British Journal ofOphthalmology 1994; 78: 560-567 Studies on a novel series of acyl ester prodrugs of prostaglandin F2a A Cheng-Bennett, M F Chan, G Chen, T Gac, M E Garst, C Gluchowski, L J Kaplan, C E Protzman, M B Roof, G Sachs, L A Wheeler, L S Williams, D F Woodward Aliergan, Inc, Departments of Biological Sciences and Medicinal Chemistry, Irvine, California, USA A Cheng-Bennett M F Chan G Chen T Gac M E Garst C Gluchowski L J Kaplan C E Protzman M B Roof G Sachs L A Wheeler L S Williams D F Woodward Correspondence to: Dr D F Woodward, Department of Biological Sciences, Allergan, Inc, 2525 Dupont Drive, PO Box 19534, Irvine, CA 92713-9534, USA. Accepted for publication 15 March 1994 Abstract A novel series of prostaglandin F2U (PGF2Z) prodrugs, with acyl ester groups at the 9, 11, and 15 positions, was prepared in order to design clinically acceptable prostaglandins for treating glaucoma. Studies involving isolated esterases and ocular tissue homogenates indi- cated that 9-acyl esters cannot provide a pro- drug since PGF2Q would not be formed as a product. In contrast, 11-mono, 15-mono, and 11, 15-diesters were converted to PGF,U in ocular tissues and could, therefore, be consid- ered as prodrugs of PGF,Q. Carboxylesterase (CE) appeared critically important for the hydrolytic conversion of those PGF2, prodrugs where the 11 or 15-OH group was esterfied and such prodrugs were not substrates for acetylcholinesterase (ACHE) or butyryl- cholinesterase (BuCHE). The enzymatic hydrolysis of PGF,,,-1-isopropyl ester was also investigated for comparative purposes. This PGF,, prodrug was a good substrate for CE, but was also hydrolysed by BuCHE, albeit at a much slower rate. The most striking feature of the enzymatic hydrolysis of PGF2,-1-isopropyl ester in ocular tissue homogenates was that it was much faster than for prodrugs esterified at the 11 and/or 15 positions. In terms of ocular hypotensive activity, all prodrugs which showed detectable conversion to nascent PGF2, were potent ocular hypotensives. Although no separation of ocular hypotensive and ocular surface hyperaemic effects was apparent for PGF,2-1-isopropyl ester, a tempo- ral separation of these effects was apparent for the novel PGF2,, ester series. This difference may reflect an unfavourably rapid conversion of PGF2,,-1-isopropyl ester in ocular surface tissues compared with anterior segment tissues. (BrJ3 Ophthalmol 1994; 78: 560-567) Prostaglandin F2, (PGF2U) has been established as an effective ocular hypotensive agent in labora- tory animals" and human subjects.56 The potency of PGF,,, may be increased by esterifying the carboxylic acid group.3 Thus, PGF,,-I- isopropyl ester has proved to be a potent ocular hypotensive in both ocular normotensive 78 and glaucomatous human volunteers.9 It has been proposed that the increased activity of PGF,2-1- esters occurs as a result of increased lipophilicity'°: the carboxylic acid moiety, which exists predominantly in the charged form at physiological pH, is transformed by esterifica- tion to a non-polar, highly lipophilic species. This greatly enhances corneal uptake which ultimately results in improved delivery of nascent PGF2, to anterior segment tissues.'0 Despite the fact that esterifying the carboxylic acid group of PGF2, improves bioavailability, it does not appear to result in a clinically acceptable ophthalmic drug because of ocular surface side effects. When considering the design of PGF2, prodrugs, there are alternatives to esterifying the carboxylic acid.' 12 PGF2U contains three -OH groups at positions 9, 11, and 15 which provide other options for synthesising ester prodrugs of PGF2a. In order to investigate alternative oppor- tunities for the design of PGF2U prodrugs with potential ophthalmic application, a novel series of PGF2U alkyl acyl esters was synthesised which comprised 9, 11 , and 15 monoesters; and 9, 11, 9, 15, and 11, 15 diesters. Three PGF2u lactones were also examined where the carboxylic acid moiety forms an internal ester with one of the -OH groups. In order to characterise these PGF2a esters the following factors received particular attention: (1) inherent pharmacological activity, (2) ester hydrolysis by esterases and ocular tissues, (3) effects on intraocular pressure, and (4) ocular surface hyperaemia. Materials and methods PHARMACOLOGICAL ACTIVITY Inherent pharmacological activity was assessed in an FP receptor preparation.'3 Mouse Swiss 3T3 fibroblasts were plated in culture flasks and were fed Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 2 mM L-glutamine, and 0 05 mg/ml gentamicin (all purchased from Gibco, Grand Island, NY, USA). Cell cultures were maintained in a humid- ified atmosphere of 95% air, 5% carbon dioxide and grown to confluency. Cells were removed from the culture flasks by a 1 minute treatment with trypsin 0-05%/0 52 mM EDTA (Gibco, Grand Island, NY, USA) at 37°C. Proteolytic activity was arrested by adding 5 ml of 10% fetal bovine serum in DMEM. The cells were consecutively washed in Hank's balanced salt solution and Maullem's buffer'4: centrifugation for the washes was performed for 15 minutes at 1200 rpm at room temperature. Cells were counted, resuspended in Maullem's buffer and incubated with 2 ,uM Fura 2/ace- toxymethyl ester in a shaking water bath for 30 minutes at 37°C. The cells were subsequently washed in Maullem's buffer as above and resus- pended at a concentration of 2 x 106 cells/ml. Aliquots of 0 5 ml cell suspension were then added to autocap microtubes to provide 106 cells per experimental determination of [Ca2+]. Fluorescence was measured in a Perkin-Elmer 560 on May 10, 2020 by guest. Protected by copyright. http://bjo.bmj.com/ Br J Ophthalmol: first published as 10.1136/bjo.78.7.560 on 1 July 1994. Downloaded from
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BritishJournal ofOphthalmology 1994; 78: 560-567
Studies on a novel series of acyl ester prodrugs ofprostaglandin F2a
A Cheng-Bennett, M F Chan, G Chen, T Gac, M E Garst, C Gluchowski, L J Kaplan,C E Protzman, M B Roof, G Sachs, L A Wheeler, L S Williams, D F Woodward
Aliergan, Inc,Departments ofBiological Sciences andMedicinal Chemistry,Irvine, California, USAA Cheng-BennettM F ChanG ChenT GacM E GarstC GluchowskiL J KaplanC E ProtzmanM B RoofG SachsL A WheelerL S WilliamsD F WoodwardCorrespondence to:Dr D F Woodward,Department of BiologicalSciences, Allergan, Inc, 2525Dupont Drive, PO Box 19534,Irvine, CA 92713-9534, USA.Accepted for publication15 March 1994
AbstractA novel series of prostaglandin F2U (PGF2Z)prodrugs, with acyl ester groups at the 9, 11,and 15 positions, was prepared in order todesign clinically acceptable prostaglandins fortreating glaucoma. Studies involving isolatedesterases and ocular tissue homogenates indi-cated that 9-acyl esters cannot provide a pro-drug since PGF2Q would not be formed as a
product. In contrast, 11-mono, 15-mono, and11, 15-diesters were converted to PGF,U inocular tissues and could, therefore, be consid-ered as prodrugs of PGF,Q. Carboxylesterase(CE) appeared critically important for thehydrolytic conversion of those PGF2, prodrugswhere the 11 or 15-OH group was esterfiedand such prodrugs were not substrates foracetylcholinesterase (ACHE) or butyryl-cholinesterase (BuCHE). The enzymatichydrolysis of PGF,,,-1-isopropyl ester was alsoinvestigated for comparative purposes. ThisPGF,, prodrug was a good substrate for CE,but was also hydrolysed by BuCHE, albeit at a
much slower rate. The most striking feature ofthe enzymatic hydrolysis of PGF2,-1-isopropylester in ocular tissue homogenates was that itwas much faster than for prodrugs esterified atthe 11 and/or 15 positions. In terms of ocularhypotensive activity, all prodrugs whichshowed detectable conversion to nascentPGF2, were potent ocular hypotensives.Although no separation of ocular hypotensiveand ocular surface hyperaemic effects was
apparent for PGF,2-1-isopropyl ester, a tempo-ral separation ofthese effects was apparent forthe novel PGF2,, ester series. This differencemay reflect an unfavourably rapid conversion
of PGF2,,-1-isopropyl ester in ocular surfacetissues compared with anterior segmenttissues.(BrJ3 Ophthalmol 1994; 78: 560-567)
Prostaglandin F2, (PGF2U) has been established asan effective ocular hypotensive agent in labora-tory animals" and human subjects.56 Thepotency ofPGF,,, may be increased by esterifyingthe carboxylic acid group.3 Thus, PGF,,-I-isopropyl ester has proved to be a potent ocularhypotensive in both ocular normotensive78 andglaucomatous human volunteers.9 It has beenproposed that the increased activity of PGF,2-1-esters occurs as a result of increasedlipophilicity'°: the carboxylic acid moiety, whichexists predominantly in the charged form atphysiological pH, is transformed by esterifica-tion to a non-polar, highly lipophilic species.This greatly enhances corneal uptake whichultimately results in improved delivery of
nascent PGF2, to anterior segment tissues.'0Despite the fact that esterifying the carboxylicacid group of PGF2, improves bioavailability, itdoes not appear to result in a clinically acceptableophthalmic drug because of ocular surface sideeffects. When considering the design of PGF2,prodrugs, there are alternatives to esterifying thecarboxylic acid.' 12 PGF2U contains three -OHgroups at positions 9, 11, and 15 which provideother options for synthesising ester prodrugs ofPGF2a.
In order to investigate alternative oppor-tunities for the design of PGF2U prodrugs withpotential ophthalmic application, a novel seriesof PGF2U alkyl acyl esters was synthesised whichcomprised 9, 11 , and 15 monoesters; and 9, 11, 9,15, and 11, 15 diesters. Three PGF2u lactoneswere also examined where the carboxylic acidmoiety forms an internal ester with one of the-OH groups. In order to characterise these PGF2aesters the following factors received particularattention: (1) inherent pharmacological activity,(2) ester hydrolysis by esterases and oculartissues, (3) effects on intraocular pressure, and(4) ocular surface hyperaemia.
Materials and methods
PHARMACOLOGICAL ACTIVITYInherent pharmacological activity was assessedin an FP receptor preparation.'3 Mouse Swiss3T3 fibroblasts were plated in culture flasks andwere fed Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal calfserum, 2 mML-glutamine, and 0 05 mg/ml gentamicin (allpurchased from Gibco, Grand Island, NY,USA). Cell cultures were maintained in a humid-ified atmosphere of 95% air, 5% carbon dioxideand grown to confluency.
Cells were removed from the culture flasks by a1 minute treatment with trypsin 0-05%/0 52mM EDTA (Gibco, Grand Island, NY, USA) at37°C. Proteolytic activity was arrested by adding5 ml of 10% fetal bovine serum in DMEM. Thecells were consecutively washed in Hank'sbalanced salt solution and Maullem's buffer'4:centrifugation for the washes was performed for15 minutes at 1200 rpm at room temperature.Cells were counted, resuspended in Maullem'sbuffer and incubated with 2 ,uM Fura 2/ace-toxymethyl ester in a shaking water bath for 30minutes at 37°C. The cells were subsequentlywashed in Maullem's buffer as above and resus-pended at a concentration of 2 x 106 cells/ml.Aliquots of 0 5 ml cell suspension were thenadded to autocap microtubes to provide 106 cellsper experimental determination of [Ca2+].
Fluorescence was measured in a Perkin-Elmer
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Studies on a novel series ofacyl ester prodrugs ofprostaglandinF2,6
LS-5 fluorescence spectrophotometer at excita-tion and emission wavelengths of 340 and 492nm, respectively with both slits at 10 nm. Foreach experimental determination 106 cells werewashed in Maullem's buffer at 1200 rpm for 5minutes, suspended in a 3 ml cuvette containing10' M eserine (Sigma, St Louis, MO, USA) anddissolved in Maullem's buffer. Stirring wasachieved by an overhead mounted paddle stirrerwith the temperature maintained at 37°C.Calibration of the Fura 2 signal was as previouslydescribed for UMR-106 cells.'4 Stock solutionsof PGF2,, and the prodrugs were prepared in0 1% polysorbate 80/10 mM TRIS.
ESTER HYDROLYSISAcetylcholinesterase (ACHE) extracted fromelectric eel, butyrylcholinesterase (BuCHE)extracted from horse serum, and carboxyl-esterase (CE) extracted from porcine liver werepurchased from Sigma (St Louis, MO, USA).Ocular tissues were obtained from New Zealandwhite rabbits (2-3 kg) and were used fresh formetabolic studies. The corneal epithelium wasremoved by gentle scraping with a scalpel, thecorneal stroma endothelium, the bulbar con-junctiva, and the iris-ciliary body were thensurgically excised. Excised tissues were kept onice, cut into small pieces and homogenised with aVirTishear homogeniser (The VirTis Co Inc,Gardiner, NY, USA) in ice cold 0-1 M phosphatebuffered saline at pH 7-4. The homogenate wascentrifuged at 3000 rpm for 30 minutes in aBeckman J22 1 centrifuge (Beckman Instru-ments, Fullerton, CA, USA) at 4°C. The super-natant was collected and stored at -70°C untiluse. Protein contents were determined by Bio-Rad assay (BioRad, Richmond, CA, USA) usingbovine serum albumin as a standard.PGF 2( prodrug metabolism was determined by
incubating [10 FtM] with esterase or ocular tissuehomogenate in 0-1 M phosphate buffer at pH 7-4and 37°C, with shaking at 100 oscillations/min inair. Aliquots (300 id) of the incubation mixturewere sampled at predetermined times through-out the experimental period and treated with200 tl of 0-3 N perchloric acid. Extraction wasperformed with 5 ml of dichloromethane. Fourml of the organic extract were removed andevaporated under a stream of nitrogen. Theresidue was reconstituted in organic solvent,injected, and analysed with high performanceliquid chromatography for concentrations ofparent prodrug, intermediate and/or PGF2,,.Three chromatographic systems were used forthe analyses of the diesters, monoesters, andPGF2,,. These consisted of a Beckman 114 pump(Beckman Instruments, Fullerton, CA, USA), aKratos 783 detector (Kratos Analytical, Ramsey,NJ, USA), and a Waters 710 WISP autoinjector(Millipore, Milford, MA, USA). The mobilephase consisted of a mixture of 0-02 M KH2PO4(pH adjusted to 3-0 with (H3PO4) and acetoni-trile. The flow rates were 1-5 ml/min. WatersNovopack ODS columns (3-9 mmx l50 mm,4 itm) were employed and effluents weremonitored using 200 nm detection. The outputfrom the ultraviolet detector was interfaced withthe Nelson chromatographic data collection
system (Perkin Elmer Nelson System, Inc,Cupertino, CA, USA). Rate constants for esterhydrolysis were derived from the slope of the lnconcentration-time plot. All metabolism experi-ments were performed in quadruplicate and therate constant is finally expressed as the mean(SEM) of the four individually determined rateconstants. They are normalised for ocular tissuehomogenate protein concentration (h/mgprotein).
IN VIVO EXPERIMENTSIntraocular pressure studies were performed inrabbits since this species mimics the response ofhuman eyes to PGF2, in that PGF2,, producespronounced ocular surface hyperaemia. NewZealand albino/Dutch belted crossbred rabbits ofboth sexes and weighing 1 5-2 5 kg were used.These rabbits had not previously received anytopical drugs. Intraocular pressure was measuredwith a pneumatonometer (Digilab) calibratedagainst the eyes ofanaesthetised rabbits by closedstopcock manometry. The correlation coefficientover a 10-30 mm Hg range was 0-98. Theanimals were acclimatised to pneumatonometryby taking unrecorded measurements beforeexperimental determinations of intraocular pres-sure. Corneal anaesthesia for tonometry wasprovided by topical application of one drop of0-05% proxymetacaine (proparacaine, Aller-gan).4 Intraocular pressure data were tabulated asthe difference between the change from baselinein intraocular pressure in test and control eyes asa convenience for depicting the large body ofdata. Statistical analysis employed Student'spaired t tests. Ocular surface hyperaemia wasassessed visually in rabbits and is described aseither present to any extent or completely absent.
All topical formulations were prepared in0-1% polysorbate 80/10 mM TRIS and adminis-tered in a 25 [tl volume: the contralateral eyereceived 25 pt1 vehicle as a control. ProstaglandinF2a 1,9,1, 11, and 1, 15 lactones were a generousgift from Upjohn (Kalamazoo, MI, USA), allother PGF2, esters were synthesised inhouse.'" 12 15-19Animal care and experimentation were per-
formed according to the ARVO resolution on theuse of animals in research.
Results
INTRACELLULAR [Ca2+] EFFECTSThe effect of the PGF2,, esters on the intracellular[Ca2 ] in Swiss 3T3 cells was used as an indicatorof inherent pharmacological activity and resultsare depicted in Figures 1-4. Standards for com-parison are provided in Figure 1 where PGF2,,produces a dose dependent increase in intra-cellular [Ca2+] which reaches maximum at I0O M(Fig IA) while PGF21-l-isopropyl ester does noteven produce a modest threshold effect until a10-5M concentration is reached (Fig 1B). Samplerecordings for these two compounds are pro-vided in Figure 2. The effect of 9, 11, and 15monoesters is depicted in Figure 3. By compar-ing dose-response curves it is apparent thatPGF2,-9-acetate retained a considerable degree of
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inherent pharmacological activity and was onlytenfold less potent than PGF2(, (Fig 3A). Incontrast, larger ester substituents at position 9resulted in less active compounds which pro-duced a clear threshold response only at 10 M(Fig 3A).
Similarly, ester substituents, such as iso-butyryl, isovaleryl, and pivaloyl at position 11 or15 resulted in compounds with inherent phar-macological activity similar to that of their9-monoester equivalents (Fig 3B, 3C). Di-esterification of PGF2,, OH groups resulted in adecrease in inherent pharmacological activity(Fig 4) such that they resembled PGF2,1-isopropyl ester in this regard. Thus, for 9, 11 (Fig4A), 9, 15 (Fig 4B), and 11, 15 (Fig 4C) diesters,no alteration in intracellular [Ca2+] was observeduntil a 10- M concentration was achieved. TheI Q I I I an tl 1 l~ IartAnfXcQ an di PCIP _1_
100 -
80 -
C 60-
Cu
) 40-
20 -
0 -
C
8 I7 --8 -7 -6 -5 -4
Log [M]Figure 3 Comparison of the effects ofPGF2,,-monoesters onintracellular [Ca2'] in Swiss 3T3 cells. The effects ofPGF2,-9-acetate (A), PGF21-9-isobutyrate (A), andPGF2,-9-pivalate (B) are shown in (A). (B) depicts theeffects ofPGF2,-11-isobutyrate (A) and PGF2a-11-pivalate. (C) depicts the effects ofPGF2,-15-isovalerate(a), and PGF2S-15-pivalate (B). Values are mean (SEM);n=3-4.
13 7, I, II.l) I3lI)J ILAvUllUb k
isopropyl, 1 1-pivaloyl diester echanges in intracellular [Ca2+] up tcnot shown).
PGF2, ESTERHYDROLYSISFigure 5 depicts two typical conce
profiles which illustrate the disalparent prodrug and the formatior
100-
CN
Figure 2 Samplerecordings ofthe Ca`+ signalin response to (A) PGF2.10- M (B) PGF2.-1-isopropyl ester 105 M.
LL
(C0.
xhibited 2'uno with determination ofmass balance. The conver-Dxhibited noM(sion ofPGF2,A--isopropyl ester to PGF2,, (Fig SA)10-'M (data and the conversion of PGF2U 9, 1 1-dipivaloyl
ester to PGF21-9-pivaloyl ester (Fig 5B) are usedto provide typical examples.Table 1 compares the rate constants for the
ntration-time hydrolysis of the monopivaloyl esters andppearance of PGF2R-1-isopropyl ester to PGF2,, in rabbit ocularof product, tissue homogenates. No detectable hydrolysis of
PGF2,,-9-pivaloyl ester was apparent. PGF2,,- 1-pivaloyl ester was hydrolysed at a slow rate in all
B tissues whereas PGF2,,-15-pivaloyl ester washydrolysed approximately one order of magni-tude faster. The most striking feature was therapid hydrolysis ofPGF2,,-1-isopropyl ester in the
O%iSiW*i4 corneal epithelium. However, in the iris-ciliary1 body and conjunctiva the hydrolysis rate for
PGF2,,-15-pivaloyl ester exceeded that forPGF2,A--isopropyl ester by approximately three-
-_ fold.(T0- ~The enzymatic hydrolysis pathways for con-
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Studies on a novel series ofacyl ester prodrugs ofprostaglandinF2,5
Figure 4 Comparison ofthe effects ofPGF2,-diesterson intracellular [Ca2'] inSwiss 3T3 cells. The effectsofPGF2,-9, lI-diacetate(A), PGF2t-9, 11diisovalerate (-), andPGF2, 9,11-dipivalate (a)are shown in (A). (B)Depicts the effects ofPGF2,,-9-15-diisobutyrate(A), PGF2,,-9, 15-diisovalerate (0), andPGF2,-9, 15-dipivalate(-). (C) depicts the effectsofPGF2,-1, 15-diisobutyrate (A),PGF2.-41, 15-diisovalerate(), and PGF2 -l, 15-dipivalate (-). Values aremean (SEM); n=3-4.
100 I A
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20
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4 -
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-8 -7 -6 -5 -4I- - I Il1 2 3 4
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B
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Figure 5 Concentration (ipm) v time profiles for (A)PGF2.-I-isopropyl ester in conjunctival homogenate and (B)PGF2,,-9, Il-dipivaloyl ester in corneal stroma homogenate.In (A) the disappearance ofPGF24-1-isopropyl ester (0) andthe appearance ofPGF2t (A) are shown with mass balancedepicted by the broken line. In (B) the disappearance ofPGF24-9, I 1-dipivaloyl ester (0) and the appearance ofPGF24-9-pivaloyl ester (A) are shown with mass balancedepicted by the broken line.
Ascarboxylic acid group did not lead to a similar
_______________________________ increase in hydrolysis of the 11-pivaloyl ester-8 -7 -6 -5 -4 moiety and the rates of conversion for the
PGF2,,-1-isopropyl, 1 1-pivaloyl diester, and theLog MIM 11-pivaloyl monoester were similar. PGF2,, for-
mation from PGF2,1 1, 11 -lactone proceeded at aof representative diesters in ocular tissue yet slower rate. The rates of PGF2,, formation pernates are depicted in Figures 6 and 7. se are reported in Table 3.ble levels of PGF2,, were not observed for Rate constants for the hydrolysis of selected1 and 9, 15-dipivaloyl esters and values PGF2,, esters by isolated esterases are given inthe rate constants for conversion to the Table 4. None of the PGF2,, esters studied were
ester (Table 2A). Enzymatic hydrolysis substrates forACHE and only PGF2,,-I-isopropyl,-1 1, 15-dipivaloyl ester was found to be esterwas hydrolysed byBuCHE, albeit at a muchc and formation of PGF2, occurred exclu- slower rate than by CE. Pivaloyl ester groups ata the 15-monoester intermediate (Fig 7). positions 11 and 15 were also hydrolysed by CE.rsis of the 1 1-pivaloyl group was, there- In contrast, CE did not hydrolyse pivaloyl esterach faster than for the 15-pivaloyl ester, groups at position 9 as indicated by an inability toin distinct contrast with the hydrolysis metabolise the 9-pivaloyl monoester or themonopivalates (Table 1). 9-monoester product of the 9, 11 and 9, 15-d, the 15-pivaloyl ester was the only dipivaloyl diesters.
detectable intermediate in ocular tissues, exceptfor a very small quantity of the 1 1-pivaloyl esterin the iris-ciliary body. In contrast with the iris-ciliary body, corneal endothelium and cornealepithelium, PGF2,. formation from PGF2,,, 15-pivalate was very rapid in the conjunctiva pre-venting accurate measurement of K2 over theexperimental period. It is also of interest to notethat although conversion of the 1 1-pivaloylmonoester to PGF2,. was slow in all tissues, thepresence of an additional ester substituent ineither the 9 or 15 position increased the rate ofhydrolysis at the 1 1-position. Esterification ofthe
INTRAOCULAR PRESSURE/OCULAR SURFACEHYPERAEMIAThe effects of PGF2,,, PGF),-1-isopropyl ester,the 9, 11, and 15-monoesters, and the diesters onrabbit intraocular pressure are summarised inTable 5. Ocular surface hyperaemia is describedas the percentage of animals which exhibitedobservable redness of the eyelid or the bulbarconjunctiva/sclera. PGF2,, and PGF2,-1-isopropylester both lowered intraocular pressure, butsignificant decreases were almost invariably
I,W_ -*- *-_..10
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associated with a total incidence ofocular surfacehyperaemia, which did not resolve until theocular hypotensive effects were either minimal orabsent. PGF2,,-9-isobutyryl and PGF2,-9-pivaloylesters both produced decreases in intraocularpressure with a similar potency to PGF2,,, butwith a reduced incidence ofocular surface hyper-aemia at 0-1%. At 1% doses the 9-monoesters
CH3 0H3C -1-
CH3 .
(1.) COOH
CH3bOH3CH-C-C OH
CH3 O
Figure 6 Enzymatichydrolysis (E-H20)pathway for PGF2 9, 11-dipivaloyl ester (compound1) and PGF2. 9, 15-dipivaloyl ester (compound2) to PGF2. 9-monopivaloylester (compound 3) +pivalic acid (in parenthesis).No conversion ofPGF2,9-monopivaloyl ester tonascent PGF2, wasdetected.
Figure 7 Enzymatichydrolysis (E-H20)pathway for PGF2, 11, 15-dipivaloyl ester (compound1). The major pathway isvia the PGF2, 15-monopivaloyl esterintermediate (compound 2)to PGF2a (compound 4)with pivalic acid liberatedat each conversion step (inparentheses). The rateconstants for this two stepprocess, K, and K2, aregiven in Table 2B. Analternative enzymatichydrolysis pathway toPGF2,, Il-monopivaloylester (compound 3) isindicated by the brokenarrow. This pathway was,however, absent in oculartissues except for the iris-ciliary body where itrepresented only a minorcomponent. PGF2,, 11-monopivaloyl ester is thenconverted to PGF2,,.
CH30
CH3-3 ..N CCOOH(2.)
OH ou CH3\CH30 CH3
also produced the initial ocular hypertensiveresponse typically produced by PGF2,, andPGF2,,-1-isopropyl ester. PGF2,, 11 and 15-monoesters potently lowered intraocular pres-sure and exhibited some separation of ocularhypotensive and ocular surface hyperaemiceffects at later time points. The 1 l-pivaloyl and15-pivaloyl esters were approximately equipo-tent to PGF2,,-1-isopropyl ester. The activity ofthe diesters examined was highly dependent onthe position of the ester groups. Diesters with afunctionality at position 9 did not profoundlylower intraocular pressure and generally exhib-ited a rather shallow dose-response profile. Incontrast, the 11, 15-diesters produced clear dosedependent changes in intraocular pressure. Alldiesters exhibited a similar level of separation
E*H20
E-H20
CH3 0H3C-Co
CH3
(3.)
z
OH OH
E CH3+ H3C-¢-COOH
CH3
CH3 0H3C C
CH3 -
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OH OH[ CH3H3C-¢-COOH
CH3
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(3.) COOH +
z~~~~~~~~
H3C--C OH
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.'E H20 E - H20
OH Of<,X5^ ~~~COOH (,
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z/OH O\ CH3
C-C-CH3 +0 CH3
CH3J
IH A4H' COOH
IH ,WOH
CH3H3C-C-COOH
CH3
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Studies on a novel series ofacyl ester prodrugs ofprostaglandinF2,5
Table 2 A Rate constants (lhlmg protein) (SD)for the hydrolysis of 9, 11 and 9, 15-dipivaloyl esters ofPGF2, in the presence ofrabbit ocular tissue homogenates. Note PGF2,,9-pivaloyl ester is the only product
Corneal epithelium Corneal stroma Iris-ciliary body Conjunctiva
between ocular hypotension and ocular surfaceredness as the monoesters.Comparison of the lactones provided more
striking differences in activity. Thus, PGF,,, 1,9-lactone was virtually devoid of ocular hypoten-sive activity, whereas both the 1, 11 and the 1, 15-lactones potently affected intraocular pressure.However, a separation of ocular hypotensive andocular surface redness effects was apparent onlyfor the 1, 1 1-lactone. PGF21-l-isopropyl, 11-pivaloyl diester was also examined as a comple-mentary 1, 1 1-diester for the 1, 1 1-lactoneinternal ester. PGF2,,-I-isopropyl, 1 1-pivaloyldiester showed a level of activity which approxi-mated that achieved for PGF2a, 1 1-pivaloyl mono-ester. Again, ocular hypotension tended to bemaintained after ocular surface hyperaemia hadresolved.
DiscussionThe novel prodrug series described herein wasdesigned to systematically evaluate the utility ofesterifying the -OH groups on the PGF2, mole-cule. Studies on their enzymatic hydrolysis, invitro pharmacological activity and effects on theeye revealed some important considerations forthe design of PGF2, prodrugs with potentialtherapeutic application in glaucoma.
In studying the enzymatic conversion of thesenovel prodrugs to nascent PGF2, we found that
Table 4 Rate constants (hlunitlml) (SD)for the hydrolysis of the PGF, prodrugs bycommercial esterases
11, 15-dipivalate * * 4-4 (0-31)* No detectable hydrolysis.
carboxylesterase (CE) was critically important inthe enzymatic hydrolysis of this novel series ofPGF2,, prodrugs where the -OH groups had beenesterified. These PGF2,, esters were substrates forCE but not ACHE or BuCHE. PGF2-I-estershave previously been described as substrates forBuCHE20 and these studies confirm previousfindings. However, PGF2,-I-isopropyl ester washydrolysed at a much faster rate by CE indicatingthat CE could be of greater importance in ocularenzymatic hydrolysis of this ester prodrug in theeye. The 11 and 15-monopivaloyl and the 11, 15-dipivaloyl PGF2, esters were also hydrolysed byCE, whereas the 9-pivaloyl ester did not appearto be hydrolysed. Since PGF2,,-1-isopropyl esterand the 11-mono, 15-mono, and 11, 15-diesterslowered intraocular pressure much more effec-tively than the PGF2, esters containing an acylgroup at position 9, CE would appear to repre-sent the target enzyme for designing PGF2,prodrugs for treating glaucoma. CE exists, how-ever, in several isoforms,22'22 and the identity ofthese different CE enzymes and their regionaldistribution in ocular tissues remains to bedetermined.The enzymatic hydrolysis rates for the
PGF2,-1-isopropyl ester and the novel PGF2Qpivaloyl esters in ocular tissues varied markedly.The inability of esterases to hydrolyse acyl esterssignificantly at position 9 was also reflected instudies with ocular tissue homogenates; 9, 11,and 9, 15-diesters were subject only to 11 and 15hydrolysis with the 9-monoester as the finalproduct. Although the rate constants for the 11and 15-monopivaloyl were similar for commer-cially available CE, PGF2 1 1-pivaloyl ester washydrolysed approximately 10 times more slowlythan PGF,a 15-pivaloyl ester in ocular tissues.Thus, the isolated CE used for these studies doesnot seem to be entirely representative of esteraseactivity in ocular tissues. Since several isozymesof CE exist, differences in CE subtype in oculartissues is a likely explanation for these variationsin hydrolysis rate. Moreover, enzymatic hydro-lysis of these PGF2, esters by lipases cannot bediscounted. Interestingly, hydrolysis of the 11-ester, but not the 15-ester moiety, is stronglyinfluenced by ester substitution at position 9.Thus, the 1 I-pivaloyl group was hydrolysedmuch more rapidly in the case of the 9, 11-diester, notably in the iris-ciliary body, whereashydrolysis of the 15-pivaloyl group in the 9, 15-diester was not similarly altered. The hydrolysisrates for the 11 and 15-monoesters were notpredictive for the pattern of enzymatic conver-sion of PGF2, 11, 15-dipivaloyl ester. In alltissues studied, hydrolysis ofthe 11, 15-diester atposition 11 was substantially faster than at posi-tion 15, with no detectable levels of the 11-pivalate ester in all but one ocular tissue homoge-nate. Thus, the order of hydrolysis for the estergroups of PGF2,,-1 1, 15-dipivalate are the con-verse of that which would be predicted fromstudies on the respective monoesters. In the caseofPGF2,,-1-isopropyl, 1 1-pivaloyl diester, hydro-lysis of the 1 1-pivaloyl ester appeared rate limit-ing, with PGF2U formation proceeding at a similarrate to that of the 1 1-monoester. PGF2 1, 11-lactone was hydrolysed more slowly. The shapeand polarity changes associated with a 1, 11-
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Table S Comparison of the effect ofPGF2, esters on intraocular pressure (mm Hg) and ocular surface hyperaemia. Intraocular pressure is given as thedifference between the changes from baseline in test and control eyes. Ocular surface hyperaemia is simply described as present or absent. n=6 or 8, *p<0.O5,**p<O.O1 according to Student's paired t test
% Animals exhibiting ocular surface hyperaemia at time (h)Doe
lactone (internal ester) may influence its ability to between the iris-ciliary body and the ocularact as a substrate for many enzymes.'2 surface tissues would be expected to be large for aThe ultimate objective in designing PGF2,, prodrug with clinical potential. This is not the
prodrugs is to design agents which lower intra- case for PGF2,- 1 -isopropyl ester where combinedocular pressure with minimal ocular surface PGF2,, formation for the corneal epithelium andhyperaemia. In order to accomplish this objec- the conjunctiva clearly exceeds that of the iris-tive, enzymatic hydrolysis must be minimised in ciliary body. For the 11-mono, 15-mono, and 11,the conjunctiva and also in the corneal epithe- 15-diesters, the conversion rate in the iris-ciliarylium so as to avoid back diffusion of nascent body exceeded that in the ocular surface tissues.PGF2,, to the tear film. The ratio offormation rate Since the uveoscieral site of action of PGF2,'23
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Studies on a novel series ofacyl ester prodrugs ofprostaglandin F2,
resides in the vicinity of the ciliary body, thesenovel agents appear to provide a step towardsidentifying a PGF29, prodrug with clinical utility.
In addition to a favourable enzymatic conver-sion profile, inherent pharmacological activity ofa prodrug should be minimal. In order to deter-mine inherent pharmacological activity, intra-cellular [Ca2+] in Swiss 3T3 cells was used as a cellpharmacology model for the FP receptor.'3 Thisis a useful system for identifying inherent PGF2,prodrug activity from a number of standpoints.In particular, the Ca2` signal is rapid responsewhich minimises the potential influence of intra-cellular CE activity. The structure-activity pro-file for the PGF29, prodrugs may be summarised asfollows. Firstly, a -COO- group appears criticalfor FP receptor stimulation: removal of theformal negative charge by esterification or lac-tonisation results in compounds which are essen-tially devoid of activity. Secondly, mono-esterification of the -OH groups can providecompounds which retain approximately 100th ofthe activity of PGF2U regardless of the position ofthe ester group. Diesterification is required forcompounds with reduced potency at the FP-receptor comparable with that of PGF2,,-I-isopropyl ester. The size of the acyl ester alsoappears important. In the case of monoesters,PGF2,-9-acetate was approximately 10 timesmore potent than PGF2,-9-isobutyrate or 9-piva-late. This may not hold for the diesters since allthe 9, 1 1-diesters were approximately equiactive.
In terms of obtaining separation betweenocular hypotensive and ocular surface hyper-aemic effects, the objective was partiallyachieved with the 11 and 15-monoesters, and the11, 15-diesters. The separation was temporal andmost apparent at later time points. The ocularhypotensive activity of the 11 and 15-monoestersand the 11, 15-diesters was comparable with thatof PGF2,A-l-isopropyl ester, despite being con-verted to PGF2t at a much slower rate in the iris-ciliary body. This suggests that rapid formationofnascent PGF22, is not necessary for providing anacceptable level of ocular hypotensive activity.The ocular hypotensive activity of PGF2,, 1, 1 1-lactone lends further support to this view. Thelactone series exhibited a rank order of potency[1, 15>11, 11>1, 9- (inactive)], which is essen-tially consistent with hydrolysis rates in previ-ously investigated biological systems. 12
In addition to ocular hypotension, an initialocular hypertensive response was also observedfor most of the PGF2,, esters. This phenomenonhas been extensively reported as prominent inrabbits and results from breakdown of the blood-aqueous barrier.2F26 Such responses to PGF29, itsesters, and its structural analogues are minimalor absent in cats, dogs, monkeys, and humansubjects. 1-9 Thus, the ocular hypertensiveresponses observed in rabbits have little or noclinical significance.Despite the absence of measurable enzymatic
hydrolysis, PGF22, 9-mono, 9, 11, and 9, 15-diesters did exhibit ocular hypotensive activity,albeit much weaker than the other esters evalu-ated. Moreover, some ocular surface hyperaemiawas apparent. The detection limit of the esterhydrolysis assay is approximately 0 3 nM/ml andthe formation of a small, undetectable level of
nascent PGF2,, may account for the activityobserved for 9-mono and 9-diesters. Regardlessofthese modest activities, emphasis on 11 and 15-esters appears preferable for the design of PGF2,prodrugs with potential clinical application.
The authors express their appreciation to J Boag for excellentsecretarial assistance and to Dr D S Chien for helpful critique.They also thank Dr Gordon L Bundy of Upjohn for providingsamples of the PGF2,, lactones used in these studies.
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