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Drug Development Research 853-62 (1986)
Pharmacokinetics and Tissue Distribution of Astemizole in the Dog Marcel Michiels, Achiel Van Peer, Robert Woestenborghs, and Jozef Heykants
Department of Drug Metabolism and Pharmacokinetics, Janssen Pharmaceutica Research Laboratories, 6-2340 Beerse, Belgium
ABSTRACT
Michiels, M., A. Van Peer, R. Woestenborghs, and J. Heykants: Pharmacokinetics and tissue distribution of astemizole in the dog. Drug Dev. Res. 853-62, 1986.
The pharmacokinetics of a single oral dose of 3H-astemizole (AST) of 1 mg/kg was studied in Beagle dogs and compared with that of a tracer dose superposed at the end of a subchronic experiment. Single and superposed doses were equally well absorbed, and elimination half-lives of AST and of its metabolites were similar at both occasions (tlla = 3-5 d). Steady-state was reached after 2-3 weeks of daily dosing. AST and its metabolite desmethylastemizole (DES-AST) were extensively distributed, mainly to well perfused tissues but only poorly to brain, muscle, and fat. All tissue levels were many times higher than the corresponding plasma concentrations. The superposed tracer dose was well stirred in the tissue compartments and showed instantaneous equilibrium with pretreat- ment levels. Tissuelplasma level ratios of AST and DES-AST remained constant with time, indicating that the elimination from tissues proceeded at a similar rate to that from plasma. The extensive distribution of AST and DES-AST is explained by a strong, but reversible tissue binding that appears to be rate-limiting for the elimination from the body.
Key words: histamine, H1-antagonist, subchronic, superposition technique, radiolabeled
INTRODUCTION
The study in experimental animals of the absorption, tissue distribution, excretion, and metabolism of astemizole (AST), an oral histamine H1-antagonist [Awouters et al., 19831, can provide a better insight into its pharmacological profile and mechanism of action and may contribute to the evaluation of data obtained in drug safety studies. The dog was suggested as
Received final version March 3, 1986; accepted March 3, 1986.
Address reprint requests to M. Michiels, Department of Drug Metabolism and Pharmacokinetics, Janssen Pharmaceutica, Turnhoutseweg 30, 2340 Beerse, Belgium.
0 1986 Alan R. Liss, Inc.
54 Michiels et al.
the animal species with a metabolic pattern and disposition kinetics for astemizole closer to man than those found in rats and guinea-pigs [Meuldermans et al., 19861. Since AST is intended for chronic therapy in man, absorption and disposition studies in animals should be performed preferably under similar dosing conditions. During chronic treatment, the disposi- tion of a substance may be altered, and one of the techniques used to detect these alterations is to study the pharmacokinetics of a radioactive dose at the beginning and at the end of a chronic study. If the kinetic processes are linear and chronic dosing does not change the disposition of the drug, its pharmacokinetic profile will be similar on both occasions [Hammer and Bozler, 1977; Colburn and Matthews, 19791. This technique was adopted to study the pharmacokinetics of astemizole in Beagle dogs after a single and a 6-week subchronic dosing. The tissue distribution and the elimination kinetics of astemizole and metabolites were studied.
METHODS Experimental Design
Four male Beagle dogs, weighing 9.5-14.8 kg, were used to study the disposition of AST in a subchronic experiment at 1 mg/kg. A solution in 0.1 M tartaric acid of 3H-AST, specifically labeled with tritium at the 2-position of the fluorobenzyl moiety [Thijssen et al., 19831 was given by gastric intubation after an overnight fast at the occasion of the first (single) and last (42nd) dose administration. The radiochemical purity of 3H-AST was 99.9% (radio- high performance liquid chromatography) and the final specific activity was 19.6 pCi/mg for the single and 28.3 pCi/mg for the last dose. Starting eight days after the first labeled dose, the dogs received daily for 41 consecutive days an oral dose of 1 mg/kg of unlabeled AST, provided as a microcrystalline powder in gelatin capsules. Venous blood samples were taken on heparin at various intervals after the single and after the 42nd radiolabeled dose and just before daily dose administration during the subchronic treatment. Urine and feces were collected for mass-balance purposes at different intervals each time at the occasion of the radioactive dose administration [Meuldermans et al., 19861. The dogs were killed (pentobar- bital sodium, 30 mg/kg IV) and autopsied, one per interval for tissue distribution 24, 48, 96 and 168 hr after the superposed tracer dose. Tissues were weighed immediately after dissection and stored at -20°C until analysis.
Determination of Radioactivity
The radioactivity of the biological samples was measured by liquid scintillation counting (Packard Tri-Carb 460 CD with automatic external standardization and conversion of cpm to dpm). Aliquots of samples were counted directly (plasma, urine), after extraction (feces) or homogenization in water (I :4,w:v) (tissues) or after previous combustion in a sample oxidizer (fecal residues, blood), (Packard Tri-Carb 306 B). Levels of total radioactivity were corrected for the presence of metabolically formed tritiated water by determining the levels of nonvolatile radioactivity (NVR) after lyophilization of the samples (Hetosicc CD 13). The detection limit was 2.5 ng-Eq/ml of plasma or 5 ng-Eq/g of wet tissue.
Determination of the Fraction of Astemizole Plus Hydroxylated Metabolites
Plasma concentrations of the fraction of AST plus hydroxylated metabolites were determined with radioimmunoassay (RIA) after extraction at pH 7.8 with heptane-isoamyl alcohol (9515, v/v) as described elsewhere [Woestenborghs et al., 19861. This fraction con- sisted mainly of astemizole and its 0-demethylated metabolite desmethylastemizole (DES- AST) [Woestenborghs et al., 19861. The detection limit was 0.1 ng-Eqlml of plasma.
Determination of Astemizole in Plasma
Plasma levels of AST after single or subchronic administration at 1 mg/kg, too low to be measured with HPLC, were determined with RIA after further separation of the hydroxy-
Disposition of Astemizole in Dogs 55
lated metabolites from the pH 7.8 extract, employing a 0.5 N sodium hydroxyde solution and extraction at pH 12 [Woestenborghs et al., 19861. The detection limit was 0.05 ng/ml of plasma.
Determination With HPLC
Tissue concentrations of AST and of DES-AST were determined with HPLC [Woesten- borghs et al., 19831. The detection limit was 50 and 100 ng/g of wet tissue for AST and DES- AST, respectively. The effluent of the HPLC column during the analysis of tissue samples for total AST or DES-AST was collected at the proper retention times, and the radioactivity corresponding with the distinct chromatographic peaks was determined by liquid scintillation counting. This allowed the determination of the contribution of the superposed (42nd) 3H-AST dose to the total tissue levels of AST or DES-AST. Concentrations of labeled AST and DES- AST were calculated on the basis of the specific activity of 3H-AST used for the last dose and the recovery of the extraction and HPLC procedure. The detection limit for the HPLC procedure with off-line radioactivity counting was 10 ng/g of wet tissue for both AST and DES-AST.
RESULTS Single-Dose Kinetics
Peak plasma levels of radioactivity corrected for tritiated water (NVR), amounting to 31.8 f 15.0 ng-Eq/ml, were attained within 2 hr (Fig. 1). Thereafter, plasma levels declined apparently biexponentially with a rapid distribution phase, lasting to about 12 hr followed by a slower elimination phase. The median area under the curve to time infinity (AUCO-,) was 1097 ng-Eq.hr/ml (range: 645-3,081). Plasma concentrations of unmetabolized AST were maximal at 0.5 hr and amounted on average to 1.86 f 0.67 ng/ml (Fig. 2). Plasma AST levels declined apparently biexponentially with sequential half-lives of 1.3 and 34 hours. The median AUCO-, amounted to 30.6 ng.hr/ml (range: 12.4-55.0), corresponding to 2.7% of total plasma radioactivity.
Superposition of Radiolabeled Dose
After subchronic dosing with unlabeled drug for 41 consecutive days, the superposed 3H-AST dose produced peak levels of radioactivity (NVR) in the range of 21-46 ng-Eq/ml at 0.5- 4 hr (Fig. 1). Thereafter, radioactivity was similarly eliminated from plasma as after the single dose. The median AUC was 470 ng-Eq.hr/ml (range: 440-1,130), which is 2.3 times lower than the corresponding value obtained after single dosing.
The median peak plasma concentrations of total AST-resulting from the radiolabeled dose and from the residual level from the pretreatment scheme-amounted to 2.70 ngiml (range: 2.24-24.6) (Fig. 2). Plasma levels of the fraction AST plus hydroxylated metabolites peaked within 4 hr after the superposed dose, representing 8.62 ng-Eqlml (range: 5.8-32). Comparing AUC’s, AST represented from 6 to 70% of the fraction of AST plus hydroxylated metabolites.
Repeated Dose Administration
Steady-state was reached within 2 to 3 weeks after the start of the subchronic experiment representing minimal levels of AST of 0.6-0.9 ng/ml (Fig. 3). Maximal levels, inferred from the last dose, were approximately two to five times higher. Minimal concentrations of the fraction AST plus hydroxylated metabolites were on average 3 ng-Eq/ml (Fig. 3), indicating that at steady-state plasma levels of DES-AST were about three times higher than those of the parent drug.
56 Michiels et al.
Dog No. L 9 2 5
i 1 J , t 0 21 L0
. Dog No. L 7 8 5
6 1 L., 0 2L 18 72 96
Dog No.6790
1 + 0 2 L L 0 72 96 120 1 L L
1 I, Hours
D o g No. LEOS
0 24 L8 72 96 120 ILL 168 H o u r s
Fig. 1 . Semilogarithmic plot of plasma concentrations of radioactivity (NVR) after a single oral dose of %AST in four Beagle dogs (0-0) or after a superposed dose in the dogs (0-0) pretreated orally with the unlabeled drug for 41 days at 1 mg/kg/day. The arrows indicate the time of sacrifice after the 42nd dose.
Tissue Distribution After Subchronic Administration
Tissue concentrations in the four dogs are reported in Table 1. The highest radioactivity levels in most tissues were observed in the second dog (48 hr). Remarkably high concentrations occurred in the liver, representing a maximal level of 13.4 pg-Eq/g, corresponding to 37.9% of the superposed dose. A peak level of 3.5 pg-Eqlg was observed in the pancreas, and further, levels of up to 1.9 pg-Eq/g occurred in kidney, lung, spleen, and some glandular tissues (Table 1). Radioactivity was poorly distributed to muscular and adipose tissue (0.1-1 pg-Eq/g) and to the brain (0.04 pg-Eqlg). The mass balance of the superposed dose, based on individual excretion data [Meuldermans et al., 19861, and the amounts of radioactivity detected in the various organs and tissues of the dogs showed a median recovery of the administered radioac- tivity of 90% (range 70-101 X).
I- m a
m f - (L
Disposition of Astemizole in Dogs 57
4 4 I 4 I 4 0 2L 68 7 2 96 120 1LL 168
0.1
2 Hours
treatment period (lmg/kg per day1 dose L 2 m
I , I 5 10 15 20 25 30 35 LO
OIJ do?1 0 3 Days
Fig. 2. Semilogarithmic plot of plasma concentrations (medians) of unchanged AST after a single (0-0) or after a superposed oral dose of 3H-AST (@-a) in four Beagle dogs pretreated orally with the unlabeled drug for 41 days at 1 mg/kg/day.
Fig. 3. Mean (& SEM) minimal plasma levels of unchanged AST (@-a) and of the fraction “AST plus hydroxylated metabolites” (0-0) in Beagle dogs during subchronic treatment at a daily oral dose of 1 mg/kg for 6 weeks.
TA
BL
E l
a. T
issu
e C
once
ntra
tions
of R
adio
activ
ity (
NV
R),
'H-A
ST,
and
'H-D
ES-
AST
in
Bea
gle
Dog
s 24
and
48 h
r af
ter
a Su
perp
osed
Tra
cer
Dos
e (4
2nd
Dos
e), a
nd C
orre
spon
ding
Res
idua
l Lev
els o
f Tot
al A
ST a
nd D
ES-
AST
, Res
ultin
g Fr
om D
aily
Ora
l Ast
emiz
ole
Adm
inis
trat
ion
at 1
mgi
kg
for
6 w
eeks
t
Plas
ma
Bra
in
Saliv
ary
glan
d Li
ver
Lung
K
idne
y A
dren
al
Hea
rt Pa
ncre
as
Thym
us
Sple
en
Lym
ph n
ode
(mes
ent.)
St
omac
h (p
ylor
ic)
Test
is
Mus
cle
Skin
Fa
t per
irena
l
Dog
no.
478
5 (4
8 hr
) -
-
Dog
no.
492
5 (2
4 hr
) N
VR
'H
-AST
'H
-DES
-AST
A
ST
DES
-AST
N
VR
'H
-AST
'H
-DES
-AST
A
ST
DES
-AST
_
__
__
__
_~
~~
_~
0.
0056
*
* 0.
0020
* 0.
0028
*
* 0.
0047
* 0.
042
0.02
8 0.
015
0.22
5 0.
295
0.03
9 0.
024
0.02
0 0.
140
**
0.75
7 0.
205
0.35
9 0.
630
1.22
1.
89
0.40
0 0.
268
I .96
2.
07
8.27
0.
465
2.78
0.
535
3.11
13
.38
1.24
4.
26
1.97
8.
79
1.34
0.
375
0.55
8 0.
665
0.78
5 1.
53
0.86
0 0.
745
1.62
1.
99
1.85
0.
I93
0.51
8 0.
215
1.54
1.
30
0.32
4 0.
484
0.57
5 1.
21
1.12
0.
435
0.48
7 1.
71
I .60
1.
80
1.35
0.
438
4.19
2.
15
0.43
2 0.
080
0.16
6 0.
165
0.33
0 0.
936
0.21
5 0.
235
0.53
5 0.
850
2.22
0.
370
0.49
3 0.
820
2.80
3.
54
1.11
1.
35
4.16
18
.8
0.36
7 0.
239
0,08
3 0.
245
0.32
5 0.
300
0.10
3 0.
111
0.18
5 0.
250
1.15
0.
415
0.49
5 0.
545
0.66
0 1.
46
0.60
0 0.
518
1.12
1.
37
0.72
9 0.
226
0.18
0 0.
725
0.74
5 0.
930
0.43
1 0.
261
I .35
1.
70
0.30
7 0.
071
0. I1
7 0.
090
0.19
5 0.
246
0.09
9 0.
068
0.23
5 0.
520
0. I7
9 0.
082
0.07
8 0.
745
0.95
5 0.
356
0.22
6 0.
120
1.51
I .
29
0.09
9 0.
039
0.05
0 0.
108
0.04
5 0.
066
**
**
0.11
8 0.
008
**
0.1
10
0.67
2 0.
056
0.08
5
0.05
5 **
0.
206
0.04
4 0.
021
0.20
0 **
**
**
0.99
7 0.
010
0.02
2 0.
075
**
**
-
'Tis
sue
conc
entra
tion:
pg-
Eqig
of
wet
tiss
ue.
*Cou
ld n
ot b
e di
ffer
entia
ted;
**,
dete
cted
.
TA
BL
E lb
. Tis
sue
Con
cent
ratio
ns of
Rad
ioac
tivity
(NV
R),
3H-A
ST, a
nd 3
H-D
ES-
AST
in B
eagl
e D
ogs
96 a
nd 1
68 h
r af
ter
a Su
perp
osed
Tra
cer
Dos
e (4
2nd
Dos
e), a
nd C
orre
spon
ding
Res
idua
l Lev
els o
f Tot
al A
ST a
nd D
ES-
AST
, Res
ultin
g Fr
om D
aily
Ora
l Ast
emiz
ole
Adm
inis
trat
ion
at l
mg/
kg
for
6 w
eeks
t
Dog
No.
479
0 (9
6 hr
) D
og N
o. 4
805
(168
hr)
N
VR
'H
-AST
'H
-DES
-AST
A
ST
DES
-AST
N
VR
3H
-AST
'H
-DES
-AS7
A
ST
DES
-AST
Plas
ma
Bra
in
Saliv
ary
glan
d Li
ver
Lung
K
idne
y A
dren
al
Hea
rt Pa
ncre
as
Thym
us
Sple
en
Lym
ph n
ode
(mes
ent.)
St
omac
h (p
ylor
ic)
Test
is
Mus
cle
Skin
**
0.02
6 0.
199
7.13
0.
515
0.78
7 0.
812
0.50
7 2.
45
0.31
6 0.
562
0.89
5 0.
247
0.22
6 0.
068
0.05
1
* **
0.16
2 0.
051
0.08
1 0.
136
0.40
0 0.
119
0.20
3 0.
070
0.03
0 0.
100
0.08
5 **
**
**
* 0.
027
0.09
7 0.
341
0.36
1 0.
223
0.40
0 0.
186
1.72
0.
165
0.32
8 0.
280
0.07
5 0.
119
**
*h
0.00
1 1*
**
**
0.39
8 0.
545
0.08
5 1.
28
0.10
2 0.
615
0.60
0 1.
50
0.99
7 2.
20
0.13
4 0.
318
0.36
1 5.
54
0.07
8 0.
342
0.03
7 0.
361
0.11
0 0.
520
**
0.07
5 **
0.
725
**
**
**
**
**
0.03
2 2.
63
7.30
0.
884
0.71
5 1.
56
0.42
3 2.
08
0.42
4 0.
618
0.60
3 0.
190
0.93
7 0.
075
0.03
8
* **
1.00
0.
100
0.33
9 0.
167
1.38
0.
150
0.18
8 0.
070
0.05
0 0.
180
0.03
5 0.
600
0.01
9 0.
012
**
0.36
0 2.
07
0.28
4 0.
145
0.13
6 0.
147
0.69
5 0.
162
0.24
0 0.
304
0.06
9 0.
289
0.02
0 0.
011
0.00
08*
1.25
0.
431
0.11
3 2.
85
0.37
0 0.
446
0.50
5 0.
800
1.97
0.
455
0.15
4 0.
300
0.38
3 3.
64
0.11
7 0.
238
0.08
8 0.
328
0.21
3 0.
500
0.02
9 0.
271
1.29
1.
17
**
**
**
**
**
0.06
0 Fa
t oer
irena
l **
**
**
**
0.
249
**
**
**
**
0.07
4
+Tis
sue c
once
ntra
tion:
pg-
Eq/g
of
wet
tiss
ue.
*, C
ould
not
be
diff
eren
tiate
d; **
, det
ecte
d.
60 Michielsetal.
At peak concentrations of the unchanged drug, emanating exclusively from the last 3H- AST dose, represented from 20 to 70% of the tissue radioactivity in most tissues (Table 1). This proportion, however, was clearly lower in the kidney, skin, fat, and in the liver (1-10%). 3H-AST was eliminated gradually from all tissues: concentrations of 3H-AST decreased to about 10% or less of the corresponding peak level within 168 hr, which allows us to estimate a half-life on the order of 60 hr for the elimination from tissues. Highest tissue concentrations of total AST (3H-labeled and unlabeled) were observed at 48 hr in the liver (2 pg/g), glandular tissues ( 2 4 . 8 pg/g), testis (1.5 pg/g), and some lymphatic tissues (1.1 pg/g). Most other tissues, including the brain, showed peak levels between 0.1 and 0.4 pg /g . AST levels in the adipose tissue did not exceed 0.05 pglg (Table 1). One-third to one-half of the total tissue concentration of the parent drug could be attributed to the last radioactive dose (Table 1). A similar elimination rate as found for 3H-AST could be estimated from total AST levels.
Concentrations of the metabolite 3H-DES-AST, originating from the radioactive dose only, were similar to those of 3H-AST in most tissues. The metabolite was poorly distributed to muscle, fat, skin, and brain but concentrations in liver, kidney, and some glandular tissues were three to seven times higher than those of the parent drug (Table 1). In the liver, up to 12 % of the superposed dose could be recovered as unconjugated 3H-DES-AST.
Except for pancreas (18.8 pg /g ) and liver (8.8 pglg), total residual levels of DES-AST ("-labeled and unlabeled) were generally about twice the corresponding levels of the parent drug. For most tissues, 25-50% of the total residual DES-AST concentration originated from the superposed radioactive dose. In brain and testis this proportion was less than 10% (Table 1).
DISCUSSION
Orally administered AST was well and rapidly absorbed in the dog; its absolute bioa- vailability could be estimated at 11% [Van Peer, unpublished results], indicating that an extensive first-pass metabolism strongly reduces its systemic availability.
Estimation of terminal half-lives of radioactivity and of AST and DES-AST was difficult owing to the interindividual variations, differences in the surviving intervals, and the very low plasma levels that were near the detection limit of the assay methods. From semilogarithmic plots of the excretion rates of the radioactivity in urine and feces against the midpoint of the collection intervals [Meuldermans et al., 19861, and from plots of the amounts of radioactivity remaining to be excreted versus time (sigma-minus plot) [Gibaldi and Perrier, 19751, a terminal half-life in a range of 66-109 hr could be calculated in this group of dogs. A similar value could be estimated from Figure 3 for unchanged drug and for the fraction of AST plus hydroxylated metabolites: upon subchronic administration of AST at 1 mg/kg/day, steady-state was reached within 2 to 3 weeks, indicative of a half-life of 3 to 5 days provided that the kinetics of AST remain linear on repeated dosing. Evidence for this assumption had to be obtained from the absorption and excretion data, since plasma levels of AST after the last tracer dose were too low to allow differentiation between steady-state levels and those from the superposed dose. As shown in Figure 1, the absorption of the radioactive dose at the end of the subchronic treatment was similar to that of a single dose, and further individual excretion and metabolism data showed similar patterns for the single and superposed dose both quanti- tatively and qualitatively [Meuldermans et al., 19861.
According to Colburn and Matthews [ 19791, alterations of pharmacokinetics upon chronic treatment can be detected on the assumption that each compartment is uniform and well stirred so that distribution and elimination processes apply equally well to the labeled tracer dose and to the preceding unlabeled doses. By differentiated measurement of AST levels specifically from the superposed dose, it was possible to measure displacement and stirring in the tissue compartment. Because of the extremely low plasma levels of AST, the latter technique could not be applied to plasma. As could be expected from the large volume of distribution (V, = 135 liter/kg) [Van Peer, unpublished results] AST and its metabolites were
Disposition of Astemizole in Dogs 61
extensively distributed to tissues, mainly to well perfused ones such as the liver, kidney, lung, and some glandular and lymphoid rich tissues. In spite of its high lipid solubility (log P = 5.60), AST was pborly distributed to fat and brain. Tissue levels, nevertheless, were always many times higher than corresponding plasma concentrations (Table 1).
Tissue levels of 3H-AST emanating exclusively from the last radioactive dose repre- sented on the average 3 0 4 % of the tissue radioactivity levels. In the kidney (10%) and liver (1-5 %) only, a clearly lower proportion of the unmetabolized drug occurred. On the other hand, one-third to one-half of the total residual levels of unmetabolized AST in tissues could be attributed to the last dose, demonstrating prompt distribution to tissues of the superposed dose and instant equilibrium with the residual drug concentrations at the level of tissue sites. Concentrations of 3H-DES-AST were in general similar to those of 3H-AST in most tissues but clearly higher in liver, kidney, and glandular tissues. In the liver, up to 12% of the superposed tracer dose was recovered as unconjugated 3H-DES-AST. However, total fecal excretion of 3H-DES-AST over 7 days hardly represented 2-5% of the radioactive dose against, e.g., 15-20% for the metabolite 6-OH-DES-AST [Meuldermans et al., 19861. Hence, it can be assumed that 0-demethylation of AST to DES-AST is just a primary rapid, though major step in the metabolic degradation of AST.
Since the parent drug and its primary metabolite DES-AST have equal pharmacological potency [Niemegeers and Awouters, unpublished results], the sum of the residual concentra- tions of either compound may be considered as a measure for drug-equivalent activity in the body. Taking the fraction of “AST plus hydroxylated metabolites” as a measure for total drug- equivalent activity in plasma, fairly equal tissue-to-plasma level ratios in a range of 1 to 100- 500 could be observed throughout the postdosing period in most tissues (Fig. 4). Highest tissue levels, as in the lung, kidney, and some glandular tissues were up to 1,500-fold the correspond- ing plasma concentration. A markedly higher ratio even was observed in the liver, adrenal, and pancreas. On the contrary, total levels of AST and DES-AST in the brain and fat were less than 50 times higher than those in plasma, which demonstrates that, in spite of their high lipophilicity, AST and its metabolite did not accumulate in brain and fatty tissue. From the persisting parallelism between plasma and tissue levels, it is obvious that for AST, DES-AST,
brain
perirenal fat
muscle
stomach
thymus
heart
spleen
lymph ncde
lung
kidney
salivary gland
testis
adrenal
liver
pancreas
10 100 1,000
rati tissuetplasma - concentration
1CJooo
Fig. 4. AST for 6 weeks. Mean tissue to plasma level ratio 1, 2, 4, and 7 days after the last dose.
Tissue distribution of AST and DESM in dogs after daily oral administration of 1 mg/kg of
62 Michiels et al.
and reasonably also for other hydroxylated metabolites, binding to and redistribution from tissues is rate-limiting for the elimination from the body. It is apparent from the observed data (Fig. I ) that the last dose is distributed and eliminated not differently from the first dose, demonstrating that the technique of superpositioning, as proposed by Hammer and Bozler [ 19771, can be applied successfully not only to compounds that are distributed rapidly through- out the organism [Colburn and Matthews, 19791 but also to compounds such as AST, which are slowly redistributed and eliminated from the body.
ACKNOWLEDGMENTS
The authors wish to thank Mr. R. Hendriks, I. Geuens, and L. Embrechts for their skillful technical assistance and Mrs. L. Geentjens and Mr. J. Van Mierlo for their help with the preparation of the manuscript.
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