- Email: [email protected]
Light-dark variations in ocular timolol concentrations following topical solution installation in the pigmented rabbit
Life Sciences, Vol. Printed in the USA
51, pp. 2025-2031
LIGHT-DARK VARIATIONS FOLLOWING TOPICAL SOLUTION
Shigehiro
Pergamon
Press
IN OCULAR TIMOLOL CONCENTRATIONS INSTILLATION IN THE PIGMENTED RABBIT
Ohdo, 1 Jaanyi
Zhu, and V~ncent
H.L.
Lee
Department
of Pharmaceutical Sciences, School of Pharmacy University of Southern California 1985 Zonal Avenue, Los Angeles, California 90033 U.S.A.
(Received
an fanal
form October
13, 1992)
Summary The ob3ective of this study was to determine whether ocular absorption of topacally applaed tamolol an the pagmented rabbat varaed wath the tame of drop inst~llataon. Twenty-five macroliters of a 0.65% timolol maleate solutaon were instilled in the pigmented rabbit eye at 0600, 0900, 1200, 1500, 1800, 2100, 2400, or 0300 hr. Timolol concentrataons an the conjunctava, sclera, corneal epithelium, corneal stroma, aqueous humor, and iris-ciliary body at 15 and 30 min post-dosang were monitored using reversed phase HPLC. Ocular timolol concentrations were higher when the drug was administered during the l~ght period (0900-1800 hr) than when at was adminastered during the dark peraod (1800-0600 hr). There exist, therefore, light-dark varaataons in the ocular absorption of topically applaed tlmolol.
The design of ocular sustained release drug delavery systems wath zero order release characteristics impllcatly assumes that ocular pharmacokinetic processes are invariant w~th time. For this reason, constant drug concentrations at the target site are usually expected from the constant drug release rate which has been preprogrammed into the delivery system. This expectation cannot be fulfilled, however, af there are time-dependent changes an any of the relevant pharmacokinetic processes. Presently, it as not known whether ocular pharmacokinetic processes follow a circadian rhythm. Work by Gregory and has coworkers (1-3) as well as by others (4-6) over the past decade has establashed the existence of a clrcadaan rhythm an the antraocular pressure of albano rabbits which varies in phase with the circadian rhythm of aqueous flow (2). Intraocular pressure as high during the dark phase and low during the light phase (1-3,5). This observation, coupled with the fact that the pharmacokinetic processes of some orally administered drugs follow a circadian rhythm (7), prompted the present study. The objective of the present study was to determine whether the tlme of drop inst111at~on influenced the concentrataons of tamolol, a nonselective ~adrenergic antagonist widely used an the treatment of glaucoma (8), in the pagmented rabbit eye at 15 and 30 min following solutaon instillataon. Both time points have been shown to lle within the absorption phase of timolol in the aqueous humor, iris-calaary body (the target tissue of timolol), and all anteraor segment tissues (9). It was not the intent of this work, however, to iEhime University
School of Medicine,
Copyright
Department
of Pharmacology,
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
Eh~me,
reserved.
Japan
2026
Variataons
elucidate the basis t h a t m i g h t exist.
in O c u l a r T i m o l o l C o n c e n t r a t a o n s
for l i g h t - d a r k
varlatlons
Materials
~n o c u l a r
Vol.
t~molol
51, No. 26,
1992
concentratlons
and M e t h o d s
Materials Male, D u t c h b e l t e d p l g m e n t e d rabblts, w e i g h i n g 2 . 0 - 2 . 5 kg, w e r e p u r c h a s e d from A m e r a c a n R a b b l t r y (Los Angeles, CA). The a n l m a l s w e r e h o u s e d in s t a n d a r d l a b o r a t o r y r a b b i t cages, w l t h food and w a t e r ad llbltum, in a l i g h t - c o n t r o l l e d r o o m at a t e m p e r a t u r e of 22 ± 1 ° C and a h u m l d l t y of 55 z 10%. They were e n t r a i n e d to a l l g h t i n g s c h e d u l e of a l t e r n a t i n g 12 hr p e r i o d s of light and d a r k (12L:I2D) for at least one w e e k p r i o r to use. L i g h t s w e r e t u r n e d on at 0600 hr and off at 1800 hr. E x p e r i m e n t s d u r i n g the d a r k p e r l o d s w e r e c o n d u c t e d u n d e r l i g h t i n g c o n d l t i o n s p r o v l d e d by B r a g h t Lab Jr. S a f e l i g h t b u l b s (Delta i, D a l las, TX), w h l c h emit a n a r r o w v i s i b l e s p e c t r u m in the far red r a n g e and w h l c h a r e u s e d p r i m a r i l y in p h o t o g r a p h l c d a r k rooms. T x m o l o l m a l e a t e and p r o p r a n o l o l HCl w e r e p u r c h a s e d from S i g m a C h e m i c a l s (St. Louls. MO). Timolol maleate (0.65%) d o s a n g s o l u t l o n s w e r e p r e p a r e d in I0 mM Tris buffer, a d j u s t e d to pM 7.4 w a t h I0 M s o d i u m h y d r o x i d e , and r e n d e r e d i s o t o n i c by a d d l n g s o d l u m chloride. Assay T ~ m o l o l was q u a n t i t a t e d u s i n g r e v e r s e d p h a s e H P L C on a B e c k m a n U l t r a s p h e r e ODS c o l u m n (25 cm x 4.6 mm, p a r t i c l e slze, 5 gm) fitted w i t h a B r o w n l e e Labs N e w g u a r d p r e c o l u m n (1.5 cm), as p r e v i o u s l y d e s c r i b e d (I0). The H P L C s y s t e m c o n s i s t e d of a S C L - 6 A s y s t e m c o n t r o l l e r , two L C - 6 A pumps, a S I L - 6 A a u t o ~ n j e c tot, a S P D - 6 A s p e c t r o p h o t o m e t r i c detector, and a C R - 3 A i n t e g r a t o r (Shimadzu I n s t r u m e n t s , B a l t a m o r e , MD). The m o b i l e p h a s e was a m l x t u r e c o n s i s t i n g of 4 p a r t s of 10% a c e t o n i t r l l e ~n m e t h a n o l and 6 parts of 0.2% t r ~ e t h y l a m l n e HCl In 5% a c e t o n l t r l l e at pH 3. T h e flow rate was 1 ml m l n -I. P r o p r a n o l o l HCl (I0 pg ml -I) s e r v e d as the internal standard. T l m o l o l was m o n i t o r e d at 294 nm. The r e t e n t i o n t z m e was 5 m i n for t a m o l o l and 12 man for p r o p r a n o l o l . The s e n s i t l v a t y of the assay was b e t t e r t h a n 1.6 ng. The ~ntra- and l n t e r r u n v a r a a t l o n s w e r e 5% and 7.5%, r e s p e c t l v e ! y . At the t a m e of assay, an a q u e o u s h u m o r sample was m ~ x e d w l t h an equal v o l u m e of a c e t o n ~ t r i l e c o n t a i n l n g 0 . 0 1 N HCl and I0 gg ml -I p r o p r a n o l o l HCI. F o l l o w i n g c e n t r i f u g a t i o n , 8 0 - 1 2 0 ~I of the s u p e r n a t a n t was i n 3 e c t e d into the HPLC. E x c i s e d t i s s u e s w e r e s o a k e d in 200 G1 of 0.6% H C l O . at 8"C for 12 hr. T h e r e a f t e r , the s a m p l e was m l x e d w i t h 0.i ml of i0 pg ml =I of p r o p r a n o l o l HCI s o l u t i o n and 0.5 ml of IM a m m o n l u m a c e t a t e b u f f e r (pH 9), e x t r a c t e d w ~ t h 5 t i m e s Its v o l u m e of d ~ e t h y l e t h e r by v o r t e x i n g for 3 mln, and t h e n c e n t r i f u g e d at 1 , 5 0 0 x g for i0 min. The u p p e r o r g a n i c layer was t r a n s f e r r e d to a 15 ml s c r e w - c a p p e d c o n l c a l c e n t r i f u g e t u b e c o n t a l n i n g 200 gl of 0.2N HCl, v o r t e x e d for 3 man, and c e n t r i f u g e d at 1,500x g for i0 min. The o r g a n l c p h a s e was d i s carded, w h a l e 1 0 0 - 1 2 0 gl of the a q u e o u s phase, c o n t a l n i n g t l m o l o l and p r o p r a n o Ioi, was i n j e c t e d into the HPLC. The e x t r a c t i o n e f f a c l e n c y of t a m o l o l from o c u l a r t i s s u e s was b e t t e r t h a n 75 ± 5%. Ocular Absorption
of T o p ~ c a l l y
Applled
Tlmolol
At 0600, 0900, 1200, 1500, 1800, 2100, 2400, or 0300 hr, 25 U1 of a d o s i n g s o l u t i o n was i n s t 1 1 1 e d d l r e c t l y o n t o the c o r n e a of one eye of e a c h r a b b i t u s i n g an E p p e n d o r f plpet, as p r e v l o u s l y d e s c r i b e d (9,10). At 15 or 30 m l n p o s t dosing, the r a b b l t was e u t h a n l z e d w i t h an o v e r d o s e of s o d i u m p e n t o b a r b l t a l s o l u t a o n (Eutha-6, W e s t e r n M e d l c a l Supply, Arcadia, CA) a d m l n i s t e r e d via a m a r g i n a l ear vein. A f t e r t h o r o u g h l y r l n s l n g the c o r n e a l and c o n j u n c t l v a l surfaces w i t h 1.17% KCl s o l u t l o n and b l o t t l n g dry, the c o r n e a l e p a t h e l l u m was s c r a p e d w i t h a No. Ii scalpel. A b o u t 1 0 0 - 2 0 0 ~I of a q u e o u s h u m o r was a s p a r a t -
Vol.
51, No.
26, 1992
Variataons
in O c u l a r T i m o l o l
Concentrations
2027
ed from t h e a n t e r i o r c h a m b e r u s i n g a 1 c.c. t u b e r c u l i n s y r i n g e fitted w i t h a 2 7 - g a u g e needle. T h e corneal s t r o m a was e x c i s e d by c u t t i n g at the c o r n e o l l m b a l margin. Since the iris and c i l i a r y bodies w e r e d i f f i c u l t to s e p a r a t e from each other, t h e y w e r e r e m o v e d as one piece. The a n t e r i o r sclera was d i s s e c t e d and t r i m m e d to r e m o v e o t h e r t i s s u e fragments. Finally, 8 to 10 m m t a n g e n t i a l s e c t i o n s of c o n j u n c t i v a w e r e d i s s e c t e d from the u p p e r and lower e y e l i d s w i t h a scalpel. D i s s e c t i o n was c o m p l e t e d w a t h i n ten manutes. All e x c i s e d t i s s u e s w e r e r i n s e d w i t h ice cold KCl solution, b l o t t e d dry, t r a n s f e r r e d to p r e - w e i g h e d m i c r o c e n t r i f u g e t u b e s c o n t a i n i n g 200 gl of 0.6% HClO4, and s t o r e d at -70 ° C. Four to eaght eyes w e r e u s e d per time poant per d o s a n g period. Statistical
Data Analysis
The S t u d e n t ' s t - t e s t was used to compare tamolol c o n c e n t r a t i o n s b e t w e e n 15 and 30 m i n u t e s at a g i v e n d o s l n g time. S t a t i s t a c a l s i g n i f i c a n c e of d i f f e r e n c e ~n t i m o l o l c o n c e n t r a t i o n s a m o n g various d o s i n g tames was t e s t e d w i t h o n e - w a y a n a l y s i s of v a r i a n c e (ANOVA) (ll). W h e r e there was a difference, m u l t i p l e c o m p a r a s o n s w e r e c o n d u c t e d among the v a r i o u s d o s i n g times u s a n g the F ~ s h e r P r o t e c t e d Least S a g n i f l c a n t D a f f e r e n c e test (ii).
Results The i n f l u e n c e of d o s i n g tame on the timolol c o n c e n t r a t i o n s in v a r l o u s a n t e r i o r s e g m e n t s at 15 and 30 m i n p o s t d o s i n g is shown an Figs. 1 and 2, respectively. O n e w a y A N O V A r e v e a l e d a s t a t i s t i c a l l y s i g n i f a c a n t d l f f e r e n c e at p < 0.05 in all the a n t e r a o r segment t a s s u e c o n c e n t r a t i o n s at 15 m i n among the v a r i o u s d o s i n g times (Fig. i). At 30 min, a s t a t i s t a c a l l y s i g n i f i c a n t difference in t i m o l o l c o n c e n t r a t i o n s among the same d o s i n g times c o u l d be d e m o n s t r a t e d only in the corneal e p i t h e l i u m (Fag. 2d), aqueous humor (Fig. 2c), and i r i s - c a l i a r y body (Fig. 2f), but not in the c o n j u n c t i v a (Fag. 2a), sclera (Fig. 2b), and corneal s t r o m a (Fag. 2e). Moreover, only in the case of the corneal e p i t h e l a u m (Fag. ld), corneal stroma (Fig. le), aqueous humor (Fig. ic), and i r i s - c i l i a r y b o d y (Fig. if) at 15 m i n and in the aqueous humor (Fig. 2c) and i r a s - c i l i a r y b o d y (Fig. 2f) at 30 m i n dad c o n s i s t e n t l a g h t - d a r k v a r i a t i o n s exist w i t h h i g h e r v a l u e s d u r i n g the laght p e r a o d (0900-1800 hr) and lower v a l u e s d u r i n g the d a r k p e r l o d (1800-0600 hr). The haghest t l m o l o l c o n c e n t r a tions w e r e o b t a i n e d at 1200 hr d o s a n g (except an the i r a s - c i l a a r y body) and the lowest at e l t h e r 0600 or 2400 hr dosing. T h e r e w a s no s t a t a s t i c a l d i f f e r e n c e in timolol c o n c e n t r a t i o n s b e t w e e n 15 and 30 m i n at most of the d o s i n g times (p > 0.05). W h e r e t h e r e was a difference, the t i m o l o l c o n c e n t r a t i o n at 15 m i n was higher than that at 30 min, e x c e p t in the i r a s - c i l i a r y body and aqueous humor. The d o s i n g t l m e y i e l d i n g t h e h i g h e s t f r e q u e n c y of statistical d i f f e r e n c e was 1200 hr, w h e n four out of six t i s s u e s / f l u i d s sampled showed a sagnaficant d a f f e r e n c e (p < 0.05).
Dascusslon Thls study d e m o n s t r a t e d that the d o s i n g time i n f l u e n c e d the timolol concent r a t i o n s in v a r i o u s a n t e r a o r segment tissues at both 15 and 30 m i n f o l l o w i n g t o p i c a l s o l u t i o n i n s t i l l a t i o n in the p i g m e n t e d rabbit. Not all v a r i a t i o n s in a n t e r i o r segment t a s s u e timolol c o n c e n t r a t i o n s w i t h d o s i n g t i m e can, however, be d e s c r a b e d as l i g h t - d a r k varaations, e v e n t h o u g h t h e y are s i g n i f i c a n t l y d i f f e r e n t at p < 0.05 by ANOVA. On the basas of a c o n s i s t e n t t r e n d in the rise and fall of drug c o n c e n t r a t i o n s w a t h d o s a n g tame, l i g h t - d a r k v a r i a t i o n s in t a m o l o l c o n c e n t r a t a o n s a p p e a r to exist in the corneal e p i t h e l i u m (Fig. id), c o r n e a l s t r o m a (Fig. le), aqueous humor (Fag. ic), and i r i s - c i l i a r y body (Fig. If) at 15 m i n p o s t - s o l u t i o n instillation, w i t h h i g h e r v a l u e s d u r i n g t h e light p e r i o d and lower v a l u e s d u r i n g the d a r k peraod. As was the case in a prelimi-
2028
Variations
in O c u l a r
T1molol
Concentratlons
vol.
51, No. 26, 1992
8
o 0 o o
E
6-
400 "
i
-
300 o 0
4
I--
c o o
i
0900
200'
h
"6 E ~-
100 •
o
200 1500 1800 21 O0 2400 0300 0600 Clock
0900 1200 1500 1 8 0 0
Hours
T
2400 0300 0600
TI
40
30
o 0
2100
C l oc k H o u r s
e
T
O O
o o
-6 "6 E
E
I-
20
10
U)
g,
O
0 0 9 0 0 1 2 0 0 1 5 0 0 1 8 0 0 2100 2400 0300 0600
0900 1200 1600 1800 2100 2400 0300 0600
Clock Hours
C l oc k H o u r s
30 E
T
l 20
o
c o 0
o O
o o
.6 -6 E
E
it.
_o
<
0900 1200 1500 1800 2100 2400 0300 0600 Clock H o u r s
I n f l u e n c e of d o s i n g (a), a n t e r i o r s c l e r a c o r n e a l s t r o m a (e), at 15 m l n f o l l o w i n g m a l e a t e solutions. e a c h d o s i n g t~me. CS, C o r n e a l stroma;
09001200150018002100240003000600 Clock H o u r s
Fag. 1 time on t i m o l o l c o n c e n t r a t i o n s an the c o n 3 u n c t a v a (b), a q u e o u s humor (c), corneal e p i t h e l i u m (d), and i r i s - c l l i a r y b o d y (f) of the p i g m e n t e d r a b b ~ t the t o p l c a l i n s t i l l a t i o n of 25 @i of 0.65% t i m o l o l Error bars r e p r e s e n t s t a n d a r d e r r o r for n = 4-8 at Key: AH, A q u e o u s humor; CE, C o r n e a l eplthelium; ICB, I r l s - c i l i a r y body.
I
Vol. 51, No. 26, 1992
o o E
T
30
A o~ ,~
T T
,T T--I--
m
2O
60"
"]-
_E
> ~ ¢J
10
~
oo
0
I,.u,I 0
I
Clock
Hours
40
bl
I T
O (J
0900 1200 1500 1800 2100 2400 0300 0600
Hours
A 20-
IIIIIIII
00900 1200 1500 1800 2100 2400 0300 0600 Clock
_s
dl
120
o o
"6
2029
150
T
40 o 0
Variatlons in Ocular Timolol Concentrations
ol
30.
u
T
o 1
o o
10
_E
I-
Iu) 0
10'
00 0 9 0 0 1 2 0 0 1 5 0 0 1600 Clock
2100 2400 0300 0600
"T
O~ ::L
0900 120015001000
Hours
Clock
,
II
Hours
30 A t~ ol ::L v
I-L_T_
2100 2400 0300 0600
T
,
20 O O
O (J
O
O O E
_s O
I--r
m o
<[ 0900 1200 1 500 1800 2100 2400 0300 0600 Clock
Hours
10 ¸ ! I
0 9 0 0 1 2 0 0 1 5 0 0 1 8 0 0 21 o o 2 4 0 0 0 3 0 0 0 6 0 0 Clock
Hours
Fig. 2 Influence of dosing time on timolol concentrations in the conjunctiva (a), anterior sclera (b), aqueous humor (c), corneal epithelium (d}, corneal stroma (e), and irls-ciliary body (f) of the pigmented rabbit at 30 min following the topical instillation of 25 ~l of 0.65% timolol maleate solutlons. Error bars represent standard error for n = 4-8 at each dosing time. Key: AH, Aqueous humor; CE, Corneal epithelium; CS, Corneal stroma; ICB, Iris-ciliary body.
2030
Varaataons
in O c u l a r
Tamolol
Concentrataons
Vol.
51, No. 26, 1992
nary study on t h e a n f l u e n c e of d o s a n g tame on o c u l a r a b s o r p t i o n of t o p i c a l l y a p p l i e d t i m o l o l (12), the h a g h e s t t i m o l o l c o n c e n t r a t i o n s at this time point w e r e a t t a i n e d at 1200 hr dosang, w h e r e a s the lowest were a t t a i n e d at 2400 hr dosing. The r a t i o of h i g h e s t to lowest timolol c o n c e n t r a t a o n s a m o n g the ant e r i o r s e g m e n t t i s s u e s / f l u i d s s a m p l e d was 3.47 ± 0.49. T h e l i g h t - d a r k v a r a a t i o n s in a n t e r i o r segment t i s s u e c o n c e n t r a t i o n s can be a t t r i b u t e d to c o r r e s p o n d i n g c h a n g e s in p r e c o r n e a l clearance, corneal p e r m e a b i l aty, or a q u e o u s humor turnover. L i g h t - d a r k v a r i a t i o n s in d r u g m e t a b o l i s m cannot be a factor sance t a m o l o l is m e t a b o l i c a l l y stable an the eye (13). Theoretlcally, h i g h t a m o l o l c o n c e n t r a t a o n s d u r i n g t h e light p e r a o d can be e x p l a a n e d by low p r e c o r n e a l clearance, hagh corneal p e r m e a b i l i t y , and low a q u e o u s h u m o r t u r n o v e r rate. A l t h o u g h t h e r e as, as yet, no i n f o r m a t a o n on c i r c a d a a n c h a n g e s an p r e c o r n e a l c l e a r a n c e and corneal p e r m e a b i l i t y in the p a g m e n t e d rabbit, the rate of a q u e o u s humor t u r n o v e r has b e e n r e p o r t e d by Smith and G r e g o r y (2) to f o l l o w a c i r c a d i a n r h y t h m w a t h low v a l u e s d u r a n g the light p e r i o d and h i g h v a l u e s d u r i n g the d a r k peraod. C o n s e q u e n t l y , high v a l u e s of t i m o l o l c o n c e n t r a t a o n s in the a q u e o u s humor (Fags. ic and 2c) and a r i s - c a l i a r y b o d y (Figs. if and 2f) d u r a n g the light p e r i o d can be attrabuted, at least in part, to c a r c a d i a n r e d u c t a o n in a q u e o u s humor t u r n o v e r rate r e p o r t e d by the above a n v e s t a g a t o r s (2). An a l t e r n a t a v e e x p l a n a t i o n for the l a g h t - d a r k v a r a a taons in t a m o l o l c o n c e n t r a t a o n s an a n t e r a o r segment t i s s u e s is b e h a v i o r a l c h a n g e s an the rabbat w h i c h m a y cause p h y s a o l o g a c a l changes. Rabbits, lake other r o d e n t s such as rats and m a c e (14), are n o c t u r n a l anamals. Thear d a f f e r entaal s e n s i t i v i t y to h a n d l a n g d u r a n g laght and d a r k p e r i o d s p r o b a b l y e x e r t s the most p r o n o u n c e d e f f e c t on p r e c o r n e a l d r u g c l e a r a n c e and aqueous humor t u r n o v e r (15,16). That l a g h t - d a r k v a r i a t i o n s an t i m o l o l c o n c e n t r a t i o n s exast an the c o r n e a l e p i t h e l a u m (Fag. Id) b u t not in t h e c o n j u n c t a v a (Fag. la), even t h o u g h b o t h t a s s u e s are b a t h e d by tears, suggests that l i g h t - d a r k v a r i a t i o n s in p r e c o r n e a l d r u g c l e a r a n c e m a y p l a y a s e c o n d a r y role to l i g h t - d a r k v a r i a t a o n s in d r u g u p t a k e and c l e a r a n c e an the c o r n e a and the c o n 3 u n c t i v a an c o n t r a b u t i n g to l a g h t - d a r k v a r a a t a o n s an drug c o n c e n t r a t a o n s an these two tassues. To summaraze, the t a m e of e y e d r o p a n s t a l l a t a o n a n f l u e n c e s the o c u l a r timoioi c o n c e n t r a t a o n s a t t a a n e d an p a g m e n t e d rabbat eye. Such d o s a n g t a m e - d e p e n d ent c h a n g e s an o c u l a r d r u g c o n c e n t r a t a o n s as an a d d i t a o n a l e x p e r a m e n t a l variable that m u s t be c a r e f u l l y c o n t r o l l e d an o c u l a r drug p h a r m a c o k a n e t a c studaes. It as also an a d d a t a o n a l factor that must be c o n s i d e r e d an p l a n n a n g d o s a n g r e g a m e n s and in d e s i g n a n g c o n t r o l l e d drug r e l e a s e systems. For timolol, the ratio of h i g h e s t to lowest v a l u e s as 3.34 ± 0.44 d u r i n g the light peraod, 1.92 ± 0.18 d u r i n g the d a r k peraod, and 3.47 ± 0.49 d u r a n g the 24-hr peraod.
Acknowledgments
tutes
Thas study was s u p p o r t e d an part by grant EY7389 from the National Instaof Health, Bethesda, M a r y l a n d and by the G a v a n S. H e r b e r t Professorship.
References i. 2. 3. 4. 5.
R.A. B R A S L O W and D.S. GREGORY, Invest. Ophthalmol. Vas. Sci. 2_88 1730-1732, 1987. S.D. S M I T H and D.S. GREGORY, Invest. Ophthalmol. Vas. Sca. 3__O0775-778, 1989. D.S. GREGORY, Invest. Ophthalmol. Vas. Sca. 3_! 715-721, 1990. J.M. ROWLAND, D.E. POTTER, and R.J. REITER, Curr. Eye Res. ! 169-173, 1981. A. BAR-ILAN, Exp. Eye Res. 39 175-181, 1984.
Vol.
51, NO. 26, 1992
6.
J.M. ROWLAND, W.K. SAWYER, J. TITTEL, and C.J. FORD, Curr. Eye Res. ~ 201206, 1986. R.H. LEVY and C.R. BANFIELD, in: "Pharmacokinetics," P.G. WELLING and F.L.S. TSE, eds., Marcel Dekker, Inc., New York, 1988. pp. 515. G.D. NOVAK, Surv. Ophthalmol. 3_!1 307-327, 1987. V.H.L. LEE, A.M. LUO, S. LI, S.K. PODDER, J.S.C. CHANG, S. OHDO, and G.M. GRASS, Invest. Ophthalmol. Vis. Sci. 3_/2 2948-2957, 1991. S.C. CHANG, D.S. CHIEN, H. BUNDGAARD, and V.H.L. LEE, Exp. Eye Res. 46 5969, 1988. J.H. ZAK, B~ostatistical AnalTsis, Prent~ce-Hall, Inc., N e w Jersey (1984). S. OHDO, G.M. GRASS, and V.H.L. LEE, Invest. Ophthalmol. Vis. Sci. 32 27902798, 1991. G.J. PUTTERMAN, J. DAVIDSON, and J. ALBERT, J. Ocular Pharmacol. 1_!1 287295, 1985. S. ARMSTRONG, J. CLARKE, and G. COLEMAN, Physiol. Behavior 2_!1 785-788, 1978. G.R. REISS, D.A. LEE, J.E. TOPPER, and R.F. BRUBAKER, Invest. Ophthalmol. Vis. Sci. 25 776-778, 1984. S. MISHIMA, A. GASSET, S.D. KLYCE,JR., and J.L. BAUM, Invest. Ophthalmol. 264-276, 1966.
7. 8. 9. i0. 11. 12. 13. 14. 15. 16.
Variatlons
in Ocular Timolol Concentrations
2031
51, pp. 2025-2031
LIGHT-DARK VARIATIONS FOLLOWING TOPICAL SOLUTION
Shigehiro
Pergamon
Press
IN OCULAR TIMOLOL CONCENTRATIONS INSTILLATION IN THE PIGMENTED RABBIT
Ohdo, 1 Jaanyi
Zhu, and V~ncent
H.L.
Lee
Department
of Pharmaceutical Sciences, School of Pharmacy University of Southern California 1985 Zonal Avenue, Los Angeles, California 90033 U.S.A.
(Received
an fanal
form October
13, 1992)
Summary The ob3ective of this study was to determine whether ocular absorption of topacally applaed tamolol an the pagmented rabbat varaed wath the tame of drop inst~llataon. Twenty-five macroliters of a 0.65% timolol maleate solutaon were instilled in the pigmented rabbit eye at 0600, 0900, 1200, 1500, 1800, 2100, 2400, or 0300 hr. Timolol concentrataons an the conjunctava, sclera, corneal epithelium, corneal stroma, aqueous humor, and iris-ciliary body at 15 and 30 min post-dosang were monitored using reversed phase HPLC. Ocular timolol concentrations were higher when the drug was administered during the l~ght period (0900-1800 hr) than when at was adminastered during the dark peraod (1800-0600 hr). There exist, therefore, light-dark varaataons in the ocular absorption of topically applaed tlmolol.
The design of ocular sustained release drug delavery systems wath zero order release characteristics impllcatly assumes that ocular pharmacokinetic processes are invariant w~th time. For this reason, constant drug concentrations at the target site are usually expected from the constant drug release rate which has been preprogrammed into the delivery system. This expectation cannot be fulfilled, however, af there are time-dependent changes an any of the relevant pharmacokinetic processes. Presently, it as not known whether ocular pharmacokinetic processes follow a circadian rhythm. Work by Gregory and has coworkers (1-3) as well as by others (4-6) over the past decade has establashed the existence of a clrcadaan rhythm an the antraocular pressure of albano rabbits which varies in phase with the circadian rhythm of aqueous flow (2). Intraocular pressure as high during the dark phase and low during the light phase (1-3,5). This observation, coupled with the fact that the pharmacokinetic processes of some orally administered drugs follow a circadian rhythm (7), prompted the present study. The objective of the present study was to determine whether the tlme of drop inst111at~on influenced the concentrataons of tamolol, a nonselective ~adrenergic antagonist widely used an the treatment of glaucoma (8), in the pagmented rabbit eye at 15 and 30 min following solutaon instillataon. Both time points have been shown to lle within the absorption phase of timolol in the aqueous humor, iris-calaary body (the target tissue of timolol), and all anteraor segment tissues (9). It was not the intent of this work, however, to iEhime University
School of Medicine,
Copyright
Department
of Pharmacology,
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
Eh~me,
reserved.
Japan
2026
Variataons
elucidate the basis t h a t m i g h t exist.
in O c u l a r T i m o l o l C o n c e n t r a t a o n s
for l i g h t - d a r k
varlatlons
Materials
~n o c u l a r
Vol.
t~molol
51, No. 26,
1992
concentratlons
and M e t h o d s
Materials Male, D u t c h b e l t e d p l g m e n t e d rabblts, w e i g h i n g 2 . 0 - 2 . 5 kg, w e r e p u r c h a s e d from A m e r a c a n R a b b l t r y (Los Angeles, CA). The a n l m a l s w e r e h o u s e d in s t a n d a r d l a b o r a t o r y r a b b i t cages, w l t h food and w a t e r ad llbltum, in a l i g h t - c o n t r o l l e d r o o m at a t e m p e r a t u r e of 22 ± 1 ° C and a h u m l d l t y of 55 z 10%. They were e n t r a i n e d to a l l g h t i n g s c h e d u l e of a l t e r n a t i n g 12 hr p e r i o d s of light and d a r k (12L:I2D) for at least one w e e k p r i o r to use. L i g h t s w e r e t u r n e d on at 0600 hr and off at 1800 hr. E x p e r i m e n t s d u r i n g the d a r k p e r l o d s w e r e c o n d u c t e d u n d e r l i g h t i n g c o n d l t i o n s p r o v l d e d by B r a g h t Lab Jr. S a f e l i g h t b u l b s (Delta i, D a l las, TX), w h l c h emit a n a r r o w v i s i b l e s p e c t r u m in the far red r a n g e and w h l c h a r e u s e d p r i m a r i l y in p h o t o g r a p h l c d a r k rooms. T x m o l o l m a l e a t e and p r o p r a n o l o l HCl w e r e p u r c h a s e d from S i g m a C h e m i c a l s (St. Louls. MO). Timolol maleate (0.65%) d o s a n g s o l u t l o n s w e r e p r e p a r e d in I0 mM Tris buffer, a d j u s t e d to pM 7.4 w a t h I0 M s o d i u m h y d r o x i d e , and r e n d e r e d i s o t o n i c by a d d l n g s o d l u m chloride. Assay T ~ m o l o l was q u a n t i t a t e d u s i n g r e v e r s e d p h a s e H P L C on a B e c k m a n U l t r a s p h e r e ODS c o l u m n (25 cm x 4.6 mm, p a r t i c l e slze, 5 gm) fitted w i t h a B r o w n l e e Labs N e w g u a r d p r e c o l u m n (1.5 cm), as p r e v i o u s l y d e s c r i b e d (I0). The H P L C s y s t e m c o n s i s t e d of a S C L - 6 A s y s t e m c o n t r o l l e r , two L C - 6 A pumps, a S I L - 6 A a u t o ~ n j e c tot, a S P D - 6 A s p e c t r o p h o t o m e t r i c detector, and a C R - 3 A i n t e g r a t o r (Shimadzu I n s t r u m e n t s , B a l t a m o r e , MD). The m o b i l e p h a s e was a m l x t u r e c o n s i s t i n g of 4 p a r t s of 10% a c e t o n i t r l l e ~n m e t h a n o l and 6 parts of 0.2% t r ~ e t h y l a m l n e HCl In 5% a c e t o n l t r l l e at pH 3. T h e flow rate was 1 ml m l n -I. P r o p r a n o l o l HCl (I0 pg ml -I) s e r v e d as the internal standard. T l m o l o l was m o n i t o r e d at 294 nm. The r e t e n t i o n t z m e was 5 m i n for t a m o l o l and 12 man for p r o p r a n o l o l . The s e n s i t l v a t y of the assay was b e t t e r t h a n 1.6 ng. The ~ntra- and l n t e r r u n v a r a a t l o n s w e r e 5% and 7.5%, r e s p e c t l v e ! y . At the t a m e of assay, an a q u e o u s h u m o r sample was m ~ x e d w l t h an equal v o l u m e of a c e t o n ~ t r i l e c o n t a i n l n g 0 . 0 1 N HCl and I0 gg ml -I p r o p r a n o l o l HCI. F o l l o w i n g c e n t r i f u g a t i o n , 8 0 - 1 2 0 ~I of the s u p e r n a t a n t was i n 3 e c t e d into the HPLC. E x c i s e d t i s s u e s w e r e s o a k e d in 200 G1 of 0.6% H C l O . at 8"C for 12 hr. T h e r e a f t e r , the s a m p l e was m l x e d w i t h 0.i ml of i0 pg ml =I of p r o p r a n o l o l HCI s o l u t i o n and 0.5 ml of IM a m m o n l u m a c e t a t e b u f f e r (pH 9), e x t r a c t e d w ~ t h 5 t i m e s Its v o l u m e of d ~ e t h y l e t h e r by v o r t e x i n g for 3 mln, and t h e n c e n t r i f u g e d at 1 , 5 0 0 x g for i0 min. The u p p e r o r g a n i c layer was t r a n s f e r r e d to a 15 ml s c r e w - c a p p e d c o n l c a l c e n t r i f u g e t u b e c o n t a l n i n g 200 gl of 0.2N HCl, v o r t e x e d for 3 man, and c e n t r i f u g e d at 1,500x g for i0 min. The o r g a n l c p h a s e was d i s carded, w h a l e 1 0 0 - 1 2 0 gl of the a q u e o u s phase, c o n t a l n i n g t l m o l o l and p r o p r a n o Ioi, was i n j e c t e d into the HPLC. The e x t r a c t i o n e f f a c l e n c y of t a m o l o l from o c u l a r t i s s u e s was b e t t e r t h a n 75 ± 5%. Ocular Absorption
of T o p ~ c a l l y
Applled
Tlmolol
At 0600, 0900, 1200, 1500, 1800, 2100, 2400, or 0300 hr, 25 U1 of a d o s i n g s o l u t i o n was i n s t 1 1 1 e d d l r e c t l y o n t o the c o r n e a of one eye of e a c h r a b b i t u s i n g an E p p e n d o r f plpet, as p r e v l o u s l y d e s c r i b e d (9,10). At 15 or 30 m l n p o s t dosing, the r a b b l t was e u t h a n l z e d w i t h an o v e r d o s e of s o d i u m p e n t o b a r b l t a l s o l u t a o n (Eutha-6, W e s t e r n M e d l c a l Supply, Arcadia, CA) a d m l n i s t e r e d via a m a r g i n a l ear vein. A f t e r t h o r o u g h l y r l n s l n g the c o r n e a l and c o n j u n c t l v a l surfaces w i t h 1.17% KCl s o l u t l o n and b l o t t l n g dry, the c o r n e a l e p a t h e l l u m was s c r a p e d w i t h a No. Ii scalpel. A b o u t 1 0 0 - 2 0 0 ~I of a q u e o u s h u m o r was a s p a r a t -
Vol.
51, No.
26, 1992
Variataons
in O c u l a r T i m o l o l
Concentrations
2027
ed from t h e a n t e r i o r c h a m b e r u s i n g a 1 c.c. t u b e r c u l i n s y r i n g e fitted w i t h a 2 7 - g a u g e needle. T h e corneal s t r o m a was e x c i s e d by c u t t i n g at the c o r n e o l l m b a l margin. Since the iris and c i l i a r y bodies w e r e d i f f i c u l t to s e p a r a t e from each other, t h e y w e r e r e m o v e d as one piece. The a n t e r i o r sclera was d i s s e c t e d and t r i m m e d to r e m o v e o t h e r t i s s u e fragments. Finally, 8 to 10 m m t a n g e n t i a l s e c t i o n s of c o n j u n c t i v a w e r e d i s s e c t e d from the u p p e r and lower e y e l i d s w i t h a scalpel. D i s s e c t i o n was c o m p l e t e d w a t h i n ten manutes. All e x c i s e d t i s s u e s w e r e r i n s e d w i t h ice cold KCl solution, b l o t t e d dry, t r a n s f e r r e d to p r e - w e i g h e d m i c r o c e n t r i f u g e t u b e s c o n t a i n i n g 200 gl of 0.6% HClO4, and s t o r e d at -70 ° C. Four to eaght eyes w e r e u s e d per time poant per d o s a n g period. Statistical
Data Analysis
The S t u d e n t ' s t - t e s t was used to compare tamolol c o n c e n t r a t i o n s b e t w e e n 15 and 30 m i n u t e s at a g i v e n d o s l n g time. S t a t i s t a c a l s i g n i f i c a n c e of d i f f e r e n c e ~n t i m o l o l c o n c e n t r a t i o n s a m o n g various d o s i n g tames was t e s t e d w i t h o n e - w a y a n a l y s i s of v a r i a n c e (ANOVA) (ll). W h e r e there was a difference, m u l t i p l e c o m p a r a s o n s w e r e c o n d u c t e d among the v a r i o u s d o s i n g times u s a n g the F ~ s h e r P r o t e c t e d Least S a g n i f l c a n t D a f f e r e n c e test (ii).
Results The i n f l u e n c e of d o s i n g tame on the timolol c o n c e n t r a t i o n s in v a r l o u s a n t e r i o r s e g m e n t s at 15 and 30 m i n p o s t d o s i n g is shown an Figs. 1 and 2, respectively. O n e w a y A N O V A r e v e a l e d a s t a t i s t i c a l l y s i g n i f a c a n t d l f f e r e n c e at p < 0.05 in all the a n t e r a o r segment t a s s u e c o n c e n t r a t i o n s at 15 m i n among the v a r i o u s d o s i n g times (Fig. i). At 30 min, a s t a t i s t a c a l l y s i g n i f i c a n t difference in t i m o l o l c o n c e n t r a t i o n s among the same d o s i n g times c o u l d be d e m o n s t r a t e d only in the corneal e p i t h e l i u m (Fag. 2d), aqueous humor (Fig. 2c), and i r i s - c a l i a r y body (Fig. 2f), but not in the c o n j u n c t i v a (Fag. 2a), sclera (Fig. 2b), and corneal s t r o m a (Fag. 2e). Moreover, only in the case of the corneal e p i t h e l a u m (Fag. ld), corneal stroma (Fig. le), aqueous humor (Fig. ic), and i r i s - c i l i a r y b o d y (Fig. if) at 15 m i n and in the aqueous humor (Fig. 2c) and i r a s - c i l i a r y b o d y (Fig. 2f) at 30 m i n dad c o n s i s t e n t l a g h t - d a r k v a r i a t i o n s exist w i t h h i g h e r v a l u e s d u r i n g the laght p e r a o d (0900-1800 hr) and lower v a l u e s d u r i n g the d a r k p e r l o d (1800-0600 hr). The haghest t l m o l o l c o n c e n t r a tions w e r e o b t a i n e d at 1200 hr d o s a n g (except an the i r a s - c i l a a r y body) and the lowest at e l t h e r 0600 or 2400 hr dosing. T h e r e w a s no s t a t a s t i c a l d i f f e r e n c e in timolol c o n c e n t r a t i o n s b e t w e e n 15 and 30 m i n at most of the d o s i n g times (p > 0.05). W h e r e t h e r e was a difference, the t i m o l o l c o n c e n t r a t i o n at 15 m i n was higher than that at 30 min, e x c e p t in the i r a s - c i l i a r y body and aqueous humor. The d o s i n g t l m e y i e l d i n g t h e h i g h e s t f r e q u e n c y of statistical d i f f e r e n c e was 1200 hr, w h e n four out of six t i s s u e s / f l u i d s sampled showed a sagnaficant d a f f e r e n c e (p < 0.05).
Dascusslon Thls study d e m o n s t r a t e d that the d o s i n g time i n f l u e n c e d the timolol concent r a t i o n s in v a r i o u s a n t e r a o r segment tissues at both 15 and 30 m i n f o l l o w i n g t o p i c a l s o l u t i o n i n s t i l l a t i o n in the p i g m e n t e d rabbit. Not all v a r i a t i o n s in a n t e r i o r segment t a s s u e timolol c o n c e n t r a t i o n s w i t h d o s i n g t i m e can, however, be d e s c r a b e d as l i g h t - d a r k varaations, e v e n t h o u g h t h e y are s i g n i f i c a n t l y d i f f e r e n t at p < 0.05 by ANOVA. On the basas of a c o n s i s t e n t t r e n d in the rise and fall of drug c o n c e n t r a t i o n s w a t h d o s a n g tame, l i g h t - d a r k v a r i a t i o n s in t a m o l o l c o n c e n t r a t a o n s a p p e a r to exist in the corneal e p i t h e l i u m (Fig. id), c o r n e a l s t r o m a (Fig. le), aqueous humor (Fag. ic), and i r i s - c i l i a r y body (Fig. If) at 15 m i n p o s t - s o l u t i o n instillation, w i t h h i g h e r v a l u e s d u r i n g t h e light p e r i o d and lower v a l u e s d u r i n g the d a r k peraod. As was the case in a prelimi-
2028
Variations
in O c u l a r
T1molol
Concentratlons
vol.
51, No. 26, 1992
8
o 0 o o
E
6-
400 "
i
-
300 o 0
4
I--
c o o
i
0900
200'
h
"6 E ~-
100 •
o
200 1500 1800 21 O0 2400 0300 0600 Clock
0900 1200 1500 1 8 0 0
Hours
T
2400 0300 0600
TI
40
30
o 0
2100
C l oc k H o u r s
e
T
O O
o o
-6 "6 E
E
I-
20
10
U)
g,
O
0 0 9 0 0 1 2 0 0 1 5 0 0 1 8 0 0 2100 2400 0300 0600
0900 1200 1600 1800 2100 2400 0300 0600
Clock Hours
C l oc k H o u r s
30 E
T
l 20
o
c o 0
o O
o o
.6 -6 E
E
it.
_o
<
0900 1200 1500 1800 2100 2400 0300 0600 Clock H o u r s
I n f l u e n c e of d o s i n g (a), a n t e r i o r s c l e r a c o r n e a l s t r o m a (e), at 15 m l n f o l l o w i n g m a l e a t e solutions. e a c h d o s i n g t~me. CS, C o r n e a l stroma;
09001200150018002100240003000600 Clock H o u r s
Fag. 1 time on t i m o l o l c o n c e n t r a t i o n s an the c o n 3 u n c t a v a (b), a q u e o u s humor (c), corneal e p i t h e l i u m (d), and i r i s - c l l i a r y b o d y (f) of the p i g m e n t e d r a b b ~ t the t o p l c a l i n s t i l l a t i o n of 25 @i of 0.65% t i m o l o l Error bars r e p r e s e n t s t a n d a r d e r r o r for n = 4-8 at Key: AH, A q u e o u s humor; CE, C o r n e a l eplthelium; ICB, I r l s - c i l i a r y body.
I
Vol. 51, No. 26, 1992
o o E
T
30
A o~ ,~
T T
,T T--I--
m
2O
60"
"]-
_E
> ~ ¢J
10
~
oo
0
I,.u,I 0
I
Clock
Hours
40
bl
I T
O (J
0900 1200 1500 1800 2100 2400 0300 0600
Hours
A 20-
IIIIIIII
00900 1200 1500 1800 2100 2400 0300 0600 Clock
_s
dl
120
o o
"6
2029
150
T
40 o 0
Variatlons in Ocular Timolol Concentrations
ol
30.
u
T
o 1
o o
10
_E
I-
Iu) 0
10'
00 0 9 0 0 1 2 0 0 1 5 0 0 1600 Clock
2100 2400 0300 0600
"T
O~ ::L
0900 120015001000
Hours
Clock
,
II
Hours
30 A t~ ol ::L v
I-L_T_
2100 2400 0300 0600
T
,
20 O O
O (J
O
O O E
_s O
I--r
m o
<[ 0900 1200 1 500 1800 2100 2400 0300 0600 Clock
Hours
10 ¸ ! I
0 9 0 0 1 2 0 0 1 5 0 0 1 8 0 0 21 o o 2 4 0 0 0 3 0 0 0 6 0 0 Clock
Hours
Fig. 2 Influence of dosing time on timolol concentrations in the conjunctiva (a), anterior sclera (b), aqueous humor (c), corneal epithelium (d}, corneal stroma (e), and irls-ciliary body (f) of the pigmented rabbit at 30 min following the topical instillation of 25 ~l of 0.65% timolol maleate solutlons. Error bars represent standard error for n = 4-8 at each dosing time. Key: AH, Aqueous humor; CE, Corneal epithelium; CS, Corneal stroma; ICB, Iris-ciliary body.
2030
Varaataons
in O c u l a r
Tamolol
Concentrataons
Vol.
51, No. 26, 1992
nary study on t h e a n f l u e n c e of d o s a n g tame on o c u l a r a b s o r p t i o n of t o p i c a l l y a p p l i e d t i m o l o l (12), the h a g h e s t t i m o l o l c o n c e n t r a t i o n s at this time point w e r e a t t a i n e d at 1200 hr dosang, w h e r e a s the lowest were a t t a i n e d at 2400 hr dosing. The r a t i o of h i g h e s t to lowest timolol c o n c e n t r a t a o n s a m o n g the ant e r i o r s e g m e n t t i s s u e s / f l u i d s s a m p l e d was 3.47 ± 0.49. T h e l i g h t - d a r k v a r a a t i o n s in a n t e r i o r segment t i s s u e c o n c e n t r a t i o n s can be a t t r i b u t e d to c o r r e s p o n d i n g c h a n g e s in p r e c o r n e a l clearance, corneal p e r m e a b i l aty, or a q u e o u s humor turnover. L i g h t - d a r k v a r i a t i o n s in d r u g m e t a b o l i s m cannot be a factor sance t a m o l o l is m e t a b o l i c a l l y stable an the eye (13). Theoretlcally, h i g h t a m o l o l c o n c e n t r a t a o n s d u r i n g t h e light p e r a o d can be e x p l a a n e d by low p r e c o r n e a l clearance, hagh corneal p e r m e a b i l i t y , and low a q u e o u s h u m o r t u r n o v e r rate. A l t h o u g h t h e r e as, as yet, no i n f o r m a t a o n on c i r c a d a a n c h a n g e s an p r e c o r n e a l c l e a r a n c e and corneal p e r m e a b i l i t y in the p a g m e n t e d rabbit, the rate of a q u e o u s humor t u r n o v e r has b e e n r e p o r t e d by Smith and G r e g o r y (2) to f o l l o w a c i r c a d i a n r h y t h m w a t h low v a l u e s d u r a n g the light p e r i o d and h i g h v a l u e s d u r i n g the d a r k peraod. C o n s e q u e n t l y , high v a l u e s of t i m o l o l c o n c e n t r a t a o n s in the a q u e o u s humor (Fags. ic and 2c) and a r i s - c a l i a r y b o d y (Figs. if and 2f) d u r a n g the light p e r i o d can be attrabuted, at least in part, to c a r c a d i a n r e d u c t a o n in a q u e o u s humor t u r n o v e r rate r e p o r t e d by the above a n v e s t a g a t o r s (2). An a l t e r n a t a v e e x p l a n a t i o n for the l a g h t - d a r k v a r a a taons in t a m o l o l c o n c e n t r a t a o n s an a n t e r a o r segment t i s s u e s is b e h a v i o r a l c h a n g e s an the rabbat w h i c h m a y cause p h y s a o l o g a c a l changes. Rabbits, lake other r o d e n t s such as rats and m a c e (14), are n o c t u r n a l anamals. Thear d a f f e r entaal s e n s i t i v i t y to h a n d l a n g d u r a n g laght and d a r k p e r i o d s p r o b a b l y e x e r t s the most p r o n o u n c e d e f f e c t on p r e c o r n e a l d r u g c l e a r a n c e and aqueous humor t u r n o v e r (15,16). That l a g h t - d a r k v a r i a t i o n s an t i m o l o l c o n c e n t r a t i o n s exast an the c o r n e a l e p i t h e l a u m (Fag. Id) b u t not in t h e c o n j u n c t a v a (Fag. la), even t h o u g h b o t h t a s s u e s are b a t h e d by tears, suggests that l i g h t - d a r k v a r i a t i o n s in p r e c o r n e a l d r u g c l e a r a n c e m a y p l a y a s e c o n d a r y role to l i g h t - d a r k v a r i a t a o n s in d r u g u p t a k e and c l e a r a n c e an the c o r n e a and the c o n 3 u n c t i v a an c o n t r a b u t i n g to l a g h t - d a r k v a r a a t a o n s an drug c o n c e n t r a t a o n s an these two tassues. To summaraze, the t a m e of e y e d r o p a n s t a l l a t a o n a n f l u e n c e s the o c u l a r timoioi c o n c e n t r a t a o n s a t t a a n e d an p a g m e n t e d rabbat eye. Such d o s a n g t a m e - d e p e n d ent c h a n g e s an o c u l a r d r u g c o n c e n t r a t a o n s as an a d d i t a o n a l e x p e r a m e n t a l variable that m u s t be c a r e f u l l y c o n t r o l l e d an o c u l a r drug p h a r m a c o k a n e t a c studaes. It as also an a d d a t a o n a l factor that must be c o n s i d e r e d an p l a n n a n g d o s a n g r e g a m e n s and in d e s i g n a n g c o n t r o l l e d drug r e l e a s e systems. For timolol, the ratio of h i g h e s t to lowest v a l u e s as 3.34 ± 0.44 d u r i n g the light peraod, 1.92 ± 0.18 d u r i n g the d a r k peraod, and 3.47 ± 0.49 d u r a n g the 24-hr peraod.
Acknowledgments
tutes
Thas study was s u p p o r t e d an part by grant EY7389 from the National Instaof Health, Bethesda, M a r y l a n d and by the G a v a n S. H e r b e r t Professorship.
References i. 2. 3. 4. 5.
R.A. B R A S L O W and D.S. GREGORY, Invest. Ophthalmol. Vas. Sci. 2_88 1730-1732, 1987. S.D. S M I T H and D.S. GREGORY, Invest. Ophthalmol. Vas. Sca. 3__O0775-778, 1989. D.S. GREGORY, Invest. Ophthalmol. Vas. Sca. 3_! 715-721, 1990. J.M. ROWLAND, D.E. POTTER, and R.J. REITER, Curr. Eye Res. ! 169-173, 1981. A. BAR-ILAN, Exp. Eye Res. 39 175-181, 1984.
Vol.
51, NO. 26, 1992
6.
J.M. ROWLAND, W.K. SAWYER, J. TITTEL, and C.J. FORD, Curr. Eye Res. ~ 201206, 1986. R.H. LEVY and C.R. BANFIELD, in: "Pharmacokinetics," P.G. WELLING and F.L.S. TSE, eds., Marcel Dekker, Inc., New York, 1988. pp. 515. G.D. NOVAK, Surv. Ophthalmol. 3_!1 307-327, 1987. V.H.L. LEE, A.M. LUO, S. LI, S.K. PODDER, J.S.C. CHANG, S. OHDO, and G.M. GRASS, Invest. Ophthalmol. Vis. Sci. 3_/2 2948-2957, 1991. S.C. CHANG, D.S. CHIEN, H. BUNDGAARD, and V.H.L. LEE, Exp. Eye Res. 46 5969, 1988. J.H. ZAK, B~ostatistical AnalTsis, Prent~ce-Hall, Inc., N e w Jersey (1984). S. OHDO, G.M. GRASS, and V.H.L. LEE, Invest. Ophthalmol. Vis. Sci. 32 27902798, 1991. G.J. PUTTERMAN, J. DAVIDSON, and J. ALBERT, J. Ocular Pharmacol. 1_!1 287295, 1985. S. ARMSTRONG, J. CLARKE, and G. COLEMAN, Physiol. Behavior 2_!1 785-788, 1978. G.R. REISS, D.A. LEE, J.E. TOPPER, and R.F. BRUBAKER, Invest. Ophthalmol. Vis. Sci. 25 776-778, 1984. S. MISHIMA, A. GASSET, S.D. KLYCE,JR., and J.L. BAUM, Invest. Ophthalmol. 264-276, 1966.
7. 8. 9. i0. 11. 12. 13. 14. 15. 16.
Variatlons
in Ocular Timolol Concentrations
2031