulative % release Profile of Micro spheres Formulations of Methotrexate

TIME (Hrs)	FC1	FC2	FC3
1	36.8	40.4	42.5
2	39.0	42.1	43.6
3	40.1	44.3	48.2
4	40.6	46.6	48.8
5	42.3	48.3	49.4
6	45.6	52.2	50.5
7	46.7	53.9	54.5
8	48.3	56.7	58.0
9	51.0	58.4	59.7
10	52.7	58.9	60.9
12	58.2	61.2	63.2
14	67.5	73.0	75.2
16	73.6	75.2	78.1
18	76.3	78.6	82.1
20	80.2	83.1	87.9
22	86.8	94.3	92.5
24	89.3	96.5	94.5

DIFFERENTIAL SCANNING CALORIMETRY (DSC) ANALYSIS:

The DSC thermo grams of methotrexate, EC and physical mixture of methotrexate and EC microcapsules are shown in (Fig .3). In order to investigate the possible interactions between the drug and polymers used, differential scanning calorimetric studies were carried out¹⁴. DSC thermogram of the formulation was compared with the DSC thermogram of the pure drug. The DSC thermogram obtained are reported in Figure-3. The pure methotrexate displayed a sharp endothermic peak at 182ºC corresponding to the melting point of the drug and a similar peak was also observed in the formulation.

Fig. 2: IN VITRO DISSOLUTION STUDIES OF METHOTREXATE MICROCAPSULES

From the DSC thermogram it was observed that the decomposition temperature of the pure drug and the formulation remained the same. Hence it can be concluded that there was no significant interaction between drug and the polymers used.

Fig. 3: DSC Thermogram of Methotrexate

SHAPE AND SIZE OF MICROCAPSULES:

The mean particle size of the obtained microcapsules containing methotrexate was determined by the optical microscopy under 45X magnification. The arithmetic mean size of microcapsules of formulation F1, F2 and F3 which shown in table and graph. The particle size range increased as the combination ratio of ethyl cellulose was increased particle size range decreases, either ratio was increased.

DIGITAL PHOTOGRAPHS:

The shape and surface morphology of methotrexate microcapsules were observed by digital photography (Fig.4) shows in plates 1 and 2. Plate 1 shows normal size of microcapsules while plate 2 shows zoomed size methotrexate microcapsules. The obtained microcapsules are round to oval in shape. The microcapsules are pale yellow in color.

1. Methotrexate microspheres

2. Zoomed View of Methotrexate Microspheres

Fig. 4: 1 SHOWS NORMAL SIZE OF MICROCAPSULES WHILE PLATE 2 SHOWS ZOOMED SIZE METHOTREXATE MICROCAPSULES

MICROMERITICS PROPERTIES¹⁰:

The micromeritics properties such as angle of repose, bulk density, tapped density and hausner’s ratio were studied (Table-3). The bulk density of microcapsules were increased i.e. F1>F2>F3. The obtained value of angle of repose ranges from 18.50 to 23.14 which indicates that all formulations are free flowing and order of angle of repose was followed F1>F2>F3.

Table-3: Micromeritic Properties of Methotrexate Microcapsules

PHYSICAL PARAMETERS	F1	F2	F3
Angle of Repose	27⁰.45’	28⁰.50’	24⁰.34’
Bulk density In g/ml	0.306	0.318	0.314
Tapped density ing/ml	0.333	0.352	0.357
Hausner’s ratio	1.08	1.104	1.135
Flow	Excellent	Excellent	Good

Results and Discussion:

The sustained release microcapsules of methotrexate were prepared by using natural polymer (ethyl cellulose) and evaluated with an aim to prevent adverse effects and increase bioavailability. It also leads to reduction in frequency of dosing which in turn improve patients’ compliance and reduce fluctuations in drug levels.

The prepared methotrexate microcapsules were subjected to various evaluation parameters such as DSC analysis, size determination by sieve and microscopic method, shape and surface determination by digital photography, micromeritics properties like bulk density, porosity, and flow property by angle of repose drug content determination and in-vitro dissolution studies.

In present work an attempt has been made to formulate sustained release micro capsule of methotrexate by using polymers, which is preferably used as a cancer agent. Microcapsules were prepared using polymer ethyl cellulose and eudragit as coating material, by emulsion solvent evaporation technique. Methotrexate meets all the ideal characteristics to formulate in the form of micro capsule sustained drug delivery system.

The compatibility evaluations were performed by DSC analysis. Studies imply that polymers are compatible with each other. There was no interaction found between polymer and drug.

The drug content was found maximum 56% in formulation F3 and order of content followed by formulations F3>F2>F1.

The mechanism of drugs release from microcapsule was dissolution followed by diffusion. Three formulations F1, F2 and F3 were able to release drug up to 24hrs and F1 released maximum drug during 24hrs. Formulation F2 and F3 were able to release drug up to 20hrs. among three formulations F1 only compiles with the USP23, test 3, which maintained the release pattern as per mention USP23 (test 3). Rests of the formulations were unable to maintain release rate as per USP23

ACKNOWLEDGEMENTS

The Authors wish to acknowledge to Dr. K. Vanitha Prakash and S.V.V Satyababu SSJ College of Pharmacy, Hyderabad, for their innovative support in present research, and the authors also acknowledge greatly Mac Mohan Pharma Ltd, Hyderabad, for providing Methotrexate gift sample.

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Received on 08.09.2011 Modified on 20.09.2011

Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1861-1864