INTRODUCTION:

Pioglitazone is an insulin sensitizing thiazolidinedione (TZD) that activates a specific nuclear receptor (Peroxisome-proliferator activated receptor-γ [PPAR-γ]) found in adipose tissue, pancreatic β-cells, vascular endothelium and macro-phages¹. PARγ is a transcription factor, which when activated by TZDs, promotes transcription of insulin-sensitive genes involved in fatty acid and glucose uptake and lipogenesis, thereby enhancing or partially mimicking selective actions of insulin.

Pioglitazone also exerts several additional beneficial effects on blood pressure, micro albumin, inflammation, endothelial dysfunction, and lipids. It is well absorbed after oral administration and exhibit an oral bioavailability of approximately 83% with peak concentrations after approximately 1.5 h after administration^2,3. It is highly bound to plasma proteins (>99%) primarily to serum albumin and undergoes metabolism mainly by CYP3A4 and CYP 2C8/9⁴.

Diabetes mellitus is a combination of heterogeneous disorders commonly presenting with episodes of hyperglycemia and glucose intolerance⁵. Today, it is well established that the development of Type 2 diabetes mellitus results from an interaction of the subjects genetic makeup and environmental influences⁶. The main defects contributing to the development of Type 2 diabetes are impaired insulin secretion and insulin resistance in peripheral tissues, such as adipose, muscles and the liver⁷.

The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors⁸.

The main reason of degradation are the chemical instability of the drug substance under the conditions of heat, humidity, solvent, pH, and light encountered during manufacture, isolation, purification, drying, storage, transportation, and/or formulation is main cause of its degradation ⁹.

Fig 1: Chemical Structure of Pioglitazone hydrochloride.

The method of analysis is based on the measuring in the near (UV) ultraviolet path of spectrum (200- 380 nm). UV spectrophotometry can be used for stress-degradation studies of Pioglitazone. The active pharmaceutical ingredient is subjected to a number of forced degradation conditions to include acidic, basic and photo conditions as per ICH guidelines¹⁰. Forced degradation should be one of the activities performed early in the development process to ensure that the method is discriminating before a lot of time, effort, and money have been expended. It is important to determine the conditions responsible to degrade the drug. Earlier publications have reported high-performance liquid chromatography (HPLC) methods for Pioglitazone^{11, 12, 13}but no report is available for degradation studies of Pioglitazone by UV. Thus, in the present study Ultraviolet-visible (UV-Vis) spectroscopy was used for the analysis of forced degradation of Pioglitazone, as it is a simple, rapid and cost effective method as compared to HPLC.

ACID/BASE STRESS TESTING:

Acid/base stress testing is used for the evaluation of forced degradation of a drug substance. This test involves degradation of a drug substance by exposure to basic or acidic medium over time to its primary degradation products. In carbonyl functional group like alcohol, imines, imides, amides (lactums), esters (lactones) aryl amines and base degradation take place by hydrolysis.

DEGRADATION BY UV LIGHT:

Many formulation or products made of synthetic or natural polymer are UV unstable. They degrade or disintegrate on its exposure to continuous sunlight. The degree of degradation or disintegration is dependent upon degree of exposure.

THERMAL (HEAT) AND HUMIDITY STRESS TESTING:

Many drugs are thermal and humidity sensitive. Thermal (Heat) and humidity may cause degradation of a substance. This test is performed by exposing the drug or formulation to thermal / humidity condition. (895d and 63603)

EXPERIMENTAL:

Material and Reagents:

API of Pioglitazone was kindly supplied as a gift sample by Aarti Drugs Ltd.( Mumbai).

Reagents:

Sodium hydroxide (NaOH), Hydrochloric acid (HCL) and Double distilled water was procured from Research lab Islampur (INDIA) 415409. All chemicals and reagents were of analytical grade (AR).

Glass wares:

All the glass material used in this research work namely test tube, beaker, measuring cylinder, funnel, pipette, volumetric flask and stirrer was made up of Pyrex glass. All the glass ware was first washed by chromic acid then rinsed with water and finally with distilled water.

Instruments:

UV-Spectrophotometer (UV- 610 Jasco) with quartz cuvettee. Weighing balance. Constant temperature water bath.

Preparation of Solution:

Following methods were used for the preparation different solutions.

Preparation of 0.1 N Hydrochloric acid solution:

0.1 N hydrochloric acid was prepared by taking 8.3 ml hydrochloric acid in 100 ml volumetric flask having purity 37% and final volume was adjusted with distilled water.

Preparation of 0.1 N sodium hydroxide solution:

Accurately weighed 4 gm of sodium hydroxide (NaOH) was taken and transferred to a 100ml volumetric flask. Small amount of water was added to it to dissolve NaOH and final volume was made up with distilled water.

Preparation of Pioglitazone Solution:

A solution containing 200ppm of Pioglitazone was prepared as per the following procedure. Accurately weighed 0.020gm of Pioglitazone was transferred into a 100ml volumetric flask. Small amount of water was added to dissolve Pioglitazone and the final volume was made up with distilled water. The absorbance of prepared solution of Pioglitazone was measured using a UV spectrophotometer at a maximum wavelength 269 nm.

PROCEDURE FOR DEGRADATION STUDIES:

For acid:

To study degradation in acid medium or to determine effect of acid on Pioglitazone, 5ml of 200ppm pioglitazone solution was taken and 5ml of 0.1N hydrochloric acid (HCL) was added to it and the solution was left for 30 minutes. After 30 minutes absorbance of solution was measured by uv spectrophotometer at maximum wavelength 269nm.

For base:

To study degradation in basic medium or to determine effect of base on Pioglitazone, 5ml of 200ppm Pioglitazone solution was taken in a test tube and 5ml of 0.1 N sodium hydroxide solution was added to it and left for 30 minutes. After 30 minutes absorbance of solution was measured by UV spectrophotometer at maximum wavelength 269nm.

For UV light:

To study degradation in UV light medium or to determine effect of uv light on Pioglitazone, 5ml of 200ppm Pioglitazone solution was taken in a test tube and add 5ml of distilled water was added to it left for 30 minutes in UV light of 320nm. After 30 minutes absorbance of solution was measured by UV spectrophotometer at 269 nm.

For thermal (Heat):

To study degradation in thermal medium or to determine the effect of heat on Pioglitazone, 5ml of 200ppm Pioglitazone solution was taken and 5ml of distilled water was added to it and the mixture was left for 30 minutes in water bath at 50⁰ C. After 30 minutes absorbance of the solution was measured by UV spectrophotometer at maximum wavelength 269 nm.

RESULT AND DISCUSSION:

Degradation studies are an essential step in the development of new drug product. Many factors are responsible for degradation of a drug or product like temperature humidity, and light. In the present research work we studied effect of various factors like acid, base, UV light and heat on Pioglitazone.

Absorbance of various parameters for the drug are represented in (Table 1) and in (Figure 2), where as the degradation pattern in percentage is shown in (Table 2) and in (Figure 3).

Table 1: Absorbance of Pioglitazone.

Degradation Parameters	Pioglitazone
Degradation Parameters	1	2	3	Average
Before	0.4460	0.4462	0.4460	0.4460
After acid	0.4420	0.4419	0.4421	0.4420
After base	1.1620	1.1621	1.1620	1.1620
After heat	0.1957	0.1957	0.1958	0.1957
After U.V	0.3686	0.3687	0.3687	0.3686

The studies showed that when Pioglitazone was subjected to 0.1N HCl, it does not exhibited significant changes in terms of availability (99.4%). Also, when the drug was subjected to 0.1 N NaOH, it showed highly significant change in terms of availability (260.54%).

Pioglitazone on its exposure to heat for 30 minutes, exhibited a decreased availability i.e. (43.88%) and on its exposure to UV light, a decreased availability of (82.65%) was observed.

Thus, from the results of our study it can be concluded that Pioglitazone do not degrade when introduced in acidic medium i.e. 0.1N HCl. (99.4 %). but degrades to greater extent when subjected to basic medium i.e. 0.1N NaOH i.e. 260.54 %. Also, when Pioglitazone was exposed to U.V light (269 nm) and heat for 30 minutes, a high degradation of 82.65% and 43.88% respectively was observed.

CONCLUSION:

According to specification given in WHO monographic for Pioglitazone, the official assay limit of the content should not less than 98% and not more than 102% of the estimated potency. From the results of our study it can concluded that Pioglitazone degrades most when exposed to the basic medium and heat but do not undergo degradation in acidic medium, whereas slight degradation occurs in the UV.

REFERENCES:

1. Waugh J, Keating G.M, Plosker G.L, Easthope S, And Robinson D.M. Pioglitazone A Review of its Use in Type 2 Diabetes Mellitus. Adis Drug Evaluation. 66 (1); (2006): 85-109.

2. Dorkhan M. And Ers F. A review of pioglitazone HCL and glimepiridein the treatment of type 2 diabetes. Vascular Health and Risk Management 3(5); (2007): 721–731.

3. Stanley S.S. Pioglitazone for the treatment of type 2 diabetes in patients inadequately controlled on insulin. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 3 (2010): 43–252.

4. Saha S.K, Chowdhury A.K.A, And Bachar S.C. Pharmacokinetics Study of Pioglitazone (30 mg) Tablets in Healthy Volunteers. Dhaka Univ. J. Pharm. Sci. 13(2); 2014: 181-186.

5. Pierol M.N, Nzaro G.M. And Njagi J.M. Diabetes mellitus–a devastating metabolic disorder. Asian Journal of Biomedical and Pharmaceutical Sciences. 4(40); (2014): 1-7.

6. Harald S. And Thomas C.W. Pioglitazone in the management of Type 2 diabetes and beyond. Drug Evaluation – Sourij and Wascher Therapy. 4(5); (2007): 517–533.

7. Richard E.P. The Early Treatment of Type 2 Diabetes. The American Journal of Medicine. 126(9A); (2013): S2-S9.

8. ICH Topic Q 1 A (R2)Stability Testing of new Drug Substances and Products. European Medicines Agency, August 2003. CPMP/ICH/2736/99.

9. Hotha K.K, Reddy S. P.K, Raju V.K. And Ravindranath L.K.Forced Degradation Studies: Practical Approch- Overview Of Regulatory Guidance And Literature For The Drug Products And Drug Substances. International Research Journal of Pharmacy. 4 (5); 2012: 78-85.

10. Food and Drug Administration, HHS International Conference on Harmonisation; Stability Data Package for Registration Applications in Climatic Zones III and IV; Stability Testing of New Drug Substances and Products; availability. Notice. Fed Regist 68. 2003, 65717-65718.

11. Sharmab S, Sharma M.C. And Chaturvedic S.C. Study of stressed degradation behavior of pioglitazone hydrochloride in bulk and pharmaceutical formulation By HPLC assay method. Journal of Optoelectronics and Biomedical Materials. 1(1); 2010: 17 – 24.

12. Ashour S, Sakur A.A. And Kudemati M. Development and Validation of Stability Indicating HPLC Method for Quality Control of Pioglitazone Hydrochloride. Canadian Chemical Transactions. 3(1); (2015): 1-11.

13. Rashmitha N, Hiriyanna S.G, Sreenivasa Rao C.H. Chandra Sekhar Reddy K, HariKiran M, Sharma H.K. And Mukkanti K. A Validated Stability Indicating HPLC method for the Determination of Impurities in Pioglitazone Hydrochloride. Scholars Research Library Der Pharma Chemica. 2(5); 2010: 426-433.

Received on 11.02.2018 Accepted on 20.03.2018

Asian J. Pharm. Ana. 2018; 8(3):125-128.

DOI: 10.5958/2231-5675.2018.00023.6

Degradation Parameters	Pioglitazone
Degradation Parameters	1	2	3	Average
Before	100 %	100.06 %	100 %	100.02 %
After acid	99.10 %	99.08 %	100.02 %	99.4 %
After base	260.53 %	260.56 %	260.53 %	260.54 %
After heat	43.87 %	43.87 %	43.90 %	43.88 %
After U.V	82.64 %	82.66 %	82.66 %	82.65 %