WO1993007853A1

WO1993007853A1 - Inhibition of odor formation and bacterial growth

Info

Publication number: WO1993007853A1
Application number: PCT/US1992/008698
Authority: WO
Inventors: George Preti; Andrew I. Spielman; Xiao-Nong Zeng; James J. Leyden; Katerina Leftheris; Kenneth J. Mckinley
Original assignee: Monell Chemical Senses Center; New York University; The Trustees Of The University Of Pennsylvania Center For Technology Transfer
Priority date: 1991-10-23
Filing date: 1992-10-13
Publication date: 1993-04-29
Also published as: IL103432A0; AU2878092A; ZA927946B; MX9206088A

Abstract

Tests indicate that topical application of a composition comprising a deodorizing amount of one or more precursor mimics to the characteristic axillary odor 3-methyl-2-hexenoic acid to a location of a warm-blooded animal can significantly inhibit and/or attenuate the intensity of body odors, such as those formed in the axillary region. Disclosed are methods for suppressing body odors by applying a deodorizing amount of one or more of the precursor mimics to such body odors, and deodorant compositions that comprise an effective amount of at least one of these mimics. The mimics are preferably acid-alanine-esters.

Description

INHIBITION OF ODOR FORMATION AND BACTERIAL GROWTH

Background of the Invention

Field of the Invention The present invention relates to a method of suppressing human body odor. More particularly, it relates to a method of utilizing a deodorant composition containing an effective amount of a synthetic mimic to the naturally- occurring precursor to the characteristic underarm odors. Description of the Prior Art •

The eccrine and apocrine sweat glands are the structures of the human body responsible for the production of sweat. Of all of the human scents, those arising in the axillae are uniquely characteristic of humans. However, this odor does not develop until puberty when the apocrine glands mature. Axillary odor is traceable to the secretions of the apocrine glands, which are attached to hair follicles. The apocrine secretion as it first emerges through the follicular opening at the skin surface is odorless. It is only when resident bacteria living on the skin surface act on the secretion that the characteristic axillary odor is generated. Consequently, the precursors for the odor are contained in the apocrine secretion.

Since modern western culture generally finds axillary odor to be offensive, a substantial amount of effort has been directed towards identifying the odoriferous chemicals in axillary odor and towards understanding the factors that control their generation. Previous analyses of both the apocrine secretion and the total axillary sweat have shown the presence of a variety of both volatile and non¬ volatile steroids. Brooksbank, "Labeling of Steroids in Axillary Sweat After Administration of H-Δ⁵-Pregnenolone and ¹C-Progesterone to a Healthy Man," 26 EXPERIENπA 1012 (1970); Brooksbank et al., "The Detection of 5α-Androst-16-en-3α-ol in Human Male Axillary Sweat," 30 EXPERIENTIA 864 (1974); Claus et al., "Occurrence of 5α-Androsten-16-en-one, a Boar Pheromone, in Man and Its Relationship to Testosterone," 68 J. ENDOCRIN., 483 (1976). It has been demonstrated that two androgens, dehydroepiandrosterone and androsterone, are secreted as their sulfates by the apocrine gland. Labows et al. , "Steroid Analysis of Human Apocrine Secretion," 34

STEROIDS 249 (1979); Labows et al., "Perspectives on Axillary

Odor," 34 J. Soc'Y COS ET. CHEM. 193 (1982); Labows, Odor

Detection, Generation and Etiology in the Axillae, in ANTI-

PERSPIRANTS AND DEODORANTS 321 (1988) . In addition to efforts to identify the odoriferous chemicals in axillary secretions, the bacteria responsible for generating the characteristic axillary odors have been studied. Labows, "Human Odors - What Can They Tell Us?," 4 PERFUMER & FLAVORBT 12 (1979), discloses that in vitro incubation of apocrine secretion and micrococci produces a sweaty odor that has been identified as isovaleric acid. Leyden et al., "The Microbiology of the Human Axilla and Its Relationship to Axillary Odor," 77 J. INVEST. DERM. 413 (1981), suggest that aerobic diphtheroids, either lipophilic or large colony diphtheroids, arethe organisms principallyresponsible for axillary odor. Both of these works are cited by Labows et al. in "Perspective on Axillary Odor," in which it is further suggested that at least one measure for controlling body odor should be directed to interfering with the bacterial production of those odors.

Numerous patents disclose the incorporation of anti¬ bacterial agents, surfactants, or odor neutralizers into deodorant compositions. Many of these are discussed and summarized in Seitz et al., Deodorant Ingredients, in ANTI- PERSPIRANTS AND DEODORANTS 345 (1988) . U. S . Patent No . 4 , 654 , 213 (Ramirez et al.) discloses antimicrobial systems that can be used in deodorants whose systems contain the magnesium sulfate adduct of 2,2'-dithiobis-pyridine-1,1'-dioxide and a water soluble zinc salt. U.S. Patent No. 4,565,693 (Marschner) discloses that zinc glycinate is a non-irritating effective deodorant that chemically neutralizes odoriferous compounds and inhibits bacterial growth. U.S. Patent No. 4,425,321 (Jacquet et al.) discloses that the biological equilibrium of the skin may be destroyed, and thus development of odors prevented, by the use of a deodorant composition containing at least one zinc or magnesium salt of a specific polyacid. It is alleged that the metal salts block development of odors by trapping the small molecules responsible for the bad odors. U.S. Patent No. 4,322,308 (Hooper et al.) discloses a deodorant composition comprising a deodorant perfume and a second deodorant other than a deodorant perfume whose composition is an effective means for inhibiting malodor development. The second deodorant can be a germicide, a zinc salt, zinc oxide, antioxidant, citrate ester, diol, or mixtures thereof. U.S. Patent No. 4,292,192 (Hooper et al.) discloses detergent bars capable of a deodorancy effect that contain 0.3 to 3% by weight of an ester of citric acid or an acetyl derivative thereof. U.S. Patent No. 4,235,873 (Packman) discloses an antiperspirant-deodorant composition and a method of suppressing odors due to the bacterial decomposition of perspiration by administeringbis-(2-pyridyl¬ l-oxide) disulfide and/or at least one adduct of bis-(2- pyridyl-1-oxide) disulfide. U.S. Patent No. 4,172,123 (Lowicki) discloses deodorizing materials that contain sparingly water soluble or water insoluble salts of unsatur- ated hydroxylated carboxylic acids having at least seventeen carbon atoms. U.S. Patent No. 3,996,807 (Curry et al.) discloses an aerosol antiperspirant or deodorant composition free from anticholinergic compounds containing hexylene glycol as a non-staining emollient substance or dispersing agent. The compositions may contain germicides. U.S. Patent Nos. 4,376,789 (Lowicki et al.) and 4,454,153 (Lowicki et al.) disclose the use of zinc salts of a hydrolyzed tricarboxylic acid from the ene-adducts of maleic anhydride to C₁₀ to C₁₈ acids, most notably, 10-undecenoic acid.

Despite the many disclosures in the art relating to deodorant compositions and deodorizing methods, current products are not sufficient to suppress odor in a significant proportion of the population, particularly during periods of "stress." Furthermore, each of the prior art deodorant methods were developed without knowledge of the structural nature of the characteristic odors or of their origin. Therefore, the prior art methods are not targeted directly at the causative, offending molecules.

Accordingly, there remains a need for new deodorant compositions and deodorant methods that are effective, safe, and economical.

Summary of the Invention

The present invention is directed to compositions and methods for suppressing odors, particularly axillary odors, of warm-blooded animals. It has now been discovered that compositions formulated to include chemical mimics of the precursors to characteristic odors have significant deodorant capacity. The methods and compositions of the present invention utilize knowledge from basic research that has revealed the structures of the characteristic axillary odors and their precursors in the apocrine secretions. In accordance with this invention, a composition comprising a deodorizing amount of at least one molecular mimic of a precursor of a body odor, such as a precursor to 3-methyl-2- hexenoic acid, is topically administered to a location of the body of a warm-blooded animal. The mimics are preferably acid-alanine-esters.

Tests indicate that the application of such a composition can significantly inhibit and/or attenuate the intensity of body odors, such as those formed in the axillary region as well as the foot, where volatile acids have also been implicated as the cause of malodor. Kanda et al. , "Eluc- idation of Chemical Compounds Responsible for Foot Malodour," 122 BRIT.J. DERM.771 (1990) . The present invention relates not only to the aforementioned method, but also to the deodorant compositions themselves. In addition, bacteriocides can be incorporated into the mimic to add a bacteriocidal effect.

Accordingly, it is an object of the present invention to provide a novel, non-irritating, highly effective deodorant compound and a method for inhibiting body odors. This and other and objects and advantages of the present invention will become apparent from the following, more detailed description.

Brief Description of the Drawinσs

FIG. 1 is a histogram of the number of colony- forming units (CFU) of total aerobes resulting from comparative forearm testing of two concentrations of a mimic of the present invention against a control and against a known antibacterial ingredient commonly used in deodorants.

FIG. 2 is a histogram of the CFU of diphtheroid bacteria resulting from comparative forearm testing of two concentrations of a mimic of the present invention against a control and against a known antibacterial ingredient commonly used in deodorants.

FIG. 3 is a histogram of the CFU of total axillary aerobes resulting from comparative axillary region testing of a mimic of the present invention against a known antibacterial ingredient commonly used in deodorants.

FIG. 4 is a histogram of the CFU of axillary diphtheroid bacteria resulting from comparative axillary region testing of a mimic of the present invention against a known antibacterial ingredient commonly used in deodorants.

Detailed Description of the Invention

The present invention provides a novel method for deodorancy by introducing one or more mimics to the naturally- occurring precursors to a body odor. One particular embodiment intoduces a mimic to the precursor of 3-methyl-2- hexenoic acid in the axillary region of humans. The preferred embodiments of this invention utilize compounds naturally occurring in axillary sweat that are combined to create precursor mimics. The mimics inhibit body odor production by competitive inhibition and/or antibiotic action and may be used singularly or in combination.

Identifying the Odorous Components of Axillary Secretions — The present invention is based on research by the named inventors in identifying the odorous components of axillary secretions and their precursors. In their studies, trained panelists performed organoleptic evaluation (smell chromatography) of the chromatographic eluate from a combined concentrated extract of 5 male donors. Areas of the chroma- togram were identified where (E)- and (Z)-3-methyl-2-hexenoic acids and other components eluted as most closely resembling the odor of concentrated whole axillary extract. Zeng et al., "Analysis of the Characteristic Odors from the Human Male Axillae," 17 J. CHEM. ECOL. 1469 (1991).

The extract was first chromatographed using a non- polar, methylsilicon phase. Reliable retention times for the volatile steroids had been previously established using this type of column. These molecules had been thought to be the compounds responsible for axillary malodor. Labows, Odor Detection. Generation and Etiology in the Axillae, in ANTI- PERSPIRANTS AND DEODORANTS 321 (1988) . This experiment showed that within the first 15 minutes of the analysis, a strong, characteristic axillary odor eluted. The odors characteristic of the volatile steroids (such as androstenol, androstenone, and androstadienone) and the pyrolysis products of dehydroepiandrosterone and androsterone sulfates elute about 40-45 minutes after analysis begins.

The identical extract was injected into a column coated with a more polar, bonded Carbowax-type phase. The characteristic axillary odors eluted over a retention time of 46-55 minutes, approximately 15-20 minutes after the sharp, acidic smells of short-chain aliphatic acids such as butyric and isovaleric acids. The volatile steroids begin to elute on this column only after 65-70 minutes. These results suggested that a yet unidentified group of compounds was responsible for the characteristic axillary odor.

Collection of the area where the characteristic axillary odors elute was performed using preparative gas chromatography. When the collected eluate was dissolved in CH₂C1₂ and treated with saturated NaHC0₃ (pH 9.9), the characteristic axillary odor was eliminated. This suggests that acidic components with 6 or more carbons (based on chromatographic retention times) carry the characteristic underarm odor.

A combined concentrated extract from the same donors was subsequently prepared and separated into acids, bases, and neutrals. Each fraction was concentrated to approximately 20 μl. The acidic concentrate had a characteristic axillary odor. The neutral and basic parts of the extract had little or no odor. Smell chromatography of the acidic fraction showed a series of components with burnt, urinous, and axillary-like qualities eluting from times 37 to 54 minutes. This suggested that a number of the compounds eluting in this time frame carried portions of the axillary odor.

Analysis by combined gas chromatography/mass spectrometry (GC/MS) of the acidic components was carried out using a Carbowax-type phase. Comparison of the data from smell chromatography with the GC/MS data was done by matching relative retention times (based on a series of ethyl esters to yield ethyl ester indices) on both the chromatograph where the smell chromatography was performed and on the GC/MS system. In addition to the mass spectral and relative retention time data, further analysis of the acidic extract was performed by combined gas chromatography/Fourier transform infrared spectroscopy (GC/FTIR) . All of the compounds identified in this mixture are listed with their corresponding ethyl ester units in Table 1. Those compounds that are thought to be important contributors to the odor (as judged by "smell chromatography") are marked with an "*". TABLE 1

Compounds Identified in the Combined Male Axillary Extract

Peak Molecular Wt. Retention Time

(Ethyl EsterUnit)

A n-hexanoic acid ' 116 12.20

B 2-methylhexanoic acid 130 12.39*

C 3-methylhexanoiσ acid 130 12.55*

D di ethylsul^one (C₂H₆S0₂) 94 12.63

E 7-C₈-lactone 142 12.79

F 4-ethylpentanoic acid 130 12.97*

G (.Z)-3-methyl-2-hexenoic acid ,¹G¹ 128 13.10*

H 2-ethylhexanoic agid 144 13.13

I n-heptanoic acid ' 130 13.22*

J 2-methylheptanoic acid 144 13.36*

K (E)-3-methyl-2-hexenoic acid ' 128 13.50*

L phenol 94 13.65

M 7-C₉-lactone 156 13.91

N n-octanoic acid ' 144 14.28*

O 2-methyloctanoic acid 158 14.41

P 4-ethylheptanoic acid 158 14.81*

Q 7-octenoic acid 14214.95* R 7-C₁₀-lactone 170 15.01 S n-tetradecanol 214 15.21 T n-nonanoic acid ' 158 15.28 U 2-methylnonanoic acid ' 172 15.38 V 4-ethyloctanoic acid 17215.64* ("goat acid")

W unsaturated C₉ acid 156 16.04*

(8-nonenoic acid?)

X n-decanoic acid '

Y 2-methyldecanoic acid T,G 172 16.28 186 16.36

Z unsaturated C₁₀ acid 170 16.46*

AA 4-ethylnonanoiσ acid ' 186 16.69*

BB 9-decenoic acid 17016.90*

CC n-hexadecanol 242 17.14

DD n-undecanoic acid ' 186 17.29

EE 4-ethyldecanoic acid ' 200 17.66

FF 10-undecenoic acid 184 17.76* tentatively assigned by mass spectral data; FT-IR spectrum corresponds to assigned structure; correspondence of mass spectrum and relative chro atographic retention times with commercially available or synthetic sample.

* = Compounds judged to be important contributors to the axillary odor.

The reconstructed ion chromatogram generated by the acidic components showed compound K to be the largest component of the mixture. Its structure has been confirmed as (E)-(trans)-3-methyl-2-hexenoic acid. The odor of this compound bears a strong resemblance to underarm odor and is an important component of the axillary "bouquet." The (23)- (cis)-isomer is also present (Peak G, Table 1) at about 1/10 the amount of the (E)-isomer. In addition to the ( )- and (E)-3-methyl-2-hexenoic acids, other unsaturated compounds appear to be present, including several compounds with terminal double bonds.

The C₈ unsaturated acid, 7-octenoic acid, is a minor component of the mixture but appears to have a high odor impact. The mass spectrum of 7-octenoic acid as well as that of a synthetic sample are identical. In addition to the similarity of the mass spectra, the relative retention times of the natural and synthetic 7-octenoic acids are identical. The GC/MS data also show two series of branched- chain acids. One homologous series consists of 2-methyl-acids beginning with 2-methyl-hexanoic acid (Peaks B, J, O, U, and Y of Table 1) . The second series possesses an ethyl group on the 4-position in the chain (Peaks F, P, V, AA, and EE of Table 1) . The 4-ethyloctanoic acid in the series (Peak V) has a "goat-like" smell and has been previously studied by others because of its low odor threshold.

To further examine the odorous components, the mixture was chromatographed on a non-polar column with and without derivatization (as trimethylsilyl esters and ethers) . The analysis without derivatization did not reveal any new components, but did show that (23)- and (E)-3-methyl-2-hexenoic acids as well as many of the other C₆-C₁₁ acids shown in Table 1 elute within the first 18 minutes. This would explain the presence of the strong axillary odors that elute in the first 15 minutes of the "smell chromatography" performed on the non- polar column. This analysis also showed the presence of only trace amounts of the volatile steroid of androstenone (< 0.5ng/μl) but no androstenol. In addition, the pyrolysis products of the two steroid sulfates (andro-stenone sulfate and dehydroepiandrosterone sulfate) were present at levels seen in previous studies (Preti et al., "HUMAN AXILLARY EXTRACTS: Analysis of Compounds from Samples Which Influence Menstrual Timing," 13 J. CHEM. ECOL. 717 (1987)).

Analysis of the trimethylsilyl derivatives (of compounds in the acidic fraction) revealed a variety of components not seen in the earlier analysis; however, a majority of the compounds are not contributors to the odors since they are higher molecular weight (C₁₂-C₁₈) straight chain saturated, branched and unsaturated acids. Several aromatic acidic compounds were identified including benzoic, phenyl- acetic, _-methylbenzoic and phenylpropanoic acids as well as p-hydroxybenzaldehyde; however, the latter compound is relatively odorless and onlyphenylacetic acid appears to lend some odor quality to the axillary odor.

The results obtained from these studies suggest that volatile C₈-C^ straight chain, branched, and unsaturated acids are the major contributors to underarm odor. Those compounds with branching or unsaturation seem to have a high odor impact. A major contributor to the axillary odor is (E)-3- methyl-2-hexenoic acid; it is both unsaturated and branched. The (_Z)-isomer, which is present at 1/10 the concentration of the (E)-structure, has an odor more like that of branched C₅ and C₆ acids.

The neutral and basic fractions possessed very little odor. The organoleptic panel that evaluated the concentrated extract, as well as its acid, base, and neutral fractions, found that the odor of the acid fraction resembled that of the entire axillary secretion extract.

Mass spectrometric evidence indicated the presence of the androstenone in the acidic fraction using the non-polar column. However, since the organoleptic evaluation of chromatographic components indicated that the important characteristic axillary odor elute with the C₆ to C_n acids and not with the steroids, the steroids are probably not important contributors to the characteristic axillary odor. The amount of (I2)-3-methyl-2-hexenoic acid present in the combined male extract described here was approximately 357ng/μl. Androstenonewas present at approximately 0.5ng/μl. No androstenol was found in the acidic fraction, even though this compound is known to be in axillary secretion extract. The latter compound may have not have been extracted into the acidic fraction. Consequently, the predominant odorous compounds in the acid fraction are the C₆-C₁₁ acids.

The Mechanism of Axillary Odor Production — It is known that the apocrine secretions contain odorless precursors that are metabolized by the axillary microorganisms to odor. Labows et al., "Perspectives on Axillary Odor," 34 J. SOC'Y COSMET. CHEM.193 (1982) . To understand the mechanism by which the axillary odors are formed, the inventors examined the apocrine gland secretion for the precursor(s) to the characteristic odors. A small amount of odorless apocrine secretion (7μl) was obtained and partitioned between water (aqueous phase) and chloroform (organic phase) . These two fractions were then hydrolyzed using 5% NaOH, concentrated and acidified. When each fraction was sampled organoleptically, the aqueous fraction possessed a distinct axillary odor; the organic extract had no odor. This raised the possibility that the axillary odors are associated with water soluble molecules.

Apocrine secretion was subsequently collected (120μl) to repeat the above procedure and to examine the water soluble components of the aqueous phase more completely. Following the partitioning of this large apocrine secretion sample between water and chloroform, the aqueous and organic phases were hydrolyzed, acidified, extracted with CHC1₃, concentrated, and analyzed by organoleptic evaluation and GC/MS. Once again, the organic phase hydrolyte had no odor while that from the aqueous phase had a strong axillary odor.

Relatively large amounts of (Z) - and (E)-3-methyl-2- hexenoic acid are seen in the aqueous phase hydrolyte

(approximately 50ng/μl (E)-isomer). Only a trace amount

(approximately 0.25ng/μl) of (E)-3-methyl-2-hexenoic acid is seen in the organic phase hydrolyte. The amounts were determined by comparison to injections of known standard solutions of (E)- and (.Z)-3-methyl-2-hexenoic acid. This finding that the water soluble components of apocrine secretion contained non-odorous molecules that could be hydrolyzedto yield themajor characteristic odor (3-methyl-2- hexenoic acid) in the axillary secretions was most unexpected. Small aliquots (approximately 7.0μl) of both the intact, non-hydrolyzed aqueous phase and the organic phase were saved for analysis by SDS polyaerylamide gel electro- phoresis. This procedure demonstrated that the water soluble, but not the lipid soluble fraction, was rich in proteins, thereby raising the possibility that the odor molecules making up the axillary characteristic odors were being secreted from apocrine glands associated with proteins.

In light of these results, the inventors began to reevaluate the protein amounts in apocrine secretion. Table 2 shows results obtained from 6 male and 3 female donors, using the Bradford assay (Bradford, "A Rapid and Sensitive Method for the Quantitation ofMicrogram Quantities of Protein Utilizing the Principle of Protein-Dye Binding," 72 ANALYT.

BlOCHEM. 248 (1976) . These results suggest that apocrine secretion contains on average 4.48 ± 3.55μg/μl of protein and that the percentage of protein in the secretion is approx¬ imately 0.5%.

TABLE 2

Protein Concentration of Apocrine Secretion from Individual Donors

Mean 4.48 + 3.56 S.D. The collection procedures for apocrine secretion were subsequently scaled up and the gel electrophoretic profile of the apocrine secretion was analyzed. This demonstrated that the apocrine secretion shows 10 protein bands, numbered in decreasing order of their apparent molecular weight. Each of the protein bands was excised from the gel, electroeluted, hydrolyzed in 5% NaOH, acidified, extracted with CHC1₃, and assayed for the presence of particular odor molecules, both organoleptically and by GC/MS. Significantly, each of two protein bands, one at apparent molecular weight of 45,000 and one at 26,000 had a distinct odor of 3-methyl-2-hexenoic acid. The presence of this acid was confirmed by GC/MS as being uniquely associated with these two protein bands. None of the hydrolytes of the other proteins or blank samples had any particular odor, nor any 3- methyl-2-hexenoic acid by GC/MS.

These results were confirmed on two other occasions with different apocrine secretion samples. Consequently, it appears that an important human axillary odor is secreted bound to proteins in some fashion.

The protein bands with apparent molecular weight of 45,000 (called apocrine secretion odor binding protein 1 — "AS0B1") and 26,000 (called apocrine secretion odor binding protein 2 — "ASOB2") were purified by electrophoresis followed by electroelution prior to amino acid analysis. The results demonstrate a novel pathway for the production of a characteristic human odor by the resident bacteria. In addition, it appears that at least one of the most abundant axillary odors is not formed on the skin surface but is actually secreted from the apocrine gland bound to particular proteins. This pathway for axillary odor production has never been reported before.

The studies have shown that the compounds causing underarm odor in humans are acidic in nature and that the largest characteristic axillary odor is bound to proteins (ASOB 1 and ASOB 2) but can be liberated from by simple hydrolysis. ASOB 1 and ASOB 2 may be classified as acyl proteins (Olson et al., "Fatty Acylation of Cellular Proteins," 261 J. BIOL. CHEM. 2458 (1986)). Proteins such as these may have an acid covalently bound to them as an ester, a thioester, or an amide (Olson et al., "Specificity of Fatty Acid Acylation of Cellular Proteins," 260 J. BlOL. CHEM. 3784

(1985)). Thus, although not intending to be bound by this theory, the inventors speculated that the compounds producing the axillary odors are, when secreted from the apocrine glands, covalently bound to the proteins ASOB 1 and ASOB 2 and that hydrolysis of those molecules free the odor-producing compounds.

Odor-Inhibiting Precursor Mimics — In the axillae, bacterial enzymes, presumably secreted by coryneform (diph¬ theroid) bacteria, recognize and interact with ASOB 1 and ASOB 2 in such a way as to release 3-methyl-2-hexenoic acid and create odor. Since the lipophilic diphtheroid bacteria have high lipase activity and require lipids as part of their growth medium, this suggests that the bacteria has a special affinity for these two proteins (i.e., non-lipids) that may be evolutionarily tied to a chemical signalling system in humans. Because of this affinity, the inventors postulated that the odor production process would be competitively inhibited by a molecule that incorporated the site on ASOB 1 and ASOB 2 where 3-methyl-2-hexenoic acid was bound. A "look alike" or mimic molecule with the binding site incorporated and present in large excess relative to the natural precursor in the axillae would inhibit odor production. Further, if the molecule bound at the active site was relatively odorless and/or bacteriocidal when released, an added benefit could be realized since bacteria would limit their own growth.

To test these postulates, the inventors first synthesized 3-methyl-2-hexenoic-alanine (I) , shown below:

I If this molecule behaves as a mimic and is hydrolyzed in a similar manner as ASOB 1 and 2, (E)- and (23)- 3-methyl-2-hexenoic acids should be released and detected by olfaction. Using techniques similar to those employed in a previous study for determining the similarity between 3- methyl-2-hexenoic acid and the entire axillary extract (Zeng et al., "Analysis of the Characteristic Odors from the Human Male Axillae," 17 J. CHEM. ECOL. 1469 (1991)), samples of this 3-methyl-2-hexenoic-alanine were incubated with standard cultures of the coryneform bacteria known to cause axillary odor. Three judges, in a blind fashion, evaluated the odor of the following samples:

(a) culture medium; (b) culture medium and coryneform bacteria; and

(c) culture medium, bacteria, and 125μg of 3-methyl- 2-hexenoic-alanine.

Each of the samples was quite odoriferous due to the presence of volatiles from the culture medium; however, each of the judges were able to correctly pick (c) as having an axillary/sweaty odor discernable above background. These results suggest that the bacteria could hydrolyze 3-methyl-2- hexenoic acid from the 3-methyl-2-hexenoic-alanine. Further, it suggests that the postulated mimic would act to competitively inhibit the endogenous odor precursor. The present invention, however, is not limited to competitive inhibitions, but rather encompasses general inhibition of body odor.

In one embodiment of the compositions of the present invention, 10-undecenoic acid was incorporated into the precursor mimic. This acid had been identified (Peak FF in Table 1, above) in the combined male extract. It is also well known as a fungicide, since it is an active ingredient in some antifungal medications and is suggested to have some antimicrobial properties (Hunting, "Properties and Uses of Undecylenic Acid and Derivatives," 96 COSMET. & TOILETRIES 29

(1981)). Thus, its inclusion as the molecule initially bound in the precursor mimic but released by the bacteria, acts to retard the bacteria themselves.

In addition to the 10-undecenoic acid, in one embodiment of the present invention, alanine, which is one of several amino acids present in eccrine sweat (Gitlitz et al., "Ion Exchange Chromatography of Amino Acids in Sweat Collected from Healthy Subjects during Sauna Bathing," 20 CLIN. CHEM.

1305 (1974)), was chosen to create a simple amido precursor mimic (10-undecenoic-alanine-methyl ester) , shown as II, below.

II

This mimic was produced as follows: Alanine methyl ester hydrochloride (5.53g, 30mmol) , 1-hydroxybenzo-triazole (4.05g, 30mmol) , 10-undecylenic acid (5.53g, 30mmol) and N- methylmorpholine (3.03g, 30mmol) were dissolved in dry tetra- hydrofurane (30ml) . The solution was stirred and cooled in an ice-water bath while dicyclohexylcarbodimide (6.40g, 31mmol) was added. Stirring was continued for one hour at 0°C and overnight at room temperature. The N,N-dicyclohexyl-urea that formed in the reaction was removed by filtration and the solvent was evaporated in vacuo. A mixture of ethyl acetate

(150ml) and a saturated solution of sodium bicarbonate in water (70ml) was added to the residue and the organic phase extracted with a 10% solution of citric acid in water (70ml) . The organic phase was again extracted with water (70ml) , then a saturated aqueous solution of sodium bicarbonate (70ml) , and then water again (70ml) . The solution was dried over anhydrous sodium sulfate, filtered, and evaporated to give a thick oil. The oil was purified by chromatography on a column of silica gel (230-400 mesh, 6θA) with hexane and ethyl acetate to give a pure product (6.7g, 83%). R_f = 0.38 in the mixture of hexane and ethyl acetate (1:1) . The melting point was 38-39.5°C.

The synthetic precursor mimic was first tested on the forearms of male volunteers (n=8) to determine if the compound was irritating to the skin. Ten (10) μl of a 1% solution (in EtOH) of the 10-undecenoic-alanine-methyl ester mimic was applied to a 5cm area of the subject's forearm and occluded for 24 hours. No irritation was noted on any of the subjects. Next, the 10-undecenoic-alanine-methyl ester mimic was tested directly against triclosan, a commonly used antibacterial ingredient used in a variety of deodorant products and soaps, to determine the mimic's antimicrobial action against each subject's axillary bacteria. Each subject had their forearms scrubbed with detergent, dried, and lOμl each of solvent (EtOH) , 1% triclosan, 1% mimic, and 2% mimic

2 placed in a different 5cm area and allowed to dry. The concentration of triclosan employed was 2-4 times the amount that is typically used in most deodorant products. Sterile cotton-tipped swabs were then dipped in sterile sampling solution (0.075 M phosphate buffer, pH 7.9, containing 0.1% Tween-80) and used to "scrub" the axillary region of the subject to harvest a representative array of axillary flora. This swab was then rubbed over one of the four forearm test sites. By repeating this procedure for each test site, the translocation of the axillary flora was accomplished for testing each product. After a 24 hour occlusion, each area was sampled for the type and number of viable axillary bacteria using a method described by Larson et al., "Composition and Antimicrobic Resistance of Skin Flora in Hospitalized and Healthy Adults," 23 J. CLIN. MlCROBlOL. 604

(1986) . Turning now in detail to the drawings; the results of this experiment are shown in Figures 1 and 2, which are histograms of the number of colony-forming units (CFU) of total aerobes (Fig. 1) or diphtheroid bacteria (Fig. 2) for each of the four test sites. These data demonstrate that both total aerobes and diphtheroids were significantly reduced in the low (1%) and high (2%) treatments with precursor mimic versus both the control site and triclosan (1%) treated site (see Tables 3 and 4) .

TABLE 3

Log of the Mean Colony Forming Units (CFU) of Total Aerobes on Forearm

Mean Std. Dev. Std. Error

TABLE 4

Log of the Mean CFU of Diphtheroids on Forearm

Mean Std. Dev. Std. E ro

An analysis of variance with repeated measures showed a significant difference between treatments in the reduction of both total aerobes (F = 17.087; p < 0.0001) and diphtheroids (F = 14.661; p < 0.0001) . Both the low and high concentrations of mimic had significantly less aerobes and/or diphtheroids present than did the control site or the triclosan-treated site.

The 10-undecenoic-alanine-methyl ester mimic was then comparatively tested in the axillae of male volunteers against triclosan. Subjects who volunteered for the study

(n=12) were asked to wash only once per day, in the morning, with Ivory soap and not to use any deodorant preparations and/or colognes in the underarm region. Two days after commencing this protocol, the axillary microflora residing in the left and right axillae of each subject was collected to measure their type and abundance. Following this sampling of microflora, the subjects remained on the protocol and were randomly assigned to receive a 1% ethanol solution of either triclosan or the 10-undecenoic-alanine-methyl ester mimic in either axillae.

As with the earlier experiment, the concentration of triclosan employed (1%) was 2-4 times that used in deodorant products. Application of the aerosoled solutions/ pump sprays was done twice a day in a double-blind fashion. The first application occurred in the morning between 8:30- 9:30 a.m., the second between 1-2 p.m. The subjects followed this protocol for four consecutive days; on the fifth day, they received the morning application, although the organoleptic evaluation of each axillae was performed in the afternoon.

The subjects were evaluated by a panel of seven judges who did not know which axillae contained triclosan and which contained mimic. Axillary odor donors were assigned a number from 1-12 and asked to present themselves to the odor judges one at a time in a rotating fashion. Donors placed their left or right hand behind their heads in succession at the request of the odor judge. Each judge sampled the donors' axillae in a counterbalanced fashion asking first for the left or right axillae, then the opposite presentation from the next donor. Each of the 7 judges sampled each axillae in succession and was required to assign one axillae or another as having a stronger odor.

Following organoleptic evaluation, the type and abundance of axillary microflora were again measured using the technique of Larson et al. (Larson et al., "Composition and

Antimicrobic Resistance of Skin Flora in Hospitalized and Healthy Adults," 23 J. CUN. MiCROBlOL. 604 (1986).) The results of these experiments are summarized in Tables 5 and 6.

TABLE 5 Organoleptic Ratings of Left and Right Axillae

More Odorous Arm More Effective Product* Subject L R (A or B) Score

*A = 1% triclosan; B = 1% synthetic precursor mimic.

TABLE 6a

Mean Colony Forming Units of Total Aerobes or Diphtheroids in Treatment Groups*

*A = triclosan; B = Precursor mimic.

As can be seen from Table 5, the organoleptic ratings show that the judges could not distinguish between the triclosan- and the mimic-treated axillae. Table 6 and the resulting histograms in Figures 3 and 4 show no significant difference in the abundance of axillary microflora.

The present invention, however, is not to be limited to this particular mimic embodiment. It is believed that a wide variety of amino acids can be used as the primary constituent to be esterized, in particular alanine, glycine, and serine, as well as dimers, trimers, and oligomers of these amino acids. The choice of this constituent will principally limited by the difficulty of synthesis.

Further, while the use of any operable bacteriocide as a substitute for R₁ in the above structure is contemplated, it is possible that such bacteriocides must be acids.

While the present invention is not so limited, the preferred precursor mimics are acid-alanine-esters having the general structure:

R, = 9-decenyl or C^^, where n > 9; R₂ = C_πH_2n.₁ where n > l; x 1

As another example of such a mimic, 10-undecenoic- alanine-dodecyl ester was also prepared, using the following steps. A solution of t-butoxy carbonyl (t-Boc)-L-alanine (5.68g, 30mmol) , dicyclo-hexylcarbodiimide (6.81g, 33mmol) , dodecanol (6.15g, 33mmol) , and4-dimethylaminopyridine (0.37g, 3mmol) in ethyl ether (120ml) was allowed to stand at room temperature overnight. The N,N-dicyclohexylurea was removed by filtration. The filtrate was washed seriatim with water (100ml) , 5% acetic acid (2 x 70ml) , and again with water (2 x 70ml) , and was dried over anhydrous sodium sulfate. The solvent was removed by water aspiration at 40°C and the concentrated oil was purified by column chromatography (silica gel) with hexane and ethyl acetate as eluents to give pure t- Boc-alanine-dodecyl ester (10.05g; 94%).

The above product was treated with 1 N solution of HC1 in ethyl ether (80ml) . After standing at room temperature for 48 hours, the crystalline hydrochloride salt was filtered, washed with ethly ether, and dried in vacuo over KOH pellets. The product melted at 106-107°C.

The purified HC1 salt (7.47g, 25.5mmol) was dissolvedwith 1-hydroxyl-benzotriazole (3.45g, 25.5mmol), 10- undecenoic acid (4.70g, 25.5mmol), and N-methyl-mopholine (2.58g, 25.5mmol) in dry tetrahydrofurane (30ml). The solution was stirred and cooled in an ice-water bath while dicyclohexylcarbodiimide (5.30g, 25.7mmol) was added to the solution with stirring. Stirring was continued for one hour at 0°C, and then overnight at room temperature. The N,N- dicyclohexylurea formed from the reaction was removed by filtration and the solvent was evaporated in vacuo. A mixture of ethyl acetate (200ml) and a saturated aqueous solution of sodium bicarbonate (70ml) was added to the residue. The organic phase was extracted with a 10% solution of citric acid in water (70ml) , water (70ml) , saturated sodium bicarbonate (70ml) , and water (70ml) . The solution was dried over anhydrous sodium sulfate, filtered, and evaporated to give 10- undecenoic-alanine-dodecyl ester. Crystallization from hot ethanol afforded a pure product melting at 72-73°C (8.85g, 82%) .

Thus, the present invention provides an inventive method for deodorancy that preferably introduces one or more of the prepared mimics of the naturally-occurring precursors to 3-methyl-2-hexenoic acid into the axillary region of humans. Nevertheless, although the inventors have named the precursors to 3-methyl-2-hexenoic acid, the proteins that have been isolated, characterized, and discussed most likely also carry several of the other acidic molecules that are present in the axillary odor bouquet. For example, hydrolysis of the total protein mix isolated from apocrine secretion yields not only large quantities of 3-methyl-2-hexenoic acid, but smaller quantities of C₆ - C₁₀ straight chain acids as well as C₈ unsaturated acid (7-octenoic acid) . Consequently, in isolation of the singular proteins and the subsequent hydrolysis of such proteins, only the 3-methyl-2-hexenoic acid is seen, although the inventors have seen in their chronamo- grams small but barely detectable amounts of the other acids. Thus, it is speculated that the two proteins are actually binding proteins for a variety of the acids present in the axillary odor mix. Embodiments of this invention can mimic the precursor to one or more of these acidic components, or other odorants, and the invention is not limited to precursor mimicking of 3-methyl-2-hexenoic acid. The present invention also contemplates inhibition of foot odors utilizing the same method of precursor mimicking.

The preferred embodiments of the precursor mimic incorporate molecules that are already present in the axillary secretions, thereby taking advantage of the non-odorous nature of C₉ to C₁₈ saturated acids and the mild anti-bacterial nature of 10-undecenoic acid. These compounds, when bound to an ester of alanine, form amides that appear to interact with the axillary bacterial enzymes and both: (a) inhibit their interaction with endogenous precursor; and (b) kill bacteria upon liberation of 10-undecenoic acid.

The bacteriocidal ability of the mimics is seen best in the experiment involving translocation of the axillary bacteria to the forearm. In this experiment the site is occluded and there are no factors, such as considerable eccrine sweating, that may remove the precursor mimic from the area. Such an effect may have contributed to the precursor 10-undecenoic-alanine-methyl ester mimic being only equal in activity with triclosan in the axillae, but markedly effective on the forearm. In addition, the axillae is a much richer environment for bacterial growth with a variety of substrates present for the bacteria to utilize. This would account for similar growth in the two axillae — a situation that could be altered by increasing the percent of mimic used.

The deodorant compositions incorporating the precursor mimics of this invention can be formulated by any of a wide variety of methods that are known in the art. The inventive composition may contain, in addition to the mimic, other known deodorant compounds, fragrances, and/or antiperspirant compounds in a non-toxic, cosmetically and/or dermatologicalϊy acceptable vehicle. The composition can be formulated in a variety of forms including sticks, roll-ons, aerosol sprays, pump sprays, creams, lotions, solutions, pads, or even in a detergent cream, solution, or bar. See Calogero, "Anti-perspirants/Deodorants," SoAP/CoSMETJCHEM. SPECIALTIES 29 (Nov. 1986) , the disclosure of which is incorporated by reference, for descriptions of typical deodorant/anti¬ perspirant compositions. Examples of non-toxic, cosmetically, or der atologically acceptable vehicles are well known and include polyhydric alcohols such as glycerine, propylene glycol, butylene glycol, polyethylene glycol; small emollient oils such as isopropyl myristate, isopropyl palmitate, octyl palmitate, fatty alcohols, fatty amides, ethoxylated or propoxylated fatty alcohols or acids, fatty glycerides or silicone; hydrocarbons; fluorinated hydrocarbons' cyclomethicones; water; and monohydric alcohols such as ethanol, isopropanol, or methanol. Further, it is obviously preferable to apply at least a deodorizing amount of one or more of the mimics to the body location to be deodorized, e.g., the axillary region. The quantity of mimic constituting a deodorizing amount, however, will vary from subject to subject and application to application and also will depend upon the other components of the deodorizing composition. Nevertheless, it is believed that greater concentrations of the inventive precursor mimics can be safely used than the concentrations of triclosan presently used in available deodorants. Triclosan is a synthetic chemical. The industry has been reluctant to incorporate it into deodorant compositions in amounts greater than about 0.25 weight percent for fear of the effects of excessive absorption into the body. The precursor mimics of the present invention, on the other hand, are generally manufactured from ingredients already present in the natural secretions of the body. Thus, there is less danger in utilizing the mimics in higher concentrations. In general, a typical deodorant composition of the types discussed above making use of the present invention would incorporate about 1-10 weight percent of the inventive precursor mimics. A preferred composition according to this invention comprises from 1-4 weight percent of the mimic, either alone or in combination with other mimics, and a most preferred composition would include about 2 weight percent of the mimic.

In one novel application of the deodorant compositions of this invention, fabrics to be used in the making of work or military uniforms, suits for use in defending against chemcial warfare, or any other clothing wherein the wearer will typically be prone to excessive sweating can advantageously be soaked in a solution including a precursor mimic in accordance with the present invention prior to use or manufacture. The mimic will then be present in the clothing to competitively inhibit the production of body odor during periods when the wearer sweats.

Thus, compositions and methods for effectively deodorizing axillary regions are disclosed. While embodiments and applications of this invention have been shown and described, it would be apparent to those skilled in the art that many more modifications are possible without departing from the inventive concepts herein. The invention, therefore, is not to be restricted except in the spirit of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. Adeodorant composition comprisingan effective deodorizing amount of at least one precursor mimic to the precursor of a body odor in a warm-blooded animal to be suppressed.

2. The deodorant composition of claim 1 wherein the precursor mimic is comprised of an ester of an amino acid, such as alanine, glycine, or serine, that is bound to an acid- amido group.

3. The deodorant composition of claim 1 wherein said one or more precursor mimics are selected from the group consisting of mimics that are acid a ido esters of the structure:

wherein R₁ is 9-decenyl or C_nH_2n_₁ where n > 9, R₂ is C_nH_2n.₁ where n > 1, and x is > l.

4. The deodorant composition of claim 1 wherein the precursor mimic includes a bacteriocidal component such as 10-undecenoic acid that is released upon interaction with bacteria resident in the odorous body portion.

5. The deodorant composition of claim 1 wherein at least one of said precursor mimics is 10-undecenoic-alanine methyl ester or 10-undecenoic-alanine dodecyl ester.

6. The deodorant composition of claim 1 wherein the precursor mimic is dissolved or suspended in combination with anon-toxic, cosmetically, ordermatologically acceptable vehicle.

7. The deodorant composition of claim 1 wherein said one or more mimics comprise from about 1 to about 10 weight percent of said composition.

8. A deodorant composition comprising:

(a) an effective deodorizing amount of at least one precursor mimic selected from the group consisting of mimics that are acid amido esters of the structure:

wherein R₁ is 9-decenyl or C^^ where n ≥ 9, R₂ is ^c _n ^H _2n-ι where n > 1, and x is ≥ 1; and (b) one or more additives selected from fragrances and antiperspirant compounds, wherein said one or more mimics comprise from about 1 to about 10 weight percent of said composition and are dissolved or suspended in combination with a non-toxic, cosmetically, or dermatologically acceptable vehicle.

9. A fabric that has been soaked in, or coated with, a deodorant composition comprising an effective amount of at least one precursor mimic to the precursor of a body odor in a warm-blooded animal to be suppressed.

10. A method of suppressing body odor such as 3- methyl-2-hexenoic acid in a warm-blooded animal comprising contacting an odorous body portion of said animal such as the axillary or foot region with the deodorant composition of any of claims 1-8. AMENDED CLAIMS

[received by the International Bureau on 8 March 1993 (08.03.93); original claim 1 amended; other claims unchanged (1 page)]

1. Adeodorant composition comprising an effective deodorizing amount of at least one precursor mimic to the precursor of a body odor in a warm-blooded animal to be suppressed, wherein the precursor mimic is comprised of an amide of an amino acid.

3. The deodorant composition of claim 1 wherein said one or more precursor mimics are selected from the group consisting of mimics that are acid amido esters of the structure:

wherein , is 9-decenyl or C,,^,,., where n > 9, R₂ is C_nH^., where n > 1, and x is > 1.

6. The deodorant composition of claim 1 wherein the precursor mimic is dissolved or suspended in combination STATEMENT UNDER ARTICLE19

Claim 1 is amended in a manner similar to an amendment recently made to the United States priority application to now include the phrase "wherein the precursor mimic is comprised of an amide of an amino acid". This amendment was made in order to more appropriately claim the invention. It is not believed that this amendment affects the specification or drawings in any way.