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Journal of Pharmacy
Research Paper
And Pharmacology

Effect of ciprofloxacin and grapefruit juice on oral

pharmacokinetics of riluzole in Wistar rats
Punna Rao Ravi, Rahul Vats and Upendra Reddy Kora
Pharmacy Department, BITS-Pilani Hyderabad Campus, Hyderabad, Andhra Pradesh, India

Keywords
ciprofloxacin; grapefruit juice;
pharmacokinetic interactions; riluzole
Correspondence
Punna Rao Ravi, Pharmacy Department, Birla
Institute of Technology and Science-Pilani
Hyderabad Campus, Jawaharnagar, Ranga
Reddy District, Andhra Pradesh 500078, India.
E-mail: rpunnarao@gmail.com,
rpunnarao@bits-hyderabad.ac.in
Received May 22, 2012
Accepted September 21, 2012
doi: 10.1111/j.2042-7158.2012.01604.x
Abstract
Objectives The objective of this study was to explore potential drug–drug/food
interactions of ciprofloxacin and grapefruit juice, known hepatic cytochrome
P450 (CYP) 1A2 inhibitors, on single-dose oral pharmacokinetics of riluzole, a
substrate of CYP 1A2 enzymes.
Methods Pharmacokinetic parameters of riluzole were determined in Wistar rats
after single-dose co-administration with ciprofloxacin and grapefruit juice.
In-vitro metabolic inhibition studies using rat and human liver microsomes and
intestinal absorption studies of riluzole in a rat everted gut-sac model were con-
ducted to elucidate the mechanism of interaction. A validated HPLC method was
employed to quantify riluzole in the samples obtained in various studies.
Key findings Co-administration of ciprofloxacin with riluzole caused signifi-
cant increase in systemic exposure of riluzole (area under the curve, maximum
plasma concentration and mean residence time were found to increase).
Co-administration of grapefruit juice with riluzole did not cause any significant
difference in the pharmacokinetic parameters of riluzole. In-vitro metabolism
studies demonstrated significant inhibition of riluzole metabolism when it was
co-incubated with ciprofloxacin or grapefruit juice. No significant change was
observed in apparent permeability of riluzole.
Conclusions Co-administration of ciprofloxacin with riluzole increases the sys-
temic levels of riluzole and thereby the oral pharmacokinetic properties of rilu-
zole while co-administration of grapefruit juice with riluzole has no significant
effect.

Introduction
Cytochrome P450 (CYP) enzymes are well known for their
participation in the oxidative and reductive metabolism of
xenobiotics including drugs.[1] CYP enzymes derived from
families 1–3 are mainly involved in the biotransformation
of most drugs.[2] It has been estimated that 90% of drug
metabolism in humans can be attributed to six main
enzymes including CYP 1A2, 2C9, 2C19, 2D6, 2E1 and 3A4/
5.[3] The CYP1 family comprises three members with CYP
1A2 being one of the major CYPs in human liver (~13–
15%).[4] CYP 1A2 metabolizes a variety of clinically impor-
tant drugs and natural and endogenous compounds, with
variable contributions to their overall elimination.[5]
Many researchers have demonstrated that drug biotrans-
formation is frequently affected by inducers or inhibitors of
CYP enzymes, which can be other drugs or chemical agents.
There have been reports on the occurrence of adverse
effects when an inducer or inhibitor of CYP enzymes was
co-administered with CYP enzymes’ substrate drugs.[6] Cur-
rently, many researchers are working to explore the possible
outcomes of CYP enzyme-mediated drug–drug or drug–
food interactions using suitable economical animal models,
including rats.
Riluzole is an anti-glutamatergic drug used in the effec-
tive treatment of several diseases including amyotrophic
lateral sclerosis (ALS),[7] Parkinson’s disease,[8] ischaemia[9]
and multiple sclerosis.[10] Riluzole is the only approved
pharmacological treatment currently available for ALS (in a
fixed dosage schedule of 50 mg twice daily) for transient
survival.[11] It shows rapid absorption (peak concentrations
occur within 1–1.5 h) with 60% oral bioavailability in

© 2012 The Authors. JPP © 2012

Royal Pharmaceutical Society Journal of Pharmacy and Pharmacology, ••, pp. ••–•• 1
CYP 1A2 inhibitors and riluzole kinetics
humans.[12] Riluzole is known to undergo extensive hepatic
metabolism, primarily by CYP 1A2 isozyme, followed by
glucuronidation. Differences in the terminal half-life (t1/2)
of riluzole have been reported between humans[13] (t1/2
12 h) and rats[14] (t1/2 9.85 h). Riluzole is also reported to be
a substrate of permeability-glycoprotein (P-gp)[15] and
breast cancer resistance protein (BCRP) transporters as well
as an inducer of BCRP on chronic administration.[16]
Riluzole is reported to exhibit dose-dependent adverse
effects such as asthenia, nausea, liver dysfunction, dizziness,
decreased lung function and vertigo in humans.[17] There-
fore, potential adverse interactions may occur when riluzole
is administered concurrently with agents that inhibit CYP
1A2 isozyme and thereby alter the time course of riluzole in
the body.
Ciprofloxacin is a broad-spectrum, synthetic antibacterial
drug used for the treatment of various bacterial infections
particularly those of the genitourinary, gastrointestinal
and respiratory tracts.[18] It has an oral bioavailability of
70%, with a mean elimination half-life of 4 h in healthy
humans.[19] Ciprofloxacin is a noted hepatic CYP 1A2
inhibitor and an ambiguous P-gp substrate.[20]
Grapefruit juice is an excellent source of many nutrients
and phytochemicals. It is widely consumed not only for its
taste and nutritive value but also because of its various
pharmacological properties. Many compounds have been
proposed to be the active ingredients in grapefruit juice.
These include both flavonoids (e.g. naringenin, naringin,
quercetin and kaempferol)[21] and non-flavanoids (e.g.
6’,7’-dihydroxybergamottin).[22] Grapefruit juice has been
reported to inhibit the activity of several CYP subfamilies
including CYP 1A2. Bergamottin and naringenin have been
identified as the active constituents in grapefruit juice
responsible for the inactivation of CYP 1A2.[23–25] The
mechanism involves irreversible and sustained inhibition of
the enzyme system, possibly accelerating the degradation of
the enzyme system.[26,27] Conflicting reports have been
found on the effects of grapefruit juice on the P-gp efflux
system, ranging from inhibition to activation.[28,29]
The effect of CYP 1A2 inhibitors and substrates on the
metabolic clearance of riluzole has already been estab-
lished in both in-vitro and in-vivo studies. Significant
drug–drug interactions have been documented in various
in-vivo pre-clinical studies involving pilocarpine[30] and
minocycline[31] upon co-administration with riluzole.
However, no reports were found on possible pharmacoki-
netic interactions between ciprofloxacin or grapefruit
juice and riluzole in either pre-clinical or clinical studies.
Therefore, this study was undertaken to investigate the
effects of ciprofloxacin and grapefruit juice on the oral
pharmacokinetics of riluzole in male Wistar rats and to
explore the possible mechanisms of drug–drug or drug–
food interaction.
2 Royal Pharmaceutical Society Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Punna Rao Ravi et al.
Materials and Methods
Riluzole and ciprofloxacin were obtained as gift sample
from Apotex Research Pvt. Ltd (Bangalore, India). Nebivo-
lol was procured from Vesta Pharmachem Pvt. Ltd (Surat,
India). Rat liver microsomes (RLMs), human liver micro-
somes (HLMs) and NADPH were procured from BD
Gentest (Woburn, MA, USA). HPLC-grade acetonitrile,
methanol, potassium dihydrogen orthophosphate
(KH2PO4) and sodium citrate were purchased from Merck
laboratories (Mumbai, India). Methylcellulose (MC)
(approx. MW = 14 000; degree of substitution = 1.5–1.9)
and Tween 80 were purchased from S.D. Fine Chem Ltd
(Mumbai, India). Milli-Q water purification system (Milli-
pore, Billerica, MA, USA) was used for obtaining high-
quality HPLC-grade water. Male Wistar rats were purchased
from Sainath agencies (Hyderabad, India). Grapefruits were
purchased from the local market in Hyderabad, India and
the juice was obtained by squeezing and filtering the edible
portion of fruit.
Pharmacokinetic studies
Male Wistar rats, 180–220 g, were used in the study. The
experimental protocol was approved by the Institutional
Animal Ethics Committee (Approval No.: IAEC-02/01–11)
dated 2nd January 2012. All rats were housed under con-
stant environmental conditions (22 ⫾ 1°C, 55 ⫾ 10% rela-
tive humidity, 12-h light–dark cycle) and were allowed free
access to food and water. The rats were fasted overnight
(12 h) before dosing and continued on fasting until 4 h
after drug administration. Thereafter, free access to rat chew
diet was provided. In all the studies, freshly prepared drug
formulations were administered by the oral route (5 ml/kg)
using gavage. Single-dose oral pharmacokinetic studies were
designed to test the effect of co-administering riluzole with
ciprofloxacin and riluzole with grapefruit juice separately,
in comparison with the pharmacokinetic profile of riluzole
alone in Wistar rats.
In the study design, all the rats were randomly divided in
to three treatment groups with five animals (n = 5) in each
group. The treatment groups were designated as: Group A
(control group), treated with riluzole (10 mg/kg suspended
in 0.5% MC) alone; Group B, co-administration of riluzole
(10 mg/kg suspended in 0.5% MC) with ciprofloxacin
(10 mg/kg suspended in 0.5% MC); Group C, co-
administration of riluzole (10 mg/kg suspended in 0.5%
MC) with grapefruit juice (2 ml/kg).
Blood samples were collected from the retro-orbital
plexus into microfuge tubes containing sodium citrate as an
anticoagulant (3.8% w/v) at pre-dose, 0.25, 0.50, 1.0, 2.0,
3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h post dose and kept on ice
until further processing. These samples were further har-
vested for plasma by centrifuging at 4°C for 10 min at
© 2012 The Authors. JPP © 2012
Punna Rao Ravi et al.
905 ¥ g and then stored at -20°C till further analysis.
Samples were analysed by a validated HPLC method for
estimation of riluzole in rat plasma.
In-vitro metabolic inhibition study
The assay was performed by incubating liver microsomes
(RLMs and HLMs, 1 mg/ml protein concentration) with
riluzole at an effective concentration of 5 mm (1.2 mg/ml)
in three different treatment conditions–control (without
inhibitors, ciprofloxacin or grapefruit juice), treatment 1
(with ciprofloxacin at an effective concentration of 5 mm)
and treatment 2 (with grapefruit juice at an effective con-
centration of 5% v/v). The reaction was initiated by the
addition of NADPH at an effective concentration of 2 mm.
Phosphate buffer (100 mm, pH 7.4) was used as the reac-
tion medium. The volume of phosphate buffer was
adjusted to get a final volume of 250 ml for reaction
mixture and to maintain the effective concentrations of
various components as mentioned above in each treatment
condition. Incubations were performed at 37°C in a
shaking water bath for 60 min. The reaction was termi-
nated by the addition of cold acetonitrile (750 ml) into the
reaction mixture at a ratio of 3 : 1. Samples were vortexed
briefly and centrifuged at 11 269 ¥ g for 15 min. The result-
ant clean supernatant fraction (50 ml) was injected and
analysed using a validated HPLC method described in the
‘HLPC analysis of riluzole in rat plasma’ section. Percent
drug remaining to be metabolized in test treatments was
compared with control. The study was performed in trip-
licate for each treatment condition.
Intestinal absorption of riluzole in rat
everted gut-sac model
The everted gut-sac studies in rats were performed using an
established method adapted from the literature.[32] Briefly,
rats were fasted overnight for 12 h but allowed free access to
water before the experiment. After anaesthetizing the rats
with urethane (1.25 g/kg, i.p.), the entire small intestine
segment was removed by cutting across the upper end of
the duodenum and the lower end of the colon and manu-
ally stripping the mesentery. The isolated intestine was care-
fully washed with the normal saline (0.9% w/v NaCl) and
ileum segment (length 5–6 cm) was rapidly removed and
gently everted over a glass rod. The everted intestine was
then slipped off the glass rod and placed in a flat Petri dish
containing perfusion buffer (40 mm Na2HPO4 (anhydrous),
26 mm NaH2PO4.2H2O and 119 mm NaCl) oxygenated with
O2/CO2 (95/5%) maintained at 37°C. One end of the
everted ileum segment was clamped and tied with a silk
suture before filling it with perfusion buffer. Buffer-filled
everted gut sacs were obtained by tying the other end with a
silk suture. The sacs were then placed in individual incuba-
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society Journal of Pharmacy and Pharmacology, ••, pp. ••–••
CYP 1A2 inhibitors and riluzole kinetics
tion chambers containing 20 ml of buffer spiked with treat-
ments at 37°C. The Papp values, expressed in cm/s, were
calculated using the following equation:
Papp = (dQ dt ) ( A.C o ) (1)
Where, dQ/dt is the rate of appearance of riluzole in the
everted gut sac (receiver compartment), Co is the initial
concentration of riluzole outside the everted gut sac (donor
compartment) and A is the total cross sectional area of
tissue.
To study the effect of ciprofloxacin/grapefruit juice on
the P-gp efflux system, and thereby its effect on permeabil-
ity of riluzole through ileum, riluzole (5 mm or 1.2 mg/ml)
was added to three different incubation chambers (chamber
A, B and C) containing everted gut sacs. Incubation
chamber A (control) contained riluzole (5 mm or 1.2 mg/ml)
only, incubation chamber B contained ciprofloxacin (5 mm)
along with riluzole (5 mm or 1.2 mg/ml) and incubation
chamber C contained grapefruit juice (25% v/v) along with
riluzole (5 mm or 1.2 mg/ml). At the end of the incubation
period (30 min), the sacs were removed separately from the
organ bath and samples were collected into different test
tubes. All samples were rapidly cooled and stored at -20°C
until analysis. The entire study was repeated in triplicate.
HPLC analysis of riluzole in rat plasma
All the plasma samples were analysed using the method
reported by our research group.[33] Briefly, an endcapped
C18 reverse-phase column (Agilent, ZORBAX Eclipse Plus,
4.6 mm ¥ 150 mm, 5 mm) equipped with a guard column of
the same packing material was used for the study. The iso-
cratic mobile phase consisted of an aqueous phase (25 mm
KH2PO4, pH adjusted to 3.5 with orthophosphoric acid)
and methanol in a ratio of 30 : 70 v/v. Buffer was filtered
through a 0.22-mm Millipore filtration membrane. The
HPLC system was stabilized for 1 h at 1 ml/min flow rate,
through baseline monitoring before actual analysis.
All the plasma samples were extracted using a simple
protein precipitation method by adding acetonitrile to
plasma samples (100 ml) in the ratio of 3 : 1 v/v. Samples
were vortexed for 2 min and centrifuged at 11 269 ¥ g for
15 min. The resultant clean supernatant fraction (50 ml) was
injected and analysed using a validated HPLC method.
Nebivolol was used as internal standard (IS) in all the
studies. Riluzole and IS were monitored at fixed wave-
lengths of 263 nm and 280 nm, respectively, with a mobile
phase flow rate of 1 ml/min. An injection volume of 50 ml
was used in the analysis of all samples.
The regression equation for the calibration curve of rilu-
zole in rat plasma matrix was Y = 0.00085 (⫾ 0.00007)
X – 0.03835 (⫾ 0.0012) (r2 = 0.999), where Y represents
the ratio of peak area of riluzole to IS, and X represents
3
CYP 1A2 inhibitors and riluzole kinetics
nominal concentrations of riluzole spiked to drug-free
plasma samples. The calibration curve was linear over the
concentration range of 50–4000 ng/ml, with a lowest limit
of quantification of 50 ng/ml.
The mean extraction recovery of riluzole, with the
protein precipitation technique, was in the range of 81.3–
85.5%. Riluzole was found to be stable (deviation within
⫾5% from nominal concentrations) in rat plasma at
various storage conditions: bench top storage at room tem-
perature for 6 h, three freeze and thaw cycles and long-term
storage at -20°C for 30 days.
Statistical analysis
In-vitro metabolic studies and intestinal absorption studies
were performed in triplicate and the data from these experi-
ments are expressed as mean ⫾ SD. Non-compartmental
pharmacokinetic analysis was performed using WinNonlin
(version 6.0; Pharsight Inc., Mountain View, CA, USA) to
determine various pharmacokinetic parameters. Analysis of
variance, followed by Dunnett’s test, was used for statistical
evaluation of the data obtained from pharmacokinetic
studies while Kruskal–Wallis test, followed by Conover test,
was used for the data obtained from in-vitro metabolic and
permeability studies to assess the significance of difference
between various treatment groups and control group.
GraphPad Prism (version 3.0; GraphPad, San Diego, CA,
USA) software was used statistical evaluation. The signifi-
cance level was set at 5%.
Results
Pharmacokinetic studies
Figure 1. shows the effect of co-administering ciprofloxacin
or grapefruit juice on the plasma concentration–time
curves of riluzole obtained in single-dose oral pharmacoki-
netic studies. Co-administration of ciprofloxacin with rilu-
zole (Group B) showed a statistically significant effect on
the oral pharmacokinetics of riluzole as determined by area
under the curve (AUC) (increased by 1.8 fold, P < 0.001,
n = 5), maximum plasma concentration (Cmax) (increased
by 1.4 fold, P < 0.01, n = 5) and mean residence time (MRT)
(increased by 1.21 fold, P < 0.05, n = 5). No statically signifi-
cant effect was observed on the time to reach maximum
plasma concentration (tmax) as compared with control group
(Group A).
Single-dose co-administration of grapefruit juice with
riluzole (Group C) did not show any statistically significant
difference in pharmacokinetic parameters of riluzole as
compared to control group (Group A). Comparative effects
of ciprofloxacin and grapefruit juice on the pharmacoki-
netic parameters of riluzole are shown in Table 1.
4 Royal Pharmaceutical Society Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Punna Rao Ravi et al.
Mean plasma concentration of RLZ (μg/ml)
2.5
Group A
Group B
2.0 Group C
1.5
1.0
0.5
0.0
0 4 8 12 16 20 24
Time (h)
Figure 1 Mean plasma concentration vs time profile of riluzole (RLZ)
obtained in oral pharmacokinetic studies. Group A (control group) was
treated with RLZ (10 mg/kg) alone, Group B was co-administered with
RLZ (10 mg/kg) and ciprofloxacin (CFX, (10 mg/kg), Group C was
co-administered with RLZ (10 mg/kg) and grapefruit juice (GFJ; 2 ml/
kg). Each data point represents the mean (⫾ SD) of five independent
determinations.
Effect of CYP 1A2 inhibitors on in-vitro
metabolism of riluzole
Results obtained from the in-vitro metabolic inhibition
studies using RLMs and HLMs are shown in Figure 2. The
mean percentage metabolism of riluzole was found to
reduce significantly in the presence of ciprofloxacin
(18.5 ⫾ 1.8%, P < 0.01) and grapefruit juice (50.8 ⫾ 1.3%,
P < 0.05) as compared with control (72.5 ⫾ 1.2%) after
60 min incubation with RLMs.
Similarly, a statistically significant reduction in riluzole
metabolism was observed in the presence of ciprofloxacin
(21.3 ⫾ 1.1%, P < 0.01) and grapefruit juice (52.5 ⫾ 1.2%,
P < 0.05) as compared with control (65.0 ⫾ 0.9%) after
60 min incubation with HLMs.
Effect of ciprofloxacin and grapefruit juice
on permeability of riluzole in rat everted
gut-sac model
A comparative profile showing the apparent permeability
(Papp) values of riluzole after 30 min incubation through rat
everted gut sacs is illustrated in Figure 3. The Papp (¥10-5)
values of riluzole were found to be 3.25 ⫾ 0.31 cm/s,
3.40 ⫾ 0.25 cm/s and 3.19 ⫾ 0.41 cm/s in chamber A (rilu-
zole alone), chamber B (ciprofloxacin + riluzole) and
chamber C (grapefruit juice + riluzole), respectively. No sta-
tistically significant difference (P < 0.05) in Papp (¥10-5)
values of riluzole was observed between the various treat-
ment groups.
© 2012 The Authors. JPP © 2012
Punna Rao Ravi et al. CYP 1A2 inhibitors and riluzole kinetics

Table 1 Effect of ciprofloxacin and grapefruit juice on the pharmacokinetic parameters of riluzole after oral administration to rats

Treatment group AUCa (mg h/ml) Cmaxa (mg/ml) tmax (h) MRTa (h)

Group A 8.46 ⫾ 1.28 1.59 ⫾ 0.15 1.0 6.51 ⫾ 0.32

Group B 15.14 ⫾ 1.82*** 2.20 ⫾ 0.14** 1.0 7.91 ⫾ 0.46*
Group C 8.74 ⫾ 1.10 1.61 ⫾ 0.11 1.0 6.67 ⫾ 0.32

AUC, area under the curve; Cmax, maximum plasma drug concentration; MRT, mean residence time; tmax, time to reach maximum plasma drug
concentration. Group A (control group) were treated with riluzole (10 mg/kg) alone; Group B were co-administered with riluzole (10 mg/kg) and
ciprofloxacin (10 mg/kg); Group C were co-administered with riluzole (10 mg/kg) and grapefruit juice (2 ml/kg).aValues are expressed as mean ⫾ SD,
n = 5.*P < 0.05,**P < 0.01,***P < 0.001 compared with Group A.

Apparent permeability (Papp) of RLZ (× 10–5 cm/s)

Mean percentage metabollsm of RLZ

80 RLM HLM
70 4.0

60 **
@@ 3.5
50 3.0
40 2.5
30 @
* 2.0
20
1.5
10
1.0
0
RLZ (5 μM) alone RLZ (5 μM) RLZ (5 μM) 0.5
+ CFX (5 μM) + GFJ (5% v/v)
0.0
Figure 2 Comparative profile showing the effect of ciprofloxacin RLZ (5 μM) alone RLZ (5 μM) RLZ (5 μM)
(CFX, 5 mM) and grapefruit juice (GFJ, 5% v/v) on metabolic stability of + CFX (5 μM) + GFJ (25% v/v)
riluzole (RLZ, 5 mM or 1.2 mg/ml) in rat and human liver microsomes
(1 mg/ml) after 60 min of incubation. Values are expressed as Figure 3 Comparative profile showing the effect of ciprofloxacin
mean ⫾ SD, n = 3. *,@P < 0.001 compared with RLZ alone using rat (CFX) and grapefruit juice (GFJ) on apparent permeability (Papp) of rilu-
liver microsomes (RLM) and human liver microsomes (HLM), respec- zole (RLZ) through rat ileum. Permeability of RLZ (5 mM or 1.2 mg/ml)
tively, **,@@P < 0.01 compared with RLZ (5 mM or 1.2 mg/ml) alone through ileum in everted gut-sac model was studied in the presence of
using RLMs and HLMs, respectively. CFX (5 mM) and GFJ (25% v/v) for 30 min at 37°C. Values are expressed
as mean ⫾ SD, n = 3.

Discussion
Single-dose oral pharmacokinetic studies were conducted to
investigate the effect of co-administering CYP 1A2 inhibi-
tors ciprofloxacin and grapefruit juice with riluzole on the
oral pharmacokinetics of riluzole. Our study was restricted
to fasted rats as significant drug–food interaction[16] has
been reported for riluzole in humans. Statistically no sig-
nificant difference was observed in AUC0–24 and AUC0-•
(data not shown) in all the treatment groups, indicating
that time points and duration of sample collection for the
studies were appropriate.
From the available literature and data from single-dose
pharmacokinetic studies of riluzole, it is evident that rilu-
zole has moderate bioavailability. The high inter-animal
variability observed in the pharmacokinetics of riluzole
could be expected due to noted polymorphism in expres-
sion of the CYP 1A2 enzyme,[5] large variations in intestinal
CYP expression[34] and poor aqueous solubility of riluzole.
Significant increase in AUC0–24, Cmax and MRT values for
riluzole in rats co-administered with ciprofloxacin suggests
that ciprofloxacin affects first-pass metabolism, hepatic
clearance and/or P-gp efflux of riluzole.
In a recent pharmacokinetic interaction study conducted
using multidrug-resistant knockout mice it was revealed
that riluzole could be a substrate of the P-gp efflux system
located at the blood–brain barrier.[31] The role of gut P-gp in
limiting the oral bioavailability of riluzole is still unknown.
Since ciprofloxacin was found to be an ambiguous P-gp
substrate in various in-vitro models,[20] the role of P-gp
interaction at an absorption level cannot be ruled out.
Therefore an everted gut-sac experiment was undertaken
to determine the contribution of P-gp in the proposed
interaction. An ileum segment of small intestine was
selected due to the relatively high P-gp expression and low
CYP expression in this region.[34] Results showed that rilu-
zole co-incubation with ciprofloxacin did not cause statisti-
cally significant enhancement in apparent permeability of
riluzole across the rat ileum, suggesting the trivial role of
P-gp. However, in this study because we have not consid-
ered the use of control inhibitors for CYP, attributing the

© 2012 The Authors. JPP © 2012

Royal Pharmaceutical Society Journal of Pharmacy and Pharmacology, ••, pp. ••–•• 5
CYP 1A2 inhibitors and riluzole kinetics
outcome of the study to P-gp modulation alone is not valid.
In this model, we could not distinguish the role of trans-
porter from metabolism.
In-vitro metabolic inhibition studies indicate a similar
trend for riluzole metabolism in liver microsomes (HLMs
and RLMs). Results indicate that there is substantial
metabolism of riluzole in the control group and statistically
significant metabolic interaction between selected drugs in
both HLMs and RLMs. This further attests to use of the rat
as an appropriate animal model for investigating this drug–
drug interaction and also suggests the role of CYP inhibi-
tion in this interaction. A significantly lower inhibitory
action (22% inhibition) of grapefruit juice on riluzole
metabolism was observed than for ciprofloxacin (55% inhi-
bition) suggesting that grapefruit juice is a weak CYP
inhibitor as compared with ciprofloxacin at selected con-
centrations. In-vitro metabolic inhibition data correlates
well with results obtained from studies on the effects of
co-administering ciprofloxacin and grapefruit juice on
pharmacokinetics of riluzole. However, we did not study
enzyme kinetics and hence the observed differences in
metabolic inhibition of riluzole could also be due to dispar-
ity in selected doses.
In-vitro metabolic inhibition study involving co-
incubation of riluzole with grapefruit juice had shown a
decrease in metabolism of riluzole suggesting that grape-
fruit juice could possibly show food–drug interaction with
riluzole even in the in-vivo study. However in the in-vivo
study, no significant difference was observed in the oral
pharmacokinetics (AUC, Cmax, tmax and MRT) of riluzole
co-administered with grapefruit juice. This could possibly
be due to the active principles of grapefruit juice (berga-
mottin and naringenin) failing to achieve inhibitory plasma
concentrations at the given dose in rats. This observation is
in close agreement with clinical studies conducted by Fuhr
and co-workers, who have evaluated the effect of grapefruit
juice on the CYP 1A2 probe substrates caffeine[35] and theo-
phylline[36] and concluded that intake of grapefruit juice
may not show clinically significant interaction with some
substrate drugs.
Grapefruit juice co-incubation failed to show significant
interaction with riluzole at absorption level in the everted
gut-sac model. The observed results could be attributed to
the high permeability of riluzole, weak interaction of grape-
fruit juice and/or riluzole with P-gp and the selected con-
centration (subinhibitory) of grapefruit juice.
To establish the drug–drug interaction potential of cipro-
floxacin on CYP 1A2 substrates, pre-clinical[37] and clinical
References xenobiotics. Cell Mol Life Sci 2001; 58:
737–747.
1. Anzenbacher P, Anzenbacherová E. 2. Wu AH. Drug metabolizing enzyme
Cytochromes P450 and metabolism of activities versus genetic variances for
6 Royal Pharmaceutical Society Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Punna Rao Ravi et al.
studies have been performed using theophylline,[38] tizani-
dine[39] and mexiletine.[40] Results from these studies showed
a significant change in the pharmacokinetic parameters
(increase in AUC, Cmax and clearance) of substrate drugs
and established ciprofloxacin as a CYP 1A2 inhibitor.
The authors attributed higher plasma exposure of these
substrates to metabolic inhibition of CYP 1A2 by co-
administered ciprofloxacin.
The results obtained from our in-vivo and in-vitro
interaction studies are in close agreement with previously
published data. Co-administration of ciprofloxacin with
riluzole increased AUC, Cmax and MRT of riluzole indicating
potential pharmacokinetic drug interaction with cipro-
floxacin. The data from our study indicates a moderate
increase in AUC (about 1.7 fold), Cmax (about 1.3 fold) and
MRT (about 1.2 fold) for riluzole after co-administration
with ciprofloxacin in rats. Though this could be insignifi-
cant when extrapolated to humans, a thorough clinical
investigation is necessary to prove the same.
Conclusions
Our study suggests that ciprofloxacin has the potential to
modulate the pharmacokinetics of riluzole by possibly
inhibiting the metabolic clearance of riluzole. The study
also shows that P-gp plays a trifling role in this interaction.
Grapefruit juice has the potential to interact with riluzole
based on the results of the study in microsomes but this was
not seen in practice. Co-administration of grapefruit juice
with riluzole had no significant effect on oral pharmacoki-
netics of riluzole. This study provides a preliminary proof
of interaction between ciprofloxacin or grapefruit juice
and riluzole, although a thorough clinical investigation is
required to extend the results to humans.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Funding
The authors are grateful to Department of Science and
Technology (DST), Science and Engineering Research
Council (SERC), New Delhi, India for funding the research
under ‘Fast Track Scheme for Young Scientists’.
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