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

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.


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 medicine 2 . 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. Bioequivalence studies may involve both in-vivo and invitro studies 3 . 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 ISSN: 2456-8058 33 CODEN (USA): UJPRA3 formulation, studies in human healthy volunteers may be required to prove that the new formulation is bioequivalent with the old one 4 . 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 optimization 5 . 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 vaginosis 5 . It is effective for dracunculiasis, giardiasis, trichomoniasis, and amebiasis 5 . It is an option for a first episode of mild-tomoderate Clostridium difficile colitis if vancomycin or fidaxomicin is unavailable 5 .

Figure 1: Chemical Structure of Metronidazole
Metronidazole is available by mouth, as a cream, and by injection into a vein 5 . 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 tablet 6 . 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/cm 2 . 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 reweighed 7 : 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 o C.six tablets were used for the determination. Accepted range for the uncoated tablet up to 30 minutes 8 .

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 o C ± 0.5 0 C .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 calculated 9 .

Data Analysis:
All dissolution data evaluated using Excel spread sheet, and the results will be plotted for each brand 6 .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 drug 11 . 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.  (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 T 2 . Correlation calculation will carried on using MINITAB14 specific statistical program. In vivo percent absorbed of reference product was calculated from equation (4): ……………. (4) Where, 0 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 ∞ respectively 13 . 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.

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.

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 100 7 . 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.

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 pvalue (>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 ISSN: 2456-8058 37 CODEN (USA): UJPRA3 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.