Methods for treatment with compositions effective against acyclovir-resistant strains of herpes viruses

The present invention relates to pharmaceutical compositions useful for the treatment of subjects suffering from an infection or disease caused by herpes virus strains that are resistant to treatment with acyclovir. In particular, it has been discovered that the low dosage amounts of 1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-5-iodouracil (FIAU) are effective in inhibiting replication of acyclovir-resistant HSV strains. The present invention is also directed to methods of the preparation of pharmaceutical antiviral compositions and to the methods of their use in the treatment of infection or disease.

1. FIELD OF THE INVENTION 
The present invention relates to pharmaceutical compositions useful for the 
treatment of human subjects suffering from an infection or disease caused 
by Herpes viruses (HSV). In particular, human subjects who suffer from 
infections caused by strains of HSV which are resistant to acyclovir 
treatment can benefit from a therapeutic regimen that includes the 
administration of the compositions of the present invention. 
2. BACKGROUND OF THE INVENTION 
The nucleoside compound 
1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-5-iodouracil (FIAU) has 
been described, for example, in U.S. Pat. No. 4,211,773, as an antiviral 
and an antitumor agent. The disclosure of this patent reference includes 
methods for the preparation of the compound FIAU and also claims 
pharmaceutical compositions comprising a general class of pyrimidine 
analogs, including FIAU, 
1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-5-iodocytosine (FIAC) or 
pharmaceutically acceptable acid addition salts thereof. Cell culture data 
showing the inhibition of Herpes Simplex Virus (HSV) replication are 
presented for a variety of nucleoside analogs. In the case of FIAC, in 
vivo data in mice inoculated with HSV-1 are also presented, showing 
improved survival rates for those test animals that received FIAC compared 
with control animals. 
Other studies involving the in vitro activity of FIAC and its primary 
deaminated uracil metabolite, FIAU, against several herpes group viruses, 
particularly herpes simplex (HSV) types 1 and 2, varicella zoster (VZV), 
and cytomegalovirus (CMV) have been performed. Although not elucidated in 
definitive detail, FIAC/FIAU (FIAC can be considered the prodrug of FIAU) 
apparently exert their effect by serving as substrates for viral DNA 
polymerase. (Cheng, Y.-C. et al. Antimicrobial Agents and Chemotherapy 
1981, 20:420-423.) The in vitro ED.sub.90 value for FIAC/FIAU against 
these viruses ranges from approximately 0.05 .mu.M for HSV-1 to about 0.5 
.mu.M for CMV. Typical cellular toxicity concentrations (ID.sub.50) are 
approximately in the 10 .mu.M range. (Lopez, C. et al. Antimicrobial 
Agents and Chemotherapy 1980, 17(5):803-806; Schinazi, R. F. et al. 
Antimicrobial Agents and Chemotherapy 1986, 29:77-84; Colacino, J. M. et 
al. Antimicrobial Agents and Chemotherapy 1983, 24:505-508; Hantz, O. et 
al. Antiviral Research 1984, 4:187-189). 
2.1. Previous Clinical Studies 
In studies designed to determine the effectiveness of FIAC against herpes 
group viruses, involving more than 100 patients, including 
immune-compromised patients, FIAC was found to be nearly completely 
absorbed following oral administration and was very rapidly converted to 
FIAU. The half-life of FIAU was sufficiently long to allow for effective 
antiviral concentrations &gt;0.2 .mu.M to be present in the plasma for 8 to 
12 hours after an oral dose of FIAC of .ltoreq.2.5 mg/kg that was not 
acutely toxic. (Feinberg, A. et al. Antimicrobial Agents and Chemotherapy 
1985, 27:733-738.) Due to the severe nature of disease in these very 
debilitated patients, high doses (.about.10-20 mg/kg-day) of FIAC were 
used and it proved very difficult and often impossible to distinguish drug 
toxicity from the natural course of these extremely ill patients. 
These studies showed that the active anti-herpetic compound in the plasma 
is FIAU. Oral FIAC, 1 mg/kg/day, was almost never detected in blood two 
hours after dosing, verifying its rapid deamination to FIAU. FIAC 1 
mg/kg/day given in 3 divided doses resulted in FIAU average peak levels of 
approximately 0.5 .mu.g/ml, were consistent between patients, and stable 
from Day 3 of dosing onward. The trough values had the same 
characteristics, except that they averaged approximately 0.05 .mu.g/ml. 
Values of FAU were low, dose dependent (.about.0.05 .mu.g/ml at 0.6 
mg/kg-day vs .about.0.10 .mu.g/ml at 1.0 mg/kg-day) and did not increase 
with continued dosing. These results confirmed that FIAU is the 
predominant active compound present in the plasma, and that neither FIAC, 
FIAU, nor their metabolites accumulated in the plasma during month long 
TID dosing. 
FIAU is the primary metabolite of FIAC and the administration of the 
metabolite simplifies the metabolism involved by eliminating the direct 
conversion of FIAC to FAC, a potentially more toxic metabolite. (Philips, 
F. S. et al. Cancer Research 1983, 43:3619-3627.) 
2.2. Acyclovir Treatment for HSV 
Despite the foregoing clinical trials, acycloguanosine, i.e., acyclovir 
(ACV), remains the principal approved treatment for mucocutaneous HSV 
infection. Acyclovir, 9-(2-hydroxyethoxymethyl) guanine, is a guanine 
derivative with an acyclic chain. It requires the herpes virus-specified 
enzyme, deoxythymidine-deoxycytidine kinase (thymidine kinase) to 
phosphorylate it intracellularly, so that the compound is "activated." The 
"activated" compound inhibits the herpes virus-specified DNA polymerase at 
least 10 times more effectively than cellular DNA polymerase. It acts as 
both inhibitor and substrate for the viral polymerase competing with GTP 
for incorporation into DNA, leading to chain termination because ACV lacks 
the 3'-hydroxyl group required for chain elongation. Since activation of 
ACV requires the herpes virus thymidine kinase, the drug is essentially 
nontoxic to uninfected cells but powerfully inhibitory to viral DNA 
synthesis in infected cells. 
In cell culture, herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) are 
very susceptible to ACV. Varicella-zoster virus (VZV) is susceptible at 
somewhat higher concentrations of the drug. The other human herpes 
viruses, EBV (Epstein Bar virus) and CMV (cytomyalovirus), neither of 
which is known to possess a gene coding for thymidine kinase, are 
nevertheless susceptible to acyclovir, albeit at much greater 
concentrations. In man, acyclovir is given intravenously, orally or 
topically and displays activity against herpes simplex viruses type-1 and 
type-2, and varicella-zoster. For example, acyclovir is very effective 
against herpes simplex keratitis. Intravenous acyclovir protects 
transplant recipients against reactivation of their latent herpes simplex. 
Acyclovir cream has been reported to shorten the healing time of recurrent 
herpes labialis. Whether delivered topically, orally, or intravenously, 
acyclovir reduces the duration of virus shedding, local pain/itching, and 
lesion healing time in primary herpes genitalis, though less persuasively 
in the more short-lived recurrent attacks, particularly in 
immunocompromised patients. 
Despite ACV's efficacy in treating the foregoing conditions, most clinical 
studies have revealed no effect against CMV (congenital CMID of infants, 
or CMV pneumonia following bone marrow transplantation), nor against EBV. 
Because of its proven efficacy and relative lack of toxicity, acyclovir has 
been considered the drug of choice for treating herpes virus infections. 
However, acyclovir-resistant mutants have emerged. In culture, the 
mutations have been located in either the gene coding for the viral 
thymidine-kinase (TK) or that for DNA polymerase. There appear to be two 
kinds of TK mutants: those failing to produce appreciable levels of TK, 
and those in which an altered TK enzyme is produced in substantial 
amounts, i.e., a TK enzyme that has an altered substrate specificity so 
that it can no longer satisfactorily phosphorylate acyclovir. Acyclovir 
resistant mutants have been isolated from man following acyclovir therapy.

3. SUMMARY OF THE INVENTION 
To the great surprise of the applicants, pharmaceutical compositions 
comprising 1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-5-iodouracil 
(FIAU, also known as fialuridine) and a pharmaceutically acceptable 
carrier have been discovered which are effective at inhibiting replication 
of HSV strains that are resistant to treatment with acyclovir. In 
accordance with the invention, such pharmaceutical formulations of FIAU or 
its prodrug, FIAC, may be utilized to treat herpes virus infections in 
human patients. A number of groups of herpes viruses may be treated in 
accordance with the invention, including but not limited to HSV-1, HSV-2, 
VZV and CMV. 
Thus, it is an objective of the present invention to provide a 
pharmaceutical composition for the treatment of infection or disease 
caused by acyclovir-resistant HSV which comprises a pharmaceutically 
acceptable carrier and an amount of FIAU sufficient to provide an 
antivirally effective dosage of FIAU in the range of about 0.05 to about 
10 mg/kg-day. Preferably, the amount of FIAU provided falls in the range 
of about 0.5 to about 5 mg/kg-day. The present invention also contemplates 
a pharmaceutical composition comprising a compound that is a prodrug of 
FIAU or a compound that is a metabolite of FIAU. In particular, FIAC can 
be used as a prodrug, in place of FIAU. Likewise, a metabolite of FIAU, 
1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)uracil (FAU), can be used 
to form at least part of the active ingredient of the pharmaceutical 
antiviral composition of the present invention. Yet other members of the 
general class of deoxyfluorinated pyrimidine nucleosides can also be used 
to form at least part of the active ingredient, including 
1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-5-methyluracil (FMAU) and 
1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-5-ethyluracil (FEAU). 
Yet another objective of the present invention is to provide a 
pharmaceutical composition for the topical treatment of infection or 
disease caused by acyclovir-resistant HSV which comprises a concentration 
of FIAU, a compound that is a prodrug of FIAU or a compound that is a 
metabolite of FIAU which concentration if tested in an in vitro plaque 
reduction assay is equivalent to 20 percent or greater of the 
concentration of FIAU, its prodrug or metabolite (in .mu.g/ml) required to 
reduce the plaque formation induced by said herpes virus strain by fifty 
percent (EC.sub.50) and a pharmaceutically acceptable carrier. 
In yet another aspect of the present invention, pharmaceutical compositions 
for the topical treatment of infection or disease caused by ACV-resistant 
HSV are disclosed which comprise FIAU, a compound that is a prodrug of 
FIAU or a compound that is a metabolite of FIAU and a pharmaceutically 
acceptable carrier, said topical compositions containing about 0.1-25% by 
weight of FIAU, its prodrug or metabolite. 
Further objects of the invention include providing compositions effective 
in a therapeutic regimen against ACV-resistant HSV while minimizing or 
eliminating the side effects associated with the administration of FIAU, 
its prodrug or metabolite, especially at the high dosages recommended in 
the prior art. 
Also contemplated by the present applicants are methods for the preparation 
of pharmaceutical antiviral compositions comprising admixing the 
antivirally active ingredients with a pharmaceutically acceptable carrier. 
In particular, pharmaceutical compositions in the form of solutions, 
suspensions, syrups, tablets, caplets or capsules are contemplated which 
are suitable for topical, oral or parenteral administration. Methods of 
treating human patients infected with ACV-resistant HSV or suffering from 
a disease caused by ACV-resistant HSV comprising the administration of the 
compositions of the present invention are likewise contemplated. Modes of 
administration include topical, oral or parenteral administration. 
Further, the period of treatment is variable, generally lasting about 3 to 
about 30 days, preferably about 7-21 days. 
Thus, these and other objects of the present invention will become apparent 
to those skilled in the art from a reading of the instant disclosure. 
4. DETAILED DESCRIPTION OF THE INVENTION 
4.1. Antiviral Formulations 
Use of pharmaceutically acceptable carriers to formulate FIAC and FIAU into 
dosages suitable for topical or systemic administration is contemplated. 
Topical preparations consisting of solutions, creams, ointments, or gels 
may be utilized. Pharmaceutical preparations for the treatment of vaginal 
infections, such as suppositories, creams, or foams may be prepared. 
Ophthalmic infection, i.e., herpes keratitis, may be treated with normal 
dosage release of FIAU, such as drops or ointments, or by implantation of 
slow release preparations on the conjuctive sac. 
Because FIAC and FIAU are stable in an acid environment, such as is found 
in the human stomach, they can be formulated readily using 
pharmaceutically acceptable carriers well known in the art, with or 
without pH buffers, into dosages suitable for oral administration. Such 
carriers enable FIAU, its prodrug or metabolite to be formulated as 
tablets, pills, capsules, liquids, gels, syrups, suspensions and the like, 
for oral ingestion by a patient to be treated for infection or disease 
caused by HSV. With proper choice of carrier and suitable manufacturing 
practice, the compositions of the present invention, in particular, those 
formulated as solutions, may also be administered parenterally, such as by 
intravenous, subcutaneous or intramuscular injection. 
Pharmaceutical compositions within the scope of the present invention 
include compositions wherein the active ingredient is contained in an 
effective amount to achieve its intended purpose. Determination of the 
effective amounts is well within the capability of those skilled in the 
art, especially in light of the detailed disclosure provided herein. 
In addition to the pyrimidine nucleosides of the present invention, these 
pharmaceutical compositions may contain suitable pharmaceutically 
acceptable carriers comprising excipients and auxiliaries which facilitate 
processing of the active compounds into preparations which can be used 
pharmaceutically. Preferably, the preparations are formulated for oral 
administration, and are in the form of tablets, dragees, and capsules. 
Alternatively, the preparations may be administered rectally, such as in 
the form of suppositories. Alternatively, solutions may be prepared for 
oral or parenteral administration. Topical preparations such as solutions, 
creams, ointments, or gels may also be utilized. The compositions of the 
present invention contain from about 0.1 to about 50 mg of FIAU, its 
prodrug or metabolite, with the balance comprising the components of the 
pharmaceutical carrier. Preferably, the compositions of the present 
invention contain 0.5, 1, 2, 4, 8, 10, 15, 20, or 30 mg of FIAU, its 
prodrug or metabolite. 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that is itself known, e.g., by means of 
conventional mixing, granulating, dragee-making, dissolving, levitating, 
emulsifying, encapsulating, entrapping or lyophilizing processes. Thus, 
pharmaceutical preparations for oral use can be obtained by combining the 
active compounds with solid excipients, optionally grinding a resulting 
mixture, and processing the mixture of granules, after adding suitable 
auxiliaries, if desired, to obtain tablets or dragee cores. 
Suitable excipients are, in particular, fillers such as sugars, including 
lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, 
for example, maize starch, wheat starch, rice starch, potato starch, 
gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, 
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If 
desired, disintegrating agents may be added, such as the cross-linked 
polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as 
sodium alginate. 
Additional auxiliaries that can be used include, but are not limited to, 
flow-regulating agents and lubricants, such as silica, talc, stearic acid 
or salts thereof such as magnesium or calcium stearate, and/or 
polyethylene glycol. Dragee cores are provided with suitable coatings. For 
this purpose, concentrated sugar solutions may be used, which may 
optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, 
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and 
suitable organic solvents or solvent mixtures. Dyestuffs or pigments may 
be added to the tablets or dragee coatings for identification or to 
characterize different combinations of active compound doses. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can 
contain the active ingredients in admixture with filler such as lactose, 
binders such as starches, and/or lubricants such as talc or magnesium 
stearate and, optionally, stabilizers. In soft capsules, the active 
compounds may be dissolved or suspended in suitable liquids, such as fatty 
oils, liquid paraffin, or liquid polyethylene glycols. In addition, 
stabilizers may be added. 
Possible pharmaceutical preparations which can be used rectally or 
vaginally include, for example, suppositories, which consist of a 
combination of the active compounds with a suppository base. Suitable 
suppository bases are, for example, natural or synthetic triglycerides, 
paraffin hydrocarbons, polyethylene glycols or higher alkanols. In 
addition, it is also possible to use gelatin rectal capsules that consist 
of a combination of the active compounds with a base. Possible base 
materials include, for example, liquid triglycerides, polyethylene 
glycols, or paraffin hydrocarbons. 
For topical preparations, the active ingredient may be formulated in an 
ointment, emulsion, cream, gel, powder, spray, foam, or topical solution. 
Water-soluble ointments, containing 0.1-10% active agent in a mixture of 
polyethylene glycols, and lipid-soluble ointments, containing a similar 
amount of active agent in petrolatum, may be prepared as preferred 
formulations for topical use. Creams containing 0.1-5% active agent and 
consisting of water (50%), emulsifying agents, such as glyceryl 
monostearate and polysorbate 80, cetyl alcohol, acting as a surface active 
agent stabilizing the emulsion, stiffening agents, such as wax 
microcrystalline, Softisan.RTM. 601 stiffening agent, paraffin and stearic 
acid, and suitable solvents, such as polyethylene glycol 300 and propylene 
glycol, may be especially useful. Other useful topical preparations are 
gels containing 0.1-5% of active agent in hydroxypropyl cellulose, serving 
as a gelling agent (4%), water (35%), alcohol (50%), and glycerin (10%). 
Topical solutions, containing 0.1-5% active agent in a mixture of alcohols 
(ethanol, 46%; polyethylene glycol-400, 44%; and propylene glycol, 5%), 
and suppositories, containing 0.1-5% active agent in polyethylene glycol 
1000 (50-98%) and polyethylene glycol 3350 (2-30%), are also viewed as 
preferred topical formulations. 
One of the preferred formulations of the compositions of the present 
invention are flavored syrups that contain about 1 to about 10 mg of 
active substance per ml of syrup. Thus, in a particular embodiment of the 
present invention, a flavored syrup comprises about 0.1 to about 1 percent 
by weight of the syrup (wt %) FIAU or FIAC, about 5-50 wt % purified water 
USP, about 5-50 wt % glycerin USP, about 5-50 wt % alcohol USP, about 5-50 
wt % propylene glycol USP. In addition, the syrup also comprises about 
0.0001-0.01 wt % of a coloring agent or combinations thereof, such as FD&C 
Red #40, FD&C Yellow #5 or FD&C Blue #1, about 0.01-0.1 wt % flavoring 
agents, such as artificial or natural Gran Marnier, orange, cherry, 
vanilla, strawberry, raspberry, lemon or chocolate flavor. Moreover, 
commercially available syrup additives, such as Syrup NF qs ad or Maltitol 
syrup, are preferably present at about 0.01 to about 0.1 wt %. 
As mentioned, previously above, pharmaceutical preparations may also be 
prepared for parenteral administration. Suitable formulations for 
parenteral administration include aqueous solutions of the active 
compounds in water-soluble form. Additionally, suspensions of the active 
compounds may be prepared as appropriate oily injection suspensions. 
Suitable lipophilic solvents or vehicles include fatty oils such as sesame 
oil, or synthetic fatty acid esters, such as ethyl oleate or 
triglycerides. Aqueous injection suspensions may contain substances which 
increase the viscosity of the suspension, such as sodium carboxymethyl 
cellulose, sorbitol, or dextran. Optionally, the suspension may also 
contain suitable stabilizers. 
Specific examples of the pharmaceutical compositions of the present 
invention are presented in further detail in the Examples Section of the 
present specification. 
4.2. Dosage Regimens 
A number of different herpes virus infections which are resistant to 
treatment with acyclovir may be treated in accordance with the invention, 
including but not limited to HSV-1, HSV-2, VZV and CMV. For example, HSV-1 
or HSV-2 infections may be treated systemically, or topically. With 
respect to topical applications, ophthalmic preparations of FIAU or its 
prodrug may be utilized for HSV infection of the cornea. Alternatively, 
suppositories may be used to treat vaginal herpes infections. In another 
embodiment, HSV infections of oral mucosa may be treated using ointment 
preparations. In any of the foregoing infections, a systemic treatment 
such as intravenous preparations could be used. 
The precise dosage amounts to be administered will be determined by routine 
experimentation. In particular, lower dosage regimens are preferred which 
range from about 0.05 to about 10 mg/kg-day. Most preferably, dosage 
levels of about 0.5 to about 5 mg/kg-day are to be administered 
continuously for about two weeks. 
Alternatively, dosage levels of active ingredient are administered to 
HSV-infected patients to achieve a steady state antivirally effective peak 
plasma concentration of FIAU, its prodrug or metabolite in the range of 
about 0.1 to about 1 .mu.g/ml. Indeed, it is anticipated that the active 
intracellular form of FIAU (or, possibly, also its metabolite, such as 
FAU) is the triphosphate. Hence, the present invention contemplates 
compositions that, when administered at certain dosage levels, provide or 
maintain intracellular amounts of the triphosphate form of the active 
ingredient which are effective to inhibit HSV. 
4.3. Summary of Results of Studies on In Vitro Activities of FIAU and FIAC 
Against ACV-sensitive and -resistant Strains of HSV-1 and HSV-2 
The antiviral activities of FIAU and FIAC against twenty four ACV-sensitive 
and -resistant strains of HSV-1 and HSV-2 were measured using a plaque 
reduction assay in human foreskin fibroblast (HFF) cells (Kern, E. R. et 
al, J. Infect. Dis. 1973, 128:290-229). This assay involved mixing serial 
two-fold dilutions of each of the three antiviral drugs in twice 
concentrated minimal essential media (MEM) with an equal volume of 1% 
agarose, applying the resulting mixture to monolayer cultures of HFF cells 
one hour after infection with 25-50 PFU of each of the virus strains, 
allowing the resulting cell culture to incubate for 72 hours, staining it 
with neutral red, counting plaques that formed as a result of infection, 
and calculating EC.sub.50 levels, which were defined as the concentration 
of drug that resulted in 50% reduction of the number of plaques in the 
control plates. As shown in Table I, FIAU and FIAC were extremely active 
against ACV-sensitive strains of HSV-1, producing EC.sub.50 values of 
0.01-0.07 .mu.g/ml; ACV gave EC.sub.50 values of 0.2-2 .mu.g/ml. Against 
ACV-resistant strains of HSV-1, FIAU and FIAC gave EC.sub.50 values of 
0.06-15.3 .mu.g/m (for three viral strains, EC.sub.50 values of 0.06-0.6 
were observed); the ACV EC.sub.50 values were greater than 40 .mu.g/ml. 
FIAU and FIAC were extremely potent inhibitors of both ACV-sensitive and 
ACV-resistant strains of HSV-2. Against ACV-sensitive strains, FIAU and 
FIAC gave EC.sub.50 values of 0.01-0.08 .mu.g/ml; ACV showed EC.sub.50 
values of 0.3-0.7 .mu.g/ml. Against ACV-resistant HSV-2 strains, ACV 
produced EC.sub.50 values in excess of 13 .mu.g/ml (primarily in excess of 
100 .mu.g/ml); FIAC and FIAU inhibited nine of these viral strains with 
EC.sub.50. in the range of 0.04-1.3 .mu.g/ml and four with EC.sub.50 
values between 3.6-24 .mu.g/ml. 
4.4. Summary of Results of Studies on Topical Treatment of Genital HSV-2 
Infections in Guinea Pigs With FIAU or FIAC and Their Relevance in the 
Treatment of Genital HSV-2 Infections in Humans 
Intravaginal inoculation of guinea pigs with HSV-2 results in a primary 
genital infection involving viral replication in the vaginal tract 
followed by the development of lesions on the vaginal external genital 
skin surface. This infection is very similar in many respects to the 
primary HSV-2 infection of humans. Studies on the topical treatment of 
HSV-2 infections have shown a good correlation between the results of 
guinea pig animal studies and subsequent human trials (Kern, E. R. 
Herpesvirus, pages 617-636; .COPYRGT.1984 Alan R. Liss, Inc.). Guinea pigs 
were thus used as an animal model system for an investigation of the 
feasibility of treatment of HSV infections with FIAU or FIAC. 
Groups of 10 weanling guinea pigs were infected intravaginally with each of 
an ACV-sensitive and an ACV-resistant HSV-2 virus preparation. 
Intravaginal inoculation with HSV-2, results in a primary genital 
infection followed by development of external vesicular lesions. The 
infected animals were treated with an ointment containing 10, 5, or 1% of 
either FIAU or FIAC, 24 hours after viral inoculation, both intravaginally 
and on the external genital skin. The response to this treatment was 
evaluated in two ways. One involved evaluation of HSV-2 replication in the 
vaginal tract by collection of vaginal secretions on days 1, 3, 5, 7, and 
10 following inoculation and measuring their viral titer in a rabbit 
kidney cell microtiter CPE assay. A second technique, which was used to 
estimate efficacy, involved the visual examination of external genital 
lesions; lesions were graded on a 0-5+ score over a period of 21 days 
after viral inoculation. The results of these viral replication and lesion 
development studies were compared with the corresponding results for 
placebo-treated and control animals in order to determine the efficacy of 
treatment of HSV-2 infections with FIAU or FIAC. 
Topical FIAU preparations containing 5% FIAU were as effective as a 
preparation containing 5% ACV in significantly reducing viral replication 
in the vaginal tract of animals infected with the ACV-sensitive strain of 
HSV-2 while 10% FIAU was more potent. 
In animals infected with an ACV-resistant strain of HSV-2 (12247), 
treatment with 10% or 5% FIAU, but not 1% FIAU, significantly reduced 
vaginal viral production. The 5% ACV preparations had no effect on vaginal 
replication of HSV-2 (12247) with respect to area under virus-titer-day 
curve or mean peak virus titer. 
Treatment of animals infected with the ACV-sensitive strain of HSV-2 with 
5% FIAC was as effective as treatment with 5% ACV in significantly 
reducing viral replication in the vagina. No significant reduction in 
viral growth was observed upon treatment with 1% FIAC in comparison to 
placebo ointment. 
In animals infected with the ACV-resistant 12247 strain of HSV-2, treatment 
with 5% FIAC significantly reduced both the area under the vaginal virus 
titer-curve and mean peak titer-curve. Treatment with 5% ACV had no effect 
on vaginal viral replication with respect to either measurement. 
The striking levels of inhibition of ACV-sensitive and -resistant strains 
of HSV by FIAU and FIAC are further demonstrated in FIGS. I and 2, which 
show the response of infection of guinea pigs by the HSV-2 strains MS 
(ACV-sensitive) and 12247 (ACV-resistant) to each of FIAU and FIAC, 
respectively, as a function of time. The four diagrams of FIG. I 
strikingly demonstrate the fact that ointments containing 5% and 10% FIAU 
are significantly more effective than ointments containing 5% ACV in 
reducing both virus titer and external lesions. The corresponding four 
diagrams of FIG. 2, with equal emphasis, demonstrate the superior effect 
of ointments containing 5% FIAC in reducing both virus titer and external 
lesions. FIG. 2 also shows that although 1% FIAC reduces the viral titer 
of HSV-2 MS less effeciently than 5% ACV, it is significantly more 
effective than ACV in reducing the severity of external lesions. 
In addition, it was found that in animals infected with the ACV-sensitive 
strain of HSV-2, treatment with 10%, 5%, or 1% FIAU, or with 5% or 1% 
FIAC, was more effective than 5% ACV in controlling the extent of genital 
lesion development as measured by the area under the lesion score-day 
curve during infection. While ACV significantly reduced lesion development 
compared to placebo-treated animals (about 50%), the effect was 
considerably less than that observed for FIAU or FIAC treated animals. 
In animals infected with the ACV-resistant 12247 strain, therapy with 1% 
FIAC, or with 5% ACV statistically reduced lesion development to a 
moderate degree. However treatment with 5% FIAC was much more effective 
than either treatment suppressing the lesion area by about 95%. Similarly, 
in animals inoculated with the ACV-resistant virus, therapy with 10%, 5%, 
or 1% of FIAU significantly reduced external lesions (by about 95% from 
control) and was considerably superior to treatment with 5% ACV, which 
suppressed the lesion score by about 50%. 
In addition to the above investigations of FIAC and FIAU, the irritancy and 
toxicity of these drugs was evaluated by treating uninfected animals with 
each of the drugs on the same schedule as the infected animals. No signs 
of any irritation of the genital area or any other type of toxicity were 
observed. 
5. EXAMPLES OF ANTIVIRAL COMPOSITIONS 
5.1. Water-Soluble FIAU Ointment 
1-100.0 mg/g 
______________________________________ 
FIAU Ointment, 5% 
Typical Component 
mg/g % % Range 
______________________________________ 
FIAU 50.0 5.0 0.1-10.0 
Propylene Glycol 
50.0 5.0 1.0-10.0 
Polyethylene Glycol- 
240.0 24.0 10.0-40.0 
3350, USP 
Polyethylene Glycol- 
660.0 66.0 40.0-80.0 
400, USP qs ad 
______________________________________ 
Excipient Rationale 
Propylene Glycol, USP--Co-solvent/Preservative; 
Possible substitutions: Glycerin, ethanol, isopropyl alcohol or other 
alcohols, polysorbates. 
Polyethylene Glycol-3350, USP--Viscosity increasing agent; Possible 
substitutions: 
Polyethylene glycol--900, 1000, 1450, 4500, 8000 or other high molecular 
weight glycols, stearyl alcohol, polyoxyl 40 stearate. 
Polyethylene Glycol--400, USP--Solvent; Possible substitutions: 
Polyethylene glycol 200, 300, 600, or other low molecular weight glycols. 
Method of Manufacture 
1. Add a portion (.about.50%) of the polyethylene glycol--400 to a suitable 
tared stainless steel mixing bowl fitted with a variable speed agitator 
and a source of heat. 
2. Add the polyethylene glycol-3350 to the bowl and warm gently to 
.about.50.degree. C. with gentle agitation. Continue stirring until the 
mixture has melted and a complete solution is obtained. 
3. Add the propylene glycol, USP and FIAU to the solution in Step 2 and 
stir to mix well. 
4. Q.S. to the final weight with polyethylene glycol-400 and blend to 
achieve a uniform, clear solution. 
5. Remove the heat and permit the solution to cool slowly, continuing 
gentle agitation until the solution has congealed to a smooth homogeneous 
ointment and the temperature is below 35.degree. C. 
5.2. FIAU Petrolatum Ointment 
1-50.0 mg/g 
______________________________________ 
FIAU Petrolatum 
Ointment, 1.0% 
Typical Component 
mg/g % % Range 
______________________________________ 
FIAU 10.0 1.0 0.1-5.0 
White Wax 50.0 5.0 1.0-10.0 
White Petrolatum 
940.0 94.0 85.0-98.9 
______________________________________ 
Excipient Rationale 
White wax--Stiffening agent; Possible substitutions: Yellow wax or beeswax, 
paraffin wax or other commercial substitutions. 
White Petrolatum--Ointment Base; Possible substitutions: Yellow Petrolatum 
or other commercial substitutes. 
Method of Manufacture 
1. Combine white wax and white petrolatum into a suitable container and 
heat gently to obtain a clear uniform mixture. 
2. Add FIAU to the mixture obtained in Step 1 and mix until thoroughly 
dispersed (.about.15 min @80.degree. C.). 
3. Remove the heat and permit the solution to cool slowly while mixing 
until the solution has congealed to a smooth homogeneous ointment and the 
temperature is below 35.degree. C. 
5.3. FIAU Cream 
1.0-50.0 mg/g 
______________________________________ 
FIAU Cream, 
1.0% 
Typical Components 
mg/g % % Range 
______________________________________ 
FIAU 10.0 1.0 0.1-5.0 
Purified Water, USP 
500.0 50.0 20.0-80.0 
Cetyl Alcohol, USP 
80.0 8.0 2.0-16.0 
Wax, 80.0 8.0 2.0-16.0 
Microcrystalline, USP 
Polysorbate 80, USP 
50.0 5.0 1.0-10.0 
Polyethylene Glycol- 
50.0 5.0 1.0-10:0 
300, USP 
Propylene Glycol, USP 
50.0 5.0 1.0-10.0 
Softisan-601 .RTM. 
60.0 6.0 1.0-12.0 
stiffening agent 
Stearic Acid, USP 
40.0 4.0 1.0-10.0 
Paraffin, USP 30.0 3.0 1.0-6.0 
Glyceryl 30.0 3.0 1.0-6.0 
Monostearate, USP 
Octoxynol 20.0 2.0 0.5-5.0 
______________________________________ 
Excipient Rationale 
Purified Water--To aid solubility of the active ingredient and in the 
formation of the oil in water cream. 
Cetyl Alcohol--Surface active agent used to stabilize the emulsions and 
imparts a smooth texture to the skin. Possible substitutions: Stearyl 
alcohol, octadecanol. 
Wax Microcrystalline, Softisan.RTM. 601, paraffin and stearic 
acid--Stiffening agents. Possible substitutions: Yellow wax, beeswax or 
other commercial substitutions. 
Polysorbate 80--Because of its hydrophilic and lyophilic characteristics it 
helps as an emulsifying agent. Possible substitutions: fatty acid esters 
of sorbitol and its anhydrides copolymerized with varying numbers of moles 
of ethylene oxide. 
Polyethylene Glycol 300--to aid solubility of the active ingredient. 
Possible substitutions: other low molecular weight glycols. 
Propylene Glycol--Solvent/Preservative. Possible substitutions: Glycerin, 
other alcohols. 
Glyceryl Monostearate--Emulsifying agent; Possible substitutions: polyol 
fatty acid esters. 
Octoxynol--Surfactant and emulsifying agent; Possible substitutions: 
Nonionic surfactants. 
Method of Manufacture 
1. Clean area and weigh out each component. 
2. Add the polysorbate 80, Octoxynol, PEG 300 and propylene glycol 
together. Add approximately 70% of the FIAU and mix. 
3. Melt the paraffin glyceryl monostearate, cetyl alcohol, stearic acid, 
wax and Softisan.RTM. 601 stiffening agent together. 
4. Heat the water (50.degree. C.) and add the remainder of the FIAU and 
stir to dissolve. 
5. Heat the old phase (3) to approximately 80.degree. C. 
6. Heat water to 70.degree. C. 
7. Place water under Ross Misier, add the oil phase and mix. As the cream 
cools, transfer to the kitchen and allow to cool to room temperature with 
gentle mixing. 
5.4. FIAU Gel 
1.0-50.0 mg/g 
______________________________________ 
FIAU Gel, 1.0% 
Typical Components 
mg/g % % Range 
______________________________________ 
FIAU 10.0 1.0 0.1-5.0 
Glycerin, USP 100.0 10.0 1.0-25.0 
Hydroxypropyl 40.0 4.0 0.5-8.0 
Cellulose, NF 
Purified Water, USP 
350.0 35.0 10.0-70.0 
Alcohol, SD40-2 qs ad 
500.0 50.0 20.0-75.0 
______________________________________ 
Excipient Rationale 
Glycerin--Co-solvent/Preservative; Possible substitutions: Propylene 
glycol, isopropyl alcohol, ethanol, methanol or other alcohols. 
Hydroxypropyl Cellulose--Gelling Agent; Possible substitutions: 
Hydroxyethyl cellulose, hydroxypropyl methycellulose, methycellulose or 
other cellulosic agents, carbomer, polyvinyl alcohol, povidone, gelatin or 
other commercial substitutions. 
Purified Water--Co-solvent. 
Alcohol, SD40-2--Co-solvent; Possible substitutions: Alcohol USP, Isopropyl 
alcohol, methanol or other alcohols. 
Method of Manufacture 
1. Combine a portion of the water and alcohol (.about.75% of each) into a 
suitable container and mix well. 
2. Add FIAU to the mixture obtained in Step 1 and mix until dissolved. 
3. Combine the remaining water and glycerin into a suitable container and 
heat to approximately 50.degree. C. 
4. Add the hydroxypropyl cellulose, slowly, to the water/glycerin mixture 
(Step 3) to obtain a smooth slurry. 
5. Add the hydroxpropyl cellulose slurry to the FIAU solution from Step 2. 
6. Q.S. to the final weight with the alcohol and continue mixing for 
approx. 30 minutes, keeping the container covered to avoid evaporation. 
5.5. FIAU Solution 
1.0-50.0 mg/ml 
______________________________________ 
FIAU Solution, 5% 
Typical Components 
mg/ml % % Range 
______________________________________ 
FIAU 50.0 5.0 0.1-5.0 
Propylene Glycol, USP 
50.0 5.0 1.0-10.0 
Polyethylene Glycol- 
440.0 44.0 10.0-80.0 
400, USP 
Alcohol, USP qs ad 
460.0 46.0 10.0-80.0 
______________________________________ 
Excipient Rationale 
Propylene Glycol, USP--Co-solvent/Preservative; Possible substitutions: 
Glycerin, isopropyl alcohol, methanol or other alcohols, polysorbates. 
Polyethylene Glycol-400--Co-solvent; Possible substitutions: Polyethylene 
glycol 200, 300, 600, or other low molecular weight glycols. 
Alcohol, USP--Solvent; Possible substitutions: Alcohol S40-2, isopropyl 
alcohol or other alcohols. 
Method of Manufacture 
1. Combine propylene glycol with polyethylene glycol in a suitable 
container and stir to obtain a uniform solution. 
2. Add a portion (.about.2/3) of the alcohol to Step 1 and mix well. 
3. Add FIAU to Step 2, rinsing the container well with a portion of the 
remaining alcohol. 
4. Q.S. to the final volume with the alcohol and mix well to obtain a clear 
solution. 
5.6. FIAU Suppositories 
Suppository dosage forms containing FIAU or FIAC suitable for vaginal or 
rectal administration as an anti-viral treatment are described. 
A typical preparation in a water soluble base is as follows: 
______________________________________ 
% Wt. gm 
______________________________________ 
Polyethylene Glycol 1000 
95 47.5 
Polyethylene Glycol 3350 
4 2.0 
FIAU 1 0.5 
______________________________________ 
Weigh the components and place the PEG-1000 in a suitable vessel equipped 
with a heat source and agitation. Heat the PEG-1000 with slow stirring to 
-50.degree. C. until the material has melted to a clear liquid. 
Maintaining the temperature at 40.degree. C., add the PEG-3350 and 
continue stirring until the mixture melts to a clear liquid. Add the FIAU 
and mix until dissolved. Remove the heat and allow the mixture to cool 
with slow agitation until the mixture begins to congeal. 
Mold the mass into suppositories using suitable mold. Package and maintain 
the finished suppositories in a cool place until use. 
Excipient Rationale 
BASE: Various ratios or mixtures of high and low molecular weight 
polyethylene glycols or other water miscible glycol polymers serve to 
dissolve the active anti-viral drug. The selection of the molecular weight 
of the polymer and ratios of these polymers permits the control and 
selection of the melting point and firmness of the resultant suppository. 
Various pharmaceutical additions, such as glycols and alcohols, water and 
gelling agents may be employed or substituted to modify the physical 
pharmaceutic characteristics of the suppository. 
Alternatives, and using the same procedure, various pharmaceutically 
acceptable oils and waves may be employed to produce lipid based 
suppositories, such as cocoa better, other natural fat material or 
synthetic high melting petrolatum bases typically employed in the 
preparation of pharmaceutic rectal suppositories. 
5.7. FIAC (Water-miscible suppository) 
______________________________________ 
Typical Component 
mg/g % Range 
______________________________________ 
FIAC 10.0 0.1 to 5.0 
Polyethylene Glycol 1000 
960.0 50 to 98 
Polyethylene Glycol 3350 
40.0 2 to 30.0 
______________________________________ 
Excipient Rationale Polyethylene Glycol 1000: Solvent. Possible 
substitutions polyethylene glycol 900, 1000, 1450 or other high molecular 
weight glycols, stearyl alcohol. Polyethylene Glycol 3350: viscosity 
increasing agent. Possible substitutions polyethylene glycol 4000, 4500, 
8000 or other high molecular weight glycols, polyoxyl 40 stearate. 
Polyoxyethylene sorbiton fatty acid esters can also be included alone or in 
combination with other wax or fatty materials. 
Method of Manufacture 
The method used to prepare the FIAU suppositories (Section 5.6) was used. 
5.8. FIAU (Water-miscible Suppository) 
______________________________________ 
Typical Component 
mg/g % Range 
______________________________________ 
FIAU 10.0 0.1 to 5.0 
Polyethylene Glycol 1000 
960.0 50 to 98 
Polyethylene Glycol 3350 
40.0 2 to 30.0 
______________________________________ 
Excipient Rationale 
Polyethylene Glycol 1000: Solvent. Possible substitutions polyethylene 
glycol 900, 1000, 1450 or other high molecular weight glycols, stearyl 
alcohol. 
Polyethylene Glycol 3350: Viscosity increasing agent. Possible 
substitutions polyethylene glycol 4000, 4500, 8000 or other high molecular 
weight glycols, polyoxyl 40 stearate. 
Polyoxyethylene sorbiton fatty acid esters can also be included alone or in 
combination with other wax or fatty materials. 
Method of Manufacture 
The method used to prepare the FIAU suppositories (Section 5.6) was used. 
5.9. FIAU Syrup 
An FIAU syrup formulation suitable for use as a pharmaceutical antiviral 
composition is described below. In particular, the syrup formulation is 
prepared by combining the components listed, with the amounts 
representative of the content of the listed component, respectively, in 1 
ml of syrup. 
______________________________________ 
FIAU 10 mg 
Glycerin, USP 0.10 ml 
Alcohol, USP 0.10 ml 
Propylene Glycol, USP 0.10 ml 
Purified Water, USP 0.10 ml 
FD&C Red #40 0.025 mg 
FD&C Yellow #5 0.010 mg 
FD&C Blue #1 0.001 mg 
Artificial Flavors 0.001 ml 
Maltitol syrup qs ad 1.0 ml 
______________________________________ 
To a suitable vessel equipped with agitation and a heat source, add the 
glycerin, alcohol and propylene glycol. Mix for approximately five minutes 
to obtain a clear, homogeneous solution, while heating to approximately 
40.degree. C. Next add the FIAU (or FIAC, as the case might be), 
maintaining agitation and rinsing well with the purified water. Mixing is 
continued for at least twenty minutes or until a clear, complete solution 
is obtained. Next, add the coloring and flavoring agents, while 
maintaining agitation for five minutes. Let the mixture cool to room 
temperature, if necessary, and bring the mixture to the final volume with 
the maltitol syrup. Mix gently to avoid incorporation of air bubbles for 
an additional 30 minutes. The batch may be tightly covered and held for 
final filtration and packaging. Filter the syrup through a 1.0 micron 
membrane filter or equivalent and package in final containers. 
The amount of the active ingredient can be adjusted so that variable 
amounts of antiviral agent can be administered per ml of syrup (e.g., 
0.25, 0.5, 1 or 5 mg of FIAU). 
Other examples of syrups, particularly flavored ones, are presented below. 
______________________________________ 
FIAU 10 mg/ml - Orange/Chocolate Flavor 
______________________________________ 
FIAU 10 mg 
Purified Water, USP 0.10 ml 
Glycerin, USP 0.10 ml 
Alcohol, USP 0.10 ml 
Propylene Glycol, USP 
0.10 ml 
FD&C Red #40 0.025 mg 
FD&C Yellow #5 0.010 mg 
FD&C Blue #1 0.001 mg 
Artificial Gran Manier 
0.0005 ml 
Artificial Chocolate 0.0005 ml 
Syrup NF qs ad 1.0 ml 
______________________________________ 
Excipient Rationale 
Vehicle: Maltitol syrup, water, glycerin, alcohol and propylene glycol; 
possible substitutions include glucose, dextrose, mannitol, saccharin, 
sucrose, sorbitol, honey, mucilages, and other flavored syrups. 
Flavors: Orange/vanilla, chocolate/grand marnier, and cherry; possible 
substitutions include raspberry, lemon, spearmint, or citric. This product 
can also contain no flavor. 
Colors: FD & C Red #40, FD & C Blue #1 and FD & C Yellow #5. Other colors 
can be substituted or the product can be without colors. 
Note: This product can contain preservatives or suitable buffering system. 
______________________________________ 
FIAC 10 mg/ml - Cherry/Orange Flavor 
______________________________________ 
FIAU 10 mg 
Purified Water, USP 0.10 ml 
Glycerin, USP 0.10 ml 
Alcohol, USP 0.10 ml 
Propylene Glycol, USP 
0.10 ml 
FD&C Red #40 0.05 mg 
FD&C Yellow #5 0.10 mg 
Natural Orange Flavor 
0.0075 ml 
Artificial Vanilla 0.0035 ml 
Maltitol syrup qs ad 1.0 ml 
______________________________________ 
Excipient Rationale 
Vehicle: Maltitol syrup, water, glycerin, alcohol and propylene glycol; 
possible substitutions include glucose, dextrose, mannitol, saccharin, 
sucrose, sorbitol, honey, mucilages, and other flavored syrups. 
Flavors: Orange/vanilla, chocolate/gran marnier, and cherry; possible 
substitutions include raspberry, lemon, spearmint, or citric. This product 
can also contain no flavor. 
Colors: FD & C Red #40, FD & C Yellow #5. Other colors can be substituted 
or the product can be without colors. 
Note: This product can contain preservatives or suitable buffering system. 
5.10. Oral Solution 
For initial investigation, FIAC will be administered as an oral solution, 
prepared prior to use. Solutions will be prepared by dissolving the FIAC 
Powder for Oral Solution (containing neat FIAC active ingredient) with 
purified water prior to administration. 
Component: FIAC, 
1-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-5-iodocytosine (neat) 
Composition: FIAC will be provided in specified pre-weighed amounts (e.g., 
from about 0.05 to about 500 mg). 
Packaging: Individually weighed doses of FIAC will be packaged in 4 oz. 
amber glass ovals (i.e., Owens-Illinois P-804) 
Closure: Owens-Illinois Clic Lock.RTM. closure 
5.10.1. Method of Preparation 
The FIAC oral solution is prepared by the following procedure: 
To each dosage unit of FIAC powder for solution: 
a. Add 100 ml of purified water USP 
b. Disperse with the aid of an ultrasonic bath (10 minutes) or by 
intermittent shaking (.about.30 minutes) 
c. Observe individual bottles visually to assure complete solution prior to 
use 
d. Maintain unused portion of prepared solution under refrigeration 
e. Note: If refrigerated--carefully observe for complete solution and 
gently warm to room temperature with agitation (ultrasonic bath may be 
used if available) to redissolve if crystals are present 
f. Discard any unused solution after one week from preparation 
5.10.2. FIAC Powder for Oral Solution 
General Methods of Manufacture and Distribution 
Clean bottle by blowing with filtered compressed air (filter 0.45 u). 
Individually weigh and record FIAC for each dose on an analytical balance 
(i.e. Sartorius or equivalent). Afterward, transfer weighed dose of FIAC 
to clean bottle and check weight for complete transfer using individual 
bottle tare on a suitable balance (i.e. Mettler top load model) or 
equivalent. Finally, cap individual bottles after checking weight and hold 
for release and labeling. 
The specifications for FIAC Powder for Oral Solution are treated as a unit 
dose due to the nature of the product. It is recognized that the resulting 
solution product is a multiple dose experimental dosage form. 
The methods of testing are drawn directly from the assay procedure for 
active substance since the experimental unit contains only neat active 
substance. 
______________________________________ 
Specifications Limits 
______________________________________ 
Appearance Crystalline powder, white to 
off-white 
Identification, HPLC 
Conforms with standard 
Solution Description 
Clear, colorless solution 
Assay for FIAC and Related Substances 
FIAC 95.0-105.0% of label claim 
FIAU 2.0% maximum 
Other Related Substances 
2.0% maximum 
______________________________________ 
5.11. One, Five and Ten Milligram Capsules 
Lower dosage range capsules were prepared as follows. Due to content 
uniformity considerations, a new technique referred to as Moisture 
Activated Dry Granulation, MADG, was used to prepare the capsule blends. 
This technique is briefly described below: 
MADG combines the ease of manufacture of a direct blend formulation with 
the advantages of a wet granulation. A modified version of the 
agglomeration step in wet granulation is utilized, however, no drying step 
is required. Briefly, this granulation technique involves blending the 
drug with an excipient ("carrier"--e.g. lactose) and a dry binder (e.g., 
PVP). This blend is then moistened using a small amount of water (1-3% of 
the formula weight). This moisture activates the dry binder and makes the 
drug adhere to the carrier particles. Moisture distributing agents (e.g. 
microcrystalline cellulose) and additional excipients are then added and 
the granulation process is complete. The resultant granulation provides 
good content uniformity as well as a free flowing blend for encapsulation 
or tabletting. 
The choice and levels of excipients for the low dosage capsules can be 
determined as needed by one skilled in the art. Also, the FIAU bulk can be 
passed through a 200 mesh screen rather than a 100 mesh screen for content 
uniformity considerations. 
5.12. FIAU Sodium Salt 
FIAU sodium salt for injection is a lyophilized sodium salt of FIAU. The 
sodium salt is prepared by allowing FIAU to react with sodium hydroxide in 
situ during preparation of the solution for lyophilization. The 
lyophilized cake is white in color. 
A clinical lot of FIAU sodium salt for injection was reconstituted for 
stability studies with Sterile Water for Injection, USP, 0.9% Sodium 
Chloride Injection, USP, and 5% Dextrose Injection, USP. The solutions 
were stored at room temperature (25.degree. C.). Chemical potency, 
impurities and degradation products were monitored by HPLC. The solution 
pH and physical appearance were also monitored. The chemical potency data 
were statistically analyzed. 
These studies support a utility time of 48 hours storage at room 
temperature (25.degree. C.) for the following diluents and concentrations, 
provided that the preconstituted vials have been stored at room 
temperature (25.degree. C.) and have not aged beyond their expiration 
date. 
Each vial was reconstituted with 2.2 ml of Sterile Water for Injection, 
USP, yielding a solution concentration of 50 mg/ml. Lower concentrations 
can be prepared with the appropriate adjustments of the respective 
components. 
Alternatively, the above solution when further diluted with Sterile Water 
for Injection, USP, 0.9% Sodium Chloride Injection, USP or 5% Dextrose 
Injection, USP, yielded a solution concentration of 0.2-5 mg/ml. 
5.13. FIAU Capsule (1-10 MG) 
______________________________________ 
Typical Components 
______________________________________ 
FIAU 3.03% 10.00 1-10 
Microcrystalline 20.0% 66.00 59-73 
Cellulose (Avicel .RTM.) 
Magnesium Sterate, USP 
0.5% 1.65 1.5-1.8 
Explotab .RTM. disintegrant 
2.0% 6.60 5.9-7.3 
Lactose, Hydrous USP qs ad 
76.0% 250.80 225-275 
Fill Weight 330 mg 
Capsule Size #1 
______________________________________ 
Excipient Rationale 
a. Diluent--microcrystalline cellulose (Avicel.RTM.), lactose, hydrous. 
Possible substitutions: Lactose, anhydrous. 
b. Lubricant--magnesium sterate. Possible substitutions: Talc, calcium 
sterate, stearic acid, magnesium salts. 
c. Disintegrant--Explotab.RTM. disintegrant. Possible substitutions: 
Starch, providone XL, sodium starch glycolate, croscarmelose, 
methylcellulose, carboxymethyl cellulose. 
Method of Manufacture 
a. Mix FIAU with an equal portion of lactose. 
b. Gradually add remainder of lactose and mix well. 
c. Combine Avicel.RTM. microcrystalline cellulose, Explotab.RTM. 
disintegrant and magnesium sterate and mix well. 
d. Combine the FIAU/lactose mixture (Step 2) with the Avicel.RTM. mixture 
(Step 3) and mix until sufficiently blended (.about.10 min. minimum). 
5.14. FIAU Tablets (1-10 mg) 
Wet Granulation/Direct Compression Method 
______________________________________ 
FIAU FIAU 
Tablet, 
Tablet, 
5 mg. 10 mg. 
Typical Components 
% % Range mg/cap mg/cap 
______________________________________ 
FIAU (5%) a 0.25-5 5 10 
Lactose (55%) a 40-70 -- -- 
PVP (15% in water) 
a 10-20 60.5 60.5 
Avicel PH101 56.5% 40-75 565 565 
microcrystalline cellulose 
Lactose 31.0% 20-50 310 310 
Explotab .RTM. 
5.0% 2-10 50 50 
disintegrant 
Sterotex K 1.0% 0.5-5 10 10 
Magnesium Stearate, 
0.5% 0.2-2 5 5 
USP 
Net Tablet Weight 200 mg 400 mg 
______________________________________ 
a = The combined percentage of the first three items is 6%. 
Excipient Rationale 
a. Diluent--lactose, hydrous. Possible substitutions: Lactose, anhydrous, 
microcrystalline cellulose (Avicel.RTM.). 
b. Lubricant--magnesium stearate; possible substitutions: Talc, calcium 
stearate, stearic acid, magnesium salts. 
c. Disintegrant--possible substitutions. Starch, providone XL, sodium 
starch glycolate, methylcellulose, carboxymethyl cellulose. 
d. Binder--Sterotex K. Possible substitutions: Starch, gelatin, sugars 
(sucrose, glucose, etc.), carboxymethylcellulose, methylcellulose, 
polyvinyl pyrrolidone. 
Method of Manufacture 
a. Mix the FIAU and lactose together, wet the mixture with the 15% Solution 
of PVP in water. 
b. Dry the granulation overnight at 40.degree. C. 
c. Pass the dried granulation through a 10 mesh screen and then through a 
20 mesh screen. 
d. Mix the magnesium stearate and sterotex K together and then add the 
explotab. 
e. Combine the two mixtures obtained in Step c and d above. 
f. Add the Avicel microcrystalline cellulose, lactose and mixture obtained 
in Step e to dry blender. 
g. Allow the mixture to blend for 15 minutes. 
h. Compress the tablet to the specified weight. 
______________________________________ 
Description 1 mg Tablet 2 mg Tablet 
______________________________________ 
Diameter 9 mm 9 mm 
Thickness 4.0 .+-. 0.5 mm 
6.0 .+-. 0.5 mm 
Hardness 3-10 kg 3-10 kg 
______________________________________ 
5.15. FIAU Tablets (1-10 mg) 
Direct Compression Method 
______________________________________ 
FIAU FIAU 
Tablet, 
Tablet, 
5 mg. 10 mg. 
Typical Components 
% % Range mg/cap mg/cap 
______________________________________ 
FIAU (100 mesh) 
2.5% 0.5-2.5 5 10 
Avicel PH101 55.0% 40-75 110 220 
microcrystalline cellulose 
Lactose, Hydrous USP 
36.0% 25-55 72 144 
Explotab .RTM. 
5.0% 2-10 10 20 
disintegrant 
Sterotex K 1.0% 0.5-5 2 4 
Magnesium Stearate, 
0.5% 0.2-2 1 2 
USP 
Net Tablet Weight 200 mg 400 mg 
______________________________________ 
Excipient Rationale 
a. Diluent--microcrystalline cellulose (Avicel.RTM.), lactose, hydrous. 
Possible substitutions: Lactose, anhydrous. 
b. Lubricant--magnesium stearate; possible substitutions: Talc, calcium 
stearate, stearic acid, magnesium salts. 
c. Disintegrant--Explotab.RTM.; possible substitutions: Starch, providone 
XL, sodium starch glycolate, croscarmelose, methylcellulose, carboxymethyl 
cellulose. 
d. Binder--Sterotex K, possible substitutions: starch, gelatin, sugars 
(sucrose, glucose, etc.), carboxymethylcellulose, methylcellulose, 
polyvinyl pyrrolidone. 
Method of Manufacture 
a. Mix the magnesium stearate and Sterotex K in a plastic bag. 
b. Add the Explotab.RTM. disintegrant to the mixture obtained in Step b. 
c. Add the FIAU to the mixture obtained in Step b. 
d. Add the Avicel PH101 microcrystalline cellulose, lactose and the mixture 
obtained in Step c in layers in the twin shell dry blender. 
e. Mix for 15 minutes. 
f. compress the tablet at the specified weight. 
______________________________________ 
Description 1 mg Tablet 
2 mg Tablet 
______________________________________ 
Diameter 9 mm 9 mm 
Thickness 3.5 .+-. 0.5 mm 
7.0 .+-. 0.5 mm 
Hardness 3-10 kg 3-10 kg 
______________________________________ 
6. In Vitro Activity of FIAU Against and FIAC Against ACV-Sensitive and 
ACV-Resistant HSV Strains 
The in vitro experiments and results outlined below demonstrate that FIAU 
and FIAC are effective against both ACV-sensitive and -resistant strains 
of HSV-1 and HSV-2. 
The following assay was used to evaluate the anti-HSV properties of FIAU, 
FIAC and ACV. 
6.1. Plaque Reduction Assay for HSV-1 and HSV-2 using Semi-Solid overlay 
Two days prior to use, HFF cells are plated into six well plates and 
incubated at 37.degree. C. with 5% CO.sub.2 and 90% humidity. One the date 
of assay, the drug is made up at twice the desired concentration in 
2.times. MEM and then serially diluted 1:5 in 2X MEM using six 
concentrations of drug. The initial starting concentration is usually 200 
.mu.g/ml down to 0.06 .mu.g/ml. The virus to be used is diluted in MEM 
containing 10% FBS to a desired concentration which will give 20-30 
plaques per well. The media is then aspirated from the wells and 0.2 ml of 
media being added to drug toxicity wells. The plates are then incubated 
for one hour with shaking every fifteen minutes. After the incubation 
period, an equal amount of 1% agarose was added to an equal volume of each 
drug dilution. This will give final drug concentrations beginning with 100 
.mu.g/ml and ending with 0.03 .mu.g/ml and a final agarose overlay 
concentration of 0.5%. The drug agarose mixture is applied to each well in 
2 ml volume and the plates then incubated for three days, after which the 
cells were stained with a 1.5% solution of neutral red. At the end of 4-6 
hr incubation period, the stain is aspirated, and plaques counted using a 
stereomicroscope at 10.times. magnification. 
6.2. Assay Results 
The results of the plaque reduction assay described above for ACV, FIAU, 
and FIAC, expressed as EC.sub.50 values, are summarized in Table I. 
TABLE I 
______________________________________ 
SUSCEPTIBILITY OF HSV ISOLATES TO 
ACV, FIAU, AND FIAC 
EC.sub.50 
(.mu.g/ml).sup.a 
Virus strain 
TK Phenotype 
ACV FIAU FIAC 
______________________________________ 
HSV-1 
E-377 positive 0.20 0.02 0.06 
SC 16 positive 0.20 0.04 0.05 
SC 16-S1 altered 91.0 0.14 0.06 
PAAr.sup.5 positive.sup.b 
2.0 0.02 0.07 
DM 2.1 deficient &gt;100 8.9 9.3 
11893 altered 40. 0.08 0.6 
13231 positive 0.30 0.01 0.04 
11359 deficient &gt;100 2.1 15.3 
13545 positive 0.78 0.01 0.04 
11360 deficient 85.0 0.03 0.50 
B-2006 deficient &gt;100 2.50 10.50 
HSV-2 
MS positive 0.70 0.01 0.07 
8705 positive 0.34 0.03 0.05 
8707 altered 34.0 0.04 0.5 
8711 deficient &gt;100 3.7 4.3 
12247 altered &gt;100 0.19 1.3 
11680 altered 13 0.03 NT.sup.c 
13386 positive 0.30 0.01 0.08 
13546 positive 0.30 0.02 0.05 
11575 partial &gt;100 3.6 24. 
11572 partial &gt;100 0.80 5.9 
11361 deficient &gt;100 0.80 22. 
AG-3 deficient &gt;100 0.09 0.30 
11785 partial 24. 0.02 NT.sup.c 
______________________________________ 
.sup.a. Plaque reduction assay in HFF cells, mean of 2 assays. 
.sup.b. Polymerase mutant. 
.sup.c. Not tested. 
7. Studies on Topical Treatment of Genital HSV-2 Infections in Guinea Pigs 
With FIAU or FIAC 
7.1. Materials and Methods 
7.1.1. Antiviral Preparations 
Ointments, such as those described in Section 5, containing 1%, 5%, or 10% 
FIAU or FIAC, and a similar preparation containing 5% ACV in polyethylene 
glycol, obtained from the Hospital Pharmacy of The University of Alabama 
at Birmingham, were used. 
7.2. Genital HSV-2 Infection of Guinea Pigs 
7.2.1. Description of the Model 
Intravaginal inoculation of weanling guinea pigs with HSV-2 results in a 
primary genital infection characterized by initial replication of virus in 
the vaginal tract followed by the development of external vesicular 
lesions. Virus titers peak on days one to three in the vaginal tract and 
gradually clear by days 7-10. The external genital lesions first appear on 
day four, peak lesion severity occurs on days 6-8, and the lesions 
generally heal by days 15-18. In this model infection using HSV-2 that is 
sensitive to ACV, treatment with topical or oral ACV against primary 
disease was completely predictive of the efficacy in subsequent human 
trials. 
7.2.2. Virus and Viral Inoculation 
In addition to wild type MS strain, a strain of HSV-2 (12247) that has an 
altered thymidine kinase and is resistant to ACV in cell culture was 
utilized for animal inoculation. Female Hartley guinea pigs (Charles 
River, Kingston, N.Y.) weighing 250-300 g were inoculated intravaginally 
(i.vag.) one hour after being swabbed for removal of vaginal secretions. 
Viral inoculation was accomplished by inserting a swab soaked with virus 
into the vaginal tract and rotating approximately six times. Animals were 
inoculated with virus preparations that titered 6.2.times.10.sup.5 plaque 
forming units (pfu) per/ml for the MS strain of HSV-2 or 
1.2.times.10.sup.5 pfu/ml for the 12247 strain of HSV-2. 
7.2.3. Treatment of Guinea Pigs 
Groups of 10 guinea pigs were treated both i.vag. and on the external 
genital skin with 0.1 ml (total of 0.2 ml per animal per treatment) of 
each preparation. Animals were treated three times daily for six days 
(FIAU) or for seven days (FIAC) beginning 24h post-viral inoculation. 
Three uninfected animals were treated with each preparation on the same 
schedule to assess local toxicity. 
7.2.4. Sample Collection, Virus Assays, and Development of Genital Lesions 
To determine the effect of treatment on HSV-2 replication in the vaginal 
tract, swabs of vaginal secretions were obtained during the primary 
infection on days 1, 3, 5, 7, and 10 after HSV-2 inoculation. The swabs 
were placed in tubes containing 2.0 ml of media, and frozen at -70.degree. 
C. until titrated for HSV. When all samples were collected, they were 
thawed, vortexed, diluted serially, and HSV-2 titers determined in rabbit 
kidney cells using a microtiter CPE assay. Another measure for determining 
efficacy of treatment is the development and severity of external genital 
lesions. Severity of lesions was graded on a 0-5+ score. The presence or 
absence and severity of lesions was recorded for 21 days after viral 
inoculation. 
7.2.5. Evaluation of Efficacy 
Infection rates, peak lesion scores, peak virus titers, areas under virus 
titer-day and lesion score-day curves between placebo-treated and 
drug-treated animals were compared using the Mann-Whitney U rank sum test. 
A p-value of 0.05 or less was considered significant. 
7.3. RESULTS 
7.3.1. Genital Irritation of FIAU or FIAC Preparations in the Guinea Pig 
There were no signs of any irritation of the genital area or any other 
toxicity in uninfected placebo-ointment, FIAU or FIAC treated animals. The 
animals remained healthy and normal in appearance throughout the 
observation period. 
7.3.2. Effect of Topical Treatment with FIAU or FIAC Preparations on 
Vaginal Viral Replication in the Guinea Pig 
The effect of topical treatment with FIAU preparations on vaginal viral 
replication is shown in TABLE III. In animals infected with the 
ACV-sensitive strain of HSV-2 (MS), therapy with 10% or 5% FIAU was as 
effective as 5% ACV in significantly reducing viral replication in the 
vaginal tract. A marginal effect was observed with 1% FIAU. 
In animals inoculated with the ACV-resistant strain of HSV-2 (12247), 
again, treatment with 10% or 5% FIAU, but not 1% FIAU, significantly 
altered vaginal virus replication. In contrast, 5% ACV had virtually no 
effect on vaginal virus titers. 
The effect of topical treatment with FIAC preparations on vaginal viral 
replication is shown in TABLE IV. In animals infected with the 
ACV-sensitive MS strain of HSV-2, therapy with 5% FIAC was as effective as 
5% ACV in significantly altering vaginal viral replication. No effect was 
observed with 1% FIAC. 
In animals infected with the ACV-resistant 12247 strain of HSV-2, only 
treatment with 5% FIAC significantly reduced vaginal virus titers. In this 
model, treatment with 1% FIAC or 5% ACV had no effect on vaginal viral 
replication. 
7.3.3. Effect of Topical Treatment with FIAU or PIAC Preparations on Lesion 
Development in the Guinea Pig 
The effect of topical treatment with the FIAU preparations on lesion 
development is summarized in TABLE V. In animals infected with the 
ACV-sensitive strain of HSV-2 (MS), therapy with 10%, 5% or 1% FIAU was 
more effective than 5% ACV in altering the course of lesion development. 
While ACV significantly affected lesion development compared to 
placebo-treated animals, the effect was considerably less than observed 
for FIAU treated animals. 
In animals inoculated with the ACV-resistant strain of HSV-2 (12247), all 
three concentrations of FIAU significantly reduced lesion scores and to a 
greater extent than ACV treatment, which was barely significant. 
The effect of topical treatment with the FIAC preparations on lesion 
development is shown in TABLE VI. While both FIAC and ACV preparations 
significantly altered lesion development in animals inoculated with the 
ACV-sensitive MS strain, therapy with 5% or 1% FIAC reduced lesion scores 
to much lower levels than treatment with 5% ACV. 
In animals infected with the ACV-resistant 12247 strain, again, therapy 
with 5% or 1% FIAC or with 5% ACV significantly reduced lesion 
development. However, treatment with 5% FIAC lowered lesion scores to a 
greater extent than 1% FIAC or 5% ACV. 
TABLE II 
______________________________________ 
EFFECT OF TOPICAL TREATMENT WITH FIAU ON VAGINAL 
VIRUS TITERS OF GUINEA PIGS INOCULATED 
INTRAVAGINALLY WITH HSV-2 STRAINS 
# Virus Virus Mean 
Positive/ 
Titer-Day Peak 
# In- Area Virus 
Treatment.sup.a 
oculated Under Curve 
P-Value 
Titer P-Value 
______________________________________ 
HSV-2, MS.sup.b 
Placebo-PBS 
10/10 31.8 -- 5.0 -- 
Placebo- 10/10 32.8 .sup. NS.sup.c 
5.3 NS 
Ointment 
FIAU 10% 10/10 8.1 0.001 4.1 &lt;0.05 
FIAU 5% 10/10 12.1 &lt;0.01 4.2 &lt;0.01 
FIAU 1% 10/10 21.5 0.07 4.8 NS 
ACV 5% 10/10 13.7 &lt;0.01 4.3 &lt;0.01 
HSV-2, 12247.sup.d 
Placebo-PBS 
10/10 33.5 -- 4.8 -- 
Placebo- 
Ointment 10/10 34.4 NS 5.3 NS 
FIAU 10% 9/9 20.5 &lt;0.05 4.6 0.01 
FIAU 5% 10/10 21.8 &lt;0.05 4.2 0.001 
FIAU 1% 10/10 31.7 NS 5.1 NS 
ACV 5% 10/10 33.2 NS 4.8 &lt;0.05 
______________________________________ 
.sup.a Topical and i.vag. treatment was initiated 24th after viral 
inoculation and was continued three times daily for 6 days. 
.sup.b ACV sensitive strain of HSV2, TK positive. 
.sup.c NS = Not Statistically Significant when compared to the appropriat 
control or placebotreated group. 
.sup.d ACV resistant strain of HSV2, TK altered. 
TABLE III 
______________________________________ 
EFFECT OF TOPICAL TREATMENT WITH FIAC ON 
VAGINAL VIRUS TITERS OF GUINEA PIGS INOCULATED 
INTRAVAGINALLY WITH HSV-2 STRAINS 
# Virus Virus Mean 
Positive/ 
Titer-Day Peak 
# In- Area Virus 
Treatment.sup.a 
oculated Under Curve 
P-Value 
Titer P-Value 
______________________________________ 
HSV-2, MS.sup.b 
Placebo-Ointment 
10/10 31.6 -- 4.9 -- 
FIAC 5% 10/10 13.4 &lt;0.01 4.6 .sup. NS.sup.c 
FIAC 1% 10/10 25.2 NS 4.5 NS 
ACV 5% 10/10 14.2 &lt;0.01 4.5 &lt;0.05 
HSV-2, 12247.sup.d 
Placebo-Ointment 
10/10 34.4 -- 4.8 -- 
FIAC 5% 10/10 21.3 0.01 4.2 &lt;0.01 
FIAC 1% 10/10 32.9 NS 4.8 NS 
ACV 5% 10/10 33.6 NS 4.8 NS 
______________________________________ 
.sup.a Topical and i.vag. treatment was initiated 24th after viral 
inoculation and was continued three times daily for 6 days. 
.sup.b ACV sensitive strain of HSV2, TK positive. 
.sup.c NS = Not Statistically Significant when compared to the appropriat 
control or placebotreated group. 
.sup.d ACV resistant strain of HSV2, TK altered. 
TABLE IV 
______________________________________ 
EFFECT OF TOPICAL TREATMENT WITH FIAU ON EXTERNAL 
LESION DEVELOPMENT IN 
GENITAL HSV-2 INFECTIONS OF GUINEA PIGS 
Lesion Score- Mean Peak 
Day Area Lesion 
Treatment.sup.a 
Under Curve 
P-value Score P-Value 
______________________________________ 
HSV-2, MS.sup.b 
Placebo-PBS 
56.4 -- 4.2 -- 
Placebo-Ointment 
60.2 .sup. NS.sup.c 
4.3 NS 
FIAU 10% 0.5 &lt;0.001 0.3 &lt;0.001 
FIAU 5% 1.0 &lt;0.001 0.5 &lt;0.001 
FIAU 1% 2.0 &lt;0.001 0.8 &lt;0.001 
ACV 5% 23.8 &lt;0.001 2.6 0.001 
HSV-2, 12247.sup.d 
Placebo-PBS 
47.7 -- 3.1 
Placebo-Ointment 
48.4 NS 3.2 NS 
FIAU 10% 2.4 &lt;0.001 0.8 &lt;0.001 
FIAU 5% 1.6 &lt;0.001 0.7 &lt;0.001 
FIAU 1% 31.8 0.01 2.9 NS 
ACV 5% 37.1 0.05 3.4 NS 
______________________________________ 
.sup.a Topical and i.vag. treatment was initiated 24th after viral 
inoculation and was continued three times daily for 6 days. 
.sup.b ACV sensitive strain of HSV2, TK positive. 
.sup.c NS = Not Statistically Significant when compared to the appropriat 
control or placebotreated group. 
.sup.d ACV resistant strain of HSV2, TK altered. 
TABLE V 
______________________________________ 
EFFECT OF TOPICAL TREATMENT WITH FIAC ON EXTERNAL 
LESION DEVELOPMENT IN 
GENITAL HSV-2 INFECTIONS OF GUINEA PIGS 
Lesion Score- Mean Peak 
Day Area Lesion 
Treatment.sup.a 
Under Curve 
P-value Score P-Value 
______________________________________ 
HSV-2, MS.sup.b 
Placebo-Ointment 
37.5 -- 3.7 -- 
FIAC 5% 1.4 &lt;0.001 0.4 &lt;0.001 
FIAC 1% 6.5 &lt;0.001 1.1 &lt;0.001 
ACV 5% 18.0 &lt;0.001 2.3 0.01 
HSV-2, 12247.sup.c 
Placebo-Ointment 
42.1 -- 2.6 -- 
FIAC 5% 2.4 &lt;0.001 0.8 &lt;0.001 
FIAC 1% 20.8 &lt;0.01 2.7 .sup. NS.sup.d 
ACV 5% 20.7 &lt;0.001 2.5 NS 
______________________________________ 
.sup.a Topical and i.vag. treatment was initiated 24th after viral 
inoculation and was continued three times daily for 6 days. 
.sup.b ACV sensitive strain of HSV2, TK positive. 
.sup.c ACV resistant strain of HSV2, TK altered. 
.sup.d NS = Not Statistically Significant when compared to the appropriat 
control or placebotreated group. 
It should be apparent to those skilled in the art that other compositions 
not specifically disclosed in the instant specification are, nevertheless, 
contemplated thereby. Such other compositions are considered to be within 
the scope and spirit of the present invention. Hence, the invention should 
not be limited by the description of the specific embodiments disclosed 
herein but only by the following claims.