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Efficacy of Topical Cobalt Chelate CTC-96 Against Adenovirus In a Cell Culture Model and Against Adenovirus Keratoconjunctivitis In a Rabbit Model

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Efficacy of Topical Cobalt Chelate CTC-96 Against Adenovirus In a Cell Culture Model and Against Adenovirus Keratoconjunctivitis In a Rabbit Model
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  BioMed   Central Page 1 of 11 (page number not for citation purposes) BMC Ophthalmology  Open Access Research article Efficacy of topical cobalt chelate CTC-96 against adenovirus in a cell culture model and against adenovirus keratoconjunctivitis in a rabbit model SethPEpstein* †1 , YevgeniaYPashinsky  †1 , DavidGershon †2 , IreneWinicov  †2 , CharlieSrivilasa †1 , KatarinaJKristic  †1  and PennyAAsbell †1  Address: 1 Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York, USA and 2 Redox Pharmaceutical Corporation, Greenvale, New York, USA Email: SethPEpstein*-seth.epstein@mssm.edu; YevgeniaYPashinsky-yevgenia.pashinsky@mssm.edu; DavidGershon-dgershon@redoxpharm.com; IreneWinicov-iwinicov@redoxpharm.com; CharlieSrivilasa-charliesrivilasa7@yahoo.com; KatarinaJKristic-KitKat570@hotmail.com; PennyAAsbell-penny.asbell@mssm.edu* Corresponding author †Equal contributors Abstract Background: Adenovirus (Ad), associated with significant morbidity, has no topical treatment. A leading CTCcompound (CTC-96), a Co III chelate, was found to have potent in vitro and in vivo antiviral efficacy against herpes viruses.In this study CTC-96 is being tested for possible anti-Adenovirus activity. Methods: The biological anti-adenovirus activity of CTC-96 in concentrations from 5 to 250 ug/ml, was evaluatedinitially by viral inactivation (viral exposure to CTC-96 followed by dilution and inoculation of cells), virucidal (viralexposure to CTC-96 and inoculation of cells without dilution) and antiviral (effect of CTC-96 on previously adsorbedvirus) plaque assays on HeLa (human cervical carcinoma), A549 (human lung carcinoma) and SIRC (rabbit corneal) cells.After verifying the antiviral activity, New Zealand White rabbits were infected with Ad-5 into: 1) the anterior cul-de-sacscarifying the conjunctiva (Group "C+"); 2) the anterior cul-de-sac scarifying the conjunctiva and cornea (Group "CC+");3) the stroma (Group "CI+"). Controls were sham-infected ("C-", "CC-", "CI-"). Other rabbits, after "CC", were treatedfor 21 days with: 1) placebo, 9x/day ("-"); 2) CTC-96, 50 ug/ml, 9x/day ("50/9"); CTC-96, 50 ug/ml, 6x/day ("50/6"); CTC-96, 25 ug/ml, 6x/day ("25/6"). All animals were monitored via examination and plaque assays. Results: In vitro viral inactivation, virucidal and antiviral assays all demonstrated CTC-96 to be effective againstAdenvirus type 5 (ad-5). The in vivo model of Ad keratoconjunctivitis most similar to human disease and producing highestviral yield was "CC". All eyes (6/6) developed acute conjunctivitis. "CI" yielded more stromal involvement (1/6) and iritis(5/6), but lower clinical scores (area × severity). Infection via "C" was inconsistent (4/6). Fifty (50) ug/ml was effectiveagainst Ad-5 at 6x, 9x dosings while 25 ug/ml (6x) was only marginally effective. Conclusion: CTC-96 demonstrated virucidal activity against Ad5 in tissue culture with HeLa, A549 and SIRC cell lines.Animal Model Development: 1) "CC" produced conjunctival infection with occasional keratitis similar to human disease;"CI" yielded primarily stromal involvement; 2) "C" consistently produced neither conjunctivitis nor keratitis.CTC Testing: 1) Conjunctivitis in all eyes; 2) Resolution fastest in "50/9" ("50/9". "50/6" > "25/6" > "-"); 3) Efficacy in "50/6" was not statistically different than "50/9"; 4) Conjunctival severity was lower in treatment groups then controls; 5) Little corneal or intra-ocular changes were noted. Published: 05 June 2006 BMC Ophthalmology   2006, 6 :22doi:10.1186/1471-2415-6-22Received: 22 August 2005Accepted: 05 June 2006This article is available from: http://www.biomedcentral.com/1471-2415/6/22© 2006 Epstein et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0   ), which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  BMC Ophthalmology   2006, 6 :22http://www.biomedcentral.com/1471-2415/6/22Page 2 of 11 (page number not for citation purposes) Background  Adenovirus is the most common external ocular viralinfection worldwide[1]. Although not permanently blind-ing, ocular adenoviral infections are associated with sig-nificant patient morbidity, including symptomatic distress, and corneal changes causing visual disturbancesthat can last months to years. About one half of the over 50 serotypes of human adenovirus are known to causeocular disease in patients[1]. Currently there are no spe-cific efficacious antiviral agents for topical or systemic treatment of Adenoviral infections[2]. The studies of the pathogenesis and treatment of ocular adenovirus infections have been limited due to the nar-row host range exhibited by human adenoviruses. It hasbeen previously determined that one serotype of humanadenovirus, adenovirus type 5 (Ad-5), has the ability toextend its host range to permit replication in the eyes of New Zealand rabbits[3,4]. These and other studies have shown human adenovirus type 5 (Ad-5) to present clini-cally within 24 to 48 hours of innoculum in rabbits[5]and last for approximately 16 days post-innoculum(mean duration of shedding)[5,6].  A number of cobalt complexes (CTC compounds) havebeen identified that exhibit potent in vitro and in vivo activity against herpes group viruses[7]. Most signifi-cantly, the mode of action of these novel compounds dif-fer from currently available antiviral nucleoside analogsand protease inhibitors[8] (and unpublished data: Redox Pharmaceutical Corporation). One CTC compound inparticular, CTC-96, has already been shown to exhibit pronounced efficacy in the topical therapy of HSV-1-induced epithelial and stromal disease in the rabbit eye[7].In this study we evaluate the efficacy of topical CTC-96against adenovirus infection in tissue culture on bothhuman and rabbit cell lines as well as against ocular ade-novirus infection in New Zealand White rabbits. Methods  Materials  Adenovirus Human Adenovirus type 5 [9-11] (AD-5) was obtained from the American Type Culture Collection (VR-5; ATCC,Manassas, VA) and propagated on human cervical carci-noma cell monolayers ["HeLa cells" (CCL-2); ATCC, Man-assas, VA]. Stabilized viral stocks grown in EMEM[Vitacells Eagle Minimum Essential Medium (30–2003; ATCC, Manassas, VA)] supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 0.1 mg/ml strepto-mycin and 2 mM L-glutamine (all from Sigma chemicalCo., St. Louis, MO) and containing approximately 9 × 10 8 plaque forming units/ml (pfu/ml) were produced andstored at -80°C for up to 1 year. Cells were infected at aMultiplicity of Infection (MOI) of 10. Eyes were infected with approximately 2 × 10 7 pfu of virus. Cells 1) Human cervical carcinoma cells [HeLa cells (CCL-2); ATCC, Manassas, VA] were grown and maintained inEMEM supplemented with FCS, penicillin, streptomycinand L-glutamine as described above.2) Human lung carcinoma cells [A549 cells (CCL-185); ATCC, Manassas, VA] were grown and maintained inHams F-12 K Medium (30–2004; ATCC, Manassas, VA)supplemented with 10% fetal calf serum, 100 units/mlpenicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine (all from Sigma chemical Co., St. Louis, MO).3) Rabbit corneal cells [SIRC (CCL-60); ATCC, Manassas, VA] were grown and maintained in EMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 0.1mg/ml streptomycin and 2 mM L-glutamine (all fromSigma chemical Co., St. Louis, MO). Pharmaceuticals Doxovir™, CTC-96 (Figure 1) was synthesized according to a standard Redox Pharmaceutical Corporation proce-dure[12]. CTC-96 solution (stable in aqueous solutions at 4°C for >6 months and for several weeks at 37°C) wasprepared from crystalline compound which is completely stable for over 2.5 years when stored desiccated at 0°C. The structure was confirmed by the manufacturer utilizing  1 H and 13 C nuclear magnetic resonance spectroscopy,mass spectrometry, and elemental analysis[12]. Using these techniques, purity was determined to be ≥  99%, with no detectable impurities[12]. Prior to use, drug con-centrations in all CTC-96 solutions were confirmed by HPLC against an independently prepared CTC-96 stand-ard. The activity of the drug solutions was confirmed in an The general structure of Doxovir™ (CTC-96) Figure 1 The general structure of Doxovir™ (CTC-96).  BMC Ophthalmology   2006, 6 :22http://www.biomedcentral.com/1471-2415/6/22Page 3 of 11 (page number not for citation purposes) anti-Herpes Simplex-1 assay[9]. Formulations of this sta-bilized active compound at the appropriate concentra-tions were prepared, filter-sterilized and supplied by themanufacturer. Samples were stored refrigerated and pro-tected from light prior to use.  Animals New Zealand White Rabbits weighing 4–5 pounds werepurchased from H.A.R.E. Industries (Hewitt, NJ). Animals were designated both SPF (specific pathogen free) and VAF (viral antigen free) and were handled in accordance with NIH guidelines. At the conclusion of each experi-ment, the rabbits were euthanized by pentobarbital injec-tion (Sleepaway, Fort Dodge Labs, Fort Dodge, IA).Rabbits were housed in the pathogen containment area(BSL-2) of the Mount Sinai Medical Center Animal Facil-ity. Newly received animals were allowed to acclimate tothe facility and to daily handling for one week prior toinfection. All rabbits were examined to verify no clinicalor culture evidence of viral disease (slit lamp biomicros-copy and viral cultures of corneal swabs followed by standardized plaque assays). Procedures I. In vitro studies A. In vitro viral inactivation assays (direct drug-virus {high titer} neutralization with post-neutralization CTC dilution)  Varying concentrations of CTC-96 or placebo were mixed with concentrated human Adenovirus type 5 (Ad5; 1.75 ×10 7 pfu) and incubated at 37°C for 60 minutes (min)under atmospheric conditions. Aliquots of the virus/drug suspension were then diluted 500 fold and plated in trip-licate on aspirated HeLa, A549 and SIRC cell monolayersto initiate infection (6–8 × 10 5 cells/cm2 in T-25 cultureflasks containing 9.9 mls fresh culture medium giving atotal volume of 10 mls with an MOI of 10). The low finalconcentration of the CTC-96 (1:500) has no inhibitory effect on Adenovirus type 5 growth. All cell lines weregrown and maintained in their appropriate medium. The cell monolayers, having been infected with the virus, were incubated for 24 hours (hr) at 37°C and 5% CO2and then washed, scraped, sonicated, centrifuged and thesupernatant serially diluted. These serial dilutions wereplated onto indicator HeLa, A549 and SIRC cell monolay-ers and adsorbed for 60 min. The supernatant was thenaspirated and a methylcellulose overlay placed over thecells which were then incubated at 37°C. Due to differ-ences in replication rates of the Adenovirus in the variouscell lines, HeLa cells were incubated for 3 days, A549 cellsfor 5 days and the SIRC cells for 7 days. All were counter-stained with 1% methylene blue, allowed to dry and theplaques counted under a phase contrast microscope in amasked fashion. B. In vitro virucidal assays (direct drug-virus neutralization without post-neutralization CTC dilution)  Varying concentrations of CTC-96 or placebo were mixed with concentrated human Adenovirus type 5 (Ad5; 1.75 ×10 7 pfu) and incubated at 37°C for 60 minutes (min).Media of the HeLa, A549 and SIRC cell monolayers (at 80% confluency) was decanted and replaced with theabove suspension. These cell monolayers, having now been infected with the virus (6–8 × 10 5 cells/cm2 in T-25culture flasks containing 9.9 mls fresh culture mediumgiving a total volume of 10 mls with an MOI of 10), wereincubated for 24 hours (hr) at 37°C and 5% CO 2 inmedium containing CTC-96 at the indicated concentra-tions and then washed, scraped, sonicated, centrifugedand the supernatant serially diluted. The serial dilutions were then plated onto indicator HeLa, A549 and SIRC cell monolayers and adsorbed for 60 minas described above. The supernatant was then aspiratedand a methylcellulose overlay placed over the cells which were then incubated at 37°C. All were counterstained with 1% methylene blue, allowed to dry and the plaquescounted under a phase contrast microscope in a maskedfashion. C. In vitro antiviral activity assays (effect of CTC-96 on virus previously adsorbed onto cells)  Adenovirus type 5 (1.75 × 10 7 pfu) was adsorbed ontopreconfluent HeLa, A549 and SIRC cell monolayers for 60min at 37°C. Various concentrations of CTC-96 or pla-cebo in medium were then added to the culture and themonolayers were subsequently incubated for 24 hr at 37°C and 5% CO 2 . Monolayers were then washed,scraped, sonicated, centrifuged, the supernatants serially diluted and ultimately stained and the plaques counted asdescribed above under the section for the viral inactiva-tion assay. D. Procedure  The biological anti-adenovirus activity of freshly solubi-lized CTC-96 was evaluated by the standard antiviral, virucidal and antiviral plaque reduction assays asdescribed above. Seven concentrations of CTC-96 wereused with each cell line and in each assay: 0, 5, 10, 25, 50,100 and 250 ug/ml. Both negative (0 ug/ml CTC-96, no Ad-5) and positive [0 ug/ml CTC-96, addition of Ad-5(MOI = 10)] controls were included. All were diluted inthe appropriate medium for each of the cell lines. E. Data analysis  All clinical and virus recovery data were analyzed for sta-tistical differences, utilizing the mean ± standard devia-tion for each of the various groups in computer-generatedtwo-tailed bivariant Student's t tests (GB-STAT, New Eng-land Software, Inc., College Station, TX, U.S.A.; SAS, SAS  BMC Ophthalmology   2006, 6 :22http://www.biomedcentral.com/1471-2415/6/22Page 4 of 11 (page number not for citation purposes) Institute Inc., Cary, NC, U.S.A.; and SPSS, SPSS Inc., Chi-cago, IL, U.S.A.)[13,14]. Individual Fisher exact tests werealso performed (GB-STAT, SAS and SPSS)[13,14], as well as an overall chi-squared analysis (GB-STAT, SAS andSPSS)[13,14] and a correlation coefficient[13,14]. Two- tailed significance was established at a confidence level of 0.05 ≤  P ≤  0.95. II. Animal studies A. Methods of viral infection (6 groups) 1. Experimentala. Installation of 20 ul containing 4 × 10 7 pfu Ad-5 intothe anterior cul-de-sac and scarifying the conjunctiva (4scratches) with a 25-gauge needle (Group "C+": 8 eyes).b. Installation of 20 ul containing 4 × 10 7 pfu Ad-5 intothe anterior cul-de-sac and scarifying the conjunctiva andcornea (8 scratches: 4 to conjunctiva, 4 to cornea) with a25-gauge needle (Group "CC+": 8 eyes).c. Injection of 20 ul containing 4 × 10 7 pfu Ad-5 into thecorneal stroma with a 25-gauge needle (Group "CI+": 8eyes).Sham-infected animals of all groups were attempted ascontrols:2. Controlsa. Installation of 20 ul containing sterile HeLa media[Eagles Minimum Essential Medium (EMEM; ATCC, Man-assas, VA) + 10% fetal calf serum (Hyclone, Logan, UT) +1% penicillin/streptomycin (Sigma Chemical Company,St. Louis, MO) + 1% l-glutamine (Sigma Chemical Com-pany, St. Louis, MO)] into the anterior cul-de-sac and scar-ifying the conjunctiva (4 scratches) with a 25-gaugeneedle (Group "C-": 8 eyes).b. Installation of 20 ul containing sterile HeLa media intothe anterior cul-de-sac and scarifying the conjunctiva andcornea (8 scratches: 4 to conjunctiva, 4 to cornea) with a25-gauge needle (Group "CC-": 8 eyes).c. Injection of 20 ul containing HeLa media into the cor-neal stroma with a 25-gauge needle (Group "CI-": 8 eyes).3. Procedure The eyes were randomly sorted into groups, examined by a masked observer (ophthalmologist) by slit-lamp biom-icroscopy [Days 1 (preinfection), 3, 5, 7, 10, 12, 15, 17,19, 23, 26, 30 post-infection (pi)] and scored using thegrading system for rabbit conjunctival disease (Table 1)derived from that srcinally described by Wander et al [15]for herpes simplex virus (HSV).Corneal swabs (Viral Culturettes, Baxter, Deerfield, IL) were taken for viral plaque assay twice per week [Days 1(preinfection), 3, 5, 7, 10, 12, 15, 17, 19, 23, 26 and 30pi]. Briefly, sterile culturettes were gently swabbed acrossthe corneas (one per cornea) and the swabbed culturettesimmediately eluted with 0.5 ml of Hanks balanced salt solution and serial 1:10 dilutions prepared for virusassays. Neither sterile culturettes nor cultures received any other manipulation. Culturettes were not frozen prior totitration. Plaque assays were performed in triplicate toensure reproducibility. B. CTC testing  1. Infection The conjunctivae and corneas were infected with 20 ulcontaining 4 × 10 7 pfu of virus into the anterior cul-de-sac and then scarified with a 25-gauge needle (8 scratches: 4to conjunctiva, 4 to cornea: exactly as in Group "CC+"above)[16].2. Treatment Groups (4 groups) The animals were sorted into treatment groups of matched disease severities on day 7 post-infection, whenall eyes showed disease signs, averaging 2–3, suggestive of acute conjunctivitis.Grouped animals were treated, both eyes identically, withinstillation of a 20 ul drop per eye of:a. Placebo treatment (formulation without active ingredi-ent), nine times per day [9x/day ("-"): 2 eyes],b. CTC-96, 50 ug/ml, 9x/day ("50/9"), 2 eyes,c. CTC-96, 50 ug/ml, six times per day [6x/day ("50/6")],2 eyes,or d. CTC-96, 25 ug/ml, 6x/day ("25/6"), 2 eyes. The formulation consisted of: 45 mg/ml mannitol, 60 ug/ml benzalkonium chloride, 2 mg/ml 2-methylimidazolein aqueous solution (pH 7.4). Drug treatments werespread out over each day (7:30 a.m. to 9:30 p.m.), for 21days of treatment (both eyes of each animal treated iden-tically).3. Procedure  BMC Ophthalmology   2006, 6 :22http://www.biomedcentral.com/1471-2415/6/22Page 5 of 11 (page number not for citation purposes)  The eyes were examined in a masked fashion by an oph-thalmologist. They were graded clinically (Table 1) andcorneal swabs (Viral Culturettes, Baxter, Deerfield, IL) were taken for viral plaque assay twice per week [Days 7(pretreatment on day of treatment initiation), 10, 14, 17,20, 25, 28, 31, 35 and 38 post-infection (pi; Days 1, 3, 7,10, 13, 18, 21, 24, 28 and 31 post-treatment initiation(pti)]. Briefly, sterile culturettes were gently swabbedacross the corneas (one per cornea) and the swabbed cul-turettes immediately eluted with 0.5 ml of Hanks bal-anced salt solution and serial 1:10 dilutions prepared for  virus assays. Neither sterile culturettes nor culturesreceived any other manipulation. Culturettes were not fro-zen prior to titration. Plaque assays were performed intriplicate to ensure reproducibility.a. Standardized Viral Plaque Assays Titers of active virions of adenovirus, scored on preconflu-ent HeLa cell culture monolayers and expressed as plaqueforming units (pfus) were determined according to estab-lished procedures[17]. Monolayers were grown and main-tained in "HeLa media" [Vitacell= s Eagle MinimumEssential Medium (30–2003; ATCC, Manassas, VA) + 10%fetal calf serum + 1% penicillin/streptomycin + 1% l-glutamine (all from Sigma chemical Co., St. Louis, MO). Three days after inoculation (at 37°C and 5% CO 2 ) withserial ten-fold dilutions of analyte, plaques had formedand the plates were stained with 1% methylene blue,allowed to dry and the plaques counted under a phasecontrast microscope in a masked fashion in that the exam-iner had no knowledge of the treatment protocol for theindividual animals. The animals were examined in aunique, random order for each examination.b. Ocular Grading SystemClinical disease was divided up into: conjunctival, cornealepithelial and stromal disease and iritis. Corneal epithe-lial disease was subdivided into SPK, epithelial adenovi-rus symptoms, pannus, and epithelial defect. Stromaldisease was subdivided into: edema, melting, neovascu-larization and infiltrate. Both the area and severity of eachsubdivision was graded individually from 0 to +4. Area of involvement was represented in increasing amounts of 25% (0 = "normal cornea", +1 = ≤  25%, +2 = >25%, ≤ 50%, +3 = > 50%, ≤  75%, +4 = > 75%, ≤  100%). Whileseverity of each subdivision was graded while the severity of each subdivision was individually graded from 0 (nor-mal cornea) to +4 (severe).c. Data Analysisaa. Clinical Data While conjunctival, corneal epithelial, stromal and iritisdata was collected clinically, for simplicity only conjunc-tival data was graphed as other findings were insignificant ( ≈ 0). Clinical scores (area × severity) were calculated andutilized for all statistical calculations.Non parametric methods of statistical analysis were usedbecause disease severity is graded by assigning numericalscores[15,16] to observed lesions. The arithmetic meansof such arbitrarily scaled scores should not be analyzed by parametic methods such as the student's t test. Instead,rank medians[18,19], a Wilcoxson-Mann-Whitney rank- sum test[19,20], as well as both Spearman[19,21] and Kendall[19,22] rank correlations, as per standard non- parametric statistics, were used for analysis of the rank nonparametric assigned scores because non-parametric statistical techniques permit evaluation of significance of chemotherapeutic efficacies when measured by changesin severity of disease signs. The results presented were con-sidered significant at a rank median of ≤  0.05 units.bb. Virus Recovery Data Viral titers (per ml) were analyzed for statistical differ-ences, utilizing the mean ± standard deviation for each of the various groups in computer-generated two-tailedbivariant Student's t tests[13,14] (GB-STAT, New England Software, Inc., College Station, TX, U.S.A.; SAS, SAS Insti-tute Inc., Cary, NC, U.S.A.; and SPSS, SPSS Inc., Chicago,IL, U.S.A.)[13,14] at all time points. Individual Fisher  exact tests[13,14] were also performed, as well as an over- all chi-squared analysis[13,14] and a correlation coeffi- cient[13,14]. Two-tailed significance was established at aconfidence level of 0.05 ≥  P ≥  0.95. Results I. In vitro studies  Viral inactivation, virucidal and antiviral efficacy studiesin tissue culture demonstrated CTC-96 to be effectiveagainst human Adenovirus type 5 in a dose-dependent fashion (Tables 1 and 2). CTC-96 at doses ≥  50 ug/mlshows high levels of antiviral efficacy in the viral inactiva-tion, virucidal and antiviral (post-infection) models,against human Adenovirus type 5 in all three cell linesused. The standardized Viral Inactivation, Virucidal and Antiviral assays showed viral yield to be almost zero at aconcentration of 50 ug/ml and virtually zero at 100 ug/mlfor the two human cell types [HeLa & A549; virucidal andantiviral: 6.67 × 10 1 pfu/flask (8-fold inactivation of viri-ons: Table 1)]. In rabbit corneal cells (SIRC), viral yieldreached undetectable levels even at 50 ug/ml (Tables 1and 2). There was a precipitous, dose dependent inhibi-tion between 25 and 50 ug/ml of CTC-96 and a modest effect at still lower concentrations (i.e. 2.47 × 10 9 pfu at 10
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