we have demonstrated , by using adenovirus type 5 , that we can reproduce human adenovirus infection in rabbit eyes and have shown both excellent antiviral activity and conjunctivitis therapy using ctc - 96 which we believe is unique as there is no effective drug against this virus and its pathology in the eye . in addition , we have shown ctc - 96 efficacy against adenovirus types 1 , 2 , 3 , 4 , 5 , and 7 in hela cells in tissue culture . since these human viruses cannot be grown in animal models , this provides an excellent indication of the effectiveness of ctc - 96 against a broad spectrum of adenovirus types . to determine ctc - 96 efficacy against several types of serotypes of adevirus the following procedure was followed : 1 . hela cells were confluent at the time of inoculation . 2 . virus dilutions were prepared from the known titers of the stock viruses ( 4 × 10 5 pfu / ml ; 4 × 10 4 / 0 . 1 ml ) of ad1 kmetz , ad2 wolf , ad3 holyfield , ad4 harris , , ad7a joseph , atcc . this virus inoculation yielded a virus infection with an m . o . i . ( multiplicity of infection ) of approximately 1 . 0 . 3 . 100 □ l of each ad serotype were inoculated onto cultures containing hela cells . 4 . during the adsorption period , doxovir concentrations of 500 , 250 , 100 , 50 , 10 , and 0 μg / ml were prepared in culture medium according to the dilution protocol . 5 . virus was adsorbed at 37 ° c . in a 5 % co 2 water - vapor atmosphere for 1 hour . 6 . after adsorption , the virus inocula were removed from all the wells and 2 wells each were overlayed with 1 ml of doxovir ( in tissue culture medium ) at concentrations of 500 , 250 , 100 , 50 , 10 , and 0 μg / ml . 7 . the plates were incubated at 37 ° c . in a 5 % co 2 water - vapor atmosphere for 24 hours . 8 . after 24 hours , the plates were washed . 9 . each well was refilled with 1 ml of fresh tissue culture medium without doxovir . 10 . the cells were scraped from the wells . 11 . the media and cells were then frozen at − 75 ° c . pending titrations . 12 . titration of duplicate samples were thawed from each ad serotype , doxovir concentration and its no drug control . 13 . viral titers were determined at each drug concentration . ctc - 96 has considerable advantages as an anti - viral drug : a ) because of its unique mode of action it is effective against herpes and hiv virus mutants which are resistant to currently used drugs ; b ) because the drug acts against two different viral targets in herpes virus the development of ctc - 96 - resistant mutants is deemed to be extremely rare ; and c ) because ctc - 96 has anti - inflammatory properties its use replaces the use of steroids in herpes virus and adenovirus therapeutics . steroids modulate the immune response in the areas where they are applied and increase tissue susceptibility to pathogens . efficacy of ctc - 96 against adenovirus types 1 , 2 , 3 , 4 , 5 , and 7 in culture anti - adenovirus activity of ctc - 96 was evaluated by standard cell culture using hela cells , a human cervical carcinoma immortalized cell line ( the usual host for laboratory grade adenovirus ) and anti - viral plaque - reduction assays . ctc - 96 has an inhibitory ( prophylactic ) effect on growth when virus is exposed to the drug prior to cell infection . fig1 shows adenovirus type 5 titers following direct exposure of the virus to ctc - 96 prior to hela cell infection . the data graphically depicted in fig1 were obtained as follows : varying concentrations of the ctc - 96 were mixed with concentrated human adenovirus , [ adenovirus type 5 ( ad5 )] and incubated at 37 ° c . for 60 minutes . aliquots were then diluted 500 fold into growth medium . hela cells were exposed to 100 μl of the diluted material to initiate infection . these monolayers were incubated for 24 hours at 37 % and 5 % co2 and then washed , scraped , sonicated , centrifuged and the supernatant serially diluted . these serial dilutions were plated onto indicator hela cell monolayers and adsorbed for 60 min , aspirated and a methycellulose overlay placed over the cells , which were then incubated for 3 days at 37 %. cultures were counterstained with 1 % methylene blue , allowed to dry and the plaques counted . results are expressed as mean ± sd ( where error bars are not visible they are contained within data point ). ctc - 96 also has a potentially therapeutic effect as can be seen by inhibition of viral growth in adenovirus infected cells , which are subsequently exposed to the drug . fig2 shows virus titers obtained after exposure of human adenovirus type 5 ( ad5 ) infected hela cells to ctc - 96 . these data were obtained as follows : adenovirus was adsorbed onto hela cell monolayers for 60 min at 37 %; serial dilutions of ctc - 96 were overlaid onto the minelayers . monolayers were then incubated for 24 hr at 37 ° c . and 5 % co2 . monolayers were then washed , scraped , sonicated , centrifuged and the supernatant serially diluted . these serial dilutions were plated onto indicator hela cell monolayers and adsorbed for 60 min , aspirated and a methylcellulose overlay placed over the cells , which were then incubated for 3 days at 37 %. cultures were counterstained with 1 % methylene blue , allowed to dry and the plaques counted . results am expressed as mean & amp ; sd ( where error bars are not visible they are contained within data point ). clinical results and plaque assay viral titers of three ctc - 96 treatment / dosing regimens of rabbit eyes infected with human adenovirus , adenovirus type 5 ( ad5 ), were evaluated . on “ day 1 ” animals were infected with human adenovirus type 5 by the installation of 10 6 pfu adenovirus according to our protocol of conjunctival and corneal scarification for the induction of keratoconjunctivitis . clinical conjunctivitis was observed in all animals by day 8 post - inoculation . animals were then randomized and the following experimental groups were treated with ctc - 96 or placebo in a double blind experiment : ( 1 ) placebo ( diluent alone ), 9 ×/ day , for 21 days : ( 4 rabbits ). ( 2 ) ctc - 96 50 μg / ml , 9 ×/ day , for 21 days : ( 4 rabbits ). ( 3 ) ctc - 96 50 μg / ml , 6 ×/ day , for 21 days : ( 4 rabbits ). ( 4 ) c t w 25 μg / ml , 6 ×/ day , for 21 bays : ( 4 rabbits ). clinical disease progression and resolution were evaluated by slit lamp microscopy on days 1 , 3 , 7 , 10 , 13 , 18 , 21 , 24 , 28 and 31 after initial drug dosing . the intensity of the keratitis was quantified using a clinical grading system ( 5 ). application of 25 μg / ml and 50 μg / ml prevented progression of disease severity . application of 50 μg / ml 6 or 9 times a day for 21 days resulted in complete resolution of clinical disease by day 21 while placebo treated animals continued to show symptoms for another 10 days . the results are depicted in fig3 which shows ctc - 96 treatment of adenovirus induced keratoconjunctivitis . the data in fig3 were obtained as follows : rabbits were infected with human adenovirus type 5 by the installation 10 6 pfu adenovirus according to our protocol of conjunctival and corneal scarification for the production of keratoconjunctivitis . on day 8 post - inoculation treatment with eye drops containing ctc - 96 or placebo was initiated . animals were examined for stromal keratitis and scored by the corneal disease scale of wander et al . ( 5 ). the following are the criteria for determination of conjunctival disease : area of conjunctival disease conjunctival severity 0 normal cornea . 0 normal conjunctiva . + 1 ≦ 25 % involved . + 1 mild conjunctival injection . + 2 & gt ; 25 %, ≦ 50 % involved . + 2 moderate conjunctival injection / chemosis . + 3 & gt ; 50 %, ≦ 75 % involved . + 3 severe conjunctival injection / chemosis . + 4 & gt ; 75 %, ≦ 100 % involved . + 4 pseudomembrane present . the efficacy of ctc - 96 treatment of rabbit eyes infected with human adenovirus , adenovirus type 5 ( ad5 ), was also evaluated by adenovirus recovery from tear film cultures adsorbed onto confluent hela cell monolayers . application of 50 μg / ml 6 or 9 times a day resulted in a rapid fall in viral presence in the eye with no detectable virus by day 13 while placebo treated eyes continued to show detectable virus until day 24 . fig4 shows adenovirus titers after treatment of rabbit eyes with ctc - 96 or placebo . these data were obtained by the following procedure : rabbits were infected with human adenovirus type 5 by the installation 10 6 pfu adenovirus according to our protocol of conjunctival and corneal scarification for the production of keratoconjunctivitis . on day 8 post - inoculation treatment with eye drops containing ctc - 96 or placebo was initiated . adenovirus recovery from tear film was evaluated by plaque assay on confluent hela cell monolayers . data are presented as average ± sd .
a method for the therapeutic and prophylactic treatment of adeviruses , more specifically , a method for the therapeutic treatment of adenovirus in a subject by topically administering an antiviral effective amount of ctc - 96 to the subject . in addition , a method for the prophylactic treatment against an adenovirus infection in a subject by topically administering a prophylactically anti - adenovirus effective amount of ctc - 96 to the subject to minimize the likelihood of the subject veing infected by the adenovirus .