Europ. J.clin. Pharmacol. 9, 319-325 (1976) © by Springer-Verlag 1976
Interindividual Differences in Chlorthalidone Concentration in Plasma and Red Cells of Man after Single and Multiple Doses* P. Collste,** M. Garle, M. D. Rawlins and F. Sj6qvist Dept. of C l i n i c a l Sweden
Received: accepted:
Pharmacology,
F e b r u a r y 11, 1975, July 19, 1975
Karolinska
and in r e v i s e d
Institutet,
form:
June
Huddinge
26,
Hospital,
Huddinge,
1975,
summary. A gas c h r o m a t o g r a p h i c m e t h o d has been e m p l o y e d to d e t e r m i n e c h l o r t h a l i d o n e in plasma and whole blood after t h e r a p e u t i c doses. R a d i o a c t i v e l y l a b e l l e d chlort h a l i d o n e was used for in v i t r o studies of the uptake of c h l o r t h a l i d o n e from p l a s m a by red blood cells. C h l o r t h a l i d o n e was m a r k e d l y c o n c e n t r a t e d in red cells and as a c o m p a r t m e n t they w o u l d a c c o u n t for at least 30% of total drug in the body after multiple doses. The ratio b e t w e e n the p l a s m a and red cell c o n c e n t r a t i o n of chlort h a l i d o n e v a r i e d b e t w e e n individuals. After a single oral dose of 50 mg in 6 h e a l t h y v o l u n t e e r s c h l o r t h a l i d o n e was e l i m i n a t e d w i t h a h a l f - l i f e of 51 to 89 hours. The a p p a r e n t v o l u m e of d i s t r i b u t i o n v a r i e d b e t w e e n 3 and 13 i/kg and the c l e a r a n c e between 53 and 145 ml/min. The m e a n s t e a d y - s t a t e plasma c o n c e n t r a t i o n s during t r e a t m e n t with a standard dose of 50 mg daily (n = 10) v a r i e d 5-fold b e t w e e n individuals. During the steady state a p p r o x i m a t e l y 50% of the d a i l y dose was e x c r e t e d u n c h a n g e d in the urine during 24 hrs. The p l a s m a levels o b s e r v e d in p a t i e n t s w e r e higher than those p r e d i c t e d from the single oral dose studies in h e a l t h y volunteers. Key words: C h l o r t h a l i d o n e , diuretics, drug p l a s m a red b l o o d - c e l l concentration, p h a r m a c o k i n e t i c s .
Chlorthalidone (Hygroton ®, Geigy), a d i u r e t i c with p h a r m a c o l o g i c a l actions similar to the thiazide diuretics, is w i d e l y used in the t r e a t m e n t of oedema and h y p e r t e n s i o n (Thomson, 1970). Few p h a r m a c o k i n e t i c studies have been performed with this drug due to the lack of a n a l y t i c a l m e t h o d s s u f f i c i e n t l y s e n s i t i v e for m e a s u r e m e n t of p l a s m a levels. T w e e d a l e and O g i l v i e (1972) studied the u r i n a r y e x c r e t i o n of chlort h a l i d o n e after oral a d m i n i s t r a t i o n of 200 mg to five h e a l t h y volunteers.
*A preliminary report of this paper was presented at the Ann. Meet. Sw. Med. Soc. Nov. 1973 **Supported by a Geigy scholarship
concentration,
protein
binding,
U r i n a r y c o n c e n t r a t i o n s were m e a s u r e d by s p e c t r o p h o t o m e t r y up to 120 hours after dosing. The h a l f - l i f e was found to be about 60 hours. W i t h a m o d i f i e d m e t h o d (Tweedale and Ogilvie, 1974) it was found that the c o n c e n t r a t i o n of chlort h a l i d o n e in red blood cells was 7-10 times g r e a t e r than that in plasma. The m e t h o d is a d e q u a t e for w h o l e blood analysis but after t h e r a p e u t i c doses of chlorthalidone plasma concentrations could not be measured. To gain further kinetic i n f o r m a t i o n about the drug a gas c h r o m a t o g r a p h i c m e t h o d has been emp l o y e d for analysis of c o n c e n t r a t i o n s in plasma and w h o l e blood after single and m u l t i p l e oral doses in h e a l t h y volunteers and patients. The in vitro distribution of 1 4 C - c h l o r t h a l i d o n e b e t w e e n plasma water, p l a s m a p r o t e i n s and red blood cells has also been studied.
320 MATERIAL
AND M E T H O D S
In vivo Studies
Plasma c o n c e n t r a t i o n s of c h l o r t h a l i d o n e were followed after a d m i n i s t r a t i o n of a single 50 mg tablet (Hygroton®, Geigy) to 6 h e a l t h y v o l u n t e e r s aged 25-31 years. The dose was taken with 1OO ml of tap w a t e r after an o v e r n i g h t fast. Venous blood was c o l l e c t e d in 10 ml h e p a r i n i s e d tubes (Vacutainer) just before the dose and after 2, 4, 8, 24, 36, 48, 72, 96, 120 and 144 hours. A f t e r c e n t r i f u g a t i o n the plasma was stored at -20oc before analysis. Ten patients being t r e a t e d w i t h c h l o r t h a l i d o n e tablets (Hygroton ®, Geigy) in doses of 50 mg daily were studied by sampling venous blood just before and 4, 8, 12, 16 and 24 hours after the m o r n i n g dose. The plasma c o n c e n t r a t i o n s of c h l o r t h a l i d o n e were measured. For 7 of these patients a 24 hour urine s p e c i m e n was c o l l e c t e d and its content of c h l o r t h a l i d o n e measured. All the patients had been treated w i t h c h l o r t h a l i d o n e for several months before this study, a l t h o u g h intake of the drug was s u p e r v i s e d for only five days beforehand. At the same time they also received p o t a s s i u m supplements (9 patients), t h e o p h y l l i n e (I patient), thyroid h o r m o n e (2 patients) and insulin (I patient). Their serum c r e a t i n i n e c o n c e n t r a t i o n s were w i t h i n normal limits (0.7 - 1.4 mg %). A further series of 3 patients treated with c h l o r t h a l i d o n e 1OO mg daily and other drugs were s t u d i e d similarly t h r o u g h o u t one d o s a g e interval, c h l o r t h a l i d o n e c o n c e n t r a t i o n s being d e t e r m i n e d both in p l a s m a and w h o l e blood.
r a d i o a c t i v i t y d e t e r m i n e d by liquid s c i n t i l l a t i o n s p e c t r o m e t r y (see below). P l a s m a p r o t e i n b i n d i n g was studied in d u p l i c a t e samples of fresh p l a s m a from 4 h e a l t h y v o l u n t e e r s by equilibration against a buffer c o n c e n t r a t i o n of c h l o r t h a l i d o n e of 0.5 ~g/ml. Plasma from one subject (D) was d i a l y s e d a g a i n s t buffer c o n c e n t r a t i o n s of 0.2, 0.5, 1.0, 2.1, 5.2, and 9.8 ~g/ml 14C-chlorthalidone, as d e s c r i b e d above. The rate of d i s t r i b u t i o n of chlort h a l i d o n e b e t w e e n p l a s m a and red blood cells was studied by adding 14C-chlorthalidone (dissolved in normal saline and 0 . 1 M sodium hydroxide, 20:1 v/v) to freshly drawn, h e p a r i n i z e d whole blood from 4 h e a l t h y v o l u n t e e r s (age 23 - 30 years) not taking any drugs. 1 4 C - C h l o r t h a l i d o n e was added to 25 ml whole blood at a c o n c e n t r a t i o n of 5 ~g/ml. The m i x t u r e was incubated in a shaking water bath at 37oc, and at various times blood was sampled and the r a d i o a c t i v i t y in whole blood and p l a s m a was m e a s u r e d (see below). In an attempt to define the m a x i m u m amount of chlorthalidone that would become d i s t r i b u t e d in red blood cells, 1 4 C - c h l o r t h a l i d o n e in 200 ~i normal saline and 0 . 1 M sodium h y d r o x i d e (20:1 v/v) was added to d u p l i c a t e 5 ml whole blood samples from 3 h e a l t h y subjects (MO, MB, PC) and one aliquot of blood bank blood (BB). The c o n c e n t r a t i o n s of drug were 5, 10, 15, 20, 25, 30 and 40 ~g/ml whole blood. A f t e r i n c u b a t i o n and m i x i n g for 75 m i n u t e s at 37oc, aliquots of whole blood were c e n t r i f u g e d and the radioa c t i v i t y of d u p l i c a t e 100 ~i samples of plasma and whole blood was m e a s u r e d (see below).
In Vitro Studies
ANALYTICAL
METHODS
P r o t e i n b i n d i n g of c h l o r t h a l i d o n e was d e t e r m i n e d by e q u i l i b r i u m dialysis (Ehrnebo et al., 1971) at 37oc of 500 ~i p l a s m a a g a i n s t 500 ~I 0 . O 1 M sodium p h o s p h a t e buffer (pH 7.4). The buffer c o n t a i n e d 1 4 C - c h l o r t h a l i d o n e labelled in p o s i t i o n s I and 3 of the iso-indoline ring s t r u c t u r e (specific a c t i v i t y 21.O ~Ci/mg), kindly p r o v i d e d by C i b a - G e i g y (Basel). Its purity was c o n t r o l l e d by t h i n - l a y e r - c h r o m a t o g r a p h y with a solvent system of xylene, toluene, isopropanol and ammonia 25% (10:10:30:20) on silica gel. In pilot studies 14C-chlorthalidone e q u i l i b r a t e d b e t w e e n p l a s m a and buffer w i t h i n 6 hours at 37oc, so these c o n d i t i o n s w e r e used t h r o u g h o u t the study. At the end of the 6 hour period of dialysis 100 ~i of both buffer and plasma were w i t h d r a w n and the
Plasma and urine c o n c e n t r a t i o n s of nonr a d i o a c t i v e c h l o r t h a l i d o n e were m e a s u r ed by gas liquid c h r o m a t o g r a p h y (Ervik and Gustavii, 1974). The m e t h o d is based on c o n v e r s i o n of c h l o r t h a l i d o n e to its t e t r a m e t h y l d e r i v a t i v e by extractive alkylation. Whole blood c o n c e n t r a t i o n s were a n a l y s e d s i m i l a r l y after d i l u t i o n of the sample w i t h 0.2 M citric acid (1:9 v/v) since p r e l i m i n a r y o b s e r v a t i o n s had shown 1OO% r e c o v e r y of c h l o r t h a l i done after this hemolysis procedure. The m e t h o d had a s e n s i t i v i t y of 5 ng chlorthalidone/ml. R a d i o a c t i v i t y in plasma from the in vitro e x p e r i m e n t s was d e t e r m i n e d in d u p l i c a t e aliquots of 1OO ~I w h i c h were added to 10 ml Instagel (Packard Instrument Company). R a d i o a c t i v i t y in whole
321
b l o o d was m e a s u r e d in d u p l i c a t e by i n c u b a t i n g 1OO ~l a l i q u o t s in 1.5 ml of a m i x t u r e of S o l u e n e 1OO (Packard I n s t r u m e n t Company), i s o p r o p a n o l (1:1 v/v) and 0.5 ml 30% h y d r o g e n p e r o x i d e in a d i s p o s a b l e glass c o u n t i n g vial for 60 m i n u t e s at room temperature. Then 15 ml of 0.5 M h y d r o c h l o r i c acid and I n s t a g e l (1:9 v/v) w e r e added, and after m i x i n g the r a d i o a c t i v i t y was m e a s u r e d by liquid s c i n t i l l a t i o n counting. All r a d i o a c t i v i t y m e a s u r e m e n t s w e r e c o r r e c t e d for q u e n c h i n g by cons t r u c t i o n of a q u e n c h c o r r e c t i o n curve using the e x t e r n a l s t a n d a r d i s a t i o n method. In some of the in ~ t r o e x p e r i m e n t s the d e g r e e of h e m o l y s i s p r o d u c e d d u r i n g the i n c u b a t i o n of w h o l e blood was d e t e r m i n e d by u t i l i z i n g the p e r o x idase a c t i v i t y of the h a p t o g l o b i n h e m o g l o b i n c o m p l e x in p l a s m a (Collste et al., u n d e r p r e p a r a t i o n ) . S p e c i m e n s s h o w i n g m o r e than 0.2% h e m o l y s i s w e r e discarded.
CALCULATIONS P h a r m a c o k i n e t i c c o n s t a n t s were d e t e r m i n e d by a s p e c i a l l y c o n s t r u c t e d comp u t e r p r o g r a m (IBM 1130, cf. A l e x a n d e r son, 1972). The p r o g r a m c a l c u l a t e d the B-slope h a l f - l i f e (t I/2B) and e l i m i n a tion rate c o n s t a n t (8) by least square r e g r e s s i o n a n a l y s i s of the t e r m i n a l m o n o e x p o n e n t i a l p o r t i o n of the p l a s m a c o n c e n t r a t i o n - t i m e curve. At least five time p o i n t s w e r e a v a i l a b l e for the c o m p u t a t i o n . The area u n d e r the p l a s m a c o n c e n t r a t i o n - t i m e curve ( A U C ) was c a l c u l a t e d by the t r a p e z o i d a l rule and e x t r a p o l a t e d to infinity. The b i o a v a i l a b i l i t y (F) of the drug was a s s u m e d to be 50% (see'Discussion') and the p l a s m a c l e a r a n c e (~Tp) was d e t e r m i n e d as: p
= Dose x F AUC
= Dose x 0.5 AUC
Red b l o o d cell c h l o r t h a ! i d o n e c o n c e n t r a t i o n (Crbc) was c a l c u l a t e d as:
Crbc = Cwb-Cpl. (1-Hcr) Hcr
w h e r e Cwb and Cpl were the w h o l e b l o o d and p l a s m a c o n c e n t r a t i o n s , r e s p e c t i v e l y , and Hcr was the h e m a t o c r i t .
RESULTS Healthy Volunteers and Patients
The p l a s m a c o n c e n t r a t i o n s of chlort h a l i d o n e in 3 of the 6 v o l u n t e e r s g i v e n a 50 mg t a b l e t of H y g r o t o n o r a l l y are shown in Fig. I. P l a s m a c o n c e n t r a tions rose r a p i d l y to r e a c h peak levels of 34 to 201 ng/ml (mean 1OO ng/ml) 2 to 4 hours after dosing. T h e r e was an initial rapid and a s u b s e q u e n t slow d e c l i n e of the p l a s m a level. F r o m 10 to 12 hours after drug a d m i n i s t r a t i o n the d i s a p p e a r a n c e of c h l o r t h a l i d o n e from p l a s m a a p p e a r e d to be m o n o - e x p o n e n t i a l . The p h a r m a c o k i n e t i c constants d e r i v e d from these studies are shown in Table I. The e l i m i n a t i o n halflives (t I/2 B) v a r i e d from 51 to 89 hours (mean 64.8 hours) and the a p p a r e n t d i s t r i b u t i o n v o l u m e (VdB) from 3 to 13 i/kg (mean 7.6 i/kg). Thus, the i n t e r i n d i v i d u a l v a r i a t i o n in e l i m i n a tion rate was 1.8-fold, w h i l s t the d i s t r i b u t i o n v o l u m e v a r i e d by a factor of 4.3. C l e a r a n c e v a l u e s (~) a v e r a g e d
Plasma concentration 3 0 0 - - chlorthalidone ng/m[ 200 -100-m
and the a p p a r e n t v o l u m e of d i s t r i b u t i o n
(Vdg) thus:
I
50--
Dose x O. 5 VdB = A U C x B 20--
Mean steady-state plasma concentrations of c h l o r t h a l i d o n e (Css) in the p a t i e n t s on m u l t i p l e dose t h e r a p y w e r e d e t e r mined accordingly: AUC ss C = ss A T w h e r e A U C s s was the area u n d e r the p l a s m a c o n c e n t r a t i o n - t i m e curve (measured by the t r a p e z o i d a l rule) d u r i n g a d o s a g e i n t e r v a l (AT).
10 ~
I
I
I
I
25
50
75
100
I
I
150
I
I
2 O0 hrs
Fig. i. Log plasma concentrations of chlorthalidone against time after 50 mg Hygroton® given as a single tablet to three healthy volunteers
322
93 ml/min, r a n g i n g from 53 to 145 ml/ min, the i n t e r i n d i v i d u a l v a r i a t i o n being 2.7-fold. The plasma c h l o r t h a l i d o n e c o n c e n t r a tion in the 10 patients r e c e i v i n g 50 mg of the drug once daily are shown in Table 2. A wide range in Css (211 ng/ml to 1138 ng/ml) and the m i n i m u m p l a s m a c h l o r t h a l i d o n e level (Cmin, 140 ng/ml to 870 ng/ml) and m a x i m u m level (Cmax, 284 ng/ml to 1372 ng/ml) was observed. A l t h o u g h Cmin was i n v a r i a b l y less than Css in all patients, the c o r r e l a t i o n
Table after 50 mg
b e t w e e n these two p a r a m e t e r s was remarkable (r = 0.996). The f l u c t u a t i o n in p l a s m a c o n c e n t r a t i o n during one dosage interval, e x p r e s s e d as the ratio b e t w e e n m a x i m a l (Cmax) and m i n i m a l c o n c e n t r a t i o n (Cmin), a v e r a g e d 2.07 w i t h a 2.5-fold i n t e r i n d i v i d u a l variation. On a v e r a g e 50% (Table 2) of the daily dose (50 mg) was e x c r e t e d in the urine in 24 hours and this f r a c t i o n v a r i e d 1.6-fold betw e e n the 7 patients studied (40-64%). The p l a s m a (C~) and whole blood (Cwb) c o n c e n t r a t i o n s oz c h l o r t h a l i d o n e in the
i. P h a r m a c o k i n e t i c c o n s t a n t s c a l c u l a t e d a s i n g l e oral dose of c h l o r t h a l i d o n e (as H y g r o t o n ®) in six h e a l t h y v o l u n t e e r s
T a b l e 3. P r o t e i n b i n d i n g of c h l o r t h a l i d o n e in d u p l i c a t e samples i n c u b a t e d f o r 6 hrs at 3 7 ° C Subject
subject
protein binding
conc.
Dose
Vd~
AUC
t i/2 8
Plasma clearance ml/min
mg/kg
i/kg
O + ~ ~g/l'hrs
hrs
GA
0.72
5.2
6805.2
66.6
61.2
A
O.5
75.6
MG
O.71
5.9
5393.3
60,8
77.3
B
O.5
74.9
MO
O.71
6.7
5300.8
68.6
78.6
MR
0.74
11.8
2883.0
61.9
144.5
chlorthalidone pg/ml
%
C
0.5
73.1
D
O.2
75.7
-"-
O. 5
75.7
"
1 .O
76.0
TR
0.59
12.9
2923.6
88.9
142.5
SS
0.67
3.2
7893.3
51.3
52.7
mean
7.6
5199.9
64.8a
92.8
SD
3.9
2071.9
11.2
40.5
-"-
2.1
76.3
51.7
39.8
17.3
43.6
"
5.2
75.7
-"-
9.8
76.0
Coeff.var.%(C.V.)
a calculated from the mean rate constant, 8. (8= O.O107 ± 0.0018 hrs; C.V. = 17.2%)
Mean value
at 0 . 5 p g / m l
Table 2. P l a s m a c o n c e n t r a t i o n s of c h l o r t h a l i d o n e in iO p a t i e n t s c h r o n i c t r e a t m e n t w i t h a dose o f 5 0 m g ( H y g r o t o n ®) daily
± S.D.
74.8 ±
during
a Patient
Age
Dose
years
mg/kg
Cmi n
Cmax
Cmax
C ss
ng/ml
ng/ml
C
ng/ml
.
mln
24 hour u r i n a r y chlorthalidone e x c r e t i o n as % of daily
EB
50
0.67
336
663
1.97
480
56
GD
58
0.53
140
284
2.02
211
42
BF
43
0.63
512
903
1.76
700
40
GL
34
0.67
232
521
2.24
364
64
AL
75
0.73
573
1372
2.39
799
48
BL
52
0.67
220
504
2.29
339
-
JM
62
0.53
229
298
1.30
271
46
LW
21
0.83
320
641
2.00
450
54
DO
72
O.61
870
1340
1.54
i138
-
MT
39
0.74
189
607
3.22
413
-
Mean
362
713
2.07
517
50
SD
227
383
0.53
284
8
a
calculated
as A U C / A T
(dosage
interval)
dose
1.2
323
3 p a t i e n t s r e c e i v i n g 100 mg of the d r u g c h r o n i c a l l y are shown in Figs. 2a-c. The c o n c e n t r a t i o n in p l a s m a m i r r o r e d that in w h o l e b l o o d d u r i n g the d o s a g e interval, the latter b e i n g on a v e r a g e 10.9 (range 8.3-14.6, subj. AA), 9.4 (range 7.7-11.6, subj. SS) and 31.O (range 23.7-35.1, subj. HJ) times higher. The m e a n red b l o o d cell c o n c e n t r a tion d u r i n g the d o s a g e interval, t a k i n g into a c c o u n t the h e m a t o c r i t values, was found to be 23.3 ~g/ml, 31.1 ~g/ml and 37.6 ~g/ml for subjects AA, SS and HJ, r e s p e c t i v e l y , w h i c h was 25,2, 23,8 and 71,3 times h i g h e r than the c o r r e s p o n d i n g m e a n s t e a d y state p l a s m a concentrations. In vitro Studies
The p r o t e i n b i n d i n g of c h l o r t h a l i d o n e in four d i f f e r e n t p l a s m a s is shown in T a b l e 3 at a c o n c e n t r a t i o n in b u f f e r of 0.5 ~g/ml. L i t t l e v a r i a b i l i t y was o b s e r v e d b e t w e e n these i n d i v i d u a l s and b i n d i n g r a n g e d from 73.1% - 75.7%. F u r t h e r m o r e , p r o t e i n b i n d i n g of chlort h a l i d o n e in one s u b j e c t (VR) a p p e a r e d to be c o n s t a n t at c o n c e n t r a t i o n s ranging from 0.2 ~g/ml to 9.8 ~g/ml. F i g u r e 3 shows the m e a n v a l u e s of the a m o u n t of c h l o r t h a l i d o n e in p l a s m a as a p e r c e n t a g e of that in w h o l e blood at v a r i o u s times after a d d i t i o n of drug to v e n o u s b l o o d from 4 h e a l t h y subjects. F r o m an initial value of 75.9 ± 3.9% (mean ± SD) at 2.8 ± 0.39 (mean ± SD) m i n u t e s the f r a c t i o n of drug in p l a s m a fell in an a p p a r e n t m o n o e x p o n e n t i a l f a s h i o n in r e l a t i o n to time, w i t h a m e a n half life of 15.2 ± 0.3 (mean ± SEM) m i n u t e s w h e n c a l c u l a t e d up to 61 minutes. There was no s i g n i f i c a n t c h a n g e b e t w e e n 60.8 ± 0.4 m i n u t e s and subsequently, the a v e r a g e v a l u e for the last 3 time p o i n t s being 5.7 ± 0.3% (mean ± SEM). This i n d i c a t e s that distribution equilibrium between plasma and the red blood cell had b e e n r e a c h e d after 60 m i n u t e s of incubation. In the four b l o o d samples s t u d i e d (Fig. 4) the c o n c e n t r a t i o n of c h l o r t h a l i d o n e in p l a s m a in r e l a t i o n to that in red cells i n c r e a s e d s i g n i f i c a n t l y w h e n the w h o l e b l o o d c o n c e n t r a t i o n exc e e d e d 20 ~g/ml. W h e n the slopes of the r e g r e s s i o n lines for the c o n c e n t r a t i o n i n t e r v a l 5-15 ~g/ml w e r e c o m p a r e d (Dixon and Massey, 1969) w i t h the slopes for the i n t e r v a l 20-40 ~g/ml there was a s i g n i f i c a n t d i f f e r e n c e (p