BIOPHARMACEUTICS & DRUG DISPOSITION

Biopharm. Drug Dispos. 21: 175 179 (2000)

Bioequivalence Evaluation of Norfloxacin 400 mg Tablets

(Uroxin and Noroxin) in Healthy Human Volunteers
Khalid A. Al-Rashooda, Khalil I. Al-Khamisa, Yoursy M. El-Sayeda, Sulaiman Al-Bellaa,
Mohd A. Al-Yamania, S. Mahmood Alamb and Ruwayda Dhamb,*
a
b

College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

Gulf Pharmaceutical Industries, Julphar, United Arab Emirates

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

* Correspondence to: Gulf Pharmaceutical Industries, Julphar 1201,

Twin Towers, PO Box 26699, Dubai, United Arab Emirates.

Copyright 2000 John Wiley & Sons, Ltd.

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,

Received 31 May 2000

Accepted 14 September 2000

176

K.A. AL-RASHOOD ET AL.

but the extent absorbed is not effected [10].

However, milk or yoghurt decrease the extent of
absorption by 40% [11]. Absorption is rapid following single doses of 200, 400, and 800 mg with
mean peak plasma levels of 0.75, 1.5, and 2.41
mg/mL, respectively [8,12 14]. The time to reach
peak plasma concentration is approximately 12
h [12]. Total protein binding is 10 15% [7,12].
Approximately 30% of the drug is metabolized
into six different metabolites with minimal antimicrobial activity than parent compound [10].
Norfloxacin is eliminated through metabolism,
biliary excretion, and renal excretion [15,16].
Urine recovery accounts for 26 32% of unchanged drug; fecal recovery accounts for another 8.3 53.3% (mean 30%) of unchanged drug
while approximately 30% of the drug is eliminated in urine and feces as metabolites [1,15
19]. The reported elimination half-life is 34 h
[8,10,13], although it has been noted to be 57 h
[12], this difference may be due to variation in
study design between investigators.

Objectives of the Study

The aim of the present study was to assess the
bioequivalence of a test formulation of norfloxacin (Uroxin 400 mg tablets, Julphar, United
Arab Emirates) relative to a reference formulation (Noroxin 400 mg tablets, Merck Sharpe &
Dohme (MSD), Netherlands) by a statistical
analysis of the pharmacokinetic parameters
AUC0 t, AUC0 , and Cmax as recommended by
the FDA.

Material and Methods

Study Products
Test product
Batch no.

Reference product

Uroxin norfloxacin
400 mg tablet
0008, expiry date: July
1998
Gulf Pharmaceutical
Industries, Julphar,
United Arab Emirates
Noroxin norfloxacin
400 mg tablets

Copyright 2000 John Wiley & Sons, Ltd.

Batch no.

HZ 15320, expiry date

October 1997
Merck Sharpe & Dohme
BV, Netherlands

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.

Drug Administration and Sample Collection

After an overnight fasting (10 h) subjects were
given single dose of either formulation (reference or test in a randomized fashion) of norfloxacin 400 mg tablet with 240 mL of water.
Food and drinks (other than water, which was
allowed after 2 h) were not allowed until 4 h
after ingestion of the tablets and then water,
breakfast, lunch and dinner were given to all
volunteers according to time schedule. Volunteers were ambulatory during the study but
prohibited from strenuous activity. Approximately, 7 mL of blood samples for norfloxacin
assay were drawn into heparinized tubes
through indwelling cannula before (0 h) and at
0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4.0, 5.0,
6.0, 8.0, 10.0, 12.0, 14.0, 16.0, and 24.0 h after
Biopharm. Drug Dispos. 21: 175 179 (2000)

177

BIOEQUIVALENCE EVALUATION OF NORFLOXACIN TABLETS

dosing. The blood samples were centrifuged at

3000 rpm for 10 min, plasma was separated and
kept frozen at 70C in coded polypropylene
tubes. After a period of 7 days study was repeated in the same manner to complete the
crossover design.

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.

Sample Preparation for HPLC Injection

A 200 mL plasma sample was taken in a glassstoppered tube and 50 mL of internal standard
(acebutolol 160 mg/mL) added and shaken on a
vortex mixer for 30 s. A total of 500 mL of
acetonitrile was added to precipitate the plasma
proteins; the mixture was again shaken for 1 min
and centrifuged at 6000 rpm for 10 min. The
supernatant was transferred to a 1.5-mL microcentrifuge tube and evaporated to dryness at
40C under a nitrogen stream. The residue was
reconstituted with 200 mL of mobile phase, followed by vortex mixing for 30 s; a 50-mL aliquot
was then injected to column and peak areas
were recorded.
Copyright 2000 John Wiley & Sons, Ltd.

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].

Results and Discussion

Norfloxacin was well tolerated by the subjects;
unexpected incidents that could have influenced
the outcome of the study did not occur. All
volunteers who started the study continued to
the end and were discharged in good health.
Both formulations were readily absorbed from
the gastrointestinal tract and norfloxacin was
measurable at the first sampling time (0.5 h) in
nearly all volunteers. The mean concentration
time profile for the two formulations is shown
by the Figure 1. All the calculated pharmacokinetic parameter values were in good agreement
with the previously reported values [119].
Biopharm. Drug Dispos. 21: 175 179 (2000)

178

K.A. AL-RASHOOD ET AL.

difference in periods, formulations or sequence,

having a p-value greater than 0.05.
90% confidence intervals also demonstrated
that the ratio of the AUC0 t or AUC0  of the
two formulations and for two periods lie within
the FDA acceptable range of 80125%. For
AUC0 t, the confidence interval was 88.0 105.5%
(for period 99.3 119.0%) and for AUC0  it was
88.7 104.7% (for period 99.2 116.9%).

Figure 1. Plasma concentrationtime profile of norfloxacin

400 mg tablets

Table 1 shows the pharmacokinetic parameters

for the two brands of norfloxacin 400 mg tablets.
For bioequivalence evaluation various statistical
modules were applied to AUC0 t, AUC0 , and
Cmax as per current FDA guidelines [25,26]. A
difference between two related parameters was
considered statistically significant for a p-value
equal to or less than 0.05 with 95% confidence
level. 90% confidence intervals for the log-transformed data were also calculated as per the FDA
guidelines [25,26].

Area Under the Curve (AUC0 t and AUC0 )

The mean and standard deviation of both
parameters for the two products were found
very close, suggesting that the plasma profiles
generated by Uroxin are comparable to those
produced by Noroxin. Analysis of variance
(ANOVA) for these parameters, after log-transformation of the data, showed no statistically
significant difference between the two formulations. ANOVA did not show any significant
Table 1. Pharmacokinetic parameters of norfloxacin tablets
Pharmacokinetic parameter

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

Values are given as 9S.D.

Copyright 2000 John Wiley & Sons, Ltd.

Peak Plasma Concentration (Cmax)

For bioequivalence evaluation, ANOVA was
performed also on Cmax values of both products.
ANOVA showed that the two formulations were
not statistically different from each other. Furthermore, there was no statistically significant
difference with regards to periods and sequences. For formulation, periods and sequence
the p-values were greater than 0.05.
90% confidence intervals also demonstrated
that ratio of the Cmax of the two formulations
was 84.5 109.3% and found within the FDA
acceptable range of 80125%.

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)

BIOEQUIVALENCE EVALUATION OF NORFLOXACIN TABLETS

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Biopharm. Drug Dispos. 21: 175 179 (2000)