TOXICI TY OF ANTIBIOTICS AND ANTIFUNGALS ON CULTURE D HUMAN CORNEAL CELLS: EFFEC T OF MIXING, EXPOSURE AND CONCENTRATION
M. BERRY, A. GURUNG and D. L. EASTY Bristol
SUMMARY effective treatment of bacterial and fungal conjuncti
Toxic effects of topical drugs may be masked by vitis or keratitis. The external eye surface may be manifestations of the disease they cure. The toxicity exposed to broad spectrum drugs or drug combina of drug mixtures has not been thoroughly studied. We tions to control multiple or unidentified infective therefore investigated cytopathic effects on primary agents. Appearance of local toxic effects of the cultures of human corneal cells of six topical anti medication, often difficult to distinguish from the microbials singly and in combinations of any two, to disease process itselC may prompt a diminution of determine the combined toxicity ranking and the drug concentration or frequency or a change of interaction between duration of exposure and concen medication. tration. Preconfluent cultures were exposed to fixed Toxicity of drug combinations, resulting from a dilutions of single drugs, or to equal-dilution mixtures summation or potentiation of individual adverse effects, of two drugs, for 7 and 14 days. Diminishing has been insufficiently studied. It is this relationship concentrations of single drugs were applied sequentially between the toxicity of a drug mixture and that of its to cultures for 14 days. The number of metabolically components that is the subject of this study. competent cells was assessed by measuring hexosami The number of all possible combinations of drugs nidase and total protein. Toxic effects depended on is too large to be exhaustively tested. We tested, substance, concentration and exposure. The scale of singly and in combinations of any two, three toxicity determined for single drugs after 7 days of antibiotics and three antifungals most frequently exposure was: gentamicin > econazole � methicillin � prescribed in the Bristol Eye Hospital: chloramphe c1otrimazole miconazole chloramphenicol. After � � nicol, gentamicin, methicillin, clotrimazole, econa 14 days this order changed: in particular chloramphe zole and miconazole. nicol showed a highly increased toxicity. The order of An in vitro system was chosen to isolate drug diminishing effects was: gentamicin> chloramphenicol toxicity from cytopathic effects of a microbial � methicillin > miconazole > econazole > c1otrima zole. A clear reduction in cytopathic effects was infection. Human corneal epithelial cell cultures observed when drug concentration was decreased were exposed to antimicrobial drugs for a period progressively only in cultures treated with gentamicin commensurate with clinical use. They were exposed or methicillin. All drug combinations were more toxic to fixed and to sequentially decreasing concentra than their components at equal dilution. Combinations tions of test substance, the latter mimicking a containing chloramphenicol ranked most toxic overall, tapering off regime. those containing econazole least. A tapering off Cytopathic effects depended on the drug(s), combination regime did not improve cell survival. concentration and duration of exposure. Rankings These in vitro toxicity data complement clinical studies of single drugs and combinations were not corre and suggest ways in which topical drugs can be chosen lated. Exposure to diminishing drug concentrations to minimise toxic effects to corneal surface. did not always improve the outcome.
Topical antimicrobial drugs are a widespread and MATERIALS AND METHODS Selection of Topical Drugs Correspondence to: Dr M. Berry, University of Bristol, Department of Ophthalmology, Bristol Ey e Hospital, Lower Twenty-four questionnaires were sent to consultants, Maudlin Street, Bristol BSI 2LX, UK. registrars and senior house officers in the Bristol Eye
Eye (1995) 9, 110-115 © 1995 Royal College of Ophthalmologists ANTI MICROBIALS IN VITRO 111
Hospital. They were asked to list in order of test substance for 3 days. On day 3 the concentration preference drugs (and combinations) for treating was halved (Cf2) for all but 6 wells, which were suspected bacterial and fungal conjunctivitis or exposed to the initial concentration for the remain keratitis. der of the experiment, as a fixed concentration comparator. On day 6 the concentration was halved Tissue Culture again (Cf4) for all but another block of 6 wells which continued to be exposed at half the initial concentra Epithelial cell cultures were established from human tion (Cf2). This process was repeated every 3 days, corneas unsuitable for transplantation from the resulting in different concentration step length UKTSSA Eye Bank in Bristol. Epithelium and a X regimes. The number of metabolically competent thin layer of underlying stroma was separated from cells was determined in all the cultures on day 21. the main bulk of the stroma by blunt dissection. Analysis of variance (ANOVA), post hoc tests and Explants 2-4 mm2 were plated on tissue culture non-parametric analysis of variance by ranks (Krus flasksin RPMI 1640 (Gibco) supplemented with 10% kal-Wallis test) were performed with Stat-View 4.02 fetal bovine serum (Gibco), streptomycin, penicillin, (Abacus Concepts, Berkeley, CA). amphotericin mixture (Sigma) and RPMI nonessen tial amino-acids (Sigma). When epithelial growths had been established explants were removed to RESULTS minimise fibroblast content of the cultures. After Thirteen of 24 questionnaires were returned. The confluence cultures were passaged and the cells used unanimous first choice of drug for the treatment of in this study. All experiments were carried out in six suspected bacterial conjunctivitis was chlorampheni replicates in tissue culture medium containing 5% col 0.5%. No drug combinations were envisaged for fetal bovine serum. this condition. For suspected bacterial keratitis gentamicin (1.5%) or a combination of gentamicin Experimental Design (1.5% or 0.3%) and methicillin 2% were equal first preferences. Suspected fungal keratitis would most Effects on Cell Proliferation. A total of 5 103 cells X often be treated with either miconazole 1%, or a per well were seeded in 96-well tissue culture plates mixture of two antifungals (e.g. econazole plus (Costar), allowed to adhere overnight at 35 °C, and miconazole), or a mixture of an antibiotic and an exposed to a fixedconcentration of test substance for antifungal (e.g. econazole plus chloramphenicol, or 7 or 14 days. Water-soluble topical solutions were clotrimazole plus gentamicin forte). diluted in tissue culture medium, lipid-soluble ones in In accordance to these preferences we chose to arachis oil (Hill Cross Pharmaceuticals). The latter study the effects of chloramphenicol 0.5% (Scher were always applied to the cell layer before tissue ring-Plough), gentamicin forte 1.5% (MoorfieldsEye culture medium to ensure penetration. Cells treated Hospital) and methicillin 2% (Bristol Eye Hospital). with arachis oil and tissue culture medium served as The antifungals chosen were econazole 1% (Moor controls for each experiment. Medium and test fields Eye Hospital), miconazole 1 % (Moorfields substance were renewed twice a week. The number Eye Hospital) and clotrimazole 1 % (Moorfields Eye of cells was evaluated by measuring hexosaminidase Hospital). activitr and total protein content (BCA Protein The number of metabolically competent cells as Assay, Pierce). The results are presented as optical evaluated by the hexosaminidase assay paralleled densities, with standard curve number of cells closely the results obtained by measuring total corresponding to that optical density on the right protein. Hexosaminidase results had, however, a hand vertical axis. wider distribution, i.e. a larger difference in optical Effects on Cell Proliferation and Migration. Cells density between the most and the least populated were plated overnight either in a dense plaque (5 x wells (Fig. 1). We chose the latter to represent the 103 cells per well) in the centre of a 1.5 cm diameter effects of treatment on cell numbers. well (Costar), or in an annulus (15 X 103 cells per well) around its perimeter, and exposed to test Effect on Cell Proliferation solutions for 14 days. The number of cells was After exposure to antimicrobials the number of cells evaluated by measuring hexosaminidase activity and depended on test substance, concentration and total protein, and well cover visualised by staining duration of exposure (Kruskal-Wallis analysis of with haematoxylin. variance by ranks, p -.2 ISO BO 40 20 10 5 4 2 2 4 5102040 BO dilution of topical solution dUutlon factor of topical solution Fig. Dependence of effect on substance and concentra Fig. Comparison between hexosaminidase and total 2. 1. tion. Analysis of variance (optical densities expressed as protein assays for evaluating population size. The box plots fraction of appropriate controls) showed a significant show the distribution of all optical densities obtained in one difference between population sizes depending on test experiment by either method. The boxes showing the means substance and concentration (p 10ES 10ES 1.4 1.4 Sxl0E4 Sxl0E4 1.2 1.2 1 � e 10E4 l0E4 ...... f e II> .S ...... J-S == -8 .!!l ] Il 01 'iltl ;:3 .S .S � l o .4 .4 Sxl0E3 Sxl0E3 .2 .2 Chloramphenicol l0E3 Gentamicin Forte l0E3 10E2 10E2 0 5 0 5 control ISO SO 40 20 10 4 2 control1S0 SO 40 20 10 4 2 dilution of topical solution dilution of topical solution � 14 days --- 7 days 10E5 1.4 10ES 1.4 5xl0E4 5xl0E4 1.2 1.2 �OJ l0E4 � l0E4 = e = e ...... -8 .S OJ -8 .S � ] � ] tl .S � .S � .4 .4 5xl0E3 SxlO8 .2 .2 Econazole 10E3 Methicillin 108 l0E2 l0E2 0 5 0 5 control1S0 80 40 20 10 4 2 control ISO SO 40 20 10 4 2 dilution of topical solution dilution of topical solution Fig. 3. Dependence of effectson duration of exposure. Fourteen days of exposure increased the toxic effects of antibiotics but not of antifungals. Kruskal-Wallis ranking: gentamicin> chloramphenicol;;.: methicillin> miconazole > econazole > clotrimazole. in diminishing order of effects: gentamicin > phenicol ;;;.: methicillin> miconazole> econazole > c1otrimazole ;;;.: econazole ;;;.: methicillin> miconazole c1otrimazole. > chloramphenicol. Exposure to combinations of two drugs (at equal After 14 days of exposure gentamicin still ranked dilution of the topical preparation) led to a dose most toxic, but the subsequent order was: chloram- dependent decrease in cell numbers compared with ANTIMICROBIALS IN VITRO 113 1.2 IOE5 1.2 IOE5 5x10E4 5xlOE4 ---- CPL+MZ -- CT+GF S -a- CT+MZ b.S -12- GF b' 10E4 1OE4 � E a; GF+MZ � -... -.0.- GF+CPL d " -<>- ME+MZ .:l.6 ." .6 � -<>- GF+EZ ...... MZ "iii " --- GF+ME "iii" -- MZ+EZ :is -M- GF+MZ 'g..4 g..4 0 .2 5xlOE3 .2 5xlOE3 IOE3 IOE3 IOE2 0 IOE2 0 SO 40 20 10 5 4 2 80 40 20 10 5 4 2 dilution of topical solution 1.2 IOE5 1.2 IOE5 5x10E4 5x10E4 � ---- CPL '" .S .8 ---- CPL+ME d -a- CPL+EZ £ " 10E '" IOE4 -a- CT+ME "" d 4 � CPL+ME " "" � GF+ME "iii" .6 -<>- CPL+MZ .6 :;l "iii -<>- ME '" ...... CT+CPL " 0 :;l ...... ME+EZ .4 '" .4 -- GF+CPL 0 -- ME+MZ .2 5xlOE3 .2 5xlOE3 IOE3 IOE3 0 IOE2 0 IOE2 SO 40 20 10 5 4 2 SO 40 20 10 5 4 2 Fig. 4. Effect of mixtures of topical solutions. Exposure to combinations of two solutions resulted in a smaller population than after exposure to either solution alone. Population size depended on treatment and concentration (ANOVA p 1.2 1.2 IOE5 lOE5 ...... 14 xlOE4 ...... 7 � .8 10E4 � .8 '" '" d d 10E4 " .IJ .6 "C:I .6 �u �'-' :p :;:J I>. .4 I>. .4 0 0 .2 xlOE3 .2 lOE3 lOE3 0 lOE2 0 IOE2 2 4 5 10 20 40 80 2 4 5 10 20 40 80 dilution of topical solution dilution of topicalsolution a b 1.2 1.2 IOE5 ...... 14 ...... 14 5x10E4 ...... 7 ...... 7 .8 � .8 �'" 10E4 .� IOE4 E " 'tl .6 "C:I .6 �'-' i :p .4 I>. 0 0 .4 .2 .2 5xlOE3 xlOE3 IOE3 IOE3 0 IOE2 0 IOE2 2 4 5· 10 20 40 80 2 4 5 10 20 40 80. dilution of topical solution dilution of topical solution c d Fig. 5. Combination of topical solutions: dependence on duration of exposure. Combinations with (a) chloramphenicol, (b) methicillin, (c) gentamicin, (d) econazole. Fourteen days of exposure increased cytopathic effects in a dilution- and substance dependent manner (ANOVA p Treatment ...... 1 .8 -0- 2 §" -+- 4 " .6 �7 =ii ..... fixed concentration (.) .4 .2 .2 0 5 10 20 40 80 160 320 640control control FIxed 2 3 6 dilution of topical solution 4 5 Treatment Fig. 6. Effect of diminishing concentrations of gentamicin (left) and methicillin (right) on cytopathic effects. A stepwise decrease in concentration was chosen to mimic a tapering off regime. All wells were exposed to the highest concentration tested for 3 days. Every 3 days the concentration was halved for all but a block of replicates which continued to be exposed to the respective higher concentration for the rest of the experiment. control (p<0.001, ANOVA; Fig. 4), except for the toxicity test. Effects on the number and metabolic combination econazole plus miconazole (Fisher's competence of corneal epithelial cells are relevant in PLSD, p = 0.05). the selection of a topical antimicrobial solution. Mixtures were more toxic than either topical It was outside the scope of this study to disentangle preparation alone: all combinations containing gen effects of pure antimicrobials from those of their tamicin were more toxic than gentamicin at the same vehicles and preservatives; the study was limited to concentration. Summing the ranks, combinations 'off the shelf' topical drugs. containing chloramphenicol were more toxic than The migration-proliferation test presented here mixtures with gentamicin (Kruskal-Wallis ANOVA lacks the element of trauma of in vitro wound by ranks). Gentamicin-containing combinations healing tests,3 and improves control over initial cell ranked equal to those with methicillin. Less toxic numbers. Comparison of effects of antiviral drugs in were ranked combinations with clotrimazole, then the two tests showed no additional effect of wound mixtures including miconazole, and last econazole ing on toxicity levels.4 The antimicrobials tested containing solutions. ranked unchanged in the proliferation and prolifer Duration of exposure significantly affected popu ation-migration test, suggesting that the main effect lation levels in cultures exposed to drug combina is on cellular proliferation. tions (Fig. 5). Econazole, miconazole and clotrimazole appeared least toxic in all in vitro tests. They are usually well Effect on Cell Migration and Proliferation tolerated by the corneal epithelium even after weeks Cells plated in a restricted area of the well covered long therapy.5,6 the naked areas and proliferated to an extent Chloramphenicol, considered a most effective depending on the test substance and concentration 'routine' topical antibiotic,7,8 produced low toxicity (ANOVA, p