Received Date: December 17, 2014; Accepted Date: February 13, 2015; Published Date: February 20, 2015
Citation: Agatonovic-Kustrin S, Morton DW, Singh R, Baboota S, Talegaonkar S (2015) Biorelevant Dissolution Studies of Pioglitazone HCL Immediate Release Tablets and the Determination of an In Vitro In Vivo Correlation. J Bioequiv Availab 7:086-089. doi: 10.4172/jbb.1000220
Copyright: © 2015 Agatonovic-Kustrin S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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In vitro dissolution testing using biorelevant dissolution media was used to the predict food effects on the absorption of Pioglitazone from immediate release tablets.Dissolution testing was carried out in three dissolution media: hydrochloric acid solution at pH 1.2, in simulated gastric fluid to simulate the gastric fasted state,and in pasteurized milk to simulate the gastric fed state. In order to validate these results, in vivo studies were conducted on young Wistar rats under fed and fasted conditions. The in vivo studies showed that food delays the onset of action of the drug significantly. The compendial dissolution test in hydrochloride solution at pH 1.2 was not able to predict the food effect. However, the biorelevant dissolution test in simulated gastric fluids predicted correctly that the release of Pioglitazone would be slower in the fed state than in the fasted state. The results demonstrate good correlation between in vitro drug release and in vivo drug absorption using the biorelevant dissolution test method.
Biorelevant dissolution, Pioglitazone, Food effects
The use of high throughput screening approaches for discovery of new compounds with pharmacological activity has resulted in the development of drugs with a wide range of physical and chemical properties. To assess whether these compounds possess adequate bioavailability following oral administration, in vitro tests that are capable of predicting in vivo performance need to be developed. The effectiveness of oral dosage forms relies on the drug dissolving in the gastrointestinal tract fluids prior to absorption into the systemic circulation. Therefore, it is important to be able to predict the rate of dissolution in vivo. However, dissolution testing in standard media does not always correlate with in vivo results because it does not adequately simulate the physiologic gastrointestinal (GI) environment. Several studies have been carried out to determine the circumstances under which dissolution testing can be predictive for in vivo performance in order to design appropriate dissolution testing factors such as composition, volume, flow-rates and mixing pattern of the fluid to simulate the GI environment [1-2]. Due the fact that the majority of drugs currently in development are poorly soluble drugs, it is important that adequate predictive dissolution testing procedures are available for use in drug development and formulation.
Drug dissolution is an important rate limiting factor in drug absorption and is directly related to the clinical response for most drugs given orally . In 1995, Amidon and coworkers introduced the biopharmaceutical classification system (BCS) of drugs to reduce the need for in vivo bioequivalence studies, by the utilization of in vitro dissolution tests as a surrogate for in vivo bioequivalence studies [2,4]. When combined with the in vitro dissolution characteristics of the drug product, the BCS takes into account three major factors: solubility, intestinal permeability, and dissolution rate, all of which govern the rate and extent of oral drug absorption from immediate release solid oral dosage forms .
According to the BCS, drugs can be divided into four classes on the basis of their aqueous solubility and permeability through gastrointestinal mucosa. Class I drugs are defined as those with high intestinal permeability which are able to dissolve readily in aqueous media over the pH range 1 to 8, while Class III are drugs with low permeability but high solubility. The absorption of class III drugs can be successfully predicted by in vitro dissolution studies because their solubility is good but permeability is poor. Thus, for Class I and III drugs, dissolution is not the rate limiting step to the oral absorption of these drugs. However, class II and IV drug compounds have limited water solubility, so the choice of dissolution medium is often important in determining the rate of dissolution due to the low solubility of these drugs in aqueous media. Moreover, the solubility of these drugs is insufficient for the whole dose to be dissolved in the gastrointestinal contents under normal conditions. Since dissolution for these substances is the rate limiting step to absorption, the media used in dissolution testing needs to closely represent the existing conditions in the gastrointestinal tract in order to achieve a meaningful in vitro/in vivo correlation [1,6]. For these types of drugs biorelevant dissolution testing with two new dissolution media, milk (3.5 % fat), and the USP simulated gastric fluid  with or without pepsin (SGF/SGFsp), are designed to reproduce fed and fasted state conditions in the GI tract . Several studies indicated that biorelevant dissolution media have strong advantages over compendia media in predicting the in vivo performance of certain drug formulations  and can serve as an in vitro surrogate for in vivo bioequivalence studies and for in vivo and in vitro correlation (IVIVC) studies [9-11].
Pioglitazone HCl (PGZ) is an oral antidiabetic agent that acts by decreasing insulin resistance and is used in the management of type II diabetes mellitus. PGZ improves sensitivity to insulin in muscle and adipose tissues and inhibits hepatic gluconeogenesis. It improves glycaemic control while reducing circulating insulin levels . It is reported that oral administration of drug in fasting state shows peak plasma concentration (Cmax) within 2 hours while food delays the time to reach peak plasma concentration (Tmax) to 3 to 4 hours but does not alter the extent of absorption . This suggests that food may affect the onset of action, Cmax, Tmax, and bioavailability of PGZ.
Due to its low permeability and low solubility, pioglitazone hydrochloride is classified as a class IV drug with limited or no in vitro/ in vivo dissolution correlation expected. Note that compounds within this class have poor bioavailability and high inter-individual variability. When attempting to develop a suitable dissolution testing procedure for oral dosage forms of class IV drugs, the selection of an appropriate biorelevant dissolution medium is critical as poor solubility is likely to have an impact on the observed IVIVCs. The aim of this work was to evaluate the performance of pioglitazone hydrochloride oral formulations using USP apparatus II and biorelevant dissolution media and to assess whether biorelevant dissolution studies can be used to successfully predict its’ bioavailability in vivo.
Chemicals and solvents
All chemicals and solvents used were of analytical or HPLC grade. PGZ was supplied by the Ranbaxy Laboratory, India. The purity and identity of the drug substance was determined in preformulation studies by assessing its physical characteristics (physical appearance, melting point and solubility) and using a number of instrumental techniques (FTIR spectral analysis, UV spectral analysis and assay by HPLC). The results obtained complied with the monograph requirements for PGZ in the Physician’s Desk Reference .
Sodium lauryl sulphate, ammonium hydroxide, alloxane monohydrate, disodium EDTA, hydrochloric acid, and methanol were purchased from SD Fine Chemicals Ltd, India. Citric acid, disodium hydrogen phosphate, 1,2-dichloroethane, acetonitrile, sodium chloride, sodium hydroxide, and potassium chloride were obtained from Merck India Ltd. Potassium dihydrogen phosphate, ortho-phosphoric acid, acetic acid, potassium hydroxide were obtained from Qualigens India Ltd. Generic PGZ immediate release tablets, Pioglar 15 (Pioglitazone, 15 mg) by Stancare (Ranbaxy Laboratories Ltd, India) were also used.
A hydrochloric acid buffer pH 1.2 (HAB pH 1.2) was prepared by adding 425.0 mL of 0.2 M hydrochloric acid to 250.0 mL of 0.2 M potassium chloride and diluting to a total volume of 1000 mL with purified water. The pH was then adjusted to 1.2 using a small amount of hydrochloric acid.
Simulated gastric fluid (SGF) (without enzyme) for the fasted state was prepared by dissolving 2.5 g of sodium lauryl sulphate (SLS) and 2.0 g of sodium chloride in HAB pH 1.2 and then making up to a total volume of 1000 mL using HAB pH 1.2. The pH was then adjusted to 1.2 with 2-3 drops of hydrochloric acid (0.01-0.05 M). Pasteurized milk was used as the dissolution medium to simulate the gastric fed condition.
A double beam UV Spectrophotometer (UV 1601 Shimadzu, Japan), HPLC (Model SPD-10AVP, Shimadzu, Japan), IR Spectrophotometer (FT/IR-410 Jasco, Japan), USP dissolution apparatus (Hanson Research SR8 plus), and differential scanning calorimeter (Pyris-6 DSC, Perkin Elmer Germany) were used.
HPLC assay conditions
HPLC analysis was carried out using a Symmetry C18 column (5μm, 250 x 4.60 mm) at a flow rate of 1.0 mL/minute at room temperature, with an injection volume of 10 μL. The mobile phase was a 10 mM potassium dihydrogen phosphate-acetonitrile (50:50 v/v) solution adjusted to pH 6.0 using a 0.1 M potassium hydroxide solution, with peak detection at λ = 225 nm. The calibration curve was obtained by plotting the peak areas against standard concentration. Good linearity (R2 = 0.999) was obtained over the investigated drug concentration range.
Dissolution of PGZ in pH 1.2 hydrochloric acid buffer (HAB pH 1.2): A 100 μg/mL solution of PGZ in HAB pH 1.2 was used as the standard stock solution for the dissolution studies in HAB pH 1.2. A series of standard solutions in the range of 2-20 μg/mL were then prepared using the standard stock solution. A calibration curve was constructed by plotting standard absorbances against their corresponding concentrations and using the least square method. The curve obeyed Beer Lambert’s law in the investigated concentration range with a high correlation coefficient (R2 = 0.997) indicating good linearity.
Dissolution tests were performed in triplicate using USP Type II dissolution test apparatus at 37 ± 0.5°C and a 50 rpm paddle speed, using 900 mL of HAB pH 1.2 as the dissolution medium. The dissolution medium was pre-warmed to the required temperature before dissolution testing commenced. Aliquots of 5 mL were periodically withdrawn at intervals of 2 min and each time the volume withdrawn it was replaced with an equivalent amount of fresh dissolution medium. The absorbance of each sample was measured at a wavelength of 225 nm and the concentration in μg/ml calculated from the calibration curve.
Dissolution of PGZ in SGF: A 100 μg/mL standard working solution was prepared by dissolving PGZ in SGF and then used to make a series of standard solutions in the range of 20-200 μg/mL using SGF. HPLC analysis was carried out using a Shimadzu chromatograph, Model SPD-10AVP with a Symmetry C18, 5μm, 250 x 4.60 mm column, at a flow rate of 1.0 mL/minute at room temperature. 10 mM potassium dihydrogen phosphate-acetonitrile (50:50 v/v) adjusted to pH 6.0 with 0.1 M potassium hydroxide was used as mobile phase. Peak detection was performed at λ = 225 nm. The calibration curve was obtained by plotting the peak areas against nominal concentration. Good linearity (R2 = 0.999) was obtained over the investigated drug concentration range.
Dissolution studies were performed in triplicate using USP dissolution Type II dissolution test apparatus at 37 ± 0.5°C and 50 rpm paddle speed using 400 mL of SGF. The dissolution medium was pre-warmed to the required temperature before dissolution testing commenced. Aliquots of 5 mL were periodically withdrawn at frequent intervals and fresh medium of SGF (pH 1.2) of 5 mL was replaced each time. The dissolution test continued for 1 hour 40 minutes.
Dissolution of PGZ in pasteurized milk: A solution of 100 μg/ mL of PGZ in anhydrous ethanol was used as a standard stock solution for dissolution in pasteurized milk. From this stock solution serial dilutions in the concentration range of 20-100 μg/mL were made. These dilutions were spiked with 2 mL of milk and the drug was extracted and reconstituted. 2 mL of milk sample was centrifuged at 3000 rpm for 30 minutes and diluted with 4.0 mL of citric acid/disodium hydrogen phosphate buffer (0.083 M/0.033 M, pH 2.8). The mixture was then rotated with 4.0 mL of 1,2-dichloroethane for 3 minutes, and centrifuged at 3000 rpm for 15 minutes. The organic layer was pipetted out and passed through a sodium sulphate drying tube and then evaporated to dryness. The dried residue was then reconstituted in mobile phase . A graph between mean peak area (y-axis) versus concentration (x-axis) was then plotted. It was found that there was a good linear relationship between mean peak area and concentration with an observed correlation coefficient of 0.999.
Dissolution studies were performed in triplicate using USP dissolution Type II dissolution test apparatus at 37 ± 0.5°C and 50 rpm paddle speed using 1000 mL of pasteurized milk. The dissolution medium was pre-warmed to the required temperature before dissolution testing commenced. Aliquots of 2 mL were periodically withdrawn at frequent intervals and fresh medium of pasteurized milk of 2 mL was replaced each time. The dissolution test continued for 1 hour 40 minutes. The concentration of PGZ in the samples was determined using HPLC with % drug release and AUC calculated using equations (1) and (2).
Amount of drug in tablets = 15 mg
Volume of dissolution medium = 900 mL
AUC = area under curve
C1 = initial concentration
C2 = final concentration
T1 = initial time
T2 = final time
In vivo bioavailability of PGZ: In vivo studies were conducted on 18 young Wistar rats, each weighing between 200-250 g. They were randomly divided into 3 groups, each group containing six rats. Rats in group I acted as a control, rats in group II were given free access to food and water, while rats in group III were fasted overnight before starting the experiment. A solution of PGZ was prepared using purified water and administered to rats via the oral route. The dose given to each individual rat was 30 mg/kg . After administering PGZ via the oral route, 0.3 mL blood samples were withdrawn at 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 minutes with the concentration of PGZ determined using HPLC  after sample extraction .
A 100 μg/mL stock ethanolic solution of PGZ was prepared and used to prepare a number of standard solutions in the concentration range of 1-10 μg/mL. Each of these standard solutions was spiked with 2 mL of plasma. There was a linear response between peak area and concentration with an observed correlation coefficient of 0.996.
2 mL of blood sample taken from the rats was centrifuged at 3000 rpm for 30 minutes to obtain plasma. Each sample of plasma was then diluted with 4.0 mL of citric acid/disodium hydrogen phosphate buffer (0.083 M/0.033 M, pH 2.8). 4.0 mL of 1,2-dichloroethane was added to the mixture with, rotated for 3 minutes, and then centrifuged at 3000 rpm for 15 minutes. The organic layer was pipetted out, passed through a sodium sulphate drying tube, and then evaporated to dryness. The dried residue was then reconstituted in a volume of the mobile phase .
PGZ is a BCS class IV drug with both poor solubility and poor permeability. One of the common methods that are used to increase the aqueous solubility of low soluble drugs is to use the salt forms of drugs in the pharmaceutical formulations. However the aqueous solubility of PGZ hydrochloride salt is still very low (0.7 mM) although it is much higher than the aqueous solubility (0.04 mM) of its corresponding freebase . Pharmacokinetic studies suggest that PGZ release and absorption occurs in the stomach , so SGF and pasteurized milk were selected as dissolution media to simulate the gastric fasted and fed conditions. To compare the dissolution profiles in biorelevant dissolution media with that of routine compendial media, the dissolution was also carried out in USP buffer solution HAB pH 1.2. The dissolution profiles for PGZ tablets are shown in Figure 1.
It was observed that there is an appreciable difference between the results obtained in HAB pH 1.2 and simulated gastric fluid (SGF). On comparing the results for in vitro fed and fasted conditions (Table 1) it can be seen that food delays the onset of action of PGZ from 16 minutes (Tmax in fasted state) to 80 min (Tmax in fed state).
% drug release
|HAB pH 1.2||14.25 ± 0.04||10.0||85.5||1354.19|
|SGF||36.72 ± 0.05||16.0||97.920||2943.174|
|Pasteurized milk||14.7 ± 0.04||80.0||98.0||2737.654|
Table 1: Comparison of the results obtained using in vitro compendial and biorelevant dissolution media.
In order to validate results from in vitro studies of PGZ, in vivo studies were conducted using 18 young Wistar rats under fed and fasted conditions. The in vitro dissolution data was then compared with the in vivo bioavailability in order to determine the most biorelevant dissolution medium (Figure 2).
The most common pharmacokinetic parameters, total area under the plasma concentration-time curve (AUC0- ∞), peak plasma concentration (Cmax) and time to reach maximum plasma concentration (Tmax), were estimated from the plasma concentration-time profiles. The AUC0- ∞ was calculated by adding the area from time zero to the last sampling time (AUC0-t) to the area from the last sampling time to infinity (AUCt-∞). The former was calculated by applying the trapezoidal rule and the latter by dividing the last measurable plasma drug concentration with the apparent elimination rate constant (ke).
On comparing various biopharmaceutical parameters obtained by in vitro dissolution studies (in compendial and biorelevant dissolution media) with in vivo results of fed and fasted conditions in rats, it was clear that the values of Cmax, Tmax and AUC for dissolution studies performed in HAB pH 1.2 (compendia dissolution media) were significantly different from the corresponding values obtained by in vivo studies (Table 2). The values of Cmax, Tmax obtained in biorelevant dissolution media in fed and fasted state were then correlated with the in vivo data for fed and fasted rats. The value of Tmax = 16 min obtained by in vitro fasted state (SGF) was similar to the value of 20 minutes obtained for in vivo fasted rats and the value of Tmax (80 minutes for in vitro simulated fed state (milk)) was identical to Tmax obtained for in vivo fed rats.
|Parameters||In fed state||In fasted state|
|AUC (mg min/ml)||620.65||625.25|
Table 2: Comparison of the results obtained in in vivofasted and fed states.
Simulation of gastrointestinal conditions for in vitro dissolution studies is essential to adequately predict the in vivo behavior of PGZ immediate release tablet formulations. To reduce the size and number of human studies required to identify a drug product with appropriate performance in both, the fed and fasted states, it is advantageous to be able to pre-screen formulations in vitro. The choice of appropriate biorelevant dissolution media for such in vitro tests is essential in order to correctly forecast the food effect in the pharmacokinetic studies of a drug product. The compendial dissolution test in hydrochloride solution at pH 1.2 was not able to predict the food effect. However, the biorelevant dissolution test in simulated gastric fluids predicted correctly that the release of pioglitazone HCl would be slower in the fed state than in the fasted state. The results demonstrate good correlation between in vitro drug release and in vivo drug absorption using the biorelevant dissolution test method.