University of Groningen Reflections on Flurbiprofen Eyedrops Van Sorge

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Reflections on flurbiprofen eyedrops van Sorge, Adriaan Alastair

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REFLECTIONS ON FLURBIPROFEN EYEDROPS REFLECTIONS ON FLURBIPROFEN EYEDROPS

RIJKSUNIVERSITEIT GRONINGEN

REFLECTIONS ON FLURBIPROFEN EYEDROPS REFLECTIONS ON FLURBIPROFEN EYEDROPS

PROEFSCHRIFT

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen, op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op maandag 2 december 2002 om 14.15 uur door

Adriaan Alastair van Sorge

geboren op 28 oktober 1944 te New Rochelle, New York, USA PROMOTORES Prof. dr. J. Zaagsma Prof. dr. W.J. Quax Prof. dr. H.W. Frijlink

CO-PROMOTOR Dr. N.J. van Haeringen

BEOORDELINGSCOMMISSIE Prof. dr. P.T.V.M. de Jong Prof. dr. J.R.B.J. Brouwers Prof. dr. H.V. Wikström

ISBN: 90-9016364-6 Verzorging proefschrift: B-Point, Karin Scheele PARANIMFEN Dr. R.F.A. Weber Dr. A.J.P.F. Lombarts

My parents, who got me started

Aty, Nina, Joline and Arlette, who let me go on

Eelco, who wouldn't let me quit

CONTENTS

Preface A historical introduction 9 Scope of thesis

Chapter 1 General introduction 17 1.1 Flurbiprofen, an overview 1.2 Cataract and caractogenesis 1.3 Cystoid macular edema 1.4 Prostanoids

Chapter 2 Rationale for using a phosphate buffer for S(+) flurbiprofen eyedrops. 43

Chapter 3 Flurbiprofen, S(+), eyedrops: formulation, enantiomeric assay, shelflife and pharmacology (1). 49

Chapter 4 Specificity of flurbiprofen and enantiomers for inhibition of prostaglandin synthesis in bovine iris/ciliary body (2). 63

Chapter 5 Flurbiprofen and enantiomers in ophthalmic solution tested as inhibitors of prostanoid synthesis in human blood (3). 73

Chapter 6 Constitutive cyclooxygenase-1 and induced cyclooxygenase-2 in isolated human iris inhibited by S(+) flurbiprofen (4). 83

Chapter 7 99mTc-Diflunisal and the human iris: topical application reveals localization (5). 95

Chapter 8 S(+) flurbiprofen and R(-) flurbiprofen. 99mTc-labeling reveals difference in stereochemistry (6). 107

Chapter 9 Alternative splicing of cyclooxygenase-1 mRNA in the human iris (7). 115

Summary/Samenvatting 123 List of publications 135 Dankwoord 137 Curriculum Vitae 143 Color pictures 147 Addendum 151 REFERENCES

1. van Sorge AA, Wijnen PH, van Delft JL, Carballosa Coré-Bodelier VMW, van Haeringen NJ. Flurbiprofen, S(+), eyedrops: formulation, enantiomeric assay, shelflife and pharmacology. Derived from Pharm World Sci 1999;21:91-5. 2. Sorge van AA, Delft van JL, Bodelier VMW, Wijnen PH, Haeringen van NJ. Specificity of flurbiprofen and enantiomers for inhibition of prostaglandin synthesis in bovine iris/ciliary body. Prostaglandins Other Lipid Mediat 1998;55:169-77. 3. Haeringen van NJ, Sorge van AA, Delft van JL, Carballosa Coré-Bodelier VMW. Flurbiprofen and enantiomers in ophthalmic solution tested as inhibitors of prostanoid synthesis in human blood. J Ocular Pharmacol 2000;16:345-52. 4. Haeringen van NJ, Sorge van AA, Carballosa Coré-Bodelier VMW. Constitutive cyclooxygenase-1 and induced cyclooxygenase-2 in isolated human iris inhibited by S(+) flurbiprofen. J Ocular Pharmacol 2000;16:353-61. 5. Sorge van AA, Etten van RJ, Rehmann CJ, Rijnders AJM, Haeringen van NJ. 99mTc- Diflunisal and the human iris: topical application reveals localization. J Ocular Pharmacol 2002;18:185-95. 6. Sorge van AA, Ruiken IWM, Janssen HWM, Haeringen NJ. S(+) flurbiprofen and R(-) flurbiprofen. 99mTc-labeling reveals difference in stereochemistry. Enantiomer 2002; Accepted pending suitable revision. 7. Dröge MJ, van Sorge AA, van Haeringen NJ, Quax WJ, Zaagsma J. Alternative splicing of cyclooxygenase-1 mRNA in the human iris. Submitted.

8 PREFACE Preface

A HISTORICAL INTRODUCTION

A simple question put forward in 1980 by one of the ophthalmologists to the hospital pharmacist led to a chain of events culminating in this thesis. The question was: "Is it possible to prepare indomethacin eyedrops?".

The principal reason for the question were reports on eye research, mainly of Japanese origin (1, 2, 3, 4, 5), indicating that use of topically applied indomethacin could prevent cystoid macular edema after lens extraction, required e.g. when a patient had acquired a senile cataract. The incidence of this complication varied between 2 and 50% but reports of 70% were known as well. The complication had been reported earlier as a newly defined vitreous syndrome following cataract surgery and was described in 1953 (6).

Just over 65 years ago it was postulated by Selye (7) that our physiological system, activated by stress, not only will try to protect and restore itself but also can derail and afflict damage. The most common responses to stress are activation of the sympathetic nervous system and of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in or accompanied by immunological changes.

The immunological defense mechanisms of the ocular surface have been reviewed in detail in 1983 (8). A review ten years earlier (9) refers to the finding by Ambache of a physiological smooth muscle stimulant as a constituent of the rabbit iris ("Irin") in 1957, and the further elucidation of its nature in 1959 (10,11).

In 1967 the synthesis of prostaglandins in the pig iris was reported (12) and in 1968 their release from bovine iris (13). Subsequently, prostaglandins were related to various ocular functions indeed (14). In 1971 a pivotal study was reported by Vane (15) demonstrating the inhibiting effect on prostaglandin synthesis as the mechanism of action of non-steroidal anti- inflammatory drugs (NSAIDs). Release of prostaglandins in the rabbit eye was shown following an acute immunological inflammatory reaction induced by a single intravitreal injection of sterile crystallized bovine serum albumin (16). This report preceded a study, also in rabbits, demonstrating that an acutely traumatized eye shows an irritative response characterized by hyperaemia of the conjunctiva and iris, miosis and disruption of the blood-aqueous barrier. One of the signs of blood-aqueous barrier disruption is an increased concentration of blood proteins in the aqueous humour. Using a relevant pharmacological model a significant reduction in protein concentration in the aqueous humour could be demonstrated by pretreating the animal with a rectal

10 Preface

dose of acetosal (acetylsalicylic acid; 600mg) (17). Stabilization of the blood-aqueous barrier in the human eye with acetosal administered orally (4 doses of 650mg; 3 before and one on completion of ocular surgery) was reported in 1975 (18). Levels of prostaglandin-like activity in aqueous humour samples correlated well with the clinical intensity of uveitis. This in contrast to patients with cataract whose aqueous humour was essentially devoid of activity when the eyes were uninflamed, and low in activity when treated with corticosteroids (19). In vitro inhibition of rabbit prostaglandin synthase systems of various organs, including the eye, by indomethacin was reported in 1974 (20). Tissue homogenates of the iris and the ciliary body (anterior uvea), the conjunctiva, the cornea and retina were prepared; spleen and kidney (medulla) were also investigated. The inhibitory effect of indomethacin was clearly demonstrated and the compound showed differential inhibitory capacity. The retinal enzymes were least susceptible to inhibition followed by iris and ciliary body (twofold more) and the conjunctiva (six fold more). This also raised the possibility that prostaglandins are involved both in external as well as internal ocular inflammation.

The potential complication reported by our ophthalmologists that could arise after cataract surgery, cystoid macular edema, seemed linked to the release of prostaglandins. Thus, in the event of adequate permeation of indomethacin through the cornea, the edema should be prevented by topical administration of eyedrops. In 1972 it has been demonstrated by application of 100 microgram radiolabelled indomethacin to the cornea (either in aqueous suspension form or in oily solution) that the drug could be detected in the cornea, aquous humor, iris, choroids and retina of the rabbit eye (21). An inflamed eye gave rise to enhanced penetration. In 1983 it was subsequently shown, by use of topically applied radiolabelled indomethacin (2% suspension in sesame oil, including 17% ethanol) on phakic and aphakic rabbit eyes, that penetration into the vitreous took place; the concentration in the vitreous was higher for the aphakic eye. Concentrations in retina and choroid were the same for both conditions, suggesting a pathway other than diffusion through the vitreous to reach these tissues. Aqueous humour concentrations were sufficient to inhibit prostaglandin synthesis in either situation (22).

Indomethacin, [1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetic acid, molecular weight 357.8 dalton, pKa 4.5, is practically insoluble in water. In aqueous buffers at pH 7.5 - 8.0 it can be rendered soluble (23). In basic solutions hydrolysis of indomethacin occurs into 5-methoxy-2-methylindolyl-3-acetic acid and 4-chloroben- zoic acid (24,25,26). These substances are pharmacologically inactive. In the European pharmacopea (1997) 4-chloro-benzoic acid is mentioned as an impurity.

11 Preface

Pharmacokinetics of indomethacin are as follows. The major route of elimination is by transformation in the liver and involves glucuronidation, O-demethylation and N- deacylation. The major (inactive) metabolites are desmethyl indomethacin, des- chlorobenzoyl indomethacin and their glucuronides. Protein binding is more than 90%. Volume of distribution is 0.12 L/kg; clearance is 1-2 mL/min/kg with a half-life of 6 hours. The compound is excreted unchanged in urine for 30%. Indomethacin was introduced into the field of ophthalmology in different types of formulations including a solution in sesame oil and an ophthalmic aqueous suspension (1,27). Concentrations in suspension eyedrops varied between 0.5% to 1% and in oily solutions from 0.1 to 1%. In the Dutch literature several formulations of indomethacin eyedrops followed the first international reports (vide supra) on the prevention of cystoid macular edema after lens extraction (28,29,30). As use expanded in the clinic, reports indicated that the prepared solutions, being a suspension or an aqueous solution, were irritating to the eye (burning sensation). A reduction in concentration was suggested from 1% to 0.2% or 0.1% to prevent this undesirable effect. In 1981 it was shown that four different indomethacin suspension eyedrops, all being 0.5% in concentration, differed in prostaglandin synthase inhibiting activity, which was attributed to the differences in physicochemical properties. It was concluded that the use of eyedrops as a suspension yields irrepro- ducible results from the pharmacokinetic point of view and gives rise to subjective complaints of irritation in the eye (31). In 1984 Indoptol®, an aqueous eyedrop suspension of 1% indomethacin, was introduced to the Dutch market and in 1986 Indocid® of comparable composition was introduced in France. In 1987 a second presentation of indomethacin followed in France in the form of Indocollyre® (0,1%), which was introduced in The Netherlands in 1994. This formulation contains indomethacin as a lyophilized (freeze-dried) product which is brought into solution by addition of a sterile borate buffer. In the international literature aqueous formulations of indomethacin eyedrops have been published (32,33,34) reflecting the need for a more suitable and reliable pharmaceutical preparation. Ongoing own research with different bases, L- Lysine, D-Lysine, L-Arginine, D-Arginine, and Tromethamol (not published), to provide an indomethacin solution with an acceptable shelflife, did not provide suitable pharmaceutical alternatives. They all were aqueous solutions in order to circumvent the irritating properties of the suspension based eyedrops and the sesame oil based solution causing blurring of vision by difference in refractive index. However, our originally introduced solution (29) without extra pharmaceutical excipients and having a concentration of indomethacin of 0.1% remained the mainstay of the eye clinic. This solution was tested in a pharmacological setting in the rabbit eye using a paracentesis model of removing the aqueous humor and measuring the influx of protein and fluorescein into the secondary aqueous humor (35). The results

12 Preface

showed, in a concentration of indomethacin as low as 0.05%, 90 - 100% pharmacological efficacy in inhibiting fluorescein and protein influx (36). The indomethacin 0.1% formulation was incorporated in the Dutch national formulary (FNA) in 1986. Inpracticalities with indomethacin in aqueous solution - no sterilization possible and a short shelflife - prompted us to investigate the possibility in formulating eyedrops based on a different NSAID. In 1990 topically applied S(+) ibuprofen was reported to be effective in a rabbit model of interleukin-1 (37) or paracentesis induced uveitis (38) at relatively elevated concentrations (0.9% and 0.8% respectively). Also with S(+) naproxen, marketed by Syntex as enantiomeric pure NSAID, an anti-inflammatory effect of eyedrops (0,5%) was demonstrated experimentally (39). In our quest for a pharmaceutically more acceptable solution of an NSAID, we turned to the USP in which a flurbiprofen ophthalmic solution is mentioned. Ophthalmic solutions of flurbiprofen, diclofenac, and indomethacin (pH 7.5), have been subjected to research in rabbit eyes to investigate the maximal effect in preventing breakdown of the blood-aqueous barrier (40). Effective doses [nmol] per eye resulting in 50% inhibition (ID50) of influx of protein and of fluorescein into secondary aqueous humor after paracentesis corresponded well for indomethacin and flurbiprofen (12 nmol for flurbiprofen, 11 nmol for indomethacin, and 8.0 nmol for flurbiprofen and 9.0 nmol for indomethacin, respectively). In a comparative test of 11 nonsteroidal anti-inflammatory compounds in 0.01% solution, using the rabbit paracentesis model, flurbiprofen proved to be the most effective, showing a half-life of the inhibitory effect of 10 hours (41). A speciality,

Ocufen®, containing 0.03% flurbiprofen sodium 2H2O (equivalent to 0.024% flurbiprofen acid), is on the market in the United States since 1987 for inhibition of intraoperative miosis (42). Ocuflur® of comparable composition, marketed in Belgium, is also indicated for use in intraoperative inhibition of miosis, treatment of inflammation as a result of surgical intervention or trabeculoplasty by laser treatment and for prevention of cystoid macular edema after cataract surgery. We embarked on a study to manufacture flurbiprofen eyedrops by protocol of june 1992. A letter of consent to aid the project (9206SO.008) was issued January 8th 1993 by the SWOR (Stichting ter bevordering van Wetenschappelijk Onderzoek in ziekenhuis Rijnstate).

REFERENCES

1. Miyake K. Prevention of cystoid macular edema after lens extraction by topical indomethacin (I). Albrecht v. Graefes Arch Klin Exp Ophthal 1977;203:81-8. 2. Mochizuki M, Sawa M, Masuda K. Topical indomethacin in intracapsular extraction of senile cataract. Jpn J Ophthalmol 1977;21:215-26. 3. Miyake K. Prevention of cystoid macular edema after lens extraction by topical indomethacin (II): a control study in bilateral extractions. Jpn J Ophthalmol 1978;22:80-94.

13 Preface

4. Miyake K, Sugiyama S, Norimatsu I, Ozawa T. Prevention of cystoid macular edema after lens extraction by topical indomethacin (III): Radioimmunoassay measurement of prostaglandins in the aqueous during and after lens extraction procedures. Albrecht v. Graefes Arch Klin Exp Ophthal 1978;209:83-8. 5. Sholiton, DB, Reinhart WJ, Frank KE. Indomethacin as a means of preventing cystoid macular edema following intracapsular cataract extraction. Am Intra-ocular Implant Soc J 1979;5:137-40. 6. Irvine SR. A newly defined vitreous syndrome following cataract surgery. Interpreted according to recent concepts of the structure of the vitreous. Am J Opthalmol 1953;36:599-619. 7. Selye H. Syndrome produced by diverse nocuous agents. Nature 1936;138:32. 8. Chandler JW, Gillette TE. Immunologic defense mechanisms of the ocular surface. Ophthalmology 1983;90:585-91. 9. Neufeld AH and Sears ML. Prostaglandin and eye. Prostaglandins 1973;4:157-75. 10. Ambache N. Properties of irin, a physiological constituent of the rabbit’s iris. J Physiol 1957;135:114-32. 11. Ambache N. Further studies on the preparation, purification and nature of irin. J Physiol 1959;146:255-94. 12. van Dorp DA, Jouvenaz GH, Struijk CB. The biosynthesis of prostaglandin in pig eye iris. Biochim Biophys Acta 1967;137:396-9. 13. Posner J. The release of prostaglandin E2 from the bovine iris. Br J Pharmacol 1968;34:163P-4P. 14. Waitzman MB. Possible new concepts relating prostaglandins to various ocular functions. Survey of Ophthalmology 1970;14:301-26. 15. Vane JR. Inhibition of prostaglandinsynthesis as a mechanism of action for aspirin-like drugs. Nature 1971;231:232-5. 16. Eakins KE, Whitelocke RAF, Perkins ES, Bennett A, Unger WG. Release of prostaglandins in ocular inflammation in the rabbit. Nature New Biology 1972;239:248-9. 17. Neufeld AH, Jampol LM, Sears ML. Aspirin prevents the disruption of the blood-aqueous barrier in the rabbit eye. Nature 1972;238:168-9. 18. Zimmerman TJ, Gravenstein N, Sugar A, Kaufman HE. Aspirin stabilization of the blood-aqueous barrier in the human eye. Am J Ophthalmol 1975;79:817-9. 19. Eakins KE, Whitelocke RAF, Bennett A, Martenet AC. Prostaglandin-like activity in ocular inflammation. BMJ 1972;3:452-3. 20. Bhattacherjee P, Eakins KE. Inhibition of the prostaglandin synthase systems in ocular tissues by indomethacin. Br J Pharmac 1974;50:227-30. 21. Hanna C, Sharp JD. Ocular absorption of indomethacin by the rabbit. Arch Ophthal 1972;88:196-8. 22. Green K, Bowman K, Luxenberg MN, Friberg TR. Penetration of topical indomethacin into phakic and aphakic rabbit eyes. Arch Ophthalmol 1983;101:284-8. 23. Katz IM. Indomethacin. Ophthalmology 1981;88:455-8. 24. Krasowska H, Krowczynski L, Bogdanik Z. The assay of indomethacin in the presence of its hydrolytic degradation products. Pol J Pharmacol Pharm 1973;1973;25:417-21. 25. Kahns, AH, Jensen, PB, Mørk N, Bundgaard H. Kinetics of hydrolysis of indomethacin and indomethacin ester prodrugs in aqueous solution. Acta Pharm Nord 1989;1:327-36. 26. Tomida H, Kuwada N, Tsuruta Y, Kohashi K, Kiryu S. Nucleophilic aminoalcohol-catalyzed degradation of indomethacin in aqueous solution. Pharm Acta Helv 1989;64:312-5. 27. Yanuzzi LA, Landau AN, Turtz AI. Incidence of aphakic cystoid macular edema with the use of topical indomethacin. Ophthalmology 1981;88:947-54. 28. Lute NP, Vyth A, De Keizer RJW. Indometacine oogdruppels 0,5%. Pharm Weekbl 1980; 115:1663-4. 29. Van Nispen tot Pannerden EBLM, Van Sorge AA. Waterige oogdruppels met indometacine in oplossing; “corpora agunt nisi soluta”. Pharm Weekbl 1981;116:386-7.

14 Preface

30. Cox HLM van der Graaf H. Indometacine-oogdruppels als oplossing. Pharm Weekbl 1981;116:387-8. 31. Oosterhuis JA, van Haeringen NJ, Glasius E, van Delft JL, Swart-van den Berg M. The effect of indomethacin on the anterior segment of the eye after paracentesis. Documenta Ophthalmologica 1981;50:303-13. 32. Bechetoille A, Chabanais JL, Jallet G, Saraux H. Contusion et perméabilité de la barrière héma- to-aqueuse à la fluorescéine. Influence d’ un pré-traitement par l’indométacine locale. J Fr Ophthalmol 1978;1:139-43. 33. Liou S-W, Yen R-J. The effect of 0.1% indomethacin eyedrops on cataract surgery. J Ocul Pharmacol 1991;7:77-81. 34. Kahan LI, Bögi J, Farkas A, Tüdos F, Imre Gy. Az Indosol – nagy terápiás hatású nemszteroid gyulladásgátló – ismertetése. Acta Pharmaceutica Hungarica 1994; 64:125-9. 35. Van Haeringen NJ, Oosterhuis JA, van Delft JL, Glasius E and Noach EL. A comparison of the effects of non-steroidal compounds on the disruption of the blood-aqueous barrier. Exp Eye Res 1982;35:271-7. 36. Van Sorge AA, Van Nispen tot Pannerden EBLM, Janssen HWM. Oogdruppels met lage concentratie indometacine: bereidingsvoorschrift en onderzoek naar de werkzaamheid. Pharm Weekbl 1986;121:1039-46. 37. Tilden ME, Boney RS, Goldenberg MM and Rosenbaum JT. The effects of topical S[+]-ibuprofen on interleukin-1 induced ocular inflammation in a rabbit model. J Ocul Pharmacol 1990;6:131-5. 38. Tjebbes GWA, van Delft JL, Barthen ER, van Haeringen NJ. d-Ibuprofen in ocular inflammation induced by paracentesis of the rabbit eye. Prostaglandins 1990;40:29-33. 39. Stampinato S, Marino A, Bucolo C, Canossa M, Bachetti T, Mangiafico S. Effects of sodium naproxen eyedrops on rabbit ocular inflammation induced by sodium arachidonate. J Ocul Pharmacol 1991,7:125-133. 40. Van Haeringen NJ, Oosterhuis JA, van Delft JL, Glasius E and Noach EL. A comparison of the effects of non-steroidal compounds on the disruption of the blood-aqueous barrier. Exp Eye Res 1982;35:271-7. 41. Van Haeringen NJ, Glasius E, Oosterhuis JA, van Delft JL. Drug prevention of blood-aqueous barrier disruption. Ophthalmic research 1983;15:180-4. 42. Anonymous. Flurbiprofen – an ophthalmic NSAID. The Medical Letter 1987;29:58-9.

15 Preface

SCOPE OF THE THESIS

The studies described in this thesis were performed to investigate and to evaluate (1) the pharmaceutical application of flurbiprofen in eyedrops and (2) the pharmacology of this non-steroidal anti-inflammatory drug - both the racemic form and the individual enantiomers -, with special reference to the constitutive and inducible cyclooxygenases, COX-1 and COX-2, respectively. Chapters 2 and 3 cover the pharmaceutical aspects of S(+) flurbiprofen eyedrops, i.e. the formulation, the analysis (including the development of an enantiomeric assay), and the chemical and enantiomeric stability under different conditions and periods of time. In Chapter 4 the specificity of flurbiprofen and its enantiomers for inhibition of

PGE2 production by COX-1 in the bovine iris/ciliary body was investigated including the possibility of chiral inversion during the period of incubation. The interaction with the COX-1 and COX-2 isozymes in whole human blood, an extra-ocular matrix, was addressed in Chapter 5. COX-1 activity was monitored by

measuring TxB2 (the stable metabolite of TxA2) production from platelets whereas

COX-2 activity was determined using PGE2 production in monocytes, following induction of this isozyme by LPS. In Chapter 6 the interaction of S(+) flurbiprofen with COX-1 and COX-2 in the human iris was studied. After LPS-treatment for 24h, substantial amounts of COX-2 immunoreactivity could be visualized for the first time in human iris/ciliary body preparations. Remarkably, S(+) flurbiprofen showed a 3,600-fold higher potency for inhibiting COX-1 compared to COX-2. Furthermore, the susceptibility of human iris COX-1 for inhibition by S(+) flurbiprofen was 70-fold higher than of COX-1 in human blood. In Chapter 7 99mTc-labeled diflunisal eyedrops were applied in the human eye in an attempt to visualize the internal structures having high(est) COX-activity. Diflunisal was used for radiolabeling instead of S(+) flurbiprofen because the labeling efficiency of the latter compound was insufficient (Chapter 8). Scintigraphic activity surrounding the pupil indeed provided clear evidence of visualization of the iris/ciliary body. In the final Chapter the occurrence of alternative splicing of COX-1 in RNA in the human iris was explored, as a possible explanation of the remarkably high affinity of S(+) flurbiprofen for COX-1 reported in Chapter 6.

16 CHAPTER 1

GENERAL INTRODUCTION Chapter 1

1.1 FLURBIPROFEN - AN OVERVIEW

Introduction Flurbiprofen, CAS registry number (Substance name) 5104-49-4, a member of the phenylalkanoic acids (1), a white (or almost white) crystalline powder, melting point 114-117°C, practically insoluble in water, but readily soluble in most organic solvents, also known as a hydratropic acid analog (2), was already subjected for evaluation of its platelet aggregation inhibiting action in 1973 (3). Chemically it is known as: 2-fluoro-α-methyl-[1,1'-biphenyl]-4-acetic acid; 2-fluoro-α-methyl-4-biphenyl-acetic acid; 2-(2-fluoro-4-biphenylyl)propionic acid; 3-fluoro-4-phenylhydratropic acid (4). In 1993 its potent anti-platelet activity was evaluated in a double-blind, placebo-controlled, multi-centre study for efficacy on preventing reinfarction and reocclusion after successful thrombolysis or angioplasty in acute myocardial infarction (5).

CH3 F CO2H

Flurbiprofen

Flurbiprofen is described in the recent editions of the United States (USP), European (EP), British Pharmacopoeia (BP) and Japanese Pharmacopoeia (JP). In the USP both flurbiprofen and its sodium salt are described in the racemic form. In the EP the racemic form is also described; however in the EP and BP monograph of flurbiprofen the existence of an enantiomer is alluded to ("and enantiomer"). The Japanese Pharmacopoeia (JPXIII) gives no hint of the chiral nature of the flurbiprofen molecule. A solution of flurbiprofen in methanol giving no optical rotation is the only description given thereof. A monograph for flurbiprofen eyedrops is mentioned in the USP as "Flurbiprofen sodium ophthalmic solution" and in the BP as "Flurbiprofen eyedrops". They contain not less than 90.0% and not more than 110.0% of the prescribed or stated amount. The sodium salt of flurbiprofen in the USP and BP is available in the dihydrate form. In the EP, five impurities for flurbiprofen are mentioned. Interestingly 4 are chiral (one chiral centre) and one diastereomeric in nature (two chiral centers).

18 General introduction

Pharmacodynamics The major pharmacological properties have already been reviewed by Adam et al. in 1975 (1). Several discriminating techniques were applied to determine the lowest effective oral dose (mg/kg) as anti-inflammatory, analgesic and antipyretic drug. In the anti-inflammatory tests three animal species were used: guinea pig, rat and mouse. In the guinea pig the UV-erythema test was employed in which the reference compound (acetylsalicylic acid, 80 mg/kg) was found to correspond to 0.25 mg/kg of flurbiprofen. In the mouse model the capillary permeability of the peritoneum was evaluated by use of a dye (Pontamine sky blue). Acetylsalicylic acid at 120 mg/kg was equivalent to 0.47 mg/kg of flurbiprofen. In the rat three methods were employed: the carageenan edema test, and two adjuvant arthritis models for the developing state and the established state. Reference compounds were, respectively, acetylsalicylic acid (81 mg/kg) in the first and indomethacin (1 mg/kg) and phenylbutazone (10 mg/kg) in the two latter models. The corresponding lowest effective dose for flurbiprofen was, 0.11 mg/kg, and 0.33 mg/kg in the two latter models. With the carageenan edema test a subgroup of rats was also tested who were bilaterally adrenalectomised to rule out any adrenocortical interference. Several conclusions were drawn from this study. Flurbiprofen was devoid of adrenocortical- stimulating properties and was one of the most potent agents of this type reported yet; at least 10 times more potent than ibuprofen. It was postulated that the mode of action in the mouse and rat was not identical to that of acetylsalicylic acid In US patent 3,755,427 (August 28th 1973) (6) it was stated that flurbiprofen was between 75 to over 100 times as potent as acetylsalicylic acid. In (2) the relative potency of various hydratropic acids were tested for their relaxing ability on guinea pig tracheal ring contraction after sensitization by rat SRS-A. Furthermore the paper not only provided information for flurbiprofen but also for the levorotary (-) and dextrorotary (+) enantiomers. It became apparent that the relaxing potency of the racemic mixure (±) was unexpectedly too low as compared to the dextrorotary component suggesting that the dextrorotary component was hindered by the simultaneous presence of the levorotary component. The putative interaction between the two enantiomers was tested by the simultaneous addition of the two separate enantiomers to the muscle bath. Reversal by the dextrorotary component was diminished by the simultaneous presence of the (-) flurbiprofen. Taking this in consideration (+) flurbiprofen was approximately 80 fold more potent than (-) flurbiprofen.

Pharmacokinetics Pharmacokinetic properties have been assessed in different species (7). In man (8), when assessed by HPLC of the racemic molecule, a two-compartment open model appeared the most appropriate for flurbiprofen. Drug absorption efficiency was found independent of the oral dose. The intact drug resides mainly in the

19 Chapter 1

peripheral and central compartments, disappearing with a terminal half life of approximately 5.5 hours. More than 99% of flurbiprofen is bound to serum proteins. The serum flurbiprofen concentrations in clinical use however show an occupancy of less than 10% of the primary binding sites. The binding site differs from that of drugs like oral anticoagulants and sulphonamides. Drug interactions will therefore not automatically occur with simultaneous use. Oxidation and conjugation are the main pathways of metabolism. More than 95% of an oral dose is excreted via the kidney within 24 hours. Forty to 47% of a daily oral dose is excreted as 2-[2-fluoro-4'-hydroxy-4-biphenylyl]propionic acid; 5% as 2- [2-fluoro-3',4'-hydroxy-4-biphenylyl]propionic acid; 20-30% as 2-[2-fluoro-3'- hydroxy-4'-methoxy-4-biphenylyl]propionic acid and 20-25%. as the parent molecule flurbiprofen. Between 65 - 85% of flurbiprofen and its metabolites are present as glucuronide and sulfate conjugates. Stereoselective HPLC of human plasma has also been performed (9). After oral administration of 25 mg of the R(-) enantiomer of flurbiprofen no indication was found that inversion to the S(+) enantiomer occurred. This was confirmed in healthy volunteers taking either 50 mg R(-) flurbiprofen or S(+) flurbiprofen (10). Several studies on the pharmacokinetics of flurbiprofen in the rat have been performed all showing that in this species a minimal amount of inversion could take place (approx. 5%), the inversion halftime being approximately half an hour (11,12,13,14). Stereoselectieve studies have been performed following the disposition of flurbiprofen in normal volunteers after a single 50 mg racemic dose (15), in healthy female subjects following oral administration of the single enantiomers of flurbiprofen, 50 mg S(+) flurbiprofen or R(-) flurbiprofen or 100 mg R(-) flurbiprofen or placebo, in a 4-way crossover design with placebo (16); in patients with end-stage renal disease undergoing continuous ambulatory peritoneal dialysis (CAPD) after administration of a single 100 mg racemic dose (17), and stereoselective disposition of racemic flurbiprofen in single and multiple dosing in uraemic patients (18). On the basis of pharmacokinetics, adjustment of flurbiprofen dosing in uraemic patients is not necessary. In CAPD patients circulating plasma levels of flurbiprofen proved 40- 50% lower than in normal subjects implying that analgesia could be less than expected in this selected group of patients. Accumulation of the 2-[2-fluoro-4'- hydroxy-4-biphenylyl]propionic acid metabolite, which has minimal anti-inflammatory activity, does occur in this group of patients but the clinical significance is not established. In patients with liver disease with ascites and in renal failure patients with a creatinine clearance of less than 10 ml.min-1, significant higher free fractions of R(-)- and S(+) flurbiprofen were detected in conjunction with lower albumin concentrations (19). An overview of the clinical pharmacokinetics of flurbiprofen and its enantiomers is presented in (20).

20 General introduction

A different model was introduced for the investigation of the pharmacokinetics of flurbiprofen enantiomers and the simultaneous inhibition of prostanoid production (21). This study was performed in healthy volunteers in whom, after receiving orally either 75 mg R(-), S(+) flurbiprofen or no medication in a randomised 3-way cross-over design, flurbiprofen pharmcokinetics were analysed by HPLC and prostanoid production was monitored by enzyme immuno assay and chemilumi- nescence assay. Here also no clinically relevant inversion of R(-) to S(+) flurbiprofen was seen. However, the study showed unexplained discrepancies in several stages of the pharmacokinetic and pharmacodynamic parameters of the flurbiprofen enantiomers. Chiral inversion of R(-) flurbiprofen to S(+) flurbiprofen has been studied in vitro to investigate the mechanism behind this phenomenon which is not shared by all 2- arylpropionic acids (22). With crude rat liver homogenates it was demonstrated that an acyl-CoA synthase enzyme in conjunction with ATP and Mg2+ is obligatory. The first step comprises the metabolic formation of a CoA thioester of R(-) ibuprofen. It has been made plausible that this step takes place in adipose tissue since after administration of R(-) flurbiprofen significant amounts of both enantiomers are found in adipose tissue. By way of a non-stereoselective racemase (epimerase) this product is converted to its S(+) ibuprofen CoA thioester. Through action of a hydrolase, S(+) ibuprofen is released from its CoA thioester form. This unidirectional enantioselective chiral inversion in man has not been reported for flurbiprofen, carprofen and ketoprofen (23,24,25). Research on the enzymatic inversion at the chiral carbon atom had been done earlier by use of deuterated ibuprofen. In that study it was noted that the R(-) isomer is the only substrate for the epimerisation reaction (26). A summary of the metabolic chiral inversion of 2-arylpropionic acid derivatives, the variations between species and the complexity that can arise due to formed chiral metabolites has been presented in (27). Biliary excretion has been examined in normal and bile-duct cannulated rats for the enantiomers of flurbiprofen after intravenous dosing of 10 mg/kg of each enantiomer (28). It was found that the fraction of enterohepatic circulation was greater for R(-) flurbiprofen than for its antipode. Although the S(+) flurbiprofen enantiomer was excreted to a greater extent in bile, reabsorption from the intestine was insignif- icant. One reason for this phenomenon may be the presumed stereoselective hydrolysis of the flurbiprofen conjugates with preference for the R(-) enantiomer. However in later similar experiments (29) enterohepatic cycling of both flurbiprofen enantiomers could be demonstrated. Glucuronidation in rat and human liver microsomes proceeds faster for the R(-) enantiomer than for its S(+) antipode. Glucuronidation is facilitated by the enzyme complex UDP-glucuronosyltransferase of which there exist several isoforms (30). However not the identity of the isoenzyme but the stereoselective interaction of the enantiomer influences the reaction velocity.

21 Chapter 1

In a review (31) on the binding of flurbiprofen to albumin in human plasma it was reported that at low therapeutic concentrations the S(+) enantiomer has a higher protein binding than its R(-) antipode. At high drug concentrations there is no meas- urable difference, however. In an ultrafiltration study done with normal volunteers the free fraction of R(-) flurbiprofen was higher than its S(+) antipode at low drug levels but similar for both enantiomers at higher drug levels. Patients with renal impairment and patients exhibiting hypoalbuminaemia have higher free fractions of flurbiprofen enantiomers than normal volunteers. Plasma protein binding of an enantiomer is not influenced by its own concentration or the presence of its antipode under clinical therapeutic conditions (32). In a model study using isolated perfused rabbit lungs it was demonstrated that flurbiprofen does not undergo pulmonary metabolism to any extent (33). As mentioned above (8) the main routes of biotransformation of flurbiprofen are through oxidation and conjugation. Oxidation has been investigated more specifically (34) for the enantiomers of flurbiprofen utilizing human liver microsomes. The most prominent oxidative metabolism route is by cytochrome P450. It was established that cytochrome P450 2C9 and its allelic variant R144C catalysed the oxidative reaction. Interestingly, there was no stereoselective preference of one enantiomer over the other. Safety for intestinal permeability changes when using the racemate or the separate enantiomers of flurbiprofen was studied in rats for which species it was established that only a minimal inversion of the R(-) enantiomer takes place. Intestinal permeability was measured by urinary excretion of 51Cr-EDTA (35). It was established that at both dosages used (1 mg/kg and 3 mg/kg for the racemic drug and half for the enantiomers) permeability was significantly different from control. R(-) flurbiprofen was safest in both dosage ranges. S(+) flurbiprofen inflicted similar damage as the racemic form. In (36) it was shown that in rats R(-) flurbiprofen gave the same increase of intestinal permeability, but the difference was that the impact on mucosal prostanoid production was smaller and not accompanied by ulcerative changes in the small intestine. Although it would seem attractive to develop therapeutic R(-) enantiomers of 2- arylproionic acids due to its supposedly lower toxicological profile it must be borne in mind that the presumed pharmacological action required for reducing inflammation is inhibition of prostaglandin synthesis. This property resides primarily, in the case of flurbiprofen, in the S(+) enantiomer for which a difference of 30 to 100 times compared to the R(-) enantiomer was established depending on the model used. Only with full metabolic inversion of a R(-) enantiomer to a S(+) enantiomer would such a therapeutic drug be a possibility. For flurbiprofen this is not the case in humans (37,38,39).

22 General introduction

In a comparative study (40) done in rabbits, the inhibitory effect on rise in intraocular pressure and increase in aqueous humor protein after topical application of arachidonic acid (5% in peanut oil) by 14 nonsteroidal anti-inflammatory inhibitors was measured. For 50% inhibition of the intraocular pressure response, flurbiprofen ranked second best with an effective concentration of approximately 0.06%. Indomethacin (suspension in water, not further specified) ranked 4th with an approx- imate concentration of 0.2%. In a short review (41) the importance of the involvement of prostaglandins to certain eye conditions is discussed. The rise in intraocular pressure and the breakdown of the blood-aqueous barrier were related to these compounds. A search for the best drug in inhibiting prostaglandin mediated diseases was called for before testing them in the human eye. The comparative in vivo inhibitory effects of flurbiprofen, indomethacin and acetylsalicylic acid, all as sodium salt solutions, have been tested in the rabbit anterior uvea and conjunctiva after topical (0.5% solutions) and intraperitoneal administration (42). In both methods of administration acetylsalicylic acid almost completely abolished prostaglandin synthesis. Flurbiprofen given intraperitoneally was more potent than indomethacin which inhibited prostaglandin synthesis only partially, even at twice the dose of acetylsalicylic acid and flurbiprofen. Topical administration revealed that acetylsalicylic acid performed well even at a dose as low as 0.01% but indomethacin and flurbiprofen performed poor. Use of flurbiprofen (0.01% and 0.1%) was evaluated in comparison to 1% pred- nisolone as an inhibitor of corneal neovascularization in New Zealand albino rabbits (43). Flurbiprofen 0.1% and prednisolon 1% were equally effective in inhibiting vessel growth. As an alternative possibility for the use of topical administration of corticosteroids a nonsteroidal anti-inflammatory drug was considered (44). Flurbiprofen was tested in a double-blind fashion to see if intraocular pressure would change and if its use could block corticosteroid induced ocular hypertension. In a selected group of patients, with known intraocular sensitivity towards corticosteroids, flurbiprofen eyedrops (0.03%) did not alter intraocular pressure following six weeks of treatment. Also pretreatment by flurbiprofen did not block corticosteroid-induced ocular hypertension. Flurbiprofen was also investigated for human use in the prevention of intraocular inflammation (45). In a randomised double-blind parallel group study, placebo or flurbiprofen (100 mg thrice daily) was given orally for 8 days starting 24 hours before routine cataract extraction. Flurbiprofen was only favoured over placebo for the resolution of corneal inflammation at day 6. Interestingly, flurbiprofen concentrations in the aqueous were detected up to 4 hours after the last dose with a con-

23 Chapter 1

centration of 0.57 mg/L (2.3x10-6M). The authors conclude that flurbiprofen may be of value in the treatment of uveitis and other kinds of intraocular inflammation. In a study (46) involving female New Zealand rabbits, eyedrop disposition was studied after application of 14C labeled flurbiprofen in a concentration of 0.03%. No metabolism was detected for flurbiprofen in the eye. The total amount present in ocular tissues (cornea, aqueous humor, iris, ciliary body, choroid and retina) in normal rabbit eyes 30 minutes after application of 50 microliter of a 0.03% solution, was 4.25%. At 6 hours this was 1.59%. Following ocular application 77.51±8.79% was found in a 24-urine collection period. Unchanged flurbiprofen accounted for 25.3±3.6%. Ocular availability was studied in female albino rabbits (47) receiving 50 microliter of 0.3% or 0.15% solution of flurbiprofen. The ocular bioavailability of the 0.3% solution was 10% and for the 0.15% solution 7%. The elimination half-life in the aqueous humor was 93 minutes which approximates the turnover rate of aqueous humor in the rabbit and indicates that drainage is the main route of elimination. Bioavailability was determined after a single dose and after multiple doses of labeled flurbiprofen in rabbit eyes using topical application (48). Multiple dosing of a flurbiprofen solution of 0.03%, every half hour three doses, gave levels in the eye high enough to prevent prostaglandin synthesis. It compared favourably to the use of a single drop of 0.1% solution possibly because of less irritation of the eye and thus less stimulation of tear flow. Peak tissue concentrations were reached between 30 minutes and 60 minutes and were 2 to 6 times higher in all tissues than seen after one drop of 0.1% solution. To determine the intraocular concentration of flurbiprofen sodium in the human aqueous humor of patients undergoing cataract surgery, samples were taken after receiving flurbiprofen sodium at selected times prior to surgery (49). Only single drop instillation was done. Samples of aqueous humor were analysed by HPLC. Flurbiprofen concentrations were detectable in the aqueous between 30 minutes to 7.25 hours after topical application. To determine if and how much drug can penetrate to the posterior segment of the eye, a study was done in white New Zealand albino rabbits, by single drop method of dosing, using paracetamol 1% in saline and bendazac lysine 0.5% in saline or other solvent (50). When comparing the data with literature data it appeared that paracetamol behaved similar to flurbiprofen as regards penetration into the aqueous humor, having a very poor entry into the vitreous and attaining higher concentrations in the retina than paracetamol. In all three eye compartments bendazac lysine permeated poorly. The data suggest an alternative entry route to the posterior segments of the eye. It appears that the lens acts as a barrier for the entry from the aqueous.

24 General introduction

In a study done by Carabaza et al. inhibition of prostaglandin synthesis was investigated using the enantiomers of three NSAIDs (ketoprofen, flurbiprofen and ketorolac), including stereoselective inhibition of inducible COX-2 (51). It became apparent that inhibition by the three enantiomer pairs is comparable for COX-1 and COX- 2. With both cyclooxygenase isoenzymes inhibition resides almost exclusively in the S(+) isomer. One of the most frequent problems encountered during cataract surgery is invol- untary pupillary constriction. In the past, several pharmacological interventions have been tried as a remedy, but without success. Albino rabbits have been used to study the effect of topical administration of indomethacin (1% aqueous solution with no further specification of buffer and pH used) and flurbiprofen (0.03% aqueous solution of sodium flurbiprofen) on this unwanted condition (52). In this set-up also local anaesthetics, capsaicin, sympathomimetic agents and an anticholinergic were involved all according to a specified protocol. Flurbiprofen demonstrated a significant inhibitory effect on miosis while topical indomethacin failed. However no single agent or combination of agents blocked the miotic response completely.

Although nonsteroidal anti-inflammatory drugs are pharmacologically effective inhibitors of cyclooxygenase activity and prostaglandin synthesis (53), cyclooxygenase-independent anti-inflammatory actions of NSAIDs are also known (54). Since it was reported that sodium salicylate and acetylsalicylic acid inhibit the action of the transcription factor nuclear factor kappa B (NF-κB), the enantiomers of flurbiprofen were tested in a zymosan-induced paw inflammation model. Although R(-) flurbiprofen does not inhibit cyclooxygenase to a significant extent, it is more potent than S(+) flurbiprofen and almost as effective as dexamethasone in this inflammatory model. Inhibition of NF-κB by R(-) flurbiprofen resulted in a reduced expression of COX-2 and tumor necrosis factor a (TNF-α). Nitric oxide formed by the inducible NO synthase (iNOS) has been implicated as a mediator of pain and tissue injury in various inflammatory and autoimmune diseases. In an in vitro model involving RAW 264.7 macrophages, it could be demonstrated that iNOS mRNA expression is equipotently suppressed by the enantiomers of flurbiprofen. S(+) flurbiprofen and R(-) flurbiprofen did not inhibit LPS induced

COX-2 mRNA expression but did inhibit LPS-induced prostaglandin E2 formation enantioselectively, with the S(+) antipode being 46 times more active than the R(-) µ µ flurbiprofen (IC50 0.0061 M and 0.28 M respectively). Collectively, these findings would suggest that the pharmacological (i.e. anti-inflammatory) activity of the flurbiprofen enantiomers is not only related to inhibition of cyclooxygenase enzyme activities but also to inhibition of transcription factor activition like NF-κB and AP-1, resulting in diminished formation of pro-inflammatory factors like iNOS and TNF-α (55,56).

25 Chapter 1

REFERENCES

1. Adam SS, McCullough KF, Nicholson JS. Some biological properties of flurbiprofen, an anti- inflammatory, analgesic and antipyretic agent. Arzneim Forsch (Drug Research) 1975;25:1786-91. 2. Greig ME, Griffin RL. Antagonism of slow reacting substance in anaphylaxis (SRS-A) and other spasmogens on the guinea pig tracheal chain by hydratropic acids and their effects on anaphylaxis. J Med Chem 1975;18:112-116. 3. Nishisawa EE, Wynalda DJ, Suydam DE Molony BA. Flurbiprofen, a new potent inhibitor of platelet aggregation. Thrombosis research 1973;3:577-88. 4. The Merck Index, Thirteenth Edition, 2001, Merck & Co., Inc. Whitehouse Station, N.J., USA. 5. Brochier ML. Evaluation of flurbiprofen for prevention of reinfarction and reocclusion after successful thrombolysis or angioplasty in acute myocardial infarction. The flurbiprofen French trial. Eur Heart J 1993;14:951-7. 6. US patent 3,755,427 (August 28th, 1973). 7. Risdall PC, Adams SS, Crampton EL, Marchant B. The disposition and metabolism of flurbiprofen in several species including man. Xenobiotica 1978;8:691-704. 8. Kaiser DG, Brooks CD, Lomen PL. Pharmacokinetics of flurbiprofen. Am J Med 1986;80:10-5 (suppl 3A). 9. Jamali F, Berry BW, Tehrani MR, Russell AS. Stereoselective pharmacokinetics of flurbiprofen in humans and rats. J Pharm Sci 1988;77:666-9. 10. Geisslinger G, Menzel-Soglowek S. Stereoselective high-performance liquid chromatographic determination of flurbiprofen in human plasma. J Chromatogr 1992;573:163-7. 11. Knihinicki RD, Day RO, Graham GG, Williams KM. Stereoselective disposition of ibuprofen and flurbiprofen in rats. Chirality 1990;2:134-40. 12. Jamali F, Berry BW, Wright MR. Dose-dependency of flurbiprofen enantiomer pharmacokinetics in the rat. J Pharm Sci 1994;83:1077-80. 13. Peskar BM, Kluge S, Peskar BA, Soglowek SM, Brune K. Effects of pure enantiomers of flurbiprofen in comparison to racemic flurbiprofen on eicosanoid release from various rat organs ex vivo. Prostaglandins 1991;42:515-31. 14. Berry BW, Jamali F. Enantiomeric interaction of flurbiprofen in the rat. J Pharm Sci 1989;78:632-4. 15. Knadler MP, Brater DC, Hall SD. Stereoselective disposition of flurbiprofen in normal volunteers. Br J Clin Pharmacol 1992;33:369-75. 16. Geisslinger G. Stereoselective disposition of flurbiprofen in healthy subjects following administration of the single enantiomers. Br J Clin Pharmacol 1994; 37:392-4. 17. Cefali EA, Poynor WJ, Sica D, Cox S. Pharmacokinetic comparison of flurbiprofen in end-stage renal disease subjects and subjects with normal renal function. J Clin Pharmacol 1991;31:808-14. 18. Knadler MP, Brater DC, Hall SD. Stereoselective disposition of flurbiprofen in uraemic patients. Br J Clin Pharmacol 1992;33:377-83. 19. Blouin R, Chaudhary I, Nishikara K, Cox S. The effects of liver and renal disease on stereoselective serum binding of flurbiprofen. Br J Clin Pharmacol 1993;35:62-4. 20. Davies NM. Clinical pharmacokinetics of flurbiprofen and its enantiomers. Clin Pharmacokinet 1995;28:100-14. 21. Oelkers R, Neupert W, Williams KM, Brune K, Geisslinger G. Disposition and effects of flurbiprofen enantiomers in human serum and blister fluid. Br J Clin Pharmacol 1997;43:145-53. 22. Knihinicki RD, Williams KM, Day RO. Chiral inversion of 2-arylpropionic acid non-steroidal anti- inflammatory drugs-1. In vitro studies of ibuprofen and flurbiprofen. Biochem Pharmacol 1989;38:4389-95. 23. Mayer JM. Stereoselective metabolism of anti-inflammatory 2-arylpropionates. Acta Pharm Nord 1990;2:197-216.

26 General introduction

24. Knadler MP, Hall SD. Stereoselective arylpropionyl-CoA thioester formation in vitro. Chirality 1990;2:67-73. 25. Wechter WJ. Drug chirality: on the mechanism of R-aryl propionic acid class NSAIDs. Epimerization in humans and the clinical implications for the use of racemates. J Clin Pharmacol. 1994;34:1036-42. 26. Wechter WJ, Loughhead DG, Reischer RJ, VanGiessen GJ, Kaiser DG. Enzymatic inversion at sat- urated carbon: nature and mechanism of the inversion of R(-) p-iso-butyl hydratropic acid. Biochem Biophys Res Commun. 1974;61:833-7. 27. Caldwell J, Hutt AJ, Fournel-Gigleux S. The metabolic chiral inversion and dispositional enantioselectivity of the 2-arylpropionic acids and their biological consequences. Biochem Pharmacol 1988;105-14. 28. Menzel S, Beck WS, Brune K, Geisslinger G. Stereoselectivity of biliary excretion of 2-arylpropionates in rats. Chirality 1993;5:422-7. 29. Eeckhoudt SL, Evrard PA, Verbeeck RK. Biliary excretion and enterohepatic cycling of R- and S- flurbiprofen in the rat. Drug Metab Dispos 1997;25:428-30. 30. Hamdoune M, Mounie J, Magdalou J, Masmoudi T, Goudonnet H, Escousse A. Characterization of the in vitro glucuronidation of flurbiprofen enantiomers. Drug Metab Dispos 1995;23:34308. 31. Lapicque F, Muller N, Payan E, Dubois N, Netter P. Protein binding and stereoselectivity of nonsteroidal anti-inflammatory drugs. Clin Pharmacokinet 1993; 25:115-25. 32. Knadler MP, Brater DC, Hall SD. Plasma protein binding of flurbiprofen: enantioselectivity and influence of pathophysiological status. J Pharmacol Exp Ther 1989; 249:378-85. 33. Hall SD, Hassazadeh-Khayyat M, Knadler MP, Mayer PR. Pulmonary inversion of 2-arylpropionic acids: influence of protein binding. Chirality 1992;4:349-52. 34. Tracy TS, Rosenbluth BW, Wrighton SA, Gonzalez FJ, Korzekwa KR. Role of cytochrome P450 2C9 and an allelic variant in the 4’-hydroxylation of (R)- and (S) flurbiprofen. Biochem Pharmacol 1995;49:1269-75. 35. Davies NM, Wright MR, Russell AS, Jamali F. Effect of the enantiomers of flurbiprofen, ibuprofen, and ketoprofen on intestinal permeability. J Pharm Sci 1996;85:1170-3. 36. Mahmud T, Somasundaram S, Sigthorsson G, Simpson RJ, Rafi S, Foster R, Tavares IA, Roseth A, Hutt AJ, Jacob M, Pacy J, Scott DL, Wrigglesworth JM, Bjarnason I. Enantiomers of flurbiprofen can distinguish key pathophysiological steps of NSAID enteropathy in the rat. Gut 1998;43:775-82. 37. Wright MR, Davies NM, Jamali F. Rationale for the development of stereochemically pure enantiomers: are the R enantiomers of chiral nonsteroidal anti-inflammatory drugs inactive? J Pharm Sci 1994;83:911-2. 38. Menzel-Soglowek S, Geisslinger G, Beck WS, Brune K. Variability of inversion of (R)-Flurbiprofen in different species. J Pharm Sci 1992;81:888-91. 39. Geisslinger G, Menzel-Soglowek S, Beck WS, Brune K. R-flurbiprofen: isomeric ballast or active entity of the racemic compound? Agents Actions Suppl 1993;44:31-6. 40. Podos SM, Becker B. Comparison of ocular prostaglandin synthesis inhibitors. Invest Ophthalmol 1976;15:841-4. 41. Podos SM. Prostaglandins, nonsteroidal anti-inflammatory agents and eye disease. Trans Am Ophthalmol Soc 1976;74:637-60. 42. Kulkarni PS, Srinivasan D. Comparative in vivo inhibitory effects of nonsteroidal anti-inflammatory agents on prostaglandin synthesis in rabbit ocular tissues. Arch Ophthalmol 1985;103:103-6. 43. Cooper CA, Bergamini MVW, Leopold IH. Use of flurbiprofen to inhibit corneal neovascularization. Arch Ophthalmol 1980;1102-5. 44. Gieser DK, Hodapp E, Goldberg I, Kass MA, Becker B. Flurbiprofen and intraocular pressure. Ann Ophthalmol 1981;13:831-3.

27 Chapter 1

45. Hillman JS, Frank GJ, Kheskani MB. Flurbiprofen and human intraocular inflammation. In Advances in prostaglandin and thromboxane research. Vol 8;1723-5:1980. Edited by B. Samuelsson, P.W. Ramwell, and R. Paoletti. Raven Press New York, USA. 46. Anderson JA, Chen CC, Vita JB, Shackleton M. Disposition of topical flurbiprofen in normal and aphakic rabbit eyes. Arch Ophthalmol 1982;100:642-5. 47. Tang-Liu DD-S, Liu SS, Weinkam RJ. Ocular and systemic bioavailability of ophthalmic flurbiprofen. J Pharmacokinet Biopharm 1984;12:611-26. 48. Anderson JA, Chen CC. Multiple dosing increases the ocular bioavailability of topically administered flurbiprofen. Arch Ophthalmol 1988;106:1107-9. 49. Ellis PP, Pfoff DS, Bloedow DC, Riegel M. Intraocular diclofenac and flurbiprofen concentrations in human aqueous humor following topical application. J Ocular Pharmacol 1994;10:677-82. 50. Romanelli L, Morrone LA, Guglielmotti A, Piccinelli D, Valeri P. Distribution of topically administered drugs to the posterior segment of rabbit eye. Pharmacol Res 1992;25: 39-40 (Suppl 1). 51. Carabaza A, Cabre F, Rotlan E, Gomez M, Gutierrez M, Garcia ML, Mauleon D. Stereoselective inhibition of inducible cyclooxygenase by chiral nonsteroidal antiinflammatory drugs. J Clin Pharmacol 1996;36:505-12. 52. Duffin RM, Camras CB, Gardner SK, Pettit TH. Inhibitors of surgically induced miosis. Ophthalmology 1982;89:966-79. 53. Versteeg HH, van Bergen en Henegouwen PMP, van Deventer SJH, Peppelenbosch MP. Cyclooxygenase-dependent signalling: molecular events and consequences. FEBS 1999;445:1-5. 54. Tegeder I, Pfeilschifter J, Geisslinger G. Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J 2001;15:2057-72. 55. Tegeder I, Niederberger E, Israr E, Gühring H, Brune K, Euchenhofer C, Grösch S, Geisslinger G. Inhibition of NF-κB and AP-1 activation by R- and S-flurbiprofen. FASEB J 2001;15:2-4; and 595- 7. To read full text: http://www.fasebj.org/ cgi/doi/10.1096/fj.00-0130fje. 56. Hinz B, Brune K, Rau T, Pahl A. Flurbiprofen enantiomers inhibit inducible nitric oxide synthase expression in RAW 264.7 macrophages. Pharm Res 2001;18:151-6.

28 General introduction

1.2 CATARACT AND CATARACTOGENESIS

Cataract Alterations in lens transparency increase with age. It seems possible that the lens stays transparent until the age of 120 years. In the fifth decade of life however approximately 65% of people will have some form of lens opacity. This can vary from small spots to complete opacification. The patient will not immediately notice the development of cataractogenesis as this process does not proceed with an overt inflammatory process nor is any pain experienced. Symptoms accompanying such a process are difficulty in reading, in recognizing faces, watching television, seeing in bright light and during driving (1,2). A simple test is available to assess visual function, the Snellen chart, but reliability warrants consideration (3). On a global scale cataract is the commonest cause of visual disability and by far the single largest cause of blindness (4). Traditional eye medicines in rural Africa inflict corneal ulcers and cause blindness in children in a quarter of cases (5). The best known medication to cause cataracts are corticosteroids (6) with evidence that phenothiazines, amiodarone, chloroquine and possibly acetylsalicylic acid also might be associated with increased risk (7). There are still limitations in the identification of the causes of cataracts, not only in developing countries but also in indus- trialised countries (8). Research is ongoing to gain a better understanding of the genetics of human cataract. It can be envisaged that knowledge of congenital cataract will provide more insight into the putative role of genes in age-related cataract (9). There is still no effective, pharmacological, remedy for established cataracts although a theoretical and experimental basis is building up to address age-onset cataractogenesis by anti-cataract agents (10). Treatment is purely surgical with an established success rate >90%. Two basic techniques are in use for management: extracapsular cataract surgery and intracapsular cataract surgery. The surgical removal of an opacified lens was first reported in 1745 (April 8th, Marseille) performed by Jacques Daviel (1693-1762). In 1752 two lectures were presented by him at the Académie Royale de Chirurgie in Paris where an account was given of 206 lens extractions. Of these 182 were successful: 88%. Almost two centuries later Sir Harold Ridley performed the first successful lens implantation (London, November 29th 1949). Innovation is still improving cataract surgery, especially by the technological advancement of extracapsular extraction and posterior-chamber intraocular lens implantation (11). These substantial improvements should become available to an increasing group of patients on the waiting list (12,13,14). Outpatient cataract sur-

29 Chapter 1

gery seems very well possible without loss of quality (15). Bilateral cataract extraction can be safely done within 48 hours (16).

Caractogenesis Age related, or senile cataract is the most common form and inflicts blindness worldwide. There are two types of age-related cataract, nuclear and cortical. One of the possibilities that has been investigated for nuclear caractogenesis is through hydroxyl radical-attack of lens proteins which causes cross-linking and protein aggregation, ultimately resulting in opacity of the lens (17). In another model it is proposed that cataract is essentially a conformational disease in which non-enzymatic modification of amino groups e.g. by sugars and steroids destabilize the lens proteins and causes conformational changes. The interaction between the amino acid of a lens protein and a sugar, well known as the Maillard reaction, will not only give rise to a colored reaction product but will also cause the protein to cross-link, aggregate and eventually to become insoluble which in turn will opacify the lens (18). In another sugar-related process it was investigated whether the polyol pathway was involved in the process of cataractogenesis. By the enzyme aldose reduc- tase glucose can be converted to sorbitol. However, when a limited amount of antioxidants is available a significant amount of hydrogen peroxide can be formed. This will give rise to the production of hydroxyl radicals and will lead to the initial stage of a "sugar" cataract (19). Another reported mechanism on the formation of cataract was the kynurenine metabolic pathway. The tryptophan metabolite, 3- hydroxykynurenine is present at elevated concentrations in the lens and is able to absorb UV radiation. However, an excessive amount in the lens has been reportedly associated with cataract formation. In the rabbit eye enzymes, leading to the formation of this metabolite, are present in the iris/ciliary body. The formed metabolite is taken up by the lens for formation of UV-filtering products. If however an excess of 3-hydroxykynurenine is present in the lens, free radical formation may occur, which will ultimately lead to tissue injury like lens opacification (20,21). Oxidative damage of lens proteins seems to be a major factor in cataract formation. A threshold of protein oxidation has been identified at which opacification will take place (22). Radiation-induced cataractogenesis will commence above 1 Gy as has been observed in survivors of the Hiroshima and Nagasaki atomic bombs (23). The lens is not a purely passive optical element but maintains an internal circulation for lens transparency. Sodium-potassium pumps have been identified in the lens as well as a major intrinsic protein belonging to the aquaporin family of water channels. Glucose is transported from the aqueous humor to the lens for energy support. It has been postulated that dysfunction of any of these links in this chain of events may ultimately lead to cataract formation (24).

30 General introduction

REFERENCES

1. Elkington AR, Khaw PT. Cataracts. BMJ 1988;296:1787-90. 2. Cotlier E. The Lens in: Adler's Physiology of the eye. Clinical application. Chapter 10; 6th Edition 1975. RA Moses, MD Editor. The CV Mosby Company ISBN 0-8016-3540-3. 3. McGraw P, Winn B, Whitaker D. Reliability of the Snellen chart (Better charts are now available). BMJ 1995;1481-2. 4. Thylefors B, Négrel A-D, Pararajasegaram R, Dadzie KY. Global data on blindness. Bull WHO 1995;73:115-21. 5. Anonymous. Traditional eye medicines: a note of concern. WHO Drug information 1995;9:152-3. 6. Butcher JM, Austin M, Mc Galliard J, Bourke RD. Bilateral cataracts and glaucoma induced by long term use of steroid eyedrops. BMJ 1994;309:43. 7. Cumming RG, Mitchell P. Medications and cataract (The blue mountains eye study). Ophthalmology 1998;105:1751-8. 8. Johnson GJ. Limitations of epidemiology in understanding pathogenesis of cataracts. Lancet 1998;351:925-6, 9. Francis PJ, Berry V, More AT, Bhattacharya S. Lens biology; development and human cataractogenesis. TIG 1999;15:1916. 10. Benedek GB, Pande J, Thurston GM, Clark JI. Theoretical and experimental basis for the inhibition of cataract. Progress in retinal and eye research. 1999;18:391-402. 11. Tielsch JM. Appropriate technology for cataract surgery. Lancet 1998;352:754-5. 12. Gray CS, Crabtree HL, Oçonnell JE, Allen ED. Waiting in the dark: cataract surgery in older people (We need a better means of assessing priorities for surgery). BMJ 1999;318:1367-8. 13. Allan B. Intraocular lens implants (Have come a long way, but the advances are not yet available to all). BMJ 2000;320:73-4. 14. Fielder AR, Watson MP, Seward HC, Murray PI. Action on cataracts should influence surgical training. BMJ 2000;321:639. 15. Javitt JC, Street DA, Tielsch JM, Wang Q, Kolb MM, Schein O, SommerA, Bergner M, Steinberg EP, on behalf of the Cataract Patient Outcomes Research team. Ophthalmology 1994;101:100-6. 16. Booth A, Coombes A, Rostron C. Bilateral cataract extraction can be safely done within 48 hours. BMJ 1999;319:579. 17. Fu S, Dean R, Southan M, Truscott R. The hydroxyl radical in lens nuclear catractogenesis. J Biol Chem 1998;273:28603-9. 18. Crabbe MJC. Cataract as a conformational disease - the maillard reaction, alpha-crystallin and chemotherapy. Cell Mol Biol 1998;44:1047-50. 19. Kubo E, Miyoshi N, Fukuda M, Akagi Y. Cataract formation through the polyol pathway is associated with free radical production. Exp Eye Res 1999;68:457-64. 20. Chiarugi A, Rapizzi E, Moroni F, Moroni F. The kynurenine metabolic pathway in the eye: studies on 3-hydroxykynurenine, a putative cataractogenic compound. FEBS1999;453:197-200. 21. Davies MJ, Truscott RJW. Photo-oxidation of proteins and its role in cataractogenesis. J Photo Biol 2001;63:114-25. 22. Boscia F, Grattagliano I, Vendemiale G, Micelle-Ferrari T, Altomar E. Protein oxidation and lens opacity in humans. Invest Ophthalmol Vis Sci 2000;41:2461-5. 23. Belkacémi Y, Touboul E, Méric JB, Rat P, Warnet JM. Cataract radio-induit: aspects phys- iopathologiques, radiobiologiques et cliniques. Cancer/Radiother 2001;5:397-412. 24. Donaldson P, Kistler J, Mathias RT. Molecular solutions to mammalian lens transparency. News Physiol Sci 2001;16:118-23.

31 Chapter 1

1.3 CYSTOID MACULAR EDEMA

In 1942 a report was published on a toxic ocular reaction. The characteristic finding was that the primary aqueous (aqueous humor obtained on a first paracentesis) did coagulate in contrast to the usual finding that coagulation only takes place in secondary aqueous (aqueous humor obtained on a second paracentesis). The nature of this phenomenon in the - as it was termed - plasmoidtoxic aqueous- of the primary aqueous, was investigated in some detail. Although no exact culprit could be defined it became clear that the increased permeability of the ciliary body was a major contributing factor in the toxic ocular reaction and that the fibrinogen system played an essential role in the coagulation process of the plasmoidtoxic aqueous (1). In 1953 a paper was published in which a complication was described following common intracapsular cataract extraction. One of the features of this complication was the development of postoperative macular changes, and of ultimate reduction of vision as a result of vitreous opacities or macular degeneration. Of the 1,068 cataract extractions 894 were intracapsular extractions. Of these 483 occurred intact; the remaining 222 showed complications varying from marked prolapse of the vitreous into the anterior chamber without rupture to late rupture of the anterior hyaloid with or without adhesions. The percentage of patients encountered with poor vision as a result of vitreous opacities or macular degeneration was found to be 2%. This was similar to the postoperative detachment of the retina after cataract surgery, as found in a total of reviewed 1,200 cases (2). In 1966 a new study was presented showing the advantage of the use of intravenous sodium fluoresceinate to detect the lesion. It was demonstrated that resolution of fluorescein leakage into the retina and optic nerve generally parallels the clinical resolution of edema of the macula and optic disc. The earlier reports of an incidence of 2% of cystoid macular edema was questioned and it was expected to be higher as experience was gained with this new staining technique (3). A review on the complication of cystoid macular edema in 1976 showed that this complication following cataract surgery was the most common and troublesome (4). The progress in surgical techniques was impressive enough to diminish the majority of complications other than cystoid maculopathy. The incidence of clinically significant cystoid macular edema remained 2 - 6%. In an attempt to grasp the etiolog- ical factors the author (A.R. Irvine) put forward the possibility of "vasoactive factors from inflammatory cells in the vitreous to penetrate the retina preferentially at the macula and disc". Of the possibilities to produce aphakic cystoid macular edema, inflammation and increased permeability were major steps in the reaction sequence. Medication seemed straight forward as to use steroids. However oral therapy proved of transient value just like periocular steroid injections and had unfavourable side effects. Topical steroid therapy was found to be ineffective. New perspectives

32 General introduction

appeared following the elucidation of the role of prostaglandins in inflammatory vascular permeability changes. However the controlled study mentioned in this review using indomethacin (orally 25 mg tid for 3 weeks) failed to demonstrate any benefi- cial effect (4). In 1985 a hypothesis was put forward for aphakic cystoid macular edema. Based on the results of a randomised double blind trial that showed a reduction in incidence of 50% for aphakic cystoid macular edema by use of an ultraviolet radiation-absorb- ing chromophore in a posterior chamber intraocular lens, it was postulated that postoperative exposure to near-ultraviolet radiation generates free radicals. These radicals would facilitate the synthesis of inflammatory mediators like prostaglandins. Prostaglandins are involved in the breakdown of blood-ocular barriers. It follows that a combination of factors like UV-A radiation and the synthesis of prostaglandins is a possibility worth testing as a contributing factor toward cystoid macular edema and therefore amenable to medical treatment (5). In an update of the pharmacological therapy it was mentioned that topical nonsteroidal anti-inflammatory agents were still not commercially available. However, topical indomethacin was mentioned as the one agent effective in the prophylaxis of angiographic aphakic cystoid macular edema. Other nonsteroidal anti-inflammatory agents and corticosteroids are mentioned but no evidence was presented other than anecdotal, not detailed enough or in number too small to evaluate statistically (6). Further studies on cystoid macular edema revealed that any disturbance of the vitreous can lead to this syndrome. In particular three possibilities are mentioned by which intraocular lenses can give rise to chronic cystoid macular edema (7). These are iris chaffing in combination with an uveitis-glaucoma-hyphema syndrome after posterior chamber intraocular lens implantation, movement of the intraocular lens with intermittent corneal touchings and the corneo-retinal inflammatory syndrome com- promising both the cornea and the retina. When one of these three situations occur intraocular lens removal is required to prevent permanent macular damage (7). Cystoid macular edema has also been described in the French literature as Irvine- Gass syndrome. An extensive treatise is presented in (8). In the German literature a report was published on the safety and efficacy of a 1% indomethacin suspension for the prevention of cystoid macular edema (also known as Irvine-Gass-Norton syndrome or Irvine syndrome). The incidence of cystoid macular edema was 1.34%. Side effects of the eyedrops, as observed in 10% of the cases, were mainly conjunctival in origin (9). Another pharmacological approach for failing visual acuity, local application of steroids and an injection of tolazoline (α- adrenergic antagonist, having some cholinergic, H2-histaminergic, and 5HT1 receptor antagonistic properties as well) in Tenon's capsule, is proposed (10). In the meantime the FDA has approved several topical NSAIDs for clinical use in ophthalmology (11). The approvals are restricted to specific indications, however;

33 Chapter 1

flurbiprofen sodium and suprofen for the prophylaxis of surgical miosis, ketorolac for the relief of itching due to allergic conjunctivitis and diclofenac for the treatment of postcataract inflammation. For intraoperative miosis no conclusive evidence has been presented that an NSAID is effective. For the prevention of postcataract surgical inflammation the NSAIDs are at least as effective and perhaps more effective than corticosteroids in preventing disruption of the blood-aqueous barrier. For cystoid macular edema the evidence is that topical NSAIDs are better than topical corticosteroids. In a Canadian report the incidence of aphakic/pseudophakic cystoid macular edema in 90 studies from 1979 to 1991 is presented using three different techniques (12). For intracapsular and extracapsular cataract extraction and the phacoemulsifi- cation technique it varied between 2 - 10%, 0 - 7.6% and 0.6 - 6.0%, respectively. However when using fluorescein angiography the incidence varied between 40 - 60%, 2.7 - 11.3% and 2.1 - 6.0%, respectively. Interestingly, aphakic cystoid macular edema occurs more frequently with intracapsular than extracapsular cataract extraction, and even less with placement of an intraocular lens in an intact capsular bag. It seems that the capsular bag encompasses properties other than just for support. The lens barrier protects, it seems, against access of inflammatory agents into the vitreous. Permanent visual impairment due to cystoid macular edema will vary between 0.5% and 2%. Treatment of clinically or angiographically proven cystoid macular edema with indomethacin decreased the incidence of cystoid macular edema; however there was no difference in visual outcome between active and placebo treated groups. No long-term effectiveness was shown yet with treatment by a NSAID. In the same report carbonic anhydrase inhibitors (e.g. acetazolamide) are mentioned as possibly effective drugs in cystoid macular edema caused by changes in the external blood-retinal barrier (retinitis pigmentosa). However, a recent report on gastric mucosa samples obtained by biopsy showed that NSAIDs (acetylsalicylic acid, indomethacin, naproxen, diclofenac and piroxicam) can activate the carbonic anhydrase isoenzymes I, II and IV (13). An extensive review was published in 1998 (14) in which the view is held that the incidence, pathogenesis and treatment of cystoid macular edema following cataract surgery are still poorly understood. Incidence of cystoid macular edema is greatest following an intracapsular cataract extraction with implantation of an iris clip lens in an older population with systemic vascular disease. Clinical characteristics of cystoid macular edema are a nonuniform distribution of the retinal intravascular fluid within the macula leading to accumulation of transudate and ultimately to a sympto- matic or asymptomatic decrease in visual acuity. Preferential leakage from perifoveal capillaries in eyes with cystoid macular edema cannot be explained yet and possibly reflects a result of an unknown capillary vitreous interaction. Inflammation, however, is the mainstay in the development of cystoid macular edema. Presumably,

34 General introduction

breakdown of the blood aqueous barrier is associated with the development of cystoid macular edema. This was established in rabbits in which the topical activity of NSAIDs in stabilizing the blood aqueous barrier following paracentesis was studied by monitoring the integrity of the barrier using anterior ocular fluorophotometry before and after paracentesis. The integrity was followed by changes in fluorescein concentrations measured after intravenous administration of fluorescein sodium in the anterior chamber of the eye. All NSAIDs studied (flurbiprofen 0.03%, 0.1% diclofenac, 0.5% ketorolac and 1% suprofen) stabilized the blood aqueous barrier after paracentesis. In a Swiss overview it was concluded that NSAIDs with potent anti-inflammatory properties allow good control of ocular inflammation, effective maintenance of mydri- asis during surgery and delay the onset of cystoid macular edema (15). A recent review on topical NSAIDs for ophthalmic use concluded that the benefit-risk ratio is still favorable when they are applied in an appropriate and judicious manner (16).

REFERENCES

1. Ayo C. A toxic ocular reaction. II On the nature of the reaction. J Immunol 1942;46:127-32. 2. Irvine SR. A newly defined vitreous syndrome following cataract surgery. Am J Ophthalmol 1953;36:599-619. 3. Gass JDM, Norton EWD. Cystoid macular edema and pailledema following cataract extraction. Arch Opthalmol 1966;76:646-61. 4. Irvine AR. Cystoid maculopathy. Surv Ophthalmol 1976;21:1-17. 5. Jampol LM. Aphakic cystoid macular edema; a hypothesis. Arch Ophthalmol 1985;103:1134-5. 6. Jampol LM. Pharmacologic therapy of aphakic and pseudophakic cystoid macular edema. Ophthalmology 1985;92:807-10. 7. Drews RC. The present understanding of cystoid macular oedema. Trans opthalmol Soc UK 1985;104:744-7. 8. Sole P et al (Expertise bibliographique). Le syndrome d' Irvine Gass. J Fr Ophthalmol 1986;1:75-83. 9. Dirscherl M, Straub W. Zur prophylaxe des zystoiden Makulaödems nach Katarkatoperationen (eine anwendungsbeobachtung van Chibro-Amuno 3). Ophthalmologica 1990;200:142-9. 10. Hruby K. Das Irvine-syndrom; diagnose, pathogenese und therapie. Fortschr Ophthalmol 1985;82:147-8. 11. Jampol LM, Jain S, Pudzisz B, Weinreb RN. Nonsteroidal anti-inflammatory drugs and cataract surgery. Arch ophthalmol 1994;112:891-4. 12. Rocha G, Deschenes J. Pathophysiology and treatment of cystoid macular edema. Can J Ophthalmol 1996;31:282-8. 13. Puscas C, Chis F, Pasca R, Pasca S, Mihaescu M, Puscas I. Gastric, vascular and antidiuretic effects of indomethacin are dependent on direct activation of carbonic anhydrase (CA). Gut 1999;45:S5:P0087. 14. Flach AJ. The incidence, pathogenesis and treatment of cystoid macular edema following cataract surgery. Tr Am Ophth Soc 1998;96:557-634. 15. Guex-Crosier Y. Anti-inflammatoires non stéroïdiens (AINS) et inflammation oculaire. Klin Monatsbl Augenheilkd 2001;218:305-8. 16. Gaynes BI, Fiscella R. Topical nonsteroidal anti-inflammatory drugs for ophthalmic use. A safety review. Drug Saf 2002;25:233-50.

35 Chapter 1

1.4 PROSTANOIDS

In 1929 it was documented that feeding of rats not only should include essential elements like amino acids, vitamins and minerals but also small amounts of unsat- urated fat. Analysis uncovered the essential fatty acid linoleic acid, an eighteen carbon atom chain with two double bonds. Screening of many types of polyunsaturated fatty acids showed arachidonic acid (20 carbon atoms and 4 double bonds) to be the most active fatty acid to prevent manifestations of nutritional deficiency. In 1930 a factor was discovered in human semen that contracted the uterus; it also lowered blood pressure. Von Euler demonstrated this to be a fatty acid and introduced in 1935 the name prostaglandin. In 1960 Bergström elucidated the struc-

ture of some prostaglandins, one of them being prostaglandin E2. In 1964 van Dorp carried out experiments in the Unilever Research laboratories (Vlaardingen) and demonstrated by use of labeled arachidonic acid that it was converted into

prostaglandin E2 by the medium of homogenized sheep seminal vessels. This finding was published in 1964 jointly with Bergström in the same journal. In 1970 Vane published his discovery that the biosynthesis of prostaglandins was inhibited by aspirin-like drugs. Four years later Samuelsson discovered the bioconversion by

platelets of arachidonic acid into thromboxane A2. Two years later prostacyclin was discovered by Vane. The first comprehensive reviews were published in 1974 summarizing results from the already expanding field of prostaglandin research (1,2). All efforts were now geared to 'visualize' the prostaglandin endoperoxide synthase enzyme (Cyclooxygenase, COX). Known features were that cyclooxygenase is a polypep- tide, homodimeric in nature (approximately 70 kDa) and in monotopic arrangement in the cell membrane. It carried two distinct functional enzyme activities, catalyzing both the bisoxygenation of arachidonic acid to its hydroperoxy arachidonate

metabolite prostaglandin G2 and consecutively catalyzing the peroxidative reduc-

tion of prostaglandin G2 to its endoperoxide H2 (3). The peroxidase activity of the enzyme complex is not affected by NSAIDs. A major advance in the field of eicosanoid research was the discovery of a second inducible cyclooxygenase isoenzyme, COX-2 (4,5). The two isoforms, COX-1 and COX-2, were believed to explain the therapeutic but also the adverse effects of the frequently used NSAIDs. By hypothesizing that the COX-1 enzyme was the constitutive enzyme, designed to be available for physiological functions, the COX- 2 enzyme was thought primarily to act in pathophysiological processes (COX dogma). An important aim would be to develop COX-2 specific NSAIDs that would aid in fighting inflammatory processes and not displaying unwanted side-effects related to inhibition of the COX-1 enzyme (6,7).

36 General introduction

Evidence was delivered that localization of the COX-1 enzyme primarily was in the smooth endoplasmic reticulum and the COX-2 enzyme in the nuclear envelope. Cell membrane receptors for prostanoids have been localized on several tissues including the eye (8,9). In studying the dynamics of inhibition by flurbiprofen and indomethacin of the human prostaglandin H synthases it became evident that these compounds influenced at least five processes, including the rate of catalytic activation, the rate of substrate turnover, the rate of autoinactivation of the enzyme complex and the association and dissociation rates of the inhibitor with the complex. Overall, indomethacin and flurbiprofen behaved similarly towards the human prostaglandin endoperoxide H synthase-1 and -2 enzymes, although the individual kinetic parameters differed (10,11). In an earlier study it was concluded that the inhibitor-enzyme complex is more stable for the flurbiprofen-prostaglandin H synthase-1 than for flurbiprofen-prostaglandin H synthase-2 complex (12). Each isoenzyme, prostaglandin endoperoxide H synthase-1 and prostaglandin endoperoxide H synthase-2, is encoded by a different gene. When activated and with adequate arachidonic acid and oxygen present, a single prostaglandin endoperoxide H synthase (COX) molecule can produce 103 molecules of prostaglandin G2, a hydroperoxide, which is catalytically reduced to its alcoholic form (PGH2) by peroxidase (13). The COX-1 and COX-2 enzymes are homodimers. Each dimer consists of three independent folding units: a membrane-binding domain, an enzymatic/catalytic domain and an epidermal growth factor-like domain. The enzymatic/catalytic domain consists of two separate, closely spaced, but inter- dependent areas encompassing 80% of the protein. The cyclooxygenase site is situated at the apex of a long hydrophobic channel in the prostaglandin endoperoxide H synthase molecule. A marked difference between COX-2 and COX-1 is the larger channel and the approximately 20% larger binding site in the former. This difference has been exploited to examine the possibility of designing selective COX-2 inhibitors (14). Although it has been suggested that the COX-1 enzyme complex is mainly active for maintenance purposes and the COX-2 enzyme, by nature of its rapid induction capabilities, for the contribution to prostaglandin related inflammation, pain and fever, implying that selective COX-2 inhibitors would benefit the patient, some doubt has arisen concerning this elegant theory (15,16). In a more recent study using a COX-1 selective inhibitor as well as COX-2 selective and non- selective inhibitors in normal and monoarthritic rats and mice with paw inflammation, it was concluded that inhibition of both COX-isoenzymes was needed for effective analgesia in inflammation (17). In studies involving the use of cyclooxygenase knockout mice it became apparent that deficiency of COX-2 had more pronounced effects on the physiological maintenance of the body than deficiency of COX-1 (18). To initiate the cyclooxygenase reaction, activation of the peroxidase active site is

37 Chapter 1

necessary (19). When activated, an aqueous insoluble, nonchiral, arachidonic acid

molecule, liberated by phospholipase A2 from the membrane phospholipids, will be 'sucked' into the hydrophobic channel where it will be converted to the

prostaglandin G2 endoperoxide. After bioconversion of arachidonic acid into the

prostaglandin G2 endoperoxide it will be transported to a reservoir type enclave formed by the dimeric cyclooxygenase enzym complex. From here transport of

prostaglandin G2 to the peroxidase catalytic site is possible, where it will be trans-

formed into prostaglandin H2 endoperoxide. Closer examination of the interaction of the NSAID flurbiprofen, a representative of the 2-phenylpropionic acid class, with the enzyme channel, reveals that the amino acid tyrosine 355, situated near the entrance of the channel, creates a local narrowing which in turn provides a handsome explanation why the S(+) flurbiprofen enantiomer of this molecule will have a better fit than its counterpart R(-) flurbiprofen (20,21). The carboxylate group of flurbiprofen as well as of indomethacin will complex with the guanidinium group of arginine 120 just like the carboxylate group of arachidonic acid. Preparation of neutral NSAIDs by transforming the carboxylate group into an ester or amide function may enhance COX-2 selectivity (22). The cyclooxygenase enzymes produce prostanoids from the polyunsaturated fatty

acid, arachidonic acid. These prostaglandins, D2, E2, F2α, I2 and thromboxane A2, act via their respective receptors to elicit various physiological reactions (23). A number of receptors have been identified (24). Prostaglandins are involved, together with histamine and bradykinine, in the local increase in vascular perme-

ability and edema in which PGE2 and PGI2 are prominently involved. It has become evident that they elicit these vascular changes as well as inflammatory pain via EP- and IP-type receptors, respectively.

In a study involving knock-out mice with a EP1 receptor deficient status, the pain- sensitivity responses were tested in two acute prostaglandin-dependent models (25). The animals' reaction was reduced by approximately 50%, the same amount of reduction that could be achieved by pharmacological interventions in wild-type mice. In determining the inhibitory effects of the flurbiprofen enantiomers on the COX-1 and COX-2 isoenzymes, it is of advantage to take the biological surroundings of the NSAID in the human body, e.g. causing protein binding, into consideration. Use of human whole blood, as an ex vivo method for quantification of the inhibitory effects of an NSAID on the synthesis of prostanoids has become an established procedure (26,27). As an unequivocal example of functional COX-1 the Ca2+-ionophore stimu-

lated platelet is used, measuring the metabolite of thromboxane A2. The lipopolysaccharide stimulated monocyte can be taken as an activity indicator of functional COX-2. Any interference by platelets is excluded by acetylation of the COX-1 enzyme by prior administration of acetylsalicylic acid.

38 General introduction

Prostaglandin levels have been measured using radioimmunoassays in 41 human eyes of patients undergoing vitrectomy (28). In 'quiet eyes' undergoing routine cataract extraction physiological prostaglandin levels of around 100 pg/ml were reported. In patients with a diagnosis of cataract and cystoid macular edema mean levels of PGI2 varied between 49 and 360 pg/ml. Induction of COX-2 mRNA can take place in the rabbit eye within three hours following glaucoma filtration surgery (29). Paracentesis however fails to induce COX-2 mRNA, possibly because of the minimal disturbance by this procedure.

Prostaglandin synthase activity exists both in vascular and avascular structures of the eye, being most abundant in the iris-ciliary body. Although the iris-ciliary body of the rabbit eye has a high functional capacity to synthesize prostanoids following paracentesis, only a transient ocular inflammatory response follows which resolves within 3 - 4 hours (30). Eicosanoid measurements in the aqueous obtained during paracentesis of the rabbit eye showed a strong and rapid rise in PGE2 levels in the aqueous humor with peak values at 20 minutes, followed by recovery to baseline within 48 hours (31).

No prostacyclin (measured as the stable metabolite 6-keto-PGF1α) was detected in the aqueous humor at the start but was markedly present at ten and twenty minutes after the initial trauma. Prostaglandin synthesis was followed shortly by an increase in aqueous humor protein, with peak levels achieved within 30 minutes after paracentesis. Both PGE2 and protein levels declined gradually to near baseline levels 48 hours after trauma.

Experimental evidence has shown that the inducible COX-2 mRNA is present in the first hours after injury and is possibly assisting in wound healing (32,33). COX-1 knockout mice do not show signs of spontaneous gastrointestinal ulceration as would have been expected when the generation of prostaglandins by the COX-1 enzyme is involved in maintenance and integrity keeping purposes (34). Classical NSAIDs still show efficacy when the COX-2 enzyme is no longer present, as shown by Western blotting (35). Administering COX-2 inhibitors then does not seem indicated. Interestingly, a COX-2 enzyme complex showed up again near resolution of inflammation and produced anti-inflammatory prostaglandins, PGD2, PGF2α and a member of the PGJ2 family (36). This is in contrast with the view hinting that cyclooxygenase-2 inhibitors might be the new approach to therapy in ocular inflammation (37). The case against the use of COX-2 inhibitors seems more compelling now that it has become clear that in the field of rheumatology a five fold higher risk of cardiac complications can occur (38,39,40,41). In a well designed study using knockout

39 Chapter 1

mice deficient of a prostacyclin receptor or a thromboxane receptor, it was shown that efficient cross talk exists between prostacyclin- and thromboxane- dependent signaling pathways (42). Inhibition of one signaling pathway might induce the other. This could account for unforeseen complications with the use of cyclooxygenase-2 inhibitors (43). Also the presence of mutations in cytochrome P450 isoenzymes that metabolize arachidonic acid could play a role in diseases involving clotting and inflammatory disorders (44). Potential drug alternatives, however, are being developed (45). Flurbiprofen is an NSAID with preferential cyclooxygenase-1 inhibiting capacity like indomethacin. Its use as a NSAID eyedrop for combatting inflammatory responses following cataract surgery therefore seems appropriate.

REFERENCES

1. Flower RJ. Drugs which inhibit prostaglandin biosynthesis. Pharmacol Rev 1974;26:33-67. 2. Flower RJ, Vane JR. Inhibition of prostaglandin biosynthesis. Biochem Pharmacol 1974;23:1439-50. 3. Merlie JP, Fagan D, Mudd J, Needleman P. Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem 1988;263:3550-3. 4. Xie W, Chipman JG, Robertson DL, Erikson RL, Simmons DL. Expression of a mitoogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 1991;88:2692-6. 5. Xie W, Robertson DL, Simmons DL. Mitogen-inducible prostaglandin G/H synthase: a new target for nonsteroidal anti-inflammatory drugs. Drug Dev Res 1992;25:249-65. 6. Vane J. Towards a better aspirin. Nature 1994;367:215-6. 7. Allen KN. Aspirin - now we can see it. Nature Medicine 1995;1:882-3. 8. Goetzl EJ, An S, Smith WL. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J 1995;9:1051-8. 9. Morita I, Schindler M, Regier MK, Otto JC, Hori T, DeWitt DL, Smith WL. Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem 1995;270:10902-8. 10. Callan OH, So O-Y, Swinney DC. The kinetic factors that determine the affinity and selectivity for slow binding of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. J Biol Chem 1996;271:3548-54. 11. So O-Y, Scarafia LE, Mak AY, Callan OH, Swinney DC. The dynamics of prostaglandin H synthases. Studies with prostaglandin H synthase 2 Y355F unmask mechanisms of time-dependent inhibition and allosteric activation. J Biol Chem 1998;273:5801-7. 12. Laneuville O, Breuer DK, Dewitt DL, Hla T, Funk CD, Smith WL. Differential inhibition of human prostaglandin endoperoxide H synthases-1 and -2 by nonsteroidal anti-inflammatory drugs. J Pharmacol Exp Ther 1994;271:927-34. 13. Kulmacz RJ. Cellular regulation of prostaglandin H synthase catalysis. FEBS letters 1998;430:154-7. 14. Brooks P, Emery P, Evans JF, Fenner H, Hawkey CJ, Patrono C, Smolen J, Breedveld F, Day R, Dougados M, Ehrich EW, Gijon-Baños J, Kvien TK, van Rijswijk MH, Warner T, Zeidler H. Interpreting the clinical significance of the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2. Rheumatology 1999;38:779-88. 15. Wallace JL. Selective COX-2 inhibitors: is the water becoming muddy? TiPS 1999;20:4-6.

40 General introduction

16. Reuter BK, Asfaha S, Buret A, Sharkey KA, Wallace JL. Exacerbation of inflammation-associated colonic injury in rat through inhibition of cyclooxygenase-2. J Clin Invest 1996;98:2076-85. 17. Mazario J, Gaitan G, Herrero JF. Cyclooxygenase-1 vs. cyclooxygenase-2 inhibitors in the induction of antinociception in rodent withdrawal reflexes. Neuropharmacology 2001;40:937-46. 18. Langenbach R, Loftin C, Lee C, Tiano H. Cyclooxygenase knockout mice. Models for elucidating isoform-specific functions. Biochemical Pharmacology 1999;58:1237-46. 19. Kiefer JR, Pawlitz JL, Moreland KT, Stegeman RA, Hood WF, Gierse JK, Stevens AM, Goodwin DC, Rowlinson SW, Marnett LJ, Stallings WC, Kurumbail RG. Structural insights into the stereochemistry of the cyclooxygenase reaction. Nature 2000;405:97-101. 20. Bhattacharyya DK, Lecomte M, Rieke CJ, Garavito RM, Smith WL. Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1. J Biol Chem 1996;271:2179-84. 21. Kurumbail RG, Kiefer JR, Marnett LJ. Cyclooxygenase enzymes: catalysis and inhibition. Curr Opinion Struct Biol 2001;11:752-60. 22. Rainsford KD. Profile and mechanisms of gastrointestinal and other side effects of nonsteroidal anti-inflammatory drugs (NSAIDs). Am J Med 1999;107 (6A) 27S-36S. 23. Tilley SL, Coffman, Koller BH. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. J Clin Invest 2001;108:15-23. 24. Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 1999;79:1193-226. 25. Stock JL, Shinjo K, Burkhardt J, Roach M, Taniguchi K, Ishikawa T, Kim H-S, Flannery PJ, Coffman Tm, McNeish JD, Audoly LP. The prostaglandin E2 EP1 receptor mediates pain per- ception and regulates blood pressure. J Clin Invest 2000;107:325-31. 26. Patrignani P, Panara MR, Greco A, Fusco O, Natoli C, Iacobelli S, Cipollone F, Ganci A, Créminon C, Maclouf J, Patrono C. Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases. J Pharmacol Exptl Ther 1994;271:1705-12. 27. Young JM, Panah S, Satchawatcharaphong C, Cheung PS. Human whole blood assays for inhibition of prostaglandin G/H synthases-1 and -2 using A23187 and lipopolysaccharide stimulation of thromboxane B2 production. Inflamm Res 1996;45:246-53. 28. Thomas MA, O'Grady GE, Swartz SL. Prostaglandin levels in human vitreous. Br J Ophthalmol 1985;69:275-9. 29. Chang MS, Tsai JC, Yang R, DuBois RN, Breyer MD, O'Day DM. Induction of rabbit cyclooxygenase 2 in the anterior uvea following glaucoma filtration surgery. Curr Eye Res 1997;16:1147-51. 30. Kulkarni PS, Srinivasan BD. Cyclooxygenase and lipoxygenase pathways in anterior uvea and conjunctiva. In: Bito LZ, Stjernschantz J, editors. The ocular effects of prostaglandins and other eicosanoids. New York Alan R Liss 1989:39-52. 31. Graff G, Brady MT, Gamache DA, Spellman JM, Yanni JM. Transient loss of prostaglandin syn- thetic capacity in rabbit iris-ciliary body following anterior chamber paracentesis. Ocul Immunol Inflamm 1998;6:227-38. 32. Mizuno H, Sakamoto C, Matsuda K, Wada K, Uchida T, Noguchi H, Akamatsu T, Kasuga M. Induction of cyclooxygenase 2 in gastric mucosal lesions and its inhibition by the specific antagonist delays healing in mice. Gastroenterolgy 1997;112:387-97. 33. Gilroy DW, Colville-Nash PR, Willis D, Chivers J, Paul-Clark MJ, Willoughby DA. Inducible cyclooxygenase may have anti-inflammatory properties. Nat Med 1999;5:698-701. 34. Morteau O, Morham SG, Sellon R, Dieleman LA, Langenbach R, Smithies O, Balfour Sartor R. Impaired mucosal defense to acute colonic injury in mice lacking cyclooxygenase-1 or cyclooxygenase-2. J Clin Invest 2000;105:469-78.

41 Chapter 1

35. Gilroy DW, Tomlinson A, Willoughby DA. Differential effects of inhibitors of cyclooxygenase (cyclooxygenase 1 and cyclooxygenase 2) in acute inflammation. Eur J Pharmacol 1998; 355:211-17. 36. Willoughby DA, Moore AR, Colville-Nash PR. COX-1, COX-2, and COX-3 and the future treatment of chronic inflammatory disease. Lancet 2000;355:646-8. 37. Masferrer JL, Kulkarni PS. Cyclooxygenase-2 inhibitors: a new approach to the therapy of ocular inflammation. Surv Opthalomol 1997;41:S35-S40. 38. Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, Davis B, Day R, Bosi Ferraz M, Hawkey CJ, Hochberg MC, Kvien TK, Schnitzer TJ, for the VIGOR study group. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. N Engl J Med 2000;343:1520-8. 39. Ray WA, Stein CM, Daugherty JR, Hall K, Arbogast PG, Griffin MR. COX-2 selective nonsteroidal anti-inflammatory drugs and risk of serious coronary heart disease. Lancet 2002;360:1071-3. 40. Jüni P, Rutjes AWS, Dieppe PA. Are selective COX 2 inhibitors superior to traditional non steroidal anti-inflammatory drugs? Adequate analysis of the CLASS trial indicates that this may not be the case. BMJ 2002;324:1287-8. 41. Jones R. Efficacy and safety of COX 2 inhibitors. New data are encouraging but the risk:benefit ratio remains unclear. BMJ 2002;325:607-8. 42. Cheng Y, Austin SC, Rocca B, Koller BH, Coffman TM, Grosser T, Lawson JA, FitzGerald GA. Role of prostacyclin in the cardiovascular response to thromboxane A2. Science 2002;296:539-4. 43. Vane JR. Back to an aspirin a day? Science 2002;296:474-5. 44. Nebert DW, Russell DW. Clinical importance of the cytochromes P450. Lancet 2002;360:1155-62. 45. Skelly MM, Hawkey CJ. Potential alternatives to COX 2 inhibitors. New molecules may overtake the COX 2 inhibitors debate. BMJ 2002;324:1289-90.

42 CHAPTER 2

RATIONALE FOR USING A PHOSPHATE BUFFER FOR S(+) FLURBIPROFEN EYEDROPS Chapter 2

RATIONALE FOR USING A PHOSPHATE BUFFER FOR S(+) FLURBIPROFEN EYEDROPS

Although the eye, eyelids and skin surrounding the eye are sensitive to external stimuli, physiological reactions due to deviations outside the near normal values for osmolality or pH are not always seen. However, in a state of ill-health or during regular use of ophthalmic preparations, this situation may be more outspoken. The active principle in the eyedrop can provoke, when not properly dissolved, an irritating or burning sensation leading to lacrimal discharge, occasional haemorrhage or endangering blinking reflexes during surgery. Lacrimal discharge will cause an unwanted dilution and drainage of medicine. Individual sensitivity may vary and physiological values of tear fluid can fluctuate, which is also dependant on the health condition of the individual eye in general, the nasal corner of the eye being the most sensitive. Even if the composition of the eyedrop approximates the ideal solution, certain (active) principles may cause discomfort to the eye. Non-irritating eyedrops should comply with: (1) sterility, (2) isotonicity and (3) pH value. Sterility is of paramount importance when an ophthalmic solution is applied to the injured eye. The character of the active ingredient will to a certain degree determine the above mentioned requirements. The osmotic value of an ophthalmic solution should reflect that of blood, corresponding to a 0,9% sodium chloride solution. Deviations from this value have been noted from 0,6% to as high as 5% sodium chloride without marked discomfort. Yet it is of prime importance to adhere as close as possible to isotonicity, as the optical integrity of the cornea can be influenced significantly by deviations thereof. In this respect the physiological term tonicity seems more appropriate than the physicochemical term osmolality. The cornea functioning as selective permeable biomembrane is better accomodating this term. The osmotic value is commonly expressed in (milli)osmol/liter (osmolarity). This can be transformed to osmolality (mosmol/kg) by dividing by the specific gravity of the solution. Eye irritation must be discerned from an allergy which requires the choice of a different pharmacological agent. Cutaneous hypersensitivity due to a particular stereoisomer has been reported (1).

There are several reasons for buffering an ophthalmic solution: · To prevent unwanted pH changes caused by hydroxyl ion release from the glass in which the solution is stored. · In case of a pH-dependent degradation of the active principle, a buffer should be used for stabilization.

44 Phosphate buffer

· In case of a pH-dependent solubility, a buffer can be used to dissolve the required amount of drug.

On the other hand there are also limitations to the use of buffers. First of all, the limited buffer capacity of the lacrimal fluid precludes the use of strong buffers outside the pH range of 6.8 - 7.6. In addition, adherence to a pH as close to the physiological pH as possible is important for preventing local precipitations of the drug and minimizing deterioration after administration.

For the formulation of S(+) flurbiprofen eyedrops, a published phosphate buffer of pH 7.4, used in an eyedrop, was chosen as a starting point (2). The specialty Ocuflur® containing racemic flurbiprofen sodium (appendix A) is produced in a citrate buffer of pH 6.45. However, the quantitative nature of the citrate buffer used was not disclosed. Analysis by HPLC revealed a citrate concentration of 5.5 gram/l (see appendix B for materials & conditions).

The buffer used by us in preparing S(+) flurbiprofen eyedrops consists of 0.022 M disodium phosphate dihydrate and 0.112 M potassium dihydrogen phoshate, resulting in approximately 0.13 M total salt concentration and an ionic strength of 0.36 M.

It should be realised, that at pH=7.4 practically all dissolved flurbiprofen (pKa= 4.22) is present in the salt form; the acidic form fraction is determined by

= Log [flurbiprofen acid/flurbiprofen salt] pKa - pH; thus % [flurbiprofen acid] = 0.066%.

The importance of this value is that because only the non-protonized form of an acid (like flurbiprofen) is able to pass the different membranes in the eye, the driving force for permeation is rather low (0.066%). It should be realized, however, that the pH of the ophthalmic solution is not the only parameter determining permeation efficacy.

The choice for a buffer applied to the ophthalmic solution is determined by the best compromise of the following issues: 1. It is convenient for patient and surgeon to stay as close as possible to the natural pH of the tear fluid (7.4). 2. As far as flurbiprofen is concerned, the solubility in aqueous solution is problematic at pH values below 7 (3), as illustrated on the next page:

45 Chapter 2

pH 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.2

3. Discomfort for the patient will not be present as long as the pH is between 6.6 - 7.8. Tolerability for the cornea is in the pH range of pH 6.6 - 8.5. Changes in permeability will occur outside the pH range of 4 and 10. 4. The permeation of flurbiprofen increases at lower pH values.

On the basis of the above considerations it follows that the optimal pH is between 7.0 and 7.4. For the present formulation the physiological pH (7.4) was chosen. The often used citrate buffer would have been less favourable, because this buffer composition has hardly any buffering capacity around pH 7.4, in clear contrast to a phosphate buffer. Interestingly, no difference in pharmacological effect could be demonstrated using ophthalmic solutions of diclofenac, indomethacin or flurbiprofen in a pH range of 5.0 - 7.5 in preventing disruption of the blood-aqueous barrier in a rabbit paracentesis model (4). In addition, the use of a phosphate buffer provides stable, single stereoisomer formulations of the NSAID ketorelac. This was in contrast to the use of acetate or citrate buffers (5).

46 Phosphate buffer

REFERENCES

1. Buzo-Sanchez G, Martin-Munoz MR, Navarro-Pulido AM, and Orta-Cuevas JC. Stereoisomeric cutaneous hypersensitivity. Ann Pharmacother 1997;31:1091. 2. van Nispen tot Pannerden EBLM, van Sorge AA. Waterige oogdruppels met indometacine in oplossing (Aqueous eyedrops with indomethacin in solution) Pharm Weekbl 1981;116:386-7. 3. Naaijkens MS and Van Sorge AA . Preparation of a 5% Flurbiprofen hydrogel. Pharmaceutical aspects. 6th Internet World Congress for Biomedical Sciences. Ciudad Real, Spain February 14th - 25th , 2000. 4. Van Haeringen NJ, Oosterhuis JA, Van Delft JL, Glasius E, Noach EL. A comparison of the effects of non-steroidal compounds on the disruption of the blood-aqueous barrier. Exp Eye Res 1982;35:271-7. 5. Brandl M, Conley D, Johnson D, Johnson D. Racemization of ketorolac in aqueous solution. J Pharm Sci 1995;84:1045-8

APPENDIX A Ocuflur® Composition according to package insert august 1995 Allergan S.A./N.V.

Flurbiprofen sodium dihydrate 0.03%, Polyvinylacohol, Thiomersal 0,005%, Sodium edetic acid, sodium chloride, sodium citrate dihydrate, sodium chloride, citric acid monohydrate, sodium hydroxide, hydrochloric acid, aqua purificata

B HPLC method of analysis Materials & Methods Column: Chrompack Organic acids 300*6.5mm conventional stainless steel Catalogue No. 28350 Eluent: 0.005 mol/l sulphuric acid Flow: 0.8 ml/min Temperature: 35°C Detector: UV 210 nm Internal Standard: oxalic acid Injection volume: 25 µL

47 48 CHAPTER 3

FLURBIPROFEN, S(+), EYEDROPS: FORMULATION, ENANTIOMERIC ASSAY, SHELFLIFE AND PHARMACOLOGY

Adriaan A. van Sorge, Peter H. Wijnen, Jan L. van Delft, Valérie M.W. Carballosa Coré-Bodelier and Nicolaas J. van Haeringen

Derived from Pharm World Sci 1999;21:91-5 Chapter 3

ABSTRACT

Aphakic cystoid macula edema, occurring after cataract extraction is ascribed to trauma-induced production of intra-ocular prostaglandins. Sufficient experimental and clinical evidence supports the use of prostaglandin synthesis inhibitors to coun- tervail this clinical condition. The active S(+) enantiomer of flurbiprofen, a prostaglandin synthesis inhibitor, has been formulated into a stereoselective, ballast free eyedrop solution in a concentration of 0.015%. Analysis by capillary zone electrophoresis shows shelflife stability up to four years at room temperature of this enantiomer. The inhibitory effect on the synthesis of prostaglandins as measured on a homogenate of bovine iris/ciliary body, remained unaffected during a shelflife period of three years.

INTRODUCTION

Following cataract surgery non-specific inflammatory responses are induced, caused by surgical trauma. In the animal model of ocular trauma it is well substantiated that the greater part of this inflammatory process is based on synthesis of prostaglandins by iris tissue (1,2). The prostaglandins are released into the aqueous humor and cause breakdown of the blood aqueous barrier, characterized by influx of plasma proteins. Diffusion of prostaglandins through the vitreous to the posterior segment of the eye, may cause breakdown of the blood retinal barrier resulting in the development of aphakic cystoid macular edema (3). Ophthalmic solutions of several nonsteroidal anti-inflammatory drugs (NSAIDs) are commercially available such as diclofenac (0.1%), indomethacin (0.1%), flurbiprofen (0.03%), ketorolac (0.5%) and a suspension of indomethacin (1.0%). Indomethacin 0.1% formulation was incorporated in the Dutch National Formulary (FNA) in 1986. Inpracticalities with indomethacin in aqueous solution - no sterilisation possibility and only a short shelflife when in solution (4-7) - prompted us to investigate the possibility in formulating an eyedrop based on flurbiprofen (8). An official monograph on Flurbiprofen sodium ophthalmic solution is mentioned in the

USP XXIII . The solubility of flurbiprofen (pKa 4.22) in its acid form is 100mg/L at pH 5.0; its sodium salt has a solubility of 400mg/L (26°C, pH 7). Shortly after it was recognized that inhibition of prostaglandin synthesis could be the main mechanism of action of NSAID's (9), analysis of the category of 2-aryl propionic acids (profens] revealed their nature to be racemic and their inhibitory activity almost exclusively residing in the S-stereoconfiguration (10). However only in 1990 the ocular effect of topically applied S(+) ibuprofen was reported in a rabbit model of interleukin-1 (11) and paracentesis induced uveitis (12] at relatively high concentrations (0.9% and 0.8% respectively). Also with S(+) naproxen, marketed

50 Flurbiprofen, S(+), Eyedrops

by Syntex as enantiomeric pure NSAID, the antiinflammatory effect in eyedrops (0,5%) was demonstrated experimentally (13). In a bovine iris/ciliary body homogenate incorporating the cyclooxygenase -1 (COX-1) enzyme, the S(+) flurbiprofen proved to be the pharmacological active moiety, showing 100 times greater potency than R(-) flurbiprofen in inhibiting prostaglandin synthesis (14). In keeping with the benefits of only using the proposedly active moiety, (S+) flurbiprofen, we investigated formulations, containing the pure enantiomers of flurbiprofen (15). We thereby avoid isomeric ballast, providing a reduction in metabolic load to the patient. Advantages would eventually be: less complex pharmacokinetic profiles (16), less complex drug interactions and uncomplicated concentration-effect relationships. The present study deals with the formulation, analysis, keeping quality and pharmacology of solutions containing 0.03% flurbiprofen, 0.015% flurbiprofen (S+) or flurbiprofen (R-) 0,015%, all three based on their acid form.

MATERIALS AND METHODS

Drugs and chemicals Flurbiprofen, (S+)flurbiprofen and (R-)flurbiprofen were purchased from Duchefa

Pharma BV (Haarlem, The Netherlands). Disodiumphosphate.2H2O, Potassium- dihydrogen-phosphate were purchased from Bufa (Uitgeest, The Netherlands). USP reference standard, USP 2-(4-Biphenylyl)propionic acid RS, (Flurbiprofen related compound A limit test; catalog number: 28576 ). Vancomycin hydrochloride was purchased from Sigma (St Louis, Mo, USA).

H3PO4, KH2PO4, TRIS and Na2HPO4 were analytical grade and obtained from JT Baker (Deventer, The Netherlands).Water for injections USPXXIII (Fresenius, 's- Hertogenbosch, The Netherlands). Water for analysis was purified in an Alpha-Q apparatus (Millipore, Bedford, MA,USA). Samples of the speciality Ocuflur® (0.03% flurbiprofen sodium, lotnumbers: 94G11 exp:01/96, 96L18 exp: 06/98 and 99K29 exp: 05/2001) were a gift from Allergan (Belgium).

Formulation of eyedrops The formulation for the 0.13M phosphate buffer, pH 7.4 and osmolality of 290 mOsmols/kg is based on previous work with indomethacin (7) Composition of buffersolution

Disodiumphosphate. 2H2O 20 gram Potassiumdihydrogenphosphate 3 gram Water for injections ad 1 Liter This solution is filtered through a 0.22 micron filter before sterilization for 15 minutes at 121°C. This buffersolution is used for preparing the eyedrops for pharmacological testing and for the shelflife procedure.

51 Chapter 3

Preparation of the racemic, (S+)- and (R-) flurbiprofen eyedrops proceeds by addition of respectively 30 mg racemic flurbiprofen and 15 mg of each of the enantiomers to 100 ml of phosphatebuffer solution. The final pH remaining unchanged at 7.4, because the relatively low concentration of flurbiprofen (1.2mM) does not burden the buffer (130mM) significantly. All flurbiprofen preparations were stored in glass containers. No preservative is added to the final solution as the eyedrops will be used prior to cataract surgery and eye-surgeons show preference to use eyedrops, if feasable, without unnecessary additions thereby avoiding possible allergic reactions. For shelflife testing, preparations were either 0.22 micron filtered, heat treated at 100°C for 60 minutes or sterilized at 121°C for 15 minutes. Preparations were stored for a period of up to 60 months, either at room temperature in subdued light or at - 20°C. Analysis was performed by capillary zone electrophoresis (CZE) on samples at t=0 and samples stored for 36, 48 and 60 months. All concentrations complied with the requirement of containing between 90 - 110% of the active moiety and were set at 100% at the start of shelflife analysis. Additional analysis was done, according to the monographs on Flurbiprofen mentioned in the USP24-NF19, European Pharmacopea 1999 and the British Pharmacopea 2000, on Flurbiprofen related substances of which 2-(4- Biphenylyl)propionic acid (Flurbiprofen related compound A limit test) is the main component.

Analytical assay A CZE is a technique which permits high separation efficiencies combined with small sample volumes. Quantitative aspects of CZE methods for enantiomeric purity testing are discussed in the literature for both basic and acidic drugs. Depending on the resolution of the peaks, limits of detection of <0.1% are shown for determination of the minor enantiomer (17-20). An applied Biosystems (San Jose, CA, USA) Model 270A-HT CZE system was used, equipped with a variable-wavelength UV absorbance detector (254 nm, 0.5 second rise time). The separations were performed in a fused silica capillary (70 cm x 50 µm inner diameter, Polymicro Technologies, Phoenix, AZ, USA) with a length of 50 cm to the detection window. The electrophoresis buffer was prepared by

adjusting a 50mM KH2PO4 solution to pH=6.0 with a 50 mM Na2HPO4 solution. The glycopeptide antibiotic Vancomycin was used as chiral selector (21-23). The selector was added to the inlet buffer only, at a concentration of 0.6mM. The separations were carried out at +15 kV, with the oven temperature set at 30°C. Samples were introduced into the capillary at the anodic end via a controlled vacuum injection system of 1 or 2 seconds corresponding to a volume of approxi-

52 Flurbiprofen, S(+), Eyedrops

mately 4 - 8 nanoliter, respectively. After sample injections the electrode and the outer surface of the fused silica capillary were dipped for 0.5 seconds in water for cleansing. Analytes are detected in the capillary near the cathode. Data were recorded using a Fisons Model VG-Multichrom system.

Enantiomeric assay Chiral separation occurs through selective complexation of the flurbiprofen enantiomers with vancomycin. From the corrected peak areas of the enantiomers, the enantiomeric ratio (E.R.) was calculated as R(-)/S(+). To determine if racemization occurred during storage conditions the E.R. was determined in all samples. The racemic drug will have an enantiomeric ratio of unity. For the S(+) and R(-) samples the impurities are calculated as percentage relative to the main optical isomer. The racemic samples under investigation were injected after appropriate dilution. Phosphate buffer, used in the formulation of the eyedrops, was injected as blank solution to rule out interference.

Quantitative assay For quantitative determination of the S(+) and R(-) samples the above described system was changed for analysis of total flurbiprofen. The CZE-buffer was stabilized to pH 7 and consisted of 40mM TRIS/H3PO4 solution without chiral selector. S(+) and R(-) flurbiprofen migrate as one peak, thus minimizing the contribution of integration errors on the quantitative results. Separations were carried out at +25 kV. Samples were analyzed in duplo and on two separate days. The racemic samples and the S(+) and R(-) samples were appropriately diluted before injection. Standards were injected at the beginning, halfway and at the end of the sample sequence. Calibration was taken into account by insertion of standards in the same concentration range during the analysis. The phosphate buffer was included as blank for investigation of possible interferences.

B A test on common impurities can be performed according to the monographs on Flurbiprofen (Sodium) described in the European Pharmacopea (1999 page 859- 61), the USP24-NF19 (effective january 2000) and the British Pharmacopea (2000 page 718) To perform a limit test on 2-(4-Biphenylyl)propionic acid (Flurbiprofen related compound A), the main impurity, a liquid chromatography system was equipped with a variable-wavelength UV absorbance detector set at 247 nm and a chrompack Inertsil 5 octadecylsilyl-3 column (catalog number CP28308). As the mobile phase, at a flow rate of 1.0 ml/min, a mixture of 5 volumes of glacial acetic acid, 35 volumes of acetonitrile and 60 volumes of water was used. Injection volume used was 20 microliter with a relative standard deviation for replicate injections

53 Chapter 3

of less than 1%. The resolution factor between the two principle peaks is larger than 1.5 (1.94) and accords with the requirements set by the British Pharmacopoeia, European Pharmacopoeia and USP24-NF19. The relative retention time for flurbiprofen related compound A to flurbiprofen is 0.87 (figures 1a and 1b). Sampling on several batches of flurbiprofen S(+) 0.015%, flurbiprofen R(-) 0.015% and of Ocuflur® (0.03% flurbiprofen sodium) was performed.

Figure 1a. Analysis of Ocuflur® (batch 96L18) for Flurbiprofen related compound A.

Figure 1b. Analysis of Flurbiprofen S(+) 0.015% (batch 28081995) for Flurbiprofen related compound A

54 Flurbiprofen, S(+), Eyedrops

Pharmacological assay Inhibition of prostaglandin synthesis by flurbiprofen was performed using bovine iris/cilliary body homogenate according to Van Sorge et al (14). In brief, 25 µLof flurbiprofen solution is added to 100 µL of iris/ciliary body homogenate, prepared from one iris/ciliary body in one ml of 0.05 M TRIS buffer pH 7.4. The enzyme reaction was stopped by heating for 3 minutes in boiling water. In the supernatant after centrifugation PGE2 was determined using an enzyme immune assay. Inhibition of

PGE2 release was calculated by the difference of PGE2 release in the absence and presence of flurbiprofen, expressed in percent of the non-inhibited release.

RESULTS

ANALYTICAL ANALYSIS

Enantiomeric analysis On injection of the phosphate buffer used in the formulation of the eyedrops no interfering peaks were detected. The E.R. of the flurbiprofen standard was 0.999 ± 0.006 (n=6) which corresponds to 50.0 ± 0.2%. Stored samples of racemic flurbiprofen displayed no change in E.R. as compared to standard. Neither a heat treated sample at 100°C and stored at roomtemperature for 60 months (E.R.: 0.996), nor a second sample of the same date sterilized for 15 minutes at 121°C and stored at room temperature (E.R.: 0.997) showed significant deviation in E.R from the standard racemic flurbiprofen (E.R. 0.999). Samples of 48 months storage at roomtemperature (E.R.: 0.996) and 24 months at -20°C (E.R.: 0.995) exhibited no change in E.R. as compared to standard. Ocuflur® (lotnumber 94G11) stored at room temperature (36 months) showed no change in E.R. (0.995). All samples of S(+) flurbiprofen (48 months or less and stored at room temperature) showed no R(-) enantiomer above limit of detection of the assay, which is approximately 0.1%.

The racemic moiety of S(+) in R(-) flurbiprofen at t=0 was determined as 0.7% ± 0.1 (n=3). In Figure 2 the electropherograms are shown for two samples of R(-) flurbiprofen, after storage at -20°C for 48 months without sterilization as well as after storage for 48 months at roomtemperature after sterilization. The presence of S(+) flurbiprofen in the R(-) storage samples (48 months at room temperature sterilized at 15 minutes 120°C) increased to 0.9% (n=2).

55 Chapter 3

Figure 2. Comparison of two superimposed electropherograms of R(-) flurbiprofen samples: (A) stored at roomtemp, sterilized; (B): not sterilized stored at -20 ºC.

Quantitative analysis S(+) flurbiprofen samples irrespective of heat treatment and storage at roomtemperature for 48 months showed a maximum decline to 93% of declared value of the active moiety. The R(-) flurbiprofen samples revealed the same degradation characteristics as those for S(+). Ocuflur® stored at roomtemperature showed a decline to 92% of the declared value after 36 months while racemic flurbiprofen samples, irrespective of heat treatment showed a maximum decline to 90% in 48 months. The type of degradation reaction involved was not investigated. In the electro- pherogram no extra signals are detected, but in the set-up used only negative charged components with UV-absorbance at 254 nm would be visible.

Analysis of Flurbiprofen related compound A (Limit test) Impurities can be found in compendial articles. According to the European Pharmacopoeia 3e Edition 2001 Flurbiprofen has five identified impurities. One of these is known as flurbiprofen related compound A and is used for the limit test. This substance in it self is a stereoisomer but is available as a racemic reference standard USP 2-(4-Biphenylyl)propionic acid RS.

56 Flurbiprofen, S(+), Eyedrops

Table 1. Flurbiprofen related compound A content in ophtalmic solutions.

Preparation Sterilized Date of manufacturing ‰ Flurbiprofen 15 min 121°C related compound A S(+)Flurbiprofen 0.015% Y 28-08-95 0,9; 1,0 N 28-08-95 1,3 ; 1,3; 0,9; 0,8 Y 08-10-93 0,5; 0,5; 0,6; 1,03 R(-)Flurbiprofen 0.015% Y 08-10-93 0,9; 0,8 N 08-10-93 0,3; 0,4 Y 28-08-95 0,2; 0,2 N 28-08-95 0,2; 0,2 Ocuflur®* Y 94G11 2,5; 2,9 Y 96L18 3,7; 3,6 Y 99K29 2,5; 2,5; 2,6; 2,3 * Flurbiprofen Sodium. 2H2O 0.03%. Y=yes, N=No. In either case the products agreed with the Pharmacopoeial limits for the known impurity Flurbiprofen related compound A (5‰), being stored at room temperature and irrespective of heat treatment.

In Table 1 the results are presented not only for the batches prepared with the flurbiprofen isomers as ophthalmic solution but also for the racemic flurbiprofen specialty Ocuflur®. The ophthalmic solutions of isomeric flurbiprofen have been analysed with the same maximum storage time as was stated under enantiomeric analysis.

PHARMACOLOGICAL ANALYSIS

Samples of flurbiprofen (R-) and flurbiprofen (S+) retained their inhibitory effect on prostaglandin synthesis during the period (36 months at room temperature) of shelflife analysis. This appeared from identical concentration inhibition curves using bovine iris/ciliary body homogenate (results not shown) as compared with freshly prepared solutions (Figure 3). The ratio of 100 in inhibitory potency for S(+) flurbiprofen vs. R(-) flurbiprofen also remained unchanged as appears from the un- -8 -6 changed IC50 of 10 M for S(+) flurbiprofen and of 10 M for R(-) flurbiprofen.

57 Chapter 3

Figure 3. Concentration-response curves for inhibition of prostaglandin synthesis in bovine iris/ciliary body homogenate by racemic flurbiprofen (∇), R(-)flurbiprofen (O), and S(+)flurbiprofen () (derived from reference 14). Concentrations of flurbiprofen are final concentrations in the assay mixture. For racemic flurbiprofen the concentration values are given in concentrations of the individual enantiomers. Each point represents the average ± S.E.M. of four to six determinations from different pools of bovine iris/ciliary homogenates.

DISCUSSION

The formulation of an eyedrop, like indomethacin based on a previously published phosphate buffer at pH 7.4 (7), is possible for flurbiprofen. Due to the racemic nature of the molecule and the current national and supranational policies (24) to require quality, safety and efficacy on medicinal products it was deemed necessary to exploit every possibility to formulate an eyedrop that was free of enantiomeric ballast and only contained the pharmacologically active moiety, S(+) flurbiprofen. The first reports on use of indomethacin as an effective eyedrop to combat macula edema after cataract surgery were reassuring but were impractical in pharmaceutical sense. Short shelflife of indometacin when in aqueous solution, uncertain- ty of the real available concentration when provided as a suspension and irritating when applied as an oily solution. The available specialties that have arrived on the Dutch market are not the satisfactory solution for this clinical entity. Indoptol® contains the irritating substance phenylethanol and also benzalkoniumchloride the latter which can react with indomethacin to form insoluble complexes that will remain unnoticed in a suspension. Indocollyre® incorporates the for the eye unpleasant substance methylparahydroxybenzoate and measures a high osmolality when brought in to solution (approximately 1500 mOsm/kg).

58 Flurbiprofen, S(+), Eyedrops

The shelflife analysis of a stereospecific ballast free flurbiprofen eyedrop formulation was carried out by CZE. To rule out pitfalls as in vitro racemization, analysis was performed of the enantiomeric ratio of flurbiprofen during the shelflife period. The observed decline in flurbiprofen was not supported by observation of degradation products in the electropherograms. This does not rule out any degradation, as only UV-active negatively charged compounds within the chosen run time would be detected. During the investigative period no evidence was found of in vitro inversion. The shelflife determination of S(+) flurbiprofen has shown the solution to be stable in glass containers for 48 months (Ocuflur® 36 months in durable plastic container) when one adheres to the limits of maximum 10% degradation. The concentration limit for the known impurity USP 2-(4-Biphenylyl)propionic acid RS has not been exceeded in any of the investigated samples. The enantiomeric inversion of R(-) flurbiprofen to the pharmacologically active S(+) flurbiprofen as observed in several species (25) has been ruled out in our assay using homogenates of bovine iris/ciliary body (14). In human blood no chiral inversion could be detected (26). Metabolism of flurbiprofen does not occur in ocular tissues as appeared from experiments in rabbits using radioactive labelled material (27).

In the pharmacological assay we investigated the potency of racemic flurbiprofen and the separate enantiomers S(+) and R(-) to inhibit the bovine iris/ciliary body cyclooxygenase-1 enzyme in producing prostaglandin E2 (14). The S(+) moiety dis- -8 played 50% prostaglandin synthesis inhibition at a concentration of 10 M (IC50). Extrapolation of in vivo data on the ocular bioavailability of ophthalmic racemic flur- -8 biprofen shows that our present data on the IC50 (10 M) for inhibition of prostaglandin synthesis by S(+) flurbiprofen fall well within the concentrations of flurbiprofen, available in aqueous humor and shown to be effective in the eye after application to the outer eye (Table 2).

Table 2. Dose/concentration relationships of racemic flurbiprofen in rabbit and human eye.

Flurbiprofen (nmol) Flurbiprofen (M) Instilled dose in aqueous humor

400* (rabbit) 800 x 10-8* -8 10 (ID50)** (rabbit) 20 x 10 *** 50**** (human) 25 x 10-8****

*Values derived from (28), **average value for protein and fluorescein influx, derived from (8), *** extrapolated from (28), **** values derived from (29)

59 Chapter 3

Investigation of the dose-response inhibition curves (8) reveals that racemic flurbiprofen with a concentration of 0.03% produces near maximal effect on the breakdown of the blood-aqueous barrier and this will also pertain to our stereospecific ophthalmic solution of 0.015%. In contrast to our study the flurbiprofen eyedrops used in clinical investigations or marketed specialities are composed of flurbiprofen sodium 0,03%. Flurbiprofen sodium complying to pharmacopoeial standards represents the dihydrate and therefore 0,03% is equivalent to only 0.024% flurbiprofen acid, or 0.012% S(+) flurbiprofen acid.

CONCLUSIONS

Analytical and pharmacological evidence is provided that eyedrops with a mere 0.015% flurbiprofen S(+) should suffice for meeting the indications for ophthalmic use and justifies its use as ballast free stereo specific drug. The shelflife of these eyedrops can be put at four years when supplied in a glass container. For a commercially available eyedrop (i.e. Ocuflur®, durable plastic eye drop container) three years is more appropriate.

Acknowledgements: Preparation of all flurbiprofen samples by Jacqueline Loos-van der Sman is greatly appreciated Stichting Wetenschappelijk Onderzoek Rijnstate (SWOR) is indebted for financial support in acquisition of Flurbiprofen enantiomers.

REFERENCES

1. Waitzman, MB. Possible new concepts relating prostaglandins to various ocular functions. Survey of Ophthalmology 1970;14:301-26. 2. Eakins KE, Whitelocke RAF, Perkins ES, Bennett A, and Unger WG. Release of a prostaglandin in ocular inflammation. Nature 1972;239:248-9. 3. Worst JGF. Biotoxizität des Kammerwassers. Eine vereinheitlichende pathologische Theorie, begründet auf hypothetische biotoxische Kammerwasserfaktoren. Klin Mbl Augenheilk 1975;167:376-84. 4. Lute NP, Vyth A, De Keizer RJW. Indometacine oogdruppels 0,5%. Pharm Weekbl 1980;115:1663-4. 5. Van Nispen tot Pannerden EBLM, Van Sorge AA. Waterige oogdruppels met indometacine in oplossing. Pharm Weekbl 1981;116:386-7. 6. Cox HLM van der Graaf H. Indometacine-oogdruppels als oplossing. Pharm Weekbl 1981;116:387-8. 7. Van Sorge AA, Van Nispen tot Pannerden EBLM, Janssen HWM. Oogdruppels met lage concentratie indometacine: bereidingsvoorschrift en onderzoek naar de werkzaamheid. Pharm Weekbl 1986;121:1039-46. 8. Van Haeringen NJ, Oosterhuis JA, van Delft JL, Glasius E and Noach EL. A comparison of the effects of non-steroidal compounds on the disruption of the blood-aqueous barrier. Exp Eye Res 1982;35:271-7.

60 Flurbiprofen, S(+), Eyedrops

9. Vane JR. Inhibition of prostaglandinsynthesis as a mechanism of action for aspirin-like drugs. Nature 1971;231:232-5. 10. Takeguchi C and Sih CJ. A rapid spectrophotometric assay for prostaglandin synthase: application to the study of non-steroidal anti-inflammatory agents. Prostaglandins 1972;2:169-84. 11. Tilden ME, Boney RS, Goldenberg MM and Rosenbaum JT. The effects of topical S(+]-ibuprofen on interleukin-1 induced ocular inflammation in a rabbit model. J Ocul Pharmacol 1990;6:131-5. 12. Tjebbes GWA, van Delft JL, Barthen ER, van Haeringen NJ. d-Flurbiprofen in ocular inflammation induced by paracentesis of the rabbit eye. Prostaglandins 1990;40:29-33. 13. Stampinato S, Marino A, Bucolo C, Canossa M, Bachetti T, Mangiafico S. Effects of sodium naproxen eyedrops on rabbit ocular inflammation induced by sodium arachidonate. J Ocul. Pharmacol. 1991,7:125-133. 14. Van Sorge AA, van Delft JL, Bodelier VMW, Wijnen PH, van Haeringen NJ. Specificity of flurbiprofen and enantiomers for inhibition of prostaglandin synthesis in bovine iris/cilliary body. Prostaglandins Other Lipid Mediat 1998;55:169-77. 15. Kean WF, Lock CJL, Howard-Lock HE. Chirality in antirheumatic drugs. Lancet 1991;338:1565-8. 16. Van Sorge AA, Essink AWG, van Delft JL and van Haeringen NJ. Pharmacokinetics of flurbiprofen enantiomers in two rabbit species. Ophthalmic Research 1997;29:S1: 033. 17. Altria KD, Walsh AR and Smith NW. Validation of a capillary electrophoresis method for the enantiomeric purity testing of fluparoxan. J Chromatogr. 1993;645:193-6. 18. Nielen MWF. Chiral separation of basic drugs using cyclodextrin-modified capillary zone electrophoresis. Anal. Chem.1993;65:885-93. 19. Guttman A and Cooke N. Practical aspects in chiral separation of pharmaceuticals by capillary electrophoresis. II Quantitative separation of naproxen enantiomers. J Chromatogr A 1994;685:155-9. 20. Altria KD, Goodall DM and Rogan MM. Quantitative applications and validation of the resolution of enantiomers by capillary electrophoresi. Electrophoresis, 1994;15:824-7. 21. Gasper MP, Bethod A, Nair UB and Armstrong DW. Comparison and modeling study of vancomycin, ristocetin A, and teicoplanin for CE enantioseperations. Anal Chem 1996; 68: 2501-14. 22. Ward TJ, Dann III C, Brown AP. Separation of enantiomers using vancomycin in a countercurrent process by suppression of electroosmosis. Chirality 1996;8:77-83. 23. Vespalec R, Billiet HAH, Frank J, Bocek P. Vancomycin as a chiral selector in capillary electrophoresis: an appraisal of advantages and limitations. Electrophoresis 1996;17:1214-21. 24. Rauws AG, Groen K. Current regulatory (draft) guidance on chiral medicinal products: canada, eec, japan, united states. Chirality 1994;6:72-5. 25. Menzel-Soglowek S, Geisslinger G, Beck WS, Brune K. Variability of inversion of R(-) flurbiprofen in different species. J Pharm Sci 1992;81:888-91. 26. Geisslinger G, Menzel-Soglowek S, Schuster O, Brune K. Stereoselective high performance liquid chromatographic determination of flurbiprofen in plasma. J Chrom 1992;573:163-7. 27. Anderson JA, Chen CC, Vita JB, Shackleton M. Disposition of topical flurbiprofen in normal and aphakic rabbit eyes. Arch Ophthalmol 1982;100:642-5. 28. Tang-Liu DD-S, Liu SS, Weinkam RJ. Ocular and systemic bioavailability of ophthalmic flurbiprofen. J Pharmacokin Biopharm 1984;12:611-26. 29. Ellis PP, Pfoff DS, Bloedow DC, Riegel M. Intraocular diclofenac and flurbiprofen concentrations in human aqueous humor following topical application. J Ocular Pharmacol 1994;10:677-82.

61 62 CHAPTER 4

SPECIFITY OF FLURBIPROFEN AND ENANTIOMERS FOR INHIBITION OF PROSTAGLANDIN SYNTHESIS IN BOVINE IRIS/CILIARY BODY

Adriaan A. van Sorge, Jan L. van Delft, Valérie M.W. Carballosa Coré- Bodelier, Peter H. Wijnen, and Nicolaas J. van Haeringen

Prostaglandins Other Lipid Mediat 1998;55:169-77 Chapter 4

ABSTRACT

In the eye prostaglandin production, as stimulated by trauma, is caused by the activity of the constitutive enzyme cyclooxygenase-1 (COX-1), present in the iris/ciliary body. For flurbiprofen, a nonsteroid anti-inflammatory drug (NSAID), used in eyedrops, the specificity of racemic flurbiprofen and the enantiomers S(+) and R(-) flurbiprofen in their inhibitory effect on COX-1 was studied, using bovine iris/ciliary body as source of the enzyme. S(+) is 100 times more potent in COX-1 inhibition than R(-). The measured effect of R(-) is not caused by metabolic inversion of inactive R(-) to active S(+) during the assay.

INTRODUCTION

Prostaglandin-like activity by vasoactive substances, termed "Irin", already was demonstrated in iris tissue of several species by Ambache (1,2). Eakins et al. (3) proved that prostaglandins were released in the aqueous humor in ocular inflammation and Miller, Eakins and Atwal (4) found that acetylsalicylate could prevent the, prostaglandin mediated, disruption of the blood-aqueous barrier after paracentesis of the anterior chamber in the rabbit. Using this paracentesis model Van Haeringen et al. (5) determined concentration-response curves of the clinical useful nonsteroid anti-inflammatory drugs (NSAIDs) indomethacin, diclofenac and flurbiprofen in eyedrops on the influx of protein and of fluorescein into the anterior chamber of rabbits. Various NSAIDs inhibit the cyclooxygenase (COX) activity of the enzyme prostaglandin G/H synthase (PGHS, E.C. 1:14.99.1), the rate limiting enzyme in the production of pro-inflammatory prostaglandins. COX exists in at least two isoforms: COX-1, the constitutive form, and COX-2 the inducible form. COX-1 was firstly characterized in sheep vesicular glands, is present in platelets, kidney, stomach and vascular smooth muscle (6,7) and in the iris/ciliary body (8). COX-2, is induced in macrophages and other migratory cells, when these are exposed to proinflammatory agents (7,9); however COX-1 also appears to be involved in prostaglandin synthesis in both control and stimulated rat macrophages (10).

Both COX-1 and COX-2 isoforms are expressed under physiological conditions in rat (11) and human (12) tissues and constitutive fetal prostaglandin synthesis is probably mediated by COX-1, whereas COX-2 is upregulated during labour in fetal membranes (13). Although COX-1 is recognized to mediate "housekeeping" functions as in the stomach and kidney, from studies using "knockout" mice, that is mice with no gene function for COX-1 or COX-2, appeared that in COX-2 deficient mice COX-1 could contribute to normal inflammatory responses (14) and

64 Bovine, iris/ciliary body

that COX-1 deficient mice have a lack of gastric ulceration (15), possibly due to as yet unknown compensatory mechanisms. The concept of the therapeutic use of NSAIDs as antiinflammatory drugs in general is based on the ability of NSAIDs to inhibit the COX-2 activity, whereas inhibition of COX-1 may explain the undesired side effects such as gastric and renal toxicity and bleeding disorders. However the inhibition of COX-1 activity even is a desired effect in the prevention of heart attacks and strokes by aspirin (16), based on the reduction of thromboxane production in platelets and in the prevention of ocular irritation during cataract extraction (17) and after therapeutic laser treatment of the anterior eye (18,19). Flurbiprofen is a racemic mixture of two isoforms: S(+) and R(-) flurbiprofen. The availability of the pure enantiomers of flurbiprofen allows the investigation of the anti-inflammatory effects of the optical isomers of this NSAID. Therefore, also with respect to the demands for ballast free stereo specific drugs, we embarked to study the effect of the enantiomers S(+) and R(-) flurbiprofen in comparison with racemic flurbiprofen on the prostaglandin E2 (PGE2) activity of bovine iris.

MATERIALS AND METHODS

Flurbiprofen and enantiomers were obtained from Duchefa Pharma (Haarlem, The Netherlands), Fresh bovine eyes were obtained in a slaughterhouse and within two hours post mortem the iris/ciliary body was removed from the eye with tweezers after resection of the cornea. The iris/ciliary body was homogenized in Tris HCl 0.05 M, pH 7.4, containing 1 mM phenylmethylsulfonylfluoride (PMSF), in a ratio of 1 iris/ciliary body per mL buffer solution, using a Potter-Elvehjem glass in glass homogeniser in melting ice. µ µ For determination of PGE2 100 l of homogenate was incubated with 25 l of phosphate buffered saline (PBS) in an 1-ml eppendorf tube at 37°C during 60 min. The effect of flurbiprofen was studied by addition of 25 µL of a solution of appropriate concentrations, prepared in PBS, reaching final concentrations of 10-4 M to 10-10M. The enzyme reaction was stopped by heating for 3 min in boiling water. Blanks were prepared by heating the tubes in boiling water, without incubation at

37°C. In the supernatant of centrifugation (30 min at 16,000 g) released PGE2 was determined using a commercially available kit for PGE2 enzyme immune analysis (Cayman Chemical Co., Ann Arbor MI, U.S.A.).

Inhibition of PGE2 release was calculated by the difference of PGE2 release in the absence and the presence of flurbiprofen expressed in percent of the non-inhibited release. In concentration-response curves the concentrations of flurbiprofen are presented as final concentrations in the test. For racemic flurbiprofen the concentrations are given in concentrations of the individual enantiomers.

65 Chapter 4

For the determination of chiral inversion by iris/ciliary body homogenate, 1 mL of homogenate was incubated with 250 µL of 10-4M R(-) or S(+) flurbiprofen in PBS at 37°C during 60 min. For the isolation of flurbiprofen enantiomers the procedure was followed as described by Geisslinger et al (20). The incubate was acidified by adding 0.1 mL of 4M hydrochloric acid, followed by extraction into 6.00 mL of ice- cooled hexane-diethylether (8:2, v/v). After centrifugation (5 min. at 1500 g), 5.00 mL of the organic layer were removed and evaporated to dryness under a gentle stream of dry nitrogen. The residue was dissolved in 1 mL PBS, acidified with phosphoric acid and extracted again with 1 mL heptane. The residue after evaporation of the organic layer was dissolved in 75 µL of buffer for capillary zone electrophoresis (CZE) and an aliquot was injected into the capillary to determine the relative concentrations of R(-) and S(+) flurbiprofen. For the analysis an Applied Biosystems (San Jose, CA, U.S.A) Model 270A-HT CZE system was used, equipped with a variable-wavelength UV absorbance detector, operated at 254 nm and a 0.5 s rise time. CZE was performed in a 70-cm x 50- µm-i.d. fused silica capillary (Polymicro Technologies, Phoenix, AZ, U.S.A.) having a length of 50-cm to the detection window. The electrophoresis buffer was prepared by

adjusting a 50 mM KH2PO4 solution to pH=6.0 with a solution of 50 mM Na2HPO4. The macrocyclic antibiotic Vancomycin was used as chiral selector (21,22). The selector was added to the inlet buffer only, at a concentration of 0.6 mM. The separations were carried out at +15 kV, with the oven temperature set at 30°C. Samples were introduced into the capillary via a controlled vacuum injection of 1 or 2 s corresponding to a volume of approximately 4-8 nL. Sample injections were followed by a 0.5-s dip in water in order to wash the electrode and the outside of the fused silica capillary. Data were recorded using a Fisons Model VG- Multichrom system. The ratio of the enantiomers, which are each others internal standard, were calculated as the relative peak areas (corrected by migration time).

RESULTS

The concentrations of PGE2 after non-inhibited release from iris/ciliary body homogenate varied from 10-20 ng/mL of assay mixture. The pharmacodynamic effects of racemic flurbiprofen, S(+) and R(-) flurbiprofen are presented in figure 1.

S(+) flurbiprofen (IC50 = 8.0 (-log M)) is equipotent in comparison to racemic flur-

biprofen (IC50 = 8.0) but is 100 times more potent than R(-) flurbiprofen (IC50 = 6.0).

In figure 2, the separation of racemic flurbiprofen is presented, showing a baseline resolution with an analysis time of 22 min. In the measurements of possible metabolic inversion of the flurbiprofen enantiomers, the optical impurity measured in the R(-) standard for S(+) was 0.7 ± 0.1% (mean ± SEM)(n=3).

66 Bovine, iris/ciliary body

Figure 1. Concentration-response curves for inhibition of the COX-1 activity of bovine iris/ciliary body racemic flurbiprofen (∇), R(-) flurbiprofen () and S(+) flurbiprofen (). Concentrations of flurbiprofen are final concentrations in the assay mixture. Each point represents the average ± S.E.M. of four to six determinations from different pools of bovine iris/ciliary body homogenates.

Figure 2. Analysis of racemic flurbiprofen by capillary zone electrophoresis, showing separation in R(-) and S(+) flurbiprofen.

67 Chapter 4

Figure 3. Electropherograms of extracted bovine iris/ciliary body homogenate after incubation for 60 min with (A) R(-) flurbiprofen 10-4 M and (B) S(+) flurbiprofen 10-4 M, showing no increase of optical antipode after incubation. Controls of standard solution of R(-) flurbiprofen already contain an impurity of 0.7% of the S(+) enantiomer.

For the S(+) standard the optical impurity of R(-) was below the detection limit of the assay (approximately 0.1%). The precision and the reproducibility of the enantiomer ratios are merely determined by integration errors (influence of noise) for the minor isomer. The measured optical enantiomer concentration of S(+) in the R(-) containing fraction isolated from the incubate was 0.69 ± 0.07% (n=6) (figure 3A) and the concentration of R(-) in the S(+) containing fraction was below the detection limit of 0.1% (n=3) (figure 3B).

DISCUSSION

The enzyme responsible for the prostaglandin production by the porcine iris/ciliary body has been characterized as COX-1, by an immunocytochemical method, using sections of tissue which were incubated with rabbit anti COX-1 polyclonal antibody and treated with goat anti-rabbit IgG, conjugated with 15-nm gold particles (8). The same authors also demonstrated that ω/ω-1 hydroxylase activity in porcine ciliary body can inactivate accumulated prostaglandins (23). In our test we obviously

measure the net result of production of PGE2 by COX-1 from arachidonate mobilized from endogenous lipids and of inactivation by ω/ω-1 hydroxylase. The pharmacodynamic profiles of inhibition by S(+) flurbiprofen and racemic flur-

biprofen of PGE2 synthesis by the iris/ciliary body are rather similar, but the curve

68 Bovine, iris/ciliary body

for R(-) flurbiprofen shows a shift of about 2 log units to the right, which means that the R(-) enantiomer possesses about 1% of the potency of the S(+) enantiomer. Kulmacz and Lands (24) found R(-) flurbiprofen not inhibitory on COX-1, purified from sheep seminal vesicles, at concentrations where S(+) flurbiprofen inhibited the enzyme for 94%. These distinctions between the two enantiomers of flurbiprofen follow the pattern observed with many other enantiomeric pairs of NSAIDs that the (+) isomer is more potent in COX-1 inhibitory action. With human recombinant PGHS-1 activity from human monocytes the inhibitory action of the (-) enantiomer of pemodolac was about 0.07% of that of the (+) enantiomer and the (-) enantiomer of etodolac was essentially inactive at concentrations up to 10-4 M (25). Also on PGHS-1 purified from sheep seminal vesicle (-) etodolac was inactive up to 3X10-4 M (26) and (-) ibuprofen was inactive up to 20 times the IC50 of the (+) enantiomer on COX-1 activity of blood platelets (27). Metabolic chiral inversion of the R(-) flurbiprofen into the S(+) enantiomer and the variation between different species has been reported, however no reports are available on the chiral inversion in bovine tissue (28). In human plasma no chiral inversion could be detected (20). We could not demonstrate any chiral inversion of the enantiomers by the bovine iris/ciliary body under the experimental conditions, using a final concentration of 10-4 M of the flurbiprofen enantiomers. Therefore the observed 1% relative inhibitory activity of the R(-) enantiomer cannot be explained by inversion to the S(+) enantiomer during the test, but must be ascribed to intrinsic inhibitory action of the R(-) enantiomer or to the small amount of S(+) flurbiprofen, present as impurity (0.7%) in the R(-) enantiomer (29). In the resolution methods used in the pharmaceutical industry to separate enantiomers from a racemate it is very difficult to remove completely the chiral impurities (30). Our results suggest that S(+) flurbiprofen might be the therapeutically relevant anti-inflammatory agent in eyedrops intended for use against conditions initially caused by activity of COX-1, including intraoperative miosis and postoperative ocular inflammation after surgery or laser treatment. R(-) flurbiprofen can be designated as unnecessary ballast with even no indication of a pro-drug property, which could be based on inversion of the less active (-) enantiomer into the far more active (+) enantiomer, as observed in other species as man.

69 Chapter 4

REFERENCES

1. Ambache, N. Properties of irin, a physiological constituent of the rabbit's iris. J. Physiol. 135:114- 132, 1957. 2. Ambache, N. Further studies on the preparation, purification and nature of irin. J. Physiol. 146:255-294, 1959. 3. Eakins, K.E., Whitelocke, R.A.F., Perkins, E.S., et al. Release of prostaglandins in ocular inflammation in the rabbit. Nature New Biol. 239:248-249, 1972. 4. Miller, J.D., Eakins,K,E. and Atwal, M. The release of PGE2-like activity into the aqueous humor after paracentesis and its prevention by aspirin. Inv. Ophthalmol. Vis. Sci. 12:939-942, 1973. 5. van Haeringen, N.J., Oosterhuis J.A., van Delft, J.L., Glasius E. and Noach, E.L. A comparison of the effects of non-steroidal compounds on the disruption of the blood-aqueous barrier. Exp. Eye Res. 35:271-277, 1982. 6. DeWitt, D.L., Meade E.A., Zeilhofer H.U. PGH Synthase isoenzyme selectivity: the potential for safer nonsteroidal antiinflammatory drugs. Am. J. Med. 95:40S-46S, 1993. 7. Mitchell, J.A., Akarasereenont, P., Thiemermann, C., Flower, R.J., Vane, J.R. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc. Natl. Acad. Sci. USA 90:11693-11697, 1994. 8. Asakura, T., Sano, N., Stichi, H. Prostaglandin synthesis and accumulation by porcine ciliary epithelium. J. Ocular Pharmacol. 8:333-341, 1992. 9. Xie, W., Robertson, D.L., Simmons DL. Mitogen-inducible prostaglandin G/H synthase: A new target for nonsteroidal anti-inflammatory drugs. Drug Dev. Res. 25:249-265, 1992. 10. Wilborn, J., De Witt, D.L., Peters-Golden, M. Expression and role of cyclooxygenase isoforms in alveolar and peritoneal macrophages. Am. J. Physiol. 268:L294-301, 1995. 11. Feng, L., Sun, W., Xia, Y., Tang, W.W., Chanmugam, P., Soyoola, E., Wilson, C.B., Hwang, D. Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch. Biochem. Biophys. 307:361-368, 1993. 12. O'Neill, G.P., Ford-Hutchinson, A.W. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett. 330:156-160, 1993. 13. Slater, D.M., Berger, L., Newton, R., Moore, G.E., Bennett, P.R. Expression of cyclooxygenase types1 and 2 in human fetal membranes at term. Am. J. Obstet. Gynecol. 172:77-82, 1995. 14. Morham, S.G., Langenbach R., Loftin, C.D., Tiano, H.F., Vouloumanos, N., Jennette, J.C., Mahler, J.L., Kluckman, K.D., Lee, C.A., Smithies, O. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 83:473-482, 1995. 15. Langenbach, R., Morham, S.G.,Tiano,H.F., Loftin,C.D.,Ghanayem,B.I.,Chulada,P.C., Mahler J.F., Lee,C.A., Goulding, E.H., Kluckman, K.D., Kim,H.S., Smithies, O. Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin- induced gastric ulceration. Cell 83:483-492, 1995. 16. Smith, J.B.., Willis, A.L. Aspirin selectively inhibits prostaglandin production in human platelets. Nature New Biol. 231:235-237, 1971. 17. Flach, A.J. Cyclo-oxygenase inhibitors in ophthalmology. Therapeutic Review. Survey Ophthalmol. 36:259-284, 1992. 18. Hotchkiss, M.L., Robin, A.L., Pollack, I.P., Quigley, H.A. Nonsteroidal anti-inflammatory agents after argon laser trabeculoplasty. A trial with flurbiprofen and indomethacin. Ophthalmology 91:969-976, 1984. 19. Pillunat, L.E., Wagner, P., Stodtmeister, R. Comparison of topically applied prostaglandin synthesis inhibitors in Nd:Yag-laser surgery. Preliminary results. Fortschr. Ophthalmol. 84:583-586, 1987. 20. Geisslinger, G., Menzel-Soglowek, S., Schuster, O., Brune, K. Stereoselective high-performance liquid chromatographic determination of flurbiprofen in plasma. J.Chrom. 573: 163-167, 1992.

70 Bovine, iris/ciliary body

21. Gasper, M.P., Berthod, A., Nair, U.B., Armstrong, D.W. Comparison and modelling study of vancomycin, ristocetin A, and teicoplanin for CE enantioseparations. Anal. Chem. 68: 2501-2514, 1996. 22. Vespalec, R., Billiet, H.A.H., Frank, J., Bocek, P. Vancomycin as a chiral selector in capillary electrophoresis: an appraisal of advantages and limitations. Electrophoresis 17:1214-21, 1996. 23. Asakura, T., Shichi, H. Cytochrome P450-mediated prostaglandin ω/ω-1 hydroxylase activities in porcine ciliary epithelial cells. Exp. Eye Res. 55:377-84, 1992. 24. Kulmacz, R.J., Lands, W.E.M. Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents. J. Biol. Chem. 260: 12572-8, 1985. 25. Glaser, K., Sung, M., O'Neill, K., Belfast, M., Hartman, D., Carlson, R., Kreft, A., Kubrak, D., Hsiao, C.-L., Weichman, B. Etodolac selectively inhibits human prostaglandin G/H synthase 2 (PGHS-2) versus human PGHS-1. Eur. J. Pharmacol.281:107-11, 1995. 26. Markey, C.M., Alward, A., Weller, P.E., Marnett, L.J. Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. J. Biol. Chem. 262: 6266-79, 1987. 27. Evans, A.M., Nation, R.L., Sansom L.N., Bochner, F., Somogyi, A.A.: Effect of racemic ibuprofen dose on the magnitude and duration of platelet cyclo-oxygenase inhibition: relationship between inhibition of thromboxane production and the plasma unbound concentration of S(+) ibuprofen. Br. J. Clin. Pharmacol. 31:131-8, 1991. 28. Menzel-Soglowek, S., Geisslinger, G., Beck,W.S., Brune, K. Variability of inversion of (R) flurbiprofen in different species. J. Pharm. Sci. 81:888-91, 1992. 29. Carabaza, A., Cabre, F., Rotlan, E., Gomez, M., Gutierrez, M., Garcia, M.L., Mauleon, D. Stereoslective inhibition of inducible cyclooxygenase by chiral nonsteroidal antiinflammatory drugs. J. Clin. Pharmacol. 36:505-12, 1996. 30. Li,Z. J. And Grant, D.J.W. Relationship between physical properties and crystal structures of chiral drugs. J. Pharm. Sci. 86:1073-8, 1997.

71 72 CHAPTER 5

FLURBIPROFEN AND ENTIOMERS IN OPHTHALMIC SOLUTION TESTED AS INHIBITORS OF PROSTANOID SYNTHESIS IN HUMAN BLOOD

Nicolaas J. van Haeringen, Adriaan A. van Sorge, Jan L. van Delft, and Valérie M.W. Carballosa Coré-Bodelier

J Ocular Pharmacol 2000;16:345-52 Chapter 5

ABSTRACT

The purpose of this study was to assess the selectivity and potency of the nonsteroidal anti-inflammatory drug (NSAID), flurbiprofen, and its enantiomers in their inhibition of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). An assay was used with freshly drawn heparinized human whole blood, incubated with 25

mM calcium ionophore A 23187 during 60 min to produce thromboxane B2 (TXB2) by activity of COX-1 in platelets. Incubation with E.Coli lipopolysaccharide (LPS)

during 24 hours produced prostaglandin E2 (PGE2) by induction of COX-2 in monocytes, suppressing any possible contribution of COX-1 activity by addition of acetylsalicylic acid. Concentration inhibition curves were determined with racemic, S(+), and R(-) flurbiprofen in final concentrations ranging from 10-3 to 10-10 M. The stereoselectivity of S(+) flurbiprofen vs. R(-) flurbiprofen, expressed as the reciprocal of

the ratio of the concentrations giving 50% inhibition (IC50), is 340 for COX-1 and 56 for COX-2. The selectivity for COX-1 vs. COX-2, expressed as the reciprocal ratio

of the IC50, was 16 for racemic, 32 for S(+), and 5.3 for R(-) flurbiprofen. Meloxicam in the same assay showed COX-2 selectivity with a ratio of 0.19.

INTRODUCTION

Cyclooxygenase (COX; prostaglandin-endoperoxide synthase, E.C.1:14.99.1), the rate limiting enzyme in the production of proinflammatory prostaglandins, exists in two isoforms: COX-1, the constitutive form, and COX-2 the inducible form. COX-1 has clear physiological functions such as in the protection of the stomach, kidney, and vessel walls, whereas COX-2 is induced by inflammatory stimuli and by cytokines in migratory and other cells, leading to inflammatory conditions. The therapeutic use of non-steroidal anti-inflammatory drugs (NSAIDs), in general, is based on their ability to inhibit the COX-2 activity, while inhibition of COX-1 might explain the undesired side effects such as gastric and renal toxicity and hematological disorders. On the basis of their inhibitory activity on COX-1 and COX-2 at least three major aspects of NSAIDs are distinguished: 1) preferential COX-1 inhibition, 2) preferential COX-2 inhibition, and 3) nonpreferential inhibition. Selectivities, varying between COX-1 preferential (1-3) and nonpreferential (1,4,5), have been reported for flurbiprofen, possibly because of methodological variations .The assay systems to investigate the potency and selectivity of NSAIDs include intact cells, broken cells, purified enzymes and microsomal preparations of recombinantly expressed enzymes. Some of the reasons for variation have been identified such as the use of animal or human cells, microsomal preparations, incubation time of the assay or protein binding of the NSAID.

74 Human blood

Whole blood, using COX-1 in platelets and induced COX-2 in monocytes represents an in vitro system as close to physiological conditions as possible. It has been shown to be a satisfactory system for testing the inhibitory action of NSAIDs on COX-1 and COX-2 activity (3,6-11) accounting for differences in plasma protein binding (12) and cellular accumulation. Platelets are known to contain COX-1 and the concentration of the enzyme remains largely stable, but two- to four-fold increases can occur in response to stimulation by hormones or growth factors (13,14). COX-2, however, cannot be brought to expression in platelets in response to lipopolysaccharide (LPS) (15). Therefore, platelets stimulated by calcium ionophore represent a useful assay system for COX-1 activity, measuring thromboxane production as the most distinguished prostanoid. Normal peripheral blood monocytes express COX-1 but can be induced to express COX-2 by LPS to 10 - 20-fold after 6-24 hours (15,16). This represents a useful assay system for COX-2 activity, measuring PGE2 or thromboxane as metabolite, when suppressing any possible contribution of COX-1 activity by addition of aspirin (acetylsalicylic acid). Aspirin acetylates platelet COX-1, thereby causing irreversible loss of its cyclooxygenase activity (17). The inactivation of COX-1 is complete by 20 min (18) and excess of aspirin itself is hydrolyzed in blood with a half-life time of 30 min (10) before expression of COX-2 by LPS is fully developed. The NSAID flurbiprofen, as used in eyedrops, is on the one hand effective against inflammatory responses of the eye (19-21) caused by activity of the COX-1 of the iris (22); on the other hand in systemic use, it is effective in arthritis (23) caused by induction of COX-2 (24,25). Flurbiprofen is a racemic mixture of S(+) and R(-) enantiomers, and its effect is largely attributed to the inhibitory action of the S(+) enantiomer, reportedly being about 100 - 10,000 times more effective than the R(-) enantiomer when measured on COX-1 or COX-2 (10,26-28). There are sufficient differences between the enzymes from different species, such that selectivity and potency established with animal enzymes is not always predictive of effects on human enzymes and no data are available on the potency or selectivity of the flurbiprofen enantiomers on COX- 1 and COX-2 in human blood. In this study, we report dose effect inhibition curves for racemic, S(+) and R(-) flurbiprofen using the human whole blood assay. In comparison the preferential COX-2 inhibitor, meloxicam, was also investigated in respect of its enzyme selectivity.

MATERIALS AND METHODS

Materials Flurbiprofen and the enantiomers were obtained from Duchefa Pharma bv, Haarlem, The Netherlands. The enantiomeric impurity of S(+) flurbiprofen was

75 Chapter 5

<0.1% and of R(-) flurbiprofen 0.7% (29). Stock solutions of flurbiprofen were prepared in a phosphate buffer pH 7.4 with a concentration of 0.03% for racemic flurbiprofen and 0.015% for the enantiomers (for details see 29). Meloxicam was a gift from Boehringer Ingelheim, Germany. Calcium ionophore A 23187, dimethylsulfox- ide (DMSO) and LPS, derived from E.Coli 0.111;B4, were obtained from Sigma (St

Louis, MO, USA). Assay kits for enzyme immune analysis of Thromboxane B2

(TXB2) and of prostaglandin E2 (PGE2) were purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.).

Measurement of COX-1 Activity For the determination of COX-1 activity 900 µl of freshly drawn heparinized human blood was incubated at 37°C with 2 µl of calcium ionophore A23187 (12.5 mM in DMSO) and 100 µl of phosphate buffered saline (PBS) for 30 min. Controls were prepared by incubation of 900 µl of blood with 2 µl of DMSO and 100 µl of PBS. The reaction was terminated by chilling quickly on ice. Plasma was separated by

centrifuging, stored at -20°C, and TXB2 levels were determined.

Measurement of COX-2 Activity For the determination of COX-2 activity, 900 µl of heparinized human blood was incubated at 37°C with 10 µl of acetylsalicylic acid (1 mg/ml in PBS), 2 µl of LPS (E.Coli 0111:B4, 5 mg/ml in DMSO), and 100 µl PBS for 24 hr. Controls were prepared by incubation of 900 µl of blood with 10 µl of acetylsalicylic acid, 2 µl of DMSO, and 100 µl PBS. The reaction was terminated by quickly chilling on ice.

Plasma was separated by centrifuging, stored at -20°C, and PGE2 levels were determined.

Enzyme Inhibition by Flurbiprofen and by Meloxicam The effect of flurbiprofen was studied by substitution of 100 µl of PBS in the assays, by 100 µl of an appropriate dilution in PBS of the ophthalmic solution, reaching final concentrations of 10-3 to 10-10 M. Meloxicam was studied using appropriate solu-

tions in PBS. Inhibition of enzyme activity was calculated by the difference of TXB2-

or PGE2 - release in the absence and the presence of flurbiprofen or meloxicam, expressed in percent of the noninhibited release. From concentration-response inhibition curves the concentration giving 50% inhibition was extrapolated and pre-

sented as IC50 .

RESULTS

Addition of calcium ionophore A23187 to human blood resulted in the generation of

large amounts of TXB2 in the plasma (128±16 ng/ml/30 min) caused by COX-1

76 Human blood

activity of the platelets. Non-stimulated controls, to which only DMSO was added, produced 5 ± 0.6 ng TXB2/ml/30 min. The production of PGE2 by LPS-challenged whole blood, caused by induction of COX-2, amounted 124 ± 19 ng/ml/24hr. Nonstimulated controls, to which only DMSO and aspirin was added, contained small amounts of PGE2 (1.3 ± 0.5 ng/ml/24hr).

Figure 1. Concentration-response curves for inhibition of the COX-1 activity of whole human blood for racemic flurbiprofen (∇), S(+) flurbiprofen () and R(-) flurbiprofen (). Concen- trations are final concentrations in the assay blood mixture. Each point represents the average ± SEM of four to six determinations from blood of different individuals.

Figure 2. Concentration-response curves for inhibition of the COX-2 activity of whole human blood for racemic flurbiprofen (∇), S(+) flurbiprofen () and R(-) flurbiprofen (). Concen- trations are final concentrations in the assay blood mixture. Each point represents the average ± SEM of four to six determinations from blood of different individuals.

77 Chapter 5

Dose response inhibition curves of racemic flurbiprofen and its S(+) and R(-) enantiomers on the activity of COX-1 and COX-2 isoenzymes in the whole blood model are presented in figures 1 and 2. Results of meloxicam in the same assay are pre-

sented in figure 3. In table 1 the IC50 values are presented together with the enzyme selectivity, expressed as the COX-2/COX-1 ratio, for racemic flurbiprofen, its R(-) and S(+) enantiomers and for meloxicam.

Figure 3. Concentration-response curves for inhibition of COX-1 ( ) and COX-2 () activity of whole human blood for meloxicam. Concentrations are final concentrations in the assay blood mixture. Each point represents the average ± SEM of four to six determinations from blood of different individuals.

Table 1. Selectivities of NSAIDs for COX-1 and COX-2 in Human Blood.

COX-1 COX-2 COX-2/COX-1 µ µ IC50 M IC50 M racemic flurbiprofen 0.14 ± 0.04 2.2 ± 0.2 16 ± 5 S(+) flurbiprofen 0.056 ± 0.030 1.8 ± 0.4 32 ± 24 R(-) flurbiprofen 19 ± 6 100 ± 32 5.3 ± 3.4 meloxicam 3.0 ± 1.1 0.56 ± 0.26 0.19 ± 0.16

IC50 are mean values derived from the concentration response inhibition curves.

78 Human blood

DISCUSSION

This is the first report to establish in the human whole blood assay the stereoselective inhibition of COX-1 and COX-2. The assays used were adapted from (3,8) and several authors of different research groups reported their results using this method, testing many NSAIDs, including racemic flurbiprofen (3,30) and meloxicam (31). The whole blood system is very useful in that in vitro it may reflect better the in vivo effectiveness of NSAIDs with contributing factors as protein-binding. The prostanoid production in this assay is measured under conditions in which arachidonic acid is generated from endogenous lipid pools rather than added in artificial- ly high (10 µM) exogenous concentrations as in other assays (28). S(+) flurbiprofen, as in general for racemic mixtures of NSAIDs, is the more potent inhibitor of both COX isoenzymes than the R(-) isomer (9,10,32,33). The enantioselectivity of S(+) flurbiprofen, expressed as the ratio of the IC50 of the R(-) and the S(+) isomer (R/S), amounts to 340 for COX-1 and 56 for COX-2. The difference in selectivity found in this study may be explained by a possible greater steric hindrance of the binding of R(-) flurbiprofen to COX-1 than to the COX-2 enzyme. The accessibility of the binding site for flurbiprofen, competing with arachidonate binding at the catalytic domain of the COX-1 structure, has been found to be more restricted than in the COX-2 structure (34-36). On the other hand the inhibitory effect measured with the R(-) isomer has been ascribed to the small amount of S(+) present as impurity in the ineffective R(-) isomer (13) Theoretically the 0.7% impurity should give a R/S ratio of about 140 and a smaller ratio might be due to metabolic inversion of the R(-) into the S(+) isomer. Metabolic inversion, however, has been demonstrated to be absent in human blood (37). Within the errors of the methods used, the inhibition curves of the various concentrations of flurbiprofen racemate and the S(+) isomer were found to run closely parallel, reflecting the two-fold (0.3 log-unit) greater concentration of the pure S(+) isomer as compared to the racemate (figures 1 and 2). These results correspond well with data obtained from eicosanoid production in ex vivo experiments in rats (26). We confirmed the selectivity of flurbiprofen towards COX-1 as found by Young et al. (3), who also used the human blood assay. The ratio COX-2/COX-1 was 16 for racemic flurbiprofen, although the IC50 values for both enzymes were about 4 times lower in the present study. The ratio COX-2/COX-1 was 32 for S(+) flurbiprofen and 5.3 for R(-) flurbiprofen, which is higher than found with guinea pig whole blood, where for S(+) flurbiprofen was found 1.0 and for R(-) flurbiprofen 0.48 (10). Genetic differences of human and guinea pig COX-2 are most likely responsible for these differences, as has also been suggested for human and murine COX-2 (38). Meloxicam has been found in a number of other assay systems to be selective towards COX-2 (6,7,39,40). Figure 3 shows that meloxicam inhibits COX-2 in

79 Chapter 5

human whole blood at concentrations that are at least 5 times lower than those -6 required to inhibit COX-1. It was also observed that meloxicam (IC50 0.56 x 10 M) -6 displayed more potency towards COX-2 than S(+) flurbiprofen (IC50 1.8 x 10 M).

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15. Hla, T., and Neilson, K. Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA 89:7384- 7388, 1992. 16. Hla, T., Ristimäki, A., Appleby, S., and Barriocanal, J.G. Cyclooxygenase gene expression in inflammation and angiogenesis. Ann. NY. Acad. Sci. 696:197-204, 1993. 17. Roth, G.J., Stanford, N., and Majerus, P.W. Acetylation of prostaglandin synthase by aspirin. Proc. Natl. Acad. Sci. USA 72:3073-3076, 1975. 18. Meade, E.A., Smith, W.L., and DeWitt, D.L. Differential inhibition of prostaglandin peroxide synthase (cyclooxygenase) isoenzymes by aspirin and other nonsteroidal anti-inflammatory drugs. J. Biol. Chem. 268:6610-6614, 1993. 19. Flach, A.J. Cyclo-oxygenase inhibitors in ophthalmology. Therapeutic Review. Surv. Ophthalmol. 36:259-284, 1992. 20. Hotchkiss, M.L., Robin, A.L., Pollack, I.P., and Quigley, H.A. Nonsteroidal anti-inflammatory agents after argon laser trabeculoplasty. A trial with flurbiprofen and indomethacin. Ophthalmology 91:969-976, 1984. 21. Pillunat, L.E., Wagner, P., and Stodtmeister, R. Comparison of topically applied prostaglandin synthesis inhibitors in Nd:Yag-laser surgery. Preliminary results. Fortschr. Ophthalmol. 84:583- 586, 1987. 22. Asakura, T., Sano, N., and Stichi, H., Prostaglandin synthesis and accumulation by porcine ciliary epithelium. J. Ocul. Pharmacol. 8:333-341, 1992. 23. Benvenuti, C., Guidoni, G., Longoni, A., and Mordini, M. Controlled study on flurbiprofen and diclofenac in the treatment of rheumatic disorders. Int. J. Tissue React. 5:61-65, 1983. 24. Xie, W., Robertson, D.L., and Simmons, D.L. Mitogen-inducible prostaglandin G/H synthase: a new target for nonsteroidal anti-inflammatory drugs. Drug Dev. Res. 25:249-265, 1992. 25. Brune, K., Menzel-Soglowek, S., and Zeilhofer, H.U. Differential analgesic effects of aspirin-like drugs. Drugs 44(Suppl. 5): 52-59, 1992. 26. Peskar, B.M., Kluge, S., Peskar, B.A., Soglowek, S.M., and Brune, K. Effects of pure enantiomers of flurbiprofen in comparison to racemic flurbiprofen on eicosanoid release from various rat organs ex vivo. Prostaglandins 42:515-531, 1991. 27. Van Sorge, A.A., van Delft, J.L., Bodelier, V.M.W., Wijnen, P.H., and van Haeringen, N.J., Specificity of flurbiprofen and enantiomers inhibition of prostaglandin synthesis in bovine iris/ciliary body. Prostaglandins OtherLip.Mediat. 55:169-177, 1998. 28. Laneuville, O., Breuer, D.K., Dewitt, D.L., Hla, T., Funk, C.D., and Smith,W.L. Differential inhibition of human prostaglandin endoperoxide H synthases-1 and -2 by nonsteroidal anti-inflammatory drugs. J. Pharmacol. Exp. Ther. 271:927-934, 1994. 29. Van Sorge, A.A., Wijnen, P.H., Van Delft, J.L., Carballosa Coré-Bodelier, V.M.W., and Van Haeringen, N.J. Flurbiprofen, S(+), eyedrops; formulation, enantiomeric assay, shelf-life and pharmacology. Pharm. World Sci. 21:91-95, 1999. 30. Santini, G., Sciulli, M.G., Panara, M.R., Padovano,R., Giamberardino, M., Rotondo, M.T., Del Soldato, P., and Patrignani, P. Effects of flurbiprofen and flurbinitroxybutylester on prostaglandin endoperoxide synthases. Eur. J. Pharmacol. 316: 65-72, 1996. 31. Patrignani, P., Panara, M.R., Sciulli, M.G., Santini, G., Renda, R., and Patrono, C. Differential inhibition of human prostaglandin endoperoxide synthase-1 and -2 by nonsteroidal anti-inflammatory drugs. J. Physiol Pharmacol. 48:4623-4663, 1997. 32. Kulmacs, R.J., and Lands, W.E.M. Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents. J Biol Chem. 260:12572-12578, 1985. 33. Evans, A.M., Nation,R.L., Sansom, L.N., Bochner, F., and Somogyi, A.A. Effect of racemic ibuprofen dose on the magnitude and duration of platelet cyclooxygenase inhibition: Relationship between inhibition of thromboxane production and the plasma unbound concentration of S(+)- ibuprofen. Br. J. Clin. Pharmacol. 31:131-138, 1991.

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34. Picot, D., Loll, P.J., and Garavito, R.M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature 367:243-249, 1994. 35. Luong, C., Miller, A., Barnett, J., Chow, J., Ramesha, C., and Browner, M.F. Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2. Nature Struct. Biol. 3:927-933, 1996. 36. Hawkey, C. J. COX-2 Inhibitors. Lancet 353:307-314, 1999. 37. Geisslinger, G., Menzel-Soglowek, S., Schuster, O., and Brune, K. Stereoselective high-performance liquid chromatographic determination of flurbiprofen in human plasma. J. Chromatogr. 573:163-167, 1992. 38. Berg, J., Fellier, H., Hartmann, M., Kremminger, P., Blaschke, H., Christoph,T., Bodenteich, A., Rovensky, F., Stimmeder, D., and Towart, R. Novel selective cyclooxygenase-(COX)-2 inhibitors of the diarylethersulfonamide type show greater inhibition on human COX-2 than on murine COX-2. Naunyn Schmiedeberg Arch. Pharmacol. 358(Suppl. 2): R716, 1998. 39. Frölich, J.C. A classification of NSAIDs according to the relative inhibition of cyclooxygenase isoenzymes. TIPS 18:30-34, 1997. 40. Engelhardt, G., Bögel, R., Schnitzler, C., and Utzmann, R. Meloxicam: influence on arachidonic acid metabolism. In vitro findings-part 1. Biochem Pharmacol 51:21-28, 1996.

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CONSTITUTIVE CYCLOOXYGENASE-1 AND INDUCED CYCLOOXYGENASE-2 IN ISOLATED HUMAN IRIS INHIBITED BY S(+) FLURBIPROFEN

Nicolaas J. van Haeringen, Adriaan A. van Sorge, and Valérie M.W. Carballosa Coré-Bodelier

J Ocular Pharmacol 2000;16:535-61 Chapter 6

ABSTRACT

The purpose of the present study was to characterize the isoforms of cyclooxygenase (COX) in the human iris before and after stimulation withlipopolysaccharide (LPS) and to determine the selectivity of the nonsteroidal anti-inflammatory drug (NSAID,) S(+) flurbiprofen, for inhibition of COX-1 and COX-2 in homogenates of this tissue. Spotblots were made of extracts of human iris in the absence and presence of LPS plus acetylsalicylic acid (aspirin). After reacting with anti-COX-1 and anti-COX-2 immunoglobulin G, the presence of both immunoreactive COX enzymes was substantiated using an indirect immunoperoxidase method. Authentic COX-1 and COX-2 were used as controls. Using an enzyme immune assay (EIA),

the production of prostaglandin E2 (PGE2) was quantified in tissue homogenates of human iris under the same conditions as described above. S(+) flurbiprofen was

added to tissue homogenates in order to determine the inhibitory effect on PGE2

production. Half maximal inhibitory concentrations (IC50) of S(+) flurbiprofen for the

PGE2 production in the tissue homogenates were determined from concentration inhibition curves. The selectivity of S(+) flurbiprofen for inhibition of COX-1 was expressed as the

ratio of IC50 for COX-2 / COX-1. Spotblots of non-stimulated iris-extracts showed positive staining for COX-1 immunoreactivity (-ir) only. After incubation with LPS plus acetylsalicylic acid, positive staining was observed for both COX-1-ir and COX-2-ir.

Concentrations of PGE2, released from homogenates of untreated iris varied from 1.5-4 ng/ml and of LPS-stimulated tissue from 10-20 ng/ml of assay mixture.

S(+) flurbiprofen inhibited PGE2 production of untreated tissue homogenates at -10 -6 an IC50 of 8x10 M, whereas in the stimulated tissue IC50 was found to be 3x10 M. The selectivity of S(+) flurbiprofen for inhibition of constitutively present COX-1, relative to the inhibition of induced COX-2, was 3,600. Our results indicate that specific expression of COX isoforms in normal human iris was substantiated at the protein level by immunoreaction on spotblots. COX-1 represents the constitutively present enzyme, and COX-2 appears after stimulation with LPS. At the functional level, S(+) flurbiprofen possesses a specificity for COX-1 in

inhibiting PGE2 production.

INTRODUCTION

The iris is the major site for prostaglandin (PG) formation in the eye. These prostanoids act through prostanoid receptors (1) to regulate smooth muscle contraction, blood-aqueous-barrier penetration and intraocular pressure. PGs are syn- thesized by a multistep pathway from arachidonic acid, which is either released

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from membrane phospholipids by phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) (2) or from intracellular triacylglycerols by triacylglycerol acylhydrolase (EC 3.1.1.3) (3). Their formation is catalyzed by the cyclooxygenase (COX) and glu- tathione-dependent peroxidase activities of PG endoperoxide synthase (EC 1.14.99.1). Two isoforms of COX have been characterized. COX-1 was initially isolated from sheep seminal vesicles (4) and is constitutively expressed in a variety of normal tissues (5) such as stomach, kidney, platelets, and in iris/ciliary body (6). However, it has been reported, that also COX-1 can make an important contribution to inflammatory responses (7). COX-2 is essentially expressed only following cell activation (8); however one exception to the low constitutive expression of COX-2-ir is the brain (9). Expression is time dependent and induced by various mediators of inflammation and bioactive agents such as lipopolysaccharides (10), cytokines (11), tumor necrosis factor (TNF) (11) and platelet activating factor (PAF) (12). COX-2 induction has been described in a variety of cells including migratory cells such as monocytes and macrophages. Also in ocular rabbit models of inflammation, COX-2 activity is found in endothelium (13) and epithelium (14) of the cornea and in iris/ciliary body (15). The elicited, immediate, production of PGs by the non-inflamed iris in vivo has been described following several experimental manipulations including paracentesis (16), mechanical stimulation (17), alkali burns (18), arachidonic acid administration (19), laser photocoagulation (20), and homogenization in vitro (21). Time related PG production has been observed after induction of uveitis with bovine serum albumin (22) or endotoxin (23). The PG synthesis in the normal iris/ciliary body has been suggested to be due to COX-1 (24) activity, being dependent solely on the availability of arachidonic acid. Induction of COX-2 may be an explanation for increased prostaglandin formation by the inflamed iris/ciliary body during long term consequences of eye injury, infection or intra-vitreal injection of endotoxin. Microsomes of rabbit iris/ciliary body synthesize increased amounts of cyclooxygenase products after intravitreal injection of endotoxin (25). The induction of COX-2 mRNA in the rabbit iris has been demonstrated within three hours following surgery of the iris (15). In contrast paracentesis failed to induce COX-2 mRNA (15). This suggests that, at least, trauma or resection of the iris is required for the appearance of COX-2 mRNA. The therapeutic use of non steroidal anti-inflammatory drugs (NSAIDs) is, in general, thought to be based on their ability to inhibit the induced COX-2 activity, being responsible for the signs of inflammation. Inhibition of COX-1 may explain the undesired side-effects, such as gastric and renal toxicity and bleeding disorders. However, in ophthalmology, during cataract extraction and laser treatment of the anterior eye, inhibition of constitutively present COX-1 accounts for the therapeutic effect of NSAIDs as prophylactic treatment to prevent miosis and ocular irritation.

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Although data are available on the potency and/or selectivity of several NSAIDs on COX-1 and COX-2 in human blood (26) , of bromfenac and nimesulide on the rabit iris/ciliary body (27), and of flurbiprofen on the bovine iris/ciliary body (24), no study has been performed on human ocular tissue. In the present study, we report the induction of COX-2 by stimulation with LPS in the human iris and dose effect inhibition curves of S(+) flurbiprofen on COX-1 and COX-2 in homogenates of human iris.

MATERIALS AND METHODS

Materials Human iris tissue with a post mortem time varying from 24-30 hrs was provided by the Cornea Bank Amsterdam. Lipopolysaccharide (LPS), derived from E.Coli 0.111;B4, was purchased from Sigma (St Louis, MO, USA). COX-1 peptide (sc- 1752 P), COX-2 peptide (sc-1745 P), goat anti-COX-1 immunoglobulin G (sc- 1752), goat anti-COX-2 immunoglobulin G (sc-1745), and horseradish peroxydase- labeled polyclonal anti-goat immunoglobulin G (anti-goat IgGHRP; sc 2020) were purchased from Santa Cruz Biotechnology Inc.(Santa Cruz, USA). S(+) flurbiprofen was obtained from Duchefa Pharma bv (Haarlem, The Netherlands). Assay kits for

enzyme immune analysis of PGE2 and enzyme activity of phospholipase A2 were from Cayman Chemical Co. (Ann Arbor MI, U.S.A.). A solution, containing 3,3'diaminobenzidine (DAB) tetrahydrochloride and H2O2 was purchased from ICN Biomedicals Inc. (Amsterdam, The Netherlands).

Stimulation of Iris Tissue with LPS For the induction of COX-2 the isolated iris, in a ratio of one tissue per 300 µl PBS, was incubated with 1 µl of a solution of LPS containing 5 mg/ml in DMSO and with 10 µl of acetylsalicylic acid (10 mg/ml in PBS) during 24 hrs at 37ºC.

Immunoprecipitation and Identification of COX-immunoreactivity Isoforms Human iris tissue was homogenized in sodium dodecyl sulfate sample buffer (125 mM Tris HCl, 4% sodium dodecyl sulfate, 20% glycerol, 1% dithiothreitol, pH6.8), boiled for 5 min and centrifuged for 5 min at 16,000 g. Nitrocellulose membrane was spotblotted with 15 µl of the supernatant and 3 µl of the control COX-1- and COX- 2 peptide, treated with blocking buffer (50 mM Tris, 0.15 M NaCl, 0.5% Tween-20, 2% non-fat dry milk, 0.02% sodium azide, pH 10) for 90 min and probed using goat anti-COX-1 or anti-COX-2 at a 1:500 dilution in blocking buffer during 90 min. After three washings of 5 min in washing buffer (10 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 8) the blot was treated with anti-goat IgGHRP at a 1:1000 dilution in blocking buffer during 30 min.

86 Human iris

After three washings of 5 min in washing buffer and one washing in buffer containing 10 mM Tris and 0.15 M NaCl staining was developed with a solution, containing DAB tetrahydrochloride and H2O2.

Measurement of Prostaglandin E2 Production The assay was performed in a modified form according to Van Sorge (24). Human iris tissue was homogenized in Tris HCl 0.05 M, pH 7.4, containing 1 mM phenylmethylsulfonylfluoride (PMSF), in a ratio of 1 iris per 400 µl buffer solution, using a Potter-Elvehjem glass in glass homogeniser in melting ice. For determination of COX activity 100 µl of homogenate was incubated with 25 µl of phosphate buffered saline (PBS) in a 1-ml Eppendorf tube at 37°C during 60 min. The effect of S(+) flurbiprofen was studied by addition of 25 µl of an appropriate dilution of the stock solution in PBS. The enzyme reaction was stopped by heating for 3 min in boiling water. Blanks were prepared by heating the tubes in boiling water, without previous incubation. In the supernatant of centrifugation (30 min at 16,000 g) released PGE2 was determined. The assay proved linear for the incubation time used and proportional to the amount of tissue-homogenate with, on average, 96% recovery. Inhibition of

PGE2 synthesis by S(+) flurbiprofen was calculated as a percentage of the activity in the presence versus the activity in the absence of the drug. The potency of the drug for COX was assessed by calculating the concentration of the drug causing 50% inhibition (IC50) of the maximal activity. The selectivity of

S(+) flurbiprofen for the two COX isoforms is expressed as the ratio of the IC50 for COX-2 versus COX-1. The higher the ratio, the more potently S(+) flurbiprofen inhibits COX-1 relative to COX-2.

Measurement of Phospholipase A2 Activity

Phospholipase A2 activity was measured in human iris tissue homogenate, prepared as described above. In a kinetic assay using diheptanoyl-thio-phosphatidyl- choline as substrate and 5,5"-dithiobis(2-nitrobenzoic acid) (DTNB) as color reagent the enzyme activity was measured at 414 nm and calculated in µmol/min/ml of homogenate, using the DTNB extinction coefficient of 10.66 mM-1.

RESULTS

The presence of COX-1 immunoreactivity (-ir) and COX-2-ir was substantiated in extracts of human iris, either untreated or following stimulation with LPS, using in vitro incubation experiments. Acetylsalicylate was added together with LPS to elim- inate any COX-1 activity in the iris by irreversible binding to the enzyme. To detect the two isoforms of COX in the homogenates of the differently treated tissues, spotblots were treated with anti-COX-1 antibody on the blotting paper, followed by

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immunoperoxidase labeling. Representative immunoblots with positive staining for COX-1-ir or COX-2-ir in both untreated and LPS-incubated iris ciliary body tissue are shown in figure 1. Controls of authentic COX-1 peptide but not COX-2 peptide showed positive staining. Treatment with anti-COX-2 produced staining of COX-2- ir only with LPS-incubated tissue and not with untreated iris tissue extract. Positive staining was detected using control COX-2 peptide but not COX-1 peptide.

Figure 1. Detection of cyclooxygenase in human iris extracts. Authentic COX-1 and COX-2 peptide and 15 µl of tissue extract were spotblotted on nitrocellulose membrane. Cyclooxygenase was detected using immunoglobulin G specific for COX-1 or for COX-2. With anti-COX-1, spots were observed in untreated iris, LPS-treated iris and with COX-1 peptide. With anti-COX-2, spots were observed in LPS-treated iris only and with COX-2 peptide.

To quantitate the functional activity of the COX isoenzymes, PGE2 production was

measured. The concentration of PGE2 produced by homogenates of normal iris tissue varied from 1.5-4 ng/ml assay mixture/hr and of iris treated with LPS from 10-

20 ng/ml of assay mixture/hr. The synthesis of PGE2, produced under these circumstances was inhibited differentially by S(+) flurbiprofen as shown in figure 2.

S(+) flurbiprofen inhibits the PGE2 production of untreated iris tissue at an IC50 of -10 -6 8 x 10 M, whereas the IC50 after LPS stimulation was found to be 3 x 10 M. The phospholipase activity in iris homogenate (43 ± 5 µmol/min/ml) was not significantly influenced in the presence of S(+) flurbiprofen in a final concentration of 10-5M.

88 Human iris

Figure 2. Concentration-response curves for inhibition of the COX-1 () activity in homogenate of untreated human iris and of COX-2 () activity in homogenate of human iris treated with LPS. Concentrations of S(+) flurbiprofen are final concentrations in the assay mixture. Each point represents the average ± SEM of four to six determinations from different pools of tissue homogenates.

DISCUSSION

The iris is the major site for prostaglandin (PG) formation in the eye. These PGs are produced after experimental manipulations but also following induction of uveitis. Whereas PG synthesis in the iris/ciliary body has been ascribed to COX-1 activity, induction of COX-2 may be an explanation for increased prostaglandin formation by the inflamed human iris such as in uveitis. In this study, not only were the involved COX-isoforms detected at the protein level, but also their functional activity was characterized using the NSAID S(+) flurbiprofen. With homogenates of untreated tissue, positive spotblot-staining for COX-1 and substantial production of PGE2 could be demonstrated. Next to the usual positive staining for COX-1-ir additional positive staining for COX-2-ir was achieved after stimulation with LPS, concomitant with greater production of PGE2. In porcine ciliary body it has been demonstrated that cytochrome P450 dependent ω/ω-1 hydroxylase activity can inactivate accumulated prostaglandins (28). In polymorphonuclear leukocytes hydrolysis of triacylglycerols by triacylglycerol hydrolase may also provide arachidonate as a source of fatty acid for COX. In our assay we obviously measured the net result of production of PGE2 by COX-1 or COX-2 from endogenous arachidonate mobilized by activation of specific hydrolases.

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The selectivity of S(+) flurbiprofen for inhibition of COX-1 with a COX-2/COX-1 ratio of 3,600 is greater than the value of 32 found using human blood as assay system (29). This discrepancy between COX-2 inhibition for S(+) flurbiprofen in ocular tissue and blood was unexpected and not readily explained. Carabaza et al. (30) found a similar great ratio for S(+) flurbiprofen, using ram seminal vesicles as -9 -6 source for COX-1, reporting an IC50 of 2X10 M, as compared to 0.47X10 M on COX-2, using the whole blood assay. Considering the complexity of the dynamics involved in inhibition of COX-1 and COX-2 by flurbiprofen, it is conceivable that intra-species differences are possibly due to differences in tissue, protein binding and assay protocols (31). Inhibition of PG production for some NSAIDs, like indomethacin and sulindac,

may also be caused by inhibition of phospholipase A2 (32,33), but not of triacygly- cerolhydrolases (32). However, flurbiprofen has been shown not to inhibit human

synovial or rat peritoneal phospholipase A2 (33) and our results show the same for

the enzyme in the iris. Therefore the observed inhibition of PGE2 production in this study might be ascribed solely to inhibitory effects on COX-1 and COX-2 and not

to inhibition of phospholipase A2. The clinical use of flurbiprofen in the inhibition of intraoperative miosis, of disruption of the blood-aqueous barrier and of cystoid macular edema is based on inhibition of the "housekeeping" enzyme, COX-1, present in the iris/ciliary body. Flurbiprofen's activity directed against inflammatory signs generated by induced COX-2, present hours after surgery or laser treatment is minor in degree (15,27). The relative contribution of other eicosanoid or non-eicosanoid pathways in mechanical blood-aqueous barrier disruption has been estimated from experiments using flurbiprofen to be less than 5% (34). We used the S(+) enantiomer of flurbiprofen, because it is the most potent isomer, like in many other enantiomeric pairs of NSAIDs. In experiments using bovine iris/ciliary body the R(-) enantiomer of flurbiprofen showed 1% of the inhibitory activity of the S(+) enantiomer (24) and in the human whole blood assay 0.3 and 2% on COX-1 and COX-2 respectively (29). With respect to the demands for ballastfree stereo specific drugs providing a reduction in metabolic load to the patient, S(+) flurbiprofen in the racemic mixture can be designated as the active agent and R(-) flurbiprofen as unnecessary ballast. Outside the field of ophthalmology, a report is available on the clinical use of S(+) flurbiprofen in dentistry (35). The NSAID S(+) ibuprofen proved pharmacologically active in the prevention of leakage of protein over the blood aqueous barrier in rabbits (36). There is no indication (37) of a pro-drug property which could be based on metabolic inversion of the less active R(-) isomer into the more active S(+) isomer, as has been observed in other species than man (38).The S(+) flurbiprofen has been formulated into a stereoselective ballast-free eyedrop solution in a concentration of 0.015% (free acid form), which is approximately half the usual

90 Human iris

concentration of commercial available specialties containing racemic flurbiprofen in the sodium salt form (39) . After systemic administration, R(-) flurbiprofen shows about one third of the antinociceptive activity of the S(+) form, indicating a central site of action independent of prostaglandin synthesis inhibition and in this respect the use of racemic flurbiprofen may be reserved for analgesic applications (40). In eye conditions resulting from prostaglandin production caused by induced COX-2, such as chronic uveitis, the concentration of S(+) flurbiprofen as selective COX-1 inhibitor in the regular eyedrops may be too low to give sufficient inhibition. The concentration of flurbiprofen in human aqueous humor has been measured as 60 ng/ml two hours after instillation of a single drop (41), which corresponds to 0.25 µM, being much µ lower than the IC50 of 3 M for COX-2 as measured in our study. The use of selective topical COX-2 inhibitors, such as meloxicam for the treatment of all forms of ocular inflammation, as advocated by Masferrer and Kulkarni (42) seems not always justified. Only the management of inflammation caused by induced COX-2 activity (15,27) may be reserved for selective COX-2 inhibitors.

Meloxicam appears to be suitable for administration as eyedrops (43). The IC50 of about 0.5 µM (26,29) for COX-2 as measured in human blood might suffice for intraocular inhibition, depending on the concentration of meloxicam reached in the aqueous humor.

Acknowledgements: The authors thank the foundation BIS (Leiden, the Netherlands) and the Cornea Bank (Amsterdam, the Netherlands) for providing human iris tissue.

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trointestinal toxicity. Gastroenterology 115:101-109, 1998. 8. Xie, W., Chipman, J.G., Robertson, D.L., Erikson, R.L., and Simmons, D.L. Expression of mitogen responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc. Natl. Acad. Sci. USA. 88:2692-2696, 1991. 9. Yamagata, K., Andreasson, K., Kaufmann, W.E., Barnes, C.A., and Worley, P.F. Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and gluco- corticoids. Neuron 11:371 - 386, 1993. 10. Akarasereenont, P., Bakhle, Y.S., Thiemermann, C., and Vane, J.R. Cytokine-mediated induction of cyclooxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide. Br. J. Pharmacol. 115:401-408, 1995. 11. Arias-Negrete, S., Keller, K., and Chadee, K. Proinflammatory cytokines regulate cyclooxygenase-2 mRNA expression in human macrophages. Biochim. Biophys. Res. Commun. 208:582- 589, 1995. 12. Bazan, N.G., Fletcher, B.S., Herschmann, H.R., and Mukherjee, P.K. Platelet-activating factor and retinoic acid synergistically activate the inducible prostaglandin synthase gene. Proc. Natl. Acad. Sci. USA 91:5252-5256, 1994. 13. Jumblatt, M.M., and Willer, S.S. Corneal endothelial repair. Regulation of prostaglandin E2 synthesis. Invest. Ophthalmol. Vis. Sci. 37:1294-1301, 1996. 14. Bazan, H.E., Tao, Y., DeCoster, M.A., and Bazan, N.G. Platelet-activating factor induces cyclooxygenase-2 gene expression in corneal epithelium. Requirement of calcium in the signal transduction pathway. Invest. Ophthalmol. Vis. Sci. 38:2492-2501, 1997. 15. Chang, M.S., Tsai, J.C., Yang, R., DuBois, R.N., Breyer, M.D., and O'Day, D.M. Induction of rabbit cyclooxygenase 2 in the anterior uvea following glaucoma filtration surgery. Curr.Eye. Res. 16:1147-1151, 1997. 16. Miller, J.D., Eakins, K.E., and Atwal, M. The release of PGE2-like activity into aqueous humor after paracentesis and its prevention by aspirin. Invest. Opthalmol. Vis. Sci. 12:939-942, 1973. 17. Cole, D.F., and Unger, W.G. Prostaglandins as mediators of the responses of the eye to trauma. Exp. Eye. Res. 17:357-368, 1973. 18. Paterson, C.A., and Pfister, R.R. Prostaglandin-like activity in the aqueous humor following alkali burns. Invest. Ophthalmol. Vis. Sci. 14:177-183, 1975. 19. Bhattacherjee, P., and Eakins, K.E. Inhibition of the ocular effects of sodium arachidonate by anti- inflammatory compounds. Prostaglandins 9:175-182, 1975. 20. Unger, W.G., Perkins, E.S., and Bass, M.S. The response of the rabbit eye to laser irradiation of the iris. Exp. Eye Res. 19:367-377, 1974. 21. Kulkarni, P.S., Fleisher, L., and Srinivasan, B.D. The synthesis of cyclooxygenase products in ocular tissues of various species. Curr. Eye Res. 3:447-452, 1984. 22. Eakins, E.K., Whitelocke, R.A.F., Perkins, E.S., Bennet, A. and Unger, W.G. Release of a prostaglandin in ocular inflammation. Nature 239:248-249, 1972. 23. Bhattacherjee, P. Release of prostaglandin-like substances by Shigella endotoxin and its inhibition by nonsteroidal anti-inflammatory compounds. Br. J. Pharmacol. 54:489-494, 1975. 24. Van Sorge, A.A., Van Delft, J.L., Bodelier, V.M.W., Wijnen, P.H., and Van Haeringen, N.J. Specificity of flurbiprofen and enantiomers for inhibition of prostaglandin synthesis in bovine iris/ciliary body. Prostag. Other Lipid Mediat. 55:169-177, 1998. 25. Kass, M.A., Holmberg, N.J., and Smith, M.E. Prostaglandin and thromboxane synthesis by microsomes of inflamed rabbit ciliary body-iris. Invest. Ophthalmol. Vis. Sci. 20:442-449, 1981. 26. Patrignani, P., Panara ,M.R., Sciulli, M.G., Santini, G., Renda, G., and Patrono, C. Differential inhibition of human prostaglandin endoperoxidase synthase-1 and -2 by nonsteroidal anti-inflammatory drugs. J. Physiol. Pharmacol. 48:623-631, 1997.

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27. Ogawa, T., Watanabe, N., and Waki, M. Role of cyclooxygenase isozyme in inflammation and pain in rabbit eye. Exp. Eye Res. 67 (Suppl. 1);S 242, 1998. 28. Asakura, T., and Shichi, H. Cytochrome P450-mediated prostaglandin w/w-1 hydroxylase activities in porcine ciliary epithelial cells. Exp. Eye Res. 55:377-384, 1992. 29. Van Haeringen, N.J., Van Sorge, A.A., Van Delft, J.L., and Carballosa Coré-Bodelier, V.M.W. Flurbiprofen and enantiomers in opthalmic solution tested as inhibitors of prostanoid synthesis in human blood. J. Ocular Pharmacol. Therap 16:345-352, 2000. 30. Carabaza, A., Cabré, F., Rotllan, E., Gómez, M., Gutiérrez, M., García, L., and Mauleón, D. Stereoselective inhibition of inducible cyclooxygenase by chiral nonsteroidal antiinflammatory drugs. J. Clin. Pharmacol. 36:505-512, 1996. 31. Callan, O.H., So, O-Y., and Swinney, D.C. The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. J. Biol. Chem. 271:3548-3554, 1996. 32. Kaplan-Harris, L., and Elsbach, P. The anti-inflammatory activity of analogs of indomethacin cor- relates with their inhibitory effects on phospholipase A2 of rabbit polymorphonuclear leukocytes. Biochim. Biophys. Acta 618:318-326, 1980. 33. Lobo, I.B., and Hoult, J.R. Groups I, II and III extracellular phospholipases A2: Selective inhibition of group II enzymes by indomethacin but not other NSAIDs. Agents Actions 41:111-113, 1994. 34. van Haeringen, N.J., Oosterhuis, J.A., van Delft, J.L., Glasius, E., and Noach, E.L. A comparison of the effects of non-steroidal compounds on the disruption of the blood-aquoeus barrier. Exp. Eye Res. 35:271-277, 1982. 35. Roszkowski, M.T., Swift, J.Q., and Hargreaves, K.M. Effect of NSAID administration on tissue levels of immunoreactive prostaglandin E2, Leukotriene B4, and (S) flurbiprofen following exy- taction of impacted third molars. Pain. 73:339-345, 1997. 36. Tjebbes, G.W.A., van Delft, J.L., Barthen, E.R., and van Haeringen, N.J. d-ibuprofen in ocular inflammation induced by paracentesis of the rabbit eye. Prostaglandins 40:29-33, 1990. 37. Geisslinger, G., Menzel-Soglowek, S., Schuster, O., and Brune, K. Stereoselective high performance liquid chromatographic determination of flurbiprofen in plasma. J. Chrom. 573:163-167, 1992. 38. Menzel-Soglowek, S., Geisslinger, G., Beck, W.S., and Brune, K. Variability of inversion of R(-) flurbiprofen in different species. J. Pharm Sci. 81:888-891, 1992. 39. Van Sorge, A.A., Wijnen, P.H., Van Delft, J.L., Carballosa Coré-Bodelier, V.M.W., and Van Haeringen, N.J. Flurbiprofen, S(+), eyedrops; Formulation, enantiomeric assay, shelflife and pharmacology. Pharm. World. Sci. 21:91-95, 1999. 40. Geisslinger, G., Ferreira, S.H., Menzel, S., Schlott, D., and Brune, K. Antinociceptive actions of R(-) flurbiprofen - a non-cyclooxygenase inhibiting 2-arylpropionic acid - in rats. Life Sci. 54:173- 177, 1994. 41. Ellis, P.P., Pfoff, D.S., Bloedow, D.C., and Riegel, M. Intraocular diclofenac and flurbiprofen concentrations in human aqueous humor following topical application. J. Ocul. Pharmacol. 10:677- 682, 1994. 42. Masferrer, J.L., and Kulkarni, P.S. Cyclooxygenase-2 inhibitors: a new approach to the therapy of ocular inflammation. Surv. Ophthalmol. 41(Suppl.20):S35-S40, 1997. 43. Stei, P., Kruss, B., Wiegleb, J., and Trach, V. Local tissue tolerability of meloxicam, a new NSAID: Indications for parenteral, dermal and mucosal administration. Br. J. Rheumatol. 35(Suppl. 1):44- 50, 1996.

93 94 CHAPTER 7

99mTc-DIFLUNISAL AND THE HUMAN IRIS TOPICAL APPLICATION REVEALS LOCALISATION

Adriaan A. van Sorge, Robert Jan van Etten, Coen J. Rehmann, Ton J. Rijnders, and Nicolaas J. van Haeringen

J Ocular Pharmacol 2002;18:185-95 Chapter 7

ABSTRACT

Following the instillation of a drug into the eye, drainage mechanisms will commence at once. In this report, an attempt was made to assess the dynamics of an instilled nonsteroidal anti-inflammatory drug (NSAID), diflunisal, labeled with 1 MBq 99mTc followed by twenty minutes of scintigraphy. The results obtained with this labeled drug were compared with instillation of the same volume and activity of 99m - TcO 4 . Although the pertechnetate anion is an excellent and innocuous indicator for detecting the external lacrimal drainage system of the eye, it cannot visualize the internal structures. A clear scintigraphic difference was noted between labeled diflunisal and the pertechnetate anion. Scintigraphic activity surrounding the pupil of the eye provides evidence of visualization of the iris/ciliary body. This seems reasonable as the cyclooxygenase enzyme is located in this structure and NSAIDs exert their mechanism of action via this complex. With this technology, visualization of some internal structures of the eye may be facilitated.

INTRODUCTION

In 1972, Rossomondo (1) published the first report on the use of sodium pertechnetate 99mTc for evaluating the lacrimal drainage system. The dose administered was 1.85MBq to 3.7 MBq (50 to 100 µCi) with a scintigraphic procedure of 25 minutes duration and excellent visualization of the external lacrimal system. Applications in other medical fields were suggested, the main point being that the anatomy of the structures involved were not altered, and important physiological parameters could possibly be solved in an elegant, non-irritating manner. Before, radiopaque dyes had been utilized for imaging the lacrimal drainage system (dacry- ocystogram) which is uncomfortable, time consuming and deforming on anatomical structures. Blanksma et al. have communicated a similar experiment with use of 99m - TcO 4 (2). Gamma scintigraphy can be of benefit in providing data (3): · on rate and extent of drug absorption · for "proof of concept" · for explaining drug and/or formulation effects · by use of sequential scintigraphic images ("time-lapse photography") · that in vivo performance is in agreement with the intended application · concerning delivery of the right amount of drug in the right place at the right time · that may overcome the poor predictability of in vitro studies Essential to the design of a radiopharmaceutical agent is the chemistry of the desired radiometaled chelate. The introduction of 99mTc as a labelling agent by chelation has changed the practice of nuclear medicine. The practice of chelation

96 99mTc-Diflunisal

in medicine is not new (4). In 1951, it was suggested that "salicylates" are capable of chelation and might, therefore, possess therapeutic properties. Aspirin aluminum is a known drug entity and incorporated in the Japanese Pharmacopoeia XIII. A very known reaction in the field of biochemistry and pharmacy is the appearance of a purple color in a weakly acid solution in the presence of a salicylate ion when ferric salts are added (5). In this manner, a quantitative estimate in a biological sample can be given in a relatively short time. Chelating ability of the ferric and ferrous cation with salicylic acid has been reported in favor of the ferric cation in a ratio of 400 to 1 (6). Spectroscopic studies have been performed on salicylic acid, salicylamide and acetylsalicylic acid as to which spatial configuration favors these drugs most. Calculations have made it plausible that the ß-form is the preferred configuration. In this orientation, the carboxylic hydroxyl group is directed towards the enolic hydroxyl group (7). Further research (8) has deepened the mechanism of complexation of 99mTc with "aspirin-like" molecules. Diflunisal (5-(2',4'-difluorophenyl)salicylic acid) inhibits the COX-1 enzyme in the same degree as sodium salicylate and approximately 20% less than flurbiprofen with the same level (80%) of COX-2 inhibition (9,10). The authors used this well defined pharmacological assay to assess the potential of S(+) flurbiprofen as a cyclooxygenase (COX) inhibitor formulated as an eyedrop (11). Acetylsalicylic acid showed the same degree of inhibition for COX-1 and COX-2 as flurbiprofen. The disadvantage of using acetylsalicylic acid, however, is its instability in solution (12). In our study, we chose the drug, diflunisal, as a representative for the mechanism of complexation as described above and sharing pharmacologic resemblance with the drug, flurbiprofen, as an NSAID in being fluorinated and possessing a biphenyl ring.

MATERIALS and METHODS

Gamma camera and computer system Gamma camera: Sopha DS7 single head, round field Energy: 99mTc, 140 keV Window: 20% Collimator: pinhole-collimator 5 mm Aperture Matrix: 128x128 Session duration: 20 minutes; dynamic images taken every 10 seconds (10 second duration) Data processing system: SMV NXT computer

97 Chapter 7

Drugs and Chemicals 99m - TcO 4 activity (sodium pertechnetate eluate) was obtained from a commercially available 99Mo/99mTc generator (Mallinckrodt). Diflunisal (product number: D-3281; lotnr:95H0528) was purchased from Sigma (St Louis, Mo, USA), Stannous Tartrate 500 microgram (BN040701; lyophilized) was purchased from IV DIRECT LTD (London, UK). The ophthalmic solution was dispensed by a Hamilton microliter syringe (50 microliter; art.nr.:3038015). Thin-layer chromatography material consisted of Whatman paper No17 (Tamson no 194K01) and ITLC-SG (Gelman no 61886); scanning was performed with a Raytest chromatogram scanner equipped with miniGITA software.

Formulation of Diflunisal Eyedrops Preparation of the diflunisal eyedrops proceeds by addition of 50 mg diflunisal to a part of the available 0.3 M sodium chloride solution. By addition of approximately 1 ml of 1M sodium hydroxide, a clear solution is obtained. The pH was adjusted between 7.0 -7.5 before adding up to 100.0 ml. The final solution was filtered through a 0.22 micron filter. All diflunisal preparations were stored in glass containers. No preservative was added.

Analytical Assay By use of HPLC combined with Diode Array Detection as described by Uges et al. (13) quality of the ophthalmic solution was assayed. In short, a reversed phase RP- 18 endcapped 5µm. 125x4mm column is used (Merck Darmstadt/Amsterdam, Catalog number: 21568); mobile phase: acetonitrile 470 ml(Lichrosolve) phosphoric acid/triëthylamine 530 ml buffer pH 3.3 is used. Content was analysed by spectrophotometric assay (Varian CARY 3) at 255 nm.

Patient Setting In this representative study, investigations were carried out in one subject (vS, 1944). Comfortably seated upright in a chair, a drop of ophthalmic solution (50 microliter) was placed into the conjunctival sac at the lateral canthus. The head was positioned with the inner canthus less than 5 mm from the pinhole collimator. Sufficient resolution could be obtained for the eye, canaliculi and lacrimal sac. Scintigraphic images were made over a 20-minute period. In two separate sessions (spaced by 1 month), the ophthalmic 99mTc-diflunisal solution and a pertechnetate sodium solution were investigated. In both instances, the left eye was used. Free passage of the lacrimonasal duct had been assured beforehand by the test of Anel. Application of the eyedrop was experienced as a transient prickling sensation.

98 99mTc-Diflunisal

Preparation of 99mTc-Diflunisal and Pertechnetate Sodium Eyedrops

99mTc-Diflunisal Eyedrops Sodium chloride 0,9% (low in oxygen content) is added to the vial containing 500 micrograms stannous tartrate. The contents of the vial are swirled cautiously. Next, the calculated amount of sodium pertechnetate activity is added (1ml=60 MBq) followed by the addition of 1ml of 0.05% diflunisal ophthalmic solution. The total contents (3 ml) is boiled for 10 minutes followed by radiochemical quality control of 99mTc-diflunisal. Labeling is set at 75 - 80% or more.

Sodium Pertechnetate Eyedrops Preparation of the sodium pertechnetate eyedrop was analogues to the 99mTc-diflunisal preparation differing in substitution by sodium chloride 0.9%. of the diflunisal solution. This solution contained 60 MBq in 3 ml solution (1 MBq in 50 microliters). Radiochemical quality control of pertechnetate was performed with purity set at 95% or more.

Analytical quality control for radiochemical purity Radiochemical purity of a radiopharmaceutical is defined as the proportion of total radioactivity in the desired compound. Analysis is performed by the two-strip mini-chromatography method as described in (14). In figure 1, the basic outline of the procedure is presented.

Figure 1. Two-strip Mini-chromatography System. On the left the Whatman no 17 and Methylethylketone solvent is used for pertechnetate separation; on the right, ITLC-SG and PBS pH 7.4 or sodium chloride 0.9% as solvent is used for quantification of bound 99mTc. Numbers 1,2,3 and 4 denote the following: 1: bound 99mTc and hydrolyzed reduced technetium; - 99m - 2: free TcO4 ; 3: hydrolyzed reduced technetium; 4: bound Tc and free TcO4 , respectively.

99 Chapter 7

A spot (10 microliter) of solution is deposited on Whatman paper No 17 and developed in methylethylketone. Pure pertechnetate will show up in the solvent front. Likewise, a spot of the same solution is deposited on the ITLC-SG strip and developed in phosphate buffered salt (PBS; pH 7.4) or 0.9% sodium chloride. The chromatography strip is developed in the chosen solvent and analyzed by a chromatography scanner recording radioactivity along the length of the strip. The percentage of radiochemical impurity is calculated from the ratio of radioactivity associated with the impurity compared with the radioactivity of the total strip.

RESULTS

ANALYTICAL ASSAY

HPLC and UV Spectrophotometry Quality control by HPLC of the ophthalmic diflunisal 0.05% solution displayed no degradation peaks (figure 2) with a content of 99.6% diflunisal measured by quantitative spectrophotometric analysis.

Figure 2. HPLC Chromatogram of a 0.05% Ophthalmic Solution of Diflunisal.

Radiopharmaceutical Quality Control

In figures 3, 4 and 5, examples of radiopharmaceutical quality control by thin-layer chromatography are depicted.

100 99mTc-Diflunisal

Figure 3. Thin-layer Chromatography of 99mTc Diflunisal on Whatman No 17 in MEK.

Figure 4. Thin-layer Chromatography of 99mTc Diflunisal on ITLC-SG in PBS pH 7.4. For legend, see Figure 1.

Figure 5. Thin-layer Chromatography of Pertechnetate on Whatman No 17 in MEK. For legend, see Figure 1.

101 Chapter 7

Labeling efficiency for diflunisal, measured by the thin-layer chromatography system as described, resulted in 77% when sodium chloride 0,9% was used and 83% when PBS pH7.4 was the solvent. Purity of pertechnetate, used in the ophthalmic solution, was 96%.

Scintigraphic Assay Figure 6 demonstrates the activity seen in frame 16 and the graphic illustration of counts versus time (whole session). Frame 16 signals the end of the eyes-closed sequence. Some activity is still left at the point of instillation of the eye drop. At the end of the sequence, all activity seems present in the lacrimonasal ductus.

Figure 6. Scintigraphic Image of the Eye Surface and the Lacrimal Sac 160 seconds after Application of 99mTc Diflunisal (frame 16). Graph represents counts versus time for whole session.

When one analyzes frame 117, residual activity (figure 7) distinct from baseline activity is present in the area of pupil and iris. This is displayed in the accompanying graph.

Comparing the frames, taken at the same time sequence, obtained with 99mTc-diflunisal and pertechnetate, a difference in scintigraphic exposure can be seen (figure 8).

102 99mTc-Diflunisal

Figure 7. Scintigraphic Image of the Area of the Iris and the Lacrimal Sac 1170 seconds after Application of 99mTc-Diflunisal (frame 117). Graph represents counts versus time for whole session.

Figure 8. Difference in Scintigraphic Exposure between Pertechnetate (left) and 99mTc- Diflunisal

Calculation of the activity as counts per pixel for the area designated as pupil, iris and conjunctiva (excluding the lacrimonasal ductus), leads to a graph (figure 9) dis- tinguishing a higher level of activity in the iris than the pupil. Correction for natural decay of 99mTc has been taken into account.

103 Chapter 7

99mTc-Diflunisal scintigraphy in selected eye structures

3,00 pupil

2,50 iris conjunctiva 2,00

1,50

1,00 counts per pixel

0,50

0,00 0 200 400 600 800 1000 1200 time (seconds) Figure 9. Display of Counts Per Pixel for 99mTc-Diflunisal in the Area of Pupil, Iris and Conjunctiva

DISCUSSION

In this study, an eye drop volume in every day practice of 50 microliters is used to illustrate the fate of the labeled NSAID diflunisal. Under the physiological conditions described, a dose of approximately 1 MBq of labeled 99mTc-diflunisal or pertechnetate generates a radiation absorbed dose to the epithelium of the lens of maximal- ly 4 x 10-11 Gy per Bq or 0.04 mGy (15). This is far below the dose known to produce radiation cataract (16). Scintigraphic evidence in the nasal cavity can be seen within one minute. As reported in the literature, closing of the eye will slow the process of drainage. After opening the eye, the tear flow will proceed at a normal physiological value of approximately 1 microliter per minute. After instillation of an eye drop, the extra volume will be rapidly eliminated in the first two minutes. This will be followed by a bi-exponential decay (initial phase of 2 - 5 minutes; basal phase from 7 minutes onward) where the tear flow will be 1.4 and approximately 1 microliter per minute, respectively. Any variation from these principles could indicate a disturbance in the delicate balance of the lacrimal system (17,18,19). In figure 9, a display of counts per pixel is given for the structures of pupil, iris and conjunctiva. The amount of labeled diflunisal that has entered the eye and is visible on the iris in frame 117 can be estimated by calculating the amount instilled, 8.35 microgram in 50 microliters, equivalent to 2900 counts. The pupil is repre- sented by 0.15 counts per pixel (total pixel count: 79) and the iris by 0.30 (total pixel count: 589). The estimated amount that has entered the eye through the cornea and is visibly attached to the iris will have to be corrected for the amount per pixel

104 99mTc-Diflunisal

that is attached to the cornea in the iris area. The latter is derived from the amount per pixel in the pupil area. The estimated amount on the iris, thus, is 0.30 - 0.15= 0.15 count per pixel. The amount of permeated labeled diflunisal is (0.15*589/2900) * 8.35µg = 0.254 microgram. This corresponds to 3% of the total amount of instilled diflunisal. Tissue concentration-time profiles for the iris-ciliary body closely resembles those for stroma-endothelium and aqueous humor. Peak concentration time for instilled pilocarpine was reached at 20 minutes (20). The initial total volume of distribution after application of an eye drop is reported to be 350 microliters. This encompasses the aqueous humor volume and the cornea (21). In this case 254 nanograms will be in 350 a microliter solution. This equates to 3x10-6M for diflunisal. For flurbiprofen, it was found that the COX-1 enzyme in the human iris is inhibited 100% by a 10-7M flurbiprofen solution (11). This will also suffice for inhibition of the COX-1 enzyme by diflunisal (9).

CONCLUSION

In vitro experiments (22,23), using a corneal perfusion device with simulated phys- iologic tear turnover as in vivo, or a three-dimensional tissue constructed to investigate drug penetration across the cornea, have been published but lack the real- time complex interplay in dynamics of tear fluid, tear turnover, aqueous humor and mechanical stress of the eyelids. This report provides scintigraphic evidence of the iris/ciliary body with use of a 99mTc-labelled NSAID, diflunisal.

Acknowledgements: The authors express their thanks to Irma Ruiken and Herman Janssen for their conscientious preparation and quality control of the radiopharmaceuticals. The staff of the Nuclear medicine department of Rijnstate is thanked for their cooperation in the scintigraphy sessions.

REFERENCES

1. Rossomondo, R.M., Carlton, W.H., Trueblood, J.H., and Thomas, R.P. A new method of evaluating lacrimal drainage. Arch. Ophthal. 88:523-525, 1972. 2. Blanksma, L.J., Schweitzer, N.M.J., Beekhuis, H., and Piers, D.A. Testing of lacrimal drainage with the aid of a gamma-ray emitting radiopharmaceutical (99mTcO4-). Doc. Ophthalmol. 42:381- 384, 1977. 3. Wilding, I., and Newman, S. Saving time in the drug development process using gamma scintigraphy. Pharm. Techno. Eur. February:26-31, 1998. 4. Schubert, J. Chelation in medicine. Sci. Am. 214:40-50, 1966. 5. Trinder, P. Rapid determination of salicylate in biological fluids. Biochem. J. 57:301-303, 1954. 6. Perrin, D.D. Stability of metal complexes with salicylic acid and related substances. Nature 182:741-742, 1958.

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7. El-Shahawy, A.S. Spectroscopic structural studies of salicylic acid, salicylamide and aspirin. Spectrochim. Acta. 44A:903-907, 1988. 8. El-Shahawy, A.S., Mahfouz, R.Z., Aly, A.A.M., and El-Zohry, M. Technetium-aspirin molecule complexes. J. Chem. Tech. Biotechnol. 56:227-231, 1993. 9. Warner, T.D., Giuliano, F., Vojnovic, I., Bukasa, A., Mitchell, J.A., and Vane, J.R. Nonsteroid drug selectivities for cyclooxygenase-1 rather than cyclooxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc. Natl. Acad. Sci. USA. 96:7563-7568, 1999. 10. Patrignani, P., Panara, M.R., Sciulli, M.G., Santini, G., Renda, G., and Patrono, C. Differential inhibition of human prostaglandin endoperoxide synthase-1 and -2 by nonsteroidal anti-inflammatory drugs. J. Physiol. Pharmacol. 48:623-631, 1997. 11. Van Haeringen, N.J., Van Sorge, A.A., and Carballosa Coré-Bodelier, V.M.W. Constitutive cyclooxygenase-1 and induced cyclooxygenase-2 in isolated human iris inhibited by S(+) flurbiprofen. J. Ocul. Pharmacol. Ther. 16:353-361, 2000. 12. Garrett, E.R. The kinetics of solvolysis of acyl esters of salicylic acid. J. Am. Chem. Soc. 79:3401- 3408, 1957. 13. Uges, D.R.A., and Conemans, J.M.H. Antidepressants and antipsychotics. In Forensic Science. Handbook of Analytical Separations, Vol. 2, Bogusz, M.J., ed., 229-257 (with special reference to pp 247-248, 2000). ISBN 0-444-82998-9. 14. Robbins, P.J. Chromatography of Technetium-99m radiopharmaceuticals: a practical guide. The Society of Nuclear Medicine, Inc., New York, USA. ISBN: 0-932004-18-0. 15. Robertson, J.S., Brown, M.L., and Colvard, D.M. Radiation absorbed dose to the lens in dacryoscintigraphy with 99mTcO4-. Radiology. 133:747-750, 1979. 16. Wilde, G., and Sjöstrand, J. A clinical study of radiation cataract formation in adult life following irradiation of the lens in early childhood. Br. J. Ophthalmol. 81:261-266, 1997. 17. Sørensen, T., and Taagehøj Jensen, F. Tear flow in normal human eyes. Determination by means of radioisotope and gammacamera. Acta Ophthalmol. 57:564-581, 1979. 18. Sørensen, T., and Taagehøj Jensen ,F. Methodological aspects of tear flow determination by means of a radioactive tracer. Acta Ophthalmol. 55:726-738, 1977. 19. Sørensen, T., and Taagehøj Jensen, F. Lacrimal pathology evaluated by dynamic lacrimal scintigraphy. Acta Ophthalmol. 58:597-607, 1980. 20. Sieg, J.E., and Robinson, J.R. Mechanistic studies on transcorneal permeation of pilocarpine. J. Pharm. Sci. 65:1816-1822, 1976. 21. Reim, M. Augenheilkunde. Ferdinand Enke Verlag, Stuttgart 1985. ISBN 3 432 94 5019. 22. Richman, J.B., Tang-Liu, D,D-S. A corneal perfusion device for estimating ocular bioavailability in vitro. J. Pharm. Sci. 79:153-157, 1990. 23. Tegtmeyer, S., Papantoniou, I., and Müller-Goymann, C.C. Reconstruction of an in vitro cornea and its use for drug permeation studies from different formulations containing pilocarpine hydrochloride. Eur. J. Pharm. Biopharm. 51:119-125, 2001.

106 CHAPTER 8

S(+) FLURBIPROFEN AND R(-) FLURBIPROFEN 99mTc-LABELING REVEALS DIFFERENCE IN STEREOCHEMISTRY

Adriaan A. van Sorge, Irma W.M. Ruiken, Herman W.M. Janssen, and Nicolaas J. van Haeringen

Accepted pending suitable revision Chapter 8

ABSTRACT

The separate enantiomers of flurbiprofen, RS-(±)-2-(2-fluoro-4-biphenylyl))-propionic acid, have been investigated for their potential in chelating with 99mTc provided by a 99Mo/99mTc generator. The used labeling procedure of a 0.05% flurbiprofen solution of the two enantiomers resulted in 1 MBq of activity in 50 microliter solution. Radiochemical quality control of the labeled compounds revealed that R(-) flurbiprofen has better chelating ability. This may indicate less steric hindrance caused by the presence of the fluorine atom in the R(-) flurbiprofen molecule.

INTRODUCTION

Flurbiprofen, belongs to the frequently prescribed and used drugs called nonsteroidal anti-inflammatory drugs (NSAIDs), often being chiral in nature (1). In the literature it has been reported that the anti-inflammatory and analgesic properties mainly reside in the dextrorotary component (2) of these drugs. This pharmacological effect is attained through inhibition of prostanoid synthesis, by the enzyme cyclooxygenase (COX) (3). As a consequence, active prostanoids cannot be formed thus giving reduction in inflammation and relief of pain. In previous studies we have established not only a marked stereoselectivity of flurbiprofen enantiomers for inhibition of COX-activity, but also preferential affinity for the constitutive COX-1 isoenzyme compared to inducible COX-2 (4,5). 99mTc-labeling of the achiral NSAID, diflunisal, (2',4'-difluor-4-hydroxy-[1,1'- biphenyl]-3-carbonic acid), gives a probe that is useful for the visualization of COX in the iris tissue of the living human eye (6). A further innovation is the labeling of ciprofloxacin, (1-cyclopropyl-6-fluoro-1,4- dihydro-4-oxo-7-(1-piperzynyl)-3-quinoline carbonic acid) with 99mTc, useful for the imaging of bacterial infections (7,8). Technetium labeling of the very potent NSAID S(+) flurbiprofen might also be useful as a probe for the localisation of COX in living tissue. In the following report labeling of the two individual enantiomers of flurbiprofen with 99mTc was undertaken.

MATERIALS AND METHODS

Drugs and Chemicals 99m - TcO 4 activity (sodium pertechnetate eluate) was obtained from a commercially available 99Mo/99mTc generator (Mallinckrodt). S(+) flurbiprofen (lotnr.: 16759) and R(-) flurbiprofen (lotnr.: 16754) were purchased from Duchefa (Haarlem, The Netherlands); the amount of S(+) flurbiprofen in R(-) flurbiprofen is 0.7% ± 0.1% whereas the R(-) flurbiprofen content in S(+) flurbiprofen is 0.1% (9). Stannous tar-

108 99mTc-S(+) flurbiprofen

trate 500 microgram (BN040701; lyophilised) was purchased from IV DIRECT LTD (London, UK); Thin-layer chromatography materials consisted of Whatman No17 paper (Tamson no.194K01) and ITLC-SG (Gelman no. 61886); scanning was performed with a Raytest chromatogram scanner equipped with miniGITA software.

Formulation of S(+) flurbiprofen and R(-) flurbiprofen Solution Preparation of the flurbiprofen enantiomeric solutions proceeds by addition of 50 mg S(+) flurbiprofen or R(-) flurbiprofen to a part of the available 0.3 M sodium chloride solution. By addition of approximately 1 ml of 1M sodium hydroxide a clear solution is obtained. The pH was adjusted between 7.0 -7.5 with hydrochloric acid 0.2M before adding up to 100.0 ml. The final solution was filtered through a 0.22- micron filter. All flurbiprofen preparations were stored in glass containers. No preservative was added.

Analytical Assay By use of HPLC combined with Diode Array Detection as described by Uges et al. (10) quality of the flurbiprofen enantiomeric solutions were assayed. In short, a reversed phase RP-18 endcapped 5µm, 125x4mm column is used (Merck Darmstadt/Amsterdam, catalogue number: 21568); mobile phase: acetonitrile 470 ml (Lichrosolve) phosphoric acid/triëthylamine 530 ml buffer pH 3.3. Injection volume: 20 microliter of the enantiomeric solutions (diluted to 1:40 in the mobile phase). Content was analyzed by spectrophotometric assay (Varian CARY 3) at 247 nm.

Preparation of 99mTc-S(+) flurbiprofen and 99mTc- R(-) flurbiprofen Solution Sodium chloride 0,9% (low in oxygen content) is added to a vial containing 500 micrograms stannous tartrate. The contents of the vial are swirled cautiously. Next the calculated amount of sodium pertechnetate activity is added (1ml=60 MBq) followed by the addition of 1ml of 0.05% S(+) flurbiprofen or R(-) flurbiprofen solution. The total content (3 ml) is boiled for 10 minutes and assayed for radiochemical purity of 99mTc-labeled flurbiprofen. Labeling is set at 85% or more.

Analytical Quality Control for Radiochemical Purity Radiochemical purity of a radiopharmaceutical is defined as the proportion of total radioactivity in the desired compound. Analysis is performed by the two-strip mini-chromatography method as described (11). In figure 1 the basic outline of the procedure is presented.

109 Chapter 8

Figure 1. Two-strip Mini-chromatography System. On the left the Whatman no 17 and methylethylketone solvent is used for pertechnetate separation, on the right ITLC-SG and PBS pH 7.4 or sodium chloride 0.9% as solvent is used for quantification of bound 99mTc. Numbers 1,2,3 and 4 denote the following, 1: bound 99mTc and hydrolysed reduced technetium; 2: free - 99m - TcO 4 ; 3: hydrolysed reduced technetium; 4: bound Tc and free TcO4 , respectively.

A spot (10 microliter) of solution is deposited on Whatman paper No 17 and developed in methylethylketone. Pure pertechnetate will show up in the solvent front. Likewise, a spot of the same solution is deposited on the ITLC-SG strip and developed in phosphate buffered saline (PBS; pH 7.4) or 0.9% sodium chloride. The chromatography strip is developed in the chosen solvent and analyzed by a chromatographyscanner recording radioactivity along the length of the strip. The percentage of radiochemical impurity is calculated from the ratio of radioactivity associated with the impurity compared with the radioactivity of the total strip.

RESULTS

Figure 2. HPLC Chromatogram of a 0.05% solution of R(-) flurbiprofen

110 99mTc-S(+) flurbiprofen

Analytical Assay HPLC and UV Spectrophotometry Quality control by HPLC of the 0.05% S(+) flurbiprofen or 0.05% R(-) flurbiprofen solution displayed no degradation peaks (figure 2) with a content of 102.2% for S(+) flurbiprofen and 99.9% for R(-) flurbiprofen measured by quantitative spectrophotometric analysis.

Radiopharmaceutical Quality Control In figures 3, 4 and 5 examples of radiopharmaceutical quality control by thin-layer chromatography are depicted.

Figure 3. Thin-layer Chromatogram of 99mTc-R(-) flurbiprofen developed in PBS pH7.4 as solvent on ITLC-SG. Peak 3 corresponds to hydrolysed reduced technetium, peak 4 to 99m - bound Tc and free TcO4

Figure 4. Thin-layer chromatography of 99mTc-S(+) flurbiprofen developed in PBS pH7.4 as solvent on ITLC-SG. Peak 3 corresponds to hydrolysed reduced technetium, peak 4 to 99m -. bound Tc and free TcO4

111 Chapter 8

Figure 5. Thin-layer chromatography of 99mTc-R(-) flurbiprofen developed in methylethylketone as solvent on Whatman No 17. Peak 1 corresponds to bound 99mTc and hydrolysed - reduced technetium, peak 2 to free TcO4 .

Labeling efficiency for 99mTc-S(+) flurbiprofen solution, measured by the thin-layer chromatography system as described, resulted in 65% when sodium chloride 0,9% and 57% when PBS pH7.4 was used as the solvent. For 99mTc-R(-) flurbiprofen solution, the efficiency accounted for 88% when sodium chloride 0,9% and 86% when PBS pH7.4 was used as the solvent.

DISCUSSION

A better chelating ability (average: 87%) was found for the chiral ligand R(-) flurbiprofen with the achiral central ion 99mTc as compared to the chelation of S(+) flurbiprofen (61%). In these experiments other chiral compounds are absent. An arte- 99m - fact in chromatographic separation of bound Tc and free TcO4 is excluded by the choice of the materials used for the analysis. The absolute configuration of S(+) flurbiprofen has been established utilising β- cyclodextrin complexation (12). Crystal structures of these complexes with R(-) flurbiprofen and S(+) flurbiprofen did reveal differences in binding to the host molecule (13). Although not directly conceivable, our result may derive from steric hindrance by the small fluorine atom present in the flurbiprofen molecule for binding of 99mTc, when one assumes that 99mTc will chelate with the carboxyl moiety of flurbiprofen However, addition of sodium pertechnetate to a stannous tartrate solution at room temperature will produce, depending on pH and the amount of Sn2+ available, a 99mTc 5+-tartrate complex in quantitative yield. Tartrate is used in radiopharmaceutical formulations as a transfer ligand, to prevent the Tc5+ from reducing further to lower 99m oxidation states, mainly to avoid the formation of TcO 2-hydrate, which is thermo-

112 99mTc-S(+) flurbiprofen

dynamically stable and will compete even in the presence of a ligand with the 99mTc- complex formation. This ligand exchange method, also termed transchelation, thus involves first the formation of a 99mTc-complex with a weak ligand and then allowing the complex to react with a second ligand that is relatively more stable. In this case a difference is seen between the S(+) isomer and the R(-) isomer in the formation of a more stable complex in favor of R(-) flurbiprofen. The 99mTc flurbiprofen complex obtained presumably is a Tc5+ complex, in which several possible structures, containing e.g. either the (Tc=O)3+ core (14), or the linear trans-oxo group (O=Tc=O)+ (15), or the bridging (O=Tc-O-Tc=O)4+ unit (16), could be present, in which either one or more flurbiprofen molecules are coordina- ted to the 99mTc-core. Hence it is both difficult and risky to make any definitive statements concerning steric hindrance of the ligands unless some structural data are available. Yet, the experimental fact showing that one isomer R(-), gives a significantly better labeling yield than the other one, S(+), under identical circumstances, points in that direction. To gain more fundamental insight in the given experimental result we intend to fol- 99m low a different approach namely the labeling of flurbiprofen with the Tc(CO)3-core according to a newly available technique (17). Some advantages thereof are the high stability expected for the resulting 99mTc 1+-complex as well as the ease of preparation and high specific activity that can be achieved.

In conclusion, the low labeling efficiency of S(+) flurbiprofen make this complex less attractive as a probe for imaging COX. Despite the greater labeling efficiency found for R(-) flurbiprofen it is unsuitable for use as a probe because the affinity of R(-) flurbiprofen for COX-1 and COX-2 is much lower (4, 5,18) than of its S(+) isomer.

REFERENCES

1. W.F. Kean, C.J.L. Lock, and H.E.Howard-Lock, Chirality in antirheumatic drugs, Lancet, 1991,338,1565. 2. J.N. Cashman, The mechanisms of action of NSAIDs in analgesia, Drugs, 1996, 52, suppl., 5,13. 3. J.R. Vane, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nature 1971, 231, 232. 4. N.J. Van Haeringen, A.A. Van Sorge, J.L. Van Delft, and V.M.W. Carballosa Coré-Bodelier, Flurbiprofen and enantiomers in ophthalmic solution tested as inhibitors of prostanoid synthesis in human blood, J. Ocul. Pharmacol. Ther., 2000, 16, 345. 5. N.J. Van Haeringen, A.A. Van Sorge, and V.M.W. Carballosa Coré-Bodelier, Constitutive cyclooxygenase-1 and induced cyclooxygenase-2 in isolated human iris inhibited by S(+) flurbiprofen, J. Ocul. Pharmacol. Ther. 2000, 16, 353. 6. A.A. van Sorge, R.J. van Etten, C.J. Rehmann, A.J.M. Rijnders, and N.J. van Haeringen, Diflunisal and the human iris Topical application reveals localisation, J. Ocul. Pharmacol. Ther. 2002, 18,185.

113 7. K.E. Britton, S. Vinjamuri, A.V. Hall, K. Solanki, Q.H. Siraj, J. Bomaji, and S. Das, Clinical evaluation of technetium-99m Infecton for the localisation of bacterial infection. Eur. J. Nucl. Med., 1997, 24, 553. 8. F.X. Sundram, W.Y. Wong, E.S. Ang, A.S. Goh, D.C. Ng and S. Yu. Evaluation of technetium-99m ciprofloxacin (Infecon) in the imaging of infection, Ann Acad Med Singapore 2000,29,699. 9. A.A. van Sorge, P.H. Wijnen, J.L. van Delft, V.M.W. Carballosa Coré-Bodelier, and N.J. van Haeringen, Flurbiprofen, S(+), eyedrops: formulation, enenatiomeric assay, shelflife and pharmacology, Pharm. World. Sci., 1999, 21, 91. 10. D.R.A. Uges, and J.M.H Conemans, Antidepressants and antipsychotics. In Forensic Science. Handbook of Analytical Separations, Vol. 2, Bogusz, M.J., (ed., pp. 229-257 (with special reference to pp. 247-248, 2000), ISBN 0-444-82998-9. 11. P.J. Robbins, Chromatography of Technetium-99m radiopharmaceuticals: a practical guide. The Society of Nuclear Medicine, Inc., New York, USA, ISBN: 0-932004-18-0. 12. K. Uekama, T. Imai, F. Hirayama, M. Otagiri, and K. Harata, X-ray crystallographic determination of the absolute configuration of (+)flurbiprofen utilizing b-cyclodextrin complexation, Chem. Pharm. Bull. 1984, 32, 1662. 13. K. Harata, F. Hirayama, T. Imai, K. Uekama, and M. Osagiri, Crystal structures of permethylated b-cyclodextrin complexes with R-(-)- and S-(+)-flurbiprofen, Chem. Lett. 1984, 1549. 14. A.M. Verbruggen, D.L. Nosco, C.G. van Nerom, G.M. Bormans, P.J. Adriaens and M.J. de Roo, Technetium-99m-L,L-ethylenedicysteine: a renal imaging agent. I. Labeling and evaluation in animals, J Nucl Med 1992, 33, 551-7. 15. P.H. Fackler, M.J. Lindsay, and M.J. Clarke ,Synthesis and structure of trans-[O2(Im)4Tc]Cl 2h2O, trans-[O2(1-meIm)4Tc]Cl 3H2O and related compounds, Inorg Chim Acta 1985, 109, 39-49. 16. F. Tisato, F Fiorenzo, U Mazzi, G Bandoli, and A Dolmella, Synthesis, characterization and elec- trochemical studies on technetium(V) and rhenium(V) oxo-complexes with N,N'-2-hydrox- ypropane-1,3-bis(salicylideneimine), Inorg Chim Acta 1989, 164, 127-35. 17. R. Alberto, R. Schibli, R. Waibel, U. Abram, A.P. Schubiger, Basic aqueous chemistry of [M(OH)2)3(CO)3]+ (M=Re, Tc) directed towards radiopharmaceutical application, Coord Chem Rev 1999, 190, 901-19. 18. A.A. van Sorge, J.L. van Delft, V.M.W. Bodelier, P.H. Wijnen, and N.J. van Haeringen, Specificity of Flurbiprofen and enantiomers for inhibition of prostaglandin synthesis in bovine iris/ciliary body, Prostaglandins Other Lipid Mediat 1998, 55, 169.

114 CHAPTER 9

ALTERNATIVE SPLICING OF CYCLOOXYGENASE-1 MRNA IN THE HUMAN IRIS

Melloney J. Dröge, Adriaan A. van Sorge, Nicolaas J. van Haeringen, Wim J. Quax and Johan Zaagsma

submitted for publication Chapter 9

ABSTRACT

In homogenates of the human iris, the non-steroidal anti-inflammatory drug (NSAID) S(+) flurbiprofen has been reported to inhibit cyclooxygenase-1 (COX-1) 70-fold more potently than in human whole blood. We hypothesized that this difference may be due to alternative splicing of COX-1 mRNA in the human iris or in whole blood. In this study, we have identified a similar COX-1 splice variant (COX-1SV) in both tissues with comparable COX-1/COX-1SV expression ratios. Therefore, we conclude

that the difference in IC50 values of S(+) flurbiprofen towards COX-1 in the human iris and human whole blood is not related to differences in the occurrence of spliced COX-1.

INTRODUCTION

Prostaglandin G/H synthase (PGHS), cyclooxygenase (COX) (EC 1.14.99.1) is a membrane bound homodimer of two 70 kDa polypeptides, catalyzing the first two steps in prostaglandin, thromboxane and prostacyclin synthesis (1). Two PGHS isoforms, referred to as COX-1 and COX-2, have now been identified and cloned, and characterized as hemoproteins possessing both cyclooxygenase and hydroperoxi- dase activity (2-4). Inhibition of the cyclooxygenase activity of PGHS is responsible for the anti-inflammatory activity of non-steroidal anti-inflammatory drugs (NSAIDs). The peroxidase activity of PGHS catalyzes oxidation of a broad range of substrates. COX-1 is constitutively expressed in various tissues (5), such as kidney, stomach, platelets, and the iris/ciliary body (6), whereas COX-2 is induced after cell activation by various mediators of inflammation and bioactive agents (7). In the human eye, the iris is the major site for producing prostaglandins, which regulate smooth muscle contraction, blood-aqueous-barrier penetration and intra-ocular pressure (8). We have previously shown in human iris homogenates that COX-1 is indeed constitutively expressed whereas COX-2 could be detected after stimulating with lipopolysaccharide (LPS) (9).

Inhibition of the COX activity towards prostaglandin E2 production could be achieved with the NSAID S(+) flurbiprofen in human iris homogenates. Remarkably, a 3600 fold stronger inhibition of COX-1 over COX-2 was observed (9). On the other hand, in separate experiments with human whole blood, S(+) flurbiprofen inhibited COX-1 and COX-2 rather similarly with a ratio of 32 (10). We hypothesized that the differential effects of S(+) flurbiprofen could be due to the presence of an alternative splice variant of the COX-1 enzyme in the iris as compared to whole blood. This seems reasonable since Diaz and coworkers reported that a splice variant of COX-1 is present in human lung fibroblasts (11). Indeed, comparison of the

IC50 values (half maximal inhibitory concentrations) for the inhibition of COX-1 by

116 COX-1 splice variant

S(+) flurbiprofen in human iris tissue and whole blood revealed a 70-fold higher potency towards COX-1 present in human iris tissue (9-10).

MATERIALS AND METHODS

Tissue collection Tissue of human irides was obtained immediately after surgery from eyes sched- uled for enucleation due to melanoma formation. After enucleation, the dissected iris tissue was snap-frozen in liquid nitrogen and stored at -80°C for subsequent RNA isolation. Human whole blood was collected in the presence of ethylenedi- aminetetraacetic acid (EDTA) and RNA extraction was performed subsequently.

RNA isolation Total RNA was isolated from iris and whole blood samples using the RNeasy mini kit and QIAamp RNA blood mini kit, respectively (Qiagen) (12). Small amounts of iris tissue (<50 mg) were homogenized in 1 ml of lysis solution using a motorized rotor- stator homogenizer. The homogenates were repeatedly centrifuged to remove tissue debris before proceeding to filtration. Further extraction steps were performed according to the manufacturer's protocol. RNA derived from this procedure was treated with DNAseI (Ambion) to remove contaminating DNA. RNA was quantified by measuring the optical density at 260 nm in triplicate.

Oligonucleotides Primers were designed as described by Diaz and coworkers (11). In short, a set of primers was designed to specifically span the COX-1SV junction: COX-1SVREV1: 5'-TTC ATG CCA AAC CTC TTG-3'; COX-1SVFOR1: 5'-GGA GAC CAT CAA GAT TGT-3' (Life Technologies). The primers amplified 405 (COX-1 mRNA) or 294 (COX-1SV mRNA) base pairs, respectively.

Reverse transcription Total RNA (1.0 µg) was reverse transcribed in a volume of 75 µl containing: 1 x

Reverse transcription buffer, 10 mM MgCl2, 1 mM dNTPs, 60 units RNasin ribonu- clease inhibitor, 30 units AMV reverse transcriptase and 1.25 µg random hexamer primers (all from Promega). Control reactions, containing neither reverse transcriptase nor RNA were run in parallel. Each subsequent PCR contained 3 µl cDNA tem- plate. Therefore, the amount of amplified PCR product was relative to a constant amount of starting RNA. To evaluate RNA quality, we performed a RT-PCR with 18S primers (18Suni1: 5'-CTA TTG CGC CGC TAG AGG TG-3'; 18Suni2: 5'-CTG AAC GCC ACT TGT CCC TC-3'; Eurogentec)

117 Chapter 9

PCR The COX-1 gene fragment was amplified using the primers COX-1SVFOR and COX-SVREV. All PCRs were performed using Pfu DNA polymerase (Stratagene, La Jolla, CA, U.S.A.). Amplification of the cDNA was performed by 40 cycles of PCR in 50 µl of Pfu DNA polymerase mixture, containing 25 pm of the COX-1SVREV1 and COX-1SVFOR1 primers. The PCR protocol had low annealing and extension temperatures: 4 min. at 94°C, followed by 10 cycles of 30 s at 94°C, 1 min 37°C and 10 min at 50°C, followed by 30 cycles in which the extension time was at first 8 min, but increased by 15 s in each cycle (first cycle: 30 s 94°C, 1 min 55°C and then 8 min 72°C). At the end, DNA production was finished with 10 min at 72°C. The PCR products were analyzed by 1.5% agarose gel electrophoresis stained with ethidium bromide. DNA size markers were from Fermentas (GeneRuler™ 100bp DNA Ladder). Restriction analysis using NcoI verified the sequence of the con- structs.

RESULTS AND DISCUSSION

The COX-1 gene is localized on the human chromosome 9q32-q33.3. The COX-1 protein is expressed constitutively in almost all mammalian tissues and is described as a housekeeping enzyme, responsible for cell-to-cell signaling, tissue homeosta- sis, and cytoprotection. More recently, Diaz et al have reported the expression of two COX-1 isoforms in human lung fibroblasts. By cloning of the cDNA, they demonstrated that the corresponding mRNA can be spliced in such a way that 111 base pairs are eliminated from exon 9, resulting in a COX-1 isoform that lacks residues 396-432 (11). Since this splicing occurs in-frame, it is expected that it will be translated into an identical protein. However, the alternative splicing results in the elimination of one of the four functionally required N-glycosylation sites at residue 409, providing a possible mechanism for differential regulation of enzymatic activity under physiological or pathological conditions (11,13). In this respect, we hypothesized that different expression levels of COX-1 and its

shorter isoform could account for the difference in IC50 values of S(+) flurbiprofen observed with human iris and human whole blood. Therefore, three human iris samples were analysed for the presence of the two transcripts variants. In addition, human whole blood (corresponding to iris 1) was screened for the presence of both transcripts as well as human blood samples from four healthy volunteers. Total mRNA was extracted from the human iris and blood samples and RT-PCR was performed subsequently. To assess whether the transcripts corresponded to COX-1 or the alternatively spliced COX-1, the cDNA was PCR amplified using a set of primers that specifically spanned the COX-1SV junction: a fragment of 405 base pairs

118 COX-1 splice variant

should be obtained from transcripts with an intact exon 9, and a fragment of 294 base pairs if exon 9 was lacking. PCR amplification revealed that both transcripts were present in the human iris tissues as well as in the whole blood samples (figure 1 (A)) as judged by the appearance of two major bands of approximately 400 and 300 base pairs, respectively. The gene fragments were further characterized by restriction analysis using NcoI. Restriction analysis would yield fragments of 285 and 120 base pairs (from the transcript of 405 base pairs corresponding to an intact exon 9) and 174 and 120 base pairs (from the transcript of 294 base pairs corresponding to the spliced variant). Figure 1 (B) illustrates that all fragments were indeed obtained, thereby confirming the correct identity of both transcripts. No significant dissimilarities were observed between the various iris and whole blood samples. Furthermore, the majority of the total COX-1 mRNA is expressed as the fully intact COX-1, whereas only a minor amount is present as the alternatively spliced variant COX-1SV. In addition, relative expression levels of COX-1 and COX-1SV in the human iris and plasma are similar.

Figure 1: (A) Analysis of mRNA from human iris tissue and whole human whole blood. 1 µg of total RNA was reverse-transcribed and amplified by PCR as described in the material and methods. The PCR product were analysed on a 1.5% agarose gel stained with ethidium bromide. Lane 1 - 3: human iris tissue; lane 4 human whole blood (corresponding to iris 1); lane 5 - 7: human whole blood references.

119 Chapter 9

Figure 1: (B) Restriction analysis of the PCR product from a human iris tissue sample (iris 3) and a human whole blood sample (whole blood reference 7). Restriction analysis was performed using NcoI. Lane 1: PCR product from human iris 3; lane 2: PCR product from human iris 3 after NcoI digestion; lane 3: PCR product from human whole blood 7; lane 4: PCR product from human whole blood 7 after NcoI digestion.

In conclusion, we have detected a splice variant of COX-1 in human irides and whole blood for the first time. Comparison of the expression levels of COX-1 and COX-1SV in the human iris and blood showed no striking differences. These find-

ings indicate that the observed difference in IC50 values of S(+) flurbiprofen towards the human iris and human whole blood does not result from an alternatively spliced COX-1 transcript. An alternative explanation may be that the iris COX-1 recognition site of S(+) flurbiprofen is able to adopt a different conformation than the blood enzyme.

Acknowledgement: Provision of tissue samples of human irides by Prof. Dr. J.E.E. Keunen (Department of Ophthalmology, Academic Medical Center Leiden, The Netherlands), Dr. P. Saeed & Prof. Dr. M.D. de Smet (Department of Ophthal- mology, Academic Medical Center Amsterdam, The Netherlands), Prof. Dr. J.M.M. Hooymans (Department of Ophthalmology, University Hospital Groningen, The

120 COX-1 splice variant

Netherlands) and Prof. Dr. W. Timens (Department of Pathology, University Hospital Groningen, The Netherlands) is greatly acknowledged. We would like to thank Mrs A. Brugman and Prof. Dr. F Muskiet (Department of Pathology and Laboratory Medicine, Groningen University Hospital, The Netherlands) for the RNA isolation from whole human blood. Dr. M. Elferink (Department of Pharmacokinetics and Drug Delivery, University of Groningen, The Netherlands) is acknowledged for her valuable advise concerning the RNA procedures of the iris samples.

REFERENCES

1. Needleman, P., Turk, J., Jakshick, B.A., Morrison, A.R., Lefkowith, J.B. (1986). Arachidonic acid metabolism. Annu Rev Biochem., 55, 69-102. 2. Hla, T., Neilson, K. (1992). Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA, 89, 7384-8. 3. Yokoyama, C., Tanabe, T. (1989). Cloning of the human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme. Biochem. Biophys. Res. Commun., 165, 888-94. 4. Hla, T. (1996). Molecular characterization of the 5.2 KB isoforms of the human cyclooxygenase- 1 transcript. Prostaglandins, 51, 81-5. 5. Simmons, D.L., Xie, W., Chipman, J.G., Evett, G.E. (1991). Multiple cyclooxygenases: cloning of a mitogen inducible form. In: Prostaglandins, Leukotrienes, Lipoxins, and PAF. Bailey J.M. ed., Plenum Press, New York, 67-78. 6. Asakura, T., Sano, N., Shichi, H. (1992). Prostaglandin synthesis and accumulation by porcin ciliary epithelium. J. Ocul. Pharmacol., 8, 333-41. 7. Xie, W., Chipman, J.G., Robertson, D.L., Erikson, R.L., Simmons, D.L. (1991). Expression of a mitogen responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc. Natl. Acad. Sci. USA, 88, 2692-6. 8. Matsuo, T., Cynader, M.S. (1993). The EP2 receptor is the predominant prostanoid receptor in the human ciliary muscle. Br. J. Ophtalmol., 77, 110-4. 9. Van Haeringen, H.J., van Sorge, A.A., van Delft, J.L., Carbalossa Coré-Bodelier, V.M.W. (2000). Flurbiprofen and enantiomers in ophthalmic solution tested as inhibitors of prostanoid synthesis in human blood. J. Ocular Pharm. Ther., 16, 345-52. 10. Van Haeringen, N.J., van Sorge, A.A., Carbalossa Coré-Bodelier, V.M.W. (2000). Constitutive cyclooxygenase-1 and induced cyclooxygenase-2 in isolated human iris inhibited by S(+) flurbiprofen. J. Ocular Pharm. Ther., 16, 353-61. 11. Diaz, A., Reginato, A.M., Jiminez, S.A. (1992). Alternative splicing of human prostaglandin g/h synthase mRNA and evidence of differential regulation of the resulting transcripts by transforming growth factor ß1, interleukin 1ß, and tumor necrosis factor a. J. Biol. Chem., 267, 10816-22. 12. Kyveris, A., Maruscak E., Senchyna, M. (2002). Optimization of RNA isolation from human ocular tissues and analysis of prostanoid receptor mRNA expression using RT-PCR. Mol. Vis., 8, 51-8. 13. Cáceres, J.F., Kornblihtt, A.R. (2002). Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Genet., 18, 186-93.

121 122 SUMMARY Summary

SUMMARY AND OUTLINE OF THIS THESIS

Surgery of the eye lens has become the treatment of choice for senile cataract. As from the first description of this therapy, there have been reports on a serious complication in the macula of the retina (macula retinae) affecting the vision of the patient. This complication, first described in 1953, has become known as cystoid macular oedema. The incidence varies between 2 and 50%, but can incidentally go up to 70%. Publications on eye research mainly of Japanese origin have described the use of topically applied indomethacin to prevent cystoid macular oedema after lens extraction. In response to these reports, in the clinics of Arnhem a question about the feasibility of preparing indomethacin eyedrops was put forward in 1980 by one the ophthalmologists to the hospital pharmacist. This question has triggered the research described in this thesis. Following these reports on the prevention of cystoid macular edema after lens extraction, several formulations of indomethacin eyedrops have been described in the Dutch and international literature. Indomethacin acts by inhibition of the synthesis of prostaglandins, mediators involved in the inflammatory reaction, that is at the basis of this complication. Indomethacin was introduced into the field of ophthalmology in different types of formulations, including solutions in sesame oil, aqueous suspensions and aqueous solutions. Concentrations in oily solutions varied from 0.1 to 1%, in suspensions from 0.5% to 1% and in aqueous solutions from 0.35 to 1%. Unfortunately, the sesame oil based solutions turned out to be less suitable, since they caused blurring of vision as a result of difference in refractive index. The aqueous solutions on the other hand, being either a suspension or a solution, were irritating to the eye (burning sensation). In order to alleviate this undesirable side effect a reduction in concentration was suggested from 1% to 0.2 or 0.1% indomethacin. In 1981 it was shown that four different indomethacin suspension eyedrops, all 0.5% in concentration, gave different prostaglandin synthesis inhibiting activities, which was attributed to differences in physicochemical properties. It was concluded that, apart from the subjective complaints of irritation of the eye, the use of eyedrops as a suspension gives rise to non-reproducible pharmacokinetic and pharmacodynamic behaviour. As a result of the afore-mentioned question, a phosphate buffered (pH 7.4) aqueous solution of indomethacin was prepared by means of the organic base meglumine, in a concentration of 0.1%. These eyedrops were introduced in clinical practice in 1981 and produced no burning sensation when applied to the patient's eye. In 1984 Indoptol ®, an aqueous suspension eyedrop of 1% indomethacin, was introduced in the Dutch market and in 1986 Indocid® of comparable composition was introduced in France. In 1987 a new presentation of indomethacin followed in

124 Summary

France as Indocollyre® 0,1%, which was introduced in The Netherlands in 1994. This formulation contains indomethacin as a lyophilized (freeze-dried) product, which is brought into solution by addition of a sterile borate buffer. Ongoing own research on our in-house developed eyedrops with different bases, L-lysine, D- lysine, L-arginine, D-arginine and tromethamol (not published), did not provide suitable pharmaceutical alternatives. In the meantime our first introduced (1981) solution of indomethacin 0.1%, without the need of extra pharmaceutical excipients, remained the mainstay of the eye clinic. This solution was tested in a pharmacological setting in the rabbit eye using a paracentesis model of removing aqueous humor and measuring the influx of protein and fluorescein into the secondary aqueous humor which reflects the breakdown of the blood-aqueous barrier. The results showed that in a concentration of indomethacin as low as 0.05%, already 90 - 100% of the pharmacological activity is obtained, as demonstrated by the inhibition of fluorescein and protein influx. Following these results our indomethacin 0.1% formulation was incorporated in the Dutch National Formulary (FNA) in 1986. Impracticalities with indomethacin in aqueous solution - no sterilisation possibility and only a relatively short shelf-life when in solution - prompted us to explore the possibility of formulating eyedrops based on a different NSAID. In 1990, topically applied S(+)ibuprofen was reported to be effective in a rabbit model of interleukin-1 or paracentesis induced uveitis at relatively elevated concentrations (0.9% and 0.8% respectively). Also with S(+) naproxen, marketed by Syntex as an enantiomeric pure NSAID, the anti-inflammatory effect of eyedrops (0,5%) was demonstrated experimentally. In our search for a pharmaceutically more acceptable solution of an NSAID - the introduction to the Dutch market of a diclofenac ophthalmic 0.1% solution (Naclof®) being imminent - we turned to the USP in which a flurbiprofen sodium ophthalmic solution is mentioned. We embarked on a study to manufacture flurbiprofen eyedrops by the protocol of June 1992. A letter of consent, with restricted financial aid, for the project (9206SO.008) was issued January 8th 1993 by the SWOR (Stichting ter bevordering van Wetenschappelijk Onderzoek in ziekenhuis Rijnstate).

The aim of this thesis was to investigate and to evaluate the pharmaceutical application of flurbiprofen in eyedrops as well as the pharmacology of this nonsteroidal anti-inflammatory drug. Flurbiprofen is a chiral molecule implying that the racemate is presumably not the preferred pharmacological form to prepare such eyedrops. Therefore it was deemed necessary to characterize the contribution of each enantiomer. At the start of the investigations, the pharmacological action of flurbiprofen was attributed to the inhibition of the cyclooxygenase enzyme (COX), which is known to be responsible for prostaglandin synthesis. In 1991 it became apparent

125 Summary

that not one but two isoenzymes are capable of synthesising prostaglandins (COX- 1 and COX-2). Our investigations became more challenging as it was postulated that this second isoenzyme would be the more relevant target for nonsteroidal anti- inflammatory drugs as its activity would be related more closely to inflammation. It was decided to study both the racemic form and the individual enantiomers of flurbiprofen for prostaglandin synthesis inhibiting activity. S(+) flurbiprofen showed a marked selectivity for inhibition of COX-1 compared to COX-2, both in an extra-ocular matrix (human whole blood) and in human iris/ciliary body preparations. Since the susceptibility of iris COX-1 for S(+)flurbiprofen was 70-fold higher than for blood COX-1 the hypothesis was raised that a splice variant of COX-1 in the human iris could be responsible for this observation. In chapter 2 the rationale for the chosen buffer in the S(+) flurbiprofen eyedrop preparation is described. Although the eye, especially the nasal corner, the eyelids and skin surrounding the eye are sensitive to external stimuli, physiological reactions due to deviations outside the normal values for osmolarity or pH are not always seen. In a state of ill-health or during regular use of ophthalmic preparations, this situation may be more outspoken, however. The active component in the eyedrop can provoke, when not properly dissolved, a prickling or burning sensation leading to lacrimal discharge, occasional haemorrhage or endangering blinking reflexes during surgery. Lacrimal discharge will cause an unwanted dilution and drainage of medicine. Individual sensitivity may vary and physiological values of tear fluid can fluctuate, dependant on the health condition of the individual eye. Eye irritation must be discerned from an allergy, which requires the choice of a different pharmacological agent. Non-irritating eyedrops should in principle comply with: (1) sterility, (2) isotonicity and (3) pH value. Sterility is of paramount importance when the ophthalmic solution is applied to the injured eye. Isotonicity with respect to the tear fluid will reduce irritation and adverse reactions of the eye. The pH value of the formulation is of utmost importance both for stability during storage and for keeping the formulation within physiological limits. During storage hydroxyl ions, released from the glass containers, can raise the pH which may endanger the stability of the active principle. On the other hand, the lacrimal fluid has only limited buffering capacity. Therefore the choice for a buffer applied to the ophthalmic solution of flurbiprofen is governed by the following issues: (1) flurbiprofen is unstable at high pH, (2) the solubility of flurbiprofen in aqueous solution is problematic at pH values below 7, (3) the natural pH of the tear fluid is 7.4 and (4) the tolerability for the patient is in the pH range of pH 6.6 - 7.8. Based on practical experience with the indomethacin eyedrops a choice was made for the phosphate buffer (pH 7.4) in the preparation of the S(+) flurbiprofen eyedrops. A citrate buffer of pH 6.45, as is employed in Ocuflur® (racemic flur-

126 Summary

biprofen sodium), was not considered favourable as it has an extreme in its buffering capacity at pH 6.5. The same choice was made later on, in a United States patent 4,996,209, describing the preparation of a single enantiomer in a phosphate buffer. Although additional components were also present in that composition, the eyedrops described in this thesis are free of other components. In chapter 3 the stability of the formulation is addressed. The active S(+) enantiomer of flurbiprofen was formulated into a stereoselective, ballast free ophthalmic solution in a concentration of 0.015%. Analysis by capillary zone electrophoresis shows shelflife stability of up to four years at room temperature of this enantiomer. The inhibitory effect of S(+) flurbiprofen on the synthesis of prostaglandins, as measured in a homogenate of bovine iris/ciliary body, remained unaffected during a shelflife period of three years after manufacture. Chapter 4 describes the pharmacological activity of different flurbiprofen preparations in isolated bovine iris/ciliary body homogenates. Concentration-response curves have been determined for S(+) flurbiprofen, R(-) flurbiprofen as well as the racemate, to inhibit PGE2 production mediated by COX-1. A significant difference between the enantiomers was established. S(+) flurbiprofen proved one hundred times more potent than R(-) flurbiprofen. It was concluded that S(+) flurbiprofen is the active component that should be incorporated in the ophthalmic solution. Using the human whole blood assay as described by Patrignani et al. (1994), in chapter 5 differences were detected between S(+) flurbiprofen, R(-) flurbiprofen and racemic flurbiprofen for inhibition of COX-1 and COX-2. COX-1 activity was monitored by measuring TxB2 (the stable metabolite of TxA2) production from the platelets, whereas COX-2 activity was determined using PGE2 production in monocytes, following induction of this enzyme by LPS. The stereoselectivity of S(+) flurbiprofen compared to R(-) flurbiprofen, expressed as the reciprocal of the ratio of the concentrations giving 50% inhibition (IC50), amounted to 340 for COX-1 and 56 for COX-2. The selectivity of racemic flurbiprofen for COX-1 versus COX-2, was 16-fold. In chapter 6 the interaction of S(+) flurbiprofen with COX-1 and COX-2 in the human iris was studied. After LPS-treatment for 24h, substantial amounts of COX-2 immunoreactivity could be visualized for the first time in human iris/ciliary body preparations. Remarkably, S(+) flurbiprofen showed a 3,600-fold higher potency for inhibiting COX-1 compared to COX-2. Furthermore, the susceptibility of human iris COX-1 for inhibition by S(+) flurbiprofen was 70-fold higher than of COX-1 in human blood. In chapter 7 the distribution of a flurbiprofen analogue in the human eye has been visualised. Technetium labelled diflunisal, sharing pharmacological and chemical resemblance with flurbiprofen as an NSAID being fluorinated and possessing a biphenyl ring, was used in an attempt to visualize COX-activity in the internal structures of the eye. The scintigraphic results obtained with this labelled drug were compared with instillation of the same volume and activity of pertechnetate. An

127 Summary

amount of 3% of instilled technetium labelled diflunisal could be localized in the eye. Activity could be visualized in the area of the iris indeed. Diflunisal was used because the labelling efficiency of S(+) flurbiprofen proved not appropriate (Chapter 8). R(-) flurbiprofen would chemically be a good labelling agent, but not pharmacologically, for obvious reasons (Chapters 5 and 6). In the final Chapter the occurrence of alternative splicing of COX-1 in RNA in the human iris was explored, as a possible explanation of the remarkably high affinity of S(+) flurbiprofen reported in Chapter 6. Indeed an alternatively spliced mRNA COX-1 splice variant (SV) could be detected in the human iris tissue from 3 patients. However, the same splice variant was also found in blood cells derived from four individuals. The amount of COX-1SV present in the human iris was not significantly different from the amount present in blood cells, implying that the occurrence of the COX-1 splice variant in the iris can not explain the observed dif-

ference in IC50 by S(+) flurbiprofen between human iris (0.8 nM) and human blood (56 nM) for COX-1 inhibition.

128 SAMENVATTING Samenvatting

SAMENVATTING

De operatieve verwijdering van de ooglens, tegenwoordig gevolgd door implantatie van een kunstlens, is vandaag de dag de voorkeurstherapie voor de behandeling van ouderdomscataract (staar). Vanaf de eerste toepassingen van deze therapie zijn ernstige bijwerkingen in de macula van het netvlies gerapporteerd, welke het gezichtsvermogen van de patiënt ernstig kunnen aantasten. Deze complicatie werd voor het eerst beschreven in 1953 en staat bekend onder de naam cystoid macula oedeem. De incidentie van deze aandoening varieert van 2 tot 50%, maar spo- radisch worden percentages van 70% gemeld. Eind jaren zeventig werd in een aan- tal Japanse publicaties het gebruik van indomethacine oogdruppels ter preventie van het cystoid macula oedeem beschreven. Op grond hiervan werd in 1980 in de klinieken van Arnhem door een van de oogartsen aan de ziekenhuisapotheker gevraagd of deze een indomethacine oogdruppel kon samenstellen, omdat deze (nog) niet in de handel was. Deze vraag was uiteindelijk de aanleiding tot het ontstaan van dit proefschrift. Na deze eerste publicaties over de preventie van cystoid macula oedeem, zijn er diverse formuleringen van indomethacine oogdruppels beschreven in de Nederlandse en internationale literatuur. Indomethacine is een remmer van de synthese van prostaglandines, een belangrijke klasse van ontstekingsmediatoren, ook bij deze aandoening. Indomethacine oogdruppels werden geïntroduceerd in verschillende formuleringen waaronder oplossingen in sesamolie, waterige suspen- sies en waterige oplossingen. Concentraties van oplossingen in olie varieerden van 0,1 tot 1,0%, in suspensie oogdruppels van 0,5% tot 1% en in waterige oplossingen van 0,35 tot 1,0%.

Helaas leidde het gebruik van olieachtige oogdruppels tot klachten over wazig zien, veroorzaakt door het verschil in brekingsindex. De waterige oplossingen daarente- gen - hetzij als suspensie, hetzij als oplossing - veroorzaakten vaak irritatie van het oog. Om van deze, hinderlijke bijwerking af te komen werd een verlaging van de indomethacine concentratie voorgesteld van 1% naar 0,2% of 0,1%. In 1981 werd aan de hand van vergelijkend onderzoek van vier indomethacine suspensie-oogdruppels (0,5%) geconstateerd, dat de mate van prostaglandinesynthese remming nogal varieerde, waarschijnlijk als gevolg van verschillen in fysisch chemische eigenschappen. Geconcludeerd werd dat het gebruik van suspensie- oogdruppels niet reproduceerbare farmacokinetische en farmacodynamische uitkomsten gaf. In antwoord op bovengenoemde vraag werd door ons met succes een waterige fosfaat-gebufferde indomethacine oplossing (pH 7,4) ontwikkeld in een concentratie van 0,1%, waarbij de indomethacine in oplossing werd gebracht met behulp

130 Samenvatting

van de organische base meglumine. Deze oogdruppel werd in 1981 in de kliniek geïntroduceerd en had geen irriterende bijwerkingen in het oog.

In 1984 werd Indoptol®, een 1% indomethacine suspensie oogdruppel, op de Nederlandse markt geïntroduceerd en 1986 werd Indocid®, met een vergelijkbare samenstelling, in Frankrijk op de markt gebracht. In 1987 volgde een nieuw ontwikkelde vorm van de indomethacine oogdruppel, Indocollyre®, in een sterkte van 0,1%. Introductie van dit product op de Nederlandse markt geschiedde in 1994. Deze formulering bevat indomethacine als gevriesdroogd product, hetgeen in oplossing gebracht kan worden door additie van een steriele boraatbuffer. Nader onderzoek van de eigen indomethacine oogdruppel met verschillende organische basen zoals L-lysine, D-lysine, L-arginine, D-arginine en trometamol leverde geen verbeterde oplossing op. De in 1981 geïntroduceerde, aseptisch bereide, gebufferde indomethacine oplossing (pH 7,4) in een concentratie van 0,1%, zonder verdere farmaceutische toevoegingen bleef dientengevolge dé oplossing in onze kliniek. Deze oplossing werd farmacologisch onderzocht middels het paracentese model, waarbij vocht aan de voorste oogkamer van het konijnenoog wordt onttrokken en de influx van eiwit en fluoresceïne in het secundair gevormde oogkamerwater (als maat voor de verbreking van de bloed-kamerwater barrière) gemeten wordt. Uit de resultaten bleek dat een concentratie van slechts 0,05% indomethacine al resul- teerde in 90 - 100% remming van eiwit- en fluoresceïne- instroom. Op grond van deze resultaten werd de door ons ontwikkelde indomethacine formulering in 1986 opgenomen in het FNA. Nadeel van deze formulering is echter, dat het hier een aseptische bereiding van een oogdruppeloplossing betreft met een beperkte houdbaarheid. Hierdoor ontstond het idee om te onderzoeken of er ook oogdruppels te formuleren waren op basis van een andere NSAID.

In 1990 werd gerapporteerd dat S(+) ibuprofen in een concentratie van respec- tievelijk 0,9% en 0,8% effectief was in de bestrijding van door interleukine-1 of door paracentese geïnduceerde uveitis. Ook van S(+) naproxen, op de markt gebracht door de firma Syntex als een zuiver enantiomeer, werd het anti-inflammatoir effect als oogdruppel (0,5%) experimenteel aangetoond. Op zoek naar een farmaceutisch meer compatibele oplossing voor een andere NSAID werd in de United States Pharmacopeia (USP) de monografie van flurbiprofen sodium ophthalmic solution aangetroffen , terwijl in Nederland de introductie van een diclofenac oogdruppel (Naclof®) op handen was. In 1992 werd het plan opgevat om flurbiprofen oogdruppels te gaan bereiden. Het project werd ter beoordeling aangeboden aan de Stichting Wetenschappelijk

131 Samenvatting

Onderzoek Rijnstate (9206SO.008) en in een brief van 8 januari 1993 werd beperkte financiële steun toegezegd. De scope van dit proefschrift betreft een onderzoek naar de farmaceutische bereiding en -eigenschappen van flurbiprofen oogdruppels en de farmacologie van dit NSAID. Flurbiprofen is een chiraal molecuul, wat inhoudt dat de racemische vorm mogelijk niet de gewenste farmacologische entiteit is voor de bereiding van oogdruppels. Derhalve werd het noodzakelijk geacht de bijdrage van elk enantiomeer afzonderlijk te onderzoeken, zodat een gefundeerde keuze gedaan kon worden voor de bereiding van een oogdruppel met de meest actieve chirale entiteit. Bij de aanvang van dit onderzoek werd aangenomen dat de farmacologische werking van flurbiprofen gebaseerd is op remming van het enzym cyclooxygenase (COX), dat verantwoordelijk is voor de synthese van prostaglandines. In 1991 werd duidelijk, dat niet één maar twee isoenzymen (COX-1 en COX-2) betrokken zijn bij de synthese van prostaglandines. Het onderzoek kreeg hierdoor een nieuwe uitdaging want er was ook geconstateerd, dat dit tweede isoenzym sterker aan ontsteking gerelateerd zou zijn - en derhalve een beter doelwit voor NSAIDs - dan COX-1. Er werd besloten het racemisch mengsel alsmede de individuele enantiomeren van flurbiprofen te onderzoeken op hun prostaglandinesynthese remmende activiteit zowel intra- als extra-oculair, en zowel op COX-1 als op COX-2. In hoofdstuk 2 wordt het gekozen buffer-systeem beargumenteerd. Ofschoon het oog, speciaal de neushoek, de oogleden en de huid rondom het oog gevoelig zijn voor externe stimuli, zijn fysiologische reacties ten gevolge van afwijkingen buiten de normaalwaarden van osmolaliteit of pH niet altijd duidelijk. Bij ziekte of bij chro- nisch gebruik van oogheelkundige preparaten kan dit echter meer op de voorgrond treden. Het farmacologische agens in een oogdruppel kan, wanneer niet goed opgelost, een prikkelende of branderige sensatie veroorzaken, hetgeen weer kan leiden tot traanproductie, soms zelfs tot bloedingen of tot gevaarlijke knipperreflex- en gedurende een operatieve ingreep. Overmatige traanproductie leidt tot ongewenste verdunning van de oogdruppel en zelfs tot het uitspoelen van het geneesmiddel. De individuele gevoeligheid kan variëren en de fysiologische waarden van het traanvocht kunnen fluctueren afhankelijk van de gezondheidsstatus van het betreffende oog. Irritatie van het oog moet echter onderscheiden worden van een allergische reactie. In het laatste geval dient een ander farmacologisch agens gekozen worden. Niet-irriterende oogdruppels moeten in principe voldoen aan eisen betreffende: (1) steriliteit (2) isotoniciteit en (3) pH waarde. Steriliteit is van eminent belang bij toepassing van oogdruppels in een beschadigd oog. Wanneer de oplossing isotoon is met het traanvocht, zullen irritaties en reacties van het oog tot een minimum beperkt blijven. De pH waarde van de formulering is van groot belang, zowel voor de stabiliteit gedurende opslag als voor het handhaven van fysiologische condities

132 Samenvatting

in het oog. Gedurende opslag kunnen hydroxyl ionen, afkomstig uit het glas van de oogdruppeldispenser, de pH omhoog brengen hetgeen de stabiliteit van het farmacologisch agens in gevaar kan brengen. Aan de andere kant heeft traanvocht slechts een beperkte buffercapaciteit. Bij de keuze voor een buffer ten behoeve van de bereiding van een flurbiprofen oogdruppel moest met de volgende gegevens rekening gehouden worden: (1) flurbiprofen is instabiel bij hoge pH, (2) de oplosbaarheid in waterige oplossingen is slecht bij pH waarden lager dan 7, (3) de normale pH waarde van traanvocht is 7,4 en (4) de tolerantie van de patiënt ligt tussen pH 6,6 tot 7,8. Op basis van de ervaring met de indomethacine oogdruppel werd voor het bereiden van de S(+) flurbiprofen oogdruppels gekozen voor een fosfaatbuffer (pH 7,4). Het specialité Ocuflur® (racemisch flurbiprofen natrium) is bereid in een citraatbuffer van pH 6,45. Dit werd door ons echter als een ongeschikt buffersysteem beschouwd, omdat voor een citraatbuffer een bovenste grens in buffercapaciteit van pH 6,5 bekend is. Een zelfde conclusie wordt overigens ook getrokken in het United States patent 4.996.209, waar een fosfaatbuffer wordt gemeld voor de samenstelling van een anti-inflammatoire S(+) flurbiprofen oogdruppel. In deze oogdruppel komen echter ook andere componenten voor. De samenstelling van de oogdruppel toegepast in dit proefschrift is vrij van die andere componenten. In hoofdstuk 3 wordt de stabiliteit van de formulering onderzocht. Van het actieve S(+) enantiomeer van flurbiprofen, is een stereoselectieve, ballastvrije oogdruppel geformuleerd in een concentratie van 0,015%. Analyse per capillaire zone electro- forese techniek toonde een houdbaarheid aan van 4 jaar bij kamertemperatuur. De prostaglandine-synthese remmende activiteit van S(+) flurbiprofen, gemeten in een homogenaat van runder iris/corpus ciliare weefsel, bleek drie jaar na bereiding onveranderd. Hoofdstuk 4 beschrijft de farmacologische activiteit van verschillende flurbiprofen bereidingen in runder iris/corpus ciliare homogenaten. Concentratie-respons cur- ven zijn gemeten voor S(+) flurbiprofen, R(-) flurbiprofen en het racemisch mengsel betreffende remming van PGE2 productie door COX-1. Een significant onderscheid in remmende werking werd vastgesteld tussen de enantiomeren, waarbij S(+) flurbiprofen honderd maal actiever bleek te zijn dan R(-) flurbiprofen. Geconcludeerd kon worden dat S(+) flurbiprofen het actieve farmacologische agens is dat onderdeel zou moeten uitmaken van een oogdruppel oplossing ten einde de prostaglandinesynthese te remmen. In hoofdstuk 5 wordt beschreven hoe, met toepassing van de methode van Patrignani et al., in humaan bloed, verschillen aangetoond werden in de remmende activiteiten van S(+) flurbiprofen, R(-) flurbiprofen en racemisch flurbiprofen op de isoenzymen cyclooxygenase-1 en cyclooxygenase-2. COX-1 activiteit werd gemeten aan de hand van TxB2 (de stabiele metaboliet van TxA2) geproduceerd door

133 Samenvatting

bloedplaatjes. COX-2 activiteit werd gemeten met behulp van PGE2 productie door monocyten na inductie van dit enzym door lipopolysaccharide (LPS). De stereose- lectiviteit van S(+) flurbiprofen ten opzichte van R(-) flurbiprofen, uitgedrukt als de

reciproke waarde van de ratio van de concentraties die 50% remming geven (IC50), liepen uiteen van 340 voor COX-1 tot 56 voor COX-2. De selectiviteit van racemisch flurbiprofen voor COX-1 versus COX-2 was 16-voudig. In hoofdstuk 6 is de interactie van S(+) flurbiprofen met COX-1 versus COX-2 in de humane iris bestudeerd. Na voorbehandeling met LPS kwam een aanzienlijke hoeveelheid COX-2 immunoreactiviteit naar voren. Verrassenderwijs toonde S(+) flurbiprofen een 3600-voudig hogere potentie tot remming van COX-1 versus COX- 2. Verder bleek de gevoeligheid van humaan iris COX-1 voor S(+) flurbiprofen 70 maal hoger dan de gevoeligheid van COX-1 in menselijk bloed. In hoofstuk 7 wordt de verdeling van een flurbiprofen analogon over het menselijk oog onderzocht. Technetium gelabeled diflunisal, een chemisch op flurbiprofen gelijkend NSAID - gefluorineerd met eveneens een bifenyl ring - werd toegepast om de COX-activiteit in de interne structuren van het oog te kunnen visualiseren. De scintigrafische resultaten van dit gelabelde farmacon werden vergeleken met die van de instillatie van eenzelfde volume en activiteit pertechnetaat anion. Een hoeveelheid van 3% van het geinstilleerde technetium gelabelde diflunisal kon in het oog worden getraceerd. COX-activiteit werd duidelijk gevisualiseerd in de regio van de iris. In dit onderzoek werd diflunisal toegepast omdat de labelingsef- ficiency van S(+) flurbiprofen aan de (te) lage kant was (hoofdstuk 8). R(-) flurbiprofen leverde weliswaar een betere labeling op, maar farmacologisch is dit het inac- tieve enantiomeer (hoofdstukken 5 en 6). In het laatste hoofdstuk is het voorkomen van een splice variant van COX-1 mRNA in de menselijke iris onderzocht als een mogelijke verklaring voor de hoge affiniteit van S(+) flurbiprofen gerapporteerd in hoofdstuk 6. Inderdaad werd een alternatieve splice variant (SV) van COX-1 aangetroffen in iris weefsel afkomstig van 3 patiënten. Echter, dezelfde splice variant werd ook gevonden in bloedcellen afkomstig van 4 vrijwilligers. De hoeveelheid COX-1SV aanwezig in de iris was niet significant verschillend van de hoeveelheid aanwezig in bloed cellen, hetgeen

impliceert dat daarmee het verschil in IC50 van S(+) flurbiprofen tussen humane iris (0,8 nM) en humaan bloed (56 nM) niet kan worden verklaard.

134 LIST OF PUBLICATIONS List of publications

1. van Sorge AA, Wijnen PH, van Delft JL, Carballosa Coré-Bodelier VMW, van Haeringen NJ. Flurbiprofen, S(+), eyedrops: formulation, enantiomeric assay, shelflife and pharmacology. Derived from Pharm World Sci 1999;21:91-5. 2. Sorge van AA, Delft van JL, Bodelier VMW, Wijnen PH, Haeringen van NJ. Specificity of flurbiprofen and enantiomers for inhibition of prostaglandin synthesis in bovine iris/ciliary body. Prostaglandins Other Lipid Mediat 1998;55:169-77. 3. Haeringen van NJ, Sorge van AA, Delft van JL, Carballosa Coré-Bodelier VMW. Flurbiprofen and enantiomers in ophthalmic solution tested as inhibitors of prostanoid synthesis in human blood. J Ocular Pharmacol 2000;16:345-52. 4. Haeringen van NJ, Sorge van AA, Carballosa Coré-Bodelier VMW. Constitutive cyclooxygenase-1 and induced cyclooxygenase-2 in isolated human iris inhibited by S(+) flurbiprofen. J Ocular Pharmacol 2000;16:353-61. 5. Sorge van AA, Etten van RJ, Rehmann CJ, Rijnders AJM, Haeringen van NJ. 99mTc-Diflunisal and the human iris: topical application reveals localization. J Ocular Pharmacol 2002;18:185-195. 6. Sorge van AA, Ruiken I, Janssen HWM, Haeringen NJ. S(+) flurbiprofen and R(-) flurbiprofen. 99mTc-labeling reveals difference in stereochemistry. Enantiomer; accepted pending suitable revision. 7. Dröge MJ, van Sorge AA, van Haeringen NJ, Quax WJ, Zaagsma J. Alternative splicing of cyclooxygenase-1 mRNA in the human iris. Submitted.

136 DANKWOORD Dankwoord

DANKWOORD

De wijze waar op dit proefschrift tot stand is gekomen is voor menigeen onbegrij- pelijk. Met slechts ƒ 1800,- aan "venture capital", verkregen via de SWOR van ziekenhuis Rijnstate, lijkt dit een onmogelijke zaak. Het kon alleen maar lukken door de enthousiaste, belangeloze medewerking van velen. Hierdoor loop ik het risico, dat ik in dit dankwoord iemand kan vergeten en daar bied ik bij voorbaat al mijn welgemeende excuses voor aan. Door mijn komst naar Arnhem kwam ik samen te werken met mijn collega's Jhr E.B.L.M. van Nispen tot Pannerden (St Elisabeth's Gasthuis; EG) en J.C. Kutsch Lojenga (Gemeente ziekenhuis). Beiden waren gedreven ziekenhuisapotheker met name door de directe lijnen tussen de kliniek en de apotheek. Hier bouwde ik verder aan mijn kennis en leerde ik nog meer in te spelen op vragen uit de kliniek, zowel verpleegkundig, organisatorisch als medisch-farmaceutisch. Het contact met de medici was zeer veelvuldig en vaak informeel, vooral in het EG waar een geza- menlijk koffiedrinken de onderlinge contacten bevorderde. Tijdens één van deze contacten kwam dr J.P. (Paul) de Haas, oogarts, met de vraag of wij een preparaat konden bereiden, dat nog niet commercieel beschikbaar was, maar volgens de literatuur wel van groot nut zou zijn bij de nabehandeling van patiënten, die een staaroperatie hadden ondergaan. Een kolfje naar de hand des ziekenhuisapothekers! Dit leidde tot de ontwikkeling van de indomethacine oogdruppels. Het was evenwel zaak, dat een en ander ook onderbouwd werd en al spoedig viel de naam van collega Nico van Haeringen, traanspecialist van wereldfaam en verbonden aan het IOI (Interuniversitair Oogheelkundig Instituut) gelokaliseerd "onder" het AMC, te Amsterdam alsmede thuis aan het LUMC te Leiden. Na diens farmacologische onderbouwing van de indomethacine oogdruppels, volgde een publi- catie en werd deze oogdruppel uiteindelijk standaard opgenomen in het Formularium der Nederlandse Apothekers (FNA). Na afloop van dit projekt verklaarde Nico zich bereid om in de toekomst mee te werken aan het farmacologisch onderbouwen/onderzoeken van eventueel nieuw te ontwikkelen oogdruppels. Het zou evenwel tot 1991 duren alvorens wij weer contact zouden hebben. Naar aanleiding van vragen en opmerkingen uit het farmaceutische en medische veld was inmiddels gebroed op een nieuwe NSAID oogdruppel. Het "oog viel" op flurbiprofen en de eerste proefnemingen volgden spoedig. Onze apothekers-assi- stenten en niet minder de vele bijna-apotheker stagiaires, wil ik hierbij hartelijk danken voor alle inspanningen die zij leverden om steeds weer een en ander te bereiden, te steriliseren enzovoort. Het was niet altijd even makkelijk, zeker niet in een tijd van fusieperikelen. Ook de analisten van ons farmaceutisch-toxicologisch laboratorium wil ik dank zeggen voor het oneindige geduld dat opgebracht werd om

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toch weer een en ander te analyseren. Het noemen van namen gaat hierbij mogelijk te ver, zo veel zijn het er geweest; een ieder heeft er zonder twijfel "last van gehad". Voor het farmacologisch onderzoek volgden wederom vele paracenteses in de konijnenstallen van het LUMC nu met flurbiprofen en diens enantiomeren als farmacologisch agens. Veel dank ben ik hierbij verschuldigd aan Jan van Delft, een expert op dit gebied. Ook Ed Barthen wil ik danken. Steeds stond hij klaar om ons te helpen, ook bij het verkrijgen en behandelen van humaan irismateriaal. Door renovatie van de dierenstallen van het LUMC werd het werkterrein verplaatst naar het IOI te Amsterdam alwaar nog enige experimenten met konijnen volgden. Dank zij toestemming van de direkteur van het IOI, Prof Paulus de Jong, mocht ik als gast-medewerker aldaar verder gaan. Hiervoor ben ik jou, Paulus, zeer erkentelijk. Spoedig evenwel werd overgestapt op de human whole blood assay. Deze techniek werd opgedaan tijdens een internationaal congres te Cannes en het was weer aan het eminent biochemisch inzicht en jouw praktische ervaring, Nico, dat wij hiermee aan de slag konden gaan. Uiteraard mijn hartelijk dank hiervoor. Door deze veranderde werkwijze verplaatsten de proefnemingen zich naar het biochemisch laboratorium, alwaar Valérie Bodelier als voortreffelijk analiste de voortgang van het onderzoek waarborgde. Ik dank haar bij deze voor het vele werk dat met toewijding en expertise is verricht. Naarmate de proefnemingen vorderden werd ook gedacht de rundermatrix te ver- vangen door die van de mens. Hierin heeft Dr Liesbeth Pels ons bijgestaan en daar ben ik haar zeer erkentelijk voor. Voorafgaand en parallel aan deze ontwikkelingen werd ook de farmaceutische kwaliteit van de flurbiprofen oogdruppels onderzocht. Toen er in de wetenschappelijke bijlage van het NRC Handelsblad een artikel over "Spiegelbeelden scheiden met een membraan" verscheen van de hand van onderzoekers verbonden aan het laboratorium van AKZO NOBEL Central Research, Dr Ir J.T.F. Keurentjes en Ir E. van Andel, waarin een nieuwe methode werd besproken om zuivere stereoiso- meren te bereiden, werd besloten contact met hen op te nemen om te bezien of deze methode voor de ziekenhuisapotheek bruikbaar kon zijn en dan in het bijzonder voor de bereiding van flurbiprofen enantiomeren. Andere enantiomeren van farmaceutisch belang, zoals bupivacaine en ketamine, werden ook tot de mogelijkheden gerekend. Uit dit contact volgde een afspraak met collega Mw Dr M. Leloux voor verdere bespreking. Ten leste werd contact gelegd met het laboratorium voor Central Research waar Peter Wijnen toestemming verleende de houdbaarheid- sproeven van de flurbiprofen isomeren aldaar te verrichten. Voor deze medewerking zijn wij AKZO NOBEL, en Peter Wijnen in het bijzonder, zeer erkentelijk. Het bijzondere is natuurlijk dat dit heeft kunnen plaatsvinden bij een multinational en ook nog in de stad Arnhem.

139 Dankwoord

Tijdens deze periode zijn eveneens congressen bezocht en voordrachten gehouden over de vorderingen, die gemaakt waren. Dit leidde tot het contact met Dr Johan Bours, eiwitexpert op oogheelkundig gebied, die veel van zijn kennis hierover heeft overgebracht. Voor deze stimulerende gesprekken, Johan, ben ik je veel dank verschuldigd. Al dit werk was zeker niet mogelijk geweest als mijn (computer)vriend van de eerste uren, Lex Dreves, mij niet geholpen had bij het leren omgaan met de nieuw geintroduceerde techniek van de computer. Ondanks zijn handicap van de ziekte cystic fibrosis, kon hij het opbrengen om mij hierbij met raad en daad bij te staan. Het was dan ook een enorme set-back hem op 30 jarige leeftijd te moeten ver- liezen. Via zijn ouders wil ik Lex alsnog hartelijk danken voor zijn ondersteuning. Ook mijn andere (computer)vrienden, Max Wolff en Carla Hulsewé, wil ik hartelijk danken voor de vele fijne gesprekken over het vorderen van het werk en het uit- zoeken van de juiste computer-configuratie, zodat dit proefschrift tot een goed einde kon worden gebracht. Vermeldenswaard en zeker een compliment, verdient de staf van onze biblio- theek. De dames Mieke Noordegraaf, Erna Altena, Anje van den Berg en Tanja van Bon zijn van grote klasse in het opzoeken en opvragen van de gezochte literatuur. Piet Huggers van het laboratorium voor pathologie dank ik voor het professioneel bewaren van de iris monsters alvorens deze te toe te passen voor onderzoek. Voorts wil ik ook de directie van ons ziekenhuis bedanken voor de mogelijkheden die onze kliniek heeft voor het doen van wetenschappelijk onderzoek. De staf en collegae ziekenhuisapothekers en Guus Essink in het bijzonder, wil ik danken voor de direkte dan wel indirekte steun bij het werk zodat ik een en ander tot een goed einde kon brengen. De oogartsen van ziekenhuis Rijnstate wil ik hartelijk danken, dat zij steeds open- stonden voor nieuwe ideeën op het gebied van de farmaceutische oogheelkunde. Aan Maarten Jonkers kan ik nu eindelijk de prachtige bijbels van de ophthalmolo- gie teruggeven! Maarten, hartelijk dank voor je geduld in deze. Ook de paranimfen, Rob Weber en Arnold Lombarts wil ik hartelijk danken, voor hun spontane toezegging te helpen daar waar mogelijk. Door deze promotie is eindelijk de cirkel rond en kunnen we alledrie terugzien op een optreden als paranimf bij elkaars promotie. Dr J.C. Verhoef, beste Hans, jou zou ik willen bedanken voor de vele uren die wij samen hebben besteed aan het hoofdstuk over de achtergronden van de gebruik- te buffer. Jouw kennis over de electrochemie nodigt uit om samen nog een hoofdstuk te schrijven over een andere benadering van de cyclooxygenase gemedieerde reactie. Hartelijk dank voor de door jou geleverde inspanning en je toezegging te willen opponeren. Bovendien bracht je mij in 2001 in kontakt met Willy H.J. Boesten, corporate scientist (scheikundige èn uitvinder) bij DSM Research, die

140 Dankwoord

bereid was een search te doen naar artikelen over de synthese van enantiomeren en die van flurbiprofen in het bijzonder. Spoedig volgde een dikke stapel literatuur, alwaar tot mijn verbazing ook een patent in terug te vinden was over het gebruik van S(+)flurbiprofen, en wel opgelost in fosfaatbuffer. Bij deze wil ik hem hiervoor hartelijk danken. Ook wil ik een woord van dank uitbrengen aan Dr Hector Knight van de firma Mallinckrodt. De bespreking van de resultaten van hoofdstuk 8 aangaande de labeling van de flurbiprofen enantiomeren heb ik bijzonder gewaardeerd. Een opzet naar een volgend onderzoek maar dan met labeling middels de IsoLink® techniek lijkt veelbelovend. Tot slot van deze historisch getinte opbouw van mijn dankwoord wil ik nogmaals Nico van Haeringen hartelijk danken voor de wetenschappelijke begeleiding. Je immer rustige maar trefzekere inschatting van het werk hebben bijgedragen aan de voltooiing van dit opus. Ook Eelco van Nispen wil ik nogmaals bedanken: reikhalzend zag hij uit naar de afronding van dit project. De Hoogleraren Prof Dr J.E.E. Keunen (LUMC), Prof Dr M.D. DeSmet (AMC), Prof. Dr. J.M.M. Hooymans (Academisch ziekenhuis Groningen), Prof. Dr. W. Timens (Academisch ziekenhuis Groningen) en Prof. Dr. F Muskiet (Academisch ziekenhuis Groningen) alsmede Mw A. Brugman (Academisch ziekenhuis Groningen) en Dr. M. Elferink (Rijks Universiteit Groningen) wil ik hartelijk danken voor hun medewerking bij het tot stand komen van het laatste hoofdstuk. In het bijzonder wil ik nog bedanken Drs Melloney Dröge wier conscientieus werk het laatste hoofdstuk praktisch mogelijk heeft gemaakt. De leden van de beoordelingscommissie, Prof Dr P.T.V.M. de Jong, Prof Dr J.R.B.J. Brouwers, en Prof Dr H.V. Wikström wil ik bedanken voor hun bereidheid het manuscript te beoordelen. De laatsten in de lange rij van personen, die ik wil bedanken voor hun bijdrage aan de tot standkoming van dit proefschrift, zijn de hoogleraren, Prof Dr H.W. Frijlink, Prof Dr W.J. Quax en Prof Dr J. Zaagsma. Zij kwamen bij mijn eerste bezoek in de zomer van 1999 tot de conclusie dat dit werk vatbaar was voor een promotie.

In het bijzonder wil ik hier de unieke begeleiding van Prof Dr J. Zaagsma ver- melden. Beste Hans, waar op (niet-academische, perifere) afstand een en ander toch wel moeilijk kan zijn is het je gelukt een bijzondere band op te bouwen. Als ziekenhuisapotheker, onkundig van de gang van zaken op het academisch pro- motievlak, heb ik het bijzonder gewaardeerd een zo plezierige ondersteuning en bejegening te mogen ontvangen. De vele doorhalingen, correcties en voorstellen middels het "ouderwetse" correctiepotlood waren altijd bestemd om tot een betere formulering te komen. Ook de meer moderne middelen als fax en e-mail zijn inten-

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sief gebruikt. Je organisatietalent heeft het mogelijk gemaakt, dat deze promotie nog dit jaar heeft kunnen plaats vinden. Hiervoor mijn oprechte dank. Ter afsluiting wil ik natuurlijk mijn ouders bedanken, die mij de mogelijkheid hebben geboden een universitaire scholing te doorlopen, hetgeen in die tijd niet altijd vanzelfsprekend was. En last but not least gaat mijn grote dankbaarheid uit naar Aty en de kinderen die zich afvroegen of dit allemaal wel de moeite waard was en het desondanks toch met groot enthousiasme hebben gesteund. Een historisch analogon is hier op zijn plaats:

"Quosque tandem A3aan abutere patientiam nostram"? s. Cicero

142 CURRICULUM VITAE Curriculum Vitae

CURRICULUM VITAE

Adriaan Alastair van Sorge (geboren 28 oktober 1944) doorliep het Gereformeerd Gymnasium (ß) gelegen aan de Keizersgracht te Amsterdam. Na de vervulling van zijn dienstplicht startte hij in 1965 met de studie Farmacie aan de Universiteit van Amsterdam. Na voltooiing van deze studie met het doctoraal examen werd in 1972 het apothekers examen met goed gevolg afgelegd. Een korte periode was hij werkzaam in de officiene apotheek maar al snel werd gekozen voor een baan in de farmaceutische industrie (R&D; Philips Duphar). Aangestoken door enthousiaste artikelen over de klinische farmacie in de Verenigde Staten van de hand van collega Armbrust, diende hij een aanvraag in bij de University of California, School of Pharmacy, te San Francisco voor het doorlopen van een Postdoctoral Pharm D residency programme in Clinical Pharmacy (1973- 1974). Deze aanvraag werd gehonoreerd. Tijdens deze studie werd eveneens ken- nisgemaakt met de klinische farmacologie onder leiding van Dr. K.L. Melmon en Dr. H.F. Morrelli. Het geheel werd afgesloten met het predikaat Pharm D. Na terugkeer uit de Verenigde Staten volgde hij de opleiding tot ziekenhuisapotheker in de Apotheek Haagse Ziekenhuizen. Tijdens deze opleiding maakte hij kennis met de interne school van Dr F. Kalsbeek (Leyenburg ziekenhuis) en werkte onder diens leiding mee aan de tweede druk van Materia Medica Selecta ("Een praktische handleiding voor het rationeel kiezen en voorschrijven van geneesmiddelen"). Tevens werd de farmacotherapeutische zorg ten behoeve van het Westeinde Ziekenhuis aan de auteur toevertrouwd. In 1976 werd de opleiding tot ziekenhuisapotheker afgesloten met een registratie onderwerp betreffende de remming van prostaglandinesynthese door niet steroide anti-inflammatoire middelen. Het werkzame leven als ziekenhuisapotheker in Arnhem werd aangevangen met een full-time functie verdeeld over het St Elisabeth's Gasthuis (hoofd Jhr E.B.L.M. van Nispen tot Pannerden) en het Gemeente Ziekenhuis (hoofd J.C. Kutsch Lojenga). Het diploma "Bevoegdheid beheer C-laboratorium" niveau 3 werd in 1979 behaald. Vanaf 1981 tot de dies finalis maakte van Sorge deel uit van de Geneesmiddelen- commissie van het Ministerie van WVC, eerst als lid en vervolgens als plaatsver- vangend voorzitter. De auteur is lid geweest van verscheidene ziekenhuisgebonden commissies. Vanaf 1987 fungeerde de auteur, met een kleine onderbreking, als opleider van apothekers in opleiding tot ziekenhuisapotheker. Inmiddels waren beide ziekenhuizen gefuseerd tot ziekenhuis De Malberg, in een twee-lokatiemodel. Met de "echte" fusie tot ziekenhuis Rijnstate in 1990, door het samengaan van De Malberg met het Diaconessenhuis, werd het team versterkt met collega dr. J.S. Meulenhoff.

144 146 COLOR PICTURES Color pictures

pH 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.2

Chapter 2. As far as flurbiprofen is concerned, the solubility in aqueous solution is problematic at pH values below 7.

Chapter 6, figure 1. Detection of cyclooxygenase in human iris extracts. Authentic COX-1 and COX-2 peptide and 15 ml of tissue extract were spotblotted on nitrocellulose membrane. Cyclooxygenase was detected using immunoglobulin G specific for COX-1 or for COX-2. With anti-COX-1, spots were observed in untreated iris, LPS-treated iris and with COX-1 peptide. With anti-COX-2, spots were observed in LPS-treated iris only and with COX-2 peptide.

148 Color pictures

Chapter 7, figure 6. Scintigraphic Image of the Eye Surface and the Lacrimal Sac 160 seconds after Application of 99mTc Diflunisal (frame 16). Graph represents counts versus time for whole session.

Chapter 7, figure 7. Scintigraphic Image of the Area of the Iris and the Lacrimal Sac 1170 seconds after Application of 99mTc-Diflunisal (frame 117). Graph represents counts versus time for whole session.

149 Color pictures

Chapter 7, figure 8. Difference in Scintigraphic Exposure between Pertechnetate (left) and 99mTc-Diflunisal

99mTc-Diflunisal scintigraphy in selected eye structures

3,00 pupil

2,50 iris conjunctiva 2,00

1,50

1,00 counts per pixel

0,50

0,00 0 200 400 600 800 1000 1200 time (seconds)

Chapter 7, figure 9. Display of Counts Per Pixel for 99mTc-Diflunisal in the Area of Pupil, Iris and Conjunctiva

150 ADDENDUM Curriculum Vitae

In deze tijd werd, ondanks de hectiek van die jaren, de grondslag gelegd voor verder onderzoek met oogdruppels. Ook het kontakt met collega Dr Nico van Haeringen, zo behulpzaam geweest bij de farmacologische onderbouwing van de inmiddels landelijk bekende indometacine oogdruppels FNA, (samen met collega Van Nispen in 1981 ontwikkeld) werd weer aangehaald. Er werd begonnen met farmacologisch onderzoek in konijnen, maar dan met flurbiprofen oogdruppels. Het onderzoek heeft zich in de beginjaren afgespeeld in het Leids Universitair Medisch Centrum. Door vernieuwing van de konijnenstallen werd het onderzoek verplaatst naar het Interuniversitair Oogheelkundig Instituut, IOI, alwaar de auteur met toestemming van Professor Paulus de Jong, direkteur van het IOI, als gast- medewerker werkte. De farmaceutische analyses van de S(+) flurbiprofen oogdruppels werden verricht in het laboratorium van de apotheek van ziekenhuis Rijnstate alsmede in de laboratoria van AKZO-NOBEL te Arnhem (P.H. Wijnen). Voor het nucleaire onderdeel werd enthousiaste medewerking verleend door de afdeling Nucleaire Geneeskunde van Ziekenhuis Rijnstate (nucleair geneeskundi- gen A.J.M. Rijnders en C.J. Rehmann). In de zomer van 1999 werd contact gezocht met de huidige promotores, de professoren W.J. Quax, J. Zaagsma en H.W. Frijlink, om te bezien of een promotie tot de mogelijkheden behoorde. Dit contact leidde tot het laatste deel van het onderzoek dat te Groningen werd verricht, alwaar de professoren Zaagsma en Quax alsmede collega Drs Melloney Dröge de aanzet gaven tot de uitwerking van het laatste hoofdstuk.

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