University of Groningen Reflections on Flurbiprofen Eyedrops Van Sorge

University of Groningen

Reflections on flurbiprofen eyedrops van Sorge, Adriaan Alastair

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record

Publication date: 2002

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA): van Sorge, A. A. (2002). Reflections on flurbiprofen eyedrops. s.n.

Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment.

Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

Download date: 02-10-2021 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 com- mence 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 rea- sonable 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 sam- ple 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 con- sisted 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 contai- ners. 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) phosphor- ic 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 per- centage 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 accompany- ing 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 pro- duce 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 indi- cate 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 visi- ble 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 encompass- es 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.

105 Chapter 7

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 gas- trointestinal 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 irra- diation 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