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Zalcitabine (2กฏ,3กฏ-dideoxycytidine; ddC), a pyrimidine
nucleoside, is highly active against human immunodeficiency
virus (HIV) and also hepatitis B virus
(HBV)[1-3]. It has been used for the treatment of HIV infection and related diseases
in combination with other antiretroviral
agents[4,5]. Zalcitabine is rapidly and extensively absorbed after oral administration,
and the primary route of elimination is renal excretion of
unchanged drug, with 60% -70% of an oral dose recovered in
urine within 24 h[5,6]. Renal clearance of zalcitabine exceeds
the glomerular filtration rate, implying that the drug
undergoes active tubular secretion in the
kidney[6,7].
Zalcitabine has been reported to interact with organic
anion transporters[8,9]. Among the isoforms of organic anion
transporters expressed in several organs and tissues,
including kidney, liver and brain, OAT1 and OAT3 play a pivotal
role in the renal excretion of a wide variety of important
therapeutics, including b-lactam antibiotics, diuretics,
hippurates and nucleoside antiviral
drugs[8-13]. Given that a considerable number of drugs and toxins can interact with
organic anion transporters, potential drug interactions via
OAT-mediated renal excretion may require close monitoring
during combination therapies involving zalcitabine.
Particular attention should be paid to interactions with commonly
prescribed or over-the-counter drugs that could affect the
efficacy and toxicity of zalcitabine. Furthermore,
considering the short plasma half-life of zalcitabine (1-3 h), the
inhibition of renal excretion of zalcitabine by the concomitant
use of OAT inhibitors may prolong systemic exposure to
zalcitabine, resulting in less frequent dosing. Therefore, in
the present study we aimed to investigate the
pharmacokinetic interactions between zalcitabine and NSAIDs (OAT
inhibitors) in rats. Because rats and humans share high
sequence homology for OAT1 (9%) and OAT3
(79%)[10], the rat was selected as an animal model for our pharmacokinetic
studies of zalcitabine.
Materials and methods
Materials Zalcitabine, naproxen, ketoprofen, and
5-bromo-2กฏ -deoxyuridine (BDU) were purchased from Sigma
(St Louis, MO, USA). All other chemicals were of analytical
grade and all solvents were of high performance liquid
chromatography (HPLC) grade.
Animal studies All animal studies were performed in
accordance with the experimental protocols approved by the
Animal Care Committee of Chosun University. Male
Sprague-Dawley rats weighing 280-300 g were obtained from Samtako
Bio Co (Osan, Korea). Rats were divided into 3 groups,
comprising 5 rats each group. Groups 1-3 were given an iv
injection of zalcitabine (20 mg/kg) with either (1) naproxen
sodium (20 mg/kg) or (2) ketoprofen (20 mg/kg) 30 min prior
to the administration of zalcitabine, or (3) no concomitant
treatment (control). Blood samples were collected from the
right femoral artery at 0, 0.083, 0.16, 0.33, 0.5, 1, 2, 4, 8, 12, and
24 h following the zalcitabine administration. Urine was also
collected at 0, 8, 12, and 24 h post zalcitabine administration
from the same group of rats. Blood samples were
centrifuged at 3 000 r/min for 10 min to obtain plasma for the HPLC
assay. Urine samples were centrifuged at 3 000 r/min for 10
min and then passed through a membrane filter (0.45 µm). All
samples were stored at -70 °C until analysis.
HPLC assay The plasma and urine concentrations of
zalcitabine were determined by an HPLC assay modified from
the method of Ibrahim and Boudinot[14]. Briefly, the internal
standard (20 µg/mL of BDU) was added to plasma samples
and then samples were deproteinized by adding acetonitrile.
After centrifugation of the samples at 3 000 r/min for 10 min,
the supernatant was completely evaporated with nitrogen
stream. The residue was reconstituted with 100 µL of the
mobile phase, and then 50 µL aliquots were injected directly
into the HPLC system. The filtered urine samples (50 µL)
were injected into the HPLC system after appropriate dilution.
The chromatographic system consisted of a pump
(LC-10AD), an automatic injector (SIL-10A) and a UV detector
(SPD-10A; Shimadzu Scientific Instruments, Japan) set at
254 nm. An octadecylsilane column (Gemini C18, 4.6 mm×250
mm, 5 µm; Phenomenex, Torrance, CA, USA) was eluted with
a mobile phase consisting of 10% methanol in phosphate
buffer (pH 6.8) at a flow rate of 1.0 mL/min. The calibration
curve from the standard samples was linear over the
concentration range of 0.1 µg/mL to 20 µg/mL. The intra-day
(n=5) and inter-day (n=5) coefficients of variation were less than
5%. The limit of detection was 0.1 µg/mL.
Pharmacokinetic analysis Non-compartmental
pharmacokinetic analysis was performed using Kinetica-4.3
(InnaPhase Corp, Philadelphia, PA, USA). The area under
the plasma concentration-time curve (AUC) was calculated
by using the linear trapezoidal method. The terminal
elimination rate constant (lz) was estimated from the slope of the
terminal phase of the log plasma concentration-time points
fitted by the method of least-squares, and then the terminal
elimination half-life (T1/2) was calculated as
0.693/lz. Total clearance (CL) was estimated by dividing dose by AUC and
the renal clearance (CLR) was determined as
CLR=Ae/AUC, where Ae (amount of unchanged drug eliminated in urine)
and AUC were measured over the same time interval.
Statistical analysis Data are expressed as mean±SD,
and analyzed using one-way analysis of variance (ANOVA),
followed by a posteriori testing with use of the Dunnett
correction. P<0.05 was considered statistically significant.
Results
As summarized in Table 1, pretreatment with naproxen 30
min prior to zalcitabine administration significantly
(P<0.05) altered the pharmacokinetics of zalcitabine in rats, compared
with the controls given zalcitabine alone. Renal clearance of
zalcitabine accounted for approximately 70% of the CL in all
cases, which is consistent with previous
reports[14]. However, renal clearance and total clearance of zalcitabine decreased
by approximately 3-4-fold in the presence of naproxen or
ketoprofen. Consequently, the AUC of zalcitabine was
significantly (P<0.05) greater than that for the controls given
zalcitabine alone (Table1, Figure 1). The terminal plasma
half-life (T1/2) of zalcitabine increased by 4-5-fold in the
presence of naproxen or ketoprofen. In the control group,
urinary excretion of zalcitabine was rapid, and approximately
84% of the dose was excreted into urine within the first 8 h.
Following the co-administration of naproxen, the urinary excretion of zalcitabine was 58%, 25%, and 6% of the
administered dose in the 8 h, 12 h and 24 h urine samples,
respectively (Figure 2). In the presence of ketoprofen, the urinary
excretion of zalcitabine was similar to that observed with the
co-administration of naproxen.
Discussion
In addition to causing changes in drug metabolism,
particularly via cytochrome P450-mediated metabolism, there is
increasing evidence suggesting that modulation of drug
transporters can cause clinically important drug interactions.
For example, the bioavailability and the intracellular
concentrations of protease inhibitors can be increased in the
presence of potent P-gp
inhibitors[15,16]. For cationic drugs,
decreases in the renal excretion of dofetilide and procainamide
by the co-administration of cimetidine can be explained by
the inhibition of organic cation transporter-mediated active
secretion in the basolateral membranes of renal proximal
tubules[17,18]. Although the majority of drug interactions have
the potential to cause adverse effects, some interactions
mediated by organic anion transporters have a positive impact,
for example combination therapy with cidofovir and
probenecid, in which the probenecid significantly reduces
the nephrotoxicity of cidofovir[19]. Therefore, transport
proteins can play an important role in many clinical drug
interactions, either negatively or positively.
The organic anion transporter family has been implicated
in the distribution of zalcitabine to the central nervous
system and the proximal tubular cells in the
kidney[8,9]. Recently, Khamdang et
al also found that NSAIDs such as ketoprofen, indomethacin, diclofenac, naproxen, and ibuprofen inhibit
organic anion uptake mediated by organic anion
transporters[12]. In particular, ketoprofen and naproxen appear to be
potent inhibitors of OAT1 and OAT3 located in the basolateral side of the renal tubular cells. Therefore, in our
study, ketoprofen and naproxen were chosen to investigate
potential drug interactions with zalcitabine. Because plasma
concentrations of zalcitabine decline very rapidly in rats,
larger doses must be administered to adequately
characterize the behavior of zalcitabine, as reported in previous
pharmacokinetic studies[7,14]. Thus, in the present study, the dose
of zalcitabine administered to rats was relatively large in
comparison to the doses given to patients in clinical trials. No
obvious toxicity was noted at the dose used in the present
study.
As illustrated in Figures 1 and 2, concurrent use of
naproxen or ketoprofen significantly altered the behavior of
zalcitabine in rats. Considering that (i) zalcitabine
undergoes active tubular secretion in the
kidneys[6,7] and that (ii) both zalcitabine and NSAIDs can interact with organic
anion transporters in the renal tubular cells[8,
9], the reduction of the CLR of zalcitabine in the presence of naproxen or
ketoprofen might result, at least in part, from the inhibition of
organic anion transporters by naproxen or ketoprofen.
Although there are potential adverse effects, these interactions
may provide a therapeutic benefit, whereby the interactions
prolong the duration of action of zalcitabine, by conferring a
longer plasma half-life, thus necessitating less frequent doses
of zalcitabine, and also a lower dose. Therefore, the clinical
significance of this finding needs to be further evaluated for
therapeutic dose levels in clinical studies.
In summary, pretreatment with naproxen or ketoprofen
prior to zalcitabine administration significantly altered the
pharmacokinetic profile of zalcitabine, implying that patients
who are being treated with NSAIDs and zalcitabine may
require close monitoring for potential drug interactions.
Acknowledgement
The authors greatly appreciate the help of Mr Ming-ji
JIN in carrying out the animal experiments.
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