Pharmaceutical compositions and their use in the treatment of conditions and diseases associated with elastin deficiency and / or aging

Abstract

Provided are pharmaceutical compositions and their use in treating or preventing an elastin deficiency-related disease in a subject in need thereof.

Description

Technical Field

[0001] This invention belongs to the field of conditions and diseases related to elastin deficiency and aging. Background Technology

[0002] Elastin is an important extracellular matrix (ECM) protein that provides tissues and organs with resilience and elasticity. It is a protein that can stretch (elongate) and retract (retract). Elastin is a major component of tissues in the body that require stretching, such as the lungs, bladder, large blood vessels, and certain ligaments. It is also present in small amounts in the skin and ear cartilage. Elastin's primary function is to allow tissues in the body to stretch and retract. For example, elastin gives arteries stretching properties, making it easier for the heart to pump blood. Elastin also allows the lungs to function like elastic bags.

[0003] Elastin is produced and secreted in the form of soluble monomeric elastin (TE). During individual development, soluble TE monomers are secreted by cells such as smooth muscle cells, fibroblasts, and endothelial cells, and assembled into highly stable, insoluble polymeric elastin fibers in the ECM under the action of enzymes (i.e., lysyl oxidase). The process of elastin fiber formation is generally referred to as elastinogenesis.

[0004] Elastin expression is primarily confined to the fetal period and early postnatal years. Elastin synthesis decreases from puberty and ceases in adulthood. Elastin has a half-life of approximately 74 years, making it one of the most stable proteins known. While elastin is a highly resilient component of the ECM, elastic fibers degenerate with age and under certain pathophysiological conditions and / or as a result of injury—a process commonly referred to as elastin dissociation. Elastin dissociation leads to a loss of elasticity and flexibility, resulting in loss of integrity and function, and also results in the release of elastin-derived peptides (EDPs). Some of these EDPs, known as elastokines, have been shown to possess biologically active properties through interaction with their homologous receptors, known as the elastin receptor complex, which is generally considered to play a role in (further) pathological development.

[0005] In the normal aging process, the gradual loss of elastin leads to tissue deterioration, resulting in organ damage and inflammation. This inflammation accelerates elastin degradation, creating a vicious cycle that contributes to certain age-related diseases. For example, the loss of elastin in blood vessels can lead to conditions such as AAA, where the vessel walls weaken and bulge, posing serious health risks, including cardiovascular disease. Increasing evidence also suggests that elastin-like peptide-1 (EDP) released during the dissociation of elastic tissue plays a role in the development of other age-related diseases and conditions through its own bioactive properties. Recent reports, for example, indicate that elastin-like peptides can induce excessive production of β-amyloid protein in Alzheimer's disease models.

[0006] Abnormalities in elastin production and / or elastic tissue dissociation can occur early in life and cause pathologies, such as due to genetic defects and acquired diseases. Well-known (hereditary) elastin deficiency-related diseases include Williams-Bueren syndrome and cutis laxa. Williams-Bueren syndrome (WBS) is a genetic disorder affecting many parts of the body and is caused by a haploinsufficiency of the ELN gene, resulting from the deletion of approximately 27 genes on the long arm of one of the two chromosomes 7, where the ELN gene is also located. Cutis laxa is a rare connective tissue disorder caused by mutations in the ELN gene, leading to a loss of skin elasticity and sagging into wrinkles. Acquired forms of cutis laxa are also known.

[0007] Given that elastin degradation is associated with various health problems and the natural aging process, it has long been a focus of attention for medical experts. However, the relative lack of success in developing interventional therapies to date is surprising. New pharmacological approaches are urgently needed to treat elastin-related diseases. One of the objectives of this invention is to meet this need. Summary of the Invention

[0008] This invention is based on the discovery that certain small molecules and combinations thereof are effective in reducing elastin loss and / or inducing or promoting the recovery of elastin deposition. Therefore, these findings strongly support the (clinical) utility of these small molecules and combinations thereof in the treatment of elastin deficiency-related diseases and in treatments aimed at slowing certain aspects of the aging process, thereby promoting the health and well-being of older adults.

[0009] More specifically, aged mice treated subcutaneously twice weekly with 1 mg / kg DCP-LA, 1 mg / kg DCP-LA + 3 mg / kg AS111793 (MMP-408), 1 mg / kg DCP-LA + 3 mg / kg alvelestat, or 1 mg / kg DCP-LA + 5 mg / kg U0126 for 12 weeks showed increased elastin expression compared to the control group, and were associated with improvements in cognitive / neurological function and the (visual) appearance of skin and fur. The combination of DCP-LA with MMP-408 and the combination of DCP-LA with alvelestat yielded particularly good results. These results support the use of these compounds and their analogues, especially combinations of such compounds, to treat and / or prevent conditions and / or diseases associated with the aging process and to promote longevity.

[0010] Therefore, according to the first aspect, a pharmaceutical composition is provided comprising a first compound selected from one group of (I), (II), (III) and (IV), and preferably a second compound selected from another group of said groups (I), (II), (III) and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors.

[0011] In a particularly preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (I) and a second compound selected from any one of groups (II), (III) and (IV).

[0012] The pharmaceutical composition is also provided, comprising a first compound selected from one group (i), (ii), (iii) and (iv) and a second compound selected from another group (i), (ii), (iii) and (iv): (i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and its salts, esters and stereoisomers; (ii) (U0126), and its salts and stereoisomers; (iii) (Alvelestat), and its salts and stereoisomers; and (iv) (MMP-408), and its salts, esters and stereoisomers.

[0013] In a particularly preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (I) and a second compound selected from any one of groups (II), (III) and (IV).

[0014] According to another aspect, a method for providing treatment to a subject in need, the method comprising administering to the subject at least one compound selected from any one of the following groups (I), (II), (III), and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors.

[0015] According to a preferred embodiment, the method includes applying to the object a first compound selected from one group of groups (I), (II), (III), and (IV), and a second compound selected from another group of groups (I), (II), (III), and (IV).

[0016] According to a more preferred embodiment, the method includes applying to the object a first compound selected from group (I) and a second compound selected from any one of groups (II), (III) and (IV).

[0017] A method of treating a subject in need is also provided, the method comprising administering to the subject a compound selected from any one of the following groups (i), (ii), (iii) and (iv): (i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and its salts, esters and stereoisomers; (ii) (U0126), and its salts and stereoisomers; (iii) (Alvelestat), and its salts and stereoisomers; and (iv) (MMP-408), and its salts, esters and stereoisomers.

[0018] According to a preferred embodiment, the method includes applying to the object a first compound selected from one group of groups (i), (ii), (iii), and (iv), and a second compound selected from another group of groups (i), (ii), (iii), and (iv).

[0019] According to a more preferred embodiment, the method includes applying to the object a first compound selected from group (i) and a second compound selected from any one of groups (ii), (iii) and (iv).

[0020] According to another aspect, in a method of providing a pharmaceutical composition for treating a subject in need, said pharmaceutical composition comprises at least one compound selected from any one of the following groups (I), (II), (III), and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors.

[0021] According to a preferred embodiment, the pharmaceutical composition for use according to the invention comprises a first compound selected from one group of groups (I), (II), (III), and (IV), and the method further comprises administering a second compound selected from another group of groups (I), (II), (III), and (IV).

[0022] According to a more preferred embodiment, the pharmaceutical composition for use according to the invention comprises a first compound selected from group (I), and the method further comprises administering a second compound selected from any one of groups (II), (III), and (IV).

[0023] A pharmaceutical composition is also provided for use in a method of treating a subject in need, wherein the pharmaceutical composition comprises compounds selected from any group (i), (ii), (iii) and (iv): (i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and its salts, esters and stereoisomers; (ii) (U0126) and its salts and stereoisomers; (iii) (Alvelestat), and its salts and stereoisomers; and (iv) (MMP-408), and its salts, esters and stereoisomers.

[0024] According to a preferred embodiment, the pharmaceutical composition for use according to the invention comprises a first compound selected from one group of groups (i), (ii), (iii), and (iv), and the method further comprises administering a second compound selected from another group of groups (i), (ii), (iii), and (iv).

[0025] According to a more preferred embodiment, the pharmaceutical composition for use according to the invention comprises a first compound selected from group (i), and the method further comprises administering a second compound selected from any one of groups (ii), (iii), and (iv).

[0026] According to another aspect, the present invention provides use of at least one compound selected from any one of the groups (I), (II), (III) and (IV) in the preparation of a medicament for use in a method of treating a subject in need: (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors.

[0027] According to a preferred embodiment, the use of a first compound selected from one group of groups (I), (II), (III), and (IV) in manufacturing a medicament for use in a method of treating a subject in need is provided, wherein the method further comprises administering a second compound selected from another group of groups (I), (II), (III), and (IV).

[0028] According to a preferred embodiment, the use of a first compound selected from group (I) in manufacturing a medicament for use in a method of treating a subject in need is provided, wherein the method further comprises administering a second compound selected from any one of groups (II), (III) and (IV).

[0029] According to a particularly preferred embodiment, use is provided of compounds selected from any of the following groups (i), (ii), (iii) and (iv) in the manufacture of a medicament for use in a method of treating a person in need: (i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and its salts, esters and stereoisomers; (ii) (U0126), and its salts and stereoisomers; (iii) (Alvelestat) and its salts and stereoisomers; and (iv) (MMP-408), and its salts, esters and stereoisomers.

[0030] According to a preferred embodiment, the use of a first compound selected from one group of groups (i), (ii), (iii), and (iv) in manufacturing a medicament for use in a method of treating a subject in need is provided, wherein the method further comprises administering a second compound selected from another group of groups (i), (ii), (iii), and (iv).

[0031] According to a preferred embodiment, the use of a first compound selected from group (i) in manufacturing a medicament for use in a method of treating a subject in need is provided, wherein the method further includes administering a second compound selected from any one of groups (ii), (iii), and (iv).

[0032] According to another aspect, a drug kit is provided, comprising a package containing multiple unit dosage forms, each unit dosage form comprising a first compound selected from any one of groups (I), (II), (III), and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors; The drug kit further includes a leaflet containing printed instructions for (self-administering) the unit dosage form to achieve any therapeutic goal as defined herein, such as treating and / or preventing conditions and / or diseases associated with the aging process and promoting longevity.

[0033] According to another aspect, a pharmaceutical kit is provided, comprising a package containing a first plurality of unit dosage forms and a second plurality of unit dosage forms, wherein the unit dosage forms constituting the first plurality of units comprise a first compound selected from one group of groups (I), (II), (III), and (IV), and the unit dosage forms constituting the second plurality of units comprise a second compound selected from another group of groups (I), (II), (III), and (IV), wherein the pharmaceutical kit optionally further comprises an insert containing printed instructions for (self-administering) the unit dosage forms contained in the kit to achieve any therapeutic goal as defined herein, such as treating and / or preventing conditions and / or diseases associated with the aging process and promoting longevity. According to a more preferred embodiment, a pharmaceutical kit is provided, wherein the first compound is selected from group (I) and the second compound is selected from any one group of groups (II), (III), and (IV).

[0034] Those skilled in the art will understand that all aspects defined herein relate to the same invention. Therefore, whenever definitions, explanations, embodiments, etc., are described in the context of one such aspect, they also apply to the other aspects, unless otherwise indicated and / or would be contrary to common sense. The various aspects of the invention described above will be explained and illustrated in more detail in the following description and examples. Attached Figure Description

[0035] Figures 1A-1E Includes vertical bar graphs showing the effects of DCP-LA, DCP-LA with Alvelestat, DCP-LA with MMP408, or DCP-LA with U0126 on gene expression levels of elastin (1A), neutrophil elastase (NE; IB), MAP2K1 (1C), MMP-12 (ID), and fibrin (IE).

[0036] Figures 2A-2D The graph shows the effects of DCP-LA (2A), DCP-LA and Alvelestat (2B), DCP-LA and MMP408 (2C), and DCP-LA and U0126 (2D) on arterial stiffness. Figure 2E Further analysis of these results is presented (multiple comparison tests).

[0037] Figures 3A-3C Includes vertical bar graphs showing the effects of DCP-LA, DCP-LA with Alvelestat, DCP-LA with MMP408, or DCP-LA with U0126 treatments on the concentrations (μm) of NAD+ (3A), NADP+ (3B), and glutathione (GSH) (3C) in blood, measured using optimized cyclic enzymatic assays and colorimetric detection.

[0038] Figure 4A and Figure 4B This includes vertical bar graphs showing the effects of DCP-LA, DCP-LA with Alvelestat, DCP-LA with MMP408, or DCP-LA with U0126 treatments on plasma lysine and valine concentrations, expressed as relative spectral intensities (AU) measured by NMR.

[0039] Figures 5A-5D Includes vertical bar graphs showing the effects of treatments with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 on hind leg raising activity in mice, which was measured using an open field assay.

[0040] Figures 6A-6D Includes vertical bar graphs showing the effects of treatments with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 on mouse activity time, which was measured using an open field assay.

[0041] Figures 7A-7D Includes vertical bar graphs showing the effects of treatments with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA with Alvelestat, DCP-LA with MMP408, or DCP-LA with U0126 on the time mice spent in the central region, which was measured using an open field assay.

[0042] Figures 8A-8D Includes vertical bar graphs showing the effects of treatments with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 on immobility time in mice, which was measured using an open field assay.

[0043] Figure 9 It is a graphical representation of the experimental setup (treatment scheme).

[0044] Figure 10 a- Figure 10e shows the effect of the EB-101 + EB-102 combination (vs. control and single treatment) on the expression of elastin pathway genes (elastin (a), neutrophil elastase (b), MMP-12 (c), MAP2K1 (d) and fibrin-5 (e)).

[0045] Figure 11 a- Figure 11 e shows the effect of the EB-102 + EB-103 combination (vs. control and single treatment) on the expression of elastin pathway genes (elastin (a), MAP2K1 (b), neutrophil elastase (c), MMP-12 (d) and fibrin-5 (e)).

[0046] Figure 12 a- Figure 12 e shows the effect of the EB-102 + EB-104 combination (vs. control and single treatment) on the expression of elastin pathway genes (elastin (a), MAP2K1 (b), neutrophil elastase (c), MMP-12 (d) and fibrin-5 (e)).

[0047] Figure 13 a- Figure 13 e shows the effects of all test combinations (vs. control and all single treatments) on the expression of genes in the elastin pathway (elastin (a), fibrin-5 (b), MMP-12 (c), MAP2K1 (d), and neutrophil elastase (e)).

[0048] Figure 14 a- Figure 14 c shows the effect of the EB-101 + EB-102 combination (vs. control and single treatment) on inflammatory markers (TNFα (a), IL-1β (b) and IL-6 (c)).

[0049] Figure 15 a- Figure 15 c shows the effect of the EB-102 + EB-103 combination (vs. control and single treatment) on inflammatory markers (TNFα (a), IL-1β (b) and IL-6 (c)).

[0050] Figure 16 a- Figure 16 c shows the effect of the EB-102 + EB-104 combination (vs. control and single treatment) on inflammatory markers (TNFα (a), IL-1β (b) and IL-6 (c)).

[0051] Figure 17 a- Figure 17c shows the effect of all test combinations (vs. control and all single treatments) on inflammatory markers (TNFα(a), IL-6(b) and IL-1β(c)).

[0052] Figure 18 a- Figure 18 f shows the effect of the EB-101 + EB-102 combination (vs. control and single treatment) on the expression of genes involved in Williams syndrome (dense protein-4 (a), BCL7B (b), NCF-1 (c), dense protein-3 (d), GTF2I (e) and RFC2 (f)) in aortic tissue samples.

[0053] Figure 19 a- Figure 19 f shows the effect of the EB-102 + EB-103 combination (vs. control and single treatment) on the expression of genes involved in Williams syndrome (dense protein-4 (a), BCL7B (b), dense protein-3 (c), NCF-1 (d), RFC2 (e) and GTF2I (f)) in aortic tissue samples.

[0054] Figure 20 a- Figure 20 f shows the effect of the EB-102 + EB-104 combination (vs. control and single treatment) on the expression of genes involved in Williams syndrome (dense protein-4 (a), BCL7B (b), NCF-1 (c), dense protein-3 (d), GTF2I (e) and RFC2 (f)) in aortic tissue samples.

[0055] Figure 21 a- Figure 21 k shows the effects of the EB-101 + EB-102 combination (vs. control and single treatment) on the expression of inflammatory markers and genes involved in Williams syndrome in the brain (IL-1(a), IL-6(b), STX1A(c), FK-506(d), F2ZD9(e), BAZB1(f), HIP-1(g), EIF-4H(h), bHLH(i), GTF2I(j) and GALNT17(k)).

[0056] Figure 22 a- Figure 22k shows the effects of the EB-102 + EB-103 combination (vs. control and single treatment) on the expression of inflammatory markers and genes involved in Williams syndrome in the brain (IL-1(a), IL-6(b), STX1A(c), FK-506(d), F2ZD9(e), GALNT17(f), BAZB1(g), HIP-1(h), bHLH(i), EIF-4H(j) and GTF2I(k)).

[0057] Figure 23 a- Figure 23 k shows the effects of the EB-102 + EB-104 combination (vs. control and single treatment) on the expression of inflammatory markers and genes involved in Williams syndrome in the brain (IL-1 (a), IL-6 (b), STX1A (c), FK-506 (d), F2ZD9 (e), GALNT17 (f), BAZB1 (g), HIP-1 (h), bHLH (i), EIF-4H (j), and GTF2I (k)).

[0058] Figure 24 a- Figure 24 f shows the effects of the EB-101 + EB-102 combination (vs. control and single treatment) on behavioral parameters (distance traveled (a), average speed (b), time at rest (c), time active (d), hind leg upright (e), and time in the central region (f)).

[0059] Figure 25 a- Figure 25 f shows the effects of the EB-102 + EB-103 combination (vs. control and single treatment) on behavioral parameters (distance traveled (a), average speed (b), time at rest (c), time active (d), time in the central region (e), and hind leg upright (f)).

[0060] Figure 26 a- Figure 26 f shows the effects of the EB-102 + EB-104 combination (vs. control and single treatment) on behavioral parameters (distance traveled (a), average speed (b), time at rest (c), time active (d), hind leg upright (e), and time in the central region (f)).

[0061] Figure 27 This demonstrates the effect of the EB-101 + EB-102 combination (vs. control and single treatment) on the tensile strength of arteries after diastole following acetylcholine treatment.

[0062] Figure 28This demonstrates the effect of the EB-102 + EB-103 combination (vs. control and single treatment) on the tensile strength of arteries after diastole following acetylcholine treatment.

[0063] Figure 29 This demonstrates the effect of the EB-102 + EB-104 combination (vs. control and single treatment) on the tensile strength of arteries after diastole following acetylcholine treatment.

[0064] Figure 30 This shows the tensile strength of arteries after vasodilation following acetylcholine treatment in aged mice versus young (untreated) mice.

[0065] Figure 31 This demonstrates the effect of the EB-101 + EB-102 combination (vs. control and single treatment) on arterial tensile strength after phenylephrine treatment.

[0066] Figure 32 This study demonstrates the effect of the EB-102 + EB-103 combination (vs. control and single treatment) on arterial tensile strength after phenylephrine treatment.

[0067] Figure 33 This study demonstrates the effect of the EB-102 + EB-104 combination (vs. control and single treatment) on arterial tensile strength after phenylephrine treatment.

[0068] Figure 34 Arterial tensile strength after norepinephrine treatment in aged mice vs. young mice (untreated).

[0069] Figure 35 A heatmap showing the results of mRNA sequencing analysis of brain samples.

[0070] Figure 36 A heatmap showing the results of mRNA sequencing analysis of aortic samples.

[0071] Figure 37 a- Figure 37 k shows the effects of all test combinations (vs. control and all single treatments) on the selected metabolomic components ((a) tryptophan; (b) histidine; (c) phenylalanine; (d) tryptophan; (e) glutamine; (f) glutamate; (g) creatine; (h) lysine; (i) fumarate; (j) citric acid and (k) succinate). Detailed Implementation

[0072] compound It will be apparent to those skilled in the art that all aspects of the present invention relate to the presence / use of one or more compounds belonging to the groups (I), (II), (III) and (IV) and / or groups (i), (ii), (iii) and (iv).

[0073] As noted earlier herein, group (I) consists of compounds capable of increasing tropoelastin expression. In a sense, a compound is considered capable of increasing tropoelastin expression if its application leads to an increase in tropoelastin mRNA or tropoelastin protein expression levels. In a preferred embodiment, group (I) consists of compounds that are activators of protein kinase Cε type (PKCε) activators and / or compounds capable of activating PKCε in suitable assays (such as those described elsewhere herein). Therefore, according to a preferred embodiment of the invention, group (I) preferably consists of PKCε activators and / or compounds capable of activating PKCε. In a sense, a compound is considered capable of activating PKCε if, for example, it enhances PKCε expression and / or activity in suitable assays (such as those described elsewhere herein). PKCε activators can be non-specific or specific activators. Specific activators achieve greater detectability of PKCε activation compared to other PKC isoforms.

[0074] A specific class of PKCε activators—DCP-LA—belongs to this class and is (therefore) considered to have specific utility in the context of this invention—is described in WO2010 / 014585 (the contents of which are incorporated herein by reference). According to WO2010 / 014585, several PUFAs (such as arachidonic acid and docosahexaenoic acid (DHA)) have been known for many years to be natural activators of PKC. WO2010 / 014585 provides certain derivatives of polyunsaturated fatty acids (PUFAs) or monounsaturated fatty acids (MUFAs) that activate PKCε at nanomolar concentrations and teaches their use in the treatment of AD, stroke, and other neurological disorders in which PKCε has neuroprotective effects.

[0075] In a preferred embodiment, the PKCε activator is selected from the group consisting of: compounds that are cis-polyunsaturated fatty acids, cis-polyunsaturated fatty acid esters, or cis-polyunsaturated fatty alcohols, wherein at least one double bond is substituted by a cyclopropane ring / group, preferably wherein all double bonds are substituted by cyclopropane; compounds that are cis-monounsaturated fatty acids, cis-monounsaturated fatty acid esters, or cis-monounsaturated fatty alcohols, wherein the double bonds are substituted by a cyclopropane ring / group; and compounds that are cis-polyunsaturated fatty acids, cis-polyunsaturated fatty acid esters, or cis-polyunsaturated fatty alcohols, wherein at least one double bond is substituted by an epoxy group. In a preferred embodiment, the fatty acid constituting or serving as the basic structure of the PKCε activator has the structure CH3(CH2)4(CH=CHCH2). x (CH2) y COOH or CH3CH2(CH=CHCH2)x(CH2) y COOH, where x is 2 to 6 and y is 2 to 6, or the structure CH3(CH2). x CH=CH(CH2) y COOH, where X and Y are odd numbers between 3 and 11. In a preferred embodiment, the fatty acid is arachidonic acid, docosapentaenoic acid, docosahexaenoic acid, eicosapentaenoic acid, linoleic acid, linolenic acid, γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid, adrenaline, oleic acid, transoleic acid, transoleyl alcohol, oleyl alcohol, myristoleic acid, palmitoleic acid, piracetamonic acid, and 1-monolinoleylrac-glycerol. In a particularly preferred embodiment, the fatty acid is linoleic acid.

[0076] In a preferred embodiment, the PKCε activator is selected from methyl docosahexaenoic acid cyclopropane, methyl eicosapenoic acid, methyl arachidonic acid cyclopropane, linoleic acid dicyclopropane, linolenic acid tricyclopropane, transoleic acid cyclopropane, oleic acid cyclopropane, and methyl benzoyl cyclopropane.

[0077] In a particularly preferred embodiment, the PKCε activator is selected from (i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) and its salts, esters and stereoisomers.

[0078] In the most preferred embodiment, the PKCε activator is 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) or its methyl ester (DCP-LA-ME), more preferably 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA).

[0079] As noted earlier in this document, group (II) consists of compounds capable of increasing elastin fiber deposition. In a sense, a compound is considered capable of increasing elastin fiber deposition if its application results in increased elastin fiber deposition in the extracellular matrix. In a preferred embodiment, group (II) consists of mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitors. In a sense, a compound is considered capable of inhibiting mitogen-activated protein kinase kinase 1 (MAP2K1) if it reduces MAP2K1 mRNA expression and / or the activity of the MAP2K1-encoded protein.

[0080] A specific class of MAP2K1 inhibitors—which are believed to have specific utility in the context of this invention—is described in US2,779,780 (the contents of which are incorporated herein by reference).

[0081] In a preferred embodiment, group (II) comprises an elastase inhibitor selected from compounds of formula (IIa) and their pharmaceutically acceptable salts. (IIa) Wherein R is a radical of 1 to 10 carbon atoms selected from the class of free radicals of hydrocarbons, monohydroxy hydrocarbons, monoamino hydrocarbons, mononitro hydrocarbons and monohalogenated hydrocarbons; more preferably, wherein R is a radical selected from phenyl, aminophenyl, especially o-aminophenyl, methyl, ethyl and hydroxyethyl.

[0082] In a preferred embodiment, group (II) comprises a MAP2K1 inhibitor selected from the following: 1,4-diamino-2,3-dicyano-1,4-bis(methyl mercapto)-butadiene; 1,4-diamino-2,3-dicyano-1,4-bis(ethyl mercapto)-butadiene; 1,4-diamino-2,3-dicyano-1,4-bis(phenyl mercapto)-butadiene; 1,4-diamino-2,3-dicyano-1,4-bis(β-hydroxyethyl mercapto)-butadiene; 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)-butadiene; 1,4-Diamino-2,3-dicyano-1,4-bis(naphthylthio)butadiene, 1,4-diamino-2,3-dicyano-1,4-bis(cyclohexylthio)butadiene; 1,4-diamino-2,3-dicyano-1,4-bis(α-amino-β-naphthylthio)butadiene, 1,4-diamino-2,3-dicyano-1,4-bis(α-hydroxy-β-naphthylthio)butadiene, 1,4-diamino-2,3-dicyano-1,4-bis(p-chlorophenylthio)butadiene, 1,4-diamino-2,3-dicyano-1,4-bis(p-nitrophenylthio)butadiene and 1,4-diamino-2,3-dicyano-1,4-bis(2-chloroethylthio)butadiene and their salts.

[0083] In a particularly preferred embodiment, the MAP2K1 inhibitor is selected from (ii). (1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene) and its salts and stereoisomers.

[0084] In the most preferred embodiment, the MAP2K1 inhibitor is (1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene).

[0085] As noted earlier herein, group (III) consists of compounds capable of reducing or inhibiting the breakdown or degradation of elastin fibers. In a sense, (e.g., in established in vitro assays) a compound is considered to inhibit elastin fiber breakdown or degradation if its application results in a decrease in the level of elastin fiber breakdown or degradation. In some embodiments, compounds selected from group (III) are elastase inhibitors or compounds capable of inhibiting elastase. In a preferred embodiment, the elastase is neutrophil elastase. Therefore, in a preferred embodiment, group (III) consists of neutrophil elastase inhibitors. In a sense, a compound is considered to inhibit (neutrophil) elastase if its application results in a decrease in the expression and / or activity of elastase. In a preferred embodiment, the compound inhibits human neutrophil elastase activity in the Human Neutrophil Elastase Quenched-FRET Assay (as described elsewhere herein).

[0086] A specific class of neutrophil elastase inhibitors—which are believed to have specific utility in the context of this invention—is described in WO2005 / 026123 (the contents of which are incorporated herein by reference).

[0087] In a preferred embodiment, group (III) comprises an elastase inhibitor selected from compounds of formula (IIIa) and their pharmaceutically acceptable salts. (IIIa) Where Y represents CR 3 Or N, preferably Y represents CR 3 ;R 1 Represents H or C1-6 alkyl; R 2 Represents a phenyl group or a 5- or 6-membered heteroaromatic ring containing 1 to 4 heteroatoms independently selected from O, S, and N; said aromatic ring is independently selected from OH, halogen, C1-6 alkyl, C1-6 alkoxy, NR 58 COR 50 COOR 51 COR 52 CONR 53 R 54 and NR 47 R 48 One to three substituents are optionally substituted; the alkyl group is replaced by OH, C1-6 alkoxy, CN or CO2R. 49 Optional further replacement; R 47 and R 48 Independently represents H, C1-6 alkyl, or C2-6 alkanoyl; R 3 Represents H or F; G 1 Represents a phenyl group or a 5- or 6-membered heteroaromatic ring containing 1 to 3 heteroatoms independently selected from O, S, and N, preferably G. 1 Represents phenyl; R 5 Represents H, halogen, C1-6 alkyl, CN, C1-6 alkoxy, NO2, NR 14 R 15 C1-3 alkyl groups substituted with one or more F atoms, or C1-3 alkoxy groups substituted with one or more F atoms; R 14 and R 15 Independently representing H or C1-3 alkyl; the alkyl group is optionally further substituted by one or more F atoms; n represents an integer 1, 2, or 3, and when n represents 2 or 3, each R 5 The functional group is selected independently, preferably R. 5 Represents Cl, CH3, CN, or CF3; R 4Represents H or C1-6 alkyl; said alkyl group optionally further substituted by OH or C1-6 alkoxy group; or R 4 The group -NR is attached to L. 4 L represents the optional inclusion of options O, S, and NR. 16 A 5- to 7-membered nitrogen heterocycle with another heteroatom; L represents bond, O, S(O). P NR 29 Or C1-6 alkyl; the alkyl group optionally incorporates elements selected from O, S, and NR. 16 The heteroatom; and the alkyl group is optionally further substituted with OH or OMe; G 2 The following monocyclic ring systems are represented: i) phenyl or phenoxy; ii) a 5- or 6-membered heteroaromatic ring containing 1 to 3 heteroatoms independently selected from O, S, and N; iii) a C3-6 saturated or partially unsaturated cycloalkyl group; or iv) a ring containing 1 to 3 heteroatoms independently selected from O, S(O). p and NR 17 One or two heteroatoms and optionally further incorporated into a carbonyl group in a C4-7 saturated or partially unsaturated heterocycle; or G 2 Represents a bicyclic ring system, wherein each of the two rings is independently selected from: i) phenyl; ii) a 5- or 6-membered heteroaromatic ring containing 1 to 3 heteroatoms independently selected from O, S, and N; iii) a C3-6 saturated or partially unsaturated cycloalkyl group; or iv) containing a ring containing a heteroatom independently selected from O, S(O). P and NR 17 One or two heteroatoms and optionally further incorporated into a carbonyl group of a C4-7 saturated or partially unsaturated heterocycle; and the two rings are fused together, either directly bonded together or by means of O, S(O). P Separated by a CH2 linking group, the monocyclic or bicyclic ring system is independently selected from CN, OH, C1-6 alkyl, C1-6 alkoxy, halogen, NR. 18 R 19 NO2, OSO2R 38 CO2R 20 C(=NH)NH2, C(O)NR 21 R 22 C(S)NR 23 R 24 , SC(=NH)NH2, NR 31 C(=NH)NH2、S(O)2R 25 SO2NR 26 R 27 C1-3 alkoxy groups substituted with one or more F atoms and those substituted with SO2R 39 NR 56 R 57Or one to three substituents of a C1-3 alkyl group substituted with one or more F atoms may optionally be further substituted; or when L does not represent a bond, G 2 It can also represent H; in each occurrence, p, q, s, and t independently represent the integers 0, 1, or 2; R 18 and R 19 Independently representing H, C1-6 alkyl, formyl, C2-6 alkylyl, S(O)tR 32 or SO2NR 33 R 34 The alkyl group is coated with halogen, CN, C1-4 alkoxy, or CONR. 41 R 42 Optional further replacement; R 25 Represents H, C1-6 alkyl, or C3-6 cycloalkyl; the alkyl group is independently selected from OH, CN, CONR. 35 R 36 CO2R 37 OCOR 40 C3-6 cycloalkyl groups, containing components independently selected from O, S(O) P and NR 43 The phenyl ring may be further substituted with one or two heteroatoms and a C4-7 saturated heterocycle containing one to three heteroatoms independently selected from O, S, and N, or with one or more substituents optionally further substituted with a 5- or 6-membered heteroaromatic ring containing one to three heteroatoms independently selected from O, S, and N; the aromatic ring may be independently substituted with halogens, CN, C1-4 alkyl, C1-4 alkoxy, OH, CONR. 44 R 45 CO2R 46 、S(O)sR 55 And one or more substituents of NHCOCH3 may optionally be further substituted; R 32 Represents H, C1-6 alkyl, or C3-6 cycloalkyl; R 16 R 17 R 20 R 21 R 22 R 23 R 24 R 26 R 27 R 29 R 31 ,R 33 R 34 R 35 R 36 R 37 R 38 R 39 R 40 R 41 R 42 R 43 R 44R 45 R 46 R 49 R 50 R 51 R 52 R 53 R 54 R 55 R 56 R 57 and R 58 Independently represents H or C1-6 alkyl.

[0088] In a preferred embodiment, group (III) comprises an elastase inhibitor selected from the following: 6-methyl-N-[4-(methanesulfonyl)benzyl]-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 5-[4-hydroxymethyl)phenyl]-6-methyl-N-[4-(methanesulfonyl)benzyl]-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 5-furan-3-yl-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6'-methoxy-2-methyl-N [4(methylsulfonyl)benzyl]-6-oxo-1[3(trifluoromethyl)phenyl]1,6-dihydro-3,3'-bipyridine-5-carboxamide; 5(2-methoxypyrimidin-5-yl)-6-methyl-N[4-(methylsulfonyl)benzyl]-2-oxo-1[3-trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 5[4(acetylamino)phenyl]-6-methyl-N[4(methylsulfonyl)benzyl]-2-oxo-1[3(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-(1H-pyrrolo-3-yl)-1-(3-trifluoro) 5-Furan-2-yl-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-2-oxo-5-phenylthio-3-yl-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-2-oxo-5-phenylthio-2-yl-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(3,5-dimethyl-iso... 5-(2,4-dimethoxy-pyrimidin-5-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(2,4-dimethoxy-pyrimidin-5-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(2,4-dioxo-1,2,3,4-tetrahydro-pyrimidin-5-yl)-6-methyl-2-oxo-1-(3-trifluoromethyl-phenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-(5-methyl-[1,3,4] (diazol-2-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-2-oxo-5-(5-propyl-[1,3,4]) Diazol-2-yl)-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; {5-[5-(4-methanesulfonyl-benzylcarbamoyl)-2-methyl-6-oxo-1-(3-trifluoromethyl-phenyl)-1,6-dihydro-pyridine-3-yl]-[1,3,4] Diazol-2-yl}-ethyl acetate; 5-(5-cyanomethyl-[1,3,4] (diazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-amino-[1,3,4] 5-(5-amino-[1,3,4]thiadiazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-amino-[1,3,4]thiadiazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-ethylamino-[1,3,4]thiadiazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; (diazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-N,N-dimethylamino-[1,3,4]) Diazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethyl-phenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-N-[4-(methanesulfonyl)benzyl]-2-oxo-5-pyrazin-2-yl-1-[3-(trifluoro-methyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-5- 4-Methanesulfonylbenzylamide; 6-Methyl-5-(1-methyl-1H-imidazol-2-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid; 6-Methyl-2-oxo-5-(1H-pyrazol-4-yl)-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid; 6-Methyl-2-oxo-5-(1H-pyrazol-4-yl)-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid; 4-Methanesulfonylbenzylamide of pyridine-3-carboxylic acid; 6-methyl-N-[4-(methanesulfonyl)benzyl]-2-oxo-5-pyrimidin-2-yl-1-[3-(trifluoro-methyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-5-(2-methyl-2H-pyrazol-3-yl)-2-oxo-1-(trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxylic acid of 4-methanesulfonylbenzylamide; 6-methyl-5-(3-methylisocyanate) (-4-yl)-N-[4-(methylsulfonyl)benzyl]-2-oxo-1-[3-(trifluoromethyl)phenyl-1,2-dihydropyridine-3-carboxamide; 6-methyl-5-(3-methyl-[1,2,4-yl]-yl]-yl]-[3-(trifluoromethyl)phenyl-1,2-dihydropyridine-3-carboxamide; 6-methyl-5-(3-methyl-[1,2,4-yl]-yl]-[3-(trifluoromethyl)phenyl-1,2-dihydropyridine-3-carboxamide ... Diazol-5-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-(3-methylisocyanate) (5-azolyl)-N-[4-(methylsulfonyl)benzyl]-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 5-(3,5-dimethylisocyanate) (4-(isopropylsulfonyl)benzyl)-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 5-(3,5-dimethylisopropyl)benzyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; (4-azolyl)-N[4-(ethylsulfonyl)benzyl]-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N[4-(cyclopropylsulfonyl)benzyl]-5-(3,5-dimethylisocyanate) (-4-yl)-6-methyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 1-(3-cyanophenyl)-5-(3,5-dimethylisocyanate) (4-azolyl)-6-methyl-N-[4-(methylsulfonyl)benzyl]-2-oxo-1,2-dihydropyridine-3-carboxamide; 1-(3-chlorophenyl)-5-(3,5-dimethyl-isocyanuric acid) (4-yl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(3,5-dimethyl-iso (-4-yl)-6-methyl-2-oxo-1-m-tolyl-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-isopropyl-[1,3,4] (diazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-[1,3,4] (diazol-2-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-hydroxy-[1,3,4]) Diazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-(5-methyl-4H-[1,2,4]triazol-3-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(4,5-dimethyl-4H-[1,2,4]triazol-3-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-methoxymethyl-[1,3,4] diazol-2-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; N-[4-(isopropylsulfonyl)benzyl]-6-methyl-5-(5-methyl-1,3,4- diazol-2-yl)-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-[4-(ethylsulfonyl)benzyl]-6-methyl-5-(5-methyl-1,3,4-) diazol-2-yl)-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-[4-(cyclopropylsulfonyl)benzyl]-6-methyl-5-(5-methyl-1,3,4-) (diazol-2-yl)-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-5-[1,3,4] Diazol-2-yl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-(propane-2-sulfonyl)benzylamide; 6-methyl-5-[1,3,4] Diazol-2-yl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-cyclopropanesulfonyl-benzylamide; 6-methyl-5-(2-methyl-1,3- (-4-yl)-N[4-(methylsulfonyl)benzyl]-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-N[4-(methylsulfonyl)benzyl]-5-(1,3-) 5-(2-amino-thiazo-4-yl)-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 5-(2-amino-thiazo-4-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(2,5-dimethyl-1,3- (-4-yl)-6-methyl-N-[4-(methylsulfonyl)benzyl]-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-5-(5-methyl-1,3-) 5-(2-amino-5-methyl-thiazolyl-4-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(2-hydroxymethyl-5-methyl-thiazolyl-4-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(2-hydroxymethyl-5-methyl-thiazolyl-4-yl)-6-methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-(5-methyl-[1,2,4-yl] (diazol-3-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-[1,2,4] Diazol-3-yl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-2-oxo-5-(1H-tetrazol-5-yl)-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-(4-methyl- (-2-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(4,5-dimethyl- 6-Methyl-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; N-(cyclohexylmethyl)-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-N-(2-morpholin-4-ylethyl)-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-1H-1,2,4-triazol-3-yl-1-[3- [-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-[2-(1H-indol-3-yl)ethyl]-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-(1-phenylethyl)-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-5-phenyl-N-(2-phenylethyl)-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl- 2-Oxo-5-phenyl-N-[(2R)-2-phenylcyclopropyl]-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-(2,3-dihydro-1H-inden-2-yl)-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-[(1-ethylpyrrolidone-2-yl)methyl]-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-N-(1-naphthylmethyl)-2-oxo- 5-Phenylacetyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-(1,3-benzodioxol-5-ylmethyl)-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide carboxamide); N-(2-chloro-4-fluorobenzyl)-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-(2-thienylmethyl)-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide;N-(2-cyclohexyl-1-en-1-ylethyl)-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-N-(4-phenoxybenzyl)-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-[(2,5-dimethyl-3-furanyl)methyl]-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N{2-[4-(aminosulfonyl)phenyl] Ethyl}-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-[4-(1H-pyrazol-1-yl)benzyl]-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-N-phenoxy-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-[(6-fluoro-4H-1,3-benzodi] [N-[(6-fluoro-4H-1,3-benzodioxin-8-yl)methyl]-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide] dihydropyridine-3-carboxamide); 6-methyl-2-oxo-5-phenyl-N-[2-(tetrahydro-2H-pyran-4-yl)ethyl]-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-[3-(1H-pyrazol-1-yl)propyl]-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-N-[(1-methyl-1H-pyrazol-4-yl)methyl]-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide ; 6-methyl-2-oxo-5-phenyl-N-[(1-phenyl-1H-pyrazol-4-yl)methyl]-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-[(5-methoxy-4-oxo-4H-pyran-2-yl)methyl]-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-(3-azacycloheptane-1-ylpropyl)-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide (N-(3-azepan-1-ylpropyl)-6- methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; N-(4-cyanobenzyl)-6-methyl-2-oxo-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-N-[3-(5-oxo-4,5-dihydro-1H-pyrazol-4-yl)propyl]-5-phenyl-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 6-methyl-5-(2-methyl-2H-pyrazol-3-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydropyridine-3-carboxamide (3-methyl-iso (5-yl)-amide; 6-methyl-5-(2-methyl-2H-pyrazol-3-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid (5-methanesulfonylmethyl-[1,2,4]) (diazol-3-ylmethyl)-amide; 6-methyl-5-(2-methyl-2H-pyrazol-3-yl)-2-oxo-1-(3-trifluoromethylphenyl)-1,2-dihydro-pyridine-3-carboxylic acid ([1,2,4] Diazol-3-ylmethyl)-amide; 6-methyl-5-(1-methyl-1H-pyrazol-5-yl)-N-{[5-(methylsulfonyl)pyridin-2-yl]methyl}-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; 5-(3,5-dimethylisocyanate) (Azol-4-yl)-6-methyl-N-{[5-(methylsulfonyl)pyridin-2-yl]methyl}-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide; and its salts.

[0089] In a particularly preferred embodiment, the elastase inhibitor is selected from (iii). (6-Methyl-5-(1-Methyl-1H-pyrazol-5-yl)-N-{[5-(methylsulfonyl)pyridin-2-yl]methyl}-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide) and its salts and stereoisomers.

[0090] In the most preferred embodiment, the elastase inhibitor is (6-Methyl-5-(1-Methyl-1H-pyrazol-5-yl)-N-{[5-(methylsulfonyl)pyridin-2-yl]methyl}-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide).

[0091] As noted earlier herein, group (IV) consists of compounds capable of reducing or inhibiting the production of elastin. In a sense (e.g., in established in vitro assays), a compound is considered to inhibit elastin production if its application results in a reduction in elastin production. In a preferred embodiment, the compounds selected from group (IV) are matrix metalloproteinase (MMP) inhibitors or compounds capable of inhibiting MMPs. In a preferred embodiment, the MMP is MMP-12. Therefore, in a preferred embodiment, group (IV) consists of MMP-12 inhibitors. In a sense (e.g., in established in vitro assays), a compound is considered to inhibit MMP or MMP-12 if its application results in a decrease in the expression of MMP or MMP-12 and / or if its application results in a decrease in the activity of MMP or MMP-12. In a preferred embodiment, the compound is capable of inhibiting MMP-12 in an MMP-12 FRET assay (as described elsewhere herein).

[0092] A specific class of MMP-12 inhibitors—which are believed to have specific utility in the context of this invention—is described in WO2008 / 057254 (the contents of which are incorporated herein by reference).

[0093] In a preferred embodiment, group (IV) comprises MMP-12 inhibitors selected from compounds of formula (IVa) and their pharmaceutically acceptable salts, esters, and hydrates. (IVa) Where R 1 It is an N-linked, free carboxyl or carboxyl-protected, natural or non-natural amino acid, or an N-linked amino acid derivative; R 2 It is a)-C(O)OR 8 Or b)-C(O)NR 8 R 9 ;R 3 It is a) H, b) -S(O) m R 8 c)-S(O) m OR 8 ,d)-S(O) m NR 8 R 9 e)-C(O)R 8 f)-C(S)OR 8 ,g)-C(S)R 8 h)-C(S)NR 8 R 9 ,i)-C(NR 8 )NR 8 R 9j) C1-10 alkyl, k) C2-10 alkenyl, l) C2-10 alkynyl, m) C1-10 haloalkyl, n) C3-10 cycloalkyl, o) C6-14 aryl, p) 3-14 membered cycloheteroalkyl, or q) 5-13 membered heteroaryl, wherein each of j)-q) is surrounded by 1-4 -ZR 10 The group may be optionally substituted; or R 3 and R 2 R 8 Parts, and the NC(=O)-O or NC(=O)-NR they are connected to. 9 Together, they form 3-14 membered cyclic urea groups or 3-14 membered cyclic carbamate groups, each of which is surrounded by 1-4 -ZR groups. 10 Groups may be optionally substituted; R 4 and R 5 Independently: a) H, b) -CN, c) -NO2, d) halogen, e) -OR 8 f)-NR 8 R 9 ,g)-S(O) m R 8 ,h)-S(O) m OR 8 i)-C(O)R 8 ,j)-C(O)OR 8 ,k)-C(O)NR 8 R 9 ,l)-C(S)R 8 m)-C(S)OR 8 ,n)-C(S)NR 8 R 9 o) C1-10 alkyl, p) C2-10 alkenyl, q) C2-10 alkynyl, r) C1-10 haloalkyl, s) C3-14 cycloalkyl, t) C6-14 aryl, u) 3-14 membered cycloheteroalkyl, or v) 5-13 membered heteroaryl, wherein each of o)-v) is surrounded by 1-4 -ZR 10 Groups may be optionally substituted; R 6 It is a) H, b) -S(O) m R 8 c)-S(O) m OR 8 ,d)-C(O)R 8 e)-C(O)OR 8 f)-C(O)NR 8 R 9 ,g)-C(S)R 8 h)-C(S)OR 8 i)-C(S)NR 8 R9 j) C1-10 alkyl, k) C2-10 alkenyl, l) C2-10 alkynyl, or m) C1-10 haloalkyl, wherein each of j)-m) is optionally substituted with a C6-14 aryl or a 5-13 heteroaryl, wherein each of the C6-14 aryl and 5-13 heteroaryl is substituted with 1-4 -ZR 10 Groups may be optionally substituted; R 8 and R 9 Each time it appears, it is independently: a) H, b) -OR 11 c)-SR 11 ,d)-S(O) m R 11 ,e)-S(O) m -OR 11 ,f)-S(O) m -NR 11 R 12 ,g)-C(O)R 11 h)-C(O)OR 11 i)-C(O)NR 11 R 12 ,j)-C(S)NR 11 XR 12 k) C1-10 alkyl, l) C2-10 alkenyl, m) C2-10 alkynyl, n) C1-10 alkoxy, o) C1-10 haloalkyl, p) C3-10 cycloalkyl, q) C6-14 aryl, r) 3-14 membered cycloheteroalkyl, or s) 5-13 membered heteroaryl, wherein each of k)-s) is surrounded by 1-4 -ZR 10 Groups may be optionally substituted; R 10 Each time it appears, it is a) halogen, b) -CN, c) -NO2, d) oxo, e) -OZR. 11 f)-NR 11 -ZR 12 ,g)-N(O)R 11 -ZR 12 ,h)-S(O) m R 11 ,i)-S(O) m OZR 11 ,j)-S(O) m NR 11 -ZR 12 ,k)-C(O)R 11 ,l)-C(O)OZR 11 ,m)-C(O)NR 11 -ZR 12 ,n)-C(S)NR 11 -ZR12 ,o)-Si(C 1-10 alkyl) 3, p) C1-10 alkyl, q) C2-10 alkenyl, r) C2-10 alkynyl, s) C1-10 haloalkyl, t) C3-10 cycloalkyl, u) C6-14 aryl, v) 3-14 membered cycloheteroalkyl, or w) 5-13 membered heteroaryl, wherein each of o)-w) is surrounded by 1-4 R 13 Groups may be optionally substituted; R 11 and R 12 Each time it appears, it is independently a) H, b) -OH, c) -SH, d) -S(O)2OH, e) -C(O)OH, f) -C(O)NH2, g) -C(S)NH2, h) -S(O) m -C 1-10 Alkyl group, i)-S(O) m -OC 1-10 Alkyl, j)-OC 1-10 Alkyl, k)-C(O)-C 1-10 Alkyl, l)-C(O)-OC 1-10 Alkyl, m)-C(S)N(C 1-10 Alkyl)2,n)-C(S)NH-C 1-10 Alkyl, o)-C(O)NH-C 1-10 Alkyl, p)-C(O)N(C 1-10 α) C1-10 alkyl, β) C2-10 alkenyl, β) C2-10 alkynyl, β) C1-10 alkoxy, β) C1-10 haloalkyl, β) C3-10 cycloalkyl, β) C6-14 aryl, β) 3-14 membered cycloheteroalkyl, or β) 5-13 membered heteroaryl, wherein each of β)-β) is surrounded by 1-4 -R 13 Groups may be optionally substituted; R 13 Each time it appears, it is a) halogen, b) -CN, c) -NO2, d) oxo, e) -OH, f) -NH2, g) -NH(C) 1-10 Alkyl), h)-N(C) 1-10 Alkyl)2,i)-S(O) m H,j)-S(O) m -C 1-10 Alkyl group, k)-S(O)2OH, l)-S(O) m -OC 1-10 Alkyl group, m)-S(O) m NH2,n)-S(O) m NH(C 1-10 Alkyl), o)-S(O) m N(C 1-10 Alkyl)2, p)-CHO, q)-C(O)-C1-10 Alkyl group, r)-C(O)OH, s)-C(O)-OC 1-10 Alkyl, t)-C(O)NH2, u)-C(O)NH-C 1-10 Alkyl, v)-C(O)N(C 1-10 Alkyl)2,w)-C(S)NH2,x)-C(S)NH-C 1-10 Alkyl, y)-C(S)N(C 1-10 Alkyl)2,z)-Si(C 1-10 Alkyl) 3, aa) C1-10 alkyl, ab) C2-10 alkenyl, ac) C2-10 alkynyl, ad) C1-10 alkoxy, ae) C1-10 haloalkyl, af) C3-10 cycloalkyl, ag) C6-14 aryl, ah) 3-14 membered cycloheteroalkyl, or ai) 5-13 membered heteroaryl; X is O, S, S(O), S(O)2 or NR 6 Y is S(O), S(O)2, or C(O); Z, each time it appears, is a) a divalent C1-10 alkyl, b) a divalent C2-10 alkenyl, c) a divalent C2-10 alkynyl, d) a divalent C1-10 haloalkyl, or e) a covalent bond; and m, each time it appears, is 0, 1, or 2. A particularly preferred embodiment is the following embodiment: wherein R 2 It is -C(O)OR 8 ; and / or where R 2 It is -C(O)NR 8 R 9 ; and / or where R 3 It is an H or C1-10 alkyl group; and / or wherein R 3 Selected from H, methyl, and ethyl; wherein R 8 Selected from H, -C(O)R 11 -C(O)OR 11 -C(O)NR 11 R 12 C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 haloalkyl, C3-10 cycloalkyl, C6-14 aryl, 3-14 membered cycloheteroalkyl, and 5-13 membered heteroaryl, wherein each of the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 haloalkyl, C3-10 cycloalkyl, C6-14 aryl, 3-14 membered cycloheteroalkyl, and 5-13 membered heteroaryl is surrounded by 1-4 -ZR groups. 10 Groups may be optionally substituted; and / or wherein R 8 Selected from H, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl and C6-14 aryl, wherein each of the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl and C6-14 aryl groups is surrounded by 1-4 -ZR groups.10 Groups may be optionally substituted; and / or wherein R 8 The group is selected from methyl, ethyl, propyl, isopropyl, isobutyl, butyl, hexyl, and butynyl, each of which is optionally substituted by 1-4 groups independently selected from the following: halogen, -S(O). m R 11 -S(O) m OZR 11 -S(O) m NR 11 -ZR 12 -C(O)R 11 -C(O)OZR 11 -C(O)NR 11 -ZR 12 C6-14 aryl and 5-13 heteroaryl; and / or R therein 8 Selected from methyl, ethyl, propyl, isopropyl, isobutyl, butyl, hexyl, 3-butynyl, 4-butynyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2-methanesulfonylethyl, 3-chloropropyl, and benzyl; and / or wherein R 8 It is a phenyl group that is optionally substituted with 1-4 groups independently selected from halogens and C1-10 alkyl groups; wherein R 8 Selected from phenyl, tolyl, fluorophenyl, and chlorophenyl; and / or R 8 and R 9 Independently selected from H, C1-10 alkyl, C2-10 alkenyl, C2-10 ynyl, C3-10 cycloalkyl, C6-14 aryl, 3-14 membered cycloheteroalkyl, and 5-13 membered heteroaryl, and each of the C1-10 alkyl, C2-10 alkenyl, C2-10 ynyl, C3-10 cycloalkyl, C6-14 aryl, 3-14 membered cycloheteroalkyl, and 5-13 membered heteroaryl is surrounded by 1-4 -ZR groups. 10 Groups may be optionally substituted; and / or wherein R 9 It is H or methyl; and / or R therein 8 Selected from H, C1-10 alkyl, C3-10 cycloalkyl, C6-14 aryl, and 5-13 heteroaryl, wherein each of the C1-10 alkyl, C3-10 cycloalkyl, C6-14 aryl, and 5-13 heteroaryl groups is surrounded by 1-4 -ZR groups. 10 The group is optionally substituted; wherein R 8 Selected from H, methyl, ethyl, isopropyl, cyclopentyl, benzyl, fluorobenzyl, phenethyl, thienylmethyl, and thienylethyl; wherein R 8 It is phenyl or 5-13 heteroaryl, each of which is surrounded by 1-4 -ZR groups. 10 Groups may be optionally substituted; and / or wherein R 8 Selected from phenyl, 2,3-dihydrobenzo[b][1,4]di Ingenyl (2,3-dihydrobenzo[b][l,4]dioxinyl group), thiophene, pyridyl and iso... Azolium group, and phenyl, thiophene, pyridinium and isopropyl groups. Each of the azole groups is independently and optionally substituted by 1-3 groups selected from the following: halogen, -OZR 11 -NR 11 -ZR 12 C1-10 alkyl and C1-10 haloalkyl; and / or wherein R 3 and R 2 R 8 Parts, and the NC(=O)-O or NC(=O)-NR they are connected to. 9 Together, they form 5-12 membered cyclic urea groups or 5-12 membered cyclic carbamate groups, each of which is surrounded by 1-4 -ZR groups. 10 The group is optionally substituted, wherein R 9 Preferably, it is H or methyl and / or a 5-12 membered cyclic urea group or a 5-12 membered cyclic carbamate group, preferably a 5-membered cyclic urea group, a 6-membered cyclic urea group, a 5-membered cyclic carbamate group or a 6-membered cyclic carbamate group.

[0094] In a preferred embodiment, group (IV) comprises an MMP-12 inhibitor selected from the following: (R)-2-(7-methoxycarbonylamino-dibenzofuran-2-sulfonylamino)-3-methyl-butyric acid, (R)-3-methyl-2-(7-(propoxycarbonylamino)dibenzo[b,d] (R)-2-(7-(isopropoxycarbonylamino)dibenzo[b,d]furan-2-sulfonamido)-3-methyl-butyric acid, (R)-3-methyl-2-(7-(phenoxycarbonylamino)dibenzo[b,d]furan-2-sulfonamido)butyric acid, (R)-2-(7-(3-ethylureo)dibenzo[b,d]furan-2-sulfonamido)-3-methyl-butyric acid, (R)-2-(7-(3-(4-fluoro-benzyl)-ureo)-dibenzofuran-2-sulfonylamino)-3-methyl-butyric acid, (R)-2-(7-(3-(4-fluoro-benzyl)-ureo)-dibenzofuran-2-sulfonylamino)-3-methyl-butyric acid, (R)-2-(7-(3-cyclopentylmethylureo) (R)-2-(7-(3-isopropylureo)-dibenzofuran-2-sulfonylamino)-3-methyl-butyric acid, (R)-3-methyl-2-(7-(3-(3,4,5-trimethoxy-phenyl)ureo)-dibenzofuran-2-sulfonylamino)-butyric acid, (R)-2-(7-(3-(3,4-difluorophenyl)ureo)dibenzo[b,d]furan-2-sulfonamido)-3-methyl-butyric acid, (R)-2-(7-(3-(4-(dimethylamino)phenyl)ureo)dibenzo[b,d]furan- (R)-3-methylbutyric acid, (R)-3-methyl-2-(7-(3-(3-phenoxyphenyl)ureo)dibenzo[b,d]furan-2-sulfonamido)butyric acid, (R)-3-methyl-2-(7-ureodibenzo[b,d]furan-2-sulfonamido)butyric acid, (R)-3-methyl-2-(7-(3-(4-(trifluoromethoxy)phenyl)ureo)dibenzo[b,d]furan-2-sulfonamido)butyric acid, (R)-2-(7-(3-(2,6-dichloropyridin-4-yl)ureo)dibenzo[b,d]furan-2-sulfonamido)-3-methylbutyric acid (R)-3-methyl-2-(7-(3-(2-(phenylthio-2-yl)ethyl)ureo)dibenzo[b,d]furan-2-sulfonamido)butyric acid, (S)-2-(7-(3-ethylureo)dibenzo[b,d]furan-2-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-methoxycarbonylamino-dibenzo[b,d]thiophene-2-sulfonamido)-3-methyl-butyric acid, (R)-2-(7-(methoxycarbonylamino)dibenzo[b,d]thiophene-2-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(isobutoxycarbonylamino)dibenzo[b,d](S)-2-(8-((2-chloroethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-((2-bromoethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(isopropoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(4-fluorophenoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-( ... (S)-3-sulfonamido)-3-methylbutyric acid, (S)-2-(8-((2-chlorophenoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methylbutyric acid, (S)-2-(8-(but-2-alkynoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methylbutyric acid, (S)-3-methyl-2-(8-(p-tolyloxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-3-methyl-2-(8-(phenoxycarbonylamino)dibenzo[b,d]furan-3- (S)-2-(8-(benzyloxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(hexyloxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-((2-fluoroethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2- (8-(ethoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(8-propoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamidobutyric acid, (S)-2-(8-(butoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-((but-3-alkynoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(3-(3,5-dimethylisocyano) (S)-3-methyl-2-(8-(3-phenylthio-3-yl)ureo)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(8-(3-phenylthio-3-yl)ureo)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-3-methyl-2-(8-(3-(3,4,5-trimethoxyphenyl)ureo)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-2-(8-(3-(3,4-difluorophenyl)ureo)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(8-(3-(3-phenoxyphenyl)ureo)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-3-methyl -2-(8-ureidodibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-2-(8-(3-(2,6-dichloropyridin-4-yl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(3-(4-(dimethylamino)phenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(8-(3-(2-(phenylthio-2-yl)ethyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-3-methyl-2-(8-(3-(4-(trifluoromethoxy)phenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butyric acid, ( (S)-2-(8-(3-cyclopentylurea)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(3-(4-fluorobenzyl)urea)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-(methoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (R)-2-(8-(methoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(3-cyclopentylurea)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(3-(2-( (S)-2-(7-(3-(4-fluorophenyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-(3-(4-fluorophenyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(3-phenylethylureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-(3-benzylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(3-(4-fluorobenzyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(3-p-tolylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(3-p-tolylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid,(s)-3-methyl-2-(7-(3-(3,4,5-trimethoxyphenyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (s)-2-(7-(3-ethylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (s)-2-(7-(3-(3,4-difluorophenyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (s)-2-(7-(3-(4-(trifluoromethyl)phenyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (s)-2-(7-(3-(4-(trifluoromethyl)phenyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (s)-2-(7-(3-(2,3-dihydrobenzo[b][1,4]di, (S)-3-methyl-2-(7-(3-phenylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(3-pyridin-4-ylureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-(3-(4-(dimethylamino)phenyl)ureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(3-pyridin-4-ylureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-(3-(3,5-dimethylisocyanate) (S)-2-(7-(methoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(methoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(ethoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(isobutoxycarbonylamino) (S)-2-(6-chloro-7-(methoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(isopropoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(p-tolyloxycarbonyl) (S)-2-(7-((4-fluorophenoxy)carbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-3-methyl-2-(7-(phenoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-((but-3-alkynoxy) ... Benzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-((2-(methylsulfonyl)ethoxy)carbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-(benzyloxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(8-(2-oxo) (S)-3-methyl-2-(8-(2-oxooxazolidin-3-yl)dibenzofuran-3-sulfonamido)butanoic acid, (S)-2-(8-(ethyl(methoxycarbonyl)amino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (R)-3-methyl-2-(7-(2-oxooxazolidin-3-yl)dibenzofuran-3-sulfonamido)butanoic acid, (S)-2-(8-((3-chloropropoxy)carbonylamino)dibenzo[b,d]furan-2-sulfonamido)butyric acid, (S)-2-(8-((3-chloropropoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (R)-2-(8-bromo-7-(methoxycarbonylamino)dibenzo[b,d]furan-2-sulfonamido)-3-methyl-butyric acid, (S)-2-(8-bromo-7-(methoxycarbonylamino)dibenzo[b,d]furan-2-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(2-oxo) (S)-2-(7-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (R)-2-(7-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (R)-2-(7-(3-(3,4-difluorophenyl)ureo)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, [b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-((2-fluoroethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-((but-2-alkynoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-((4-fluorophenoxy)carbonylamino) (S)-3-methyl-2-(7-(3-phenylthio-3-ylureido)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-3-methyl-2-(7-(3-(2-(phenylthio-2-yl)ethyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-3-methyl-2-(7-(3-(2-(phenylthio-2-yl)ethyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butyric acid, (S)-3-methyl-2-(7-ureido) (S)-2-(7-(3-(3,4-difluorophenyl)ureo)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(ethyl(methoxycarbonyl)amino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(8-(2-oxo-1,3-) (S)-3-methyl-2-(8-(2-oxo-l,3-oxazinan-3-yl)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-3-methyl-2-(7-(2-oxo-l,3-oxazinan-3-yl)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-3-methyl-2-(7-(2-oxo-l,3-oxazinan-3-yl)dibenzo[b,d]furan-3-sulfonamido)butanoic acid (S)-2-(7-(methoxycarbonyl(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-(methoxycarbonyl(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(ethoxycarbonyl(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(isopropoxycarbonyl(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S) (S)-2-(7-(((4-fluorophenoxy)carbonyl)(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(1,3,3-trimethylureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-2-(7-(3-ethyl-1-methylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(3-benzyl-1-methylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(3-benzyl-1-methylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(3-benzyl-1-methylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (d)-thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-2-(7-(3-benzyl-1,3-dimethylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (S)-3-methyl-2-(7-(2-oxoimidazolidine-1-yl)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (S)-3-methyl-2-(7-(2-oxotetrahydropyrimidin-1(2H)-yl)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid, (R)-2 (R)-(7-(methoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (R)-2-(7-(ethoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (R)-2-(7-(3-cyclopentylureo)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butyric acid, (R)-3-methyl-2-(7-(3-phenylureo)dibenzo[b,d]thiophene-3-sulfonamido)butyric acid; and their salts and esters.

[0095] In a particularly preferred embodiment, the MMP-12 inhibitor is selected from... (iv), and its salts, esters and stereoisomers.

[0096] In the most preferred embodiment, the MMP-12 inhibitor is selected from... (iv) ((S)-2-(8-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid), and its salts, esters and stereoisomers.

[0097] In the most preferred embodiment, the MMP-12 inhibitor is (iv) ((S)-2-(8-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid).

[0098] As used herein, the term "salt" has its conventional meaning and includes acid addition salts and base salts of the compounds mentioned. Exemplary pharmaceutically acceptable minerals or organic acids or bases are listed in Tables 1-8 of the Handbook of Pharmaceutical Salts (PH Stahl and CG Wermuth (eds.), VHCA, Zurich 2002, pp. 334-345).

[0099] As used herein, the term "ester" has its conventional meaning and specifically includes derivatives of the described compounds obtained by esterification of the carboxylic acid group of the mentioned compounds with alkyl alcohols (such as short-chain aliphatic alcohols, such as C1-C2 alcohols, C3 alcohols, or C4 alcohols), specifically ethyl esters or methyl esters.

[0100] Unless otherwise expressly stated, if a compound is available in optional tautomer, regio isomer, and / or stereoisomer forms, all optional isomers are covered within the scope of the claimed subject matter. For example, when a compound is described as a specific optical isomer, D- or L-, it is intended to cover both optical isomers herein. For example, if a compound is described as having one of two tautomer forms, it is intended to cover both tautomers herein. Thus, the compounds provided herein may be enantiomerically pure, stereoisomers, or mixtures of diastereomers. The compounds provided herein may contain a chiral center. Such a chiral center may be (R) or (S) configurations, or a mixture thereof. Similarly, the compounds provided herein may contain cis-trans geometrical isomers.

[0101] Pharmaceutical Composition It will be apparent to those skilled in the art that one or more compounds are preferably provided and / or used in the form of a pharmaceutical composition.

[0102] According to a first specific aspect, a pharmaceutical composition is provided comprising a first compound selected from a group of groups (I), (II), (III) and (IV) as defined above herein, and a second compound preferably selected from another group of groups (I), (II), (III) and (IV).

[0103] In a particularly preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (I) and a second compound selected from any one of groups (II), (III), and (IV). In an even more preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (I) and a second compound selected from any one of groups (III) and (IV). In the most preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (I) and a second compound selected from group (IV).

[0104] In a particularly preferred embodiment, the pharmaceutical composition comprises a first compound selected from any one of groups (i), (ii), (iii), and (iv) as defined above herein, and a second compound preferably selected from another group of groups (i), (ii), (iii), and (iv).

[0105] In a particularly preferred embodiment, the pharmaceutical composition disclosed herein comprises a first compound selected from group (i) and a second compound selected from any one of groups (ii), (iii), and (iv). In an even more preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (i) and a second compound selected from any one of groups (iii) and (iv). In the most preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (i) and a second compound selected from group (iv).

[0106] As used herein, the term "pharmaceutical composition" has its conventional meaning and refers to a pharmaceutically acceptable composition.

[0107] As used herein, the term "pharmaceutically acceptable" has its conventional meaning and refers to compounds, substances, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for contact with the tissues of mammals (especially humans) without excessive toxicity, irritation, allergic reactions, or other problematic complications, in proportion to a reasonable benefit / risk ratio.

[0108] In some embodiments, the pharmaceutical composition disclosed herein comprises a first compound and a second compound in a molar ratio of 1:10 to 10:1. In some embodiments, the pharmaceutical composition comprises a first compound and a second compound as defined above herein, in a molar ratio (first compound: second compound) in the range of 1:100 to 100:1, such as in the range of 1:5 to 50:1; in the range of 1:20 to 20:1; in the range of 1:10 to 10:1; in the range of 1:8 to 8:1; in the range of 1:6 to 6:1; in the range of 1:5 to 5:1; in the range of 1:4 to 4:1; in the range of 1:3 to 3:1; or in the range of 1:2 to 2:1, such as a ratio of approximately 1:1. Each possibility represents a separate embodiment of the invention.

[0109] In a preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (I) as defined above herein and a second compound selected from any of groups (II), (III) and (IV) as defined above herein, wherein the molar ratio (first compound: second compound) is in the range of 1:100 to 100:1, such as in the range of 1:5 to 50:1; in the range of 1:20 to 20:1; in the range of 1:10 to 10:1; in the range of 1:8 to 8:1; in the range of 1:6 to 6:1; in the range of 1:5 to 5:1; in the range of 1:4 to 4:1; in the range of 1:3 to 3:1; or in the range of 1:2 to 2:1, such as a ratio of about 1:1.

[0110] In a preferred embodiment, the pharmaceutical composition comprises a first compound selected from group (i) as defined above herein and a second compound selected from any of groups (ii), (iii) and (iv) as defined above herein, wherein the molar ratio (first compound: second compound) is in the range of 1:100 to 100:1, such as in the range of 1:5 to 50:1; in the range of 1:20 to 20:1; in the range of 1:10 to 10:1; in the range of 1:8 to 8:1; in the range of 1:6 to 6:1; in the range of 1:5 to 5:1; in the range of 1:4 to 4:1; in the range of 1:3 to 3:1; or in the range of 1:2 to 2:1, such as a ratio of about 1:1.

[0111] In some embodiments, as disclosed above, the first compound or a combination of the first compound and the second compound present in the pharmaceutical composition constitutes at least 95% by weight, at least 98% by weight, at least 99% by weight, or at least 99.9% by weight of the active agent (one or more) of the pharmaceutical composition.

[0112] In embodiments, the pharmaceutical composition typically comprises a first compound in a relative amount of at least 0.001 wt.% (such as at least 0.005 wt.%, at least 0.01 wt.%, at least 0.05 wt.%, or at least 0.1 wt.%, at least 0.5 wt.%, or at least 1 wt.%) based on the total composition and / or in a relative amount of no more than 25 wt.% based on the total weight of the pharmaceutical composition, such as no more than 15 wt.%, no more than 10 wt.%, no more than 7.5 wt.%, no more than 5 wt.%, or no more than 2.5 wt.%.

[0113] In embodiments, the pharmaceutical composition typically includes a second compound (if present) in a relative amount of at least 0.001 wt.% (such as at least 0.005 wt.%, at least 0.01 wt.%, at least 0.05 wt.%, or at least 0.1 wt.%, at least 0.5 wt.%, or at least 1 wt.%) based on the total composition and / or in a relative amount of no more than 25 wt.% based on the total weight of the pharmaceutical composition, such as no more than 15 wt.%, no more than 10 wt.%, no more than 7.5 wt.%, no more than 5 wt.%, or no more than 2.5 wt.%.

[0114] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier, adjuvant, or excipient.

[0115] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0116] As used herein, the terms “carrier,” “adjuvant,” or “excipient” refer to any pharmaceutically acceptable component of a pharmaceutical composition that is not an active agent and is administered together with the pharmaceutically active ingredient. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation excipient, or simply a sterile aqueous medium, such as saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxymethyl ethyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, and cellulose acetate; powdered astragalus gum; malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, and agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions and phosphate buffer solutions; and other non-toxic and compatible substances used in pharmaceutical preparations. Some non-limiting examples of substances that may serve as carriers in this document include sugars, starches, cellulose and their derivatives, powdered astragalus gum, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifiers, and other non-toxic, pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium dodecyl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions considered herein.Suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, such as those described in The Merck Index, 13th Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th Edition (2004); and the “Inactive Ingredient Guide,” US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (the entire contents of which are incorporated herein by reference). Examples of pharmaceutically acceptable excipients, carriers, and diluents used in the compositions of the present invention include distilled water, physiological saline, Ringer's solution, glucose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18. th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21 stEd., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005) (each of which is incorporated herein by reference in its entirety). The compositions described in this invention can also be included in artificially created structures such as liposomes, ISCOMS, slow-release particles, and other mediators that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes used with the peptides described in this invention are formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is typically determined by factors such as liposome size and blood stability. For example, Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, reviews various methods that can be used to prepare liposomes, and see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0117] The carrier may comprise approximately 0.1% to approximately 99.99999% of the total weight of the pharmaceutical composition presented herein.

[0118] According to a particularly preferred embodiment of the invention, the pharmaceutical composition is preferably provided in a unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable for administration to a human subject, each unit containing a predetermined amount of active substance, which, when used in combination with any suitable drug carrier (one or more) and / or excipient (one or more), produces a calculated desired therapeutic effect. Exemplary, non-limiting unit dosage forms include tablets, caplets, capsules (e.g., hard or soft capsules), lozenges, films, strips, gelcapsules, and any metered volume of solution, suspension, syrup, or elixir, or similar dosage forms, which may be, for example, contained in vials, syringes, applicator devices, sachets, nebulizers, micropumps, etc. According to a particularly preferred embodiment of the invention, the unit dosage form is a unit dosage form suitable for oral administration. Most preferably, it is a solid unit dosage form, such as a tablet or capsule.

[0119] As used herein, the term "fixed-dose combination" has its conventional meaning and refers to a combination of two or more drugs or active ingredients in a defined dose, presented and so administered in a single dose unit (e.g., tablet or capsule). As used herein, the term "free-dose combination" has its conventional meaning and refers to a combination of two drugs or active ingredients administered simultaneously but as two different dose units.

[0120] Therapeutic and non-therapeutic indications As noted earlier in this article, the present invention provides the following methods: - Methods for treating or preventing elastin deficiency-related diseases in individuals who require them; - Methods for treating and / or preventing conditions and / or diseases associated with the aging process in individuals who require them; and / or - Methods to promote longevity among those who need it; The method includes applying to the object at least one compound selected from any one of the groups (I), (II), (III) and (IV) as defined herein.

[0121] According to a preferred embodiment, the method includes applying to the object a first compound selected from one group of groups (I), (II), (III), and (IV), and a second compound selected from another group of groups (I), (II), (III), and (IV).

[0122] According to a more preferred embodiment, the method includes applying to the object a first compound selected from group (I) and a second compound selected from any one of groups (II), (III) and (IV).

[0123] The present invention also provides a method for treating and / or preventing conditions and / or diseases associated with the aging process and / or promoting longevity in subjects in need, the method comprising administering to the subject a compound selected from any group of groups (i), (ii), (iii) and (iv) as defined herein.

[0124] According to a preferred embodiment, the method includes applying to the object a first compound selected from one group of groups (i), (ii), (iii), and (iv), and a second compound selected from another group of groups (i), (ii), (iii), and (iv).

[0125] According to a more preferred embodiment, the method includes applying to the object a first compound selected from group (i) and a second compound selected from any one of groups (ii), (iii) and (iv).

[0126] The terms “treat,” “treating,” or “treatment,” when used in connection with a specific disease or symptom (e.g., “method of treating a disease…”), refer to and / or encompass the cure, relief, or elimination of said disease and / or accompanying symptoms, reduction of disease severity, stabilization (i.e., non-deterioration) of disease state, delay or slowing of disease progression, improvement of disease state, and extension of survival (compared to expected survival without treatment). Treatment does not necessarily mean a complete cure of the disease, impairment, or condition. To be effective treatment, the compositions available herein need only reduce the severity of the disease, impairment, or condition, reduce the severity of associated symptoms, or provide an improvement in the quality of life for the patient or subject. The terms “treat,” “treating,” or “treatment,” when used with regard to a patient or subject (e.g., “method of treating a subject”), generally refer to the act of administering a therapeutic compound to said patient or subject, regardless of the therapeutic and / or preventative purpose.

[0127] As used herein, the terms “prevent,” “preventing,” or “prevention” refer to and / or encompass the delay, prevention, suppression, or inhibition of the occurrence / onset of a disease, disorder, or condition. As used in accordance with the subject matter described in this invention, the term “prevention” refers to a preventative process of exposing a subject to the compositions or preparations described in this invention prior to the occurrence / onset of a disease / disorder. The term “suppression” is used to describe a condition in which a disease / disorder process has begun, but obvious symptoms of the condition have not yet appeared. Thus, an individual’s cells may have the disease / disorder, but external signs of the disease / disorder have not yet been clinically recognized. In either case, the term “prevention” can be used to encompass both prevention and suppression.

[0128] As used herein, the term "elastin deficiency-related disease" refers to any disease characterized by, including, involving, propagated by, enhanced by, caused by, promoted by, or manifested as: increased elastin degradation, decreased elastin expression, decreased elastin levels, decreased elastin stability, decreased elastin deposition, or any combination thereof.

[0129] In some embodiments, elastin deficiency-related diseases are characterized by or include decreased expression of primorelastin (including mRNA transcription, protein translation, or both), reduced elastin fiber deposition, increased elastin fiber breakdown, increased production of elastic factors, or any combination thereof.

[0130] As used herein, the term “elasticity factor (one or more)” encompasses bioactive elastin peptides (one or more) – which are the result of the hydrolytic degradation of elastin proteins.

[0131] In some implementations, elastin deficiency-related diseases are age-related diseases.

[0132] In some implementations, the probability of having or being susceptible to elastin deficiency-related diseases is related to the subject's age.

[0133] In some implementations, elastin deficiency-related diseases are selected from: arteriosclerosis, peripheral vascular disease, angina pectoris, ischemia, cardiac ischemia, stroke, myocardial infarction, reperfusion injury, emphysema, Williams-Beuren syndrome, restenosis after angioplasty, hypertension, cardiovascular inflammation, stiff skin syndrome, MASS syndrome, age-related macular degeneration (ARMD3), familial thoracic aortic aneurysm (AAT10), autosomal recessive cutis laxa (ARCL), established atherosclerotic cardiovascular disease (ASCVD), arterial tortuosity syndrome, abdominal aortic aneurysm (AAA), supravalvular aortic stenosis (SVAS), and autosomal dominant cutis laxa type 1. ADCL1), acromicric dysplasia, echinophysic dysplasia, Williams-Beuren syndrome, Marfan syndrome (MFS), Werner-Marfan syndrome (WMS), congenital contracture arachnoidosis (CCA), pseudoxanthoma (PXE), Costello syndrome, arterial tortuosity syndrome, and isolated lens ectopia (ECTOL).

[0134] In a particularly preferred embodiment, the elastin deficiency-related diseases are selected from: abdominal aortic aneurysm (AAA), supravalvular aortic stenosis (SVAS), autosomal dominant cutischameleon type 1 (ADCL1), acromial dysplasia, happy achondroplasia, Williams-Beuren syndrome, Marfan syndrome (MFS), Werner-Marfan syndrome (WMS), congenital contracture arachnoidosis (CCA), pseudoxanthoma (PXE), Costello syndrome, arterial tortuosity syndrome, and isolated ectopic lens (ECTOL).

[0135] In some embodiments, the method of the present invention is effective and / or intended to increase the expression, stability and activity of elastin in the subject.

[0136] In some embodiments, the method of the present invention is effective and / or intended to increase the elasticity of the aorta of the object and / or reduce the stiffness of the aorta of the object.

[0137] As those skilled in the art will understand based on the teachings of this invention, the treatment and / or prevention of conditions and / or diseases associated with the aging process specifically includes delaying the progression of such diseases or conditions, slowing the progression of such diseases or conditions, and stopping the progression of such diseases or conditions.

[0138] Conditions and / or diseases associated with the aging process generally include, for example, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, immune system diseases, and skin-related diseases or conditions.

[0139] In a preferred embodiment of the invention, the method described herein is provided to treat and / or prevent conditions or diseases associated with the aging process, wherein the disease or condition is characterized by, involves, is driven by, is enhanced by, is caused by, is promoted by, or is manifested as: increased elastin degradation, decreased elastin expression, decreased elastin levels, decreased elastin stability, decreased elastin deposition, or any combination thereof.

[0140] In a preferred embodiment of the invention, the method described herein is provided to treat and / or prevent conditions or diseases associated with the aging process, wherein said conditions or diseases are characterized by decreased expression of elastin (including mRNA transcription, protein translation, or both), reduced elastin fiber deposition, increased elastin fiber degradation, increased production of elastic factors, or any combination thereof.

[0141] As used herein, the term “elasticity factor (one or more)” encompasses bioactive elastin peptides (one or more) – which are the result of the hydrolytic degradation of elastin proteins.

[0142] In a preferred embodiment of the invention, the method described herein is provided for treating and / or preventing conditions or diseases associated with the aging process, wherein the diseases or conditions are selected from neurodegenerative diseases, cardiovascular diseases, mitochondrial diseases, fragility, musculoskeletal diseases, and skin diseases.

[0143] In a preferred embodiment of the present invention, the neurodegenerative disease is selected from spinocerebellar ataxia, spinal muscular atrophy, Huntington's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, prion disease, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and dementia, specifically age-related dementia.

[0144] In a preferred embodiment of the present invention, the cardiovascular disease is selected from hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, valvular heart disease, myocarditis, aortic disease, peripheral artery disease, thromboembolic disease, venous thrombosis, coronary artery disease, atherosclerosis, and pulmonary hypertension.

[0145] In a preferred embodiment of the present invention, the mitochondrial disease is selected from stroke, atherosclerosis, hypertension, chronic inflammation, type 2 diabetes, insulin resistance, cardiovascular disease, non-alcoholic fatty liver disease (NAFLD), and β-cell dysfunction.

[0146] In a preferred embodiment of the present invention, the fragility and musculoskeletal diseases are selected from frailty, osteoporosis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gouty spondylitis, osteoopenia tendinopathy, degenerative disc diseases, sarcopenia, fracture (susceptibility), musculoskeletal pain, atrophy, and malnutrition.

[0147] In a preferred embodiment of the present invention, the skin disease is selected from impaired wound healing, alopecia, wrinkles, age spots, thinning skin, xeroderma pigmentosum, xerosis, pruritus, eczematic dermatitis, purpura, chronic venous insufficiency, and congenital dyskeratosis.

[0148] In a preferred embodiment of the present invention, the method provided herein is a method for promoting longevity.

[0149] In a preferred embodiment of the present invention, the method provided herein is a method for preventing aging.

[0150] In some embodiments of the invention, methods are provided for treating objects as defined herein, wherein the methods are non-therapeutic methods, such as cosmetic methods, for example for improving skin appearance, for preventing visible signs of aging of the skin, for preventing and / or reducing wrinkles, for improving and / or maintaining skin smoothness, for improving and / or maintaining skin firmness, for improving and / or maintaining skin elasticity, for skin rejuvenation, and / or for maintaining youthful skin.

[0151] In some embodiments, the method of the present invention is effective and / or intended to increase the expression, stability and activity of elastin in the subject.

[0152] Treatment plan As used herein, the terms "administering," "administration," and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in reasonable medical practice. One aspect of the subject matter of this invention is the oral administration of a therapeutically effective amount of a composition of the subject matter of this invention to a patient in need. Other suitable routes of administration may include parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal administration. The dosage will depend on the recipient's age, health status, and weight, the type of concurrent treatment (if any), the frequency of treatment, and the nature of the desired effect.

[0153] In some embodiments, application includes local application. In some embodiments, application includes subcutaneous injection into the subject. In some embodiments, application includes systemic application.

[0154] In some embodiments, the routes of administration of the pharmaceutical compositions disclosed herein include intravenous, intramuscular, subcutaneous, or oral delivery. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injection, such as intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other injection method known in the art. In some embodiments, the pharmaceutical compositions of the present invention are administered via percutaneous, oral, rectal, vaginal, topical, nasal, inhalation, and ocular treatment. Furthermore, it is also desirable to introduce the pharmaceutical compositions of the present invention via any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter (e.g., attached to a reservoir).

[0155] In embodiments of the present invention, the first compound and the second compound may be administered separately via the same route of administration or via different routes of administration.

[0156] In embodiments of the present invention, the first compound and the second compound may be applied individually or in combination via the same route of administration.

[0157] In embodiments of the invention, the first compound to be administered to a subject is formulated in a pharmaceutical composition, such as a pharmaceutical composition as defined herein. In a particularly preferred embodiment, the first compound to be administered is formulated in a unit dosage form, such as a unit dosage form as defined herein.

[0158] In embodiments of the invention, the second compound to be administered to a subject is formulated in a pharmaceutical composition, such as a pharmaceutical composition as defined herein. In a particularly preferred embodiment, the second compound to be administered is formulated in a unit dosage form, such as a unit dosage form as defined herein.

[0159] In embodiments of the present invention, the first compound and the second compound may be formulated separately in two different pharmaceutical compositions or two different unit dosage forms.

[0160] In embodiments of the present invention, the first compound and the second compound may be co-formulated in a pharmaceutical composition, such as the pharmaceutical composition defined above herein. In embodiments of the present invention, the first compound and the second compound may be co-formulated in a unit dosage form, such as the unit dosage form defined above herein.

[0161] The term "therapeutic effective amount" refers to a concentration of at least one compound or any combination thereof disclosed herein, which, when standardized to body weight (BW), is effective in treating a disease or disorder. The term "therapeutic effective amount" refers to the amount that, at the necessary dosage and for the necessary duration, effectively achieves the desired therapeutic or preventative outcome. A physician with ordinary skills can easily determine the effective amount of the required bioactive agent and prescribe that amount. The exact dosage form and regimen will be determined by the physician based on the patient's condition. The dosage administered will depend on the recipient's age, health status and weight, the type of concurrent treatment (if any), the frequency of treatment, and the nature of the desired effect.

[0162] In some embodiments, the method of the present invention includes applying a first compound and a second compound at a molar ratio of 1:10 to 10:1. In some embodiments, the method includes applying a first compound and a second compound as defined herein at a molar ratio (first compound: second compound) in the range of 1:100 to 100:1, such as in the range of 1:5 to 50:1; in the range of 1:20 to 20:1; in the range of 1:10 to 10:1; in the range of 1:8 to 8:1; in the range of 1:6 to 6:1; in the range of 1:5 to 5:1; in the range of 1:4 to 4:1; in the range of 1:3 to 3:1; or in the range of 1:2 to 2:1, such as a ratio of approximately 1:1. Each possibility represents a separate embodiment of the invention.

[0163] In a preferred embodiment, the method includes applying a first compound selected from group (I) as defined herein and a second compound selected from any of groups (II), (III) and (IV) as defined herein at a molar ratio (first compound: second compound) in the range of 1:100 to 100:1, such as in the range of 1:5 to 50:1; in the range of 1:20 to 20:1; in the range of 1:10 to 10:1; in the range of 1:8 to 8:1; in the range of 1:6 to 6:1; in the range of 1:5 to 5:1; in the range of 1:4 to 4:1; in the range of 1:3 to 3:1; or in the range of 1:2 to 2:1, such as a ratio of approximately 1:1.

[0164] In a preferred embodiment, the method includes applying a first compound selected from group (i) as defined herein and a second compound selected from any one of groups (ii), (iii), and (iv) as defined herein at a molar ratio (first compound: second compound) in the range of 1:100 to 100:1, such as in the range of 1:5 to 50:1; in the range of 1:20 to 20:1; in the range of 1:10 to 10:1; in the range of 1:8 to 8:1; in the range of 1:6 to 6:1; in the range of 1:5 to 5:1; in the range of 1:4 to 4:1; in the range of 1:3 to 3:1; or in the range of 1:2 to 2:1, such as a ratio of approximately 1:1.

[0165] In some embodiments of the invention, the method includes administering the first compound once or twice daily, most preferably once daily. In further embodiments of the invention, the method includes administering the second compound once or twice daily. It will be understood that, for patient convenience and / or patient compliance, it is preferable to administer the first and second compounds in combination or to administer one compound shortly after the other. In a particularly preferred embodiment of the invention, treatment includes administering a single unit dosage form comprising the first and second compounds once, twice, three or four times daily, preferably once, twice or three times daily, more preferably once or twice daily, and most preferably once daily. According to the invention, treatment includes administering the first compound, preferably in combination with the second compound, according to the above-defined regimen, for a period of at least one month, at least three months, at least four months, at least six months, at least nine months, at least one year, at least two years, at least three years, at least five years, at least ten years, at least twenty years, or at least thirty years, preferably in combination with the second compound. There is no specific upper limit; treatment may continue as long as it is deemed beneficial to the overall health and well-being of the subject (as determined by a qualified healthcare professional), for example, for the remainder of the subject's life.

[0166] Patients awaiting treatment The term "object" refers to a living organism, typically a mammal, specifically a human object. In one embodiment of the invention, the object is a male human. In another embodiment of the invention, the object is a female human.

[0167] In a further preferred embodiment of the invention, the subject is an individual who is over 35 years old, over 40 years old, over 45 years old, over 50 years old, over 55 years old, over 60 years old, over 65 years old, or over 70 years old.

[0168] In a particularly preferred embodiment of the invention, the subject is a person suffering from and / or diagnosed with an elastin deficiency-related disease as defined above herein.

[0169] In some implementations, the subject is a subject that suffers from and / or has been diagnosed as suffering from reduced elastin expression (including mRNA transcription, protein translation, or both), reduced elastin fiber deposition, increased elastin fiber degradation, increased elastic factor production, or any combination thereof.

[0170] In a further preferred embodiment of the invention, the subject is a subject who is considered to be at risk of developing or suffering from elastin deficiency-related diseases as defined above herein (typically at risk above average).

[0171] In a preferred embodiment of the invention, the subject is a person suffering from one or more conditions known to have a causal and / or epidemiological association with the occurrence of diseases associated with elastin deficiency as defined above herein.

[0172] In a further preferred embodiment of the invention, the object is an individual with a genetic predisposition to develop elastin deficiency-related diseases as defined above herein.

[0173] In a further preferred embodiment of the invention, the object is an object that is prone to developing "elastin deficiency-related diseases" as defined above herein due to lifestyle / habit factors.

[0174] In some embodiments, the object is characterized by exhibiting decreased levels of elastin expression (including mRNA transcription, protein translation, or both), decreased levels of elastin fiber deposition, increased elastin fiber degradation, increased production of elasticity factor, or any combination thereof. The term "decreased" in this context typically refers to a level below a predetermined reference value, such as 10% below such a predetermined reference value, or optionally at least 20%, 30%, 40%, 50%, 60%, 70%, or 75% below said predetermined reference value. It will be understood that the term "predetermined reference value" refers to a threshold distinguishing between normal, non-pathological, and pathological states, where values ​​above the threshold indicate a normal, non-pathological endotype, and values ​​below the threshold indicate a pathological endotype (and vice versa, depending on the selected parameter). Typically, such a "threshold" or "cutoff value" can be determined experimentally, empirically, or theoretically. In embodiments of the invention, the pathological state is characterized by a decrease in a certain parameter (compared to a healthy, non-pathological state). The predetermined reference value may be, for example, the mean, average, or median established in a sufficient number of normal, non-pathological reference groups, or it may be the 50th percentile cutoff point, 25th percentile cutoff point, 20th percentile cutoff point, 15th percentile cutoff point, or 10th percentile cutoff point established in a sufficient number of normal, non-pathological reference groups. In the context of this invention, the term "increase" generally refers to a level above a predetermined reference value, such as at least 10% above the predetermined reference value, or optionally at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 75% above the predetermined reference value, wherein the predetermined reference value may be the mean, average, or median established in a sufficient normal, non-pathological reference population, or it may be the 50th percentile cutoff point, 75th percentile cutoff point, 80th percentile cutoff point, 85th percentile cutoff point, or 90th percentile cutoff point established in a sufficient normal, non-pathological reference population.

[0175] Methods for identifying proteins of interest (including peptides or polypeptides), such as elastin, are common and include flow cytometry, immunohistochemical staining of tissue slices or sections, Western blotting, ELISA, radioimmunoassay (RIA), antibody microarrays, etc., all of which will be readily apparent to a person of ordinary skill in the field of biochemistry.

[0176] Various methods are known in the art for measuring the expression levels of one or more genes, such as by amplification of nucleic acids (e.g., PCR, isothermal method, rolling circle method, etc.) or by quantitative in situ hybridization.

[0177] RT-qPCR: RT-qPCR is a commonly used technique for measuring RNA abundance, in which reverse transcription (RT) is followed by real-time quantitative PCR (qPCR). Reverse transcription first generates template DNA from RNA. This single-stranded template is called cDNA. The cDNA template is then amplified in the quantification step, during which the fluorescence emitted by labeled hybridization probes or insertion dyes changes as the DNA amplification process proceeds. Quantitative PCR produces measurements of increases or decreases in the original RNA copy number and has been used to attempt to determine changes in gene expression in cancer tissue compared to comparable healthy tissue.

[0178] RNA-Seq: RNA-Seq utilizes recently developed deep sequencing technologies. Generally, RNA masses (total or fractionated, such as poly(A)+) are converted into a cDNA fragment library, with adaptors ligated at one or both ends. Each molecule (whether amplified or not) is then sequenced in a high-throughput manner to obtain short sequences from one end (single-end sequencing) or both ends (paired-end sequencing). Depending on the DNA sequencing technology used, reads are typically 30-400 bp. In principle, any high-throughput sequencing technology can be used for RNA-Seq. After sequencing, the resulting reads are either aligned to a reference genome or reference transcript, or de novo assembled to produce a genome-scale transcriptomic map consisting of both the transcriptional structure and / or expression level of each gene, without a genome sequence. Direct RNA sequencing can also be applied to avoid artifacts and biases introduced by reverse transcription.

[0179] Microarrays: Microarray technology can be used to assess gene expression levels. In this method, polynucleotide sequences of interest (including cDNA and oligonucleotides) are arranged on a substrate. The arranged sequences are then contacted under conditions suitable for specific hybridization with detectably labeled cDNA generated from RNA of the test sample. As with RT-PCR, the RNA source is typically total RNA—isolated from tumor samples, and optionally from normal tissue of the same patient as an internal control, or from cell lines. RNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples. For archived formalin-fixed tissues, cDNA-mediated annealing, selection, extension, and ligation can be performed using the DASL-Illumina method. As a non-limiting example, PCR-amplified cDNA to be measured is applied to the substrate in a dense array. Microarray analysis can be performed using commercially available equipment, following the manufacturer's protocols (such as using Affymetrix GenChip technology or Incyte's microarray technology).

[0180] Methods for measuring the stiffness of tissues, such as the aorta, are common and will be readily apparent to those skilled in the art, as exemplified below in the Examples section.

[0181] In some embodiments of the invention, particularly those where the method has a non-therapeutic purpose (such as cosmetic purposes), the subject is a healthy individual, or an individual who has not yet been diagnosed with any disease or pathology, or an individual who has not yet been diagnosed with any disease or pathology requiring treatment to increase elastin expression, improve elastin stability, and / or increase elastin activity. In some embodiments, the subject is a healthy human subject, preferably a healthy adult male or female human aged 18-65 years.

[0182] The drug kit of the present invention According to another aspect, a drug kit is provided, comprising a package containing a plurality of unit dosage forms, each unit dosage form comprising a first compound selected from any of the following groups (I), (II), (III), and (IV) as defined herein (e.g., selected from any of the groups (i), (ii), (iii), and (iv) as defined above herein), wherein the drug kit further comprises an insert containing printed instructions for (self-administering) the unit dosage forms to achieve any therapeutic goal as defined herein, such as for: - To treat or prevent elastin deficiency-related diseases in individuals who require it; - To treat and / or prevent conditions and / or diseases associated with the aging process in individuals who require it; and / or - Promote longevity among those who need it.

[0183] Another aspect of the invention relates to a pharmaceutical kit comprising a package containing a first plurality of unit dosage forms and a second plurality of unit dosage forms, wherein the unit dosage forms constituting the first plurality of units comprise a first compound selected from one group of groups (I), (II), (III), and (IV), and the unit dosage forms constituting the second plurality of units comprise a second compound selected from another group of groups (I), (II), (III), and (IV), wherein the pharmaceutical kit optionally further comprises an insert containing printed instructions for (self-)administering the unit dosage forms contained in the kit to achieve any therapeutic goal as defined herein, such as for: - To treat or prevent elastin deficiency-related diseases in individuals who require it; - To treat and / or prevent conditions and / or diseases associated with the aging process in individuals who require it; and / or - Promote longevity among those who need it.

[0184] According to a more preferred embodiment, a drug kit is provided, wherein the first compound is selected from group (I) and the second compound is selected from any one of groups (II), (III) and (IV).

[0185] Another aspect of the invention relates to a pharmaceutical kit comprising a package containing a first plurality of unit dosage forms and a second plurality of unit dosage forms, wherein the unit dosage forms constituting the first plurality of units comprise a first compound selected from one group of groups (i), (ii), (iii), and (iv), and the unit dosage forms constituting the second plurality of units comprise a second compound selected from another group of groups (i), (ii), (iii), and (iv), wherein the pharmaceutical kit optionally further comprises an insert containing printed instructions for (self-)administering the unit dosage forms contained in the kit to achieve any therapeutic goal as defined herein, such as for: - To treat or prevent elastin deficiency-related diseases in individuals who require it; - To treat and / or prevent conditions and / or diseases associated with the aging process in individuals who require it; and / or - Promote longevity among those who need it.

[0186] According to a more preferred embodiment, a drug kit is provided, wherein the first compound is selected from group (i) and the second compound is selected from any one of groups (ii), (iii) and (iv).

[0187] According to embodiments of the invention, a pharmaceutical kit includes a container, such as a cardboard box, containing one or more blister packs, said one or more blister packs containing a plurality of solid unit dosage forms (preferably a plurality of tablets as defined above herein), said solid unit dosage forms comprising the first compound. In a preferred embodiment of the invention, the pharmaceutical kit includes at least 5, at least 8, at least 10, at least 12, or at least 15 of said unit dosage forms comprising the first compound, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of said unit dosage forms comprising the first compound. In a particularly preferred embodiment of the invention, the kit includes one or more other blister packs containing a plurality of solid unit dosage forms (preferably a plurality of tablets as defined above) as described herein, the solid unit dosage forms including the second compound. For example, the drug kit may include at least 5, at least 8, at least 10, at least 12, or at least 15 of the unit dosage forms including the second compound, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the unit dosage forms including the second compound.

[0188] According to the present invention, the pharmaceutical kit includes an insert inserted into the container, typically a patient information insert containing printed information, which may include a description of the form and composition of the unit dosage forms included in the kit, instructions for the intended therapeutic indications of the product, instructions for use of the product, and information and warnings regarding adverse effects and contraindications related to use. Those skilled in the art will understand based on the information presented herein that the insert, as part of the kit according to the present invention, will typically contain information regarding therapeutic indications, uses, treatment regimens, etc., as described above with respect to the treatment methods of the present invention. In a particularly preferred embodiment of the invention, the insert contains printed instructions for repeated (self-administered) unit dosage forms to achieve the following purposes: - To treat or prevent elastin deficiency-related diseases in individuals who require it; - To treat and / or prevent conditions and / or diseases associated with the aging process in individuals who require it; and / or - Promote longevity among those who need it.

[0189] Various precautions Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are exemplary only and are not intended to be limiting.

[0190] Other embodiments of the invention and its full scope will become apparent from the detailed description given below. However, it should be understood that while preferred embodiments of the invention are indicated, the detailed description and specific examples are given by way of example only, and various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.

[0191] As used herein, the term “about” when combined with a value refers to a reference value plus or minus 10%. For example, a length of about 1000 nanometers (nm) means a length of 1,000 nm ± 100 nm.

[0192] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the,” include plural references unless the context clearly specifies otherwise. Thus, for example, a reference to “polynucleotide” includes a plurality of such polynucleotides, while a reference to “polypeptide” includes one or more polypeptides and their equivalents known to those skilled in the art, and so on. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a priori basis for the use of exclusive terms such as “only,” “just,” or “negatively” in conjunction with the description of the claim elements. It should also be noted that the term “or” is generally used in its broadest sense, meaning “and / or,” unless the context clearly specifies otherwise.

[0193] In cases where agreements such as "at least one of A, B, and C" are used, such a structure is generally intended to be understood by a person skilled in the art in the sense of the agreement (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, systems having only A, only B, only C, having A and B together, having A and C together, having B and C together, and / or having A, B, and C together, etc.). A person skilled in the art will further understand that any antonymous conjunctions and / or phrases presenting two or more optional terms, whether in the specification, claims, or drawings, should be understood to include the possibility of including one of these terms, any one of these terms, or both of these terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0194] It should be understood that, for clarity, certain features of the invention described in a single embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to the invention are specifically encompassed by the invention and disclosed herein, just as each combination is individually and explicitly disclosed herein. Furthermore, all sub-combinations of various embodiments and their elements are specifically encompassed by the invention and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0195] Other objects, advantages, and novel features of the invention will become apparent to those skilled in the art upon examination of the following embodiments, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the invention, as described above and claimed in the following claims, is experimentally supported in the following embodiments.

[0196] The terms “comprise” and its variations, such as “comprises” and “comprising”, as used herein should be interpreted in an open, inclusive sense, meaning that the described implementation includes the described features, but does not exclude the presence of other features, provided that such features do not render the implementation impossible.

[0197] As used herein, expressions such as "one embodiment," "specific embodiment," and "embodiment" should be interpreted as meaning that a specific feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Therefore, such expressions appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. For example, it is also clearly contemplated that certain features of this disclosure described herein in the context of individual embodiments may be combined in a single embodiment.

[0198] List of implementation methods Further, particularly preferred embodiments of the present invention include the following: 1. A pharmaceutical composition comprising a combination of at least two compounds selected from the group consisting of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), , , This includes any salt thereof, any stereoisomer thereof, or any combination thereof.

[0199] 2. The pharmaceutical composition of embodiment 1, wherein the composition comprises: (i) the 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), including any salt thereof, any stereoisomer thereof, or any combination thereof; and (ii) at least one compound selected from the group consisting of: , , This includes any salt thereof, any stereoisomer thereof, or any combination thereof.

[0200] 3. A pharmaceutical composition comprising at least two compounds selected from the following: a first compound capable of increasing elastin expression, a second compound capable of increasing elastin fiber deposition, a third compound capable of reducing or inhibiting elastin fiber breakdown or degradation, and a fourth compound capable of reducing or inhibiting the production of elasticity factors.

[0201] 4. A method for preparing a pharmaceutical composition according to any one of embodiments 1 to 3, comprising mixing a compound as defined in any one of embodiments 1 to 3 with a pharmaceutically acceptable carrier.

[0202] 5. A pharmaceutical composition according to any one of embodiments 1 to 4, used for the prevention or treatment of elastin deficiency-related diseases.

[0203] 6. A combination therapy for the prevention or treatment of elastin deficiency-related diseases in subjects of need, comprising at least two compounds selected from the following: a first compound capable of increasing primorelalin expression, a second compound capable of increasing elastin fiber deposition, a third compound capable of reducing or inhibiting elastin fiber breakdown or degradation, and a fourth compound capable of reducing or inhibiting the production of elasticity factor.

[0204] 7. The combination therapy according to embodiment 5, wherein any one of the following: a. The first compound includes a mitogen-activated protein kinase 1 (MAP2K1) inhibitor; b. The second compound comprises a protein kinase Cε type (PKCε) activator; c. The third compound includes an elastase inhibitor; d. The fourth compound includes a matrix metalloproteinase (MMP) inhibitor; and e. Any combination of (a) to (d).

[0205] 8. A combination therapy according to embodiment 7, wherein the combination comprises the PKCε activator, and any one of the following: the MAP2K1 inhibitor, the elastase inhibitor, and the MMP inhibitor.

[0206] 9. The combination therapy used according to embodiment 7 or 8, wherein the elastase is neutrophil elastase.

[0207] 10. A combination therapy according to any one of embodiments 7 to 9, wherein the MMP is MMP-12.

[0208] 11. A combination therapy according to any one of embodiments 6 to 10, wherein the at least two compounds are individually formulated in two different pharmaceutical compositions.

[0209] 12. A combination therapy according to any one of embodiments 6 to 10, wherein the at least two compounds are formulated in a single pharmaceutical composition.

[0210] 13. A combination therapy according to any one of embodiments 6 to 12, wherein any one of the at least two compounds is selected from: small molecules, nucleic acids, peptides, polypeptides, antibodies, peptidomimetic peptides, carbohydrates, and lipids.

[0211] 14. A combination therapy according to any one of embodiments 7 to 13, wherein the PKCε activator comprises or is composed of the following: 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA).

[0212] 15. A combination therapy according to any one of embodiments 7 to 14, wherein the MAP2K1 inhibitor comprises or is composed of the following: This includes any salt thereof, any stereoisomer thereof, or any combination thereof.

[0213] 16. A combination therapy according to any one of embodiments 7 to 15, wherein the elastase inhibitor comprises or is composed of the following: This includes any salt thereof, any stereoisomer thereof, or any combination thereof.

[0214] 18. A pharmaceutical composition comprising at least one compound selected from the group consisting of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), , , The pharmaceutical composition, including any salt thereof, any stereoisomer thereof, or any combination thereof, is used to treat or prevent elastin deficiency-related diseases in subjects of need.

[0215] 19. A pharmaceutical composition for use according to embodiment 18, comprising a combination of at least two compounds selected from: 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), , , This includes any salt thereof, any stereoisomer thereof, or any combination thereof.

[0216] 20. A pharmaceutical composition for use according to embodiment 19, wherein the composition comprises: (i) the 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), including any salt thereof, any stereoisomer thereof, or any combination thereof; and (ii) at least one compound selected from: , , This includes any salt thereof, any stereoisomer thereof, or any combination thereof.

[0217] 21. A pharmaceutical composition for use according to embodiment 20, comprising (i) and (ii) in a molar ratio of 1:10 to 10:1.

[0218] 22. A pharmaceutical composition for any of the uses of embodiments 18 to 19, further comprising a pharmaceutically acceptable carrier.

[0219] 23. A pharmaceutical composition for any one of embodiments 18 to 22, wherein the elastin deficiency-related disease is selected from: arteriosclerosis, peripheral vascular disease, angina pectoris, ischemia, myocardial ischemia, stroke, myocardial infarction, reperfusion injury, emphysema, Williams Beuren syndrome, post-angioplasty restenosis, hypertension, cardiovascular inflammation, scleroderma, MASS syndrome, age-related macular degeneration (ARMD3), familial thoracic aortic aneurysm (AAT10), autosomal recessive cutischameleon syndrome (ARCL), established atherosclerotic cardiovascular disease (ASCVD), arterial tortuosity syndrome, abdominal aortic aneurysm (AAA), supravalvular aortic stenosis (SVAS), autosomal dominant cutischameleon type 1 (ADCL1), acromiocele, happy chondrodysplasia, Williams-Beuren syndrome, Marfan syndrome (MFS), Williams-Marfan syndrome (WMS), congenital contracture arachnoidosis (CCA), pseudoxanthoma (PXE), Costello syndrome, arterial tortuosity syndrome, and isolated ectopic lens (ECTOL).

[0220] 24. A pharmaceutical composition for any of the uses of embodiments 18 to 23, wherein the treatment comprises: increasing the expression, stability, activity, or any combination thereof of elastin in the subject.

[0221] 25. A pharmaceutical composition for any of the uses of embodiments 18 to 24, wherein the treatment comprises increasing the elasticity of the aorta of the subject or reducing the stiffness of the aorta in the subject.

[0222] 26. A kit comprising a pharmaceutical composition according to any one of embodiments 1 to 3, packaged in a separate container, said pharmaceutical composition being used in combination to treat or prevent elastin deficiency-related diseases.

[0223] 27. A method for treating or preventing diseases related to elastin deficiency, comprising administering to a subject in need an effective amount of a pharmaceutical composition according to any one of embodiments 1 to 3.

[0224] Example In general, the nomenclature used in this paper and the laboratory procedures used in this invention encompass molecular, biochemical, microbiological, and recombinant DNA technologies. Such technologies are explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Vol. I-III, Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series," Vol. I-4, Cold Spring Harbor Laboratory Press, New York (1998). The methodologies described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology", Volumes I-III, Coligan, JE, ed. (1994); Stites et al.(eds.), "Basic and Clinical Immunology" (8th edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of these are incorporated by reference. Other general references are provided throughout this document.

[0225] In all examples, the designation EB-101 is used to denote the compound Alvelestat (i.e., 6-methyl-5-(1-methyl-1H-pyrazol-5-yl)-N-{[5-(methylsulfonyl)pyridin-2-yl]methyl}-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2-dihydropyridine-3-carboxamide) known under INN; the designation EB-102 is used to denote DCP-LA (i.e., 8-[2-(2-pentyl) (-Cyclopropylmethyl)-Cyclopropyl]octanoic acid); the code EB-103 is used to represent the compound also known as MMP-408 (i.e., (S)-2-(8-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butyric acid); and the code EB-104 is used to represent the compound known as U-0126 (i.e., 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene).

[0226] Example 1 In vivo / in vitro studies were conducted to demonstrate that small molecules or combinations thereof, as disclosed herein, are suitable for treating elastin loss and for inducing or promoting the recovery of elastin deposition.

[0227] Materials and Methods Determination of purified PKCε Recombinant PKC (1 ng α or ε isoform) was mixed with the test compound in the presence of 10 μmol histone, 5 mM CaCl2, 1.2 μg / μl phosphatidyl-L-serine, 0.18 μg / μl 1,2-dioctanoyl-sn-glycerol (DAG), 10 mM MgCl2, 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8 μg / ml aprotinin, 8 μg / ml leucopeptide, and 2 mM benzalkonium chloride. 0.5 μg Ci [ y32[P] ATP. Incubate the mixture at 37°C for 15 min, with a total volume of 10 μL. Spot the reaction mixture onto a 1 × 2 cm strip of cellulose phosphate paper (Whatman P81) and immediately wash twice with 0.5% H3PO4 (1 hr each time) to stop the reaction. Count the cellulose phosphate strip using a scintillation counter. In some experiments, phosphatidylserine, diacylglycerol, and / or calcium were removed.

[0228] Protein kinase C assay Rat hippocampal cells were cultured and scraped with 0.2 ml homogenization buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaF, 1 μg / ml leucopeptide and 0.1 MMPMSF), and homogenized by sonication (5 seconds, 10W) ​​in a Marsonix microprobe sonicator. To measure PKC, 10 μl of cell homogenate or purified PKC isoenzyme (purchased from Calbiochem) was incubated at 37°C for 15 min with 10 μM histone, 4.89 mM CaCl2, 1.2 μg / μl phosphatidyl-L-serine, 0.18 μg / μl 1,2-dioctanoyl-sn-glycerol, 10 mM MgCl2, 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8 μg / ml aprotinin, 8 μg / ml leucopeptide, and 2 mM benzidine. 0.5 μCi [ γ32 P] ATP is measured by adsorption onto cellulose phosphate. 32 P-phosphoprotein formation, as previously described. Nelson and Alkon, J. Neurochemistry. 1995; 65: 2350-57. For measurements of activation of one or more test compounds, PKC activity was measured in the absence of diacylglycerol and phosphatidylserine, as described by Kanno et al.; while PKC δ, ε, η, and μ were measured without the addition of EGTA and CaCl2, as described by Kanno et al., J. LipidRes. 2006; 47: 1146-50. Low concentrations of CaCl2 were used. 2+ Because of high Ca 2+ It interacts with the PKC phosphatidylserine binding site and prevents activation. 1,2-Diacylglycerol was omitted for the measurement of bryostatin activation.

[0229] Human neutrophil elastase quenching FRET assay This assay used human neutrophil elastase (HNE) purified from serum (Calbiochem art. 324681; Ref. Baugh, RJ et al., 1976, Biochemistry. 15, 836-841). HNE was stored at -20°C in 50 mM NaOAc, 200 mM NaCl, pH 5.5, supplemented with 30% glycerol. The protease substrate used was Elastase Substrate V Fluorogenic, MeOSuc-AAPV-AMC (Calbiochem art. 324740; Ref. Castillo, MJ et al., 1979, Anal. Biochem. 99, 53-64). The substrate was stored in DMSO at -20°C. The assay was performed as follows: The test compound and control were added to a black 96-well plate (Greiner 655076). First, 1 μL of 100% DMSO was added, followed by 30 μL of HNE in assay buffer containing 0.01% Triton X-100. The assay buffer consisted of 100 mM Tris (pH 7.5) and 500 mM NaCl. The enzyme and compound were incubated at room temperature for 15 minutes. Then, 30 μL of substrate was added to the assay buffer. After incubation at room temperature for 30 minutes, 60 μL of stop solution (140 mM acetic acid, 200 mM sodium chloroacetate, 60 mM sodium acetate, pH 4.3) was added to terminate the reaction. Fluorescence was measured on a Wallac 1420Victor 2 instrument with the following settings: excitation 380 nm, emission 460 nm. Using a model 205, the IC50 was determined using Xlfit curve fitting. 50 When tested in the above screening, the inhibition of human neutrophil elastase activity, IC50... 50 Compounds with values ​​less than 30 μM are considered to have useful therapeutic properties.

[0230] MMP-12 FRET assay The compounds were tested in the MMP-12 FRET assay as follows. Assay buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM CaCl2, and 0.005% Brij-35), purified human MMP-12 enzyme, and various concentrations of the test compound (prepared by serial dilution of the stock solution in 100% DMSO) were added to the wells of a black polystyrene 96-well plate. The plate was incubated at room temperature for 30 minutes. The enzymatic reaction was initiated by adding the substrate MCA-Pro-Leu-Gly-Leu-Dpa(DNP)-Ala-Arg (final concentration 20 μM) containing the fluorescent group (7-methoxycoumarin, MCA) and 2,4-dinitrophenyl (DNP). The final concentration of DMSO in the assay was 10%. The reaction was monitored at room temperature for 30 minutes using a fluorescence plate reader (λ). ex 325 nm, λ em The initial rate of the cleavage reaction was determined using an inhibitor concentration of 395 nm. The plot of inhibitor concentration versus initial cleavage rate was fitted with the following equation: y = V max ×(l-(x n / (K n + x n ))), where x = inhibitor concentration, y = initial rate, V max =Initial rate without inhibitors, n = slope factor, and K = IC50 of the inhibition curve. 50 IC 50 A value less than 30 μM indicates useful therapeutic efficacy.

[0231] Animal experiments Twelve-month-old aged male C57BL / 6JRj mice were obtained from Janvier Labs and acclimatized for two weeks at the animal facility of the Department of Pharmacology, University of Oxford, where all subsequent treatments were performed. Mice were housed under controlled temperature (23°C) and lighting (12h light / 12h dark cycle) conditions and had free access to water and standard feed (Harlan Research Laboratories) or HCD (58% fat, 25% carbohydrates, Research Diets 12231D). Animal care procedures complied with the Institutional Animal Care Committee guidelines, and all procedures were reviewed and approved by the UK Home Office and the Department of Pharmacology, University of Oxford. Animals were treated with subcutaneous injections of the aforementioned medications twice weekly, according to the protocol shown in the table below. Animal euthanasia and tissue collection At the end of the 12-week treatment period, animals were anesthetized with 4% isoflurane and then cervical dislocation was performed. The neck was then removed, and blood was collected from the trunk into 500 μl Eppendorf tubes pre-filled with 100 µl of 0.5 M EDTA. Next, aortic tissue was dissected for use as needed. For RNA and protein extraction, the aortic tissue was dissected and immediately flash-frozen in liquid nitrogen and stored at -80°C for future analysis. For arterial arteriography, the protocol is as follows.

[0232] Biochemical analysis RNA extraction RNA was extracted from primary cultured astrocytes according to the manufacturer's protocol, following the NucleoSpin® RNA tissue RNA purification protocol provided in the Macherey-Nagel (Macherey-Nagel, Düren, Germany) kit.

[0233] Reverse transcription cDNA synthesis was performed using total RNA (9 μL). The RNA extracted from the sample was reverse transcribed into cDNA using the Cloned AMV First-Strand cDNA Synthesis Kit (Invitrogen™) in a Biometra T-gradient thermal cycler following the protocol provided. The sample was diluted 1:1 or 1:2 with DEPC-treated water, depending on the amount of RNA extracted.

[0234] Components of reverse transcriptase reaction Quantitative real-time PCR (qPCR) For each PCR reaction, use 2 μL of cDNA and Platinum. TM SYBR TM Green qPCR SuperMix-UDG w / ROX (Invitrogen TMThe kit was used according to the manufacturer's instructions. For lower RNA concentrations, the DEPC water in the PCR reaction mixture could be replaced to increase the cDNA amount to 11 μl. The standard qPCR protocol consisted of the following: an initial cycle of 2 min at 50°C, followed by a cycle of 2 min at 95°C, then 40 cycles (including 15 s at 95°C to 1 min at 60°C) ending with a final cycle (15 s at 95°C to 20 s at 60°C and 15 s at 95°C). Expression values ​​were normalized by the geometric mean of several housekeeping genes, namely peptidyl prolyl isomerase A (PPIA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-actin. Oligonucleotide primers were used for the detection of specific cDNAs.

[0235] Protein blot Sample preparation After quantifying proteins according to the Lowry protein assay, 120 μl of each tissue lysate was mixed with 40 μl of 4 × SDS-2-mercaptoethanol and denatured at 95 °C for 10 min. All samples were diluted with 1 × SDS (4 × SDS, diluted with 1 × CLB) to the lowest protein concentration to be measured.

[0236] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Using commercially available acrylamide gels, in a vertical mini-gel electrophoresis system (Mini-PROTEAN) ® In TetraVertical Electrophoresis Cell (Bio-Rad), proteins were separated by size at 100V for 2.05 h. Acrylamide gels containing separating and stacking gels were then prepared. The chambers were filled with SDS electrophoresis buffer (containing 3.03 g Tris base, 14.4 g glycine, and 1 g SDS per liter of H2O). Precision Plus Protein was loaded into the first well of the stacking gel. TM The standard (Bio-Rad) was used, while the other wells were loaded with 50 to 100 μg of protein.

[0237] Semi-dry protein transfer Following SDS-PAGE, the separated proteins were transferred from the separating gel to Immobilon-P (Millipore) membranes using a semi-dry protein transfer method. The membranes and filters were immersed in Towbin blot buffer (3.03 g Tris base, 14.41 g glycine, 1 g SDS in 800 ml, and 200 ml methanol (MeOH), pH 8.2–8.4) and stacked on Hoefer membranes. ® SemiPhor TM In a TE-70 electrophoretic blot system (Amersham Pharmacia Biotech). The transfer will run at 100 V for 1.5 hours.

[0238] Immunostaining Use 5% bovine serum albumin (BSA, Sigma-Aldrich) ® The proteins now transferred to the membrane were blocked, and immunostaining was performed overnight at 4°C with antibodies against elastin, neutrophil elastase, MMP12, fibrin 5 / DANCE, and MAP2K1. The next day, the membrane was washed with 1× PBS and incubated at room temperature (RT) with horseradish peroxidase (HRP) secondary antibody for 1 h. The HRP-enhanced chemiluminescent substrate SuperSignal was diluted 1:4 in H2O with FUSION SOLO S (Vilber). ® West Femto-Substrate (Thermo Scientific TM This makes the secondary antibody that binds to the corresponding protein visible.

[0239] Immunohistochemistry Three proximal thoracic aortas were randomly selected from each group, and ≥9 tissue sections / aorta were generated using a cryostat. Tissue sections (10 µm) were generated every 100 µm from the aortic root to the aortic arch. The three sections corresponding to the area of ​​greatest dilation between the sinotubular junction and the aortic arch were analyzed. Multiple high-magnification images of each section were acquired using ImageJ.20, and these images were then aggregated into a single image of the entire section.

[0240] For histological analysis, the distal portion of the aortic arch (between the left subclavian artery and the origin of the descending aorta) was fixed in formalin and embedded in paraffin (FFPE), then cut into 5 μm thick sections. Staining was performed after dewaxing and rehydration until water was obtained.

[0241] The above-described immunostaining protocol was used to perform standard immunohistochemical staining for neutrophil elastase, MMP12, fibrin / DANCE5, and MAP2K1 on aortic sections. The sections were incubated overnight at 4°C with antibodies specific to these four antibodies, followed by incubation with goat anti-mouse IgG conjugated with Alexa488 (Molecular Probes, Eugene, OR, USA) at room temperature for 60 min. After staining with 4',6-diamidinyl-2-phenylindole (DAPI), the fluorescently labeled cells were visualized using a confocal scanning laser microscope (Axiovert / LSM510; Carl Zeiss, Oberkochen, Germany).

[0242] Measuring arterial stiffness and elasticity Contraction of isolated arterial (aortic, mesenteric, and pulmonary) rings was measured using a small vessel wire myograph (Model 420A, DanishMyo Technology, Aarhus, Denmark). Tension measurements (in mN) were normalized to the arterial ring length (in mm) to allow for comparison of data obtained from rings of slightly different lengths. Tissue was stimulated with blue light of varying intensities (0.02–7.21 mW·mm⁻²). Unless otherwise specified, the pulse duration was 7 min.

[0243] In the absence of PE, the relationship between tension and blue light intensity can be fitted by the following form of the Hill equation: T = Tss1 + (EL 50 Light intensity) s, where "T" is the tension measured at a given light intensity (normalized to the arterial ring length), "Tss" is the maximum asymptotic tension reached, and "EL" is the maximum asymptotic tension reached. 50 “ is the light intensity that causes half-maximal tension, and “s” is the slope coefficient.

[0244] result Elastin is an important extracellular matrix (ECM) protein that provides tissues and organs with resilience and elasticity. It is a major protein in stretchable tissues and is particularly important for maintaining the cardiovascular system, as well as the lungs and skin. Aging leads to elastin degradation, resulting in decreased tissue function and being associated with a variety of age-related diseases.

[0245] Current proof-of-concept indicates that increased elastin expression ( Figure 1AThe arterial tonic strength of aged mice was functionally enhanced compared to a saline control (Figure 2). As those skilled in the art will understand, arterial stiffness above 1800 μM stretch is not physiologically relevant. The improved arterial stiffness—approximately 600 to 1200 μM stretch—is attributed to increased expression, stability, activity, or any combination thereof of elastin in mice. Indeed, this could be of great significance for treating diseases resulting from elastin loss and associated declines in elastin content and quality. Where one or more treatment methods are available, the inventors propose employing one or more molecular pathways to achieve this goal, including activation of PKC, inhibition of neutrophil elastase, and matrix metalloproteinases. Indeed, the currently proposed treatment methods are significant for, but not limited to, abdominal aortic aneurysms, supravalvular aortic stenosis, Marfan syndrome, and cutis laxa.

[0246] Example 2 Further in vivo studies will be conducted in mice to investigate the effects of small molecules or combinations thereof, as disclosed herein, on certain aspects of the aging process.

[0247] Materials and Methods Animal experiments: All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the Animal Welfare and Ethical Review Body at Oxford University. Animals were housed under standard laboratory conditions (12-hour light / dark cycle, lights on at 7:00 AM; 21 ± 1°C; humidity 50 ± 5%) with free access to food and water. All experimental conditions were established and maintained in accordance with the European Community Council Directive on the Care and Use of Laboratory Animals (2010 / 63 / EU) and the ARRIVE Guidelines (http: / / www.nc3rs.org.uk / arrive-guidelines). The following drug combination was used to treat 12-month-old aged C57BL / 6JRj mice twice weekly for 12 weeks: Behavior At the end of the 12-week treatment period, prior to tissue collection, behavior was measured using an open field test (OFT). The OFT apparatus consisted of four equal-sized quadrants (l × w × h; 50 cm × 50 cm × 50 cm) to allow for the simultaneous testing of four mice. The open field was defined as 25% of the total area of ​​each quadrant (l × w; 25 cm × 25 cm). Mice were placed in the upper left corner of each quadrant in a random order, and video was recorded for 5 minutes. During the experimental intervals, the floor and walls were wiped with 75% ethanol to prevent the influence of odor and coprophagia on mouse behavior. All behavioral parameters except hind-leg uprighting were measured using ANY-Maze software (Stoelting Co., IL, USA), which was manually counted in a blinded manner and defined as the time at which a mouse stood upright (supported by a wall or free-standing) using both hind legs.

[0248] Fur Analysis At the end of the 12-week treatment period, the color and quality of the mouse fur were analyzed before tissue collection.

[0249] tissue collection Following a 12-week treatment period, mice were anesthetized with isoflurane gas. After confirming the absence of plantar reflexes, whole blood was collected via cardiac puncture into EDTA (0.1 M, pH 8)-coated tubes and centrifuged at RT for 30 min (10 min, 2000 x g). Tissue was perfused via the heart with heparinized saline until the liver was depleted of blood. The entire brain was removed and flash-frozen in isopentane on dry ice, and liver samples were collected and immediately flash-frozen on dry ice. All tissues and plasma were stored at -80°C for subsequent gene expression.

[0250] Arterial arterial kinetics - stress-strain curve At the end of the experiment, mice (n=5) were euthanized, and the artery was dissected from the surrounding tissue. A 2 mm ring was dissected and fixed in a 5 mL myograph containing physiological saline solution (composition: 119.0 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 24.9 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM CaCl2, 11.1 mM glucose). The myograph was ventilated with a 95% O2 / 5% CO2 mixture, and the temperature was maintained at approximately 37°C throughout the experiment. Changes in force generation after a cumulative stretch of 100 μm were detected using a force transducer, and data were acquired using LabChart 7 software (ADInstruments).

[0251] Arterial arterial kinetics - systole and relaxation RNA extraction and reverse transcription RNA was isolated from 15 to 20 mg of aortic or brain tissue according to the manufacturer's instructions [RNeasy® Mini Kit (Cat No. / ID: 74106)]. Liver and PFC RNA were eluted in nuclease-free water, and 1000 ng of RNA was converted to cDNA using the Applied Biosystems High Capacity cDNA Conversion Kit, following the manufacturer's instructions. The reverse transcription step was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher, cat. 4368813), following the manufacturer's instructions.

[0252] qPCR Real-time qPCR was performed on samples using a SsoAdvanced Universal SYBR Green Supermix (BioRad) and a RocheLightCycler 480, repeated twice (25 ng / well). It has been previously demonstrated that transcripts of acute-phase reactions are translated into proteins (Campbell et al., 2003). Melting curve analysis was performed after the reaction to ensure primer specificity. Ct values ​​were normalized to control housekeeping genes GAPDH or β-actin and expressed as a fold change relative to saline controls using the Δ-ΔCT (2-ΔΔCT) method. Oligonucleotide primers were used for the detection of specific cDNAs.

[0253] Analysis of NAD and glutathione metabolites in frozen blood Analysis was performed by NADMed (Helsinki, Finland; www.nadmed.com). A proprietary extraction procedure was used to extract NAD and glutathione metabolites from frozen blood in a single step, and each metabolite was measured individually using optimized cyclic enzymatic assays and colorimetric detection. For stability, the NADMed extraction method uses an extraction solution representing a non-buffered complex mixture of an organic solvent in water. The sample was injected into the pre-warmed extraction solution to force proteins to unfold and release non-covalently bound metabolites into solution. All pH- and redox-sensitive metabolites remained stable, including both reduced and oxidized glutathione. After protein removal, total GSH and GSSG were measured individually using the supernatant. GSH was defined as the difference between the total GSH pool and the GSSG value. Similarly, total NAD and NAD+ were measured individually using the supernatant, as were total NADP and NADP+. NADH was defined as the difference between total NAD and NAD+. GSH is defined as the difference between total GSH and GSSG.

[0254] Data Analysis All statistical analyses were performed using GraphPad Prism 9 software. Any outliers were identified and removed using the GraphPad Outlier Calculator. Normality was tested using the Shapiro-Wilk normality test; if the test failed, a logarithmic transformation was applied before statistical analysis. Two-way ANOVA was used to assess the primary effects of treatment (TLR agonist administration) on behavior, relative mRNA expression, and spectral intensity. Multiple comparisons were performed post-hoc using Tukey's test if a significant interaction between treatment and sex was found. Significance thresholds were set as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, and all results are expressed as mean ± standard error of the mean (SEM). All correlations were analyzed using Pearson correlation analysis and the false discovery rate (FDR) was adjusted.

[0255] NMR metabolomics Sample processing Metabolites were extracted from plasma samples as previously described (Radford-Smith et al., 2022). Samples were kept on dry ice, and 8 μL of ice-cold acetonitrile (50%) was added per mg of sample. The samples were then vortexed and centrifuged at 5060 × g for 5 min at 4 °C. The supernatant was rapidly frozen on dry ice, lyophilized overnight, and stored at -80 °C until the day of NMR analysis. When ready for analysis, the lyophilized samples were placed at room temperature and resuspended in 550 μL of NMR buffer (75 mM sodium phosphate buffer, prepared in D2O, pH 7.4). Plasma samples were also thawed at RT, and 150 μL was added to 400 μL of NMR buffer.

[0256] NMR experiment All prepared samples were then placed in 5 mm NMR tubes and measured at 16.4 T using a 700 MHz Bruker AVII spectrometer equipped with a 1H (13C / 15N) TCI cryoprobe, as previously described (Chan et al., 2020). Plasma and liver spectra were acquired using spin-echo sequences (Carr-Purcell-Meiboom-Gill [CPMG]) with 32 data acquisitions at an acquisition time of 1.5 s, a relaxation delay of 2 s, and fixed receiver gain. Brain spectra were acquired using nuclear overhauser effect spectroscopy (NOSEY) sequences, as previously described (Chan et al., 2020). All samples were measured within 9 h after thawing.

[0257] NMR data processing In Topspin 4.1.4 (Bruker), spectra were phase-corrected, baseline-corrected, and calibrated using the lactate biphasic peak at δ = 1.33 ppm as a reference. Each resonance signal was manually binned using ACD / Labs Spectrus Processor Academic version 12.01 (Advanced Chemistry Development, Inc.) to eliminate all noise from the analysis. The integrals of these bins were summed, normalized, and exported to R. Metabolite assignment was performed using a combination of in-house databases, literature reviews, and 2D total correlation spectroscopy (TOCSY) experiments.

[0258] Spectral Intensity Statistical analysis univariate All statistical analyses were performed using GraphPad Prism 9 software. Any outliers were identified and removed using the GraphPad Outlier Calculator. Normality was tested using the Shapiro-Wilk normality test; if the test failed, a logarithmic transformation was applied before statistical analysis. Two-way ANOVA was used to assess the primary effects of treatment (TLR agonist administration) on behavior, relative mRNA expression, and spectral intensity. Multiple comparisons were performed post-hoc using Tukey's test if a significant interaction between treatment and sex was found. Significance thresholds were set as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, and all results are expressed as mean ± standard error of the mean (SEM). All correlations were analyzed using Pearson correlation analysis and corrected for false discovery rate (FDR).

[0259] Multivariable Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on the metabolome using the ROPLS package in R to determine whether differences existed between groups. Variable importance in projection (VIP) scores were used to identify which metabolites were most important in driving the distinction between groups. Then, univariate analysis was performed on the bins with the highest VIP scores (before the inflection point when comparing all VIP scores) to understand the direction of metabolite variation, as described above.

[0260] result This study was conducted to demonstrate that the small molecules or combinations thereof disclosed herein are suitable for treating age-related diseases and (age-related) neurodegenerative conditions. Significant improvements were observed in mice treated with the small molecules or combinations thereof according to the invention compared to untreated mice.

[0261] Fur Analysis Compared with untreated mice and mice treated with DPC-LA, Alvelestat, MMP-408, or U0126, mice treated with DCP-LA + Alvelestat, DCP-LA + MMP-408, and DCP-LA + U0126 showed significantly enhanced fur color (from mottled gray to solid black), better fur quality, and less fur damage.

[0262] Analysis of NAD and glutathione metabolites in frozen blood Nicotinamide adenine dinucleotide (NAD) plays a dual role in both its oxidized (NAD+) and reduced (NADH) forms. It is both a redox coenzyme and a co-substrate (NAD+) of enzymes involved in deacylation reactions, mono- and poly-ADP ribosylation, and the production of adenine second messengers. NAD+ and NADH can be phosphorylated by NAD+ kinase (NADK) to NADP and NADPH, respectively. NADP and NADPH play similar roles in metabolic pathways to NAD+ and NADH.

[0263] The NAD(P)+ / NAD(P)H ratio is an important indicator of intracellular redox status and plays a crucial role in the regulation of key metabolic pathways. It has been shown that NAD(P)(H) status affects cellular signaling and metabolism, energy homeostasis, mitochondrial biosynthesis, oxidative stress responses, and DNA maintenance and repair. Furthermore, the progression of age-related diseases such as obesity, non-alcoholic fatty liver disease, neurodegenerative diseases, mitochondrial diseases, cardiovascular diseases, and muscle atrophy or malnutrition is also associated with alterations in NAD+-related metabolites.

[0264] Blood acts as a buffer for NAD+ and its metabolites. Aging leads to a decrease in blood NAD+ levels, with levels reaching approximately 20 uM in the blood of mice older than 12 months. However, blood NAD+ levels alone are insufficient to indicate overall metabolic health and must be correlated with behavioral and other markers.

[0265] Current research indicates that treatment of 12-month-old mice with a combination of DCP-LA + MMP-408 and DCP-LA + U0126 twice weekly for 12 weeks resulted in a significant decrease in serum NAD+ levels. Figure 3A Similarly, treatment with a combination of DCP-LA + MMP-408 and DCP-LA + U0126 resulted in a significant decrease in NADP+ levels in the blood. Figure 3B This decrease in blood levels indicates increased tissue uptake of NAD+ and NADP+, suggesting increased tissue energy utilization and counteracting catabolistic stress caused by aging. Elevated blood glutathione (GSH) levels ( Figure 3CThis further supports this positive effect on aging. Given that GSH performs important functions such as antioxidant and cell repair, the levels of GSH in the blood of mice treated with the DCP-LA + MMP-408 and DCP-LA + U0126 combinations were significantly increased compared to untreated mouse blood levels. Figure 3C This further supports the positive effects of combined treatments on aging.

[0266] NMR metabolomics Aging is associated with impaired protein metabolism. Compared to younger individuals, the levels of amino acids in the blood are generally lower. However, due to muscle loss and tissue damage, the concentrations of amino acids such as lysine and valine in the blood are higher than in younger individuals.

[0267] Current data indicate that treatment of 12-month-old mice with a combination of DCP-LA + MMP-408 and DCP-LA + U0126 twice weekly for 12 weeks resulted in a decrease in blood lysine levels. Figure 4A Similarly, treatment of 12-month-old mice with the DCP-LA + U0126 combination twice a week for 12 weeks resulted in a significant decrease in blood valine levels. Figure 4A Compared to control mice, mice treated with DCP-LA alone and those treated with DCP-LA + Alvelestat also showed a decreasing trend in lysine and valine levels. These results suggest reduced muscle loss, decreased aging-related catabolic stress, and increased tissue autophagy, thus supporting better longevity.

[0268] Behavior Aging is associated with various behavioral changes mediated by brain structure and networks. Age-related behavioral changes have been demonstrated through comparisons between young and older animals. Multiple studies have reported declines in behavioral performance related to locomotor activity and anxiety-like behaviors in older mice compared to younger mice.

[0269] To measure the behavior of mice at the end of the 12-week treatment period, an open field test (OFT) was used. The open field test is an experimental test used to determine an animal's general level of motor activity, anxiety, and exploratory willingness. Current data show that mice treated with the small molecule or combination thereof according to the present invention exhibited improvements in behavioral markers in tests assessing mobility and anxiety compared to untreated control mice.

[0270] Hind-leg uprighting behavior, as explored by rodents standing on their hind legs, provides a measure of activity and anxiety in rodents. Compared to control mice, 12-month-old mice treated with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA + Alvelestat, DCP-LA + MMP-408, and DCP-LA + U0126 showed significantly increased hind-leg uprighting behavior. Figure 5A , Figure 5B , Figure 5C , Figure 5D This indicates an increase in activity level and a decrease in anxiety level.

[0271] Decreased activity levels and increased anxiety levels—potentially associated with aging—lead to reduced movement. Twelve-month-old mice treated with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA + MMP-408, and DCP-LA + U0126 were more active (expressed as activity time in seconds) compared to control mice. Figure 6A , Figure 6B , Figure 6C , Figure 6D This indicates an increase in activity level and a decrease in anxiety level.

[0272] Decreased activity levels and increased anxiety levels led to a preference for staying near the playground walls, while increased activity levels and decreased anxiety levels were associated with longer time spent in the central area of ​​the playground. Compared to control mice, 12-month-old mice treated with MMP-408, U0126, DCP-LA + MMP-408, and DCP-LA + U0126 did indeed spend less time in the central area. Figure 7C , Figure 7D This behavior indicates an increased level of activity and a decreased level of anxiety.

[0273] Compared with control mice, 12-month-old mice treated with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA + Alvelestat, and DCP-LA + MMP-408 showed significantly reduced immobility time. Figure 8A , Figure 8B , Figure 8C , Figure 8D This behavior indicates an increased level of activity and a decreased level of anxiety.

[0274] Example 3 Further in vivo studies in mice will be conducted to investigate the effects of small molecules or combinations thereof disclosed herein on changes in gene expression in the elastin synthesis pathway; changes in gene expression in inflammatory cytokines; changes in gene expression involved in Williams syndrome; behavior; in vivo motion recording; and metabolomics.

[0275] Materials and Methods Animal experiments and treatment 12-month-old aged C57BL / 6JRj mice were used according to the methods shown in Table 3.1 (and in...). Figure 9 (Visualized) Treat with saline or any combination of drugs twice a week for up to 12 weeks.

[0276] Table 3.1: Processing All procedures were conducted in accordance with the UK Animal (Scientific Procedures) Act 1986 and approved by the Oxford University Animal Welfare and Ethics Review Board. The animals were housed in standard laboratory conditions (12-hour light / dark cycle, lights on at 7:00 AM; 21 ± 1°C; humidity 50 ± 5%), with free access to food and water.

[0277] Behavior Behavior was measured using an open field test (OFT) 20 h post-injection, prior to tissue collection. The OFT apparatus consisted of four equal-sized quadrants (l × w × h; 50 cm × 50 cm × 50 cm) to allow simultaneous testing of four mice. The open field was defined as 25% of the total area of ​​each quadrant (l × w; 25 cm × 25 cm). Mice were placed in the upper left corner of each quadrant in random order, and video was recorded for 5 minutes. During the experimental intervals, the floor and walls were wiped with 75% ethanol to prevent the influence of odor and coprophagia on mouse behavior. All behavioral parameters except hind-leg uprighting were measured using ANY-Maze software (Stoelting Co., IL, USA), which was manually counted in a blinded manner and defined as any time a mouse stood upright (supported by a wall or self-supporting) using both hind legs.

[0278] tissue collection Following a 12-week treatment period, mice were anesthetized with isoflurane gas. After confirming the absence of plantar reflexes, whole blood was collected via cardiac puncture into EDTA (0.1 M, pH 8)-coated tubes and centrifuged at RT for 30 min (10 min, 2000 x g). Tissue was perfused via the heart with heparinized saline until the liver was depleted of blood. The entire brain was removed and flash-frozen in isopentane on dry ice, and liver samples were collected and immediately flash-frozen on dry ice. All tissues and plasma were stored at -80°C for subsequent gene expression.

[0279] RNA was isolated from aortic samples. RNA was isolated from 10 to 15 mg aortic samples according to the manufacturer's instructions [RNeasy® Mini Kit (Cat No. / ID: 74106)]. Aortic RNA was eluted in nuclease-free water, and 1000 ng of RNA was converted to cDNA using the Applied Biosystems High Capacity cDNA Conversion Kit, following the manufacturer's instructions. The reverse transcription step was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher, cat. 4368813), following the manufacturer's instructions.

[0280] 8.5 µl of RNA isolated from aortic samples was treated with RNase-free DNase I (Thermo Fisher Scientific, Cat. EN0521) at 37 °C for 10 min, followed by stopping the reaction with 1 μl of reaction buffer at 70 °C for 2 min. RNA quality and concentration were analyzed using the Agilent RNA 6000 Pico kit (Cat. 5067-1513).

[0281] RNA isolation from the brain hemispheres Total RNA was extracted from frozen brain hemispheres using the miRNeasy Mini kit (Qiagen, Germany, Cat. 217004). Each sample was aliquoted into three equal-sized fractions and homogenized in 800 μl of Qiazol with Lysing Matrix Z (Cat. 116961500) using a Bead Beater FastPrep24 5G (MPBiomedicals™). Homogenates from each sample were combined, and 400 μl was used for further separation (according to the manufacturer's instructions), while the remaining liquid was stored at -80°C. After incubation at room temperature for 10 min, 140 μL of chloroform was added to the lysate, followed by centrifugation at 12,000 g for 15 min at 4°C. 350 μL of the upper aqueous phase was precisely transferred to a miRNeasy mini-column, and the RNA was precipitated with ethanol and then automatically washed with RPE and RWT buffers in a Qiacube liquid handling robot. Finally, total RNA was eluted in 30 µL of nuclease-free water and stored at -80°C until further analysis. RNA quality and concentration were analyzed using the Agilent RNA 6000 Nano kit (Cat.5067-1511).

[0282] mRNA sequencing analysis Library preparation was performed using the QuantSeq 3' mRNA-Seq V2 Library Prep Kit FWD and Unique Dual Indices (Lexogen). For RNA isolated from brain tissue, the standard input protocol was followed, while DNase I-treated aortic RNA was prepared according to the manufacturer's instructions using a low-input / low-quality protocol. Briefly, reverse transcription was performed using the reverse transcription mixture provided in the QuantSeq kit, followed by second-strand synthesis using the second-strand synthesis mixture provided in the kit. The resulting cDNA was purified with magnetic beads and amplified by PCR. To determine the number of amplification cycles, the PCR add-on and reamplification kit V2 (Lexogen) was used. Therefore, brain tissue RNA was amplified using 15 PCR cycles, while aortic RNA was amplified using 22 or 24 cycles. The amplified cDNA was then purified again to remove any contaminants. The quality of the prepared libraries was assessed using the Agilent High Sensitivity DNA Kit (Cat. 5067-4626). An equimolar merge consisting of all sequencing libraries was prepared and sequenced on an Illumina NextSeq 2000. NGS data were processed using the meND pipeline.

[0283] qPCR Real-time qPCR was performed on the samples using a SsoAdvanced Universal SYBR Green Supermix (BioRad) and a RocheLightCycler 480, repeated twice (25 ng / well). It has been previously demonstrated that transcripts of the acute phase response are translated into proteins (Campbell et al., 2003). Melting curve analysis was performed after the reaction to ensure primer specificity. Ct values ​​were normalized to the housekeeping gene GAPDH and expressed as a fold change relative to the saline control using the Δ-ΔCT (2-ΔΔCT) method. Primer sequences are shown in Table 3.2.

[0284] Table 3.2: Primer Sequences Data Analysis All statistical analyses were performed using GraphPad Prism 9 software. Any outliers were identified and removed using the GraphPad Outlier Calculator. Normality was tested using the Shapiro-Wilk normality test; if the test failed, a logarithmic transformation was applied before statistical analysis. Two-way ANOVA was used to assess the primary effects of treatment (TLR agonist administration) on behavior, relative mRNA expression, and spectral intensity. Multiple comparisons were performed post-hoc using Tukey's test if a significant interaction between treatment and sex was found. Significance thresholds were set as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, and all results are expressed as mean ± standard error of the mean (SEM). All correlations were analyzed using Pearson correlation analysis and corrected for false discovery rate (FDR).

[0285] Myograph After dissecting the blood vessels and removing any connective and adipose tissue, they were cut into 2 mm loops. PA loops and aortic loops were threaded onto two parallel pins in the 5 ml chamber of a multi-myograph system (Danish Myo Technology, Model 610M). Because the mesenteric resistance artery was too small (100-200 μm in diameter), wire kinesiography was used instead. The mesenteric artery (MA) loop was threaded onto two stainless steel wires (40 μm in diameter), which were mounted in the same 5 ml kinesigraph chamber of the multi-myograph system. The blood vessel was immersed in 5 ml of PSS and continuously ventilated at 37°C. In terms of kinesigraph setup, briefly, one mounting pin / arm is held in place and attached to the force transducer, while the other is attached to a micrometer and allows adjustment of the vessel diameter. The force transducer—capable of detecting force changes as small as 0.01 mN—connects to a computer via an analog-to-digital interface (National Instruments, USB-6221-BNC).

[0286] After the vessels were installed, basal tension was applied. It has been shown that vascular vasoreactivity depends on the degree of stretching (Schubert et al., 1999). Therefore, it was important to determine the amount of basal tension applied to each type of vessel and to ensure the use of the inner circumference that provides maximum response. The amount of basal tension applied to each type of vessel was defined as the tension required to achieve maximum response to 50 mM KCl. The procedure was as follows: the vessels were subjected to a 50 mM KCl challenge at low tension (approximately 1 mN) until sustained contraction was observed. Then, using a micrometer, the vessels were subjected to further KCl challenges with progressively increasing tension until maximum response was achieved. After performing this procedure, it was found that 4 mN, 6 mN, and 10 mN of tension were required to achieve maximum KCl response for the PA, MA, and 2 mm aortic rings, respectively; therefore, these basal tensions were applied to each type of vessel at the start of the experiment. After applying basal tension, the vessels were allowed to equilibrate for 30–45 min, with the bath solution replaced with fresh PSS every 15–20 min.

[0287] After the equilibration period, the viability of the preparation was tested by challenging the blood vessel with 50 mM KCl. The blood vessel was exposed to 50 mM KCl for 6 min, and then rinsed with fresh PSS solution. This process was repeated twice or until a sustained KCl response was observed. The blood vessel was considered viable if i) maximum contraction was observed within 5 min after KCl application, and ii) stable baseline tension was observed between each KCl challenge.

[0288] After flushing the final KCl reaction, place the blood vessel to re-establish baseline tension before starting any subsequent experiments.

[0289] Vascular contractility was assessed by increasing the concentration of phenylephrine (PE; 1 μM), an α-adrenergic agonist. After PE reached a stable maximum contraction, acetylcholine was applied to the blood vessel at progressively increasing concentrations to measure the diastolic response until stable diastole was achieved. Data from each experiment were fitted to the Hill equation and extrapolated to EC. 50 Value. Also, EC 50 Value converted to pEC 50 Values ​​are used for statistical comparison of the data. For presentation purposes, the results of each experimental group are pooled together and the average concentration response data are plotted. Where permissible, the Hill equation is used to curve-fit the average data.

[0290] mRNA sequencing analysis RNAseq showed that elastin therapy resulted in transcriptome-wide changes in both the brain and aorta. Heatmaps of the two tissues (respectively...) Figure 35 and Figure 36The results showed that the EB-102-101 combination produced the most dramatic differential gene expression compared to the control. Kegg pathway analysis revealed that the pathways most significantly affected by EB-102-101 treatment were aminoacyl-tRNA biosynthesis (adjusted p=6.6E-7), ribosomes (p=3.5E-6), COVID-19 (p=0.00014), amyotrophic lateral sclerosis (p=0.003), Alzheimer's disease (p=2.5E-2), and Huntington's disease (p=0.035). Pathways significantly affected but not passing multiple comparisons included neurodegenerative disease-multiple disease pathways, thermogenic pathways, Parkinson's disease pathways, autophagy pathways, and oxidative phosphorylation pathways, among others. It is important to note that some of the key genes affected in these pathways may still have significant biological importance. Kegg pathway analysis of the aorta revealed more significant changes. After multiple comparison correction, the following pathways ranked among the top 68 significantly altered pathways: protein infectious diseases (p=1.2E-50), Parkinson's disease (p=1.2E-50), oxidative phosphorylation (p=3.3E-49), diabetic cardiomyopathy (p=2.2E-46), and Alzheimer's disease (p=2.6E-43). Of particular interest were observed in the longevity pathway, with significant impacts on insulin pathway (p=1.6E-6), AMPK signaling (p=1.7E-6), HIF-1 signaling (p=6.7E-4), cellular senescence (p=7.3E-3), mTOR signaling (p=1.6E-2), autophagy (p=1.6E-2), mitophagy (p=2.1E-2), and longevity regulatory pathways (p=2.2E-2). Notably, NFkB was significantly downregulated in EB-101-102 mice compared to the control (p=0.01 after multiple comparison correction). Similar pathway effects were observed in other groups in the aorta, but to a lesser extent. In the brain, only two genes remained significantly altered compared to the control in EB-102-103, while no genes remained significantly altered compared to the control in the EB-102 single-treatment group.

[0291] Analysis of NAD and glutathione metabolites in frozen blood Analysis was performed by NADMed (Helsinki, Finland; www.nadmed.com). A proprietary extraction procedure was used to extract NAD and glutathione metabolites from frozen blood in a single step, and each metabolite was measured individually using optimized cyclic enzymatic assays and colorimetric detection. For stability, the NADMed extraction method uses an extraction solution representing a non-buffered complex mixture of an organic solvent in water. The sample was injected into the pre-warmed extraction solution to force proteins to unfold and release non-covalently bound metabolites into solution. All pH- and redox-sensitive metabolites remained stable, including both reduced and oxidized glutathione. After protein removal, total GSH and GSSG were measured individually using the supernatant. GSH was defined as the difference between the total GSH aggregate and the GSSG value.

[0292] NMR Metabolomics - Sample Acquisition Whole blood was collected into EDTA tubes via cardiac puncture. After 30 minutes at room temperature (RT), the tubes were centrifuged at 1500 xg for 10 minutes at RT. The plasma was then flash-frozen on dry ice and immediately transferred to a -80°C freezer. On the day of analysis, the plasma samples were thawed at room temperature and mixed by pipetting. 75 μL of plasma was then added to 475 μL of 75 mM sodium phosphate buffer (pH 7.4) prepared in D2O and transferred to 5 mM borosilicate glass NMR tubes (Norell 502-7) using a glass pipette. All NMR samples were prepared on the day of analysis. NMR experiments were performed using a 950 MHz NMR spectrometer equipped with a TCI cryoprobe (Department of Biochemistry, University of Oxford) at the parameters previously described (see: Probert et al. Plasma Nuclear Magnetic Resonance Metabolomics Discriminates Between High and Low Endoscopic Activity and Predicts Progression in a Prospective Cohort of Patients With Ulcerative Colitis. J Crohns Colitis. 2018;12(11):1326-37. pmid:30016408).

[0293] NMR metabolomics - Analysis Process each plasma sample as previously described. 11H NMR spectroscopy (see: Dunstan et al. Unique pathways downstream of TLR-4 and TLR-7 activation: sex-dependent behavioral, cytokine, and metabolic consequences. Front Cell Neurosci. 2024;18. doi:10.3389 / fncel.2024.1345441). In summary, spectra were phase-corrected, calibrated, and baseline-corrected in TopSpin 4.1.4 (Bruker). Each resonance signal was manually binned using ACD / Labs Spectrus Processor Academic version 12.01 (Advanced Chemistry Development, Inc.), and the integrals were summed, normalized, and exported to R for analysis. Principal component analysis (PCA) and (orthogonal) partial least squares discriminant analysis ((O)PLS-DA) (using the ropls package) were used to investigate the metabolomics characteristics of each group. Projected Importance (VIP) scores and loadings were used to identify which metabolites were most important for driving intergroup differentiation. Univariate analyses were then performed on bins with VIP scores above 1.5 (or before the inflection point when comparing all VIP scores) to understand the direction of metabolite variation between groups. Metabolites were specified using 2D spectroscopy, incorporation of reference compounds, literature values, or reference to internal databases.

[0294] result Changes in gene expression in the elastin synthesis pathway Figure 10 (ae)- Figure 13 (ae) shows the effects of single treatments of EB-101, EB-102, EB-103 and EB-104, and specifically their combinations (compared to control), on elastin, neutrophil elastase, MMP-12, MAP2K1 and fibrin-5.

[0295] These data show that the Alvelestat (EB-101) + DCP-LA (EB-102) combination resulted in a 4-fold increase in elastin expression in the aorta of mice treated for 12 weeks. Simultaneously, MAP2K1 increased 4-fold and fibrin-5 increased 8-fold. Both are markers of elastin synthesis. Neutrophil elastase and macrophage elastase increased 2-fold and 4-fold, respectively. This is presumably a compensatory mechanism for elastase inhibition. All these increases were statistically significant compared to control and to treatment with single therapy. Notably, none of the single therapies resulted in a significant increase in the expression of any of the genes in question.

[0296] The combination of MMP-408 (EB-103) and DCP-LA (EB-102) resulted in a 2-fold increase in elastin expression in the aorta of mice treated for 12 weeks. Simultaneously, MAP2K1 and fibrin-5 both increased 2-fold, and fibrin-5 increased 8-fold. Both are markers of elastin synthesis. Neutrophil elastase and macrophage elastase (MMP-12) each increased 4-fold. This is presumably a compensatory mechanism for elastase inhibition. All of these increases were statistically significant compared to control and to treatment with single therapy, except for MAP2K1. Notably, none of the single therapies resulted in a significant increase in the expression of any of the genes in question.

[0297] The combination of MMP-408 (EB-103) + DCP-LA Alvelestat (EB-101) + DCP-LA (EB-102) resulted in a 2-fold increase in elastin expression in the aorta of mice treated for 12 weeks. Simultaneously, MAP2K1 and fibrin-5 both increased 2-fold, and fibrin-5 increased 8-fold. Both are markers of elastin synthesis. Neutrophil elastase and macrophage elastase (MMP-12) each increased 4-fold. This is presumably a compensatory mechanism for elastase inhibition. All of these increases were statistically significant compared to control and compared to treatment with single therapy, except for MAP2K1. Notably, none of the single therapies resulted in a significant increase in the expression of any of the genes in question.

[0298] The combination of U0126 (EB-104) and DCP-LA (EB-102) resulted in a 3-fold increase in elastin expression in the aorta of mice treated for 12 weeks. Simultaneously, MAP2K1 and fibrin-5 both increased 2-fold, and fibrin-5 increased 6-fold. Both are markers of elastin synthesis. Neutrophil elastase and macrophage elastase (MMP-12) increased 2-fold and 3-fold, respectively. All of these increases were statistically significant compared to control and compared to treatment with single therapy, except for MAP2K1. Notably, none of the single therapies resulted in a significant increase in the expression of any of the genes in question.

[0299] Changes in the expression of inflammatory cytokine genes Figure 14 (ac)- Figure 17 (ac) shows the effects of single treatments of EB-101, EB-102, EB-103 and EB-104, and specifically their combinations (compared to control), on the inflammatory markers TNFα, IL-1β and IL-6.

[0300] These data show that the Alvelestat (EB-101) + DCP-LA (EB-102) combination led to an increase in TNF expression, but this was not statistically significant, while IL-6 was unaffected by any treatment. Alvelestat (EB-101) monotherapy increased IL-1β tenfold.

[0301] The combination of MMP-408 (EB-103) and DCP-LA (EB-102) did not affect TNF. Similar to EB-101 + EB-102, MMP-408 monotherapy increased IL-1β by 3-fold. Interestingly, the combination therapy reduced IL-6 by 60%. This is consistent with reduced elastin degradation.

[0302] The combination of U0126 (EB-104) and DCP-LA (EB-102) resulted in a 0.5-fold increase in TNF expression, but this was not statistically significant, while IL-6 was unaffected by any treatment. Alvelestat (EB-101) monotherapy resulted in a 2-fold increase in IL-1β.

[0303] Changes in gene expression of genes involved in Williams syndrome Figure 18 (af)- Figure 20 (af) shows the effects of single treatments of EB-101, EB-102, EB-103 and EB-104, and specifically their combinations (compared to control), on the expression of genes involved in Williams syndrome (dense protein-4, BCL7B, NCF-1, dense protein-3, GTF2I, RFC2) in aortic tissue samples.

[0304] Compared to a 0.8-fold increase with EB-101 alone, Alvelestat (EB-101) + DCP-LA (EB-102) treatment resulted in a 1.5-fold increase in dentin 4 expression. Monotherapy and dual therapy resulted in a decrease in dentin 3 expression, with EB-101 reducing it by 60%, and EB-102 + EB-101 and EB-102 as monotherapy reducing it by 25%. EB-102 treatment reduced BCL7B by 60%, while EB-101 alone increased it by 1.5-fold. Dual treatment had no effect. EB-101 increased GTF2I expression by 2-fold. Dual therapy with EB-102 + EB-101 and EB-102 monotherapy reduced RFC2 by 50%. NCF-1 was unaffected by any treatment.

[0305] The combination of MMP-408 (EB-103) and DCP-LA (EB-102) increased the expression of filamentin-3 and filamentin-4 by 4-fold, while increasing BCL7B expression by 1-fold. All treatments reduced RFC2 expression by 50%. MMP-408 (EB-103) + DCP-LA (EB-102) reduced NCF-1 expression by 50%. EB-103 monotherapy increased BCL7B expression by 1-fold.

[0306] The U0126 (EB-104) + DCP-LA (EB-102) combination reduced micrin-3 expression by 70% and increased micrin-4 expression by 1.5-fold. Dual therapy increased BCL7B expression by 2.5-fold, EB-104 monotherapy increased it by 1-fold, and EB-102 monotherapy reduced it by 70%. Both monotherapy treatments reduced RFC2 by 50%, while the U0126 (EB-104) + DCP-LA (EB-102) dual therapy showed a slight reduction—20%. EB-104 monotherapy increased GTF2I by 1-fold, while EB-102 monotherapy and U0126 (EB-104) + DCP-LA (EB-102) reduced its expression by 50%. U0126 (EB-104) + DCP-LA (EB-102) increased NCF-1 by 0.5-fold, while both monotherapy treatments showed a moderate reduction—25%.

[0307] The genes in the brain that are affected in Williams syndrome.

[0308] Figure 21 (ak)- Figure 23(ak) shows the effects of single treatments of EB-101, EB-102, EB-103 and EB-104, and specifically their combinations (compared to control), on the expression of genes involved in Williams syndrome in the brain (STX1A, FK-506, F2ZD9, GALNT17, BAZB1, HIP-1, bHLH, EIF-4H and GTF2I) and on inflammatory markers (IL-1 and IL-6).

[0309] Dual treatment with EB-101 + EB-102 and single treatment with EB-101 increased BAZB1 by 0.5-fold. Dual treatment also increased HIP-1 by 1.5-fold, while EB-101 increased it by 5-fold, and EB-102 increased it by 2-fold. EB-101 also increased F2ZD9 expression by 1-fold. Dual treatment upregulated bHLH by 0.5-fold, while single treatment with EB-101 upregulated it by 1.5-fold. STX1A, GALNT17, GTF2I, IL-1, and IL-6 were unaffected by treatment.

[0310] Dual treatment with EB-103 and EB-102 significantly increased F2ZD9 by 1x, HIP-1 by 4x, EIF-4H by 1x, bHLH by 3x, FK-506 by 4x, and BAZ1B by 25%. EB-103 alone increased STX1A by 0.5x, HIP-1 by 5x, EIF-4H by 2x, and bHLH by 1x.

[0311] EB-104 + EB-102 and EB-104 monotherapy showed similar results, significantly increasing BAZ1B by 30%, HIP-1 by 6-fold, EIF-4H by 1-fold, F2ZD9 by 1-fold, and FK-506 by 3-fold, while dual therapy increased GALNT17 by 40%.

[0312] Behavior Figure 24 (af)- Figure 26 (af) shows the effects of single treatments of EB-101, EB-102, EB-103, and EB-104, and specifically their combinations (compared to control), on behavioral parameters (distance traveled, average speed, time at rest, time at activity, time with hind legs upright, and time in the central region).

[0313] In the open field test, behaviors indicative of activity and anxiety were measured. All treatments increased walking distance, with EB-101 alone showing the greatest increase, while EB-102 alone and the dual treatment showed similar walking distances. This result was similar to and consistent with the measurement of average speed. This was consistent with the results on immobility time, with mice treated with EB-101 alone showing the shortest immobility time. Anxiety measures, such as hind leg uprighting and time spent in the central region, also showed similar results. Mice treated with the dual treatment showed the longest time in the central region, indicating more exploratory behavior and less anxiety, with a slightly lower increase in hind leg uprighting compared to EB-101 alone, but still higher than EB-102 alone.

[0314] Mice in the EB-103 + EB-102 cohort showed the least variation across all measures. Compared to controls, all mice treated with single or dual methods exhibited increased hind-leg uprighting behavior.

[0315] Mice treated with EB-104 exhibited the longest walking distance and longest activity time. Hind-leg uprighting behavior was increased in all treatment groups. Interestingly, both the EB-104 treatment group and the EB-102 + EB-104 treatment group showed reduced time spent in the central region.

[0316] Real-time myocardial recording Figures 27-29 The study showed the effects of single treatments with EB-101, EB-102, EB-103, and EB-104, as well as their combinations (compared to control), on the intensity of arterial contractility after acetylcholine treatment. Figure 30 The image shows a comparison of arterial contractility intensity in aged mice and young mice (without any treatment according to the invention) after treatment with acetylcholine.

[0317] Figures 30-32 The study showed the effects of single treatments with EB-101, EB-102, EB-103, and EB-104, and specifically their combinations (compared to control), on arterial systolic strength after phenylephrine treatment. Figure 33 The image shows a comparison of arterial contractility intensity in aged mice and young mice (not treated according to the invention) after phenylephrine treatment.

[0318] In response to phenylephrine, aortas isolated from mice treated with EB-101 + EB-102 showed significantly higher contractile strength. This increase was significantly higher than in either of the single-treatment groups. Interestingly, after acetylcholine treatment to relax the arteries, EB-101 + EB-102 maintained the highest tensile strength, while the EB-101-treated group showed lower tensile strength than the control.

[0319] Interestingly, after stimulation with phenylephrine, the aortas isolated from mice treated with EB-103 + EB-102 showed significantly higher contractile strength compared to the two single-treatment groups. After relaxation with acetylcholine, EB-103 + EB-102 maintained the highest tensile strength, while the EB-102-treated group showed the lowest tensile strength.

[0320] The EB-104 + EB-102 processing group showed similar results to the EB-103 + EB-102 processing group.

[0321] Compared to 6-week-old young mice, the tensile strength of the aorta in mice treated with all three combinations was similar to or better than that in aortas extracted from young mice. The results after relaxation were identical for all mice except for EB-103 + EB-102.

[0322] Metabolomics Figure 37 (ak) shows the effects of single treatments of EB-101, EB-102, EB-103 and EB-104, and specifically their combinations (compared to control), on selected metabolomics parameters ((a) tryptophan; (b) histidine; (c) phenylalanine; (d) tryptophan; (e) glutamine; (f) glutamate; (g) creatine; (h) lysine; (i) fumarate; (j) citric acid and (k) succinate.

[0323] Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to assess plasma metabolomic differences between mice receiving a saline control solution and those receiving single or combined elastin therapy. OPLS-DA score plots showed significant differences between pairs of interest, indicating that treatment with small elastin-modified molecules produced different metabolic profiles. Univariate analysis of metabolites driving this difference between the treatment and control groups was performed on the metabolite bin with a VIP score of 1.5 or higher. Generally, the treatment group showed increased plasma glutamine, glutamate, lysine, succinate, citrate, creatine, and valine (not shown), while plasma phenylalanine, histidine, tryptophan, fumarate, and acetic acid (not shown) were decreased in the treatment group. Significant differences existed between the single and combined groups, such as a more dramatic increase in plasma glutamine and citrate after treatment with EB-102-103 compared to treatment with EB-102 or 103 alone; however, the biological significance of these changes will be determined in future studies using disease models or, in fact, in determining metabolite levels in young mice.

[0324] discuss This invention demonstrates for the first time that PKCε activators exhibit a synergistic effect when used in combination with neutrophil elastase inhibitors, MMP-12 inhibitors, or MEK1 / 2 inhibitors. At the biological level, 12-month-old aged mice receiving the drug combination for 12 weeks showed good tolerance with no side effects. The mice exhibited remarkable longevity and restored coat color. Most importantly, the aorta isolated from these mice showed a several-fold increase in elastin expression, along with several-fold increases in markers of elastin synthesis. This increase was well complemented by increased aortic tensile strength following stress-strain experiments, as well as increased contractile strength upon norepinephrine stimulation. Upon relaxation with acetylcholine, the aorta maintained its tension without becoming excessively relaxed or loose. Interestingly, these results were comparable to those in 6-week-old young mice, which were 15-16 months old at the end of the experiment. The results of mice receiving the combination therapy were consistently superior to those receiving any single therapy within the combination. In observing mouse behavior, the mice showed superior performance in an open field compared to controls. Although the behavioral outcomes of dual treatment at each measurement are not always superior, the combination therapy showed better overall outcomes in mice when considering the results for the aorta, the effects on fur, and the RNA-Seq and metabolomics results. RNA-Seq results showed significant effects on multiple pathways, ranging from neurodegenerative disorders to diabetic neuropathy, autophagy, and oxidative phosphorylation. Taken together, all results indicate that the restoration of elastin in the extracellular matrix has a significant impact not only on the aorta, a major elastin expresser, but also on the entire organism.

[0325] According to the present invention, in some embodiments, targeting multiple pathways can provide a synergistic effect, thereby opening up possibilities for treatment with high / increased efficacy and low / reduced side effects.

[0326] Although the invention has been specifically described, those skilled in the art will understand that various changes and modifications can be made. Therefore, the invention should not be construed as limited to the specifically described embodiments, and the scope and concept of the invention will be more readily understood by referring to the appended claims.

Claims

1. A pharmaceutical composition comprising a first compound selected from one group of the following groups (I), (II), (III) and (IV) and a second compound selected from another group of said groups (I), (II), (III) and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors.

2. The pharmaceutical composition according to claim 1, wherein: Group (I) consists of protein kinase Cε type (PKCε) activators; Group (II) consists of inhibitors of mitogen-activated protein kinase 1 (MAP2K1); Group (III) consists of neutrophil elastase inhibitors; and / or Group (IV) consisted of MMP-12 inhibitors.

3. The pharmaceutical composition according to claim 1 or 2, comprising a first compound selected from group (I) and a second compound selected from any one of groups (II), (III) and (IV).

4. The pharmaceutical composition according to claim 1 or 2, comprising a first compound selected from one group (i), (ii), (iii) and (iv) and a second compound selected from another group (i), (ii), (iii) and (iv): (i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and its salts and esters; (ii) (U0126), and its salts; (iii) (Alvelestat), and its salts; and (iv) (MMP-408), and its salts and esters.

5. The pharmaceutical composition according to any one of the preceding claims, comprising 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) or a salt or ester thereof. Or a combination of its salts.

6. A pharmaceutical composition, in a method of treating a subject in need, wherein the pharmaceutical composition comprises a first compound selected from any one of the following groups (I), (II), (III) and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors; The method further includes applying a second compound selected from another group of groups (I), (II), (III), and (IV). The method described therein is: - Methods for treating or preventing elastin deficiency-related diseases in individuals who require them; - Methods for treating and / or preventing conditions and / or diseases associated with the aging process in individuals who require them; and / or - Methods to promote longevity among those who need it.

7. The pharmaceutical composition for use according to claim 6, wherein: Group (I) consists of protein kinase Cε type (PKCε) activators; Group (II) consists of inhibitors of mitogen-activated protein kinase 1 (MAP2K1); Group (III) consists of neutrophil elastase inhibitors; and / or Group (IV) consisted of MMP-12 inhibitors.

8. The pharmaceutical composition for use according to claim 6 or 7, wherein the pharmaceutical composition comprises a first compound selected from group (I), and wherein the method further comprises administering a second compound selected from any one of groups (II), (III), and (IV).

9. A pharmaceutical composition for use according to any one of claims 6-8, wherein the pharmaceutical composition comprises compounds selected from any one of the following groups (i), (ii), (iii) and (iv): (i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and its salts; (ii) (U0126), and its salt; (iii) (Alvelestat), and its salts; and (iv) (MMP-408), and its salts and esters.

10. The pharmaceutical composition for use according to claim 9, wherein the method further comprises administering a second compound selected from another group of groups (i), (ii), (iii) and (iv).

11. The pharmaceutical composition for use according to claim 10, comprising a first compound selected from group (i), and the method further comprising administering a second compound selected from any one of groups (ii), (iii), and (iv).

12. A pharmaceutical composition for use according to any one of claims 6-11, comprising 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) or a salt or ester thereof; wherein the method further comprises administration Or its salt.

13. A pharmaceutical composition for use according to any one of claims 6-12, wherein the pharmaceutical composition comprises the first compound and the second compound.

14. The pharmaceutical composition for use according to any one of claims 6-13, wherein the condition or disease associated with the aging process is selected from neurodegenerative diseases, cardiovascular diseases, fragility diseases, musculoskeletal diseases, and skin diseases.

15. The pharmaceutical composition for use according to claim 14, wherein the condition or disease associated with the aging process is a neurodegenerative disease selected from Alzheimer's disease, Parkinson's disease, and dementia.

16. The pharmaceutical composition for use according to claim 14, wherein the condition or disease associated with the aging process is a cardiovascular disease selected from coronary artery disease, atherosclerosis, and pulmonary hypertension.

17. The pharmaceutical composition for use according to claim 14, wherein the condition or disease associated with the aging process is a frail disease selected from frailty, osteoporosis, and sarcopenia.

18. The pharmaceutical composition for use according to claim 14, wherein the condition or disease associated with the aging process is selected from skin diseases such as impaired wound healing, alopecia, wrinkles, age spots, thinning of the skin, xeroderma pigmentosum, and congenital dyskeratosis.

19. A method of treating a subject in need, the method comprising administering to the subject at least one compound selected from any one of the groups (I), (II), (III), and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors; And the method further includes applying a second compound selected from another group of groups (I), (II), (III) and (IV), The method described therein is: - Methods for treating or preventing elastin deficiency-related diseases in individuals who require them; - Methods for treating and / or preventing conditions and / or diseases associated with the aging process in individuals who require them; and / or - Methods to promote longevity among those who need it.

20. Use of at least one compound selected from any one of groups (I), (II), (III) and (IV) in the preparation of a medicament for use in a method of treating a subject in need: (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors; And the method further includes applying a second compound selected from another group of groups (I), (II), (III) and (IV), The method described therein is: - Methods for treating or preventing elastin deficiency-related diseases in individuals who require them; - Methods for treating and / or preventing conditions and / or diseases associated with the aging process in individuals who require them; and / or - Methods to promote longevity among those who need it.

21. A non-therapeutic or cosmetic method of treating a subject, the method comprising applying to the subject at least one compound selected from any one of the groups (I), (II), (III), and (IV): (I) Compounds that can increase the expression of elastin; (II) Compounds that can increase elastin fiber deposition; (III) Compounds that can reduce or inhibit the breakdown or degradation of elastin fibers; and (IV) Compounds that can reduce or inhibit the production of elasticity factors; And the method further includes applying a second compound selected from another group of groups (I), (II), (III) and (IV). The methods described therein are non-therapeutic or cosmetic methods.

22. The non-therapeutic or cosmetic method of claim 20, wherein the method is for improving skin appearance, for preventing visible signs of aging of the skin, for preventing and / or reducing wrinkles, for improving and / or maintaining skin smoothness, for improving and / or maintaining skin firmness, for improving and / or maintaining skin elasticity, for revitalizing the skin, and / or for maintaining youthful skin.

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