Development and Evaluation of Sustain Release Pioglitazone Microsphere

 

Anand Patel*, Dr. C. C. Patil, Dr R. V. Kulkarni, Dr. Vijaykumar A, Prashant Jorapur, Akshay Deshpande, Swapna W

B.L.D.E.A’s SSM College of Pharmacy, Vijayapura B.LD.E University Campus-586101

 

ABSTRACT:

The current aim of this work was to development and evaluation of sustain release pioglitazone microsphere. The microspheres were prepared by solvent evaporation method using polymers such as Ethyl cellulose, HPMC, Eudragit RL100. The prepared microspheres were characterized for particle size analysis, DSC, FTIR, XRD, DEE, in-vitro drug release and in vivo antidiabetic study. The in vitro drug release study suggested that the microspheres were capable of releasing drug up to 24 hours depending upon the formulation variables; The drug release was slow from the microspheres which were formulated with ethylene cellulose and eudragit RL100 as compared to those prepared with HPMC &Ethylene cellulose, HPMC & Eudragit RL100. The average particle size was in the range of 120-210 µm. Drug entrapment efficiency of pioglitazone loaded microspheres found in the range of 73.63% to 88.32%. The DSC analysis and X-ray diffraction study indicated that the drug uniformly dispersed in amorphous state in molecular level. The in-vivo anti-diabetic activity reveals that, the pure drug pioglitazone has shown maximal reduction in blood glucose at 2 hr and then reduction in blood glucose was decrease. But with the rat treated with p3 microsphere, the reduction in glucose level was initially slowly as compared to pure pioglitazone drug up to 2 hrs but it was slowly increased to 52.49% at 12 hr, suggesting the sustained and uniform release of pioglitazone over longer period from microsphere.

 

 

 

1.1 INTRODUCTION:

Primary objectives of sustain drug delivery system are to ensure the safety and to improve efficiency of drug as well as patient compliance. Pioglitazone is an oral anti diabetic agent belonging to the class of thiazolidinediones that acts primarily by decreasing insulin resistance. It is used in the management of type 2 diabetes mellitus. It improves sensitivity to insulin in muscle and adipose tissue and inhibits hepatic gluconeogenesis also improves glycemic control while reducing circulating insulin levels. The most common adverse effect of Pioglitazone are respiratory tract infection, headache, sinus infection, muscle pain, sore throat.

 

 

Due to side effects of pioglitazone a sustained release medication is required to get prolonged effect with reduced fluctuations in drug plasma concentration levels. Mucoadhesive microspheres can provide the sustained release of drug and advantage for pioglitazone in the management of type-2 diabetes with high margin of safety and reduced side effects.

 

1.2 ESTIMATION OF PIOGLITAZONE MICROSPHERE:

Spectrophotometric method based on the measurement of absorbance at  269 nm of UV region in 0.1 N HCL of PH 1.2. Phosphate buffer of PH 7.4 were used for the estimation of pioglitazone.

 

Prepration of standard curve of pioglitazone in PH 1.2 (0.1 N HCL):

100 mg of pioglitazone was weighed and dissolved in 40 ml methanol and volume was made upto 100 ml with 0.1N HCl. This was primary stock solution containing 1000µm/ml. From this stock solution,1ml was pipette out and transferred in to a 100 ml volumetric flask and volume was made up to100 ml with 0.1 N HCl  which contained the concentration of 1µg/ml (second stock solution ).from this second stock solution aliquots equivalent to 2-10 µg(2,4,6,8,and 10ml were pipette out in series of 100 ml volumetric flask and volume was made up to 10 ml with 0.1 N HCl .the absorbance of these solution was measured against the 0.1 N HCl  as blank at 269 nm using UV –visible double beam spectrophotometer. then calibration curve was plotted taking concentration in µg/ml on X-axis and absorbance on Y-axis¹.

 

Prepration of standard curve of pioglitazone in PH 7.4 Phosphate Buffer:

100 mg of pioglitazone was accurately weighed and dissolved in 40 ml methanol and volume was made upto 100 ml with 7.4 phosphate buffer. This was primary stock solution containing 1000µm/ml. From this stock solution,1ml was pipette out and transferred in to a 100 ml volumetric flask and volume was made up to100 ml with 7.4 phosphate buffer which contained the concentration of 1µg/ml (second stock solution ).from this second stock solution aliquots equivalent to 2-10 µg(2, 4, 6, 8 and 10ml) were pipette out in series of 100 ml volumetric flask and volume was made up to 10 ml withs7.4 phosphate buffer .the absorbance of these solution was measured against the 7.4 phosphate buffer  as blank at 263 nm using  UV –visible double beam spectrophotometer. Then calibration curve was plotted taking concentration in µg/ml on X-axis and absorbance on Y-axis².

 

1.3 Prepration of pioglitazone microspheres by solvent evaporation method:

pioglitazone microspheres were prepared based on solvent evaporation technique. Weighed quantities of drug and polymers were dissolved in mixture of dichloromethane and ethanol (1:1 solvent ratio) at room temperature. This solution was poured into 100 ml distilled water containing 0.5% of PVA solution. The resultant emulsion was stirred with a propeller type agitator at 900 rpm for 2-3 hour to allow the volatile solvent to evaporate. The microspheres formed were filtered, washed with water and dried overnight at room temperature.

 

1.4 The prepared microspheres were evaluated by the following parameter:

1. Particle size analysis:

Particle size analysis was carried out by using optical microscopy. About 1000 microsphere  Randomly selected and their sizes were determined by using optical microscope fitted with standard  micrometer scale³.

 

2. Scanning electron microscopy study:

The surface morphology of the microsphere was analysed by means of scanning electron microscope (Model JSM 5400, Jeol, Tokyo,Japan). The microspheres were previously fixed on a brass stub using double-sided adhesive tape and then were made electrically conductive by coating, in a vacuum, with a thin layer of platinum (3–5 nm), for 100 s and at 30 W³.

 

3.Differential scanning calorimetric analysis:

by using Differential scanning calorimeter, DSC thermogram of microspheres was recorded (DSC 823 Mettler Toledo, Japan). Samples were accurately weighed onto aluminum pans and then thermetically sealed with aluminum lids. Thermogram were obtained at a scanning rate of 10°C/min conducted over a temperature range of 30-300°C in theenvironment of liquid nitrogen⁴.

 

4.Fourier transform infrared spectroscopy analysis:

Interaction between drug–polymer was studied by infrared spectroscopy using FTIR spectrometer (shimadju, Jasco-Japan) with diffuse reflectance principle. Sample preparation involved mixing the sample with potassium bromide (KBr), triturating in glass mortar and finally placing in the sample holder. The spectrum was scanned over a frequency range 4000-400 cm-1⁴.

 

5. X-ray diffraction studies:

Powder X-ray diffractometric analyses were performed using an X-ray diffractometer (Rigaku, D-Max/2200). Diffraction of raw crystals and microspheres of pioglitazone were measured. Before measuring the X-ray powder diffractogram, the microspheres were crushed into powder because they were too big to measure. The data were collected in the continuous scan mode using step size of 0.01O 2θ. The scanned 2 range was 2θ to 35O⁵.

 

 

 

 

Table.1: Formulation Table Of Pioglitazone Microsphere By Solvent Evaporation Method

Drug (mg)	P1	P2	P3	P4	P5	P6	P7	P8	P9
Pioglitazone	100	100	100	100	100	100	100	100	100
Ethyle cellulose	150	200	250	150	200	250	-	-	-
HPMC	150	200	250	-	-	-	150	200	250
Eudragit RL100				150	200	250	150	200	250
DCM (ml)	5	5	5	5	5	5	5	5	5
Ethanol (ml)	5	5	5	5	5	5	5	5	5
PVA 10%	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Stirring speed (RPM)	500	500	500	500	500	500	600	700	800

6. Drug encapsulation efficiency:

100 mg of dried microsphere were weighted accurately and drug was extracted from microspheres by digesting for 24 hrs with 100 ml of (pH 7.4). During this period the suspension was agitated. After 24 hrs the suspension was centrifuged at 2000 rpm for about 3 minutes. The solution was filtered through 0.45 mm membrane filter, and the filtrate was analyzed for drug content at 263 nm. Entrapment efficiency was calculated according to equation.

 

Drug             =  Practical drug content/therotical drug content X 100⁶.

entrapment

efficiency

 

7. In-vitro drug release study:

The release rate of pioglitazone from microspheres was determined using USP dissolution testing apparatus I (Basket type). The dissolution test was performed using 900 ml of 0.1N HCl, at 37±0.5°C and 100 rpm. Microspheres equivalent to 100 mg of pioglitazone were used for the test. A 5 ml sample solution was withdrawn from the dissolution apparatus for 1 h, and there after every 1h upto 12h. Samples were replaced by its equivalent volume of dissolution medium. The samples were filtered through Whatmann filter paper and solutions after appropriate dilution were analyzed at 269 nm by UV Spectrophotometer.

 

8. In vivo-pharmacodynamic study:

Wistar rats ( 150-250 gm) of either sex were selected for the study. They were housed in standard polypropylene cages and kept under controlled room temperature. The rats were given standard laboratory diet and water. Protocols were approved by the Institutional Animal Ethics Committee, Registered under CPCSEA.

 

Streptozotocin treatment:

The study was performed in streptozotocin induced diabetic rats. The rats were made diabetic by intraperitoneal injection of freshly prepared streptozotocin solution at a dose of 60 mg/kg dissolved in water for injection. After 48 hrs of streptozotocin administration, the rat  with blood glucose level of 180 mg/dL of more were considered diabetic and were used in the study. The diabetic rats were divided into 4 groups of 6 rats each and treated as follows,

 

Group 1: Normal control rats,

Group 2: Diabetic control rats, 

Group 3: Standard rats and,

Group 4: Test Rats

 

Blood samples were collected from retro-orbital plexus of each rats under mild anesthesia at 0, 2, 4, 6, 8 and 12 hrs after administration and they were analyzed for blood glucose.

The in-vivo anti-diabetic activity reveals that, the pure drug pioglitazone has shown maximal reduction in blood glucose at 2 hr and then reduction in blood glucose was decrease. But with the rat treated with p3 microsphere, the reduction in glucose level was initially slowly as compared to pure pioglitazone drug up to 2 hrs but it was slowly increased to 52.49% at 12 hr, suggesting the sustained and uniform release of pioglitazone over longer period from microsphere.

 

RESULT:

 

Figure. 01: Standard calibration curve of  pioglitazone in pH 1.2 buffer

 

Figure .02: Standard calibration curve of pioglitazone in pH 7.4  buffer

 

 

 

 

Figure. 03: Scanning electron microscopic photographs of (A)P4,  (B)P9, Microspheres.

Table. 02:- Average size and drug entrapment efficiency (DEE) of pioglitazone microspheres.

Serial. No	Microspheres code	Average size (µm)	DEE (%)
1	P1	120+ 3.4	80.85+0.90
2	P2	130+ 2.7	83.41+0.93
3	P3	140+ 2.8	84.58+1.12
4	P4	130 + 2.7	82.10+0.99
5	P5	140+ 2.7	76.21+1.22
6	P6	150+ 2.7	84.7+0.79
7	P7	140+ 2.7	77.78+0.89
8	P8	130+ 2.7	78.11+0.93
9	P9	120+ 2.7	77.15+0.93

The values are average of three determinations. +  indicates SD values:

 

 

 

 

 

 

 

Figure 04: Infrared spectroscopy of pioglitazone, P3 Formulation.

         

Figure 05:- DSC thermograms of pioglitazone (A), Dummy of P3 (B) and drug loaded P3 microspheres (C).

 

 

 

 

 

Figure 06; X-Ray Diffractograms of pioglitazone (A), Drug loaded microspheres P6 (B) Dummy P6 (C) Pure Drug

 

 

Figure 07 - In-vitro drug release study of pioglitazone microsphere

 

 

Figure 08; Reduction of blood glucose in experimental animals treated with pioglitazone and P3 microspheres.

 

Figure 09: Blood glucose levels of different groups of experimental animals

 

DISCUSSION:

The sustain release pioglitazone microsphere were prepared by solvent evaporation methods from polymers Ethyl cellulose, HPMC, and Eudragit RL100, The prepared sustain release microspheres were evaluated for different physicochemical test such as particle size, drug entrapment efficiency, in-vitro drug release behaviors. Preliminary experiments conducted on formulation of the microspheres showed that Eudragit RL100/HPMC/of greater than 1:1:1, temperature of 20-30 ⁰C stirring rate of 500-1200 rpm should be used in order to obtained  microspheres of adequate characteristics. The mean particle size of the microspheres was  range 120+2.6 µm to 210+4.6 µm. The drug entrapment efficiency of prepared microspheres was carried by swelling method and results are noted table no 01. The drug entrapment efficiency was found to be in the range of 76.21% to 84.58 % The drug entrapment efficiency increases with increase in the concentration of polymer.

 

The Drug-Polymer compatibility was confirmed by infrared Spectrophotometric study Figure no 04. FT-IR studies indicated that the principal peaks of the plain drug are not altered in the spectra of formulation. The DSC analysis has been one of the most widely used calorimetric technique to characterise the physical state of drug in the formulations. Fig no-05 depict the DSC thermogram of pioglitazone (A), drug free P3 (B), microspheres  formulation P3 (C). The DSC thermogram of Pioglitazone exhibited a single sharp endothermic peak at 89⁰C due to its melting transition temperature. Finally the thermograms showed no such characteristic peak, indicating that the drug was uniformly dispersed at the molecular level in microspheres.

 

The X-ray diffractograms  of pure drug, drug free microspheres and drug loaded microspheres are presented in Fig no 06. pioglitazone has shown characteristic intense peak between the 2Ө of 10.35 and 32 due to its crystalline nature. Where as in case of drug free microspheres and drug loaded microspheres, no intense peaks related to drug were noticed between 2Ө of 10.35 and 32 and both the  diffractograms are identical. This indicates that drug is amorphously dispersed after entrapment in the microspheres.

 

The dissolution profile of pioglitazone are given in figure no 07; data are presented in above table & figure shows results of in-vitro drug release. A 95.11, 92.57, 90.45, 93.97, 83.91, 78.83, 94.08, 91.56, 89.42 drug was released from P1, P2, P3, P4, P5, P6, P7, P8, P9 formulations respectively at the end of 24 hour, whereas results shows the controlled and sustained drug released at the extended period of time.

 

CONCLUSION:

In this work, an attempt was made to prepare and evaluate pioglitazone loaded microspheres using HPMC, Eudragit RL100 and Ethyl cellulose by solvent evaporation method. The drug entrapment efficiency of the prepared microspheres was found to be in the range of 76.21 % to 84.58 %. The DSC and XRD analyses indicate that the drug was uniformly dispersed in an amorphous state in the microspheres. The in vitro drug release study indicated that the microspheres were capable of releasing drug up to 24 hours depending upon the formulation variables. The drug release was slow from the microspheres which were prepared with Eudragit RL100 as compared to those prepared with HPMC and Ethyl cellulose. Drug release mechanism followed non-Fickian transport. The in-vivo anti-diabetic activity reveals that, the pure drug pioglitazone has shown maximum reduction in blood glucose at 2 hrs and then the reduction in blood glucose was decreased. But with the rats treated with P3 microspheres, the reduction in glucose level was initially slow as compared to pure pioglitazone up to 2 hrs, but it was slowly increased to 52.49 % at 12^th hr, suggesting the sustained and uniform release of pioglitazone over longer period from microspheres.

 

REFERENCE:

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4.     Pande AV, Vaidya PD, Arora A, Madhura V. Dhoka. In-vitro and In-vivo evaluation of ethyl cellulose based floating microspheres of Cefpodoxime proxetil,Int J Pharm Biomed Res 2010, 1(4), 122-128.

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Accepted on 30.08.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(12): 5348-5354.