Europ. J.clin. Pharmacol. 9, 155-159 (1975) © by Springer-Verlag 1975
Pharmacokinetics of the Anticonvulsant Drug Clonazepam Evaluated from Single Oral and Intravenous Doses and by Repeated Oral Administration A. Berlin 1 and H. DahlstrSm 2 I
Karolinska
Received:
Pharmacy,
October
Stockholm,
11,
1974,
and 2Roche Products
accepted:
May
Inc.,
Sk~rholmen,
Sweden
5, 1975
Eight h e a l t h y v o l u n t e e r s were given single i.v. and oral doses of c l o n a z e p a m (2 mg). The d i s p o s i t i o n curves after i.v. a d m i n i s t r a t i o n showed a bie x p o n e n t i a l decline and the data were applied to a t w o - c o m p a r t m e n t open model. The volume of d i s t r i b u t i o n ((Vd) B) ranged between 1.5 and 4.4 i/kg and the plasma half-life (ti/2) between 19 and 60 hours. A b s o r p t i o n after oral a d m i n i s t r a t i o n was fast, with peak plasma c o n c e n t r a t i o n s w i t h i n 4 hours in all subjects. Five of the subjects r e c e i v e d repeated oral doses of c l o n a z e p a m 0.5 mg bid for 15 days. The plasma level d u r i n g steady state (estimated as Cmi n w i t h i n the dose interval) could be p r e d i c t e d from the constants A, B, e and B o b t a i n e d in the single dose study with a c o e f f i c i e n t of v a r i a t i o n of 6%. The plasma h a l f - l i v e s after c e s s a t i o n of the subchronic dosing were of the same m a g n i t u d e as after single doses. Summary.
Key words: A n t i c o n v u l s a n t s , chromatography, man.
benzazepines,
clonazepam,
C l o n a z e p a m (Fig. I) is a member of the b e n z o d i a z e p i n e series of drugs which has been shown to have a n t i c o n v u l s a n t properties (Gastaut, 1970). Its d i s p o s i t i o n has been studied after i.v. and oral a d m i n i s t r a t i o n to man using the 1 4 C labelled compound (Eschenhof, 1973), and after oral a d m i n i s t r a t i o n (Kaplan eta!., 1974). The aim of the p r e s e n t study was to investigate the p h a r m a c o k i n e t i c properties of c l o n a z e p a m in healthy volunteers after d i f f e r e n t routes of administration, and to study the plasma levels of the drug following repeated oral doses.
MATERIALS
O 2 N I ~ c
[
Fig. i. Chemical structure of clonazepam
pharmacokinetics,
gas
AND M E T H O D S
Subjects and Drug Administration
F o u r females and four males, aged 22 to 59 years, p a r t i c i p a t e d in the study as paid volunteers. They had given their informed consent to it. Routine laboratory analyses (Hb, WBC, urinalysis, and p r e g n a n c y tests) were carried out before the experiment. Kidney and liver function were normal a c c o r d i n g to their clinical histories. Single oral doses of c l o n a z e p a m 2 m~ were a d m i n i s t e r e d as tablets (Rivotril @ 4 x 0.5 mg, Roche, Basle, Switzerland). All subjects were given a c o n t i n e n t a l b r e a k f a s t two hours after the oral dose. The single i.v. doses of 2 mg were given as a rapid injection (Rivotril ®, 1 mg/ml. For s u b c h r o n i c a d m i n i s t r a t i o n of clonazepam, Rivotril ® tablets (0.5 mg) were a d m i n i s t e r e d at 8 a.m. and 8 p.m. for 15 days.
156 Blood Samples and Analysis of Clonazepam
In the single i.v. dose e x p e r i m e n t s b l o o d samples were c o l l e c t e d after 10 and 30 min, I, 2, 4, 8, 12, 24, 48, 72 and 96 hours. The same s a m p l i n g times were used after the single oral dose e x c e p t for o m i s s i o n of the 10 m i n sample. In the m u l t i p l e dose e x p e r i m e n t s b l o o d samples were taken p r i o r to the m o r n i n g d o s e on days 10, 11, 12, and 15, and on seven o c c a s i o n s over 80 hours of the p o s t - s t e a d y state period of e l i m i n a t i o n . The o b s e r v e d steady state c o n c e n t r a t i o n s w e r e c a l c u l a t e d as the m e a n of v a l u e s o b t a i n e d on Days 10, 11, 12 and 15 and so they r e f e r to the m i n i m u m c o n c e n t r a t i o n (Cmi n) w i t h i n the dose interval. All b l o o d samples were c e n t r i f u g e d w i t h i n six hours and the p l a s m a deep frozen at -18 ° C p e n d i n g a n a l y s i s of clonazepam. A m o d i f i c a t i o n of a m e t h o d p r e v i o u s l y d e s c r i b e d for d i a z e p a m (Berlin et al., 1972) was u s e d for a n a l y s i s of clonazepam. To p e r m i t d e t e r m i n a t i o n of low p l a s m a levels, 0.5 - I ml p l a s m a was used and the final e x t r a c t was e v a p o r a t ed to about 100 ~i. The sample was subj e c t e d to gas c h r o m a t o g r a p h i c a n a l y s i s using a V a r i a n 1400 Gas C h r o m a t o g r a p h e q u i p p e d w i t h a 63Ni e l e c t r o n c a p t u r e detector, and an OV-17 column (3% on Gas C h r o m Q, 80/1OO mesh) w i t h g r i s e o f u l v i n as i n t e r n a l standard. The ret e n t i o n times for c l o n a z e p a m and g r i s e o f u l v i n w e r e 6.9 and 8.7 min, r e s p e c t i v e ly. A m i n o r p e a k o c c u r r e d in p l a s m a from u n t r e a t e d s u b j e c t s at 5.9 min, but it did not i n t e r f e r e w i t h the q u a n t i t a t i v e d e t e r m i n a t i o n . No peaks o c c u r r e d at the r e t e n t i o n times of c l o n a z e p a m or the i n t e r n a l standard.
Pharmacokinetic Calculations
The single i.v. p l a s m a c o n c e n t r a t i o n data were i n t e r p r e t e d a c c o r d i n g to the t w o - c o m p a r t m e n t open m o d e l f i r s t desc r i b e d by T e o r e l l (1937), and later d i s c u s s e d by, for e x a m p l e Riggs (1963), R i e g e l m a n et al. (1968) and G i b a l d i et al. (1969). The p l a s m a c o n c e n t r a t i o n curve a f t e r a single i.v. dose can be d e s c r i b e d by the e q u a t i o n C = A
- e
-~.t
+ B
e
-B.t
where C is the p l a s m a c o n c e n t r a t i o n at time t; and ~ and B are rate c o n s t a n t s d u r i n g the initial r a p i d and t e r m i n a l slower phase of the curve. A and B are the c o n t r i b u t i o n of the r e s p e c t i v e e x p o n e n t i a l s at t = O.
The c o n s t a n t s A, B, e and B were d e t e r m i n e d u s i n g an i t e r a t i v e least square p r o g r a m on a PDP-8 M i n i d a t o r . The areas 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 curves (AUC) were e s t i m a t e d u s i n g the t r a p e z o i d a l rule, with the i n f i n i t e area c a l c u l a t e d as Ctn/k, w h e r e Ctn represented the last d e t e r m i n a t i o n on the E-phase of the e l i m i n a t i o n curve, and k was the slope of this curve. B i o a v a i l a b i l i t y (F) was c a l c u l a t e d as the ratio of the areas under the p l a s m a c o n c e n t r a t i o n curves after oral and i.v. a d m i n i s t r a t i o n . The v o l u m e of d i s t r i b u t i o n ((V d) ~) was e s t i m a t e d a c c o r d i n g to G i b a l d i et al. (1969) as D Vd = ~.
(AUC)
i.v.
where D was the dose g i v e n intravenously. The rate c o n s t a n t kei, w h i c h r e f l e c t s e l i m i n a t i o n from the c e n t r a l compartment, can be c a l c u l a t e d from A, B, e and B (Rescigno and Segre, 1966). Since B r e f l e c t s o v e r a l l e l i m i n a t i o n from the body and kel e l i m i n a t i o n from the central c o m p a r t m e n t , the ratio B/tel will give i n f o r m a t i o n on the p e r c e n t a g e of drug a v a i l a b l e for e l i m i n a t i o n in the c e n t r a l c o m p a r t m e n t at any time (Gibald i e t al., 1969). P l a s m a c o n c e n t r a t i o n s after m u l t i p l e oral doses were p r e d i c t e d by m o d i f i c a t i o n of the e q u a t i o n s for a o n e - c o m p a r t m e n t (Boxer et al., 1948) and a t h r e e - c o m p a r t m e n t m o d e l (Kaplan et al~, 1973). For a t w o - c o m p a r t m e n t m o d e l the plasma c o n c e n t r a t i o n (C) f o l l o w i n g the n'th dose at time t is given by the e q u a t i o n A t _ A t (e-a °At) n C =
B t - B t (e-~'At) n +
I - e -~.At
I - e -~'At
w h e r e At is the dose i n t e r v a l and A t and B t are the c o n t r i b u t i o n s from the resp e c t i v e e x p o n e n t i a l s at time t a f t e r a single dose.
R E S U L T S AND D I S C U S S I O N Analytical Procedure
The s t a n d a r d d e v i a t i o n of the gas c h r o m a t o g r a p h i c p r o c e d u r e for c l o n a z e p a m was found to be 5, 6, and 8% at p l a s m a c o n c e n t r a t i o n s of I (n = 17), 8 (n = 22), and 20 (n = 25) ng/ml, respectively. The h i g h e r SD at h i g h e r p l a s m a levels m i g h t be due to d e c r e a s e d l i n e a r i t y of the r e s p o n s e of the e l e c t r o n c a p t u r e d e t e c t o r as the a m o u n t of c l o n a z e p a m injected increased. However, when the
I57 Table I. Pharmacokinetic parameters found after administration of clonazepam to eight healthy volunteers Single i.v. dose 2 m g
Repeated oral doses
Single oral dose 2 mg
0.5 mg x 2, At=f2 h
I o
-i
g
A
F
27
49
B
F
22
75
18.0
5.9
C
F
47
57
19.2
8.3
D
F
59
68
28.8
12.8
0.802
E
M
34
80
3,0
5.3
0.238
F
M
58
74
13.1
14.3
O~765
G
M
52
7O
9.7
6.8
H
M
33
70
-
.~
~
o
0 .
0.0290
24
589
2.39
0.O371 b
19
675
1.15
22.1
4
11.5
0.387
0.0158
44
412
4.1C
0.O196
35
231
0.56
7,1
4
4.5
t,070
0.0175
4o
492
4,0~
0.0180
39
560
1.14
19.0
4
0.0210
33
630
2.22
0.0196
35
530
0.84
23.6
1
0.0147
47
383
4.44
O.0186
41
611
1.60
13.2
2
O.0254
27
593
1.79
0.0240
29
500
0.84
17.0
2
0.205
0.0116
60
604
4.08
O.O166
42
498
O.82
13.2
1
-
0.0200
35
963
1.48i
0.0212 c
33
841
0.87
19,8
2
12.O
35
4.6
33
7.1
7.1
42
8.4
8.6
31
11.6
32
13.1
a
F = bioavailability b c
B = 25.8 ng/ml B = 17.5 ng/ml
ng/rnl 40, 3O 2O
10
2 I I I 036912
F i g . 2. I n t r a v e n o u s of clonazepam 2 mg
(,)
and
__,...
oral
24 (A) p l a s m a
t 36
....
1
concentrations
plasma c o n c e n t r a t i o n e x c e e d e d 20 ng/ml the final e x t r a c t was d i l u t e d to permit analysis w i t h i n the linear range of the detector. The s e n s i t i v i t y of the m e t h o d was 0.5 ng/ml under the conditions described.
I 72
_
48 curves
t __ 96 h
in subject D a f t e r
A. Intravenous Administration.
single doses
In all
sub-
jects the d i s p o s i t i o n
Single Doses
curves showed a hie x p o n e n t i a l decline after i.v. administration, w i t h the initial d i s t r i b u t i o n phase lasting b e t w e e n 8 and 10 hours. The data were a n a l y s e d a c c o r d i n g to a t w o - c o m p a r t m e n t open model. In their study on the p h a r m a c o k i n e t -
All the data about the v o l u n t e e r s and the various p h a r m a c o k i n e t i c p a r a m e t e r s has been p r e s e n t e d in Table I. Fig. 2 shows an example of the d i s p o s i t i o n curves (Subject D, s e m i l o g a r i t h m i c plot).
ics of diazepam, Kaplan et al. ( 1 9 7 3 ) used a three-compartment model for interpretation of the results. This rethe quires very frequent sampling during f i r s t 30 m i n a f t e r a d m i n i s t r a t i o n , which was not done in the present study.
158 In s u b j e c t s A and H, s l i g h t irregul a r i t i e s in the i.v. d i s p o s i t i o n curves p r e v e n t e d a c c u r a t e e s t i m a t i o n of the c o n s t a n t s A, B, and a. In t h e s e subjects data from the single oral dose e x p e r i m e n t s was used for p r e d i c t i o n of s t e a d y state levels. The h i g h e s t m a x i m u m c o n c e n t r a t i o n was found in s u b j e c t D (36.8 n g / m l at 10 min). All s u b j e c t s s h o w e d m a r k e d sedation for a p p r o x i m a t e l y 30 m i n after the i.v. dose. The p l a s m a h a l f - l i v e s (ti/2) e s t i m a t e d from the ~ phase of the elim i n a t i o n curve r a n g e d b e t w e e n 24 and 60 hours, and the total v o l u m e of dist r i b u t i o n ((Vd) ~) r a n g e d b e t w e e n 1.8 and 4.4 I/kg. The m e a n ratio b e t w e e n 8 and kel was found to be 0.43 (cf. Table 2). K a p l a n et al. (1973) s h o w e d that for diazepam, r e d i s t r i b u t i o n from the deep p e r i p h e r a l c o m p a r t m e n t was the rate l i m i t i n g step in the o v e r a l l e l i m i n a t i o n . A l t h o u g h the r e d i s t r i b u t i o n of c l o n a z e p a m will i n f l u e n c e the m a g n i t u d e of 8, it is not the rate l i m i t i n g step in e l i m i n a t i o n , since k 2 1 > k e l in all cases, as s h o w n in T a b l e 2. Clonazepam showed rapid a b s o r p t i o n and peak p l a s m a levels were a c h i e v e d w i t h i n four hours in all B. Oral Administration.
15
subjects. The m a x i m u m p l a s m a levels r a n g e d b e t w e e n 7.1 and 23.6 ng/ml. All s u b j e c t s were a f f e c t e d for a p p r o x i m a t e l y three hours after drug a d m i n i s t r a t i o n , w i t h s l u r r e d speech, d i f f i c u l t y in coo r d i n a t i o n and sleepiness. Subchronic Administration
The i n t e r d a y v a r i a t i o n s in the five s u b j e c t s in this part of the study were n e g l i g i b l e , e x c e p t in s u b j e c t H, who r e c e i v e d i n c o r r e c t m e d i c a t i o n on Day 12. In his case the mean of the concent r a t i o n s d e t e r m i n e d on days 10 and 11 was used instead. Table 2. Individual rate constants for clonazepam distribution Subjects
k12
k21
kel
8/kel
B
0.295
O.107
0.O57
0.28
C
0.752
0.335
0.056
0.31
D
0.562
O.261
0.064
0.33
E
0.095
O.157
0.022
0.66
F
0.379
0.411
0.047
0.54
G
O.125
0.091
0.026
0.45
-
H
/
.o10 /~ c
yt/,~//
i
vU
y.- 1.25+ 0.85×
r = 0,978
_ 5
_
,
0 0
!
5
I
I
10
15
(Cmin)predicted
Fig. 3. Relationship between predicted and observed steady state concentrations steady state
(Cmin) during
159 There was a two-fold variation in steady state concentrations (estimated as C m i n w i t h i n t h e d o s e i n t e r v a l ) b e t ween the five subjects. The concentrations achieved after multiple oral doses w e r e in g o o d a g r e e m e n t w i t h t h o s e p r e d i c t e d f r o m s i n g l e d o s e s , as s h o w n in T a b l e I a n d F i g . 3. T h e m a x i m u m d e v i a t i o n f r o m t h e e x p e c t e d v a l u e w a s 12% ( S u b j e c t s B a n d H) a n d t h e c o e f f i c i e n t of v a r i a t i o n w a s 6% a t t h e m e a n p r e dicted plasma level. The possibility of prediction of p l a s m a l e v e l s w i t h t h i s a c c u r a c y c o u l d b e o f g r e a t v a l u e in clinical practice. The plasma half-lives after cessation of s u b c h r o n i c administration of clonaz e p a m w e r e of t h e s a m e m a g n i t u d e as those found after single doses (except in s u b j e c t A), w h i c h s u g g e s t s t h a t d r u g metabolizing e n z y m e s in t h e l i v e r w e r e not induced after administration of clonazepam. However, this requires confirmation in m o r e p r o l o n g e d studies in p a t i e n t s .
Acknowledgements. The authors express their thanks to Miss Inger Wallin for skilful technical assistance and to the volunteers for taking part in the study.
REFERENCES Berlin, A., Siwers, B., Agurell, S., Hiort, A., Sj~qvist, F., Str6m, S.: Determination of bioavailability of diazepam in various formulations from steady state plasma concentration data. Clin. Pharmacol. Ther. 13, 733-744 (1972) Boxer, G.E., Jelinek, V.C., Tompsett, R., Du Bois, R., Edison. A.: Streptomycin in the blood; chemical determinations after single and repeated intramuscular injections. J. Pharm. exp. Ther. 92, 226-235 (1948)
Eschenhof, E.: Untersuchungen ~ber das Schicksal des Antikonvulsivums Clonazepam im Organismus der Ratte, des Hundes und des Menschen. Arzneimittel-Forsch. 23, 390-400 (1973) Gastaut, H.: Propri~t~s anti-~pileptiques exceptionelles d'une benzodiazepine nouvelle, le Ro 5-4023. Vie m~d. 51, 5175-5788 (1970) Gibaldi, M., Nagashima, R., Levy, G.: Relationship between drug concentration in plasma or serum and amount of drug in the body. J. pharm. Sci. 58, 193-197 (1969) Kaplan, S.A., Jack, M.L., Alexander, K., Weinfeld, R.E.: Pharmacokinetic profile of diazepam in man following single intravenous and oral and chronic oral administrations. J. pharm. Sci. 62, 1789-1796 (1973) Kaplan, S.A., Alexander, K., Jack, M.L., Puglisi, C.V., de Silva, J.A.F., Lee, T.L., Weinfeld, R.E.: Pharmacokinetic profiles of clonazepam in dog and humans and of flunitrazepam in dog. J. pharm. Sei. 63, 527-532 (1974) Rescigno, A., Segre, G.: Drug and tracer kinetics. Waltham, Mass.: Blaisell 1966 Riegelman, S., Loo, J.C.L., Rowland, M.: Shortcomings in pharmacokinetie analysis by conceiving the body to exhibit properties of a single compartment. J. pharm. Sci. 57, 117-123 (1968) Riggs, D.S.: The mathematical approach to physiological problems. Baltimore, Md.: Williams & Wilkins 1967 Teorell, T.: Kinetics of distribution of substances administered to the body. II. The intravascular modes of administration. Arch. intern. Pharmacodyn. 57, 226-240 (1937)
Dr. A. Berlin Astra L~kemedel AFE S - 151 85 S~dert~lje Sweden
Pharmacokinetics of the Anticonvulsant Drug Clonazepam Evaluated from Single Oral and Intravenous Doses and by Repeated Oral Administration A. Berlin 1 and H. DahlstrSm 2 I
Karolinska
Received:
Pharmacy,
October
Stockholm,
11,
1974,
and 2Roche Products
accepted:
May
Inc.,
Sk~rholmen,
Sweden
5, 1975
Eight h e a l t h y v o l u n t e e r s were given single i.v. and oral doses of c l o n a z e p a m (2 mg). The d i s p o s i t i o n curves after i.v. a d m i n i s t r a t i o n showed a bie x p o n e n t i a l decline and the data were applied to a t w o - c o m p a r t m e n t open model. The volume of d i s t r i b u t i o n ((Vd) B) ranged between 1.5 and 4.4 i/kg and the plasma half-life (ti/2) between 19 and 60 hours. A b s o r p t i o n after oral a d m i n i s t r a t i o n was fast, with peak plasma c o n c e n t r a t i o n s w i t h i n 4 hours in all subjects. Five of the subjects r e c e i v e d repeated oral doses of c l o n a z e p a m 0.5 mg bid for 15 days. The plasma level d u r i n g steady state (estimated as Cmi n w i t h i n the dose interval) could be p r e d i c t e d from the constants A, B, e and B o b t a i n e d in the single dose study with a c o e f f i c i e n t of v a r i a t i o n of 6%. The plasma h a l f - l i v e s after c e s s a t i o n of the subchronic dosing were of the same m a g n i t u d e as after single doses. Summary.
Key words: A n t i c o n v u l s a n t s , chromatography, man.
benzazepines,
clonazepam,
C l o n a z e p a m (Fig. I) is a member of the b e n z o d i a z e p i n e series of drugs which has been shown to have a n t i c o n v u l s a n t properties (Gastaut, 1970). Its d i s p o s i t i o n has been studied after i.v. and oral a d m i n i s t r a t i o n to man using the 1 4 C labelled compound (Eschenhof, 1973), and after oral a d m i n i s t r a t i o n (Kaplan eta!., 1974). The aim of the p r e s e n t study was to investigate the p h a r m a c o k i n e t i c properties of c l o n a z e p a m in healthy volunteers after d i f f e r e n t routes of administration, and to study the plasma levels of the drug following repeated oral doses.
MATERIALS
O 2 N I ~ c
[
Fig. i. Chemical structure of clonazepam
pharmacokinetics,
gas
AND M E T H O D S
Subjects and Drug Administration
F o u r females and four males, aged 22 to 59 years, p a r t i c i p a t e d in the study as paid volunteers. They had given their informed consent to it. Routine laboratory analyses (Hb, WBC, urinalysis, and p r e g n a n c y tests) were carried out before the experiment. Kidney and liver function were normal a c c o r d i n g to their clinical histories. Single oral doses of c l o n a z e p a m 2 m~ were a d m i n i s t e r e d as tablets (Rivotril @ 4 x 0.5 mg, Roche, Basle, Switzerland). All subjects were given a c o n t i n e n t a l b r e a k f a s t two hours after the oral dose. The single i.v. doses of 2 mg were given as a rapid injection (Rivotril ®, 1 mg/ml. For s u b c h r o n i c a d m i n i s t r a t i o n of clonazepam, Rivotril ® tablets (0.5 mg) were a d m i n i s t e r e d at 8 a.m. and 8 p.m. for 15 days.
156 Blood Samples and Analysis of Clonazepam
In the single i.v. dose e x p e r i m e n t s b l o o d samples were c o l l e c t e d after 10 and 30 min, I, 2, 4, 8, 12, 24, 48, 72 and 96 hours. The same s a m p l i n g times were used after the single oral dose e x c e p t for o m i s s i o n of the 10 m i n sample. In the m u l t i p l e dose e x p e r i m e n t s b l o o d samples were taken p r i o r to the m o r n i n g d o s e on days 10, 11, 12, and 15, and on seven o c c a s i o n s over 80 hours of the p o s t - s t e a d y state period of e l i m i n a t i o n . The o b s e r v e d steady state c o n c e n t r a t i o n s w e r e c a l c u l a t e d as the m e a n of v a l u e s o b t a i n e d on Days 10, 11, 12 and 15 and so they r e f e r to the m i n i m u m c o n c e n t r a t i o n (Cmi n) w i t h i n the dose interval. All b l o o d samples were c e n t r i f u g e d w i t h i n six hours and the p l a s m a deep frozen at -18 ° C p e n d i n g a n a l y s i s of clonazepam. A m o d i f i c a t i o n of a m e t h o d p r e v i o u s l y d e s c r i b e d for d i a z e p a m (Berlin et al., 1972) was u s e d for a n a l y s i s of clonazepam. To p e r m i t d e t e r m i n a t i o n of low p l a s m a levels, 0.5 - I ml p l a s m a was used and the final e x t r a c t was e v a p o r a t ed to about 100 ~i. The sample was subj e c t e d to gas c h r o m a t o g r a p h i c a n a l y s i s using a V a r i a n 1400 Gas C h r o m a t o g r a p h e q u i p p e d w i t h a 63Ni e l e c t r o n c a p t u r e detector, and an OV-17 column (3% on Gas C h r o m Q, 80/1OO mesh) w i t h g r i s e o f u l v i n as i n t e r n a l standard. The ret e n t i o n times for c l o n a z e p a m and g r i s e o f u l v i n w e r e 6.9 and 8.7 min, r e s p e c t i v e ly. A m i n o r p e a k o c c u r r e d in p l a s m a from u n t r e a t e d s u b j e c t s at 5.9 min, but it did not i n t e r f e r e w i t h the q u a n t i t a t i v e d e t e r m i n a t i o n . No peaks o c c u r r e d at the r e t e n t i o n times of c l o n a z e p a m or the i n t e r n a l standard.
Pharmacokinetic Calculations
The single i.v. p l a s m a c o n c e n t r a t i o n data were i n t e r p r e t e d a c c o r d i n g to the t w o - c o m p a r t m e n t open m o d e l f i r s t desc r i b e d by T e o r e l l (1937), and later d i s c u s s e d by, for e x a m p l e Riggs (1963), R i e g e l m a n et al. (1968) and G i b a l d i et al. (1969). The p l a s m a c o n c e n t r a t i o n curve a f t e r a single i.v. dose can be d e s c r i b e d by the e q u a t i o n C = A
- e
-~.t
+ B
e
-B.t
where C is the p l a s m a c o n c e n t r a t i o n at time t; and ~ and B are rate c o n s t a n t s d u r i n g the initial r a p i d and t e r m i n a l slower phase of the curve. A and B are the c o n t r i b u t i o n of the r e s p e c t i v e e x p o n e n t i a l s at t = O.
The c o n s t a n t s A, B, e and B were d e t e r m i n e d u s i n g an i t e r a t i v e least square p r o g r a m on a PDP-8 M i n i d a t o r . The areas 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 curves (AUC) were e s t i m a t e d u s i n g the t r a p e z o i d a l rule, with the i n f i n i t e area c a l c u l a t e d as Ctn/k, w h e r e Ctn represented the last d e t e r m i n a t i o n on the E-phase of the e l i m i n a t i o n curve, and k was the slope of this curve. B i o a v a i l a b i l i t y (F) was c a l c u l a t e d as the ratio of the areas under the p l a s m a c o n c e n t r a t i o n curves after oral and i.v. a d m i n i s t r a t i o n . The v o l u m e of d i s t r i b u t i o n ((V d) ~) was e s t i m a t e d a c c o r d i n g to G i b a l d i et al. (1969) as D Vd = ~.
(AUC)
i.v.
where D was the dose g i v e n intravenously. The rate c o n s t a n t kei, w h i c h r e f l e c t s e l i m i n a t i o n from the c e n t r a l compartment, can be c a l c u l a t e d from A, B, e and B (Rescigno and Segre, 1966). Since B r e f l e c t s o v e r a l l e l i m i n a t i o n from the body and kel e l i m i n a t i o n from the central c o m p a r t m e n t , the ratio B/tel will give i n f o r m a t i o n on the p e r c e n t a g e of drug a v a i l a b l e for e l i m i n a t i o n in the c e n t r a l c o m p a r t m e n t at any time (Gibald i e t al., 1969). P l a s m a c o n c e n t r a t i o n s after m u l t i p l e oral doses were p r e d i c t e d by m o d i f i c a t i o n of the e q u a t i o n s for a o n e - c o m p a r t m e n t (Boxer et al., 1948) and a t h r e e - c o m p a r t m e n t m o d e l (Kaplan et al~, 1973). For a t w o - c o m p a r t m e n t m o d e l the plasma c o n c e n t r a t i o n (C) f o l l o w i n g the n'th dose at time t is given by the e q u a t i o n A t _ A t (e-a °At) n C =
B t - B t (e-~'At) n +
I - e -~.At
I - e -~'At
w h e r e At is the dose i n t e r v a l and A t and B t are the c o n t r i b u t i o n s from the resp e c t i v e e x p o n e n t i a l s at time t a f t e r a single dose.
R E S U L T S AND D I S C U S S I O N Analytical Procedure
The s t a n d a r d d e v i a t i o n of the gas c h r o m a t o g r a p h i c p r o c e d u r e for c l o n a z e p a m was found to be 5, 6, and 8% at p l a s m a c o n c e n t r a t i o n s of I (n = 17), 8 (n = 22), and 20 (n = 25) ng/ml, respectively. The h i g h e r SD at h i g h e r p l a s m a levels m i g h t be due to d e c r e a s e d l i n e a r i t y of the r e s p o n s e of the e l e c t r o n c a p t u r e d e t e c t o r as the a m o u n t of c l o n a z e p a m injected increased. However, when the
I57 Table I. Pharmacokinetic parameters found after administration of clonazepam to eight healthy volunteers Single i.v. dose 2 m g
Repeated oral doses
Single oral dose 2 mg
0.5 mg x 2, At=f2 h
I o
-i
g
A
F
27
49
B
F
22
75
18.0
5.9
C
F
47
57
19.2
8.3
D
F
59
68
28.8
12.8
0.802
E
M
34
80
3,0
5.3
0.238
F
M
58
74
13.1
14.3
O~765
G
M
52
7O
9.7
6.8
H
M
33
70
-
.~
~
o
0 .
0.0290
24
589
2.39
0.O371 b
19
675
1.15
22.1
4
11.5
0.387
0.0158
44
412
4.1C
0.O196
35
231
0.56
7,1
4
4.5
t,070
0.0175
4o
492
4,0~
0.0180
39
560
1.14
19.0
4
0.0210
33
630
2.22
0.0196
35
530
0.84
23.6
1
0.0147
47
383
4.44
O.0186
41
611
1.60
13.2
2
O.0254
27
593
1.79
0.0240
29
500
0.84
17.0
2
0.205
0.0116
60
604
4.08
O.O166
42
498
O.82
13.2
1
-
0.0200
35
963
1.48i
0.0212 c
33
841
0.87
19,8
2
12.O
35
4.6
33
7.1
7.1
42
8.4
8.6
31
11.6
32
13.1
a
F = bioavailability b c
B = 25.8 ng/ml B = 17.5 ng/ml
ng/rnl 40, 3O 2O
10
2 I I I 036912
F i g . 2. I n t r a v e n o u s of clonazepam 2 mg
(,)
and
__,...
oral
24 (A) p l a s m a
t 36
....
1
concentrations
plasma c o n c e n t r a t i o n e x c e e d e d 20 ng/ml the final e x t r a c t was d i l u t e d to permit analysis w i t h i n the linear range of the detector. The s e n s i t i v i t y of the m e t h o d was 0.5 ng/ml under the conditions described.
I 72
_
48 curves
t __ 96 h
in subject D a f t e r
A. Intravenous Administration.
single doses
In all
sub-
jects the d i s p o s i t i o n
Single Doses
curves showed a hie x p o n e n t i a l decline after i.v. administration, w i t h the initial d i s t r i b u t i o n phase lasting b e t w e e n 8 and 10 hours. The data were a n a l y s e d a c c o r d i n g to a t w o - c o m p a r t m e n t open model. In their study on the p h a r m a c o k i n e t -
All the data about the v o l u n t e e r s and the various p h a r m a c o k i n e t i c p a r a m e t e r s has been p r e s e n t e d in Table I. Fig. 2 shows an example of the d i s p o s i t i o n curves (Subject D, s e m i l o g a r i t h m i c plot).
ics of diazepam, Kaplan et al. ( 1 9 7 3 ) used a three-compartment model for interpretation of the results. This rethe quires very frequent sampling during f i r s t 30 m i n a f t e r a d m i n i s t r a t i o n , which was not done in the present study.
158 In s u b j e c t s A and H, s l i g h t irregul a r i t i e s in the i.v. d i s p o s i t i o n curves p r e v e n t e d a c c u r a t e e s t i m a t i o n of the c o n s t a n t s A, B, and a. In t h e s e subjects data from the single oral dose e x p e r i m e n t s was used for p r e d i c t i o n of s t e a d y state levels. The h i g h e s t m a x i m u m c o n c e n t r a t i o n was found in s u b j e c t D (36.8 n g / m l at 10 min). All s u b j e c t s s h o w e d m a r k e d sedation for a p p r o x i m a t e l y 30 m i n after the i.v. dose. The p l a s m a h a l f - l i v e s (ti/2) e s t i m a t e d from the ~ phase of the elim i n a t i o n curve r a n g e d b e t w e e n 24 and 60 hours, and the total v o l u m e of dist r i b u t i o n ((Vd) ~) r a n g e d b e t w e e n 1.8 and 4.4 I/kg. The m e a n ratio b e t w e e n 8 and kel was found to be 0.43 (cf. Table 2). K a p l a n et al. (1973) s h o w e d that for diazepam, r e d i s t r i b u t i o n from the deep p e r i p h e r a l c o m p a r t m e n t was the rate l i m i t i n g step in the o v e r a l l e l i m i n a t i o n . A l t h o u g h the r e d i s t r i b u t i o n of c l o n a z e p a m will i n f l u e n c e the m a g n i t u d e of 8, it is not the rate l i m i t i n g step in e l i m i n a t i o n , since k 2 1 > k e l in all cases, as s h o w n in T a b l e 2. Clonazepam showed rapid a b s o r p t i o n and peak p l a s m a levels were a c h i e v e d w i t h i n four hours in all B. Oral Administration.
15
subjects. The m a x i m u m p l a s m a levels r a n g e d b e t w e e n 7.1 and 23.6 ng/ml. All s u b j e c t s were a f f e c t e d for a p p r o x i m a t e l y three hours after drug a d m i n i s t r a t i o n , w i t h s l u r r e d speech, d i f f i c u l t y in coo r d i n a t i o n and sleepiness. Subchronic Administration
The i n t e r d a y v a r i a t i o n s in the five s u b j e c t s in this part of the study were n e g l i g i b l e , e x c e p t in s u b j e c t H, who r e c e i v e d i n c o r r e c t m e d i c a t i o n on Day 12. In his case the mean of the concent r a t i o n s d e t e r m i n e d on days 10 and 11 was used instead. Table 2. Individual rate constants for clonazepam distribution Subjects
k12
k21
kel
8/kel
B
0.295
O.107
0.O57
0.28
C
0.752
0.335
0.056
0.31
D
0.562
O.261
0.064
0.33
E
0.095
O.157
0.022
0.66
F
0.379
0.411
0.047
0.54
G
O.125
0.091
0.026
0.45
-
H
/
.o10 /~ c
yt/,~//
i
vU
y.- 1.25+ 0.85×
r = 0,978
_ 5
_
,
0 0
!
5
I
I
10
15
(Cmin)predicted
Fig. 3. Relationship between predicted and observed steady state concentrations steady state
(Cmin) during
159 There was a two-fold variation in steady state concentrations (estimated as C m i n w i t h i n t h e d o s e i n t e r v a l ) b e t ween the five subjects. The concentrations achieved after multiple oral doses w e r e in g o o d a g r e e m e n t w i t h t h o s e p r e d i c t e d f r o m s i n g l e d o s e s , as s h o w n in T a b l e I a n d F i g . 3. T h e m a x i m u m d e v i a t i o n f r o m t h e e x p e c t e d v a l u e w a s 12% ( S u b j e c t s B a n d H) a n d t h e c o e f f i c i e n t of v a r i a t i o n w a s 6% a t t h e m e a n p r e dicted plasma level. The possibility of prediction of p l a s m a l e v e l s w i t h t h i s a c c u r a c y c o u l d b e o f g r e a t v a l u e in clinical practice. The plasma half-lives after cessation of s u b c h r o n i c administration of clonaz e p a m w e r e of t h e s a m e m a g n i t u d e as those found after single doses (except in s u b j e c t A), w h i c h s u g g e s t s t h a t d r u g metabolizing e n z y m e s in t h e l i v e r w e r e not induced after administration of clonazepam. However, this requires confirmation in m o r e p r o l o n g e d studies in p a t i e n t s .
Acknowledgements. The authors express their thanks to Miss Inger Wallin for skilful technical assistance and to the volunteers for taking part in the study.
REFERENCES Berlin, A., Siwers, B., Agurell, S., Hiort, A., Sj~qvist, F., Str6m, S.: Determination of bioavailability of diazepam in various formulations from steady state plasma concentration data. Clin. Pharmacol. Ther. 13, 733-744 (1972) Boxer, G.E., Jelinek, V.C., Tompsett, R., Du Bois, R., Edison. A.: Streptomycin in the blood; chemical determinations after single and repeated intramuscular injections. J. Pharm. exp. Ther. 92, 226-235 (1948)
Eschenhof, E.: Untersuchungen ~ber das Schicksal des Antikonvulsivums Clonazepam im Organismus der Ratte, des Hundes und des Menschen. Arzneimittel-Forsch. 23, 390-400 (1973) Gastaut, H.: Propri~t~s anti-~pileptiques exceptionelles d'une benzodiazepine nouvelle, le Ro 5-4023. Vie m~d. 51, 5175-5788 (1970) Gibaldi, M., Nagashima, R., Levy, G.: Relationship between drug concentration in plasma or serum and amount of drug in the body. J. pharm. Sci. 58, 193-197 (1969) Kaplan, S.A., Jack, M.L., Alexander, K., Weinfeld, R.E.: Pharmacokinetic profile of diazepam in man following single intravenous and oral and chronic oral administrations. J. pharm. Sci. 62, 1789-1796 (1973) Kaplan, S.A., Alexander, K., Jack, M.L., Puglisi, C.V., de Silva, J.A.F., Lee, T.L., Weinfeld, R.E.: Pharmacokinetic profiles of clonazepam in dog and humans and of flunitrazepam in dog. J. pharm. Sei. 63, 527-532 (1974) Rescigno, A., Segre, G.: Drug and tracer kinetics. Waltham, Mass.: Blaisell 1966 Riegelman, S., Loo, J.C.L., Rowland, M.: Shortcomings in pharmacokinetie analysis by conceiving the body to exhibit properties of a single compartment. J. pharm. Sci. 57, 117-123 (1968) Riggs, D.S.: The mathematical approach to physiological problems. Baltimore, Md.: Williams & Wilkins 1967 Teorell, T.: Kinetics of distribution of substances administered to the body. II. The intravascular modes of administration. Arch. intern. Pharmacodyn. 57, 226-240 (1937)
Dr. A. Berlin Astra L~kemedel AFE S - 151 85 S~dert~lje Sweden
