Doc Ophthalmol (2008) 116:177–191 DOI 10.1007/s10633-007-9081-x

ORIGINAL RESEARCH ARTICLE

Retinal safety of a new fluoroquinolone, pradofloxacin, in cats: assessment with electroretinography

Andre Messias Æ Florian Gekeler Æ Alfred Wegener Æ Klaus Dietz Æ Konrad Kohler Æ Eberhart Zrenner

Received: 1 June 2007 / Accepted: 10 September 2007 / Published online: 2 October 2007 Springer-Verlag 2007

Abstract Purpose To investigate the safety of a Pradofloxacin showed no effects in respect to rod b- new fluoroquinolone, pradofloxacin, on the cat retina wave, Vmax, k and maximum scotopic a-wave using electroretinogram. Methods Ganzfeld ERGs (P [ 0.05). Oscillatory potentials, cone ERG and were recorded in 40 cats treated orally for 23 days in flicker were also unaltered (P [ 0.05). Rod b-wave 4 groups: CTRL (n = 9): placebo-vehicle; PRADO30 was undetectable after treatment in ENRO30 group,

(n = 10): pradofloxacin 30 mg/kg/day; PRADO50 Vmax was reduced to 10.5% of the baseline (n = 14): pradofloxacin 50 mg/kg/day; and ENRO30 (P \ 0.05), accompanied by an increase of k by (n = 7): enrofloxacin at toxic doses of 30 mg/kg/day. 1 log cd s/m2 (P \ 0.05). Oscillatory potentials, ERG was performed before treatment and once cone b-wave amplitude and 30 Hz flicker amplitude weekly during the treatment period. An extended were reduced to 8.3%, 58.9% and 37.4% of the ISCEV protocol with addition of 8 steps of increasing baseline, respectively (P \ 0.05). Effects were also luminance in dark adapted condition was carried out seen in OCT and retinal histology starting within one to assess: Vmax (saturated scotopic b-wave amplitude) week after the start of treatment and thereafter and k (luminance inducing Vmax/2). OCT and retinal remaining stable. Conclusion Pradofloxacin at 6 and histological changes were also investigated. Results 10 times the recommended doses was shown to have no retinal toxic effects in cats, neither on rod or cone function with ERG. A. Messias (&) F. Gekeler E. Zrenner Center for Ophthalmology, University of Tu¨bingen, Schleichstraße 12-16, 72076 Tubingen, Germany Keywords Cat Electroretinography e-mail: [email protected] Enrofloxacin Fluoroquinolone Pradofloxacin Retina Toxicity A. Wegener Department of Ophthalmology, University of Bonn, Ernst-Abbe-Strasse 2, 53127 Bonn, Germany Introduction K. Dietz Department of Medical Biometry, University of Tu¨bingen, Westbahnhofstraße 55, 72070 Tubingen, Fluoroquinolones (FQ) offer many potential benefits Germany of antibacterial agents: possible oral and parenteral application; a broad spectrum of activity including K. Kohler gram-negative and gram-positive aerobes, and newer Center for Regenerative Biology and Medicine, University of Tu¨bingen, Paul-Ehrlich-Straße 15, generations possessing activity against anaerobes and 72076 Tubingen, Germany even mycobacteria; good tissue penetration; adequate 123 178 Doc Ophthalmol (2008) 116:177–191 rates of clearance; all combined with a favorable treated with enrofloxacin at up to 6 and 10 times the safety profile [1–3]. recommended dosage. The major ocular side effects Pradofloxacin is a new compound developed for described are mydriasis, retinal atrophy, attenuated the management of bacterial infections in dogs and retinal vessels, increased reflectivity of the tapetum, cats, which has been shown to have the highest abnormal electroretinogram (ERG) responses and therapeutic efficacy combined with high potential for apparent loss of vision. Recent experimental studies restricting the emergence of resistance in comparison in healthy cats confirmed the ocular and systemic with other six veterinary FQs in vitro [4], and a recent changes induced by toxic dosage of enrofloxacin multicentre study in vivo showed that pradofloxacin (50 mg/kg/day) [9]. In contrast, post-approval safety has an efficacious therapy comparable to amoxycil- studies performed by the manufacturer reported no lin/clavulanic acid for deep bacterial pyoderma in adverse retinal effects, including in ERG evaluations, dogs [5]. in the 8 cats receiving 5 mg/kg (label doses), daily for This new FQ is distinguished from enrofloxacin by 21 days (Abraham, A.; 2000—report number: 75240. two structural elements: a bicyclic amine, pyrroli- Bayer Corporation Agricultural Division, Animal dine-piperidine, replacing the ethyl-piperazine Health—Shawnee Mission, KS USA). moiety located at position C7 of enrofloxacin; and a The safety of ocular (topical and/or intra-vitreal) cyano group attached to position C8 (Fig. 1). The administration of ofloxacin [10–12], ciprofloxacin substitution at position C8 also differentiates prado- [10], evofloxacin [13], norfloxacin [14], trovafloxacin floxacin from its analog for human use, moxifloxacin. [15], moxifloxacin [16] and garenoxacin [17] has Enrofloxacin was synthesized in 1980, introduced been investigated, and the results lead to the conclu- to the market in 1988, and was the first FQ approved sion that FQs show varied affinity for retinal tissues for veterinary use. This compound differs from its and there are a marked differences among compounds counterpart for human use and major metabolite, for safe doses for ocular administration. ciprofloxacin, by the presence of an ethyl group on The exact mechanisms of FQ ocular side effects the para-position of the piperazinyl ring (Fig. 1). This have not been well delineated. Since the early compound has a broad spectrum of activity against observations that melanin binding of drugs, such as gram-positive and gram-negative bacteria as well as chloroquine (CQ), influences ocular toxicity [18], against Mycoplasma spp. and possesses useful phar- drug accumulation in pigmented tissues has also been macokinetic and tissue distribution properties in suggested to be related to the FQ ocular damages, as various animals species [6]. there is evidence of the FQs having high affinity for During the past 19 years, enrofloxacin has been melanin and other pigmented tissues [19–21]. Ono broadly used for the management of diseases in dogs and Tanaka showed that different FQs vary in their and cats with rare relevant side effects at the affinity and capacity to bind with melanin; they also recommended dosage rate. However, the presence discussed that the inclusion of a basic aliphatic amino of a flexible dosage range in the United States to group at position C7 may play a critical role address more severe pathogens has stimulated the diminishing the FQ interaction with melanin [22]. usage of higher doses by veterinarians [7, 8]. Gelatt In a recent study in mice, Gao et al. showed that et al. [8] observed ocular side effects in 17 cats moxifloxacin, a fourth-generation FQ, can be safely

Fig. 1 Chemical structure O O of veterinary FQs F COOH F COOH 4 4 enrofloxacin and 6 6 pradofloxacin and their 7 H 7 counterpart for human use 8 1 8 1 N N H N N compounds ciprofloxacin N and moxifloxacin (modified N R from Wetzstein [4]) R H

Enrofloxacin: R=H52 C Pradofloxacin: R=CN

Ciprofloxacin:R=H Moxifloxacin:R=OCH3 123 Doc Ophthalmol (2008) 116:177–191 179 administrated intra-vitreously for treatment of thereafter for 4 weeks (1W, 2W, 3W and 4W, endophthalmitis [23]. This would offer potential respectively): advantages over the treatment schema recommended by the The Endophthalmitis Vitrectomy Study [24], because the FQ bacterial coverage permits mono ERG measurement therapy, can be an alternative for patients with penicillin allergies, and would avoid retinal side The ERG protocol developed for the purpose of this effects induced by the antibiotics proposed in this study is based on the international standard for study, as for example, retinal infarction caused by electroretinography (ISCEV) [26]. The protocol was amikacin even at therapeutic concentrations [25]. extended to improve the robustness of ERG assess- In our study, we aimed to investigate effects of ment, minimize variability, and yield additional high doses (6 and 10· the recommended dose) of the information on retinal function. newer compound pradofloxacin on the feline retina, Cats were kept in a dark room for at least 2 h for and to describe the changes induced by toxic dark adaptation before anesthesia, which was per- administration (6· the recommended dose) of enro- formed by intramuscular injection of 1–2 mg/kg body floxacin, using ERG. weight xylazine and 10 mg/kg body weight ketamine. Pupillary dilatation was performed with 1 drop of tropicamide. Methods ERG responses were recorded in both eyes simul- taneously by means of JET contact electrodes on the Animals and treatment corneas (Microcomponents SA). Subcutaneous nee- dles in the skin near the lateral canthus of both eyes Forty domestic short hair cats HsdCpb:CADS, 20 were used as references; ground electrode was placed females and 20 males, were included in the study. in the tail. Electrode impedance was checked before Body weight was: 3.5–4.5 kg in males and 2.5– and after each measurement and was less than 5 kX 3.5 kg in females. Cats were healthy, non-pregnant at 25 Hz. Eyes were stimulated using a Ganzfeld and non-lactating; ages at the beginning of the study LED stimulator (ColorDome; Diagnosys LLC, Lit- ranged from 10 to 12 months. Animals were kept tleton, MA). Flashes of white light (6,500 K) with a under a 12 h light–dark cycle. duration of 4 ms were delivered in 9 steps of Pure test substances were filled into gelatinous increasing luminance (0.0001, 0.0003, 0.001, 0.003, capsules and administrated orally by gavage without 0.01, 0.03, 0.1, 0.3 and 10 cd s/m2) with 30 s inter sedation. Treatment was performed by dividing the stimulus interval (ISI) in the dark adapted stage. animals into four groups: Group treated with prado- After 10 min of light adaptation with a back- floxacin at 30 mg/kg body weight per day ground light of 17 cd/m2, light adapted ERG (PRADO30; n = 10), group treated with pradofloxa- recordings were performed in 3 steps with increasing cin at 50 mg/kg body weight per day (PRADO50; luminance flashes of red light (to enhance cone n = 14), group treated with toxic doses of enroflox- isolation) of 1, 3 and 10 cd s/m2 (15 s ISI) followed acin at 30 mg/kg body weight per day (ENRO30; by a 30 Hz white flicker stimulus of 10 cd s/m2. n = 7), and a control group vehicle-treated (CTRL; Responses were amplified (band pass filter: n = 9). 0.3–300 Hz) and stored for off-line analysis (using the Espion; Diagnosys LLC, Littleton, MA) after averaging of 6 up to 40 individual measurements at Procedures each step depending on the signal/noise ratio.

The experiments were performed in accordance with the ARVO Statement for the Use of Animals in OCT Ophthalmic and Vision Research. The following procedures were performed one day The retinal thickness, structure of retinal layers, and before starting the treatment (baseline) and weekly geometry of the papilla were investigated by means 123 180 Doc Ophthalmol (2008) 116:177–191 of STRATUS OCT (Stratus OCT, Carl Zeiss, Ger- defined as the minimum response point between 0 many) in the animals of all groups. and 60 ms for dark adapted ERG, and between 0 and 30 ms for light adapted ERG. The b-wave amplitude was calculated as the difference between the a-wave Blood samples amplitude and the maximum between 50 and 120 ms for the dark adapted phase and between 25 and 60 ms Samples of blood were obtained in 10 cats of group for light adapted records. The scotopic b-wave was PRADO30, in 10 cats of group PRADO50 and in 4 digitally filtered by the evaluation software using a cats of group ENRO30. Sampling was made at 1, 2, 4, fast Fourier transform (FFT) algorithm (low pass 24 h after administration on treatment days 1 and 21. filter, Fc \ 50 Hz), to remove oscillatory potentials (OP) before fitting. Data retrieved by the computer program was checked manually to ensure that the Retinal histology proper peaks were detected. Interrelation between dark adapted b-wave ampli- After the last treatment day, cats were sacrificed and tude and stimuli luminance (Eq. 1) was modeled eyes were then enucleated. A large full-thickness using the Naka-Rushton function [27] that yields incision was made in the cornea, and the eye was fixed three parameters: Vmax is the asymptotic (maximum immediately in 4% formaldehyde in 0.1 M phosphate b-wave amplitude); k is the necessary luminance buffer (pH 7.4). After 15 min in the fixative, the lens reaching 50% of Vmax, which is a mark of dark was removed and the eye was cut along the cornea- adapted sensitivity, and n is a exponential that is optic nerve axis into halves. Gross examinations of the related to the slope at the linear phase (Eq. 1). tissues were performed. Tissues were further fixed V In VðIÞ¼ max ð1Þ overnight in 4% paraformaldehyde and 0.5% glutaral- In þ kn dehyde in 0.1 M phosphate buffer (pH 7.4). Tissues were then embedded in resin, sectioned at a thickness Leading edges of the saturated a-wave elicited by the dark adapted maximum response (white— of 1 lm, and stained with Richardson’s dye. A light 2 microscope was used for histological examinations. 6,500 K flash—10 cd s/m ) were analyzed using a Electron microscopic examination was also per- rod activation model proposed by Pugh and Lamb formed; results to be published elsewhere. [28]. The model is characterized by a parameter A, the amplification constant, which may be expressed as the product of physical and biochemical factors of the transduction cascade. Data evaluation hi 0 2 f ðtÞ¼amax 1 exp A ðÞt teff ð2Þ Evaluation software was developed (Delphi 7.0— Borland 2002) to automatically determine a- and b- Here, f(t) is the cGMP-activated current expressed wave amplitudes and implicit times. The a-wave was as a fraction of its dark value, amax is the saturated

10 21st day

3 st 1 day

ntration ( mg/l) PRADO30 PRADO50 ENRO30 1 st n=1021 day n=10 21st day n=4 0.3 1st day 1st day 0.1

Serum conce 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 Time (hours) Time (hours) Time (hours)

Fig. 2 Serum concentration plotted against time after oral small differences were found between serum concentrations 1 h administration of pradofloxacin at 30 (PRADO30) and 50 mg/kg after administration, however enrofloxacin showed longer half- (PRADO50) and enrofloxacin at 30 mg/kg (ENRO30). Only life times 123 o ptaml(08 116:177–191 (2008) Ophthalmol Doc Table 1 Dark adapted rod b-wave and maximum response b-wave amplitude, b-wave implicit time and a-wave amplitude (mean ± SEM) Group Baseline Treatment period 1 week 4 weeks b- Amplitude b- Imp. a- Amplitude b- Amplitude b- Imp. a- Amplitude b- Amplitude b- Imp. a- Amplitude (lV) Time (ms) (lV) (lV) Time (ms) (lV) (lV) Time (ms) (lV)

Rod b-wave CTRL 168.4 ± 7.2 79.5 ± 0.9 171.1 ± 8.1 80.4 ± 1.2 159.1 ± 10.9 80.2 ± 0.9 PRADO30 159.0 ± 12.4 81.3 ± 1.3 186.1 ± 20.7 78.4 ± 0.6 176.7 ± 14.5 80.6 ± 1.3 PRADO50 177.8 ± 10.7 80.3 ± 0.9 185.5 ± 16.3 82.0 ± 0.8 194.0 ± 8.6 81.4 ± 1.2 ENRO30 177.6 ± 23.5 78.4 ± 1.6 * * * * Dark adapted maximum response CTR 303.8 ± 22.9 36.0 ± 2.1 109.5 ± 6.3 299.9 ± 25.4 33.0 ± 0.8 122.8 ± 5.6 311.7 ± 27.2 34.8 ± 0.7 120.7 ± 9.1 PRADO30 317.3 ± 26.2 37.3 ± 2.4 121.2 ± 10.4 321.9 ± 32.7 40.7 ± 3.6 127.2 ± 11.1 291.3 ± 27.4 36.3 ± 2.2 115.3 ± 10.5 PRADO50 313.6 ± 16.1 38.0 ± 4.5 120.8 ± 7.9 314.3 ± 16.7 37.8 ± 4.1 130.3 ± 8.8 300.4 ± 16.3 36.2 ± 1.5 124.4 ± 6.7 ENRO30 372.1 ± 41.1 35.8 ± 3.8 118.3 ± 17.3 17.4 ± 6.0 37.2 ± 3.0 46.2 ± 6.2 44.9 ± 8.8 56.0 ± 1.0 8.2 ± 2.0 *No b-wave determined (under minimum amplitude)

0 Table 2 Parameters of Naka-Rushton function (dark adapted b-wave amplitude): k and Vmax ; a-wave activation constant, parameter from the Pugh and Lamb model: A ; and dark adapted OP (absolute area nder the curve) from maximum response (measured at 10 cd s/m2) (mean ± SEM) Group Baseline Treatment period 1 week 4 weeks

0 0 0 K log Vmax A OP k log Vmax A OP K Log Vmax A OP cd s/m2 (lV) (U/s2) (lV · ms) cd s/m2 (lV) (U/s2) (lV · ms) cd s/m2 (lV) (U/s2) (lV · ms)

CTRL –2.47 ± 0.05 367.5 ± 15.2 51.91 ± 1.75 430.27 ± 21.50 –2.47 ± 0.02 375.2 ± 23.8 64.2 ± 7.16 469.53 ± 66.67 –2.42 ± 0.04 368.2 ± 24.7 357.21 ± 23.64 54.89 ± 7.94 PRADO30 –2.49 ± 0.04 333.0 ± 23.6 54.48 ± 3.57 403.55 ± 68.18 –2.50 ± 0.04 392.5 ± 35.2 62.14 ± 8.82 426.87 ± 70.83 –2.57 ± 0.06 343.7 ± 30.1 327.92 ± 53.06 48.81 ± 5.44 PRADO50 –2.49 ± 0.05 378.9 ± 23.0 58.49 ± 3.24 399.49 ± 78.90 –2.41 ± 0.06 418.6 ± 35.1 67.73 ± 3.60 388.91 ± 82.18 –2.55 ± 0.03 381.1 ± 17.9 334.99 ± 32.07 55.18 ± 6.15 ENRO30 –2.40 ±0.07 410.2 ± 49.6 56.32 ± 5.14 314.85 ± 75.58 –1.84 ± 0.15 48.5 ± 12.2 4.94 ± 1.01 18.10 ± 6.18 –1.54 ± 0.07 47.4 ± 11.0 41.73 ± 12.37 0.79 ± 0.15

123 U—Number of photoizomerizations per rod 181 182 Doc Ophthalmol (2008) 116:177–191

0 a-wave amplitude, teff is a brief delay, and A is the All function fittings were iteratively performed product of an amplification constant (A) and the with standard lest square fit algorithm in JMP-IN number of photoisomerizations per rod produced by version 5.1. the flash (A0 = –1/2 · A · A—from the original model proposed by Pugh and Lamb) and is expressed in A/s2 (A = estimated number of photoisomeriza- Statistical analysis tions per rod). Once the primary parameters, amax and teff, were determined for each animal at baseline, The averages of quantitative ERG data of right and parameter A0 was the only one that varied for fittings left eyes were used for statistical analysis. A quotient of the treatment period responses. between the 4 periods of treatment and the baseline OP were obtained from the maximal dark adapted was calculated dividing results obtained in each one response by means of a FFT as a band pass of the 4 treatment weeks by individual baseline. This frequency filter. The frequency interval used was quotient was used for the comparison between from 75 Hz up to 300 Hz. Area under the OP curve experimental groups at each time point. A statistically was determined between a- and b-wave implicit significant effect was defined as a difference in a times (Fig. 6B). factor of 1 (a = 0.95, Student’s t-distribution). A linear function of the light adapted b-wave amplitude in function of the logarithm of stimulus luminance was fitted and the necessary luminance to Results produce b-wave amplitude of 25 lV (fixed threshold) was determined. General clinical findings Flicker (30 Hz) response phase was determined by the delay time of the first peak (average of 50 cycles); Animals treated with pradofloxacin at 30 mg/kg (6· results are shown in percent of the period time recommended dose) showed no weight loss during 1 : whereas flicker amplitude was taken as the treatment period. At 50 mg/kg (10· recommended 30 Hz average of the first 4 cycles amplitude. dose), only slight weight loss was observed in 3 males

Fig. 3 Example of dark Baseline 1 Week 4 Weeks adapted ERG responses at increasing stimulus luminance (8 steps—from CTRL 0.0001 to 0.3 cd s/m2) performed before (Baseline), 1 week and 4 weeks after treatment begin. Plots are from one cat of control (untreated) group (CTRL), one cat PRADO30 treated with pradofloxacin at 30 mg/kg bw/day (6· the recommended dose) (PRADO30), one cat treated with pradofloxacin at 50 mg/kg bw/ day (10· the PRADO50 recommended dose) (PRADO50) and one cat treated with enrofloxacin at 30 mg/kg bw/day (6· the 200 µV recommended dose) (ENRO30) 100 ms ENRO30

123 Doc Ophthalmol (2008) 116:177–191 183

(up to 150 g during last week). Additional symptoms Serum concentration and drug half life were vomiting (nearly all animals; at 30 and 50 mg/kg), increased salivation (some animals) and diarrhea (one Serum concentrations of enrofloxacin and pradofloxa- animal). cin, given at 30 mg/kg, reached similar concentrations In contrast, all animals treated with the toxic doses in the first 1 h after oral administration, 5.44 ± 2.06 and of enrofloxacin (30 mg/kg—6x recommended dose) 7.11 ± 0.85 mg/l, respectively (P = 0.1326). However, lost weight (up to 500 g per week). And nearly all enrofloxacin showed a higher serum concentration at cats sporadically presented with symptoms indicative 24 h after treatment: 2.63 ± 0.20 and 0.14 ± 0.13 mg/l, of systemic toxicity: vomiting and increased saliva- respectively (P \ 0.05), due to a longer half life during tion, piloerection, reduced mobility, staggering or the entire treatment period in comparison to pradoflox- uncoordinated gait, sitting in prone abdominal acin: 14.01 ± 4.06 and 2.77 ± 0.79 h, respectively position, extension spasms, accelerated and labored (P \ 0.05). breathing, and breathing sounds; one female cat was Pradofloxacin administrated at 50 mg/kg demon- sacrificed due to moribund condition. strated only slightly higher concentrations than at

baseline 1 week 4 weeks AB b-wave aplitude (µV) normalized Vmax normalized 1/log10 k 400 1 1 300 200 0.5 0.5 100 0 CTRL 0 0

400 1 1 300 200 0.5 0.5 100 PRADO30 0 0 0

400 1 1 300 200 0.5 0.5 100 PRADO50 0 0 0

400 1 1 Vmax 300 200 0.5 0.5 ENRO30 100 k 0 0 0 .0001 .001 .01 .1 BL 1W 2W 3W 4W BL 1W 2W 3W 4W luminance (cd.s/m²) Treatment period Treatment period

Fig. 4 (A) Examples of the V log I function (Naka-Rushton) function, plotted against the treatment duration, ‘‘BL’’ is the of the b-wave (peak to peak amplitude) from one cat of control baseline and 1W, 2W, 3W and 4W are the 4 weeks of (untreated) group (CTRL), one cat treated with pradofloxacin treatment, respectively. Circles are the mean of the ratio at 30 mg/kg bw/day (6· the recommended dose) (PRADO30), between treatment week and baseline. Error bars represent one cat treated with pradofloxacin at 50 mg/kg bw/ day (10· mean ± the 97.5% quantile from the Student’s t-distribution the recommended dose) (PRADO50) and one cat treated with with numbers of cats per group –1 as degrees of freedom. enrofloxacin at 30 mg/kg bw/day (6· the recommended dose) There is no statistically significant difference between base- (ENRO30). Unfilled circles represent the b-wave at the lines and the various periods of treatment in groups CTRL, baseline; filled squares are from the responses after one week PRADO30 and PRADO50 neither in Vmax nor in k, and high of treatment and unfilled squares after 4 weeks. (B) Normal- differences found in group ENRO30 in both parameters ized Vmax and sensitivity (k): parameters of Naka-Rushton 123 184 Doc Ophthalmol (2008) 116:177–191

ABNormalized response (a(t) / amax) Normalized A’ baseline 0 1 week 4 weeks 1.5

1 -0.5 CTRL

0.5

-1 0

0 1.5

-0.5 1 PRADO30 0.5

-1 0

0 1.5

-0.5 1 PRADO50 0.5 -1 0 0 1.5

-0.5 1 ENRO30 0.5

-1 0 BL 1W 2W 3W 4W 0 4128 16 Time (ms) Treatment period

Fig. 5 (A) maximum a-wave response, elicited by a flash of model30. (B) Development of the amplification constant A0 10 cd s/m2, normalized with individual baseline. Plots are from plotted against the treatment duration, ‘‘BL’’ is the baseline and one cat of control (untreated) group (CTRL), one cat treated 1W, 2W, 3W and 4W are the 4 weeks of treatment, with pradofloxacin at 30 mg/kg bw/day (6· the recommended respectively. Circles are the mean of the ratio between dose) (PRADO30), one cat treated with pradofloxacin at treatment week and baseline and error bars represent mean ± 50 mg/kg bw/ day (10· the recommended dose) (PRADO50) the 97.5% quantile from the Student’s t-distribution with and one cat treated with enrofloxacin at 30 mg/kg bw/day (6· numbers of cats per group as degrees of freedom. There is no the recommended dose) (ENRO30). Unfilled circles represent statistically significant difference between baselines and the the a-wave at the baseline; filled squares are from the responses various periods of treatment in groups CTRL, PRADO30 and after one week of treatment and unfilled squares after 4 weeks, PRADO50 and high differences found in group ENRO30 the lines represent the best fit addressed for the Pugh-Lamb

30 mg/kg 1 h after treatment: 10.86 ± 1.30 and indicate that there is saturation of drug absorption in the 7.01 ± 1.30, respectively, and also 24 h after treatment: range of the 30 mg/kg dose. Similar results were 0.31 ± 0.12 and 0.14 ± 0.13, respectively, which may obtained at the 21st day of treatment (Fig. 2).

123 Doc Ophthalmol (2008) 116:177–191 185

Dark adapted ERG intensified to 5% of baseline in the second week, staying constant until the fourth week (Table 1, b-Wave amplitude (rod ERG, maximum response and Fig. 5). Similar effects were seen for constant A’, Naka-Rushton function) which was reduced to 11% after the first week and to 1% from the second week onwards (Table 2, Fig. 5). Dark adapted rod b-wave amplitude and implicit time, scotopic maximum response b-wave amplitude and implicit time, and parameters Vmax, k, and n were Oscillatory potentials (OP) not altered in groups PRADO30, PRADO50 or CTRL in the 4 weeks after treatment. Rod b-wave amplitude There was no statistically difference between OP area was undetectable in ENRO30, and a decrease to under the curve at the baseline and any of the approximately 5% of the baseline in the scotopic treatment periods in CTRL, PRADO30 and PRA- maximum response (P \ 0.05). Vmax was approxi- DO50. However, in group ENRO30 OPs were mately 10% of baseline (P \ 0.05) and these effects reduced to approximately 6% of the baseline after were constant during the whole treatment period. 1 week staying constant until the 4th week of Parameter k increased by approximately 0.6 log-unit treatment (Table 2, Fig. 6). (P \ 0.05) at the first week of treatment and 1 log- unit from the second treatment week on (Tables 1 and 2; Figs. 3 and 4). Light adapted ERG

Cone b-wave a-Wave amplitude and activation constant (A) The luminance necessary to produce 25 lV of cone There was no statistically significant difference in the b-wave amplitude at the baseline remained a-wave amplitude or A between baseline and any of unchanged in CTRL, PRADO30 and PRADO50 over the treatment periods in CTRL, PRADO30 and the entire treatment period (P [ 0.05). In contrast, in PRADO50. In the ENRO30 group, the a-wave ENRO30, it was augmented by 6 log-units after amplitude was diminished to 39% of baseline after 1 week (P \ 0.05), staying constant until the fourth the first week of treatment. This reduction was week of treatment (Table 3, Fig. 7).

Fig. 6 (A) normalized absolute area under the OP curve of dark quantile from the Student’s t-distribution with numbers of cats adapted maximum response elicited by a flash of 10 cd s/m2 per group –1 as degrees of freedom. There is no statistically filtered with digital Fourier transform (FFT) (from 75 to 100 Hz), significant difference between baselines and the various periods plotted against the treatment period in weeks, ‘‘BL’’ is the of treatment in groups CTRL, PRADO30 and PRADO50, and baseline and 1W, 2W, 3W and 4W are the 4 weeks of treatment, high differences (* factor 0.2 after 1 week of treatment) in respectively. Circles are the mean of the ratio between treatment group ENRO30 week and baseline. Error bars represent mean ± the 97.5% 123 186 Doc Ophthalmol (2008) 116:177–191

30 Hz flicker

The 30 Hz flicker amplitude remained unchanged in

CTRL, PRADO30 and PRADO50 in treatment Flicker phase % of period

period. However, in ENRO30, there was a reduction ) (mean ± SEM) V)

to 34% of the baseline amplitude and 10% cycle l Hz 1 delay (phase shifting) was seen after 1 week of 30 treatment, staying constant until the fourth week (Table 3, Fig. 8). Flicker amplitude (

OCT 2 V threshold

Optical coherence tomography allowed to clearly l log cd s/m distinguishing between the two compounds. Retinal 25 thickness in cats on both dosages of PRADO stayed constant over the treatment period, whereas it was significantly reduced in all cats receiving 30 mg of ENRO30 (6· the recommended dose). In addition Flicker phase % of period also the retinal vessel diameter was markedly reduced

in ENRO30 treated cat eyes (results will be published V) l in elsewhere).

Histology Flicker amplitude ( V (threshold); 30 Hz flicker amplitude and phase (% of period time l

Histological examination with light microscopy did 2 not reveal any retinal abnormality in control eyes V threshold

(animals treated with drug vehicle). In eyes of cats l 1 week 4 weeks log cd s/m treated 23 days with pradofloxacin at 50 mg/kg, no 25 retinal abnormality was observed in any retinal area (Fig. 9). In contrast, eyes from cats treated with a toxic overdose of enrofloxacin showed degradation and reduction of the photoreceptor layer, with evidence of Flicker phase % of period

loss of photoreceptor inner and outer segments. The V) inner nuclear layer as well as the ganglion cell layer l showed no obvious degenerative changes and appeared intact (Fig. 9). Flicker amplitude (

Discussion 2

The leading edge of the dark-adapted b-wave is a V threshold l log cd s/m measurement of the extracellular field potential that 25 primarily arises from depolarizing rod bipolar cells in Light adapted luminance necessary to produce b-wave amplitude of 25 response to dim flashes of light. Modeling the b-wave amplitude versus luminance interrelation with the Group Baseline Treatment period CTRLPRADO30 0.73PRADO50 0.62 ± ± 0.08 0.12 0.75ENRO30 ± 0.11 0.95 45.7 ± 48.6 ± 0.27 ± 2.4 3.2 41.2 ± 2.3 48.6 ± 75.24 8.5 75.93 ± ± 0.49 0.74 76.67 ± 0.56 1.04 0.64 ± ± 0.31 0.15 74.05 0.80 ± ± 1.35 0.13 7.22 46.4 ± 50.4 ± 1.54 ± 5.7 3.1 46.2 ± 3.6 17.1 ± 2.6 76.19 73.7 ± ± 0.31 0.61 74.67 ± 0.62 0.71 0.81 ± ± 0.14 0.12 0.88 ± 0.15 84 ± 0.67 47.8 45.1 ± ± 4.3 6.01 2.3 ± 41.4 1.13 ± 2.6 75.42 ± 17.9 0.61 75.9 ± ± 76.17 3.5 0.52 ± 0.36 89.72 ± 2.63 classical Naka-Rushton [27] hyperbolic function Table 3 123 Doc Ophthalmol (2008) 116:177–191 187 yields a reliable marker for rod (dark adapted) Pradofloxacin treatment caused no changes on sensitivity (k). Additionally, an analysis of the dark cone function (neither cone threshold nor flicker adapted saturated a-wave derives the activation amplitude or phase); in contrast, cone function was constant (A0), parameter from the model of rod markedly impaired after treatment with toxic doses of activation proposed by Pugh and Lamb [28] that enrofloxacin. permits evaluation of the rod transduction cascade. In addition, no morphological alterations were Cats showed unchanged k and A0 after treatment evident in histology or OCT with pradofloxacin, with 6 and 10 times the recommended dose of while high dosage enrofloxacin caused retinal degen- pradofloxacin for 23 days, which provides strong eration evident in histology and OCT. evidence of the non-toxicity of this compound on the The general clinical observations found in this rod pathway. In contrast, cats treated with toxic doses study (weight loss, gastrointestinal and motoric of enrofloxacin showed alterations in these parame- dysfunctions) confirm that the doses of enrofloxacin ters, beginning as soon as one week after treatment used here was in the toxic range, since this findings started. Oscillatory potentials demonstrated similar are seldom observed in administration using producer results, indicating that the inner retina also was not recommended doses. The toxic effects of high doses affected by pradofloxacin. of enrofloxacin on the visual system were first noted

Normalized sensitivity threshold AB(1/Logcd.s/m²) Baseline 4 weeks 1.5 CTRL 1

0.5 0 1.5 PRADO30 1

0.5 0 1.5 PRADO50 1

0.5 0 1.5 ENRO30 40 µV 1 20 ms 0.5 0 BL 1W 2W 3W 4W Treatment period

Fig. 7 (A) Example of light adapted ERG responses at plotted in function of the treatment period in weeks, BL is the increasing stimulus luminance (3 steps 1, 3 and 10 cd s/m2) baseline and 1W, 2W, 3W and 4W are the 4 weeks of performed before (Baseline—BL) and after 4 weeks (4W) in treatment, respectively. Circles are the mean of the ratio treatment period. Plots are from one cat of control (untreated) between treatment week and baseline. Error bars represent group (CTRL), one cat treated with pradofloxacin at 30 mg/kg mean ± the 97.5% quantile from the Student’s t-distribution bw/day (6· the recommended dose) (PRADO30), one cat with numbers of cats per group –1 as degrees of freedom. treated with pradofloxacin at 50 mg/kg bw/ day (10· the There is no statistically significant difference between base- recommended dose) (PRADO50) and one cat treated with lines and the various periods of treatment in groups CTRL, enrofloxacin at 30 mg/kg bw/day (6· the recommended dose) PRADO30 and PRADO50, and high differences (* factor 0.2 (ENRO30). (B) Normalized b-wave amplitude threshold after after 1 week of treatment) in group ENRO30 linear interpolation of amplitude against log of luminance 123 188 Doc Ophthalmol (2008) 116:177–191 in cats with diffuse retinal degeneration [29]. Gelatt enrofloxacin-induced retinal degeneration may not be et al. [8] described lesions caused by enrofloxacin idiosyncratic but a concentration-dependent adverse administration limited to the retina with diffuse effect. retinal degeneration, primarily affecting the outer On the other hand, the molecular structural differ- nuclear and photoreceptor layers, and, focal areas of ence between enrofloxacin and pradofloxacin hypertrophy and proliferation of the retinal pigment (position C7 of the basic quinolone ring—Fig. 1) epithelium in the peripheral and central retina. may be responsible for a strong dissimilarity in One main issue concerning why retinal changes melanin affinity between the two compounds (enro- are induced by elevated dosages of enrofloxacin but floxacin [[ pradofloxacin) [22]. This might lead to not by pradofloxacin relates to the different pharma- less drug accumulation of pradofloxacin within the cokinetics profiles of these compounds. In this study cellular lysosomes, including retinal pigment epithe- we showed that that these FQs reach similar serum lium cells, than enrofloxacin. Therefore, it could be concentrations 1 h after oral treatment in cats, hypothesized that different melanin binding properties however, enrofloxacin has approximately 7 times between the two compounds might explain the retinal higher half- life than pradofloxacin. The association toxic effect of enrofloxacin at elevated doses. between high peak enrofloxacin serum concentrations A long term administration of compounds with following high doses administration, and drug accu- high melanin affinity is usually necessary to induce mulation in geriatric or renally impaired cats with ocular toxicity in humans, e.g. CQ (0.25 g or more, retinal degeneration [8] may in fact indicate that daily) [30] or hydroxychloroquine (HCQ) (6.5 mg/kg

A B Normalized Amplitude Normalized Phase (%) Baseline 4 weeks 30 1 CTRL 20

0.5 10

0 0 30 1 PRADO30 20

0.5 10

0 0 30 1 PRADO50 20

0.5 10

0 0 30 40 µV 1 ENRO30 20 20 ms 0.5 10

0 0 BL 1W 2W 3W 4W BL 1W 2W 3W 4W Treatment period Treatment period

Fig. 8 (A) Example of light adapted 30 Hz flicker responses period in weeks, BL is the baseline and 1W, 2W, 3W and 4W (10 cd s/m2) performed before (Baseline—BL) and after are the 4 weeks of treatment, respectively. Circles are the mean 4 weeks (4W) in treatment period. Plots are from one cat of of the ratio between treatment week and baseline. Error bars control (untreated) group (CTRL), one cat treated with represent mean ± the 97.5% quantile from the Student’s t- pradofloxacin at 30 mg/kg bw/day (6· the recommended dose) distribution with numbers of cats per group –1 as degrees of (PRADO30), one cat treated with pradofloxacin at 50 mg/kg freedom. There is no statistically significant difference between bw/ day (10· the recommended dose) (PRADO50) and one cat baselines and the various periods of treatment in groups CTRL, treated with enrofloxacin at 30 mg/kg bw/day (6· the PRADO30 and PRADO50 neither in amplitude nor in phase, recommended dose) (ENRO30). (B) Normalized amplitude and high differences were found in group ENRO30 in and phase (% of period) plotted in function of the treatment amplitude and phase, in group ENRO30 123 Doc Ophthalmol (2008) 116:177–191 189

Fig. 9 Example of retinal sections from one cat of groups retina of cats of group ENRO30 showed diffuse retinal CTRL (treatment with compound vehicle), PRADO50 (prado- degeneration, primarily affecting the outer nuclear and floxacin at 50 mg/kg—10· the recommended doses) and photoreceptor layers and retinal pigment epithelium. RPE: ENRO (enrofloxacin at 30 mg/kg—6· the recommended pigment epithelium; POS: photoreceptor outer segment; PIS, doses), 21 days after treatment start. Retina of the cat in group photoreceptor inner segment; ONL: outer nuclear layer; INL: PRADO50 showed no morphological alterations, while the inner nuclear layer daily) for at least 5 years [31]. The low dosage [34, 35], and that the molecular side chain constituent associated with the mentioned treatment does not on position 7 of the FQ ring has the strongest normally produce acute noticeable ocular changes, as influence on the degree of GABA binding strength. is also the case with administration of enrofloxacin Later, determining field potentials in hippocampus within the recommended treatment doses and dura- slices after administration of a wide range of FQs in tion. Furthermore, the retinal effects observed after rats, Schumuck et al. [36] suggests the N-methyl-D- long periods of CQ or HCQ treatment might be relate aspartate receptor as the probable target of the CNS to the lysosomal drug accumulation that would side effects, and that the excitatory potency is highly possibly occur acutely at higher doses of dependent on the FQ chemical structure. enrofloxacin. In summary, some FQ, as for example enroflox- Alternatively, Leblanc et al. proposed that mela- acin, at toxic dosages seem to initiate a whole nin binding and retinal toxicity were two separate cascade of metabolic and morphologic changes in the entities, the latter being related to the intrinsic retina that can be detected with ERG. Important toxicity of the compound rather than its ability in questions in this respect are whether toxic effects melanin binding [32]. In an experimental study in would be equally observed in pigmented and non- albino rats, Duncker and Bredehorn showed that pigmented animals, if retinal degeneration is revers- after withdrawal of chloroquine, retinal lipidosis ible, and up to which stage of retinal damage is abated, whereas the degeneration of the photorecep- reversibility possible. tor cell layer progressed [33]. However, in this The new veterinary FQ undergoing development, study, retinal functional changes were observed only pradofloxacin, was demonstrated to have no toxic after 12 weeks of treatment and major effects were effects on feline retinas, and should be considered for related to the neuroretina (i.e. ganglion and Mueller veterinary treatment in cases when a FQ is indicated. cells, with few changes in photoreceptors histology) The evidence of retinal safety of pradofloxacin rather than in outer retina, which suggests a confirms that this FQ structural model may be suited different toxic mechanism than that seen with for future applications in human ophthalmology. high-dose enrofloxacin that occurs already 1 week after treatment onset. Acknowledgements The authors acknowledge the expert Other mechanisms to explain retinal toxicity of technical assistance of Mrs. Heike Laser-Junga during performance of the optical coherence tomography and her FQs may arise from influences on retinal neurotrans- very valuable expertise in OCT data analysis, and Mrs Sylvia mitter systems. It has been postulated that FQs may Bolz for histological preparations. We also thank Dr. Frank inhibit binding at the GABAA receptors in the CNS Kro¨tlinger, Dr. Frank Langewische and Dr. Markus Edingloh

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