Manual ofOculotoxicity Testingof Drugs

Edited by Otto Hockwin, Bonn/Fed. Rep. GermanyKeith Green, Augusta, GA/USALionel F. Rubin, Philadelphia, PA/USA

With contributions byC. Baumstark-Khan, G. Bessems f» J- Bours, H. F. Edelhauser,R. Garraffo, K. Green, O. Hockwin, E. M. Jackson, I. Korte,Ph. Lapalus, S. Lerman, C. B. Millard, U. Miiller-Breitenkamp,M. V. Riley, H. Rink, L. F. Rubin, L. Salminen, D. M. Schiavo,J.Schmidt, C. Schmitt, R. C. Tripathi, B.J. Tripathi, J. L. Ubels,A. Wegener, I. Weisse and E. Zrenner

164 figures and 33 tables

| Gustav Fischer VerlagSEMPER m Stuttgart • Jena • New York 1992

ContentsAuthors' addresses VII

Foreword IX

Part I: General introduction and routine methods 1

Chapter 1:Animal testing in biomedical researchE. M. Jackson 3

1 Biomedical research 31.1 History 31.2 Current scope of ophthalmological research 31.3 The impact of the Animal Rights Movement on biomedical

research 42 The Animal Rights Movement 42.1 The current movement 52.2 Industry response to the current movement 53 Alternatives to animal testing 53.1 Tissue culture methods 63.2 The Limulus Amebocyte Lysate (LAL) model 64 Legal/regulatory aspects of biomedical research 75 The future of biomedical research 7

Chapter 2:Special topics about the use of laboratory animals in toxicology — anophthalmoscopic assessmentD. M. Schiavo 9

1 Introduction 92 Environmental factors affecting laboratory animals 93 Some factors affecting drug absorption and formulation of eye

preparations • . . . 104 Important factor regarding oculotoxicity 115 Methods and grading system of ocular toxicity 116 Photographic illustration of ocular toxicity affecting various

laboratory animals — (Various compound classes) 137 Good laboratory practice — Ophthalmology 177.1 Regulatory guidelines 177.2 Ophthalmoscopic examinations 188 Suggested standard ophthalmic examinations procedures for

detection of ocular abnormalities, data recording-photomicrographs 18

Chapter 3:Comparative anatomy of the eyeL. F. Rubin 21

1 Orbit 212 Extraocular muscles 223 Eyelid 23

XII Contents

4 Lacrimal system 255 Conjunctiva 276 Nictitating membrane (third eyelid) 287 Cornea 298 Sclera 319 Iris 3110 Ciliary body 3311 Choroid 3412 Vitreous 3513 Retina 3714 Optic nerve 3915 Lens 41

Chapter 4:Comparative physiology and biochemistry of the eyeM. V. Riley and K. Green 45

1 Background 452 Physiology 452.1 Tear film 462.2 Corneal physiology 462.3 Wound healing 472.4 Intraocular pressure 482.5 Inflammation 482.6 Aqueous humor production 492.7 Outflow pathways 502.8 Iris 512.9 Blood flow 522.10 Lens 532.11 Vitreous humor 542.12 Retina 542.13 Aging effects 553 Biochemistry 563.1 Tear film 563.2 Cornea 573.3 Wound healing and alkali burns 583.4 Aqueous humor 593.5 Ciliary body and iris 613.6 Lens 613.7 Vitreous humor 633.8 Retina 634 Conclusion 654.1 Areas where data is lacking 654.2 Drug effects on physiology and biochemistry 65

Chapter 5:Current methods of eye examination in vivoL. F. Rubin 81

1 Examination area and restraint 812 Ophthalmic examination 82

Contents XIII

3 Examination procedures 843.1 Ophthalmic stains 843.2 Diagnosis of tear production abnormality 843.3 Esthesiometry 853.4 Corneal endothelial visualization: Specular microscopy 863.5 Measurement of corneal thickness: Pachymetry 863.6 Corneal and conjunctival cytology 873.7 Anterior chamber paracentesis 883.8 Examination of the drainage angle: Gonioscopy 883.9 Measurement of intraocular pressure 893.9.1 Manometry 903.9.2 Tonometry 903.9.3 Tonography 933.10 Measurement of episcleral venous pressure 943.11 Measurement of aqueous flow in rabbits 943.12 Slit lamp biomicroscopy 943.13 Scheimpflug photography 983.14 In vivo photomicrography of the lens 983.15 Examination of the ocular fundus 983.15.1 Directophthalmoscopy 983.15.2 Indirect ophthalmoscopy 1003.16 Fundus photography 1013.17 Fluorescein angiography 1033.18 Electroretinography 1043.19 Refraction 1053.20 Ultrasonography 106

Chapter 6:New non-invasive methods to evaluate ocular drug efficacy and side effectsS. Lerman 109

1 Introduction 1092 Ocular photosensitization 1093 Lens fluorescence I l l4 Evaluation of anticataract drug therapy 1135 Human lens fluorescence aging index 114

Chapter 7:Ocular pharmacokineticsPh. LapalusandR. G. Garraffo 119

1 Pharmacokinetic study: main data evaluation 1201.1 Absorption 1201.1.1 Routes of administration 1201.1.2 Topical administration 1211.1.3 Absorption data for topical administration 1231.1.4 Significance of absorption with other administration routes 1231.1.5 Conclusions 1241.2 Distribution 1241.2.1 Distribution phase 124

XIV Contents

1.2.2 Distribution parameters 1241.3 Metabolism 1251.3.1 Metabolism phase 1251.3.2 Metabolism study 1251.4 Elimination 1251.4.1 Elimination characteristics 1251.4.2 Elimination parameters (half-life, elimination constant, clearance) . . 1251.5 Conclusions 1262 Ocular pharmacokinetic modeling 1262.1 Main data 1262.2 Example of a non-compartmental model 1262.3 Practical strategy of pharmacokinetic data modeling 1273 Experimental and clinical models for kinetic studies 1273.1 In vitro models and applications 1273.2 In vivo models 1273.2.1 Normal eyes 1273.2.2 Pathological eyes 1273.2.3 Example of experimental infectious model 1284 Drugs ocular targets — relation kinetic-efficacy and/or toxicity 1284.1 Conjunctiva as a target for treatment of allergic and inflammatory

conjunctivitis 1294.2 Cornea as a target for drug toxicity 1294.2.1 For drug toxicity 1294.2.2 For drug efficacy 1294.3 Uvea as a target for efficacy of antiglaucoma drugs 1304.4 Lens as a target for drugs preventing or inducing lens opacities 1304.5 Vitreous as a target for drug efficacy 1304.6 Retina as a target for drug toxicity 1305 Example of drug ocular kinetics: Antibiotics 1315.1 Kinetics of topically administered antibiotics 1325.2 Systemic or intra vitreous administration 1325.3 Example of systemic quinolones 1336 Conclusion: A proposal of pharmacokinetic guidelines for drugs used

in ocular diseases and for ocular diffusion of systemic administereddrugs 134

Chapter 8:Microscopical examination of the eyeI. Weisse 137

1 Preparation techniques 1371.1 General remarks 1371.2 Light microscopy 1381.2.1 Fixation 1381.2.2 Fixed specimen 1401.2.3 Tissue processing 1401.2.4 Sectioning and staining 1411.2.5 Lens and histology 1411.3 Transmission Electron Microscopy (TEM) 1421.3.1 Fixation 143

Contents XV

1.3.2 Tissue processing 1441.3.3 Sectioning and staining 1441.4 Scanning Electron Microscopy (SEM) 1451.4.1 Fixation 1451.4.2 Tissue processing 1462 Structural differences 1483 Age-dependent changes 154

Chapter 9:Spectroscopic methods to evaluate drug toxicity in ocular tissueS. Lerman 165

1 Introduction 1652 Chemical structure and absorption characteristics 1653 Optical spectroscopy 1654 Localization and photobinding of 3H-8 Methoxypsoralen to intra-

ocular tissues 1664.1 Rat lens studies . 1664.2 Monkey studies 1684.3 Allopurinol therapy and cataractogenesis in humans 1694.4 A method to monitor reductase inhibitors in the lens 1704.5 NMR spectroscopy 1714.6 NMR pulse relaxation studies on the normal aging and cataractous

lens 1744.7 Sorbitol generation and its inhibition by sorbinil in the aging normal

human and rabbit lens and human diabetic cataracts 175

Chapter 10:Species differences relevant for ocular toxicity studiesL.F.Rubinandl. Weisse 177

1 Albinism 1771.1 Melanin drug-binding 1771.1.1 Ocular consequences of albinism 1771.1.2 Interactions with ocular pigment 1781.1.3 Reactions of ocular tissues to exogenous compounds in albino and

pigmented animals 1791.1.4 Choice of experimental animal 1811.2 Phototoxic retinopathy 1812 Tapetum lucidum — an additional tissue for oculotoxicity 185

Part II: Systems, models and methods to test drug toxicity 193

Chapter 11:Models and methods for testing toxicity with tear fluid, cornea, conjunctivaH. F. EdelhauserandJ. L. Ubels 195

1 Toxicity testing and the tear fluid 1951.1 Collection of rabbit lacrimal gland fluid 1961.2 Isotretinoin and lacrimal gland function 196

XVI Contents

2 Toxicity testing and the conjunctiva 1982.1 Gobletcells 1982.2 Surface area of ratio conjunctiva to cornea 1993 Toxicity testing and the cornea 2003.1 Epithelium 2003.2 Measurement of corneal epithelial healing rates 2003.3 Evaluation of toxicity using corneal epithelial healing 2013.4 Measurement of corneal thickness 2043.5 Endothelium 2053.6 In vivo specular microscopy 2063.7 Morphometric analysis 2073.8 In vitro specular microscopy 2093.9 Anterior chamber infusion studies 2113.10 Draizetest 213

Chapter 12:Models and methods for testing toxicity of aqueous humor, iris and ciliarybodyK. Green 219

1 Aqueous humor 2191.1 In vivo 2191.1.1 Methods 2191.1.2 Models 2201.1.3 Test procedures 2211.2 Invitro 2261.2.1 Methods 2261.2.2 Models 2271.2.3 Test procedures 2282 Iris 2292.1 Invivo 2292.1.1 Methods 2292.1.2 Models 2302.1.3 Test procedures 2313 Ciliary body 2323.1 Invivo 2323.2 Invitro 2333.2.1 Methods 2333.2.2 Models 2333.2.3 Test procedures 233

Chapter 13:Models and methods for testing toxicity with ocular pressureK. Green 243

1 Methods 2432 Models 2483 Test procedures 248

Contents XVII

Chapter 14:Models and methods for testing toxicity: LensO. Hockwin, A. Wegener, G. Bessems f, J. Bours, I. Korte, U. Mu'ller-Breitenkamp, ]. Schmidt and Ch. Schmitt 255

1 Introduction 2552 In-vivo lens examinations 2552.1 Lens transparency 2552.1.1 Slit lamp microscopy 2552.1.2 Scheimpflug image and evaluation 2602.1.3 Retroillumination photography 2642.2 Lens fluorescence 2652.3 Biometry 2662.4 Refraction and accommodation 2672.4.1 Definition 2672.4.2 Drug-dependent alterations of refraction and accommodation 2672.4.3 Diagnostics 2673 Post-mortem lens examinations 2683.1 Microscopy 2683.1.1 Light microscopy 2693.1.2 Electron microscopy 2723.2 Biochemistry 2763.2.1 Introduction 2763.2.2 Lens wet weight 2773.2.3 Heterogeneity of lens metabolism 2793.2.4 Lens proteins 2843.2.5 Determination of enzyme activities 2903.2.6 Acid-soluble compounds 2934 Testing the subliminal potential for co- or syncataractogenesis 2944.1 Introduction 2944.2 Principles 2974.3 Animal models '. . . 2974.3.1 Naphthalene cataract 2974.3.2 Diabetic cataracts in rats 3004.3.3 UV-B cataracts in rats 3014.3.4 X-ray cataracts in rats and rabbits 3024.3.5 Anaesthesia of laboratory animals 3044.4 Grouping of animals 3044.5 Summary and recommendations 3055 Special aspects of lens examinations 3065.1 Species differences 3065.1.1 Morphologic and physiologic particularities of the eyes of some

laboratory animals 3065.1.2 Norm variations and spontaneous changes of the eye 3065.1.3 Use of albinotic animals in oculotoxicity tests 3085.1.4 Lens metabolism and range of accommodation 3085.2 Age and lens properties 3095.3 Pharmacokinetics and lens 3105.4 The test on the permeability of the lens capsule 310

XVIII Contents

Chapter 15:Models and methods for testing toxicology with vitreousL. Salminen 3191 Introduction 3192 Abnormal vitreous 3203 Clinical examination of vitreous 3213.1 Ophthalmoscopy and slit lamp examination 3214 Ultrasonography 3225 Fluorophotometry 3236 Drug toxicity testing with vitreous 3236.1 Introduction 3236.2 Vitreous toxicity of systemically administered drugs 3246.2.1 Drug penetration into the vitreous 3246.2.2 Drug testing with vitreous 3246.3 Vitreous toxicity of ocularly administered drugs 3246.3.1 Drug penetration into the vitreous 3246.3.2 Drug testing with an intact eye 3256.4 In travitreal injections 3256.5 Vitreous and related disease models treated with intravitreal injec-

tions 3267 Recommendations 326

Chapter 16:Tests of retinal function in drug toxicityE. Zrenner 3311 Basic considerations of drug toxicity in the retina 3311.1 Structure and function of the retina 3321.2 The retinal pigment epithelium 3321.3 Photoreceptors 3331.4 Bipolar cells, horizontal cells, amacrine cells 3341.5 Ganglion cells 3351.6 Glia cells 3361.7 Summary 3362 Electrophysiological testing 3362.1 The electroretinogram 3382.1.1 Rod responses 3382.1.2 Cone responses 3392.1.3 Consideration of the origin of the electroretinogram 3402.1.4 Other ERG components 3412.1.5 Standardized electroretinography 3422.2 The electrooculogram 3432.3 The visually evoked cortical potential 3442.4 The arterially perfused cat eye 3463 Psychophysical methods 3473.1 Visual acuity and contrast vision 3473.2 Dark adaptation 3483.3 The cone-flicker threshold during dark adaptation 3483.4 Tests of glare sensitivity 3493.5 Color vision testing 350

Contents XIX

3.6 Spectral sensitivity functions 3533.7 Transient tritanopia 3544 Conclusion 356

Chapter 17:Methods and models for testing retinal toxicity: Morphologic and metabolicaspects of toxicityR. C. Tripathi, B.J. TripathiandC. B. Millard 3631 Introduction 3632 Morphologic changes: Clinical methods 3642.1 Ophthalmoscopy 3642.1.1 Direct ophthalmoscopy 3642.1.2 Indirect ophthalmoscopy 3642.1.3 Scanning laser ophthalmoscopy 3652.2 Biomicroscopy 3652.2.1 Goldmann fundus contact lens 3662.2.2 Hrubylens 3662.2.3 ElBayadilens 3662.2.4 78and90Dlens 3662.3 Fluorescein angiography 3672.4 Fundus photography 3673 Morphological changes: Non-clinical methods 3693.1 Gross examination 3693.2 Histology 3693.3 Histochemical analysis 3703.4 Phase-contrast and interference microscopy 3713.5 Electron microscopy 3713.5.1 Transmission electron microscopy (TEM) 3723.5.2 Scanning electron microscopy (SEM) 3733.5.3 Electron probe microanalysis 3734 Metabolic changes . 3734.1 Lipid separation . 3734.1.1 Thin-layer chromatography (TLC) 3744.1.2 Gas-liquid chromatography (GLC) 3744.1.3 Column chromatography 3744.2 Protein separation 3754.2.1 Isoelectric focusing 3754.2.2 SDS-PAGE applications 3754.2.3 Two-dimensional analysis 3764.2.4 Receptor protein isolation 3765 Tissue culture studies 377

Part III: Prospective models for in vitro/in vivo testing 383

Chapter 18:Drug toxicity testing with new delivery systemsS. Lertnan 3851 Liposome drug delivery studies 3852 Miniosmotic pump drug delivery system 386

XX Contents

Chapter 19:Cell culture methodsH. Rink and C. Baumstark-Khan 389

1 Introduction 3891.1 General considerations 3891.2 Eye lens specific considerations 3892 Cell culture systems 3902.1 Advantages 3902.2 Limitations 3902.3 Celllines 3912.3.1 Mycoplasmatest 3922.3.2 Chromosome distribution 3922.3.3 Growth rate 3922.3.4 Morphology 3932.4 Testcriteria 3932.4.1 Observation of morphological alterations 3942.4.2 Dose effect curves 3942.4.3 Dye exclusion 3952.4.4 Enzyme release 3952.4.5 Mitotic index/Division index 3952.5 Drug application 3962.5.1 Solubility 3962.5.2 Drug intermediates as toxic agents 3962.5.3 Time of drug application 3962.5.4 Period of observation 3962.6 Special tests 3962.6.1 Enzyme release 3962.6.2 Protein composition 3962.6.3 Protein synthesis 3962.6.4 Nucleotides 3972.6.5 Oxygen and substrate consumption 3982.6.6 Sulphydryl groups 3982.6.7 Unscheduled DNA-synthesis 3982.6.8 Sister chromatid exchange 3982.6.9 Cell transformation 3992.6.10 Cytoskeleton 3992.6.11 Membrane lipids 3992.7 Long term treatment 3992.7.1 Mitotically arrested cultures 3992.7.2 Repeated treatment 400

Chapter 20:Lens culture systems7. Korte 403

1 Introduction 4031.1 Suitability of the lens for cultivation 4031.2 Suitability of the cultivated lens for drug testing 4032 Preliminary examinations 404

Contents XXI

2.1 Prescreening with lens homogenates 4042.2 Permeability of the lens capsule for the drug in question 4052.2.1 Permeability of the isolated lens capsule 4052.2.2 Determination of the test substance within the intact lens 4053 Lens organ culture 4053.1 General considerations 4053.2 Culture systems 4063.3 Designs for lens organ cultures 4063.4 Choice of the method 4073.4.1 General remarks on the method 4073.4.2 Paired assay 4083.4.3 Medium, recommendations 4084 Test parameter 4084.1 Macroscopical appearance 4084.2 Alterations of lens wet weight 4094.3 Microscopical appearance 4094.4 Rate of mitosis 4094.5 Biochemical analyses 4094.5.1 Protein fractions 4094.5.2 Ion concentration 4094.5.3 Carbohydrate breakdown 4105 Conclusions 412

Index 415