This invention relates to alcohol-containing compositions which are useful in treating virus infections and inflammatory diseases of the skin and membranes, including burns, laceration damage and acute injuries. More specifically the present invention relates to a naarrow class of oaliphatic straightchain saturated monohydric alcohols which have from 20 to 26, preferably 22 to 26, carbons in the chain.
It is well-known that certain selected alcohols have some physiological activity. It is known, for example, that 1-triacontanol stimulates the growth of plants, see, e.g. Ries, Stanley K. and Sweeney, Charles C., U.S. Pat. No. 4,150,970. Interestingly, the C-30 aalcohol triacontanol appears to possess this physiologicl activity and that the C-28 and C-32 do not possess such physiological activity, or at least have very much less physiological activity in plant growth, see, e.g., the patents and publications of Ries et al., ibid, and of Ashmead, Harvey H., Weleber, Andrew J., Laughlin, Robert G., Nickey, Donald O. & Parker, Dane. K, and Ohorogge, Alvin J.
Triacontanol has also been reported to accelerate the decomposition of sewage and reduce H.S, Starr, Jerry, U.S. Pat. No. 4,246,100.
Beeswax comprises, inter alia, esters of long-chain aliphatic alcohols having chain lengths in the area of interest, and it is known to obtain such alcohols by hydrolysis of beeswax. Beeswax has been used sicne antiquity in a great variety of cosmetic and thereapeutic applications, as a bse for lipstick, in lotions and creams, as an emollient and as a constituent in therapeutic products for topical and membrane application. Various constituents of beeswax and products derived from beeswax have also been used in cosmetic and therapeutic applications. For example, Slimak, Karen M., U.S. Pat. No. 4,793,991, describes a hypoallerginic cosmetic comprising single plant source beeswax. Gans, Eugen, Nacht, Sergio and Yeung, David have described the use of the non-polar saturated straight chain C-21 to C-33 hydrocarbon fraction of beeswax in the treatment of inflammatory skin disorders, U.S. Pat. No. 4,623,667.
The mechanism of the rather diverse and upredictable physiological effects of the vaarious alcohols are, at best, poorly understood and studies are not generally definitive. There appears to some interaction of certain nalkanols with lipid bilayer membranes, Westerman, PW, Pope, JM, Phonphok, N., Dan, JW, dubro, DW, Biochim Biophys Acta (NETHERLANDS) 939, 64-78 (1988), and studies have been conducted respecting the partitioning of long-chain alcohols into lipid bilayers, Franks NP & Lieb WR, Proc. Natl. Acad. Sci. USA 83 5116-20 (1986); cholesterol solubility of n-alkanols, Pal S. & Moulik SP, Indian J Biochem Biophys 24 24-8 (1987); neurological effects of certain long-chain alcohols, Natarajan V & Schmid HH, Lipids 12 128-30 (1977); Snider SR, Ann Neurol 16 723 (1984); Borg J, Toazara J. Hietter H, Henry M, Schmitt G, Luu B, FEBS Lett 213 406-10 (1987).
Levin, Ezra reported that tetracosanol, hexacosanol, octacosanol and triacontanol aand their esters improved physical performance of athletes and disclosed compositions comprising such alcohols and esters in vegetable oil bases for oral ingestion, U.S. Pat. No. 3,031,376.
An incidental disclosure of a composition intended for topical application comprising a major portion liquified gaseous propellant and a minor portion of a mixiture of C-12 to C-30 fatty alcohols which were used simply to mark the areas of application of the aerosol is contained in U.S. Pat. No. 3,584,115 to Gebhart.
Clark, U.S. Pat. No. 4,670,471 discloses the use of triacontanol, in a suitable carrier, as a treatment for inflammatory disorders such as herpes simplex, eczema, shingles, atopic dermatitis, psoriasis, etc. Clark performed experiments with the compositions of the type disclosed by Gebhart, U.S. Pat. No. 3,584,115 comprising an aerosol and a mixture of triacontanol and palmitic acid, which Clark indicates to be as effective as pure triacontanol, and concluded that the aerosol carrier destroyed the effect of triacontanol and that a hydrophilic carrier for triacontanol was necessary to achieve the desired anti-inflammatory effect. There is some reason to believe that Clark's composition was simply saponified beeswax which would contain triacontanol and palmitic acid, as Clark indicates, but which would also contain, as substantial constituents, hexacosanolic acid nd various hydrocarbons. Results of gas chromatographic-mass spectrum analysis of various compositions believed to have been used by Clark were not definitive, but suggested that at least some such compositions were very complex mixtures, some of which may be lower alkanes, esters, acids or alcohols. Whether or not these were found by Clark to be effective anti-inflammatory compositions is not known. McKeough, Mark & Spruance, SL evaluated the efficacy of 5 percent triacontanol in a branch chain ester base in the treatment of HSV-1 dorsal cutaneous infection in guinea pigs and concluded that the active ingredient in triacontanol is the long chain hydrocarbon (unpublished report in the file of U.S. Pat. No. 4,670,471).
Revici, Emanuel, Sherwood, Bob E., Benecke, Herman P., Rice, John M., and Geisler, Richard W., U.S. Pat. No. 4,513,008, disclose a method of inactivating developed virus using C-20 to C-24 polyunsaturated acids, aldehydes or alcohols having 5-7 double bonds, and references disclosures by Sands et al. (Antimicrobial Agents and Chemotherapy 15, 67-73 (1979)), antiviral acivity of C-14 to C-20 unsaturated alcohols having 1-4 double bonds, C-20 tetraenyl alcohol having low activity, Snipes et al., (Antimicrobial Agents and Chemotherapy 11, 98-104 (1977) and Symp. Pharm. Effects Lipids (AOCS Monograph No. 5) 63-74 (1978) even lower antiviral activity for saturated long-chain alcohols.
Katz, Martin & Neiman, Herbert M, U.S. Pat. No. 3,592,930 disclose a medicant vehicle containing from 15 to 45 parts of saturated fatty alcohol from 16 to 24 carbons, along with glycol solvent, plasticizer, penetrant and adjuvant which is used as a carrier for oantibiotics, steriods, antihistamines, etc.
Ryde, Emma Marta & Ekstedt, Jan Erik, U.S. Pat. No. 3,863,633 disclose a composition for topical treatament of the eye which comprises a lipophilic substance, a hydrophilic swellable polymer and from 10 to 80 percent C-12 to C-22 surface active alcohols such as 1-docossanol, 1-hexadecanol, 1-octadecanol and 1-eicosanol which serve as a stabilizer for the mixture.
The content of othe prior art and the corresponding skill of the art, relative to topically administered compositions, may be summarized as follows: Short-chain alcohols, i.e. under about 16 carbons, tend to be irritants while longer chain alcohols, particularly the aliphatic alcohols tend to be non-irritating (Katz et al., supra). 1-Triacontanol, a 30-carbon unsaturated aliphatic alcohol, in a suitable hydrophilic carrier has (or may have depending upon the precise compositions used by Clark) value in treating inflammatory conditions of the skin (Clark, supra). Shorter chain C-10 to C-14 aliphatic alcohols demonstrate low level in vitro virucidal characteristics, while C-18 alcohols show noo discernable virucidal activity in vitro (Snipes, supra). Polyunsaturated C-20 to C-24 alcohols inactive enveloped virus (Revici et al., supra). C-16 to C-24 aliphatic alcohols are useful as stabilizers in carrier compositions for drugs having diverse physiological activity.
Respecting aliphatic alcohols, one would predict from the studies of Snipes and Clark that, in the continuum of aliphatic alcohols from C-10 to C-30 virucidal activity, at a very low level, may appear (if in vitro studies may be used to predict in vivo results) in C-10 to C-14 alcohols (which would also be irritants as reported by Katz), that virucidal activity disappears inthe C-16 to C-28 range and than appears uniquely (if Clark's compositions were pure triacontanol or mixtures of triacontanol with palmitic acid as he indicates) with the C-30 alcohol 1-triacontanol, which has been shown to have unique physiological effects in plant treatment.
Even considering the possible ambiguity of Clark's compositions, one would not predict any significant virucidal activity for aliphatic alcohols in the C-20 through C-28 chain-length.
Notwithstanding the negative teachings of the prior art, the present invention comprises compositions and methods for topical treatment of inflammatory diseases, including virus-induced inflammation, burns, laceration damage and acute injuries, in which the active constitute consists essentially of C-20 to C-26, and preferably C-22 to C-26 aliphatic alcohols, e.g. docosanol, tetraccosanol and hexacosanol.
The present invention is embodied in a method treating inflammatory and viral skin diseases, such as may result, for example, from virus infection, burns, lacerations and acute injuries, comprising application of a composition consisting of one or more of C-20 to C-26 aliphatic alcohols, preferably one or more alcohols selected from the group consisting of 1-docosanol, 1-tetracosanol and 1-hexacosanol in a physiologically compatible carrier.
The compositions suitable for use in this invention consists essentially of a carrier which is physiologically compatible with the skin and membrane tissues of the patient, i.e. non-irritating, and which is substantially inactive physiologically (except for possible emollient properties) and, as the physiologically active composition, one or more C-20 to C-26 aliphatic alcohols, e.g. one or more of 1-eiconol, 1-docosanol, 1-tetracosanol and 1-hexacosanol.
The method may be carried out using compositions inwhich the sole physiologically active agent(s) is the C-20 to C-26 aliphatic alcohol, or comparable compositions which may also include other physiologically active constituents which do not interfere with the efficacy of the C-20 to C-26 alcohols.
The composition of the carrier is not critical so long as the carrier is non-irritating to skin and membranes and is substanatially free from physiological effect, e.g. has no physiological effect other than be an emollient.
An exemplary composition for use in this invention would be similar to that disclosed by Katz, et al. in U.S. Pat. No. 3,592,930 without the addition of any other physiologically active constituent, e.g. a mixiture of C-20 to C-26 alcohols, preferably one or more of the alcohols 1-docosanol, 1-tetracosanol and 1-hexacosanol, a glycol solvent such as propylene glycol, and, if desired, a plasaticizer such as glycerol or a polyethylene glycol having a molecular weight of from 800 to 20,000.
A suitable carrier may comprise white petrolatum, stearyl alcohol, isopropyl myristate, sorbitan monooleate, propylelne glycol, water and a detergent such as polyoxyl stearate mixed for form a stable cream. The active alcohols, e.g. one or more of 1-docosanol, 1-tetracosanol and 1-hexacosanol is added to the carrier in amounts from about 0.1 to about 25 percent by weight, typically in the range of from 1 to 5 percent. Higher concentrations of the active alcohol(s) may be used but no increase in efficacy results from concentrations above about 15 to 25 weight percent. The concentration of the active alcohol(s) is not critical, but optimum efficacy coupled with efficient use of the active ingredient would be found in the 1 to 5 weight percent range.
Another suitable composition for use in the method of this invention would be a cream formulated of water, white petrolatm, isopropyl myristate, lanolin alcohols, mineral oil and cetylstearyl alcohol into which from 1 to 5 percent of C-20 to C-26 alcohols, e.g. one or more alcohols selected from the group consisting of 1-docosanol, 1-tetracosanol and 1-hexacosanol has been intimately mixed.
An alternative suitable composition for use in this invention may be formulated of stearyl alcohol, petrolatum, water and mineral oil stabilized with a detergent such as sodium lauryl sulfate and may include a preservative such as methylparaben or propylparaben, and an effective amount, typically from about 0.1 to 5 percent by weight of one or more alcohols selected from the group consisting of 1-docosanol, 1-tetracosanol and 1-hexacosanol.
In all cases, suitale preservatives, such as ethylene diamine tetraacetate salts, methylparaben, propylparaben, etc., may be added to prevent bacterial and fungal growth. Penetrants, such as a zone, may also be added if desired.
The method of the present invention will require application to the inflamed area of skin or membrane of compositions, such as those described above as merely exemplary, in which the active ingredient consists essentially of one or more aliphatic alcohols having from 20 to 26 carbons in the aliphatic chain, an exemplary composition comprising one or more alcohols selected from the group consisting of 1-docosanol, 1-tetracosanol and 1-hexacosanol. Three to 6 appliccations of the ointment or ocream per day will, in most cases, be expected to produce prompt relief from the itching, discomfort associated with such diseases and promote healing of damaged tissues within a few days to a few weeks.
The method described is useful in treating a wide variety of viral and inflammatory diseases, examples of which include herpes, simplex, eczema, shingles psoriasis, atopic dermatitis, and in treating inflammation resulting from burns, lacerations and acute injuries.
It will be readily understood from the foregoing that the essential constituent(s) of the compositions useful in the present method is one or more aliphatic alcohols having from 20 to 26 carbons in the aliphatic chain of the alcohol(s), and that the composition of the carrier is non-critical and subject to great variation.
This invention is useful in treating virus-induced inflammatory diseases of humans and other animals, and inflammation resulting from burns, lacerations and acute injuries.
FIGS. 1 through 3B and FIGS. 6A and 6B pertain to experiments involving herpes simplex virus type 1 (HSV-1), while FIGS. 4 and 5 and FIGS. 7 through 9 involve herpes simplex virus type 2 (HSV-2).
FIG. 1 presents the comparative activities of Formulation I (n-docosanol 10.0 wt. %; sucrose stearates 11.0 wt. %; sucrose cocoate 5.0 wt. %; mineral oil 8.0 wt. %; propylene glycol 5.0 wt. %; 2-ethyl-1,3-hexanediol 2.7 wt. % and purified water 58.3 wt. %), three different preparations of Formulation II (same as Formulation I except 5 wt. % sucrose stearates was replaced with sucrose distearate and ethyl hexanediol was replaced with an equivalent amount of polyoxypropylene-15-stearyl ether) and ZOVIRAX (acyclovir; Burroughs Wellcome Co., Research Triangle Park, NC; a treatment of HSV infections which inhibits activity of viral DNA polymerase) in inhibiting HSV-1-induced cutaneous lesions in hairless guinea pigs.
FIG. 2 presents the comparative activities of Formulation I, Formulation II, and Formulation IA (n-docosanol 10.0 wt. %; sucrose stearates 11.0 wt. %; sucrose cocoate 5.0 wt. %; mineral oil 8.0 wt. %; propylene glycol 5.0 wt. %; benzyl alcohol 2.7 wt. % and purified water 58.3 wt. %).
FIG. 3A shows a comparison of activities of Formulation I versus Formulation III (n-docosanol 10.0 wt. %; sucrose stearates 5.0 wt. %; mineral oil 8.0 wt. %; propylene glycol 5.0 wt. %; benzyl alcohol 2.7 wt. %; and purified water 58.3 wt. %).
FIG. 3B depicts data comparing the activities of certain modifications of these formulations in which the relative surfactant concentrations have been modified from that of Formulation I. Modifications of surfactant concentrations were found to have appreciable deleterious effects on the extent of drug activity.
FIG. 4 depicts data showing the dose-response relationship of Formulation III for the inhibition of HSV-2 induced cutaneous lesions in hairless guinea pigs.
FIG. 5 graphically represents data showing that n-docosanol containing cream based upon a sucrose ester surfactant system (Formulation III) also inhibits HSV-24induced cutaneous lesions in hairless guinea pigs.
FIG. 6A graphically depicts data that demonstrates that n-docosanol, formulated as a suspension using the surfactant Pluronic F-68, also inhibits HSV-1 induced vesicles when applied before vesicles are present. The suspension formulation did not contain any of the excipients of n-docosanol containing cream including benzyl alcohol.
FIG. 6B graphically depicts data that demonstrates that n-docosanol, formulated as a suspension in nonionic surfactant Pluronic F-68, also inhibits HSV-1 induced vesicles when applied after vesicles are present. The suspension formulation did not contain any of the excipients of n-docosanol containing cream including benzyl alcohol.
FIGS. 7 through 13 depict data elucidating the pharmacology of n-docosanol.
FIG. 7 depicts data showing that n-docosanol inhibits acyclovir-resistant HSV-2. Vero cells were cultured in 35-mm wells (6×105 cells per well) in medium alone (=none) or in the presence of the indicated concentration of acyclovir, n-docosanol-Pluronic F-68 suspension or control suspension (Pluronic F-68 only). The cultures were inoculated 24 hours later with 150 PFU of either wild-type HSV-2 or an acyclovir-resistant laboratory isolate from the wild-type HSV-2 that was plaque purified and passaged in 20 μg/ml acyclovir 44 hours later, the plates were incubated, fixed, stained, and scored for numbers of plaques. The data presented are means of plaques scored from duplicate cultures. The percent inhibition observed in cultures treated with acyclovir or n-docosanol relative to untreated control cultures is denoted in parentheses.
FIG. 8 depicts data showing the dose response of the topical emulsion formulation of n-docosanol on cutaneous HSV in guinea pigs. The backs of hairless guinea pigs were cleaned and inoculated with purified HSV-2 by puncture of the skin with a tattoo instrument. Two hours after virus inoculation, the inoculation sites were either untreated or treated with 100 μl of n-docosanol-containing cream or control vehicle; the sites were similarly treated 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 vesicle number derived from duplicate sites per determination. The numbers in parentheses depict percent inhibition of vesicle number at treated sites as compared to the untreated sites.
FIG. 9 depicts data showing that HSV-2 remains on the surface of n-docosanol treated Vero cells for prolonged times. Vero cells were cultured as described in the legend to FIG. 7 and incubated overnight. The cultures were then chilled to 4° C., inoculated with 100 PFU of HSV-2, and incubated 3 hours at 4° C. At time zero the cultures were washed with medium, inoculated with fresh medium (containing the indicated inhibitor) and incubated at 37° C. At each indicated time period, the cultures were washed with citrate buffer (pH 2.5) and reinoculated with fresh medium (lacking inhibitor). After a total of 44 hours incubation the cultures were stained and scored for HSV-2-induced plaques. The data are expressed as geometric means and standard errors derived from triplicate cultures per group.
FIG. 10 depicts data showing that radioactive metabolites of n-[14C]docosanol display the properties of phosphatidylcholine and phosphatidylethanolamine. A portion (0.5 ml) of the methanol eluate of the silica lipid fractionation was evaporated under nitrogen, resuspended in 20 μl chloroform:methanol (3:2; v:v) and spotted on a silica thin layer chromatography (TLC) sheet. After development with chloroform:methanol:acetic acid:water (60:50:1:4; v:v:v:v), the positions of standards were determined by staining with iodine vapors and the cpm per fraction determined by scintillation spectrometry after cutting the plastic-backed sheet into 5 mm strips.
FIG. 11 depicts data showing that n-[14C]-Docosanol is metabolized more by Vero cells than by MDBK cells. Vero or MDBK cells were plated as described. n-[14C]-docosanol was added to 6 mM (0.24 mM Tetronic 908) and the cultures were incubated 72 hours at 37° C./CO2. Cells were extracted and analyzed on TLC with hexane:diethyl ether:acetic acid (20:30:1; v:vv) as the developing solvent. With this solvent system the polar phosphatides remain at the origin. The position of migration of n-[14C]-docosanol is indicated. Duplicate plates were treated with an identical suspension lacking the radioactive label, and the numbers of cells in these duplicate plates were determined by counting cells excluding trypan blue with a hemocytometer.
FIG. 12 depicts data showing that n-docosanol inhibits in vivo Friend virus induced leukemia and viremia. Adult BALB/c mice were injected intravenously with 75 spleen focus-forming units of FV. Treated groups were injected intravenously with the indicated doses of n-docosanol or Pluronic vehicle alone on the same day as virus inoculation and once daily for the next 3 days. After 10 days, half of the animals in each group were sacrificed and examined for leukemic foci in their spleens (panel A). The remaining mice were retained 10 more days and bled for viremia determinations (panel B). Viremia was measured using the X-C plaque assay. Briefly, primary fibroblast cultures were derived by digestion of 14-day BALB/c embryos with trypsin and culturing in DMEM plus 10% fetal calf serum. After 72 hours, the cells were transferred into 16-mm dishes (105/well), pretreated with 5 μg/ml polybrene and then infected with 75 X-C plaque-forming units of Friend virus stock or dilution of test plasma. After incubation for 7 days, the cultures were irradiated and overlaid with X-C cells (3×105/well). Three days later, the cultures were washed, stained, and scored for plaques of multinucleated giant cells. The data presented are geometric means and standard errors of splenic foci or X-C plaque-forming units derived from three animals per group.
FIG. 13 depicts data showing that n-docosanol inhibits in vitro replication of HIV-1 in cultures of PHA/IL-2-stimulated human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were cultured in medium containing 1 μg/ml PHA plus 5 units/ml IL-2 alone or also containing 100 μg/ml PFA, the indicated dosage of n-docosanol/Pluronic F68, or the amount of Pluronic F-68 control vehicle contained in the high dose of n-docosanol/Pluronic F-68. After overnight incubation, the cultures were inoculated with HIV-1 at a multiplicity of infection of 1 virion/cell. After 24 hours incubation at 37° C., the cultures were washed and inoculated with fresh medium containing PHA and IL-2, but lacking inhibitor. Replication of HIV-1 was determined 4 days later by quantitation of viral antigens by a p24-specific ELISA for HIV-1.
FIG. 14 illustrates the Kaplan-Meier distributions for time-to-healing for treatment of acute HSL using n-docosanol 10 wt. % cream. Time-to-healing was measured from initiation of treatment until the date and time of the clinic visit at which complete resolution of all local signs and symptoms was clinician determined.
FIG. 15 provides a graphical depiction of HSV-1 inhibition in hairless guinea pigs with PEG formulations.
FIG. 16 provides a graphical depiction of HSV-2 inhibition in hairless guinea pigs with PEG formulations.
FIG. 17 provides a graphical depiction of HSV-2 vesicle numbers in hairless guinea pigs.
FIG. 18 provides a graphical depiction of HSV-2 inhibition in Hartley guinea pigs.
FIG. 19 provides a graphical depiction of HSV-2 vesicle numbers in Hartley guinea pigs.
FIGS. 20a and 20b show the inhibition of HSV-1 increases when cells are incubated with n-docosanol before viral addition and this inhibitory effect has a half-life of approximately 3 h. (A) Vero cells were plated and incubated with 9 mM n-docosanol, the corresponding control vehicle or no addition for 0, 3, 6, or 24 h prior to the addition of HSV-1. The viral plaque assay was continued and the number of p.f.u. determined. The data are expressed as % inhibition compared to wells receiving no treatment. (B) Vero cells were plated, n-docosanol or the corresponding control vehicle was added and cells were incubated at 37° C. in 10% humidified CO2. After 21, 24, 25, 26, and 27 h (6, 3, 2, 1, and 0 h before the addition of HSV-1), media containing drug was removed and the cells were washed with media. After a total of 27 h, HSV-1 was added to all wells. Two hours later virus-containing media was removed and replaced with fresh media lacking virus or drugs. The cultures were incubated and processed for determination of the number of HSV-induced plaques as in (A).
FIG. 21 shows the uptake of HSV-1(KOS)gL86 into HEp-2 cells when incubated in n-docosanol-treated cells. After attachment of HEp2 cells to culture wells, n-docosanol-vehicle, vehicle alone, or no agent (control) was added. Five to six hours after infection, the cells were processed, X-gal was added, and the absorbance at 600 nm was determined.
FIG. 22 provides a graph demonstrating that n-docosanol suspended with Tetronic 908 inhibits the entry of HSV-2 (333) into CHO-IEβ8 cells. CHO-IEβ8 cells were seeded into 24-well plates. After cell attachment, heparin, n-docosanol-vehicle, vehicle alone, or no agent (control) was added. Five to six hours after infection, the cells were processed, X-gal was added, and the absorbance at 600 nm was determined.
FIG. 23 provides a graphic depicting experimental results for n-Docosanol-treated NC-37 human B cells, the cells exhibiting decreased fusion with octadecyl rhodamine B chloride-labeled HSV-2. NC-37 human B cells were inoculated in the presence of 15 mM n-docosanol, the corresponding concentration of Tetronic 908 (0.1 mM) or without addition. Cells were harvested and R-18-labeled HSV-2 was added to aliquots in the presence of compounds at their original concentration. Following incubation at 37° C. for the times indicated, cells were fixed and fluorescence intensity determined by FACScan.
The following description and examples illustrate a preferred embodiment of the present invention in detail. Those of skill in the art will recognize that there are numerous variations and modifications of this invention that are encompassed by its scope. Accordingly, the description of a preferred embodiment should not be deemed to limit the scope of the present invention.
10%) Docosanol Cream
To prepare a cream, n-docosanol (98% pure; M. Michel and Co., New York, N.Y.), a water-insoluble compound, is mixed at 80° C. with sucrose cocoate, sucrose stearates, sucrose distearate, mineral oil, propylene glycol and polyoxypropylene-15-stearyl ether. Water was added and mixed in to finish the cream.
A cream can also be formed by adding all the materials except n-docosanol to water to form the cream 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, i.e., n-eicosanol, n-heneicosanol, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, 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 ester(s) comprise about 3 wt. % or more, preferably about 10 wt. % 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 0-5 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-docosanol 5-10 wt. % (or n-Docosanol in mixture with at least one other long chain aliphatic alcohol having from 20 to 28 carbon atoms, i.e., n-eicosanol, n-heneicosanol, n-tricosanol, n-tetracosanol, n-pentacosanol, n-hexacosanol, n-heptacosanol, and n-octacosanol); sucrose stearates 6 wt. %; sucrose cocoate 5 wt. %; sucrose distearate 5 wt. % (with the proviso that at least one sucrose ester be present and that sucrose ester(s) 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. % (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-1,3hexanediol 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 preferred in certain embodiments, for example, in compositions intended for repetitive topical application.
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 FIGS. 1 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 benzyl alcohol as co-solvent excipient, as described below.
In certain especially preferred embodiments providing stable compositions, only one or two 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 preservative effectiveness.
The differences in Formulation III as compared with Formulation I include the replacement of 2-ethyl-1,3-hexanediol with benzyl alcohol, a well-known preservative and co-solvent with a long history of safe use and compendial status. The liquid nature and like functions of benzyl alcohol 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 on 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 properties of the formulation.
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 in the guinea pig herpes model was verified on repeated occasions.
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, i.e., 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 42° C. or 24 freeze-thaw cycles all samples remained as off-white creams. There was no evidence of syneresis or phase separation. Based on the above visual inspection, the Formulation III of 10 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 life is more than adequate for a commercial n-docosanol containing cream. Thus, while certain n-docosanol formulations are unstable, specific formulations, Formulation 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 polyoxyalkylene-based ethers may also be found to be substitutable for polyoxypropylene-15-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-docosanol cream is that of Formulation III containing 10 wt. % n-docosanol, 5 wt. % sucrose stearates, 8 wt. % mineral oil NF, 5 wt. % propylene glycol USP, 2.7 wt. % benzoyl alcohol NF and 69.3 wt. % purified water.
Long-term stable cream preparations that contain effective amounts of n-docosanol alone or in 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 formulation that have a shelf-life 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 virus-induced and inflammatory diseases of the skin or membranes of an animal, including the treatment of humans, are provided. The ingredients of the cream include n-docosanol, alone or in mixture with other normal long chain (C-20 to C-28) 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 penetration of the n-docosanol into the affected area of the skin or membrane and co-act with the ester in forming a stable carrier for the physiologically active alcohol (s).
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 polyalcohols, and include both saccharides and disaccharides, disaccharide-based esters being preferred. High molecular weight polyhydric 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 catalogs, e.g., McCutcheon's directories, Volume 1-EMULSIFIERS & DETERGENTS, and Volume 2-FUNCTIONAL MATERIALS, (McCutcheon's Division, The Manufacturing Confectioner Publishing Co., Glen Rock, N.J., USA, 1993). Sucrose-fatty acid esters are preferred. Sucrose stearate and sucrose distearate are nonionic surfactants that are preferred for use in n-docosanol cream formulations to emulsify the oil and aqueous phases of the cream. These surfactants have a non-irritating nature, which makes them particularly preferred 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, Del. under the tradenames Brij, Myrj, and Span) demonstrate superior properties as a surfactant for n-docosanol.
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-docosanol cream formulations. Together with the n-docosanol, it forms the liquid phase of preferred cream formulations. Mineral oil has a long history of safe use in topical products and may perform such functions as acting as an emollient, e.g., by acting as a barrier to transdermal water loss, and to improve the texture of topical products.
Certain of the pharmacological studies were conducted using suspensions that are more compatible with the cells used in these studies but which are not suitable for use as topical pharmaceutical preparations in certain embodiments as they may lack the body and stability required for effective topical treatment.
A generally preferred cream formulation of certain embodiments includes, by weight based on the total weight of the final cream formulation, n-docosanol, typically about 5 to about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 wt. %, more preferably about 6, 7, 8, or 9 wt. % to about 11, 12, 13, 14, or 15 wt. %, most preferably about 10.0%; sucrose stearates, typically about 0 to about 11, 12, 13, 14, or 15 wt. %, preferably about 1, 2, or 3 wt. % to about 4, 5, 6, 7, 8, 9, or 10 wt. %; and/or sucrose cocoate; typically about 0 to about 11, 12, 13, 14, or 15 wt. %, preferably about 1, 2, or 3 wt. % to about 4, 5, 6, 7, 8, 9, or 10 wt. %; and/or sucrose distearate typically about 0 to about 11, 12, 13, 14, or 15 wt. %, preferably about 1, 2, or 3 wt. % to 4, 5, 6, 7, 8, 9, or 10 wt. %; at least one sucrose ester or an equivalent sugar-based ester comprising typically 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 oil NF typically about 3 wt. % to about 15 wt. %, preferably about 4, 5, 6, or 7 wt. % to about 9, 10, 11, or 12 wt. %, most preferably about 8.0 wt. %; a glycol, e.g., propylene glycol USP or equivalent, typically 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. %; an emollient glycol ether, e.g., polyoxypropylene-15-stearyl ether, or benzyl alcohol, typically about 0 to about 3.5, 4, 4.5, or 5 wt. %, preferably about 0.5, 0.75, 1, 1.24, 1.5, 1.75, 2, 2.25, 2.5, or 2.6 wt. % to about 2.75, 2.8, 2.9, or 3 wt. %, most preferably about 2.7 wt. %; and water typically about 40, 41, 42, 43, or 44 wt. % to about 70, 71, 72, 73, 74, 75,76, 77, 78, 79, or 80 wt. %, preferably about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 wt. % to 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69 wt. %, most preferably about 69.3 wt. %. Within this general formulation, many specific formulations can be prepared which will be stable and which will exhibit the therapeutic effect noted based upon the data presented above, the teachings of the specification and the guidelines provided in the specification. Thus, an effective topical therapeutic composition wherein the therapeutically active material consists essentially of n-docosanol, alone or in mixture with normal long chain (C-20 to C-28) aliphatic alcohols may be prepared.
The formulations may be used in the manufacture of pharmaceuticals and also in the treatment of human and animal patients.
To confirm in an experimental model the efficacy of n-docosanol cream on HSV-induced lesions, and to compare its activity to that of ZOVIRAX, hairless guinea pigs were inoculated with 1×105 PFU of HSV-1, and then treated with either n-docosanol-containing or control cream, or ZOVIRAX ointment. The n-docosanol creams were constructed as described. The control cream was constructed in a similar manner except stearic acid was substituted for n-docosanol. Treatment was started either 2 or 48 hours after virus inoculation. The sites were evaluated for vesicle formation, defined as a pus-filled blister, at the indicated time points.
FIG. 1 presents the comparative activities of Formulation I and three different preparations of Formulation II as well as ZOVIRAX. Formulation I and Formulation II of n-docosanol creams both showed greater inhibitory power than ZOVIRAX ointment.
FIG. 2 presents the comparative activities of Formulation I, Formulation IA and Formulation II. Significant inhibition of HSV-1-induced lesions was demonstrated for all three formulations.
FIG. 3 shows a comparison of activities of Formulation III versus Formulation I and also depicts certain modifications of these formulations in which the relative surfactant concentrations have been modified from that of Formulation I.
Modifications of surfactant concentrations were found to have appreciable deleterious effects on the extent of drug activity. Formulation III was shown to have potent inhibitory power for HSV-I-induced lesions.
Volunteer patients with recurrent oral or genital HSV I or II infections have also been treated with topical n-docosanol-containing cream at various stages of an acute herpes outbreak. When treatment is initiated during the prodromal stage, n-docosanol cream may abort further progression of the infection (i.e., prevent vesicle formation). When treatment is started after vesicle formation has already occurred, n-docosanol cream may shorten the time for healing (i.e., complete re-epithelialization) of such herpes lesions.
The selection of 10 wt. % n-docosanol in the formulation was tested in a dose-response study in the hairless guinea pigs. The sites on the backs of hairless guinea pigs were inoculated with HSV-2 as described previously. The sites were treated with 1, 5, 10, and 20 wt. % n-docosanol formulations. A vehicle control containing no n-docosanol was also included in the study. The results, illustrated in FIG. 4, show that after 72 hours of virus inoculation the untreated sites exhibited an average of 41 vesicles. Treatment with 20 wt. % and 10 wt. % n-docosanol containing cream inhibited vesicle number by 50% and 60%, respectively. Creams containing 1 wt. % and 5 wt. % n-docosanol were less effective than the 10 wt. % preparation. The control vehicle was without appreciable inhibitory effect.
It has been observed that the level of n-docosanol in the cream may play a role in the physical appearance, stability, and efficacy of n-docosanol cream. A comparison of creams containing 5, 10, and 20 wt. % n-docosanol was conducted. In general, it was observed that the viscosity of the product varied directly with the concentration of n-docosanol in the formulation (FIG. 4). The 5 wt °% formulation had the lowest viscosity with lotion-like appearance and had a tendency to separate into phases. The 20 wt. % formulation had the highest viscosity, was difficult to rub in and had a tendency to leave a white residue on human skin. Complete removal of n-docosanol 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 cosmetically most pleasing, rubbing in easily and not leaving any residue on human skin. The results indicate that in addition to its function as an active ingredient, n-docosanol also functioned as a thickening agent and an emulsion stabilizer in the creams tested. In vivo studies with hairless guinea pigs showed that the 10 wt. % formulation had better efficacy than 5 wt. % or 20 wt. % formulations. While the 10% formulation was preferred for most applications, in certain embodiments, however, a formulation containing less than 10 wt. %, e.g., 5 wt. % or less n-docosanol may be preferred, while in other embodiments a formulation containing more than 10 wt. %, e.g., 20 wt. % or more n-docosanol may be preferred.
Since it has been reported that benzyl alcohol had some antiviral activity under certain circumstances, (Farah, A. E. et al., U.S. Pat. No. 4,200,655) a formulation of a preferred embodiment was tested to determine if benzyl alcohol acts as an antiviral reagent in the formulation. The cream containing benzyl alcohol and n-docosanol (10 wt. % n-docosanol cream) and the cream containing benzyl alcohol 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 HSV-2. The sites were treated as indicated in FIG. 5 and evaluated for vesicle formation at 48, 56, 72 and 78 hours after virus inoculation. There was an average of 44 vesicles in the untreated sites at the 48-hour time point, which remained relatively constant up to 72 hours after infection. At the 78-hour time point, resolution of the lesions became evident and by 96 hours post-inoculation vesicles were no longer visible. Treatment with n-docosanol cream inhibited vesicle number by 50-60% at the 48-56-hour time points, and by a slightly higher amount at the 72-78-hour points of analysis. Treatment with the control vehicle was without appreciable effect on vesicle number at any time point. Untreated and treated sites were excised and processed for viral culture. The presence of vesicles was directly correlated with the presence of infectious virus regardless of treatment or time of assay (not shown). Thus, vesicle number is an appropriate indicator for disease state in the studies described herein. Additionally, the cream and the placebo were tested in a phase II pilot study comprising sixty-eight patents with herpes labialis. The result of the double blind trial showed that early application of n-docosanol cream cut the duration of the episodes nearly in half. The treated groups' average outbreak period was 3.4 days, while the placebo group had outbreaks averaging 6.6 days.
The above results demonstrate that the presence of n-docosanol in the formulation is responsible for significant antiviral action.