I
`
`Current
`kye
`Research
`
`Volume 9 number 11 1990
`
`Corneal and conjunctival/scleral penetration of p-aminoclonidine, AGN 190342, and
`clonidine in rabbit eyes
`
`Du-Shieng Chien, James J.Homsy , Charles Gluchowskil and Diane D.-S.Tang-Liu
`
`Departments of Pharmacokinetics and 'Chemical Sciences, Allergan Pharmaceuticals, 2525 Dupont Drive,
`Irvine, C A 92715, USA
`
`ABSTRACT
`The ocular penetration pathways o f three
`a,-adrenergic
`agents (p-ami noclonidine,
`AGN 190342, and clonidine) were Investlgated i n
`rabbits both i n v i t r o and i n vlvo. The corneal
`permeabilities of the compounds correlated
`p o s i t i v e l y with t h e i r octanol/water d i s t r i b u t i o n
`coefficients. The ocular drug absorptlon v i a
`corneal and conjunctival/scleral penetration
`routes was evaluated separately a f t e r drug
`perfusion i n vivo.
`I n most cases,
`the corneal
`route was the major pathway f o r the intraocular
`drug absorption. However, the conjunctival/
`scleral penetration pathway was the predominant
`pathway f o r the delivery o f p-aminoclonidine,
`the
`least l l p o p h i l i c compound among the three drugs,
`t o the c i l i a r y body. The drug concentration i n
`the i r i s was contributed mainly by the corneal
`route and correlated well with drug
`l i p o p h i l i c i t y .
`
`INTRODUCTION
`The cornea i s the major b a r r i e r for drug
`absorption after topical i n s t i l l a t i o n t o eyes.
`Only a small p o r t i o n o f the ophthalmic dose
`(~10%) penetrates the cornea and reaches the
`intraocular tissues. Drug absorption v i a the
`conjunctiva i s generally regarded as non-
`productive f o r intraocular drug bioavai l a b i l i t y .
`However several studies suggest t h a t conjunctival/
`scleral penetration may be important i n d e l i v e r i n g
`poorly absorbed drugs t o intraocular tissues
`(1-5).
`Ahmed and Patton demonstrated t h a t
`conjunctlval / s c l e r a l penetration was the major
`route f o r d e l i v e r i n g i n u l i n t o the i r i s and
`c f l i a r y body (4.5).
`The p o s s i b i l i t y o f the
`conjunctiva/sclera being an ocular d e l i v e r y route
`f o r peptides and proteins has also drawn much
`attention (6,7). The importance o f conjunctival/
`scleral penetration pathway for the small
`
`Received on May 7, 1990; accepted on 26 October 1990
`
`molecules was recently reported f o r &blockers
`and t i m o l o l prodrugs (8,9).
`The al-agonists
`such as phenylephrine
`have been widely used for the mydrlatic a c t i v i t y
`i n cataract surgery and i n r o u t l n e eye examination
`(10). Clonidine and AGN 190342 ( a l s o known as
`UK 14,304) are selective a2-adrenergic
`agonists and lower intraocular pressure (IOP)
`(11-14). Many of the a-agonists cause systemic
`side effects after topical use, p r i m a r i l y due to
`t h e i r penetration across the blood-brain b a r r i e r
`(15-17).
`Presumably, the l i p o p h i l i c drugs can
`penetrate across the blood-brain b a r r i e r more
`r e a d i l y than the hydrophilic ones and cause
`s l g n l f l c a n t central e f f e c t s . The compound,
`p-amlnoclonidine, more polar than clonidine and
`i s a l s o a potent ocular hypotensive
`AGN 190342,
`drug, but i t does not cause sedation side e f f e c t
`f o l l o w i n g i n s t i l l a t i o n (18-20).
`I n developing a-adrenergic compounds f o r
`i r i s and c i l i a r y body are the
`ophthalmic use,
`major t a r g e t tissues f o r drug d e l i v e r y . These
`tissues contain an abundance of the adrenoceptors
`mediating the ocular adrenerglc a c t i v i t y
`the primary objective i n
`(10,21,22).
`Therefore,
`the design of a-adrenergic drugs should be t o
`t a r g e t drug d e l i v e r y to irls and c i l i a r y body.
`This study used a corneal well to separately
`measure the i n v i v o drug penetration across the
`cornea and conjunctiva/sclera f o r three
`a -adrenergic agonists (p-aminoclonidine,
`2
`AGN 190342, and clonidine) i n r a b b i t eyes (23).
`Their chemical structures are presented i n
`Figure 1. These compounds vary i n b a s i c i t y (pKa)
`
`1051
`
`Page 1 of 9
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`SLAYBACK EXHIBIT 1034
`
`

`

`Current
`Eye
`Research
`
`p-Aminoclonidine
`
`Br
`
`H
`
`AGN 190342
`(UK 14,304)
`
`CI
`/
`
`H
`
`H
`
`\
`CI
`Clonidine
`
`Chemical structures of p-aminoclonidine,
`Figure 1.
`AGN 190342, and clonidine.
`
`The dependence of drug
`and lipophiliclty.
`penetration across different ocular surface
`membranes on the drug distribution coefficient was
`i nvestigated.
`
`MATERIALS AND METHODS
`Materials
`p-Aminoclonidine HCl and clonidine HCl were
`purchased from Sigma Chemicals (St. Louis, MO).
`AGN 190342 was synthesized in-house according t o a
`procedure previously described (24). Tritium-
`labeled p-aminoclonidine, AGN 190342, and
`clonidfne HCl were obtained from New England
`Nuclear (Boston, MA) with specific activities o f
`58.2. 63.0, and 73.2 Ci/mmol, respectively. The
`radiochemical purity (>98%) of each drug was
`verified by TLC or HPLC procedures as recommended
`
`by supplier (New England Nuclear, Boston, MA)
`before use. All other chemicals used were of
`analyt i cal or reagent grade.
`Female New Zealand rabbits (Vista Rabbi try,
`Vista, CAI, weighing 2.2 to 3.0 kg, without
`visible eye damage, were used. All experiments
`were conducted according to the Guiding Principles
`in the Care and Use of Animals of NIH.
`Determi nation of di s tr i but ion coef f i c i en t ( D O
`The octanol lwater di stri button coeff icients of
`the agonists were determined by dissolving each
`compound in 10 mM phosphate buffer (pH 7.4)
`presaturated with n-octanol.
`The drug solution
`was agitated with n-octanol (presaturated wl th
`aqueous phosphate buffer) in a separatory funnel.
`The drug solution was allowed t o sit at 2OoC for
`at least 12 hours t o reach distribution
`equilibrium. The drug concentration in the
`aqueous phase before (Cb) and after (Caf mixing
`with the octanol phase was determined by HPLC
`assays.
`The distribution coefficient (DC) was
`calculated by:
`
`V w
`x -Po-
`
`DC s ---cz--
`Cb-Ca
`where V o and Vw were the volume of n-octanol and
`water phases, respectively.
`Determlnation of corneal permeabi 1 1 ty
`The apparent corneal permeability (Papp) of
`the drug was determined using Ussing perfusion
`chambers in vitro as previously described ( 2 5 ) .
`Rabbits were sacrified by intravenous injection of
`e
`an overdose of T-61 euthanasia solution
`(Hoechst-Roussel Agri-Vet Company, Somervi 1 le,
`NJ).
`The eyes were enucleated before the corneas
`were excised and the corneas mounted between the
`perfusion chambers as described previously (25).
`Two and one-half ml of glutathione bicarbonated
`Ringer's solution (GBR) at pH 7.4 were added to
`the receptor chamber (facing the corneal
`An equal volume of GBR
`endothelial side).
`solution containing the non-radiolabeled drug was
`immediately added t o the donor chamber (facing the
`corneal epithelial side). A mixture of
`O,:CO, (95:5) was bubbled through the
`
`Page 2 of 9
`
`SLAYBACK EXHIBIT 1034
`
`

`

`~~~
`
`perfusion f l u i d i n both chambers a t a r a t e of
`three to f i v e bubbles per second d u r i n g t h e
`A t l e a s t s i x corneas were used t o
`experiment.
`determine the corneal permeabi 1 i t y for each
`compound.
`A l i q u o t s (50 p l ) were sampled from the
`receptor f l u i d every 30 minutes for f o u r hours and
`analyzed by HPLC for the drug content.
`The
`temperature of t h e chamber f l u i d s was maintained
`a t 35OC. A f t e r each sampling, an equal volume
`of f r e s h GBR s o l u t i o n was immediately added t o t h e
`receptor chamber t o maintain a constant volume.
`The hydration l e v e l of the wet cornea was
`determined a t t h e end o f each experiment and was
`79 2 2% ( W I W ) .
`Reversed phase HPLC assays were used t o
`The HPLC system
`q u a n t i f y the drug content.
`included an u l t r a s p h e r e ODS column (4.0 mm x 15
`cm, 5 pm, Beckman), a WISP autosampler (Waters),
`a solvent d e l i v e r y pump (114M, Beckman), and a UV
`The mobile
`detector (Spectroflow 783, Kratos).
`phase was aqueous acetoni tri l e (20-60% v/v)
`containing 5 mM sodium heptanesulfonic a c i d a t
`pH 3.5.
`The flow r a t e o f mobile phase was 1.0-
`The drug concentration was monitored
`1.5 ml/min.
`The r e t e n t i o n times for the drugs of
`a t 254 nm.
`i n t e r e s t were between 3 t o 10 minutes.
`The q u a n t i t y o f drug present i n the acceptor
`The slope
`f l u i d (q) was p l o t t e d versus t i m e ( t ) .
`(Aq/At) of the drug amount-time curves was
`determined by least-square
`l i n e a r regression.
`apparent p e r m e a b l l i t y c o e f f i c i e n t (Papp) was
`calculated by d i v i d i n g the slope o f the drug
`amount-time curve by the corneal surface area (A,
`-1.089 cm2) and the i n i t i a l drug
`concentration (Co) i n the donor f l u i d .
`
`The
`
`Aq/At
`Papp (cm/sec)= ------
`TcoTTAJ-------
`
`Measurement of corneal and c o n j u n c t i v a l / s c l e r a l
`p e n e t r a t i o n
`The o c u l a r drug absorption v i a corneal and
`c o n j u n c t i v a l / s c l e r a l p e n e t r a t i o n routes were
`separately conducted i n v i v o using a mechanical
`The r a d i o l a b e l e d drug
`blocking technique (3.23).
`
`Current
`Eye Research
`
`(-200 ng/ml) was f r e s h l y dissolved i n a 10 mM
`phosphate b u f f e r e d s a l i n e (PBS). Rabbits
`underwent general anesthesia w i t h an intramuscular
`i n j e c t i o n o f ketamine (35 mg/kg) and acepromazine
`(5 mg/kg).
`A p l a s t i c corneal w e l l , 12 mm
`i n
`diameter (0.d.) w i t h an 0.8 mm w a l l thickness, was
`secured along the corneoscleral j u n c t i o n of t h e
`The l e f t
`r i g h t eye by a cyanoacrylate adhesive.
`eye of each r a b b i t remained undisturbed.
`Two groups ( A and B) of r a b b i t s were used.
`I n
`group A. 0.3 m l of t h e drug s o l u t i o n was a p p l i e d
`i n the w e l l for corneal drug absorption.
`I n group
`6, 0.4 m l of the drug s o l u t i o n was
`i n s t i l l e d i n t o
`the o u t s i d e area of t h e w e l l ( c o n j u n c t i v a l
`t o measure t h e drug absorption through
`surface)
`the c o n j u n c t i v a and i t s underlying s c l e r a .
`I n
`group 6,
`t h e e y e l i d s were opened by a
`microdissecttng r e t r a c t o r (Roboz S u r g i c a l
`Instrument) to m a i n t a i n the drug s o l u t i o n i n t h e
`c o n j u n c t i v a l sac.
`I n both groups,
`the drug
`s o l u t i o n was replaced every 10 minutes t o maintain
`a constant drug concentration exposed t o the
`o c u l a r surface throughout t h e experiment.
`No
`leakage o f the drug s o l u t i o n through the glue-seal
`was observed.
`The o c u l a r surface t i s s u e s were perfused w i t h
`the drug s o l u t i o n for 60 minutes i n o r d e r to
`A t the end o f
`achieve d i s t r i b u t i o n e q u i l i b r i u m .
`the experiment, the drug s o l u t i o n on t h e o c u l a r
`surface was c o l l e c t e d and the corneal w e l l was
`The o c u l a r surfaces were thoroughly
`removed.
`r i n s e d w i t h normal s a l i n e and g e n t l y b l o t t e d d r y .
`The animal was then s a c r i f i c e d using T-61@
`euthanasia s o l u t i o n . Approximately 200 p1 of
`aqueous humor was a s p i r a t e d fol lowed by the
`d i s s e c t i o n o f the conjunctiva, cornea,
`i r i s , lens,
`c i l i a r y body, and the a n t e r i o r p o r t i o n of the
`sclera. A l l t i s s u e s except aqueous humor were
`combusted by a t l s s u e o x i d i z e r (Packard).
`The
`t o t a l r a d i o a c t i v i t y i n a l l samples was analyzed by
`l i q u i d s c i n t i l l a t i o n counting.
`The t i s s u e drug
`concentration a f t e r e i t h e r corneal o r c o n j u n c t i v a l
`dosing was c a l c u l a t e d by c o n v e r t i n g the t o t a l
`r a d i o a c t i v i t y t o the amount o f drug per gm or m l
`o f t i s s u e . The t o t a l drug concentration i n o c u l a r
`
`1053
`
`Page 3 of 9
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`SLAYBACK EXHIBIT 1034
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`

`

`Current
`Eye Research
`
`tissues, i f the e n t i r e o c u l a r surface had been
`exposed t o the dose, was the sum of drug
`concentrations a f t e r both corneal and
`con j u n c t i Val / s c l era1 dos i ng routes.
`Drug p a r t i t i o n i n g i n ocular surfaces and
`i ntraocular ti ssues
`The p a r t i t i o n c o e f f i c i e n t of the drug between
`the aqueous s o l u t i o n and the ocular tissues was
`dependent upon t h e physicochemical p r o p e r t i e s of
`the biophase and t h e drug. Assuming r a p i d
`d i s t r i b u t i o n e q u i l i b r i u m i n o c u l a r tissues.
`drug p a r t i t i o n c o e f f i c i e n t s i n v i v o were
`calculated as the r a t i o of drug concentrations i n
`the surface t i s s u e s (cornea and conjunctiva) to
`the dosing s o l u t i o n , or the r a t i o o f drug
`concentrations i n i r i s (or c i l l a r y body) to
`aqueous humor.
`
`t h e
`
`RESULTS
`Table 1 summarizes t h e molecular weights, pKa
`values, octanol /water d i s t r i b u t i o n c o e f f i c i e n t s ,
`and the corneal p e r m e a b i l i t i e s of c l o n i d i n e ,
`The
`AGN 190342. and p-aminoclonidine.
`d l s t r i b u t i o n c o e f f i c i e n t of c l o n i d i n e (3.31) was 2
`and 30 f o l d higher than t h a t of AGN 190342 and
`p-ami noc 1 oni d i ne , respect 1 v e l y .
`C1 o n i d i ne
`penetrated r a b b i t cornea approximately 4 and 83
`f o l d f a s t e r than AGN 190342 and p-aminoclonidine,
`r e s p e c t i v e l y .
`Ocular t i s s u e concentrations of
`p-am1 nocloni d i ne, AGN 190342, and c l o n i d i ne
`f o l l o w i n g d i f f e r e n t r o u t e s o f dosing are shown i n
`With drug exposure o n l y to the corneal
`Table 2.
`surface,
`the h i g h e s t drug concentration was
`observed i n the cornea,
`f o l l o w e d by aqueous humor
`
`Molecular weights, pKa, octanol/water d i s t r i b u t i o n c o e f f i c i e n t s ,
`Table 1.
`corneal p e r m e a b i l i t i e s , and drug p a r t i t i o n c o e f f i c i e n t s between s o l u t i o n and
`o c u l a r tissues (cornea and conjunctiva). and between aqueous humor and
`i r i s / c i l i a r y body o f p-aminoclonidine, AGN 190342, and c l o n i d i n e .
`
`p-Ami nocl oni d i ne
`
`AGN 1 90342
`
`C1 o n i d i ne
`
`Molecular Weight
`
`PKa
`
`245.1
`
`9.31
`
`292.2
`
`7.52
`
`230.1
`
`8 . 1 1 ~ 3 ~ 4
`
`D i s t r i b u t i o n C o e f f i c i e n t 5
`
`0.11 k 0.01
`
`1.48 fr 0.09
`
`3.31 fr 0.89
`
`Corneal Permeabilities6
`(X106 cm/sec 1
`
`0.44 2 0.15
`
`9.81 fr 1.29
`
`36.30
`
`1.45
`
`CorneaIDrug Sol u t i o n 7
`
`0.04 fr 0.01
`
`0.29 fr 0.04
`
`Conjunctiva/Drug Solution7 0.03 fr 0.02
`Iri s/Aqueous Humor8
`
`1.48
`
`C i 1 i a r y Body/Aqueous Humor8
`
`0.81
`
`0.06 fr 0.01
`
`1.02
`
`0.31
`
`0.38 fr 0.06
`
`0.02 * 0.01
`
`1.20
`
`0.22
`
`I Reference 26.
`pKa was determined from p o t e n t i o m e t r i c t i t r a t i o n method a t 2OOC.
`3 Reference 27.
`Reference 28.
`5 D i s t r i b u t i o n c o e f f i c i e n t (n-octanol /phosphate b u f f e r , 1OmM) was determined
`a t pH 7.4 and 20°C, mean fr SD, n=6-9.
`6 Mean fr SD, n=4.
`Mean
`SD, n=6-9.
`8 P a r t i t i o n c o e f f i c i e n t s were c a l c u l a t e d from mean value o f t h e t o t a l drug
`concentration absorbed v i a corneal and c o n j u n c t i v a l I s c l e r a l
`routes.
`
`1054
`
`Page 4 of 9
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`SLAYBACK EXHIBIT 1034
`
`

`

`Current
`Eye
`Research
`
`and i r i s . The c o n c e n t r a t i o n s i n the c i l i a r y body,
`lens, c o n j u n c t i v a , and s c l e r a were low f o r a l l
`t h r e e drugs. The d r u g c o n c e n t r a t i o n i n t h e o c u l a r
`t i s s u e s was h i g h e s t for c l o n i d i n e . the most
`l i p o p h i l i c of t h e t h r e e compounds, f o l l o w e d by
`AGN 190342 and p-ami n o c l o n i d i ne, t h e 1 e a s t
`l i p o p h i l i c .
`When the drug s o l u t i o n was d i r e c t l y perfused
`o n t o the c o n j u n c t i v a l surface,
`t h e drug
`concentration i n t h e c o n j u n c t i v a and s c l e r a was
`s i g n i f i c a n t l y increased and the drug
`concentrations i n t h e cornea, aqueous humor, and
`i r i s ( b u t n o t i n t h e c i l i a r y body) decreased t o a
`small f r a c t i o n o f those observed w i t h corneal
`dosing. AGN 190342 showed a higher drug
`
`c o n c e n t r a t i o n than c l on! d i ne and p-ami nocl o n i d i ne
`i n most o c u l a r t i s s u e s . The t o t a l t i s s u e drug
`concentrations absorbed v i a t h e corneal and
`c o n j u n c t i v a l / s c l e r a l r o u t e s f o l l o w e d a d i f f e r e n t
`rank o r d e r between these t h r e e compounds
`(Table 3 ) . These r e s u l t s show t h a t the drug
`a b s o r p t i o n i n c o n j u n c t i v a and s c l e r a became more
`s i g n i f i c a n t for t h e p o l a r p-aminoclonidine than
`for c l o n i d i n e or AGN 190342.
`Figures 2 and 3 compare the drug
`concentrations i n t h e i r i s and c i l i a r y body,
`r e s p e c t i v e l y .
`I n t h e i r i s , t h e l i p o p h i l i c
`c l o n i d i n e showed t h e h i g h e s t drug c o n c e n t r a t i o n
`(13.9 ng/g) among t h e t h r e e drugs,
`f o l l o w e d by
`AGN 190342 (7.8 ng/g) and p-aminoclonidine
`
`Table 2. Ocular drug c o n c e n t r a t i o n (ng/ml o r ng/g) o f p-aminoclonidine, AGN 190342, and c l o n i d i n e
`absorbed v i a corneal and c o n j u n c t i v a l I s c l e r a l r o u t e s 1
`
`Tissue
`Concentration P-Aminoclonidine
`
`AGN 190342
`
`C l o n i d i n e
`
`P-Aminoclonidine
`
`AGN 190342
`
`C l o n i d i n e
`
`Corneal Route
`
`C o n i u n c t i v a l / S c l e r a l Route
`
`Con j unc ti va
`
`Scl e r a
`
`Cornea
`
`Aqueous Humor
`
`I r i s
`
`C i 1 i a r y Body
`
`Lens
`
`0. 2302
`(0.212)
`
`0.164
`(0.155)
`
`8.032
`(2.598)
`
`0.554
`(0.136)
`
`0.657
`(0.269)
`
`0.170
`(0.080)
`
`0.016
`(0.003)
`
`0.414
`(0.157)
`
`0.471
`(0.118)
`
`58.490
`(7.119)
`
`7.542
`(0.887)
`
`7.295
`(0.951)
`
`1.506
`(0.481 1
`
`0.265
`(0.052)
`
`0.678
`(0.172)
`
`0.712
`(0.217)
`
`76.471
`(12.129)
`
`11.399
`(1 .709)
`
`13.411
`(2.494)
`
`2.126
`(0.71 1)
`
`0.815
`(0.151)
`
`5.985
`(3.230)
`
`2.069
`(1.537)
`
`1.459
`(0.505)
`
`0.038
`(0.015)
`
`0.216
`(0.085)
`
`0.312*
`(0.235)
`
`0.004
`(0.002)
`
`10.894
`(1.301)
`
`3.369
`(0.796)
`
`4.104
`(1.720)
`
`0.131
`(0.087)
`
`0.541
`(0.259)
`
`0.839
`(0.372)
`
`0.010
`(0.004)
`
`4.057
`(2.022)
`
`1.223*
`(0.734)
`
`2.209
`(0.647)
`
`0.126
`(0.063)
`
`0.459
`(0.154)
`
`0.369
`(0.178)
`
`0.010
`(0.003)
`
`~~~
`
`f o r 60 minutes.
`1 Rabbit eyes were perfused w i t h drug s o l u t i o n (-200 ng/ml)
`2 Mean ? SD, n=6-8.
`3 *Not s t a t i s t i c a l l y d i f f e r e n t (p>O.l) when compared t o t h e d a t a o b t a i n e d from t h e corneal r o u t e .
`
`1055
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`

`Current
`Eye
`Research
`
`Table 3. Total drug concentration of
`p-aminoclonidine, AGN 190342, and clonidine in
`ocular tissues after a 60 minute perfusion.
`
`X from
`Total Drug
`Concentration Corneal
`Route
`(ng/ml or ng/g)
`
`% from
`Conjunctival/
`Scleral Route
`
`p-Aminoclonidine
`6.22
`Conjunctiva
`2.23
`Sclera
`9.49
`Cornea
`Aqueous Humor
`0.59
`Iris
`0.87
`0.48
`Ci 1 iary Body
`Lens
`0.02
`
`ACN 190342
`Conjunctiva
`Sclera
`Cornea
`Aqueous Humor
`Iris
`Ciliary Body
`Lens
`
`11.31
`3.84
`62.59
`7.67
`1.84
`2.35
`0.28
`
`Clonidine
`Conjunct i va
`Sclera
`Cornea
`Aqueous Humor
`Iris
`Ciliary Body
`Lens
`
`4.74
`1.94
`78.68
`11.53
`13.87
`2.50
`0.83
`
`3.1
`7.3
`84.6
`93.6
`75.3
`35.3
`80.0
`
`3.7
`12.3
`93.4
`98.3
`93.1
`64.2
`96.4
`
`14.3
`36.8
`97.2
`98.9
`96.7
`85.2
`98.8
`
`96.3
`92.7
`15.4
`6.4
`24.7
`64.7
`20.0
`
`96.3
`87.7
`6.6
`1.1
`6.9
`35.8
`3.6
`
`85.7
`63.2
`2.8
`1.1
`3.3
`14.8
`1.2
`
`The drug concentration of clonidine
`(0.9 ng/g).
`in the ciliary body was similar to that of
`AGN 190342 (-2.4 ng/g) despite their different
`lipophillcity. Both compounds showed
`approximately 4 times higher drug concentration
`than p-aminoclonidine in the ciliary body. The
`total iris drug concentrations of clonidine,
`AGN 190342. and p-aminoclonidine were
`
`1056
`
`-
`.
`5 12
`c - 9
`c
`0 .-
`s
`=
`ii
`
`P)
`P)
`
`c
`d)
`0
`c
`
`P)
`
`15
`
`-r
`
`
`
`
`
`
`
`9
`
`6
`
`3
`
`0
`
`p-Aminoclonidine
`
`AGN 190342
`
`Clonidine
`
`Figure 2. Drug concentrations (ng/g) in iris after
`a 60 minute perfusion. Data are expressed as mean
`Drug absorption via corneal
`standard error.
`drug absorption via conjunctivallscleral
`route;
`total drug absorption in iris.
`
`3
`
`-
`
`T
`
`p-Aminoclonidine
`
`AGN 190342
`
`Clonidine
`
`Figure 3. Drug concentrations (ng/g) in ciliary
`body after a 60 minute perfusion.
`Data are
`expressed as mean
`standard error.
`Drug
`absorption via corneal route; H drug absorption
`via conjunctivallscleral route; H total drug
`absorption in ciliary body.
`
`approximately 5.6. 3.3, and 1.8 fold higher than
`those in the ciliary body, respectively. The drug
`absorption in the iris was mainly contributed by
`the corneal route (75-9773 for all three drugs
`(Table 3). The corneal route was the major
`pathway o f drug absorption in the ciliary body for
`clonidine and AGN 190342 (>64%). The
`conjunctival/scleral route was important for
`p-aminoclonidine (-65%).
`
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`

`The p a r t i t i o n c o e f f i c i e n t s ( t i s s u e l w a t e r ) a r e
`summarized i n Table 1.
`The cornea/water p a r t i t i o n
`c o e f f i c i e n t c o r r e l a t e d p o s i t i v e l y t o the drug
`l i p o p h i l i c i t y , approximately 2.5-8.7
`f o l d l e s s
`than t h e i r corresponding octanol /water
`d i s t r i b u t i o n coefficients.
`There was no obvious
`r e l a t i o n s h i p between the p a r t i t i o n i n g i n t o the
`conjunctiva and the drug l i p o p h i l i c i t y .
`AGN 190342 showed approximately twice the
`conjunct i valwater p a r t i t ion coeff i c i ent than
`c l o n i d i n e and p-aminoclonidine.
`The p a r t i t i o n
`c o e f f i c i e n t o f t h e drug i n t o the conjunctiva was
`approximately 1.3-19.1
`f o l d l e s s than t h a t i n t o
`the cornea.
`The drug p a r t i t i o n i n g from aqueous humor i n t o
`the i r i s and c i l l a r y body are a l s o shown i n
`Table 1. The drug p a r t i t i o n C o e f f i c i e n t s i n t o the
`i r i s o f c l o n i d i n e , AGN 190342, and
`p-aminoclonidine ranged between 1 and 1.5, higher
`than the drug p a r t i t i o n c o e f f i c i e n t s into the
`I n c i l i a r y
`cornea, conjunctiva, and c i l i a r y body.
`body, p-aminoclonidine showed a higher
`drug
`p a r t i t i o n c o e f f i c i e n t (-0.8) than AGN
`90342 and
`f o l d ,
`c l o n i d i n e by approximately 2.5 and 3.6
`r e s p e c t i v e l y .
`Drug d i f f u s i o n between cornea and
`c o n j u n c t i v a / s c l e r a was observed for a1
`compounds (Table 2). S i g n i f i c a n t drug
`concentrations were detected i n the c o n j u n c t i v a
`and s c l e r a when the drugs were perfused onto the
`corneal surface. The l a t e r a l d i f f u s i o n from the
`cornea t o conjunct1 va/sc l e r a c o r r e 1 ated p o s i t 1 v e l y
`with the drug l i p o p h i l i c i t y . Clonidine had the
`highest drug concentrations i n the conjunctiva and
`s c l e r a among these three compounds.
`f o l l o w e d by
`AGN 190342 and p-aminoclonidine. When t h e drug
`was perfused o n t o the c o n j u n c t i v a l surface, drug
`t r a n s f e r t o the cornea from c o n j u n c t i v a / s c l e r a was
`a l s o noted. AGN 190342 demonstrated more l a t e r a l
`than p-ami nocloni d i ne and c l o n i d i ne .
`diffusion
`The s c l e r a l drug absorption v i a
`corneal-scleral
`l a t e r a l d i f f u s i o n and
`conjunctival I s c l e r a l p e n e t r a t i o n are compared i n
`Figure 4.
`The study r e s u l t s show t h a t l a t e r a l
`diffusion from cornea t o s c l e r a c o n t r i b u t e d
`
`three
`
`Current
`Eye Research
`
`5
`
`T
`
`p-Aminoclonidine
`
`AGN 190342
`
`Clonidine
`
`i n s c l e r a
`F i g u r e 4. Drug concentrations (ng/g)
`a f t e r a 60 minute p e r f u s i o n . Data a r e expressed as
`mean
`standard e r r o r .
`Drug absorption v i a
`from cornea; H drug absorption
`l a t e r a l d i f f u s i o n
`v i a c o n j u n c t i v a l / s c l e r a l route;
`a b s o r p t i o n i n s c l e r a .
`
`approximately 35% o f t h e t o t a l drug absorption I n
`the s c l e r a for c l o n i d i n e ; however, i t o n l y
`c o n t r i b u t e d l e s s than 15% o f the t o t a l drug
`absorption for p-aminoclonidine and AGN 190342.
`
`DISCUSSION
`the corneal well was used to
`I n t h i s study,
`determine t h e o c u l a r drug absorption v i a corneal
`and c o n j u n c t i v a l / s c l e r a l pathways i n vivo.
`Although t h i s study design does n o t t r u l y evaluate
`the o c u l a r pharmacokinetics o f the drug f o l l o w i n g
`t o p i c a l dosing, i t does provide a simple and
`steady-state c o n d i t i o n t o examine the p e n e t r a t i o n
`mechanism v i a corneal and c o n j u n c t i v a l l s c l e r a l
`routes by continuous p e r f u s i n g drug s o l u t i o n o n t o
`the o c u l a r membranes.
`This system i s completely
`devoid o f complication by precorneal f a c t o r s such
`as b l i n k i n g ,
`t e a r turnover, and drug washout.
`The study r e s u l t s show t h a t t h e corneal
`p e r m e a b i l i t y c o r r e l a t e d posi t i v e l y with the drug
`l i p o p h i l i c i t y . A t pH 7.5, approximately 22% and
`50% o f c l o n i d i n e and AGN 190342, r e s p e c t i v e l y , a r e
`present as t h e unionized f r e e base I n the s o l u t i o n
`i s unionized.
`w h i l e o n l y 1.7% of p-aminoclonldine
`The unionized drug presumably penetrates corneal
`membrane f a s t e r than t h e i o n i z e d one, so t h a t t h e
`
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`

`Current
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`
`corneal permeability of p-amlnoclonidine i s 82
`times less than that of clonidine and AGN 190342.
`The drug concentration of p-aminoclonidine in iris
`and ciliary body was approximately 10 fold less
`than that of clonidine. Our data indicate a
`posi tlve correlation between intraocular drug
`concentration and drug 1 lpophi 1 ici ty, a1 though
`other factors such as drug clearance from the eye
`a1 so affect ocular tissue concentrations.
`Figure 5 describes the possible pathways of
`drug absorption and distribution into the
`intraocular tissues. For the adrenergic agents
`tested in this study, the drug in aqueous humor
`primarily comes from the corneal penetration
`before its distribution to other intraocular
`tissues. The drug in cornea also diffuses
`laterally into the sclera. The intraocular drug
`absorption was solely dependent upon the drug
`penetrating across the cornea, and therefore the
`
`I ti-'
`
`Ocular Drug Absorption
`
`Precorneal Tear
`
`I
`
`I
`
`I
`
`I
`
`Conjunctiva
`
`intraocular bioavailability of the drugs
`correlated positively with their corneal
`permeabilities.
`Conjunctival absorption might result In
`systemic drug absorption via the conjunctival and
`episcleral blood vessels and ocular drug
`absorption via the underlying sclera. Our data
`demonstrate that the conjunctival lscleral
`penetration is not as discriminatory to the drug
`1 1 pophi 1 1 city as compared to the corneal route.
`Three compounds showed the similar drug
`concentrations in irls and ciliary body via the
`conjunct i Val /scl era1 absorption. The simi 1 ar
`results for p-blockers were reported that the
`conjunctival/scleral penetration was less
`sensitive t o changes in drug lipophilicity as
`compared t o the corneal route (8.9). It was also
`indicated that the likely route of drug access to
`the intraocular tissues for hydrophilic compounds
`i s through conjunctival/scleral pathway. Our
`study results show that the conjunctival/scleral
`route i s the major pathway for the drug absorption
`of hydrophilic p-aminoclonidine into ciliary body
`as shown in Table 3.
`The use of the corneal well allowed us t o
`detect the magnitude of lateral drug diffusion
`between cornea and sclera. The lateral drug
`diffusion from cornea t o sclera can be important
`for drug absorption into the ciliary body because
`of the tissue adjacency. Our study results
`demonstrate the existence of lateral diffusion for
`all three drugs; however, the importance of
`lateral diffusion on intraocular drug absorption
`needs to be further investigated.
`
`Aqueous Humor
`
`Sclera
`
`I
`
`?i
`I
`
`L*:++,l
`
`I
`
`+
`
`Ciliary Body
`
`\I/
`Systemic Circulation
`
`* d /
`
`Figure 5. Pathways o f ocular drug absorption.
`4 Corneal route i s the major penetration
`pathway for the most drug absorption after topical
`insti 1 lation; --+Conjunctival /scleral route
`becomes important for the hydrophilic
`compounds. .--------> Corneal-scleral lateral diffusion
`i s also observed.
`
`ACKNOWLEDGEMENTS
`The authors greatly thank Dr. Ming-Fai Chan for
`synthesizing AGN 190342.
`
`CORRESPONDING AUTHOR
`Diane D.-S.
`Tang-Liu. Ph.D.
`Department of Pharmacokinetics
`A1 1 ergan Pharmaceut i cal s
`2525 Dupont Drive
`Irvine, California 92715, USA
`
`1058
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`

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