CA2421026C - Viral inhibition by n-docosanol - Google Patents

Abstract

This invention relates to topical therapeutic preparations and methods for treating viral and inflammatory diseases and for reducing the pain of topical inflammation of skin and mucous membranes. The preparations include creams containing n-docosanol.

Description

~~~~t- c~ S o ~: .3, ~ ~ 9 . , , .
AVANIR.089VPC ' PATENT
VIRAL INHIBITION BY n-DOCOSANOL
Field of the Invention This invention relates to topical therapeutic preparations and methods for treating viral and inflammatory diseases and for reducing the pain of topical Inflammation of skin and mucous membranes. The preparations include creams containing n-docosanol.
Back4round of the lnvenfron Most antiviral therapeutic compounds block various specific viral genetic replicative mechanisms within infected target cells. These approaches have drawbacks including toxk~ty to host cells, induction of drug-resistant viral sub-strains, and the potential to act as mutagens andlor teratogens for host cells.
Consequently, the search for new antiviral compounds that provide efficacious therapy, without such deleterious consequences to the host, is of paramount importance.
Recurrent oral-facial herpes simplex (recurrent herpes simplex labialis, HSL) is a common disease estimated to occur in 20 to 40 percent of the Unfted States' population.
(Higgins CR, Schotreld JK, Tatnall FM, Leigh IM J. Med. Virol. Suppl. 1:22-6, 1993). A main feature of the disease is the ability of herpes simplex virus {generally type 1 [HSV-1~ to remain latent prior to erupting in response to such stimuli as stress, sunlight, fever, respiratory tract infections, and menstruafron. (Spruance SL, in Clinical management of herpes viruses, Sacks SL, Straus SE, Whitley RJ, Gtiffiths PD, editors, Amsterdam: IOS Press, p. 3-d2, 1995). Episodes that do not progress beyond the papule have been referred to as aborted or.nonlesional episodes. Classical lesions are those that progress to the vesiculo-ulcerative stage prior to healing.
HSL is self-limiting with healing normally occumng in 7 to 10 days. (Spruance SL, Overal JC, Kem E, Krueger GG, Pliam V, Miller W New Engl. J. Med. 297:69-75, 1997; Spruanoe SL Semin. in Dermatol.
11:200-6,1992; and Shafran SD, Sacks SL, Aoki FY, Tyrtell DL, Schlech WF 3b, Mendelson J, Rosenthal D, et aL J. Infect. Dis. 176:78-83, 1997). Lesions evolve rapidly with maximum lesion severity often occurring within 8 hours of onset. (Spruance SL, Wenerstrom G. Oral Surg. 58:667-71, 1984). The window of time for therapeutic treatment is therefore small and it is essential that antiviral therapies be administered early.
Antiviral therapies initiated at the papule or later stages cannot significantly affect lesion severity or the frequency of aborted lesions.
Compounds that exert antiviral activities without being potentially detrimental to the infected host have been identified and have shown some promising results. The oral antiviral medication valacyclovir hydrogen chloride is used to suppress genital herpes outbreaks, and for the treatment of recurrent outbreaks of genital herpes. In the late 1970's, for example, Snipes and colleagues (Snipes W, Person S, Keller G, Taybr W, Keith A Anfimicrob. Agents Chemother. 11:98-104 {1977); Sands J, Auperin D, Snipes W
Antimicrob. Agents Chemofher. 15:67-73 {1979)) reported a series of studies demonstrating such activities for both. saturated and unsaturated alcohols of moderate chain lengths. Optimal antiviral activity was observed with 10-12 carbon-long saturated alcohols; less antiviral activity was observed with alcohols 14-18 carbons long, and alcohols of higher chain lengths were oat tested. -While significant antlvlral activity was observed with C-10 and G12 alcohols, these compounds also exhibited cytotoxic and hemolytic effects. Similar observations were made with unsaturated alcohols and 5 monoglycerides, peak activity occuning with a C-18 alcohol containing three double bonds. Subsequentty, Clark and colleagues (Clark LL, U. S. Patent No. 4,670,471 (1987); McBride PT, dark LL, Krueger GG J.
Invest. DermatoL 89:380-383 (1987)) concluded that the 30 carbon-long saturated alcohol, triacontanol, was active as an anti-herpes agent. however, since tissue culture studies demonstrated that triacontanol lacked direct antiviral activity, it was speculated that the apparent anti-herpes activity observed in animal studies might reflect an immunomodulatory effect of this compound.
As early as 1974, n-docosanol was reported to have systemic therapeutic value.
For example, Debat, U. S. Paient No. 4,186,211, reported that 1-docosanol when taken orally was therapeutically effective In the treatment of enlargement of the prostate gland. Similar work was reported a decade later by Yamamoto et al., e.g., U. S. Patent No. 4,624,966, who, incorrectly as to chemical nomenclature, listed n-docosanol as a 15 polyphenyl compound and described the peroral or parenteral administration of n~focosanol In therapy.
Compounds longer than 18 carbons have been examined to ascertain if they might exhibit topical antiviral or inflammatory activity {Katz et al., PCT Applicatan No. WO
97/18434). Studies in our laboratory testing the antivlral properties of n-docosanol were favorable (Katt, DH, U.
S. Patent No. 4,874,794).
n-Docosanol inhibits in vitro a broad spectrum of lipid-enveloped viruses including HSV-1 and HSV
20 2, cytomegalovirus, varlcella zoster virus, and human herpes virus 6. (Kalz DH, Maroelletti JF, Khalil MH, Pope LE, Katz LR. Proc. Nafl. Acad. Sci. USA 88:10825-9, 1991; Katz DH, Marcelletti JF, Pope LE, Khalil MH, Kay LR, McFadden R, Ann, NY Acad. Scl. 724:472-88, 1994; Maroelletti JF, Pope LE, Khalil MH, McFadden RR, Katz LR, Katz DH. Drugs of the Future 17:879-82, 1992; Pope LE, Marcelletti JF, Katz LR, Katz DH J. Upld Res. 37:2167-78, 1996; and Pope LE, Marcelletti JF, Katz LR, Lin JY, Katz DH, Parish ML, 25 Spear PG Antivir. Res. 40:85-94,1998). Its mechanism of action is novel:
following cellular incorporation and metabolic conversion, n-docosanol inhibits one or more steps of viral entry, blocking nuclear localization and subsequent replication of the virus. More recent experiments indicate that rrdocosanol may exert anti-HSV
activity predominantly by interfering with the process of viral fusion with the host cell. (Pope LE, Marcelletti JF, Katr LR, Lin JY, Katz DH, Parish ML, Spear PG Antivir. Res. 40:85-94,1998). In July 2000, n-docosanol 30 10 wL °~ cream was approved by the U.S. Food and Drug AdminisUation as an OTC topical treatment for recurrent oral-facial herpes simplex infections (trade name Abreva'").
Tt~e preparation of stable, eificac'rous n-docosanol-containing topical formulations presents a challenge. While creams and ointments of certain conventional formulations may be adequate for preliminary evaluations, certain excipients may be detrimental to the activity of n~docosanol. For example, penetration 35 enhancers are often used as excipients in such formulations, but the effect on stabilizing activity of exclpients in topical formulations may not be accurately predicted. Azone, reported by RaJadhyaksha, for example, is an excellent penetration enhancer but has not been known as a stabilizing constituent in cream formulations.
Sucrose esters of coconut fatty acids have been formulated as penetration enhancers, Cheng et aL, U. S. Patent No. 4,865,848, and other patents. Cheng et al., do not suggest, however, any cream stabilization 5 resulting from these materials, nor is there any reason to infer such stabilization from the Cheng ef al. patents.
literature on such compounds does not suggest these materials as being particularly effective in stabilizing C-20 to C-28 aliphatic alcohol-containing creams.
Summary of the invention The preparation of stable, efficacious n~docosanol-containing topical formulations presents a 10 challenge. While creams and ointments of certain conventional formulations may be adequate for preliminary evaluations, certain excipienis may be detrimental to the activity of n-docosanol. Therefore, there fs a need for reproducibly effective formulations of n-docosanol that are stable for long periods of time, physiologicaAy acceptable and suitable for topical application to skin and membranes.
In a first embodiment, a therapeutic cream is provided for application to skin and mucous 1S membranes in the treatment of viral and inflammatory diseases including about 10 wt. % n-docosanol; about wt. % of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. % light mineral oil; about 5 wt. % propylene glycol; about 2.7 wt. % benzyl alcohol; and about 69.3 wt. % water.
In a second embodiment, a method of treating viral infections and inflammations of skin and mucous 20 membranes is provided including applying to the skin or mucous membranes a stable therapeutic topical cream including about 10 wt. % n-docosanol; at~out 5 wt. °~ of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt.
% light mineral oil; atxxJt 5 wt.
propylene glycol; about 2.7 wt. % benzyl akohol; and about 69.3 wt. °.6 water.
In a third embodiment, a method of reducing the pain of a surface inflammation of skin and mucous 2S membranes is provided including applying to the Inflamed surface a composition including about 10 wt. % n docosanol; about 5 wt. % of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. % light mineral oil; about 5 wt.
% propylene glycol; about 2.7 wt.
benzyl alcohol; and about 69.3 wt. 9~° water.
In a fourth embodiment, the use of a composition including about 10 wt.
°~ n-docosand; about 5 wt.
30 % of a stearaie selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. °~ light mineral oil; about 5 wt. % propylene glycol; about 2.7 wt. ~° benzyl alcohol; and about 69.3 wt. 9~o water, in the preparation at a medicament for treatment of viral infections and inflammation of the skin or mucous membranes is provided.
In a fifth emt~odiment, the use of a composition Including about 10 wt. % n-docosanol; about 5 wt %
35 of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. 9'° light mineral og; about 5 wt. % propylene glycol; afx'ut 2.7 Hrt. 9'° benzyi alcohol; and about 69:3 wt. % water, in the preparation of a medicament for reducing the pain of a surface inflammation of -the skin or mucous membranes is provided.
In a sixth embodiment, a therapeutic cream is provided for application to skin and membranes in the treatment of viral and inflammatory diseases hcluding sugar based ester surfactant, greater than about 5 wt.
n-docosanol, mineral oil, an emoil(ent co-solvent, and water.
In a first aspect of the sixth embodiment, the cream is stable at temperatures of at least 40°C for a period of at least three months and after repeated freeze-thaw cycles.
In a second aspect of the sixth embodiment, the sugar based ester surfactant is selected from the group consisting of sucrose cocoate, sucrose stearates, and sucrose distearate.
In a thud aspect of the sixth embodiment, the sugar based ester surfactant Includes at least one compound selected from the group of sucrose esters consisting of sucrose coooate, sucrose stearates and sucrose distearate, wherein sucrose ester (s) include about 3 wt. % or more of the cream. In another.aspact the sucrose esters) Include about 5 wt. 9'0 or more of the cream.
15 In a fourth aspect of the sixth embodiment, the emollient co-solvent is selected from the group consisting of polyoxypropylene stearyi ether, ethyl hexanedai, and benzyl alcohol, or combinations thereof.
In a fifth aspect of the slxih embodiment, the n~ocosanol includes at least approximately 10 wt. % of the cream.
In a seventh embodiment, a stable, efficacious therapeutic cream is provided wherein a prindpal 20 therapeutic composition consists essentially of n~docosanol, and wherein the cream base including one or more compounds selected from the group consisting of sucrose cocoate, sucrose stearates and sucrose distearate and one or more compounds selected from the group consisting of potyoxypropylene stearyl ether, ethyl hexanediol, and benryl alcohol.
In a first aspect of the seventh embodiment, sucrose esters) Include at least approximately 5 wt °Y°
25 of the cream.
In a second aspect of the seventh embodiment, the n-docosanol includes at least approximately 10 wt. °~6 of the cream.
In a third aspect of the seventh embodiment, the therapeutic cream has the formulation: n~ocosanol making up from 5 to 15 wt. ~° of the total cream; sucrose stearates making up from 0 to 15 wt. ~o of the total 30 cream; sucrose cocoate making up from 0 to 10 wt. % of the total cream;
sucrose distearate making up from 0 to 10 Hrt. % of the total cream; wish the proviso that at least one sucrose ester be present and make up at feast about 3 wt, % of the total composition; mineral oil making up from 3 to 15 by weight of the total cream;
benzyl alcohol making up from 0.5 to 10 wt. % of the total cream; and water making up from 40 to 70 wt. % of the total cream--4_ In an eighth embodiment, a method of treating viral infections and inflammations of skin and mucous membranes is provided including applying a stable therapeutic topical cream wherein- the therapeutically active composition consists essentially of n.docosanol, and wherein the cream base consists essentially of sugar based ester surfactant, at least orre lorg chain aliphatic ~cohol having from 24 to 28 carbon atans S selected from the group consisting of n-eicosanol, n-heneicosanol, n-tricosanol, n-tetracosanol, n-peniacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol, or mixtures then3of, mineral oil, an emollient co-solvent, and water.
In a first aspect of the eighth embodiment, n-docosanol includes more than one-half of the long chain aliphatic alcohols In a ninth embodiment, a method of treating viral infections and inflammations of skin and mucous membranes Is provided including applying a topical cream having the formulation: n~Jocosanot about 5-20 wt.
%; sucrose stearates about 0-15 wt. %; sucrose cocoate about 0-90 wt. %;
sucrose distearate about 0-10 wt.
%, with the proviso that at least one sucrose ester be present and, wherein sucrose esters) include about 3 wl. % or more of the cream; mineral oil about 3-15 wt. %; propylene gfycd about 2-10 wl. %;
polyoxypropylene-15 stearyl ether about 0-5 wt. °~; benzyl a~ohol about 0.5-5 wt. %; with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amo~t of at least about 1 wt. %; and water about 40-70%.
In a first aspect of the ninth embodimenk sucrose esters) include about 5 wl.
% or more of the cream.
!n a tenth embodiment, an anti-inflammatory and antiviral cream is provided having the tormdatbn:
n-docosanof about 5-20 wt. %; sucrose stearaies about 0-15 wt. %; sucrose cocoate about 0-10 wt. %;
sucrose distearate about 0-10 wt. %, with the proviso that at least or>e sucrose ester be present and wherein sucrose ester (s) include about 3 wt. °~ or more of the cream, mineral oil about 3-15 wt. %; propylene glycol about 2-10 wt. %; polyoxypropylene stearyl ether about 0-5 wt. %; benzyl ak:ohol 0-5 wt. %; with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of about 1 wt. % or more; and water about 40-70 wt. %.
In a first aspect of the tenth embodiment, sucrose esters) Include about 5 wt.
°~ or more of the cream.
In an eleventh embodiment, a method of reducing the pain of a surface inflammation of the skin or membrane is provkied including applying to the inflamed surface a composition of n-d~osanol In a physiologically compatble carrier, said n-docosanol including from about 5 to about 25 wt. % of said composition.
In a first aspect of the eleventh embodiment, the physalogically compatible cartier is a cream base that includes one or more compounds selected from the group consisting of sucrose toccata, sucrose n r:
stearates and sucrose distearate and one or more compounds selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol.
Various embodiments of this invention provide use of a composition consisting essentially of sugar-based ester surfactant, greater than about 5 wt. % n-docosanol, mineral oil, an emollient co-solvent and water, for preventing the onset of lesions caused by recurrent herpes simplex labialis.
Various embodiments of this invention provide use of n-docosanol, a sugar-based ester surfactant, mineral oil, an emollient co-solvent and water in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, wherein the composition comprises greater than about 5 wt. % n-docosanol.
Various embodiments of this invention provide a composition consisting essentially of sugar-based ester surfactant, greater than about 5 wt. % n-docosanol;
mineral oil, an emollient co-solvent and water, in the preparation of a medicament for preventing the onset of lesions caused by recurrent herpes simplex labialis.
Various embodiments of this invention provide use of a composition consisting essentially of n-docosanol and a cream base comprising one or more compounds selected from the group consisting of sucrose cocoate, sucrose stearates, and sucrose distearate, and one or more compounds selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol, for preventing the onset of lesions caused by recurrent herpes simplex labialis.
Various embodiments of this invention provide use of n-docosanol and a cream base comprising one or more compounds selected from the group consisting of sucrose cocoate, sucrose stearates and sucrose distearate and one or more compounds selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, wherein the composition consists essentially of said n-docosanol and a cream base.
Various embodiments of this invention provide a composition consisting essentially of n-docosanol and a cream base comprising one or more compounds selected from the group consisting of sucrose cocoate, sucrose stearates, and sucrose distearate, and one or more compounds selected from the group consisting of polyoxypropylene -5 a-stearyl ether, ethyl hexanediol, and benzyl alcohol, for preventing the onset of lesions caused by recurrent herpes simplex labialis.
Various embodiments of this invention provide use of a composition having the formulation: n-docosanol comprising from 5 to 15 wt. % of the total composition;
sucrose stearates comprising from 0 to 15 wt. % of the total composition;
sucrose cocoate comprising from 0 to 10 wt. % of the total composition; sucrose distearate comprising from 0 to 10 wt. % of the total composition; at least one sucrose ester comprising at least about 3 wt. % of the total composition; mineral oil comprising from 3 to 15 wt. % of the total composition; benzyl alcohol comprising from 0.5 to 10 wt. % of the total composition; and water comprising from 40 to 70 wt. % of the total composition; for preventing the onset of lesions caused by recurrent herpes simplex labialis.
Various embodiments of this invention provide use of n-docosanol, at least one sucrose ester, mineral oil, benzyl alcohol and water, and optionally sucrose stearates, sucrose cocoate, and sucrose distearate in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composing having the formulation: n-docosanol comprising from 5 to 15 wt. % of the total composition;
sucrose stearates comprising from 0 to 15 wt. % of the total composition;
sucrose cocoate comprising from 0 to 10 wt. % of the total composition; sucrose distearate comprising from 0 to 10 wt. % of the total composition; at least one sucrose ester comprising at least about 3 wt. % of the total composition; mineral oil comprising from 3 to 15 wt. % of the total composition; benzyl alcohol comprising from 0.5 to 10 wt. % of the total composition; and water comprising from 40 to 70 wt. % of the total composition.
Various embodiments of this invention provide a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition having the formulation: n-docosanol comprising from 5 to 15 wt. % of the total composition;
sucrose stearates comprising from 0 to 15 wt. % of the total composition;
sucrose cocoate comprising from 0 to 10 wt. % of the total composition; sucrose distearate comprising from 0 to 10 wt. % of the total composition; at least one sucrose ester comprising at least about 3 wt. % of the total composition; mineral oil comprising from 3 to 15 wt. % of the total composition; benzyl alcohol comprising from 0.5 to 10 wt. % of the total composition; and water comprising from 40 to 70 wt. % of the total composition.
-Sb-Various embodiments of this invention provide use of a composition consisting essentially of n-docosanol and a cream base, wherein the cream base consists essentially of sugar-based ester surfactant, at least one long chain aliphatic alcohol having from 20 to 28 carbon atoms selected from the group consisting of n-icosanol, n-henicosanol, n-tricosanol, n-tetracosanol; n-pentacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol, or mixtures thereof, mineral oil, an emollient co-solvent, and water, for preventing the onset of lesions caused by recurrent herpes simplex labialis.
Various embodiments of this invention provide use of n-docosanol and a cream base in the preparation of a medicament for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of n-docosanol and the cream base, and wherein the cream base consists essentially of sugar-based ester surfactant, at least one long chain aliphatic alcohol having from 20 to 28 carbon atoms selected from the group consisting of n-icosanol, n-henicosanol, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol, or mixtures thereof, mineral oil, an emollient co-solvent, and water.
Various embodiments of this invention provide a composition for preventing the onsets of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of n-docosanol and a cream base, and wherein the cream base consists essentially of sugar-based ester surfactant, at least one long chain aliphatic alcohol having from 20 to 28 carbon atoms selected from the group consisting of n-icosanol, n-henicosanol, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol, or mixtures thereof, mineral oil, an emollient co-solvent, and water.
Various embodiments of this invention provide use of a topical cream having the formulation: n-docosanol about 5-20 wt. %; sucrose stearates about 0-15 wt. %;
sucrose cocoate about 0-10 wt. %; sucrose distearate about 0-10 wt. %, with the proviso that at least one sucrose ester be present and, wherein sucrose esters) comprise about 3 wt. % or more of the cream; mineral oil about 3-15 wt. %; propylene glycol about 2-10 wt. %;
polyoxypropylene-15 stearyl ether about 0-5 wt. %; benzyl alcohol about 0.5-5 wt. %;
with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of at least about 1 wt. %; and water about 40-70 wt. %, for preventing the onset of lesions caused by recurrent herpes simplex labialis.
-Sc-Various embodiments of this invention provide a topical cream for preventing the onset of lesions caused by recurrent herpes simplex labialis, the cream having the formulation: n-docosanol about 5-20 wt. %; sucrose stearates about 0-15 wt. %;
sucrose cocoate about 0-10 wt. %; sucrose distearate about 0-10 wt. %, with the proviso that at least one sucrose ester be present and, wherein sucrose esters) comprise about 3 wt. % or more of the cream; mineral oil about 3-15 wt. %; propylene glycol about 2-10 wt. %;
polyoxypropylene-15 stearyl ether about 0-5 wt. %; benzyl alcohol about 0.5-5 wt. %;
with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of at least about 1 wt. %; and water about 40-70 wt. %.
Various embodiments of this invention provide use of an anti-inflammatory and antiviral cream for preventing the onset of lesions caused by recurrent herpes simplex labialis, the cream having the formulation: n-docosanol about 5-20 wt. %;
sucrose stearates about 0-15 wt. %; sucrose cocoate about 0-10 wt. %; sucrose distearate about 0-10 wt. %, with the proviso that at least one sucrose ester be present and wherein sucrose ester (s) comprise about 3 wt. % or more of the cream; mineral oil about 3-15 wt. %;
propylene glycol about 2-10 wt. %; polyoxypropylene stearyl ether about 0-5 wt. %;
benzyl alcohol about 0-5 wt. %; with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of about 1 wt. % or more; and water about 40-70 wt. %.
Various embodiments of this invention provide an anti-inflammatory and antiviral cream for preventing the onset of lesions caused by recurrent herpes simplex labialis, the cream having the formulation: n-docosanol about 5-20 wt. %; sucrose stearates about 0-15 wt. %; sucrose cocoate about 0-10 wt. %; sucrose distearate about 0-10 wt.
%, with the proviso that at least one sucrose ester be present and wherein sucrose ester (s) comprise about 3 wt. % or more of the cream; mineral oil about 3-15 wt. %; propylene glycol about 2-10 wt. %; polyoxypropylene stearyl ether about 0-5 wt. %; benzyl alcohol about 0-5 wt. %; with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of about 1 wt. % or more; and water about 40-70 wt. %.
Various embodiments of this invention provide use of a composition consisting essentially of about 10 wt. % n-docosanol; about 5 wt. % of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. % light mineral oil; about 5 wt. % propylene glycol; about 2.7 wt. %
benzyl alcohol;
-5 d_ and about 69.3 wt. % water, for preventing the onset of lesions caused by recurrent herpes simplex labialis.
Various embodiments of this invention provide use of n-docosanol, stearate, light mineral oil, propylene glycol, benzyl alcohol and water in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of about 10 wt. % n-docosanol; about 5 wt.
of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. % light mineral oil; about 5 wt.
% propylene glycol; about 2.7 wt. % benzyl alcohol; and about 69.3 wt. % water.
Various embodiments of this invention provide a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of about 10 wt. % n-docosanol; about 5 wt. % of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. % light mineral oil; about 5 wt. % propylene glycol; about 2.7 wt. %
benzyl alcohol;
and about 69.3 wt. % water.
-Se-Brl~~on of the Drawir>gs Figures 1 through 3B and Figures 6A and 6B pertain to experiments invahi~g herpes siriplex virus type t (HS1I 1j, whtie Figures 4 and 5 and F'~gures 7 through 9 involve herpes simplex vlnrs type 2 (HSV ~.
Figure 1 presents the comparative activities of Formulation ( (n~docosanol 10.0 wt. %; sucrose stearates 11,.0 wt. %; sucrose c~oate 5.0 wt. %; mineral oil 8.0 wt. %;
propylene glycol 5.0 wt 9'0; 2-ethyl-1,3-hexanediol 2.7 wt. % and purified water 58.3 wt. %j, three different prep~atans of Formulation ll (same as Formulation i except 5 wt. % s~rose stearates was replaced with sucrose dtsfearata and ethyl hexar~edioi was replaced with an equivalent amount of polyoxypropylene-15-sfearyi ether) and ZOVII(acycdovir;
Burtoughs Welicoms Co., Research Triangle Park, NC; a treatment of HSV
infections which inh~ifs activity of .
viral DNA polymerasej in inhibiting HSV 1-krduced cutar~ous lesions in hairless guinea p(gs.
Figure 2 presents the comparative activities of Formulation 1, Formulation il, and Formulation IA (rr docosanol 10.0 wt. %; sucxose stearates 11.0 wt. %; sucrose cxrcoate ,5.0 wt.
%; mineral 08 8Ø wt. %;
I5 propylene glycol 5.0 wt. %; benzyl alcohol 2.7 wt. % and purified water 58.3 wt: %j.
Figure 3A shows a comparison of activities of Formulation I versus Formulation III (rt~locosanol 10.0 wt: %; sucrose stearabes .5.0 wt %; mineral oil 8.0 wt. ~o; propylene glycol 5.0 wt. %; benzyl alcohol 2.7 wt %;
and purified water 58.3 wt. %j.
Figure 38 depicts data cornpari~g the activities of certain modifications of these formulations in which the relative surfactant concentrations have been modified from that of Fortnulatan L tdoditicatfons of surfac~nt ~ncer~ations were found to have appreciable deleterious effects on the extent of drrg actinrily.
Figure 4 depicts data showier the dose relationship of Formulation III for the ~hibition of HSV-2 induced cutarreous lesi~rs in hakless guinea pigs.
Figure 5 graphically represents data showing that n~iocosanol corttaining cream based upon a sucrose ester surfactant system (Formulation III) also inhibits HSV-2-induced cutaneous lesions in hairless 9u~ Pi9s.
Figure 6A graphically depicts data that demonstrates that n-docosanol, formulated as a suspension using the s~factanf Pluronic*F-68, also inhibits HSV 1 induced vesicles when applied before v~eskles are present. The suspension formulation did not contain any of the excipienis of ~docosan~ containing cream including benzyl ak~hol.
Figure 6B graphically depicts data that demonstrates that n-docosanol, fortnu~ted as a suspension in nonionic surfactant Pluronic'~'F-68, also inhibits HSV-1 induced vesicles when applied after vesicles are present. TI~ suspension formulation did not contain any of the excipierrts of rrdocosanol containing cream includ~g berrzyl akrohol.
Figures 7 through 13 depict data elucidating the pham~acology of rr~iocosanol.
*Trademark _6_ Figure 7 dep'~cts data showing that n~docos~oi inhibits acydovtr-resistant HSV
2. Vero cells were c~tured in 35-mm wells {6 x 106 cells per weld in medium alone (= norrej or in tha presence of the ~dicated concentration of acyclovir; n~docosanol-Pturonid~F-68 suspension or contra suspension (Pluronie*F-68 only).
The cultures were inoculated 24 hours later with 150 PFU of either wild-type HS1I 2 or an acydov~~esistant S laboratory isolate from the wild-type HSV.2 th~ was plaque purified ~d passaged fi 20 Hglml ac~OVir 44 hours later, the plates were incubated, fixed, stained, and scored for numbers of plaques. The data presented are means of plaques scon;d from duplicate cultures. The percent inhibition otrserved in cultures treated with acydovir or n-docosanol relative to untreated control cultures is denoted in parentheses.
Figure 8 depicts data showing the dose response of the topkal emul~on formulation of n~docosanoi 10 on cutaneous HSV in guinea pigs. The backs of hairless guinea pigs were cleaned and tnor~lated with purified HSV 2 by puncture of the skin with a tattoo instrument. Two hours after virus inoculation, the inoculatfon sites were either untreated or treated with 100 pl of n~locosanol-containing cream or control vehicle; the sites were similarly treed 24, 30, 48, 52; and 56 hours after virus inoculation. Vesicle number per site was determined at the indicated time points. The data are expressed as means and standard errors of 15 vesicle number derived from duplkafe sues per de~rmination: The numbers in parentheses depkt p~oeM
inhibition of vesicle number at treated sites as compared to the uM~ed sites.
Figure 9 depicts data showing chat HSV 2 remains on the surface of n-docosanol treated Vero oelis for prolonged times. Vero cells were cultured as described In the legend bo Figure 7 and incubated ovem~ht.
The cultures were then chilled 60 4°C, inoculated with 100 PFt) of HSV
2, and Incubated 3 hours at 4°C. At 20 time zero the cultures were washed with medium, inoculated with fresh tnediufi (corrteining the Indkabed inhlbitarj and incubated at 37°C. At each indicated time period, the cultures wr3re wastu:d with citrab buffer (pH 2.5j and reinoculated with fresh medium {lacking inhibitor). Aver a total of 44 holes tncubarion the cultures were stained and scored for HSV-2-induced plaques. The data are expressed as geomefic means and st~dard errors derived from Mplicate cultures per group.
25 Figure 10 depicts data showing that radioactive metabolites of n.{~C)anoi display the properties of phosphatidylcholine and phosphatidylethanotamine. A por5on (0.5 mlj of the methand eluate of the silica lipid fractionation was evaporated under tirtrogen, resuspend~ in 20 W chiorofortnarrethanol (3:Zi v:vj and spotted on a silica thin layer chromafiography (TLCy sheet. After development with chloroform:
methanol:acedc actd:water (60:50:1:4; v:v:v:v), the positions of standards were determined by sialning with 30 , iodine vapors and the rpm per fraction determined by sctnttilatlon spectrometry ~ter arfting the plastrc~
backed street into 5 mm strips:
figure 11 depicts data showing that n-[aCj~docosanol is metabolized nmre by Vero ceAs fhan by MDBK cells. Vero or MDBK cells were plated as described, n-ICJ-docosano) was added to 6 mM (0.24 mM
Tetromic*908j and the cultures were incr~ated 72 hours et 37°CJCOi.
Cells were extracted and analyzed on 35 TLC with hexane:diethyi ether:acetic acid (20:30:1; vv:vj as the developing sohrent. With this solvent system *Trademark _~_ the polar phosphatides remain a~ the origin. The posftion of migrafjon of n-[~C]-docosaral is indicated.
Duplicate plates were treated with an fdentk~t suspension lacking the radioactive label, and the numbers of cells in these duplicate plates were determined by counting cells excluding trypan blue with a hemocythmater.
Figure 12 depicts data show~g that rr~ocosanol inhibits In vlvo Friend virus induced leukemia ~d vuemia. Adult BALBIc mice were injected intravenously with 75 sptean focxis-forming units of FV. Treated grows were injected intravenously with the indicated doses of n~docosanol or Plunonic~"lrehide aloha on the .
same day as virus inoculation and once daily for the next 3 days. After 10 days, haif.of the animals in each group were sacrificed and examined for leukemic foci in their spleens (penal A). The remaining mice were retained 10 more days and bled for virerrria determinations (panel B). Viremla was measvaed using the X C
plaque assay. Briefly, primary flbrobiast cultures were derived by digestion of 14-day BALBk embryos with trypsin and culturing In DMEM plus i0% fetal calf senun. After 72 hours, the tens were transferred irrth 1& .
mm dishes (1061we11), pretreated with 5 ~glml polybrene and then infected with 75 X-C plaque-forming units of Friend virus stock or d~ution of test plasma. After incubation for 7 days, the eultur~es were irradiated and overlaid with X-~ cells (3 x 105tweu}. Three days later, the cultures were washed, stained, and scored for plaques of muitinucleated giant cells. The data presented are geometric means and standard errors of sptenic foci or X-C plaque-forming units. derived finKrr three anktrals per group.
Figure 13 depicts data showing that rr~ocosanol inhtbits in vitro replication of Ht1! 1 in culdu!es of ' PHAJiL-2-strmulated human peripheral blood mononuclear cells. Human peripheral blood mononuclear cet~
were cuBured in_ medium containing 1 ~glmi PHA plus 5 unitslml IL-2 alone or also containing 100 Pglml PFA, the indicated dosage of n~docosanoflPluronic'"f-68, or the amount of Plutoni~F-68 control vehicle c4ntained in the high dose of n-docosanoUPiuroni~'F-68. After overnight incubation, the cultures were inoculated wfth HIV 1 at a multiplicty of infection of 1 vlrioNcell. After 24 hours incubation at 37°C, the cultures were washed and inoculamd with fresh medium containing PHA aril IL-2, but lacking inhibitor. Replication of HIV 1 was determined 4 days later by quantitation of viral aiogens by a p24-spedfic ELiSA for HIV 1.
Figure 14 illustrates the iCaplan-Meter distributions for time-to-hearing for treatment of acute HSL
usm9 n~locosanol 10 wt. % cream. Time-to-healing was measured from initiation of treahnent until the date and time of the clinic visit at which complete resolution of all kxat signs and symptoms was clinidan ___~ __ _._ _..__, . ___-._ .--_-__. __ . _~. ___._. _. _ mrined:
Figure i5 provides a graphical depiction of HSV 1 inhibition in hairless guinea pigs with PEG
formulations.
Frgrae 16 provides a graphical depiction of HSV 2 inhibition in hairless guinea pigs with PEG
fotrrrulations.
Figure 17 provides a graphical depiction of HSV 2 vesicle numbers in hakiess guinea pigs.
Figure 18 provides a graphical depiction of HSV 2 inhibition In Hartiey guinea pigs. .
Figure 19 provides a graphical depiction of HSV 2 vesicle numbers in Harttey gdnea pigs.
*Trademark.
_g_ Fgures ZOa and 20b r ilx fnhibiflorr of HSV ~ increases den cage ane incuba~d wllh n-docosanoi before viral addition and this inhibitory effect has a half-Ufe ~
ap~aoama~dy 3 h. (I~ Vero ~Ils were plated and incubated with 9 mM n-docosarrol, the corresponding control veh~le or no addItian for 0, 3, 6, or 24 h prior to the addition of HSV 1. The viral plaque assay was continued and fhe number of p.fu.
determined. The data ~e expressed as % inhibi0on compared to wells receiving no UBabneM. (B) Vero calls were pEated, n-docosan0l d~ the corresponding rxmbd vehicle Was added and ceAs were iru~afed et 37°C in 10% humidiUed COi. After 21, 24, 25, 26, and 2T h (6, 3, 2,1, and 0 h before the add~or~ of HSV-1), media containing drug was removed and the cells were washed with media. After a total of n h, HSV 1 was added to as wells. Two hours later virus-containing media was removed and replaced with fresh media lacking virus ZO or drugs. The cultures were incubated and processed for determination ~
ttre number of HSV-induced.
piques as In (J~.
Figure 21 shows the uptake of HSV 1(KOS}g186 into HF.p-2 cells when incubated kr n-docosanol b~eated ceAs. After attachment of HEp-2 cells to culture wells, n-docosanol-vehicle, vehicle alone, or no agent (control) was added. Fve to six hours after infection, the cells were processed, X-gal was added, and the absorbance at 600 nm rues de~mined.
F~ure 22 provides a graph demonstrating that n-do~rrol suspended with Tetronic*90B inhibits.lhe entry of HSV 2 1333) Into CNO-IE~8 cells. CHO-(E(38 cells ware seeded Into 24-well play, Affsr ceN
attachment, heparin, n-docosanol-vehicle, vehicle alone, or no agent (conbnlj was added. Fhre bo six hours after infection, the cells were processed,, X-gal was added, and the absorbance at 600 nm was deterrrrirred.
Figure 23 provides a graphic depicting experimental results for n-DocQSanoi-treated NC-9T human $
cells, the cell exh~iting deaeased fusion with odadecyl ttrodam~e 8 cHorlds-labeled HSV 2. NG3T
human.8 cells were inoculated in tl~ presence of 13 mM n-docosanol, the comespond~ concentration of Tetroni~908 (0.1 rnM} or without addition. CeAs were harvested and R 18 labeled HSV 2 was added to aliquots in the presence of compounds at their original concentration.
Fohowing incubation of 37°C for the times indica~d, cells were fiixed and ttuorescenoe interrsky detemrined by FACScan.
Deta~ed Des '~~ ,P~~d E~od, The following description and examples illustrate a preferred embodiment of the present Invention in deta~: Those of skill in the art will recognize that there are numerous variations and modifications of this invention that are encompassed by rTs scope. Accordingly, the descript~n of a pr~efan~ed embodiment should not be deemed to imit the scope of the present Invention.
10 % Docosanoi Cream To prepare a cream, rndocosand (98°/o pure; M. Michei and Co., New York, NY), a war-irrsoiubfe compound, is mbced at~ 80°C with sucrose cocoate, sucrose: sGearates, sucrose distearate, mineral orl, PropY~e g~ a~ P~IroxYPropY~-15-stearyl ether Water was added and mixed h io fir~sh the cream.
*Trademark A cream can also be formed by adding au the materials except n-docosanol to water to form the cieam base and blending the n~docosanol into the cream base.
The following proportions were found to be generally suitable: n-Docosanol 5-25 wt. % (or n-docosanol in mixture with at least one other long chain aliphatic alcohol having from 20 to 28 carbon atoms, S i.e., n~icosanol, n-heneicosanol, n-Uicosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-hepiacosanol, and n~octacosanol); sucrose stearates 0-15 wt. %; sucrose cocoate 0-10 wt. %; sucrose distearate 0-10 wt. % (with the proviso that at least one sucrose ester be present and that sucrose esters) comprise about 3 wt. % or more, preferably about 10 wt. 9'° of the total composition); mineral oil NF 3-15 wt.
°~; propylene glycol USP 2-10 wt. °~; polyoxypropylene-15-stearyl ether 0-5 wt %; benzyl alcohol NF 0~ wt.
°~ (with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of 2 wt. %); purified water 40-70 wt. °~. However, in certain embodiments other proportions may be preferred.
The following proportions were found to be generally optimal: n-Docosanoi 5-10 wt. % (or n-docosano! in mixture with at least one other long chain aliphatic alcohol having from 20 to 28 carbon atoms, i.e., n~eicosanol, n-heneicosanoi, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, end n~ctacosanol); sucrose stearates 6 wt. %; sucrose cocoate 5 wt. %; sucrose distearate 5 wt. 9'° (with the proviso that at least one sucrose ester be present and that sucrose esters) comprise about 3 wt. % or more, preferably about 10 wt. % of the total composition); mineral oil NF 8 wt. %; propylene glycol ' USP 5 wt. %; polyoxypropylene-15 stearyl ether 2-3 wt. °~; benzyl alcohol NF 2-3 wt. °r6 (with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of 2 wt. %); purified water 55 60 wt. %. However, in certain embodiments other proportions may be preferred.
A formulation containing 2-ethyl-1,3-hexanediol instead of polyoxypropylene stearyl ether or benzyl alcohol and sucrose esters was also found to be effective. However, a component other than 2-ethyl-1,3-hexanediol may be pneferted in certain embodiments, for example, in compositions intended for repetitive topical application.
An n-docosanot composition of a preferred embodiment (FORMULATION I) is described in Table 1 below:

n-0OCOSANOL FORMULATION t INGREDIENT WT. % ~ FUNCTIOt~URAT14NALE

n-Docosaral 10.0 Active drug substance Sucrose Stearates11.0 Emulsifier, Emollient Sucrose Cocoate5.0 Emulsifier, Emollient Mineral Oil 8.0 Emollient NF

Propylene Glycol5.0 Co-solvent, humectant, USP skin-feel modifier, auxiliary preservative 2-Ethyl-1,3-hexanediol2.7 Co-solvent, auxiliary preservative Pur'rfred-water58.3 Vehicle medi~un qs-ad--This n~ocosanol cream was sufficiently stable for more than a short period of time to pemtit the canying out of a comprehensive series of animal therapy trials in which the n~iocosanol was found to be consistently active in the animal herpes model (Figures 1 through 3) and was used for the initial Phase I
S human cllnlcal studies which showed it to be safe and tolerable. In oertaln countries outside of the United States, 2-ethyl-l,3fiexanediol may potentially be unacceptable for repetitive use. Therefore, in another embodiment it was preferred to substitute polyoxypropylene-15-stearyl ether for 2-ethyl-1,3-hexanedlo~ In equivalent amounls (2.7 wt. %), and 5 wL % of the sucrose stearates were replaced with 5 wt. % sucrose distearate. The resulting n~ocosanol composition (Formulation II) composition is described in Table 2, below:

n-DOCOSANOL FORMULATION II
INGREDIENT WT. h FUNCTIONIRATIONALE

n-0ocosanol 10.0 Active drug substance Sucrose Stearates 6.0 Fanulsi6er, Emollient Sucrose Cocoate 5.0 Emulsifier, Emollient Sucrose Distearate 5.0 Emulsifier, Emollient Mineral Oil NF 8.0 Emollient Propylene Glycol 5.0 Co-solvent, humectant, USP skin-feel modifier, auxiliary preservative Polyoxypropylene-15 2.7 Co-solvent, auxiliary Stearyl Ether preservative Purified water qs 58.3 Vehicle medium-ad This modified Formulation II succeeded in providing physical stability to the final drug product and performed well in the guinea pig herpes animal model (see Figures i and 2).
This formulation failed the USP
preservative effectiveness test, however. Therefore, the formulation is only suitable for use in applications wherein passing the USP preservative effectiveness test is not necessary, i.e., certain non-human applications. Improved microbiological stability was achieved by replacing polyoxypropylene-15-stearyl ether with benryl alcohol as co-solvent excipient, as described below.
In certain especially preferred embodiments providing stable compositions, only one or two 20 surfactants of the classes described are used, wherein the surfactants are present in amounts of about 5 wt.
%. The ability to use a limited number of types of surfactants and lower amounts of surfactant to produce stable creams was an unexpected and desirable result of our laboratory work.
Excessive surfactant is not desirable because excess surfactant increases the potential for irritation at levels of surfactants above 5 wt.

%. in addition, formulations with excessive amounts of nonionic surfactants frequently have problems with proservative effectiveness.
Utilizing several surfactant blends, with hydrophilic-lipophilic balance (HLB) values ranging from 9.0 to 13.0, a variety of n~ocosanot creams were formulated and then screened for optimal emulsion quality, physical characteristics, drug efficacy and accelerated physical stability.
Although most pharmaceutical emulsions are based on binary surfactant Mends to optimize the HLB, test results revealed that sucrose stearates alone perform as well as or better than other surfactant blends in the improved rr~doc~anol formula.
An n~iocosanol formulation having such a surfactant blend (Formulation III) is as follows:

n-DOCOSANOL (FORMULATION Ill) INGREDIENT WT. % FUNCTIONIRATIONALE

n-Docosanol 10.0 Active drug substance Sucrose Stearates5.0 Emulsifier, Emollient Mineral 0(I 8.0 Emollient NF

Propylene Glycol5.0 ~ Co-solvent, humectant, USP skin-feel modifier, auxiliary preservative Benzyl Alcohol 2.7 Co-solvent, auxiliary NF preservative Purified water 69.3 Vehicle medium qs ad The differences in Formulation Ill as compared with Formulation I include the re~acement of 2-ethyl-1,3-hexanediol with benzyl a~ohol, a well-known preservative and co-solvent with a long history of safe use and compendia) status. The liquid nature and like functions of benzyi a~ohol make it a rational and low risk replacement for ethyl hexanediol. The total surfactant level was reduced to 5 wt. % active with no change in the pharmaceutical characteristics of the product, no negative effect an the quality of emulsion based on microscopic examination, and no loss of physical stability in accelerated testing. Sucrose cocoate was omitted from the formulation without substantially affecting the propeNes of the formulation.
The seam can be made by heating and addition of ingredients, or by a more preferred method of 2U combining oil-soluble ingredients and heating them separately from the water soluble components. The hot oil-soluble components are then added to the hot water phase while mixing vigorously. Table 4 summarizes certain evaluated formulations.

FORMULATIONS (WT. 9'° COMPOSITION) INGREDIENTS I II IA III FUNCTION!

RATIONALE

n-Docosanol 10.0 10.0 ~ 10.0 Active i)rug Substance ~ 10.0~
~

Sucrose Stearates11.0 6.0 11.0 5.0 Emulsifier, emollient Sucrose Cocoa~e 5.0 5.0 5.0 Emulsifier, emolGenf Sucrose Distearate 5.0 Emulsifier, emollient Mineral Oil NF 8.0 8.0 8.0 8.0 Emollient Propylene Glycol5.0 5.0 5.0 5.0 Co-solvent, auxrllary preservative 2-Ethyl-1,3-hexanedlol2.7 Co-solvent, auxiliary preservative Polyoxypropylene-15- 2.7 Co-solvent, auxiliary stearyl preservative ether Benzyl Alcohol 2.7 2.7 Co-solvent, preservative NF

Water 58.3 58.3 58.3 69.3 Vehicle medium The n-docosanol Formulation III passed accelerated physical stability screening (storage at 42°C, freeze-thaw cycles) and also passed the USP preservative effectiveness test.
Drug efficacy 1n the guinea pig herpes model was verified on repeated occasions.
5 To monitor stability, the n-docosanol cream formulations were stored, variously, at room temperature (30°C), at elevated temperature (42°C), and under freeze-thaw conditions In polypropylene jars. The freeze-thaw samples were subjected to 48 hours of freeze-thaw cycles, Le., 24 hours at freezing temperature (-15°C) and 24 hours at ambient room temperature. The cream samples, stored under the respective conditions, were visually inspected for physical stability at various time points. After 12 months at 30°C or 3 months at 10 42°C or 24 freeze-thaw cycles ah samples remained as off-white creams. There was no evidence of syneresis or phase separa#ion. Based on the above visual Inspection, the Formulation III
of f0 wt. °~ n-docosanol cream was considered to be physically stable when stored under any of the stated conditions.
The exact shelf life of Formulation III has not been determined but experience suggests that shelf Iffe is more than adequate for a commeroial n-docosanol containing cream. Thus, while certain n-docosanol 15 formulations are unstable, specific formulations, FormulaC~on III being preferred, have been found to be both stable and efficacious.
Those skilled in the art of formulating creams of hydrophobic and hydrophilic compounds will recognize that certain substitutions may be preferred In certain embodiments.
Glycerol or another glycol may be preferred, with some adjustments in ratios, in place of propylene glycol, for example. Other 20 polyoxyalkylene-based ethers may also be found to be substitutable for polyoxypropylene-f 5-stearyl ether.
The relative proportions of the sugar-based esters may be varied considerably, so long as the total amount of sugar-based ester present is sufficient to stabilize the n~docosanol. This amount Is preferably from about 5 to about 25 wt. %, although the minimum and maximum amounts have not been determined with precision.

In a particularly preferred embodiment, the formulation for n~ocosanol cream is that of Fomtutafion Ill containing i0 wL °~ n~locosanol, 5 wt: % sucrose stearates, 8 wt: %
mineral ofl NF; 5 wt. % propyler>e glycol USP, 2.7 wl. % benzyl alcohol NF and 69.3 wt. % purffied wa#er.
Long-term stable cream preparations that contain effective amounts of n~docosanol alone a in 5 mixture with other such alcohols have been prepared, and the pharmacology of these compounds has been elucidated. In preferred embodiments, long-term stable topical creams formulaUon that have a shelf fife of greater than a year under normal handling conditions, i.e., is stable for a year or more at room temperatures and will withstand repeated freeze-thaw cycles, suitable for use in treating vines-induced and inflammatory diseases of the skin or membranes of an animal, including the treahnent of humans, are provided. The 10 . ingredients of the cream include n~iocosanol, alone or in mixture with other normal long chalri (G20 to G28j aliphatic alcohols, as the physiologically active ingredient, water, oil, an ester of a sugar and a fatty acid, the ester being physiologically inert or capable of being metabolized by the body, and an emollient to assist in peneVation of the n~iocosanol into the affected area of tf~e skin or memtxane and co-act with the ester in forming a stable cartier for the physiologically active alcohol (sj.
15 The sugar-based esters include a sugar moiety having a molecular weight of greater than about 150 and preferably above 250 and a fatty acid ester moiety having a molecular weight of about 150 or higher, and preferably above 250. The ester has a molecular weight of about 400 or higher.
Sugars, as the term is used here, are sweet or sweetish carbohydrates that are ketonic or aldehydic derivatives of higher pofyalcohols, and include both saccharides and disaccharides, disaccharide-based esters being preferred. High molecular 20 weight polyhydrlc alcohols may be substituted for the more traditional sugars. Examples of such esterified sugar-based surfactants can be found in the chemical literature generally and in various catabgs, e.g., McCutcheon's directories, Volume 1-EMULSIFIERS 8 DETERGENTS, and Volume 2-MATERIALS, (McCutcheon's Division, The Manufacturing Confectioner Publishing Co., Glen Rock, NJ, USA, 1993). Sucrose-fatty acid esters are preferred. Sucrose stearate and sucrose distearate are nonionic 25 surfactants that are preferred for use in n~Jocosanol cream formulations to emulsif)r the o~ and aqueous phases of the cream. These surfactants have a non-imtating nature, which makes them particularly preferted for treating, e.g., blisters caused by herpes virus. Sucrose stearates, when compared to conventional surfactants (such as surfactants marketed by ICI Americas of Wilmington, DE
under the hadenames i3rij, Myrj, and Span), demonstrate superior properties as a surfactant for n~dacosanol.
30 Propylene glycol Is preferred for use in n~docosanol cream formulations as having a long history of safe use in topical formulations. One of the uses of propylene glycol in cream formulations is as a humectant to give a smooth supple feeling to the skin. Mineral oil is also preferred for use in n~iocosanol cream formulations. Together with the n~locosanol, it forms the fiquld phase of preferred cream formulations.
Mineral oil has a tong history of safe use in topical products and may perform such functions as acting as an 35 emollient, e.g., by acting as a barrier to transdermal water loss, and to improve the texture of topical products.

Certain of the pharrruuological studlas ire fed uskrg s~pensior~s Drat ere more come witir the cells used in these studies but which era not suitable for use as topic( pharmaceuutical preparaSOns in certain embodiments as they may lack the body and stability required for effective.topical treatment.
A gener~ly preferred cream fomnulaSOn of certain embodirtreMs includes, bY
weft based on the tote) weight of the 5nal cream formulation, rHfocosanol, iypicaily about 8 to about 16,17,18,19, 20, 21, 22, 23, 24, ~ 25 wt. %, more preferably about 6, T, 8, or 8 wt. % ~ about 11, 12,13, i4; or 15 wt. %; most pn3ferably about 10.0%; sucrose .stearates, typically about O o about 11,12,13,14, ar 15 wt. %, preferably about 1, 2, or 3 wt. % to about 4, 5, 6, 7, 8, 9, or 10 wt %; andlor sucxose cocoa; typkally about 0 to about 11, 12,13,14, ~ 15 wt. %, preferably about 1, 2, or 3 wt. % to about 4, 5, 6, 7, 8, 9. or 10 wt %; andlor sucrose distearate typ~ally about 0 to about 11,12,13,14, or 15 wt. %, preferably about 1, 2, or 3 vut. % to 4, 5, 6, 7, 8, 9, or 10 wt. °~; at feast one sucrose ester or an equ'rvaient sugar-based saber comprishg iypical~ at least about 3 %, preferably about 4 wt. % to about 6, 7, 8, 9,10,11;12,13,14, or 15 wt %, most preferably about 5.0 wt % of the.total composition; oil, e.g., mineral 07 NF typically about 3 wt % to about l5 wt. 96, prggr~r about 4, 5, 6, or-7 wt: % to about 9,10,11, or 12 wt. %, mgt prey about 8.0 wt. %; a glycol, e.g, propylene glycol USP or equivalent, typicaAy about 2 wt. % to about 8, 9, or 10 wt. %, preferably about 3 or 4 wt. % to about 6 or 7 wt. %, most preferably about 5.0 wt. %; ~ emollient glycol ether, e.g.,, polyoxypropylerre-15-saryl ether, or benryi a~ohd, typically about 0 b~ abort 3.5, 4, 4:5, or 5 rrt. %, preferably about 0.5, 0.75,1,1.24,1.5,1.75, 2, 2.25, 2.5, or 2.6 wt. % ~ about 2.75, 2:8, 2.9, or 3 wt. %, roost preferably about 2.7 wt %; and water typically about 40, 41, 42, 43, or 44 wt.
% to about 70, 71, 72, 73, 74, 2fl 75,76; 77, 78, 79, ~ 80 wt. %, preferab~r about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 wt. % b 56, 57, 58; 59, 60, 61, 62, 63, 64, 65, 66, fi7, 68, or 69 wt. %, most preferably about 69.5 wt. %. Within thts general fomwl~ion, many speafic formulations cart ' be prepared which will be stable and which wUl exhd~it the therapeutic effect noted t>a~d upon the data presented above, die teachings of the specification and the guidelines provided in the spectficafion: Thus, an effective topical therapeutic composition wtrerein the therapeutically active rtmt~iai essentialgr of n-docosanol, akme or in mixdue wHh nomnat bog drekr (C~20 to G28) aliphatic aloohole may be prepared.
The formulations may be u~d in the manufacture of pharmaceuticals and also in the treatment of human and animal p~rrts.
a nts .
. To confirm in an experimental animal model the efficacy of n~dacosanol cream oit.HSV~uced ~~, ~ ~ ~~ ~ ~ of ZOVIRA~"'hatrless guinea pps were inoarfabad widr 1 x 108-PFU
of HS111, and then treated with either n~docosanol-containing or control cream, or zOVli'~iA7~oiniment. The n-docosarai creauns wee caastnrcted as described. The control cream was co~ructed ~r a simiHar mamer except stearlc acid was substituted for ndocosanot. Treatment was started erTtrer 2 or 48 hours after vinrs *Trademark inoculation. The sites were evaluated for vesicle formation, defined as a pus-fled blister, at the indicated time Figure 1 presents the comparative activities of Formulation I and three different preparations of Formulation U as wen as ZOVIRAX:~Formulation I and Formulation II of n~iocosanoi creams both showed greater inhibitory power than ZOVIRA7~5intment.
Figure 2 presents the comparative activities of Fonnulatton i, Fomtulation IA
and Formulation II.
Signifecant inhibition of HSV-1-induced lesions was demonstrated form three formula5ons.
Figure 3 shows a comparison of activities of Formulation ill versus Formulation I and also depicts certairi modifications of these formulations in which the relative surfactant concentrations have been rrw~fied from that of Formulation i.
Modifications of surfactant concentrations were found to have appreciable deleterious etfec~ on ~e extent of drug activity. Formulation ill was shown to have patent inhibitory powerfar HSV-I-induced lesions.
Volunteer patients with recurrent oral ar genital HSV I or il infections have also been treated with topical n~docosanot-containing cream. at various stages of an acute herpes outbreak. When treatment is initiated during the prodromal stage, n~docosanol cream may abort further fan of the infectiotr (i:e.;
prevent vesicle fvrtnation). Nihen treatment is started after .vesicle fom>atian has already axurred, n-docosanol cream may shorten the time for healing (te., complete ra-epithekalization) of such herpes lesions.
The selection of 10 wf. % n-docosanol in the formulatan was tested in a dose-response study kr the hairless guinea pigs. The sites on the backs of hairless guinea pigs were inoculated with HS112 as des<x~ed previously. The sites were treated with 1, 5, 10, and 20 wt. .% n-dooosanol formulations. A vehicle oordnol containing no n~docosanol was also included in the study. The results, iAustrated in Figure 4, show that after T2 hours of virus inoculation the untreated sites exhibited an average of 41 vesicles. Treatment with 20 vvt. %
and 10 HA. 9'o ndocosanol containing cream inhibited vesicle number by 50% and 60%, respectively, t containing 1 wt. % and b wt. % n-docosanol were less effective than the 10 wt.
% preparation. The control vehicle was whhout appreciable inhibitory effect.
1t has been observed.ihat the level of n-docosanol in the cream may play a rote in the physkal appearance, stability, and effcacy of n-docosanol cream. A comparison of creams containing 5, t0, and 20 wt. % n-docosanot was conducted. In gen~ai, it was observed that the viscosity of the product varied directly with the concentration of n~docosanol in the formulation (Figure 4). The 5 wt.
% formulation had the lowest viscosity with lotion-like appearance and had a tendency to separate into phases. The 20 wt. ~o forrnuiation , had the highest viscosity, was diffiarlt to rub in and had a tendency to leave a white residue on human akin.
Complete removal of n-docosanoi from the cream resulted in a watery, lotion type formulation that underwent phase separation after overnight storage at room temperature. The 10 wt. %
formulation was physically stable and cosmetic~ly most pieas'mg, rubbing in easily and not leaving any residue on human skin. The rests indicate that in addition to its function as an active ingredient, n-dooosanol also functioned as a thickening *Tr~demark _16_ agent and an emulseon stabilizer in the creams tested. In vlvo studio with hairless guinea pigs showed that the 10 wt. % formulaban had be~6er ef fiCacy than 5 wt. % or 20 wt. %
formulaf~s. WtSle the 10% famulaifon was preferred for most applications, in certain embodiments, however, a formulation.cor>tainh>g less than 10 wt. %, e.g., 5 wt. % or less n~locosanol may be preferred, while in other embodGnents a formulaBon S containing more than 10 wt. %, eg., 20 wt. % ~ more rr~ocosanol may be .
Since it has bean reported that benzyl alcohol had ,some antivlral aotjvityr under certain, .
c'ucumstances, (Farah, A. E. ef al, U. S. Patent No. 4,200,655) a tortnulation of a preferred embodimer~ was ~ determine if benzyl alcohol acts as an antivir~ reagerd ~ the formulation.
The cream cordaintng benzyl alcohol and rr~ocosanol (10 wt. % n~docosanol cream) arid the.cneam cordaining benzyl a~olxr) alone (placebo) were tested on HSV-2 induced cutaneous lesions in the hairless guinea pigs. Sites on the backs of guinea pigs were inoculated with I~V 2. The sites vuere treated ~ irr~caled in Figure 5 arxi avak~fied ton vesicle formation at 48, 56i 72 and 18 hours after virus inoculation. There was an average of 44 in the untreated sites at the 48-hour time point; which remained relatively constant up to 72 hours otter infedfon.
At the 78-hour tone poird, resohrtio<r of the lesions became evident and by 96 fmrxs posWnoa~n rays were no longer vis~ie.. Tneatsment with n-dooosanol cream inhibited treslcle rwmber by 50-6096 at the 48~66-hour time points, and by a slightly h~her amount at the 72-78~hour points of analysis. Treatment with the contra vehicle was wiUwut appreciable effect on aside number at any time point. Untreated ~d treated sites were excised and pmxcessed f~ viral culture. The pteserrce of vesicles was directly cone~sd with the presence of infectious virus regardless of treatment or time of assay (not shown). Thus, veside'number Is as appropria~ tndkator for disease stabs in the stur5es described herein.
Additionally. the cram and the ~aoebo were tested in a, phase A pilot study comprising sixty-eight patients with herpes labialis. The result of tire double blind trial showed that early app~cation of n~docosanol cream cut the duration of the episodes really M
h~f. The treated groin' averse oulbne~c period was 3.4 days, vrfwle the piaoebo group had oufbrea~
averaging t?.6 days , The above results demonstrate that the presence of ndocosanol in the formulation is responsible for significant aMivirat ac~On.
The antiviral activity of n~docosanol has also been dem~onsUated in a susp~sion formulation of n-docosanol in the nonionic surtactarri Pluronic'~F-68 which did not contain any of the excipienfs of 10 wt. % n-dacogarwl seam forrrruia8on itrc~rding berrzyt alcohol. The results, sumrnatfzed ~ Figure 6, demonstaae two important points. F~sst, as shown in panes A, a suspension formulation of nil In Plur~ani~'F-88 also inhibits HSV-1 induced vesicles when applied, 2 hog after virus infection, as observed with the cream ibtomwiatwn. Thus, the untreated sites exhibited an average of 74 ~re,~Cles at 48 hours atler vlrtrs, but only 28 vesicles were observed h the sites treated with n-docosanoUPIuroni~F-88 (63%
inhibition). Trent with ZOVIRAX~an FDA-approved treatmera for certain HSV infections in humane, was also aesodated with decreased veskle number, but ass so than wAh rHiooosand.
*Trademark _17_ Continued tnesimerrt with rtdocosanoi rued in many fewer vesicles at the 72 hour time. post also.
The vehicle control for the n~locosanoi preparation was without effect at either time point The second major print delved from F~ure 6 is that n~ooos~ol hastens resdu0ort of HSV 1 induced disease even when administered after veskles have emerged (Panel B}.
The various sites exhibited roughly eq~ent nurr~er of vesicles at the 48fiour tkrre point, v~ich would be expected s~tce none had been treated by that time. Vesfde numbers decreased in the untreated sites from a mean of 73 ve~cles at 48 hours b 43 vesicles at 72 hours. Treat<ner>t wkh ZOVIRA~was associated wlNt a mode~ly hashed dis~e r~esdution at T2 hours (27 ves'~les, a 37% decrease versus the untreated sites), whkh is consistent with other expetimerrts of a sim~ar design. Importantly, app~calion of n~looosanoIIF-68 yl~amdy hastened a resolution as shown by the 77°~ inhibition of vesicle number when compared with the untreated group. The same condusioirs were obtained using the caeam formulation in expetim~ts of a similar.des~n. This dernonstrates that n~docosanol need not be administered prophylactkally to after the HSV induced course of arsease.
'fhn~e safety and tolerance .studies were conducted in healthy Caucasian male and fenu~e volunteers. A total of. TB healthy volunteers were exposed io drug. The safety studies hdicated that the formulation of n-docosanoi 10 wt.~% cream does not cause phototoxlcity, but is a mihi Iximary irtitant which also has the potenti~, a~eit in low incidence, to cause alierg~ sensitization (1 subject of the 78 exposed experierroed contact dermadtjss).
Two clinical efficacy studies plus trials have been completed. Study A was a randomized, double blkrd, placebo~controNed study in ~xty-three patients (male and female} with nrcurrent herpes labia6s. AB of the thirty-one n-docosanol 10 wt. % cream-treated patients in the herpes labialis stay, Study A, completed their b~trrmrrk Mro of chose thbtlwne patients repor~d a homing or stinging serasat~n after ~piication of the cream.
No clinically sigridicant chatrges ~ cGnicat.laboratory values tblood ~, hema~ofogy, and urine analysis) were revealed in eHher study. Study B was a randomized, double bend, piac;ebo-controlled trial 1n forty female patients with recurrent herpes genitalia. AU of the fio n~docosanol 10 wt. % crearn treated patients In rite genital study, Study B, completed their treatment wntrout repotdng arty dntg-t~elated adverse erer~.
Study A
Sixty five patients (aged 1&60) took part in the Study A, thirt~two patients were initi~a~y rar>domaed to receive 10 vrt: % n~docosanol cream, and thirty-three were initiaOy randomized to receive pl~ebo cream.
Treatment was patient-initiated, and treatment inittatlon was deflrlerl as 'early" if the treatment started at prodrome or erythema stage and as'late' if started at the papule stage or later. Two patferrts were ~gxduded from the analysis. Of the sixty-three evaluable patients, twenty-two were entered into the crossover phase of the study. In addil<.on, thirteen patierds treated more than one episode with the same study madiceOOn.
*Trademark ~l8_ Therefore, a total of ninety-eight herpes episodes - forty-eight treated with 10 wL % n~dooosanol cream and _ fihy heated with placebo cream - were analyzred. _ The results of Study A are summarized according to first treatment episodes, crossover treatments and all treatment episodes combined in Table 5.

STUDY A: TIME TO HEALING (DAYS) OF
RECURRENT HERPES LABIALIS EPISODES
Part A. Analyrsis of first episodes n-0OCOSANOL PLACEBO

YIfT.
%

MEAN SD {n) MEAN SD (n) Early treatment 2.5 2.4 (10) 6.8 4.2 (4) Late treatment 6.8 3.2 (21) 7.3 2.T (29) All treatments 5.4 3.6 (31} 7.3 2.8 {32}

Part B. AnaNsis of crossover st,~rdv n-DOCOSANOL PLACEBO

WT.
%

MEAN SD (n) MEAN SD (n) Eariy treatment2.7 2.2 (7) 7.0 (1) Late treatment 5.6 2.1 (15) 8.0 2.6 (21) All treatments 4.7 2.5 (22) 8.0 2.5 (22) Part C. Analysis of alt treatment episodes in the study n-DOCOSANOL PLACEBO

WT.
%

MEAN SD (n) MEAN SD (n) Early treatment 3.4 3.0 (13) 6.7 3.9 (7) Late treatment 6.5 2.7 (35) 7.4 2.7 (43) AU treatments 5.7 3.1 (48) 7.3 2.9 (50) I f I I

Thirty~ne patients treated their first episode of herpes labialis with 10 wt.
°~ n-docosanol and thirty-two with placebo (Part A). Ten patients in the n~ocosanol group and four in the placebo group were classified as early treatments. Mean healing time in the early-treatment n~docosanol group was 2.5 days, a reduction in mean heating time of 4.3-4.8 days compared with the other treatment modalities. This difference was statistically significant {P=0.0001) in favor of n~docosanol. In the late treatment cohort, n-docosanol reduced mean healing time in the first episodes by 0.5 day, which was not statistically significant.

Of the twenty-two patients entered into the aoss-over study, the number who had treated their lesions early in both -parts of the study (seven using n-docosanol in the crossover phase and one using placebo) was too small for meaningful statistical analysis (Part B). However, a substantial number (fifteen using n-docosanol in the crossover phase and twenty-one using placebo) had treated their lesions late, ~tus S allowing for intro-patent comparison in this respect Analysis of variance of the results of late treatment revealed a significant difference in favor of n~iocosanol (P = 0.03).
Evaluating the data from all ninety-eight treatment episodes of Study A
together (single episodes, cross-over episodes and additional episodes with the same medication) reveals a statistically signifccant (P =
0.02) reduction in mean overall healing time of 1.6 days in n-docosanol-treated (5.7 days) versus placebo-10 treated (7.3 days) patients (Part C). In the total twenty episodes classed as early treatments, topical n-docosanol reduced mean healing time by 3.3 days (P = 0.05). Finally, when effectiveness of early treatment with n-docosanol was compared to all other treatment modalities, mean healing time In the early treatment n-docosanol group (3.4 days) differed quite significantly from the range of 6.5 to 7.4 days in the other groups;
this difference was highly significant in favor of n-docosano( (P = 0.0002).
The differences between late 15 treatment with n~docosanol 10 wt. % and early and late placebo treatment were not significant As demonstrated by the data summarized in Table 5, early treatment with 10 wt.
% n-docosanol cream (in the prodromal or erythema stage) produced a highly significant shortening of healing time compared with that obtained with the other treatments. In addition, late treatment, started after lesions had appean;d, resulted in a statistically significant reduction in heating time in the n~locosanol-treated group In the crossover 20 portion of the study, though not in the other analyses.
Studv BB
Sixty temale patients with recurrent herpes genitalia entered the study while symptom-free and not in a prodromal stage. Thirty subjects were initially randomized to receive 10 wt.
~ n-docosanol cream and thirty to receive placebo cream in this patient-initiated trial for the treatment of early-stage herpes genitalia 25 recurrences. Forty-four patients initiated treatment and returned to the clinic with a herpeGc episode; twenty-two of these patients received n~tiocosanol and twenty-two received placebo.
The mean time to healing in the sixteen evaluable n-docosanol patients was 4.7 days t1.9, ranging from 1.8 to 8.6 days; for the eighteen evaluable placebo patients, healing was complete within a mean of 5.1 days t2.3, ranging from 1.7 to 10.4 days. The difference was not statistically significant (p = 0.5827, t-test).
30 Patients with non-genital lesrons, who were noncompliant or had dosing interruptions, who had prodrome with no observable episode, or who had concurrent yeast infection, were considered nonevaluable. When all patients are Included, the mean time to heating of the n-docosanol group was 5.5 days t2.5, ranging from 1.8 to 9.8 days. For the placebo group, healing was achieved in a mean of 4.7 days t2.3. Healing time in this group ranged from 1.7 to 10.4 days. There was no statistically significant difference in the mean time to 35 healing between the two treatment groups (p = 0.2703, t-test).

There was also no statistically significant difference between treatment groups when patients were stratified according to stage of the lesions (prodrome; erythema, or papule) when the treafit-ent was Initiated:
The average healing time based on patient ratings was similar to the clinicians' (5.6 days for all ntiocosanol patients versus 4.5 for all placebo patients).
Three pain analyses were conducted, based on patients' self assessment of pain: time to sustained 'no pain'; time to first "no pain'; and time to first n~tluction of pain.
Time to sustained 'no pain' was measured from the time of first pain ei application to the time when 1) pain was scored as'no pain" for a minimum of 2 consecutive recordings; and 2) during the remainder of the episode, additional pain recordings were no more frequent and severe than two separated episodes of two consecutive recordings of "mild" pain. Time to first'no pain' was defined as the interval from first pain at application bo the first recording of'no pain." Time to first reduction in pain was measured from the time of first pain at application to the first time when a decrease in pain level was noted, re~d~ to the previous assessment. Several patients were excluded from these analyses because of either lack of pain wtfhin the first 24 hours, or noncompliance in reporting pain.
The fifteen evatuable patients treated with n~docosanol achieved a sustained response of'no path"
sooner than the fourteen evaluable placebo patients; a mean of 3.2 days X1.9 for n-docosanol pafients compared to 4.1 days t2.5 for placebo patients. The n-dooosanol patients also achieved 'no pain' sooner than the placebo patients. The n-docosanol patients first recorded "no pain' a mean of 2.6 days t2.1 after pain onset, while the placebo patients first reported 'no pain' a mean of 3.4 days f2.1 after pain onset.
Among the evaluable n-docosanol patients, the first reduction in pain, relative to pain at the preceding application, occurred at a mean of 1.2 days t1..0 after pain onset. First reduction in pain occurred In the placebo patients at a mean of 1.8 days t1.4. These differences were not statisticaAy significant (p = 0.2775, 0.325, and 0.1757, respectively, t-test). Patients with non-genital lesions, who were noncomptlant or had dosing interruptions, prodrome with no observable episode, and concurrent yeast infection, were considered nonevaluable.
In preferred embodiments, a method of reducing the pain of a surface inflammation of the sftin or membrane Including applying to the inflamed surface a composition of n-docosanol, optionally in combination with at least one long chain aliphatic a~ohol having from 20 to 2ti carbon atoms selected from the group consisting of n-eicosanol, n~heneicosanol, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexaeosanol, n-heptacosanol, and n~etacosanol, or mixtures thereof, in a physiologically compatible carrier, said alcohd including from about 5 to about 25 wt. % of said composition. Preferably, the physiologically compatible carrier is a cream base that includes one or more compounds selected from the group consisting of sucrose cocoate, suaose stearates and sucrose distearate and one or more compounds selected from the group consisting of polyoxypropylene stearyl ether ethyl hexanediol and benzyt alcohol.

While no statistically sigrNfiicant differences were noted ~ Study B. in time-to-heallng t~tween patients who received 10 wt. % n~docosanot cream and those who received placebo cream, a fraud tbwards reduced time-to-heaGng among the evaluable patients treated with n-docosanol was observed. Three different pain analyses alt showed a more rapid resolution of pain in the subjects who received n~tocosarat 10 wt. %
5. cream, though none of the differences were statistically significant. The inabilitic to detect significance in this study may reflect, in part, (1) the small study population; (2) differences at study enter between the fwo study groups with respect to the natural history' of herpes genffaalis lesions; and (3) an unequal distribution between the two groups of lesional stage at episode and treatment initiation.
in additwn to the clinical studies, several studies were carried out to elucidate the ptiarrnacology of n~docosanol. These studies resulted in the data depicted in Figures 7 through 13, and are discussed bebw.
An appropriate forniulation which allowed acceptable delivery of the n-docosanoi bo bidogic~ .
systems was developed. InitFally, this was accomplished by formul~ing a suspension of the n-do~nol molecule in the inert and nontoxic nonionic surfactant, Pluronic~-68. Such suspensions are homogeneous, consisting of n-docosanol containing particles averaging 0.10 microns in diameter. Suspended ~ this way, n-docosanol exerts inhibitory activity in vitro against type 1 and 2 herpes simplex virus (HSV) infedivhy of simian and human cell lines. n-DocosanollPluronic*suspensions are equally effective against wilddype and acyclovir-resistant rtotants of HSV.
Thus, as shown in Figara T, Panel A, acyclovir and n~docosanol irdtibit plaque fomratbn by wild type HSV 2 equally. Figure T, Panel B illustrates that an acyciovir-resistant HSV 2 mutant is not inhibited by acyclovir, but is ~hibited by n~ocosanol. The Pluronic*surfactant alone lacks ~y antiviral activity. Host cell toxicftir was not observed with n~focosarbl at concentra~ons as huh as 3 mM.
Extensive studies designed to delineate the mechanism by which n-docosanol exerts ils antiviral activity were conducted. The collective implications of the results of the studies are the the compound appears to interfere with one or more of the common pathways of viral enhy into the Celt and migration to the nucleus of infected target cells. The key points of evidence supporting this notion can be summarized as follows: (a) the compound has no direct yiricidal activit)r, since virus can be mixed with a n~dorrosarwl suspension, then recovered from the suspension and shown to ret~n nomral infectrvify; (b) although the compound does not interfere with binding of herpes virus to HSV-specific receptors on target'ceUs, HSV
virions which have bound to fiarget calf receptors in the presence of n-dooosanol remain on the oeil surface for a prolonged time period; and (c) subsequent migration to the cell nucleus of virus which has been internal is inhibited, as measured by detectable HSV core and envelope protein, numbers of cells expressing the immediate early protein, (CP-4, and secondary plaque assays.
The. delay in virus irrremalizatioh described above is illustrated in the experiment summarized in Figure 9. tn this experiment, HSV-2 was incubated with Vero cells in the absence or presence of n~do~nol at 4°C to allow for receptor binding of the virus. At the end of 3 hours, all cultures ware washed and then *Trademark reputed at 37°C in order to initiate the viral entry process: At 20 minute interval thereafter, the various cultures were exposed ~ pH 3.0 agate buffer, conditions which remove and inactivate surface-!round, but not internalized, HSV vMons, and tt~n re-cultured the -tuh 44 hog palled required ~ develop optimal HSV
plaques. All cultures exposed to citrate buffer at time-0 fa0ed ~ develop plaques, as expelled.
As shown by the uppermost Ones on the graph, trderrralization of HSV 2 is virtuaay complete wflhin 20 minutes after the shift to 37°C in the untreated and Pluronic*control treated atitures. Iri contrast, irrbemalization of HSV ~ the n~doc~arrol heated culdmes was less than 4096 complete by 20 mlrut~ and required more than i hour to reach completion. These results clearly indicate that the kinetics of viral fusion andtor transmembrane migration are delayed in same way by n~iocosatbl.
Even alher intemafriatian reaches compteGon in rrdocosanoi~reated ceAs, subsequent viral migration to the cell nucleus is significantly inhibited.. Thus, the amounts of both HSV core and envelope protein antigens detectable by EUSJ4, as we9 as the numbers of inieded ceAs expressir~ the krb~arxx~ar HSV-specific immediate-early protein, ICP-4, by immunofluorescence, are reduced by more than 80%.
FinaOy, the repifcation of irdectrous virbns as measured in second~r piaqne assay cures is markedly diminished by 99% a more in n-docosanot-treated cells.
To summarize, dre presence of rrdocosanol has no effect on the initial steps of v(rai binding, but consllerably delays entry of virus into ~e target ceA cytoplasm through some yet-to.be~debrmined mechanism. in addition, the process of migration to, and localization in, the nucleus is sut~stantial~ blocked;
having the ultimate effect of a marks decrease k1 producli~ viral repAcafion.
In order to betEer define the predse mechanism by which n-dor~sanol exerts its arrtlvirai activity, the cellular uptake, dlstnbution, and rrmtabofism of n~do~sanol from surfactant-~bOfred sr~pe~nsions has bin studied. The results of such studies have provided some interesting insight into ~e metaboOc basis of the compound's antivira! action. It has been shown that radfoactivety .I~eled rr~aosand is progressively Incorporated hto cukured Verso cells, readdng a peals uptake per ceo between 6 and f 2 hours aiber exposrme.
The process is irreversible, since once the compound is cell-associated it cannot be removed even with extensive wash~g with cesium bromide, whtctr effectively removes nonspedflcally assodatad cel4borurd particles.
Second, at saturating concenir~ions, less than 1% of the total n-dooosarrot added to cuihues becomes cell-assadafed win 24 hours. None~eless, this corresponds to neatly 8 x 109 mdecutes per cefl, 3.0 an amount which approximates the number of lipid molecules typically found in plasma membranes. .
The fact that such a small fraction of n~docos~roi in the suspension added to cultures.bacanes ceN-associated indicates that the actual trioactlve dose is oniers of magnitude less than the amount of drug added to the aritures.
Cenular distn'butron studies examining subceliutar fractions recovered by differential centrifugation of ~ sonication disrupted cells demonstrated that after 12 hours of exposrme 75%
of the radioactive compound ~
*Trademark contained tn ceA membranes, and less than 1 % is associated with nuclear fractions; the balance of radioacflvity is associated with the soluble cytoplasmic fractlmr.
Analyses of the metabo~C conversions of n.docosanol have shorm that the compound is progressively metabolized to polar compounds, whkh were demonstrated by ttdn layer chromatiography to be phosphatides, generated either via anabolic (ether linkages) or catabolic (oxidative) reactions. Figure 10 demorutrates a thin layer chromatographic analysis of a mettaewl e~ted (plwsphatlde~gj fiction from a silica get cokrmn of an extract of n~locosanoW~eated hero cells.
Nonmetabdized n~docosaral was previously eluted from the silica with chloroform. As shown, approximately 82%
of the counts rttigrated in the region of phosptratidylcholkre and 38% migrated ~ the r~ion of ptrosphatldylethanolamine.
1 U Our studies have also documented that such metabolic conversions can be blocked by aplxopriate metabolic inhibitors. Thus, the affective energy poisons sodium azide and 2~deoxyglucose reduce both uptake of n~docosanol by Vero oe~s by !i~% and metabolic conversion krto, polar metebo~ by 8016. 8 is probable . .
that the combination of sodium azide and 2-deoxyglucose matnly inhibits cellular uptake of n~docosanol by inhibit'mg endocyfosis; however other mechanisms of uptake, including an energy-dependent firsfon .
mecharirsm, or a passive diifus'ron mechanism facilitated by the subsequent energyd~endeM metarv of n~iocosanoi, could also be inhibited by these energy poisons.
An interesting aspect of these studies is die indkation of a possible role for the polar me~bo~ of n~docosmrol in the amivkal ao5vity of the car~ound. n has recently been demonstrated that of mouse flbroblasts to polyethylene glycol-induced fusion correlated wfth.an Increase in both free fatty ~cohols and an elevation ki glyoerkies, irrdudirrg an ether-linked cor~oond that woukl be analogous b the products obtained via metabolic conversion of n.docosanol ass described atx~.
Expedmer~ts were conducted to investigate the possibility that the enzymatic conversion of n-docosarroi is a rr Prerequisite for its antivirat activity. The rosults of such studies have demor>gtrated, firstly, flrat the rate and e~dent of metabolic conversion, but not that of.
ceAular uptake, of n~docosan~ to its polar metabolites is determined by the nature of the surfactant used to suspend the compound and, indeed,.
that effiaency of m~abor~c conversion directly oonelatwwiih the magnitude of ~riral acHvt<y of n-docosanol.
~An initial step in conducting such studies involved switching to a different surfactant fix suspending anal. Tetronic~J08 is ck~seiy rela~d ~ Ptmonic*F68; tom are Wick copolymers of ethyleire oxide acrd propylene oxide. However, whereas Pluronic*is a bifunctbnal polymer wlUt a molecular weight of 8,400, letroni~908 is a tetrafunctlonal copolymer, produced by adding propyAene oxide aced ethylene oxide to ethyler~diamine and resulting Jn a moleade with an average molecular weight of 25,000. Among other thkrgs, when Vero c;eiis are exposed to equivalent doses of n~iocosanol suspended in Tetronic'"~ersus Pluronic~;'the rate and extent of metabofrsm of the compoernd to polar metabo&tes is s(gniticarrtly higher with the Tetroni~*
than the Piuronic'FSUSpensiat. The Mtal uptake of radioacflve n~dooosand was equivalent from die taro Trademark different suspension formu~tions; only tiv: met~o~c con~rsion differed sign)~ar~y. Cort~el~ng witit tide higher metabolic conversion from Tetronic~than Piuronic~suspenslons is the Ending that tile EDao for inhtbitiorr of HSV replication by n.docosanoi is 5-10 mM in Tetroni~*and approx(rnafely 3 times higher in Pluror~' This appears bo relate to the 3-fold higher iev~s of meiab~c conversion in cells treated with n~locosand in Tetronic*
To eliminate the possib~iitr that these findings are pecul~r fa the Vero caw culture system, a.
reaprocai analysis was made, taking advantage of the fact that, relative to Vero cells, the epithelial-like bovine kidney cell I(ne, MDBK, exhibits an interesting apparent ~esistanoe ~
the anti-HSV of n~
docosanol. This difference is significant in that n~docosanol is 3-4~oid more effective in inhibitir~ HSV
. induced plaques in Vero cans than in MpBK cells. A comparison of total cellular uptake and relative metabolism showed that both the tote) amount of n.docosarbl uptake and the relive amourd of .me~boGc conversion were 3-4 times higher in Vero than in MDBK cells. The combined effect of decreased uptake and decreased metabolism in MDBK versus Vero ceAs is gr~hk:ally ~lustrated 1n Figure 1t, whch shows Ihat after 72 hours, Vero cells contain almost 4-told higher amounts of the phosphatide metabolite, which remains at the origin in ~~ solvent system. Of the counts that are metaboAzed (n two cans tines, the relative amounts in die major classes of ~Sphatides that are formed, PhosPhatrdY~holine and phosphatidyied~ano~mmine, are not different in the two coil lines. Moreover, pulse.chase experiments showed that both lines eventually concert all of ~e incorpora~d courts into the more polar form, . Such. results suggest that MDBK cells may effectively regu~te uptake andlor metabolism of n-docosanoi through a feedback type mecum that is either less effedtve or nonaperative in Vero cells.
Consistent with the mechanistic abservatlons summarized above, it was predicted that n~locx~rot would have potential for interfering an'th a variety of d'rfiaront vln>ses, specifically those which corrtarcr Lipid ~
their outer envelopes and which use fusion nisms for entering susceptible target cps. Table 6 summarizes the human and murine Gpld-enveloped viruses that have been shown to be susceptible.to the anti~rat activfty of n~docosarrol.

SPECTRUM OF ANTnJIRA! ACTIVITY OF n-DOCOSANOL
AGAINST LIPID-ENVELOPED VIRUSES
HUMAN VIRUSES MURINE VIRUSES
~~

He s Sim tax-18~ . C m alovirus .

Varioella Zosber Friend Leukemia Vines Virus Human H svirus-B LP-BM5 Virus Res al Vinrs C om ~ alovicus .

Influenza A

HN=1 *Trademark Every lipid-enveloped virus tested can be effectively blocked by this drug. n-Docosanol has anti-retroviral activity troth In vJtro and >rr vnro. A fomnulation possessing aMi-retroviral activity and tacking toxiGty has substantial usefulness in treating a variety of retroviral diseases in humans and domestic animals.
Notwithstanding the implications for treatment of AIDS, availability of a treatment regimen for diseases caused S by retrovlnrses like feline leukemia virus, bovine leukemia vinis, as well HTLV 1 and-2 has substantial benefits in humanitarian terms. Studies have established that n-docosanol inhibits replication of murtrre reiroviruses In vJtro and in viw.
Initial studies focused on the murine Friend leukemia virus (Ft/; 8).
Itroculat~on of aduti mice with Ft/
results in the induction of a leukemia of erythroid progenitors, specrflcaily the basophilic erythroblast. This erythroleukemia is characterized by the rapid profrferation of virus-infected erythroid cells, viremia, irnmunosuppression, and ultimately death of the animal. Intravenously injected Ft/ will circulate through hematopoietic organs, such as the spleen, and infect erythroid cetis. ff such infected spleens are fixed on day .
14 after vinrs injection, discrete macroscoptc nodules can be seen on the surface of the organ; these represent clones of leukem~ tails and form the basis of the spleen focus assay.
The experiment summarized in Figure 12 itiustrates that n~ocosanoi inhibits Friend Virus-induced leukemia and viremia in adult mice injected intravenously with 75 focus-forming units of Friend Virus. Treated groups were injected intravenously with the varying doses of n~ocosarrol or Pluroni~F-fib vehicle alone intravenously on the same day as virus Inoculation and once daily for the next 3 days. Affer 10 days, half of the animals in each group were sacrificed and examined for the presence of leukemic foci in their spleens, while the remaining animals were retained for 10 addiCrona[ days to monitor viremia. Treatment with n-docosanol exerted a very clear dose-related inhibitory affect on both the development of leukemic fod, shown in Panel A, and the development of vkemia, shown in Panel B. In contrast, treatment with comparable amounts of the Pluronic Fj68 vehicle alone as control exerted no discernible effect. It is believed that these results reflect the inhibitory activity of n~docosanol on viral replication, since corotiary M vitro studies have documented a very potent activity of this drug against replication of Friend V'ws in primary embryo flbroblast cultures. n-Docosanoi inhibits in vitro replication of Hlll 1 and human trerpes virus 6.
The initial studies an HIV were conducted in collaboration with a U. S.
National Ir~stituutes of Health Laboratory and one of several experiments of this type is summarized in Figure 13. Normal human peripheral blood mononuclear cells were activated with 1 uglml PHA plus 5 unitslmi of IL-2 in medium alone or in the presence of n-docosanol, Piuronic'~F-88 control vehicle, or phosphonoformic aad (PFA). The next day, the cultures were inoculated with HIV t and examined 4 days later for evidence of viral replication by detec~on of the p24 viral antigen. Substantial levels of tilt/ 1 replication occurred in the control-treated ~ cultures, comparable to that observed in the untreated group. As shown, n-docosanol exhibited a dose-n~lafed inhibitory activity against HIV-1 in cultures of PHAlIL 2-stimulated human peripheral blood mononuclear cells:
*Trademark _26-Activity at the highest dose wes comparable to that observed with the very potent antivi<a) compound, phosphonofocmic acid (PFA).._ To determine whether rrdocosanoi 10 wL % cream (docosanol) was efficacious compared tao placebo for the topical treatment of episodes of acute FiSL, two identical clinic-initiated, double-blind, placebo s controlled studies were conducted at a total of 21 sites. Od~erwise healthy adults, with documented histories of HSL, were randomized to n~ocosanol or polyethylene glycol placebo and initiated therapy in the ptodrotne or etythema stage of an episode. Treatment was 5 times daily unti healing occurred (the crust iep off spontaneously or there was no longer evidence of an active lesion) with twice dally visits.
Each gram of n~iocosanol 10 wL % cream contained 100 rr~ niiocosanol formulated into a white, 10 non-greasy, moisturizing cream that was easily applied and readily disappeared into skin and mucous membranes. The composition included rr-docosanol 10.0 wt °h, sucrose stearate and sucrose distearate 5 wt. %, light mineral oil NF 8.0 wt. °Io, propylene glycol USP 5.0 wt.
%, benzyl alcohol NF 2.7 wt %, and purified water USP 69.3 wt. %. The composition is marketed under license from Avanir Pharmaceuticals under the tradename ABREVA~' by GIaxoSmithKline of Research Triangle Park, NC.
A placebo formulation I 5 lacking n-docosanol but containing PEG provided a medication similar in appearance to rr~locosanol 10 wt.
cream. The PEG formulation was identical to chat utilized previously as a vehicle for t~ical acyclovir and as a placebo for topical HSL trials and was chosen in consultation with FDA. (See Spruance SL, Wenerstrom G.
Oral Surg. 58:667-71, 1984; Spruance SL, Schipper LE, Overall JC, et al. J.
lnlect. Dis.146:85-90,1982; and Fidctian AP, Ivanyi L. tint J. DermatoL 109:321-6, 1983). in this instance it was not p~sible to use the 20 vehicle of the cream as placebo, because the active drug substance, rHdocosanol at a 10 wt. °lo concentration, is a major contributor to the consistency of the cream.
Removing it produces a watery vehicle clearly unsurtabie as a control for a blinded study.
Patients were recruited at twenty one sites including university clirircs, private practices, and public health facilities across the U.S. Eight sftes were assigned to study #O(i and thirteen sites were assigned to 25 study #47. All sites were included in the combined study, designated ~06I07. No single site enrolled more than twelve percent of the total study population in the combined study or more than twenty tour percent in the individual studies. These sites recruited mace and female immunocompetent patients 18 years of age or older who presented for clinical assessment within 12 hours of noticing the onset of prodrome or erythema.
By patient history, signs and symptoms must not have been present for more than 12 hours, and on clirmcal 30 examination, the episode must not have progressed beyond the ery<hema stage. Patients, detertnfned to be healthy otherwise, must have had a clinical history of HSL with at least two recurrences during tin; past 12 mon>hs. The most recent previous episode must have healed at least 14 days prior io screening. Institutional Review Board approval for all sites was obtained for the protocol and the informed consent document. All patients were properly informed of the study purpose and risks and a signed consent form was obtained prior 35 to their enrollment.

Subjects agreed not to use cosmetics on or around the mouth during the treatment period. Women - of childbearing potential were to be practicing an established method of birthcontrol and were not to be pregnant as determined by a negative urine test at enrollment Subjects with known allergies to topical cosmetics were excluded as were those with lesions above the pares, below the chin, or inside the mouth.
5 The use of any investigational drug during or within 30 days prior to the study and the use of an approved antiviral agent, topical corticosteroid, or any other non-specific therapy for HSL during or within seven days prior to the study were not allowed. Concomitant use of systemic corticosteroids or other drugs known to induce immune stimulation or Immune suppression was also not allowed.
The study was a mult3center; randomized, double-blind, placebo-controlled, parallel group, clinic 10 initiated, early-treatment study to compare and evaluate the safety, efficacy, and tolerance of topical n-docosanol with a placebo in a population of patients with acute recurrences of HSL. Treatment was initiated within 12 hours of episode onset with symptoms in the prodrome or erythema stage and prior to the papule stage. Subjects were randomized in a double-blind fashion by site in blocks of four to receive either n-docosanol or placebo treatment. At study entry, the first application of study medication was to be made by I S the subject at the clinic. Subsequent applications were to be made by the subject during normal waking hours., Study medication was to be applied to the lesion area five times per day until healing for a maximum of 10 days. Subjects were instructed to re-apply study medication after heavy exercise, showering, or bathing. These extra applications were not counted as scheduled. Subjects kept a daily diary of study medication application times.
20 Subjects were required to report for twice daily assessments by the investigator or other trained clinician for the first seven days. Clinic visits could not be closer together than 6 hours or longer apart than 16 hours. The initial treatment area was marked on a diagram in the case report form (CRF) at the baseline clinical assessment Localized signs and symptoms at the treatment area were documented at each visit, including prodromelerytherna, papule, vesicle, user, crust, or healed skin (with or without residual erylhema), 25 and subject reports of pain, burring, itching, or tingling. Subjects with HSL episodes that did not abort or heal within seven days were also followed once per day for Days 8 to 10. HSL
episodes that did not abort or heal within 10 days discontinued treatment and were again assessed at the point of lesion abortion, healing, or adverse experience. All baseline and efficacy and safety parameters were clinician~ietermined.
The primary efficacy endpoint (time-to-healing) was calculated from the date and time of therapy 30 initiation until the date and time of the cliroc visit at which complete resolution of all local signs and symptoms was documented, i.e., the lesion had aborted or complete healing had occurred (censored at Day 10), thereby including patients both with classical episodes and with aborted episodes.
(The time of the Gnat Day 10 visit was used for primary endpoint analysis in subjects censored at Day 10.) For patients experiencing classical episodes, complete healing was defined as "the absence of crust, with no evidence of active Lesion, whether or not there were any residual post-lesion skin changes which might include erythema, flaking, or slight a~metry~ _ _ Secondary endpoints included the time from treatment initiation bo 1} complete healing of classical episodes (episodes which progressed to the vesicular or later stages; censored at Day 10); 2) e~sode 5 abortion, 3) complete cessation of pain; and 4) the proportion of aborted episodes, defined as episodes which did not progress beyond the papule stage. Abated episodes were considered healed at the time of the clinic visit where cessation of HSL-related signs or symptoms was reported.
Safety and tolerance of topical n-docosanol 10 wt. % cream were determined by adverse experience reports and assessment of clinical laboratory variables.
10 The sample size for the combined study was based on data from prior clinical studies. The combined study was planned to have 700 evaluable patients (350 per group), that would allow the detection of a 13-hour mean difference between treated and placebo groups with 82%
power. The two sub-studies were also analyzed separately.
Statistical methodologies were outlined in the protocol. The intent; to-treat (ITT) population Included 15 all patients who received medication and had at least one treatment evaluation. The efficacy evaluable population was protocol adherent and applied at least 809'0 of scheduled doses. Protocol deviations were evaluated prior to study unblinding. The safety evaluable population included aY those who use at least one application of study medication.
Demographic and medical history data were tabulated by treatment group and descriptive statistics 20 were used for continuous variables. Frequencies and proportions were used for categorical vat.
Baseline variables such as signs and symptoms, location of prodrome, current experience, and lesion stage were compared for homogeneity between randomized treatment groups using either ~alysis of variance or Cochran-Mante4-Haenszel tests. (See Agresti A. An introduction to categorical data analysis. New York:
Wiley 1996; pp. 60-d). Descriptive statistics for baseline vital signs were calculated.
25 For the primary efficacy analyses all patients who had at least one post baseline efficacy assessment were included. Time-to-event distributions were estimated by Kaplan-Meter product-limit estimates. (Kaplan EL, Meter P. J. Am. Stat. Assoc. 53:457-81, 1958). Time-to-eveni dlsMbuGons were oanpared be#ween treatments ushg the Gehan generalization of the Wiicoxon test, stralifled by site. (Gehan E.A Biometrika 52:203-23, 1965). In consultation with FDA, the Generalized Wi~oxon test was chosen 30 because it has good power when the effects of treatment are expec~d early in the treatment period.
Confidence intervals (hours of difference) were obtained by numerical inversion of the stratified Wilcoxon test Lesion assessments from participants whose Lesions were unhealed at 10 days were censored at that point The percentage of aborted episodes is presented by latest stage at baseline visit.
Possible adjustment for Important baseline covariabes was identified in the protocol. Because tire 35 Generalized Wlcoxon test does not readily allow for adjustments for covariates, proportional hazards regression (Cox regression) was used as a means of gauging whether covariate adjustment would have an effect on the p-value for treatment. All p-values reported represent the unadjusted analysis.
In the combined study, seven hundred forty-three subjects were randomized at twenty one U.S.
sites. Three hundred seventy-three individuals were randomized to receive n-docosanol while tluse hundred 5 seventy were randomized to receive placebo, Three n~Jocosanol-treated and three placebo-treated patients (0.8°~ of the study population) did not return to the clinic afker the irwtial visit. These six patients were included in the safety analysis, however, per protocol design, they were excluded from the intent-to-treat efficacy population. The efficacy evaluable population was nearly identical to the intent-to-treat population - 97.4%
of randomized patients were efficacy evaluable. As such, only the data from the 1TT population are discussed.
In sub-study #06 eight sites randomized three hundred seventy patients, one hundred eighty-five to n~ocosano! and one hundred eighty-flue to placebo. In sutrstudy #07 thirteen si0es randomized three hundred seventy three patients,188 to n-docosanol and to placebo.
Patient demographic and baseline characteristics for the ITT populaflon of the combined study are 15 presented in Table 7. The demographics of the individual studies were similar arid are not shown. There were no significant differences between treatments in race, age, orirequency of HSL recurrences. The mean age of study patients was 37 years with a range of 18 to 80 years. Minor gender differences were identified.
The majority of study participants were female and Caucasian, however, males comprised a smaller proportion of the n-docosanol recipients compared to placebo recipients (25°r6 versus 33%, respectively;
20 p=0.01). At enrollment all recurrent episodes were less than 12 hours in duration. Between 75 and 80% of patients presented for treatment with erythema, with the remainder presenting with prodrome only. This distribution was similar in both treatment groups (See also Table 10). Pain reported at baseline also did not differ between treatment groups.
Past experience with HSL as obtained by patient report at the baseline visit is also surtunarized in 25 Table 7 for the combined study. Between treatment groups there were no statistically significant differences in the time since first onset of HSL or the time since the last HSL episode, the number of episodes ar the previous year, the proportion of participants who usually experience localized prodrome or the duration of the most recent HSL episode. n-Docosanoi recipients, however, reported a bnger historical mean episode duration compared with placebo recipients (9.5 versus 8.4 days, respectively;
p=0.02). This statistical 30 difference was also observed in study #O6 (10.1 days and 8.4 days, respectively; p=0.01). The mean duration of the most recent previous episode (10.0 versus 8.4 days; p=0.02, n-docosanol versus piacet~o) was also statistically different in study #06. No treatment group differences in HSL
history were observed in study #07.
Where statistical differences were observed in study demographics between treatment groups, Cox regression analysis was utilized to assess the covarfate affect.

This was an experienced HSL population. Participants reported a median of flue episodes in the past 12 months with a mean HSL history greater than 20 years. More than 99% of participants reported that they rbrmally experience prodromal symptoms prior to their HSL ep'~sodes.
The mean number of applications for the n~docosanol group was 24.1 and the mean number for the placebo group was 25.7. Treatment compliance was assessed by comparing the number of applications actually made to the number that should have been made and averaged 99.2% in the n~iOCOSar~ol group and 98.6% in the placebo groups. There were no statistically significant differences between treatment groups with respell to the number of applications or Compliance.

PATIENT CHARACTERISTICS AND HISTORICAL INFORMATION FOR ITT POPULATION COMBINED

Docosanol placebo Parameter _. N=370 N=367 P-v~ue' Gender - -0.007 Male 91 (24.6%)122 (33.2%~

Female 279 75.4% 245 66 8%

Race , NSb Caucasian 348 (94.1%)345 (94.0%) Black 10 (2.7%) 13 (3.5%) ' 2 (0.5%) 1 (0.3%) Hispanic 8 (2.2%) 4 (1.1%) Age (year;) 2 0.5% 4 1.1 %

NS
N

Mean (SD) 37.2 (12.8)37.4 Ran (13.4) Stage of Lesion at Baseline 80 NS
Prodrome 71 (19.2%)80 (21.8%) E hems 299 80.8% 287 78.2%
Average Episode Duration from 0 Patient History (days) 016 N 370 367 .
Mean (SD) 9.5 (4.2) 8.4 (3.7) R

Duration of Most Recent Previous NS
Episode (days) Mean (SD) 9.1 (5.0) 8.2 (4.2) Ran a 1- 60 1. 30 Time since Last Onset of Oral-Facial Herpes Simplex NS
(months) Mean (SD) 3.0 (2.2) 3.0 (22) Time since First Onset of Oral-Facial Herpes Simplex NS
(years) Mean (SD) 22.4 (13.8)21.4 Ran a (13.2) Number of Episodes In Past 12 Months NS
N

Mean (SD) 5.2 (3.7) 5.1 (3.1) Ran a Does Patient Experience Localized Prodrome?

NS
No 3 (0.8%) 1 (0.3%) Yes 367 99.2~6366 99.7%
aP-varno frfr r~lnnnr....,i ....

~~b~~w~ ra~p~~~G~o~~ uvrn ~ocnran.mante~-Haenszel test adjusted for site. P-value for continuous,parameters from analysis of variance model with effects for treatment, site, and site-by-treatment interaction.
"Not significant Efficacy data are summarized for both the combined study and each sub-study in Table 8: Only the combined study results are discussed )n the text. The vast majority of participants healed during the l0~ay treatment period (91% of n~docosanol recipients and 90°~ of placebo recipients). Kaptan-Meier curves for times to healing are displayed in Figure 14. The median time-to-complete-healing for all lesions was 4.08 days for n~ocosanol recipients versus 4.80 days for placebo recipients, a difference of 15% (p=0.008; 95%
CI 2, 22 h). The distribution of healing times also favored n~Jocosanol treatment at the 25th and 75th percentiles.
Covariate adjustment utilizing proportional hazards regression for.
differences in the number of males had no affect on the p~value for time-to-heallng; however, for historical episode duration the p-value decreased (i.e., became more significant).

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h Approximately 60 to 659'0 of subjects developed classical episodes. The difference in time-to-healing (Table 8~ was statisticaNy shorter in the n-docosanol-versus the placebo-treated groups (p=O.Q2; 95% CI 1, 24.5 h). For this endpoint, larger differences were observed at the 25th and 75th peroentiles (-19 h) than et the median (1 h).
Values for the time-to-cessation of individual lesion stages for classical episodes are displayed in Table 9. The median for time-to-cessaflon of vesicles was approximately 2.1 days and the median (or time-to-cessation of hard crusts was approximately 5.8 days. Neither was statistically different between treatment 8roups.
However, the median time-to-cessation of the ulcerlsoff crust stage was shorter in the n-docosanol group (3.61 versus 3.94 days; p<O.OOi; 95% Ci 8, 25 h).

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G. 'S ~ o '~ ~ o ~ Q-A total of seven hundred five {96°~) of the seven hundred thirty seven patienks in the ITT group, equally distributed between placebo and n~ocosanol-treated populations, experienced lesion pain andlor burning, itching, or tingling during the study. Median times to complete cessation of pain andJa burning, Itching, or tinglfig tOr all participants (Table 9) was 2.18 days for n-docosanol recipients versus 2.74 days !or placebo recipients (approximately 20% reduction; p=0.002; Cf 3,16.5 h).
Results for patients with aborted episodes by stage at baseline are summarized ~ Table 10. For all subjects, a trend (not statistically different) toward more aborted episodes was identified with 39.7% n-docosanol recipients experiencing aborted episodes versus 34.1 % placebo recipients (p =0.109; CI for odds ratio 0.95,1.73).
For sub-study #06, ~ subjects who began treatment with erylhema, 34.3% of n-docosanol recipients versus 23.3% of placebo recipients (p=0.048; CI 1.00, 2.75) experienced aborted episodes. The times to episode abortion were rapid and not different between treatment groups.

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cn ~ ~ a Adverse experiences were quantitatively and qualitatively similar between n-docosand-treated and placebo-treated patients. At least one adverse experience was reported by 19.6% (731373) of n~ocosanol recipients and 18.9% (70!370) of placebo recipients for the combined study population. Headache, which was reported by 5.9% of patients in each treatment group, was the most common adverse experience. With the exception of application site reaction (2.1 % of the n~docosanol group and 1.9°h of the placebo group) and herpes simplex outside of the treatment area (2.49'° of the n~~docosanol group and 1.4°~ of the placebo group), all adverse experiences were reported by less than 29'0 of the patients in either treatment group. Two patients, (one patient in each group) were withdrawn from the study due to adverse experiences of rash and herpes simplex outside the treatment area, respectively. There were no statistically significant differences between treatment groups with respect to change from baseline in either hematology or clinical chemistry parameters.
This trial with n~ocosanol 10% cream demonstrates clinical efficacy of early clinic-Initiated therapy of recurrent HSL. The combined study analysis showed statistically significant reductions In time-to-complete-healing, time to-complet~healing of classical episodes, cessation of the most active infectious lesion stage (u~erlsoft crust) and cessation of all HSV symptoms. Median time-io-healing was the primary efficacy parameter and was reduced by 0.72 day compared with placebo. The times-to-healing of classical lesions and the times-to-cessation of ulcerlsoft crust were also significantly reduced. The ulcedsoft crust stage represents the peak period of viral replication and inflammation, which may explain its sensitivity of response.
The statistical differences idenlfied in the individual sub-studies {#06 and #a7) were slightly less robust than in the combined study, reflective of fewer participants. The sub-studies were similar in treatment effects to the combined study and to each other. Consistency of the results across the sub-studies was analyzed utilizing various methods of analysis and measures of effect including Proportional Odds Regression, Proportional Hazards Regression, and Log-Logistic Regression models (results not shown) in addition to the Generalized Wilcoxon reported here. The estimated treatment effects are very similar regardless of the measure of effect used. Furthermore, confidence Intervals for treatment effects computed are almost completely overlapping.
The combined analysis approach for the sub-studies was planned by the protocol. The two studies combined represented a cohort size approximately half that reported for each of two topical penciclovir cream studies in HSL; nevertheless, the studies demonstrated clinical and statistical significance for n~ocosanol against both the healing and symptom components of HSL. (For a discussion of topical penciclovir cream studies in HSL, see Spruance SK, Rea TL, Thoming C, Tucker R, Saltzman R, Boon R JAMA 277:1374-9,1997; and Rabom GW
36th lnferscience Conference on Antimicrobiai Agents and Chemotherapy, New Orleans, 1996). Using this early, clinic-initiated model with twice daily observations, a cohort of 700 to 800 patients appears sufficient to demonstrate these key efficacy components of HSL treatment In contrast, demonstration of lesion prevention may require a larger patient population. Despite the interesting trends in favor of n-docosanol treatment, this study was not sufficiently powered to demonstrate lesion prevention at the rates observed and, unfortunately, to date, lesion prevention has never been unequivocally demonstrated.
Nevertheless, clinic-initiated treatment prior to lesion onset clearly offers the potential for demonstration of this treatment benefit (given the proper cohort size) where if exists. No other reported study design truly provides the opportunity to demonstrate such effects since a high proportion of patier>ts who seN-Inlf~ate therapy In the prndromal phase may actuaDy have eariy, established lesions prior to commencement of therapy. (Spruance SL, Overal JC, Kem E, Krueger GG, Pram V, Miller W
New Engl. J. Med. 297:69-75,1997; and Spruance SL Semin. ~ Dermatd. 11:200-6,1992).
Penciclovir cream 1% is curnently available by prescription for the topical treatment of recurrent herpes simplex labialis. Based on infomration from the product insert for penciclovir cream, In the US multlcenter study more than twice the size of the current study, Spruance ef al. demonstrated that penciclovir-heated patients experienced a significantly shorter mean time to healing with a 0.5 day difference (4.5 versus 5.0 days; p<0.001).
(See Spruance SK, Rea TL, Thoming C, Tucker R, Saltzman R, Boon R JAMA
277:1374-9, 1991). Lesion pain was reduced, as demonstrated by an approximately half-day reduction in the mean duration of lesion pain (3.9 versus 4.4 days; p<0.001). Spruance et al, reported viral shedding was reduced by penciclovlr as demonshated by changes over the shedding period followed (vesicle and ulcer/soft crust), although no differences In median times to loss of viral shedding were observed (3.0 versus 3.0 days). The difficulty In demonstrating an antiviral effect with penciclovir cream, given the large numbers of subjects tested, suggests that viral cultures must be aggressively obtained 3n order to make this the sensitive ethcacy marker It has been in studies of herpes genitalis.
(See Diaz-Mitoma F, Ruben M, Sacks SL, MacPhersom P, Caissie G. J. Clip.
MicrobioL 34:657-63,199fi; Sacks SL, Aoki FY, Diaz-Mitoma F, Sellors J, Shafran SD JAMA 276:44-9, 1996; and Sacks SL, Tyrrell DL, Lawee D, Schlech W, Gill MJ, Aoki FY, ef al. J. Intel. Dis. 164:665-72,1991).
Aggressive viral culturing has often not been pursued In HSL because of a possible effect on delaying healing, which may, in tum, contrrbute to the lade of sensitivity of this parameter in HSL studies. Accordingly, viral cultures were not perfomred in the current studies.
As observed in the penciclovir studies, the placebo treated time of about 5 days in these studies is shorter than the reported natural history of HSL lesion healing of 7 to 10 days. (Spruance SL, Overal JC, Kem E, Krueger GG, Pliam V, Miller W New EngL J. Med. 297:69-75, 1997; Spruance SL.
Semln. In DermatoL 11:200-6, 1992; and Shafran SD, Sacks SL, Aoki FY, Tyrrell DL, Schlech WF 3~, tufendelson J, Rosenthal D, et al. J. lnled.
Dis. 176:7ti-83, 1997). This raises suspicions of a placebo effect, which has treen well recognized in HSL.
(Spruance SL In: Clinical management of herpes viruses, Sacks SL, Straus SE, Whitley RJ, Griffiths PD, editors, Amsterdam; IOS Press, p. 3-42, 1995; and Guinan ME, MacCalman J, Kem ER, Overall JC Jr., Spruance SL
JAMA 243:1059-61,1980). Placebo effects often occur with dermatological products, resulting not only from the psychological effects typically associated with placebo-treatment, but also due to simply covering the lesion which itself alters the physiology of untreated skin. (Placebo effects are discussed In Chaput de Saintonge DM, Herxheimer A Lancet 344:995-$,1994).
Although the effect of n-docosanol 10 °~ craam In HSL may appear modest, the self-limiting nature of the disease makes decreased duration of almost a day (18 h) significant to patients. Additionally, the apparent magnitude of the clinical effect may be lessened by what appears to be a substantial placebo effect in the treatment of HSL, as discussed above. Reduced healing time is accompanied by relief of pain andlor burning, itching or tingling, also important to patients. The time of the most severe stage (u~edsoft crust) of the !es'ron is significantly reduced, a medically important effect that has not been reported previously. Its approval as an over the-counter (OTC) product allows it to be applied early in the course of an episode where it is rtwst likely to be effective.
Docosanol appears to inhibit viral entry Into host cells by inhibiting the normal process of viral fusion with the cell's plasma membrane thus blocking enUy and subsequently limiting viral replication, n-Docosanol and its metatwlites do not interact directly with viral proteins or nucleic acids.
AccoMingly, the emergence of dnrg-resistant HSV is uni~cely. Because of the different mode of action tiom antivlral nucleosides, resistance to n-docosanol would not diminish the effectiveness of other topical or systemic antivirals, even if it were shown to exist. In addition, the unique mechanism of action suggests that combination therapy with antiviral nucleosides is worthy of consideration.
In summary, n~docosanol 10 % cream was shown to be effective in this clinic-initiated, placebo controlled, clinical trial in early HSi_. This treatment reduced episode duration overall, duration of those episodes that developed into classical lesions, and the duration of all lesion symptoms. Based on these studies, treatment with n-docosanol 10 wt % cream should be initiated as early as possible in the course of HSL.
A study was conducted to examine the in vivo efficacy of 1096 docosanol in two formulations using guinea pig models. Clinical trials have demonstrated efficacy of docosanol 10%
cream ('doc') in the treatment of herpes simplex labialis (HSL) leading to FDA approval in July 2000. Docosanol exhibits antiviral activity in vitro, but inconsistent results have been reported in animal HSV models.
Hairless and Hartley (shaved and treated with Nair ) guinea pigs were inoculated at 6-8 sites on the back with HSV-1 (3 x 10 PFU Maclntyre or KOS) or HSV-2 (2 x 10 PFU MC) with an electric tattoo marker or by scratching with a 20 gauge needle. Treatments with doc or vehicles ('veh') began 12 hours later and continued 3-4 times daily. At the peak of viral liter, animals were sacrificed, lesions removed and virus titer determined by assessing cytopathic effect of homogenates in Vero cells.
In hairless guinea pigs antiviral activity of doc against HSV-1 and HSV-2 was shown by reduced veslde number following inoculation with a tattoo marker {e.g., for HSV-2 mean veh =
21.4 t 2.4 vs mean doc = 12.7 t 1.9; p<0.01) and reduced viral titers (e.g., for HSV-1 mean log veh = 4.0 vs mean log doc = 3.(i; p<0.001 and for HSV-2 mean log veh = 5.84 vs mean log doc = 4.84; p=0.002) for both inoculation methods. There was no reduction in lesion score or size following inoculation by scarificaGon. In Hardey guinea pigs HSV-1 and HSV-2 vesicle numt~ers were inhibited (e.g., HSV-2 mean bg veh = 26.7 t 8 vs mean log doc = 7.7 t 4.8; p<0.002).
Inhibition of viral titer was less than in hairless animals but was statistically significant in some experiments (HSV-1 mean log veh = 6.38 vs mean log doc = 5.79; p<0.001 and for HSV-2 mean log veh = 5.47 vs mean log doc =

5.07; p=0.06). Formulation differences were noted.
Doc reduces HSV-1 and HSV-2 viral tilers in hairless guinea pigs and vesicle numbers in both hairless and Hartley guinea pigs. The difference in efficacy in the two models may explain previous varying results with doc in animal models despite its demonstrated clinical efficacy. The hairless model, including inoculation with a tattoo gun, may be a better model of clinical HSV infections.
As discussed above, n-docosanol is a saturated 22-carbon primary alcohol that inhibits HSV replication in tissue culture. See, e.g., Katr et al., 'Antiviral activity of 1-docosanol, an inhibitor of Ilpid-enveloped viruses including herpes simplex," Pnx. Nafi: Acad. Sci. (1991) 88:10825-9; and Pope et al., 'The anti-herpes simplex virus activity of n-docosanol includes inhibition of the viral entry process,"
Ant'ivir. Res. 40:85-94 (1998). It has also been demonstrated to shorten the duration of disease in experimental animals. See Marcellettl et al., "Docosanol' 17:879-82 (1992). Clinical trials have demonstrated efficacy of docosanol 10°~ cream (doc) in the treatment of herpes simplex labialis. See Habbema et al., "n-Docosanol 10%
cream in the treatment of recurrent herpes labialis; Acta i7erm. VenereoJ. 76:479-81 (1996). In a large double-blind study, the median time-to-healing in the three hundred seventy docosanol-treated patients was 4.1 days, 18 hours shorter than observed in the 367 placebo-treated patients (p=0.008). See Sacks, et al., 'Clinical efficacy of topical docosanol 10°~ cream for herpes simplex IabiaGs: A multicenter, randomized, placebo-controlled trial; J. Amer. Acad. Dorm. 45:222-30 (2001). The docosanol group also exhibited reduced times from treatment initiation to: 1) cessation of pain and all other symptoms (itching, burning andlor tingling, p=0.002); 2) complete healing of classical lesions (p=0.023); and 3) cessation of the ulcerlsoft crust stage of classical lesions (p<0.001). FDA
approved docosanol 10% cream as an over-the counter treatment for cold sores in July 2400. Docosanol inhibits In vitro a broad spectrum of Ilpfd-enveloped viruses including HSV-1 and HSV-2, cytomegalovirus, varicella zoster virus and human herpes virus.
Data suggest that after ceGular incorporation and metabolic conversion, docosanol inhibits v'rcal entry by inhibiting viral fusion with the host ceG, blocking nuclear localization and subsequent replication of virus. See Pope et al., 'AnU-herpes simplex virus activity of n-docosanol correlates with intracellular metabol~ conversion of the drug," J.
Cipid Res. 77:2167-78 (1996). This mechanism of action is different from that of other avaBable treatment options for herpes infections, where antiviral activity results from inhibition of DNA
synthesis. See Elion, 'Acyclovir:
discovery, mechanism of action, and selectivity; J. Met. Virol.1:2-6 (1992).

Docosanol and acyclovir were prepared in two types of formulations: a cream fonnulatlon and a polyethylene glycol-(PEG) based ointment. The compositions of both formulations are Ilsted in Table 11s (Composition of Docosanol and Acydovir Creams) and Table 11b (ComposiUon of Docosanol and Acyclovir in PEG).
Tab 11a InyradloM Formdaeon (x wVwj Doeoaanol Acyclovlr l/ohlc~ t0% Croam b7rr Cnata oocooanot o 10 0 Acycbvir 0 0 5 Cream axctplenb t00 90 95 Table 11b InErodbni Formulstlon ~5f, yvlwj PE6 Docoaanot Acydovlr Vehldo 10% In b% fn P~EO PFO

0ocosarbl 0 10 0 Acyclovlr 0 0 5 Hairless and Hartley guinea pigs (Crt:(HA)BR) were obtained from Charles River laboratories. They were quarantined 7 days before use and fed diet and water ad libihrm. The animals were individually caged and housed under strict pathogen-free conditions. Two strains of HSV-1 (Kos strain and Maclntyre strain) and the MS stain of HSV-2 were used. The virus was a cell culture preparation that had been pre-titered in guinea pigs prior to use in these experiments.
Prior to inoculation the haired guinea pigs were shaved with an electric razor, dampened with warts water, then Nair depilatory cream was applied for 3-4 minutes to remove the remaining hair. The backs of both hairless and haired animals were then washed with warm water and thoroughly dried. In Inoculation Method 1, the backs of guinea pigs were marked into a grid of 8 squares and within each area a 10 mm diameter lesion (wound) was induced by applying virus to the skin and scarifying the area with 10 light vertical and horizontal scratches using a 20 gauge inoculation needle. In Inoculation Method 2, a grid of six squares was drawn with a marking pen.

Each square was inoculated with 50-75 I volume of virus with an electric tattoo gun (Spaulding and Rogers, Inc., Voorheesville, NY). The instrument was triggered 80 times at each inoculation site with the dial set at 17.
With Inoculation Method 1 (scarification) the length and width of each lesion (wound) is measured, and the lesion is assigned a score daily that ranges from 0 (normal) to 4 (maximal). These measurements were made S up until the time of sacrifice on Day 4 {Hariley guinea pig). Vesicles may form within the lesion, but these were not counted. With the tattoo inoculation method {Method 2), discrete vesicles are formed, and there is no wound between the vesicles. With this inoculation method vesicles were counted and recorded, and the total involved area was not determined.
Animals were sacr~ced at the peak of viral titer. Skin containing each lesion was removed from the sacrificed animals and homogenized in an approximately 10% wlv suspension in MEM con#aining 2% FBS, 0.180 NaHCOa, and 50 glml gentamicin. Serial dilutions were assayed in triplicate wells in 96-weQs plates containing a 24-h monolayer of Vero cells. The plates were sealed, incubated for 7 days at 37°C, and then examined under a microscope for discernible viral cytopathic effect.
For the experiment described in Table 12, Students t-test was used to compare mean lesion size and mean lesion virus titers. Ranked sum analysis was used to evaluate lesion score. For all other experiments one-way Analyses of Variance (ANOVA) for treatment as the factor were performed separately for each study. If the ANOVA was significant, Then the least squares (LS) means for lesion viral titers and vesicle numbers were calculated and unadjusted multiple comparisons were performed testing for differences in these means for al( pairs of treatments.
T ba le 12 Effect of Topical Therapies on HSV-1 Induced Lesions in Hairless Guinea Pigs Mean Moan Alleen Tresttnentt.edon t~slon Yaus Scare 8ise Tltsr * * SO Day 4 SD b I *

D

sox Docosenol (PEG) 0.70.30.8,x,0.932.9=14.540.818.43.712"

lox Docoeend /C~eant) 0.8 0.8 34.2 43.3 38 t 1.3"
r 0.3 18.1 18.3 0.3 5x AcycloWr fPEG) 0.80.30.810.325.813.635.015.74.01.1"

S~ Acyclovir (cream) 0.4 0.4 13.2 13.6 3.1 0.8""' ~0.2 t 0.2 10.3'"' t 9.0"'"
~' CreamVehtcle~0.5020.510.324,011.529.3=15.74.9=0.8 PEG Vehicle0.7;020.810.333.7114.838.0,+r13.74.70.8 Untreated0.8020.8 28.2=9.538.8114.15.00.7 X0.3 'Od x 4 be~lnNng 12 h poet virus exposure 'P<0.05 "P<0.01 ""P<0.001 ocmpared to appraprlate placebo 'P<0.05 "P<0.01 "'P<0.001 compared to untreated controls The formulations prepared are listed In Tables 11a and 11b above. All samples were subjected to analytical testing prior to experimental use. The cream formulation with docosanol is a white, odorless, non-staining and non-water soluble cream. In the abserx;e of docosanol, the cream vehicle and 59b acyclovir in cream vehicle have watery, lotion-like consistencies. The PEG vehicle is a clear, water-soluble ointment that becomes white in formulations containing docosanol and acyclovir.
The formulations listed in Tables 11a and 11b were evaluated for efficacy In the treatment of cutaneous lesions induced by HSV-1 in hairless models using Inoculation Method 1 (scarification). Topical treatment began IO 12 hours later and continued every 8 hours for a total 10 treatments.
Lesion size and severity were assessed on Day 2 and Day 3 of infection. On Day 4 each lesion was exdsed and assayed for viral content.
The results are summarized in Table 12. Lesion size and score were not inhibited by docosanol in cream or ointment or by acyclovir ointment. It has been reported that greater inhibition of lesion size and severity is generally observed if treatment Is continued past Day 4, and since guinea pigs in this study were sacrificed on Day 4 for determination of viral content, it was not unexpected that effects on lesion size and severity were not observed. The virus titer reduction data indicated that docosanol treatment in both vehicles reduced the mean virus titedgram by approximately 1 logo when compared to the untreated control mean. This difference was statistically significant (p< 0.01}. Docosanol in PEG reduced the viral titer by 1.0 logo and acyclovir in PEG
reduced the viral titer by 0.7 logo. The differences between acyclovir and docosanol were not statistically sign~cant.
Based on the results in Table 12, and because the PEG vehicle is similar in consistency to that of docosanol in PEG, PEG formulations were selected for further study. The results of tests in hairless guinea pig with inoculation of virus with Method 2 are illustrated in Figure 15. Viral titer levels were statisticany significantly reduced compared to vehicle treated sites (mean logso = 4.0) by docosanol (mean logo = 3.5) and acyclovir (mean kyo = 3.0).
Hairless guinea pigs were inoculated with the Maclntyre strain HSV-1 (60 uJ 5 x 10 PFUImI) with tattoo inoculation on each of 8 sites on the dorsal surface. Treatments were started twelve hours post Inoculation and were repeated 3 times per day for 3 days. Lesion skin was collected on Day 4, at 12 hours after the last treatment and assayed for viral content. The treatment groups were doc = 1096 docosanol in PEG (10 sites), acy = 5°~
acyclovir in PEG (10 sites), veh = PEG vehicle (10 sites), and none ~= no treatment (8 sites).
Similar observations were made following inoculation of hairless guinea pigs with HSV-2 (MS strain).
HSV-2 viral titer levels were statistically significantly reduced compared to PEG vehicle treated sites (mean logsa =
5.8) by a topical treatment with docosanol in PEG (mean logo = 5.2) and acyclovlr in PEG (mean logro = 5.0).
Viral tilers were reduced 7596 (docosanol) and 78% (acyclovir) following topical treatment (Figure 16). The numbers of vesicleJsite on Day 3 and Day 4 after infection were also assessed and are shown in Figure 17.
Hairless guinea pigs were inoculated with the MS strain of HSV-2 {60 ~I 1 x 10 PFUIm~ ut~izing a tattoo gun as described in Materials and Methods on each of 6 sites on the dorsal surface. Treatments started twelve hours post inoculation and were repeated every 8 hours for 3 days. Vesicle numbers were counted on Days 3 and 4. Lesion skin was collected on Day 4, 12 hours after the last treatment and assayed for viral content. All treatments were applied to 9 sites except that only 3 sites received no treatment.
The disease duration in the hairless guinea pig models is 4-5 days after inoculation with virus. The duration of disease with the haired guinea pig is 8-9 days. The longer disease duration provides two advantages:
1) it better represents the disease course in humans of 8 to 10 days for herpes labiaf~s and 7 to 10 days for herpes genitaf~s in both men and women and 2) it provides a larger window to observe a therapeutic effect. See Spruance 'The natural history of recurrent oral-fadal herpes simplex virus infection,"
Semin. Dermatol. 11:200-6 {1992);
Whitley et al. "Herpes simplex virus infections," >_ar~cet 357:1513-18 {2001).
The model has the drawback, 2S however, in that Nair treatment, followed by shaving irritates the skin, thus exacerbating any sensitivity to the applied formulations. Cream formulation vehicle resulted in severe irritat'ron in iha Hariley guinea pig model mak(ng It impossible to Interpret results of doco~anol cream treatment to the apprti~riate veh~le. This IMtatlon does dot occur in the hairless model. _'' ' ' ' . , Inoculation of HSV-2 with tattoo gun re5elts In the development of discrete lesions that evolve over,tlme ., ..,,; , , ;
up uritaf Day 6 with complete resolution by Day .9, ~In the treatment studies, animals~were sacr~ced on Day 6 for ~f; :..
determinaGop_,of peak viral titer levels. Vesicle numbers were recorded up until the time of sacrifice. Tueatment begari f2 hours after inoculation and was repeated four times per day on Day 1 through Day 3 and three times per day on Day 4 and Day 5 for a total of 19 treatments. Skin samples were collected on Day 6 fior analysis of virus titers. Mean viral titer per lesion is shown in Figure 18 far each treatment. Statistical infom~ation is summarized in the labia below the figure. Vesicle numbers observed on Day 3 through Day 5 are shown in Figure 19, which produced the same pattern of results as lesion viral titers.
Hartley guinea pigs were inoculated with the MS strain of HSV-2 (60 ~I 2.9 x 10 PF(Jlml) utilizing a tattoo gun as described in Materials and Methods on each of 6 sites on the dorsal surface. Treatments started twelve hours post inoculation and were repeated 4 timeslday for 3 days and three tunes a day for two days. Vesicle numbers were counted on Days 3, 4, and 5. Lesion skin was collected on Day 6, at 12 hours a(ier the last treatment and assayed for viral content. Each treatment was applied to 9 sites.
Previous studies with docosanol had not included viral titer measurements, but rather focused on decreased disease duration as assessed by counting the number of vesicles until healing occ~ned. The fas6er healing times observed in the earlier studies could have resulted from mechanisms of actions not related to an antiviral activity. The results from this study established that docosanol-containing formulations result in decreased viral content in the skin of guinea pigs Infected with HSV-1 and HSV-2 in both the hairless (compared to vehicle or untreated sites) and Hartley guinea pig models (compared to untreated sites). The inhit~tion observed is approximately equivalent to that observed with acyclovir and statistically significant differences between the two treatments were not generally observed.
The anti-HSV activity of a topically applied compound is highly dependent upon the topical vehicle used.
Sae Sidwell et al., "Effect of vidarabine in DMSO vehicle on type 1 herpesvirus-induced cutaneous lesions in laboratory animals," Chemofher. 33:141-50 (198. It appears that both PEG and cream vehicle worked relatively 20 well for delivery of docosanol and acyclovir, although other vehicles could potentially enhance the antiviral activity.
The differing results in the two models may be a result of the imtation induced in the chemicauy depilated and shaved skin. Irritation induces inflammation that may alter the healing rate. Decreased viral titer levels were observed following docosanol treatment In hairless and Hartley guinea pigs, but statistical significance compared to vehicle treated sites was more reproducibly demonstrated in the hairless model. Decreased viral titer per lesion also corelated with decreased vesicle numbers although the magnitude of the effect was less when vesicle numbers were evaluated.
The hairless guinea pig model with iraculafion with a tattoo gun provided reproducible evidence of effectiveness of docosanol formulations in the treatment of cutaneously induced herpes lesions and provides more reproducible results than the Hartley guinea pig mode(. The results of this study estabf~sh that docosanol inhibits replication of HSV in these model systems to an extent approximately equivalent to that of acyclovlr ointment, suggesting that its efficacy in the treatment of cold sores may result from its antiviral actrvlty.
n-Docosanol formulated as n-docosanol 10% cream was studied for the topical treatment of herpes simplex infectious. Efficacy in reducing the healing time of recurrent oral-facial herpes simplex infections has been demonstrated in Phase II and in Phase f 11 placebo controlled r,~nical trials. Positive results vrere also obtained in a Phase III pilot study using n-docosanol 10°~ cream as. a topical treatment for cutaneous Kaki's sarcoma lesions in HIV-) positive patients. n-Dooosanol topical cream prevented vaginal transmission of SIVmac25t In rfiesus macaques, suggesting that the compound has antirnicrobial functior>s the may be useful as a prophylactic io prevent the transmission of HN iri humans.
n-Docosanot exhib~s antiviral ac8vlty in vitro against a wide range of lipid-enrreloped viruses.
Suscepiibie human viruses include HS111 and HSV 2 {including acydovlr resistant strains and cUnicai iso~tes), influenza A, respiratpry syncytial vinrs, cytomegalovirus, variceAa zoster virus, human herpesvirus 6 and HN-1.
The IDso values (concentration where 50% inhibition is observed) ranged from 3 to 12 mM ~r these susceptible viruses. Non-enveloped viruses and enveloped viruses that are endocytased have an apparent resistarrce'to the effects of n-docosanot. Far in vitro efficacy studies, the insoluble n-docosano) is formulated by suspending the molecule in the inert and non-toxic surfactant Pturonic*F-88, a block copolymer of polyethylene oxide aril polypropylene oxide, or a related molecule, Tetronic 908. The relatively high concentrations of trdornosanol required for in vitro activity maybe a resttit of the physiochemical nature of the surfactant-shabilized particles.
However, because n-docosanoi concentrat'rons as high as 300 mM are not cytotoxic, the theraper~ic index thr the drug is favorable.
Studies, generally conducted with HSV, demonstrated that n-docosarrol does not directly Inactivate virus since virus preparations can be mixed with the compound without loss of iMectivity. Instead, the drug apparently modifies the target cell in a manner chat inhibits viral replication. Studies have demonstrated omit radiolabeled n-docosanol is extensively Incorporated into host cells and metabolized to phosphoGpids with the chromatographic properties of phosphatidylcholine and phosphatidylethanolamlne: Furthermore, conditions that increase the amo<rnt of n-docosanol metabolism increase the amount of antivirai activity, suggesting that this intracellular metabolic conversion of the drug is requkeed for antfviral activity.
n-Docosanol inhibit HSV-induced plaque formation and production of viral part(des as Judged th a secondary plaque assay. It also inhrbtts, as determined by ELISA, the production of HSV core and envelope proteins and the ntnnber of cells expressing the intranudear HSV-I specific Immediate-early protein. These observations suggested that n-docosanot Interferes with an early step in HSV
hfection.
Studies were conducted to investigate the mechanism of action for the anti-HSV
activfty of n-docosanol utilizing (i) an HSV recombinant virus which expresses (i-galactosidase on entry of the viral genome into the nucleus of a susceptible host cell; {2) a hit cell transformed to express (3-gala~ctosidase upon entry of iiSV virion proteins into the ceA; and (3) HSV 2 fluerescentiy labeled with octadecyl rhodamine t3 chiortde.
n-Docosanoi {98% pure; M. Michel, New York) was suspended iii Tetronic 908 (poloxamine 908, Mw 25000; BASF; Parsippany, NJ) generally as follows. 7etronic~908 was diluted to 1.8 mM In 3TC sterile. satkre, and *Trademark ~g the solution was then heated to 50°C. n-Docosanol was added to 300 mM
to the Tetronic in saline and the mixture was sonicated (Branson 450 sonifier; Danbury, CT) for 21 min at an Initial output of 65 W; this warms the mixture to 86°C. The resulting suspension consists of very' fine globular particles with au averse size of 0.1 microns as measured by transmission electron microscopy.
S Heparin and NP-40 were obtained from Sigma (St. Louis, MO) and octadecyl rhodamine B from Molecular Probes (Eugene, OR). Anti~gD neutralizing monoclonal antibody (III-174) was generated. Plaque reduction assays were typically performed in Vero cells (African Green monkey kidney; ATCC no. CCL-81). The HEp-2 (human epidermoid carcinoma; ATCC na. CCL-23), cell line and NG37 human B cells (ATCC No.
CCL214) were obtained from the American Type Culture Collection. The CHO-IEp8 ceu line was developed. It 10 was selected by transfection of Chinese hamster ovary cells (CHO-KI; ATCC
no. CCL-61) with a plasmld carrying a puromycin (Pur) selectable marker and lacZ under control of the HSV-I ICP4 promoter. The cell line was selected In Pur and screened for expression of (i-galactosldase after HSV
Infection but not h the absence of infection.
The Maclntyre strain of HSV-I (VR-539) and the M5 strain of HSV-2 (VR-540) were obtained from the 15 American Type Culture Collection. HSV-2 (333), a wild-type strain, was obtained from Dr Fred Rapp. Stock preparations were titered for levels of plaque-forming units (PFU) in Vero cells and stored frozen at-80°C. HSV-I(KOS)gL86 is a replication-defective mutant in which the gL ORF is replaced with lacZ under control of the CMV
promoter. This mutant is propagated in gL-expressing Vero cells and is fully infectious but can undergo only one rourxi of replication in non-complementing cells.
20 Cultured cells were placed in 35-mm wells (2 ml; 3 x 105 cellslml) In DMEM
containing L-gtucamine, pennstrep.(cDMEM) and supplemented with 5% FCS. n-Docosanol or the corresponding control vehicle (lacking n-docosano!) was added at the outset of the culture. All cultures were then inoculated with 175 p.f.u. of HSV-1 or HSV-2.
The cultures were incubated for an additional 42-44 h, washod once with fresh medium, stained and 2S fixed (the stainingKxative consists of 1.25 mglml of carbol-fuchsin plus 2.5 mglml of methylene blue in methanol) and then scored for HSV-induced plaques using a dissecting microscope (10 x magnification). The data are averages of duplicate cultures, which varied by no more than 5-1096.
Twenty-four hours before infection, cultured cells were seeded into 24-well (16-mm) plates at 2.5 x 10g cellslwell in 0.5 ml cDMEM supplemented with 10% fetal bovine serum (FES).
After cell attachment (4-6 h later) 30 heparin, n-docosanol-surfactant, or surfactant alone was added to the cells fn 0.5 mi DMEM I 10°I° FES. The agents were dissolved in the medium at two times the desired final concentrafron. For infection, 0.7 ml of medium was removed from each well and 25 W of virus suspension was added to the remaining 0.3 ml to give a virus dose of at least 20 p.f,u.lcell The places were rocked at 37°C for 3 h and then put in a 37°C C0~ incubator for another 2-3 h.
At 5-6 h after infection, the cells were fixed with PES containing 2%
formaldehyde and 0.2%
glutaraldehyde, washed, then permeabilized with 0.02°~6 NP-00, 0.01 %
deoxycholate and 2 mM MgClz. After 5 washing again. 5-bromo-4~fibro-3indolyl-p-galactopyranoside (X-gad was added for development of blue product. The substrate was removed end replaced with 50% glycerol. Plates wero photographed. To quantNy the . amount of color in each well the glycerol was removed from all wells that were then washed 3 x with distilled H~.
DMSO (0.6 m~ was added to solubilize the dye and, after transferring 100 W of each sample well from the 24-well plate to a 96-well plate, the ODsoo was recorded using a 96-well plate reader.
10 The HSV envelope was labeled with octadecyl rhodamine B chloride (R-18). NC-37 human B cells were inoculated at 2,5 x 706 cellslml, 25 ml per flask. Cells were incubated overnight at 37°C with no addition or in the presence of 15 mM n-docosanol or the corresponding concentration of Tetronic 908, Cells were harvested by centrifugation and resuspended to 1x106 cellshnl. Aliquots (0.2 ml in test tubes) were chilled for 20 min at 4°C
before the addition of 100 ~I R-18 labeled HSV-2. After 3 h at 4°C, 3 ml media containing n-docosanol or Tetronic 15 908 at the original concentrations were added and the samples were incubated at 37°C for various t&nes. Cells were centrifuged at 4°C, washed with saline, centrifuged, and resuspended in 10% fortna6n in saline (3 ml). The cells were washed with saline and resusperxied in PBS containing 109ro FCS.
Fluorescence Intensity was measured by using a fluorescence-activated cep sorter (FACScan; Becton-Dickinson).
The active form of the drug has a finite lifetime in the coil membrane with a half life of approximately 3 h.
20 Antiviral activity is increased In target cells incubated with n-docosanol prior to the addition of HSV. This is illustrated in Fig. 20a which shows the effect of incubation time of Vero cells with 9 mM n-docosanol on inhibition of HSV-I-induced plaques, In this experiment, 9 mM n-docosanol inhibited plaque torrnation in Vero cells 28%
when added simultaneously with, or 3 h prior to vints addition; this was increased when cells were treated with drug 6 h prior to inhibition, but the greatest inhibition occurred in cells treated 24 h before HSV-I addition.
25 Intermediate time intervals were not examined.
To establish the length of time Vero cells remain resistant to HSV rcrfection after optimal time of incubation with n-docosanol, Vero cells were incubated with 9 mM n-docosanol for 21~27 h. Media containing unincorporated drug was then removed and replaced with fresh media. Drug was not replaced. HSV 1 was added immediately, or following a 1-, 3-, or B-h period of incubation at 37°C. Two hours following addition of HSV-1, 30 excess virus was removed and the plaque reduction assay was continued es described above. As shown in Fig.
20b, the an6vira! activity observed (% inhibition plaque fonnation) decreased gradually as the time between drug removal and viral addition increased. With a 3-h Interval between drug removal and HSV-I addition, 5090 of the inhibitory activity was lost; with a 6-h interval no inhltHtion of HSV I
plaque fom>ation was observed.

Att~hment of HSV I to spedfic cell surface receptors is unaffected in n-docosanof-tn~ated ceAs. Previous studies have .verified that attachment of HS111 to specific cell surface roceptors is unaffected In n-docrosanol-treated cells: Vero cell incubated with 15 mM n-docosanol bound normal levels of [~H~HSV-I added ai 2 p.f.u.lcell. Heparin inhibited this interaction 9fi%. The specificity of the binding assay was cor~rmed using mouse HSV-I immune sera which reduced binding by 96% compared to normal mouse sera whkh did not inhibit [~H]HSV
binding.
Production of Gi-gal is inhibited In n-docosanol-treated HF.p-2 cells infected with HSV-I(KOS}gLBfi. To investigate the effects of n-docosanol treaimant on entry of HSV alto target cells, a viral constyct HSV-1 (KOS)gL86 was utilized. In this n:plicaiion-defective mutant, in which tacl expression is under control of the CMV promoter, ~-galactosidase is expn3ssed after entry of the va~af genome into a susceptible host cell nucleus:
Addition of X-gal results in the ~velopment of blue color proportional to the numt~er of cells infected. The intensity of the signal is inhibited by agents such as heparin which block viral binding (see Fig. 21) or agents whkh prevent entry including neutralizing monoclonal antkodies to gD, an HSV-spedfic prote~
required forestry. This signal is not inhibited by acyclovir or other agents that inhibit DNA replic~iion.
The effect of n-docosanoi treatment of HEp-2 cells at doses ranging from 0.9 fio 9.9 mM (0.333.3 mgim~
on the entry of HSV-1(KOS)gL88 was examined. HEp-2 cells were Incubated for 24 h with the indicated concentrations of n-docosanol suspended in Telronic*908 prior to addition of the mutant virus. At 5-fi h after infection the cells were faced and permeabHized and X-gal was added. n-Da~anoi treatment resulted in the visibly apparent production of fewer blue calls at n-docosano! concentrations as low as 4 mM. Almost no color development occurred in cells treated wHh 8 and 10 mM n-docosanol, respectively. To quantify the ir~hibiBon of viral infectivity, the substrate within the HEp-2 cells was solubiGzed by the addition of DMSO and the ODsoo was recorded as shown in Fig. 21. The IDso for n-docosarwl was approxrnately 7 mM, mughty equivalent to the IDsu values, 4 and 9. mM, for Inhibition of HSV production and plaque formation, respectively, in Vero. cells. The vehicle, Tetronic'~908, without n-docosanol, was not inhibitory to viral entry. in fact, treatment of celb with equivalent volumes of vehide enhanced the blue color development as much as 40%. Heparin was examked.at . ---~~~atians between-1 and 10-uglml; inhibition appeared to be complete at 8 ~glmL. These. results established that the HSV genome does not effectively enter the nucleus In n-docosanot-treated ceAs: Combined with the failure of n-docosanol to inhibit viral attachment, this experiment indicates tha# a step of viral entry is bkxked by n docosanol treatment and that this event occurs subsequent to viral attachmerd but prior to nuclear entry of the viral genome.
n-0ocosanol inhibits HSV 2(333) iniediviiy of CHO-IEpB cells. To further r>amow the point of inhitation of viral entry in n-docosanol-treated cells, the effects of the drug on entry of HSV 2 Into CHO-IE~iB cells selected by transfection of CHO cells with a plasmid carrying a Pur selectable marker and lacl rmde~ control of the HSV-1 *Trademark -51-ICP4 pr~rwter was investigated. In this tail Ikre, ~i-gal expression is inducal upon entry of HS11 vaion prot~ms into the veil, an event which occurs immediately upon viral entry into the cellular cytoplasm and which is not dependent on virion transport to the nudeus. Color development is proportional to the number df cells inie~d and, as in the previous assay, Is y irrhibUed by agents such as heparin vrtdCh block viral attachcra~t ~d by agents which inhibit entry {such as anbbodles to gD) but not by. acyclovir and other Inhibitors of DNA
rep~a5on.
BAs iilrutrated in Fig. 22, n-docosanoi inhibited j3-galactosidase expression In this assay. Whereas treatment of CHO.IE~B cells with vehicle abne resulted in a slight incr~se ~
OD~oo, (~ 10%~ n-doa~sanof treatment of ceAs results in a concentration~dependent decrease in the color development signifying Infected cells.
In thts experiment 30 mM n-dooosanol inhibited color production 40% compared to unOreaied cells and 55%
compared 1o Tebmnic~'908-treated cells. The maximal observed tnhib(tion in mmparisfln to caAs was approximately 75%. This, in combination with the lack of inhibition in the binding assay, narrows a pdrrt of , inhibition to an event after viral aitachrrmnt but prior to release of virion proteins and mani(estaSOrr of VP16 transactivator adivky (an immediate post-entry event not dependent on virion transport to the ntxleus), n-Docosanol-treated NG37 human B~caBs exhibit decreased fusion with odadecyl rhodamfne B daoride~
labeled HSV 2. Because of the seiecUvtty of the inhibitory effects of n~docosanol f~ Upid-enveloped fasion-dependent viruses and the absence of virlddai effects, we considered the possibiUty that n-doeosartol may inhibU
viral entry by albedng I~get coil membrerres to prevent effective fusion of vkal parNdes wilt target ails. To investigate the effects of n-docosanol on NSV fusion vHth ce~ular membranes we conducted 8uorescen~
dequenching assays. The membranes of intact HSV-2 viriiorrs were labe~d wife odadecyf rhodamine chloride (i~
18) and added to human B cells. In this model, if viral fusion with the cellular membrane occurs, the tightly packed rhodamine molecules diffuse into the larger memtu~e of the host veil. This relieves fluorescerroe sel~que and causes an increase in signal intensity.
NG37 human B cells were treated w'rfh 15 mM n-docosanol 24 h before the add'tion of R-18 labeled HSV 2. As shown in Fig. 23, this corxentrabon of n-docosand iN~ibbed the rela~re increase in fluorescenoe .
intensity_occumog._wifh._viraUoell_fusion..b~!_approximately_..SQ%__compared_to _cells. receiving_no treatrnent.
Treatment of.NG87 ceAs wflh Tetroni~'conbol suspensans was Trot dboty, and instead caused a increase In fluorescerroe Intensity, reminiscent of the observaflon made with the ~-gal_ expressU~g systems discussed above (Fgs: 21 and 22). Compared to the effect observed wi8r the T~roni~Or~trd alone, n-dooosanoi .
inhrbibed the fluorescent response by as much as 7896. n-Doaosanol was not Inhibiloty if added only during the fusion process; a prior incubation period of the canpound with cells was necessary. This is eonsisfent with the requirement for metabolle conv~erslon in the anEiuiral process. The observation ale ~blishes chat the ~esenoe of n-docosanol does not itse~ quench or otherwise inhibit fluorescence. Anti-gD monoclonal arr6body (a spedflc 'Trademark .:52.. .

inhibitor of penei<ation) at a 1:40 dilution completely blocked the increase in fluorescence signal (not shown) confirming that the experimental protocol is an appropriate measure of viral penetration. These rasuns indicate that fusion of HSV viral particles to the host membranes is significantly inhibited in n-docosanol-treated cells.
Mosi available antiviral therapeutic compounds block replicafron processes shared by the virus and 5 infected target cell and hence are toxic, mutagenic, andlor teratogenic and can potentially Induce drug-resistant viral mutant substrains. Therefore, the identification of new antiviral compounds, parflcularly those with new mechanisms of action, is important. The 22-carbon, saturated, primary alcohol, n-docosanol, lacks any toxic, mutagenic, or teratogenic properties. In contrast to the mode of action of conventional antiviral agents, the predominant mechanism for the an0-HSV activity of n-docosanol appears to be inhibition of fusion between the 10 plasma membrane and the HSV envelope and, as a result, the blocking of entry and subsequent viral replication.
The mechanism of action explains the effectiveness of n-docosanol against all bested Ilpid-enveloped viruses that employ fusbn as the sole or major means of entry into the cell and contrasts its mode of action to other antiviral agents that target a single viral protein. Based on this mechanism of action the emergence of HSV strains resistant to the anfiviral effects of n-docosano! may be unlikely.
15 Previous results had suggested that n~iocosanol may be specific for lipid-enveloped viruses, and that lipid~nveloped viruses which primarily enter ceN by fusion with the plasma membrane are effectively blocked by n-docosanol. In contrast, the drug generally exerts no detectable activity against viruses that are either non-enveloped, or are enveloped and endocytosed. One exception to this general pattern is influenza A, an enveloped virus that has been reported to enter cells via receptor-mediated endocytosis but which is effectively inhibited by 20 n-docosanol. The reasons for this anomaly are currently unclear.
The in vitro doses (mM) required for aniiviral inhibition with n-docosanol are high compared to results with existing therapeutic compounds such as acyclovir. Thfs may result from the nature of fhe surfactant-stab~ized suspensions of n-docosanol. Due to the insolubiltty of n-docosanol, the particles are thermodynarnlcally stable, making transfer to cultured cells an inefficient process. As detem~ined using radiolabeled n~iocosanol, less than I
25 out of 1000 molecules of n-docosanol added to culture enters the cell.
Optimal inhibition of viral replication was observed In Vero cell cultures to which HSV~was added &24 h after addition of n-docosanol. This observation can be explained by a time-dependent uptake and metabolism of n-docosanol by host cells, an event apparently required for antiviral activity. The rate of this metabolic conversion in vivo is likely to be faster than that observed in the artificial milieu of the tissue culture system, especially 30 considering the thermodynamic stability of the surfactant-stabilized particles. The gradual loss of resistance to HSV in n-docosanol-treated cells reported herein would also be predicted due to rapid turnover not only of a required lipid metabolite but of the plasma memtxane itself which is constantly being internalized and replaced.

However, even with this rapid turnover, viral entry was reduced for several hours following removal of uninoorporated drug.
Furthermore, the topically applied cream remains on the skin surface acting as a constant resenroirof n docosanol. Available data demonstrated that n-docosanoi exerts an effect on the host cell that Inhlbiis early 5 events in viral replication bul does not Inhibft the amount of HSV which attaches b cells. The effed of n~looosano) on progressively earlier events in viral entry was therefore examined.
Penetration of HSV-t(KOS)gle6 into HEp-2 calls was inhibited by n-docosanol with a concentration dependence (IDso = 7 mM) roughly equivalent to Inhibition of HS1I 1 or HSV 2 production (IDso = 4 mM) ~ plaque formation (IDso = 9 mM) in Vero cells (Fig 21) confirming that n-docosanol inhib3ls an early event h the viral 10 replication cycle. The inhibitory activity of n-docosanol on )i-galactosidase expression must counteract the apparent stimulatory action of the vehicle alone, the mechanism for which is unclear. n-Docosanol inhibition of HSV 2 entry was also evidenced by reduced release Into treated cells of virlon-associated regulatory prMeins (Fig. 22). n-Docosanol treatment caused as much as an 80% reduction In the expression of (i-galactosidase in target cells containing a stably transfected Iac1 gene under control of an HSV
immediate early promoter (IGP4).
15 This observation, in combination with the lack of inhibition of veal attachmer~ ~ rrdocosarml-tread cells, confirms that n-docosanol blocks an event occumng after viral attachment but prior to release of tegument proteins. This is an immediate post~entry event and is not dependent upon virion localization In the nucleus. The inhibitory concentrations were higher than that generally required for in vitro anti-HSV activity. Additional early events in viral replication may also be Inhibited try n~docosanof.
20 n-Docosanol appears to inhibit the biophysical process of virallcell fusion. The fusion-dependant dequenching of octadecyl rhodamine B chloride, inserted into the NSV envelope was significantly inhibited in n-docosanol-heated cells (Fig. 23). The concentration dependence of fluorescence inhib'dion correlated to chat observed for Inhibition of HSV-1 replication by n-docosanol in other in vitro assays. Incorporation of n~locosanol, or its metabolites, and resulting perturbations of normal membrane compc~itan may alter the biophysical 25 properties of the plasma membrane in such a way as to inhibft fusion of attached virions. The compound may inhlbR the function of normally occurring cellular mediators of entry.
Inhibition of fusion between the plasma membrane and the HSV envelope, and the subsequent lack of repllcative events, may be the predominant mechanism for the anti-HSV activity of n~dooosanol. This mechanism of action may be generally applicable to the spectrum of viruses susceptible to the inhibitory effect of n~docosanol.
30 The above description discloses several methods and materials of the present invention. This Invention Is susceptible to modifications In the methods and materials, as well as alterations in the fabrication methods and equipment. Such modificati~s will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it coverall modifications and alternatives coming within the true scope and spirit of the invention as embodied in the attached claims.

Claims (41)

WHAT IS CLAIMED IS:

1. Use of a composition consisting essentially of sugar-based ester surfactant, greater than about 5 wt. % n-docosanol, mineral oil, an emollient co-solvent and water, for preventing the onset of lesions caused by recurrent herpes simplex labialis.

2. Use of n-docosanol, a sugar-based ester surfactant, mineral oil, an emollient co-solvent and water in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, wherein the composition comprises greater than about 5 wt. % n-docosanol.

3. The use of claim 1 or 2, wherein the composition is stable at temperatures of at least 40°C for a period of at least three months and after repeated freeze-thaw cycles.

4. The use of claim 1, 2, or 3, wherein the sugar-based ester surfactant is selected from the group consisting of sucrose cocoate, sucrose stearates and sucrose distearate.

5. The use of claim 1, 2, or 3, wherein the sugar-based ester surfactant comprises at least one compound selected from the group of sucrose esters consisting of sucrose cocoate, sucrose stearates and sucrose distearate, wherein sucrose ester(s) comprise about 3 wt. % or more of the composition.

6. The use of claim 5, wherein sucrose ester(s) comprise about 5 wt. % or more of the composition.

7. The use of any one of claims 1-6, wherein the emollient co-solvent is selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol, or combinations thereof.

8. The use of any one of claims 1-7, wherein the n-docosanol comprises at least approximately 10 wt. % of the composition.

9. A composition consisting essentially of sugar-based ester surfactant, greater than about 5 wt. % n-docosanol, mineral oil, an emollient co-solvent and water, in the preparation of a medicament for preventing the onset of lesions caused by recurrent herpes simplex labialis.

10. The composition of claim 9 wherein the composition is stable at temperatures of at least 40°C for a period of at least three months and after repeated freeze-thaw cycles.

11. The composition of claim 9 or 10, wherein the sugar-based ester surfactant is selected from the group consisting of sucrose cocoate, sucrose stearates and sucrose distearate.

12. The composition of claim 9 or 10, wherein the sugar-based ester surfactant comprises at least one compound selected from the group of sucrose esters consisting of sucrose cocoate, sucrose stearates and sucrose distearate, wherein sucrose ester(s) comprise about 3 wt. % or more of the cream.

13. The composition of claim 12 wherein sucrose ester(s) comprise about 5 wt. % or more of the composition.

14. The composition of any one of claims 9-13, wherein the emollient co-solvent is selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol, or combinations thereof.

15. The composition of any one of claims 9-14, wherein the n-docosanol comprises at least approximately 10 wt. % of the composition.

16. Use of a composition consisting essentially of n-docosanol and a cream base comprising one or more compounds selected from the group consisting of sucrose cocoate, sucrose stearates, and sucrose distearate, and one or more compounds selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol, for preventing the onset of lesions caused by recurrent herpes simplex labialis.

17. Use of n-docosanol and a cream base comprising one or more compounds selected from the group consisting of sucrose cocoate, sucrose stearates and sucrose distearate and one or more compounds selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, wherein the composition consists essentially of said n-docosanol and a cream base.

18. The use of claim 16 or 17, wherein the cream base comprises sucrose ester(s) which comprise at least 5 wt. % of the composition.

19. The use of claim l6, 17, or 18, wherein the n-docosanol comprises at least approximately 10 wt. % of the composition.

20. A composition consisting essentially of n-docosanol and a cream base comprising one or more compounds selected from the group consisting of sucrose cocoate, sucrose stearates, and sucrose distearate, and one or more compounds selected from the group consisting of polyoxypropylene stearyl ether, ethyl hexanediol, and benzyl alcohol, for preventing the onset of lesions caused by recurrent herpes simplex labialis.

21. The composition of claim 20 wherein the cream base comprises sucrose ester(s) which comprise at least 5 wt. % of the composition.

22. The composition of claim 20 or 21, wherein the n-docosanol comprises at least approximately 10 wt. % of the composition.

23. Use of a composition having the formulation: n-docosanol comprising from 5 to 15 wt. % of the total composition; sucrose stearates comprising from 0 to 15 wt. % of the total composition; sucrose cocoate comprising from 0 to 10 wt. %
of the total composition; sucrose distearate comprising from 0 to 10 wt. % of the total composition;
at least one sucrose ester comprising at least about 3 wt. % of the total composition;
mineral oil comprising from 3 to 15 wt. % of the total composition; benzyl alcohol comprising from 0.5 to 10 wt. % of the total composition; and water comprising from 40 to 70 wt. % of the total composition, for preventing the onset of lesions caused by recurrent herpes simplex labialis.

24. Use of n-docosanol, at least one sucrose ester, mineral oil, benzyl alcohol and water, and optionally sucrose stearates, sucrose cocoate, and sucrose distearate in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composing having the formulation: n-docosanol comprising from 5 to 15 wt. % of the total composition; sucrose stearates comprising from 0 to 15 wt.
% of the total composition; sucrose cocoate comprising from 0 to 10 wt. % of the total composition; sucrose distearate comprising from 0 to 10 wt. % of the total composition;
at least one sucrose ester comprising at least about 3 wt. % of the total composition;
mineral oil comprising from 3 to 15 wt. % of the total composition; benzyl alcohol comprising from 0.5 to 10 wt. % of the total composition; and water comprising from 40 to 70 wt. % of the total composition.

25. A composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition having the formulation: n-docosanol comprising from 5 to 15 wt. % of the total composition; sucrose stearates comprising from 0 to 15 wt.
% of the total composition; sucrose cocoate comprising from 0 to 10 wt. % of the total composition; sucrose distearate comprising from 0 to 10 wt. % of the total composition;
at least one sucrose ester comprising at least about 3 wt. % of the total composition;
mineral oil comprising from 3 to 15 wt: % of the total composition; benzyl alcohol comprising from 0.5 to 10 wt. % of the total composition; and water comprising from 40 to 70 wt. % of the total composition.

26. Use of a composition consisting essentially of n-docosanol and a cream base, wherein the cream base consists essentially of sugar-based ester surfactant, at least one long chain aliphatic alcohol having from 20 to 28 carbon atoms selected from the group consisting of n-icosanol, n-henicosanol, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol, or mixtures thereof, mineral oil, an emollient co-solvent, and water, for preventing the onset of lesions caused by recurrent herpes simplex labialis.

27. Use of n-docosanol and a cream base in the preparation of a medicament for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of n-docosanol and the cream base, and wherein the cream base consists essentially of sugar-based ester surfactant, at least one long chain aliphatic alcohol having from 20 to 28 carbon atoms selected from the group consisting of n-icosanol, n-henicosanol, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol, or mixtures thereof, mineral oil, an emollient co-solvent, and water.

28. The use of claim 26 or 27, wherein the composition comprises n-docosanol in an amount more than one-half of the amount of long chain aliphatic alcohols in the composition.

29. A composition for preventing the onsets of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of n-docosanol and a cream base, and wherein the cream base consists essentially of sugar-based ester surfactant, at least one long chain aliphatic alcohol having from 20 to 28 carbon atoms selected from the group consisting of n-icosanol, n-henicosanol, n-tricosanol, n-tetxacosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol, or mixtures thereof, mineral oil, an emollient co-solvent, and water.

30. The composition of claim 29, wherein the composition comprises n-docosanol in an amount more than one-half of the amount of long chain aliphatic alcohols in the composition.

31. Use of a topical cream having the formulation:
n-docosanol about 5-20 wt. %;
sucrose stearates about 0-15 wt. %;
sucrose cocoate about 0-10 wt. %;

sucrose distearate about 0-10 wt. %, with the proviso that at least one sucrose ester be present and, wherein sucrose ester(s) comprise about 3 wt. % or more of the cream;
mineral oil about 3-15 wt. %;
propylene glycol about 2-10 wt. %;
polyoxypropylene-15 stearyl ether about 0-5 wt. %;
benzyl alcohol about 0.5-5 wt. %;
with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of at least about 1 wt. %; and water about 40-70 wt. %, for preventing the onset of lesions caused by recurrent herpes simplex labialis.

32. The use of claim 31 wherein sucrose ester(s) comprise about 5 wt. % or more of the cream.

33. A topical cream for preventing the onset of lesions caused by recurrent herpes simplex labialis, the cream having the formulation:
n-docosanol about 5-20 wt. %;
sucrose stearates about 0-15 wt. %;
sucrose cocoate about 0-10 wt. %;
sucrose distearate about 0-10 wt. %, with the proviso that at least one sucrose ester be present and, wherein sucrose ester(s) comprise about 3 wt. % or more of the cream;
mineral oil about 3-15 wt. %;
propylene glycol about 2-10 wt. %;
polyoxypropylene-15 stearyl ether about 0-5 wt. %;
benzyl alcohol about 0.5-5 wt. %;
with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of at least about 1 wt. %; and water about 40-70 wt. %.

34. The topical cream of claim 33 wherein sucrose ester(s) comprise about 5 wt. % or more of the cream.

35. Use of an anti-inflammatory and antiviral cream for preventing the onset of lesions caused by recurrent herpes simplex labialis, the cream having the formulation:
n-docosanol about 5-20 wt. %;
sucrose stearates about 0-15 wt. %;
sucrose cocoate about 0-10 wt. %;
sucrose distearate about 0-10 wt. %, with the proviso that at least one sucrose ester be present and wherein sucrose ester (s) comprise about 3 wt. % or more of the cream;
mineral oil about 3-15 wt. %;
propylene glycol about 2-10 wt. %;
polyoxypropylene stearyl ether about 0-5 wt. %;
benzyl alcohol about 0-5 wt. %;
with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of about 1 wt. % or more; and water about 40-70 wt. %.

36. The use of claim 35, wherein sucrose ester(s) comprise about 5 wt. % or more of the cream.

37. An anti-inflammatory and antiviral cream for preventing the onset of lesions caused by recurrent herpes simplex labialis, the cream having the formulation:
n-docosanol about 5-20 wt. %;
sucrose stearates about 0-15 wt. %;
sucrose cocoate about 0-10 wt. %;
sucrose distearate about 0-10 wt. %, with the proviso that at least one sucrose ester be present and wherein sucrose ester (s) comprise about 3 wt. % or more of the cream;
mineral oil about 3-15 wt. %;
propylene glycol about 2-10 wt. %;
polyoxypropylene stearyl ether about 0-5 wt. %;
benzyl alcohol about 0-5 wt. %;
with the proviso that either polyoxypropylene stearyl ether or benzyl alcohol be present in an amount of about 1 wt. % or more; and water about 40-70 wt. %.

38. The cream of claim 37, wherein sucrose ester(s) comprise about 5 wt. % or more of the cream.

39. Use of a composition consisting essentially of about 10 wt. % n-docosanol;
about 5 wt. % of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. % light mineral oil;
about 5 wt. %
propylene glycol; about 2.7 wt. % benzyl alcohol; and about 69.3 wt. % water, for preventing the onset of lesions caused by recurrent herpes simplex labialis.

40. Use of n-docosanol, stearate, light mineral oil, propylene glycol, benzyl alcohol and water in the preparation of a composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of about 10 wt. % n-docosanol; about 5 wt. % of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof;
about 8 wt.
% light mineral oil; about 5 wt. % propylene glycol; about 2.7 wt. % benzyl alcohol; and about 69.3 wt. % water.

41. A composition for preventing the onset of lesions caused by recurrent herpes simplex labialis, the composition consisting essentially of about 10 wt. % n-docosanol; about 5 wt. % of a stearate selected from the group consisting of sucrose monostearate, sucrose distearate, and mixtures thereof; about 8 wt. % light mineral oil;
about 5 wt. % propylene glycol; about 2.7 wt. % benzyl alcohol; and about 69.3 wt. %
water.