Exp . E.111: RM. ( rn8+) 38. 1~1 -19+
`
`Comparison of the Ocular Hypotensive Efficacy of
`Eicosanoids and Related Compounds
`LASZLO z. BITO
`Ophthalmology Research, Department of Ophthalmology, Columbia University ,
`College of Pl.y,sir,ians and Surgeons, New York, NY 10032, U.S.A.
`
`(Received 29 April 198.1 and accepted 1 September 1983 , New York)
`
`It has recently hflf!n iihown that prostaghi.ndin (PG)E2 or F2« reduces the intraocular pressure
`(IOP) of caw ll>n<l primates when applied topically to the eye in very small doses, and that reduced
`TOP can be maint ained in these species as long a.~ the topical application of one of these PGs is
`repeated daily or twice daily. In the present study the ocular hypotensive efficacy and some of
`the ocular side-effects of 15 eico!lanoids and related compounds, especially derivatives of PGF ~·
`were eompared an<l were also compared to some clinically used ocular hypotensive agt:nts.
`Derivatives of PGF:., were found that had short-tt:rm and long-term ocular hypotensi\·e potencies
`some 10- to 5()-fold greater than PG!'h itself.
`Kf.y wnrdis: prostagla.ndins; pro11taglandin analogues; prostaglandin esters; PGF h ; eicosanoids:
`t imolol: carbachol; ocular hypot-ensi vcs ; cat; glaucoma.; ocular therapeutics ; intraocular pressure ;
`miosis.
`
`1. Introduction
`
`Prostaglandins (PGs) have generally heen regarded as ocular hypertensive agents and
`potent mediators of the ocular irritative and/or inflammatory response (for review,
`see Eakins, 1973, 1976, 19ii; Podos, .Becker and Kass, 19i3). However, a low dose
`{5 µg per eye) of topically applied PGF 2a has been shown to reduce, rather than
`increase intraocular pressure (IOP) in rabbits (Camras, Bito and Eakins, 1977),
`although higher doses (25-200 µg per eye) of PGF za and, especially, PGE2 have indeed
`been found to cause an increase in IOP and breakdown of the blood-aqueous barrier.
`Thus, at least in rabbits, there is an unacceptably narrow margin between the
`potentially therapeutic and pathogenic doses of these autacoids. Furthermore, rapid
`development of subsensitivity or tachyphylaxis to repeated topical applications of a
`hypotensive <lose of PGF2:r precluded long-term maintenance of reduced IOP in
`rabbits (.Sito, Draga, .Blanco and Camras, 1983a).
`In contrast, it has recently been shown that topical application of PGE2 or PGF2:x
`maintains reduced IOP in cats and Rhesus monkeys as long as a hypotensive dose
`of one of these PCs is applied at least once a day (Bito et al., 1983a). Although PCs
`of the E series are reputed to produce more severe ad•:erse effects i11 the mammalian
`eye than PGs of the F series (Beitch and Eakins, 1969 ; Eakins, 197i), topical PGE2
`treatment for up to nine months caused only minimal flare and cellular responses in
`the anterior chamber of cat eyes and produced no serious local or systemic side effects
`{Bito, Srinivasan, Baroody and Schubert, 1983b).
`In evaluating this potential new class of ocular hypotensive agents, it must be noted
`that the previously studied naturally occurring PGs may not be the best suited for
`clinical use. PGE 2 , for example. is not sufficiently stable in aqueous solution, while
`corneal penetration of the much more stable PGF2:1 is limited by its hydrophilic nature
`(Bito and Baroody, 1982). The present investigation was undertaken to evaluate the
`
`Plea~e address correspondenee to Dr La.s-do Z. Bito. Columbia t·nh·ersity, College of P & S, 630 West
`168th Stret>t, Xew York. XY 1om2. U.S.A.
`
`OClt.J-48:3,5/s.l/020181 + 14- S03.00/ 0
`
`,© 1984 Academic· Pre~~ Inc (London) Limited
`
`Micro Labs Exhibit 1049
`Micro Labs v. Santen Pharm. and Asahi Glass
`IPR2017-01434
`
`

`

`182
`
`L. 1/,. BITO
`
`ocular hypotensive efficacy of a wide spectrum of ei<'osanoids representing cla;;ses of
`compo unds with <lifferent physical, chemieal and biological properties.
`
`2. Materials and Methods
`
`Cats of mixed breeds and of eit her sex (2·5-4·0 kg) were traine<l daily for four to se,·en days
`to accept handling, periodic restraint in animal boxes and the perform ance of tonometry with
`only a topical anesthetic (Alcninc; Alcon Laboratories. Inc .. Fort, Worth, TX). 8ome cats
`had been trained and u1:W<l in a previous investigation (Bito et al., 1983a) that ended more
`than two months prior to this series of experiments. The eyes of all animals were examined
`by slit-lamp; only cats that showed no sign of ocular inflammation were used.
`Each PG, PG isomer or PG analogue was dissoln~d in vehicle solution and 50 µI wa;, applied
`topically to one eye of three to 1,1ix ell.ts in each experiment. The contralateral eyes which sen-ed
`as controls were untreated or given an equal volume of the vehicle solution alone. The
`following compounds were dissolved in 0·02°.0 Xa/'.0 3 in water: PGE2 , PGF2:1 free acid,
`PGF, 1
`, PG1'\p, PGD2, prostacyclin, 15-ket,o-PGFi.., 16.16-di methyl PGF2:o, and the PG
`analogue U-44069 ((15S ]·hydroxy-9ct, I la:· [epoxymethan,1]prnsta-tiZ, 13E-dienoic acid). The
`other PG analogue (Merck L-644, 122; 4-{3-[3-[2-( 1-hydroxycyclohexyl)ethyl)-4-oxo-2-thia(cid:173)
`zolidinyl)propyl}benzoic acid) was dissolved in water with a molar excess of .XaHC03 • The
`tromethamine sal ts of PGFb a-nd PGF2,,. were dissolved in normal saline, except for the
`highest concentration of PGF:,,-trometham(ne ( 100011.g per eye), which was dissoh·ed in
`dist illed water. PGF~ .. methyl. ethyl. and isopropyl esters n-ere each dissolved in peanut oil
`(Sigma Chemical Co., St Louis, MO), as was PGF22-free acid, in some experiments, to allow
`direct comparison of its effects to those of the PG esters. PGF2'1 ethyl and ispropyl esters
`were prepared from PGF2.:r obtained from The rpjohn Comp,,n~·- Kalamazoo, )II. Carbachol
`(750 µg per eye carbamylcholine chloride; Sigma) was <.Jissoh
`... n distilled water. An aqueous
`solut ion of timolol maleate powder 54·4 mg ml- 1 ()Ierck) wa,; !,re pared to yield a concentration
`of -10 mg ml- 1 of timolol equivalent, and was applied in a. ·:rl: 2,1 ;tl aliquot. For the lower
`dose (500 µg) . two 50 µl aliquots of a commercially aYailabie st, tion of 5 mg mJ- 1 timolol
`equh·alents (Timoptic; Merck) were applie(l I min apart. Tht' dost's of the PGF2.,- and
`PG1'\p-tromethamine salt are presented as PGF2:,, free-acid equivalents ( I ·34 mg of the salt
`equals 1 mg of free acid). All other doses are presented as total weight per volume. without
`correction for differences in molecular weights.
`·
`)lost compounds were dissolved in vehicle solution immediateh· before use and discarded
`t hereafter. Exceptions include the PGF2:.-tromethamine salt an~I the PGF2., esters. which
`were stored frozen for several weeks. In one experiment . 200 µg m1- 1 of PGF2:1 methyl-ester
`in peanut oil was allowed to stand in the dark for three months at room temperature before
`it was retested on a group of cats.
`Before the first application of each drug, one drop of 0·5 °·0 alca.ine was applied topically
`to obtain corneal anesthesia and TOP was measured with a floating-tip pneumatic tonometer
`(Pneumotonograph; Alcon). T he shortest median-lateral pupillary diameter of each eye was
`measured in dim light ( < 10 lux), using a mm-scale pupil gauge, and slit-lamp examinations
`were made. Cellular response and aqueous flare in the anterior chamber were evaluated by
`the same person throughout the series of experiments.
`Aqueous humor flare was rated according to the extent of the T yndall effect visible with
`the slit-lamp: none = O; barely visible using a bright slit = 0·5; easily visible -= 1; moderate
`to dense = 2; and dense with fibrin clots = 3. Cellular response was rated: no cell present = O;
`Yery few cells seen during a through examination of all regions of the anterior chamber = 0·5:
`some cells easily visible without a thorough examination = l ; many cells in ea.ch slit-lamp
`field = 2 ; and cells densely dispersed or contiguous (clumps) = 3. In most cases, animals were
`treated with one dose of a drug and examined periodically for 24 hr thereafter. Pupil
`diameters were measured under the same conditions as the initial measurement every 30 min
`for the first 3 hr and at 4, 5. 6 and 24 hr. lOP was measured 1, 2. 3. 6 and 2-1 hr. Slit-lamp
`examination of the a.ntR.rior chamber was repeated. in most cases. at 6 and :?-1 hr.
`In multiple-dose experiments sets of cats recei,·ed topiC'al application of the same dose of
`the same drug (PGI<\,.-tromethaminesalt, PGF2, methyl. ethyl orisopropyl ester. :\lerck L-644.
`122, or timolol) at 24-hr intervals for four days. In these cases. pupil diameters and IOPs
`
`...
`
`Micro Labs Exhibit 1049-2
`
`

`

`O<TL.-\R HYPOTE~St\·E POTE~C1E8 OF EICOS . .\XOIDS
`
`183
`
`werf> measured at I.~- 3. 6 and 24 hr after the first anrl fourth trf>atment-11. and at 6 and 24 hr
`a.ftcr the second and third treatments : slit·lamp examinations were performed at 6 and 24 hr
`after earh t.reatment.
`
`3. Results and Discussion
`
`Short-term ocular hypotensive efficacy
`Ocular hypotensive potency was found to vary considerably among the eicosanoids,
`their derivatives, analogues and metabolites. The fact that thP. initial metabolite of
`PG}'z,., 15-keto-PGF2x, and the isomer of PGF <n, PGF zp were less potent in reducing
`!OP than PGF22 itself (Table 1) is consistent with the fact that these compounds are
`less potent than POF2a in other organ systems (Anggard, 1966). The relative ocular
`hypotensive potencies of the three compounds suggest that the hypotensive effects
`of eicosanoids are mediat-ed by stereospecific 'receptors•.
`Of the five PGs surveyed (.PGE2 , PG1'\<X, PGD2, PG1 2, and PGF2,.), PGE2 showed
`the greatest ocular hypotcnsivc potency. Although PGI2 has been reported to be more
`potent than PGE 2 in many biological systems (Vane, 1978), it was found to be
`ineffective in reducing lOP at comparable doses. It should be noted, however, that
`because the half-life of PGI 2 in aqueous solution 1s only a few minutes (Gryglewski,
`Bunting, Moncada, Flower and Vane, 1976), only a small fraction of the PGI 2 , even
`when dissolved in its vP.hicle within 1 min prior to its topical application, can be
`expected to reach intraocular target sites before inactivation.
`The finding that PGE 2 has an apparently greater ocular hypotensive potency than
`PGF2,. or its tromethamine salt should not be taken as an indication that E-type PGs
`are better sui tcd for clinical use as ocular hypo tensive agents than F-type PGs. Indeed,
`intracameral administration of PGEs has been reported to produce more severe
`adYerse ocular effects than PGFs, including transient ocular hypertension, breakdown
`of the blood-aqueous barrier, and iridial hyperemia (Beitch and Eakins, 1969;
`Waitzman and King, 1967). Furthermore, PGEs, but not PGFs have been shown to
`have adverse effects on retinal electrical activities when administered intravitreally
`in large doses, particularly in animals pretreated with a PG transport inhibitor
`(Wallenstein and Bito, 1977 ; Siminoff and Bito, 1982), Lastly, PG Es are more labile
`in aqueous solution than PGFs (Roseman, Sims and Stehle, 1972). Thus PGs of the 1''
`series must be regarded as potentially more promising therapeutic agents for the
`long-term treatment of glaucoma. For this reason, most of the remaining compounds
`sdected for this study are related to PGF2a.
`PG analogues that are protected against enzymatic inactivation as a result of steric
`hindrance at the site of attack of 15-hydroxy-PG-dehydrogenase, the first step in the
`enzymatic inactivation of E and F PGs (Anggard and Samuelsson, 1966; Nakano,
`Anggard and Samuelsson, 1969), have been shown to be more potent than the parent
`PGs in other biological systems (Wiqvist, Martin, Bygdeman and Green, 1975). In our
`study, the representative of such sterically protected analogues, 16,16-dimethyl-PGF 2!t
`(Hansson a.nd Granstrom, 1976), showed no evidence of enhanced hypoterisive efficacy
`as compared to either PGF ui or its tromethamine salt (Table I). This finding is not
`surprising in that intra.ocular tissues cannot effectively metabolize PCs (Eakins, Atwal
`and Bhattacherjee, 1974; Bi to and Baroody, 1974}; thus, protection against metabolism
`is not expected to increase ocular hypotensive potency. Further studies on this
`sterically hindered derivative were not done at this time because only a limited supply
`of this analogue was available to us. Furthermore, in the absence of a substantial
`
`Micro Labs Exhibit 1049-3
`
`

`

`'l'All[.V. I
`Compari,wm of the ocular hypoten:_;ive potency* of eicosanoids and other drug.~ six hours after unilateral lo7Jica.l ap71liaition .to tlte /dine. P.!JI!.
`
`1()()0
`
`fiOO
`
`100
`
`50
`
`10
`
`5
`
`~-:,
`
`______ , ... _ __
`
`oc
`,I..
`
`Topically applied dose in µg per eye
`
`- 11 ±2-4
`(6)
`
`- 9 ±2·1
`(4)
`-:l±O·O
`(0)
`
`-8±1·:l
`(to)
`-2 ± 1·1
`(II)
`
`-4±o-!l
`( l 1}
`
`- 6±3·8
`(2)
`- 1± 0·5
`(7 )
`- 1±0·6
`(Ii)
`0± I ·Ii
`(4)
`
`PG1<;2
`
`PCr'i,2
`
`POD-;
`
`PGl1
`
`PGF;a
`
`PGJ<\at
`
`PG F ia ·trometha.mine
`
`PGF2p
`
`PG l<'zp·tromethamine
`
`I 5-kl'tO-PG l•\a
`
`16,16-dimethyl PGI<\:x
`
`POJ•'a ,mithyl t,11!Airt
`
`PG Fia ethyl el!tert
`
`I 'U 1•\a i>H>lll'Hfl,YI 1•14l1wt
`
`U-HOu!lt
`
`Mernk L-1144, 122§
`
`'L'imolol
`
`-
`
`Cilrluwhol (7!i0 µg)
`
`I ± 0·8
`((i)
`
`- 1±0·8
`(HJ
`l ± Mi
`{HJ
`
`-
`
`- <J·a±,>-4
`(Ii)
`
`- 7 ± 1'1
`(U)
`-2±1 ·1
`(7)
`- 2 ±0·8
`(6)
`0 ± 0·7
`(4)
`- 1± 0·3
`(H)
`-1 ±0·4
`(4)
`- 4±0·7
`(11 J
`-2 ± 2·(1
`(4J
`I ± 0·8
`(6)
`! ± (Hi
`(4)
`
`-
`
`- 1± (>-!)
`(!i)
`-1 ±2·!i
`(Ii)
`
`-6±1>-7
`(3)
`
`-
`
`l ±O·H
`(8)
`
`-:3±0·6
`( 16)
`
`-1±0·!)
`{14)
`
`O±<M
`(ta)
`
`I ±(H)
`(4)
`0±0·3
`(4)
`
`- :'i±(Ml
`( Iii)
`- 5 ± 0·!)
`(18)
`- 4 ± 0·!1
`ti~)
`
`0±0·7
`(3)
`- 3±0·8
`(12 )
`- 2±1·7
`(H)
`0±0·7
`(12)
`
`-2±1·1
`(8)
`- 2± 1·7
`(11)
`- 1±2·~
`(H)
`
`0 ± 0·7
`( 1-t)
`I ± 0·7
`(Ii)
`ll ± IMI
`(Ii)
`
`~
`N
`c.:,
`
`.--3
`0
`
`________ .,.
`
`• Mnnn ( IOl'.,x11) - (IOl'.,001J in mrnH~±!i.K.M., (1•J.
`t I 11 pn1mut oil.
`t ( l !i8}-hydl'Oxy-Ha, I la·(epoxy1m•.thano) prrnlt1i-fi1/., I :11,:-11i1:11oic 1i1:id.
`§ 4-{:\-\ :1-l t -( 1-hyclroxycyclt>hexyl)cthyl]·-l·Hxo-2-thia;,.olidinyl lpropyl) lumwic; 1ieitl.
`
`Micro Labs Exhibit 1049-4
`
`

`

`•
`
`u
`
`O<TL .\H HYPOTJ,;X:·;Jn; POT~:X<"IEK OF EIC'OS.-\'.\'OJD:--
`
`185
`potentiation of ocular hypotension , protection of PGs against metabolism in the rest
`of the hody. including the lungs, must he regarded as an unneceRRary riRk since such
`protection will enhance delivery of active forms of these compounds to other organ
`svsterrn;. and hence would increase the likelihood of .1dverse svstemic side effects.
`Studies in our laboratory have shown that the corneal epithelium is an effecti\'e
`permeability barrier to PGF2a (Bito and Baroody. l982 ). While penetration of PC Fi~
`through the sclera of the isolated globe was found to be relatively unrestricted, th is
`route of penetration must be hindered in the in situ eye by the conjunctiva. These
`observations suggest that the relative rates of their penetration through the outer
`coats of the globe is another important criterion in the selection of PG analogues for
`topical ophthalmic use. It has been well established that the permeability of the cornea
`to compounds of relatively low molecular weight depends largely on their lipid
`solubility. In the present study, PGF2~ methyl , ethyl and isopropyl esters were used
`to represent more lipid-soluble PG analogues. Since these compounds are virtually
`insoluble in water, they were dissolved in peanut oil, a vehicle that has been used
`clinically for an ophthalmic preparation of diisopropylfluorophosphate (Leopold and
`Comroe, 1946).
`As a result of their lipid solubility , the PGF2a: esters are likely to cross the corneal
`epithelium more readily than the more hydrophilic parent compound. Once such esters
`have crossed the epithelial barrier, esterases in the cornea (Lee, )lorimoto and
`Stratford, 1982) can be expected to liberate the hydrophilic free acid which, in turn,
`will diffuse tlirough the corneal stroma with little further hinderance. Because there
`is no e,·idence that the three PGF 2:x esters have greater efficacy per se, it is likely that
`their relatively high ocular hypotensive potencies (Table I) can indeed be attributed
`to greater penetration through the corneal epithelium, followed by de-esterification.
`Increased efficacy of delivery to intra.ocular tissues, combined with effect ive systemic
`and pulmonary metabolism , bot h of which apply to these PGF2:z esters, offer the
`important therapeutic advantage of reducing t he possibility of adverse effects on other
`organ systems.
`While all three .PGPz.,: esters were found to reduce IOP at much lower doses than
`PGF22 or its tromethamine salt, the methyl ester may not be best suited for long-term
`human ·use, since its hydrolysis results in the release of methyl alcohol. whose
`metabolites are known to be toxic to the eye. Although hypotensive doses of PGF2~
`methyl ester will yield only trace amounts of methyl alcohol per treatment, even such
`small quantities may be cause for concern when daily treatment must be given for
`several years. Thus, other lipid-soluble PGF 24 esters, such as the ethyl or isopropyl
`esters, even if they are not more potent ocular hypotensivcs than the methyl ester,
`may be more appropriate for long-term ocular use.
`Another important factor in the choice of an appropriate PG ester is its relative
`rate of hydrolysis whi_ch, in most biological systems, is determined by the molecular
`size and steric configuration of the ester, and by the enzymatic profile of the tissues
`in question. Delayed hydrolysis after penetration into the epithelial surface can be
`expected to result in a slower release of the free acid from the cornea. Thus the use
`of higher molecular weight or sterically hindered esters, either alone or in combination
`with more readily hydrolyzable forms, can be expected to increase the duration of t,he
`hypotensive activity of these derivatives.
`The PC analogues U-44069 and L-644, 122 were found to be less effect ive ocular
`hypotensive agents thatn PGE2 or P GF 2a·tromethamine salt, and were much less
`effecti\·e than t he PGF22 esters in reducing IOP six hours after their topical
`
`Micro Labs Exhibit 1049-5
`
`

`

`lilfi
`
`L.Z.B(TO
`
`application. Timolol , at a dose of 1000 p.g per eye , its commercial ophthalmic
`preparation (Timoptie, 0·;') 0,0 ) applie<l in two consecutiYe 50 µI volumes, and carbachol
`at the dose of 7,>0 µg per eye yielded only negligible IOP reductions as compared to
`that obtained six hours after the topical application of only 10 µg per eye of any of
`the three PGF2:,; esters (Ta.hie I). This implies that, in the cat, these POF2~ esters have
`at least 50 times greater hypotensivc potency than timolol or carbachol. Considering
`tha't t he therapeuticdoseofallother hypotensive agents are also in thcrangeofO·l-8 %
`while the concentration of the PGF 2:r esters required to yield the effective dose when
`applied in a typical t herapeutic volume of 50 µI per treatment is only 0·01-0·02% ,
`the PGF2 .. esters must be regarded as potentially more potent ocular hypotensive
`agents than any of the currently used anti-glaucoma drugs.
`Previous reports have already dealt with the effects of some topically applied
`eicosanoi<ls on the IOP of other species. Thus, in Rhesus (Bito et al., 1983a) and in
`Owl monkeys (Camras and Bito, 1981) PGF~ doses of 10~1000 µg per eye were shown
`to reduce IOP with little or no side effects. In contrast, topical application of even
`much lower doses of PGE 2 or PGF2~ to rabbit eyes caused an immediate and sustained
`rise in lOP that was followed by the development of flare in the anterior chamber as
`well as other signs of ocular irritation and inflammation (Camras, et al., 197i).
`Although lower doses of PGE 2, and especially PGFu., reduced the IOP of rabbits
`temporarily (Camras et al., 1977), such reduction could not be mainta ined even for
`a few days, since daily application of PG1'\.2 resulted in a gradual decrease in its ocular
`hypotensive efficacy that could not be overcome by increasing the d ose of PGF2:r.
`Indeed, at a time that tachyphylaxis to the hypo tensive effect of 5 µg of PGF u had
`developed, a higher dose (10 µ,g per eye) caused an increase, rather than a decrease
`in IOP (Bito ct al., 1983a).
`Kulkarni and Srinivasan (1 982) also examined the effects of topically administered
`eicosanoids on the eyes ofrahbits. Several members of the group tested, such as PG E 2 •
`6-keto PGE 1, P012 , and 6-keto PGF1;2 caused a transient unilateral increase in IOP
`at a dose of 10 µg per eye while, at the same dose, the stable endoperoxide analogue,
`l:-46619, was reported to cause a small bilateral IOP increase. Only in one instance.
`100 min after the topical application of 0· 1 µg of PGE 2, did these authors obsen·e a
`slight, b ut statistically significant decrease in IOP. It is apparent, therefore, that the
`rabbit ~ye is much more senstive to eicosanoids than eyes of other species and that
`PGs have a predominantly hypertensive, rather than hypotensive effect on the rabbit
`eye. These atypical responses of the rabbit eye t o PGs are not entirely surprising in
`,·iew of recent d emonstrations that the responses of the rabbit eye to many forms of
`ocular irritat ion, some of which are known to be PG-mediated in that species, are
`quantitatively, or even qualitatively, different from the ocular responses of other
`,·ertebrates to the same agents (Bito and Klein , 1981; Klein and Bito, 1983) .
`
`.llaintained ocular hypotensive efficacy
`The data presented in Table I clearly indicate that the esters of PGF2:x, as measured
`six hours after their topical application, are much more potent ocular hypotensives
`than any of the other su hstances studied. However , from the point of view of potential
`therapeutic use for the treatment of chron ic glaucomas, the ability of these esters
`to maintain lowered IOP during the course of prolonged daily treatment is a much
`more important criterion. Clearly, initial hypotensive efficacy is irrelevant in this
`regard if the long-term effects of a compound are blocked by tachyphylaxis or
`drug-induced subsensitivity.
`
`Micro Labs Exhibit 1049-6
`
`

`

`O<TL..\ R HY POTE-;:s-:,:J \' E POTEXCI Efi OF 1-~H'OlsAXO I l>:-i
`
`18i
`
`0
`
`2
`
`3
`
`4
`
`Days
`Fm. I. The effects of four consecutive daily application:! of different doi!es of PGF22 methyl-ester [(a)
`I; (h) 2·5; (c) 5 and (d) 10 µ.g/eye per treatment) on the intra.ocular pressure of cats as compared to the
`effeds of similar treatment with IO µg PGF2~ trometha.mine salt {e). One eye of each animal was treated
`with these PGs ( e ) a.s indicated with the arrows while the contrnla.teral eyN< !Ser\'ed a., untreated controls
`{ O ). The points represent ov~ans and the limits ± 1 s.1::. ~I.; n = 6.
`
`Our results show that during the course of four coni-ecutive daily treatments the
`hypotensive efficacy of POF2:i esters is not diminished but, in fa.ct, appears to be
`enhanced. For example, a threshold ocular hypotensive dose of PGF22 methyl ester
`(2·5 µg per eye) maintained reduced IOP for up to 24 hr after the fourth treatment,
`but not after the first one (Fig. 1 ). The maintained hypotensive effect of this ester was
`even more pronounced when the daily dose was increased to 5 or 10 µg per eye (Fig.
`1). Similar potentiation of maintained !OP reduction was apparent during daily
`application of PGF2:Z ethyl ester (.Fig. 2). Potentiation was not obi:;crved during daily
`treatment with 2·5 µg per eye of PGF zo: isopropyl ester, since this ester maintained
`some !OP reduction for 24 hr even after the first treatment. The longer duration of
`action following the first treatment with ispropyl ester may be explained on the basis
`that this ester is hydrolyzed more slowly, and hence is released from the cornea at
`a slower rate than the other two esters. Although further experiments will clearly have
`to be done to compare the relative duration and hypotensive efficacy of such esters,
`the present findings suggest that the isopropyl ester provides more effective long-term
`maintenance of IOP reduction than either methyl or ethyl ester. The PG analogue,
`L-664, 122, did not show a consistent IOP reduction during the four-day treatment.
`In contrast, timolol showed no tendency to potentiate its ocular hypotensive effects
`during the four days oftreatement. The reduction oflOP 24 hr after the second topical
`
`Micro Labs Exhibit 1049-7
`
`

`

`188
`
`L. 7, 8 fTO
`
`( o )
`
`i
`.
`Ob.
`
`I
`
`· ·<>-·· Control
`-
`Treated
`I
`i
`
`I
`
`~
`
`t:...,....__.
`
`••
`
`J,-~-- -- ~----~ r
`· r
`~r .... 1-& ·::_:_t·-;·-· i.i-··-.!
`
`I
`.
`-----rt-
`· r··
`• 2·5 J.49 PGF20 isopropyl ester
`
`.
`
`I
`~ .
`
`1
`I
`I
`..
`~(cl.,~ - - - ' • ____ J,
`
`20
`
`20
`
`15
`3 0
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`QJ
`
`O'
`I
`E
`..s
`'5
`CJ)
`</I
`~
`a.
`0 ·s
`
`<..)
`0
`~
`~
`
`25
`
`20
`
`15
`30
`
`25
`
`20
`
`25
`
`20
`
`~~J.49 Merc\\L-644,1~
`
`' (d)
`
`I
`
`l
`
`:
`
`~~.:.:-··¼:¢- ~ ~
`
`Q
`5001-'9 Timolol (timoptic)
`LL. .. --1--
`.i....--'---'----'--
`--'----'--
`i (e}
`I
`
`; 25 t~
`ZO f 1000,.g Timolol moleote
`
`--'--i
`
`0
`
`3
`
`4
`
`2
`
`Ooys
`
`Fw. 2. Comparison of the IOP effects of four consecutive daily topical applications of three ei<'osanoid
`deri\·atives: (a) 2·5 µg PGF22 ethy]-{lster; (b) 2·5 PG Fu isopropyl-ester; (c) 100 ,ug of the PG analogue.
`:\Jerek L644, 122 ; and (d) 500 µg of a. clinically ul>t'd anti-glau<'oma agent . timolol ; (e) 1000 µg timolol
`maleate. 8ee also legend to Fig. I. Treated ( e ) a.nd untreated e~;e controls ( 0).
`
`application of lOOO µg per eye of timolol appears to be greater than that after the third
`or fourth treatment. These findings a.re consistent with previous observations of the
`inability oftimolol to maintain a reduced IOP in cats (B. K . Colasanti, pers. comm.).
`
`1'he stability of PGF2~ esters
`~..\.nother important consideration in evaluating a potential drug for long-term use
`as a topical ocular hypotensive agent is its stability in an appropriate vehicle solution.
`The stability of PG!t\(1 esters dissolved in peanut oil was found to be excellent. One
`batch of 0·02% PGF2~ methyl ester was tested on the eyes of six cats 30 min after
`it had been dissolved in peanut oil. This solution was then kept at room temperature
`for over three months (93 days) and ultimately retested on six cats. Six hours after
`its initial application, the fresh preparation had produced a mean IOP reduction of
`5·2±0·8 mm Hg; three months later, the same preparation produced a decrease in IOP
`of 6·5 ± 1 ·7 mm Hg.
`
`~lliotic effect8 of eicosanoids
`Two hours after their topical application, all biologically active F-type PGs in doses
`from 1 to 1000 µg per eye were found to reduce pupillary diameter by 2-6 mm. The
`most effective miotic agent was PGFz:x isopropyl-ester which, at a dose of 1 µg per
`eye. reduced the mean pupillary diameter by 4·i ±0·3 mm as compared to the
`
`Micro Labs Exhibit 1049-8
`
`

`

`O(TL:\R HYP OTEXHf VF. POTF.XCIE8 OF' EI C08.-\XOTD::;
`
`189
`
`TA!il,F. II
`The extent of miosis at tu:o lwu1·s, anterior chamber fl.are at six hour8, a:nd cells at
`24 hr rifler topical application of eir.osanoid8 or other drugs
`
`Drug
`
`PGE2
`
`PGF 1,.
`PGD2
`PGI 2
`PG}\,,
`
`PGF u -trometha,mine
`
`F2p
`F,,1-trornt!thaminc
`
`15-keto PC:F2"
`
`16.16-dimethyl PGl-\,.
`
`PGF2" methyl ester
`
`PG}\" ethyl ester
`
`PGF1~ isopropyl ester
`
`l."-44069
`)lerck L-6-14. 122
`
`Timnlol
`
`Ca.rbachol
`
`l>o8e
`µg per eye
`
`10
`50
`100
`500
`100
`100
`100
`500
`5
`JO
`50
`100
`100
`100
`500
`50
`100
`500
`
`5
`1
`2·5
`5
`10
`1
`2·5
`5
`10
`J
`2·5
`5
`10
`100
`100
`500
`500
`IOOO
`750
`
`(n)
`
`(8)
`(3)
`(6)
`(6)
`(6)
`(4)
`(6)
`{5)
`(13)
`( 14)
`(Hi)
`(6)
`(6)
`(6)
`(6)
`(4)
`(4)
`(4)
`(:l)
`(3)
`(1 4)
`(18)
`(8)
`(16)
`(6)
`(tl)
`(6)
`(18)
`(6)
`(6)
`(12)
`(12)
`(5)
`(6)
`(6)
`(6)
`(6)
`(6)
`
`Pupil
`{cont)-(expJ
`mm
`
`0·3±0·:2
`2·0±0·0
`0±0
`3·0±0·8
`0±0
`0±0
`2·0 ±0·4
`3·i±l·2
`2·6±0·9
`3·8±1·0
`-!-3±0·5
`4·6±0·6
`0±0
`0±0
`0±0
`0±0
`0±0
`1·5± 1·5
`0±0
`1·0±0·6
`1·0 ± O·-l
`5·4±0·4
`-H±0·4
`54±0·6
`2·3±0·6
`5·0±0·-l
`5·6±0·5
`6·0±0·4
`•Vi±0·3
`5·9±0·9
`5·3±06
`5·9±0·4
`0±0
`0-5±0·2
`0±0
`2·8±0·3
`2·8±0·6
`3·2±0·7
`
`Flare•
`
`0±0
`0·9±0·2
`0·2±0·2
`0·9±0·1
`0·3±0·1
`0·3±0·1
`0·5±0
`2·1 ±0·2
`0·3 ±0-2
`0-2 ±0· 1
`0·2±0·1
`0·4±0·2
`0±0
`0·2±0·1
`0·9±0·2
`0±0
`0±0
`0±0
`0·3±0·2
`0·5±0
`0·6±0·2
`0·5±0·2
`0·8±0·1
`0·7 ±0·1
`0·9±0·2
`0·9±0-1
`1·3±0·3
`1·0±0-2
`1·1 ±0·2
`0·8±0·1
`1·2±0·2
`!·0±0·2
`0·6±0·2
`0·;}±0
`0·6 ±0·2
`0· 1±0·1
`O·i±0·2
`0±0
`
`Cells•
`
`,._ ... _,,, ___
`
`0·5±0·2
`O·i ±0<~
`0±0
`0·6±0·2
`0±0
`0±0
`0±0
`Hi±0-4
`0±0
`0±0
`0·2±0·2
`0·2±0·2
`0±0
`0±0
`0·8±0·3
`0±0
`0±0
`0±0
`0±0
`0±0
`0±0
`0·2±0·2
`0·8±0·3
`05±0·2
`0·5±0·:2
`0·2±0·1
`J·3±0·4
`0·8±0·2
`0·2±0·2
`0 ±0
`0·4±0·2
`1·0± 0·3
`0·2±0·1
`0·1±0·1
`0±0
`0·2±0·2
`0±0
`0±0
`
`• See )Iaterials and Methods for criteria. of rating the extent of eellular and flare responses in the
`,interior chamber.
`
`contralateral control eye (Table II). The development of maximal miosis was more
`rapid after the topical application of t he esters than after the application of
`PGF 2.2-tromethamine salt, and the period of maximum pupillary constriction tended
`to be longer (Fig. 3). Since the PG.Fu esters were at least 10 times more potent as
`miotic agents than the PGFu·tromethamine salt, duration of effect of the same
`dose could not be compared.
`\\'hen considering both the extent and the duration of pupillary constriction, the
`ethyl and the isopropyl esters appeared to be more effecti\'e miotics than the methyl
`
`Micro Labs Exhibit 1049-9
`
`

`

`tf)I)
`
`I.. 7.. BITO
`
`ester (Fig :3). Because the dosps of all eieosanoid:s used, \vith t he exception of the
`tromethami ne salt of P<.: l·\:z, are reportt'd as the actual weight applied to thE' eye. the
`larger moll'cular weight ethyl and isopropyl ester:,; reprE'gent ~lightly smaller amounts
`of applied PGF2x free acid. These con:,;iderations support the assumption that delivery
`of the bi ologically active POF~ to the anterior uvea was more effective after topical
`application of the PGFb t-sters than after topical application of the same dose of PGF2:z
`or its trornethamine salt.
`
`,>· • --'v• • . ..; ... · \~
`
`I O r--~?"~ ~~--
`8 L' ~..:-.;---.. ~- .,
`--0 · -- Control
`6; ( o J
`-
`Treoted
`4"
`fO µg PGF2c tromet hom,ne
`2 ~
`
`Hoors
`
`Fm. 3. The effects of (a) 10 and (b), 50 /tg of PGF1~-tromf'thamint> :salt or 5 µg PGF22 csters on the
`shortest(' horizon ta.I ') pupilla.ry diameter of cat eyes [(c) methyl ester; (u) ethyl e~ter and (e) isopropyl
`t-ster). One .. ye of each animal was treatl'd with a singlt' topical dose of eico~anoid ( e) while the eon(cid:173)
`tralatera.1 eyes sen·ed as untrc-at.t-tl controls (0). The points represent mt-,ins and the limits ± I s . ~ .. ,1.:
`II= 6.
`
`In contrast, PGF2,a yielded no miotic response, even after topical application of 100
`or 500 µg per eye, and the initial.metabolite of PGF2:x , 15-keto PGFi:x· caused no miosis
`at doses up to 100 µg per eye, and only a slight miosis at a dose of 500 µg per eye.
`This is consistent with the fact that these compounds have only very weak biological
`activity as compared to PGF2:i (Anggard, 1966). The PG analogues u-44069 and L-64-1,
`122, at a dose of 100 µg per eye, yielded little or no miosis (Table II ), indicating that
`t hese compounds are either delivered less effectively to intraocular tissues after their
`topical application or t hat they have less effect on the 'PGF Za: receptors' of t hese
`tissues.
`It has already been shown that topical application of the F, but not the E. PGs
`causes strong miosis in the eyes of cats (Stern and Bito, 1982: Bito et al. , 1983a).
`However, none of several eicosanoids administered intravitreally or topically in doses
`sufficient to produce other ocular effects had similar miotic effects on rabbit eyes
`(Kulkarni and Srinivasan, 1982 ; Bito, Nichols and Baroody , 1982}, and neither E nor
`
`Micro Labs Exhibit 1049-10
`
`

`

`<HTL • .\R HYPOTEXM\'1•.: POTE'.\CIES OF E ICO~AXO J D:-;
`
`l91
`
`F PGs ,vere found to have an appre('iable miotic effect on Rhesus monkey eyes (Stern
`and Bito. 1 !)82: Bito et al. , 1983a). Purthermorc. preliminary observations (Bito, L. Z.
`and Srinivasan, B. D., unpubl. obs.) indicate that PGF2" is also without rniotic effect
`on human eyes. It appears, therefore. that the extent of mio::.is induced in feline eyes
`by PGF2" may not be relevant to the miotic potential of these drugs in other species.
`including humans. None the les..,;;;, the relative miotic potency of these derivatives on
`cat eyes may be a useful criterion for the relative ocular penetration of derivatives
`of F-type PGs.
`
`Biom·icroscopic observations on the anterior segment of cat eyes