Effect of itraconazole on the pharmacokinetics of diclofenac in beagle dogs.
ABSTRACT The objective of this study was to investigate the potential effect of itraconazole on the pharmacokinetics of diclofenac potassium in beagle dogs after oral coadministration. Five male beagle dogs received a single oral 50 mg dose of diclofenac potassium alone in phase I, and along with a single oral 100 mg dose of itraconazole in phase II. Blood samples obtained for 8.0 hours post dose were analysed for diclofenac concentration using a validated high performance liquid chromatography (HPLC) assay method. The area under plasma concentration-time curve (AUC(0ââ)), maximum plasma concentration (C(max)), time to reach C(max) (T(max)) and elimination half-life (t(1/2)), were calculated for diclofenac before and after itraconazole administration. The coadministration of itraconazole with diclofenac potassium has resulted in a significant reduction in AUC(0ââ) and C(max) of diclofenac, which was about 31 and 42%; respectively. No statistically significant differences were observed for T(max) and t(1/2) of diclofenac between the two phases. Therefore, it could be concluded that oral coadministration of itraconazole may have the potential to affect the absorption of diclofenac as indicated by the significant reduction in its AUC and C(max) in beagle dogs.
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ABSTRACT: The aim of the present work was to characterize the electrophysiological effects of the non-steroidal anti-inflammatory drug diclofenac and to study the possible proarrhythmic potency of the drug in ventricular muscle. Ion currents were recorded using voltage clamp technique in canine single ventricular cells and action potentials were obtained from canine ventricular preparations using microelectrodes. The proarrhythmic potency of the drug was investigated in an anaesthetized rabbit proarrhythmia model. Action potentials were slightly lengthened in ventricular muscle but were shortened in Purkinje fibers by diclofenac (20 µM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl(2) application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 µg/kg) significantly lengthened the QT(c) interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QT(c). Diclofenac alone did not induce Torsades de Pointes ventricular tachycardia (TdP) while TdP incidence following dofetilide was 20%. However, the combination of diclofenac and dofetilide significantly increased TdP incidence (62%). In single ventricular cells diclofenac (30 µM) decreased the amplitude of rapid (I(Kr)) and slow (I(Ks)) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (I(Ca)) was slightly diminished, but the transient outward (I(to)) and inward rectifier (I(K1)) potassium currents were not influenced. Diclofenac at therapeutic concentrations and even at high dose does not prolong repolarization markedly and does not increase the risk of arrhythmia in normal heart. However, high dose diclofenac treatment may lengthen repolarization and enhance proarrhythmic risk in hearts with reduced repolarization reserve.PLoS ONE 01/2012; 7(12):e53255. · 3.73 Impact Factor
S c i Pharm
Open Ac c ess
Effec t of Itrac onazole on the
Pharmac okinetic s of
Dic lofenac in Beagle Dogs
Fahad I. AL-JENOOBI
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia.
Sci Pharm. 2010; 78: 465–471
May 19th 2010
Accepted: May 19th 2010
Received: March 13th 2010
This article is available from: http://dx.doi.org/10.3797/scipharm.1003-10
© Al-Jenoobi; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
The objective of this study was to investigate the potential effect of itraconazole
on the pharmacokinetics of diclofenac potassium in beagle dogs after oral
coadministration. Five male beagle dogs received a single oral 50 mg dose of
diclofenac potassium alone in phase I, and along with a single oral 100 mg dose
of itraconazole in phase II. Blood samples obtained for 8.0 hours post dose
were analysed for diclofenac concentration using a validated high performance
liquid chromatography (HPLC) assay method. The area under plasma
concentration-time curve (AUC0–∞), maximum plasma concentration (Cmax), time
to reach Cmax (Tmax) and elimination half-life (t1/2), were calculated for diclofenac
before and after itraconazole administration. The coadministration of
itraconazole with diclofenac potassium has resulted in a significant reduction in
AUC0–∞ and Cmax of diclofenac, which was about 31 and 42%; respectively. No
statistically significant differences were observed for Tmax and t1/2 of diclofenac
between the two phases. Therefore, it could be concluded that oral
coadministration of itraconazole may have the potential to affect the absorption
of diclofenac as indicated by the significant reduction in its AUC and Cmax in
Interaction • Pharmacokinetic parameters • Plasma concentration • Absorption • HPLC
466 F. I. Al-Jenoobi:
Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic
properties. It is widely used in management of mild to moderate pain particularly when
inflammation is also present as in cases of rheumatoid arthritis, osteoarthritis,
musculoskeletal injuries and some postoperative conditions [1–3]. Its pharmacological
effects are believed to be due to blocking the conversion of arachidonic acid to
prostaglandins by inhibiting cyclo-oxygenase enzymes .
Diclofenac is almost completely absorbed after oral administration. However, due to its
first-pass hepatic metabolism, only about 50% of the absorbed dose is systematically
available [5–8]. The major metabolite of diclofenac in human is 4'-hydroxydiclofenac, which
is mainly formed by cytochrome P4502C9 (CYP2C9) enzyme [9–10]. The minor diclofenac
metabolites are formed by several enzymes including CYP3A4 . About 99% of the drug
is bound to human plasma proteins, mainly albumin [12, 13]. The potassium salt of
diclofenac was found to be particularly useful for quick pain relief compared to the sodium
salt because of its higher solubility in the stomach acidic medium .
Itraconazole is a triazole antifungal agent that is used for a number of indications including
systemic and superficial mycosis . It has been shown to interact with many drugs
mainly by inhibiting their CYP3A4-mediated metabolism and/or multidrug resistance
protein 1 (MRP1)-mediated transport [16–19]. In addition, itraconazole is highly bound to
plasma proteins, primarily albumin and therefore, may interact with other albumin highly
bound drugs [15, 20].
A literature search revealed that pharmacokinetic interactions between itraconazole and
diclofenac have never been investigated. In fact, there is a very limited published
information about the pharmacokinetic interactions between triazole antifungals and
NSAIDs in general . Coadministration of itraconazole with diclofenac may be
considered for the treatment of certain fungal infections in cases of rheumatoid arthritis,
musculoskeletal injuries and some postoperative conditions. Therefore, it is important to
investigate the potential interaction between the two drugs. The aim of this study was to
investigate the effect of itraconazole on the pharmacokinetics of diclofenac in vivo using
beagle dogs as an animal model.
Diclofenac potassium 50 mg tablets (Cataflam®, Novartis Pharma, Egypt) were purchased
from the market while itraconazole 100 mg capsules (Sporanox®, Janssen-Cilag, Beerse,
Belgium) were obtained from the pharmacy of King Khalid University Hospital. Diclofenac
and flufenamic acid (internal standard) analytical powders were purchased from Sigma (St.
Louis, MO, USA). Acetonitrile was obtained from BDH, England, UK and glacial acetic acid
from Polysciences Inc. Warrington, PA, USA. All other reagents were of analytical grade.
The protocol of the in vivo study in beagle dogs was approved by the Experimental
Animals Care Centre of College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
Sci Pharm. 2010; 78: 465–471.
Effect of Itraconazole on the Pharmacokinetics of Diclofenac in Beagle Dogs 467
Five healthy male beagle dogs weighing 10.2–13.6 kg were used. The study was
conducted in two phases with one week wash-out period. The dogs were fasted for 12 hrs
before drug administration and continued fasting for 2 hrs post dose but allowed free
access to water. Each dog was administered one tablet of 50 mg diclofenac potassium
alone (phase I) or with a 100 mg itraconazole capsule (phase II). No other medications
were taken during the study period. Venous blood samples (3.0 ml) were taken from the
femoral vein into heparinized tubes before drug administration (to serve as a blank) and at
0.25, 0.50, 0.75, 1.00, 1.50, 2.00, 3.00, 4.00, 6.00 and 8.00 hr after drug administration.
Samples were centrifuged immediately at 5000 rpm for 10 min. and the separated plasma
samples were kept at −20 °C for analysis.
Assay of diclofenac in plasma
The plasma concentration of diclofenac was determined by a modified high-performance
liquid chromatography (HPLC) assay method  using Shimadzu HPLC system (Kyoto,
Japan) that is composed of a liquid chromatograph pump (Model LC-20A), a UV detector
(Model SPD-20A), a degasser (Model DGU-20A), a communication bus module (Model
CBM-20A) and an autosampler (Model SIL-20A). The drug and internal standard were
eluted from Nucleosil 5 µm C-18 column (150 mm x 4.6 mm, MACHEREY-NAGEL GmbH
& Co. KG, Germany) at an ambient temperature using a mobile phase of acetonitrile and
water (50:50 % v/v, adjusted to pH 3.3 with glacial acetic acid) at a flow rate of 1.5 ml/min
with a UV detection at 280 nm. To 0.25 ml dog plasma samples, an aliquot of 20 µl internal
standard (0.1 µg/ml flufenamic acid) was added followed by shaking on a vortex mixer for
30 sec. Precipitation of serum proteins was achieved by addition of 500 µl cold acetonitrile.
The mixture was shaken again on a vortex mixer for 1 min., and centrifuged for 5 min. at
10000 rpm. The supernatant was transferred to an autosampler vial for injection in HPLC.
Calculation of pharmacokinetic parameters
Maximum plasma concentration (Cmax) and the time to reach it (tmax) were obtained directly
from plasma data. Elimination half-life (t1/2) was calculated as 0.693/Kel, where Kel is the
elimination rate constant obtained from the slope of the terminal exponential phase. The
total area under plasma concentration time curve (AUC0–∞) was calculated as the sum of
AUC0–8hr and AUC8hr–∞, where AUC0–8hr was determined by the trapezoidal rule method
and AUC8hr–∞ as the last plasma concentration divided by Kel.
The significance of the differences between plasma concentrations of diclofenac at each
sampling time and the pharmacokinetic parameters of treatment group versus control were
evaluated using Student’s paired t-test. P value ≤ 0.05 was taken as the criterion for
statistically significant difference.
Results and Discussion
Figure 1 shows the plasma concentration of diclofenac in beagle dogs following oral
administration of 50 mg diclofenac potassium tablet alone (control) or with 100 mg
itraconazole capsule. It was noticed that the plasma concentration of diclofenac was
significantly affected by the presence of itraconazole upon oral coadministration to beagle
dogs. This finding was supported by the significant reduction in the Cmax and AUC0–∞ after
Sci Pharm. 2010; 78: 465–471.
468 F. I. Al-Jenoobi:
itraconazole administration (about 42% and 31%; respectively, Table 1). No statistically
significant differences (P > 0.05) were observed for the values of tmax and t1/2 after
itraconazole treatment compared to control, which indicated similar times to reach
maximum concentration and similar elimination rate constants.
Itraconazole is a known inhibitor of CYP3A subfamily of enzymes and most of the reported
pharmacokinetic interactions that are caused by this drug are believed to be mainly due to
inhibition of CYP3A. However, the observed interaction in this study can not be explained
by inhibition of drug metabolism simply because such inhibition would result in an increase
in the AUC and plasma concentration of the affected drug rather than a decrease as
observed with diclofenac in this study. In fact, CYP3A subfamily is not a major contributor
to diclofenac elimination both in human and in beagle dog [5, 6, 9, 10]. In addition,
alteration of diclofenac elimination, in general, does not seem to play a major role in the
observed interaction as strongly indicated by the lack of significant itraconazole effect on
the t1/2 of diclofenac [Table 1].
Displacement of diclofenac from its plasma binding sites by itraconazole is unlikely to be a
major cause for this observed interaction. This is supported by the finding of Hynninen and
colleagues who have shown that itraconazole could significantly reduce the Cmax and
AUC0–∞ of meloxicam in human subjects without affecting the unbound plasma meloxicam
Fig. 1. Mean plasma concentration (± SE) of diclofenac in beagle dogs following oral
administration of 50 mg diclofenac potassium tablet alone (control) or with 100
mg itraconazole capsule (n=5). *P < 0.05.
The results obtained from this study suggest that itraconazole reduces the plasma
concentration of diclofenac by interfering with its gastrointestinal absorption. Impairment of
absorption by itraconazole has been suggested for the first time by Hynninen and
Sci Pharm. 2010; 78: 465–471.
Effect of Itraconazole on the Pharmacokinetics of Diclofenac in Beagle Dogs 469
coauthors to explain itraconazole effect on the plasma concentration of another NSAID;
meloxicam . However, the exact mechanism of such interaction is not clear.
Appropriate mechanistic studies that involve suitable intestinal absorption models may
help to clarify the mechanism(s) behind this interaction.
In conclusion, itraconazole was shown in this study to significantly reduce the Cmax and
AUC0–∞ of diclofenac in beagle dogs suggesting that it has the potential to decrease the
intensity of diclofenac pharmacological effect. The exact mechanism of this interaction is
not clear but the results suggest an alteration in diclofenac absorption by itraconazole. In
addition, results obtained from this study warrant further investigation in human subjects to
evaluate the clinical relevance of this interaction.
Table 1. Pharmacokinetic parameters of diclofenac in beagle dogs (mean ± SD) following
oral administration of 50 mg diclofenac potassium tablet alone (control) or with 100 mg of
itraconazole capsule (n=5). *P < 0.05.
6.28 ± 3.46
1.15 ± 0.60
25.43 ± 6.18
2.10 ± 0.37
3.65 ± 2.22*
1.94 ± 1.23
17.65 ± 5.09*
1.98 ± 0.07
The author would like to thank the College of Pharmacy Research Center, King Saud
University for the financial support of this study (grant number: CPRC-147).
The author declares no conflict of interest.
The institutional and (inter)national guide for the care and use of laboratory animals was
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Sci Pharm. 2010; 78: 465–471.