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Antimicrobial Agents and Chemotherapy, September 1998, p. 2405-2409, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Pharmacokinetics of Pentoxifylline and Its Metabolites in Healthy
Mice and in Mice Infected with Candida
albicans
Kenneth
Miller,1,
Arnold
Louie,2
Aldona L.
Baltch,2,*
Raymond P.
Smith,2
Patrick J.
Davis,4 and
Morris A.
Gordon3
Albany College of
Pharmacy,1
Stratton Veterans Affairs
Medical Center and Albany Medical College,2 and
Mycology Laboratory of the Wadsworth Center for
Laboratories and Research, New York State Department of Health,
Albany, New York,3 and
College of
Pharmacy, University of Texas, Austin, Texas4
Received 6 October 1997/Returned for modification 22 March
1998/Accepted 10 June 1998
 |
ABSTRACT |
Pentoxifylline has immunomodulatory properties and has been shown
to decrease organ damage and improve survival in animals with
gram-negative sepsis or endotoxemia. This effect is mediated by a
reduction in endotoxin-induced production of tumor necrosis factor
alpha (TNF-
) by the host. In earlier studies, we observed an
unexpected increase in mortality in mice infected with Candida albicans that were given pentoxifylline even though
concentrations of TNF- in serum were not affected. The current study
was designed to determine whether the pharmacokinetics of
pentoxifylline and its metabolites were altered in C. albicans-infected mice and, if so, whether these changes could
have contributed to the increased mortality. Noninfected mice and mice
infected with C. albicans were treated with pentoxifylline
(60 mg/kg of body weight) intraperitoneally every 8 h. Serum was
collected from animals after one (day 0), four (day 1), or seven (day
2) injections of pentoxifylline or saline (controls). The first dose
was administered 6 h after C. albicans infection.
Serum was pooled. Concentrations of pentoxifylline and
metabolites I, IV, and V were determined by capillary gas chromatography. Renal function and hepatic profiles were assessed. Pharmacokinetic parameters (maximum concentration of pentoxifylline in
serum, half-life, and area under the concentration-time curve from
0 h to infinity [AUC0-
]) for all noninfected mice were similar and did not differ from those for day 0-infected mice. For day 1-infected mice, values of these three
pharmacokinetic parameters for pentoxifylline and metabolite I were
increased two- to fourfold over values for noninfected and day
0-infected mice. For metabolites IV and V, the AUC0-
was increased approximately eightfold over control values. In addition,
day 1-infected mice demonstrated evidence of renal and hepatic
dysfunction. In summary, C. albicans infection produced
marked changes in the pharmacokinetics of pentoxifylline and its
metabolites in the mice. The high concentrations of pentoxifylline and
its metabolites in serum attained in infected mice may have contributed
to the increased mortality of mice with systemic candidiasis.
| |
INTRODUCTION |
Pentoxifylline is one of several
methylxanthine compounds that has immunomodulatory properties
(10). In vitro, pentoxifylline pretreatment can attenuate
the production of interleukin 1 and tumor necrosis factor alpha by
human mononuclear cells in response to bacterial endotoxin
(12). In addition, it can decrease endotoxin-mediated migration, adherence, and production of superoxide radicals by phagocytic cells (13). In vivo, pentoxifylline improves the outcome for animals challenged with high-level inocula of gram-negative bacilli or endotoxin, suggesting that pentoxifylline or related compounds might be of value in treating sepsis (6, 7). These observations have led to its extensive use in animal models of sepsis,
burns, and traumatic shock (4).
In a series of experiments to determine the role of cytokine activation
in systemic Candida albicans infection, we observed decreased survival rates in pentoxifylline-treated mice
(5). However, pentoxifylline administration did not
affect the production of tumor necrosis factor alpha or interleukin 6 in these infected mice. It was possible that altered pharmacokinetics
of pentoxifylline and its metabolites in systemic C. albicans infection were responsible for the shortened survival
times. However, a review of the literature failed to provide
information about the metabolism and clearance of pentoxifylline in
infected versus healthy animal models. Therefore, the purpose
of this study was to determine the pharmacokinetics, including
clearance, of pentoxifylline in a murine model of systemic candidiasis
in an attempt to define a possible pharmacologic cause for the
unexpected results of our previous studies (5).
| |
MATERIALS AND METHODS |
C. albicans.
Strain 88-689-6 was isolated from the
blood of a neutropenic patient. The microorganism was maintained on
Sabouraud dextrose agar (BBL Microbiology Systems, Cockeysville, Md.)
at 22°C until use. For each study, two or three colonies of C. albicans were subcultured onto potato dextrose agar (BBL) and
incubated at 35°C for 48 h. A fungal suspension was prepared
with sterile, pyrogen-free, phosphate-buffered saline (PBS; Gibco-BRL
Inc., Grand Island, N.Y.) and quantified with a hemocytometer.
Morphologic examination demonstrated that >99% of the organisms were
blastoconidia. The viability of the yeast was found to be >95% by
trypan blue exclusion and quantitative cultures. The endotoxin
concentration in the fungal suspension was found to be <0.05 endotoxin
units (EU) per ml by a competitive endotoxin enzyme-linked
immunosorbent assay (PyroChek competitive lipopolysaccharide ELISA;
ALerCHEK, Portland, Maine).
Mice.
Female, 18- to 20-g NYLAR white mice were raised at
the Animal Research Facility of the Wadsworth Center for
Laboratories and Research (Griffin Laboratories, Guilderland, N.Y.).
These outbred Swiss mice were housed in hanging metal cages and
received food and water ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committees of the respective institutions.
Pentoxifylline.
Pentoxifylline powder was provided by
William Novick, Jr. (Hoechst-Roussel, Somerville, N.J.). The potency of
the drug was confirmed by the manufacturer just prior to the initiation
of this study. Pentoxifylline was dissolved in sterile, pyrogen-free water to produce a stock solution at a concentration of 20 mg/ml. The
stock solution was passed through a 0.45-µm-pore-size filter (Lida
Manufacturing, Kenosha, Wis.). The drug was further diluted to the
desired concentrations with sterile, pyrogen-free PBS and was used
immediately. The solutions contained less than 0.25 EU of endotoxin per
ml, as determined by Limulus amebocyte lysate assay
(Whittaker M. A. Bioproducts, Walkersville, Md.).
Systemic candidiasis model.
Mice were injected intravenously
with 106 CFU of C. albicans or PBS in a 0.2-ml
volume. Mice received 60 mg of pentoxifylline per kg of body weight
intraperitoneally (i.p.) at 8-h intervals beginning 6 h after the
intravenous injection of C. albicans or PBS. In a previous
study (5), this pentoxifylline regimen resulted in a
statistically significant decrease in mean survival of NYLAR mice
systemically infected with C. albicans. The volume of each i.p. injection was 0.1 ml. Animals were assessed prior to each injection for mortality. Previous studies demonstrated that mice died
within 4 h after becoming moribund; therefore, moribund mice were
sacrificed.
Measurement of serum levels of pentoxifylline and its metabolites
by capillary gas chromatography.
The method used in the assay of
plasma pentoxifylline and metabolites I, IV, and V was described by
Burrows (2). The procedure included a single extraction of
all species from plasma into chloroform, derivative formation (the
trifluoracetyl derivative in the case of metabolite I and the methyl
ester derivatives in the case of metabolites IV and V and their
internal standard), and analysis by capillary gas chromatography. An
analog of pentoxifylline (Hoechst EH79-0254) was employed as the
internal standard for pentoxifylline and metabolite I, while
1-(5'-carboxypentyl)-3,7-dimethylxanthine (Hoechst-Roussel) was used as
the internal standard for measuring metabolites IV and V.
Standard curves were generated for pentoxifylline, metabolite I, and
metabolite IV in the range of 5 to 2,000 ng/ml, and for
metabolite V in
the range of 25 to 10,000 ng/ml. Capillary gas
chromatography was
performed with a Varian model 3700 gas chromatograph,
modified for
split-injection capillary column analysis and fitted
with a thermionic
radiation-specific (nitrogen/phosphorus) detector.
The separation was
conducted with a DB-5 phenylmethyl column (30
by 0.32 [inside
diameter] mm; Varian model JW123503-20) fitted
with a precolumn glass
insert guard column and packed with quartz
fiberglass (CapSaver;
N-Phase, Inc.). The linearities, sensitivities,
and percent
coefficients of variation at high and low concentrations
of
pentoxifylline and metabolites I, IV, and V are given in Table
1.
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TABLE 1.
The linearities, sensitivities, and percentages of
coefficient variation at high and low concentrations of
pentoxifylline and its metabolites I, IV, and V
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|
Pharmacokinetic study.
To determine the pharmacokinetics of
pentoxifylline and metabolites in noninfected and infected mice, three
or four animals in each group were sacrificed by CO2
asphyxiation at 0, 5, 15, 30, 60, and 90 (infected animals only) min
after the first (day 0), fourth (day 1), and seventh (day 2) doses of
pentoxifylline. The first dose of drug was administered 6 h after
mice were injected with C. albicans or PBS. None of the
infected mice survived to day 2. Blood was collected by cardiac
puncture and allowed to clot at 4°C. At each time point the serum was
separated from the clot by centrifugation, pooled, and stored at
70°C until analyzed.
Pharmacokinetic analysis.
Pharmacokinetic parameters were
determined by standard techniques. The concentration-time data for
pentoxifylline and its three metabolites were analyzed separately. The
elimination rate constant (kel) was determined
by linear regression of the log concentration-time data, and the
half-lives (t1/2) were calculated as
t1/2 = 0.693/kel. The
t1/2 of metabolites IV and V could not be
determined in any treatment group because concentrations were essentially unchanging over the measurement interval.
The linear-trapezoidal rule was used to calculate the area under the
concentration-time curve to the last measured value
(AUC
),
and the residual area, AUC
res, was
calculated by dividing the
last measured concentration
(
C) by
kel. The total
area under
the curve (AUC
0-) = AUC
+ AUC
res.
| |
RESULTS |
Pharmacokinetics.
The maximum plasma concentration (Cmax)
of pentoxifylline occurred at the first sampling time, 5 min after
injection. For the noninfected mice on days 0, 1, and 2 and
the infected mice on day 0, the Cmax,
t1/2, and AUC0- values for
pentoxifylline were similar, but the Cmax,
t1/2, and AUC0- values were
higher on day 1 in infected mice (Table
2). In all treatment groups,
pentoxifylline concentrations declined over time in a log-linear
fashion (Fig. 1). The pharmacokinetic
parameters for noninfected mice on all three treatment days were
similar and, thus, were combined to obtain an estimate of the
within-treatment variability of pentoxifylline pharmacokinetics. The
pharmacokinetic parameters for noninfected mice are reported as the
means ± standard deviations.
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TABLE 2.
Pharmacokinetics, including AUC,
t1/2, and Cmax, of
pentoxifylline and its metabolites for noninfected mice and mice
infected with C. albicans on days 0, 1, and 2 after
fungal infectionsa
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FIG. 1.
Natural logarithm of concentrations of pentoxifylline in
serum over time after i.p. dosing. Well, uninfected mice; inf.,
infected mice. All sera for each time point and group were pooled.
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|
The
Cmax of metabolite I in all treatment groups
also occurred at the first sampling time. The
Cmax,
t1/2, and
AUC
0- values for metabolite I on days 0, 1, and 2 for
noninfected mice
and on day 0 for infected mice were similar, but all
parameters
were higher for the day 1-infected mice (Table
2). Metabolite
I concentrations declined
in a log-linear fashion in all treatment
groups (Fig.
2). The pharmacokinetic parameters of
metabolite
I for noninfected mice on all treatment days were also
combined
and reported as the means ± standard deviations.
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FIG. 2.
Natural logarithm of concentrations of pentoxifylline
metabolite I in serum over time after i.p. dosing. Well, uninfected
mice; inf., infected mice. All sera for each time point and group were
pooled.
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|
Metabolite IV (1-[4-carboxybutyl]-3,7-dimethylxanthine),
which is important in humans (
1,
11), was present in very
low
concentrations in all treatment groups (Fig.
3). The time to
Cmax (
Tmax) varied in the
noninfected mice, occurring at 5 min in two
groups and at 30 min in one
group. It was not possible to obtain
an accurate estimate of the
t1/2 of metabolite IV in any treatment
group. The values of AUC
0- of metabolite IV for the
day
1-infected group were dramatically higher than those for the
noninfected and day 0-infected mice (Table
2).
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FIG. 3.
Natural logarithm of concentrations of pentoxifylline
metabolite IV in serum over time after i.p. dosing. Well, uninfected
mice; inf., infected mice. All sera for each time point and group were
pooled.
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The
Tmax of metabolite V, the major urinary
metabolite of pentoxifylline in humans (3-carboxypropyl), occurred at 5 min in
two noninfected-mouse groups and at 15 min in one
noninfected-mouse
group. For day 0-infected mice, metabolite
V concentrations and
pharmacokinetic parameters were similar to those
for noninfected
mice. In day 1-infected mice, metabolite V levels
increased throughout
the sampling protocol so that
Cmax,
t1/2, and
AUC
0- could not be accurately determined (Fig.
4). However, the AUC
for
metabolite V for the day 1-infected mice was almost 10-fold
greater
than the AUC
0- for the noninfected and day
0-infected mice, and the
Cmax for the day
1-infected mice was
more than 3-fold higher than those for the
noninfected and day
0-infected mice (Table
2).
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FIG. 4.
Natural logarithm of concentrations of pentoxifylline
metabolite V in serum over time after i.p. dosing. Well, uninfected
mice; inf., infected mice. All sera for each time point and group were
pooled.
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Organ function.
Values of serum creatinine, lactate
dehydrogenase, alanine transaminase, aspartate transaminase, and
alkaline phosphatase, measured in aliquots of drug concentration
samples, revealed that in day 0-infected mice, renal and hepatic
functions were still within normal limits while in day 1-infected mice,
renal and hepatic functions were significantly compromised, indicating
organ injury (Table 3).
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TABLE 3.
Mean serum creatinine, lactate dehydrogenase, alanine
transaminase, aspartate transaminase, and alkaline phosphatase levels
in noninfected mice and mice infected with C. albicans
before and during pentoxifylline administration
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| |
DISCUSSION |
Pentoxifylline was eliminated very rapidly after absorption
in both the infected and noninfected mice. The
t1/2 of both pentoxifylline and its metabolites
in noninfected mice were within the ranges reported for healthy mice by
other investigators (3, 9). There was also an increase in
the AUCs for pentoxifylline (fourfold), metabolite I (threefold), and
metabolites IV and V (greater than fourfold) in the infected mice
(Table 2). These large increases in the AUCs of pentoxifylline and its
metabolites in infected mice resulted from decreased hepatic and renal
elimination due to the impaired liver and kidney functions, and from
increased pentoxifylline absorption following i.p. administration. Raju et al. reported a 29% bioavailability for i.p. administration compared
to that for subcutaneous administration for healthy mice (8). These authors speculated that this low i.p.
bioavailability was due to extensive "first pass" hepatic
metabolism of pentoxifylline. In our study, increased pentoxifylline
bioavailability resulting from decreased hepatic elimination during the
first pass through the liver would have contributed to an increase in
both the AUC and Cmax of pentoxifylline in the
infected mice. Pentoxifylline metabolite formation would also have
increased as a result of the increased bioavailability of
pentoxifylline in the infected mice.
Murine pentoxifylline pharmacokinetics differ among various mouse
strains, resulting in differences in the extent of metabolite formation. This study and that of Honess et al. (3) found
measurable concentrations of metabolites I, IV, and V at comparable
doses. Conversely, Raju et al. (8) were not able to detect
metabolite IV in plasma following i.p. or subcutaneous administration
of pentoxifylline. Honess et al. (3) found that metabolite I
and IV concentrations were approximately 1/10 that of pentoxifylline, while in the present study metabolite I and IV concentrations were 1/4
and 1/100, respectively, those of pentoxifylline. Raju et al.
(8) found that metabolite I levels were about one-fourth those of pentoxifylline, while metabolite V concentrations were somewhat higher than those of pentoxifylline. Metabolite V
concentrations were of the same order of magnitude as pentoxifylline
concentrations in all three studies. These differences in the
metabolite patterns of pentoxifylline, particularly pharmacologically
active metabolite I, caution against any extrapolation of the
physiological effects of pentoxifylline among mouse strains, unless the
pharmacokinetics and metabolic profiles are known.
Did the changes in the pharmacokinetics of pentoxifylline and its
metabolites caused by the C. albicans infection contribute to a reduction in mouse survival time and greater mortality? The magnitudes of the AUCs of pentoxifylline and metabolites I and V for
the infected mice would only be achieved for noninfected mice with
doses greater than 240 mg/kg, assuming dose-independent pharmacokinetics. Honess et al. reported that single i.p. doses of up
to 200 mg/kg were well tolerated by healthy mice but that 400 mg/kg was
highly toxic (3). However, in our study, the day 1-infected
mice received three doses of pentoxifylline, and as the C. albicans infection progressed, liver and kidney functions became
progressively impaired. Thus, with each successive dose of
pentoxifylline different pharmacokinetics were exhibited,
reflective of successively increasing doses. The combination of the
infection-induced organ function impairment and the resulting
increasing systemic exposure to pentoxifylline and its metabolites
likely was responsible for the shorter survival times and greater
mortality of the infected animals.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Infectious
Diseases (111D), Stratton VA Medical Center, 113 Holland Ave.,
Albany, NY 12208. Phone: (518) 462-3311, ext. 3080. Fax: (518)
462-3350. E-mail: baltch.aldona{at}albany.va.gov.
Present address: American Association of Colleges of Pharmacy,
Alexandria, VA 22314-2841.
| |
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Antimicrobial Agents and Chemotherapy, September 1998, p. 2405-2409, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
