Laboratoire de Pharmacocinétique Clinique (S.L.L., F.B.) and
Laboratoire de Pharmacologie (P.P., G.C.), Faculté de Pharmacie,
Montpellier, France; and Agence Française de Sécurité
Sanitaire des Produits de Santé (R.K.d.R., P.-A.B.), Vendargues,
France
Sodium tungstate has been found to correct hyperglycemia in insulin-
and noninsulin-dependent models of diabetes when administered in
drinking fluid with a low degree of toxicity; thus, it provides a
potential treatment for diabetes. In the present report,
pharmacokinetic studies with sodium tungstate were carried out in the
Sprague-Dawley rat and beagle dog. This drug was administered either
i.v. (8.97 mg/kg in rat; 25 and 50 mg/kg in dog) or orally in the form
of solution (35.9 and 107.7 mg/kg in rat; 25 and 50 mg/kg in dog). Tungsten was quantified using an inductively coupled plasma method. Pharmacokinetic parameters were estimated using a population approach. Sodium tungstate followed first order kinetics, and plasma
concentration-versus-time data were adequately described by a
two-compartment model. In rat, bioavailability was high (92%), whereas
it was lower in dog (approximately 65%). The total volume of
distribution expressed by unit of body weight was much higher when the
animal was smaller (0.46 l/kg in rat versus 0.23 l/kg in dog). The
total body clearance normalized by weight, 0.19 l/h/kg in rat versus
0.043 l/h/kg in dog, changed as for the volume of distribution. The
elimination half-life was two times higher in dog (approximately 4 h) than in rat (approximately 1.7 h). In the range of 35.9 to
107.7 mg/kg after oral administration in rat and 25 to 50 mg/kg after
oral and i.v. administration in dog, tungsten plasma concentrations increased in proportion to dose.
 |
Introduction |
Transition
metal derivatives have recently been found to possess antidiabetic
activity. Vanadate was first described as able to lower blood glucose
in insulin-deficient rats (Heyliger et al., 1985
) and in animal models
of insulin resistance (Brichard et al., 1989). However, vanadate
treatment was associated with significant side effects, including
digestive troubles, which might be explained by its low bioavailability
(10-20%). The antidiabetic activities of other transition metal
derivatives such as chromium, molybdate, or selenate (Fillat et al.,
1992; Battell et al., 1998; Anderson, 2000) have also been
demonstrated. However, their toxicity decreases their interest for
clinical use. Recently, sodium tungstate was found to correct
hyperglycemia in insulin- and noninsulin-dependent models of diabetes
when administered at a dosage of 169 to 418 mg/kg/day in drinking fluid
for 15 days with low degree of toxicity (Barbera et al., 1994, 1997)
and should be tested clinically. A biokinetic model for systemic
tungsten in humans was recently published by Leggett (1997). This model
is based on experimental data on the biokinetics of radiotungsten in
laboratory animals and information on the kinetics of molybdenum or
other physiological analogs of tungsten in humans and laboratory
animals. An elimination half-life (t1/2
elim) of 12 to 14 h was reported by Mason et al. (1989) in sheep. Experimental results concerning the distribution of
tungsten between plasma and red blood cells (RBCs) have been published.
In beagle dogs receiving 181W as sodium
tungstate, the ratio of concentration plasma to RBCs was approximately
3 during the first 24 h (Aamodt, 1973). Higher RBC-to-plasma
ratios for radiotungsten have been determined in rodents than in larger
animals (Kaye, 1968). In rats and mice, RBC-to-plasma ratios of
approximately 9 and 14 were found at 3 to 4 days after administration.
In sheep, [185W]tungstate was not protein bound
(Mason et al., 1989).
This study was conducted to determine the pharmacokinetic profile of
sodium tungstate after i.v. and oral administration in two different
species: rat and dog. Individual pharmacokinetic parameters were
estimated using an empirical Bayes' methodology. The linearity of the
kinetics was also investigated for different doses administered i.v. or
orally. Our results indicate that after oral administration of 35.9 to
107.7 mg/kg sodium tungstate in rat and after oral and i.v.
administration of 25 to 50 mg/kg sodium tungstate in dog, tungsten
plasma concentrations increased in proportion to dose and that the
bioavailability of sodium tungstate was relatively high (65% in dog
and 92% in rat).
| |
Materials and Methods |
Compound.
Sodium tungstate was obtained from Carlo Erba (Val
de Reuil, France). Tungsten as the sodium salt was administered to
animals in an aqueous solution containing 0.9% sodium chloride for the i.v. route and in distilled water for the oral route.
Animals.
This study was conducted in male Sprague-Dawley
rats and in male beagle dogs.
Rats.
Two hundred sixteen Sprague-Dawley rats weighing 316 to 532 g (IFFA CREDO, L'Arbresle Cedex, France) at age 10 weeks
were used (one animal per time point). Animals underwent an
acclimatization of a minimum of 2 weeks before treatment. They were
group housed in stainless steel cages with suspended wire-mesh floors
(maximum of three rats per cage). The rats were fed a standard
laboratory rodent diet (UAR sterile food, Usine d'Alimentation
Rationnelle, Villemoisson, Epinay s/Orge, France) and allowed free
access to drinking water. Rats were fasted overnight (12 h) before drug administration and then weighed.
Dogs.
Six male beagle dogs (Harlan, Grannat, France)
weighing 11.5 ± 0.24 kg were housed individually during the study
in metabolism stainless steel cages with access to pelleted food (400 g/day/animal; Usine d'Alimentation Rationnelle). Tap water was
distributed ad libitum. The animals underwent an acclimatization period
of 10 days before the experiment.
The dogs were fasted for 24 h before each experiment and then
weighted. The day of administration, food was distributed 12 h
after drug intake. The same animals were dosed i.v. and orally; both
administration routes were investigated after at least a 15-day
wash-out period.
For all animals, the housing rooms had controlled environmental
conditions with temperature and relative humidity of approximately 18-21°C and 40 to 70%, respectively, and artificial lighting, alternating on a 12-h light/dark cycle.
Drug Administration.
Single i.v. (8.97 mg/kg) and oral (35.9 and 107.7 mg/kg) doses of sodium tungstate were administered to each
rat. For which, three different solutions of sodium tungstate were
prepared on the day of administration: one solution in 0.9% sterile
isotonic saline (4.5 mg/ml) for i.v. administration and two solutions
in distilled water (3.6 and 10.8 mg/ml) for oral administration. These
solutions were used to treat animals, under the administered volume of
2 ml/kg (i.v.) and 10 ml/kg (oral).
Dogs received four different treatments: two i.v. doses (25 and 50 mg/kg) and two oral doses (25 and 50 mg/kg) of sodium tungstate. The
vehicles used were 0.9% sterile isotonic saline (0.1 ml/kg b.wt.) for
i.v. administration and double distilled water (1 ml/kg b.wt.) for oral administration.
For i.v. administration, the dose was administered over 1 min into the
tail vein in the rat and the cephalic vein in the dog. For oral
administrations, the drug was given by gavage through stomach tubing
using a polypropylene catheter.
Blood Sampling.
In rat, blood samples were collected (one
sample per rat) at the following time points (six animals per time
point): 5, 10, 15, and 30 min and 1, 2, 4, 8, 12, 16, and 24 h
after drug administration. Untreated animals were used for basal
tungsten level determination. Two minutes before sampling, rats were
anesthetized with diethylether and then sacrificed by section of the
carotid artery. Total blood was collected in heparinized polypropylene
tubes (0.1 ml sodium heparinate per tube).
In dog, blood samples (8 ml) were collected from a superficial vein of
the forelimbs into polypropylene tubes coated with sodium heparinate
before the oral and i.v. doses; after 5, 10, 15, 30, 45, and 60 min and
2, 4, 8, 12, 16, 24, and 36 h for the i.v. route; and after 10, 20, 40, and 60 min and 2, 4, 8, 12, 16, 24, and 36 h for the oral route.
Tubes containing blood samples were immediately and gently agitated to
prevent coagulation and then centrifuged at 2000g for 20 min. Plasma was removed, transferred into two polypropylene tubes, and
then stored at 
20°C until assay.
Assay Method.
Concentrations were expressed in tungsten
metal. Tungsten plasma concentrations were determined using an
inductively coupled plasma emission spectrometric method at a
wavelength of 207.91 nm (Poucheret et al., 2000). Samples (500 µl)
were directly nebulized; each determination was performed in replicate
(n = 5). Calibration curves were obtained in the range
134 to 1300 ng/ml. Precision ranged from 0.4 to 17%, and accuracy was
between 89 and 105%. Dilution has no influence on the performance of
the method, which could then be used to quantify plasma samples
containing up to 90 µg/ml. The limit of quantification was 100 ng/ml,
it was defined as the lowest drug concentration that can be
determined with an accuracy of 100 ± 20% and a relative standard
deviation of 
20% on a day-to-day basis. Using quality control
samples at this level, the precision averaged 17%.
Population Pharmacokinetic Analysis.
Individual
pharmacokinetic parameters were estimated using an empirical Bayes'
methodology (Sheiner et al., 1972). In this analysis, population
characteristics of the parameters to be estimated were used as prior
information to estimate each individual pharmacokinetic parameter.
In rat, a preliminary analysis was carried out using the Pk-fit
software (version 1.1.4, 1999; RDPP, Montpellier, France), to compute
pharmacokinetic parameters from the average concentration values at
each time points. Such an analysis allowed us to estimate the initial
pharmacokinetic parameters that will be used in the population analysis
and to choose the pharmacokinetic model. Individual pharmacokinetic
parameters were then determined using a population approach and the
baseline-corrected plasma concentrations.
In dog, plasma concentration-versus-time data were first analyzed
using a noncompartmental approach (Pk-fit software). Such an analysis
allowed us to verify that drug concentration increased in proportion to
dose. Then, despite the few number of dogs, the data obtained in each
dog from the whole treatment (i.v. plus oral administration) were
analyzed using a population approach. Such an analysis 1) avoided a
possible bias in the estimation of the
t1/2 elim.
Indeed, at low doses, 36 h after administration, tungsten
concentrations were below the limit of quantification of the analytical
method in the majority of animals, so the
t1/2 elim could not be estimated with the same
accuracy at low and high doses. 2) It allowed us to use the same model
after i.v. and oral routes (the distributive phase being poorly
detectable after oral administration, so using a classic approach, this
phase cannot be accurately fitted). 3) It allowed a better estimation
of individual pharmacokinetic parameters, including bioavailability.
Moreover, this approach allowed us to confirm that the selected model
in dog properly fit the entire set of available data.
The population analysis was performed using the P-PHARM computer
program (version 1.4, SIMED, Créteil, France). Details concerning the mathematical presentation of the implemented algorithm have been
previously provided (Gomeni et al., 1994; Mentré and Gomeni, 1995). The population estimation algorithm used in P-PHARM is an
EM-type procedure (Dempster et al., 1977) that computes the maximum
likelihood estimates using an iterative procedure. For the expectation
step (E step), for each individual, the individual parameters are
estimated (bayesian estimate) given the current population parameters
and the individual data. For the maximization step (M step), the
population parameters are estimated by maximum likelihood given the
current estimates (E step) of the individual parameters.
The E and M steps are iterated up to the convergence of the algorithm.
The EM algorithm iterates until the fractional changes of the fixed,
random, and residual error variance parameters between two consecutive
iterations became lower than 0.01.
Base Structural and Statistical Models.
In rat, the model
was chosen according to the results of the preliminary analysis
performed on the average concentration values at each time point. Thus,
the basic pharmacokinetic parameters (
) considered in the population
analysis are clearance (1 = CL), initial
volume of distribution (2 = V),
transfer rate constants (3 = k12 and 4 = k21), absorption rate constant
(5 = ka), and bioavailability (6 = F).
In dog, preliminary analysis was performed to 1) compare one-, two-,
and three-compartment models, 2) to evaluate the presence of a lag time
(tlag) after oral administration, and
3) to choose between zero order and first order input rate. This
analysis revealed that the two-compartment model with first-order
absorption rate with a tlag
statistically improved the fit on the basis of the examination of
Akaike's information criterion (Yamaoka et al., 1978) and of the
objective function (Gomeni et al., 1994). Inspection of weighted
residual and individual predicted-versus-observed concentration plots
were also used in part to select the appropriate structural model. A
one- or a three-compartment provided biased parameter estimates and did
not statistically improve the objective function.
The basic pharmacokinetic parameters () considered in the population
analysis are clearance (1 = CL), initial
volume of distribution (2 = V),
transfer rate constants (3 = k12 and 4 = k21), absorption rate constant
(5 = ka), lag
time (6 = tlag), and bioavailability
(7 = F) .
To evaluate the statistical models, an homoscedastic model and an
heteroscedastic model were considered. For the two species studied, the
residual distribution showed that the error variance was better
described by an heteroscedastic model (proportional to the estimated
values of the predictions).
The posterior individual pharmacokinetic parameter estimates were then
assumed to arise from a normal or log-normal distribution characterized
by a population mean and an interindividual variance. On the basis of
the examination of the objective function and of the inspection of
weighted residuals and individual predicted-versus-observed concentration plots, results indicate that in rat, for all parameters, the probability distribution of the random effect parameters
[sigma(CL), sigma(V),
sigma(k12),
sigma(k21),
sigma(ka), sigma(F)] was
better described by normal distribution. In dog, for CL and
V, the probability distribution of the random effect
parameters was better described by log-normal rather than normal
distribution; for the other parameters (k12,
k21,
ka,
tlag, and F), a normal
distribution was used.
The t1/2 elim, area under the plasma
concentration-time curve (AUC), and the steady-state volume of
distribution (Vd) were calculated as
follows:
|
(1)
|
with
![k<SUB><UP>el</UP></SUB>=[<UP>CL</UP>/F]/[V/F]](/content/vol294/issue2/fulltext/714/img002.gif)
|
(2)
|
![<UP>AUC</UP>=[<UP>dose</UP>×F]/<UP>CL</UP>](/content/vol294/issue2/fulltext/714/img003.gif)
|
(3)
|
|
(4)
|
From this model, the plasma concentration of tungsten at any
time can be easily computed using the empirical Bayes' estimate of the
pharmacokinetic parameters.
Consistency Check of Pharmacokinetic Parameter Estimates.
To
check the error model assumptions and the distribution on the estimated
population pharmacokinetic parameters, P-PHARM estimates the expected
concentrations (Cexp) for each
individual in the population and computes appropriate statistical tests
to evaluate the distribution properties of the differences between the
expected and the observed data. For each concentration, a standardized
concentration prediction error (SCPE) is calculated as follows:
SCPE = [Cobs Cexp]/S.D.(Cexp),
where Cobs represents the observed
concentrations, and S.D.(Cexp)
represents the estimated standard deviation for the expected values
computed using all sources of random variability, including the
residual error.
To assess the posterior distribution properties of the residuals and
the individual parameters, the t test was used to compare the mean of SCPE with zero; the Kolmogorov-Smirnov test was used to
compare the sampled distribution to the expected one
[N(0,1)] (Sokal and Rohlf, 1969).
Statistical Evaluation.
Results in the text are presented as
mean ± S.D.
In rat, differences in pharmacokinetic parameters (CL,
Vd,
t1/2 elim) were
compared across the three treatment groups by using the Kruskal-Wallis
test (Siegel and Castellan, 1988). The effect of the administered dose
(35.9 versus 107.7 mg/kg sodium tungstate) was also assessed by
comparing AUC.
In dog, a two-way ANOVA was performed to assess the effect of the
administered dose on parameters determined by noncompartmental approach
(i.e., AUC after i.v. administration of 25 and 50 mg/kg sodium
tungstate, and Cmax and AUC after oral
administration of 25 and 50 mg/kg sodium tungstate).
Before the statistical analyses, CL,
Vd,
Cmax, and AUC were log-transformed;
Cmax and AUC were normalized to the
same administered dose. A 5% level of statistical significance was used.
| |
Results |
Pharmacokinetics in Rat.
The mean endogenous tungsten
concentration was 137 ng/ml. Semilogarithmic plots of the mean plasma
concentration-time curve after i.v. bolus injection of 8.97 mg/kg and
oral administrations of 35.9 and 107.7 mg/kg sodium tungstate are shown
in Fig. 1. Data were consistent with a
two-compartment model. Twelve hours after i.v. administration and
24 h after oral administration of 35.9 mg/kg, concentrations
returned to baseline value (i.e., 137 ng/ml). After oral
administration, absorption was rapid (approximately 2 h).
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Fig. 1.
Mean tungsten plasma concentration-versus-time curve
after i.v. (8.97 mg/kg) and oral (35.9 and 107.7 mg/kg) administrations
of sodium tungstate in rat.
|
|
A total of 176 concentrations were used to compute population
parameters (Table 1). From the population
characteristics, it can be seen that CL was the parameter that
exhibited the lowest coefficient of variation (CV = 27.4%) and
k12 was the parameter that exhibited
the highest (CV = 41.9%). Bioavailability averaged 92%.
From the empirical Bayes' estimate of the individual pharmacokinetic
parameters, mean pharmacokinetic parameters, according to the three
treatment groups, are given in Table 2.
CL, Vd, and
t1/2 elim did
not differ statistically between treatments. No significant
relationship was found between weight and CL or between weight and
Vd. AUC averaged 26.5 ± 2.3 mg/l × h after i.v. administration of 8.97 mg/kg sodium tungstate
and 111.2 ± 10.1 and 326.9 ± 31.0 mg/l × h after oral
administration of 35.9 and 107.7 mg/kg sodium tungstate, respectively.
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TABLE 2
Mean (±S.D.) posterior pharmacokinetic parameters of sodium tungstate
in male rat computed using the population approach
|
|
Bioavailability was 95% (CV = 9.5%) after oral administration of
35.9 mg/kg and 88% (CV = 14.8%) after oral administration of
107.7 mg/kg. No dose dependence was observed, suggesting linear kinetics.
Pharmacokinetics in Dog.
The mean baseline tungsten
concentration value was 129 ng/ml. After oral administration, the
absorption was rapid (tmax, 1-2 h).
Semilogarithmic plots of the plasma concentration-time curve after i.v.
bolus injections of 25 and 50 mg/kg sodium tungstate and oral
administrations of the same doses are shown in Fig.
2.
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Fig. 2.
Mean tungsten plasma concentration-versus-time curve
after i.v. (25 and 50 mg/kg) and oral (25 and 50 mg/kg) administrations
of sodium tungstate in dog.
|
|
Preliminary analysis performed by noncompartmental approach revealed
that after i.v. and oral administrations, concentrations increased in
proportion to dose. AUC averaged 297.2 ± 34.9 and 647.4 ± 70.7 mg/l × h after i.v. administration of 25 and 50 mg/kg, respectively; after oral administration of the same doses, they were
177.0 ± 53.3 and 431.0 ± 93.7 mg/l × h, respectively.
A total of 351 concentrations were used to compute population parameters.
The parameter estimates given by the model are summarized in Table
3. From the population characteristics,
it can be seen that k21 was the
parameter that exhibited the lowest coefficient of variation (CV = 19.1%) and tlag was the parameter
that exhibited the highest (CV = 73%). Mean bioavailability was
66% (57-74%).
Individual pharmacokinetic parameters are presented in Table
4.
A typical posterior individual fitting is illustrated in Fig.
3.
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Fig. 3.
Typical posterior individual fit after administration
of sodium tungstate. Dose 1, 25 mg/kg i.v. Dose 2, 50 mg/kg i.v. Dose
3, 25 mg/kg oral. Dose 4, 50 mg/kg oral.
|
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Consistency Check of Pharmacokinetic Parameter Estimates.
Under the assumption of a correct regression model and unbiased
parameter estimates, the Kolmogorov-Smirnov test showed that the SCPE
distribution was not significantly different from a normal distribution
with unitary variance and that the mean SCPE value was not
significantly different from zero (Student's t test).
The goodness of fit was shown by the analysis of the scatterplot of the
posterior predicted values versus the individual observed concentrations (Fig. 4) and by the
frequency distribution histogram of the normalized residuals, which
reveals a distribution very close to the expected one (normal with zero
mean and unitary variance; Fig. 5).
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Fig. 4.
Scatterplot of predicted concentrations (bayesian
estimates) versus observed concentrations with the unitary slope in the
population group. A, rat. B, dog.
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| |
Discussion |
The aim of the this study was to determine the
pharmacokinetic profile of sodium tungstate after single i.v. or oral
(gavage) administration in two different species: rat and dog. In these two species, doses have been chosen according to the maximum tolerated dose for each route of administration. In dog, side effects occurred at
doses higher than 50 mg/kg for both oral or i.v. routes. In rat, the
highest oral dose of 107.7 mg/kg did not produce any apparent side
effects and was close to the doses used in efficacy studies performed
by Barbera et al. (1994, 1997). In these two published studies, sodium
tungstate (154-205 mg/kg/day in healthy rats and 169-418 mg/kg/day in
diabetic rats) was administered in drinking water. However, the maximum
i.v. tolerated dose was lower in rat than in dog (8.97 mg/kg).
The results found in this study indicated that absorption of sodium
tungstate when administered in solution form was rapid (tmax = 1-2 h). In rat,
bioavailability was high (approximately 92%). However, lower results
of 40 to 70% were found after the administration of radiotungsten as
tungstate (Ballou, 1960; Fleshman et al., 1966; Kaye, 1968), whereas
uptake of radiotungsten administered as tungstic acid was only 1%
(Ballou, 1960). Cardin and Mason (1976) found that the maximum rate of
transport of tungstate through the small intestine of the rat, as
studied in vitro using the everted sac technique, occurs in the lower
ileum. Molybdate, tungstate, and sulfate will be readily transported
across the lower ileum in the rat by a common transport system subject
to competitive inhibition. In dog, the bioavailability averaged 65%.
These results are higher than that of 25% reported by Aamodt (1975)
after the intragastric administration of a weakly acidic aqueous
suspension of tungstic oxide in beagle dog.
In both species, over the sampling times monitored, plasma
concentration profiles versus time were compatible with a
two-compartment model and first-order kinetics. These results were in
accordance with those found by Leggett (1997). In rat, after oral
administration of 107.7 mg/kg sodium tungstate, mean
concentration-versus-time curve showed a double-peak phenomenon during
the input process. However, further investigations carried out on rat
after repeated administrations (data not shown) did not confirm such a
result. The total volume of distribution
(Vd = 0.46 l/kg in rat versus 0.23 l/kg in dog) and the total body clearance (0.19 l/h/kg in rat versus
0.043 l/h/kg in dog) normalized by body weight were both much higher
when the animal was smaller. No relationship occurred between weight
and CL or Vd. The
t1/2 elim was
two times higher in dog (approximately 4 h) than in rat
(approximately 1.7 h). These results confirmed the findings of
Leggett (1997) that rats appear to excrete tungsten at a much higher
rate than do larger animals. This relatively high excretion rate could
be related to the unusually low requirements of the rat for the
chemically similar element molybdenum (Higgins et al., 1956). After
oral administration of 35.9 to 107.7 mg/kg sodium tungstate in rat and
after oral and i.v. administration of 25 to 50 mg/kg sodium tungstate
in dog, tungsten plasma concentrations increased in proportion to dose.
These pharmacokinetic properties indicate that tungsten is relatively
unique among pharmacologically active metals. Indeed, although tissue
storage was not specifically studied in the present work, the short
t1/2 elim of
sodium tungstate suggests a rather limited tissue accumulation.
Moreover, a major difference between tungstate and vanadate is the
bioavailability. Vanadate bioavailability is still a matter of debate;
it was reported in rat to vary from 5% (Conklin et al., 1982; French
and Jones, 1993; Nielsen, 1988) to a maximum of 30% (Bodgen et al.,
1982; Setyawati et al., 1998; Wiegmann et al., 1982). This low
bioavailability was associated with digestive side effects (e.g.,
cramps and diarrhea) in correlation with the pharmacological activity
of the metal on the digestive tract (Goldfine et al., 1995;
Soulié et al., 1996) and led to the development of organomineral
compounds (Yuen et al., 1997). In this perspective, the high
bioavailability of sodium tungstate will certainly constitute a major advantage.
In conclusion, this report represents valuable information about the
pharmacokinetics of sodium tungstate in rat and dog. Because this study
was conducted in healthy animals, it was not possible to correlate
tungsten plasma levels to a pharmacological response. Future studies
will explore pharmacodynamic/pharmacokinetic relationships in diabetic animals.
We gratefully acknowledge C. Ballongue (Agence Française
de Sécurité Sanitaire des Produits de Santé,
Vendargues, France) for technical support.
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Abstract
Introduction
) in rats and effect of Al(OH)3.
J Toxicol Environ Health
10:
233-245[Medline].
