INTRODUCTION:

Flutamide is a potent nonsteroidal anti androgen that is used in the treatment of advanced prostate cancer. It blocks the androgen receptors on the cancer cells and inhibits the androgen dependent cell growth. The usual oral dose of flutamide is 250 mg three times daily. Its oral absorption is rapid and it attains peak plasma concentration in 1 h after a single dose¹. The drug has high first pass hepatic metabolism and low elimination half life (5-6 h). Low functionality of flutamide is due to its rapid metabolism, less active metabolites (hydroxyl flutamide) and low bioavailability. Low bioavailability may be due to its poor wetability and low aqueous solubility.

Moreover high dose of Flutamide produces hepatotoxicity. Treatment with flutamide may cause a variety of side-effects including diarrhea, tiredness, impotence, enlargement of male breast and liver malfunction. In order to decrease the frequency of drug administration and also the incidence of adverse effects, a sustained release formulation of flutamide is desirable. Preparation of nanoparticles is a method that makes it possible to increase the bioavailability, reduce the incidence and severity of adverse effects, especially gastrointestinal disorders and hepatic impairment².

Nanoparticles are defined as particulate dispersions or solid particles with a size in the range of 10 - 1000nm³. Due to large surface to volume ratio, the nano-scale structures have unique properties and dissolution behaviours which are expected to avoid the unwanted side effects. Sustained release of the drug from the nanoparticles maintains the therapeutic concentration for long durations.

In the present study, Chitosan (CS) was chosen as the material for the particle matrix. Chitosan is a biocompatible, bioactive, and biodegradable polymer and widely reported for preparing micro and nanoparticles. Because of its cationic charge, biocompatibility and low toxicity, chitosan has been used as a vehicle for delivery of various category drugs. Chitosan nanoparticles were prepared by the ionotropic gelation process based on the interaction between the negative groups of sodium tripolyphosphate (STPP) and the positively charged amino groups of chitosan⁴. Sodium tripolyphosphate (STPP) was used to prepare chitosan nanoparticles, because it is nontoxic, multivalent and is able to gelate through ionic interaction between positively charged amino groups of chitosan and negatively charged STPP.

Hence, the present study was aimed to formulate Flutamide loaded chitosan – sodium tripolyphosphate (STPP) nanoparticles using ionic gelation method. These nanoparticles were characterized for its physiochemical properties and in vitro release studies.

MATERIALS AND METHODS:

Flutamide loaded chitosan nanoparticles (F1 to F6) were prepared by Ionotropic gelation method. The formulated nanoparticles were evaluated for external morphological characters, particle size analysis, zeta potential, drug content, entrapment efficiency and in-vitro release studies.

Methods:

Preformulation Studies:

Organoleptic Properties:

Pure drug Flutamide was evaluated for organoleptic properties such as appearance and colour.

Solubility Analysis:

The solubility of Flutamide was checked in water. The solubility was confirmed by analyzing the sample by quantitative determination using UV spectroscopy at a wave length of 228nm.

Melting Point Determination:

Melting point of the obtained drug sample was determined; as it is a first indication of purity of the sample. The presence of relatively small amount of impurity can be detected by lowering as well as widening in the melting point range.

Identification of Pure Drug:

FTIR spectroscopy was used for identification of pure drug.

Determination of λ _max:

Preparation of Stock Solution:

Accurately weighed 10mg of Flutamide was transferred in a 100ml volumetric flask. To the flask, methanol was added in small proportion (20ml) to dissolve Flutamide. The volume was made up to 100ml with phosphate buffer pH 7.4 to get a concentration of 100μg/ml.

Determination of λ _max:

20μg/ml solution of Flutamide was prepared with above stock solution and the solution was scanned in UV - Vis spectrophotometer from 400 - 200nm to determine the λ max.

Compatibility studies:

A successful formulation of a stable and effective dosage form depends on selection of excipients that are added to promote the consistent release and bioavailability of the drug and protect it from degradation. If the excipients are new and not been used in formulation containing the active substance, the compatibility studies are mandatory. This was confirmed by infrared light absorption scanning spectroscopy (IR) studies. Infra red spectra of pure drug and mixture of formulations were recorded by dispersion of drug and mixture of formulations in suitable solvent (KBr) using Fourier Transform Infrared Spectrophotometer⁵.

Formulation Development:

Calibration of standard curve:

Preparation of Standard Solution of Flutamide: Accurately weighed 100mg of Flutamide was dissolved in 100ml of pH 7.4 phosphate buffer solution which is having concentration of 1000µg/ml (1mg/ml). 10ml of this solution was further diluted up to 100ml with 7.4 pH phosphate buffer to give a solution of Concentration 100 µg/ml.

Preparation of Working Solution:

From the above stock solution different concentrations (1 to 10µg/ml) were prepared by transferring 1 to 10ml into 10ml volumetric flasks and the final volume was made up with pH 7.4 phosphate buffer. From each concentration sample was taken and the absorption was measured at 228nm by using UV spectrophotometer and pH7.4 phosphate buffer as a blank. The calibration curve of absorbance against concentration was plotted.

Preparation of NP’s by Ionic gelation method: Flutamide nanoparticles were prepared by ionic gelation method. Where the chitosan was cross linking with anionic sodium tri poly phosphate. Chitosan was dissolved in aqueous solution of acetic acid (1%v/v) at various concentrations. 10mg STTP was dissolved in 10ml of deionised water and added dropwise to the 10ml chitosan solution containing 10mg flutamide under magnetic stirring at a speed of 1500rpm. After 2 hours of crosslinking, nanoparticles were isolated by centrifugation at 12,00rpm and 5ºC for 30 minutes, and subsequently washed several times with water. The particles were freeze dried and stored in dry conditions at 25ºC. The nanoparticles were prepared with different concentration of chitosan, STTP and acetic acid.

The formulated nanoparticles were evaluated for external morphological characters, particle size analysis, zeta potential, drug content, entrapment efficiency and in-vitro release studies.

Table-1: Formulation table

S. No	Formulation Code	Flutamide (mg)	Chitosan (mg)	STPP (mg)	Acetic Acid (%)
1	F1	10	10	10	1
2	F2	10	20	10	1
3	F3	10	30	10	1
4	F4	10	40	10	1
5	F5	10	50	10	1
6	F6	10	60	10	1

RESULTS AND DISCUSSION:

Preformulation Studies:

Oraganoleptic Properties:

Buff to yellow colour, unpleasant smell, very fine crystalline powder.

Solubility Studies:

Flutamide was low soluble in water and freely soluble in organic solvents. The solubility in water was found to be 0.0094mg/ml.

Melting Point Determination:

After performing capillary method melting point of Flutamide found in the range of 111 - 113ºc.

Identification of Pure Drug:

FT-IR spectroscopy was used to determine the functional group present in the pure drug sample. The FTIR spectrum of pure Flutamide has shown the characteristic peaks at 3355, 1709, 1509, 1314 and 654 cm−¹. The absorption bands between 3250 and 3400 cm-¹was indicates presence of –NH stretching. The wave numbers observed at 1709 and 1314 may be assigned to the C=O and C-N bonds respectively and the sharp peak occurred at 1509 and 654 indicates presence of N=O and C–F₃ group attached to C=C. IR Spectra of Flutamide is as follows:

Fig-1: IR Spectra of Flutamide

Determination of λmax:

The Flutamide with pH 7.4 PBS was scanned in UV - Vis spectrophotometer from 400 – 200nm to determine the λmax. The λmax was found to be at 228nm, so the calibration curve of Flutamide was developed at this wavelength.

Drug - Excipient

Compatibility Studies:

Fourier Transform Infra Red Spectroscopy (FTIR): The interaction studies were carried out to ascertain any kind of interaction of drug with the excipients used in the preparation of polymeric nanoparticles. Physical mixture of Flutamide and each selected excipients were prepared in the 1:1 w/w ratio gently blending with spatula at room temperature. The blends were considered homogeneous mixture when the mixture is used for IR analysis.

Fig-2: IR Spectra of Flutamide + Chitosan + STPP

The FTIR spectra of Flutamide were recorded on a FTIR multiscope spectrophotometer (Brooker) equipped with spectrum 11.0.0.0449 software using KBr pellet method. The spectrum for each sample was recorded over than 600 - 4000 cm^-1. The FTIR spectra of the pure drug and formulations were shown in Figures 3-4.

Inference:

The FTIR spectrum of formulations had shown characteristic absorption bands which were comparable with absorption bands of pure drug. The results illustrated that, there were no chemical instabilities in drug – excipient combinations.

Preparation of Nanoparticles:

Nanoparticles of Flutamide were prepared by Ionotropic gelation technique using Chitosan and STPP polymers in varying concentration ratio like 1:1 to 6:1. Total six batches were formulated (F1, F2, F3, F4, F5 and F6) and all the formulations were investigated for various parameters like particle size, Zeta potential, Scanning electron microscopy (SEM), Drug content, Entrapment efficiency, Percentage yield, in vitro drug release and drug release kinetics.

Characterization of Prepared NPS:

Size Analysis:

Six formulations have been developed by varying the concentration of chitosan and the effect of particle size has been determined. The concentration ratio of chitosan and STPP 3:1 the particle size was found to be 148 nm. The formulations of varying the concentration of chitosan to STPP such as 1:1, 2:1, 4:1, 5:1, 6:1 was developed and particle size was found to be 190 nm, 195 nm 220 nm, 284 nm and 317 nm. The results indicate that the particle size increases with increased concentration ratio of chitosan and STPP. Variations in particle size to increase the concentration are due to agglomeration of the particles. The formulation F3 showed minimum particle size of 148 nm compare to other formulations.

Fig-3: Mean particle size analysis of different formulations of nanoparticles

Surface Charge (Zeta Potential):

The electrostatic repulsion between particles with the same electric charge prevents the aggregation of the particles. The zeta potential values of formulation F3 was in negative and this demonstrated that the anionic surface of drug delivery system would provide improved targeting ability as compared to the cationic carrier. The zeta potential of the formulation F3 with a concentration of chitosan 3:1 was found to be - 46.4, which implies that it is having good Stability.

Fig-4: Zeta potential of nanoparticles formulation F3

Scanning Electron Microscopy (SEM):

SEM analysis of the F3 showed that the nanoparticles are hollow spherical structure with a large central cavity in which Flutamide was loaded. The outer surface of the nanoparticles was smooth and shell of the nanoparticles also showed some porous structure. SEM analysis of formulations F3 is shown in Figure 8.

Fig-5: SEM analysis of Nanoparticle formulation F3

Drug Content:

The drug content was evaluated for all the formulations and it was observed that the nanoparticles obtained from F1 formulation showed maximum drug content (87.9± 1.2%) and F6 showed minimum drug content (69.5± 7.2%). The drug content was decreased with increase in chitosan concentration. This may be due to loss of drug during manufacturing stage or increase in entrapment efficiency, so that drug is not available for estimation. This result indicated that there was no drug loss by manufacturing process or by excipients used in the formulation.

Entrapment Efficiency:

Prepared nano formulations were estimated for entrapment efficiency. Drug entrapment efficiency varied from 55.50±2.4 to 86.30±3.6%. This result indicated that drug entrapment efficiency increased with increasing concentration of polymer up to 0.3% (F3). After that, there was no significant increase in entrapment efficiency. This may be due to unavailability of drug for entrapment. This can be attributed to fact that higher extent of polymer resulted in formation of a more rigid network structure which prevent the leaching out of drug during preparation of nanoparticles. The optimum efficiency was based on the drug content and polymer usage. From drug content and entrapment efficiency results chitosan nanoparticles F3 were considered as optimum trials.

Percentage Yield:

The percentage yield of nanoparticles prepared by ionotropic gelation method was recorded and it was determined by collected the nanoparticles and weighed. The measured weight was divided by the initial dry weight of starting materials, which were used for the preparation of the nanoparticles. The percentage yields of nanoparticles of all formulations were in the range of 48.24±1.24 to 86.13±1.37%. It was found that when concentration of chitosan increased, the percentage yields also increased. The formulation F6 showed maximum percentage yield of 86.13±1.37% compare to other formulations

In vitro release studies:

From the in-vitro release studies of Flutamide loaded chitosan nanoparticles (F1–F6), it was observed that release profiles in pH 7.4 phosphate buffer were found to have very good controlled efficacy. The drug release depends upon concentration of the polymer matrix and increase in polymer concentration produced much more time for release of drug for all formulations.

High polymer concentration (≥0.4% chitosan–F4 to F6) showed slow drug release for more than 24 h. Low polymer concentration (≤0.2% chitosan–F1 to F2) showed quick drug release within short period.

Hence the formulations (F1 to F2, F4 to F6) were considered to be not satisfactory for controlled delivery of Flutamide either by quick release or over retarding. Flutamide nanoparticles prepared with 0.3% chitosan (F3) showed controlled and sustained drug release for a period of 24 h. The percentage cumulative drug release of F3 at the end of 24 h was found to be 95.85± 0.54%.

Fig-6: In vitro drug release studies

CONCLUSION:

The particle size and zeta potential of optimized formulation (F3) was found to be 148 nm and – 46.4, which indicates that the formulation was having good stability. The nanoformulations were designed for sustained release of the drug for a period of 24 h and this may reduce the frequency of dosing, thereby minimizing the occurrence of side effects. From this results, it was concluded that F3 formulation was considered to be the best formulation and serves as a potential formulation for the treatment of prostate cancer.

ACKNOWLEDGMENT:

The presenting authors are thankful to Management and Principal of Vijaya Institute of Pharmaceutical Sciences for Women, Vijayawada for their valuable support in carrying out this work.

REFERENCES:

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Received on 16.03.2020 Modified on 29.04.2020

Research J. Pharm. and Tech 2021; 14(3):1668-1672.

DOI: 10.5958/0974-360X.2021.00296.1