Micro Labs Exhibit 1061
`Micro Labs v. Santen Pharm. and Asahi Glass
`IPR2017-01434
`
`

`

`1
`
`4,599,353
`
`USE OF EICOSANOIDS AND THEIR
`DERIVATIVES FOR TREATMENT OF OCULAR
`HYPERTENSION AND GLAUCOMA
`
`The invention described herein was made in the
`course of work under U.S. Public Health Service Re-
`search Grant Numbers EY 00333 and EY 00402 from
`the National Eye Institute, Department of Health and
`Human Services.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`In primates, intraocular pressure is measured with a
`tonometer. A normal reading for a healthy, adult pri-
`mate eye would be in the range 14 to 24 mm Hg.[See
`generally DeRousseau,C. J. and Bito, L. Z., EXP. EYE
`RES.32:407-417 (1981); Kornblueth, W., et al., ARCH.
`OPHTHALMOL.72: 489-490 (1964).] An increase of
`about 4 to 7 mm Hg. above the average reading for a
`specific subject would be indicative of ocular hyperten-
`sion.
`
`20
`
`Glaucoma, an eye disorder afflicting various mam-
`mals, including primates, is characterized by increased
`intraocular pressure (ocular hypertension). In man, such
`ocular hypertension results from an imbalance between
`the rate of secretion of aqueous humorby theciliary
`epithelium into the anterior and posterior chambers of
`the eye and the rate of outflow or drainage of the aque-
`ous humor from the anterior and posterior chambers,
`primarily via the canal of Schlemm.It is generally be-
`lieved that obstruction of aqueous humordrainageis the
`primary cause of the imbalance.
`Chronic glaucoma typically results in slow, progres-
`sive loss of visual fields, and, if not controlied, ulti-
`mately in blindness. Initial treatment usually involves
`topical application of miotics, particularly pilocarpine
`and carbachol. If treatment with miotics is not effective,
`systemic administration of carbonic anhydrase inhibi-
`tors may be employed. If such approaches are unsuc-
`cessful, the glaucoma may haveto be treated by sur-
`gery.
`The treatment of human glaucoma with miotics is
`unsatisfactory for several reasons. The miotics may
`destroy a patient’s night vision or cause ciliary muscle
`spasms. Moreover, long-term use of miotics may result
`in the developmentof tolerance (tachyphylaxis) to the
`miotics, necessitating the use of progressively higher
`doses. Finally, miotics may cause discomfort or other
`undesirable side effects. Long-term use of carbonic
`anhydraseinhibitors has likewise been found unsatisfac-
`tory. Such use may produce adverse systemic results or
`lead to the developmentof cataracts.
`Eicosanoids and their derivatives include numerous
`biologically useful compounds. For example, the pros-
`taglandins (PGs), a group of eicosanoids which contain
`cyclical fatty acids, are known to possess diverse bio-
`logical activities. Originally isolated as lipid-soluble
`extracts from sheep seminal vesicles and human seminal
`fluid, prostaglandins have now been found in most
`mammalian tissue, although in lesser concentrations.
`Activities of prostaglandins include stimulation of
`smooth muscle, dilation of small arteries, bronchial
`dilation, lowering of blood pressure, inhibition of gas-
`tric secretion, of lipolysis and of platelet aggregation,
`and induction of labor, abortion and menstruation.
`It has been previously believed that administration of
`PGs, particularly PGE2, increases intraocular pressure
`based uponthe results of studies involving intracameral
`
`35
`
`40
`
`45
`
`65
`
`2
`and intravitreal injection of PGs into mammalian eyes.
`Accordingly, most research in this area focused on the
`use of prostaglandin antagonists rather than prostaglan-
`dins per se in the treatment of glaucoma.
`More recently, studies of the effect of exogenous
`administration of PGs in cannulated and uncannulated
`rabbit eyes showed that topical aand intravitreal appli-
`cation of about 25 to 200 wg. PGE2 or PGE2q per eye
`produced a short hypertensive phase, followed by hy-
`potony. [Camras, C. B., Bito, L. Z. and Eakins, K. E.,
`INVEST. OPHTHALMOL.VIS. SCL, 16:1125-1134
`(1977)] However, a small dosage of PGF29, about 5 wg,
`topically applied on rabbit eyes, produced a long period
`of hypotony, without any significantinitial rise in intra-
`ocular pressure. Id. Other studies have shown that rab-
`bits produce tolerance or tachyphylaxis to intracamer-
`ally or topically administered PGs.
`[Eakins, K. E.,
`EXP. EYE RES., 10:87 (1970); Beitch, B. R. and Ea-
`kins, K. E., BRIT. J. PHARM., 37:158 (1969); Bito, L.
`Z. et al., ARVO, 22(No. 3):39 (1982)]
`In addition, studies on species variations in ocular
`irritative and inflammatory response have shown that
`vertebrates such as primates and birds, which depend
`primarily on vision for sensory input, have more com-
`plex eye structures than rabbits, including more sophis-
`ticated ocular defense mechanisms. Accordingly, the
`eyes of primates and birds respondto topical application
`of chemicalirritants in a manner unlike those of rabbits.
`This phenomenon maybe dueto the fact that the ciliary
`processes in rabbits are morphologically different from
`those of other species. In rabbits, there are abundant
`iridial ciliary processes which are uniquely susceptible
`to breakdown,e.g., by neuronalirritation or paracente-
`sis, and deterioration of the blood-aqueous barrier. This
`propensity for breakdown appears to have an important
`protective function for rabbits which have highly ex-
`posed eye globes. Because of its exaggerated ocular
`irritative response, the rabbit has been widely used in
`studies of the role of PGs in ocular inflammation. In
`contrast, primates show a qualitatively different re-
`sponse to paracentesis: protein entry through the canal
`of Schlemm rather than breakdownofthe ciliary pro-
`cesses. [Raviola, EXP. EYE RES.25 (Supp.):27 (1977)].
`Accordingly, use of the rabbit eye as a model for pri-
`mates has been discredited except in ocular inflamma-
`tion studies. [Bito, L. Z. and Klein, E. M., EXP. EYE
`RES. 33:403-412 (1981); Klein, E. M. and Bito, L. Z.,
`PROC. INT. SOC. EYE RES.1:65; Klein, E. M. and
`Bito, L. Z., INVEST. OPHTHALMOL.VIS. SCI. 20
`(Supp.):33 (1981)].
`SUMMARYOF THE INVENTION
`
`A methodfor treating glaucoma and ocular hyperten-
`sion in primatesis disclosed comprising topical! adminis-
`tration of an effective amount of an eicosanoid to the
`afflicted eye. Repeated application, preferably daily,
`provides long-term reduction of intraocular pressure,
`without development of tachyphylaxis. Eicosanoids
`which may be employed for purposes of the present
`invention include prostaglandins and their derivatives,
`for example, PGE2, PGF2a andtheir derivatives. C1 to
`Cs alkyl esters of PGF2g, particularly PGF2,-methyl-
`ester, are presently preferred.
`Pharmaceutical preparations in accordance with the
`present invention comprise effective amounts of eicosa-
`noids and an ophthalmically acceptable carrier. Suitable
`carriers include sterile saline solution, peanut oil and
`mineral oil.
`
`Micro Labs Exhibit 1061-2
`
`Micro Labs Exhibit 1061-2
`
`

`

`3
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`4,599,353
`
`Ocular hypertension and glaucoma can be controlled
`in afflicted primates by topical application ofeffective
`amounts of eicosanoids. Periodic application of eicosa-
`noids reduces elevated intraocular pressure levels to
`normal values which continue during the course of
`treatment without development of
`tachyphylaxis.
`Treatments are preferably applied daily.
`Of the family of eicosanoids, prostaglandins (PGs)
`have been found particularly effective. In particular,
`PGE) and PGF2¢, and derivatives thereof have pro-
`vided long-term effectiveness. Daily application of
`PGE2 and PGF»,or their derivatives in amounts vary-
`ing from about 0.01 yg to about 1000 wg, per eye has
`been found effective In monkeys the preferred ranges
`would be 0.1-500 yg;
`in man the preferred ranges
`would be 0.1-1000 pg.
`Lipid soluble PGE2 and PGF2q derivatives are par-
`ticularly preferred for use in treatment of ocular hyper-
`tension. Such lipid solubility permits more ready pene-
`tration of the protective layers of the primate eye andit
`has been found that smaller quantities of such com-
`pounds can be used than non-liquid soluble PGs. In
`particular, C1 to Cs alkyl esters of PGF2q, such as
`PGF, methyl! ester, PGF2, ethyl ester, PGF29 isopro-
`pyl ester, and PGF2, isobutyl ester, would be suitable
`liquid soluble PGF2¢ derivatives. Such liquid soluble
`compoundsare effective in lower amounts, e.g. from
`about 0.01 to about 100 yg per eye. In man the preferred
`range would be from about 0.1 to 100 yg, particularly
`between about 1 pg to 50 pg.
`Physiologically acceptable salts of PGF2a and PGE,
`or their derivative can also be employed. In particular,
`. PGF, tromethamine would besuitable for use in treat-
`ment of intraocular hypertension. Other suitable salts
`would include PGF2¢ in sodium carbonate.
`invention
`Compositions according to the present
`would generally comprise effective amounts of an
`eicosanoid or an eicosanoid derivative and an ophthal-
`mically compatible carrier. Suitable ophthalmically
`acceptable carriers include sterile saline solution, an
`anhydrous peanutoil or a mineraloil. If prostaglandins
`and their derivatives are used, as noted above, the quan-
`tities topically applied to the primate eyeare relatively
`small. Accordingly, compositions according to the
`present
`invention will generally be about 0.01% to
`2.0% solutions of PGs (or PG equivalents if PG deriva-
`tives are used). Compositions according to the present
`invention containing PGF, and PGF2, tromethamine,
`and sodium salts of PGF2q may be employedinsterile
`saline solutions. The hyrophobic esters of PGF
`(methyl ester, ethyl ester, etc.) may be employed in
`sterile anhydrous peanutoil.
`EXPERIMENT1
`
`Thefirst experimentis also reported in Camras, C. B.
`and Bito, L. Z, CURRENT EYE RESEARCH
`1:205-209 (1981),
`the disclosure of which is hereby
`incorporated by reference into the present application.
`Five normal owl monkeys (Aotustrivirgatus), 3 males
`and 2 females; 0.8 to 1.0 kg, and one female with unilat-
`eral angle recession glaucoma were conditioned to ac-
`cept handling, restraint, and tonometry without anes-
`thesia. The intraocular pressure (IOP) of both eyes was
`measured over a one-year period at random intervals,
`but at least once each month. One drop of 0.5% propar-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`40
`
`45
`
`55
`
`60
`
`65
`
`4
`acaine hydrochloride (Alcaine; Alcon Corp., Forth
`Worth, TX) was applied to the eye before IOP was
`measured with a floating tip pneumatic tonometer
`probe attached to a pressure transducer and a recorder.
`Each animal wasplaced in the supine position on the lap
`of the investigator and 2 or 3 IOP measurements, each
`several seconds in duration, were taken. The best
`steady-state segments of the IOP tracings were read and
`averaged. Pupillary diameter was measured in normal
`room light with a pupil gauge. Anterior chamberflare
`and cellular invasion were determined byslit lamp ex-.
`amination.
`The tromethamine salt of PGF2q was dissolved in
`physiological saline to yield PGF2_ concentrations of
`20, 40, 80 or 200 mg/ml. In each experiment5 pl of one
`of these solutions was applied to one eye of each mon-
`key. The eyes were rinsed 3 to 5 min. later with 2 to 4
`mis of saline. An equal volume (5 pl) of saline was
`similarly applied to the contralateral control eyes fol-
`lowed by rinsing. Measurements of IOP, pupillary di-
`ameter, and slit lamp evaluation of aqueous flare and
`cellular content of the anterior chamber were made at
`various intervals after PGF2, application.
`RESULTS
`
`Normal Owl Monkey
`Topical application of 0.2 mg of PGF2_ to one eye
`(left eye in 2 and right eye in 3 animals) of the 5 normal .
`owl monkeysdid notresult in significant effects on the
`IOP as comparedto the baseline IOP of the treated eye
`or the simultaneously measures IOP in the contraleteral
`eye. However, topical application of 1 mg of PGF2, to
`the left eye of these animals 4 to 14 days after the first
`trial resulted in a prolonged hypotonyin the treated eye
`compared with the contralateral eye. In 3 of the 5 eyes
`this hypotony was preceded by a 2-3 mm Hgrise in
`IOP occurring 15 min after treatment and showing
`borderline significance compared with the contralateral
`eye. A prolonged hypotony was also observed when
`the same dose of PGF29 was applied 6 days later to the
`contralateral (right) eyes of these monkeys, or whenit
`was applied 18 days later to the originally treated eyes.
`Although the extent of ocular hypotension in the
`treated eye was about the sameafter each application of
`1.0 mg of PGF2a, the significance of the IOP differences
`between treated and contralateral eyes was reduced on
`subsequent PG application because of an apparent con-
`tralateral hypotensive effect. These IOP effects on the
`untreated contralateral eyes were not due to diurnal
`variations since tonometry done over a 24-hr period on
`the eyes of these same animals after bilateral saline-
`treatment or after unilateral treatment with a low dose
`of (0.2 mg) PGF2¢ did not showsignificant lowering of
`IOP.
`One half hour after topical application of 1.0 mg
`PGF, there was an average of 2.00.3 mm pupillary
`miosis compared to the contralateral control eyes. A
`gradual return to normal pupil size (4.80.2 mm) oc-
`curred over the next 18 hr. Slight aqueous flare was
`present in 4 of 5 eyes between 2 and 12 hr after the
`topical application of 1.0 mg of PGF2,. At 48 hr, a few
`cells were observed in 3 of the 5 treated, but in none of
`the control eyes. There was no apparent correlation
`between JOP reduction and the presence of flare and
`cells in the anterior chamber, i.e., the ocular hypoten-
`sion was not associated with a notable inflammatory
`response.
`
`Micro Labs Exhibit 1061-3
`
`Micro Labs Exhibit 1061-3
`
`

`

`5
`
`4,599,353
`
`Glaucomatous Owl Monkey
`Whenpurchased, one female monkey had eyes exhib-
`iting a marked anisocoria with the right pupil being a
`consistent 2 mm larger than the left. Gonioscopic exam-
`ination of the right eye revealed angle recession. The
`mean of 46 IOP measurements taken over a period of
`one year was 47.2++0.7 and 24.5+0.6 mm Hg for the
`right and left eyes respectively. Eleven months before
`this study on the effects of PGF2a, topical application of
`1% pilocarpine reduced the IOP by 4 mm Hgin theleft
`eye, but raised the IOP of the right eye by 16 mm Hg.
`Oxotremorine (0.05%) also increased the IOP of the
`right eye.
`Within 20 min after application of 1.0 mg of PGF2to
`the right eye of this owl monkey, IOP dropped from an
`average pretreatment value of 50 mm Hg to 32 mm Hg,
`followed by a more gradual decline during the next 12
`hr, ultimately reaching a value similar to that of the
`control eye and as low as 14 mm Hg. The IOP ofthe
`two eyes then remained similar for about 3 days, fol-
`lowed by a gradual return in the right eye to pretreat-
`ment IOP levels of 50 mm Hg.
`there was
`During this period of normotension,
`marked clearing of the corneal hazeofthe right eye, but
`this haze reappeared as the IOP rose to its baseline
`values in the 40-50 mm Hg range. However,for several
`weeks thereafter, the IOP of this eye appeared to be
`much morelabile than it was before the PGF29 applica-
`tion.
`
`EXPERIMENT 2
`
`Fourteen cats of either sex (2.5 to 3.5 kg) and two
`female rhesus monkeys (Mucaca mulatta; 3.8 and 4.0 kg)
`werelightly tranquilized with 5-10 mg/kg of ketamine
`(Ketaset; Bristol-Myers Co., Syracuse, NY). Such doses
`of ketamine were found to tranquilize rhesus monkeys
`without significantly altering their IOP. The monkeys
`were kept in primate chairs throughout each experi-
`ment.
`One drop of 0.5% proparacaine hydrochloride (A1-
`caine; Alcon Corp., Fort Worth, TX) was applied to
`each eye and IOPs were measured with a Pneumon-
`tonograph (Alcon Corp.) which was calibrated on the
`eyes of several species, including rhesus monkeys. New
`animals were accustomed to the tonometer by taking
`several readings the day before they were to be used in
`an experiment. Several sets of baseline readings were
`taken 0.5-1 hr before each experiment and the best
`steady state readings were averaged. Pupillary diame-
`ters were measured in normal room light with a pupil
`gauge. In cats, the nasotemporal (shorter) diameter was
`always recorded. In several experiments, the pupillary
`diameters of cats were re-measured in total darkness,
`using infrared illumination and an infrared image con-
`verter. Anterior chamber flare and cellular invasion
`were determined by slit
`lamp examination. A 50-yl
`aliquot of a solution containing one of several concen-
`trations of prostaglandin Ez (PGEz2), converted to its
`soluble sodium salt with the addition of an equimolar
`amount of Na2COs3, or the tromethaminesalt of prosta-
`glandin Fre (PGF2¢; The Upjohn Co., Kalamazoo,
`Mich.), both dissolved in saline, was topically applied to
`one eye of each cat or monkey. An equal volume of
`physiological saline was applied to the contralateral
`eye. In one set of experiments, two cats were pretreated
`with 10 mg/kg of indomethacin (Sigma Chemical Co.,
`St. Louis, MO)injected i-p. at 24, 16 and 2 hrs. prior to
`
`ho 0
`
`25
`
`30
`
`45
`
`35
`
`60
`
`65
`
`6
`the topical administration of the PG solution; two other
`animals received no such pretreatment. All of the drugs
`were madejust prior to their administration. In another
`experiment, both eyes of a set of four cats were treated
`with 125 pl of 0.5% atropine (Isopto atropine, Alcon
`Corp.) 20 min. prior to administration of the PG solu-
`tion. In all cases, measurements of IOP, pupillary diam-
`eter and slit lamp examinations for flare and cellular
`invasion of the anterior chamber were madeat various
`intervals up to 72 hr after the application of PGs.
`Because ofthe limited availability of rhesus monkeys,
`different doses of PGs were tested on each eye of two
`animals in a random sequence. At least seven days
`elapsed between any two applications of PG-containing
`solution to the same eye. Cats were re-used to a much
`morelimited extent; only one PG solution was tested on
`each eye of most cats, allowing at least one week be-
`tween eachtest. In some cases, an eye which showed no
`observable response or only a moderate response to a
`low dose of PG wasused for a secondtime, but not less
`than two weeks after it was first treated with a PG
`solution.
`
`Cat Results
`
`Topical application of up to 1000 wg of PGE»to the
`cat eye produceda significant decrease in IOP with the
`maximum reduction, as compared to the IOP of the
`contralateral eye, occurring between 1 to 8 hr after PG
`administration. The greatest and most prolonged hypo-
`tensive response was observed in eyes given 500 pg of
`PGE. In eyes which were subjected to less frequent
`tonometry, the IOP remained 6 mm Hg below baseline
`for 48 hr; this hypotension was not preceded by an
`initial hypertensive phase. In contrast, topical applica-
`tion of 1000 pg of PGE» produced a distinct initial
`ocular hypertension between 0.25 and 2 hr followed at
`6 hr by a maximum decrease of 11.7 mm Hg below the
`IOP of the contralateral control eye. Topical applica-
`tion of the same doses of PGF2_ produced IOP re-
`sponsessimilar in magnitude and duration to those pro-
`duced by PGE).
`Topical administration of 1.0 wg of PGF29 caused a
`threshold miotic response, decreasing the pupillary
`diameter by an average of 1.5 mm, from 11 mm to 9.5
`mm at 1 hr. An approximately one-half maximal miotic
`response occurredafter the topical application of 5 pg
`of PGF2, with a decrease in pupillary diameter of over
`5 mm at 2 hr. A dose of 100 pg of PGF2, produced an
`apparently maximum miotic response (9.5 mm decrease
`in pupillary diameter) within 2 hr, which not exceeded
`in extent or duration in eyes treated with a ten-fold
`greater dose (1000 wg) of PGF2,. Topical pretreatment
`of cat eyes with 0.5% atropine, which wassufficient to
`block the pupillary light reflex, did not affect the miotic
`potency of topically applied PGF29. The administration
`of similar doses of PGE) resulted in far more moderate
`miotic responses. The threshold miotic dose of PGE?
`was 100 wg and even a 100-fold greater dose produced
`only a sub-maximal decrease in pupillary diameter
`(from 10 mm to 2.5 mm), followed by rapid re-dilation.
`In one experiment, in which 2 out of 4 cats were
`pretreated with indomethacin (10 mg/kg i.p.) prior to
`the topical application of PGE, no difference in either
`the miotic or IOP response was observed between in-
`domethacin-pretreated and controlcats, indicating that
`the IOP lowering effect of PGE2 was not due to the
`stimulation of the synthesis of PGs and/orrelated cy-
`clo-oxygenase products from endogenous precursors.
`
`Micro Labs Exhibit 1061-4
`
`Micro Labs Exhibit 1061-4
`
`

`

`4,599,353
`
`8
`TABLE 2a
`Extent and duration of IOP reduction in rhesus monkeys induced by
`the topical application of PGF2a or PGE).
`Intraocular Pressure
`——___(mmHg)
`:
`Max.
`Baseline
`Reduction
`(OD)
`(OS)
`(exp—cont)
`
`Prosta-
`glandin
`dose/eye
`PGE2g
`100 pg
`
`500 pg
`
`1000 pg
`
`Eye
`
`A*(OS)
`A (OD)
`B (OD)
`A (OD)
`A (OS)
`B (OD)
`B (OS)
`A (OS)
`B (OS)
`B (QD)
`
`23
`24
`27
`25
`24
`26
`21
`25
`28
`25
`
`24
`26
`28
`25
`25
`26
`21
`25
`28
`26
`
`-—7
`—5
`—8
`—6
`—8
`—8
`—8
`-9
`—6
`—2
`
`PGE?
`100 pg
`
`-7
`25
`25
`A (OS)
`—7
`25
`24
`A (OD)
`—4
`26
`26
`B (OS)
`*A and B refer to the two monkeysused in this experiment
`
`7
`Several sets of cats had their pupillary diameters mea-
`sured in both normal room light and complete darkness
`(with the aid of an infrared image converter) at the time
`when they showed a maximum pupillary constriction.
`The pupils of both eyes dilated slightly in complete
`darkness (by 1 to 3 mm) as compared totheir diameters
`in room light, but the difference between the pupillary
`diameters of the PG-treated and the contralateral con-
`trol eyes was only minimally affected.
`Flare was not observed undercarefulslit lamp exami-
`nation in any ofthese cats at any time after the topical
`application of up to 1000 ug of PGF2a. However, some
`flare was observed in the anterior chamber of most cats
`2-18 hr after the topical application of 100 or 500 yg of
`PGE, but not after the application of 10 ug of PGE2.
`
`Rhesus Monkey Results
`
`Topical application of 100, 500, or 1000 pg of PGF2a
`to the eyes of rhesus monkeys produced a significant
`decrease in IOP within 2 hr; application of a much
`lower dose, 10 wg, did not have a similar effect. While
`insignificant initial increases in IOP were observed fol-
`lowing application of 100 or 500 yg of PGF2a, 1000 pg
`of PGF2q produced a brief (<30 min) initial IOP in-
`crease of 8 mm Hg, followed by a more prolonged
`decrease in IOP to 5 mm Hgbelowbaseline. The appli-
`cation of 100 wg of PGE2 or PGF2g produced very
`similar IOP effects, with maximum decreases of 5 and 6
`mm Hg, respectively. The IOP of eyes treated with
`PGE, however, returned to baseline values more grad-
`ually than eyes which received PGF2,. With both PGs,
`somereduction in IOP was maintained for 3 to 10 hr.
`No miosis was observed in rhesus eyes after the topi-
`cal application of any of the PGF2_ doses used here.
`However, 100 pg of PGE2 produced a small butsignifi-
`cant and brief decrease (3 mm) in pupillary diameter,
`followed by re-dilation to near baseline values by 2 hr
`after PG administration. Noflare or cellular invasion of
`the anterior chamber of this species was detectable by
`careful slit lamp examination at any timeafter the topi-
`cal application of 100 pg of PGE2 or up to 1000 pg of
`PGF.
`Tables 1 and 2 summarize results obtained in Experi-
`ment2.
`
`
`TABLE1
`
`Comparison of maximum IOP reduction 3
`to 6 hrafter unilateral topical application of
`various doses of PGE, or PGF2a. to cat _eyes.*
`Mean difference (exp-cont)
`in IOP (mm Hg)
`
`PGE2
`4.5 +21
`~12.0 + 14
`—13.8 + 0.8
`—11.8 + 3.6
`
`PGF2a
`—4.8 +11
`—8.8 + 0.8
`—9.7 + 0.3
`113 + 24
`
`Dose
`pg/eye
`10
`100
`500
`1000
`
`*]OP was measured at 3, 4 and 6 hrafter the topical application ofthe indicated dose
`of PGE, or PGF2a.Thelargest negative value IOPex,) ~ IOPgon:) observed for
`each animal during these three measurements was used in all cases to calculate the
`means.
`
`10
`
`_ 5
`
`20
`
`35
`
`35
`
`60
`
`65
`
`.
`
`Duration of
`> 50% IOP
`reduction (hr.)
`
`3
`3
`4
`5
`3
`6
`5
`5
`5
`5
`
`5
`6
`4
`
`EXPERIMENT3
`
`Fourteen cats of mixed breeds and ofeither sex (2.5 to
`3.5 kg) were trained daily for 4-7 days to accept han-
`dling, periodic restraint in animal boxes and tonometry
`withoutthe use of general anesthesia. One drop of 0.5%
`proparacaine hydrochloride (Alcaine, Alcon Corp.,
`Fort Worth, TX) was applied topically to each eye and
`IOPs were measured using a floating-tip pneumatic
`tonometer (pneumotonograph; Alcon Corp.). Pupillary
`diameters (naso-temporal) were measured in normal
`room light and/or in dim light with a millimeterruler.
`All eyes were examined with a slit-lamp and only ani-
`mals which showed no signs of ocular inflammation
`were includedin this study.
`A 50-1 aliquot of 0.2 mg/ml Na2CO3 in saline or a
`saline solution containing 100 or 500 pg of prostaglan-
`din E2 (PGE2) or F2 (PGF2) was topically applied to
`one eye of each animal typically at 24-hr intervals, but
`in some cases at 12-, 48-, or 72-hr intervals. An equal
`volumeof vehicle solution was applied to the contralat-
`eral eye. Based on the prior experiment (Experiment2),
`the dose of PGEapplied at each treatment throughout
`the 7-month period was 100 pg/eye, with the exception
`of the 100th day of treatment when 500 pg was applied
`to the experimental eyes of these animals. This high
`PGE) dose, however, resulted in the development of
`pronounced flare in the anterior chamber of every
`treated eye and was therefore not applied again. An-
`otherset of 6 cats received unilateral topical application
`of 100 or 500 pg/eye of PGF2. for shortertime periods.
`IOPs and pupil diameters were measured, in most cases,
`every day at approximately 9 AM (just before the morn-
`ing PG treatment), and on most daysat 1, 3, 4 and 6 hr
`after the morning treatment. Whentreated twice daily,
`the second treatment was given between 9 and 10 PM.
`The protocolincluded rinsing of the tonometer probe in
`saline solution between each IOP reading in order to
`minimize the chances of transferring topically applied
`PGs from the experimental to the control eyes of these
`animals. Slit-lamp examinations were performed 4 to 5
`hr after some PG applications and anterior chamber
`flare and cellular invasion were rated.
`Similar experiments were also performed on two 5- to
`7-year-old female rhesus monkeys. Both of these ani-
`
`Micro Labs Exhibit 1061-5
`
`Micro Labs Exhibit 1061-5
`
`

`

`4,599,353
`
`9
`mals had been used intermittantly in ocular drug studies
`over the previous 3 years, most recently to establish the
`single dose of topically applied PGF2, required to re-
`duce IOP in this species (Experiment 2). However,
`neither animal had been used in any study for 3 months
`prior to the experiments described here. Both animals
`were restrained in primate chairs throughout the pres-
`ent experiment. One animal required light tranquiliza-
`tion with Ketamine HC! (Ketaset; Bristol Labs., Syra-
`cuse, NY; 20-30 mg/kg im.),
`in addition to topical
`anesthesia (Alcaine), before each IOP reading. The
`other animal cooperated sufficiently to permit tonome-
`try to be performed under topical anesthesia only. One
`eye of each animal was treated twice daily (between 9
`and 10 AM and between 4:30 and 10 PM) for 6 days
`with 50 pl of a solution containing 100 pg of PGF2o.
`Starting on the 7th day the dose was increased to 500
`pg/eye per treatment for 12 days with the exception of
`the 9th day, when only the morning treatment was
`given, and the 10th day, when the animals received no
`treatment. Beginning on the 25th day, each PGF2g dose
`was increased to 1000 ppg/eye for 5 days. IOP readings
`were typically taken immediately before the morning
`treatment and at 2, 4 and 6 hr thereafter.
`Thefree acid of PGE2 was converted to its more
`water-soluble sodium salt with the addition of equimo-
`lar amounts of Na2CQ3in saline just before each treat-
`ment. The more water-soluble and highly stable trome-
`thamine salt of PGF2_ was periodically made up in
`saline and refrigerated for use over several days.
`Intraocular Pressure Results
`
`Baseline tonometry, taken thrice daily for 4-7 days
`prior to treatment, indicated no significant difference
`between the IOPs of the left and right eyes of cats.
`Within 1 hr after the unilateral topical application of 100
`pe PGE? (0.2% solution) to cat eyes, the IOP of the
`treated eyes was significantly (<0.01; paired t-test)
`lower than baseline. Although somereturn toward the
`pretreatment IOP level was observed by6 hr, the IOP
`of the PGE2-treated cat eyes remained significantly
`(p<0.02 lower even 24 hr after the first PGE2 applica-
`tion than the pretreatment baseline IOP of these eyes or
`the concurrently measured IOP of the contralateral,
`saline-treated eyes. A second application of 100 ug of
`PGE:to the same eyes immediately following the 24-hr
`IOP reading produced a more gradual decrease in IOP;
`however, the magnitude of the maximal and maintained
`hypotensive effects, observed respectively at 3 and 24
`hr after the second treatment, were greater than those
`achieved after the first PGEapplication.
`The lowest 9 AM IOP value was measured 24 hr
`following the fourth treatment and was maintained at
`approximately this low level for the subsequent 3 days
`of this treatment regimen, although further IOP reduc-
`tions were observed within thefirst 2 hr after each daily
`PG application. Between the 7th and 10th days and the
`105th and 123rd days following the initial PG applica-
`tion, the eyes of these cats were treated with the same
`dose (100 wg/eye) of PGE2 twice daily, producing a
`greater decrease in IOP than typically observed during
`the daily treatment periods. During twice daily treat-
`ment, IOP fluctuations between PG applications were
`minimal.
`The IOPs of the contralateral control eyes showed
`some fluctuations which, for the most part, were much
`smaller in extent and less consistent than the IOP reduc-
`tions observed in the treated eyes; someofthese fluctua-
`
`10
`tions, however, appeared to be temporally associated
`with, although somewhat delayed as compared to, the
`PG-induced IOP reduction in the treated eye.
`When PGE)treatment of these cat eyes was sus-
`pended for 72 hours between the 10th and 13th, 14th
`and 16th, and 115th and 118th days, a significant in-
`crease in the 9 AM IOPofthe experimental eyes was
`observed. When, beginning on the 20th day, these cats
`received one PGE2 treatment every other day over a
`period of 10 days, the IOP ofthe treated eyes was main-
`tained for several days below the level measured prior
`to the first PG application and, for the mostpart, signifi-
`cantly below the concurrently measured IOP of the
`contralateral eye. When once-daily treatment was re-
`sumed between days 30 and 99, and from day 118 to the
`end of the 7-month treatment period reported here, the
`IOP of the experimental eye was maintained below that
`of the control eye. On the 100th day of treatment, a
`single application of 500 wg/eye of PGE?resulted in a
`further reduction in the IOP of the experimental eye.
`However, this high dose of PGE2 causedthe develop-
`mentofsignificantflarein the anterior chamberof these
`eyes and therefore was not applied again.
`Qualitatively similar results were obtained following
`topical application of 100 pg of PGF2, to the right eye
`of a different set of six cats. Four hours after the first
`PGF2¢ application, the IOP of the treated eyes dropped
`significantly (p<0.05) from the baseline of 2341.6 to
`171.1 mm Hg and remained reduced throughout the
`7-day treatment period. The IOPs of the contralateral
`eyes of these animals showed considerable fluctuations;
`in fact, 24 hr after the first PGF2, treatment, the IOP of
`the contralateral eye was reduced almost as muchas the
`treated eye. When daily treatment of the same eyes with
`a higher dose of PGF2q (500 pg/eye) was initiated 12
`days after the last treatment with 100 wg of PGF2, a
`greater decrease in JOP was observed and this decrease
`could be maintained throughout this treatment period.
`Topical application of 100 ug of PGF2¢ to eyes of
`rhesus monkeys produced a decrease in the IOP of the
`experimental eye. The maximum IOP reduction ob-
`served within 6 hr after the first topical application of
`100 ng of PGF2q was only slightly greater than that
`measured after the 3rd, 5th, 9th or 11th twice-daily
`application of the same dose. The lowest IOP measure-
`ment obtained within 6 hr after the first application of
`500 pg of PGF2q was equal both to that obtained after
`the first application of 100 ug of PGF2_ and to those
`obtained after subsequent applications of 500 wg of
`PGF2. However, after a 4-day break, increasing the
`PGFregimen to 1000 ug/eye applied twice daily for
`5 days, the IOP of the experimental eye was reduced
`only to a level slightly less than that obtained following
`administration of 500 wg of PGF2. This indicates that
`the optimal PGF2,_ dose for IOP reduction in this spe-
`cies is between 100 and 1000 pg/eye. Results similar to
`those reported above were obtained on the second rhe-
`sus monkey. How