Enhancement of Solubility of Leflunomide with b-cyclodextrin inclusion complexation

 

P. R. Mahaparale¹*, V. P. Thorat²

¹Government College of Pharmacy, Osmanpura Aurangabad, Maharashtra 431005.

²Siddhi College of Pharmacy Chikhali, Pune, Maharashtra 411062.

 

ABSTRACT:

Leflunomide is a Non-steroidal Anti-Inflammatory drug, which is poorly water soluble. In present study attempt has been made to prepare and characterize inclusion complexes of drug with Beta Cyclodextrin (b-CD). The phase solubility analysis indicated the formation of a 1:1 molar inclusion complex of drug with b-CD. Apparent stability constant (K_C) was 674 K^-1 for b-CD complex. The inclusion complexes were prepared by three different methods viz. coprecipitation, kneading and microwave irradiation method. The prepared complexes were characterized using dissolution study, FTIR and X-ray diffractometry. The inclusion complex prepared with b-CD by kneading method exhibited greatest enhancement in solubility (18 mg/100ml) and fastest dissolution (T₉₀ = 29 min.) of Leflunomide.

 

 

 

INTRODUCTION:

Rheumatoid arthritis (RA) is the most common form of inflammatory arthritis (autoimmune disease) affecting approximately 0. 5 to 1% of the population. Leflunomide is drug with immunomodulatory and anti-inflammatory properties that has shown promising results in terms of efficacy and safety for the treatment of this crippling autoimmune disease and is a welcome addition to the roster of anti-rheumatoid drugs. It has very low water solubility (2. 4 mg/ 100 ml at 25°C), which limits its dissolution and consequently its bioavailability. Thus it is important to enhance the solubility and dissolution rate of Leflunomide to improve its oral bioavailability.

 

Many approaches such as modification of drug crystal form, addition of cosolvents, addition of surfactants, inclusion complexes with cyclodextrins (CD) may be used to increase solubility, dissolution and bioavailability of drug.

 

The objective of the present study is to investigate the possibility of improving the solubility and dissolution rate of leflunomide by complexation with b-CD. The complexes of leflunomide with β-CD were prepared by using different methods like Kneading, co-precipitation, microwave irradiation. Formation of complex was confirmed by phase solubility analysis, Fourier transform infrared (FTIR), dissolution study.

 

MATERIALS AND METHODS:

Materials:

Leflunomide (Alembic Pharmaceuticals Ltd, Baroda), β-Cyclodextrin (Signet Chemical Corporation, Mumbai) were used. All other chemicals and reagents used were of analytical grade.

 

Methods:

Phase solubility analysis for Leflunomide:

Phase solubility studies were performed to determine stoichiometric proportions of Leflunomide with β-CD. This data was used to determine stability constant of complexes. Phase solubility study was performed as per Higuchi and Connors. For this, stock solution of 0. 01M β-CD was prepared separately using distilled water. These stock solutions were diluted with distilled water to give molar solutions in the range of 0. 002 to 0. 01 M for β-CD¹. Five ml of each molar solution was filled in screw-capped vials and the excess quantity of drug was added to each vial separately. The vials were kept for shaking at ambient temperature for 48 hrs using a lab shaker (Remi). The supernatant solutions were collected carefully and filtered using Whatman filter paper (No. 41). The concentration of drug in filtered solutions was determined using UV visible spectrophotometer. No change in l max of drug was found after complexation with cyclodextrins, hence absorbances of resultant solutions were recorded at 259. 5nm, which is l max of drug. From slope and intercept value (S₀) of phase solubility curve stability constant (Kc) was determined².

 

Kc = Slope / [S₀ (1- Slope)]

 

Preparation of Physical mixture and Inclusion complexes:

Physical mixture method:

The required molar (1:1) quantities of drug and β-CD were weighed accurately and mixed together thoroughly in a mortar with vigorous trituration for about an hour. These mixtures were then passed through sieve no. 44 and finally were stored in airtight containers till further use³.

 

Preparation of inclusion complexes:

Inclusion complexes of Leflunomide were prepared by following different methods.

1) Kneading method:

The required quantities of drug and β-CD were weighed accurately in 1:1 molar ratio. A homogeneous paste was prepared in a mortar by adding water: methanol mixture (1:1) in small quantities with continuous kneading for three hours. An appropriate quantity of water: methanol mixture (1:1) was added further to maintain suitable consistency of paste⁴. This paste was dried in hot air oven at 45°- 50°C for 24 hours. The dried complexes were then powdered and passed through sieve no. 44 and stored in airtight containers till further use.

2) Coprecipitation method:

The required molar (1:1) quantities of drug and β-CD were dissolved in methanol and water respectively. The solution of drug was added drop wise into β-CD solution. The contents were continuously stirred for 6 hours and finally were dried at 45°- 50°C for 48 hours, collected and stored in airtight containers till further use.

3) Microwave irradiation method:

The required molar (1:1) quantities of drug and β-CD were weighed accurately and transferred to round bottom flask. Minimum amount of solvent mixture (Methanol: Water, 1:1 v/v) was added to this. The mixture was reacted for two minutes at 60⁰C in the microwave oven. After reaction was complete, adequate amount of solvent mixture was added to remove the residual free drug and β-CD. Precipitate obtained was filtered using Whatman filter paper (No. 41) and dried in vacuum oven at 40°C for 48 hrs⁵.

 

Characterization of Leflunomide inclusion complexes:

Inclusion complexes were characterized using following analytical techniques.

1)    Drug content estimation:

The quantities of inclusion complex equivalent to 10mg of Leflunomide were dissolved in Water: Methanol mixture (1:1). Appropriate dilutions were made and drug content of each complex was calculated from UV absorbance recorded at lmax 275. 5nm.

 

2)    Solubility studies:

Solubility study was performed according to method reported by Higuchi and Connors. Excess quantities of inclusion complexes were added to 25ml distilled water taken in stoppered conical flasks and mixtures were shaken for 24 hrs in rotary flask shaker. After shaking to achieve equilibrium, 2 ml aliquots were withdrawn at 1 hr intervals and filtered through Whatman filter paper no. 41. The filtrate was analysed spectrophotometrically at 259.5nm. Shaking was continued until three consecutive readings were same.

 

3)    IR spectral analysis:

Infra-red spectra of drug and inclusion complexes were recorded by KBr method using Fourier Transform Infrared Spectrophotometer (FTIR-8400s).

 

4)    X – ray diffraction (XRD) study:

The X-ray diffraction pattern of the selected inclusion complexes was compared with that of the pure Leflunomide. This was done by measuring 2ø in the range of 4 to 50° with reproducibility of ±0. 001⁰ on a X ray diffractometer (Philips). The XRD patterns were recorded automatically using rate meter with time constant of 2 × 10² pulse/second and with the scanning speed of 2° (2ø)/min⁶.

 

5)    Dissolution study of Leflunomide and its inclusion complexes:

Dissolution of inclusion complexes (equivalent to 20mg of Leflunomide) was studied using USP XXII six station dissolution apparatus (Type II). The dissolution was carried out in 900ml of 0. 1N HCl at 37 ± 0. 5°C at 50 rpm. Aliquots of 10ml were withdrawn periodically and replaced with 10ml of fresh dissolution medium. The concentrations of drug in samples were determined by measuring absorbance at 259. 5nm. Cumulative percent drug released was determined at each time point. Pure drug was used as a control. The t₉₀ (time required for 90% dissolution of drug) (Table 1) of various solid dispersions was calculated⁷.

 

Table 1: Comparison of various parameters for Leflunomide inclusion complex

*LF- Leflunomide, PM- Physical mixture, CP- Coprecipitation method, KN- Kneading method, MW- Microwave irradiation method

 

RESULTS AND DISCUSSION:

Phase solubility analysis of Leflunomide:

Phase solubility study was done to determine the stoichiometric proportion of Leflunomide with complexing agent β-CD. The phase solubility analysis indicated formation of a 1:1 molar inclusion complex of drug with b-CD. Apparent stability constant (K_C) was 674 K^-1 for b-CD complex.

 

Characterization of Leflunomide inclusion complex:

All inclusion complexes prepared by different methods such as kneading method, coprecipitation method and microwave irradiation method were found to be free flowing powders.

1) Estimation of drug content:

Physical mixture and inclusion complexes showed 94- 100 % drug content. (Table 1)

2) Solubility study:

The saturation solubility of inclusion complexes of Leflunomide with β-CD are indicated in Table 1. Cyclodextrin and their derivatives have proved to be powerful solubilizers for many poorly water-soluble drugs by forming inclusion complexes. In present work, significant enhancement in the solubility of drug was observed for all inclusion complexes with β-CD. The findings indicate that inclusion complexes prepared with β-CD showed greater enhancement in solubility. The inclusion complexes prepared by different methods showed different saturation solubility values. This may be because of variable degree of complexation. Inclusion complexes prepared by kneading method showed higher saturation solubility than those prepared by other methods^8,9.

3) IR spectral analysis:

IR Spectra of pure drug and inclusion complexes of Leflunomide with β-CD prepared by different methods are given in Fig. 1. The spectra of inclusion complexes and physical mixtures of components revealed disappearance of characteristic peaks of aromatic C-H stretching, N-H stretching and Amide at 3250 cm^-1, 3400 cm^-1 and 1670 cm^-1respectively. Therefore, it suggests that vibrating and bending movements of guest molecule that is Leflunomide were restricted due to formation of inclusion complex. It may be aromatic ring portion of Leflunomide, which has been included into the cavity of Beta cyclodextrin. IR spectra thus suggest the possibility of formation of inclusion complexes^10,11.

4) X – ray diffraction (XRD) study:

The inclusion complexes of drug prepared with β- CD by kneading method showed highest enhancement in solubility and fastest dissolution profile than any other methods (Table 1). Hence the same inclusion complex was characterized further by XRD study. Leflunomide, β-CD and inclusion complexes of drug with β-CD prepared by kneading method were subjected to XRD analysis (Fig 2).

 

Fig. 1: IR spectra of inclusion complexes of Leflunomide with b- CD prepared by different methods

Drug- Leflunomide, b CD- Beta cyclodextrin, PM- Physical mixture, CP- Coprecipitation method, KN- Kneading method, MW- Microwave irradiation method

 

Fig. 2: XRD pattern for Leflunomide (pure), b- CD (pure) and their inclusion complex prepared by kneading method

 

Many broad peaks of very low intensity were observed which indicates pure drug existed as microcrystalline particles. However no sharp peaks were detected. The X-ray diffraction pattern for inclusion complexes was characterized by complete absence of any diffraction peaks for the drug, suggesting probable transformation of microcrystalline form into an amorphous state^{12, 13}.

5) Dissolution study of Leflunomide and its complexes:

The inclusion complexes of Leflunomide with β-CD produced pronounced enhancement in its dissolution. From Table 1, T₉₀ value for physical mixture and inclusion complexes was found to be less than pure drug. Thus β-CD helps in enhancing the dissolution rate of Leflunomide (Fig. 3).

 

Fig. 3: Dissolution profiles of Inclusion complexes of Leflunomide and Beta Cyclodextrin prepared by different methods

 

CONCLUSIONS:

Phase solubility data suggests 1:1 molar complex formation of Leflunomide with β-CD (A_L type curve). All complexes showed increase in saturation solubility and dissolution than drug alone. The enhancement in dissolution profiles has been attributed to formation of an inclusion complex in the solid state and reduction in the crystallinity of the product as confirmed by XRD study. The inclusion complexes of drug prepared with β- CD by kneading method showed highest enhancement in solubility (18mg/100ml) and fastest dissolution profile (T₉₀ = 29 min). Kneading method is of particular interest for industrial scale preparation as it has major advantage of shorter reaction time and higher yield of product. However further in vivo studies are needed to correlate the effect of increasing solubility of Leflunomide with its bioavailability.

 

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Received on 31.01.2020            Modified on 08.03.2020

Accepted on 11.04.2020         © RJPT All right reserved