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ABSTRACT: A bioequivalence study of two oral formulations of 400 mg norfloxacin was carried out in
18 healthy volunteers according to a single dose, two-sequence, cross-over randomized design at College
of Pharmacy, King Saud University, Riyadh, Saudi Arabia, jointly with King Khalid University Hospital.
The two formulations were: Uroxin (Julphar, United Arab Emirates) as test and Noroxin (Merck
Sharpe & Dohme, BV, Netherlands). Both test and reference formulations were administered to each
subject after an overnight fasting on 2 treatment days separated by 1 week wash-out period. After
dosing, serial blood samples were collected for a period of 24 h. Plasma harvested from blood, was
analysed for norfloxacin by a sensitive, reproducible and accurate HPLC method. Various pharmacokinetic parameters including AUC0 t, AUC0 , Cmax, Tmax, T1/2, and Kel were determined from plasma
concentrations for both the formulations and found to be in good agreement with reported values.
AUC0 t, AUC0 , and Cmax were tested for bioequivalence after log-transformation of data. No significant difference was found based on ANOVA; 90% confidence interval for test/reference ratio of these
parameters were found within a bioequivalence acceptance range of 80 125%. Based on these statistical
inferences, it was concluded that Uroxin is bioequivalent to Noroxin. Copyright 2000 John Wiley &
Sons, Ltd.
Key words: bioequivalence evaluation; human volunteers; norfloxacin
Introduction
Bioequivalence of two formulations of the same
drug comprises equivalence with respect to the
rate (Cmax) and extent of absorption (AUC) especially in conventional drug formulations [1]. In
the present study bioequivalence of two norfloxacin formulations was evaluated by comparing these pharmacokinetic parameters.
Norfloxacin is a fluoroquinolone antibacterial
agent suitable for oral administration, primarily
indicated in urinary tract infections and gonorrhea [2,3]. Norfloxacin exerts broad-spectrum
bactericidal effects via inhibition of the essential
bacterial enzyme DNA gyrase; the drug has significant activity against gram-positive and gramnegative organisms, and Pseudomonas [46].
Three specific events occur at the molecular level
(in E. coli cells): (a) inhibition of the ATP-dependent DNA supercoiling reaction catalyzed by
DNA gyrase, (b) inhibition of the relaxation of
supercoiled DNA, and (c) promotion of doublestranded DNA breakage [7].
Norfloxacin is an effective treatment for uncomplicated and complicated urinary tract infections. Other places in therapy include
gastrointestinal infections due to its pronounced
activity against pathogens responsible for most
diarrheal diseases (Salmonella, Shigella, Campylobacter, Yersinia and E. coli ) [7].
Norfloxacin is 3040% absorbed after oral administration [810]. Food may delay absorption,
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Reference product
Uroxin norfloxacin
400 mg tablet
0008, expiry date: July
1998
Gulf Pharmaceutical
Industries, Julphar,
United Arab Emirates
Noroxin norfloxacin
400 mg tablets
Batch no.
Study Subjects
Eighteen healthy adult male volunteers participated in this comparative study at King Khalid
University Hospital, and College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia.
Their mean age was 34.99 7.9 years with a
range of 2150 years and mean body weight
was 78.499.2 kg with a range of 6090 kg. On
the basis of medical history, clinical examination
and laboratory investigation (haematology,
blood biochemistry, and urine analysis), no subject had a history or evidence of hepatic, renal,
gastrointestinal or haematologic deviations or
any acute or chronic diseases or drug allergy.
Upon completion of study, the physical examination and selected clinical laboratory measurements were repeated. The subjects were
instructed to abstain from taking any medication
and xanthine containing foods for at least 2
weeks prior to and during the study period. No
milk or dairy products were served during the
study. Informed consent was obtained from the
subjects after explaining the nature and purpose
of the study. The study protocol was approved
by the College of Medicine, Research Center
(CMRC), King Saud University, Riyadh.
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Chromatographic Conditions
Plasma samples were analyzed for norfloxacin
according to an HPLC method [20]. All solvents
used were of HPLC grade; norfloxacin and acebutolol hydrochloride (internal standard) were
obtained from Sigma Chemical Co. (St. Louis,
MO, USA).
The HPLC system was from Waters Associates
(Milliford, MA, USA), which consisted of a solvent delivery pump (M-501), a system controller
(M-721), a data module (M-730) and an autosampler (Wisp-712) in combination with a model
740 scanning fluorescence detector. Chromatographic separation was performed using Novapak C-18 (4 mm, 150 mm 3.9 mm). The mobile
phase consisted of 14% acetonitrile in buffer
solution. The aqueous phase was prepared by
mixing 2 g of citric acid, 2 g of sodium acetate,
and 1 mL of triethylamine in 1 L of Milli-Q
water. The mobile phase was eluted at a flow
rate of 1.2 mL/min, and effluent was monitored
at excitation and emission wavelengths of 330
and 440 nm, at attenuation 2 and gain 10.
Quantitation was achieved by measurement of
the peak area ratio of the drug to the internal
standard.
Pharmacokinetic Analysis
Pharmacokinetic analysis was performed by
means of model independent method using MSExcel software. The maximum norfloxacin concentration (Cmax) and the corresponding peak
times (Tmax) were determined by the inspection
of the individual drug plasma concentration
time profiles. The elimination rate constant (Kel)
was obtained from the least-square fitted terminal log linear portion of the plasma concentration time profile. The elimination half-life (T1/2)
was calculated as 0.693/Kel. The area under the
curve to the last measurable concentration
(AUC0 t ) was calculated by the linear trapezoidal rule. The area under the curve extrapolated to infinity (AUC0 ) was calculated by
equation AUC0 t + Ct /Kel, where Ct is the last
measurable concentration.
Statistical Analysis
For the purpose of bioequivalence analysis
AUC0 t, AUC0 , and Cmax were considered as
primary variables. Two-way analysis of variance
(ANOVA GLM model [21] SAS Institute, NC,
USA) for crossover design was used to assess
the effect of formulations, periods, sequences
and subjects on these parameters. A difference
between two related parameters was considered
statistically significant for a p-value equal to or
less than 0.05. The 90% confidence intervals of
the ratio test/reference (T/R) were calculated according to various reported methods [22 24].
178
Uroxin 400 mg
tablets (test)
Noroxin 400 mg
tablets (reference)
AUC0t
(ng/mL h)
AUC0
(ng/mL h)
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
Kel (h1)
6040.2092184.24 6204.3692112.38
6309.9092163.44 6489.47 9 2113.58
1299.949534.08
1.569 0.76
4.927 90.468
0.1429 0.016
1337.219 480.40
1.44 9 0.74
5.004 9 0.56
0.140 90.01
Conclusion
Statistical comparison of the AUC0 t, AUC0 ,
and Cmax clearly indicated no significant difference between Uroxin 400 mg tablets and
Noroxin 400 mg tablets in any of the calculated
pharmacokinetic parameters. The confidence intervals for the ratios of mean AUC0 t, AUC0 ,
and Cmax indicated that these values are entirely
within the bioequivalence acceptance range of
80125% (using log-transformed data).
On the basis of the plasma levels of the 18
subjects completing this study (see Figure 1), the
mean relative bioavailability of test product
(Uroxin 400 mg tablets) was 98.90% for AUC0 t,
98.50% for AUC0 , and 102.60% for Cmax.
Based on the above pharmacokinetic and
statistical results of this study, we can conclude
that Uroxin, manufactured by Gulf Pharmaceutical Industries, United Arab Emirates is bioequivalent to Noroxin, manufactured by MSD,
Netherlands and that both products can be considered equally effective in medical practice.
Biopharm. Drug Dispos. 21: 175 179 (2000)
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