Evaluation of voriconazole anti-Acanthamoeba polyphaga in vitro activity, rat cornea penetration and efficacy against experimental rat Acanthamoeba keratitis

Evaluation of voriconazole anti-Acanthamoeba polyphaga in vitro activity, rat cornea penetration... Abstract Background Acanthamoeba keratitis (AK) is a sight-threatening infectious disease. Its effective and safe medical therapy remains highly debated. Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically to treat human AK cases. Objectives To measure anti-Acanthamoeba polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of eye-drop and oral voriconazole. Methods A. polyphaga was maintained in axenic cultures. In vitro, amoebicidal and cysticidal activities of voriconazole were measured using an XTT assay. AK lesions of Sprague Dawley rats were scored from grade 0 to grade 3. For 21 days, from day 7 post-infection, voriconazole (1% solution) eye drops were instilled or voriconazole was administered by gavage (60 mg/kg/day). After killing, superficial corneal epithelium scrapings were cultured and analysed by PCR, and eye-globe histology was performed. Cornea and plasma concentrations were determined using 2D HPLC separation and tandem MS. Results In vitro, voriconazole inhibited trophozoite proliferation with an IC50 value of 0.02 mg/L and an IC90 value of 2.86 mg/L; no cysticidal effect was found. In AK rats, eye drops reduced clinical worsening from day 7 to day 14 post-infection and oral voriconazole was not effective. Voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations, which resulted in lower plasma concentrations, whilst oral voriconazole resulted in lower cornea concentrations. Conclusions Present data underline the need for high-frequency eye-drop instillation regimens for efficient AK therapy. Introduction Although its occurrence is low, human Acanthamoeba keratitis (AK) is a severe, sight-threatening condition, often associated with contact-lens wearing, with a substantial proportion of patients requiring further optical or therapeutic keratoplasty or enucleation. Acanthamoeba castellanii and Acanthamoeba polyphaga are the most common species to cause keratitis.1 The efficacy and safety of currently available medical treatments are highly debated. Biguanides are commonly used as first-line treatment, with polyhexamethylene biguanide (PHMB) and chlorhexidine previously established as the most successful cysticidal agents in vitro.2 Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically in human AK cases.3–5 The aim of this study was to measure anti-A. polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of topical and oral voriconazole. Materials and methods Acanthamoeba From axenic A. polyphaga cultures (isolate ATCC 50495, Rockville, MD, USA) grown at 30°C in 20 cm2 flasks in Peptone Yeast Extract Glucose Broth (PYG) medium, 80% trophozoite suspensions were obtained by refrigerating flasks in ice-water baths. To obtain encystment, washed trophozoites were resuspended in high salt encystment medium (250 mM NaCl, 4.6 mM MgSO4 and 0.36 mM CaCl2) as previously described.6 Voriconazole preparations Voriconazole solutions were prepared with sterile injection water using 10 mg/mL (1% w/v) intravenous voriconazole lyophilisate (Vfend, Pfizer, Paris, France) for in vitro studies and eye drops and 200 mg Vfend tablets for oral treatment. In vitro evaluation of voriconazole amoebicidal and cysticidal activities Trophozoites (103/100 μL/well) were allowed to adhere to 96-well flat-bottom microplates (2 h, 37°C) and voriconazole was added for 72 h (final concentrations of 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 20 and 40 mg/L). Trophozoite viability was measured using an XTT assay (Sigma-Aldrich, Saint-Quentin-Fallavier, France) (final concentration of 0.3 mg/mL, incubation for 6–24 h at 37°C). After subtracting the 630 nm absorbance value from corresponding 490 nm values, for wavelength correction of optical defects, results were expressed as the percentage of control (medium alone) culture well values. To evaluate cysticidal activity, voriconazole was added for 24 or 48 h (final concentrations of 5, 10, 50, 100 and 200 mg/L) in microplates containing 103 cysts/100 μL/well and maintained at 37°C for 5 days, i.e. until >80% trophozoite confluence in control wells. Trophozoite reversion was evaluated using XTT colorimetry after incubation for 2, 4 or 6 h at 37°C and absorbance reading and correction as above. In control wells, >90% trophozoite reversion was microscopically verified. Experimental A. polyphaga rat keratitis and voriconazole treatment All procedures were performed according to regulations of the French Ministry of Research after approval of the ad hoc Ethics Committee (No. 00755.02). In 150 g male Sprague Dawley specific-pathogen-free rats (Janvier, Le Genest-Saint-Isle, France), housed two per cage, AK was obtained as described.7 Eyes were examined weekly using a slit lamp by the same experienced investigator and keratitis lesions were scored. The following grade scheme was used: grade 0, no corneal opacity; grade 1, corneal opacity visible only using oblique slit beam; grade 2, corneal opacity visible using retro-illumination but not sufficient to obscure iris details; or grade 3, corneal opacity visible using retro-illumination and obscuring iris details.8,9 Nineteen rats with left eye AK received voriconazole (1% solution) eye drops daily for 21 days in both eyes. Instillation sequences are summarized in Table 1. Orally treated AK rats received voriconazole by gavage (60 mg/kg/day, two daily doses). Table 1. Rat instillation sequences of 1% voriconazole eye drops and voriconazole cornea concentration measured in the right non-infected cornea Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) p.i., post-infection. Table 1. Rat instillation sequences of 1% voriconazole eye drops and voriconazole cornea concentration measured in the right non-infected cornea Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) p.i., post-infection. Ophthalmic microbiological analyses and histology On day 28 post-infection (p.i.), the rats were euthanized, blood samples were collected and superficial corneal epithelium scrapings were obtained for cultures (non-nutrient agar plates with Escherichia coli suspension overlay, examined for viable Acanthamoeba every week for 1 month). Acanthamoeba spp. quantitative PCR was performed in a Light Cycler 2.0 (Roche Diagnostics), using primers and probe as described by Qvarnstrom et al.10 The reaction mix contained 1× FastStart DNA master mix (Roche Diagnostics, Meylan, France), 0.2 μM primers, 0.2 μM probe, 3.5 mM MgCl2 and 10 μL of DNA in a total reaction volume of 25 μL. Cycling parameters were 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 30 s at 63°C. Eye globes of killed animals were punctured, placed in a 10% buffered formaldehyde solution and paraffin-embedded and 3 mm sections were stained with haematoxylin–eosin. Cornea and plasma voriconazole concentrations during topical and oral treatments in AK rats At the end of each instillation regimen as above, right (non-infected) cornea samples were thawed and weighed, and 100 μL of voriconazole-d5-surcharged methanol (internal standard) was added to each sample as an extraction/precipitation reagent. After vortex-mixing, tubes were incubated for 30 min at room temperature and sonicated for 15 min before centrifugation (16 000 g for 5 min). For plasma samples, each 50 μL aliquot was mixed with 100 μL of extraction/precipitation reagent, vortex-mixed and centrifuged in a like manner. Supernatants were transferred to HPLC vials and 1 μL was injected for quantification in a 2D HPLC separation and tandem MS system consisting of a 3200 QTRAP tandem mass spectrometer equipped with a TurboIonSpray source (AB Sciex, Framingham, USA). Sample clean-up was carried out online in a perfusion column preceding an analytical octadecyl column connected to the mass spectrometer. Quantification was performed by multiple reaction monitoring (MRM) in positive ion mode with voriconazole-d5 as the internal standard. The assay, conducted in accordance with US FDA bioanalytical guidelines,11 resulted in lower and upper quantification thresholds of 0.005 and 0.01 μg/mL, respectively, for both cornea and plasma samples. Data analysis Statistics were performed using contingency analysis (Fisher’s exact tests). A P value <0.05 was considered significant. Results In vitro voriconazole amoebicidal activity Optimal XTT staining to evaluate trophozoite viability was obtained after 24 h incubation (data not shown). In three triplicated independent experiments, voriconazole inhibited trophozoite proliferation in a concentration-dependent manner; incubation with 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 20 and 40 mg/L voriconazole resulted in inhibition percentages of 44.4 ± 1.3, 55.3 ± 3.5, 67.9 ± 22.5, 81.6 ± 26.0, 77.5 ± 10.3, 93.5 ± 3.1, 98.6 ± 1.4, 96.6 ± 4.5 and 100 ± 0.7 (mean ± SD), respectively, corresponding to an average IC50 value of 0.02 mg/L and an average IC90 value of 2.86 mg/L. After cyst incubation with 5, 10, 50, 100 and 200 mg/L voriconazole for 24 or 48 h, reversion to trophozoites was similar to that of untreated controls (>90%, P > 0.05). Voriconazole efficacy against A. polyphaga rat keratitis As shown in Table 2, worsening of clinical symptoms from day 7 to day 14 p.i. was observed in fewer rats in the eye-drop group (topically treated with 1% voriconazole from day 7 to day 28 p.i.) than in the untreated control group (P = 0.001), and clinical infection similarly worsened in both groups from day 14 to day 28 p.i. (P > 0.05). In orally treated rats, worsening of clinical infection was similar to that of untreated controls from day 7 to day 28 p.i. (P > 0.05). Topical or oral voriconazole did not affect day 28 p.i. culture, PCR or histology positivities for A. polyphaga (Table S1, available as Supplementary data at JAC Online). Table 2. Evaluation of the clinical efficacy of eye-drop and oral voriconazole treatments against A. polyphaga rat keratitis symptoms Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 a Grades were evaluated at day 14 p.i. b Ratios of animals with grade worsening from day 7 to day 14 p.i. * P < 0.01 compared with the untreated group. Table 2. Evaluation of the clinical efficacy of eye-drop and oral voriconazole treatments against A. polyphaga rat keratitis symptoms Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 a Grades were evaluated at day 14 p.i. b Ratios of animals with grade worsening from day 7 to day 14 p.i. * P < 0.01 compared with the untreated group. Cornea and plasma voriconazole concentrations in AK rats treated with eye drops and oral voriconazole As shown in Table 1, voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations. Voriconazole cornea concentrations were higher after topical administration hourly and every 2 h than after administration every 4 h (P = 0.005, respectively). Following oral twice-a-day administration of 60 mg/kg/day for 21 days, mean cornea and plasma voriconazole concentrations averaged 1.72 ± 1.79 ng/mg (as ng/μL cornea, considering that 90% of cornea tissue is composed of water)12 and 3.29 ± 2.27 mg/L in 10 rats (mean ± SD), respectively. Discussion The voriconazole Acanthamoeba IC90 evaluation (2.86 mg/L) in this study compares with previously reported values of 1–52.85 mg/L and strong inhibition observed over 40 mg/L.13,14 No cysticidal activity was detected, consistent with the reported cyst resistance of clinical isolates at 1 and 10 mg/L concentrations.15 In other discrepant studies, however, cysticidal activity was obtained at 5–15 mg/L or at higher concentrations for collection strains and clinical isolates, respectively.16,17 Differences in Acanthamoeba strains and methodologies (such as staining) may account for such variations. In an AK rat model, topical and oral voriconazole doses were determined according to previous studies in humans.18 Eye-drop treatment from day 7 p.i. reduced AK lesion worsening from day 7 to day 14 p.i. without later effects, whereas oral voriconazole therapy did not modify clinical lesions until day 28 p.i. In both untreated and treated infected eyes, Acanthamoeba persistence was established on day 28 p.i. by culture, PCR and histology. For the first time, to our knowledge, corneal voriconazole concentrations were measured in non-infected eyes of voriconazole-treated AK rats to suitably document concentrations during infection. At the end of a 3-day hourly topical instillation sequence, cornea concentrations were in the same range as the estimated amoebicidal IC90 and were markedly reduced after switching to instillations every 2 h. While using 1000 × more concentrated eye drops, resulting cornea concentrations were not proportionally increased compared with values obtained in normal and Paecilomyces lilacinus keratitis rabbits, respectively,19 and appeared to depend more on instillation frequency than on voriconazole dose, consistent with an absence of the corneal ‘reservoir’ effect, as reported in human aqueous humour, and reminiscent of the need for high instillation frequency to maintain voriconazole corneal concentrations above the MIC90 for most fungal species.20 Accordingly, corneal voriconazole concentrations were found to be equivalent, after a regimen of eye drops every 2 h, to that in the aqueous humour of humans,20 and elevated aqueous humour concentrations were rapidly obtained after eye instillation in humans.18 In topically treated rats, plasma concentrations were close to those in healthy horses21 and presumably lower than those associated with adverse side effects in humans.22 Owing to ethical constraints, one eye of each rat was infected. Thus, voriconazole was quantified in non-infected cornea since infected corneas were used for microbiological/histological assessments. Since concentrations in infected corneas likely exceed those in non-infected ones, this may represent a limitation for extrapolating our results to infected corneas.12 Reminiscent of reports of aqueous humour in humans, in which voriconazole concentration was half of that in plasma 3 h after oral administration,23 the oral regimen resulted in cornea concentrations lower than the IC90. Unwanted side effects may, however, prevent the use of high systemic doses of voriconazole, which also exhibits high inter- and intra-individual variabilities in residual concentrations and non-linear pharmacokinetic characteristics.22 The present data seem to underline the need for high voriconazole corneal concentrations for AK therapy. Therefore, further studies are necessary to investigate other routes of administration to achieve higher corneal concentrations and to study the cysticidal effect of voriconazole on different Acanthamoeba strains and its efficacy in association with other drugs. Acknowledgements We are grateful to Auda Alsafandi and Sarah Vazirnejad for technical support and for helpful discussion over the course of the in vitro study. Funding Financial support was obtained from the University of Rouen, France. R. R. is a postdoc fellow supported by the University of Rouen, France and the Normandie region, France. Transparency declarations None to declare. Author contributions J. G., L. L. G. and L. F. designed the study, J. G., L. L. G., P. C., S. L., E. C., C. A., F. D., A. F., R. R. and L. F. performed the experiments, J. G., L. L. G., M. M. and L. F. analysed and interpreted the results, J. G. and L. L. G. wrote the draft manuscript, J. J. B. revised the manuscript and M. M. and L. F. approved the final version of the manuscript. Supplementary data Table S1 is available as Supplementary data at JAC Online. References 1 Dart JK , Saw VP , Kilvington S. Acanthamoeba keratitis: diagnosis and treatment update 2009 . Am J Ophthalmol 2009 ; 148 : 487 – 99.e2 . Google Scholar CrossRef Search ADS PubMed 2 Elder MJ , Kilvington S , Dart JK. A clinicopathologic study of in vitro sensitivity testing and Acanthamoeba keratitis . Invest Ophthalmol Vis Sci 1994 ; 35 : 1059 – 64 . Google Scholar PubMed 3 Cabello-Vilchez AM , Martín-Navarro CM , López-Arencibia A et al. Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru . Parasitol Res 2014 ; 113 : 755 – 9 . Google Scholar CrossRef Search ADS PubMed 4 Bang S , Edell E , Eghrari AO et al. Treatment with voriconazole in 3 eyes with resistant Acanthamoeba keratitis . Am J Ophthalmol 2010 ; 149 : 66 – 9 . Google Scholar CrossRef Search ADS PubMed 5 Tu EY , Joslin CE , Shoff ME. Successful treatment of chronic stromal Acanthamoeba keratitis with oral voriconazole monotherapy . Cornea 2010 ; 29 : 1066 – 8 . Google Scholar CrossRef Search ADS PubMed 6 Sykes DE , Band RN. Polyphenol oxidase produced during encystation of Acanthamoeba castellanii . J Protozool 1985 ; 32 : 512 – 7 . Google Scholar CrossRef Search ADS PubMed 7 Vasseneix C , Gargala G , François A et al. A keratitis rat model for evaluation of anti-Acanthamoeba polyphaga agents . Cornea 2006 ; 25 : 597 – 602 . Google Scholar CrossRef Search ADS PubMed 8 Polat ZA , Walochnik J , Obwaller A et al. Miltefosine and polyhexamethylene biguanide: a new drug combination for the treatment of Acanthamoeba keratitis . Clin Exp Ophthalmol 2014 ; 42 : 151 – 8 . Google Scholar CrossRef Search ADS PubMed 9 Ruddell TJ , Easty DL. Drug therapy in a murine model of Acanthamoeba keratitis . Eye 1995 ; 9 : 142 – 3 . Google Scholar CrossRef Search ADS PubMed 10 Qvarnstrom Y , Visvesvara GS , Sriram R et al. Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri . J Clin Microbiol 2006 ; 44 : 3589 – 95 . Google Scholar CrossRef Search ADS PubMed 11 US Department of Health and Human Services, FDA . Guidance for Industry, Bioanalytical Method Validation. http://www.gmp-compliance.org/guidemgr/files/4252FNL.PDF. 12 Cahane M , Ben SGJ , Barequet IS et al. Human corneal stromal tissue concentration after consecutive doses of topically applied 3.3% vancomycin . Br J Ophthalmol 2004 ; 88 : 22 – 4 . Google Scholar CrossRef Search ADS PubMed 13 Rocha-Cabrera P , Reyes-Batlle M , Martín-Navarro CM et al. Detection of Acanthamoeba on the ocular surface in a Spanish population using the Schirmer strip test: pathogenic potential, molecular classification and evaluation of the sensitivity to chlorhexidine and voriconazole of the isolated Acanthamoeba strains . J Med Microbiol 2015 ; 64 : 849 – 53 . Google Scholar CrossRef Search ADS PubMed 14 Schuster FL , Guglielmo BJ , Visvesvara GS. In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri . J Eukaryot Microbiol 2006 ; 53 : 121 – 6 . Google Scholar CrossRef Search ADS PubMed 15 Sunada A , Kimura K , Nishi I et al. In vitro evaluations of topical agents to treat Acanthamoeba keratitis . Ophthalmology 2014 ; 121 : 2059 – 65 . Google Scholar CrossRef Search ADS PubMed 16 Martín-Navarro CM , López-Arencibia A , Arnalich-Montiel F et al. Evaluation of the in vitro activity of commercially available moxifloxacin and voriconazole eye-drops against clinical strains of Acanthamoeba . Graefes Arch Clin Exp Ophthalmol 2013 ; 251 : 2111 – 7 . Google Scholar CrossRef Search ADS PubMed 17 Iovieno A , Miller D , Ledee DR et al. Cysticidal activity of antifungals against different genotypes of Acanthamoeba . Antimicrob Agents Chemother 2014 ; 58 : 5626 – 8 . Google Scholar CrossRef Search ADS PubMed 18 Neoh CF , Leung L , Chan E et al. Open-label study of absorption and clearance of 1% voriconazole eye drops . Antimicrob Agents Chemother 2016 ; 60 : 6896 – 8 . Google Scholar CrossRef Search ADS PubMed 19 Sponsel W , Chen N , Dang D et al. Topical voriconazole as a novel treatment for fungal keratitis . Antimicrob Agents Chemother 2006 ; 50 : 262 – 8 . Google Scholar CrossRef Search ADS PubMed 20 Vemulakonda GA , Hariprasad SM , Mieler WF et al. Aqueous and vitreous concentrations following topical administration of 1% voriconazole in humans . Arch Ophthalmol 2008 ; 126 : 18 – 22 . Google Scholar CrossRef Search ADS PubMed 21 Clode AB , Davis JL , Salmon J et al. Evaluation of concentration of voriconazole in aqueous humor after topical and oral administration in horses . Am J Vet Res 2006 ; 67 : 296 – 301 . Google Scholar CrossRef Search ADS PubMed 22 Dolton MJ , Mikus G , Weiss J et al. Understanding variability with voriconazole using a population pharmacokinetic approach: implications for optimal dosing . J Antimicrob Chemother 2014 ; 69 : 1633 – 41 . Google Scholar CrossRef Search ADS PubMed 23 Hariprasad SM , Mieler WF , Holz ER et al. Determination of vitreous, aqueous, and plasma concentration of orally administered voriconazole in humans . Arch Ophthalmol 2004 ; 122 : 42 – 7 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Evaluation of voriconazole anti-Acanthamoeba polyphaga in vitro activity, rat cornea penetration and efficacy against experimental rat Acanthamoeba keratitis

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Abstract

Abstract Background Acanthamoeba keratitis (AK) is a sight-threatening infectious disease. Its effective and safe medical therapy remains highly debated. Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically to treat human AK cases. Objectives To measure anti-Acanthamoeba polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of eye-drop and oral voriconazole. Methods A. polyphaga was maintained in axenic cultures. In vitro, amoebicidal and cysticidal activities of voriconazole were measured using an XTT assay. AK lesions of Sprague Dawley rats were scored from grade 0 to grade 3. For 21 days, from day 7 post-infection, voriconazole (1% solution) eye drops were instilled or voriconazole was administered by gavage (60 mg/kg/day). After killing, superficial corneal epithelium scrapings were cultured and analysed by PCR, and eye-globe histology was performed. Cornea and plasma concentrations were determined using 2D HPLC separation and tandem MS. Results In vitro, voriconazole inhibited trophozoite proliferation with an IC50 value of 0.02 mg/L and an IC90 value of 2.86 mg/L; no cysticidal effect was found. In AK rats, eye drops reduced clinical worsening from day 7 to day 14 post-infection and oral voriconazole was not effective. Voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations, which resulted in lower plasma concentrations, whilst oral voriconazole resulted in lower cornea concentrations. Conclusions Present data underline the need for high-frequency eye-drop instillation regimens for efficient AK therapy. Introduction Although its occurrence is low, human Acanthamoeba keratitis (AK) is a severe, sight-threatening condition, often associated with contact-lens wearing, with a substantial proportion of patients requiring further optical or therapeutic keratoplasty or enucleation. Acanthamoeba castellanii and Acanthamoeba polyphaga are the most common species to cause keratitis.1 The efficacy and safety of currently available medical treatments are highly debated. Biguanides are commonly used as first-line treatment, with polyhexamethylene biguanide (PHMB) and chlorhexidine previously established as the most successful cysticidal agents in vitro.2 Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically in human AK cases.3–5 The aim of this study was to measure anti-A. polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of topical and oral voriconazole. Materials and methods Acanthamoeba From axenic A. polyphaga cultures (isolate ATCC 50495, Rockville, MD, USA) grown at 30°C in 20 cm2 flasks in Peptone Yeast Extract Glucose Broth (PYG) medium, 80% trophozoite suspensions were obtained by refrigerating flasks in ice-water baths. To obtain encystment, washed trophozoites were resuspended in high salt encystment medium (250 mM NaCl, 4.6 mM MgSO4 and 0.36 mM CaCl2) as previously described.6 Voriconazole preparations Voriconazole solutions were prepared with sterile injection water using 10 mg/mL (1% w/v) intravenous voriconazole lyophilisate (Vfend, Pfizer, Paris, France) for in vitro studies and eye drops and 200 mg Vfend tablets for oral treatment. In vitro evaluation of voriconazole amoebicidal and cysticidal activities Trophozoites (103/100 μL/well) were allowed to adhere to 96-well flat-bottom microplates (2 h, 37°C) and voriconazole was added for 72 h (final concentrations of 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 20 and 40 mg/L). Trophozoite viability was measured using an XTT assay (Sigma-Aldrich, Saint-Quentin-Fallavier, France) (final concentration of 0.3 mg/mL, incubation for 6–24 h at 37°C). After subtracting the 630 nm absorbance value from corresponding 490 nm values, for wavelength correction of optical defects, results were expressed as the percentage of control (medium alone) culture well values. To evaluate cysticidal activity, voriconazole was added for 24 or 48 h (final concentrations of 5, 10, 50, 100 and 200 mg/L) in microplates containing 103 cysts/100 μL/well and maintained at 37°C for 5 days, i.e. until >80% trophozoite confluence in control wells. Trophozoite reversion was evaluated using XTT colorimetry after incubation for 2, 4 or 6 h at 37°C and absorbance reading and correction as above. In control wells, >90% trophozoite reversion was microscopically verified. Experimental A. polyphaga rat keratitis and voriconazole treatment All procedures were performed according to regulations of the French Ministry of Research after approval of the ad hoc Ethics Committee (No. 00755.02). In 150 g male Sprague Dawley specific-pathogen-free rats (Janvier, Le Genest-Saint-Isle, France), housed two per cage, AK was obtained as described.7 Eyes were examined weekly using a slit lamp by the same experienced investigator and keratitis lesions were scored. The following grade scheme was used: grade 0, no corneal opacity; grade 1, corneal opacity visible only using oblique slit beam; grade 2, corneal opacity visible using retro-illumination but not sufficient to obscure iris details; or grade 3, corneal opacity visible using retro-illumination and obscuring iris details.8,9 Nineteen rats with left eye AK received voriconazole (1% solution) eye drops daily for 21 days in both eyes. Instillation sequences are summarized in Table 1. Orally treated AK rats received voriconazole by gavage (60 mg/kg/day, two daily doses). Table 1. Rat instillation sequences of 1% voriconazole eye drops and voriconazole cornea concentration measured in the right non-infected cornea Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) p.i., post-infection. Table 1. Rat instillation sequences of 1% voriconazole eye drops and voriconazole cornea concentration measured in the right non-infected cornea Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) Voriconazole eye-drop regimen Voriconazole cornea concentration (last day of eye-drop sequences), mean ± SD first instillation sequence (frequency/duration) subsequent instillation sequence (frequency/duration) hourly (13 times a day, 8 am to 8 pm)/ 3 days none 5.6 ± 4.4 ng/mg (5 rats) (day 10 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/ 11 days 2.38 ± 1.6 ng/mg (5 rats) (day 17 p.i.) hourly (13 times a day, 8 am to 8 pm)/ 3 days every 2 h (7 times a day, 8 am to 8 pm)/11 days then every 4 h (4 times a day, 8 am to 8 pm)/ 7 days 0.32 ± 0.15 ng/mg (9 rats) (day 28 p.i.) p.i., post-infection. Ophthalmic microbiological analyses and histology On day 28 post-infection (p.i.), the rats were euthanized, blood samples were collected and superficial corneal epithelium scrapings were obtained for cultures (non-nutrient agar plates with Escherichia coli suspension overlay, examined for viable Acanthamoeba every week for 1 month). Acanthamoeba spp. quantitative PCR was performed in a Light Cycler 2.0 (Roche Diagnostics), using primers and probe as described by Qvarnstrom et al.10 The reaction mix contained 1× FastStart DNA master mix (Roche Diagnostics, Meylan, France), 0.2 μM primers, 0.2 μM probe, 3.5 mM MgCl2 and 10 μL of DNA in a total reaction volume of 25 μL. Cycling parameters were 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 30 s at 63°C. Eye globes of killed animals were punctured, placed in a 10% buffered formaldehyde solution and paraffin-embedded and 3 mm sections were stained with haematoxylin–eosin. Cornea and plasma voriconazole concentrations during topical and oral treatments in AK rats At the end of each instillation regimen as above, right (non-infected) cornea samples were thawed and weighed, and 100 μL of voriconazole-d5-surcharged methanol (internal standard) was added to each sample as an extraction/precipitation reagent. After vortex-mixing, tubes were incubated for 30 min at room temperature and sonicated for 15 min before centrifugation (16 000 g for 5 min). For plasma samples, each 50 μL aliquot was mixed with 100 μL of extraction/precipitation reagent, vortex-mixed and centrifuged in a like manner. Supernatants were transferred to HPLC vials and 1 μL was injected for quantification in a 2D HPLC separation and tandem MS system consisting of a 3200 QTRAP tandem mass spectrometer equipped with a TurboIonSpray source (AB Sciex, Framingham, USA). Sample clean-up was carried out online in a perfusion column preceding an analytical octadecyl column connected to the mass spectrometer. Quantification was performed by multiple reaction monitoring (MRM) in positive ion mode with voriconazole-d5 as the internal standard. The assay, conducted in accordance with US FDA bioanalytical guidelines,11 resulted in lower and upper quantification thresholds of 0.005 and 0.01 μg/mL, respectively, for both cornea and plasma samples. Data analysis Statistics were performed using contingency analysis (Fisher’s exact tests). A P value <0.05 was considered significant. Results In vitro voriconazole amoebicidal activity Optimal XTT staining to evaluate trophozoite viability was obtained after 24 h incubation (data not shown). In three triplicated independent experiments, voriconazole inhibited trophozoite proliferation in a concentration-dependent manner; incubation with 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 20 and 40 mg/L voriconazole resulted in inhibition percentages of 44.4 ± 1.3, 55.3 ± 3.5, 67.9 ± 22.5, 81.6 ± 26.0, 77.5 ± 10.3, 93.5 ± 3.1, 98.6 ± 1.4, 96.6 ± 4.5 and 100 ± 0.7 (mean ± SD), respectively, corresponding to an average IC50 value of 0.02 mg/L and an average IC90 value of 2.86 mg/L. After cyst incubation with 5, 10, 50, 100 and 200 mg/L voriconazole for 24 or 48 h, reversion to trophozoites was similar to that of untreated controls (>90%, P > 0.05). Voriconazole efficacy against A. polyphaga rat keratitis As shown in Table 2, worsening of clinical symptoms from day 7 to day 14 p.i. was observed in fewer rats in the eye-drop group (topically treated with 1% voriconazole from day 7 to day 28 p.i.) than in the untreated control group (P = 0.001), and clinical infection similarly worsened in both groups from day 14 to day 28 p.i. (P > 0.05). In orally treated rats, worsening of clinical infection was similar to that of untreated controls from day 7 to day 28 p.i. (P > 0.05). Topical or oral voriconazole did not affect day 28 p.i. culture, PCR or histology positivities for A. polyphaga (Table S1, available as Supplementary data at JAC Online). Table 2. Evaluation of the clinical efficacy of eye-drop and oral voriconazole treatments against A. polyphaga rat keratitis symptoms Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 a Grades were evaluated at day 14 p.i. b Ratios of animals with grade worsening from day 7 to day 14 p.i. * P < 0.01 compared with the untreated group. Table 2. Evaluation of the clinical efficacy of eye-drop and oral voriconazole treatments against A. polyphaga rat keratitis symptoms Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 Group of rats Grade 0a Grade 1a Grade 2a Grade 3a Grade worseningb Untreated (n = 10) 1/10 1/10 4/10 4/10 9/10 Eye-drop voriconazole (n = 10) 0/10 4/10 5/10 1/10 1/10* Oral voriconazole (n = 9) 0/9 1/9 5/9 3/9 4/9 a Grades were evaluated at day 14 p.i. b Ratios of animals with grade worsening from day 7 to day 14 p.i. * P < 0.01 compared with the untreated group. Cornea and plasma voriconazole concentrations in AK rats treated with eye drops and oral voriconazole As shown in Table 1, voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations. Voriconazole cornea concentrations were higher after topical administration hourly and every 2 h than after administration every 4 h (P = 0.005, respectively). Following oral twice-a-day administration of 60 mg/kg/day for 21 days, mean cornea and plasma voriconazole concentrations averaged 1.72 ± 1.79 ng/mg (as ng/μL cornea, considering that 90% of cornea tissue is composed of water)12 and 3.29 ± 2.27 mg/L in 10 rats (mean ± SD), respectively. Discussion The voriconazole Acanthamoeba IC90 evaluation (2.86 mg/L) in this study compares with previously reported values of 1–52.85 mg/L and strong inhibition observed over 40 mg/L.13,14 No cysticidal activity was detected, consistent with the reported cyst resistance of clinical isolates at 1 and 10 mg/L concentrations.15 In other discrepant studies, however, cysticidal activity was obtained at 5–15 mg/L or at higher concentrations for collection strains and clinical isolates, respectively.16,17 Differences in Acanthamoeba strains and methodologies (such as staining) may account for such variations. In an AK rat model, topical and oral voriconazole doses were determined according to previous studies in humans.18 Eye-drop treatment from day 7 p.i. reduced AK lesion worsening from day 7 to day 14 p.i. without later effects, whereas oral voriconazole therapy did not modify clinical lesions until day 28 p.i. In both untreated and treated infected eyes, Acanthamoeba persistence was established on day 28 p.i. by culture, PCR and histology. For the first time, to our knowledge, corneal voriconazole concentrations were measured in non-infected eyes of voriconazole-treated AK rats to suitably document concentrations during infection. At the end of a 3-day hourly topical instillation sequence, cornea concentrations were in the same range as the estimated amoebicidal IC90 and were markedly reduced after switching to instillations every 2 h. While using 1000 × more concentrated eye drops, resulting cornea concentrations were not proportionally increased compared with values obtained in normal and Paecilomyces lilacinus keratitis rabbits, respectively,19 and appeared to depend more on instillation frequency than on voriconazole dose, consistent with an absence of the corneal ‘reservoir’ effect, as reported in human aqueous humour, and reminiscent of the need for high instillation frequency to maintain voriconazole corneal concentrations above the MIC90 for most fungal species.20 Accordingly, corneal voriconazole concentrations were found to be equivalent, after a regimen of eye drops every 2 h, to that in the aqueous humour of humans,20 and elevated aqueous humour concentrations were rapidly obtained after eye instillation in humans.18 In topically treated rats, plasma concentrations were close to those in healthy horses21 and presumably lower than those associated with adverse side effects in humans.22 Owing to ethical constraints, one eye of each rat was infected. Thus, voriconazole was quantified in non-infected cornea since infected corneas were used for microbiological/histological assessments. Since concentrations in infected corneas likely exceed those in non-infected ones, this may represent a limitation for extrapolating our results to infected corneas.12 Reminiscent of reports of aqueous humour in humans, in which voriconazole concentration was half of that in plasma 3 h after oral administration,23 the oral regimen resulted in cornea concentrations lower than the IC90. Unwanted side effects may, however, prevent the use of high systemic doses of voriconazole, which also exhibits high inter- and intra-individual variabilities in residual concentrations and non-linear pharmacokinetic characteristics.22 The present data seem to underline the need for high voriconazole corneal concentrations for AK therapy. Therefore, further studies are necessary to investigate other routes of administration to achieve higher corneal concentrations and to study the cysticidal effect of voriconazole on different Acanthamoeba strains and its efficacy in association with other drugs. Acknowledgements We are grateful to Auda Alsafandi and Sarah Vazirnejad for technical support and for helpful discussion over the course of the in vitro study. Funding Financial support was obtained from the University of Rouen, France. R. R. is a postdoc fellow supported by the University of Rouen, France and the Normandie region, France. Transparency declarations None to declare. Author contributions J. G., L. L. G. and L. F. designed the study, J. G., L. L. G., P. C., S. L., E. C., C. A., F. D., A. F., R. R. and L. F. performed the experiments, J. G., L. L. G., M. M. and L. F. analysed and interpreted the results, J. G. and L. L. G. wrote the draft manuscript, J. J. B. revised the manuscript and M. M. and L. F. approved the final version of the manuscript. Supplementary data Table S1 is available as Supplementary data at JAC Online. References 1 Dart JK , Saw VP , Kilvington S. Acanthamoeba keratitis: diagnosis and treatment update 2009 . Am J Ophthalmol 2009 ; 148 : 487 – 99.e2 . Google Scholar CrossRef Search ADS PubMed 2 Elder MJ , Kilvington S , Dart JK. A clinicopathologic study of in vitro sensitivity testing and Acanthamoeba keratitis . Invest Ophthalmol Vis Sci 1994 ; 35 : 1059 – 64 . Google Scholar PubMed 3 Cabello-Vilchez AM , Martín-Navarro CM , López-Arencibia A et al. Voriconazole as a first-line treatment against potentially pathogenic Acanthamoeba strains from Peru . Parasitol Res 2014 ; 113 : 755 – 9 . Google Scholar CrossRef Search ADS PubMed 4 Bang S , Edell E , Eghrari AO et al. Treatment with voriconazole in 3 eyes with resistant Acanthamoeba keratitis . Am J Ophthalmol 2010 ; 149 : 66 – 9 . Google Scholar CrossRef Search ADS PubMed 5 Tu EY , Joslin CE , Shoff ME. Successful treatment of chronic stromal Acanthamoeba keratitis with oral voriconazole monotherapy . Cornea 2010 ; 29 : 1066 – 8 . Google Scholar CrossRef Search ADS PubMed 6 Sykes DE , Band RN. Polyphenol oxidase produced during encystation of Acanthamoeba castellanii . J Protozool 1985 ; 32 : 512 – 7 . Google Scholar CrossRef Search ADS PubMed 7 Vasseneix C , Gargala G , François A et al. A keratitis rat model for evaluation of anti-Acanthamoeba polyphaga agents . Cornea 2006 ; 25 : 597 – 602 . Google Scholar CrossRef Search ADS PubMed 8 Polat ZA , Walochnik J , Obwaller A et al. Miltefosine and polyhexamethylene biguanide: a new drug combination for the treatment of Acanthamoeba keratitis . Clin Exp Ophthalmol 2014 ; 42 : 151 – 8 . Google Scholar CrossRef Search ADS PubMed 9 Ruddell TJ , Easty DL. Drug therapy in a murine model of Acanthamoeba keratitis . Eye 1995 ; 9 : 142 – 3 . Google Scholar CrossRef Search ADS PubMed 10 Qvarnstrom Y , Visvesvara GS , Sriram R et al. Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri . J Clin Microbiol 2006 ; 44 : 3589 – 95 . Google Scholar CrossRef Search ADS PubMed 11 US Department of Health and Human Services, FDA . Guidance for Industry, Bioanalytical Method Validation. http://www.gmp-compliance.org/guidemgr/files/4252FNL.PDF. 12 Cahane M , Ben SGJ , Barequet IS et al. Human corneal stromal tissue concentration after consecutive doses of topically applied 3.3% vancomycin . Br J Ophthalmol 2004 ; 88 : 22 – 4 . Google Scholar CrossRef Search ADS PubMed 13 Rocha-Cabrera P , Reyes-Batlle M , Martín-Navarro CM et al. Detection of Acanthamoeba on the ocular surface in a Spanish population using the Schirmer strip test: pathogenic potential, molecular classification and evaluation of the sensitivity to chlorhexidine and voriconazole of the isolated Acanthamoeba strains . J Med Microbiol 2015 ; 64 : 849 – 53 . Google Scholar CrossRef Search ADS PubMed 14 Schuster FL , Guglielmo BJ , Visvesvara GS. In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri . J Eukaryot Microbiol 2006 ; 53 : 121 – 6 . Google Scholar CrossRef Search ADS PubMed 15 Sunada A , Kimura K , Nishi I et al. In vitro evaluations of topical agents to treat Acanthamoeba keratitis . Ophthalmology 2014 ; 121 : 2059 – 65 . Google Scholar CrossRef Search ADS PubMed 16 Martín-Navarro CM , López-Arencibia A , Arnalich-Montiel F et al. Evaluation of the in vitro activity of commercially available moxifloxacin and voriconazole eye-drops against clinical strains of Acanthamoeba . Graefes Arch Clin Exp Ophthalmol 2013 ; 251 : 2111 – 7 . Google Scholar CrossRef Search ADS PubMed 17 Iovieno A , Miller D , Ledee DR et al. Cysticidal activity of antifungals against different genotypes of Acanthamoeba . Antimicrob Agents Chemother 2014 ; 58 : 5626 – 8 . Google Scholar CrossRef Search ADS PubMed 18 Neoh CF , Leung L , Chan E et al. Open-label study of absorption and clearance of 1% voriconazole eye drops . Antimicrob Agents Chemother 2016 ; 60 : 6896 – 8 . Google Scholar CrossRef Search ADS PubMed 19 Sponsel W , Chen N , Dang D et al. Topical voriconazole as a novel treatment for fungal keratitis . Antimicrob Agents Chemother 2006 ; 50 : 262 – 8 . Google Scholar CrossRef Search ADS PubMed 20 Vemulakonda GA , Hariprasad SM , Mieler WF et al. Aqueous and vitreous concentrations following topical administration of 1% voriconazole in humans . Arch Ophthalmol 2008 ; 126 : 18 – 22 . Google Scholar CrossRef Search ADS PubMed 21 Clode AB , Davis JL , Salmon J et al. Evaluation of concentration of voriconazole in aqueous humor after topical and oral administration in horses . Am J Vet Res 2006 ; 67 : 296 – 301 . Google Scholar CrossRef Search ADS PubMed 22 Dolton MJ , Mikus G , Weiss J et al. Understanding variability with voriconazole using a population pharmacokinetic approach: implications for optimal dosing . J Antimicrob Chemother 2014 ; 69 : 1633 – 41 . Google Scholar CrossRef Search ADS PubMed 23 Hariprasad SM , Mieler WF , Holz ER et al. Determination of vitreous, aqueous, and plasma concentration of orally administered voriconazole in humans . Arch Ophthalmol 2004 ; 122 : 42 – 7 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Mar 27, 2018

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