R¹ is a carbon-carbon single bond or a straight chain or branched chain alkylene; R² is a carbon-oxygen single bond, or a straight chain or branched chain alkylene; R³ is a straight chain or branched chain alkyl, a cycloalkyl, an alkcycloalkyl, an alkenyl, an aryl or an aralkyl; each R⁴ is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkcycloalkyl, cycloalkyl, alkoxy, alkcycloalkoxy, cycloalkoxy, acyl, acyloxy and halogen; and each R⁵ independently alkyl, or in the case of the acetals of Formula

II the two R⁵S together with the atoms to which they are attached form a heterocycloalkyl. Also provided by the present invention are methods of treating rosacea by topically administering the rosacea-effective agent and one or more compounds of Formula I, II, and/or III. The methods provide that either the rosacea-effective agent is topically administered to the patient followed by topical administration of the oxy group-bearing aromatic aldehyde, the two materials are applied in the opposite order or the rosacea- effective agent and the oxy group-bearing aromatic aldehyde are topically administered simultaneously.

In addition, the invention provides kits for coadminstration of the aldehyde and the rosacea-effective agent as separate topical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : A schematic diagram illustrating inflammatory processes in the skin and showing the relationship of inflammation to the release of various proteins.

Figure 2: A repeat of Figure 1 illustrating those inflammatory processes which are effectively treated using the present invention.

Figure 3 and Figures 4A and 4B: Bar graphs which show the effects of aldehydes employed in the compositions of this invention on interleukin 1 (IL-l)-induced prostaglandin E2 (PGE₂) expression in dermal fibroblasts.

Figure 5: A bar graph which shows the effects of aldehydes employed in the compositions of this invention on tetradecanoyl phorbol acetate (TPA)-induced PGE₂ expression in keratinocytes.

Figure 6: A table shows the effects of aldehydes employed in the compositions of this invention and other related compounds on expression levels of various proteins in fibroblasts challenged with IL-I or UV light. Figure 7: A table which shows the effects of aldehydes employed in the compositions of this invention and other related compounds on expression levels of various proteins in keratinocytes challenged with TPA or UV light.

Figure 8A, 8B₅.9 A, 9B; 1OA, 1OB, HA and HB: Bar graphs of data tabulated in Fig. 6. Figures 12A, 12B, 13A, 13B, 14A and 14B: Bar graphs of data tabulated in Fig. 7. Figures 15A and 15B: Bar graphs of data obtained in Example 9 of an in vivo test of lotion Formulation 1 in the treatment of rosacea.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

When describing the aromatic oxy group-bearing aldehyde compounds and the rosacea effective agents, employed in the cosmetic and pharmaceutical compositions, kits and methods of this invention, the following terms have the following meanings:

"Aromatic aldehyde" refers to compounds that contain an aryl ring and an aldehyde group or an aldehyde group protected as an acetal or hemiacetal pendent from the ring.

"Acyl" refers to the group -C(O)R where R is hydrogen, alkyl or aryl. When R is hydrogen this is a "formyl", when R is CH₃ this is "acetyl".

"Acyloxy" refers to the group -O- Acyl. "Alkyl" refers to monovalent saturated aliphatic hydrocarbon groups preferably having from 1 to about 20 carbon atoms, more preferably from 1 to 12, even more preferably 1 to 8 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, π-butyl, isobutyl, tert-butyl, «-hexyl, n-octyl, tert-octyl and the like. The term "lower alkyl" refers to alkyl groups having 1 to 6 carbon atoms and especially 1 to 4 carbon atoms.

"Substituted alkyl" refers to an alkyl group, preferably of from 1 to about 20 carbon atoms, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, cycloalkyl, cycloalkoxy, acyl, aminoacyl, amino, aminocarbonyl, cyano, halogen, hydroxyl, carboxyl, keto, thioketo, alkoxycarbonyl, thiol, thioalkoxy, aryl, aryloxy, nitro, -OSO₃H, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO₂- alkyl, -SO₂-substituted alkyl, -SO₂-aryl, and mono- and di-alkylamino, mono- and di- arylamino, and unsymmetric di-substituted amines having different substituents selected from alkyl, substituted alkyl and aryl, and where appropriate, pharmaceutically acceptable salts thereof. "Alkenyl" refers to monovalent unsaturated aliphatic hydrocarbon groups having from 1 to 20 carbon atoms and preferably 1 to 6 carbon atoms and 1 to 2 and especially 1 olefinic unsaturation.

"Alkylene" refers to divalent saturated aliphatic hydrocarbon groups preferably having from 1 to 20 carbon atoms and more preferably 1 to 6 carbon atoms which can be straight chain or branched. This term is exemplified by groups such as methylene (-CH₂-), ethylene (-CH₂CH_2-), the propylene isomers (e.g. -CH₂CH₂CH₂- and - CH(CH₃)CH₂-) and the like.

"Alkcycloalkyl" refers to -alkylene-cycloalkyl groups preferably having from 1 to 20 carbon atoms in the alkylene moiety and from 3 to 8 carbon atoms in the cycloalkyl moiety. Such alkcycloalkyl groups are exemplified by -CH₂-cycloproρyl, -CH₂- cyclopentyl , -CH₂CH₂-cyclohexyl, and the like.

"Alkcycloalkoxy" refers to -O-alkylene-cycloalkyl groups preferably having from 1 to 20 carbon atoms in the alkylene moiety and from 3 to 8 carbon atoms in the cycloalkyl moiety. Such alkcycloalkyl groups are exemplified by -OCHrcyclopropyl, - OCH₂-cyclopentyl , -OCH₂CH₂-cyclohexyl, and the like.

"Alkoxy" refers to the group "alkyl-O-". Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec- butoxy, /t-pentyloxy, 7?-hexyloxy, 1,2-dimethylbutoxy, and the like. "Alkoxycarbonyl" refers to the group -C(O)OR where R is alkyl.

"Aminocarbonyl" refers to the group -NRC(O)R where each R is independently hydrogen or alkyl.

"Aminoacyl" refers to the group -C(O)NRR where each R is independently hydrogen or alkyl. "Aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like. Unless otherwise constrained by the definition for the individual substituent, such aryl groups can optionally be substituted with from 1 to 3 substituents selected from the group consisting of alkyl, alkoxy , alkaryloxy, alkenyl, alkynyl, amino, aminoacyl, aminocarbonyl, alkoxycarbonyl, aryl, carboxyl, cycloalkoxy, cyano, halo, hydroxy, nitro, trihalomethyl, thioalkoxy, and the like, and where appropriate, pharmaceutically acceptable salts thereof. "Aralkyl" refers to the group -alkylene-aryl groups and is most typically benzyl.

"Aryloxy" refers to -O-aryl groups wherein "aryl" is as defined above. "Carboxyl" refers to the group -C(O)OH. "Cyano" refers to the group -CN.

"Cycloalkyl" refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings, including fused and bridged ring systems, which can be optionally substituted with from 1 to 3 alkyl groups. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

"Cycloalkoxy" refers to -O-cycloalkyl groups. Such cycloalkoxy groups include, by way of example, cyclopentyloxy, cyclohexyloxy and the like.

The term "rosacea-effective agent" refers to several categories of agents. One category is agents that are pharmaceuticals or cosmeceuticals that have been shown to be effective in treating rosacea, but may have adverse event profiles that would benefit from the addition of one or more aldehydes of the invention. These agents can include agents that were not given approval or are not currently in use because of adverse effects profiles, but have been demonstrated to have effective activity against rosacea. These include, but are not limited to clindamycin, doxycycline, erythromycin, minocycline hydrocholoride, tetracycline hydrochloride, azelaic acid, sodium sulfacetamide, nicotinamide, benzoyl peroxide, salicylic acid, adapalene, isotretinoin, tazarotene, tretinoin and metronidazole, some or all of which have certain adverse effect profiles with include dryness, itchiness, flaking of the skin, peeling erythema. In one embodiment, the rosacea-effective agent is metronidazole.

Another category includes agents that are not effective alone in treating certain patients' rosacea but that are effective when combined with one or more aldehydes of the invention. It is contemplated that this group of agents includes, but is not limited to, trimethoprim-sulfamethoxazole, methotrexate, dapsone, primaquine, chloroquine, and oral prednisone.

"Isolated," when used to define the state of purity of the aromatic aldehyde compounds used in the practice of this invention, means that the aromatic aldehyde has been substantially freed of (i.e. at least about 90% and especially at least about 95% freed of) or separated from related feedstocks, co-products, or in the case of naturally-occurring mixtures, related materials with which the aldehyde appears in nature.

"Pharmaceutically-acceptable topical carrier" and equivalent terms refer to an inactive liquid or cream vehicle capable of suspending or dissolving the aromatic aldehyde and the rosacea-effective agent and having the properties of being nontoxic and noninflammatory when applied to the skin. This term is specifically intended to encompass carrier materials approved for use in topical cosmetics. Representative carriers include water, oils, both vegetable and mineral, cream bases, lotion bases, ointment bases and the like. These bases include suspending agents, thickeners, penetration enhancers, and the like. Their formulation is well known to those in the art of cosmetics and topical pharmaceuticals. Additional information concerning carriers can be found in Part 8 of Remington's Pharmaceutical Sciences, 17^th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

"Therapeutically effective dose" refers to a dose of a composition of this invention which, when applied topically to the skin of a patient afflicted with rosacea results in an observable improvement in the patient's condition. This is also referred to herein as a "effective rosacea-treating amount."

"Topical", when used to define a mode of administration, refers to a material that is administered by being applied to the skin. "Topically effective" refers to a material that, when applied to the skin, produces a desired pharmacological result either locally at the place of application or systemically as a result of transdermal passage of an active ingredient in the material.

B. Compositions

1. The Oxy Group-bearing Aromatic Aldehydes

The formulations of the present invention comprise oxy group-bearing aromatic aldehydes compounds of Formula I as well as their acetal and hemiacetal equivalents shown in Formulas II and III. At this time, the base aldehydes of Formula I are preferred. Preferably, in the aromatic aldehyde compounds of Formula I above, R¹ is selected from the group consisting of a carbon-carbon single bond, methylene and ethylene. More preferably, R¹ is a carbon-carbon single bond.

Preferably, R² is selected from the group consisting of a carbon-oxygen single bond, methylene and ethylene. More preferably, R² is a carbon-oxygen single bond.

Preferably, R³ is a 2 to 6 carbon alkyl.

The four R⁴S are most commonly hydrogen, alkyl or alkoxy. hi this case, generally at least about two of the R⁴S are hydrogen.

Preferably, each R⁵ is independently alkyl, or in the case of the acetals of Formula II, the two R⁵S together with the atoms to which they are attached form a heterocycloalkyl. More preferably each of the R⁵S together with the atoms to which they are attached form 1,4-dioxacyclopentanyl or a substituted 1,4-dioxacyclopentanyl.

An especially preferred group of compounds of Formula I are those in which R¹ is a carbon-carbon single bond; R² is a carbon-oxygen single bond located in the 4 position on the aromatic ring relative to the aldehyde functionality, R³ is a 2 to 6 carbon alkyl and at least two R⁴S are each hydrogen.

In another of its preferred composition aspects, this invention is directed to cosmetic and pharmaceutical compositions comprising a suitable carrier and at least one additional rosacea-effective agent and one or more of the following oxy group-bearing aromatic aldehyde compounds: 2-ethoxybenzaldehyde, 4-allyloxy-benzaldehyde, A- ethoxybenzaldehyde, propoxybenzaldehyde, 4-butoxybenzaldehyde, A- pentyloxybenzaldehyde, and 4-hexyloxybenzaldehyde.

A particularly preferred aldehyde of the invention is 4-ethoxybenzaldehyde (4-EB).

2. The Additional Cosmetically or Pharmaceutically Effective Agent

While not being limited to any one theory it is believed that the combination therapy employing the aldehydes described herein will be helpful to reduce the redness, itching or other irritation that often results from rosacea-effective agents. It is also contemplated that the combination of the rosacea-effective agent and the aldehyde may give the combination superior benefits than either material alone. As discussed above, the preferred rosacea-effective agent is metronidazole which has the following structure:

While the mechanism of action of metronidazole is unknown, metronidazole is known to have an anti-inflammatory effect on patients with rosacea.

C. General Synthetic Procedures

The aromatic aldehydes employed in the compositions, kits and methods of this invention are either known compounds or are compounds that can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e. reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

For example, such compounds are readily prepared by acylation of the corresponding aryl compound with the appropriate acyl halide under Friedel-Crafts acylation reaction conditions. Additionally, the formyl compounds, i.e. those compounds where R⁴ is hydrogen, can be prepared by formulation of the corresponding aryl compound using, for example, a disubstituted formamide, such as N-methyl-N-phenylformamide, and phosphorous oxychloride (the Vilsmeier-Haack reaction), or using Zn(CN)₂ followed by water (the Gatterman reaction). Numerous other methods are known in the art for preparing such aryl carbonyl compounds. Such methods are described, for example, in I. T. Harrison and S. Harrison, Compendium of Organic Synthetic Methods, Wiley, New York, 1971, and references cited therein.

Certain aromatic aldehyde compounds of Formula I can also be prepared by alkylation of the corresponding aryl hydroxy compound (e.g. 4-hydroxybenzaldehyde and the like). This reaction is typically conducted by contacting the aryl hydroxy compound witή a suitaoie oase, sucn as an alkali or alkaline earth metal hydroxide, fluoride or carbonate, in a inert solvent, such as ethanol, DMF and the like, to deprotonate the hydroxyl group. This reaction is generally conducted at about 0°C to about 50°C for about 0.25 to 2 hours. The resulting intermediate is then reacted in situ with about 1.0 to about 2.0 equivalents of an alkyl halide, preferably an alkyl bromide or iodide, at a temperature of from about 25⁰C to about 100⁰C for about 0.25 to about 3 days.

Additionally, various aromatic aldehydes of Formula I can be prepared by reduction of the corresponding aryl nitriles. This reaction is typically conducted by contacting the aryl nitrile with about 1.0 to 1.5 equivalents of a hydride reducing agent, such as LiAlH(OEt)₃, in an inert solvent such as diethyl ether, at a temperature ranging from about -78° to about 25⁰C for about 1 to 6 hours. Standard work-up conditions using aqueous acid then provides the corresponding aryl aldehyde.

The aromatic aldehydes of Formula II and III employed in the compositions and methods are either known compounds or compounds that can be prepared from known compounds by conventional procedures. The hemiacetals can be formed by either acid or base catalyzed reaction of the corresponding aldehyde with and alcohol. If a single equivalent of the alcohol is added to the carbonyl, the hemiacetal is formed. Addition of 2 equivalents of an alcohol to the carbonyl produces the acetal. Acetal formation is acid catalyzed and is typically conducted by adding 1 mole of aldehyde and a 0.1 mole of aldehyde and a 0.1 mole of CaCl₂ to 1.9 mole of ethanol. The reaction mixture is held at room temperature for 1 to 2 days. Standard work up conditions provide the acetal protected aromatic aldehyde.

D. Pharmaceutical and Cosmetic Compositions and Their Use

The compositions containing a combination of oxy group-bearing aromatic aldehydes and rosacea-effective agent are administered in the form of pharmaceutical or cosmetic compositions. In one embodiment, the agents are coadministered as two separate compositions. Preferably, the agents are coadministered as a single composition. Such compositions can be prepared in manners well known in the pharmaceutical and cosmetic arts.

Generally, the compositions of this invention are administered in a cosmetic amount or a therapeutically effective dose. The amount of the aldehyde compound and rosacea-effective agent actually administered in therapeutic settings may typically be determined by a physician, in the light of the relevant circumstances, including the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's rosacea symptoms, and the like, hi cosmetic settings the amount to be applied is selected to achieve a desired cosmetic effect.

The cosmetic and pharmaceutical compositions of this invention are to be administered topically. In a primary application, this leads to the aldehyde and the other active agent working upon and treating the skin.

In such compositions, the total amount of the one ore more aromatic aldehyde compounds is usually a minor component (from about 0.001 to about 20% by weight or preferably from about 0.01 to about 15% by weight), and the rosacea-effective agent(s) is present in similar amounts and especially from about 0.2 to about 15% by weight and especially from about 0.5 to about 10% by weight, with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form. This proportions apply to compositions containing only one or both of the aromatic aldehyde and the rosacea-effective agent.

Topical cosmetic forms and topical pharmaceutical dosing forms can include lotions, shampoos, foams, soaks, gels, creams, ointments and pastes. Lotions commonly employ a water and oil base. Gels are semi-solid emulsions or suspensions. Creams generally contain a significant proportion of water in their base, while ointments and creams are commonly more oily.

Liquid forms, such as lotions suitable for topical administration or suitable for cosmetic application, may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, thickeners, penetration enhancers, and the like. Solid forms such as creams or pastes or the like may include, for example, any of the following ingredients, water, oil, alcohol or grease as a substrate with surfactant, polymers such as polyethylene glycol, thickeners, solids and the like. Liquid or solid formulations may include enhanced delivery technologies such as liposomes, microsomes, microsponges and the like. The above-described components for liquid, semisolid and solid topical compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17^th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

The aldehyde-containing compositions of this invention can also be administered in sustained release transdermal forms or from transdermal sustained release drug delivery systems. A description of representative sustained release materials can be found in the incorporated materials in Remington's Pharmaceutical Sciences.

When it is desired to provide the one or more aromatic aldehyde as a first composition and the one or more rosacea-effective agents as a separate second composition, the two compositions are preferably and most conveniently provided as a kit suitable to effect and facilitate proper coadministration. Such kits comprise a first container containing a composition of this invention containing the one or more aromatic aldehydes and a second container containing the composition of the invention containing the one or more rosacea- effective agents.

In one embodiment the kit includes instructions informing the patient of the amounts of one or both of the two compositions to apply and/or how to apply them.

In another embodiment the kit includes instructions the patient of the order in which to apply the two compositions.

In another embodiment the kit provides one or both of the compositions in a unit dosage form which delivers an amount of the composition(s) to be effective treating rosacea (for example from about 0.25 to about 40.0 cc and especially 0.5 to about 30.0 cc and particularly about 1 to about 30.0 cc of either or both compositions).

The following formulation example illustrates representative cosmetic and pharmaceutical compositions of this invention and their use.

Formulation 1 -Aldehyde-Containing Skin-Treating Cream for Rosacea

A commercial mineral oil-water cold cream base is obtained. To 100 grams of this base, 1.0 gram of a compound of Formula I, 4-ethoxybenzaldehyde and 2.0 grams of niacinamide is added with continuous mixing and stirring to yield a cosmetic or pharmaceutical cream composition. This composition includes the following: deionized water (57.6% by weight); glycerin (4.0%); phenonip (1.0%); propylene glycol (5.0%); transcutol (3.2%); jojoba oil (3.5%); isocetyl alcohol (2.0%); isocetyl stearate (3.5%); mineral oil (3.0%): 4-ethoxybenzaiαenyαe i,i.υ%); isostearyl palmitate (3.0 %); PEG-7 glyceryl cocoate (2.0%); Glycereth-7 (2.0%); POLYS ORB ATE-20™ (0.2%); cetyl ricinoleate (1.0%); glyceryl stearate/PEG-100 stearate (4.0%); and SEPIGEL™ 305 (1.5%); and niacinamide (2.0%).

E. Utility and Dosing

The compositions and methods of this invention can be used topically to treat rosacea.

In these applications the cosmetic and pharmaceutical compositions are administered topically to achieve a desired cosmetic effect or a topical therapeutic effect. In one aspect of the invention, the aldehyde and the rosacea-effective agent are co-formulated and may be administered to the patient from a single vial or other container. In other aspects of the invention, the aldehyde is first applied and then the rosacea-effect agent is applied (or vice versa), and thus the aldehyde and the rosacea- effective agent are separately formulated. In these uses the dose levels or application levels can be expressed in terms of the amount of active aromatic aldehyde and other active ingredients delivered to the skin. For example, 1 to about 5 doses or applications per day, each containing from about 0.001 grams to about 1 gram of active aldehyde and similar amounts of the other active ingredients. Alternatively, dose levels can be expressed in terms of the volume of formulated composition administered. For example, 1 to about 5 doses or applications per day, each containing from about 1 to about 30 grams of composition containing from about 0.01 % to about 10% by weight of active aldehyde and especially from 0.02% to about 15% by weight, and similar amounts of the rosacea-effective agent.

F. Biology and Testing

The examples include a number of in vitro studies to investigate the ability of the aldehydes used in these combination products to block various inflammatory processes in the skin. For these studies primary human keratinocytes and dermal fibroblast cell strains have been used as well as THP-I monocytes and the Jurkat T-cell derived cell line. The in vitro experiments used to assess the anti-inflammatory activities of the aldehydes were selected on the basis of current knowledge about the skin inflammatory process. Fig. 1 depicts the events involved in cutaneous inflammation.

Inflammation in the skin is characterized by itching, pain, redness, swelling and, frequently, rough and flaky skin. These symptoms result from changes in blood flow to the site of inflammation, increased vascular permeability, the migration of immune cells from the circulation into the tissue, and the release of soluble mediators including cytokines, prostaglandins and chemokines.

A common finding in inflammation is that cells in the skin respond to inflammatory stimuli by activating either one of two intracellular signaling pathways (or in some cases both pathways). These pathways are commonly referred to as the Stress

Activated Kinase (SAK) pathway and the NF-kB pathway. The SAK pathway leads to the activation of the AP-I transcription factor, which then binds to and activates several inflammatory genes including COX-2, IL-6 and MCP-I. Activation of the NF-kB pathway results in NF-kB protein translocation to the nucleus and activation of NF-IcB driven inflammatory genes such as IL-8, MMP-I, TNF-α and the adhesion molecule, VCAM-I. Interestingly, many inflammatory genes including IL-I have promoter elements that bind both AP-I and NF-kB transcription factors and are thus regulated to some extent by both signaling pathways. The screening assays are designed to determine which pathway is blocked by the compound under investigation, or if both pathways are effectively inhibited. A compound with the capacity to block the transcription of inflammatory genes regulated by each of these pathways will likely provide significant anti-inflammatory effects when applied topically. For each putative anti-inflammatory compound under consideration the initial screening program concentrates on the following target sites for intervention:

Inhibiting the production of IL-I and PGE-2 in UVR or Tetradecanoyl . Phorbol Acetate-treated keratinocytes.

Inhibiting the production of PGE-2 in UVR treated dermal fibroblasts. Inhibiting the induction of PGE-2 in IL-I treated fibroblasts: Because one of the mostcommon activators of skin inflammation is sunlight, specifically UVB radiation, the determination of a compound's ability to block the induction of pro-inflammatory PGE-2 by UVR in both keratinocytes and fibroblasts , represents a logical first step in the screening process. In addition, because skin inflammation is often triggered by contact with chemical irritants or allergens, the use of

TPA, which is known to trigger an inflammatory response in the skin, provides an additional model tor tήe analysis of anti-inflammatory activities of test compounds. Finally, because IL-I is one of the most important mediators and propagators of inflammation and is rapidly induced by an inflammatory stimulus, such as UVR, determining the ability of a potential anti-inflammatory compound to block either the production or action of IL-I is a critically important initial screening study. As shown in Figs. 1 and 2, by blocking IL-I production from keratinocytes, not only is the activation of fibroblasts suppressed but the activation of mast cells is also blocked thus preventing the release of histamine and other inflammatory mediators. Furthermore, inhibition of IL-I production in the skin would prevent the activation of a large number of inflammatory genes that are stimulated solely by IL-I. These include COX-2, MMP-I, and a variety of cytokine and chemokine genes.

For all of the initial screening studies described herein, cells in culture are exposed to the appropriate agonist, (i.e. UVR, TPA or IL-I) and then incubated in medium for 24 or 48 hours in the presence or absence of the compound under investigation. At 24 and 48-hour time points, medium from the cells is removed and assayed for a number of inflammatory mediators by ELISA.

Only primary keratinocyte and fibroblast cell strains were used, not immortalized cell lines, for the screening studies. The use of normal cells from the skin increases the probability that results from in vitro studies will be predictive of effects of a given compound when applied topically. Aldehydes that are found to completely (100 %) suppress PGE-2 induction at a concentration of 100 micromolar or less are then subjected to more demanding response studies including the following sequence of experiments:

1. Assessment by ELISA of a compound' s ability to block a variety of UVR, TPA, or IL-I induced inflammatory mediators in keratinocytes and fibroblasts including IL-6, TNF- a, IL-8, and MMP-I .

2. Assessment by ELISA of a compound' s ability to block the production and secretion of inflammatory mediators by monocytes (THP-I monocyte line) stimulated by lipopolysaccharide (LPS) and by T lymphocytes (Jurkat cells) stimulated with an antibody ligand that activates the cells. The use of RPA (ribonuclease protection analysis) to determine if a compound is acting at the gene level to suppress the activity of specific inflammatory genes stimulated by exposure of cells to various agonists including UVR, IL-I, TPA₅ or LPS

(lipopolysaccharide). Cutanix has developed a customized RPA "cocktail" for keratinocytes, fibroblasts, 1 -cells, and monocytes to simultaneously measure the expression of cell-type specific inflammatory genes in cells stimulated with UVR, IL-I, TPA or LPS in the presence or absence of the compound under investigation.

The use of microarray gene analysis to simultaneously examine the effect of any compound on the expression of more than 5,500 genes specific for cells present in the skin. The gene arrays used were purchased from Research Genetics and provide read-outs on genes known to be expressed in the skin.

The aldehydes can suppress a number of pro-inflammatory mediators and

Fig. 2 identifies some of the events that are likely inhibited by the aldehydes in vivo (shown by the circled X).

EXAMPLES

The following examples are provided to further describe the invention and are not intended as limitations on the scope of the invention which is defined by the appended claims .

EXAMPLE 1

An initial in vitro experiment was conducted to demonstrate the activity of the aromatic aldehyde, 4-ethoxybenzaldehyde, ("4-EB") as a component of a topically- administered pharmaceutical or cosmetic combination product of this invention.

For this experiment, human skin fibroblasts were seeded into 12 well culture dishes at a density of 80,000 cells/wells in tissue culture medium and left overnight to attach to the dish. The next day, medium was removed and replaced with fresh medium containing either 1% ethanol as a diluent control, IL-I at a concentration of 500 picograms/ml, or IL-I plus 4-EB at either 250μM or 500μM. Cells were incubated for an additional 24 hours and at this time, the medium was removed and assayed by ELISA for the presence of PGE-2 in the culture medium. The results show that IL=I caused a 17.8 fold increase in PGE-2 (control = 727 ρg/10⁶ cells: IL-I = 12,976 ρg/10⁶ cells). However, cells treated with either concentration of 4-EB showed a complete inhibition of the IL-I induction of PGE-2. The percent inhibitions are as follows: 4-EB, 100 %, 6% and 10 % at 50μM, lOμM and 1 μM. EXAMPLE 2

Subsequent studies were carried out to determine the dose-response of human skin fibroblasts to 4-EB. 4-EB completely blocked the IL-I induction of PGE-2 at lOOμM, blocked 82% of the PGE-2 induction at 50μM, and blocked 35% at a concentration as low as lOμM. The results of the study are provided graphically in Fig. 3 A.

EXAMPLE 3

Similar in vitro studies as those described in Example 1 were run using human skin keratinocytes. The experimental set up was the same as described for Example 2, but replacing IL-I with tetradecanoyl phorbol acetate (TPA) at a concentration of 32 nM as the agonist. The percent inhibitions are as follows: 4-EB₅ 94.9 % and 79.9 % at lOOμM and 50μM.

EXAMPLE 4

Subsequent in vitro experiments were conducted to demonstrate the activity of other aromatic aldehydes compared to the 4-ethoxybenzaldehyde, ("4-EB") as topically- administered pharmaceuticals and cosmetics. The compounds tested were 2- ethoxybenzaldehyde (2-EB), 3-ethoxybenzaldehyde (3-EB), and 4-methoxybenzaldehyde (4-MB).

For this experiment, human skin fibroblasts were seeded into 12 well culture dishes at a density of 80,000 cells/wells in tissue culture medium and left overnight to attach to the dish. The next day, medium was removed and replaced with fresh medium containing either 1 % ethanol as a diluent control, IL-I at a concentration of 500 pico grams/ml, or IL-I plus one of the compounds under investigation at a concentration of 1, 10, 50 or lOOμM. Cells were incubated for an additional 24 hours and at this time, the medium was removed and assayed by ELISA for the presence of PGE-2 in the culture medium. The results show that IL-I cause a 4 to 22 fold increase in PGE-2.

Percent inhibitions as shown in the detailed results in Fig. 4A) are as follows: 2-EB, 82.9% and 58.9% at lOOμM and 50μM; 3-EB, 41.2% and 42.6% at lOOμM and 50μM; 4-EB, 81.5% at lOOμM. Concentrations of 10 or 50μM 4-MB did not appear to inhibit the IL-I induced production of PGE-2 in the fibroblasts. Percent inhibitions as shown in the detailed results of Fig. 4B) are as follows: 4-MB, 13.6 % and 16.2 % at 50μM and lOμM.

EXAMPLE 5

Similar in vitro studies as those described in Example 4 were run using human skin keratinocytes. The experimental set up was the same as described for Example 5 but replacing IL-I with tetradecanoyl phorbol acetate (TPA) at a concentration of 32 nM as the agonist. The compounds tested were 2-ethoxybenzaldehyde (2-EB), and 3- ethoxybenzaldehyde (3-EB) and 4-ethoxybenzaldehyde (4-EB) in concentrations of either 10, 50, or lOOμM. The results show that TPA caused a 3.5 fold increase in PGE-2. However, treatment with any of these compounds blocked PGE-2 production by at least 50% .

The percent inhibitions as shown in the detailed results in Fig. 5 are as follows: 2-EB₅ 83%, 76.6% and 55.2% inhibition at lOOμM, 50μM and lOμM; 3-EB, 76.7% and 57.7% at lOOμM and 50μM; 4-EB, 94.9% and 79.9% at lOOμM and 50μM.

EXAMPLE 6

In vitro experiments were conducted to demonstrate the activity of a series of aromatic aldehydes as agents in topically-administered pharmaceuticals and cosmetics. The compounds tested and the measured results are tabulated in Fig. 6 and shown graphically in Figs. 8-11. These data include results for aldehydes of Formula I and also include results for other related compounds.

For this experiment, human skin fibroblasts were seeded into 12 well culture dishes at a density of 80,000 cells/wells in tissue culture medium and left overnight to attach to the dish. The medium was then replaced with PBS for a challenge with either UV-light or with EL-I . After irradiation or introduction of IL-I, the PBS was removed and culture medium containing the appropriate compound (or DMSO for controls) was then added and the cells cultured for an additional 24 hours. At that time, the medium was removed and assayed by ELISA for the presence of PGE-2, IL- 1 , IL-6, IL-8, or MMP- 1 in the culture medium. The levels oi proxein in the conditioned medium were measured and reported as percent relative to diluent controls.

IL-I Challenge On the second day, the medium was removed and replaced with fresh medium containing either 1% ethanol as a diluent control, IL-I at a concentration of 500 picograms/ml, or IL-I plus one of the compounds under investigation at a concentration of 10O₅ 10, or lμM.

UV-Iight Challenge

On the second day, the medium was removed and replaced with fresh PBS for irradiation. The fibroblasts were then irradiated with 50 mJ of UVB. UVB irradiation was obtained by illuminating the samples with an FS-20 sunlamp through the lids of the multi-well plates in order to filter out the UVC radiation. After irradiation the PBS solution was removed and replaced with a solution containing either 1% ethanol as a diluent control, or one of the aldehyde compounds at a concentration of 100, 10, or lμM. The cells were incubated for another 24 hours and the medium was then removed for the ELISA assays and the cells were counted.

EXAMPLE 7

Similar in vitro studies as those described in Example 6 were run using human skin keratiiiocytes. The experimental set up was the same as described for Example 6. The products assayed by ELISA for the presence of PGE-2, IL-I, IL-6, IL-8, MMP-I or TNF-O! in the culture medium.

For the cells challenged by a biochemical agonist, IL-I was replaced with tetradecanoyl phorbol acetate (TPA) at a concentration of 32 nM. When UV-light was used to challenge the cells, they were exposed to 75 mJ of UVB, obtained by illuminating the samples with an FS-20 sunlamp through the lids of the multi-well plates in order to filter out the UVC radiation.

The compounds tested were in concentrations of either 100, 10, or lμM, and the protein expression levels are reported in percent inhibition relative to control treated cells. The measured percent inhibitions are tabulated in Fig. 7 and shown graphically in Figs. 12-14.

EXAMPLE 8

Because of the marked anti-inflammatory effects seen when 4-EB was used in human fibroblast cell culture models, in vivo studies were carried out to determine if topically applied 4-EB could block an inflammatory response in humans. While the details provided herein are for a specific compound, the same tests can be used on any of the aromatic aldehydes of the present invention.

Formulation 1, containing 4-EB, was used. This lotion was then tested by Franz cell percutaneous absorption analysis to determine how much 4-EB could penetrate human skin over a 24 hour period. The lotion formulation above provided a flux rate of 4-EB through human skin of 30-50 micrograms/hour.

This lotion was then tested to determine if it could prevent an inflammatory response when applied topically to human skin. For this study a lab volunteer was irradiated on a quarter sized spot on the inner forearm with 60-80 mJ of UVB light (a sunlamp). This dose was sufficient to cause a highly visible red erythema response. Immediately following irradiation on both arms, one arm was treated with the above 4-EB lotion while the other arm was treated with the same lotion formulation but with no 4-EB. Within 2-6 hours after irradiation the vehicle-treated arm developed a pronounced red erythema response at the site of irradiation while the 4-EB lotion treated spot did not. Even the next_. day, 14 hours post-irradiation, the spot treated with 4-EB showed no redness. This study demonstrates that topically applied 4-EB has marked anti-inflammatory activity.

EXAMPLE 9

Rosacea Clinical Study

Thirty subjects with mild to moderate rosacea were treated either with lotion of Formulation 1 containing 1% w 4-EB (20 subjects) or with a control lotion with the active material removed. The study was randomized and double blinded. During their first visit, patients were evaluated using 4 measurements of disease: 1) erythema, 2) desquamation (peeling), 3) uneven skin tone, and 4) dermatitis. The clinician also provided an "Overall Severity" score which ranged from 1-6 with 6 being the most severe level of overall disease. After evaluation patients were sent home with either the test lotion or the control lotion and told to apply it morning and evening for two weeks. They then returned to the clinic for a two-week evaluation and at that time received more product for an additional two weeks. At four weeks, both the clinician and the subjects evaluated the severity of their disease.

Of the 30 rosacea patients that started the study, 28 completed the four- week period. None of the subjects, including those who dropped out, experienced any irritation or other adverse effect from the product. The bar graph of Fig. 15A summarizes the percentage improvement in "Overall Severity" for the test lotion treated group at 4 weeks. As can be seen, the severity of rosacea decreased in 13/18 subjects (72%). Average improvement amount those responding was 68% (49% for all patients). This is a statistically significant result.

The bar graph of Fig. 15B summarizes the percentage improvement in "Overall Severity" for the control lotion treated group at 4 weeks. As can be seen, the severity of rosacea decreased in 6/10 subjects (60%) but increased in 3/10 (30%). Average overall improvement was 15 % which is not a significantly significant result.

The test lotion also achieved another important statistical threshold in the rosacea study. The degree of improvement in the test lotion treated group was significantly better than the degree of improvement in the control treated group (p=0.05) using both

Wilcoxon and Analysis of Variance statistics. These results are of sufficient quality to meet regulatory standards for drug efficacy and clearly establish the ability of 4- ethoxybenzaldehyde to suppress skin inflammation in humans.

Rosacea is a difficult disease to treat because of the severity of skin inflammation and vasodilation. Considering that a 2% formulation of 4-EB has been shown to be more effective in blocking UV-induced erythema than the 1% formulation used in this clinical study, a higher strength version of the test lotion may provide even greater efficacy in treating rosacea.

These results show that a lotion containing aromatic aldehyde could be used beneficially together with a rosacea-effective agent.

Claims

WHAT IS CLAIMED IS:

1. A composition for treating rosacea comprising: a pharmaceutically or cosmetically acceptable topical carrier, and as active ingredients, the combination of at least one oxy group-bearing aromatic aldehyde compound of Formula I₅ II or III:

wherein

R¹ is a carbon-carbon single bond or a straight chain or branched chain alkylene;

R² is a carbon-oxygen single bond or a straight chain or branched chain alkylene; R³ is selected from the group consisting of a straight chain alkyl, branched chain alkyl, cycloalkyl, alkenyl, alkcyclo alkyl, aryl and aralkyl; each R⁴ is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkcycloalkyl, cycloalkyl, alkoxy, alkcycloalkoxy, cycloalkoxy, acyl, acyloxy and halogen; and each R⁵ is independently alkyl, or in the case of the acetals of Formula II, the two R⁵S together with the atoms to which they are attached form a heterocycloalkyl; and at least one additional rosacea-effective agent.

2. The composition according to Claim 1 wherein R¹ is a carbon-carbon single bond.

3. The composition according to Claim 1 wherein R¹ is a straight chain alkylene.

4. The composition according to Claim 1 wherein R² is a carbon-oxygen single bond.

5. The composition according to Claim 2 wherein R² is a carbon-oxygen single bond.

6. The composition according to Claim 1 wherein R³ is a straight chain alkyl.

7. The composition according to Claim 5 wherein R³ is a straight chain alkyl.

8. The composition of Claim 1 wherein the aldehyde compound is of Formula I and is selected from the group consisting of 2-ethoxybenzaldehyde, 4- allyloxybenzaldehyde, 4-ethoxybenzaldehyde, 4-propoxybenzaldehyde, 4- butoxybenzaldehyde, 4-pentyloxybenzaldehyde, and

4-hexyloxybenzaldehyde.

9. The composition of Claim 1 wherein the composition is a cosmetic composition.

10. The composition of Claim 8 wherein the composition is a cosmetic composition.

11. The cosmetic composition of Claim 10 wherein the carrier is a liquid carrier or a cream carrier.

12. The composition of Claim 1 wherein the composition is a pharmaceutical composition and the aldehyde compound is present in a pharmaceutically effective rosacea-treating amount.

13. The composition of Claim 8 wherein the composition is a pharmaceutical composition and the aldehyde compound is present in a pharmaceutically effective rosacea-treating amount.

14. The pharmaceutical composition of Claim 13 wherein the carrier is a topical carrier.

15. The pharmaceutical composition of Claim 13 wherein the composition is a transdermal pharmaceutical composition and the aldehyde compound is present in a transdermally effective amount.

16. The transdermal composition of Claim 15 in a sustained release dosage form.

17. The composition of Claim 1 or 8 wherein the at least one additional rosacea- effective agent is selected from the group consisting of metronidazole, clindamycin, doxycycline, erythromycin, minocycline hydrocholoride, tetracycline hydrochloride, azelaic acid, sodium sulfacetamide, nicotinamide, benzoyl peroxide, salicylic acid, adapalene, isotretinoin, tazarotene, and tretinoin.

18. The composition of claim 17, wherein the agent is metronidazole.

19. The composition of Claim 1 or 8 wherein the at least one additional rosacea- effective agent is selected from the group consisting of trimethoprim- sulfamethoxazole, methotrexate, dapsone, primaquine, chloroquine, and oral prednisone.

20. A method for treating rosacea which method comprises topically applying to a human a cosmetically effective rosacea-treating amount of a cosmetic composition of Claim 10.

21. A method for treating a patient with rosacea, which method comprises topically administering to said patient a therapeutically effective rosacea- treating amount of the topical pharmaceutical composition of Claim 12.

22. A method for treating a patient with rosacea, which method comprises topically administering to said patient a therapeutically effective rosacea- treating amount of the topical pharmaceutical composition of Claim 17.

23. A method for treating a patient with rosacea, which method comprises topically administering a first composition comprising a topical carrier and an effective amount of an aldehyde of Formula I, II, or III which are defined herein, and a second composition comprising a topical carrier and an effective amount of a rosacea-effective agent.

24. The method of claim 23, wherein said method comprising first administering to the patient rosacea-effective agent and then administering the aldehyde.

25. The method of claim 23, wherein said method comprises first administering to the patient the aldehyde and then administering the rosacea-effective agent.

26. A kit for treating rosacea comprising a container containing a composition comprising a rosacea-effective agent, a container containing a composition containing an aromatic aldehyde of formula I, II, or III and instructions for the topical administration of the two compositions.