Formulation Development and Evaluation of Sustained Release Tablets of Mercaptopurine

Introduction

A sustained drug delivery system (SR) is a delivery of drug system that can achieve a slow releasing of a drug over a longer time of drug release. Due to the flexibility of the oral route, more attention was drawn to the dosage form of SR. Administration of a drug in single dose is released over a long duration of time and is more beneficial than repeated doses. The main goal is to attain a steady plasma concentration of the drug in the blood to improve patient compliance, also to improve the drug effectiveness for its use. The development of a matrix tablet as an extended release (SR) is the least complex approach to developing SR dosage forms. The excipients used in formulating matrix systems are the use of hydrophilic and hydrophobic polymers ² .

Matrix tablets:

Matrix tablets are designed for controlled drug release entity in which the drug release is in accurate manner. Drug release is followed by mechanisms to control dissolution and diffusion. Drugs having different solubility, release of drugs can be controlled by using hydrophilic polymers, hydrophobic agents, or plasticizers. 6-Mercaptopurine (6-MP) is a drug used to treat leukemia, pediatric non-Hodgkin's lymphoma, true polycythemia, psoriatic arthritis, and bowel disease like inflammatory (such as Crohn's disease and ulcerative colitis). 6-MP is a purine antagonist and belongs to an antihistamine. Metabolites that inhibit biosynthesis or the use of natural cellular metabolites. 6-MP is a water insoluble purine analogue, 10-50% of the administered drug when t reaches the systemic circulation and undergoes rapid inhibition. The limiting factor in using 6-MP is its very short plasma half-life 4 (60 to 120 minutes). In this research work, a formulation was designed, developed and evaluated for prolonged drug delivery of 6-mercaptopurine by improving solubility, and thus bioavailability as well as half-life.

Materials and Methods

Materials

Mercaptopurine is gift sample received from Sai laboratories, Pune, Maharashtra. pectin and magnesium stearate were purchased from central drug house pvt ltd, Hydroxypropylmethylcellulose 4KM were purchased from yarrow chemicals pvt ltd, talc were purchased from Loba chemicals pvt ltd and microcrystalline cellulose were purchased from Ozone international. All other chemicals and solvents were used as analytical grade.

Method

Preparation of Matrix tablets:⁵

A wet granulation process was used to prepare Mercaptopurine matrix tablets using HPMC K4M and pectin as polymers. Various formulations have been prepared with wet granulation technology. The powder was sieved through 80 mesh. The requisite quantity of Mercapturine, HPMCK4M and pectin as polymers were mixed with MCC as diluents with a binder (5% w / v cornstarch) in sufficient quantities. When forming a suitable cohesion, the damp mass of powder was sieved through a 22/44 mesh, and then granules were formed. The resulted granules were kept for drying in a hot air oven and dried. The dried granules were sieved through 44 mesh.

A fine granules of 10% were added. The granules were finally mixed with Talc as glidant and Magnesium stearate as lubricants respectively. The compression of granules was done with a rotary tablet punching machine. The tablet weight was 500 mg with strength of 50 mg Mercaptopurine drug contained in each tablet.

Evaluation parameters:

Evaluation parameters for granules: The evaluation of granules were done for the following parameters.

Angle of repose⁶

The finely weighed powder is fed into a funnel. The funnel height of the was set at a distance of 2 cm from the flat surface. The powder is poured freely over the funnel and the powder was made to flow through the funnel over the surface of the ground where the pile of powder comes into contact with the bottom end of the funnel. The cone diameter of powder was measured and the angle found or calculated using the equation:

ϴ = tan^-1h / r

Bulk density and tap density⁸

A certain amount of granules from each formulation was transferred to a graduated 10 ml cylinder. The initial volume was observed, and then the cylinder was tapped. Tapping was performed until no further volume changes were observed. Bulk density and tapped bulk density were calculated using following formula; 

BD= Wt. of the powder / Vol. of powder

TD = Wt. of the powder / Tapped vol. of powder

Percentage Compressibility or Carr’s index:⁹

Carr’s index was determined by using the following formula:

Carr’s Index (%) = TD / TD- BD x 100

Powder grade according to Carr’s Index

Hausner’s ratio:

Hausner ratio is an assessment of ease of powder flow. Hausner’s ratio can be calculated by the following equation,

Hausner’s ratio = Tapped density / Bulk density 

Hasuner’s value:

Lower the Hausner ratio i.e less than 1.25 shows better powder flowing property than higher one. more than 1.25. The formulated tablets were evaluated for the following parameters.

Hardness:

Tablets are required some strength to withstand the mechanical impact of handling during packaging and shipping. It is expressed in kg/cm² .Ten tablets were randomly selected from all formulations, hardness was examined, and finally the values were averaged.

Friability¹¹.

A Roche friability tester was used to determine the fraiability of tablets. In friabilator placed 20 pre-weighted tablets. “The friabilator was rotated for 4 minutes at a speed of 25 rpm. After 100 cycles, the tablets were removed from the machine and re-weighed.” The fractionation ratio was determined by the following formula.

% Friability = Initial wt- Final wt × 100/ Initial wt

Weight variation:¹²

To study the variation in weight, the tablets were weighed individually and weight of 20 tablets was noted one by one by the help of an electronic scale. Their average weight (WA) of tablets was calculated. The change in weight is calculated in percentage as follows. 

% weight variation = (WA–WI) 100 / WA

Percentage deviation in tablets weight

Thickness¹³

The tablets thickness was measured. Total 10 tablets from each formulation were randomly taken and measured thickness.

Uniformity of drug content¹³

Around 10 tablets were randomly selected from each formulation, the tablets were powdered in a mortar and a accurate weight of tablet was taken from the powdered mixture. Then the sample was placed in 100 ml volumetric flasks containing pH 7.4 phosphate buffers up to the mark and kept overnight. Then the solution was filtered and made appropriate dilution. The drug content of each tablet was determined using UV-visible spectroscopy.

Dissolution study in simulated gastric and intestinal fluids¹⁶

A dissolution study was conducted with the USP-2 Rotary dissolution apparatus. Dissolution was performed at 37.0 + 0.5 ° C at a speed of 100 rpm. ‘The drug was released from the tablets in 900 mL of 0.1N HCl of pH 1.2 medium for about 2 hour, then in the phosphate buffer of a pH 7.4 for the remaining hours until the completion of the test.’ At every specified time interval 5 ml of sample was taken and fresh same volume was replaced. The drug which was released through dissolutio was analyzed with a UltraViolet visible spectrophotometer at 311 nm. The drug release data were superimposed on different kinematic release models to determine which model best matches the release profiles obtained.

1)Zero order kinetic

2) The Higuchi Model

3) Korsmeyer-Peppas Model 

Stability studies¹⁷

Stability studis have been conducted at 40 ° C and 75%RH for three months. The evaluation of formulation was made for drug content and physical parameters after 90 days.

X-Ray diffraction studies (XRD)¹⁸

A Philips X-ray diffractometer, PW-171, was used with CuKa irradiation filtered by Cu-NF to record spetra. For the internal standard Quartz was used. An attachment of X-ray powder diffraction scale was made to a digital graphics set and a computer with a 25 KV / 20 Ma Cu-NF tube as the CuKa radiation source in the range of 0-50⁰

Fourier transform infrared spectroscopy (FTIR)¹⁴

A pellet is made by crushing samples with KBr under hydraulic pressure, and then the spectra were noted in 400 to 4000 cm^-1 range.

Differential scanning calorimetric analysis¹⁵

The differential scanning calorimeter measurement of Mercaptopurin, physical mixtures and embedding complexes was performed using a differential scanning calorimeter (DSC) Q2000 V24.2 Build 107. The mass of the empty pan and the reference pan was taken into account to calculate the heat flux. The mass of the sample ranged from 3 to 10 + 0.5 mg and was placed in sealed aluminum trays. The refrigerant used was liquid nitrogen. Samples were cleared at 10 ° / min from 20 ° to 300 ° C.

Results

Tablets of mercaptopurine were prepared by wet granulation method using various polymers like hydroxypropyl methylcellulose 4KM, pectin in different proportion. The evaluation process for tablets were carried out for precompression evaluation parameters like bulk density, tap density, angle of repose, carrs index and hausner’s ratio and post compression evaluation parameters like appearance, thickness, hardness, friability, weight variation, drug content, and In vitro drug release, short term stability and drug-excipients interaction.

Total weight of each tablet: 500mg

Mercaptopurine sustained release tablet were formulated by using Pectin (F1, F2, F3), HPMC4KM (F4, F5, F6), in combination of both the polymers (F7, F8, F9). Bulk density and tapped density of the granules are determined. The value of these two parameters ranges in between 0.320 to 0.360gm/cc and 0.327 to 0.480gm/cc respectively. By Hausner ratio and Carr’s index it is concluded that the granules has good packing ability which are in the range of 9.5 to 15.78% and 1.00 to 1.23 respectively. The granules were evaluated for angle of repose which is in the range of 22.890 to 26.190. All the granules showed good flow properties, because the value of angle of repose was less than 30^o . The hardness of the prepared sustained release tablet range lies in between 4.8 kg/cm² to 5.8 kg/cm² . Hardness of the tablets increases as the concentration of pectin increases in the formulation.The friability value of the tablets lies in the range of 0.69% to 0.80%. <1% suggests good physical strength to hold out while handling and transportation. The range of tablets thickness is in between of 3.038 to 3.612.

Discussion

Drug content

Evaluation of drug content in the tablets was determined which lies in range of 94.69% to 98.0%. Weight variation in the prepared tablets were done, the results were in the range of 500 to 504 mg which lies within the IP limit.

Incompatibility

The FTIR spectra of Mercaptopurine as shown (in fig-4,5) in the characteristic peak at about 3430cm^-1, 1275cm^-1, 1155cm^-1.The same characteristic peak with slight variations also found in the spectra of combination of drug and polymers.

It indicates that the drug is in compatible with the polymers. An XRD of pure drug shows peaks in between ϴ of 220 to 300 due its crystalline nature, and XRD of F9 formulation also shows same characteristic peak. Hence the drug is crystalline in the formulations.

The DSC curve for the pure drug, Mercaptopurine is shown in fig-1 and DSC curve of formulation F9 shown in fig-2. Pure powdered Mercaptopurine showed a melting endotherm at 170.60C. The DSC graph of F9 formulation exhibits endothermic peaks at 193 near to the pure drug temperature. This indicates a small degree of interaction between drug and polymers in the formulation.

In vitro studies

“In vitro release study was carried out in USP dissolution apparatus type II(Basket method) at 100 rpm in 0.1 N HCL(1.2pH) for 2 hours and phosphate buffer(7.4pH) till end of the study. The dissolution profiles shown in figure (9) from the dissolution profile it is evident that designed formulation have displayed in range of 70.76 to 96.82 % that is F1(96.82), F2(94.81), F3(90.78), F4(79.5), F5(75.28), F6(70.76), F7(89.77), F8(88.34), F9(72.78). The results indicate that the prepared tablets were released the drug up to 12 hours depending upon the formulation variables.”The data of release of drug in media were adjusted as per the Zero order release, First order release, ‘Higuchi’s equation’ and Peppas equation are calculated. The calculated values are shown in the table. The calculated slope values are more than 0.5 in all cases suggested that the mechanism of drug release followed by non –Fickian transport. Based on the result of evaluation data of all 9 formulations, F1 (96.82), F2 (94.81), F3 (90.78), and F7 (89.77) were optimized because of their sustained release property. And this concludes that as concentration of Pectin increases the drug release decreases and in combination of HPMCK4M and pectin that is 70% pectin and 30% HPMC4KM also shows maximum drug release.

Among these 4 formulations F1 found to be optimized formulation in this present work.

All the formulations F1, F2, F3, F4, F5, F6, F7, F8, and F9 release the drug by zero order kinetics. To know the diffusion mechanism the slope values of peppas was calculated for all the formulations was in the range of 0.507 to 0.734. The calculated slope values are the more than 0.5 in all the cases suggesting that the release of drug was by non-fickian method of diffusion.

The drug content did not show any changes in values after 3 months of stability study at 40+2^o C/75+5%RH which shows that the tablet formulations are stable.

Conclusion

“Sustained release matrix tablets of Mercaptopurine were formulated by the wet granulation technique by using polymers like Pectin and Hydroxypropylmethylcellulose 4KM in different proportion. The formulated tablets were evaluated for drug content, hardness, friability, Fourier transform infrared spectroscopy, differential scanning colorimetry, In vitro drug release study, X-ray diffraction, drug content. All the formulated tablets were in good quality with no capping and chipping. In all the tablet formulation 5%w/v starch paste is used as a binding agent which has showed acceptable hardness of prepared formulation. The values of friability were less than 1% indicates good physical strength to withstand the rigours of handling and transportation.” “FTIR study and DSC indicated that there is no drug excipients interaction.XRD peaks showed the drug is crystalline form in the formulation.” Formulations of F1, F2, F3, F7 were made optimal due to their good quality of strength and being able to extend the drug release from the matrix more than the 12 hrs period. As the concentration of pectin increases the drug release decreases and in combination of pectin and HPMC4KM also showed good sustained release property. 

Acknowledgments

Authors are thankful to BLDEA’S SSM College of Pharmacy and research centre Vijayapur, for giving the opportunity and providing required necessary facilities for carrying out this work.