Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

IN VITRO EQUIVALENCE STUDIES OF COMMERCIALLY AVAILABLE CEFUROXIME

AXETIL TABLETS UNDER BIOWAIVER CONDITIONS
Omar Sarheed*, Ramesh KVRNS, Shahnaz Usman, Fasiha Shah.
RAK College of Pharmaceutical Sciences, RAK Medical and Health Sciences University, Ras AlKhaimah, UAE.
ARTICLE INFO
Article history
Received 22/11/2014
Available online
16/12/2014

Keywords
In Vitro Equivalence,
Cefuroxime Axetil,
Biowaiver,
Dissolution.

ABSTRACT
World Health Organization (WHO) recommends biowaivers for immediate-release solid oral
products employing dissolution testing as a surrogate for in vivo bioequivalence studies.
Cefuroxime is a semi-synthetic antibiotic belonging to the cephalosporin group. The drug is
poorly soluble in water. In the present investigation, the in vitro equivalence of tablets
containing cefuroxime axetil , for commercially available in Ras AlKhaimha, UAE under
biowaiver conditions and the innovator product, was conducted. The dissolution profiles of
cefuroxime axetil products were determined using the USP dissolution paddle method. Both
products are rapidly dissolving, but they do not meet the criteria for dissolution profile
similarity, f1 and f2. This may be attributed to the formulation variations between the two
products. Therefore, in vivo bioequivalence studies are required to ascertain therapeutic
equivalence. Assessment of different generic products available in the market is very
important to ensure that generic drugs being sold can be used interchangeably with the
branded products.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as Omar Sarheed et al. In Vitro Equivalence Studies of Commercially Available Cefuroxime Axetil
Tablets Under Biowaiver Conditions. Indo American Journal of Pharm Research.2014:4(12).

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Corresponding author
Omar Sarheed
RAK Medical & Health Sciences University,
Ras Al Khaimah, UAE.
sarheed@rakmhsu.ac.ae

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INTRODUCTION
Cefuroxime is an oral semi-synthetic antibiotic and the first commercially-available second-generation cephalosporin to be
widely used in therapy (1). Cefuroxime axetil is the acetoxyethyl ester prodrug of cefuroxime as shown in Figure 1. It has proven in
vitro antibacterial activity against several gram-positive and gram-negative organisms (2).
The World Health Organization (WHO) has promoted the use of generic drugs in order to make the cost of medicines
affordable especially for the developing countries (3). A generic drug is defined as pharmaceutical product,, that is manufactured
without a license from the innovator company and marketed after the expiry date of the patent. It is usually intended to be
interchangeable with an innovator product when they are pharmaceutically and therapeutically equivalent (4).
Bioequivalence (BE) studies are commonly used to assess therapeutic equivalence. They involved many methods such as
pharmacokinetics (PK) studies and pharmacodynamics (PD) studies. However, these studies are often costly and require human
volunteers. Alternatively, the Biopharmaceutics Classification System (BCS) can be used for biowaivers which is the absence clinical
bioequivalence testing in humans.
Cefuroxime axetil is class II drug according Biopharmaceutics Classification System (BCS) and has a poor aqueous
solubility. Cefuroxime is a weak acid with pKa of 2.5 (5).
The number of generic drug products registered in the United Arab Emirates is increasing day by day. Those products are
used interchangeably. So to ensure the quality of the products, equivalence studies are necessary to evaluate the quality of different
formulations available in the market. There are no reports on the in vitro equivalence for cefuroxime axetil in the United Arab
Emirates.
The aim of this work was to investigate if there are any differences between various commercially available cefuroxime
axetil tablets through the evaluation of in vitro dissolution profiles. Two brands of immediate-release solid dosage form cefuroxime
axetil tablets were obtained from pharmacies in Ras AlKhaimah, United Arab Emirates for this study. The selection based on survey.

Degassing of Dissolution Medium

According to the USP, each dissolution medium was heated to about 41C and then filtered under vacuum through a 0.45-m
membrane into a suitable filtering flask equipped with stirring device. Further the flask was sealed and the vacuum continued with
stirring for additional five minutes. The temperature of dissolution medium does not fall below 37 C prior the initiation of the test.

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MATERIALS AND METHODS

Materials
Innovator cefuroxime axetil 500 mg (Zinnat) used as reference product, and a generic version (test product) were evaluated.
They were purchased from a local pharmacy in Ras AlKhaimah, United Arab Emirates.
Reagents hydrochloric acid, sodium hydroxide, potassium phosphate monobasic, sodium acetate and acetic acid were analytical grade
and purchased from Sigma-Aldrich (UK).
Hydrochloric acid- (pH 1.2), acetate- (pH 4.5) and phosphate buffers (pH 6.8) were used as the dissolution media as specified
in the United States Pharmacopoeia (6).
Hydrochloric acid buffer at pH 1.2 was prepared by mixing 50 mL of 0.2 M potassium chloride with 85 mL of 0.2 M HCl
and sufficient distilled water to produce 10 L. Acetate buffer at pH 4.5 was prepared by dissolving 2.99 g of sodium acetate in 14 mL
of 2N glacial acetic acid and mixed with sufficient distilled water to produce 10 L. Phosphate buffer (pH 6.8) was prepared by mixing
50 mL of monobasic potassium phosphate with 22.4 mL of 0.2 M NaOH and sufficient distilled water to produce 10 L.
Pure cefuroxime axetil powder (Cefuroxime Axetil USP, SigmaAldrich, UK) was dissolved in methanol and then diluted
the dissolution medium to make a series of standard calibration solutions with different concentrations for development of a
calibration curve using a UV spectrophotometer at 278 nm.

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Figure 1: Chemical structure of cefuroxime axetil.

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Dissolution Testing
In vitro dissolution was carried out via USP Apparatus 2,paddle type (Copley, UK) at a speed of 55 rpm in 900 mL of the
dissolution media USP mentioned in above and maintained at 37 0.5 C using a water bath fitted with a variable speed stirrer and
heater (Erweka DT 600, Frankfurt, Germany). The selection of paddle rotation speed is based on the dissolution method of cefuroxime
axetil in the United States Pharmacopoeia (6).
Samples of 5 mL were taken manually at 5, 10, 20, 30, 45 and 60 min and replaced with an equal volume of fresh medium to
maintain a constant dissolution volume. The samples were filtered with 0.8 micrometer syringe filter, and the absorbance was
measured at 278 nm using a UV spectrophotometer (Shimadzus UV-1800 spectrophotometer). Each profile is the average of six
individual tablets.
Data Analysis
Drug release data had been analyzed using model-independent approaches which was proposed by Moore and Flanner (7). It
is the simplest mathematical model to compare the dissolution profile using two factors, f1 and f2 and provides a single number to
describe the comparison of dissolution profile data. This approach is endorsed by the FDA and the European Agency for Evaluation of
Medicinal Products (EMEA) as criteria for assessment of similarity between two dissolution profiles.
Fit Factors
According the FDA (8), the difference factor (f1) calculates the percent (%) difference between the two curves at each time point
and is a measurement of the relative error between the two curves:

The compared dissolution curves are considered similar and bioequivalent, if f1 is between 0 and 15.
Whereas the similarity factor (f2) is defined as a logarithmic reciprocal square root transformation of the sum of squared error and
is a measurement of the similarity in the percent (%) dissolution between the two curves.

where Rt and Tt are the cumulative percentage dissolved at each of the selected n time points of the reference and test product
respectively and n is the number of time points. Values of 50 or above (50100) ensure similarity (difference 10%) of the curves.

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RESULTS AND DISCUSSION

United Arab Emirates included generic drugs in the national formulary. The inclusion of such items reduces the expenditure
on a healthcare system. To ensure the quality of the generic items compared to brand items, bioequivalence studies are required as an
evidence-based method to prove the therapeutic efficacy. The in vitro bioequivalence is recommended by the WHO for Class I that
are very rapidly dissolving (85% drug dissolved within 15min) or are rapidly dissolving (85% drug dissolved within 30min) (4). In
the case of cefuroxime axetil (Class II) which is poorly soluble drug, the in vitro bioequivalence is also recommended by WHO (4) if
the drug molecule is rapidly dissolving.
The potential of biowaiver extension for BCS class II drugs, if they meet the following criteria:
1. They are weak acid properties and high solubility at pH 6.8 but not at pH 1.2 and pH 4.5 (9).
2. The dosage form is rapidly dissolving (85% in 30 min or less) in pH 6.8 buffer (only).
3. The test product exhibits similar dissolution profiles, as determined by the f2 value or equivalent statistical evaluation, to those of the
reference product at the three pH values (pH 1.2, 4.5, and 6.8).
Furthermore the excipients in the products containing class II drug should critically evaluated as they affect the solubility and
the permeability (10). For poorly water soluble drug such as cefuroxime axetil, the addition of sodium lauryl sulfate (SLS), as a
wetting agent, to the formulation may be required to improve the in vivo solubilization when poorly soluble drugs used. In this study,
both the innovator and the generic products contain SLS as shown in the Table 1.
Both products can be considered rapidly dissolving at pH 6.8 as they release greater than 85% with 30 minutes (Table 2).

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Statistical Analysis
Results are expressed as mean S.D. for replicate samples. The statistically significant difference among the groups was
determined by one-way analysis of variance (ANOVA) using statistical software (Version 17; Minitab Inc., Coventry, UK). Statistical
significance was considered at a level of p < 0.05.

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Table 1: Excipients Contained in (1) Reference and (2) Test Products.

Excipient
Microcrystalline cellulose
Croscarmellose sodium, Type A
Sodium lauryl sulfate
Hydrogenated vegetable oil
Silica colloidal anhydrous
Methylhydroxypropyl cellulose
Propylene glycol
Methyl parahydroxybenzoate
Propyl parahydroxybenzoate
Opaspray white M-1-7120J [containing titanium
dioxide (E171) and sodium benzoate (E211)}]
Colloidal silicon dioxide
Sodium starch glycolate
Magnesium stearate
Opaglos

Evaluated Product
1, 2
1, 2
1, 2
1
1
1
1
1
1
1
2
2
2
2

Table 2: The Percentage of Dissolution for Evaluated Products at pH 6.8.

Medium
pH 6.8

Reference Product
% dissolved % dissolved
( ) 20 min
( ) 30 min
85.5
87.8

Test Product
% dissolved % dissolved
( ) 20 min
( ) 30 min
88.7
91.3

Table 3: The Dissolution Test Results.

pH 6.8

Test Product
% dissolved
( )
66.4
85.8
93.3
93.8
93.2
93.6
61.0
79.4
90.0
92.3
91.2
91.0
60.9
76.1
88.7
91.3
90.0
89.6

RSD
(%)
6.8
10.0
7.9
8.2
9.3
6.8
6.2
4.1
3.2
4.2
3.4
3.0
8.3
7.0
5.0
4.0
4.1
4.3

Dissolution at pH 1.2
Dissolution profile at pH 1.2, 4.5 and 6.8 is shown in Figures 2-4.
Figure 2 shows the comparative dissolution profile of the reference and the test products at pH 1.2. Both the reference and the
test products were rapidly dissolved as shown in Table 3.However the products are not similar (f2 = 44 and f1 = 16).Based on fit factors
method as they are not fulfilling the FDA criteria which requires the f1 values up to 15 and f2 values greater than 50 to ensure
equivalence of the dissolution curves. This dissimilarity might be attributed to the amount of SLS present in the products. The
difference in the types of excipients used in the formation may play role in the dissimilarity between the two products. However, such
difference between the reference and the test products may not necessarily lead to in vivo bioinequivalence especially for class II drugs
such cefuroxime axetil where the drug permeability is high (11,12).

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pH 4.5

Time (min)
5
10
20
30
45
60
5
10
20
30
45
60
5
10
20
30
45
60

RSD
(%)
2.7
6.1
7.0
7.0
6.6
8.5
3.0
2.6
3.1
3.7
3.4
4.2
5.8
3.2
2.6
3.6
3.3
4.2

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Medium
pH 1.2

Reference Product
% dissolved
( )
48.0
65.9
81.8
86.5
87.8
88.4
55.3
72.0
86.0
89.7
90.8
90.5
51.1
69.3
85.5
87.8
89.3
87.6

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The dissolution method validity is confirmed by the calculation of the relative standard deviation for all time points fulfilling
all requirements (20% for first time point, 10% for other time points).

Figure 2: Dissolution profiles of reference and test products at pH 1.2.

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Figure 3 : Dissolution profiles of reference and test products at pH 4.5.

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Dissolution at pH 4.5 and pH 6.8

Figure 3 and 4 show the comparative dissolution profile of the reference and the test products at pH 4.5 and 6.8, respectively.
Both products at the pH 4.5 and at pH 6.8 are rapidly dissolving (85% drug release in 30 min or less). Also both products are similar
with fit factors (f2 = 61 and f1 = 8) at pH 4.5. At pH 6.8, f2 = 59 and f1 = 8 which make the products similar. This might be as a result of
increasing total solubility of cefuroxime axetil as the gastric pH increase and/or enhanced solubilization by the presence of surfactant
(SLS). The dissolution method is valid as shown in Table 3.
The dissolution data is suggesting that the formulation and/or manufacturing process parameters have an impact on the dissolution
profile in the GIT. This issue is crucial especially for poorly soluble molecules such as cefuroxime axetil of which its solubility is ratelimiting for the absorption.

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Figure 4: Dissolution profiles of reference and test products at pH 6.8.

CONCLUSION
The evaluated product of cefuroxime does not fulfill biowaiver criteria for drug products containing BCS Class II. Both
products are rapidly dissolving, but they do not meet the criteria for dissolution profile similarity, f1and f2. The dissolution profile
of the test product is similar to that of the reference product in pH 4.5 and 6.8 buffers but not similar in pH 1.2 buffer using the paddle
method at 55 rpm. Thus, these products are considered in vitro inequivalent. For the future, in vivo bioequivalence studies are required
to ascertain therapeutic equivalence the future work. Additionally, in vitro equivalence studies need to include factors that influence
solubility and permeability to ensure that it simulates the in vivo conditions.

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ACKNOWLEDGMENT
The authors express their gratitude to the Vice Chancellor of RAK Medical & Health Sciences University and the Dean of
RAK College of Pharmaceutical Sciences for the encouragement and facilities provided. The authors also would like to thank Mr.
Mohammad Quamrul Islam, Mohammed Bassam Shehada, Mohammed Sariah Haroosh Mossa and Noor Anwar Al Halabi for their
assistance.

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11. Shohin IE., Kulinich JI., Ramenskaya GV., Vasilenko GF., Evaluation of in vitro equivalence for drugs containing BCS class II
compound ketoprofen, Dissolution Technologies, 2002: 17: 1: 26-29.
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