A PROCESS OF EVALUATING FLUTAMIDE LOADED POLY CAPROLACTONE NANOPARTICLES

Abstract

To prepared and evaluated polymeric Nanoparticles (NPs) of Flutamide by nanoprecipitation method and factorial design. The influences of various formulation components such as polymer, organic phase volume and surfactant on the characteristics of nanoparticles were investigated. Methods: The (Polycaprolactone) PCL loaded with drug was evaluated for surface morphology, surface charge, particles size, encapsulation efficiency, drug content and in vitro release studies. FT-IR studies was indicated no interaction between the drug and polymer. Results: The results of drug release study of nanoparticles may fit with different kinetic equations. The particle size varied from 128 to 317 nm and zeta potential was in negative and its value found to be - 46.4 mv. Drug content of Flutamide loaded PCL nanoparticles between 74 % ± 0.72 to 92 % ± 0.53. The minimum and maximum entrapment efficiencies were found to be of 75 % ± 0.66 and 92 % ± 0.70. The percentage yields of all formulations were in the range of 46.05 % ± 1.56 to 86.78% ± 1.32. The In-vitro drug release followed zero order with sustained behavior for a period of 24 hr. Results of accelerated storage conditions of optimized formulation revealed that no significant changes in formulation F2. Conclusion: The present investigation opens new frontiers in developing Flutamide NPs for targeting delivery to the prostate for the prostate cancer treatment. Which also overcome the problems associated with conventional formulations like multiple dose therapy, poor patient compliance and high treatment cost.

Specification

Description:FIELD OF THE INVENTION
[0001] The present invention is related to evaluate polymeric nanoparticles (NPs) of flutamide more particularly using phosphate flutamide, polycaprolactone and pluronic F-127.

BACKGROUND
[0002] Flutamide is a nonsteroidal pure antiandrogen that acts by inhibiting the uptake and/or binding of dihydrotestosterone to the target cell receptor, thus interfering with androgen action. Flutamide is well absorbed orally and extensively metabolized; its active metabolite, 2-hydroxyflutamide, is formed rapidly and excreted almost entirely by the kidneys. Clinical studies in prostate cancer patients have demonstrated efficacy with flutamide monotherapy in patients who had received no prior treatment, in untreated patients with combined androgen blockade concomitantly with a luteinizing hormone-releasing hormone (LHRH)-agonist, and in relapsed patients. A randomized, placebo-controlled trial demonstrated a 26 percent increase in median survival for patients treated with leuprolide plus flutamide compared with leuprolide plus placebo. When given as monotherapy and in combination with an LHRH-agonist, flutamide is well tolerated. The usual adverse effects are gynecomastia and mild diarrhea when given as a single agent. In combination with an LHRH-agonist, hot flashes, loss of libido, impotence, mild nausea and vomiting, gynecomastia, and diarrhea are commonly reported. However, only diarrhea occurred more frequently in patients treated with leuprolide plus flutamide than in those treated with leuprolide plus placebo. Flutamide is indicated in combination with an LHRH-agonist (e.g., leuprolide) as initial therapy in metastatic (stage D2) prostate cancer. The usual dose is 250 mg po tid given at eight-hour intervals and started concurrently with the LHRH-agonist. Formulary addition is recommended.

[0003] A novel particulate delivery matrix based on ionically crosslinked casein (CAS) nanoparticles was developed for controlled release of the poorly soluble anticancer drug flutamide (FLT). Nanoparticles were fabricated via oil-in-water emulsification then stabilized by ionic crosslinking of the positively charged CAS molecules below their isoelectric point, with the polyanionic crosslinker sodium tripolyphosphate. With the optimal preparation conditions, the drug loading and incorporation efficiency achieved were 8.73% and 64.55%, respectively. The nanoparticles exhibited a spherical shape with a size below 100 nm and a positive zeta potential (+7.54 to +17.3 mV). FLT was molecularly dispersed inside the nanoparticle protein matrix, as revealed by thermal analysis. The biodegradability of CAS nanoparticles in trypsin solution could be easily modulated by varying the sodium tripolyphosphate crosslinking density. A sustained release of FLT from CAS nanoparticles for up to 4 days was observed, depending on the crosslinking density. After intravenous administration of FLT-CAS nanoparticles into rats, CAS nanoparticles exhibited a longer circulation time and a markedly delayed blood clearance of FLT, with the half-life of FLT extended from 0.88 hours to 14.64 hours, compared with drug cosolvent. The results offer a promising method for tailoring biodegradable, drug-loaded CAS nanoparticles as controlled, long-circulating drug delivery systems of hydrophobic anticancer drugs in aqueous vehicles.

SUMMARY
[0004] Polymeric Nanoparticles (NPs) of Flutamide were successfully prepared and evaluated by nanoprecipitation method and factorial design. The influences of various formulation components such as polymer, organic phase volume and surfactant on the characteristics of nanoparticles were investigated. Aesthetic results proved that formulation F2 containing 50 mg PCL and 10 ml acetone with 1 % pluronic F127 produced the most ideal nanoparticles with encapsulation efficiency and controlled drug release within 24 hr. This suggest that, the administration of F2 nanoparticles at low dose at once daily, as the rate of release of prepared Flutamide nanoparticles is less than the half-life of free Flutamide (8 hr). Therefore, this investigation opens new frontiers in developing Flutamide NPs for targeting delivery to the prostate for the prostate cancer treatment. Which also overcome the problems associated with conventional formulations like multiple doses, high treatment cost and poor patient compliance.

DESCRIPTION OF THE FIGURES:
[0005] Table-1 illustrates 23 Factorial design of Nanoparticles.

[0006] Figure 1: illustrates IR spectra of flutamide.

[0007] Figure 2: illustrates λ max of flutamide.

[0009] Figure 3: illustrates IR spectra of flutamide.

[0010] Figure 4: illustrates IR Spectra of Flutamide + PCL +Pluronic F 127.

[0011] Figure 5: illustrates Size analysis of Nanoparticles formulations F1 - F8.

[0012] Figure 6: illustrates mean particle size analysis of nanoparticle formulations F1 - F8.

[0013] Figure 7: illustrates scanning electron micrographs of nanoparticles formulations F1 - F8.

[0014] Figure 8: illustrates zeta potential of nanoparticles formulation F7.

[0015] Figure 9: illustrates % Drug Content of nanoparticle formulations F1 - F8.

[0016] Table 2: illustrates Drug Entrapment Efficiency.

[0017] Table 3: illustrates percentage yield.

[0018] Fig. 10: Invitro Drug Release of Formulations. [A] Low Polymer 10 mg [B] High Polymer 50 mg [C] Low Organic Phase Volume 10 ml [D] High Organic Phase Volume 30 ml [E] 1% Pluronic F127 [F] 1.5% Pluronic F127.

[0019] Table 5: illustrates accelerated stability studies of optimized formulations F2.

DETAILED DESCRIPTION OF THE INVENTION:

[0020] Flutamide was purchased from Yarrow Chem Products, Mumbai. Polycaprolactone and Pluronic F-127 purchased from Coastal chemical Limited, Visakhapatnam and all other ingredients used were of analytical grade obtained from obtained from Qualigens Fine chem, Mumbai.

[0021] Organoleptic Properties: Flutamide was evaluated for organoleptic properties such as appearance and colour. Solubility Analysis: The solubility of Flutamide was checked in water and it was confirmed by quantitative determination using UV spectroscopy at 228 nm. Melting Point Determination: Melting point is a first indication of purity of the sample and the presence of small amount of impurity can be detected by lowering or widening the melting point range. Identification of Pure Drug: FTIR spectroscopy was used for identification of Flutamide.

[0022] Determination of λ max: Preparation of Stock Solution: 10 mg of Flutamide was transferred in a 100 ml volumetric flask and methanol was added in small proportion (20ml) to dissolve Flutamide. The volume was made up to 100 ml with PBS pH 7.4 to get 100 μg/ml concentrations. Determination of λ max: 20 μg/ml Flutamide was prepared from above stock solution and the solution was scanned between 400 - 200 nm to determine the λ max.

[0023] Compatibility study: A stable and effective dosage form was formulated depends on selection of excipients that are promote the drug release and bioavailability and protect it from degradation. If the excipients not were used in formulation containing the active drug, the compatibility study is mandatory.

[0024] Drug - polymer compatibility studies by FTIR: IR spectra of Flutamide and formulation mixture were recorded by dispersion of drug and polymer in suitable solvent using Fourier Transform Infrared Spectrophotometer (FTIR). A base line was made using dried KBr and then the spectra of mixture of drug, polymer mixture and KBr were recorded on FTIR [9].

[0025] Calibration of standard curve: In 100 ml of pH 7.4 phosphate buffer solution 100 mg of accurately weighed Flutamide was dissolved. 10 ml of this solution was further diluted with 7.4 pH phosphate buffer to get 100 µg/ml solutions. From the above stock solution different concentrations (2 to 20 µg/ml) were prepared with pH 7.4 phosphate buffer. From each concentration sample was taken & the absorption was measured at 228 nm by using UV spectrophotometer and pH 7.4 phosphate buffer as a blank. The calibration curve was plotted and the regression equation and correlation coefficient were determined.

[0026] 23 Factorial Design: The optimization phase was carried out statistically using 23 factorial design in which the polymer concentration, stabilizer and organic solvent volume were kept at two different levels. The eight formulations were categorized in to four groups (given below) for ease of analysis and comparison.



Group I: All variables at low level (Formulation F1).
Group II: Any one of three variables at high level (Formulations F2, F3 & F5).
Group III: Any two of three variables at high level (Formulations F4, F6, & F7).
Group IV: All three variables at high level (Formulation F8).

[0027] Nanoprecipitation method: Flutamide loaded PCL nanoparticles were prepared by the nanoprecipitation method [10]. This method involves the precipitation of polymer from an organic solvent and diffusion of organic solvent into aqueous phase in the presence of a surfactant. PCL and Flutamide were dissolved in acetone and this solution was added to pluronic F-127 in PBS pH 7.4 at 1 ml/min speed using syringe under magnetic stirring conditions. The obtained suspension was stirred at 500 rpm for 2 hr to evaporate acetone. The suspension was cooling centrifuge at 11,000 rpm for 30 minutes to remove precipitants and supernatant was collected, lyophilized and stored at 4°C. Repeated the procedure by varied in the concentrations of polymer, stabilizer and organic solvent volume to optimize the formulation. A blank nanoparticle was prepared by the same procedure, but excluding Flutamide.

[0028] CHARACTERISATION OF PREPARED NP's: The obtained formulations of Flutamide loaded PCL nanoparticles were characterized for following parameters. Particle Size Analysis & Surface Morphology: Particle Size Analysis: Particle size measurement was carried out by Photon Correlation Spectroscopy (PCS) (Malvern Instruments). Samples were diluted with ultra purified water, and measured at 25° and 90° scattering angles, recorded for 180 sec and the mean diameters for all samples were obtained by cumulative analysis in triplicate. The morphological study of nanoparticles was carried out using scanning electron microscopy (SEM) (Tecnai 20 G2 S TWIN). SEM was employed to understand the arrangement and orientation of molecules within the nanoparticle to determine its behavior and stability.

[0029] Surface Charge determination: Nanoparticles were characterized for Zeta potential (ζ) using a Zeta Sizer [11]. Entrapment Efficiency: The Entrapment Efficiency (EE %) also known as Association Efficiency [12]. Was studied by centrifuging the drug loaded nanoparticles at a high speed of 3500 - 4000 rpm for 30 min and the supernatant was assayed for free drug concentration using spectrophotometer. Entrapment efficiency was then calculated as below:

EE %=(Actual amount of drug added - Un bound drug (Free drug) )/(Actual amount of drug added)× 100

[0030] Drug content: Free drug in the supernatant (drug content) was estimated by centrifuging the nanoparticles suspension at 15,000 rpm for 40 min at 25°C. Concentration of Flutamide present in the supernatant was determined by UV - spectrophotometer at 228 nm [13].

[0031] Percentage yield: It was determined by the equation [12]

Percentage yield=(Weight of dried nanoparticles recovered )/(Sum of initial dry weight of starting material )×100


[0032] In vitro release studies: In vitro release profile of the prepared nanoparticle suspensions were studied by diffusion method using an artificial membrane. In the donor compartment nanosuspension containing known concentration of drug (20 mg) was placed and in the receptor compartment pH 7.4 phosphate buffer was placed and constantly agitated using a magnetic stirrer at 100 rpm and 37 °C. 0.5ml samples were withdrawn from the receptor compartment for estimation of released drug and replaced with similar volume of buffer. This study was carried out for 24 hr, and the concentration of drug released was determining the absorbance at 228 nm using UV spectrophotometer [10].

[0033] Kinetic modelling: To understand the kinetics and mechanism of drug release from optimized formulation F2, the results of in vitro release study were fitted with zero order, first order and Higuchi's model [14]. Stability study: The stability study was carried for the formulation F2. The stability was determined in terms of its drug content, entrapment efficiency and in vitro drug release. The formulation was incubated for a period of 6 months at 37 ± 1°C [10] and at specified time intervals; the suspension was centrifuged at 4,000 rpm for 1 hr. The amount of drug present in the supernatant was determined by UV - Vis spectrophotometer at 228 nm.

[0034] 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 0.0094 mg/ml. Melting Point Determination: The melting point of pure drug Flutamide was determined in range of 111 - 113oC by capillary method. Identification of Pure Drug: FTIR spectrum of Flutamide has showed the 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 indicate presence of N = O and C - F3 group attached to C = C.

[0035] Determination of λmax: The Flutamide with pH 7.4 PBS was scanned between 400 - 200 nm with UV - Vis spectrophotometer and the λmax was found to be at 228 nm. Hence the standard curve of Flutamide was developed at 228 nm.

[0036] Drug - Excipient Compatibility Studies: Fourier Transform Infra-Red Spectroscopy (FTIR): FTIR studies used to determine the interaction between drug and excipients used in nanoparticle preparation. The mixture of drug and excipients were prepared in 1:1 w/w ratio and used for IR analysis. The IR spectra of Flutamide was recorded over than 600 - 4000 cm-1 using brooker spectrophotometer with KBr pellet method. Inference: IR spectra of formulation had showed absorption peaks which were comparable with absorption peaks of pure drug. The results indicated that, no chemical interaction between drug and excipients.

[0037] Standard Graph of Flutamide: The standard curve of Flutamide was done by using pH 7.4 PBS as the medium and the maximum absorbance was found at 228 nm. The standard graph was constructed between 2 to 20 µg/ml concentrations. Absorbances were examined under UV - visible spectrophotometer at an absorption maximum of 228 nm. The standard graph was constructed by taking the absorbance on Y - axis and concentrations on X - axis. Drug concentration and absorbance followed linear relationship the curve obeyed Beer - Lambert's law and the correlation coefficient value (R2) is 0.998.

[0038] PREPARATION OF NANOPARTICLES: Nanoprecipitation method was used for preparation of Flutamide loaded nanoparticles. The formulations were designed by 23 factorial design, which contained three variables like polymer concentration, stabilizer and organic solvent volume at two levels. Total eight batches were formulated (F1 - F8) and all the formulations were investigated for various parameters.

[0039] CHARACTERISATION OF PREPARED NPs: Size Analysis: Eight formulations were developed by varying the concentrations of polymer, stabilizer and volume of organic solvent at two levels and the effect on particle size was determined. Formulation F7 showed minimum particle size of 128 nm and the formulation F2 showed maximum particle size of 317 nm. The results indicate that the particle size increased with increasing polymer (PCL) concentration of 10 & 50 mg due to agglomeration of the particles [15]. The particle size decreased with increasing organic phase volume of 10 & 30 ml and the particle size decreased with increasing pluronic F-127 from1 - 1.5%.

[0040] Scanning Electron Microscopy (SEM): SEM image F1 shown formed nanoparticles at an average diameter of 194 nm with a minute amount of wrinkles on the surface. While image F2 shown two defferent sizes with less uniformity. The image also displays a large amount of larger nanoparticles surrounding the smaller nanoparticles with average diameter of 317 nm. On observing image F3 and F4 showed nanoparticles at an average diameter of 190 nm and 217 nm. Image F5 showed a complete spherical shaped nanoparticle at an average diameter of 148 nm. Image F6 showed the nanoparticles at an average diameter of 297 nm that have minimal wrinkles or porous structure on the exterior. Image F7 showed fully formed spherical shape smooth nanoparticles with an average diameter of 128 nm. SEM image F8 shown nanoparticles at an average diameter of 211 nm that have some wrinkles or porous structure on the exterior.

[0041] Surface Charge (Zeta Potential): The zeta potential of the formulation F7 was found to be - 46.4, which implies that it is having good stability. The negative zeta potential values of formulation F7 can be attributed to the presence of uncapped end ionized carboxyl groups of the polymer at the particle surface [16].

[0042] Drug Content: The nanoparticles obtained from F2 formulation showed maximum drug content (92 % ± 0.53) and F7 showed minimum drug content (74% ± 0.72). Drug content was increased with increasing polymer concentration of 10 & 50 mg. It was decreased with increased acetone volume of 10 & 30 ml and decreased significantly with increasing amount of surfactant pluronic F 127.

[0043] Entrapment Efficiency: On increasing the polymer concentration increasing (10 & 50 mg) entrapment efficiency was observed, whereas increase in acetone volume (10 & 30 ml) has slightly decreased the entrapment efficiency and increase in surfactant pluronic F127 (1% to 1.5%w/v) has significantly decreased entrapment efficiency. Drug entrapment efficiency varied from 75% ± 0.66 to 92% ± 0.70. The formulation F2 stabilized with 1% w/v pluronic F127 and 1:1 ratio of organic phase: aqueous phase volume showed maximum entrapment efficiency 92% ± 0.70.

[0044] Percentage Yield: The percentage yield of nanoparticles prepared by nanoprecipitation 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. It was found that when concentration of PCL increased, the percentage yields also increased. It was decreased with increased organic phase acetone volume and increased significantly with increasing amount of surfactant pluronic F 127. The percentage yields of nanoparticles of all formulations were in the range of 46.05 % ± 1.56 to 86.78% ± 1.32. The formulation F6 containing 50 mg PCL and 1:1 ratio organic: aqueous phase volume with 1.5 % pluronic F 127 showed maximum percentage yield of 86.78% ± 1.32 compare to other formulations.

[0045] In vitro release studies: The Flutamide PCL nanoparticles have exhibited fast release in the first 4 hr and slow / continuous sustained release in the next 20 hr. The fast release of drug was attributed to surface associated drug, where as the slow release was attributed to the drug entrapment in the nanoparticles. The drug release was slowed down due to the increased viscosity and diffusion path as the polymer ratio was increased. This was because of. High polymer concentration (50 mg PCL - F2, F4, F6 and F8) showed slow drug release for more than 24 hr (Figure B). Low polymer concentration (10 mg PCL - F1, F3, F5 and F7) showed almost complete drug release within 24 hr (Figure A). Increase in organic phase volume increases the rate of release of drug (Figure D) due to increased diffusivity and hydrodynamics at the interface. A slight rise in drug release was noticed Pluronic F127 concetration was increased (Figure F). The size of nanoparticle place a major role in drug release, smaller the size of nanoparticles higher the drug release rates. The formulation F7 containing particle size of 128 nm had shown maximum drug release of 98.23 % ± 1.24 compare to other formulations. The Flutamide nanoparticles (F2) prepared with 50 mg PCL, 10 ml acetone and 1 % pluronic F127 had shown better controlled and sustained drug release of 77.42 % ± 1.32 with in 24 hr. From the results of entrapment efficiency (92 %), drug content (92 %) and in vitro release studies (77.42 %) formulation F2 was considered as optimized trial.

[0046] Kinetic Modelling of Drug Release: The exact mechanism of drug release of F2 formulation was subjected to kinetic models. The R2 value of zero order and first order was found as 0.939 & 0.923 respectively. This result suggests that the drug released by zero order kinetics. The R2 value of Higuchi's and peppas diffusion equation was obtained as 0.993 and 0.710 respectively. This result suggests that the drug released followed diffusion mechanism.

[0047] Accelerated Stability Studies: The optimized formulation F2 was sealed in an aluminum foil and subjected to stability studies. The result showed that there was no significant changes occurs during storage after 90 days. , Claims:CLAIMS:
I/WE CLAIM:
1.A process of flutamide loaded poly caprolactone nanoparticles by factorial design, comprising:
a phosphate buffer solution;
a flutamide;
a 100 ml of pH 7.4 phosphate buffer solution was dissolved in 100 mg of accurately weighed
flutamide;
a 10 ml of this solution was further diluted with 7.4 pH phosphate buffer to get 100 μg/ml solutions;
whereby the stock solution different concentrations (2 to 20 μg/ml) were prepared with pH 7.4
phosphate buffer;
whereby from each concentration sample was taken and the absorption was measured at 228 nm
by using UV spectrophotometer and pH 7.4 phosphate buffer as a blank;
the optimization phase was carried out statistically using 23 factorial design in which the polymer
concentration, stabilizer and organic solvent volume were kept at two different levels;
flutamide loaded PCL nanoparticles were prepared by the nanoprecipitation method;
wherein this method involves the precipitation of polymer from an organic solvent and diffusion
of organic solvent into aqueous phase in the presence of a surfactant;
whereby PCL and flutamide were dissolved in acetone and this solution was added to pluronic f-
127 in pbs pH 7.4 at 1 ml/min speed using syringe under magnetic stirring conditions;
whereby the obtained suspension was stirred at 500 rpm for 2 hr to evaporate acetone;
the suspension was cooling centrifuge at 11,000 rpm for 30 minutes to remove precipitants and
supernatant was collected, lyophilized and stored at 4°c;
whereby repeated the procedure by varied in the concentrations of polymer, stabilizer and organic
solvent volume to optimize the formulation; and a blank nanoparticles were formed.

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