Pharmaceutical compositions and use thereof in treatment of conditions and diseases related to elastin deficiency and / or aging

Abstract

Pharmaceutical composition and use of same in treatment or prevention of an elastin deficiency related disease in a subject in need thereof are provided.

Description

PHARMACEUTICAL COMPOSITIONS AND USE THEREOF IN TREATMENT OF CONDITIONS AND DISEASES RELATED TO ELASTIN DEFICIENCY AND / OR AGINGFIELD OF INVENTION

[0001] The present invention is in the field of conditions and diseases related to elastin deficiency and aging.BACKGROUND

[0002] Elastin is a key extracellular matrix (ECM) protein that provides resilience and elasticity to tissues and organs. It’s a protein that can stretch out (extend) and shrink back (recoil). Elastin is a major component of tissues in the body that require stretchiness, like the lungs, bladder, large blood vessels and some ligaments. Smaller amounts exist in the skin and ear cartilage. Elastin’s main function is to allow tissues in the body to stretch out and shrink back. For examples, elastin gives arteries stretchy characteristics that make it easier for the heart to pump blood. Elastin also allows lungs to work like an elastic bag.

[0003] Elastin is produced and secreted as a soluble monomer tropoelastin (TE). During ontogenetic development, the soluble monomer TE is secreted by cells, such as smooth muscle cells, fibroblasts, and endothelial cells, and assembled into highly stable, insoluble, polymeric elastin fibers in the ECM by the enzyme lysyl oxidase. The process of generating the elastin fibers is often referred to as elastinogenesis.

[0004] TE expression is largely restricted to the fetal phase and the early postnatal years. From adolescence onward, elastin synthesis decreases and ceases in adults. The half-life of elastin is approximately 74 years, which makes it one of the most stable proteins known. Although elastin is a highly resilient component of the ECM, degeneration of elastic fiber, a process often referred to as elastolysis, occurs with age and under certain pathophysiological conditions and / or as a consequence of injury. Elastolysis, can lead to loss of tissue elasticity, flexibility and thus, integrity and functionality, and also results in the release of elastin-derived peptides (EDPs). Some of these EDPs, known as elastokines, have been shown to have bioactive properties through interaction with their cognate receptor called elastin receptor complex, which are generally considered to play a role in the development of (further) pathology.

[0005] In the normal aging process, the gradual loss of elastin leads to the deterioration of tissues, causing organ damage and inflammation. This inflammation accelerates elastin degradation, creating a vicious cycle, contributing to (certain) age-related diseases. For instance, the loss ofelastin in blood vessels can result in conditions like AAA, where vessel walls weaken and bulge, posing significant health risks, cardiovascular diseases. A growing body of evidence also suggests that the EDPs released in elastolysis, through their own bioactive properties, play a role in the development of (other) age-related diseases and conditions. Recently, for instance, elastinlike polypeptides have been reported to induce overproduction of beta-amyloid in a model of Alzheimer's disease.

[0006] Abnormalities in elastinogenesis and / or elastolysis can occur, and cause pathology, earlier in life, e.g. as a consequence of hereditary defects as well as acquired disease. Well-known examples of (hereditary) elastin deficiency related disease include Williams Bueren syndrome and cutis laxa. Williams-Beuren syndrome (WBS), is a genetic disorder that affects many parts of the body and is caused by haploinsufficiency of the ELN date, as a consequence of the deletion of about 27 genes from the long arm of one of the two chromosome 7s, where the ELN gene is also located. Cutis laxa is a rare connective tissue disorder, caused by mutations in the ELN gene, in which the skin becomes inelastic and hangs loosely in folds. Acquired forms of cutis laxa are also known to occur.

[0007] Considering that the degradation of elastin, linked with a variety of health issues as well as the natural aging process, has long been on the radar of medical experts, the relative lack of success, to date, in developing interventional therapies is striking. There is a compelling need for new pharmacological approaches in the treatment of elastin-related diseases. An objective of the present invention is to meet that need.SUMMARY

[0008] The present invention, generally speaking, is predicated on the finding that certain small molecules and small molecule combinations, are highly efficaceous in reducing elastin loss and / or inducing or promoting recovery of elastin deposition. The findings, as presented herein, thus provide strong support for the (clinical) utility of these small molecules, and their combinations, in (a.o.) the treatment of elastin deficiency related disease as well as in treatments aimed at slowing down certain aspects of the aging process, thereby promoting health and well being of the aged.

[0009] More in particular, aged mice, which were twice a week subcutaneously treated with 1 mg / kg DCP-LA, 1 mg / kg DCP-LA + 3 mg / kg ASH 1793 (MMP-408), 1 mg / kg DCP-LA + 3 mg / kg alvelestat or 1 mg / kg DCP-LA + 5 mg / kg U0126 for a period of 12 weeks, show an increase in elastin expression and in , correlating with improvements in cognitive / neurological functioning and in the (visual) appearance of the skin and coat, as compared to control groups.Particularly good results were obtained with the combination of DCP-LA and MMP-408 and with the combination of DCP-LA and alvelestat. These results support the use of these compounds, and analogues thereof, especially combinations of such compounds, in the treatment and / or prevention of conditions and / or diseases related to the aging process and the promotion of longevity.

[0010] Hence, according to a first aspect, there is provided a pharmaceutical composition comprising a first compound that is selected from one of the following groups (I), (II), (III) and (IV) and, preferably, a second compound that is selected from another one of said groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production.[Oi l] In a particularly preferred embodiment, the pharmaceutical composition comprises a first compound that is selected from said group (I), and a second compound that is selected from any one of said groups (II), (III) and (IV).

[0012] Also provided is the pharmaceutical composition comprising a first compound selected from one of the following groups (i), (ii), (iii) and (iv) and a second compound selected another one of said groups (i), (ii), (iii) and (iv):(i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and salts, esters and stereoisomers thereof;s thereof; andsalts, esters, and stereoisomers thereof.

[0013] In a particularly preferred embodiment, the pharmaceutical composition comprises a first compound that is selected from said group (i), and a second compound that is selected from any one of said groups (ii), (iii) and (iv).

[0014] According to another aspect, there is provided a method of treating a subject in need thereof, said method comprising the administration to said subject of at least one compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production.

[0015] According to a preferred embodiment, the method comprises the administration to said subject of a first compound that is selected from one of said groups (I), (II), (III) and (IV), and a second compound that is selected from another one of said groups (I), (II), (III) and (IV).

[0016] According to a more preferred embodiment, the method comprises the administration to said subject of a first compound selected from said group (I) and a second compound that is selected from any one of said groups (II), (III) and (IV).

[0017] Also provided is a method of treating a subject in need thereof, said method comprising the administration to said subject of a compound selected from any of the following groups (i), (ii), (iii) and (iv):(i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and salts, esters, and stereoisomers thereof;(U0126), and salts and stereoisomers thereof;

[0018] According to a preferred embodiment, the method comprises the administration to said subject of a first compound that is selected from one of said groups (i), (ii), (iii) and (iv), and a second compound that is selected from another one of said groups (i), (ii), (iii) and (iv).

[0019] According to a more preferred embodiment, the method comprises the administration to said subject of a first compound selected from said group (i) and a second compound that is selected from any one of said groups (ii), (iii) and (iv).

[0020] According to another aspect, there is provided a pharmaceutical composition for use in a method of treating a subject in need thereof, said pharmaceutical composition comprising at least one compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production.

[0021] According to a preferred embodiment, said pharmaceutical composition for use in accordance with the invention comprises a first compound that is selected from one of said groups (I), (II), (III) and (IV) and the method further comprises the administration of a second compound that is selected from another one of said groups (I), (II), (III) and (IV).

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

[0023] Also provided is a pharmaceutical composition for use in a method of treating a subject in need thereof, said pharmaceutical composition comprising a compound selected from any of the following groups (i), (ii), (iii) and (iv):(i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and salts, esters, and stereoisomers thereof;(ii) (U0126) and salts and stereoisomers thereof;

[0024] According to a preferred embodiment, said pharmaceutical composition for use in accordance with the invention comprises a first compound that is selected from one of said groups (i), (ii), (iii) and (iv), and the method further comprises the administration of a second compound that is selected from another one of said groups (i), (ii), (iii) and (iv).

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

[0026] According to another aspect, there is provided the use of at least one compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production; in the manufacture of a medicament for use in a method of treating a subject in need thereof.

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

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

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

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

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

[0032] According to another aspect, there is provided a a pharmaceutical kit comprising a package containing a plurality of unit dosage forms, said unit dosage forms comprising a first compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokine production; wherein said pharmaceutical kit further comprises a leaflet containing printed instructions to (self) administer said unit dosage forms in order to accomplish any of the therapeutic objectives as defined herein, such as to treat and / or prevent a condition and / or disease related to the aging process and to promote longevity.

[0033] According to another aspect, there is provided a 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 making up said first plurality comprise a first compound that is selected from one of said groups (I), (II), (III) and (IV), and the unit dosage forms making up said second plurality comprise a second compound that is selected from another one of said groups (I), (II), (III) and (IV), wherein said pharmaceutical kit optionally further comprises aleaflet containing printed instructions to (self) administer the unit dosage forms contained in the kit, in order to accomplish any of the therapeutic objectives as defined herein, such as to treat and / or prevent a condition and / or disease related to the aging process and to promote longevity. According to a more preferred embodiment, there is provided the pharmaceutical kit wherein the first compound is selected from said group (I) and the second compound is selected from any one of said groups (II), (III) and (IV).

[0034] As will be understood by those skilled in the art, the aspects as defined here above all concern the same invention. Consequently, whenever definitions, explanations, embodiments, etc. are described herein in the context of one such aspect, these are equally applicable to said other aspects, unless something else is indicated and / or this would go against common sense. The various aspects of the invention as described above will be explained and illustrated in more detail in the following detailed description and examples.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figures 1A-1E include vertical bar graphs showing the effect of DCP-LA, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 on gene expression levels of Elastin (1A), Neutrophil elastase (NE; IB), MAP2K1 (1C), MMP-12 (ID), and Fibulin (IE).

[0036] Figures 2A-2D are graphs showing the effect 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 presents a further analysis (multiple comparison tests) of these results.

[0037] Figures 3A-3C include vertical bar graphs showing the effect of DCP-LA, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 treatment on NAD+ (3 A), NADP+ (3B) and glutathione (GSH)(3C) concentration in blood (pm) as measured using optimized cyclic enzymatic assays with colorimetric detection.

[0038] Figures 4A and Figure 4B include vertical bar graphs showing the effect of DCP-LA, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 treatment on Lysine and Valine concentration in plasma as represented by NMR-measured relative spectral intensities (AU).

[0039] Figures 5A-5D include vertical bar graphs showing the effect of DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 treatment on mice rearing activity as measured by the Open Field Test.

[0040] Figures 6A-6D include vertical bar graphs showing the effect of DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 treatment on mice active time as measured by the Open Field Test.

[0041] Figures 7A-7D include vertical bar graphs showing the effect of DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 treatment on time spent by mice in the central zone as measured by the Open Field Test.

[0042] Figures 8A-8D include vertical bar graphs showing the effect of DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA and Alvelestat, DCP-LA and MMP408, or DCP-LA and U0126 treatment on mice immobile time as measured by the Open Field Test.

[0043] Figure 9 graphic representation of the experimental set-up (treatment schemes).

[0044] Figure lOa-lOe shows the effects of the EB-101+EB-102 combination (vs. controls and mono-treatments) on elastin pathway gene expression (elastin (a), neutrophil elastase (b), MMP- 12 (c), MAP2K1 (d) and Fibulin-5 (e)).

[0045] Figure lla-lle shows the effects of the EB-102+EB-103 combination (vs. controls and mono-treatments) on elastin pathway gene expression (elastin (a), MAP2K1 (b), neutrophil elastase (c), MMP-12(d) and Fibulin-5 (e)).

[0046] Figure 12a-12e shows the effects of the EB-102+EB-104 combination (vs. controls and mono-treatments) on elastin pathway gene expression (elastin (a), MAP2K1 (b), neutrophil elastase (c), MMP-12(d) and Fibulin-5 (e)).

[0047] Figure 13a-13e shows the effects of all tested combinations (vs. control and all monotreatments) on elastin pathway gene expression (elastin (a), Fibulin-5 (b), MMP-12 (c), MAP2K1 (d) and neutrophil elastase (e).

[0048] Figure 14a-14c shows the effects of the EB-101+EB-102 combination (vs. controls and mono-treatments) on inflammatory markers (TNFa (a), IL-ip (b), and IL-6 (c)).

[0049] Figure 15a-15c shows the effects of the EB-102+EB-103 combination (vs. controls and mono-treatments) on inflammatory markers (TNFa (a), IL-ip (b), and IL-6 (c)).

[0050] Figure 16a-16c shows the effects of the EB-102+EB-104 combination (vs. controls and mono-treatments) on inflammatory markers (TNFa (a), IL-ip (b), and IL-6 (c)).

[0051] Figure 17a-17c shows the effects of all tested combinations (vs. control and all monotreatments) on inflammatory markers (TNFa (a), IL-6 (b), and IL-ip (c)).

[0052] Figure 18a-18f shows the effects of the EB-101+EB-102 combination (vs. controls and mono-treatments) on the expression of genes involved in Williams syndrome (Claudin-4 (a), BCL7B (b), NCF-1 (c), Claudin-3 (d) GTF2I (e) and RFC2(f)) in aortic tissue samples.

[0053] Figure 19a-19f shows the effects of the EB-102+EB-103 combination (vs. controls and mono-treatments) on the expression of genes involved in Williams syndrome (Claudin-4 (a), BCL7B (b), Claudin-3 (c), NCF-1 (d), RFC2 (e) and GTF2I (f)) in aortic tissue samples.

[0054] Figure 20a-20f shows the effects of the EB-102+EB-104 combination (vs. controls and mono-treatments) on the expression of genes involved in Williams syndrome (Claudin-4 (a), BCL7B (b), NCF-1 (c), Claudin-3 (d) GTF2I (e) and RFC2(f)) in aortic tissue samples.

[0055] Figure 21a-21k shows the effects of the EB-101+EB-102 combination (vs. controls and mono-treatments) on inflammatory markers and expression of genes involved in Williams syndrome (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)) in the brain.

[0056] Figure 22a-22k shows the effects of the EB-102+EB-103 combination (vs. controls and mono-treatments) on inflammatory markers and expression of genes involved in Williams syndrome (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)) in the brain.

[0057] Figure 23a-23k shows the effects of the EB-102+EB-104 combination (vs. controls and mono-treatments) on inflammatory markers and expression of genes involved in Williams syndrome (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)) in the brain.

[0058] Figure 24a-24f shows the effects of the EB-101+EB-102 combination (vs. controls and mono-treatments) on behavioral parameters (distance travelled (a), mean speed (b), immobile time (c), active time (d) rearing (e), and time in center zone (f)).

[0059] Figure 25a-25f shows the effects of the EB-102+EB-103 combination (vs. controls and mono-treatments) on behavioral parameters (distance travelled (a), mean speed (b), immobile time (c), active time (d) time in center zone (e) and rearing (f)).

[0060] Figure 26a-26f shows the effects of the EB-102+EB-104 combination (vs. controls and mono-treatments) on behavioral parameters (distance travelled (a), mean speed (b), immobile time (c), active time (d) rearing (e), and time in center zone (f)).

[0061] Figure 27 shows the effects of the EB-101+EB-102 combination (vs. controls and monotreatments) on arterial tensile strength upon relaxation by acetylcholine treatment.

[0062] Figure 28 shows the effects of the EB-102+EB-103 combination (vs. controls and monotreatments) on arterial tensile strength upon relaxation by acetylcholine treatment.

[0063] Figure 29 shows the effects of the EB-102+EB-104 combination (vs. controls and monotreatments) on arterial tensile strength upon relaxation by acetylcholine treatment.

[0064] Figure 30 shows the arterial tensile strength upon relaxation by acetylcholine treatment in old vs. young mice (untreated).

[0065] Figure 31 shows the effects of the EB-101+EB-102 combination (vs. controls and monotreatments) on arterial tensile strength upon phenylephrine treatment.

[0066] Figure 32 shows the effects of the EB-102+EB-103 combination (vs. controls and monotreatments) on arterial tensile strength upon phenylephrine treatment.

[0067]

[0068] Figure 33 shows the effects of the EB-102+EB-104 combination (vs. controls and monotreatments) on arterial tensile strength upon phenylephrine treatment.

[0069] Figure 34 shows the arterial tensile strength upon phenylephrine treatment in old vs. young mice (untreated).

[0070] Figure 35 shows a heatmap visualizing the results of mRNA sequencing analysis of brain samples.

[0071] Figure 36 shows a heatmap visualizing the results of mRNA sequencing analysis of aortic samples.

[0072] Figure 37a-37k shows the effects of all tested combinations (vs. control and all monotreatments) on selected metabolome components ((a)tryptophan; (b) histidine; (c) phenylalanine; (d) tryptophan; (e) glutamine; (f) glutamate; (g) creatine; (h) lysine; (i) fumarate; (j) citrate and (k) succinate).DETAILED DESCRIPTIONCompounds

[0073] As will be apparent to those skilled in the art, all aspects of the present invention entail the presence / use of one or more compounds that belong to the groups referred to as groups (I), (II), (III) and (IV) and / or groups (i), (ii), (iii) and (iv).

[0074] As indicated herein before, group (I) consists of compounds that are capable of increasing tropoelastin expression. In one sense a compound is considered capable of increasing tropoelastin expression if its administration results in an increase in tropoelastin mRNA or tropoelastin protein expression levels. In preferred embodiments, group (I) consists of compounds that are activators of protein kinase C epsilon type (PKCs) activators and / or compounds capable of activating PKCs, e.g. in suitable assay, such as the ones described herein elsewhere. Hence, in accordance with preferred embodiments of the invention, group (I) preferably consists of PKCs activators and / or compounds capable of activating PKCs In one sense, a compound is considered capable of activating PKCs if it enhances expression and / or activity of PKCs, e.g. in suitable assay, such as the ones described herein elsewhere. The PKCs activator may be a non-specific or specific activator. A specific activator activates PKCs to a greater detectable extent than other PKC isoforms.

[0075] A specific class of PKCs activators, to which DCP-LA belongs and which are (thus) considered to be of particular utility in the context of the present invention, are described in WO 2010 / 014585, the contents of which are incorporated herein by reference. According to WO 2010 / 014585, some PUFAs, such as arachidonic acid, and Docosahexaenoic acid (DHA) have been known for many years to be natural activators of PKC. WO 2010 / 014585 provides certain derivatives of polyunsaturated fatty acids (PUFA) or monounsaturated fatty acids (MUFA) that activate PKCs at nanomolar concentrations, and teaches to use them for the treatment of AD, stroke, and other neurological diseases in which PKCs is neuroprotective.

[0076] In preferred embodiments said PKCs activators are selected from the group of compounds that are a c / .s-polyunsaturated fatty acid, a c / .s-polyunsaturated fatty acid ester or a cis- polyunsaturated fatty alcohol in which at least one of the double bonds is replaced by a cyclopropane ring / group, preferably wherein all of the double bonds are replaced by a cyclopropane; compounds that are a cv.s-monounsaturated fatty acid, a c / .s-m on oun saturated fatty acid ester, or a c / .s-m on oun saturated fatty alcohol in which the double bond is replaced by a cyclopropane ring / group; and compounds that are a c / .s-polyunsaturated fatty acid, a cis- polyunsaturated fatty acid ester or a c / .s-polyunsaturated fatty alcohol, in which at least one of the double bonds is replaced with an epoxyl group. In preferred embodiments the fatty acid that forms or is part of the basic structure for said PKCs activator, has the structure CH3(CH2)4(CH=CHCH2)x(CH2)yCOOH or CH3CH2(CH=CHCH2)x(CH2)yCOOH wherein x is between 2 and 6, and y is between 2 and 6 or the structure CH3(CH2)xCH=CH(CH2)yCOOH wherein X and Y are odd numbers between 3 and 11. In preferred embodiments said fatty acid is arachidonic acid, docosapentaenoic acid, docosahexaenoic acid, eicosapentaenoic acid, linoleic acid, linolenic acid, y- linoleic acid, a-linolenic acid, eicosatetraenoic, adrenic acid, oleic acid, elaidic acid, elaidic alcohol, oleyl alcohol, myristoleic acid, palmitoleic acid, vernolic acid, and 1-monolinoleyl rac-glycerol. In particularly preferred embodiments said fatty acid is linoleic acid.

[0077] In preferred embodiments said PKCs activators are selected from the group of cyclopropanated docosahexaenoic acid methyl ester, eicosapentaenoic acid methyl ester, cyclopropanated arachidonic acid methyl ester, linoleic alcohol dicyclopropane, linolenyl alcohol tricyclopropane, elaidic alcohol cyclopropane, elaidic acid cyclopropane, oleyl alcohol cyclopropane and vernolic acid methyl ester cyclopropane.

[0078] In particularly preferred embodiments, said PKCs activators are selected from (i) 8-[2- (2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) and salts, esters, and stereoisomers thereof.

[0079] In a most preferred embodiment, the PKCs activator is 8-[2-(2-pentyl- cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) or the methyl ester thereof (DCP -LAME), most preferably it is 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP- LA).

[0080] As indicated herein before, group (II) consists of compounds that are capable of increasing elastin fiber deposition. In one sense a compound is considered capable of increasing elastin fiber deposition if its administration results in an increase of elastin fiber deposits in the extracellular matrix. In preferred embodiments, group (II) consists of mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitors. In one sense, a compound is considered capable of inhibiting mitogen-activated protein kinase kinase 1 (MAP2K1) if it reduces mRNA expression of MAP2K1 and / or activity of the MAP2K1 -encoded protein.

[0081] A specific class of MAP2K1 inhibitors, which are considered to be of particular utility in the context of the present invention, are described in US2,779,780, the contents of which are incorporated herein by reference.

[0082] In preferred embodiments group (II) consists of elastase inhibitors selected from the group consisting of compounds of formula (Ila) and pharmaceutically acceptable salts thereof,wherein R is a radical of 1-10 carbon atoms selected from the class consisting of hydrocarbon, monohydroxyhydrocarbon, monoaminohydrocarbon, mononitrohydrocarbon and monohalohydrocarbon radicals; more preferably wherein R is a radical selected from phenyl, aminophenyl, especially o-aminophenyl, methyl, ethyl and hydroxy ethyl.

[0083] In preferred embodiments group (II) consists of MAP2K1 inhibitors selected from the group consisting of l,4-diamino-2,3-dicyano-l,4-bis(methylmercapto)-butadiene; 1,4-diamino- 2,3-dicyano-l,4-bis(ethylmercapto)-butadiene; l,4-diamino-2,3-dicyano-l,4- bis(phenylmercapto)-butadiene; l,4-diamino-2,3-dicyano-l,4-his(beta-hydroxyeth- ylmercapto)butadiene; 1,4-diamino- 2,3-dicyano-l,4-bis(o-aminophenylmercapto)butadiene;

[0084] l,4-diamino-2,3-dicyano-l,4-bis(naphthylmercapto)butadiene, l,4-diamino-2,3- dicyano-l,4-bis(cyclohexylmercapto)butadiene; l,4-diamino-2,3-dicyano-l,4-bis(alpha-amino- beta-naphthylmercapto)butadiene, l,4-diamino-2,3-dicyano-l,4-bis(alpha-hydroxy-beta- naphthylmercapto)butadiene, l,4-diamino-2,3-dicyano-l,4-bis( / ?-chlorophenylmercapto)butadiene, l,4-diamino-2,3-dicyano-l,4-bis( / ?-nitrophenylmercapto)butadiene, and 1,4-diamino- 2,3-dicyano-l,4-bis(2-chloroethylmercapto)butadiene and salts thereof.

[0085] In particularly preferred embodiments, the MAP2K1 inhibitor is selected from (ii)(l,4-diamino-2,3-dicyano-l,4-bis(o-aminophenylmercapto)butadiene) and salts and stereoisomer thereof.

[0086] In a most preferred embodiment, the MAP2K1 inhibitor is(l,4-diamino-2,3-dicyano-l,4-bis(o-aminophenylmercapto)butadiene).

[0087] As indicated herein before, group (III) consists of compounds that are capable of reducing or inhibiting elastin fiber breakdown or degradation. In one sense a compound is considered capable of inhibiting elastin fiber breakdown or degradation if its administration results in reduced levels of elastin fiber breakdown or degradation, e.g. in an e.g. in an established in vitro assay. In some embodiments, the compound selected from group (III) is an elastase inhibitor or a compound capable of inhibiting elastase. In preferred embodiments, said elastase is neutrophil elastase. Hence, in a preferred embodiment, group (III) consists of neutrophil elastase inhibitors. In one sense, a compound is considered capable of inhibiting (neutrophil) elastase if its administration results in reduced expression and / or activity of elastase. In a preferred embodiment the compound inhibits human neutrophil elastase activity in a Human Neutrophil Elastase Quenched-FRET Assay as described herein elsewhere.

[0088] A specific class of neutrophil elastase inhibitors, which are considered to be of particular utility in the context of the present invention, are described in WO 2005 / 026123, the contents of which are incorporated herein by reference.

[0089] In preferred embodiments group (III) consists of elastase inhibitors selected from the group consisting of compounds of formula (Illa) and pharmaceutically acceptable salts thereof,wherein Y represents CR3or N, preferably Y represents CR3; R1represents H or Cl -6 alkyl; R2represents phenyl or a five- or six-membered heteroaromatic ring containing 1 to 4 heteroatoms independently selected from O, S and N; said aromatic ring being optionally substituted by 1 to 3 substituents selected independently from OH, halogen, Cl -6 alkyl, Cl -6 alkoxy, NR58COR50, COOR51, COR52, CONR53R54and NR47R48; said alkyl being optionally further substituted by OH, Cl -6 alkoxy, CN or CO2R49; R47and R48independently represent H, Cl -6 alkyl or C2-6 alkanoyl; R3represents H or F; G1represents phenyl or a five-or six-membered heteroaromatic ring containing 1 to 3 heteroatoms independently selected from O, S and N, preferably G1represents phenyl; R5represents H, halogen, Cl -6 alkyl, CN, Cl -6 alkoxy, NO2, NR14R15, Cl -3 alkyl substituted by one or more F atoms or Cl -3 alkoxy substituted by one or more F atoms; R14and R15independently represent H or Cl -3 alkyl; said alkyl being 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 R5group is selected independently, preferably R5represents Cl, CH3, CN or CF3; R4represents H or Cl -6 alkyl; said alkyl being optionally further substituted by OH or Cl -6 alkoxy; or R4and L are joined together such that the group-NR4L represents a 5 to 7 membered azacyclic ring optionally incorporating one further heteroatom selected from O, S and NR16; L represents a bond, O, S(O)P, NR29or Cl -6 alkyl; said alkyl optionally incorporating a heteroatom selected from O, S and NR16; and said alkyl being optionally further substituted by OH or OMe; G2represents a monocyclic ring system selected from: i) phenyl or phenoxy, ii) a 5 or 6 membered heteroaromatic ring containing one to three heteroatoms independently selected from O, S and N, iii) a C3- 6 saturated or partially unsaturated cycloalkyl, or iv) a C4-7 saturated or partially unsaturated heterocyclic ring containing one or two heteroatoms independently selected from O, S(O)p and NR17and optionally further incorporating a carbonyl group; or G2represents a bicyclic ring system in which each of the two rings is independently selected from: i) phenyl, ii) a 5 or 6 membered heteroaromatic ring containing one to three heteroatoms independently selected fromO, S and N, iii) a C3-6 saturated or partially unsaturated cycloalkyl, or iv) a C4-7 saturated or partially unsaturated heterocyclic ring containing one or two heteroatoms independently selected from O, S(O)Pand NR17and optionally further incorporating a carbonyl group; and the two rings are either fused together, or are bonded directly together or are separated by a linker group selected from O, S(O)Por CH2, said monocyclic or bicyclic ring system being optionally further substituted by one to three substituents independently selected from CN, OH, Cl -6 alkyl, Cl -6 alkoxy, halogen, NR18R19, NO2, OSO2R38, CO2R20, C(=NH)NH2, C(O)NR21R22, C(S)NR23R24, SC(=NH)NH2, NR31C(=NH)NH2, S(O)2R25, SO2NR26R27, Cl-3 alkoxy substituted by one or more F atoms and Cl-3 alkyl substituted by SO2R39, NR56R57or by one or more F atoms; or when L does not represent an bond, G2may also represent H; at each occurrence, p, q, s and t independently represent an integer 0,1 or 2; R18and R19independently represent H, Cl -6 alkyl, formyl, C2-6 alkanoyl, S(O)tR32or SO2NR33R34; said alkyl group being optionally further substituted by halogen, CN, Cl-4 alkoxy or CONR41R42; R25represents H, Cl-6 alkyl or C3-6 cycloalkyl; said alkyl group being optionally further substituted by one or more substituents selected independently from OH, CN, CONR35R36, CO2R37, OCOR40, C3-6 cycloalkyl, a C4-7 saturated heterocyclic ring containing one or two heteroatoms independently selected from O, S(O)Pand NR43and phenyl or a 5 or 6 membered heteroaromatic ring containing one to three heteroatoms independently selected from O, S and N; said aromatic ring being optionally further substituted by one or more substituents selected independently from halogen, CN, Cl-4 alkyl, Cl-4 alkoxy, OH, CONR44R45, CO2R46, S(O)sR55and NHCOCH3; R32represents H, Cl-6 alkyl or C3-6 cycloalkyl; R16, R17, R20, R21, R22, R23, R24, R26, R27, R29, R31,R33, R34, R35, R36, R37, R38, R39, R40, R41, R42, R43, R44, R45, R46, R49, R50, R51, R52, R53, R54, R55, R56, R57and R58independently represent H or Cl-6 alkyl.

[0090] In preferred embodiments group (III) consists of elastase inhibitors selected from the group consisting of: 6-methyl-N-[4-(methylsulfonyl)benzyl]-2-oxo-5-phenyl-l-[3-(trifluoromethyl)phenyl]-l,2-dihydropyridine-3 carboxamide; 5[4(hydroxymethyl) phenyl]-6- methyl-N [4(m ethyl sulfonyl) benzyl]-2-oxo-l [3(trifluoromethyl) phenyl]-l,2-dihydropyridine- 3 -carboxamide; 5-furan-3-yl-6-methyl-2-oxo-l-(3-trifluoromethylphenyl)-l,2-dihydro- pyridine-3 carboxylic acid 4-methanesulfonyl-benzylamide; 6'-methoxy-2-methyl-N [4(m ethyl sulfonyl) benzyl]-6-oxo-l [3(trifluoromethyl) phenyl] l,6-dihydro-3,3'-bipyridine-5- carboxamide; 5(2-methoxypyrimidin-5-yl)-6-methyl-N [4(m ethyl sulfonyl) benzyl]-2-oxo- l [3(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 5[4(acetylamino) phenyl]-6- methyl-N[4(methylsulfonyl) benzyl]-2-oxo-l[3(trifluoromethyl) phenyl]-l,2-dihydropyridine-3- carboxamide; 6-methyl-2-oxo-5-(lH-pyrrol-3-yl)-l-(3-trifluoromethylphenyl)-l,2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 5-furan-2-yl-6-methyl-2-oxo-l-(3- trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl- benzylamide; 6-methyl-2-oxo-5-thi ophen-3 -yl-1 -(3 -trifluoromethylphenyl)- 1, 2-dihydro- pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-2-oxo-5-thiophen-2-yl-l- (3 -trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl- benzylamide; 5-(3,5-dimethyl-isoxazol-4-yl)-6-methyl-2-oxo-l-(3-trifluoromethylphenyl)-l,2- dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 5-(2,4-dimethoxy- pyrimidin-5-yl)-6-methyl-2-oxo-l-(3-trifluoromethylphenyl)-l,2-dihydro-pyridine-3- carboxylic acid 4-methanesulfonyl-benzylamide; 5-(2,4-dioxo-l,2,3,4-tetrahydro-pyrimidin-5- yl)-6-methyl-2-oxo-l -(3 -trifluorom ethyl-phenyl)- 1,2-dihy dro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 6-methyl-5-(5-methyl-[l,3,4] oxadiazol-2-yl)-2-oxo-l-(3- trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl- benzylamide; 6-methyl-2-oxo-5-(5-propyl-[l,3,4] oxadiazol-2-yl)-l-(3-trifluoromethylphenyl)-1.2-dihy dro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; {5-[5-(4- methanesulfonyl-benzylcarbamoyl)-2 -methyl -6-oxo-l -(3 -tri fluoromethyl-phenyl)-l, 6-dihydro- pyridin-3-yl]-[l,3,4] oxadiazol -2 -yl} -acetic acid ethyl ester; 5-(5-cyanomethyl-[l,3,4] oxadiazol -2 -yl)-6-methyl-2-oxo-l-(3-trifluoromethylphenyl)-l,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-amino-[l,3,4] oxadiazol-2-yl)-6-methyl-2-oxo-l-(3- trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl- benzylamide; 5-(5-amino-[ 1,3,4] thiadiazol-2-yl)-6-methyl-2-oxo-l-(3-trifluoromethylphenyl)-1.2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-ethylamino-[1,3,4] oxadiazol -2 -yl)-6-methyl-2-oxo-l -(3 -trifluorom ethylphenyl)- 1,2-dihy dro-pyridine-3 - carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-N,N-dimethylamino-[l,3,4] oxadiazol -2- yl)-6-methyl-2-oxo-l -(3 -trifluorom ethyl-phenyl)-l,2-dihydro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 6-methyl-N-[4-(methylsulfonyl) benzyl]-2-oxo-5-pyrazin-2-yl-l- [3-(trifluoro-methyl)phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-5-oxazol-2-yl-2- oxo-1 -(3 -trifluorom ethylphenyl)- 1,2-dihy dro-pyridine-3 -carboxylic acid 4-methanesulfonyl- benzylamide; 6-methyl-5-(l-methyl-lH-imidazol-2-yl)-2-oxo-l-(3-trifluoromethylphenyl)-l,2- dihy dro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-2-oxo-5-(l H- pyrazol -4-yl)- 1 -(3 -trifluorom ethylphenyl)- 1,2-dihy dro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 6-methyl-N-[4-(methylsulfonyl) benzyl]-2-oxo-5-pyrimidin-2- yl-1 -[3 -(trifluoro-methyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-5-(2-methyl- 2H-pyrazol-3-yl)-2-oxo-l -(trifluorom ethylphenyl)- 1,2-dihy dro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 6-methyl-5-(3-methylisoxazol-4-yl)-N [4-(m ethyl sulfonyl)benzyl]-2-oxo-l -[3 -(trifluoromethyl) phenyl- 1,2-dihydropyri dine-3 -carboxamide; 6-methyl-5- (3-methyl-[l,2,4] oxadiazol-5-yl)-2-oxo-l-(3-trifluoromethylphenyl)-l,2-dihydro-pyridine-3- carboxylic acid 4-mathanesulfonyl-benzylamide; 6-methyl-5-(3-methylisoxazol-5-yl)-N [4- (m ethyl sulfonyl) benzyl]-2-oxo-l -[3 -(trifluoromethyl) phenyl]- 1,2-dihydropyri dine-3- carboxamide; 5-(3,5-dimethylisoxazol-4-yl)-N [4-(isopropyl sulfonyl) benzyl]-6-methyl-2-oxo- 1 -[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 5-(3,5-dimethylisoxazol-4- yl)-N [4-(ethyl sulfonyl) benzyl]-6-methyl-2-oxo-l-[3-(trifluoromethyl) phenyl] -1,2- dihydropyridine-3 -carboxamide; N [4-(cyclopropylsulfonyl) benzyl]-5-(3,5-dimethylisoxazol-4- yl)-6-methyl-2-oxo-l -[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; l-(3- cyanophenyl)-5-(3,5-dimethylisoxazol-4-yl)-6-methyl-N-[4-(methylsulfonyl) benzyl]-2-oxo- 1,2-dihydropyri dine-3 -carboxamide; l-(3-chlorophenyl)-5-(3,5-dimethyl-isoxazol-4-yl)-6- methyl-2-oxo-l, 2-dihydro-pyri dine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 5-(3,5- dimethyl-isoxazol-4-yl)-6-methyl-2-oxo-l-m-tolyl-l,2-dihydro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 5-(5-isopropyl-[l,3,4] oxadiazol-2-yl)-6-methyl-2-oxo-l-(3- trifluoromethylphenyl)-l,2-dihydro-pyridine-3-carboxylic acid 4-methanesulfonyl- benzylamide; 6-methyl-5-[l,3,4] oxadiazol -2 -yl)-2-oxo-l -(3 -trifluoromethylphenyl)- 1,2- dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-hydroxy-[l,3,4] oxadiazol -2 -yl)-6-methyl-2-oxo-l -(3 -trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-(5-methyl-4H-[l,2,4] triazol-3-yl)-2-oxo-l-(3- trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-methylsulfonyl-benzylamide; 5-(4,5-dimethyl-4H-[l,2,4] triazol-3-yl)-6-methyl-2-oxo-l -(3 -trifluorom ethylphenyl)- 1,2- dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 5-(5-methoxymethyl- [1,3,4] oxadiazol -2 -yl)-6-methyl-2-oxo-l -(3 -trifluoromethylphenyl)- 1, 2-dihydro-pyri dine-3- carboxylic acid 4-methanesulfonyl-benzylamide; N-[4-(isopropylsulfonyl) benzyl]-6-methyl-5- (5-methyl-l,3,4-oxadiazol-2-yl)-2-oxo-l-[3-(trifluoromethyl) phenyl]- 1,2-dihydropyri dine-3- carboxamide; N-[4-(ethylsulfonyl) benzyl]-6-methyl-5-(5-methyl-l,3,4-oxadiazol-2-yl)-2-oxo- l-[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; N [4-(cyclopropylsulfonyl) benzyl]-6-methyl-5-(5-methyl-l,3,4-oxadiazol-2-yl)-2-oxo-l-[3-(trifluoromethyl) phenyl]-l,2- dihydropyridine-3 -carboxamide; 6-methyl-5-[l,3,4] oxadiazol-2-yl-2-oxo-l-(3- trifluoromethylphenyl)-l,2-dihydro-pyridine-3 -carboxylic acid 4-(propane-2-sulfonyl)- benzylamide; 6-methyl-5-[l,3,4] oxadiazol -2 -yl-2-oxo-l-(3 -trifluorom ethylphenyl)- 1, 2-dihydro- pyri dine-3 -carboxylic acid 4-cyclopropanesulfonyl-benzylamide; 6-methyl-5-(2-methyl-l,3- oxazol-4-yl)-N [4-(m ethyl sulfonyl) benzyl]-2-oxo-l-[3-(trifluoromethyl) phenyl]-l,2- dihydropyri dine-3 -carboxamide; 6-methyl-N [4-(m ethyl sulfonyl) benzyl]-5-(l,3-oxazol-4-yl)-2-oxo-1 -[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 5-(2-amino-thiazol-4- yl)-6-methyl-2-oxo-l -(3 -trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 5-(2,5-dimethyl-l,3-oxazol-4-yl)-6-methyl-N [4- (m ethyl sulfonyl) benzyl]-2-oxo-l -[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3- carboxamide; 6-methyl-5-(5-methyl-l,3-oxazol-4-yl)-N [4-(m ethyl sulfonyl) benzyl]-2-oxo-l- [3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 5-(2-amino-5-methyl-thiazol- 4-yl)-6-methyl-2-oxo-l -(3 -trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 5-(2-hydroxymethyl-5-methyl-thiazol-4-yl)-6-methyl-2-oxo-l- (3 -trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-m ethanesulfonylbenzylamide; 6-methyl-5-(5-methyl-[l,2,4] oxadiazol-3-yl)-2-oxo-l-(3-trifluoromethylphenyl)-1.2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 6-methyl-5-[l,2,4] oxadi azol -3 -y 1-2 -oxo-1 -(3 -trifluorom ethylphenyl)- l,2-dihydro-pyndine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; 6-methyl-2-oxo-5-(lH-tetrazol-5-yl)-l-(3- trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl- benzylamide; 6-m ethyl-5 -(4-m ethyl-oxazol-2-yl)-2-oxo-l -(3 -trifluorom ethylphenyl)- 1,2- dihydro-pyridine-3 -carboxylic acid 4-methanesulfonyl-benzylamide; 5-(4,5-dimethyl-oxazol-2- yl)-6-methyl-2-oxo-l -(3 -trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid 4- methanesulfonyl-benzylamide; N-(cyclohexylmethyl)-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-N-(2-morpholin-4- ylethyl)-2-oxo-5-phenyl-l-[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6- methyl-2-oxo-5-phenyl-N-lH-l,2,4-triazol-3-yl-l-[3-(trifluoromethyl) phenyl]-l,2- dihydropyridine-3 -carboxamide; N-[2-(lH-indol-3-yl) ethyl]-6-methyl-2-oxo-5-phenyl-l-[3- (trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-(l- phenylethyl)-l -[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-2- oxo-5-phenyl-N-(2-phenylethyl)-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine-3- carboxamide; 6-methyl-2-oxo-5-phenyl-N-[(2R)-2-phenylcyclopropyl]-l-[3-(trifluoromethyl)phenyl]-l,2-dihydropyridine-3-carboxamide; N-(2,3-dihydro-lH-inden-2-yl)- 6-methyl-2-oxo-5-phenyl-l -[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; N-[(l-ethylpyrrolidin-2-yl)methyl]-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl)-phenyl]-1.2-dihydropyridine-3-carboxamide; 6-methyl-N-(l-naphthylmethyl)-2-oxo-5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; N-(l,3-benzodioxol-5- ylmethyl)-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl)phenyl]-l,2-dihydropyridine-3- carboxamide; N-(2-chloro-4-fluorobenzyl)-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-(2-thienylmethyl)-l-[3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; N-(2-cyclohex-l-en-l- ylethyl)-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine-3- carboxamide; 6-methyl-2-oxo-N-(4-phenoxybenzyl)-5-phenyl-l-[3-(trifluoromethyl) phenyl]- l,2-dihydropyridine-3 -carboxamide; N-[ (2,5-dimethyl-3-furyl) methyl]-6-methyl-2-oxo-5- phenyl-l[3(trifluoromethyl)phenyl]-l,2-dihydropyridine-3-carboxamide; N{2[4-(aminosulfonyl) phenyl] ethyl}-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl)-phenyl]-l,2- dihydropyridine-3 -carboxamide; 6-methyl-2-oxo-5-phenyl-N-[4-(lH-pyrazol-l-yl) benzyl]-! - [3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-N-phenoxy- 5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2-dihydro-pyridine-3-carboxamide; N-[ (6-fluoro- 4H-l,3-benzodioxin-8-yl) methyl]-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2- dihydropyridine-3 -carboxamide; 6-methyl-2-oxo-5-phenyl-N-[2-(tetrahydro-2H-pyran-4-yl) ethyl]-l-[3-(trifluoromethyl)-phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5- phenyl-N-[3-(lH-pyrazol-l-yl) propyl]-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine-3- carboxamide; 6-methyl-N-[ (l-methyl-lH-pyrazol-4-yl) methyl]-2-oxo-5-phenyl-l-[3- (trifluoromethyl)-phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-2-oxo-5-phenyl-N-[ (1- phenyl-lH-pyrazol-4-yl) methyl]-l-[3-(trifluoromethyl)-phenyl]-l,2-dihydropyridine-3- carboxamide; N-[ (5 -m ethoxy -4-oxo-4H-pyran-2-yl) methyl]-6-methyl-2-oxo-5-phenyl-l-[3- (trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; N-(3-azepan-l-ylpropyl)-6- methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; N- (4-cyanobenzyl)-6-methyl-2-oxo-5-phenyl-l-[3-(trifluoromethyl) phenyl]-l,2-dihydropyridine- 3 -carboxamide; 6-methyl-2-oxo-N-[3-(5-oxo-4,5-dihydro-lH-pyrazol-4-yl) propyl]-5-phenyl-l- [3 -(trifluoromethyl) phenyl]-l,2-dihydropyridine-3-carboxamide; 6-methyl-5-(2-methyl-2H- pyrazol-3-yl)-2-oxo-l -(3 -trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid (3- methyl-isoxazol-5-ylmethyl)-amide; 6-methyl-5-(2-methyl-2H-pyrazol-3-yl)-2-oxo-l-(3- trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid (5-methanesulfonylmethyl- [1,2,4] oxadiazol-3-ylmethyl)-amide; 6-methyl-5-(2-methyl-2H-pyrazol-3-yl)-2-oxo-l-(3- trifluorom ethylphenyl)- l,2-dihydro-pyridine-3 -carboxylic acid ([1,2,4] oxadiazol-3-ylmethyl)- amide; 6-methyl-5-(l -methyl -lH-pyrazol-5-yl)-N-{ [5-(methylsulfonyl)pyri din-2 -yl]methyl}-2- oxo-l-[3-(trifluoromethyl)phenyl]-l,2-dihydropyridine-3-carboxamide; 5-(3,5- dimethylisoxazol-4-yl)-6-methyl-N-{ [5-(methylsulfonyl) pyridin-2-yl] methyl }-2-oxo-l-[3- (trifluoromethyl) phenyl]-!, 2-dihydropyridine-3-carboxamide; and salts thereof.

[0091] In particularly preferred embodiments, the elastase inhibitor is selected from (iii)(6-methyl-5-(l -methyl-lH-pyrazol-5-yl)-N-{[5-(methylsulfonyl)pyri din-2 -yl]methyl }-2-oxo-l- [3-(trifluoromethyl)phenyl]-l,2-dihydropyridine-3-carboxamide) and salts and stereoisomer thereof.

[0092] In a most preferred embodiment, the elastase inhibitor is(6-methyl-5-(l -methyl-lH-pyrazol-5-yl)-N-{[5-(methylsulfonyl)pyri din-2 -yl]methyl }-2-oxo-l- [3-(trifluoromethyl)phenyl]-l,2-dihydropyridine-3-carboxamide).

[0093] As indicated herein before, group (IV) consists of compounds capable of reducing or inhibiting elastokines production. In one sense a compound is considered capable of inhibiting elastokines production if its administration results in a reduced production of elastokines e.g. in e.g. in an established in vitro assay. In preferred embodiments, the compound selected from group (IV) is a matrix metalloproteinase (MMP) inhibitor or a compound capable of inhibiting MMP. In preferred embodiments said MMP is MMP-12. Hence, in a preferred embodiment, group (IV) consists of MMP-12 inhibitors. In one sense, a compound is considered capable of inhibiting MMP or MMP-12 if its administration results in reduced expression of MMP or MMP-12 respectively and / or if its administration results in reduced activity of MMP or MMP-12 respectively, e.g. in an established in vitro assay. In a preferred embodiment the compound is capable of inhibiting MMP-12 in an MMP-12 FRET Assay, as described herein elsewhere.

[0094] A specific class of MMP-12 inhibitors, which are considered to be of particular utility in the context of the present invention, are described in WO 2008 / 057254, the contents of which are incorporated herein by reference.

[0095] In preferred embodiments group (IV) consists of MMP-12 inhibitors selected from the group consisting of compounds of formula (IVa) and pharmaceutically acceptable salts, esters and hydrates thereof,

[0096] Wherein R1is an N-linked, free carboxyl or carboxyl-protected, natural or non-natural amino acid, or an N-linked amino acid derivative; R2is a) -C(O)OR8or b) -C(O)NR8R9; R3is a) H, b) -S(O)mR8, c) -S(O)mOR8, d) -S(O)mNR8R9, e) -C(O)R8, f) -C(S)OR8, g) -C(S)R8, h) - C(S)NR8R9, i) -C(NR8)NR8R9, j) a Cl -10 alkyl group, k) a C2-10 alkenyl group, 1) a C2-10 alkynyl group, m) a Cl -10 haloalkyl group, n) a C3-10 cycloalkyl group, o) a C6-14 aryl group, p) a 3-14 membered cycloheteroalkyl group, or q) a 5-13 membered heteroaryl group, wherein each of j) - q) optionally is substituted with 1-4 -Z-R10groups; or R3and the R8portion of R2, taken together with N-C(=0)-0 or N-C(=O)-N-R9to which they are attached, form a 3-14 membered cyclic urea group or a 3-14 membered cyclic carbamate group, each of which optionally is substituted with 1-4 -Z-R10groups; R4and R5independently are a) H, b) -CN, c) - NO2, d) halogen, e) -OR8, f) -NR8R9, g) -S(O)mR8, h) -S(O)mOR8, i) -C(O)R8, j) -C(O)OR8, k) - C(O)NR8R9, 1) -C(S)R8, m) -C(S)OR8, n) -C(S)NR8R9, o) a Cl -10 alkyl group, p) a C2-10 alkenyl group, q) a C2-10 alkynyl group, r) a Cl -10 haloalkyl group, s) a C3-14 cycloalkyl group, t) a C6-14 aryl group, u) a 3-14 membered cycloheteroalkyl group, or v) a 5-13 membered heteroaryl group, wherein each of o) - v) optionally is substituted with 1-4 -Z-R10groups; R6is a) H, b) -S(O)mR8, c) -S(O)mOR8, d) -C(O)R8, e) -C(O)OR8, f) -C(O)NR8R9, g) -C(S)R8, h) - C(S)OR8, i) -C(S)NR8R9j) a Cl -10 alkyl group, k) a C2-10 alkenyl group, 1) a C2-10 alkynyl group, or m) a Cl-10 haloalkyl group, wherein each of j) - m) optionally is substituted with a C6- 14 aryl group or a 5-13 membered heteroaryl group, wherein each of the C6-14 aryl group and the 5-13 membered heteroaryl group optionally is substituted with 1-4 -Z-R10groups; R8and R9, at each occurrence, independently are a) H, b) -OR11, c) -SR11, d) -S(O)mRn, e) -S(O)m-ORn, f) -S(O)m-NRnR12, g) -C(O)Rn, h) -C(O)ORn, i) -C(O)NRnR12, j) -C(S)NR1 XR12, k) a Cl-10 alkyl group, 1) a C2-10 alkenyl group, m) a C2-10 alkynyl group, n) a Cl-10 alkoxy group, o) a Cl- 10 haloalkyl group, p) a C3-10 cycloalkyl group, q) a C6-14 aryl group, r) a 3- 14 memberedcycloheteroalkyl group, or s) a 5-13 membered heteroaryl group, wherein each of k) - s) optionally is substituted with 1-4 -Z-R10groups; R10, at each occurrence, is a) halogen, b) -CN, c) -NO2, d) oxo, e) -O-Z-R11, f) -NRn-Z-R12, g) -N(O)Rn-Z-R12, h) -S(O)mRn, i) -S(O)mO-Z- R11, j) -S(O)mNRn-Z-R12, k) -C(O)Rn, 1) -C(O)O-Z-Rn, m) -C(O)NRn-Z-R12, n) -C(S)NRn-Z- R12, o) -Si(Ci-io alkyl)s, p) a Cl-10 alkyl group, q) a C2-10 alkenyl group, r) a C2-10 alkynyl group, s) a Cl-10 haloalkyl group, t) a C3-10 cycloalkyl group, u) a C6-14 aryl group, v) a 3-14 membered cycloheteroalkyl group, or w) a 5-13 membered heteroaryl group, wherein each of o)- w) optionally is substituted with 1-4 R13groups; R11and R12, at each occurrence, independently are a) H, b) -OH, c) -SH, d) -S(O)2OH, e) -C(O)OH, f) -C(O)NH2, g) -C(S)NH2, h) -S(0)m-Ci-io alkyl, i) -S(0)m-OCi-io alkyl, j) -OC1-10 alkyl, k) -C(0)-Ci-io alkyl, 1) -C(0)-OCi-io alkyl, m) - C(S)N(Ci-io alkyl)2, n) -C(S)NH-Ci-io alkyl, o) -C(0)NH-Ci-io alkyl, p) -C(0)N(Ci-io alkyl)2, q) a Cl-10 alkyl group, r) a C2-10 alkenyl group, s) a C2-10 alkynyl group, t) a Cl-10 alkoxy group, u) a Cl-10 haloalkyl group, v) a C3-10 cycloalkyl group, w) a C6-14 aryl group, x) a 3-14 membered cycloheteroalkyl group, or y) a 5- 13 membered heteroaryl group, wherein each of h)- y) optionally is substituted with 1-4 -R13groups; R13, at each occurrence, is a) halogen, b) -CN, c) -NO2, d) oxo, e) -OH, f) -NH2, g) -NH(Ci-io alkyl), h) -N(Ci-io alkyl)2, i) -S(O)mH, j) -S(O)m- C1-10 alkyl, k) -S(O)2OH, 1) -S(0)m-OCi-io alkyl, m) -S(O)mNH2, n) -S(0)mNH(Ci-io alkyl), o) - S(0)mN(Ci-io alkyl)2, p) -CHO, q) -C(0)-Ci-io alkyl, r) -C(O)OH, s) -C(0)-OCi-io alkyl, t) - C(O)NH2, U) -C(0)NH-CI-IO alkyl, v) -C(0)N(Ci-io alkyl)2, w) -C(S)NH2, x) -C(S)NH-Ci-io alkyl, y) -C(S)N(Ci-io alkyl)2, z) -Si(Ci-io alkyl)3, aa) a Cl-10 alkyl group, ab) a C2-10 alkenyl group, ac) a C2-10 alkynyl group, ad) a Cl-10 alkoxy group, ae) a Cl-10 haloalkyl group, af) a C3-10 cycloalkyl group, ag) a C6-14 aryl group, ah) a 3-14 membered cycloheteroalkyl group, or ai) a 5-13 membered heteroaryl group; X is O, S, S(O), S(O)2, or NR6; Y is S(O), S(O)2, or C(O); Z, at each occurrence, is a) a divalent Cl-10 alkyl group, b) a divalent C2-10 alkenyl group, c) a divalent C2-10 alkynyl group, d) a divalent Cl-10 haloalkyl group, or e) a covalent bond; and m, at each occurrence, is 0, 1, or 2. Particularly preferred embodiments are those wherein R2is -C(O)OR8; and / or wherein R2is -C(O)NR8R9; and / or wherein R3is H or a Cl-10 alkyl group; and / or wherein R3is selected from H, a methyl group, and an ethyl group; wherein R8is selected from H, -C(O)Rn, -C(O)ORn, -C(O)NRnR12, a Cl-10 alkyl group, a C2-10 alkenyl group, a C2-10 alkynyl group, a Cl-10 haloalkyl group, a C3-10 cycloalkyl group, a C6-14 aryl group, a 3-14 membered cycloheteroalkyl group, and a 5-13 membered heteroaryl group, wherein each of the Cl-10 alkyl group, the C2-10 alkenyl group, the C2-10 alkynyl group, the Cl-10 haloalkyl group, the C3-10 cycloalkyl group, the C6-14 aryl group, the 3-14 membered cycloheteroalkyl group, and the 5-13 membered heteroaryl group optionally is substituted with 1-4 -Z-R10groups;and / or wherein R8is selected from H, a Cl-10 alkyl group, a C2-10 alkenyl group, a C2-10 alkynyl group, and a C6-14 aryl group, wherein each of the Cl-10 alkyl group, the C2-10 alkenyl group, the C2-10 alkynyl group, and the C6-14 aryl group optionally is substituted with 1-4 -Z- R10groups; and / or wherein R8is selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, an isobutyl group, a butyl group, a hexyl group, and a butynyl group, each of which optionally is substituted with 1-4 groups independently selected from a halogen, - S(O)mRn, -S(O)mO-Z-Rn, -S(O)mNRn-Z-R12, -C(O)Rn, -C(O)O-Z-Rn, -C(O)NRn-Z-R12, a C6-14 aryl group, and a 5-13 membered heteroaryl group; and / or wherein R8is selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, an isobutyl group, a butyl group, a hexyl group, a 3 -butynyl group, a 4-butynyl group, a 2-fluoroethyl group, a 2- chloroethyl group, a 2-bromoethyl group, a 2-m ethanesulfonylethyl group, a 3 -chloropropyl group, and a benzyl group; and / or wherein R8is a phenyl group optionally substituted with 1-4 groups independently selected from a halogen and a Cl-10 alkyl group; wherein R8is selected from a phenyl group, a tolyl group, a fluorophenyl group, and a chlorophenyl group; and / or wherein R8and R9independently are selected from H, a Cl-10 alkyl group, a C2-10 alkenyl group, a C2-10 alkynyl group, a C3-10 cycloalkyl group, a C6-14 aryl group, a 3-14 membered cycloheteroalkyl group, and a 5-13 membered heteroaryl group, and each of the Cl-10 alkyl group, the C2-10 alkenyl group, the C2-10 alkynyl group, the C3-10 cycloalkyl group, the C6- 14 aryl group, the 3-14 membered cycloheteroalkyl group, and the 5-13 membered heteroaryl group optionally is substituted with 1-4 -Z-R10groups; and / or wherein R9is H or a methyl group; and / or wherein R8is selected from H, a Cl-10 alkyl group, a C3-10 cycloalkyl group, a C6-14 aryl group, and a 5-13 membered heteroaryl group, and each of the Cl-10 alkyl group, the C3- 10 cycloalkyl group, the C6-14 aryl group, and the 5-13 membered heteroaryl group optionally is substituted with 1-4 -Z-R10groups; wherein R8is selected from H, a methyl group, an ethyl group, an isopropyl group, a cyclopentyl group, a benzyl group, a fluorobenzyl group, a phenethyl group, a thienylmethyl group, and a thienylethyl group; wherein R8is a phenyl group or a 5-13 heteroaryl group, each of which optionally is substituted with 1-4 -Z-R10groups; and / or wherein R8is selected from a phenyl group, a 2,3- dihydrobenzo[b][l,4]dioxinyl group, a thienyl group, a pyridyl group, and an isoxazolyl group, and each of the phenyl group, the thienyl group, the pyridyl group, and the isoxazolyl group optionally is substituted with 1-3 groups independently selected from halogen, -O-Z-R11, -NRn-Z-R12, a Cl-10 alkyl group, and a Cl-10 haloalkyl group; and / or wherein R3and the R8portion of R2, taken together with N-C(=0)-0 or N-C(=O)-N-R9to which they are attached, form a 5-12 membered cyclic urea group or a 5-12 membered cyclic carbamate group, each of which optionally is substituted with 1-4 -Z-R10groups, wherein R9preferably is H or a methyl group and / or wherein the 5-12 membered cyclic urea group or the 5-12 membered cyclic carbamate group preferably is 5-membered cyclic urea group, a 6-membered cyclic urea group, a 5-membered cyclic carbamate group, or a 6-membered cyclic carbamate group.

[0097] In preferred embodiments group (IV) consists of MMP-12 inhibitors selected from the group consisting of (R)-2-(7-methoxycarbonylamino-dibenzofuran-2-sulfonylamino)-3-methyl- butyric acid, (R)-3-methyl-2-(7-(propoxycarbonylamino)dibenzo[b,d]furan-2- sulfonamido)butanoic acid, (R)-2-(7-(isopropoxycarbonylamino)dibenzo[b,d]furan-2- sulfonamido)-3-methyl-butanoic acid, (R)-3-methyl-2-(7-(phenoxycarbonylamino)dibenzo[b,d]furan-2-sulfonamido)butanoic acid, (R)-2-(7-(3- ethylureido)dibenzo[b,d]furan-2-sulfonamido)-3-methylbutanoic acid, (R)-2-(7-(3-(4-fluoro- benzyl)-ureido)-dibenzofuran-2-sulfonylamino)-3-methyl-butyric acid, (R)-2-(7-(3- cyclopentylmethylureido)-dibenzofuran-2-sulfonylamino)-3-methyl-butyric acid, (R)-2-(7-(3- isopropylureido)-dibenzofuran-2-sulfonylamino)-3-methyl-butyric acid, (R)-3-methyl-2-(7-(3- (3,4,5-trimethoxy-phenyl)-ureido)-dibenzofuran-2-sulfonylamino)-butyric acid, (R)-2-(7-(3- (3,4-difluorophenyl)ureido)dibenzo[b,d]furan-2-sulfonamido)-3-methyl-butanoic acid, (R)-2- (7-(3-(4-(dimethylamino)phenyl)ureido)dibenzo[b,d]furan-2-sulfonamido)-3-methyl -butanoic acid, (R)-3-methyl-2-(7-(3-(3-phenoxyphenyl)ureido)dibenzo[b,d]furan-2-sulfonamido)- butanoic acid, (R)-3-methyl-2-(7-ureidodibenzo[b,d]furan-2-sulfonamido)butanoic acid, (R)-3- methyl-2-(7-(3-(4-(trifluoromethoxy)phenyl)ureido)dibenzo[b,d]furan-2-sulfonamido)butanoic acid, (R)-2-(7-(3-(2,6-dichloropyridin-4-yl)ureido)dibenzo[b,d]furan-2-sulfonamido)-3-methyl- butanoic acid, (R)-3-methyl-2-(7-(3-(2-(thiophen-2-yl)ethyl)ureido)dibenzo[b,d]furan-2- sulfonamido)butanoic acid, (S)-2-(7-(3-ethylureido)dibenzo[b,d]furan-2-sulfonamido)-3- methyl-butanoic acid, (S)-2-(7-methoxycarbonylamino-dibenzo[b,d]thiophene-2-sulfonamido)- 3 -methyl-butanoic acid, (R)-2-(7-(m ethoxy carbonylamino)dibenzo[b, d]thiophene-2- sulfonamido)-3 -methyl-butanoic acid, (S)-2-(8-(isobutoxycarbonylamino)dibenzo[b,d]furan-3- sulfonamido)-3 -methyl-butanoic acid, (S)-2-(8-((2- chloroethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2- (8-((2-bromoethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(8-(isopropoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(8-((4-fluorophenoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl- butanoic acid, (S)-2-(8-((2-chlorophenoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3- methyl-butanoic acid, (S)-2-(8-(but-2-ynyloxycarbonylamino)dibenzo[b,d]furan-3- sulfonamido)-3 -methyl-butanoic acid, (S)-3-methyl-2-(8-(p-tolyloxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-3 -methyl -2-(8-(phenoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-2-(8- (benzyloxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(8- (hexyloxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(8- ((2 -fluoroethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl -butanoic acid, (S)-2-(8-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl -butanoic acid, (S)-2-(8-(ethoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)- 3-methyl-2-(8-propoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamidobutanoic acid, (S)-2-(8- (butoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(8- ((but-3-ynyloxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl -butanoic acid, (S)- 2-(8-(3-(3,5-dimethylisoxazol-4-yl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl- butanoic acid, (S)-3-methyl-2-(8-(3-thiophen-3-yl)ureido)dibenzo[b,d]furan-3- sulfonamido)butanoic acid, (S)-3-methyl-2-(8-(3-(3,4,5- trimethoxyphenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-2-(8-(3-(3,4- difluorophenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-3-methyl- 2-(8-(3-(3-phenoxyphenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-3- methyl-2-(8-ureidodibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-2-(8-(3-(2,6- dichloropyridin-4-yl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2- (8-(3-(4-(dimethylamino)phenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl -butanoic acid, (S)-3-methyl-2-(8-(3-(2-(thiophen-2-yl)ethyl)ureido)dibenzo[b,d]furan-3- sulfonamido)butanoic acid, (S)-3-methyl-2-(8-(3-(4-(trifluoromethoxy)phenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-2-(8-(3- cyclopentylureido)dibenzo[b,d]furan-3-sulfonamido)-3 -methyl-butanoic acid, (S)-2-(8-(3-(4- fluorobenzyl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(8- (m ethoxy carbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3 -methyl-butanoic acid, (R)-2- (8-(methoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl -butanoic acid,(S)-2-(7-(3-cyclopentylureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl -butanoic acid, (S)-3-methyl-2-(7-(3-(2-(thiophen-2-yl)ethyl)ureido)dibenzo[b,d]thiophene-3- sulfonamido)butanoic acid, (S)-2-(7-(3-(4-fluorophenyl)ureido)dibenzo[b,d]thiophene-3- sulfonamido)-3 -methyl-butanoic acid, (S)-3-methyl-2-(7-(3- phenethylureido)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid, (S)-2-(7-(3- benzylureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(7-(3-(4- fluorobenzyl)ureido)dibenzo[b,d]thiophene-3-sulfonamido)-3 -methyl-butanoic acid, (S)-3- methyl-2-(7-(3-p-tolylureido)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid, (S)-3-methyl-2-(7-(3-(3,4,5-trimethoxyphenyl)ureido)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid, (S)-2-(7-(3-ethylureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl -butanoic acid, (S)-2-(7-(3-(3,4-difluorophenyl)ureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl- butanoic acid, (S)-3-methyl-2-(7-(3-(4-(trifluoromethyl)phenyl)ureido)dibenzo[b,d]thiophene- 3-sulfonamido)butanoic acid, (S)-2-(7-(3-(2,3-dihydrobenzo[b][l,4]dioxin-6- yl)ureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butanoic acid, (S)-3-methyl-2-(7-(3- phenylureido)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid, (S)-2-(7-(3-(4-(dimethylamino)phenyl)ureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl- butanoic acid, (S)-3-methyl-2-(7-(3-pyridin-4-ylureido)dibenzo[b,d]thiophene-3- sulfonamido)butanoic acid, (S)-2-(7-(3-(3,5-dimethylisoxazol-4-yl)ureido)dibenzo[b,d]thiophene-3-sulfonamido)-3- methyl-butanoic acid, (S)-2-(7-(methoxycarbonylamino)dibenzo[b,d]thiophene-3- sulfonamido)-3 -methyl-butanoic acid, (S)-2-(7-(ethoxycarbonylamino)dibenzo[b,d]thiophene- 3 -sulfonamido)-3 -methyl-butanoic acid, (S)-2-(7-(isobutoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(6-chl oro-7-(m ethoxy carbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3 -methyl- butanoic acid, (S)-2-(7-(isopropoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3- methyl-butanoic acid, (S)-3-methyl-2-(7-(p-tolyloxycarbonylamino)dibenzo[b,d]thiophene-3- sulfonamido)butanoic acid, (S)-2-(7-((4-fluorophenoxy)carbonylamino)dibenzo[b,d]thiophene-3 -sulfonamido)-3 -methyl-butanoic acid, (S)-3-methyl-2-(7-(phenoxycarbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid, (S)-2-(7-((but-3- ynyloxy)carbonylamino)dibenzo[b,d]thiophene-3-sulfonamido)-3 -methyl-butanoic acid, (S)-3- methyl-2-(7-((2-(methylsulfonyl)ethoxy)carbonylamino)dibenzo[b,d]thiophene-3- sulfonamido)butanoic acid, (S)-2-(7-(benzyloxycarbonylamino)dibenzo[b,d]thiophene-3- sulfonamido)-3 -methyl-butanoic acid, (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)dibenzo[b,d]furan-2-sulfonamido)butanoic acid, (S)-2-(8-((3- chloropropoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (R)- 2-(8-bromo-7-(methoxycarbonylamino)dibenzo[b,d]furan-2-sulfonamido)-3-methyl -butanoic acid, (S)-2-(8-bromo-7-(methoxycarbonylamino)dibenzo[b,d]furan-2-sulfonamido)-3-methyl- butanoic acid, (S)-3-methyl-2-(7-(2-oxooxazolidin-3-yl)dibenzo[b,d]furan-3- sulfonamido)butanoic acid, (R)-2-(7-(methoxycarbonylamino)dibenzo[b,d]furan-3- sulfonamido)-3-methyl-butanoic acid, (R)-2-(7-(3-(3,4-difluorophenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(7- (methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(7- ((2 -fluoroethoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl -butanoic acid,(S)-2-(7-((but-2-ynyloxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(7-((4-fluorophenoxy)carbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl- butanoic acid, (S)-3 -methyl-2-(7-(3 -thi ophen-3 -ylureido)dibenzo[b,d]furan-3 - sulfonamido)butanoic acid, (S)-3-methyl-2-(7-(3-(2-(thiophen-2- yl)ethyl)ureido)dibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-3-methyl-2-(7- ureidodibenzo[b,d]furan-3-sulfonamido)butanoic acid, (S)-2-(7-(3-(3,4- difluorophenyl)ureido)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(7- (ethyl(methoxycarbonyl)amino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl-butanoic acid, (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-oxooxazolidin-3-yl)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid, (S)-2- (7-(methoxycarbonyl(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl -butanoic acid, (S)-2-(7-(ethoxycarbonyl(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl- butanoic acid, (S)-2-(7-(isopropoxycarbonyl(methyl)amino)dibenzo[b,d]thiophene-3- sulfonamido)-3-methyl-butanoic acid, (S)-2-(7-(((4- fluorophenoxy)carbonyl)(methyl)amino)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl- butanoic acid, (S)-3-methyl-2-(7-(l,3,3-trimethylureido)dibenzo[b,d]thiophene-3- sulfonamido)butanoic acid, (S)-2-(7-(3-ethyl-l-methylureido)dibenzo[b,d]thiophene-3- sulfonamido)-3-methyl-butanoic acid, (S)-2-(7-(3-benzyl-l- methylureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butanoic acid, (S)-2-(7-(3- benzyl-l,3-dimethylureido)dibenzo[b,d]thiophene-3-sulfonamido)-3-methyl-butanoic acid, (S)- 3-methyl-2-(7-(2-oxoimidazolidin-l-yl)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid, (S)-3-methyl-2-(7-(2-oxotetrahydropyrimidin-l(2H)-yl)dibenzo[b,d]thiophene-3- sulfonamido)butanoic acid, (R)-2-(7-(methoxycarbonylamino)dibenzo[b,d]thiophene-3- sulfonamido)-3 -methyl-butanoic acid, (R)-2-(7-(ethoxycarbonylamino)dibenzo[b,d]thiophene- 3 -sulfonamido)-3 -methyl-butanoic acid, (R)-2-(7-(3-cyclopentylureido)dibenzo[b,d]thiophene- 3 -sulfonamido)-3 -methyl-butanoic acid, (R)-3-methyl-2-(7-(3- phenylureido)dibenzo[b,d]thiophene-3-sulfonamido)butanoic acid; and salts, and esters thereof.

[0098] In particularly preferred embodiments, said MMP-12 inhibitors are selected from, and salts, esters and stereoisomer thereof.

[0099] In most preferred embodiments, said MMP-12 inhibitors are selected from(methoxycarbonylamino)dibenzo[b,d]furan-3- sulfonamido)-3 -methyl-butanoic acid), and salts, esters and stereoisomer thereof.

[0100] In most preferred embodiments, the MMP-12 inhibitor is(methoxycarbonylamino)dibenzo[b,d]furan-3- sulfonamido)-3 -methyl-butanoic acid).

[0101] The term ‘salt’ as used herein has its conventional meaning and includes the acid addition and base salts of the referenced compounds. Exemplary pharmaceutically acceptable mineral or organic acids or bases are as listed in Tables 1-8 in Handbook of Pharmaceutical Salts, P. H. Stahl and C. G. Wermuth (eds.), VHCA, Zurich 2002, pp. 334-345.

[0102] The term ‘ester’ as used herein has its conventional meaning and includes, in particular, derivatives of the recited compounds that result from the ester formation between a carboxylic acid group of the referenced compound and a an alkyl alcohol, such as a short chain aliphatic alcohol, such as a Ci- C2-, C3-, or C4-alcohol, in particular ethyl ester or methyl ester.

[0103] Unless specifically stated otherwise, where a compound may assume alternative tautomeric, regioisomeric and / or stereoisomeric forms, all alternative isomers, are encompassed within the scope of the claimed subject matter. For example, when a compound is described as aparticular optical isomer D- or L-, it is intended that both optical isomers be encompassed herein. For example, where a compound is described as having one of two tautomeric forms, it is intended that both tautomers be encompassed herein. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. The compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configurations, or may be a mixture thereof. Similarly, the compounds provided herein may contain cis-trans geometric isomers.Pharmaceutical Compositions

[0104] As will be apparent to those skilled in the art, the or more compounds are preferably provided and / or used in the form of a pharmaceutical composition.

[0105] According to a first particular aspect, there is provided a pharmaceutical composition comprising a first compound that is selected from one of the groups (I), (II), (III) and (IV), as defined herein before, and, preferably, a second compound that is selected from another one of said groups (I), (II), (III) and (IV).

[0106] In particularly preferred embodiments, the pharmaceutical composition comprises a first compound that is selected from said group (I), and a second compound that is selected from any one of said groups (II), (III) and (IV). In even more preferred embodiments, the pharmaceutical composition comprises a first compound that is selected from said group (I), and a second compound that is selected from any one of said groups (III) and (IV). In a most preferred embodiment, the pharmaceutical composition comprises a first compound that is selected from said group (I), and a second compound that is selected from said group (IV).

[0107] In particularly preferred embodiments, said pharmaceutical composition comprises a first compound that is selected from any one of the groups (i), (ii), (iii) and (iv), as defined herein before, and, preferably, a second compound that is selected from another one of said groups (i), (ii), (iii) and (iv).

[0108] In particularly preferred embodiments, the pharmaceutical composition as disclosed herein comprises a first compound that is selected from said group (i), and a second compound that is selected from any one of said groups (ii), (iii) and (iv). In even more preferred embodiments, the pharmaceutical composition comprises a first compound that is selected from said group (i), and a second compound that is selected from any one of said groups (iii) and (iv). In a most preferred embodiment, the pharmaceutical composition comprises a first compound that is selected from said group (i), and a second compound that is selected from said group (iv).

[0109] The term ‘pharmaceutical composition’ as used herein has its conventional meaning and refers to a composition which is pharmaceutically acceptable.

[0110] The term ‘pharmaceutically acceptable’ as used herein has its conventional meaning and refers to compounds, materials, compositions and / or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit / risk ratio.

[0111] In some embodiments, the pharmaceutical composition as disclosed herein comprises the first compound and the second compound in a molar ratio between 1 : 10 and 10: 1. In some embodiments, the pharmaceutical composition comprises the first and second compound as defined herein before in a molar ratio (first compound : second compound) within the range of 1 : 100 to 100: 1, such as within the range of 1 :5 to 50: 1; within the range of 1 : 20 to 20: 1; within the range of 1 : 10 to 10: 1; within the range of 1 :8 to 8: 1; within the range of 1 :6 to 6: 1; within the range of 1 :5 to 5: 1; within the range of 1 :4 to 4: 1; within the range of 1 :3 to 3: 1; or within 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.

[0112] In preferred embodiments, the pharmaceutical composition comprises a first compound selected from group (I) as defined herein before and a second compound selected from any one of groups (II), (III) and (IV) as defined herein before, in a molar ratio (first compound : second compound) within the range of 1 :100 to 100: 1, such as within the range of 1 :5 to 50: 1; within the range of 1 : 20 to 20: 1; within the range of 1 : 10 to 10: 1; within the range of 1 :8 to 8: 1; within the range of 1 :6 to 6: 1; within the range of 1 :5 to 5:1; within the range of 1 :4 to 4: 1; within the range of 1 :3 to 3: 1; or within the range of 1 :2 to 2: 1, such as a ratio of approximately 1 : 1.

[0113] In preferred embodiments, the pharmaceutical composition comprises a first compound selected from group (i) as defined herein before and a second compound selected from any one of groups (ii), (iii) and (iv) as defined herein before, in a molar ratio (first compound : second compound) within the range of 1 :100 to 100: 1, such as within the range of 1 :5 to 50: 1; within the range of 1 :20 to 20: 1; within the range of 1 : 10 to 10: 1; within the range of 1 :8 to 8:1; within the range of 1 : 6 to 6 : 1 ; within the range of 1 : 5 to 5 : 1 ; within the range of 1 :4 to 4 : 1 ; within the range of 1 :3 to 3: 1; or within the range of 1 :2 to 2: 1, such as a ratio of approximately 1 : 1.

[0114] In some embodiments, the first compound or the combination of the first compound and the second compound that may be present in the pharmaceutical composition as disclosed hereinabove, constitutes at least 95%, at least 98%, at least 99%, or at least 99.9% by weight, of the active agent(s) of the pharmaceutical composition.

[0115] In embodiments, the pharmaceutical composition typically comprises the first compound in a relative amount of at least 0.001 wt.%, based on the total composition, 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.% and / or in a relative amount of not more than 25 wt.% based on the total weight of the pharmaceutical composition, such as not more than 15 wt.%, not more than 10 wt.%, not more than 7.5 wt.%, not more than 5 wt.% or not more than 2.5 wt.%.

[0116] In embodiments, the pharmaceutical composition typically comprises the second compound, if present, in a relative amount of at least 0.001 wt.%, based on the total composition, 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.% and / or in a relative amount of not more than 25 wt.% based on the total weight of the pharmaceutical composition, such as not more than 15 wt.%, not more than 10 wt.%, not more than 7.5 wt.%, not more than 5 wt.% or not more than 2.5 wt.%.

[0117] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, or excipient.

[0118] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0119] As used herein, the term “carrier”, “adjuvant”, or “excipient” refers to any pharmaceutically acceptable component of a pharmaceutical composition that is not the active agent and with which a pharmaceutically active ingredient is administered. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as com starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxymethyl ethylcellulose, hydroxypropyl methylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, 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 glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer’s solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives,powered tragacanth, 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), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, 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 contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer’s solution, dextrose 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 are 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, 18thEd., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21stEd., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0120] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0121] In accordance with particularly preferred embodiments of the invention, the pharmaceutical composition is preferably provided in unit dosage form. The term “unit dosage form” refers to a physically discrete unit that is suitable for administration to a human subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with any suitable pharmaceutical carrier(s) and / or excipient(s). Exemplary, non-limiting unit dosage forms include a tablet, caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gelcap as well as any metered volume of a solution, suspension, syrup or elixir or the like, which may be contained, for instance in a vial, syringe, applicator device, sachet, spray, micropump etc. In accordance with particularly preferred embodiments of the invention, the unit dosage form is a unit dosage form that is suitable for oral administration. Most preferably, it is a solid unit dosage form, such as a tablet or capsule.

[0122] The term ‘fixed dose combination’ as used herein has its conventional meaning and refers to a combination of defined doses of two or more drugs or active ingredients presented in a single dosage unit (e.g. a tablet or a capsule) and administered as such. The term ‘free dose combination’ as used herein has its conventional meaning and refers to a combination of two drugs or active ingredients administered simultaneously but as two distinct dosage units.Therapeutic and non-therapeutic indications

[0123] As indicated herein before, the present invention, in one aspect, provides methods of:- treating or preventing an elastin deficiency related disease in a subject in need thereof;- treating and / or preventing conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promoting longevity in a subject in need thereof; said method comprising the administration to said subject of at least one compound selected from any one of groups (I), (II), (III) and (IV) as defined herein.

[0124] According to a preferred embodiment, the method comprises the administration, to said subject, of a first compound that is selected from one of said groups (I), (II), (III) and (IV), and a second compound that is selected from another one of said groups (I), (II), (III) and (IV).

[0125] According to a more preferred embodiment, the method comprises the administration to said subject of a first compound that is selected from said group (I) and a second compound that is selected from any one of said groups (II), (III) and (IV).

[0126] Also provided is a method of treating and / or preventing conditions and / or diseases related to the aging process and / or promoting longevity in a subject in need thereof, said method comprising the administration to said subject of a compound selected from any of groups (i), (ii), (iii) and (iv) as defined herein.

[0127] According to a preferred embodiment, the method comprises the administration, to said subject, of a first compound that is selected from one of said groups (i), (ii), (iii) and (iv), and a second compound that is selected from another one of said groups (i), (ii), (iii) and (iv).

[0128] According to a more preferred embodiment, the method comprises the administration, to said subject, of a first compound selected from said group (i) and a second compound that is selected from any one of said groups (ii), (iii) and (iv).

[0129] The terms "treat", "treating" or "treatment", when used in conjunction with a specific disease or symptom (for example: “method of treating disease ...”) refer to and / or encompass curing, alleviating or abrogating said disease and / or accompanying symptoms, diminishing extent of disease, stabilizing (i.e. not worsening) the state of disease, delaying or slowing of disease progression, ameliorating the disease state, prolonging survival (as compared to expected survival without treatment), etc. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life. The terms “treat”, “treating” or “treatment”, when used in relation to a patient or subject (for example: “method of treating a subject”), typically refers to the act of administering a therapeutic compound to said patient or subject for whatever therapeutic and / or prophylactic purpose.

[0130] The terms "prevent", "preventing" or "prevention", as used herein, refer to and / or encompass the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition. As used in accordance with the presently described subject matter, the term “prevention” relates to a process of prophylaxis in which a subject is exposed to the presently described compositions or formulations prior to the induction or onset of the disease / disorder process. The term “suppression” is used to describe a condition wherein the disease / disorder process has already begun but obvious symptoms of the condition have yet to manifest. Thus, the cells of an individual may have the disease / disorder, but no outside signs of the disease / disorder have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression.

[0131] As used herein, the term “elastin deficiency related disease” refers to any disease characterized by, comprising, involving, propagated by, enhanced by, induced by, promoted byor manifested by increased elastin degradation, reduced elastin expression, reduced elastin levels, reduced elastin stability, reduced elastin deposition, or any combination thereof.

[0132] In some embodiments, the elastin deficiency related disease is characterized by or comprises reduced tropoelastin expression (including mRNA transcription, protein translation, or both), reduced elastin fiber deposition, increased elastin fiber breakdown, increased elastokines production, or any combination thereof.

[0133] As used herein, the term “elastokines(s)” encompasses bioactive elastin peptide(s) being the result of proteolytic degradation of elastin.

[0134] In some embodiments, the elastin deficiency related disease is an age-related disease.

[0135] In some embodiments, the probability of being afflicted with or predisposition to developing an elastin deficiency related disease is increased in correlation with the age of a subject.

[0136] In some embodiments, the elastin deficiency related disease is selected the group consisting of: arteriosclerosis, peripheral vascular disease, angina, 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), Autosomal dominant cutis laxa type 1 (ADCL1), Acromicric dysplasia, Geleophysic dysplasia, Williams-Beuren syndrome, Marfan syndrome (MFS), Weill-Marchesani syndrome (WMS), Congenital contractual arachnodactyly (CCA), Pseudoxanthoma elasticum (PXE), Costello syndrome, Arterial tortuosity syndrome, and Isolated ectopia lends (ECTOL).

[0137] In a particularly preferred embodiment, the elastin deficiency related disease is selected the group consisting of: Abdominal aortic aneurysm (AAA) Supravalvular aortic stenosis (SVAS), Autosomal dominant cutis laxa type 1 (ADCL1), Acromicric dysplasia, Geleophysic dysplasia, Williams-Beuren syndrome, Marfan syndrome (MFS), Weill-Marchesani syndrome (WMS), Congenital contractual arachnodactyly (CCA), Pseudoxanthoma elasticum (PXE), Costello syndrome, Arterial tortuosity syndrome, and Isolated ectopia lends (ECTOL).

[0138] In some embodiments, the method of the invention is effective in and / or intended for increasing expression of elastin, increasing stability of elastin and increasing activity of elastin, in the subject.

[0139] In some embodiments, the method of the invention is effective in and / or intended for increasing elasticity of the aorta and / or reducing stiffness of the aorta of the subject.

[0140] As will be understood by those skilled in the art, based on the present teachings, the treatment and / or prevention of conditions and / or diseases related to the aging process includes, in particular, delaying the progression of such diseases or conditions, slowing down the progression of such diseases or conditions, halting the progression of such diseases or conditions.

[0141] Conditions and / or diseases related to the aging process generally include, e.g., neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, immune system diseases and skin related diseases or conditions.

[0142] In preferred embodiments of the invention, methods as described herein are provided for treating and / or prevention a condition or disease related to the aging process, wherein said disease or condition is characterized by, involving, propagated by, enhanced by, induced by, promoted by or manifested by increased elastin degradation, reduced elastin expression, reduced elastin levels, reduced elastin stability, reduced elastin deposition, or any combination thereof.

[0143] In preferred embodiments of the invention, methods as described herein are provided for treating and / or prevention a condition or disease related to the aging process, wherein said disease or condition is characterized by reduced tropoelastin expression (including mRNA transcription, protein translation, or both), reduced elastin fiber deposition, increased elastin fiber breakdown, increased elastokines production, or any combination thereof.

[0144] As used herein, the term “elastokines(s)” encompasses bioactive elastin peptide(s) being the result of proteolytic degradation of elastin.

[0145] In preferred embodiments of the invention, methods as described herein are provided for treating and / or prevention a condition or disease related to the aging process, wherein said disease or condition is selected from the group consisting of neurodegenerative diseases, cardiovascular diseases, mitochondrial diseases, fragility, musculoskeletal diseases and skin diseases.

[0146] In preferred embodiments of the invention said neurodegenerative disease is selected from the group consisting of 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, in particular age-related dementia.

[0147] In preferred embodiments of the invention, said cardiovascular disease is selected from the group consisting of hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, valvular heart disease, carditis, aortic disease, peripheral artery disease,thromboembolic disease, venous thrombosis, coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

[0148] In preferred embodiments of the invention, said mitochondrial disease is selected from the group consisting of stroke, atherosclerosis, hypertension, chronic inflammation, Type 2 diabetes mellitus, insulin resistance, cardiovascular diseases, nonalcoholic fatty liver disease (NAFLD), [3-cell dysfunction.

[0149] In preferred embodiments of the invention said fragility and musculoskeletal disease is selected from the group consisting of frailty, osteoporosis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout spondylarthritis, osteopenia tendinopathy, degenerative disc diseases, sarcopenia, (sensitivity) to fractures, musculoskeletal pain, atrophy, dystrophy.

[0150] In preferred embodiments of the invention, said skin disease is selected from the group consisting of impaired wound healing, alopecia, wrinkles, senile lentigo, skin thinning, xeroderma pigmentosum, xerosis, pruritis, eczematic dermatitis, purpura, chronic venous insufficiency and dyskeratosis congenita.

[0151] In preferred embodiments of the invention, the method provided herein is a method of promoting longevity.

[0152] In preferred embodiments of the invention, the method provided herein is a method of preventing aging.

[0153] In some embodiments of the invention, methods of treating a subject as defined herein are provided, wherein said method is a non-therapeutic method, such as a cosmetic method, e.g. for improving the appearance of the skin, for protecting the skin from the visible signs of aging, for preventing and / or reducing wrinkles, for improving and / or preserving smoothness of the skin, for improving and / or preserving firmness of the skin, for improving and / or preserving elasticity of the skin, for skin rejuvenation and / or for maintaining a youthful skin.

[0154] In some embodiments, the method of the invention is effective in and / or intended for increasing expression of elastin, increasing stability of elastin and increasing activity of elastin, in the subject.Treatment regimens

[0155] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration caninclude parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0156] In some embodiments, administering comprises local administering. In some embodiments, administering comprises subcutaneously injecting, in the subject. In some embodiments, administering comprises systemic administering.

[0157] In some embodiments, the route of administration of the pharmaceutical composition disclosed herein comprises an intravenous route, an intramuscular route, a subcutaneous route, or an oral delivery route. The route of administration of the pharmaceutical composition will depend on the disease or condition in need of treatment. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. In some embodiments, the pharmaceutical composition of the invention is administered via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.

[0158] In embodiments of the invention, the first compound and second compound may be separately administered, via the same route of administration or via distinct routes of administration.

[0159] In embodiments of the invention, the first compound and second compound may be administered via the same route of administration, either separately or conjunctly.

[0160] In embodiments of the invention, the first compound to be administered to the subject is formulated in a pharmaceutical compositions, such as the pharmaceutical compositions as defined herein before. In particularly preferred embodiments, the first compound to be administered is formulated as a unit dosage form, such as a unit dosage form as defined herein.

[0161] In embodiments of the invention, the second compound to be administered to the subject is formulated in a pharmaceutical compositions, such as the pharmaceutical compositions as defined herein before. In particularly preferred embodiments, the second compound to be administered is formulated as a unit dosage form, such as a unit dosage form as defined herein.

[0162] In embodiments of the invention, the first compound and second compound may be separately formulated in two distinct pharmaceutical compositions or two distinct unit dosage forms.

[0163] In embodiments of the invention, the first compound and second compound may be coformulated, in one pharmaceutical compositions, such as the pharmaceutical compositions as defined herein before. In embodiments of the invention, the first compound and second compound may be co-formulated, in one unit dosage form, such as the unit dosage form as defined herein before.

[0164] The term “therapeutically effective amount” refers to the concentration of at least one compound as disclosed herein, or any combination thereof, that is normalized to body weight (BW) and is effective to treat a disease or disorder in a subject. The term “a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A physician of ordinary skill can readily determine and prescribe the effective amount of the bioactive agent required. The exact dosage form and regimen would be determined by the physician according to the patient’s condition. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0165] In some embodiments, the method of the invention comprises the administration of the first compound and the second compound in a molar ratio between 1 : 10 and 10: 1. In some embodiments, the method comprises the administration of the first and second compound as defined herein before in a molar ratio (first compound : second compound) within the range of 1 : 100 to 100: 1, such as within the range of 1 :5 to 50: 1; within the range of 1 :20 to 20: 1; within the range of 1 : 10 to 10:1; within the range of 1 : 8 to 8 : 1 ; within the range of 1 : 6 to 6 : 1 ; within the range of 1 :5 to 5: 1; within the range of 1 :4 to 4:1; within the range of 1 :3 to 3: 1; or within 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.

[0166] In preferred embodiments, the method comprises the administration of a first compound selected from group (I) as defined herein before and a second compound selected from any one of groups (II), (III) and (IV) as defined herein before, in a molar ratio (first compound : second compound) within the range of 1 :100 to 100: 1, such as within the range of 1 :5 to 50: 1; within the range of 1 : 20 to 20 : 1 ; within the range of 1 : 10 to 10:1; within the range of 1 : 8 to 8 : 1 ; within the range of 1 :6 to 6: 1; within the range of 1 :5 to 5:1; within the range of 1 :4 to 4: 1; within the range of 1 :3 to 3: 1; or within the range of 1 :2 to 2: 1, such as a ratio of approximately 1 :1.

[0167] In preferred embodiments, the method comprises the administration a first compound selected from group (i) as defined herein before and a second compound selected from any one of groups (ii), (iii) and (iv) as defined herein before, in a molar ratio (first compound : second compound) within the range of 1 :100 to 100: 1, such as within the range of 1 :5 to 50: 1; within therange of 1 :20 to 20: 1; within the range of 1 : 10 to 10: 1; within the range of 1 :8 to 8:1; within the range of 1 : 6 to 6 : 1 ; within the range of 1 : 5 to 5 : 1 ; within the range of 1 :4 to 4 : 1 ; within the range of 1 :3 to 3: 1; or within the range of 1 :2 to 2: 1, such as a ratio of approximately 1 : 1.

[0168] In certain embodiments of the invention, the method comprises the administration of the first compound once or twice daily, most preferably once daily. In further embodiments of the invention, the method comprises the administration of the second compound once or twice daily. It will be understood that, with a view to convenience for and / or compliance by the patient, it is preferred that the first and second compound are administered conjunctly or very shortly after another. In one particularly preferred embodiments of the invention, the treatment comprises the administration of a single unit dosage form comprising the first compound and the second compound once, twice, three times or four times a day, preferably one, twice or three times a day, more preferably once or twice a day, most preferably once a day. In accordance with the invention, the treatment comprises the administration of the first compound, preferably in combination with the second compound, in accordance with the above-defined regimens, during 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 year, at least three year, at least 5 year, at least 10 year, at least 20 year, at least 30 year. There is no particular upper limit; treatment may be continued for as long as it is deemed beneficial to the subject’s overall health and well-being (as determined by appropriately qualified healthcare professional), e.g. for the rest of the subject’s life.Subjects to be treated

[0169] The term “a subject " refers to a living organism, typically a mammal, in particular a human subject. In one embodiment of the invention, the subject is human male. In another embodiment of the invention, the subject is human female.

[0170] In further preferred embodiments of the invention, the subject is a subject being over 35 years of age, over 40 years of age, over 45 years of age, over 50 years of age, over 55 years of age, over 60 years of age, over 65 years of age or over 70 years of age.

[0171] In particularly preferred embodiments of the invention, the subject is a subject that is suffering from and / or has been diagnosed with an elastin deficiency related disease as defined herein above.

[0172] In some embodiments, the subject is a subject that is suffering from and / or has been diagnosed as suffering from reduced tropoelastin expression (including mRNA transcription,protein translation, or both), reduced elastin fiber deposition, increased elastin fiber breakdown, increased elastokines production, or any combination thereof.

[0173] In further preferred embodiments of the invention, the subject is a subject that is considered to be at risk, typically at above-average risk, of attracting or developing an elastin deficiency related disease as defined herein above.

[0174] In preferred embodiments of the invention, the subject is a subject suffering from one or more conditions known to bear a causal and / or epidemiological correlation with the occurrence of an elastin deficiency related disease as defined herein above.

[0175] In further preferred embodiments of the invention, the subject is a subject that is genetically predisposed to develop an elastin deficiency related disease as defined herein above.

[0176] In further preferred embodiments of the invention, the subject is a subject prone to develop an “elastin deficiency related disease” as defined herein above, as a consequence of lifestyle / habitual factors.

[0177] In some embodiments, the subject is characterized by displaying reduced tropoelastin expression level (including mRNA transcription, protein translation, or both), reduced elastin fiber deposition level, increased elastin fiber breakdown, increased elastokines production, or any combination thereof. The term “reduced”, in this context, typically refers to a level that is below a predetermined reference value, such as 10% below such a predetermined reference value, or alternatively at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 75% below said predetermined reference value. It will be understood that the term “pre-determined reference value” refers to a threshold value that distinguishes the normal, non- pathological state from a pathological state, where values above the threshold are indicative of the normal, non-pathological endotype and values below the threshold are indicative of the pathological endotype (or vice versa, depending on the parameter selected). Typically, such a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. In embodiments of the invention wherein the pathological state is characterized by a decrease in a certain parameter (compared to a healthy, non-pathologic state), the predetermined reference value may e.g. be the average, mean or median value as established in a normal, non-pathological reference population of sufficient size, or it may be the 50thpercentile cut-off point, the 25thpercentile cut-off point, the 20thpercentile cut-off point, the 15thpercentile cut-off point or the 10thpercentile cut-off point as established in a normal, non-pathological reference population of sufficient size. The term “increased”, in the present context, typically refers to a level that is above a predetermined reference value, such as at least 10% above said predetermined reference value, or alternatively at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, or at least 75% above said a predetermined reference value, wherein the predetermined reference value may be the average, mean or median value as established in a normal, non-pathological reference population of sufficient size, or it may be the 50thpercentile cut-off point, the 75thpercentile cut-off point, the 80thpercentile cut-off point, the 85thpercentile cut-off point or the 90thpercentile cut-off point as established in a normal, non- pathological reference population of sufficient.

[0178] Methods for determining proteins (including peptides or polypeptides) of interest, such as, in particular, elastin, are common and include flow cytometry, immunohistochemical staining of tissue slices or sections, western blot, ELISA, radioimmunoassay (RIA) assays, an antibody microarray, and the like, all of which would be apparent to one of ordinary skill in the art of biochemistry.

[0179] Numerous methods are known in the art for measuring expression levels of a one or more gene such as by amplification of nucleic acids (e.g., PCR, isothermal methods, rolling circle methods, etc.) or by quantitative in situ hybridization.

[0180] RT-qPCR: A common technology used for measuring RNA abundance is RT-qPCR where reverse transcription (RT) is followed by real-time quantitative PCR (qPCR). Reverse transcription first generates a DNA template from the RNA. This single-stranded template is called cDNA. The cDNA template is then amplified in the quantitative step, during which the fluorescence emitted by labeled hybridization probes or intercalating dyes changes as the DNA amplification process progresses. Quantitative PCR produces a measurement of an increase or decrease in copies of the original RNA and has been used to attempt to define changes of gene expression in cancer tissue as compared to comparable healthy tissues.

[0181] RNA-Seq: RNA-Seq uses recently developed deep-sequencing technologies. In general, a population of RNA (total or fractionated, such as poly(A)+) is converted to a library of cDNA fragments with adaptors attached to one or both ends. Each molecule, with or without amplification, is then sequenced in a high-throughput manner to obtain short sequences from one end (single-end sequencing) or both ends (pair-end sequencing). The reads are typically 30-400 bp, depending on the DNA-sequencing technology used. In principle, any high-throughput sequencing technology can be used for RNA-Seq. Following sequencing, the resulting reads are either aligned to a reference genome or reference transcripts, or assembled de novo without the genomic sequence to produce a genome-scale transcription map that consists of both the transcriptional structure and / or level of expression for each gene. To avoid artifacts and biases generated by reverse transcription direct RNA sequencing can also be applied.

[0182] Microarray: Expression levels of a gene may be assessed using the microarray technique. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are arrayed on a substrate. The arrayed sequences are then contacted under conditions suitable for specific hybridization with detectably labeled cDNA generated from RNA of a test sample. As in the RT-PCR method, the source of RNA typically is total RNA isolated from a tumor sample, and optionally from normal tissue of the same patient as an internal control or 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 tissue cDNA-mediated annealing, selection, extension, and ligation, DASL-Illumina method may be used. For a non-limiting example, PCR amplified cDNAs to be assayed are applied to a substrate in a dense array. Microarray analysis can be performed by commercially available equipment, following manufacturer’s protocols, such as by using the Affymetrix GenChip technology, or Incyte’s microarray technology.

[0183] Methods for measuring stiffness of a tissue, such as the aorta, are common and would be apparent to one of ordinary skill in the art, such as exemplified herein below in the Example Section.

[0184] In some embodiments of the invention, typically in those embodiments wherein the method has a non-therapeutic purpose, such as a cosmetic purpose, the subject is a healthy individual or an individual that has not been diagnosed with any disease or pathology or an individual that has not been diagnosed with any disease or pathology requiring treatment aimed at increasing expression of elastin, increasing stability of elastin and / or increasing activity of elastin. In some embodiments, the subject is a healthy human subject, preferably a healthy adult male or female human of 18-65 years of age.Pharmaceutical kits of the invention

[0185] According to another aspect, there is provided a a pharmaceutical kit comprising a package containing a plurality of unit dosage forms, said unit dosage forms comprising a first compound selected from any one of the following groups (I), (II), (III) and (IV) as defined herein, e.g. from any one of groups (i), (ii), (iii) and (iv) as defined herein before, wherein said pharmaceutical kit further comprises a leaflet containing printed instructions to (self) administer said unit dosage forms in order to accomplish any of the therapeutic objectives as defined herein, such as to:- treat or prevent an elastin deficiency related disease in a subject in need thereof;- treat and / or prevent conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promote longevity in a subject in need thereof.

[0186] Another aspect of the invention is directed 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 making up said first plurality comprise a first compound that is selected from one of said groups (I), (II), (III) and (IV), and the unit dosage forms making up said second plurality comprise a second compound that is selected from another one of said groups (I), (II), (III) and (IV), wherein said pharmaceutical kit optionally further comprises a leaflet containing printed instructions to (self) administer the unit dosage forms contained in the kit, in order to accomplish any of the therapeutic objectives as defined herein, such as to:- treat or prevent an elastin deficiency related disease in a subject in need thereof;- treat and / or prevent conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promote longevity in a subject in need thereof.

[0187] According to a more preferred embodiment, there is provided the pharmaceutical kit wherein the first compound is selected from said group (I) and the second compound is selected from any one of said groups (II), (III) and (IV).

[0188] Another aspect of the invention is directed 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 making up said first plurality comprise a first compound that is selected from one of said groups (i), (ii), (iii) and (iv), and the unit dosage forms making up said second plurality comprise a second compound that is selected from another one of said groups (i), (ii), (iii) and (iv), wherein said pharmaceutical kit optionally further comprises a leaflet containing printed instructions to (self) administer the unit dosage forms contained in the kit, in order to accomplish any of the therapeutic objectives as defined herein, such as to:- treat or prevent an elastin deficiency related disease in a subject in need thereof;- treat and / or prevent conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promote longevity in a subject in need thereof.

[0189] According to a more preferred embodiment, there is provided the pharmaceutical kit wherein the first compound is selected from said group (i) and the second compound is selected from any one of said groups (ii), (iii) and (iv).

[0190] In accordance with embodiments of the invention, the pharmaceutical kit comprises a container, such as a cardboard box, holding one or more blister packs, said one or more blister packs containing a plurality of solid unit dosage forms as defined herein before, preferably a plurality of tablets as defined herein before, comprising said first compound. In preferred embodiments of the invention, the pharmaceutical kit comprises at least 5, at least 8, at least 10, at least 12 of at least 15 of said unit dosage forms comprising the first compound, e.g. 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 particularly preferred embodiments of the invention, the kit holds one or more further blister packs, said one or more further blister packs containing a plurality of solid unit dosage forms as defined herein before, preferably a plurality of tablets as defined herein before, comprising said second compound, e.g. the pharmaceutical may kit comprise at least 5, at least 8, at least 10, at least 12 of at least 15 of said unit dosage forms comprising the second compound, e.g. 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 second compound.

[0191] In accordance with the invention, the pharmaceutical kit comprises a leaflet inserted into the container, typically a patient information leaflet containing printed information, which information may include a description of the form and composition of the unit dosage forms contained in the kit, an indication of the therapeutic indications for which the product is intended, instructions as to how the product is to be used and information and warnings concerning adverse effects and contraindications associated with the use. It will be understood by those of average skill in the art, based on the information presented herein, that the leaflet that is part of the kit according to the invention, will typically contain the information concerning the therapeutic indications, uses, treatment regimens, etc. as described here above in relation to the methods of treatment of the present invention. In particularly preferred embodiments of the invention, the leaflet contains printed instructions to repeatedly (self)administer the unit dosage forms in order to:- treat or prevent an elastin deficiency related disease in a subject in need thereof;- treat and / or prevent conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promote longevity in a subject in need thereof.Miscellaneous

[0192] 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 the inventionpertains. Although methods and materials similar or equivalent to those described herein can 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, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0193] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0194] As used herein, the term “about” when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0195] It is noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. It should also be noted that the term “or” is generally employed in its broadest sense, that is as meaning “and / or” unless the content clearly dictates otherwise.

[0196] In those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of “A” or “B” or “A and B.”

[0197] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0198] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0199] The expression “comprise” and variations thereof, such as, “comprises” and “comprising” as used herein should be construed in an open, inclusive sense, meaning that the embodiment described includes the recited features, but that it does not exclude the presence of other features, as long as they do not render the embodiment unworkable

[0200] The expressions “one embodiment”, “a particular embodiment”, “an embodiment” etc. as used herein should be construed to mean that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of such expressions in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. For example, certain features of the disclosure which are described herein in the context of separate embodiments are also explicitly envisaged in combination in a single embodiment.NUMBERED EMBODIMENTS

[0201] Further particularly preferred embodiments of the invention include the following:1. A pharmaceutical composition, comprising a combination comprising at least two compounds selected from the group consisting of: 8-[2-(2-pentyl-cyclopropylmethyl)-any salt thereof, any stereoisomer thereof, or any combination thereof.2. The pharmaceutical composition of embodiment 1, wherein said combination comprises: (i) said 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), including any salt thereof, any stereoisomers thereof, or any combination thereof; and (ii) at least one compoundany salt thereof, any stereoisomer thereof, or any combination thereof. 3. A pharmaceutical composition comprising at least two compounds selected from the group consisting of: a first compound capable of increasing tropoelastin 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 elastokines production.4. A process for the preparation of a pharmaceutical composition according to any one of embodiments 1 to 3, which comprises admixing compounds as defined in any one of embodiments 1 to 3, with a pharmaceutically acceptable carrier.5. The pharmaceutical composition according to any one of embodiments 1 to 4, for use in prevention or treatment of an elastin deficiency related disease.6. A combination therapy for use in prevention or treatment of an elastin deficiency related disease in a subject in need thereof, comprising at least two compounds selected from the group consisting of: a first compound capable of increasing tropoelastin 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 elastokines production.7. The combination therapy for use according to embodiment 5, wherein any one of: a. said first compound comprises a mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor; b. said second compound comprises a protein kinase C epsilon type (PKCs) activator; c. said third compound comprises an elastase inhibitor; d. said fourth compound comprises a matrix metalloproteinase (MMP) inhibitor; and e. any combination of (a) to (d).8. The combination therapy for use according to embodiment 7, wherein said combination comprises said PKCs activator, and any one of said: MAP2K1 inhibitor, elastase inhibitor, and MMP inhibitor.9. The combination therapy for use according to embodiment 7 or 8, wherein said elastase is neutrophil elastase.10. The combination therapy for use according to any one of embodiments 7 to 9, wherein said MMP is MMP-12.11. The combination therapy for use according to any one of embodiments 6 to 10, wherein said at least two compounds are separately formulated in two distinct pharmaceutical compositions.12. The combination therapy for use according to any one of embodiments 6 to 10, wherein said at least two compounds are formulated in a single pharmaceutical composition.13. The combination therapy for use according to any one of embodiments 6 to 12, wherein any one of said at least two compounds is selected from the group consisting of: a small molecule, a nucleic acid, a peptide, a polypeptide, an antibody, a peptidomimetic, a carbohydrate, and a lipid.14. The combination therapy for use according to any one of embodiments 7 to 13, wherein said PKCs activator comprises or consists of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]- octanoic acid (DCP-LA).15. The combination therapy for use according to any one of embodiments 7 to 14, wherein said MAP2K1 inhibitor comprises or consists, including any salt thereof, any stereoisomer thereof, or any combination thereof.16. The combination therapy for use according to any one of embodiments 7 to 15, whereinsaid elastase inhibitor comprises or consists of including any salt thereof, any stereoisomer thereof, or any combination thereof. 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),, y , y , or any combination thereof, for use in treatment or prevention of an elastin deficiency related disease in a subj ect in need thereof.19. The pharmaceutical composition for use according to embodiment 18, comprising a combination comprising at least two compounds selected from the group consisting of: 8-[2-(2- pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA),any combination thereof.20. The pharmaceutical composition for use according to embodiment 19, wherein said combination comprises: (i) said 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid(DCP-LA), including any salt thereof, any stereoisomers thereof, or any combination thereof; and f:, y , y , or any combination thereof.21. The pharmaceutical composition for use according to embodiment 20, comprising said (i) and said (ii) in a molar ratio between 1 : 10 and 10: 1.22. The pharmaceutical composition for use according to any one of embodiments 18 to 19, further comprising a pharmaceutically acceptable carrier.23. The pharmaceutical composition for use according to any one of embodiments 18 to 22, wherein said elastin deficiency related disease is selected from the group consisting of: arteriosclerosis, peripheral vascular disease, angina, 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 (ARMD), 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), Autosomal dominant cutis laxa type 1 (ADCL1), Acromicric dysplasia, Geleophysic dysplasia, Williams-Beuren syndrome, Marfan syndrome (MFS), Weill-Marchesani syndrome (WMS), Congenital contractual arachnodactyly (CCA), Pseudoxanthoma elasticum (PXE), Costello syndrome, Arterial tortuosity syndrome, and Isolated ectopia lends (ECTOL).24. The pharmaceutical composition for use according to any one of embodiments 18 to 23, wherein said treatment comprises: increased expression, stability, activity, or any combination thereof, of elastin in said subject.25. The pharmaceutical composition for use according to any one of embodiments 18 to 24, wherein said treatment comprises increased elasticity of aorta of said subject or reduced stiffness of aorta in said subject.26. A kit comprising in separate containers in a single package pharmaceutical compositions according to any one of embodiments 1 to 3, for use in combination to treat or prevent elastin deficiency related diseases.27. A method of treating or preventing elastin deficiency related diseases comprising administering to a subject in need an effective amount of the pharmaceutical composition according to any one of embodiments 1 to 3.EXAMPLES

[0202] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols inMolecular Biology” Volumes I-III Ausubel, R. M., 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”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. 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, J. E., ed. (1994); “Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8thEdition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), “Strategies for Protein Purification and Characterization - A Labor“tory Course Manual" CSHL Pres” (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0203] In all examples, the code EB-101 is used to denote the compound known under the INN Alvelestat (i.e. 6-methyl-5-(l-methyl-lH-pyrazol-5-yl)-N-{[5-(methylsulfonyl)pyridin-2- yl]methyl}-2-oxo-l-[3-(trifluoromethyl)phenyl]-l,2-dihydropyridine-3-carboxamide); the code 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 denote the compound also known as MMP-408 (i.e. (S)-2-(8-(methoxycarbonylamino)dibenzo[b,d]furan-3-sulfonamido)-3-methyl -butanoic acid); and the code EB-104 is used to denote the compound known as U-0126 (i.e. l,4-diamino-2,3- dicyano-l,4-bis(o-aminophenylmercapto)butadiene).EXAMPLE 1

[0204] An in / ex vivo study was conducted to show that the small molecules, or their combinations, as disclosed herein, are suitable for treating elastin loss, and induce or promote recovery of elastin deposition.Materials and MethodsPurified PKC Epsilon assay

[0205] Recombinant PKC (1 ng of alpha or epsilon isoform) is mixed with test compound in the presence of 10 micromolar histones, 5 mM CaCh, 1.2 pg / pl phosphatidyl-L-serine, 0.18 pg / pl 1,2-dioctanoyl-sn -glycerol (DAG), 10 mM MgC12, 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8 pg / ml aprotinin, 8 pg / ml leupeptin, and 2 mM benzamidine. 0.5 micro Ci [y32P]ATP is added. The incubation mixture is incubated for 15 min at 37 degrees in atotal volume of 10 microliters. The reaction is stopped by spotting the reaction mixtures on 1x2 cm strips of cellulose phosphate paper (Whatman P81) and immediately washing twice for 1 hr in 0.5% H3PO4. The cellulose phosphate strips are counted in a scintillation counter. In some experiments, phosphatidylserine, diacylglycerol, and / or calcium were removed.Protein kinase C assay

[0206] Rat hippocampal cells are cultured and scraped in 0.2 ml homogenization buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaF, 1 pg / ml leupeptin, and 0.1 mM PMSF) and homogenized by sonication in a Marsonix micro-probe sonicator (5 sec, 10W). To measure PKC, 10 pl of cell homogenate or purified PKC isozyme (purchased from Calbiochem) is incubated for 15 min at 37°C in the presence of 10 pM histones, 4.89 mM CaCh, 1.2pg / pl phosphatidyl-L-serine, 0.18pg / pl 1 ,2-dioctanoyl-sn-glycerol, 10 mM MgC12 , 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4 mM EGTA, 4% glycerol, 8 pg / ml aprotinin, 8 pg / ml leupeptin, and 2 mM benzamidine. 0.5pCi [y32P]ATP is added and32P -phosphoprotein formation is measured by adsorption onto phosphocellulose as described previously. Nelson and Alkon, J. Neurochemistry. 1995; 65: 2350- 57. For measurements of activation by test compound(s), PKC activity is measured in the absence of diacylglycerol and phosphatidylserine, as described by Kanno et al., and PKC 5, a, p, and p are measured in the absence of added EGTA and CaCh , as described by Kanno et al., J. Lipid Res. 2006; 47: 1146-50. Low concentrations of Ca2+are used because high Ca2+interacts with the PKC phosphatidylserine binding site and prevents activation. For measurements of bryostatin activation, 1,2-diacylglycerol is omitted.Human Neutrophil Elastase Quenched-FRET Assay

[0207] The assay uses Human Neutrophil Elastase (HNE) purified from serum (Calbiochem art. 324681; Ref. Baugh, R. J. et al., 1976, Biochemistry. 15, 836-841). HNE is stored in 50 mM NaOAc, 200 mM NaCl, pH 5.5 with added 30% glycerol at -20°C. The protease substrate used is Elastase Substrate V Fluorogenic, MeOSuc-AAPV-AMC (Calbiochem art. 324740; Ref. Castillo, M. J. et al. , 1979, Anal. Biochem. 99,53-64). The substrate is stored in DMSO at -20°C. The assay additions are as follows: Test compounds and controls are added to black 96-well flatbottom plates (Greiner 655076), 1 L in 100% DMSO, followed by 30 yL HNE in assay buffer with 0.01% TritonX-100. The assay buffer constitution was: 100 mM Tris (pH 7.5) and 500 mM NaCl. The enzyme and the compounds were incubated at room temperature for 15 minutes. Then 30 pl substrate in assay buffer is added. The assay is stopped after 30 minutes incubation at room temperature by adding 60 pl stop solution (140 mM acetic acid, 200 mM sodiummonochloroacetate, 60 mM sodium acetate, pH 4.3). Fluorescence is measured on a Wallac 1420 Victor 2 instrument at settings: Excitation 380 nm, Emission 460 nm. IC50 values are determined using Xlfit curve fitting using model 205. When tested in the above screen, compounds giving IC50 values for inhibition of human neutrophil elastase activity of less than 30 pM, are indicative of useful therapeutic properties.MMP-12 FRET Assay

[0208] Compounds are tested in an MMP-12 FRET assay as follows. To each well of black polystyrene 96-well plate is added assay buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM CaCh and 0.005% Brij-35), purified human MMP-12 enzyme, and varied concentrations of test compounds (prepared by serial dilution of a stock solution in 100% DMSO). The plates are incubated at room temperature for 30 minutes. The enzymatic reactions are initiated by addition of a substrate, MCA-Pro-Leu-Gly-Leu-Dpa(DNP)-Ala-Arg, containing a fluorescent group (7- methoxycoumarin, MCA) and a 2,4-dinitrophenyl group (DNP), to a final concentration of 20pM. The final DMSO concentration in the assay is 10%. The reaction was monitored for 30 minutes at room temperature and the initial rate of the cleavage reaction is determined using a fluorescence plate reader (XeX: 325 nm, Xem: 395 nm). Plots of the inhibitor concentration vs. the initial cleavage rate are fit to the following equation: y = Vmax*(l-(xn / (Kn+ x11))), whereby x = inhibitor concentration, y = initial rate, Vmax = initial rate in the absence of inhibitor, n = slope factor, and K = IC50 for the inhibition curve. IC50 values of less than 30 pM, are indicative of useful therapeutic properties.Animal Experiments

[0209] Aged male C57BL / 6JRj mice are obtained from Janvier Labs at 12 months of age and habituated for 2 weeks at the animal facility of the department of Pharmacology at the University of Oxford, where all subsequent treatments are performed. The mice are housed under conditions of controlled temperature (23 °C) and illumination (12-h light / 12-h dark cycle) and allowed ad libitum access to water and standard chow (Harlan Research Laboratories) or HCD (58% fat, 25% carbohydrates, Research Diets 1223 ID). Animal care procedures are within institutional animal care committee guidelines, and all procedures are reviewed and approved by the Home Office of the United Kingdom and University of Oxford, Department of Pharmacology. Animals are treated with subcutaneous injections of the drugs mentioned above twice a week using the protocol in the table below.Animal Sacrifice and Tissue Collection

[0210] At the end of the 12 weeks treatment period, animals are anaesthetized using 4% isoflurane followed by cervical dislocation. Necks are then detached, and trunk blood is collected in a 500 pl Eppendorf tube previously filled with 100 pl 0.5M EDTA. Next, aortic tissue is isolated for its appropriate use. For RNA and protein extraction aorta tissue is isolated and immediately snap-frozen in liquid nitrogen and stored in -80 °C for future analysis. For arterial myography, the protocol is mentioned below. Biochemical AnalysisRNA Extraction

[0211] RNA extraction from primary cultured astrocytes followed the NucleoSpin® RNA protocol for RNA purification from tissue delivered within the kit from Macherey -Nagel. (Macherey -Nagel, Duren, Germany) following the manufacturer’s protocol.Reverse Transcription

[0212] Total RNA (9 pl) is used for cDNA synthesis. RNA extracted from the samples was reverse-transcribed into cDNA by First-Strand cDNA Synthesis Reaction using the Cloned AMV First-Strand cDNA Synthesis Kit (InvitrogenTM) following the protocol provided within the kit in a T-Gradient Thermocycler (Biometra). Depending on the amount of extracted RNA, samples are diluted 1 : 1 or 1 :2 with DEPC-treated water.Components of Reverse Transcriptase ReactionQuantitative real-time PCR (qPCR)

[0213] For each PCR reaction 2 pl cDNA are used with the Platinum™ SYBR™ Green qPCR SuperMix-UDG w / ROX (Invitrogen™) kit following the manufacturer’s protocol. For lower RNA concentration, the amount of cDNA could be increased up to 11 pl replacing the DEPC water in the PCR reaction mix. The standard protocol for qPCR consists of an initial cycle at 50 °C for 2 min, followed by one cycle of 95 °C for 2 min, continued for 40 cycles containing of 95 °C for 15 s to 60 °C for 1 min and finished with a final cycle of 95 °C for 15 s to 60 °C for 20 s and 95 °C lasting 15 s. Expression values are normalized to the geometric mean of several housekeeping genes peptidyl prolyl isomerase A (PPIA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta actin (P-actin). For detection of specific cDNA, oligonucleotide primers are used.Western BlotSample preparation

[0214] After protein quantification according to Lowry protein assay, 120 pl of each tissue lysate are mixed with 40 pl 4* SDS-2 -Mercaptoethanol and denatured at 95 °C for 10 min. All samples are diluted with lx SDS (4x SDS diluted with lx CLB) to the lowest protein concentration measured.Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

[0215] Proteins are separated by size via commercially available acrylamide gels in a vertical mini gel electrophoresis system (Mini-PROTEAN® Tetra Vertical Electrophoresis Cell; BioRad) under 100 V for 2.05 h. The acrylamide gel containing the separating and stacking gels are prepared after. The chamber is filled with SDS electrophoresis buffer (3.03 g Tris Base, 14.4 g Glycine and 1 g SDS / 51 per litre H2O). The first well of the stacking gel is loaded with Precision Plus Protein™ Standards (Bio-Rad) while the other wells are loaded with 50 to 100 pg of protein.Semi dry protein transfer

[0216] After SDS-PAGE the separated proteins are transferred from the separating gel to a Immobilon-P membrane (Millipore) by semi dry protein transfer. Membrane and filters are soaked in Towbin blotting buffer (3.03 g Tris Base, 14.41 g glycine and 1 g SDS in 800 ml and 200 ml Methanol (MeOH), pH 8.2 - 8.4) and stacked in a Hoefer® SemiPhor™ Electrophoresis Blotter TE-70 (Amersham Pharmacia Biotech). The transfer will run for 1.5 h at 100 V.Immuno-Staining

[0217] The proteins, now transferred to the membrane, are blocked with 5 % Bovine Serum Albumin (BSA, Sigma-Aldrich®) and immune-stained first with antibodies against Elastin, Neutrophil Elastase, MMP12, Fibulin5 / DANCE, MAP2K1 at 4 °C, overnight. The following day membranes are washed with 1 x PBS and incubated in a horseradish peroxidase (HRP) secondary antibody for Ih at room temperature (RT). The secondary antibody bound to the respective protein are made visible by a 1 :4 dilution of the HRP enhanced-chemiluminescent-substrate SuperSignal® West Femto- Substrate (Thermo Scientific™) in H2O using a FUSION SOLO S (Vilber).Immunohistochemistry

[0218] Three proximal thoracic aortas per group are selected randomly and >9 tissue sections per aorta are generated using a cryostat. Tissue sections (10 pm) are generated from the aortic root to the aortic arch at 100-pm intervals. Three sections corresponding to the region of largest dilation between the sinotubular junction, and the arch are analyzed. Multiple high-magnification images are acquired of each section that are subsequently aggregated to a single image of the entire section using ImageJ.20.

[0219] For histological analyses, the distal part of the aortic arch (between the left subclavian artery and the beginning of the descending thoracic aorta) are fixed with formalin and embeddedin paraffin (FFPE), then cut into 5 pm thick sections. Staining is performed after dewaxing and rehydration up to water.

[0220] Standard immunohistochemistry is performed for Neutrophil Elastase, MMP12, Fibulin / DANCE5, and MAP2K1 using the immunostaining protocol mentioned above, performed on sections of the aorta, where the sections are reacted with an antibodies specific for Neutrophil Elastase, MMP12, Fibulin / DANCE5, and MAP2K1 overnight at 4 °C followed by a goat anti-mouse IgG conjugated with Alexa 488 (Molecular Probes, Eugene, OR, USA) for 60 min at room temperature. After staining with 4’,6-diamidino-2-phenylindole (DAPI), fluorescence-labeled cells are visualized with a confocal scanning laser microscope (Axiovert / LSM510; Carl Zeiss, Oberkochen, Germany).Measuring Arterial Stiffness and Elasticity

[0221] Contraction of isolated artery (aortic, mesenteric, and pulmonary) rings is measured using a small vessel wire myograph (model 420A, Danish Myo Technology, Aarhus, Denmark). Tension measurements (in mN) are normalized for the artery ring length (in mm) to enable comparison of data obtained from rings of slightly different length. Tissue is stimulated with blue light of various intensities (0.02-7.21 mW mm-2). Duration of the pulses is 7 min, unless stated otherwise.

[0222] The tension versus blue light intensity relationships in the absence of PE is fitted with the Hill equation of the form: T=Tssl+(EL5olight intensity)s, where ‘T’ is the tension (normalized for length of the artery ring) measured at a given light intensity, ‘T ss’ is the maximal asymptotic tension reached, ‘EL50’ is the light intensity causing half-maximal tension, and ‘s’ is the slope coefficient.Results

[0223] Elastin is a key extracellular matrix (ECM) protein that provides resilience and elasticity to tissues and organs. It is the dominant protein in extensible tissues and is especially important to preserve the cardiovascular system, as well as lungs and skin. Degradation of elastin due to aging leads to reduced tissue function and is associated with various age-related diseases.

[0224] The current proof-of-concept indicates an increase in elastin expression (Fig. 1A) as well as a functional increase of tensile strength of the arteries of aged mice compared to a saline control (Fig. 2). As will be understood by those skilled in the art, the arterial stiffness above 1800 pM stretch is not physiologically relevant. The improved arterial stiffness around 600 to 1200 pM stretch is attributed to increased expression, stability, activity, or any combination thereof,of elastin in the mice. Indeed, this could have major implications in treating diseases that occur due to elastin loss as well as related decline in elastin amount and quality. With one or multiple therapeutic approaches, the inventors suggest to utilize one or multiple molecular pathways to achieve this, inclusive of activation of PKC, inhibition of neutrophil elastase and matrixmetalloproteinases. Indeed, the currently suggested therapeutic approach has implications for abdominal aortic aneurysm, supravalvular aortic stenosis, Marfan syndrome, and Cutis Laxa, to name a few, but not limited thereto.EXAMPLE 2

[0225] A further in vivo study was conducted in mice to study the effects of the small molecules, or their combinations, as disclosed herein on certain apsects of the aging process.Materials and methodsAnimal Experiments:

[0226] All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986, and were approved by the Animal Welfare and Ethical Review Body of the University of Oxford. Animals were kept in standard laboratory housing conditions (12 h light / dark cycle, lights on at 7am; 21 ± 1°C; humidity 50 ± 5%) with access to both food and water ad libitum. All experimental conditions were set up and maintained in accordance with the European Communities Council Directive for the care and use of laboratory animals (2010 / 63 / EU) and the ARRIVE guidelines (http: / / www.nc3rs.org.uk / arrive-guidelines). Aged C57BL / 6JRj mice of 12 months old were treated with drug combinations for 12 weeks twice weekly as follows:Behaviour

[0227] The Open Field Test (OFT) was used to measure behaviour at the end of the 12-week treatment period, prior to tissue collection. The OFT apparatus consisted of four quadrants of equal dimensions (1 * w x h; 50 cm * 50 cm x 50 cm) to enable four mice to be tested simultaneously. The open field was defined as 25% of the total area of each quadrant (1 x w; 25 cm x 25 cm). Mice were placed in a randomised order in the top left comer of each quadrant and were video recorded for 5 min. 75% ethanol was used to wipe the floors and walls in between experiments to prevent the influence of odorants and coprophagy on mouse behaviour. ANY- Maze software (Stoelting Co., IL, USA) was used to measure all behavioural parameters except rearing, which was counted manually whilst blinded and was defined as any time a mouse was raised on its two hindlegs (supported by walls or free-standing).Coat analysis

[0228] At the end of the 12-week treatment period, prior to tissue collection mice coats were analyzed for color and quality.Tissue Collection

[0229] Mice were anaesthetised under isoflurane gas after 12 weeks of treatment. After checking for the absence of a pedal reflex, cardiac puncture was used to collect whole blood in EDTA (0.1 M, pH8)-coated tubes and centrifuged after 30 min at RT (10 min, 2000xg). Tissue was transcardially perfused with heparinised saline until the liver was clear of blood. Whole brains were extracted and snap-frozen in isopentane on dry ice and liver samples were collected and immediately snap-frozen on dry ice. All tissue and plasma was stored at -80°C for subsequent gene expression.Arterial Myography-Stress-Strain Curve

[0230] At the end of the experiment, mice (n =5) were euthanized and arteries were dissected free from the surrounding tissue and one 2 mm ring is dissected and mounted in 5 mL myographs containing physiological saline solution (composition: 119.0 mM NaCl, 4.7 mM KC1, 1.2 mM MgSO 4 , 24.9 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 , 11.1 mM glucose) aerated with 95% 0 2 / 5% CO 2 gas mix, warmed and maintained at approximately 37 ° C throughout the experiment. Changes in force production following 100 pm cumulative stretches were detected by force transducers and data captured using Lab Chart 7 software (ADInstruments).Arterial Myography-Contraction and RelaxationRNA Extraction and Reverse Transcription

[0231] RNA was isolated from 15 to 20 mg of aorta or brain tissue according to manufacturer’s instructions [RNeasy® Mini Kit (Cat No. / ID: 74106)]. Liver and PFC RNA was eluted in nuclease-free water and 1000 ng was converted to cDNA with the Applied Biosystems High Capacity cDNA conversion kit, following manufacturer’s instructions. The reverse transcription step was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher, cat. 4368813) using manufacturer’s instructions. qPCR

[0232] Real-time qPCR was performed with samples in duplicate (25 ng / well) using the SsoAdvanced Universal SYBR Green Supermix (BioRad) with the Roche LightCycler 480. Transcripts of the acute phase response have previously been shown to be translated to protein (Campbell et al., 2003). Upon reaction completion, melt curve analysis was carried out to ensure primer specificity. The Ct values were normalised to the housekeeping gene, GAPDH or beta actin, and were represented as the fold change relative to saline controls using the delta-delta CT (2-A A CT) method. For detection of specific cDNA, oligonucleotide primers are used.NAD and glutathione metabolite analysis from frozen blood

[0233] Analysis was carried out by NADMed (Helsinki, Finland; www.nadmed.com). NAD and glutathione metabolites were extracted from frozen blood in a single step using a proprietary extraction procedure, and each metabolite was measured individually using optimized cyclic enzymatic assays with colorimetric detection. For stability, the NADMed extraction method utilizes an extraction solution representing a non-buffer complex mixture of organic solvents in water. Samples are injected into a pre-warmed extraction solution to force protein unfolding and release non-covalently bound metabolites into solution. All pH and redox sensitive metabolites remain stable, including reduced and oxidized glutathione. After protein removal, supernatants are used for separate measurements of total GSH and GSSG. GSH is determined as the difference of total GSH pool and GSSG values. Similarly, supernatants are used for separate measurements of total NAD and NAD+, and for separate measurements of total NADP and NADP+. NADH is determined as the difference of total NAD and NAD+. GSH is determined as the difference of total GSH and GSSG.Data Analysis

[0234] All statistical analyses were carried out using the GraphPad Prism 9 software. Any outliers were identified using the GraphPad Outlier Calculator and were removed accordingly. The Shapiro-Wilk normality test was used to test for normality and log transformation was applied for the statistical analysis in instances that the test was not passed. Two-way analysis of variance (ANOVA) was used to assess a main effect of treatment (TLR agonist administration) on behaviour, relative mRNA expression, and spectral intensity. Post-hoc Tukey’s tests were then used for multiple comparisons if there was a significant interaction between treatment and sex. The significance threshold was set to *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and all results were presented as mean ± standard error of the mean (SEM). All correlations were performed via Pearson correlational analyses and were corrected for false discovery rate (FDR).NMR metabolomicsSample processing

[0235] Metabolites were extracted plasma samples as previously described (Radford-Smith et al., 2022). Samples were kept on dry ice before adding 8 uL / mg sample of ice-cold acetonitrile (50%). Samples were then vortexed for mixing and centrifuged at 5060 x g for 5 min at 4°C. Supernatants were snap frozen on dry ice, lyophilised overnight and stored at -80°C until the day of NMR. When ready for analysis, lyophilised samples were placed at room temperature and resuspended in 550 uL of NMR buffer (75 mM sodium phosphate buffer prepared in D2O, pH 7.4). The plasma samples were also allowed to thaw at RT and 150 uL was added to 400 uL NMR buffer.NMR experiments

[0236] All prepared samples were then placed in a 5 mm NMR tube and measured using a 700- MHz Bruker AVII spectrometer operating at 16.4 T, equipped with a 1H (13C / 15N) TCI cryoprobe, as described previously (Chan et al., 2020). Plasma and liver spectra were acquired using a spin-echo sequence (Carr-Purcell-Meiboom-Gill [CPMG]), 32 data collections, an acquisition time of 1.5 s, a relaxation delay of 2 s, and a fixed receiver gain. Brain spectra were acquired with a Nuclear Overhauser Effect Spectroscopy (NOSEY) sequence as previously described (Chan et al., 2020). All samples were run within 9-h of being thawed.NMR Data Processing

[0237] Spectra were phased, baseline-corrected and referenced to the lactate doublet at 5 = 1.33 ppm in Topspin 4.1.4 (Bruker). ACD / Labs Spectrus Processor Academic Edition 12.01(Advanced Chemistry Development, Inc.) was used to manually bin each resonance signal, excluding all noise from analysis. The integrals of these bins were sum normalised and exported to R. Metabolites assignment was conducted through a mix of in-house databases, literature reviews, and 2D total correlation spectroscopy (TOCSY) experiments.

[0238] Spectral intensityStatistical analysisUnivariate

[0239] All statistical analyses were carried out using the GraphPad Prism 9 software. Any outliers were identified using the GraphPad Outlier Calculator and were removed accordingly. The Shapiro-Wilk normality test was used to test for normality and log transformation was applied for the statistical analysis in instances that the test was not passed. Two-way analysis of variance (ANOVA) was used to assess a main effect of treatment (TLR agonist administration) on behaviour, relative mRNA expression, and spectral intensity. Post-hoc Tukey’s tests were then used for multiple comparisons if there was a significant interaction between treatment and sex. The significance threshold was set to *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and all results were presented as mean ± standard error of the mean (SEM). All correlations were performed via Pearson correlational analyses and were corrected for false discovery rate (FDR).Multivariate

[0240] The ROPLS package in R was used for orthogonal partial least squares discriminant analysis (OPLS-DA) of the metabolomes to determine whether there were any between group differences. Variable importance in projection (VIP) scores were used to detect which metabolites were most important for driving separation between groups. Bins with highest VIP scores (before a point of inflexion when comparing all VIP scores) then underwent univariate analyses to understand the direction of change of metabolites, as described above.Results

[0241] This study was conducted to show that the small molecules, or their combinations as disclosed herein, are suitable for treating aging-related diseases as well as (age-related) neurodegenerative conditions. Compared to untreated mice, significant improvements were observed in mice treated with the small molecules or their combinations according to the invention.Coat analysis

[0242] Compared to untreated mice and mice treated with either DPC-LA, Alvelestat, MMP-408 or U0126, mice treated with DCP-LA + Alvelestat, DCP-LA + MMP-408 and DCP-LA + U0126 showed a markedly enhanced coat color (from spotted gray to all black) as well as a better coat quality with less coat damage.NAD and glutathione metabolite analysis from frozen blood

[0243] Nicotinamide adenine dinucleotide (NAD) plays a dual role as a reduction-oxidation (redox) co-enzyme, in its oxidized (NAD+) and reduced (NADH) forms, and as a co-substrate (NAD+) for enzymes involved in deacylation, mono- and poly-ADP-ribosylation, and the creation of adenine-based second messengers. NAD+ and NADH can be phosphorylated to NADP and NADPH, respectively, by NAD+ Kinases (NADKs). NADP and NADPH fulfil similar roles as NAD+ and NADH in the metabolic pathway.

[0244] The NAD(P)+ / NAD(P)H ratio is an important indicator of the intracellular redox state and plays a critical role in the regulation of key metabolic pathways. NAD(P)(H) status has been shown to affect cell signaling and metabolism, energy homeostasis, mitochondrial biogenesis, oxidative stress responses and DNA maintenance and repair. Furthermore, the progression of age-related diseases, such as obesity, non-alcoholic fatty liver disease, neurodegeneration, mitochondrial and cardiovascular diseases, and muscle atrophy or dystrophy, has been linked to alterations in NAD+-related metabolites.

[0245] The blood acts as a buffer for NAD+ and its metabolites. Aging leads to lower NAD+ levels in blood reaching around 20 uM in aged mice over 12 months of age. However, blood NAD+ levels alone are not indicative for overall metabolic health, and correlation to behaviour and other markers are necessary.

[0246] The current study indicates that treatment of 12 month-old mice with the combination of DCP-LA + MMP-408 and DCP-LA + U0126 for 12 weeks twice weekly led to a significant reduction in NAD+ levels in blood (Figure 3 A). Similarly, the treatment with the combination of DCP-LA + MMP-408 and DCP-LA + U0126 led to a significant reduction in NADP+ levels in blood (Figure 3B). This reduction in blood levels is indicative for higher uptake of NAD+ and NADP+ by the tissue, therewith pointing towards higher energy use by tissues and counteracting catabolic stress due to aging. This positive effect on aging is further supported by the higher glutathione (GSH) levels in blood (Figure 3C). Given the fact that GSH fulfills vital functions such as antioxidant and cellular repair functions, the significant increase of GSH levels in blood of mice treated with the combination of DCP-LA + MMP-408 and DCP-LA + U0126, ascompared to GSH levels in blood of untreated mice (Figure 3C) further supports the positive effect of combined treatment on aging.NMR metabolomics

[0247] Aging is associated with impaired protein metabolism. There is a general decline in levels of amino acids in blood compared to younger age. However, due to muscle loss and tissue damage, amino acids such as lysine and valine show increased concentrations in blood compared to younger age.

[0248] The current data indicates that treatment of 12 month-old mice with the combination of DCP-LA + MMP-408 and DCP-LA + U0126 for 12 weeks twice weekly led to a significant reduction in lysine levels in blood (Figure 4A). Similarly, the treatment of 12 month-old mice with the combination of DCP-LA + U0126 for 12 weeks twice weekly led to a significant reduction in valine levels in blood (Figure 4A). Lysine and valine levels as well showed a trend of reduction in mice treated with DCP-LA only and DCP-LA + Alvelestat as compared to control mice. These results are indicative for less muscle loss, reduced aging-associated catabolic stress and higher autophagy in tissue, which supports better longevity.Behaviour

[0249] Aging is associated with various behavioral changes that are mediated by brain structures and networks. In rodents, age-related changes in behavior have been demonstrated by comparisons of young and old-aged animals. A number of studies have reported that compared to young mice, aged mice exhibit declines in behavioral performance related to locomotor activity and anxiety-like behavior.

[0250] To measure behavior of the mice at the end of the 12-week treatment period the Open Field Test (OFT) was employed. The open field test is an experimental test used to assay general locomotor activity levels, anxiety, and willingness to explore in animals. Current data shows improved behavioral markers in tests assessing mobility and anxiety in mice treated with small molecules or their combinations according to the invention, as compaired to untreated control mice.

[0251] Rearing behavior, in which rodents stand on their hind legs to explore, provides a measure of mobility and anxiety in test rodents. 12 month-old mice treated with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA + Alvelestat, DCP-LA + MMP-408 and DCP-LA + U0126 showed markedly increased rearing behavior compared to control mice (Figure 5A, 5B, 5C, 5D). This is indicative for increased levels of mobility and decreased levels of anxiety.

[0252] Reduced levels of mobility and increased levels of anxiety, which can be associated with aging, results in less locomotionl2 month-old mice treated with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA + MMP-408 and DCP-LA + U0126 are more active (as represented by active time in seconds) compared to control mice (Figure 6A, 6B, 6C, 6D). This is indicative for increased levels of mobility and decreased levels of anxiety.

[0253] Reduced levels of mobility and increased levels of anxiety results in a preference to stay close to the walls of the field, while increased levels of mobility and reduced levels of anxiety is correlated with more time spent in the center zone of the field. 12 month-old mice treated with MMP-408, U0126, DCP-LA + MMP-408 and DCP-LA + U0126 did spent less time in the center zone compared to control mice (Figure 7C, 7D). This behavior is indicative for increased levels of mobility and decreased levels of anxiety.

[0254] 12 month-old mice treated with DCP-LA, Alvelestat, MMP-408, U0126, DCP-LA + Alvelestat and DCP-LA + MMP-408 showed markedly reduced immobile time compared to control mice (Figure 8A, 8B, 8C, 8D). This behaviour is indicative for increased levels of mobility and decreased levels of anxiety.EXAMPLE 3

[0255] A further in vivo study was conducted in mice to study the effects of the small molecules, or their combinations, as disclosed herein, on gene expression changes in the elastin synthesis pathway; gene expression changes in inflammatory cytokines; changes in expression of genes involved in Williams Syndrome; behavior; live myopgraphy and metabolomics.Materials and methodsAnimal Experiments and Treatments

[0256] Aged C57BL / 6JRj mice of 12 months old were treated with saline or any drug combination as indicated in Table 3.1 (and visualized in Figure 9), for 12 weeks twice weekly.Table 3.1 : treatments

[0257] All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986, and were approved by the Animal Welfare and Ethical Review Body of the University of Oxford. Animals were kept in standard laboratory housing conditions (12 h light / dark cycle, lights on at 7am; 21 ± 1°C; humidity 50 ± 5%) with access to both food and water ad libitum.Behaviour

[0258] The Open Field Test (OFT) was used to measure behaviour at 20 h post-injection, prior to tissue collection. The OFT apparatus consisted of four quadrants of equal dimensions (1 x w x h; 50 cm x 50 cm x 50 cm) to enable four mice to be tested simultaneously. The open field was defined as 25% of the total area of each quadrant (1 x w; 25 cm x 25 cm). Mice were placed in a randomised order in the top left corner of each quadrant and were video recorded for 5 min. 75% ethanol was used to wipe the floors and walls in between experiments to prevent the influence of odorants and coprophagy on mouse behaviour. ANY-Maze software (Stoelting Co., IL, USA) was used to measure all behavioural parameters except rearing, which was counted manually whilst blinded and was defined as any time a mouse was raised on its two hindlegs (supported by walls or free-standing).Tissue Collection

[0259] Mice were anaesthetised under isoflurane gas after 12 weeks of treatment. After checking for the absence of a pedal reflex, cardiac puncture was used to collect whole blood in EDTA (0.1 M, pH8)-coated tubes and centrifuged after 30 min at RT (10 min, 2000xg). Tissue was transcardially perfused with heparinised saline until the liver was clear of blood. Whole brains were extracted and snap-frozen in isopentane on dry ice and liver samples were collected and immediately snap-frozen on dry ice. All tissue and plasma was stored at -80°C for subsequent gene expression.RNA Isolation from aorta samples

[0260] RNA was isolated from 10 to 15 mg of aorta samples according to manufacturer’s instructions [RNeasy® Mini Kit (Cat No. / ID: 74106)]. Aorta RNA was eluted in nuclease-freewater and 1000 ng was converted to cDNA with the Applied Biosystems High Capacity cDNA conversion kit, following manufacturer’s instructions. The reverse transcription step was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher, cat. 4368813) using manufacturer’s instructions.

[0261] 8,5 pl of RNA isolated from aorta samples were treated for 10 minutes at 37°C with RNase-free DNase I (Thermo Fisher Scientific, Cat. EN0521) before reaction was stopped using 1 pl reaction buffer for 2 minutes at 70°C. RNA quality and concentration was analyzed via Agilent RNA 6000 Pico Kit (Cat. 5067-1513).RNA isolation from brain hemispheres

[0262] Total RNA was extracted from snap-frozen brain-hemispheres using the miRNeasy Mini Kit (Qiagen, Germany, Cat. 217004). Each sample was divided into 3 equally sized sections and homogenized in 800 pl Qiazol each with Lysing Matrix Z (Cat. 116961500 ) using a Bead Beater FastPrep24 5G (MP Biomedicals™). Homogenates of each sample were pooled together and 400 pl were used for further isolation according to the manufacturer’s instruction, while remaining liquid was stored at -80°C. After incubation at room temperature for 10 minutes, 140 pL chloroform were added to the lysates followed by cooled centrifugation at 12,000g for 15 minutes at 4°C. Precisely 350 pL of the upper aqueous phase were transferred to a miRNeasy mini column, and RNA was precipitated with ethanol followed by automated washing with RPE and RWT buffer in a Qiacube liquid handling robot. Finally, total RNA was eluted in 30 pL nuclease free water and stored at -80°C until further analysis. RNA quality and concentration was analyzed via Agilent RNA 6000 Nano Kit (Cat. 5067-1511). mRNA Sequencing Analysis

[0263] The QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with Unique Dual Indices (Lexogen) was used for library preparation. For RNA isolated from brain tissue, the standard input protocol was followed, while DNase Ltreated aorta RNA was prepared according to the low input / low quality protocol as per the manufacturer’s instructions. In brief, reverse transcription was performed using the QuantSeq kit’s reverse transcription mix, followed by second strand synthesis using the second strand synthesis mix provided in the kit. The resulting cDNA was purified using magnetic beads and amplified by PCR. To determine amplification cycle numbers, the PCR add-on and Reamplification Kit V2 (Lexogen) was applied. Accordingly, brain tissue RNA were amplified using 15 PCR-cycles, while aorta RNA was amplified with either 22 or 24 cycles. Amplified cDNA was then purified again to remove anyremaining contaminants. The quality of the prepared libraries was assessed using an Agilent High Sensitivity DNA Kit (Cat. 5067-4626). An equimolar pool consisting of all sequencing libraries was prepared and sequenced on an Illumina NextSeq 2000. NGS data were processed using the meND pipeline. qPCR

[0264] Real-time qPCR was performed with samples in duplicate (25 ng / well) using the SsoAdvanced Universal SYBR Green Supermix (BioRad) with the Roche LightCycler 480. Transcripts of the acute phase response have previously been shown to be translated to protein (Campbell et al., 2003). Upon reaction completion, melt curve analysis was carried out to ensure primer specificity. The Ct values were normalised to the housekeeping gene, GAPDH, and were represented as the fold change relative to saline controls using the delta-delta CT (2-A A CT) method. Primer sequences can be found in Table 3.2. Table 3.2: Primer sequencesData Analysis

[0265] All statistical analyses were carried out using the GraphPad Prism 9 software. Any outliers were identified using the GraphPad Outlier Calculator and were removed accordingly. The Shapiro-Wilk normality test was used to test for normality and log transformation was applied for the statistical analysis in instances that the test was not passed. Two-way analysis of variance (ANOVA) was used to assess a main effect of treatment (TLR agonist administration) on behaviour, relative mRNA expression, and spectral intensity. Post-hoc Tukey’s tests were then used for multiple comparisons if there was a significant interaction between treatment and sex. The significance threshold was set to *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and all results were presented as mean ± standard error of the mean (SEM). All correlations were performed via Pearson correlational analyses and were corrected for false discovery rate (FDR).Myography

[0266] Dissected blood vessels, cleaned of any connective and adipose tissue, were cut into 2 mm rings. PA and aorta rings were threaded onto two parallel pins in a 5 ml chamber of a multimyograph system (Danish Myo Technology, Model 610M). As the mesentery resistance arteries were considerably smaller in size (100-200 pm diameter), wire myography was employed instead. Mesenteric artery (MA) rings were threaded onto two stainless steel wires (40 pm diameter), which were mounted in 5 ml myograph chambers of the same multi-myograph system. Vessels were bathed in 5 ml PSS, continually aerated at a 37 °C. In terms of the myograph setup, in brief, one of mounting pins / arms remains in a fixed position and is attached to a force transducer, while the other is attached to a micrometer and allows for the adjustment in vessel diameter. The force transducer, which is able to detect changes in force, as small as 0.01 mN, is connected to a computer via an analogue to digital interface (National Instruments, USB-6221- BNC).

[0267] Once the vessels were mounted a basal tension was applied. The vasoreactivity of vessels has been shown to be dependent on the degree of stretch (Schubert et al., 1999). It is, therefore, important to define the amount of basal tension applied to each type of vessel and ensure that an internal circumference, providing a maximum response, is used. The amount of basal tension applied to each type of vessel was defined as the tension required to achieve a maximum response to 50 mM KC1. The procedure was carried out as follows: vessels were subjected to 50 mM KC1 challenges, under low tension (~1 mN), until a consistent contraction was observed. The vessels were then subjected to further KC1 challenges under increasing increments in tension, using the micrometer, until a maximum response was achieved. Performing this procedure, it was found that 2 mm artery rings of PA, MA and aorta required tensions of 4 mN, 6mN and lOmN, respectively, to achieve a maximum KC1 response, thus these basal tensions were applied to each type of vessel at the start of the experiments. Once the basal tension had been applied the vessels were allowed to equilibrate for 30-45 min, replacing the bath solution with fresh PSS every 15- 20 min.

[0268] Following the equilibration period, the viability of the preparations was tested by challenging the vessels with 50 mM KC1. Vessels were exposed to 50 mM KC1 for 6 min followed by washout with fresh PSS. This process was repeated a further two times or until a consistent KC1 response was observed. Vessels were deemed viable if i) a maximum contraction was observed within 5 min of KC1 application and ii) a steady baseline tension was observed between each KC1 challenge.

[0269] Upon washout of the final KC1 response the vessels were left to re-establish baseline tension before any subsequent experiments were begun.

[0270] Contractility of the vessels was assessed with increasing phenylephrine concentration (PE; 1 pM), an alpha adrenergic agonist. A steady maximum contraction to PE was reached, then increasing concentrations of acetylcholine were applied to the vessel to measure the relaxation response until a steady relaxation was reached. The data obtained from each experiment were fit to the Hill equation and EC50 values were extrapolated. The EC50 value\s were also converted to pEC50 values, which were used for statistical comparison of the data. For display purposes results from each set of experiments were pooled together and mean concentration response data were plotted. Where possible, a curve was fitted to the mean data using the Hill equation. mRNA Sequencing Analysis

[0271] RNAseq revealed transcriptome-wide changes in both the brain and aorta following Elastin therapies. The heatmaps for both tissues (Figure 35 and Figure 36 respectively) revealthat the EB-102-101 combination yielded the most exaggerated differential expression of genes relative to controls. Kegg pathway analysis revealed the most impacted pathways following EB- 102-101 treatment were aminoacyl tRNA biosynthesis (adjusted p=6.6E-7), ribosome (p=3.5E- 6), COVID-19 (p= 0.00014), Amyotrophic Lateral Sclerosis (p= 0.003), Alzheimer Disease (p=2.5E-2), and Huntington Disease (p= 0.035). Pathways that were significantly impacted but did not survive multiple comparison include pathways of neurodegeneration - multiple diseases, thermogenesis, Parkinson Disease, autophagy, and oxidative phosphorylation, to name a few. These are important to note as some of the key genes impacted in these pathways may still have important biological significance. Kegg pathway analysis of the aorta showed more dramatic changes, with the following pathways as the top few of 68 significantly altered pathways following correction for multiple comparisons: Prion disease (p=1.2E-50), Parkinson disease (p=1.2E-50), Oxidative phosphorylation (p=3.3E-49), Diabetic Cardiomyopathy (p=2.2E-46), and Alzheimer Disease (p=2.6E-43). Of interest in the longevity field, significant impact is seen on the insulin pathway (p=1.6E-6), AMPK signalling (p=1.7E-6), HIF-1 signalling (p=6.7E-4), cellular senescence (p=7.3E-3), mTOR signalling (p=1.6E-2), autophagy (p=1.6E-2), mitophagy (p=2.1E-2), and longevity regulating pathways (p=2.2E-2). Of particular note, NFkB was significantly downregulated in EB-101-102 mice relative to controls (p=0.01 following correction for multiple comparisons). In the aorta, the other groups show similar pathway impacts, but to a lesser extent. In the brain, only two genes remained significant in EB- 102- 103 compared to controls and there were no genes in the EB-102 single treatment group that remained significantly altered compared to controls.NAD and glutathione metabolite analysis from frozen blood

[0272] Analysis was carried out by NADMed (Helsinki, Finland; www.nadmed.com). NAD and glutathione metabolites were extracted from frozen blood in a single step using a proprietary extraction procedure, and each metabolite was measured individually using optimized cyclic enzymatic assays with colorimetric detection. For stability, the NADMed extraction method utilizes an extraction solution representing a non-buffer complex mixture of organic solvents in water. Samples are injected into a pre-warmed extraction solution to force protein unfolding and release non-covalently bound metabolites into solution. All pH and redox sensitive metabolites remain stable, including reduced and oxidized glutathione. After protein removal, supernatants are used for separate measurements of total GSH and GSSG. GSH is determined as the difference of total GSH pool and GSSG values.NMR Metabolomics - Sample collection

[0273] Whole blood was collected via cardiac puncture into EDTA tubes. After 30 minutes at room temperature (RT), the tubes were centrifuged at 1500 xg for 10 minutes, RT. The plasma was then snap frozen on dry ice and transferred immediately into a -80°C freezer. On the day of analysis, plasma samples were thawed at room temperature and mixed by pipetting. 75uL of plasma was then added to 475uL of 75 mM sodium phosphate buffer prepared in D2O (pH 7.4) and transferred to a 5mm borosilicate glass NMR tube (Norell 502-7) using a glass pipette. All NMR samples were prepared on the day of analysis. The NMR experiments were acquired using a 950 MHz NMR spectrometer equipped with a TCI cryoprobe (Department of Biochemistry, University of Oxford) under parameters described previously (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(l l):1326-37. pmid:30016408NMR Metabolomic - analysis

[0274] 1H NMR spectra from each plasma sample was processed as previously described (See: Dunstan et al. Unique pathways downstream of TLR-4 and TLR-7 activation: sex-dependent behavioural, cytokine, and metabolic consequences. Front Cell Neurosci. 2024; 18. doi: 10.3389 / fncel.2024.1345441). Briefly, spectra were phased, referenced, and baseline- corrected in TopSpin 4.1.4 (Bruker). ACD / Labs Spectrus Processor Academic Edition 12.01 (Advanced Chemistry Development, Inc.) was used to manually bin each resonance signal and the integrals were sum normalised and exported to R for analysis. Principle Component Analysis (PCA), as well as (Orthogonal) partial least squares discriminatory analysis ((O)PLS-DA) using the ropls package were used to investigate metabolomic signatures of each group. Variable importance in projection (VIP) scores and loadings were used to detect which metabolites were most important for driving separation between groups. Bins a VIP score above 1.5 (or before a point of inflexion when comparing all VIP scores) then underwent univariate analyses to understand the direction of change of metabolites between groups. Metabolites were assigned using 2D-spectroscopy, spiking with reference compounds, literature values, or reference to inhouse databases.ResultsGene Expression Changes in the Elastin Synthesis Pathway

[0275] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and , in particular, of their combinations (versus control) on elastin, neutrophil elastase, MMP-12, MAP2K1 and Fibulin-5 are shown in Figures 10(a-e) - 13 (a-e).

[0276] As can be derived from these figures, Alvelestat (EB-101) + DCP-LA (EB-102) combination has led to a five-fold increase in elastin expression in aortas of mice treated for 12 weeks. In parallel MAP2K1 was increased by five-fold and Fibulin-5 was increased by 9 fold. Both are markers of elastin synthesis. Neutrophil Elastase and Macrophage Elastase were increased by three fold and five-fold respectively. This is expected as a compensatory mechanism to elastase inhibition. All these increases were statistically significant compared to the control and to treatment with single therapy. It is notable that none of the single therapies have caused any significant increase in expression of any of the genes in question.

[0277] MMP-408 (EB-103) + DCP-LA (EB-102) combination has led to a three-fold increase in elastin expression in aortas of mice treated for 12 weeks. In parallel MAP2K1 and Fibulin 5 were both increased by three-fold and Fibulin-5 was increased by 9 fold. Both are markers of elastin synthesis. Neutrophil Elastase and Macrophage Elastase (MMP-12) were increased by five-fold each. This is expected as a compensatory mechanism to elastase inhibition. All these increases were statistically significant compared to the control and to treatment with single therapy except for MAP2K1. It is notable that none of the single therapies have caused any significant increase in expression of any of the genes in question.

[0278] MMP-408 (EB-103) + DCP-LA Alvelestat (EB-101) + DCP-LA (EB-102) combination has led to a three-fold increase in elastin expression in aortas of mice treated for 12 weeks. In parallel MAP2K1 and Fibulin 5 were both increased by three-fold and Fibulin-5 was increased by 9 fold. Both are markers of elastin synthesis. Neutrophil Elastase and Macrophage Elastase (MMP-12) were increased by five-fold each. This is expected as a compensatory mechanism to elastase inhibition. All these increases were statistically significant compared to the control and to treatment with single therapy except for MAP2K1. It is notable that none of the single therapies have caused any significant increase in expression of any of the genes in question.

[0279] U0126 (EB-104) + DCP-LA (EB-102) combination has led to a four-fold increase in elastin expression in aortas of mice treated for 12 weeks. In parallel MAP2K1 and Fibulin 5 were both increased by three-fold and Fibulin-5 was increased by seven fold. Both are markers of elastin synthesis. Neutrophil Elastase and Macrophage Elastase (MMP-12) were increased by three-fold and four-fold respectively / All these increases were statistically significant comparedto the control and to treatment with single therapy except for MAP2K1. It is notable that none of the single therapies have caused any significant increase in expression of any of the genes in question.Gene Expression Changes in Inflammatory Cytokines

[0280] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and, in particular, of their combinations (versus control) on inflammatory markers, TNFa , IL-ip, and IL-6 are shown in Figures 14(a-c) - 17 (a-c).

[0281] As can be derived from these figures, Alvelestat (EB-101) + DCP-LA (EB-102) combination has led to an increase of TNF expression, without any statistical significance, while IL-6 was not affected by any of the treatments. IL-ip was increased 10-fold by the single therapy with Alvelestat (EB-101).

[0282] MMP-408 (EB-103) + DCP-LA (EB-102) combination did not affect TNF. Similar to EB-101 + EB-102, IL-ip was increased 4 foldby the single therapy with MMP-408. Interestingly, the combination therapy has led to a 60% decrease in IL-6. This is consistent with reduced elastin degradation.

[0283] U0126 (EB-104) + DCP-LA (EB-102) combination has led to an increase of TNF expression by 1.5 fold , without any statistical significance, while IL-6 was not affected by any of the treatments. IL-ip was increased 3 -fold by the single therapy with Alvelestat (EB-101)Gene Expression Changes in Genes involved in Williams Syndrome

[0284] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and, in particular, of their combinations (versus control) on the expression of genes involved in Williams Syndrome (Claudin-4, BCL7B, NCF-1, Claudin-3, GTF2I, RFC2), in aortic tissue samples, are shown in Figures 18(a-f) - 20(a-f).

[0285] Alvelestat (EB-101) + DCP-LA (EB-102) treatment has led to a 2.5 fold increase in Claudin 4 expression compared to a 1.8 fold increase with EB-101 alone. Claudin 3 expression was reduced in expression by single and double therapy, 60% reduction by EB-101 and 25% reduction by EB-102 + EB-101 and EB-102 alone as a single therapy. BCL7B was reduced by 60% with EB-102 treatment and increased by 2.5 fold by EB-101 alone. The double treatment did not have any effect. EB-101 increased GTF2I expression by 3-fold. RFC2 was 50% reduced by the double therapy of EB-102 + EB-101 as well as the single therapy of EB-102. NCF-1 was unaffected by any of the treatments.

[0286] MMP-408 (EB-103) + DCP-LA (EB-102) combination increased Claudin-3 and Claudin- 4 expression by 5 fold each and BCL7B expression by 2 fold. All treatments reduced the expression of RFC2 by 50%. MMP-408 (EB-103) + DCP-LA (EB-102 reduced NCF-1 expression by 50%. BCL7B was increased by 2 fold with EB-103 single therapy.

[0287] U0126 (EB-104) + DCP-LA (EB-102) combination reduced Claudin-3 expression by 70% and increased Claudin-4 expression by 2.5 fold. BCL7B expression was increased 3.5 fold with the double therapy, 2 fold by EB-104 single therapy and 70% reduced by EB-102 alone. Both single treatments have reduced RFC2 by 50% while U0126 (EB-104) + DCP-LA (EB-102) double treatment showed a mild reduction of 20%. EB-104 alone increased GTF2I by 2 fold while single EB-102 treatment and U0126 (EB-104) + DCP-LA (EB-102) reduced its expression by 50 percent. U0126 (EB-104) + DCP-LA (EB-102) increased NCF-1 by 1.5 fold, while both single treatments showed a modest 25% reduction.Genes that are affected in the brain in Williams syndrome.

[0288] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and, in particular, of their combinations (versus control) on the expression of genes involved in Williams Syndrome (STX1A, FK-506, F2ZD9, GALNT17, BAZB1, HIP-1, bHLH, EIF-4H and GTF2I) as well as on inflammatory markers (IL-1 and IL-6), in the brain, are shown in Figures 21(a-k) - 23(a-k).

[0289] BAZB1 was increased by 1.5 fold with the double treatment EB-101 + EB-102, as well as the single treatment of EB-101. The double treatment also increased HIP-1 by 2.5 fold while EB-101 increased it by 6 fold, and EB-102 by 3-fold. EB-101 also increased F2ZD9 expression by 2 fold. bHLH was upregulated by 1.5 fold by the double treatment, and 2.5 fold by the single treatment of EB-101. STX1A, GALNT17, GTF2I, IL-1 and IL-6 were unaffected by the treatments.

[0290] The double treatment by EB-103 + EB-102 increased F2ZD9 by 2 fold, HIP-1 by five fold and EIF-4H by 2-fold, bHLH by 4 fold and FK-506 by 5 fold and BAZ1B by 25% significantly. EB-103 alone increased STX1A by 1.5 fold, HIP-1 by 6 fold, EIF-4H by 3 fold and bHLH by 2 fold.

[0291] EB-104 + EB-102 and EB-104 single treatment showed similar results, they increased BAZ1B significantly by 30%, HIP-1 by 7 fold, EIF-4H by 2 fold, F2ZD9 by 2 fold, FK-506 by 4 fold while the double treatment increased GALNT17 by 40%.,Behavior

[0292] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and, in particular, of their combinations (versus control) on behavioral parameters (distance travelled, mean speed, immobile time, active time, rearing, and time in center zone) are shown in Figures 24(a-f) - 26(a-f).

[0293] In an open field test, behaviors indicative of mobility and anxiety were measured. All treatments have increased the distance traveled, with EB-101 alone showing the most increase and EB-102 alone as well as the double treatments showing similar distance traveled. The result is similar to and consistent when mean speed is measured. This is consistent with the results of immobile time, where mice treated with EB-101 alone showed the least immobile time. Anxiety measures such as rearing and time in center zone have also shown similar results. The double treated mice showed the most time in the center zone indicating more exploratory behavior and less anxiety, with slightly lower increase in rearing behavior compared to EB-101 alone but still higher than EB-102 alone.

[0294] Mice in the EB-103 + EB-102 cohort have shown the least changes in all measures. Rearing behavior was increased in all single and double treated mice compared to the controls.

[0295] EB-104 treated mice had the highest distance traveled and active time. Rearing behavior was increased among all treatment groups. Interestingly both EB-104 treated and EB-102 + EB- 104 treated groups had reduced time in center zone.Live Myography

[0296] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and, in particular, of their combinations (versus control) on arterial contractile strength upon relaxation by acetylcholine treatment are shown in Figures 27-29. In Figure 30, a comparison is shown for the arterial contractile strength upon treatment with acetylcholine in old versus young mice (without any treatment according to the present invention).

[0297] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and, in particular, of their combinations (versus control) on arterial contractile strength upon phenylephrine treatment are shown in Figures 30-32. In Figure 33, a comparison is shown for the arterial contractile strength upon phenylephrine treatment in old versus young mice (without any treatment according to the present invention).

[0298] Aortas isolated from mice treated with EB-101 + EB-102 showed significantly higher contractile strength in response to phenylephrine. Such increase is significantly higher than both single-treated groups. Interestingly, upon relaxation of the arteries by acetylcholine treatments,EB-101 + EB-102 maintained the highest tensile strength while EB-101 treated groups showed lower tensile strength than the controls.

[0299] Interestingly, upon stimulation with phenylephrine, aortas isolated from mice treated with EB-103 + EB-102 showed significantly higher contractile strength compared to both singletreated groups. Upon relaxation with acetylcholine, EB-103 + EB-102 maintained the highest tensile strength while EB-102 treated groups showed the lowest tensile strength.

[0300] EB-104 + EB-102 treated groups showed similar results to EB-103 + EB-102 treated groups.

[0301] In comparison to younger mice at 6 weeks old, aortas from mice receiving all three combination treatments were either similar to or superior in tensile strength to aortas extracted from younger mice. The results are the same upon relaxation with the exception of EB-103 + EB-102Metabolomics

[0302] The effects of the EB-101, EB-102, EB-103 and EB-104 mono-treatments and, in particular, of their combinations (versus control) on selected metabolomics parameters ((a)tryptophan; (b) histidine; (c) phenylalanine; (d) tryptophan; (e) glutamine; (f) glutamate; (g) creatine; (h) lysine; (i) fumarate; (j) citrate and (k) succinate) are shown in figures 37(a-k).

[0303] Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to assess plasma metabolome differences between mice receiving the saline control solution and mice receiving either single or combination elastin therapies. The OPLS-DA scores plots show clear separation between each of the pairs of interest, indicating that treatment with the elastinmodifying small molecules generates a distinct metabolic signature. Univariate analyses of the metabolites that were responsible for driving such separation between treatment and control groups was performed on metabolite bins with VIP scores above 1.5. Broadly, the treatment groups displayed increases in plasma glutamine, glutamate, lysine, succinate, citrate, creatine, and valine (not shown), while plasma phenylalanine, histidine, tryptophan, fumarate and acetate (not shown) were among the metabolites that were decreased in the treatment groups. There are notable differences between single and combination groups, such as a more exaggerated increase in plasma glutamine and citrate following treatment with EB-102- 103 compared to EB-102 or 103 alone; however, ascertaining the biological significance of such changes will be determined in future studies using disease models or indeed, determining the metabolite levels in younger mice.Discussion

[0304] The present results show for the first time the synergistic effect of the use of a PKCs activator in combination of either a neutrophil elastase inhibitor or an MMP-12 inhibitor or a MEK1 / 2 inhibitor. On an organism level, aged mice at 12 months receiving the drug combinations for 12 weeks have tolerated the drugs well with no side effects. The mice have shown striking longevity effects with recovered coat color. Most importantly, aorta isolated from the mice have shown a multiple fold increase in elastin expression, along with markers of elastin synthesis. This increase was well complemented by an increase of aortic tensile strength following a stress-strain experiment, in addition to an increase in contractile strength when stimulated by phenylephrine. When relaxed with acetylcholine, the aortas kept their tension and have not become exceedingly relaxed or loose. Interestingly, the results are comparable to those of a young mice at 6 weeks old, while the treated mice were 15-16 months old at the end of the experiment. The results in mice receiving the combination therapy were always superior to those receiving either single therapy of each combination. When looking at mice behavior, the mice have shown superior performance in an open field compared to controls. Despite the fact that the behavior results were not always superior with the double treatment in each measurement, when taken together with the results of the aorta, the effects on the coat as well as the RNA-Seq results and metabolomics results, combination therapies have better overall outcomes for the mice. RNA-Seq results have shown a significant influence on a wide range of pathways from neurodegenerative disorders to diabetic neuropathy, autophagy, and oxidative phosphorylation, to name a few. When taken together, all the results show a pronounced effect of the recovery of elastin in the extracellular matrix not just on the aorta as a major expressor of elastin but on the organism as a whole.

[0305] According to the present invention, in some embodiments, the targeting of multiple pathways may provide a synergistic effect, which opens up possibilities for treatments with high / increased efficacy and low / reduced side or adverse effects.

[0306] While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

Claims

CLAIMS1. Pharmaceutical composition comprising a first compound that is selected from one of the following groups (I), (II), (III) and (IV) and a second compound that is selected from another one of said groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production.

2. Pharmaceutical composition according to claim 1, wherein: group (I) consists of protein kinase C epsilon type (PKCs) activators; group (II) consists of mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitors; group (III) consists of neutrophil elastase inhibitors; and / or group (IV) consists of MMP-12 inhibitors.

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

4. Pharmaceutical composition comprising according to claim 1 or 2, comprising a first compound selected from one of the following groups (i), (ii), (iii) and (iv) and a second compound selected another one of said groups (i), (ii), (iii) and (iv):(i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and salts and esters thereof;salts and thereof;eof.

5. 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; in combination withor a salt thereof.

6. Pharmaceutical composition for use in a method of treating a subject in need thereof, said pharmaceutical composition comprising a first compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production; wherein the method further comprises the administration of a second compound that is selected from another one of said groups (I), (II), (III) and (IV),wherein the method is a method of:- treating or preventing an elastin deficiency related disease in a subject in need thereof;- treating and / or preventing conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promoting longevity in a subject in need thereof.

7. Pharmaceutical composition for use according to claim 6, wherein: group (I) consists of protein kinase C epsilon type (PKCs) activators; group (II) consists of mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitors; group (III) consists of neutrophil elastase inhibitors; and / or group (IV) consists of MMP-12 inhibitors.

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

9. Pharmaceutical composition for use according to any one of claims 6-8, said pharmaceutical composition comprising a compound selected from any of the following groups (i), (ii), (iii) and (iv):(i) 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), and salts and thereof;thereof; andsalts and esters thereof.

10. Pharmaceutical composition for use according to claim 9, wherein the method further comprises the administration of a second compound that is selected from another one of said groups (i), (ii), (iii) and (iv).

11. Pharmaceutical composition for use according to claim 10, comprising a first compound selected from said group (i), and the method further comprises the administration of a second compound that is selected from any one of said groups (ii), (iii) and (iv).

12. 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 the administration ofsalt thereof.

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

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

15. Pharmaceutical composition for use according to claim 14, wherein said condition or disease related to the aging process is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, and dementia.

16. Pharmaceutical composition for use according to claim 14, wherein said condition or disease related to the aging process is a cardiovascular disease selected from the group consisting of coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

17. Pharmaceutical composition for use according to claim 14, wherein said condition or disease related to the aging process is a fragility disease selected from the group consisting of frailty, osteoporosis, and sarcopenia.

18. Pharmaceutical composition for use according to claim 14, wherein said condition or disease related to the aging process is a skin disease selected from the group consisting of impaired wound healing, alopecia, wrinkles, senile lentigo, skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

19. Method of treating a subject in need thereof, said method comprising the administration to said subject of at least one compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production; and wherein the method further comprises the administration of a second compound that is selected from another one of said groups (I), (II), (III) and (IV), wherein the method is a method of:- treating or preventing an elastin deficiency related disease in a subject in need thereof;- treating and / or preventing conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promoting longevity in a subject in need thereof.

20. Use of at least one compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production; in the manufacture of a medicament for use in a method of treating a subject in need thereof; and wherein the method further comprises the administration of a second compound that is selected from another one of said groups (I), (II), (III) and (IV), wherein the method is a method of:- treating or preventing an elastin deficiency related disease in a subject in need thereof;- treating and / or preventing conditions and / or diseases related to the aging process in a subject in need thereof; and / or- promoting longevity in a subject in need thereof.

21. Non-therapeutic or cosmetic method of treating a subject, said method comprising the administration to said subject of at least one compound selected from any one of the following groups (I), (II), (III) and (IV):(I) compounds capable of increasing tropoelastin expression;(II) compounds capable of increasing elastin fiber deposition;(III) compounds capable of reducing or inhibiting elastin fiber breakdown or degradation; and(IV) compounds capable of reducing or inhibiting elastokines production; and wherein the method further comprises the administration of a second compound that is selected from another one of said groups (I), (II), (III) and (IV), wherein said method is a non-therapeutic method or a cosmetic method22. Non-therapeutic or cosmetic method according to claim 20, wherein said method is a method for improving the appearance of the skin, for protecting the skin from the visible signs of aging, for preventing and / or reducing wrinkles, for improving and / or preserving smoothness of the skin, for improving and / or preserving firmness of the skin, for improving and / or preserving elasticity of the skin, for skin rejuvenation and / or for maintaining a youthful skin.

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