Dose Ranging Pharmacokinetics and Brain Distribution

of Norfloxacin Using Microdialysis in Rats

SANDRINE MARCHAND, MARYLORE CHENEL, ISABELLE LAMARCHE, CLAUDINE PARIAT, WILLIAM COUET
Equipe Medicaments Anti-infectieux et Barrie`re Hematoencephalique, Pole Biologie Sante, Medecine-Sud, Niveau 1, 40,
Avenue du Recteur Pineau, 86022 Poitiers Cedex, France

Received 18 April 2003; revised 19 June 2003; accepted 19 June 2003

ABSTRACT: The purpose of this study was to investigate the effect of dose on norfloxacin
pharmacokinetics and distribution into the brain extracellular fluid (ECF), in freely
moving rats. Unbound concentrations of norfloxacin in hippocampus were determined by
microdialysis after an i.v. bolus dose of 12.5, 25, 50, 100, or 150 mg/kg in rats. In vivo
recovery of norfloxacin was determined by retrodialysis by calibrator. Among three
fluoroquinolones (enoxacin, pefloxacin, and ciprofloxacin) selected as potential calibrators, ciprofloxacin was selected as the best one. Maximum ECF brain norfloxacin
concentrations are rapidly obtained but the ECFbrain/plasma areas under curves (AUC)
ratios are low and independent of dose with a mean value of 8.2
5.8%. By contrast,
norfloxacin systemic pharmacokinetics was nonlinear, with total plasma clearance
decreasing significantly from 23.0
3.4 to 14.4
3.8 mL/min/kg when dose increased
from 12.5 to 150 mg/kg. 2003 Wiley-Liss, Inc. and the American Pharmacists Association
J Pharm Sci 92:24582465, 2003

Keywords:

norfloxacin; microdialysis; CNS; fluoroquinolones; pharmacokinetics

INTRODUCTION
Central nervous system (CNS) side effects represent the second most common type of adverse
events following therapy with fluoroquinolones
(FQs).1 They vary in severity, and include headache, dizziness, agitation, sleep disorders, psychosis, and, in rare occasions, convulsions.2 These
side effects depend upon two characteristics, FQs
ability to reach the receptors involved in this
excitatory activity at the central nervous system
level (pharmacokinetic contribution), and ability
to interact with these receptors (pharmacodynamic contribution). These two determinants of the
CNS toxicity are highly variable between FQs.
Using an in vivo approach with determination of
drug concentration within the cerebrospinal fluid
Correspondence to: William Couet (Telephone: 33-5-49-4543-79; Fax: 33-5-49-45-43-78; E-mail: wcouet@univ-poitiers.fr)
Journal of Pharmaceutical Sciences, Vol. 92, 24582465 (2003)
2003 Wiley-Liss, Inc. and the American Pharmacists Association

2458

(CSF) at the onset of maximal seizures, we have

previously observed that these two characteristics
have a tendency to compensate for each other.3
Pefloxacin distributes extensively within the CSF
but high concentrations are necessary to induce
seizures. By contrast, the CSF norfloxacin distribution is much more limited, but it induces
seizures at much lower concentrations. The blood
brain barrier (BBB) or/and bloodcerebrospinal
fluid barrier (BCSF) plays a major role in controlling the CNS toxicity, and it is therefore important to investigate the CNS distribution of these
compounds. This has been partially done in vitro4
as well as in vivo.58 FQs lipophilicity seems to be
a major determinant of their CNS distribution.4,5
However, at the onset of maximal seizures the
CCSF/CU ratios estimated for norfloxacin was
equal to 45%, that is four to five less than that
of enoxacin (20%), although the two compounds
have virtually similar log-D values.8 Efflux transport systems could be involved in the CNS distribution of some fluoroquinolones. It was shown

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in vitro using Caco-2 and LLC-PK1 cell lines that

sparfloxacin9 and levofloxacin10 are transported
by P-glycoprotein, and it was suggested that
in vivo P-glycoprotein may partially contribute
to the limited brain distribution of sparfloxacin,11
grepafloxacin,12 and of a new quinolone antibacterial agent, HSR-903.13 However, norfloxacin has not been found to be a glycoprotein-P
substrate.11 Other anionic transporters such as
multidrug resistance-associated protein were suggested to be involved in the limited brain distribution of HSR-903 and grepafloxacin.12,13
The involvement of active transport systems
raises the issue of nonlinearity, and previously
published studies have suggested nonlinear CSF
distribution of ciprofloxacin and norfloxacin in
rats.5,14 Saturation of efflux system should lead to
a more than proportional drug exposure to the
brain as doses increase, which may become relevant during massive drug ingestion and could
exacerbate CNS toxicity. Drug pharmacokinetic
profiles in brain and blood have been analyzed
from a theoretical point of view and compared to
experimental data obtained with intracerebral
microdialysis.15 For many drugs, unbound brain
concentrations are much lower than the corresponding plasma concentrations but equilibrium
between brain and plasma is very rapid and halflife in brain is similar to that in plasma.15 Such
behavior can be explained by more efficient efflux
out than influx into the brain, possibly due to cerebrospinal bulk flow but according to the authors,
most likely to active transport out of the brain.15
The aim of the present study was therefore to
investigate the linearity of the CNS distribution
of norfloxacin using intracerebral microdialysis,
after intravenous bolus administrations of various
doses including those responsible for CNS side
effects (12.5, 25, 50, 100, or 150 mg/kg) to awake
freely-moving rats.

EXPERIMENTAL
Chemicals
Ciprofloxacin hydrochloride and pefloxacin methanesulfonate were respectively provided by Bayer
Pharma (Puteaux, France) and Rhone Poulenc
Rorer (Alfortville, France). Enoxacin and norfloxacin were obtained from Sigma (Saint-Quentin
Fallavier, France). A norfloxacin salt was prepared as previously described.3 Solvents, including
water, were of analytical grade.

2459

Animals
Male Sprague-Dawley rats from Depres Breeding
Laboratories (St. Doulchard, France), weighing
293
22 g were used. The animals were placed
in wire cages in a 12-h lightdark cycle for a
minimum of 5 days before the beginning of
experiment to adjust to the new environment.
During this period, they had free access to food
(UAR A04, U.A.R. Laboratories, Villemoissonsur-Orge, France) and water. Ethical approval
was obtained from the Animal Ethics Committee
of the Faculty (BHE 2000/12/AA).
Animals Surgery
Implantation of Blood Femoral Cannulas
The day before experiment, rats were anaesthetized by a intraperitoneal injection of sodium
pentobarbital (60 mg/kg) (Sanofi Laboratories,
Libourne, France). Two polyethylene catheters, a
first one with a small diameter (i.d.: 0.26 mm, o.d.:
0.61 mm; Phymep, Paris, France) connected to a
larger one (i.d.: 0.58 mm, o.d.: 0.96 mm, Harvard,
Les Ulis, France) were implanted in the left
femoral vein for drug administration and in the
left femoral artery for blood samples collection.
A heparinized saline solution (100 UI/mL) was
maintained into cannulae to prevent clotting.
Implantation of Microdialysis Probes
The day before experiment, the anaesthetized
rats (sodium pentobarbital, 60 mg/kg) were placed on the stereotaxic instrument (David Kopf
Instruments, Tujunga, CA) and then connected
to a CMA 150 temperature controller (CMA
microdialysis, Phymep, Paris, France). A midline
incision was made to expose the skull and a
microdialysis CMA/12 guide cannula was implanted into the left dorsal hippocampus with
coordinates: lateral 4.8 mm, anterior 5.6 mm,
ventral 4.7 mm relative to bregma.16 The guide
cannula was fixed to the skull by two screws
and two types of dental cement. A first cement
with aqueous solvent (Aquacem, Promodentaire,
Gonesse, France) was used directly in contact
with the skull and a second cement with organic solvent (Heliotone, Promodentaire, Gonesse,
France) was then used to finalize the assembling.
The animals were placed in thermostated chamber until their first signs of movement. They were
allowed to recover for 24 h before the beginning of
the experiment. Food was withdrawn 12 h before

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MARCHAND ET AL.

the experiment, but animals had free access to

water.
Experimental Design
Three fluoroquinolones, enoxacin, pefloxacin, and
ciprofloxacin, were a priori selected as potential
calibrators of norfloxacin based upon their structural or/and lipophilicity characteristics.
Choice of an Adequate Calibrator Using the
Retrodialysis Method In Vivo
The day of the experiment, a CMA/12 (0.5  3 mm,
CMA microdialysis, Phymep, Paris, France)
microdialysis probe was inserted into dorsal
hippocampus. The CMA/12 probe was first perfused at a flow rate of 1 mL/min with Ringer solution
for 1.5 h to stabilize the system and to obtain
blank samples (CMA 100 microdialysis pump,
Phymep, Paris, France). The Ringer medium with
a pH equal to 7.4, consisted of 145 mM NaCl,
0.6 mM KCl, 1.0 mM MgCl2, 1.2 mM CaCl2,
0.2 mM ascorbic acid, in a 2 mM potassium phosphate buffer.17 After the blank period, a retrodialysis period was started by changing the blank
perfusion solution to a Ringer containing either
norfloxacin (400 nM) and enoxacin (15,000 nM)
(n 5), norfloxacin (400 nM), and pefloxacin
(400 nM) (n 4) or norfloxacin (400 nM) and
ciprofloxacin (400 nM) (n 5). After starting the
perfusion of Ringer containing fluoroquinolones, a
2-h period of equilibration was performed before
collecting samples at 30-min intervals during 6 h.
The microdialysate samples were collected automatically by a CMA/140 Microfraction collector
(CMA microdialysis, Phymep, Paris, France). The
concentration of each fluoroquinolone was determined in the perfusate (Cin) and in dialysates
(Cout) by HPLC. The in vivo relative recovery by
loss was expressed as a percentage (RLin vivo) and
calculated as (1):
RLin vivo Cin  Cout =Cin   100

Effect of Concentration on In Vivo

Retrodialysis Loss
This was tested only with ciprofloxacin. The day
of the experiment, a CMA/12 microdialysis probe
was inserted into dorsal hippocampus. The CMA/
12 probe was first perfused at a flow rate of 1 mL/
min with Ringer solution for 1.5 h to stabilize
the system and to obtain a blank sample. After
the blank period, a Ringer solution containing

norfloxacin or ciprofloxacin was perfused at two

rising concentrations separated by a 2-h equilibration period. Three different concentrations
were tested: 200, 400, and 800 nM. After equilibration and for each level of concentration,
samples were collected at 30-min intervals during
2 h. The concentration of each fluoroquinolone
was determined in the perfusate (Cin) for each
level of concentration and in dialysates (Cout) by
HPLC. The in vivo relative recovery by loss (RL)
was calculated according to eq. 1.
Comparison of Norfloxacin and Ciprofloxacin
Probe Recovery by Gain and Loss In Vitro
For in vitro recovery by gain determination, a
CMA/12 microdialysis probe was placed in a
20-mL bath containing Ringer solution at 378C
with norfloxacin and ciprofloxacin (final concentration 400 nM). The probe was perfused with
blank Ringer solution at a flow rate of 1 mL/min
using a CMA 100 microdialysis pump (Phymep,
Paris, France). After equilibration time, the microdialysate samples were collected for 6 h at 30-min
intervals. The concentration of norfloxacin and
ciprofloxacin were determined in Ringer medium
surrounding the probe (Cm) and in dialysates
(Cout) by HPLC. The relative in vitro recovery by
gain was expressed as a percentage (RGin vitro)
and was calculated as (2):
RGin vitro Cout =Cm  100

The same microdialysis probe was then placed

in a 20-mL bath containing Ringer blank solution
to determine the in vitro recovery by loss. The
probe was perfused with Ringer solution containing norfloxacin and ciprofloxacin (final concentration: 400 nM) at a flow rate of 1 mL/min. After
equilibration the microdialysate samples were
collected for 6 h at 30-min intervals. The concentration of norfloxacin and ciprofloxacin were
determined in the perfusate (Cin) and in dialysates
(Cout) by HPLC. The relative recovery by loss
(RL)in vitro was calculated according to eq. (1).
CNS Distribution of Norfloxacin
The day of the experiment, a CMA/12 microdialysis probe was inserted into dorsal hippocampus.
The CMA/12 probe was first perfused at a flow
rate of 1 mL/min with Ringer solution for 1.5 h to
stabilize the system and to obtain a blank sample.
A retrodialysis period was then started by changing the blank perfusion solution to a Ringer
containing calibrator (ciprofloxacin: 400 nM). A

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period of 2 h equilibration was performed before

the bolus administration of norfloxacin via the left
femoral vein. Rats received norfloxacin at either
12.5 (n 4), 25 (n 6), 50 (n 5), 100 (n 5), or
150 mg/kg (n 4) dose (corresponding, respectively, to 35.1, 70.3, 141, 281, and 422 mmol/kg).
The norfloxacin solution was prepared by dissolving an adequate amount of norfloxacin salt
in NaCl 0.9% so that the final volume of administration was set to 1 mL. Arterial blood
samples were drawn at 0, 5, 15, 30, 60, 180, 300,
and 420 min via left femoral artery cannula.
The plasma was separated by centrifugation
(3000 rpm, 10 min, 48C) and frozen at 208C
until the analysis. Microdialysate samples were
collected for 7 h by fractions corresponding
to 30-min intervals, and analyzed the day of
experiment.

Sample Analysis
Microdialysis Samples
Ten microliters of the microdialysis samples were
directly injected onto a Kromasil C18 column
(5-mm particles, 250  3 mm i.d., Varian, Les Ullis,
France). The chromatographic system consisted
of a Shimadzu LC-10AT pump and a Waters
717 plus Autosampler connected to a fluorescence
detector (Waters 474). Data were recorded and
processed using a Waters 746 integrator. All FQs
were analyzed at same wavelengths (lex 280 nm,
lem 445 nm). The flow rate was ranging from
0.5 to 1 mL/min and the mobile phase consisted of
0.1 M aqueous citric acid solution containing 4
to 8% (v/v) acetonitrile and 10 mM tetra-butylammonium perchlorate. The between-day variability was characterized each day of the analysis
during all retrodialysis experiments, and was
always less than 10%.
The limit of quantification of norfloxacin in
microdialysates was 25 nM with a CV% and an
accuracy (n 10) respectively equal to 2.5 and
101.3% for an injected volume of 10 mL. During
infusion experiments, the between-day variability
of ciprofloxacin was characterized at 400 nM by a
coefficient of variation and accuracy respectively
equal to 6.6% and 100.0% (n 32) with calibration
curve concentrations ranging from 100 to 800 nM.
The between-day variability of norfloxacin was
characterized at 25, 200, and 400 nM by a coefficient of variation respectively equal to 16.7%
(n 16), 6.8% (n 15), and 3.8% (n 14), and accuracy respectively equal to 93.9% (n 16), 99.6%

2461

(n 15), and 101.3% (n 14). The calibration curve

used to quantitative norfloxacin in microdialysates ranged from 25 to 800 nM.
Plasma Samples
The chromatographic system used to analyze
norfloxacin in plasma samples consisted of a
Shimadzu LC-6A pump, a Waters 710A Autosampler connected to a fluorescence detector
(Waters 470) (lex 280 nm, lem 445 nm). Data
were recorded and processed using a Waters 746
integrator. The flow rate was 0.8 mL/min and the
mobile phase consisted of 0.1 M aqueous citric
acid solution containing 8% (v/v) acetonitrile
and 10 mM tetra butyl ammonium perchlorate.
Plasma samples (50 mL) were diluted by addition
of 200 mL of perchloric acid (0.1 N). The mixture
was then centrifuged (10,000 rpm, 5 min) and
20 mL of the supernate were injected onto the
column. For the 100-mg/kg and 150-mg/kg norfloxacin doses, initial plasma samples (sampling
times: 5, 15, 30, and 60 min) were appropriately
diluted with blank plasma. The calibration curve
used for the determination of norfloxacin plasma
concentration ranged from 0.78 to 100 mM. The
within-day variability of the method was characterized at two concentrations: 100 and 1.56 mM
with coefficients of variation (n 6) respectively
equal to 8.8 and 4.5% and accuracy (n 6) respectively equal to 100.4 and 103.1%. The betweenday variability of the method was characterized
each day of the analysis at two different concentrations: 50 and 3.125 mM with coefficients of
variation respectively equal to 8.4% (n 19) and
9.5% (n 16) and accuracy respectively equal to
96.3% (n 19) and 100.8% (n 16).
Pharmacokinetic Analysis
Plasma Data
Norfloxacin pharmacokinetic parameters were
determined in each individual rat by a noncompartmental approach. Total body clearance (CLT)
was calculated as CLT Dose/AUCplasma, where
AUCplasma is the total area under the plasma
concentration versus time curve. The AUCplasma
was calculated using the trapezoidal rule; the
area remaining under the curve after the last
measured concentration, C(last), was determined
from C(last)/k. The rate constant k and its corresponding half-life (t1/2 plasma) were estimated by
least squares fit of data points (log concentration
time), in the terminal phase of the decline. The

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MARCHAND ET AL.

steady state volume of distribution (Vss) was obtained from (Dose  AUMCplasma)/(AUCplasma)2,
where AUMCplasma is the total area under moment
curve.
Brain Extracellular Fluid Concentrations
Unbound concentrations of norfloxacin in hippocampus were obtained by correcting measured
dialysate concentrations by the mean recovery
by loss of ciprofloxacin estimated during the 7-h
experiment. Norfloxacin pharmacokinetic parameters in brain were determined in each individual
rat by a noncompartmental approach. AUCECF
was calculated using the trapezoidal rule; the
area remaining under the curve after the last
measured concentration, C(last), was determined
from C(last)/k. The brain half-life (t1/2 brain) was
estimated by least squares fit of data points (time,
log concentration), in the terminal phase of the
decline.

From these initial experiments, ciprofloxacin

was selected as the most appropriate calibrator.
Subsequent experiments were therefore performed with this calibrator only, first to test a
concentration effect on the relative loss of norfloxacin and ciprofloxacin in vivo, and second to compare the in vitro dialysance of norfloxacin and
ciprofloxacin.
Effect of Norfloxacin and Ciprofloxacin
Concentrations on In Vivo Recovery by Loss
The recovery by loss (RLin vivo) of norfloxacin and
ciprofloxacin were independent of concentrations over the range investigated (200800 nM)
(ANOVA, p > 0.05) and respectively ranged from
9.0
0.9 to 13.1
2.5% and from 7.0
1.3 to 13.5

0.8%. Moreover, no significant difference was observed between the relative loss of norfloxacin and
ciprofloxacin at the various concentrations tested
(p > 0.05, ANOVA analysis of variance).

Statistical Analysis
The in vivo relative recoveries by loss of norfloxacin, ciprofloxacin, pefloxacin, and enoxacin were
compared by a paired t-test ( p < 0.05). Parametric
one-way analysis of variance after applying the
Bartletts test to check for homogeneity of variance, were used to compare the impact of concentration on recovery loss. The in vitro recovery by
gain and loss of norfloxacin and ciprofloxacin were
compared by unpaired t-test ( p < 0.05). Between
doses, comparisons of plasma CLT, t1/2 and Vss
were performed using a parametric one way analysis of variance, followed when appropriate (for
clearance), by post hoc tests on different contrasts
with the Scheffe procedure. The significance level
was fixed at p < 0.05. Results are presented as
mean
SD.

RESULTS
Recovery by Loss of Fluoroquinolones In Vivo
The relative recovery by loss (RL) of norfloxacin in
five rats over a period of 6 h was compared with
those of enoxacin, pefloxacin. or ciprofloxacin perfused simultaneously into the probes placed in the
hippocampus. Only norfloxacin and ciprofloxacin
exhibited similar recovery by loss when perfused
simultaneously ( p < 0.05, paired t-test) with mean
ratios norfloxacin/ciprofloxacin ranging between
0.94
0.05 (Rat 3, n 12) and 1.07
0.05 (Rat 2,
n 12).

Probe Recovery by Gain and Loss In Vitro

At a flow rate of 1 mL/min, RG and RL of norfloxacin and ciprofloxacin in vitro appeared stable
over a 6-h period with mean in vitro recoveries
ranging between 32 and 37%. There was no statistical difference between the RG and the RL for
either compound ( p > 0.05, unpaired t-test). Furthermore, the RG of norfloxacin was not statistically different from the RL of ciprofloxacin
( p > 0.05, unpaired t-test) and the ratio of the
loss of ciprofloxacin and recovery of norfloxacin
was determined and equal to 1.04
0.13.
Norfloxacin Brain Distribution Studies
Norfloxacin brain ECF concentrations were much
less than the corresponding plasma concentrations, but the highest ECF concentrations of
norfloxacin were observed in the first (030 min)
dialysate. Then norfloxacin concentrations in
ECF and plasma decayed in parallel with time.
Norfloxacin ECF brain concentrations after
12.5 mg/kg i.v. bolus were most often lower than
the quantification limit. Typical examples are presented on Figure 1.
Pharmacokinetic parameters obtained for each
dose are presented in Table 1. A statistically significant effect of dose was observed for clearance,
with a difference between the mean of the two
lower doses (12.5 and 25 mg/kg) and the mean of
the three others (50, 100, and 150 mg/kg doses)

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2463

Figure 1. Norfloxacin ECF brain and plasma concentrations in representative rats

following respectively 12.5, 25, 50, 100, and 150 mg/kg (35.1, 70.3, 141, 281, and 422 mmol/
kg) i.v. bolus doses. (*) Norfloxacin concentrations in hippocampus extracellular fluid;
(&) Norfloxacin concentrations in plasma. Norfloxacin concentrations in hippocampus
extracellular fluid following 12.5 mg/kg i.v. bolus were not detectable.

being significantly significant. The steady state

volume of distribution (Vss) had a tendency to
decrease when dose increased but this trend was
no statistically significant. The apparent elimination half-life slightly increased with dose but again
without reaching the level of significance (Table 1).
Half-lives were not statistically different between plasma (155
82 min, n 24) and brain
(178
110 min, n 20) and the AUCECF Brain/
AUCplasma ratio was independent of dose, and
equal on average to 8.2
5.8% (n 20) (Table 1).

DISCUSSION
In microdialysis studies, the in vivo recovery
is most often accomplished by the no-net flux
method18 or by retrodialysis with an appropriate

calibrator.19 A major limitation with the no-net

flux method is that it necessitates to reach steady
state both in plasma and brain and cannot provide
information about the CNS rate of distribution.
Retrodialysis was therefore preferred for investigating norfloxacin CNS distribution. An ideal
calibrator is one that demonstrates the same
dialysance as the compound of interest. Therefore, a related compound with a comparable
molecular weight, degree of ionisation and lipophilicity may serve as a potential calibrator.19
However, careful a priori choice of a calibrator
does not preclude proper a posteriori validation,
in particular by conducting simultaneous in vitro
and in vivo retrodialysis with the calibrator and
the test compound.19,20
Ciprofloxacin appeared to be the best calibrator. The relative in vivo recoveries of norfloxacin

Table 1. Norfloxacin Plasma and Brain Pharmacokinetic Parameters

Doses
(mg/kg)
Group
Group
Group
Group
Group

1 (n 4)
2 (n 6)
3 (n 5)
4 (n 5)
5 (n 4)

12.5
25
50
100
150

Doses
(mmol/kg)

t1/2 plasma
(min)

CLTa
(mL/min/kg)

Vss
(mL/kg)

AUCECF Brain/
AUCplasma (%)

t1/2 brain
(min)

35.1
70.3
141
281
422

143
20
126
45
160
72
168
31
189
68

23.0
3.4
24.2
8.0
16.3
5.0
14.7
4.0
14.4
3.8

3580
407
3533
594
2991
816
3002
298
3160
952

NA
12.3
5.9
8.7
7.3
3.9
1.0
6.7
4.1

NA
135
78
140
60
299
151
141
17

NA: Data not available. Norfloxacin concentrations in brain ECF were lower than the quantification limit.
a
Statistically different by parametric one way analysis of variance (p < 0.05).
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MARCHAND ET AL.

and ciprofloxacin at a flow rate of 1 mL/min

varied between probes from 6.97
1.59% to
17.49
1.20% (mean
SD), but for any individual probe the in vivo recovery was essentially
stable with time, as previously observed with
other compounds including AZT,19 fluconazole,20
gabapentin,21 morphine,22 and codeine.23 To avoid
outliers that may occur within these low range of
recoveries, the recovery by loss of ciprofloxacin was
estimated as an average value obtained by making
the arithmetic mean of the individual estimates.
Differences between the two procedures were
most often negligible except for the initial dialysates when the recovery estimate was probably
overestimated.
Norfloxacin CNS distribution was investigated
in the hippocampus, which was selected as an area
of interest because this seizure-prone brain region
is recognized as being involved in the regulation of
the brain excitability and has been suggested as
playing a role in fluoroquinolones induced convulsions.24 No sign of CNS toxicity was observed
following the four lowest doses (12.5, 25, 50, and
100 mg/kg), but rats who had received the 150 mg/
kg dose presented sudden shakes occurring most
often at about 30 min postdosing and lasting for 2
to 3 h. The two extreme values of this dose ranging
study, 12.5 and 150 mg/kg, were therefore selected
for analytical sensitivity and toxicity constraints,
respectively.
Norfloxacin ECF brain concentrations were
much lower than the corresponding plasma concentrations (Figure 1 and Table 1). Considering
that norfloxacin plasma protein binding is limited
and on average close to 20%3 the AUC ratios between ECF and unbound plasma concentrations
should still be much less than unity. Equilibrium
between brain and plasma was very rapid and halflife in brain was similar to that in plasma. These
characteristics have already been observed with
many other compounds and suggest a more efficient efflux out than influx into the brain, most
likely due to the presence of active efflux transport
systems.15 ECF brain to plasma AUC ratios, corresponding to the influx to the brain over efflux
from the brain clearances ratio, did not vary significantly with dose (Table 1), although the mean
value obtained after the 25 mg/kg dose was apparently higher than after larger doses. Interestingly, an increased ratio at low doses is just the
opposite of what would be expected in the presence
of saturable active efflux transporters. The previous observation was due to relatively high AUC
ratios obtained in several rats, with close inspec-

tion of these data not revealing any particular

problem. Overall, the low ECF brain to plasma
AUC ratios observed in the present study do not
prove but are consistent with the existence of
active efflux transport systems. The linear CNS
distribution with dose would then simply mean
that these transport systems are not saturated
in the wide range of norfloxacin concentrations
encountered during this study.
Although it has no apparent effect on its CNS
distribution, the sixfold increase of dose was accompanied by an approximately 1.7-fold, statistically significant and unexplained reduction of
norfloxacin systemic clearance. It has been shown
that norfloxacin clearance in rats had a tendency
to decrease (from 3.75
1.30 L/h/kg to 2.66

0.06 L/h/kg) as the dose increased (from 42 mg/kg
to 60 mg/kg), which was attributed to a saturation of metabolic pathways or renal secretion.25
However, no metabolite has been detected in this
situation and because the contribution of renal
clearance to total clearance is limited to 15%,25 a
saturation of renal secretion cannot have a major
impact on total clearance. Biliary or/and intestinal
secretion leading to recirculation phenomenon
may be observed with FQs. These phenomenon
involve active, and therefore, potentially saturable
transport systems and could therefore contribute
to this apparent nonlinearity.26,27 However, and
more importantly, they should hamper the interpretation of clearance estimates obtained from the
Dose/AUCplasma ratio.
In conclusion, this study has demonstrated that
the CNS diffusion of norfloxacin in rats was linear
in a wide range of doses administered, whereas its
systemic pharmacokinetics was not.

ACKNOWLEDGMENTS
This article is dedicated in the memory of our
colleague Serge Bouquet, deceased on January
14th 2003. This work was supported by grant
from the French National Academy of Medicine.
The authors gratefully acknowledge the laboratory assistance of Agnes Audurier-Devignes.

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