IN VITRO-IN VIVO BIO-EQUIVALENCE CORRELATION STUDY OF METRONIDAZOLE, AND ITS BRANDS OF IMMEDIATE RELEASE TABLET UNDER BIO-WAIVER CONDITIONS

Ahmed EM¹, Ibrahim ME¹, Magbool FF²*

¹College of Pharmacy, Department of Pharmaceutical Chemistry, Ribat National University, Khartoum, Sudan.

²Department of Pharmaceutical Chemistry, Omdurman Islamic University, Sudan

ABSTRACT

Objective: The aim of present study is to examine the in-vitro in-vivo correlation (IVIVC) of immediate release product. Metronidazole 500mg and its brands of immediate release dosage forms. Metronidazole is clearly classified into BCS class I, and could be evaluated under bio waiver conditions.

Methods: The in vitro parameters employed were hardness, weight uniformity, friability, disintegration time, absolute drug content, dissolution rate (in 0.1 N Hydrochloric acid, phosphate buffer and acetate buffer at 37ºC), and dissolution efficiencies were also analyzed. The in-vitro dissolution study was performed on the brands, according to FDA,USP  dissolution profile in three different PH (1.2), (4.5), and (6.8) at37ºC, using the USP apparatus II,  then f1, f2 were determined for the time intervals of 10, 15, 30, 45 and 60 minutes, and dissolution efficiencies were calculated.  MINITAB 14 statistical program used for in vitro in vivo correlation, level A was done for reference product.

Results: A non linear relation was established which is typical for immediate release formulation, of class 1. There was significant relationship between in vitro and in vivo data of reference metronidazole product, Correlation and distribution of data with correlation coefficient (r=0.724, 0.837, 0.707), nonlinear relationship with p-value (>0.05) =(0.167, 0.098, 0.182), there is no out lines, no lake of fits at P-Values=0.0040, 006, 0.026.

Conclusion: Study concluded that there is no linear correlation between percent of drug released and percent of drug absorbed ,this may be due to uncontrollable gastric emptying rate for class one Metronidazole.

Keywords: Bioavailability, bioequivalence, biopharmaceutical classification system, bio-waiver correlation.

INTRODUCTION

Bio-equivalence:

Is defined as “the absence of significant differences in the extent and rate to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study” ¹. If two medicines are bioequivalent there is no clinically significant difference in their bioavailability. In vitro testing, preferably based on a documented "in-vitro/in-vivo correlation". May sometimes provide the same indication of bioequivalence between two pharmaceuticals. Bioequivalence is determined based on the relative bioavailability of the innovator medicine versus the generic medicine². It is measured by comparing the ratio of the pharmacokinetic variables for the innovator versus the generic medicine where equality is 1. Bioequivalence studies focus on the release of drug from dosage form, formulation and subsequent absorption into the systemic circulation. Bio-equivalence studies may involve both in-vivo and in-vitro studies³.

In Vivo in Vitro Correlations:

Formulation design, development and optimization of the product is an integral part of manufacturing and marketing of any therapeutic agent which is indeed a time consuming and costly process. Optimization processes may require alteration in formulation compositions, manufacturing processes and equipments. If these types of changes are applied to a formulation, studies in human healthy volunteers may be required to prove that the new formulation is bioequivalent with the old one⁴. Certainly, implementation of these requirements not only halts the marketing of the new formulation but also increases the cost of the optimization processes. It would be, desirable, therefore, to develop in vitro tests that reflect bioavailability data. A regulatory guidance for both immediate- and modified-release dosage forms has been, therefore, developed by the FDA to minimize the need for bioavailability studies as part of the formulation design and optimization⁵.

IVIVC can be used in the development and optimization of pharmaceuticals to reduce the number of human studies during the formulation development.

Correlation Definitions:

The term correlation is frequently employed within the pharmaceutical and related sciences to describe the relationship that exists between variables. Mathematically, the term correlation means interdependence between quantitative or qualitative data or relationship between measurable variables and ranks ². From biopharmaceutical standpoint, correlation could be referred to as the relationship between appropriate in vitro release characteristics and in vivo bioavailability parameters, Two definitions of IVIVC have been proposed by the USP and by the FDA^3,4.

Metronidazole, is an antibiotic and antiprotozoal medication5. It is used either alone or with other antibiotics to treat pelvic inflammatory disease, endocarditis, and bacterial vaginosis⁵. It is effective for dracunculiasis, giardiasis, trichomoniasis, and amebiasis⁵. It is an option for a first episode of mild-to-moderate Clostridium difficile colitis if vancomycin or fidaxomicin is unavailable⁵.

Figure 1: Chemical Structure of Metronidazole

Metronidazole is available by mouth, as a cream, and by injection into a vein⁵. The aim of present study is to examine the in-vitro in-vivo correlation of Metronidazole 500mg and its brands of immediate release dosage forms.

MATERIALS AND METHODS

Metronidazole reference standard USP, Mfg. August 2015, Exp. July 2020(Azal Industries, Khartoum, Sudan), and three different brands of Metronidazole tablets 500 mg obtained from local market. The brands under study were selected based on frequency of prescription, DW and Methanol 99.8% (Sharlau, Spain).

Physical Test:

Uniformity of Weight Test:

Twenty randomly selected tablets were weighed. The average weights were determined. The tablets were weighed individually and the percentage of deviation of its weight from the average weight was determined for each tablet⁶. The deviation of individual weight from the average weight should not exceed the limit given in Table 1.

Hardness Test:

The hardness of ten tablets randomly selected from each batch were determined on an automatic tablet hardness tester. The crushing strength of uncoated tablets is accepted within 4-8 kg/cm².

Friability Test:

Twenty tablets previously freed of dust were weighed together before transferring to a frabilator set to run for 4 min at 25 r.p.m. Thereafter they were removed, dusted and reweighed⁷:

Where;

 Wi is the initial weight and Wf the final weight of the tablets.

Disintegration Time Test:

According to official monograph determination of disintegration time for uncoated tablets was adopted using a disintegrating apparatus and the medium was distilled water at 37±1^oC.six tablets were used for the determination. Accepted range for the uncoated tablet up to 30 minutes⁸.

Absolute Drug Content:

Five pre-weighed tablets were crushed; the equivalent weight of a tablet was weighed out and dissolved in 500 ml of 0.1M NaoH in a volumetric flask, and filtered. The absorbance reading was determined using UV-visible spectrophotometer at 319nm.

In Vitro Dissolution Test:

Volume of 900 ml of each buffer was employed. Dissolution testing was performed using Tablet Dissolution Tester (USP Apparatus 2) at 75 rpm for class I, test and reference products, temperature will be adjusted to 37^oC ± 0.5 ⁰C .Twelve dosage units of each product test and reference were evaluated in the three media. Sample aliquots of 10 ml were taken manually with syringes. Samples were withdrawn at specified time intervals (10, 15, 30, 45, and 60 min) and replaced with 10 ml of appropriate medium. With drawn samples were filtered using 0.45-μm Millipore Filters, then 5 ml taken after filtration by volumetric pipette (3ml taken when use HCL buffer solution, and 1ml taken in case of acetate and phosphate buffer, and diluted to 50 ml). A UV–visible spectrophotometer was used to analyze dissolved drug in dissolution testing. Scanning of wavelength done in each buffer, and spectrum recorded between 200-800nm, and percentage % of drug dissolved calculated⁹.

Buffers Preparation:

Simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and acetate buffer pH (4.5) were prepared according to instructions in USP test solution. All media were prepared without enzymes, as follow:

1. Simulated Gastric Fluid (SGF) pH (1.2):

To prepare hydrochloric acid 0.1N, 8.5 ml was taken from concentrated HCL (37%) and volume completed to 1000 ml by distilled water.

6. Simulated Intestinal Fluid (SIF) pH (6.8):

Potassium phosphate monobasic KH₂PO₄ 0.2 M was prepared by dissolving 27.22 g in water, and volume diluted to 1000 ml by distilled water. Then sodium hydroxide 0.2 M prepared by dissolving 8g in water and volume diluted to 1000 ml by distilled water. 250 ml from Potassium phosphate monobasic KH₂PO₄ 0.2 M was placed into 200 ml volumetric flask, also 112 ml taken from sodium hydroxide 0.2M and volume completed to 1000 ml with distilled water¹⁰.

4. Acetate Buffer pH (4.5):

Firstly acetic acid 0.2N was prepared from concentrated acetic acid 99.93%. 116 ml was taken and diluted with distilled water. Then 2.99 g of sodium acetate (NaC₂H₃O₂) taken, and placed in 1000 ml volumetric flask,14ml from acetic acid was added and volume completed to 1000 ml by distilled water.

Preparation of Standard Stock Solutions:

Standard stock solutions of Metronidazole in HCL, phosphate and acetate buffers were prepared by dissolving 500 mg of standard in 100 ml volumetric flask using HCL, acetate and phosphate buffers as solvents to give concentration of 5 mg/ml, one ml taken by volumetric pipette in 100 ml volumetric flask to give concentration of 50μg/ml, using 50 ml volumetric flask to give serial concentration of standard curveError! Reference source not found..

Data Analysis:

All dissolution data evaluated using Excel spread sheet, and the results will be plotted for each brand⁶.Average of % content of active pharmaceutical ingredient (API) dissolved in each media of 12 tablets will be taken and a plot of % of ( API) dissolved against time will be drawn to represent the dissolution profile .The dissolution profile for local brand will be compared to that of the reference drug¹¹. If they are similar the similarity factor, f2 equal to or more than 50.This means that they are equivalent, if it’s less than 50 they are not equivalent.

f1 = {[3t=1n | Rt - Tt| ]/[3t=1n Rt ]}C……… (1)

f2 = 50 C log {[1+(1/n)3t=1n ( Rt - Tt )2 ] -0.5C 100}……… (2)

Similarity factor f2 has been adopted by FDA and the European Agency for the Evaluation of Medicinal Products (EMEA) by the Committee for Proprietary Medicinal Products (CPMP) as a criterion to compare the similarity of two or more dissolution profiles. Similarity factor f2 is included by the Centre for Drug Evaluation and Research (CDER) intheir guidelines such as guidance on dissolution testing of immediate release solid oral dosage forms^7, and guidance on Waiver of In-Vivo Bioavailability and Bioequivalence Studies for Immediate Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System⁷.The area under the dissolution-time curve method was used in calculating the dissolution efficiency (DE), and this was calculated at 30 min .The higher the dissolution efficiency (DE) is, the better the release efficiency of the tablets’ active ingredient, according to equation (3):

Where %D is the percentage dissolved at time t, % D (max) is the maximum dissolved at the final time T, and AUC(0-T )is the area under the curve from zero to time T².

Correlation calculation will carried on using MINITAB14 specific statistical program.

In vivo percent absorbed of reference product was calculated from equation (4):

Where, denotes the fraction of drug absorbed at time t, Ct is the plasma drug concentration at time t, Kel is elimination rate constant, AUC0-t and AUC0-∞ are the area under the plasma concentration– time profile curve at time t and ∞ respectively¹³. Then the values of percent of drug released were plotted against the percent of drug absorbed for reference products of Metronidazole using MINITAB14 analysis program to find out the relationship between data (correlation). Amount of drug released in the three different pH was plotted against amount of drug absorbed.

RESULTS AND DISCUSSION

A summary of the results of weight uniformity, hardness, friability, disintegration and assay are shown in Table1 and Table 2. Weight uniformity may serve as a pointer to amount of the active pharmaceutical ingredient (API) contained in the formulation. All the brands complied with the compendial specification for weight uniformity.

Table 1: Weight uniformity of atenolol tablets

Hardness is referred to as non-compendial test. The hardness or crushing strength assesses the ability of dosage form to withstand handling without fracturing or chipping, It can also influence other parameters such as friability and disintegration. Hence, the dosage formsof all brands were satisfactory for hardness.  Friability test is used to evaluate the tablets resistance to abrasion. Friability is now included in the United States Pharmacopeia as a compendia test. The compendial specification for friability is less or equal to 1%. Friability for all brands of Metronidazole were  below 1%.

Figure 2: Dissolution profile of metronidazole (pH 4.5)

Figure 3: Dissolution profile of metronidazole in pH (6.8)

Disintegration is the process of breaking of tablets in the liquid. Disintegration is a crucial step for immediate release dosage forms because the rate of disintegration affects the dissolution and subsequently the therapeutic efficacy of the medicine.

Table 2: Quality control results of Metronidazole

%F=Friability, DT=Disintegration time

A drug will be released rapidly as the dosage forms disintegrate. British Pharmacopeia specifies that uncoated tablets should disintegrate within 15 min and film coated tablet disintegrate within 30 min while USP specification for disintegration is 30 min for both uncoated and film coated tablets. All the brands were complied with both BP and USP specifications for disintegration as maximum disintegration time¹⁴.  Potency is the average amount of the active ingredient present per tablet. All the brands complied both BP and USP specification, as USP specification is that the content of active ingredient should not be less than 90% and not more than 110% while BP specifies that the content should not be less than 95% and not more than 105%.

The results of dissolution studies are graphically represented in the dissolution profile figures. All dissolution data are based on the actual drug content of the test dosage form as calculated from the assay results. All the Metronidazole brands released <90% drug in acidic media (pH 1.2) within 30 min, and PH (4.5). Amount released in phosphate buffer PH (6.8) were about 84% for reference drug and 91.4%,86.5% for test brands, This may be due to the pH depended solubility of Metronidazole.

Table 3: F1 and F2 Values

Analysis of Dissolution Data: To compare the dissolution profiles of the brands, a model independent approach of difference factor f1 and similarity factor f2 were employed.

Figure 4: Metronidazole correlation in pH (1.2)

Difference factor f1 is the percentage difference between two curves at each point and is a measurement of the relative error between the two curves. The similarity factor (f2) is a logarithmic reciprocal square root transformation of the sum of squared error and is a measurement of the similarity in the percent (%) dissolution between two curves.

Two dissolution profiles to be considered similar and bioequivalent, f1 should be between 0 and 15 while f2 should be between 50 and 100⁷.

Figure 5: Metronidazole correlation in pH (4.5)

Figure 6: Metronidazole correlation in pH (6.8)

All the values for f2 and f1shown in tables 29 for metronidazole, as mentioned in previous tables, all brands f2 values were more than 50 and f1 values were less than 15. Which mean that all brands are equivalent with the innovator brand.

In-vitro AUC in three pH (1.2),(4.5),(6.8)  for class I product were found three times in vivo  bioequivalence AUC calculated before, which is acceptable result because the in-vitro dissolution studies were carried out in ideal conditions without any factors that could affect their performance, such as volunteers internal biological inconsistency.

Dissolution efficiency (DE) was also employed to compare the drug release from various brands. The reference and the test product can said to be equivalent if the difference between their dissolution efficiencies are within appropriate limits (±10%, which is often used) ².

Dissolution efficiency of all the brands (class I) differed by less than 10% from the innovator brand. So, we can say that all the brands are pharmaceutically equivalent with the innovator brand.

Table 4: Dissolution efficiency for Metronidazole brands

Table 5: Relative dissolution efficiency of Metronidazole brands

In Vivo in Vitro Correlation Data Analysis:

As IVIVC is a predictive mathematical model describing the relationship between variables (an in vitro property of a dosage form and a relevant in vivo response). According to MINITAM 14 statistical program, there was significant relationship between in vitro and in vivo data of reference metronidazole product, Correlation and distribution of data with correlation coefficient (r=0.724, 0.837, 0.707), nonlinear relationship with p-value (>0.05)=(0.167, 0.098 , 0.182), there is no out lines, no lake of fits at P-Values =0.0040, 006, 0.026. By analysis of variance (ANOVA) the data points have significant relationship with p-value (> 0.05) for the three pH (1.2), (4.5), (6.8) respectively. Estimating the uncertainty in predicted correlation between in vitro and in vivo data was also performed. The interval is represented by the curved lines on either side of the regression line and gives an indication of the range within which the ‘true’ line might lie. Note that the confidence interval is narrowest near the center (the point x, y) and less certain near the extremes.

Using MINITAM 14 statistical program, there was significant relationship between in vitro and in vivo data of reference Atenolol product, Correlation and distribution of data with correlation coefficient (r= 0.798, 0.815, 0.967), nonlinear relationship with p-value (>0.05)=(0.106, 0.93, 0.009), there is no out lines, no lake of fits at P-Values=0.106, 0.040, 0.056 (>0.05) for the three pH (1.2,4.5,6.8) respectively. Estimating the uncertainty in predicted correlation between vitro and vivo data. The interval is represented by the curved lines on either side of the regression line and gives an indication of the range within which the ‘true’ line might lie. Note that the confidence interval is narrowest near the center (the point x, y) and less certain near the extremes. By applying analysis of variance (ANOVA) for the dissolution data using MINITAB 14 we concluded that the test products are bioequivalent to reference products of metronidazole and atenolol and could be interchangeable.

CONCLUSION

The bio waiver study has emphasized that pharmaceutical equivalence indicate that product have same drug molecule with approximately same pattern of dissolution release profile. By making fine turning in bioequivalent study we can reduce the time, cost, avoid Ethical, Ethnical consideration by unnecessary exposure of healthy subjects to medicines and finally to market the quality generic drug product. By applying level An in-vivo in-vitro correlation, we might concluded that there is no linear correlation between percent of drug released and percent of drug absorbed, this may be due to uncontrollable gastric emptying rate for class one Metronidazole. Metronidazole is an immediate release formulations. As dissolution is not a rate-limiting step in IR products, the fraction of drug absorbed against the fraction of drug released profile would be non-linear type which was obtained in our present study. So it may be concluded that the In vitro-In vivo correlation is well established and justified for reference formulation by level A correlation.

By applying analysis of variance (ANOVA) for the dissolution data using MINITAB 14 we concluded that the test products are bioequivalent to reference products of Metronidazole and could be interchangeable.

ACKNOWLEDGEMENT

The authors are thankful to wish to Azal Industries, Khartoum, Sudan, for providing the gift sample of Metronidazole (standard).

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHOR'S CONTRIBUTION

All authors have worked equally for this work.

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