Methods for modulating pigmentation by angiotensin-converting enzyme 2 modulation

Abstract

A cosmetic method for modulating pigmentation in a subject includes administering a modulator of angiotensin-converting enzyme 2 (ACE2 modulator) to the subject. The ACE2 modulator can be an inhibitor of angiotensin-converting enzyme 2 (ACE2 inhibitor), in which case, the ACE2 inhibitor can be administered to increase pigmentation in the subject. The ACE2 modulator can also be an activator of angiotensin-converting enzyme 2 (ACE2 activator), in which case the ACE2 activator can be administered to decrease pigmentation in the subject. The treatment of inflammatory skin disease can also be achieved by inhibition of angiotensin-converting enzyme 2.

Claims

1. A method for the treatment of an inflammatory skin disease, the method comprising administering an inhibitor of angiotensin-converting enzyme 2 (ACE2 inhibitor) to a subject in need thereof.

2. The method according to claim 1, wherein the ACE2 inhibitor inhibits the carboxypeptidase digestion of α-melanocyte stimulating hormone (α-MSH).

3. The method according to claim 1, wherein the ACE2 inhibitor leads to one or more effects selected from the group consisting of: elevated levels of α-MSH.sub.1-13 in subjects who have received ACE2 inhibitor treatment compared to subjects who have not received said treatment, elevated levels of one or more melanins in subjects who have received ACE2 inhibitor treatment compared to subjects who have not received said treatment, and elevated expression of one or more melanogenic genes in subjects who have received ACE2 inhibitor treatment compared to subjects who have not received said treatment.

4. The method according to claim 1, wherein the inflammatory skin disease is selected from the group consisting of protoporphyria, psoriasis, vitiligo, acne, solar urticaria (SU), Hailey-Hailey disease, Polymorphous light eruption (PLE), alopecia areata and overexposure of skin to ultraviolet (UV) radiation (sun burn).

5. The method according to claim 1, wherein the treatment reduces the risk of a subject to overexposure of the skin to ultraviolet (UV) radiation (sun burn).

6. The method according to claim 1, wherein the treatment reduces the risk of a subject to melanoma.

7. The method according to claim 1, wherein the ACE2 inhibitor is administered topically.

8. The method of claim 4, wherein the protoporphyria is erythropoietic protoporphyria (EPP).

Description

FIGURES

(1) The invention is further described by the following figures. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

(2) FIG. 1: Melanogenesis gene expression in the skin of ACE-KO mice on FVB/N and C57131/6 genetic background compared to respective controls (upper panels) and coat colour of ACE2-knockout mice on FVB/N genetic background (upper group; lower group: FVB/N controls) (lower panel).

(3) FIG. 2: Melanogenesis in B16F10 melanoma cells stimulated with α-MSH.sub.1-13 and α-MSH.sub.1-12. *** p<0.001 compared to control without α-MSH, +++, +p<0.001, p<0.05 α-MSH.sub.1-12 compared to the same concentration of α-MSH.sub.1-13.

(4) FIG. 3: Inhibition of the melanogenic effect of α-MSH.sub.1-13 (upper panel), but not of α-MSH.sub.1-12 (lower panel), by human recombinant ACE2 in B16F10 melanoma cells. The specific ACE2-inhibitor MLN-4760 inhibits the effect of ACE2. **, p<0.01; ***, +++p<0.001

(5) FIG. 4: ACE2 limits the efficiency of αMSH.sub.1-13 in human skin; Tyrosinase expression. Tyrosinase (melanin synthesizing enzyme) mRNA expression was measured in human abdominal skin biopsies after treatment with vehicle, 10 nM αMSH.sub.1-13, 10 nM αMSH.sub.1-13+100 nM of the ACE2-Inhibitor MLN4760, or 100 nM MLN4760 alone. As expected, αMSH.sub.1-13 stimulates tyrosinase expression. This effect is increased by ACE2 inhibition. ACE2 inhibition alone already stimulates tyrosinase expression. These results indicate that ACE2 limits the efficiency of αMSH.sub.1-13 in human skin. *p<0.05.

(6) FIG. 5: ACE2 limits the efficiency of αMSH.sub.1-13 in human skin; Fontana-Masson staining in human abdominal skin biopsies. Melanin was stained black by Fontana-Masson staining in human abdominal skin biopsies after treatment with vehicle, 10 nM αMSH.sub.1-13, or 10 nM αMSH.sub.1-13+100 nM of the ACE2-Inhibitor MLN476. As expected, αMSH.sub.1-13 increases melanin levels. This effect is further increased by ACE2 inhibition. These results again indicate that ACE2 limits the efficiency of αMSH.sub.1-13 in human skin.

(7) FIG. 6: ACE2 plays a limiting role on melanin synthesis in mouse skin. Melanin was isolated from hair of C57131/6 (B16) wildtype (WT) and ACE2-deficient (KO) mice and quantified by spectrophotometry (405 nm). The melanin content is higher in the absence of ACE2 confirming a limiting role of ACE2 on melanin synthesis in mouse skin. *p<0.05.

(8) FIG. 7: DIZE inhibits the stimulating effect of aMSH by activating ACE2 or increasing its expression. B16F10 mouse melanoma cells were incubated with 1 nM aMSH, 10 ng/μl diminazene aceturate (DIZE, ACE2 activator) or a combination of both for 48 hours and the expression of the melanin-synthesizing enzyme tyrosinase (TYR) was quantified by real-time RT-PCR; **p<0.01; *p<0.05.

(9) FIG. 8: DIZE stimulates ACE2 expression in B16F10 mouse melanoma cells. B16F10 mouse melanoma cells were incubated with 10 ng/μl diminazene aceturate (DIZE, ACE2 activator) for 48 hours and the expression of ACE2 was quantified by real-time RT-PCR; *p<0.05. DIZE stimulates ACE2 expression in these cells

EXAMPLES

(10) The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

(11) Generation and Characterisation of ACE2-KO mice

(12) The inventors have investigated the renin-angiotensin system and in particular its protective arm. This arm comprises ACE2, generating the peptide angiotensin(1-7), which interacts with its receptor Mas to exert antihypertensive, antihypertrophic, and antiinflammatory actions in the cardiovascular system (Bader, 2013, Rentzsch et al., 2008).

(13) Since also PRCP can generate angiotensin(1-7), the inventors generated and characterized besides ACE2-KO mice (Rabelo et al., 2016; Motta-Santos et al., 2016; Nunez-Souza et al., 2016) also mice lacking PRCP (Maier et al., 2017). These mice were used to analyze which enzyme may degrade α-MSH in the skin.

(14) The inventors have found that the skin of ACE2-KO mice overexpresses several melanogenic genes, such a tyrosinase, Trp1, Trp2 and GPNMB, compared to the skin of control mice (FIG. 1). Accordingly, ACE2-KO mice on an albino background (FVB/N) were not completely white as their controls but showed a cream coat colour (FIG. 1). These findings demonstrate that ACE2 degrades α-MSH, and that mice lacking the enzyme therefore exhibit increased skin concentrations of the peptide visualized by the increased melanogenic activity. This is also supported by the fact, that ACE2 is a carboxypeptidase preferentially cutting peptide substrates with a proline residue at the penultimate position (Vickers et al., 2002), as it is the case for α-MSH.

(15) The inventors have shown that the expected ACE2 digestion product of α-MSH1-13, α-MSH1-12, is at least 100 times less active in a cell culture assay for melanogenesis using B16F10 mouse melanoma cells (FIG. 2).

(16) Additionally, the inventors could verify that the addition of human recombinant ACE2 to α-MSH1-13, but not to α-MSH1-12, reduces its melanogenic activity in this cell culture model (FIG. 3). In wild-type mice, the inventors further confirmed previous studies in humans (Ham-ming et al., 2004, Grzegrzolka et al., 2013) showing that ACE2 is expressed in the skin.

(17) It appears ACE2 directly degrades α-MSH ACE2. Not to be bound by theory, ACE2 may however not directly degrade α-MSH, but may only indirectly activate its action on melanocytes.

(18) ACE2 Limits the Efficiency of αMSH1-13 in Human Skin:

(19) As shown in FIGS. 4 and 5, Tyrosinase (melanin synthesizing enzyme) mRNA expression was measured in human abdominal skin biopsies, and Melanin was stained black by Fontana-Masson staining in human abdominal skin biopsies. Biopsies were treated with vehicle, 10 nM αMSH1-13, 10 nM αMSH1-13+100 nM of the ACE2-Inhibitor MLN4760, or 100 nM MLN4760 alone.

(20) As expected, αMSH1-13 stimulates tyrosinase expression. This effect is increased by ACE2 inhibition. ACE2 inhibition alone already stimulates tyrosinase expression.

(21) Also, as expected, αMSH1-13 increases melanin levels. This effect is further increased by ACE2 inhibition. These results again indicate that ACE2 limits the efficiency of αMSH1-13 in human skin.

(22) ACE2 Plays a Limiting Role on Melanin Synthesis in Mouse Skin:

(23) As shown in FIG. 6, Melanin was isolated from hair of C57131/6 (B16) wildtype (WT) and ACE2-deficient (KO) mice and quantified by spectrophotometry (405 nm). The melanin content is higher in the absence of ACE2 confirming a limiting role of ACE2 on melanin synthesis in mouse skin.

(24) Further experimentation is ongoing to further support and elucidate the present invention.

(25) DIZE Inhibits the Stimulating Effect of αMSH by Activating ACE2 or Increasing its Expression:

(26) As shown in FIGS. 7 and 8, DIZE inhibits the stimulating effect of αMSH, probably by activating ACE2 or increasing its expression in B16F10 mouse melanoma cells. B16F10 mouse melanoma cells were incubated with 1 nM aMSH, 10 ng/μl diminazene aceturate (DIZE, ACE2 activator) or a combination of both for 48 hours and the expression of the melanin-synthesizing enzyme tyrosinase (TYR) was quantified by real-time RT-PCR. DIZE inhibits the stimulating effect of αMSH.

(27) Furthermore, B16F10 mouse melanoma cells were incubated with 10 ng/μl diminazene aceturate (DIZE, ACE2 activator) for 48 hours and the expression of ACE2 was quantified by real-time RT-PCR. DIZE stimulates ACE2 expression in these cells.

(28) We further expect XNT to provide a similar effect in assays of this kind.

(29) Measurement of α-MSH and Degradation Products in the Skin of ACE2-KO, PRCP-KO, and Wild-Type Mice by LC-MS:

(30) An LC/MS method is carried out to distinguish between α-MSH.sub.1-13 and α-MSH.sub.1-12, the putative degradation product generated by ACE2. Skin and other organs of ACE2-KO, PRCP-KO and wild-type mice are isolated and the content of these peptides is measured. Degradation may be performed with or without an agent of the invention (ACE2 modulator). We expect to observe degradation of alpha MSH by ACE2 incubation, and subsequent inhibition or activation of this activity in the presence of ACE2 inhibitors or activators, respectively.

(31) α-MSH Degradation Studies in vitro:

(32) MSH.sub.1-13 is incubated with commercially available recombinant human ACE2 and the resulting products are analyzed by LC/MS. Degradation may be performed with or without an agent of the invention (ACE2 modulator). We expect to observe degradation of alpha MSH by ACE2 incubation, and subsequent inhibition or activation of this activity in the presence of ACE2 inhibitors, or activators, respectively.

(33) Effect of ACE2 Inhibition on Skin and Hair Pigmentation in vivo:

(34) Agouti-coloured wild-type and ACE2-KO mice are partially shaved and the ACE2 inhibitor, MLN-4760 (IC.sub.50: 0.5 nM), is applied to the skin at a 10 mM concentration for 3 consecutive days. Then some animals are sacrificed and the concentration of α-MSH.sub.1-13 and α-MSH.sub.1-12 and the expression of melanogenesis genes is determined at the site of application and compared to another untreated part of the skin. Some animals remain alive and the colour of the regrown hair is determined.

(35) Incubation of Human Skin Biopsies with ACE2 Activators (E.G., DIZE) in Tissue Culture Inhibits Melanin Synthesis and Reduces Melanin Content:

(36) Human abdominal skin biopsies are commercially available. They will be incubated for 5 days with 3 different concentrations of the ACE2 activator diminazene aceturate (DIZE) or XNT. For each concentration 6 different skin discs will be applied. Afterwards, the skin samples will be cut in 2 pieces and melanin content will be measured in one part by a spectrophotometric method. For this purpose, the tissue will be powdered in liquid nitrogen and then incubated in 1 M NaOH and 10% DMSO for 24 h. After centrifugation the melanin content will be measured using an absorbance reader at 475 nm and normalized to the total tissue protein concentration. Moreover, expression of melanogenesis genes, such as for tyrosinase, Trp1, and Trp2, will be quantified by real-time qPCR in RNA isolated from the other part of the skin samples. We expect a dose-dependent decrease in melanin content and melanogenesis gene expression by DIZE or XNT treatment.

(37) Injection of ACE2 Inhibitors (E.G., MLN4760) into Mice Stimulates Melanin Synthesis in the Skin and Increases Melanin Content:

(38) The ACE2 inhibitor MLN4760 will be injected into the skin of mice at 3 different concentrations daily for 4 days. Afterwards the skin is isolated and cut in 2 pieces. Melanin content will be measured in one part by a spectrophotometric method. For this purpose, the tissue will be powdered in liquid nitrogen and then incubated in 1 M NaOH and 10% DMSO for 24 h. After centrifugation the melanin content will be measured using an absorbance reader at 475 nm and normalized to the total tissue protein concentration. Moreover, expression of melanogenesis genes, such as for tyrosinase, Trp1, and Trp2, will be quantified by real-time qPCR in RNA isolated from the other part of the skin samples. We expect a dose-dependent increase in melanin content and melanogenesis gene expression by MLN4760 treatment.

(39) Injection of ACE2 Activators (e.g., DIZE) into Mice Inhibits Melanin Synthesis in the Skin and Reduces Melanin Content:

(40) The ACE2 activator DIZE or XNT will be injected into the skin of mice at 3 different concentrations daily for 4 days. Afterwards the skin is isolated and cut in 2 pieces. Melanin content will be measured in one part by a spectrophotometric method. For this purpose, the tissue will be powdered in liquid nitrogen and then incubated in 1 M NaOH and 10% DMSO for 24 h. After centrifugation the melanin content will be measured using an absorbance reader at 475 nm and normalized to the total tissue protein concentration. Moreover, expression of melanogenesis genes, such as for tyrosinase, Trp1, and Trp2, will be quantified by real-time qPCR in RNA isolated from the other part of the skin samples. We expect a dose-dependent decrease in melanin content and melanogenesis gene expression by DIZE or XNT treatment.

(41) Topical Application of ACE2 Inhibitors (E.G., MLN4760) to the Skin of Mice and Human Volunteers Stimulates Melanin Synthesis, Increases Melanin Levels, and Tans the Skin:

(42) The ACE2 inhibitor MLN4760 will be topically applied to the skin of C57BL/6 mice at 3 different concentrations daily for 4 days. Afterwards the skin is isolated and cut in 2 pieces. Melanin content will be measured in one part by a spectrophotometric method. For this purpose, the tissue will be powdered in liquid nitrogen and then incubated in 1 M NaOH and 10% DMSO for 24 h. After centrifugation the melanin content will be measured using an absorbance reader at 475 nm and normalized to the total tissue protein concentration. Moreover, expression of melanogenesis genes, such as for tyrosinase, Trp1, and Trp2, will be quantified by real-time qPCR in RNA isolated from the other part of the skin samples. We expect a dose-dependent increase in melanin content and melanogenesis gene expression by MLN4760 treatment.

(43) The ACE2 inhibitor MLN4760 will be topically applied to the skin of Caucasian volunteers at 3 different concentrations daily for 4 days. Afterwards the skin pigmentation is quantified by a Skin Pigmentation Analyzer. We expect a dose-dependent increase in skin pigmentation by MLN4760 treatment.

(44) Topical Application of ACE2 Activators (E.G., DIZE) to the Skin of Mice and Human Volunteers Inhibits Melanin Synthesis, Reduces Melanin Levels, and Lightens The Skin:

(45) The ACE2 activator DIZE or XNT will be topically applied to the skin of C57BL/6 mice at 3 different concentrations daily for 4 days. Afterwards the skin is isolated and cut in 2 pieces. Melanin content will be measured in one part by a spectrophotometric method. For this purpose, the tissue will be powdered in liquid nitrogen and then incubated in 1 M NaOH and 10% DMSO for 24 h. After centrifugation the melanin content will be measured using an absorbance reader at 475 nm and normalized to the total tissue protein concentration. Moreover, expression of melanogenesis genes, such as for tyrosinase, Trp1, and Trp2, will be quantified by real-time qPCR in RNA isolated from the other part of the skin samples. We expect a dose-dependent decrease in melanin content and melanogenesis gene expression by DIZE or XNT treatment.

(46) The ACE2 activator MLN4760 will be topically applied to the skin of Caucasian volunteers at 3 different concentrations daily for 4 days. Afterwards the skin pigmentation is quantified by a Skin Pigmentation Analyzer. We expect a dose-dependent decrease in skin pigmentation by DIZE treatment.

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