COMPARATIVE IN VITRO BIO-EQUIVALENCE EVALUATION OF SIX BRANDS OF AMOXICILLIN CAPSULE MARKETED IN DUTSE, JIGAWA STATE, NIGERIA

ABSTRACT

Comparative in vitro bio-equivalence study of biopharmaceutics class I and III drugs had gained prominence in recent times. In vitro bio-equivalence offered many benefits compared to conventional in vivo bio-equivalence studies, due to its reduced cost and time of product release as well as avoiding unnecessary use of human volunteers.This study is aimedat evaluating and comparing the in vitro bio-equivalence of branded and generic amoxicillin capsules available in Dutse, Jigawa State, Nigeria. The samples were randomly selected and evaluated for quality control studies via BP 2009 and USP 2009 specifications.Four UVSpectrophotometry methods for the determination of amoxicillin in simulated physiological media (pH 1.2, 4.5, 6.8, and 7.4), were developed and validated according to ICH guideline. Dissolution testing was conducted using USP apparatus I, sink volume of 900 ml, temperature 37± 0.5 oC and 100 rpm, samples were withdrawn at an interval of 5, 15, 25, 35 and 45 minutes respectively. Bio-equivalence of the samples were compared using different statistical methods; the difference factor (f1), similarity factor (f2) and the dissolution efficiency (% D.E.). From the result of quality control studies, all the brands were found to passed identification test as their IR spectra were superimposable with reference amoxicillin spectrum. Four of the six brands (A, B, C and E) passed the assay test,(90-120) USP 2009 while, brands D and F failed. The uniformity of weight test all the brands passed with percentage mean deviation <7.5% (BP 2009).All the brands disintegrated in less than 15 minutes as required by BP 2009.The four methods developed for the determination of amoxicillin have λmax. 229nm for buffer solutions(pH 1.2 and 4.5) and 228nm for (pH 6.8 and 7.4).The calibration curves were linear at the concentration range of 10-60 μg/ml their correlation co-efficient were 0.999, 0.997, 0.997 and 0.996 for buffer solutions (pH 1.2, 4.5, 6.8 and 7.4) respectively. A regression equation of y= 0.0133x+0.1847, y=0.0137x+0.1967, y=0.0143x+0.2113, and y=0.0162x+0.0807for (pH 1.2, 4.5, 6.8 and 7.4) respectively. The percentage recoveries of the developed methods were within the official range of 98-102%. Likewise, the intra-day and inter-day precision were within normal range of co-efficient of variation<15% (2.05% ,3.46%,2.48% ,0.66% and 9.15%,9.00%,5.82%,0.66%) for the simulated media(pH1.2,4.5, 6.8 and 7.4) respectively. The dissolution profiles obtained for each medium was subjected to bio-equivalence comparison. The result of f1 for brands B and E were similar and within the acceptable range of ≤15 in each pHs. Similarly, the f2 values were ≥50 in pH 1.2 and 4.5 for B and pH 4.5 for E while in pH 6.8 and 7.4 for B and pH 1.2, 6.8 and 7.4 for E were below the acceptable range thus, failed f2 test. Also the % D.E. values of each of the simulated pHs for brands B and E were within the acceptable limit of ±10 %. So, from the result they are considered bio-equivalent with A. While, brands C, D and F failed both f1, f2 and % D. E. comparison as their values were outside the accepted range. Also, the analysis of variance (ANOVA) and dunnett multiple comparison results further, confirmed the observed difference among the brands obtained using f1, f2 and % D.E methods at (p<0.05).Therefore, brands C, D and F, are not bio-equivalent with A. About 60 % of the samples may not be considered bio-equivalent with innovator (A). CHAPTER ONE

1.1 INTRODUCTION

Bio-equivalence evaluation using in vivo pharmacokinetic parameters are often assumed to be the gold standard to established product bio-equivalence (BE) of immediate release solid oral dosage forms(Polli et al., 2008).However, in vitro studies are sometimes better than in vivo studies in assessing BE of immediate release solid oral dosage forms due to the fact that in vitro studies serve as the better method that lead to reduce costs, directly assess product performance, offers benefits in terms of ethical considerations (Polli et al., 2008).In vitro studies directly assess product performance than do conventional human pharmacokinetic BE studies, since in vitro studies focus on comparative drug absorption from the two products (Arleneet al., 2014). Alsoin vivo BE testing suffers from complications due to its indirect approach (Polli et al., 2008).Regarding ethical considerations, in vitro studies better embrace the principle ―No unnecessary human testing should be performed’ and can result in faster product development (Polli et al., 2008). Dissolution of solid oral dosage is preceded by disintegration prior to being absorbed into blood circulation to be made bioavailable at the site (s) of a drug action (Kassaye and Genete, 2013). Therefore, drug filled in a capsule shell is released rapidly as the capsule shell disintegrates; essential step for immediate release oral dosage forms because the rate of disintegration affects the dissolution and subsequently the therapeutic efficacy of the medicine (Kassaye and Genete, 2013).Dissolution is the main in vitro method used in quality control and of recent to determine bio-equivalence between certain drug products(Arlene et al., 2014). Hence, dissolution procedure has played many roles including its contribution in drug development, quality assurance, and investigation of similarity between the different brands of the same active pharmaceutical ingredients (APIs) formulation (Arlene et al., 2014).

Amidon et al., (1995) proposed the biopharmaceutics drug classification system (BCS) as a schematic scientific framework for correlating in vitro drug product dissolution with in vivo bioavailability based on the belief that drug dissolution and gastrointestinal permeability are the major parameters controlling rate and extent of drug absorption.The BCS classified drug products into four classes according to their aqueous solubility and intestinal permeability; Class I: HIGH solubility / High permeability, Class II: LOW solubility / High permeability, Class III: HIGH solubility / LOW permeability, and Class IV: LOW solubility / LOW permeability (WHO, 2005). Furthermore, BCS is used for bio-waiver of in vivo studies, which means that in vivo bioavailability and/or bio-equivalence studies may be waived (not considered necessary for product approval) (FDA, 2015b). Instead of conducting expensive and time consuming in vivo studies, a dissolution test could be adopted as the substitute for the decision as to whether the two pharmaceutical products are equivalent (Ferrazetal., 2007).The rate and extent of drug absorption from the gastrointestinal (GI) tract are very complex and are affected by various factors; including physicochemical factors (e.g. pKa, solubility, stability, diffusivity, lipophilicity, polar and non-polar surface area, presence of hydrogen bonding, particle size, and crystal form), physiological factors (e.g., GI pH, GI blood flow, gastric emptying, small intestinal transit time, colonic transit time, and absorption mechanisms), and factors related to the dosage form (e.g., tablet, capsule, solution, suspension, emulsion, and gel) (Dahan and Amidon, 2008 ; Yu et al.,1996). Despite these complexities, the work of Amidon et al., (1995) revealed that the fundamental events controlling oral drug absorption are the permeability of the drug through the GI membrane and the solubility/dissolution of the drug dose in the GI environment (Dahan et al., 2009). These key parameters are characterized in the Biopharmaceutics Classification System (BCS) by three dimensionless numbers:

I. Absorption number (An),

II. Dissolution number (Dn) and

III. Dose number (Do).

These numbers take into account both physicochemical and physiological parameters and are fundamental to the oral absorption process (Lobenberg and Amidon, 2000; Martinz and Amidon, 2002).Class I drugs exhibit a high absorption number and a high dissolution number. Therefore, the rate limiting step is drug dissolution and if dissolution is very rapid then gastric emptying rate becomes the rate determining step (Amidon et al.,1995). Hence, rate of absorption is higher than rate of excretion e.g. Amoxicillin, Metoprolol, Diltiazem (Amidon et al.,1995). Class II drugs have a high absorption number but a low dissolution number (Amidon et al.,1995). In vivo drug dissolution is then a rate limiting step for absorption except at a very high dose number (Amidon et al.,1995). The absorption for class

II. drugs is usually slower than class I and occurs over a longer period of time e.g. Mefenamic acid, Nifedipine (Amidon et al.,1995). Class III drugs, permeability is rate limiting step for drug absorption (Cheng,et al., 2004). These drugs exhibit a high variation in the rate and extent of drug absorption (Jantratid et al., 2006). Since the dissolution is rapid, the variation is attributable to alteration of physiology and membrane permeability rather than the dosage form factors. e.g. Cimetidine, Neomycin B, Captopril (Amidon et al.,1995). Class IV drugs exhibit a lot of problems for effective oral administration (Amidon et al.,1995). Fortunately, extreme examples of class IV compounds are the exception rather than the rule and are rarely developed and reach the market. Nevertheless, a number of class IV drugs do exist. e.g.

Taxol, Griseofulvin.

Amoxicillin has been classified as the BCS class I which is highly soluble and highly permeable active pharmaceutical ingredients (API) and to release ≥85 % or more of their drug in 30 min (rapid release) or 15 min (very rapid release) (WHO, 2006b). BCS guidelines are approved by USFDA, WHO, and EMEA (European Medicines Agency) (Lipka and Amidon, 1999; FDA, 1995a). BCS class I drug products, should met these conditions to supportin-vivo biowaiver:

The drug substance is highly soluble.

The drug substance is highly permeable.

The drug product (test and reference) is rapidly dissolving, and

The product does not contain any excipients that will affect the rate or extent of absorption of the drug.

While class III drug products should have;

The drug substance is highly soluble.

The drug product (test and reference) is very rapidly dissolving and

The test product formulation is qualitatively the same and quantitatively very similar, e.g., falls within scale-up and post-approval changes (SUPAC) immediate release level 1 and 2 changes, in composition to the reference.

The recommended methods for determining solubility, permeability, and in vitro dissolution are;

The solubility class boundary is based on the highest strength of an immediate release drug product that is the subject of a bio-waiver request (WHO, 2005). A drug substance is considered highly soluble when the highest strength is soluble in 250 ml or less at 37± 0.5 OC of aqueous media over the pH range of 1-6.8 (Krämer et al, 2005). The 250 ml volume was obtained from BE study protocols that recommend administration of a drug product to fasting human volunteers with a glass (about 8 ounces) of water (FDA, 2015b).

The permeability class is based indirectly on the extent of absorption (fraction of dose absorbed, not systemic BA) of a drug substance in humans, and directly on measurements of the rate of mass transfer across human intestinal membrane(Krämer et al, 2005). Alternatively, other systems capable of predicting the extent of drug absorption in humans can be used (e.g., in situ animal, in vitro epithelial cell culture methods). A drug substance is considered to be highly permeable when the extent of absorption in humans is determined to be 85 percent or more of an administered dose based on a mass balance determination (along with evidence showing stability of the drug in the GI tract) or in comparison to an intravenous reference dose(Krämer et al, 2005).

An immediate release drug product is considered rapidly dissolving when 85 percent or more of the labeled amount of the drug substance dissolves within 30 minutes, using United States Pharmacopeia (USP) Apparatus I at 100 rpm or Apparatus II at 50 or 75 rpm when appropriately justified in a volume of 900 ml or less in each of the following media: (1) 0.1 N HCl or simulated gastric fluid without enzymes; (2) a pH 4.5 buffer; and (3) a pH 6.8 buffer or simulated intestinal fluid without enzymes (FDA,2015b). Moreover, formulations of the same active ingredient can lead to different therapeutic effects in terms of their dissolution profile (Jinginger et al.,1998; Vanitasgar et al., 2012). In effect, poor dissolution of active ingredient can result in low bioavailability which may lead to therapeutic in effectiveness initially and subsequently lead to spread of resistance (Benouda et al.,2009; Chadli et al., 2005). Defects in the formulation or manufacturing process may be responsible for the development of generic drugs of poor quality (Ghorab et al.,2012). An in vitro in vivo correlation (IVIVC) has been defined by the Food and Drug Administration (FDA) as a predictive mathematical model describing the relationship between an in vitro property of a dosage form and an in vivo response(Sakore and Chakraborty, 2011). Generally, the in vitro property is the rate or extent of drug dissolution or release while the in vivo response is the plasma drug concentration or amount of drug absorbed (FDA, 1995b).The purpose of IVIVC is to use drug dissolution results from two or more products to predict similarity or dissimilarity of expected plasma drug concentration(Sakore and Chakraborty, 2011).Before considering relating in vitro results to in vivo, it is imperative to know how to establish similarity or dissimilarity of in vivo response i.e. plasma drug concentration parameters. An important method of establishing similarity or dissimilarity of plasma drug concentrations profile is commonly known as bio-equivalence testing(Sakore and Chakraborty, 2011).There are guidances and standards set for establishing bio-equivalence between drug profiles and products (FDA,1995a). For the IVIVC perception, dissolution is proposed to be a surrogate of drug bioavailability. Thus, dissolution standard may be necessary for the in vivo waiver (Amidon et al., 1995).So far, The FDA has implemented the BCS system to allow waiver of in vivo BA/BE testing of immediate release solid dosage forms for class I, high-solubility, high permeability drugs (Blume and Schug, 1999). As for class III (high-solubility low-permeability) drugs, as long as the drug product does not contain agents and/or excipients that may modify intestinal membrane permeability, in vitro dissolution test can ensure BE (Cheng et al.,2004).

1.2 Statement of the Research Problem

More than 25 % of drugs in the developing countries are said to be counterfeited or substandard, 50% of which are said to be beta-lactams(Kelesidis and Falagas, 2015). Amoxicillin is one of the most prescribed beta-lactam penicillin drugs with numerous generics available, this has been accompanied with diverse problems of which the most serious is the prevalence of substandard generics and fake drug products. Consequently, healthcare providers and patients are mostly concerned when selecting one brand from among several generic brands of the same drug during the treatment regime (Almeri et al., 2012).In vivo BE studies are costly and time consuming involving the use of human volunteers (Polli et al., 2008).

1.3 Justification

Amoxicillin is one of the first line drug in Nigeria as contained in the standard treatment guideline of the Federal Ministry of Health in the treatment of upper respiratory tract infections, tonsillitis, typhoid, as well as other indications.Owing to this, there is availability of numerous generics in the market leading to concern on selection among generics available to interchange with the innovator brand(STG, 2008).Hence, the need to evaluate the quality and bio-equivalence of the numerous generics available. BCS asbetter alternative to in vivo studies, in terms of cost, time utilization and avoidance of unnecessary use of human volunteers can be used to evaluate bio-equivalence of various generics against innovator brand.

1.4 Research Hypothesis

1.4.1 Null hypothesis

There is no statistically significant difference in the pharmaceutical, chemical, dissolution profile and in vitro bio-equivalence between the generic and innovator brand of Amoxicillin capsules available in Dutse, Jigawa State, Nigeria.

1.4.2 Alternate hypothesis

There is statistically significant difference in the pharmaceutical, chemical, dissolution profile and in vitro bio-equivalence between the generics and innovator brand of Amoxicillin capsules available in Dutse, Jigawa State, Nigeria.

1.5 Aim and Objectives of the Study

1.5.1 Aim

The aim of this study is to evaluate the bio-equivalence of six brands of amoxicillin using in vitro dissolution profile as surrogate to in vivo bio-equivalence studies.

1.5.2 Objectives

The objectives of the study are to; randomly select six different brands of amoxicillin using systematic random sampling.

Conduct quality control studies on amoxicillin capsules sampled (BP and USP, 2009).

Develop and validate UV methods for determination of amoxicillin in simulated physiological media pH 1.2, 4.5, 6.8 and 7.4 determine the dissolution profiles of the individual brands in each of the simulated physiological media.

Evaluate the bio-equivalence of the six brands using difference factor (f1), similarity factor (f2) and dissolution efficiency (D.E.).