KR102429810B1 - Chemical Inhibitors of Sebocyte Function - Google Patents

Abstract

The present invention relates generally to the field of cosmetic and therapeutic compounds. More specifically, the invention relates to those described herein as "Dapa compounds" useful for the treatment of, for example, acne, seborrheic dermatitis, and disorders (such as diseases) of sebaceous gland cell function, including "oily skin" and other cell proliferative disorders. to certain 1-dialkylphosphorylalkanes. The present invention also relates to topical dermatological compositions formulated for delivery of Dapa compounds, and the use of such compounds and compositions, for example, in cosmetic or dermatological therapy.

Description

Chemical Inhibitors of Sebocyte Function

The present findings relate generally to the field of cosmetic and therapeutic compounds. More specifically, the invention relates to those described herein as "Dapa compounds" useful for the treatment of, for example, acne, seborrheic dermatitis, and disorders (such as diseases) of sebaceous gland cell function, including "oily skin" and other cell proliferative disorders. to certain 1-dialkylphosphorylalkanes. The present invention also relates to topical dermatological compositions formulated for delivery of Dapa compounds, and the use of such compounds and compositions, for example, in cosmetic or dermatological therapy.

Rowsell and Spring described Phosphine oxides having a physiological cooling effect. US 4,070,496. 1978] described that trialkylphosphine oxides have a "physiological cooling action".

The sebaceous gland unit [PSU] is an epidermal invagination found on most surfaces of the human body, and is composed of hair follicles, sebaceous glands, and pilaris. The sebaceous glands produce an oily/waxy substance called sebum that is pushed into the hair follicles and then pushed over the surface of the skin. PSU is distributed throughout the skin except for the palms and soles of the feet, and the density of PSUs is particularly high in the scalp, face, and upper body. Sebaceous glands are also found in the hairless areas of the eyelids, nose, penis, labia majora, and nipples (glabrous skin) [Hinde. et al. A practical guide for the study of human and murine sebaceous in situ. Exp. Dermatol. 22, 631-7 (2013)].

The main cells of the sebaceous gland are the sebaceous gland cells. When sebaceous gland cells mature and rupture (holocrine secretion), cellular components form sebum, which is predominantly lipid, such as triglycerides, free fatty acids, wax esters, squalene, cholesterol and cholesterol esters, and other cellular products. Do [Picardo et al. Sebaceous gland lipids. Dermatoendocrinol. 1, 68-71 (2009)]. Sebum protects the skin from overhydration and helps regulate body temperature.

Sebum dysfunction is associated with skin disorders such as acne, oily skin, and seborrheic dermatitis [Zouboulis. Acne and sebaceous gland function. Clin. Dermatol. 22, 360-6 (2004)]. These conditions have a high prevalence [eg, for acne, ~14 million visits per year in the United States; For oily skin, a prevalence of ~26% in Chinese women, confirmed by Sebumeter measurements] is associated with a perceived decrease in quality of life [Nouveau-Richard et al. Oily skin: specific features in Chinese women. Skin research and technology: 13, 43-8 (2007); Wu et al. A preliminary investigation of the impact of oily skin on quality of life and concordance of self-perceived skin oiliness and skin surface lipids (sebum). Int. J. Cosmet. Sci. 35, 442-7 (2013)]. Currently, the only drugs that directly target sebaceous gland cell function are androgen receptor antagonists such as cyproterone acetate, and possibly to the extent of isotretinoin. Although antagonists of melanocortin receptor 5 have been proposed, they have not yet been used clinically [Zhang et al. Melanocortin-5 receptor and sebogenesis. Eur. J. Pharmacol. 660, 202-6 (2011)].

Acne, oily skin, and seborrheic dermatitis affect the appearance of facial skin and are a source of shame and distress. The main pathogenesis factor that promotes acne is increased sebum production from the sebaceous gland unit [PSU] induced by androgenic action on sebaceous gland cells. Other factors in pathogenesis are excessive keratinization of the ducts, colonization of PSU by Propionibacterium acnes), and inflammation [Cunliffe et al. Comedone formation: etiology, clinical presentation, and treatment. Clin. Dermatol. 22, 367-74 (2004)]. Oily skin [seborrhea] is a common cosmetic problem that occurs when the giant sebaceous glands produce excess sebum that makes the skin look shiny and shiny. Seborrheic dermatitis is characterized by skin redness, peeling, and shine, and is most commonly seen on the scalp, nasolabial folds, ears, eyebrows and chest. The prevalence of seborrheic dermatitis is not precise, but may affect up to 1-5% of the adult population [Schwartz et al. Seborrhric dermatitis: an overview. Am. Fam. Physician 74, 125-30 (2006)].

A therapeutic agent that interferes with sebaceous gland cell function and reduces sebum secretion would be beneficial for disorders of sebum function such as acne, oily skin, and seborrheic dermatitis.

It is an object of the present invention to use certain compounds described herein as "Dapa compounds" useful for the treatment of disorders (such as diseases) of sebaceous gland cell function, including, for example, acne, seborrheic dermatitis, oily skin, and other cell proliferative disorders. To provide 1-dialkylphosphorylalkane.

Another object of the present invention is to provide topical dermatological compositions formulated for the delivery of Dapa compounds and the use of such compounds and compositions, for example in cosmetic therapy or dermatological therapy.

For the purpose of the present invention, there is provided a topical composition for the treatment of a mammal having acne, oily skin, or seborrheic dermatitis and in need thereof, the treatment comprising topically administering the composition, The composition has the agent dispersed in a dermatologically acceptable vehicle, and the administered agent has the structure:

wherein R ₁ and R ₂ are isopropyl and R ₃ is an alkyl chain having 10 to 12 carbon atoms.

The agent is 1-diisopropylphosphoryldecane.

The agent is 1-diisopropylphosphorilundecane.

The agent is 1-diisopropylphosphoryldodecane.

The agent is present in the composition at a concentration of 0.001 to 5 mg/mL.

The composition is as claimed in claim 1 , wherein the dermatologically acceptable vehicle is an aqueous liquid, gel, lotion, cream or ointment.

The composition includes a water or saline solution.

The composition is delivered onto an applicator, swab, wipe, pad, or small paper towel.

The composition is administered via a manually-actuated nebulizer.

The composition is administered to the skin surface in a unit volume of 0.05 to 0.5 mL.

The mammal has acne, oily skin, or seborrheic dermatitis.

The treatment is to reduce the synthesis of lipids in the mammal.

Treatment is to reduce sebum secretion.

The treatment is to reduce the proliferation of somatic cells in the mammal.

Furthermore, for the purpose of the present invention, there is provided a treatment for reducing malignant and uncontrolled somatic cell proliferation in a mammal.

The reduction in sebum secretion from the forehead skin of a human subject (as measured by a sebumometer) after application of certain 1-dialkylphosphorylalkane (sometimes referred to herein as "Dapa") is described below. The mechanism of action of Dapa agonists was then investigated in cultured human sebaceous gland cells. Surprisingly, some of Dapa showed a potent inhibitory effect on sebaceous gland cell function. For one Dapa compound [1-12], an inhibitory effect on sebaceous gland cell function was observed at less than 30 μg/mL. It is noted that the amphiphilic properties of Dapa enhance efficacy. Although the exact cellular target mediating these effects has not yet been characterized, these Dapa chemicals formulated for delivery to the skin are believed to be useful in the modulation of disorders related to sebum secretion, and other disorders of lipid synthesis and cell proliferation.

1 is a graph showing the efficacy of 1-diisopropylalkane for the TRPM8 receptor assay. In the ordinate, potency is plotted relative to l-menthol. On the abscissa, the number of carbons in the n -alkane is listed.
2 is a graph showing sebum secretion after topical application of 20 mg/mL of 1 to 7 to the forehead skin of a volunteer subject [N=4 or 5 per group]. The amount of sebum was measured with a sebum meter [CK Electronic, Cologne, Germany].
3 is a graph showing the sebaceous gland cell viability [ordinate] as a percentage of the control as a function of the concentration of mM of the test substance [abscissa] measured by the MTT assay. In the graph, 1-7 [circle] did not inhibit sebaceous gland cell viability but 1-8 [square] strongly inhibited at 1 mM.
Figure 4 is a graph showing the sebaceous gland cell viability [ordinate] as a percentage of the control group according to the concentration [abscissa] of the test substance, measured by the MTT assay. In the graph, three test substances, 1-10, 1-11 and 1-12, all strongly inhibited sebaceous gland cell viability at 0.10 μM.
5 shows photographs of thin layer chromatography plates of lipids extracted from sebaceous gland cells incubated with various test substances. In the presence of insulin growth factor [IGF-1], lipids are increased, as measured by gel staining, and are inhibited by cis- and trans-retinoic acid (RA). As can be seen from the picture, 1-10, 1-11, and especially 1-12 lower the amount of squalene.
6 shows the basic structure of an amphiphilic material with a hydrophilic “polar head” and a hydrophobic [lipophilic] “tail”. Preferred embodiments 1-11 and 1-12 are amphiphiles. Dissec-butyl groups [eg 2-7] cover more polar heads than isopropyl groups [eg 1-8], resulting in loss of bioactivity.
7 shows examples of 1-11 at 10 10 mg/mL in distilled water in a vial before [7A] and after [7B] manual shaking. The foaming phenomenon seen in 7B is characteristic of surfactants or "surfactants" and is caused by the amphiphilic structure.

Selected chemical agents called Dapa compounds were synthesized, and their skin cooling action and activity on an ion channel called TRPM8 were investigated. Two analogues, 1-7 and 1-8, applied to the forehead skin at a concentration of 20 mg/mL and a volume of 0.08 to 0.15 mL, induce strong localized cooling and refreshing sensation lasting several hours, although not changing the skin temperature. One was given The skin was fresh, dry and cold. After application of 1-7, skin temperature was measured and showed no change. It was noted that sebum secretion of the forehead skin in human subjects was inhibited by topical application of 1-7. The seborrheic properties of 1-7 and 1-8 were then investigated in the laboratory on sebaceous gland cells isolated from culture. 1-8 were shown to have activity in inhibiting sebaceous gland cell viability and sebum synthesis. Dapa compounds [and other compounds] were then screened for sebaceous gland cells using cell viability (MTT assay) and ³ H-thymidine incorporation as indices of sebaceous gland cell function. Surprisingly, compounds 1-10, 1-11, and 1-12 were shown to have potent inhibitory activity on sebaceous gland cell function. For example, the synthesis of squalene, a characteristic lipid of skin cells, was strongly inhibited by 1-12. Compounds 1-10, 1-11, and 1-12 have low potency against TRPM8: therefore their activity on sebaceous gland cells was not related to the TRPM8 receptor. Further investigation of the chemical structures of 1-10, 1-11, and 1-12 revealed that the non-ionic amphiphilic character of these molecules confers sebaceous gland cell-inhibitory activity, where longer chain alkanes selectively molecules suggests that it makes the Therefore, a set of compounds with novel mechanisms of action to reduce sebum production are identified. In the sections that follow, these observations are described in detail.

In summary, several Dapa agonists have been shown to have potent [sub-micromolar] inhibitory effects on human sebaceous gland cell function in culture. One of the Dapa-compounds, coded 1-12, showed activity at 30 μg/mL. This potent activity was unexpected and 1-12 have the potential to be utilized to ameliorate "oily skin", acne, and seborrheic dermatitis. Such direct drug action on human sebaceous gland cells has not been previously described. Preferred embodiments are believed to be useful in the treatment of disorders of sebaceous gland function, and other cellular proliferative and lipid synthesis disorders.

Example

Study 1. Chemical Synthesis and TRPM8 Activity

The present invention relates to certain 1-dialkylphosphorylalkanes described herein as "Dapa compounds". As noted previously, Rowsell and Spring [Phosphine oxides having a physiological cooling effect. US 4,070,496. 1978] described that trialkylphosphine oxides have a "physiological cooling action". Lunkenheimer et al. synthesized dimethyl- and diethyl-phosphoryl alkanes and evaluated their physical and chemical properties related to micelle formation [ surface chemistry of n -alkyl-dimethyl and n -alkyl-diethyl phosphine oxides. On the absorption properties of pure aqueous solutions, Colloids and Surfaces 22, 215-224 (1987)].

Dapa compounds of interest herein have the following general chemical structure:

wherein R ₁ and R ₂ are isopropyl, or n -propyl, and R ₃ is an alkyl chain having 10 to 18 carbon atoms.

In a preferred embodiment for the inhibition of sebaceous gland cell function, R ₁ and R ₂ are isopropyl. Therefore, the preferred embodiment is 1-diisopropylphosphorylalkane, wherein R is n- decanyl, n- undecanyl, and n- dodecanyl.

"Dapa" is an abbreviation for 1-dialkylphosphorylalkane. Other synonyms for this chemical group include trialkylphosphine oxides or 1-dialkylphosphinoylalkanes. Phosphorus is pentavalent in Dapa. R ₃ , the third alkyl group in the general formula, can be a number from 4 to 12 and correspond to butyl, pentyl, hexyl, heptyl, octyl, nonyl, decanyl, undecanyl and dodecanyl side chains, respectively. R ₃ alkanes are in a linear or “straight” form, with a phosphoryl group attached to the primary position of the carbon chain. Individual compounds in Tables 1 and 2 are labeled "1-x" for a series of diisopropyl or "2-x" for disec-butyl, where x is the numerical identifier of the number of carbon atoms in R ₃ . . Therefore, 1-7, 1-8, and 1-12 are 1-diisopropylphosphorylheptane, 1-diisopropylphosphoryloctane, and 1-diisopropylphosphoryldodecane, respectively, and 2-6 and 2-7 are 1-disec-butyl-phosphorylhexane and 1-disec-butylphosphorylheptane, respectively.

The Dapa compound was synthesized by the following general method: 100 mL (23.7 g, ˜200 mmol) of isopropylmagnesium chloride (or, alternatively, sec-butylmagnesium chloride for dicec-butyl derivatives) was obtained from Acros as tetra Obtained as a 25% solution in hydrofuran (THF) and placed under nitrogen in a 500 mL flask (equipped with a stir bar). A solution of diethylphosphite in THF (D99234 from Aldrich; 8.25 g in 50 mL, 60.6 mmol) was added dropwise. After approximately 30 minutes, the reaction mixture was warmed to boiling point. After the reaction mixture was stirred for an additional 30 min, an appropriate solution of n- alkyl iodide in THF (from TCI, 60 mmol in 20 mL) was added dropwise. The reaction mixture was then stirred overnight at room temperature. The reaction mixture was diluted with water, transferred to a separatory funnel, acidified with acetic acid (~10 mL) and extracted twice with ether. The ether layer was washed with water and evaporated (RotaVap Buchi, bath temperature 40° C.). The light brown oil was distilled under high vacuum. The final product confirmed by mass spectrometry was a colorless or transparent liquid with a slight pale yellow color. Tables 1 and 2 list some of the Dapa compounds synthesized and tested.

The in vitro effect of the test article was first evaluated in the cloned hTRPM8 channel. Test compounds were sent to ChanTest Corporation (14656 Neo Parkway, Cleveland, OH 44128, USA) for assay. Test cells were Chinese Hamster Ovary (CHO) cells stably transfected with human TRPM8 cDNA. The test solution and L-menthol reference were prepared by diluting the stock solution in HEPES-buffered saline (HBPS). Substances were evaluated at 8 concentrations (0.03 to 300 μM), n=4 replicates per measurement. For Fluorescence Imaging Plate Reader (FLIPRTETRA ^™ ) assay, cells were seeded per well in a 384-well black wall, flat clear-bottom microtiter plate (type: BD Biocoat Poly-D-Lysine Multiwell Cell Culture Plate). Approximately 30,000 cells were plated. Cells were incubated overnight at 37° C. to reach a near confluent monolayer, suitable for use in fluorescence assays. The test procedure involves removing the growth medium and adding 40 μL of HBPS containing Fluo-8 for 30 minutes at 37° C., adding 10 μL of a test compound, vehicle, or control solution in HBPS to each well, and adding Fluo- Readings were for 4 minutes using an 8 calcium kit and a FLIPRTETRA ^™ instrument (Molecular Devices, Sunnyvale, Calif.). Subsequently, the compounds were tested by David Andersson at King's College, London, UK using standard published procedures [Andersson et al. TRPM8 activation by menthol, icilin, and cold is differentially modulated by intracellular pH. J. Neurosci. 24, 5364-9 (2004)].

Concentration-response data were analyzed via the FLIPR control software with the FLIPR system (MDS-AT) by fitting the Hill equation as follows: response = base + (maximum base) / 1 + (x half / x) ^rate , where "base" is the reaction at low concentrations of the test compound; "Maximum" is the maximum response at high concentrations; "half x" is the EC ₅₀ (the concentration of the test compound that results in half the maximum activity); "Speed" is the Hill coefficient. A simple 1-to-1 coupling model was assumed to create a nonlinear least squares method. The same data set was further analyzed using GraphPad Prism 6 software (San Diego, California) to obtain 95% confidence intervals.

Tables 1 and 2 show the structures of the compounds tested for TRPM8 activation. The results of the tests are shown in Table 3 and FIG. 1 . It can be seen that the more potent compounds are 1-7, 1-8, and 1-9 in diisopropyl and 2-6 and 2-7 in disec-butyl. 1-12 have very low activity against TRPM8.

1 is a graph showing the efficacy of 1-diisopropylalkane in the TRPM8 receptor assay. In the ordinate, potency appears proportional to l-menthol. On the abscissa, the number of carbons in the n-alkane is listed.

Compounds tested for TRPM8 activation, skin and sebaceous gland cells code R or R ₃ chemical name rescue 1-5 n- C ₅ H ₁₃ 1-Diisopropylphosphorylpentane

1-6 n- C ₆ H ₁₃ 1-Diisopropylphosphorylhexane

1-7 n- C ₇ H ₁₅ 1-Diisopropylphosphorylheptane

1-8 n- C ₈ H ₁₇ 1-Diisopropylphosphoryloctane

1-9 n- C ₉ H ₁₉ 1-Diisopropylphosphorylnonane

1-10 n- C ₁₀ H ₂₁ 1-Diisopropylphosphoryldecane

1-11 n- C ₁₁ H ₂₃ 1-Diisopropylphosphorylundecane

1-12 n- C ₁₂ H ₂₅ 1-Diisopropylphosphoryldodecane

Compounds tested for TRPM8 activation, skin and sebaceous gland cells code R or R ₃ chemical name rescue 2-4 n- C ₄ H ₉ 1-Disec-butylphosphorylbutane

2-5 n- C ₅ H ₁₁ 1-Disec-butylphosphorylpentane

2-6 n- C ₆ H ₁₃ 1-Disec-butylphosphorylhexane

2-7 n- C ₇ H ₁₅ 1-Disec-Butylphosphorylheptane

2-8 n- C ₈ H ₁₇ 1-Disec-butylphosphoryloctane

EC ₅₀ for TRPM8 and Relative Efficacy for l-Menthol code EC ₅₀ μM 95% confidence period Relative Efficacy l-menthol 3.8 2.5 to 5.6 1.0 1-5 5.6 4.4 to 7.2 0.7 1-6 2.4 1.5 to 4.0 1.6 1-7 0.7 0.5 to 1.0 5.4 1-8 0.7 0.5 to 1.0 5.4 1-9 0.9 0.4 to 2.5 4.0 2-4 14.5 7 to 29 0.3 2-5 1.7 1.0 to 2.9 2.2 2-6 0.8 0.5 to 1.3 4.7 2-7 1.1 0.6 to 2.3 3.4 2-8 1.3 0.7 to 2.3 2.9

Study 2: cooling for the skin , tissue temperature, and sebum secretion

When certain Dapa compounds are applied to the skin of the cheekbones and forehead, a feeling of coolness/coolness can be felt and the intensity of the sensation can be quantified [Wei. 1-Di-isopropyl-phosphinoyl-alkanes as topical agents for the treatment of sensory discomfort. US 2015/0164924]. You can set the intensity of the sensation to 0, 1, 2 or 3: 0 is no change; 1 is slightly cool or cool; 2 is a clear sign of coolness or coolness; and 3 is strong coolness or coolness. The recording interval is 5 to 15 minutes and continues until at least two consecutive zeros are obtained. The onset of drug action is defined as the time until coolness intensity 2 is reached. Active compounds herein, such as 1-7, cause cooling 1 minute after application. The duration of the Dapa-compound is defined as the end time minus the onset time. The end of drug action is defined herein as the time when the intensity falls below 2 after previously exceeding 2.

The test compound was applied to the forehead and cheekbone skin using cotton gauze (0.4 g, rectangular, 50 mm x 60 mm; CS-being, Daisan Cotton, Japan). Test compounds were used at a concentration of 20 mg/mL in distilled water. The results are summarized in Table 4. From the data presented above, it can be seen that, among these compounds, 1-7 and 1-8 induced coolness/cooling in the cheekbone/forehead skin.

Cooling properties of molecules applied to cheekbone/forehead skin code R carbon atom start (min) quality of sensation Duration (hours) 1-5 5 11 ~1 dynamic 0.5 1-6 6 12 ~1 dynamic 1.3 1-7 7 13 ~1 dynamic - ice cold 3.2 1-8 8 14 ~1 cold - as cold as ice 4.0 1-9 9 15 ~2 coolness 2.0 2-4 4 12 ~1 coolness 0.3 2-5 5 13 ~1 coolness 1.1 2-6 6 14 ~2 ice 1.5 2-7 7 15 ~2 ice 2.4 2-8 8 16 5 ice 5.6

The molecules in Tables 1 and 2 activate the TRPM8 ion channel. To determine whether the sensation of heat extraction was accompanied by a change in tissue temperature, 1-7 were applied to the forehead skin of subjects (N = 5) (with a wipe at a concentration of 20 mg/mL in distilled water) and the skin temperature was recorded. did. Subjects noted a cooling effect of 1-7 lasting 30 to 45 minutes on the forehead skin, but skin temperature was not affected by 1-7. 1-7 evoked a refreshing and powerful feeling of coolness that invigorates the test subject: this effect is accompanied by a refreshing, dry feeling.

Skin temperature of human forehead after 1-7, 20 mg/mL hour Temperature (℃) control 1-7 before application 37.3 37.4 0 minutes 37.2 37.4 15 minutes 37.5 37.5 30 minutes 37.1 37.1 45 minutes 37.4 37.2 60 minutes 37.0 37.1

Sebum is an oily substance secreted by the sebaceous glands. Adhering to the hair follicles, these glands are abundant on the face and scalp. In teenage years, for example, increased secretion of sebum associated with androgen stimulation is associated with acne. Sebum is also increased in oily skin and in seborrheic dermatitis. Herein, sebum secretion was measured using a sebum meter, a standardized instrument based on a matt tape coming into contact with the skin, and then an oil stain photometer [CK Electronic, Cologne, Germany].

The baseline level of sebum secretion in 9 human subjects was 188±19 μg/cm ² . On the forehead, after topical application of 1-7 at 20 mg/mL, sebum secretion was significantly reduced as shown in FIG. 2 . Previously, this pharmacological effect was not observed with Dapa-compounds such as 1-7. This was a new and unexpected observation.

2 is a graph showing sebum secretion after topical application of 1-7, 20 mg/mL to the forehead skin of volunteer subjects [N=4 or 5 per group]. The amount of sebum was measured with a sebum meter [CK Electronic, Cologne, Germany].

Study 3. Effects of 1-7 and 1-8 on sebaceous gland cells

The activities of 1-7 and 1-8 on immortalized sebaceous gland cell lines were investigated in the laboratory of Professor Appointe using the methods previously described [Im et al. Epigallocatechin-3-gallate suppresses IGF-I-induced lipogenesis and cytokine expression in SZ95 sebocytes. J. Invest. Dermatol. 132, 2700-8 (2012)]. Proliferation of sebaceous gland cells was measured using the MTT assay, by examination of micrographs of cells in culture, and by ³ H-thymidine incorporation. The effect of the test agent on lipogenesis was further studied by Oil Red O staining and analysis of the lipid composition of the cells. Studies were also conducted on signaling pathways for lipogenesis [PPAR and SREBP-1, Trivedi et al. Peroxisome proliferator-activated receptors increase human sebum production. J. Invest. Dermatol. 126, 20029 (2006)].

The MTT assay is a colorimetric assay for cell viability. MTT [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide], a yellow tetrazole dye, is converted to purple formazan by NADPH-dependent oxidoreductase in living cells. is returned When the number of viable cells decreases, the enzyme activity and the amount of purple decrease, which can be measured spectrophotometrically. When viable cells were incubated in the presence of 1-8 [0.1 mM], micrographs of the cell culture also revealed interspersed voids, confirming the loss of viable cells.

3 is a graph showing the sebaceous gland cell viability [ordinate] as a percentage of the control according to the mM concentration of the test substance [abscissa], measured by the MTT assay. In the graph, 1-7 [circle] did not inhibit sebaceous gland cell viability, but 1-8 [square] strongly inhibited it at 1 mM.

To determine whether functional sebum production was affected by 1-8, sebaceous gland cells were stimulated with IGF-1 at 50 ng/mL. IGF-1 is an “insulin-growth factor” and is a protein that exerts hormone-like activity in increasing cellular functions [Smith et al. IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway. J. Invest. Dermatol. 128, 128693 (2008)]. Oil Red O [Sudan Red 5B] is a diazo dye used to stain neutral triglycerides and lipids on cells. Studying the micrographs of sebaceous gland cells revealed that lipids were increased in the cells after IGF-1 application, and this increase was inhibited in cells treated with 1-8 [0.1 mM]. This inhibition is further confirmed by analytical measurements of lipids, using thin layer chromatography and gas-chromatography. Sebum-specific lipids [Picardo et al., 2009] squalene and wax esters were selectively reduced by 1-8. This drug action of 1–8 may be particularly important for anti-acne effects, as squalene peroxide is believed to be an instigator of tubular cell proliferation, an event leading to clogging of hair follicles and formation of comedones. can Blockage of sebum excretion can then lead to bacterial invasion of sebum and production of irritants that induce inflammatory manifestations of acne [Cunliffe et al. 2004].

There are intracellular biochemical receptors and markers of sebaceous gland cell activation and sebum production. Peroxisomal proliferator-activated receptor [PPAR] agonists increase sebum production in isolated sebaceous gland cells [Trivedi et al. (2006)]. This class of receptors is reduced after 1-8 applications. Similarly, sterol-responsive binding protein [SBEP-1] linked to lipogenesis in sebaceous gland cells was decreased by 1-8. These effects were specific for cellular receptors such as TRMP8 and markers of sebum production, as shown in that actin was not affected by 1-8.

This series of studies clearly shows that 1-8 potently inhibits sebaceous gland cell function at the cellular/sebaceous gland cell level at 0.1 mM. Although 1-7 decreased sebum secretion in the forehead skin of volunteers, they did not show the above cellular effect. This is the first time that 1-dialkylphosphorylalkane has been shown to be a specific inhibitor of sebaceous gland cell function.

Study 4. Preferred Embodiments of Super Efficacy

1-7 and 1-8 are sensory coolants of the skin and activators [agonists] of the TRPM8 ion channel protein. As can be seen from the data in Table 3, Figure 1 and Table 6, there is no correlation between the efficacy of TRPM8 activation and the inhibitory effect on sebaceous gland cell function when the various agonists are compared, and therefore these two events is not related The indicator used for direct comparison was sebaceous gland cell viability measured in the MTT assay followed by ³ H-thymidine incorporation assay.

Analysis of structurally related analogs of Dapa showed that compounds 1-10, 1-11, and 1-12 showed the highest activity and inhibited sebaceous gland cell viability. In Figure 4, it can be seen that 1-10, 1-11, and 1-12 have 10,000-fold greater activity than 1-8 in the MTT sebaceous gland cell assay. In addition, 1-12, the most potent analog, was the only compound that clearly inhibited ³ H-thymidine incorporation into sebaceous gland cells. 1-12 were fully effective at 0.01 μM equivalent to 30 μg/mL. 1-12 was also effective in inhibiting IGF-1 stimulated production of squalene in cultured sebaceous gland cells. Compounds 1-10, 1-11, and 1-12 were tested at three concentrations and the dose-response data for the MTT assay is shown in FIG. 4 .

Figure 4 is a graph showing the sebaceous gland cell viability [ordinate] as a percentage of the control group according to the concentration [abscissa] of the test substance, measured by the MTT assay. In the graph, three test substances, 1-10, 1-11, and 1-12, all strongly inhibited sebaceous gland cell viability at 0.10 μM.

5 shows photographs of thin layer chromatography plates of lipids extracted from sebaceous gland cells incubated with various test substances. In the presence of insulin growth factor [IGF-1], lipids are increased, as measured by gel staining, and inhibited by cis- and trans-retinoic acid (RA). As can be seen from the picture, 1-10, 1-11, and especially 1-12 lower the amount of squalene.

The potent inhibitory effects of 1-10, 1-11, and 1-12 on cell growth are unexpected and novel. Although the range of cell types in which this drug action is exhibited is still unknown, the data clearly indicate that these compounds may be useful in proliferative skin diseases such as psoriasis, neurofibromatosis, diabetic milk fat necrosis, and in the manifestation of acne in general. Furthermore, these compounds may be useful in the control and management of malignant and uninhibited somatic cell proliferation [cancerous lesions] and in the inhibition of lipid synthesis.

Test substance for sebaceous gland cell viability as measured by MTT assay and ³ H-thymidine incorporation into cells code Molecular Weight MTT viability at 0.1 _μM ³ H-thymidine incorporation at 10 _μM 1-7 232

100%N.S.,

100% N.S.,

100% 1-8 246 ~60% N.S.,

78% 1-9 260 ~60% N.S.,

85% 1-10 274

5% N.S.,

100% 1-11 288

5% N.S.,

88% 1-12 302

5% ~40% 2-5 232 N.S.,

100% N.S.,

98% 2-6 246 N.S.,

98% N.S.,

100% 2-7 260 N.S.,

80% N.S.,

85% 2-8 274 ~55% N.S.,

90%

Study 5. Amphiphilicity Preferred Embodiments and Mechanism of Action The Dapa compounds investigated herein are "amphiphilic", ie they have hydrophilic and hydrophobic [lipophilic] properties at each end of the molecule. Many amphiphiles have "surfactant" or "surface-active" properties and are used in soaps and detergents. Surfactants impart a cleansing and lathering action to the shampoo and are designed to remove sebum from the hair shaft. Surfactants lower the surface tension between two phases, such as liquid-liquid, liquid-solid, and allow a solid surface to be "wetted" or coated with a liquid more readily. In addition to lowering the surface tension, surfactants can form spherical aggregates (eg, of 40 to 100 molecules) called "micelles", trap oil droplets into the center of the micelles and further purify. can be improved

6 shows the basic structure of an amphiphilic material with a hydrophilic “polar head” and a hydrophobic [lipophilic] “tail”. Preferred embodiments 1-11 and 1-12 are amphiphiles. Disec-butyl groups [eg 2-7] cover more polar heads than isopropyl groups [eg 1-8] and reduce hydrophilicity, resulting in loss of bioactivity.

7 shows examples of 1-11 at 10 10 mg/mL in distilled water in a vial before [7A] and after [7B] manual shaking. The foaming phenomenon seen in 7B is characteristic of surfactants or "surfactants" and is caused by the amphiphilic structure.

The ability of preferred embodiments 1-11 to lower the surface tension of water is shown in FIG. 7 . Fresh water will not foam because the water molecules adhere more strongly to each other than to air. However, when a surfactant is added, the surface tension of the liquid is reduced and can itself entrap air and form bubbles or lather. Foaming is enhanced when the liquid is shaken vigorously.

Preferred Dapa embodiments described herein can be classified as non-ionic surfactants because the Dapa molecules have no charge in solution. Standard surfactants used in shampoos are usually anionic [eg sodium lauryl sulfate]. Cationic surfactants include, for example, quaternary ammonium salts such as benzalkonium chloride used as antimicrobial agents. Non-ionic surfactants include ethoxylates (such as polysorbate 80 or sorbitan oleate) and propoxylates (such as PPG-10 cetyl ether). The ability of dimethyl- and diethyl-phosphorylalkanes to act as surfactants and form micelles has been recognized by physical chemists [eg, Lunkenheimer et al. (1987)], 1-dialkyl-phosphorylalkanes are not used for commercial use as surfactants or detergents.

At present, it is not yet clear whether the preferred embodiments affect sebaceous gland cell viability by surfactant action or by selective and specific mechanism of action, such as interfering with enzymatic activity, so an appropriate term to call Dapa embodiments is an amphiphilic substance, not a surfactant. Lesnik et al. [Agents that cause enlargement of sebaceous glands in hairless mice. I. Topical Agents. Archives Dermatol. 284:100-105 (1992) investigated the effects of cationic and anionic surfactants, solvents, and emulsifiers on the skin of hairless mice and found that the number of sebaceous gland cells was invariably increased after chemical exposure: This effect is contrary to what might be expected from the results presented herein.

In Figure 5, 1-12 appears to strongly reduce squalene production by sebaceous gland cells. Squalene is a 30-carbon long-chain molecule important for lipid synthesis in skin cells. The distribution of lipids in human sebum is 30-50% glycerides, 15-30% free fatty acids, 26-30% wax esters, 12-20% squalene, and 3-6% cholesterol. In young adults with acne, sebum production is increased on average by 59% with a 2.2-fold increase in the squalene content of the lipid [Pappas et al. Sebum analysis of individuals with and without acne. Dermato-Endocrinology 1:3, 157-164, June (2009)]. Agents that interfere with squalene physiology in sebaceous gland cells are likely to have distinct inhibitory effects on sebum production. Since the structure of 1-12 resembles the amphiphilic properties of anionic fatty acids, it is possible that the mechanism of action of 1-12 in the reduction of sebaceous gland cell viability is inhibition of lipid synthesis.

As a result of analyzing the structure-activity relationship of the preferred embodiments, it is shown that the inhibitory effect on sebaceous gland cells is increased when the longest alkane chain has 10 carbon atoms or more. It should be noted that 2-8 has an activity greater than 2-6 or 2-7 [Table 6], so even here, extension of the alkane chain by 1 carbon number increases the efficacy. As exemplified by the absence of activity of 2-6 and 2-7 molecules on sebaceous gland cell viability, whereas 1-9 with carbon number equal to 2-7 are active, the isopropyl group is sec -Preferred for butyl.

An increase in the number of carbons attached to the phosphorus atom may decrease the hydrophilic properties of the amphiphilic material. The anti-sebaceous gland cell activity of single or doubly substituted methyl, ethyl, and n -propyl analogs in phosphorylalkanes has not yet been studied. Such information may be specified in the general formula below:

where R ₁ and R ₂ =methyl, ethyl, isopropyl, or n- propyl, and R ₃ is the same as an alkyl side chain having 10 to 18 carbon atoms.

manufactured goods

One embodiment of the invention is an article of manufacture comprising the topical formulation of the invention in a suitable container with labeling and instructions for use. The container may be a reservoir, a gel contained within a reservoir tube, or a manually-actuated nebulizer with a suitable eyelet size connected to a small paper towel suitably sealed with a water impermeable wrap.

Preferably, the description is, for example, a pamphlet or packaging label packaged with the formulation of the invention. The labeling description states that the topical formulation of the invention is administered to the skin surfaces of the face, scalp, and trunk rich in sebaceous gland units in an amount sufficient to treat or reduce sebum secretion and seborrhea, and the signs and symptoms thereof, for a sufficient period of time. explain how. Labeling instructions are an important aspect of the invention, in that the composition must be approved for sale by the US Food and Drug Administration (US FDA) before it can be approved for any particular use. Part of that process involves providing a label that will ultimately accompany the marketed pharmaceutical composition. Preferably, the label includes dosage and administration instructions, composition of the topical formulation, clinical drug properties, drug resistance, pharmacokinetic properties, absorption, bioavailability, and contraindications.

Claims (15)

A pharmaceutical composition for the treatment of a mammal having acne, oily skin or seborrheic dermatitis and in need thereof, comprising:
A pharmaceutical composition which is administered topically and has an agent dispersed in a dermatologically acceptable vehicle, wherein the administered agent has the structure:

Wherein R ₁ and R ₂ are isopropyl (isopropyl) and R ₃ is an alkyl chain of 10 to 12 carbons. The pharmaceutical composition according to claim 1, wherein the agent is 1-diisopropylphosphoryldecane. The pharmaceutical composition according to claim 1, wherein the agent is 1-diisopropylphosphorylundecane. The pharmaceutical composition according to claim 1, wherein the agent is 1-diisopropylphosphoryldodecane. The pharmaceutical composition of claim 1 , wherein the agent is present in the composition at a concentration of 0.001 to 5 mg/mL. The pharmaceutical composition according to claim 1, wherein the dermatologically acceptable excipient is an aqueous liquid, gel, lotion, cream or ointment. The pharmaceutical composition according to claim 1, wherein the composition comprises water or a saline solution. The pharmaceutical composition of claim 1, wherein the composition is delivered as an applicator, swab, wipe, pad or towelette. The pharmaceutical composition of claim 1 , wherein the composition is administered via a manually operated nebulizer. The pharmaceutical composition according to claim 1, wherein the composition is administered to the skin surface in a unit volume of 0.05 to 0.5 mL. The pharmaceutical composition according to claim 1, wherein the mammal has acne, oily skin or seborrheic dermatitis. The pharmaceutical composition of claim 1 , wherein said treatment is for reducing the synthesis of lipids in a mammal. The pharmaceutical composition according to claim 1, wherein said treatment is for reducing sebum secretion. The pharmaceutical composition of claim 1 , wherein said treatment is for reducing proliferation of somatic cells in a mammal. The pharmaceutical composition of claim 1 , wherein said treatment is for reducing proliferation of malignant and uninhibited somatic cells in a mammal.

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