Reduction of intraocular pressure by
`prostaglandins applied topically to
`the eyes of conscious rabbits
`
`Carl B. Camras, Laszlo Z. Bito, and Kenneth E. Eakins
`
`Topically applied prostaglandins (PG's) in the dose range of 25 to 200 fig/eye caused a pro-
`longed (15 to 20 hr.) ocular hypotony (as much as 7 mm. Hg below control values) in the
`conscious rabbit following the well-known initial hypertensive phase. The aqueous humor
`protein concentration was elevated, and the ascorbic acid concentration was decreased during
`the initial hypertony, but both approached normal during the first half of the hypotensive
`phase. This biphasic intraocular pressure (IOP) response was dose-dependent and was also
`observed after intravitreal PG administration. A reduction of IOP (as much as 7 mm. Hg)
`lasting for 12 hr. or more was observed following the topical application of a very low dose
`(5 iig) of PGFta which was insufficient to cause an initial increase in IOP. Neither pretreat-
`ment with indomethacin nor sympathetic denervation diminished the biphasic IOP effect of
`PGEt, suggesting that neither de novo synthesis of PG's nor release of endogenous norepi-
`nephrine was responsible for the hypotony. Assay of aqueous humor for PG's showed a high
`level of PG activity after 30 to 60 min. and a small residual activity 6 to 18 hr. following
`PG application. The hypotensive phase was associated with a reduction in outflow resistance
`as measured in either anesthetized or freshly killed animals. The present experiments suggest
`that exogenous administration of low doses of certain PG's or their analogues may aid in
`the treatment of ocular hypertension and that endogenous PG synthesis may, in some cases,
`contribute to or actually mediate the profound hypotony that often follows ocular trauma
`and inflammation.
`
`Key words: PGE2, PGF^, prostaglandins, intraocular pressure, uveitis, inflammation (ocular),
`hypotony
`(ocular), glaucoma, sympathetic denervation,
`indomethacin, outflow resistance,
`aqueous humor, ascorbic acid.
`
`the Departments of Ophthalmology and
`From
`Pharmacology, Columbia University, New York,
`N. Y.
`This work was supported by United States Public
`Health Service grants EY 00402 and EY 00457
`from the National Eye Institute.
`Submitted for publication May 10, 1977.
`Reprint requests: Laszlo Z. Bito, Ph.D., Ophthal-
`mology Research, Columbia University College
`of Physicians and Surgeons, 630 West 168 St.,
`New York, N. Y. 10032.
`
`• rostaglandins (PG's), administered in-
`tracamerally,
`topically, or intravenously,
`have been shown to reproduce most of the
`characteristic signs of acute ocular inflam-
`mation, including entry of plasma proteins
`into the aqueous humor and a rise in intra-
`ocular pressure (IOP). Increased levels of
`PG's were found in the aqueous humor of
`patients with untreated acute anterior
`
`1125
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 1/10
`
`Santen/Asahi Glass Exhibit 2003
`Micro Labs v. Santen Pharm. and Asahi Glass
`IPR2017-01434
`
`

`

`1126 Camras, Bito, and Eakins
`
`Invest. Ophthalmol. Visual Sci.
`December 1977
`
`uveitis1 and of rabbits at the height of the
`inflammatory response induced by the intra-
`vitreal injection of either bovine serum
`albumin (BSA)2 or bacterial endotoxin.3
`These and other observations suggest that
`PCs are important mediators in die break-
`down of the blood-aqueous barrier and the
`associated increase in IOP during the initial
`phase of ocular inflammation.4
`The ocular hypertensive phase of uveitis
`is followed by a more prolonged hypoten-
`sive phase that lasts for 1 to 2 weeks fol-
`lowing endotoxin- or x-irradiation-induced
`uveitis and for 1 to 2 months following
`BSA-induced uveitis.5> 6 Furthermore, ocular
`hypotony characteristically accompanies
`chronic uveitis in humans. In contrast to
`the initial hypertensive phase, the mecha-
`nism and the possible chemical mediators of
`the prolonged reduction in IOP have not
`been studied in detail.
`The intracameral administration of PGEi
`or PGE2 into rabbit eyes has been shown to
`produce a dose-dependent reduction of
`IOP following the initial hypertensive re-
`sponse.7 These experiments were carried
`out in cannulated eyes of anesthetized ani-
`mals; thus the duration of the hypotension
`could not be determined. In the same study,
`it was observed that when the ocular hy-
`pertension caused by exogenous PGEX or
`PGE2 was blocked by pretreatment with
`polyphloretin phosphate, the hypotensive
`action of these PG's remained. The possibil-
`ity of a dual effect of PG's on intraocular
`fluid dynamics is also suggested by a PG-
`induced increase in total outflow facility
`during the initial hypertensive phase.8-10
`In the present experiments, the complete
`time course of PG-induced hypertension
`and hypotension was studied in conscious
`rabbits. The relation between the dose of
`topically applied PG's and the IOP effects
`was established to determine whether a
`dose of PG's could be selected which would
`reduce IOP without an initial hypertensive
`response. In addition, the time course of
`PG-induced alterations in PG, ascorbic acid,
`and protein concentrations in the aqueous
`humor was correlated with the time course
`
`of IOP effects. The IOP response to PG's
`was also studied on
`indomethacin-pre-
`treated animals and on sympathetically
`denervated eyes in an attempt to elucidate
`the mechanism by which PG's reduce IOP.
`
`Methods
`New Zealand white (albino) rabbits (2 to 4
`kg.) were placed in rabbit boxes in order to ac-
`custom them to handling and restraint before they
`were used in these experiments.
`Measurement of IOP. Following topical appli-
`cation of 0.5 percent Ophthaine solution (E. R.
`Squibb & Sons, Inc., Princeton, N. J.), the IOP
`of the conscious animals was measured with a
`pneumatic floating-tip tonometer1" calibrated on
`the cannulated rabbit eye by the open-stopcock
`method. At least two IOP measurements were
`made on each eye within 1 hr. prior to PG admin-
`istration. Following the PG administration, IOP
`was measured at varying intervals for up to 22
`to 30 hr.
`Drug administration
`Prostaglandins. In the first series of experiments,
`the IOP effects of three different modes of PGE=
`administration were compared.
`1. A 25 fig amount of PGE: (The Upjohn Co.,
`Kalamazoo, Mich.) in 50 fi\ of phosphate buffer
`(50 mM, pH 7;4) was dropped onto the cornea
`of the experimental eye, the contralateral control
`eye receiving 50 fi\ of phosphate buffer.
`2. A 25 fig amount of the sodium salt of PGE2
`(made by adding sodium carbonate to PGE2 dis-
`solved
`in ethanol according
`to
`the procedure
`provided by The Upjohn Co.) in 5 fi\ of saline
`(0.9 percent sodium chloride) was dropped onto
`the cornea of the experimental eye and rinsed off
`3 to 4 min. later with 2 to 4 ml. of saline; 5 fi\
`of the vehicle solution (ethanol and sodium car-
`bonate in saline) was similarly applied to the
`contralateral control eye and rinsed.
`3. A 10 fig amount of PGE* in 10 fi\ of 10 per-
`cent ethanol was injected into the center of the
`vitreous body of the experimental eye; 10 fi\ of 10
`percent ethanol was similarly injected
`into the
`contralateral control eye.5 The globe was rinsed
`with saline after the needle was withdrawn.
`Best results were obtained with the second mode
`of administration (see Results); therefore, in all
`subsequent experiments, PGE; or PGF^
`(ob-
`tained as the tromethamine salt; The Upjohn Co.)
`was applied topically in a 5 fi\ volume, followed
`3 to 4 min. later by rinsing of the cornea and
`conjunctival cul-de-sac with 2 to 4 ml. of saline.
`Indomethacin. The sodium salt was prepared
`immediately prior
`to use by adding sufficient
`sodium carbonate to a 20 mg./ml. indomethacin
`(Merck, Sharp & Dohme, Rahway, N. J.) suspen-
`sion to obtain a clear solution; 50 mg./kg. free
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 2/10
`
`

`

`Volume 16
`Number 12
`
`IOP reduction by topical prostaglandins 1127
`
`acid equivalent was injected intraperitoneally 1
`hr. prior to PG administration.31 The effectiveness
`of
`this pretreatment was demonstrated by the
`absence of an ocular hypertensive response to 50
`/ul of 1.0 percent sodium arachidonate applied
`topically to the contralateral control eye at least
`6.5 hr. after indomethacin pretreatment.
`Superior cervical ganglionectomy. Eight rabbits
`were anesthetized with Chloropent (3 ml./kg.,
`intravenously; Fort Dodge Laboratories, Fort
`Dodge, Iowa) before unilateral removal of the
`superior cervical ganglion together with approxi-
`mately 1 cm. of preganglionic sympathetic fiber.
`After 24 hr. all animals showed the characteristic
`ipsilateral iridial hyperemia and ocular hypotony
`(3 to 8 mm. Hg below control). Seven to 14 days
`later, the IOP was measured both before and up
`to 24 hr. after administration of 50 ng of PGE2
`to both eyes.
`Aqueous humor composition. Forty-four rabbits
`were killed with an overdose of sodium pento-
`barbital (Nembutal; Abbott Laboratories, North
`Chicago, 111.) 0.5 to 24 hr. after topical applica-
`tion of 50 /ig of PGE2 or PGF2a; the aqueous
`humor was removed from the PG-treated and the
`contralateral control eyes. The protein and the
`ascorbic acid concentrations of individual control
`and experimental aqueous samples were deter-
`mined.5 Pooled aqueous samples from two to six
`eyes were extracted for PG assay,12 and the PG
`content of these extracts was measured either with
`bioassay on the rat stomach strip preparation13
`to determine total PG-like activity or with radio-
`immunoassay (Clinical Assays, Inc., Cambridge,
`Mass.) to determine PGF?a levels.
`Measurement of gross outflow resistance. Fol-
`lowing topical application of 50 jug of PGE2, gross
`outflow resistance was measured simultaneously
`on both the experimental and the contralateral
`infusion14 during
`control eyes by constant-rate
`the hypotensive phase (i.e., 5 to 12 hr. after
`application) or during recovery from hypotony
`(i.e., 17 to 19 hr. after application). The IOP's
`were monitored manometrically with Sanborn
`267b pressure
`transducers
`in conjunction with
`Sanborn 500A carrier preamplifiers and a multi-
`channel Model 350 rectilinear recorder. In these
`experiments, the outflow resistance was determined
`on animals anesthetized with Chloropent (3 ml./
`kg., intravenously) or, in some cases, on animals
`killed with
`an overdose of Chloropent or
`Nembutal.
`
`Results
`Topical application or intravitreal injec-
`tion of PGE2 resulted in a biphasic IOP
`response: a relatively short initial hyper-
`tensive phase followed by a prolonged hy-
`potony (Fig. 1). The extent of the hyper-
`
`(A) TOPICAL
`
`I 25 pq E2 in 50 pi
`
`(B) TOPICAL (rinsed)
`
`(C) INTRAVITREAL
`
`30
`
`20
`
`10
`
`30
`
`20
`
`10
`40
`
`30
`
`20
`
`10
`
`Q_
`O
`
`10
`
`20
`
`30
`
`Hours
`Fig. 1. Effects of three different modes of PGE2
`administration on
`the IOP of
`the PG-treated
`((_#_#_»_) and contralateral control (-O-O-O-)
`eyes of rabbits. A, 25 ng of PGE2 in 50 n\ of
`phosphate buffer (50 mM, pH 7.4) was applied
`topically to the experimental eye of 8 rabbits.
`The contralateral control eye received 50 /A of
`phosphate buffer. B, 25 ng of PGE2 in 5 n\ of
`normal saline was applied topically to the experi-
`mental eyes of four rabbits, which were then
`rinsed with saline. The contralateral control eye
`received 5 pi of vehicle solution and was also
`rinsed. C, 10 /ug of PGE= in 10 fil of 10 percent
`ethanol was injected intravitreally into the experi-
`mental eye of six rabbits. Ten microliters of 10
`percent ethanol was similarly injected
`into the
`contralateral control eye. The points represent
`means and the limits ± 1 S.E.M.
`
`tensive response seemed to depend on the
`volume in which the PGE2 was applied to
`the cornea, since topical application of 25
`ixg of PGE2 in 50 ^1 of buffer solution
`caused a smaller rise in IOP than the same
`amount of PGE2 in 5 ^1 of saline solution
`(Fig. 1, A vs. B). Furthermore, with the
`second mode of application (see Methods),
`using the smaller volume of a more con-
`centrated solution and/or rinsing the con-
`junctival sac eliminated
`the significant
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 3/10
`
`

`

`1128 Camras, Bito, and Eakins
`
`Invest. Ophthalmol. Visual Set.
`December 1977
`
`(A)
`o— 1x25yg PGE2 to O.S.
`•— 2x25]jg PGE2 to O.D.
`
`PGF9
`
`I
`
`-10
`
`L
`
`10
`
`EE co
`
`Ot
`
`-H
`I
`
`a. o
`X0?
`O
`
`10r
`
`6x25pg PGE2
`
`(B)
`
`O.D., O.D.
`35 O.S. only
`
`30
`
`25
`
`I 20
`
`15
`
`Oi
`
`—i
`
`15
`
`10
`
`XEJ
`
`
`
`- 5
`
`-10
`
`o
`0.°
`
`2 a
`
`>
`
`10
`
`20
`
`30
`
`Hours
`Fig. 2. Effects of repeated topical PGE^ applica-
`tion on the IOP of rabbits. A, 25 /tg of PGE2 in a
`5 id volume was applied to both eyes of five rab-
`bits. After 3.5 hr., a second dose of 25 ng of PGE2
`was applied to the right eyes only of each animal.
`The left eye received 5 id of vehicle solution at
`this time. B, 25 ng of PGE, in a 5 jul volume was
`applied six consecutive times at hourly intervals
`to one eye of six rabbits. Five microliters of ve-
`hicle solution were applied to the contralateral
`control eyes at the same time. The points repre-
`sent means or mean differences [(IOP of the PG-
`treated eye) - (IOP of the contralateral control
`eye)] measured at the same time; the limits are
`± 1 S.E.M.
`
`hypertensive effect seen in the control eye
`following the first mode of PG application
`(Fig. 1, A vs. B). Although intravitreal
`injection of PGE2 produced a similar bi-
`phasic IOP response, the onset of the hy-
`potony was more variable, as indicated by
`the larger standard errors during the slower
`transition from
`the hypertensive to the
`hypotensive phase (Fig. 1, C). For this
`reason, the second mode of PG application
`was used in all subsequent experiments.
`When a second dose of PGE2 was ap-
`plied to rabbit eyes during the hypotonic
`phase—3.5 hr. after the first PG applica-
`
`Hours
`Fig. 3. Time course of the IOP response to various
`doses of PGF2a. Each dose of PGF2a was applied
`topically in 5 ^1 of saline to one eye of four to
`six rabbits. The contralateral control eye received
`5 M\ of vehicle solution. Points represent mean dif-
`ferences (for explanation of IOPexp - IOPcom see
`legend of Fig. 2) and the limits ± 1 S.E.M.
`
`tion—there was a significant second rise in
`the IOP followed by an accentuated hypot-
`ony as compared with the contralateral
`eyes which received only a single dose of
`PGE2 (Fig. 2, A). When six identical doses
`(25 /xg in 5 fil each) of PGE2 were applied
`at hourly intervals, the duration of the hy-
`pertonic phase was increased, and the
`ocular hypotony was accentuated (Fig. 2,
`B, vs. Fig. 1,B).
`A biphasic IOP response was also ob-
`served following topical application of high
`doses of PGF2ft (Fig. 3, C and D). At a
`dose of 50 /xg, PGF2a produced a smaller
`initial rise in IOP than the same dose of
`PGE2 even though the hypotony produced
`by both PG's was similar both in extent and
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 4/10
`
`

`

`Volume 16
`Number 12
`
`IOP reduction by topical prostaglandins 1129
`
`20
`
`50pg PGE2
`
`A) IOP
`
`B) Protein
`
`c 10o
`
`O»
`
`—i
`I
`Q.
`X
`
`g-,0
`
`40
`
`20
`
`Co
`.x
`CD
`
`oa
`
`30
`
`20
`
`10
`Hours
`Fig. 4. Comparison of the time course of the IOP
`response (A) with the time course of changes in
`aqueous humor protein (B) and ascorbic acid (C)
`concentrations after topical application of 50 /*g
`of PGE- to one eye. IOP values are based on 17
`rabbits. Protein and ascorbic acid concentrations
`were determined on individual aqueous samples
`from each eye of four to six rabbits for each
`point; each eye was used for one aqueous sample
`only. The points represent means (for explanation
`of IOP«xP - IOPcont see legend of Fig. 2) and
`the limits ± 1 S.E.M.
`
`treated eyes decreased rapidly but were
`still slightly elevated during the onset of
`the hypotensive phase. At 12 hr. after ad-
`ministration of PGF2« or PGE2, the aqueous
`humor of the treated eyes contained clearly
`detectable amounts of PGF2« or PGE2 as
`determined by immunoassay or bioassay,
`respectively. On the other hand, PG activ-
`ity could not be detected in the control
`eyes by either technique at 12 hr. or any-
`time thereafter. After 24 hr., PG's could
`not be detected in the aqueous humor of
`either the experimental or the control eyes.
`
`duration (Fig. 3, C, vs. Fig. 4, A). At a
`dose of 5 fig of PGF2«, a prolonged hypot-
`ony was obtained without any significant
`initial rise in IOP (Fig. 3, B).
`For some of these experiments, the differ-
`ences between the IOP's of the two eyes—
`as a function of time after the application
`of PG or the vehicle solution—are reported,
`since this expression eliminates animal-to-
`animal variations in the pre-existing IOP,
`changes in the physiological state of the
`animals, diurnal IOP variations, and tonom-
`etry-induced IOP effects. As in the case of
`PGE2, when PGF2« was applied at any of
`the doses in a small volume (5 ju.1) to one
`eye, the contralateral control eye did not
`show a significant initial increase in IOP.
`The possibility that the contralateral control
`eyes show small reductions in IOP after
`PG administration to the experimental eyes
`cannot be ruled out at this time. The study
`of such contralateral effects would require
`the use of much larger numbers of animals,
`including untreated controls, to distinguish
`among drug effects, circadian variations,
`and tonometry-induced changes in IOP.
`The aqueous humor protein concentra-
`tion was increased 30- to 50-fold 2 hr. after
`the administration of 50 fig of PGE2 (Fig.
`4, B), and the ascorbic acid concentration
`was decreased by 30 percent (Fig. 4, C).
`The onset of these changes approximated
`the initial rise in IOP. Both of these param-
`eters began to return toward normal at the
`onset of the hypotensive phase and showed
`almost complete recovery during the first
`half of this period (Fig. 4).
`PG levels in the aqueous humor were
`measured at varying time intervals follow-
`ing the topical application of 50 fig of
`PGF2a (Fig. 5, A) or PGE2 (Fig. 5, B).
`With the use of either radioimmunoassay
`for PGF2« levels or bioassay for total PG-
`like activity, the PG concentrations were at
`their highest level 0.5 hr. after topical PG
`administration, corresponding in time to the
`maximal rise in IOP. At this time, small but
`clearly measurable amounts of PG's were
`also detected in the contralateral control
`eyes. The high levels of PG's in the PG-
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 5/10
`
`

`

`1130 Camras, Bito, and Eakins
`
`Invest. Ophthalmol. Visual Sci.
`December 1977
`
`— O - - NORMAL
`— • —
`INDOMETHACIN
`(50mg/kgI.R)
`
`50
`
`40
`
`30
`
`20
`
`XEE
`
`a.
`o
`
`1 0 L
`
`20
`
`25
`
`15
`10
`HOURS
`Fig. 6. Comparison of the IOP response to PGE2
`(50 Mg) applied topically to one eye of seven
`normal and six indomethacin-pretreated rabbits.
`The points represent means and the limits ± 1
`S.E.M.
`
`Discussion
`The results presented here show that in
`addition to their well-known ocular hyper-
`tensive effects, topically applied PCs also
`cause a significant and prolonged reduction
`in IOP. This hypotony is not a compensa-
`tory response to the initial hypertensive
`phase, since it was also observed with a
`dose of PGFoa too low to cause any sig-
`nificant initial rise in IOP. This finding is
`consistent with the observation that intra-
`cameral injection of PGEi or PGE2 lowered
`IOP in cannulated eyes of anesthetized
`rabbits when the hypertensive effect was
`blocked by pretreatment of the eye with
`polyphloretin phosphate.7 Bhattacherjee
`and Hammond15 referred to preliminary
`results indicating that "250 ng. of PGEi do
`not raise but in fact reduced ocular tension
`by a small degree (approx. 1-2 mm. Hg),"
`but neither the route of administration nor
`the duration of the effect was stated.
`The present observation that a low dose
`of topically applied PGF2« can lower IOP
`by as much as 7 mm. Hg for 12 hr. or more
`without a significant initial rise in IOP sug-
`gests that suitable doses of certain PCs or
`their analogues may be useful in the thera-
`peutic control of ocular hypertension. In
`
`160
`
`120
`
`80
`
`4 0
`
`80
`
`4 0
`
`A)
`
`(Immunoassay)
`
`• Topical PGF 2 a
`(50pg in 5 JJI)
`O Control Eye
`
`B) PGE2 Equivalent (Bioassay)
`• Topical PGE2
`(50jjgin 5pi)
`O Control Eye
`
`3O<
`
`D
`
`3 < E
`
`CL
`H-
`
`oo
`
`>
`c
`
`-A--7
`
`16
`
`20
`
`24
`
`12
`Hours
`Fig. 5. Levels of PG in the aqueous humor fol-
`lowing topical PG application at a dose of 50 /xg.
`A, PGF2n levels at various times after PGF2a appli-
`cation were measured by radioimmunoassay on
`aqueous humor samples pooled for each point
`from either the PG-treated or the contralateral
`control eyes of three rabbits. B, PG-like activity at
`various times after PGE= application was deter-
`mined by bioassay on one or two aqueous humor
`samples pooled for each point from either the PG-
`treated or the contralateral control eyes of two to
`six rabbits.
`
`Two experiments were carried out to de-
`termine whether endogenous PCs or cate-
`cholamines contribute to the ocular hypo-
`tensive effects of exogenously administered
`PCs. Neither indomethacin pretreatment
`(Fig. 6) nor chronic sympathetic denerva-
`tion (Fig. 7) diminished the biphasic IOP
`response to 50 jug of PGE2.
`During the time of maximal hypotony,
`the gross outflow resistance of the PG-
`treated eye was 40 to 50 percent that of
`the contralateral control eye. Moreover, this
`decrease in outflow resistance persisted
`when the animals were killed prior to
`the measurement (Fig. 8). As the outflow
`pressure was returning to the control value
`18 hr. after PG administration, the outflow
`resistance also approached
`the control
`value.
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 6/10
`
`

`

`IOP reduction by topical prostaglandins 1131
`
`100
`
`O
`
`80
`
`8 60
`
`i 40
`
`Oc
`
`c
`£ 20
`
`MAX HYPOTONY RECOVERY
`Fig. 8. Graphic comparison of calculated outflow
`pressure (IOP-PV) with measured outflow resistance
`(R) during maximal hypotony (5 to 12 hr. after
`topical application of 50 /tg of PGE, to one eye)
`and during recovery from hypotony (17 to 19
`hr. after PG application). The outflow resistances
`of the PG-treated and the contralateral control
`eyes of 11 anesthetized (R«nvo) or freshly killed
`(Rdead) rabbits were measured as described in
`the text. The percent change in outflow pressure,
`[(IOP - Pv)exp/(IOP - PT)cont] x 100, was cal-
`culated with the mean IOP before PG treatment,
`during maximal hypotony, and during recovery
`(based on the 17 rabbits shown in Fig. 4, A);
`episcleral venous pressure (P,,) was assumed to
`be 9 mm Hg.14
`
`the present experiments could not be ac-
`counted for by a maintained breakdown of
`this barrier, since the aqueous humor pro-
`tein concentration returned toward normal
`during the first half of the hypotony. The
`fact that a significant increase in IOP re-
`sulted when a second dose of PGE2 was ap-
`plied to the eye during the hypotonic phase
`also suggests that the blood-aqueous barrier
`was functional. If the barrier were broken
`down, a second dose of PG's would not
`show a further increase in IOP.17
`The long-term hypotony observed in
`these experiments cannot be accounted for
`by damage to the secretory mechanisms or
`reduced blood flow to the ciliary processes,
`since the aqueous humor ascorbic acid con-
`centration, which depends on both secre-
`tory processes and ciliary blood flow,18
`returned toward its normal level during
`maximal hypotony. These considerations
`
`Volume 16
`Number 12
`
`50
`
`- 50pg PGE2
`
`40
`
`30
`
`20
`
`X
`
`0.
`O
`
`INNERVATED
`—o~
`— • — SYMPATHECTOMIZED
`
`25
`
`20
`
`15
`10
`HOURS
`Fig. 7. Comparison of the IOP response to PGEj
`(50 fig) applied topically to both the sympathec-
`tomized and the contralateral normally innervated
`eyes of eight rabbits. The points represent means
`and the limits ± 1 S.E.M.
`
`this respect, it is interesting to note that
`PGF2«, administered by intrauterine or in-
`travenous injections to pregnant women to
`induce abortions, has been reported to
`cause a reduction in IOP.1G However, the
`duration of this PG-induced ocular hypot-
`ony was not determined.
`It has been suggested that the decreased
`gross outflow resistance observed during
`PG-induced ocular hypertension reflected
`an increased pseudofacility due to the
`breakdown of the blood-aqueous barrier.8'10
`The work of Masuda and Mishima10 showed
`that the increased rate of aqueous flow,
`which reached a peak at 1.5 hr. after the
`administration of a very high dose (100
`ju,g) of PGEl5 returned to essentially normal
`by 4 hr. During this phase of increased
`aqueous formation, there was an increase
`of pseudofacility in the eyes of these ure-
`thane-anesthetized rabbits. None of these
`studies included measurement of outflow
`facility, flow, or pseudofacility more than
`4 hr. after PG administration. Thus the
`conclusions of these authors refer only to
`the hypertonic phase which is clearly as-
`sociated with a breakdown of the blood-
`aqueous barrier.
`The PG-induced hypotony observed in
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 7/10
`
`

`

`1132 Camras, Bito, and Eakins
`
`Invest. Ophthalmol. Visual Sci.
`December 1977
`
`indicate that the reduction in IOP could
`not be due to alterations in secretory mech-
`anisms or to pseudofacility, and hence they
`must be primarily attributed to a reduction
`in true outflow resistance. This conclusion
`was further supported by the fact that the
`decreased outflow resistance measured in
`the present study persisted in the eyes of
`dead rabbits.
`The possibility that the hypotony de-
`pends on the de novo synthesis of PCs or
`other cyclo-oxygenase products such as en-
`doperoxides, thromboxane A2, or prostacy-
`clin can be ruled out because the hypoten-
`sive phase was not diminished by systemic
`pretreatment with indomethacin, an effec-
`tive inhibitor of cyclo-oxygenase.19'20 Fur-
`thermore, it is unlikely that the IOP effect
`of PCs is related to local sympathetic ac-
`tivity or to the release of endogenous cate-
`cholamines, since the time course of the
`IOP response was very similar in both
`normally innervated and sympathetically
`denervated eyes. In fact, the only difference
`was a faster transition from the hyperten-
`sive to the hypotensive phase in the dener-
`vated eye.
`Since small amounts of PG activity could
`be detected in the aqueous humor by either
`immunoassay or bioassay for 6 to 18 hr.
`after topical PG application, the hypoten-
`sive phase may result from the continual
`presence of PCs in the eye. Although more
`detailed studies will be required to deter-
`mine the statistical and physiological sig-
`nificance of these residual PG levels, it
`should be noted that the aqueous humor
`levels may underestimate true bioavailabil-
`ity of the exogenous PCs at the receptor
`sites, since the anterior uvea, for example,
`was shown to accumulate PCs against a
`several-fold concentration gradient.21
`We must also consider the possibility
`that the prolonged hypotonic effects of
`PCs is due to the accumulation of a second
`autocoid. PCs are known to exert their ef-
`fects in many biological systems by altering
`intracellular cyclic AMP levels.22 PCs were,
`in fact, shown to increase the cyclic AMP
`content of several ocular tissues in vitro, in-
`
`cluding the sclera-trabecular ring, iris-
`ciliary body, and cornea,23 and intracameral
`injection of cyclic AMP was shown to in-
`crease outflow facility in the rabbit eye.24
`Therefore, following exogenous PG admin-
`istration, the prolonged residual PG levels
`in the eye may cause an even longer in-
`crease in the cyclic AMP content of intra-
`ocular tissues and/or fluids, which in turn
`may account for the long-term increase in
`outflow facility.
`The release of a second messenger could
`also provide an explanation for the hypo-
`tonic effect of intravitreally injected PG. It
`was shown that intravitreally injected PCs
`do not effectively reach the anterior cham-
`ber as a result of the absorptive transport
`activity of the ciliary processes.21 Therefore
`it could be argued that PCs could not
`reach the chamber angle from the vitreous
`and hence could not have an effect on true
`outflow facility. On the other hand, accum-
`ulation of PCs in the ciliary processes may
`cause the release of cyclic AMP or other
`endogenous autocoids, which in turn could
`reach the trabecular meshwork region by
`bulk flow through the posterior and anterior
`chambers or by diffusion
`through
`the
`stroma of the ciliary body and the iris. In
`addition, PCs may reach the outflow angle
`by the latter route, since PCs, which are
`accumulated by the ciliary body, were
`shown not to be effectively metabolized by
`this tissue.25
`Severe trauma may cause irreversible
`changes in the secretory processes, resulting
`in prolonged and sometimes irreversible hy-
`potony. However, the reversible hypotony
`that is observed following more moderate
`forms of uveitis, e.g., those induced by bac-
`terial endotoxin or cataractogenic doses of
`x rays,5-6 may be mediated by the continual
`release of PCs. It was shown that ocular in-
`flammation or uveitis can damage the trans-
`port processes of the anterior uvea which
`normally remove PCs from the intraocular
`fluids.5 Interference with this active trans-
`port mechanism may contribute to the ac-
`cumulation of PCs within the aqueous
`humor during uveitis, since PCs are not
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933302/ on 08/30/2017
`
`IPR Page 8/10
`
`

`

`Volume 16
`Number 12
`
`IOP reduction by topical prostaglandins 1133
`
`effectively metabolized by intraocular tis-
`sues.-6 In the absence of such absorptive
`transport processes, endogenously produced
`PCs could exert long-term hypotensive ef-
`fects.
`The long-term reduction of IOP induced
`by twice daily topical epinephrine treat-
`ment was recently shown to be blocked by
`pretreatment of the rabbits with indometha-
`cin,15 suggesting that the epinephrine-in-
`duced hypotony may be dependent upon
`the endogenous synthesis of PCs. The
`present demonstration that PCs can cause
`a long-term reduction of IOP in normal
`conscious rabbits supports this interpreta-
`tion and suggests that the use of very low
`doses of certain PCs or PG analogues may
`provide a more direct therapeutic approach
`to the control of ocular hypertension.
`
`We thank R. Baroody, A. S. Zaragoza, E. V.
`Salvador, M. E. Reitz, and D. W. Garnick for
`their assistance and Dr. John Pike of The Upjohn
`Co., Kalamazoo, Mich., for the supply of prosta-
`glandins. Indomethacin was kindly provided by
`Merck, Sharp & Dohme Research Laboratories,
`Rahway, N. J.
`REFERENCES
`1. Eakins, K. E., Whitelocke, R. A. F., Bennett,
`A., and Martenet, A. C : Prostaglandin-like
`activity in ocular inflammation, Br. Med. J.
`3:452, 1972.
`2. Eakins, K. E., Whitelocke, R. A. F., Perkins,
`E. S., Bennett, A., and Unger, W. C : Re-
`lease of prostaglandins in ocular inflammation
`in the rabbit, Nature New Biol. 239:248,
`1972.
`3. Bhattacherjee, P.: Release of prostaglandin-
`like substances by Shigella endotoxin and its
`inhibition by non-steroidal anti-inflammatory
`compounds, Br. J. Pharmacol. 54:489, 1975.
`4. Eakins, K. E.: Prostaglandin and non-prosta-
`glandin mediated break down of the blood-
`aqueous barrier. In Bito, L. Z., Davson, H.,
`and Fenstermacher, J., editors: The Ocular
`and Cerebrospinal Fluids, Fogarty Interna-
`tional Center Symposium, Exp. Eye Res. 25
`(Suppl.): in press, 1977.
`5. Bito, L. Z.: The effects of experimental
`uveitis on anterior uveal prostaglandin trans-
`port and aqueous composition, INVEST. OPH-
`THALMOL. 13:959, 1974.
`6. Worgul, B. V., Bito, L. Z., and Merriam,
`G. R., Jr.: Intraocular inflammation produced
`by X-irradiation of the rabbit eye, Exp. Eye
`Res. 25:53, 1977.
`
`7. Starr, M. S.: Further studies on the effect
`of prostaglandin on intraocular pressure in
`the rabbit, Exp. Eye Res. 11:170, 1971.
`8. Kass, M. A., Podos, S. M., Moses, R. A., and
`Becker, B.: Prostaglandin Ei and aqueous
`humor dynamics, INVEST. OPHTHALMOL. 11:
`1022, 1972.
`9. Green, K., and Kim, K.: Pattern of ocular
`response
`to
`topical and systemic prosta-
`glandin, INVEST. OPHTHALMOL. 14:36, 1975.
`10. Masuda, K., and Mishima, S.: Effects of
`prostaglandins on inflow and outflow of the
`aqueous humor in rabbits, Jpn. J. Ophthal-
`mol. 17:300, 1973.
`11. Podos, S. M., Becker, B., and Kass, M. A.:
`Indomethacin blocks arachidonic acid-induced
`intraocular pressure, Prosta-
`elevation of
`glandins 3:7, 1973.
`12. Unger, W. G., Stamford, I. F., and Bennett,
`A.: Extraction of prostaglandins from human
`blood, Nature New Biol. 233:336, 1971.
`13. Vane, J. R.: A sensitive method for the assay
`of 5-hydroxy tryptamine, Br. J. Pharmacol.
`Chemother. 12:344, 1957.
`14. Sears, M. L.: Outflow resistance of the rabbit
`eye: technique and effects of acetazolamide,
`Arch. Ophthalmol. 64:823, 1960.
`15. Bhattacherjee, P., and Hammond, B. R.: Effect
`of indomethacin on the ocular hypotensive
`action of adrenalin