WO2016030445A1 - Aminoguanidines of formula (i) for use in the treatment of fibrosis - Google Patents

Abstract

The present invention relates to certain benzylideneaminoguanidines for the treatment of fibrosis. In some preferred embodiments, the fibrosis is pulmonary fibrosis.

Description

AMINOGUANIDINES OF FORMULA (I) FOR USE IN THE TREATMENT OF

FIBROSIS

The present invention relates to certain benzylideneaminoguanidines for the treatment of fibrosis.

Tissue consists of cells and the extracellular space in between them. This space is primarily composed of a meshwork of proteins, e.g. collagen fibrils and proteoglycans such as decorin, versican and biglycan. Together these constitute the extracellular matrix (ECM). Sustaining tissue homeostasis is critical for maintaining the biochemical and structural support of cells provided by the ECM. Under normal conditions, wound healing or tissue repair is a tightly regulated process. Damaged connective tissue is replaced through synthesis of extracellular matrix (ECM) proteins produced by e.g. activated fibroblasts or myofibroblasts [1 , 2]. In fibrotic diseases, the regulatory mechanism is disrupted leading to excessive ECM synthesis and tissue fibrosis. The fibrotic tissue disrupts the physiological tissue structure, leading to organ dysfunction and contributes to morbidity and increased mortality of affected patients. Fibrotic disease of the lung, such as idiopathic pulmonary fibrosis (IPF), is a chronic disease with unmet medical needs and a mortality rate of 50% within 3-5 years after diagnosis [3]. The aetiology of fibrotic diseases is still unknown. However, some factors are associated with the development of fibrosis such as an ongoing autoimmune disease, exposure to radiation, chemotherapy and inhalation of asbestos.

Even though the exact mechanisms leading to pathological fibroblast and myofibroblast activation are not completely understood, an important mediator of fibrosis is the pro- fibrotic transforming growth factor beta-1 (TGF-βΙ ) [4], which promotes differentiation of fibroblasts into myofibroblasts with contractile properties, a cell type featuring increased expression of alpha-smooth muscle actin (a-SMA) along with enhanced production of ECM proteins such as proteoglycans [5-7]. Biglycan and decorin, belonging to the family of small leucine-rich proteins (SLRPs) of proteoglycans, has e.g. been shown to enhance cell migration in human lung fibroblasts following exposure to the core proteins [8]. Interestingly, decorin, in comparison to biglycan, showed a separate effect on cytoskeletal rearrangements with significantly increased expression of a-SMA, possibly connecting decorin to lung fibroblast activation. The myofibroblast is abundant in so- called fibroblastic foci and it is recognized as a hallmark in certain types of pulmonary fibrotic diseases such as idiopathic pulmonary fibrosis [9].

Although being an important neurotransmitter in the CNS, serotonin (5- hydroxytryptamine (5-HT)) is mainly found in the periphery where it is involved in e.g. regulation of vascular tone, platelet aggregation, immune response and bowel peristalsis [10, 1 1]. Peripheral 5-HT is mainly produced by the enterochromaffin cells of the gut and it is derived form tryptophan in a reaction catalysed by tryptophan hydroxylase-1 (Tph-1 ). Under physiological conditions, the level of free plasma 5-HT is low and strictly regulated by specific 5-HT transporters present on the surface of e.g. platelets. Extracellular 5-HT is effectively transported into platelets where it is either degraded by 5-HT degrading enzymes or stored in dense granules [12]. When platelets encounter, e.g. endothelial damage they become activated and aggregate, and release 5-HT resulting in an increase in local concentration [13].

To date, fourteen human serotonergic receptors by which 5-HT has its action have been identified [14]. The 5-HT₂ receptor family consists of 3 subtypes: 5-HT₂A, 5-HT₂B and 5- HT₂c which have repeatedly been implicated in detrimental progressions of several diseases such as cardiovascular diseases. The 5-HT_2B receptor has been linked to pulmonary artery hypertension (PAH) and the phenotype of the 5-HT_2B receptor knockout mice shows its importance for heart development [15, 16]. The 5-HT₂ receptors have also been shown to be involved in e.g. fibrosis, inflammation and pain [17-25].

It is well known that platelet-derived 5-HT is critical for normal wound healing where it stimulates both vasoconstriction and vasodilation, influences inflammatory responses and promotes formation of a temporary scar. However, repeated injury or an

unbalanced 5-HT signalling can have detrimental effects and promote aberrant wound healing resulting in tissue fibrosis [26]. Recent studies support the idea that 5-HT₂ receptors have an important role in fibrotic disease by regulating production of pro- fibrotic mediators and modifying cell differentiation and activation [19, 23, 26]. 5-HT has been proposed to induce the expression of TGF-βΙ [19, 27]. Dermal fibroblasts from patients diagnosed with systemic sclerosis have a dose-dependent increase of TGF-βΙ mRNA in response to 5-HT as well as an increased expression of 5- HTR2B [19].

In support of an important role of 5-HT2 receptors in fibrosis, it has been found that IPF patients have an increased expression of the 5-HT₂A and 5-HT₂B receptors in the lung with primary localizations at the bronchial and vascular smooth muscle cells, and bronchial and alveolar epithelial cells [23]. The receptors have also been shown to be expressed in lungs of bleomycin-treated animals; and inhibition of the receptors with terguride (a 5-HT₂A and 5-HT₂B receptor antagonist) resulted in marked attenuation of established bleomycin-induced lung fibrosis [23]. In an earlier study, it was shown that lung 5-HT content was increased in bleomycin-induced lung fibrosis and that inhibition of the 5-HT_2A and 5-HT_2B receptors promoted an anti-fibrotic environment [28].

It has now been found that certain benzylideneaminoguanidines are capable of inhibiting myofibroblast differentiation and/or proteoglycan production and are therefore of use in the treatment of fibrosis. These benzylideneaminoguanidines are capable of interacting with one or more of the three serotonergic receptors 5HT_2A, 5HT_2B and 5HT_2C.

It is therefore an object of the invention to provide certain benzylideneaminoguanidines for the treatment of fibrosis. In one embodiment, therefore, the invention provides a compound of general Formula I:

or a tautomer, diastereomer or enantiomer thereof, wherein n is 0, 1 , 2 or 3, wherein at least one of R1 , R2, R3, R4 and R5 is -S-R, wherein R is selected from alkyl having 1 to 5 carbon atoms, cycloalkyl having 3 to 6 carbon atoms, and aryl having 6 to 10 carbon atoms, and wherein the other R1 , R2, R3, R4 and R5 groups are the same or different and are selected from hydrogen, halogen, alkyl having 1 to 5 carbon atoms, alkoxy having 1 to 5 carbon atoms, hydroxy, cyano, nitro, trifluoroalkyl, amide, alkylamino having 2 to 6 carbon atoms, benzoyloxy, nitroxy, phenyl and sulpho; or a pharmacologically-acceptable salt thereof, for the treatment of fibrosis. In the compounds of Formula I, at least one of R1 , R2, R3, R4 and R5 is -S-R.

For example, 1 or 2, preferably only 1 , of R1 -R5 is -S-R.

Preferably, R1 or R2 is -S-R. More preferably, only R1 or only R2 is -S-R. R is selected from alkyl having 1 to 5 carbon atoms, cycloalkyl having 3 to 6 carbon atoms, and aryl having 6 to 10 carbon atoms.

As used herein, the term alkyl includes straight- and branched-chain hydrocarbon groups. Preferably, the "alkyl having 1 to 5 carbon atoms" is a lower alkyl such as methyl, ethyl, propyl or iso-propyl. Most preferably, alkyl is methyl.

The term alkoxy includes straight- and branched-chain alkoxy groups. Preferably, the "alkoxy having 1 to 5 carbon atoms" is a lower alkoxy such as methoxy, ethoxy, propoxy or iso-propoxy, most preferably methoxy.

In some embodiments, at least one of R1 -R5 is halogen. For example, 1 , 2 or 3, preferably only 1 , of R1 -R5 may be halogen. The term halogen includes fluoro, chloro, bromo and iodo. Preferably, the halogen is fluoro or chloro. Preferably, R1 or R2 is halogen.

In some particularly-preferred embodiments, (i) R1 is -S-R and R2 is halogen; or (ii) R1 is halogen and R2 is -S-R.

In some preferred embodiments, R3 is alkoxy, preferably methoxy.

Preferably R4 and R5 are both H. In some embodiments, R3, R4 and R5 are all H.

Preferably, the trifluoroalkyi is trifluoromethyl, trifluoroethyl, trifluoropropyl or trifluoroiso- propyl.

The term "alkylamino" refers preferably to groups having 2-6 carbon atoms, particularly dialkylamino groups, and most preferably dimethylamino or diethylamino. n is 0, 1 , 2 or 3, preferably 0 or 1 . This linking group, when present, may be saturated or unsaturated. The following are preferred compounds of the invention:

N-(2-Methylthio-3-chlorobenzylideneamino)guanidine

N-(2-Chloro-4-Methoxy-3-methylthiobenzylideneamino)guanidine

N-(2-Chloro-3-Methoxy-4-methylthiobenzylideneamino)guanidine

N-(2-Chloro-3,4-dimethylthiobenzylideneamino)guanidine

N-(2-Methylthio-3-chlorophenylpropylideneamino)guanidine

N-(2-Chloro-4-Methoxy-3-methylthiophenylpropylideneamino)guanidine

N-(2-Chloro-3-Methoxy-4-methylthiophenylpropylideneamino)guanidine, and

N-(2-Chloro-3,4-dimethylthiophenylpropylideneamino)guanidine; or a pharmacologically-acceptable salt thereof. In some embodiments, the following compounds are particularly preferred:

(i) N-(2-methylthio-3-chlorobenzylideneamino)guanidine or a pharmacologically- acceptable salt thereof (referred to herein as COMP2); and

(ii) N-(2-chloro-4-methoxy-3-methylthiobenzylideneamino)guanidine (referred to herein as COMP1 ) or a pharmacologically-acceptable salt thereof.

The compounds of Formula (I) have basic properties and, consequently, they may be converted, if desired, to their therapeutically-active pharmaceutically-acceptable acid addition salts by treatment with appropriate acids, e.g. inorganic acids such as

hydrochloric, hydrobromic, hydroiodic, sulphuric, nitric and phosphoric acid, or organic acids such as acetic, propanoic, glycolic, lactic, malonic, succinic, fumaric, tartaric, citric, palmoic or para-toluene-sulphonic acid.

Conversely, the salt form may be converted into the free base form by treatment with alkali.

The invention also provides a pharmaceutical composition comprising:

(i) a compound as defined herein; and

(ii) one or more pharmaceutically-acceptable adjuvants, carriers or excipients, for use in the treatment of fibrosis.

The invention also provides for the use of a compound as defined herein in the manufacture of a medicament for the treatment of fibrosis. The invention also provides a method of treating fibrosis comprising administering an effective amount of a compound as defined herein to a patient in need thereof.

Fibrosis is characterised by excessive connective tissue deposition. Examples of fibrosis include pulmonary fibrosis (Interstitial Lung Disease, Idiopathic pulmonary fibrosis, Chronic Obstructive Pulmonary Fibrosis, etc.), liver fibrosis, heart fibrosis, kidney fibrosis, skin fibrosis and scleroderma. Preferably, the fibrosis is lung fibrosis or liver fibrosis, e.g. fibrotic disease of the lung or the liver. Suitable forms of pharmaceutical preparation for administration include for example tablets, capsules, solutions, syrups, and emulsions. The content of the pharmaceutically effective compound(s) in each case should desirably be in the range from 0.1 to 5 wt.%, of the total composition.

The preparations maybe administered orally in the form of a tablet, as a powder, as a powder in a capsule (e.g. a hard gelatine capsule), as a solution or suspension. It is preferable if the compounds of Formula (I) are administered orally. Suitable tablets may be obtained, for example, by mixing the active substance(s) with known carriers, diluents or excipients, such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatine, lubricants such as magnesium stearate or talc and/or agents for delaying release, such as carboxymethyl cellulose, cellulose acetate phthalate, or polyvinyl acetate. The tablets may also comprise several layers.

Coated tablets may suitably be prepared by coating cores produced similarly to the tablets with substances normally used for tablet coatings, for example collidone or shellac, gum arabic, talc, titanium dioxide or sugar. The tablet coating may consist of a number of layers to achieve delayed release, possibly using the excipients mentioned above for the tablets.

Syrups containing the active substances or combinations thereof according to the invention may additionally contain a sweetener such as saccharin, cyclamate, glycerol or sugar and a flavour enhancer, e.g. a flavouring such as vanillin or orange extract. They may also contain suspension adjuvants or thickeners such as sodium

carboxymethyl cellulose, wetting agents such as, for example, condensation products of fatty alcohols with ethylene oxide, or preservatives such asp-hydroxybenzoates.

Capsules containing one or more active substances or combinations of active substances may for example be prepared by mixing the active substances with inert carriers such as lactose or sorbitol and packing them into gelatine capsules.

Suppositories may be made for example by mixing with carriers provided for this purpose, such as neutral fats or polyethyleneglycol or the derivatives thereof. Excipients which may be used include, for example, water, pharmaceutically-acceptable organic solvents such as paraffins (e.g. petroleum fractions), vegetable oils (e.g.

groundnut or sesame oil), mono- or polyfunctional alcohols (e.g. ethanol or glycerol), carriers such as e.g. natural mineral powders (e.g. kaolins, clays, talc, chalk), synthetic mineral powders (e.g. highly dispersed silicic acid and silicates), sugars (e.g. cane sugar, lactose and glucose), emulsifiers (e.g. lignin, spent sulphite liquors,

methylcellulose, starch and polyvinylpyrrolidone) and lubricants (e.g. magnesium stearate, talc, stearic acid and sodium lauryl sulphate). The composition is preferably administered orally or by inhalation.

For oral administration, the tablets may contain, in addition to the above-mentioned carriers, additives such as sodium citrate, calcium carbonate and dicalcium phosphate together with various additives such as starch, preferably potato starch, gelatine and the like. Moreover, lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used at the same time for the tabletting process. In the case of aqueous suspensions, the active substances may be combined with various flavour-enhancers or colourings in addition to the excipients mentioned above. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : The in vitro experiment set-up.

Figure 2: HFL-1 cells immunostained for the 5-HT₂B receptor. HFL-1 cells (A) untreated (x20 magnification), (B) treated 24h with TGF-βΙ 10ng/ml + 5-HT 1 μΜ (x10

magnification), (C) untreated and permeabilized (x20 magnification), were stained with DAPI (nuclear staining, blue) and anti-5-HT2B receptor antibody (red).

Figure 3: Production of a-SMA in HFL-1 cells treated with fibrotic stimuli and 5-HT₂B receptor antagonists.

(A) The relative quantity of intracellular a-SMA, quantified with Western Blot, was assessed after 24h in HFL-1 cells treated with fibrotic stimuli (10 ng/ml TGF-P±5-HT 1 or 10 μΜ. (B) The relative quantity of intracellular a-SMA in HFL-1 cells was evaluated after treatment with 5-HT_2B receptor antagonists, COMP1 (10μΜ) and COMP2 (10μΜ). The antagonists were added either 1 h prior to (1 h pretreatment) or simultaneous with (no pretreatment) addition of fibrotic stimuli (TGF-βΙ 10ng/ml + 5-HT 1 μΜ) (B). All samples were normalized to control; cells cultured in medium without added stimuli (A) or cells cultured in 10ng/ml TGF-βΙ +Ι μΜ 5-HT (B), n=4-5.

(C) HFL-1 cells immunostained for a-SMA. The protein was detected in stress fibres after 24h treatment with TGF-βΙ 10ng/ml + 5-HT 1 μΜ (green: anti-a-SMA antibody, blue: DAPI).

Statistical analysis: one sample t-test, p<0.05 vs control.

Figure 4: Proteoglycan production in fibrotic stimulated HFL-1 cells treated with COMP1 and COMP2. Cell culture medium from HFL-1 cells cultured in the presence of medium (control), TGF-βΙ 10ng/ml ± 5-HT 1 μΜ (A) or co-cultured with TGF-βΙ 10ng/ml + 5-HT 1 μΜ and COMP1 or COMP2 were quantified for total amount proteoglycans (B) or total amount decorin (C). The relative amount of newly synthesized sulfated [³⁵S] labeled proteoglycans, analyzed with densitometry, normalized to total protein content (mg) is presented as mean percent total proteoglycan (PG) content per mg protein vs control (untreated) n=4-5 (A) or (TGF-βΙ 10ng/ml + 5-HT 1 μΜ) n=3-4 (B,C).

Statistical analysis: one sample t-test, p<0.05 vs control.

Figure 5: Lung tissue remodeling after p.o. treatment with 5-HT₂B receptor antagonists in BLM-treated mice. Lung segments from bleomycin-treated mice were analyzed for lung tissue density after p.o. treatment with 5-HT₂B receptor antagonists using

hematoxylin/eosin staining. Tissue density was shown as positive stained tissue area vs total area (mm²), % + SD. Statistical analysis; 1 way ANOVA Kruskal Wallis, Dunn's Multiple comparison test: *p<0.05, **p=0.01 vs saline, n=5-6.

EXAM PLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

EXAMPLE 1

MATERIALS AND METHODS Human cells

Human foetal lung fibroblasts (HFL-1 ) were obtained from American type culture collection (ATCC, Manassas, VA, U.S., cat.no. CCL-153). HFL-1 cells were cultured in Minimal Essential Medium (MEM), supplemented with 10U Penicillin, 0.1 mg/ml

Streptomycin (Sigma Aldrich, St Louis, MO, USA) and 1 % Glutamine, at 37°C, 5-10 % CO2. The cell culture medium also contained 10% foetal clone III serum (FCIII) (Thermo Scientific, Waltham, MA, USA, cat. no. SH30109.03 ).

In vitro experiments

In vitro experiments were performed in Dulbecco's Modified Eagle Medium (DMEM) with the same supplementation as for MEM, with exceptions for serum content, at 10% CO2, 37°C. For further details see Figure 1. The 5-HT2 receptor antagonists used in this study were provided by AnaMar AB (Lund, Sweden). The selectivity of the antagonists COMP1 and COMP2 for the 5-HT₂B receptor are given in Table 1. Table 1. Binding profiles of 5-HT? receptor antagonists: COMP1 and COMP2

The K, and IC₅₀ of the compounds were examined in Chinese hamster ovary (CHO) cells transfected with human recombinant 5-HT₂ receptors. IC50 = half maximal inhibitory concentration; Ki = inhibition constant; 5-HT = serotonin. 5-HT2A Ki 5-HT2B Ki 5-HT2C Ki 5-ΗΤ2Α 5-ΗΤ2Β 5-HT2C

IC50 IC50 IC50 ΙΡ1 hum ΙΡ1 hum GTP hum

COMP1 1 .01 μΜ 0.045 μΜ 0.31 μΜ 7.54 μΜ 0.082 μΜ 5.1 1 μΜ

COMP2 0.46 μΜ 0.026 μΜ 0.1 1 μΜ 0.84 μΜ 0.029 μΜ 0.65 μΜ qRT-PCR

HFL-1 cells cultured 24h with or without fibrotic stimuli (TGF-βΙ 10ng/ml ± 5-HT 1 μΜ, 10μΜ) were extracted for total RNA. The cell lysate RNA was isolated using

QiaShredder columns and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. The RNA amount and purity was measured with a

Nanophotometer (Implen GmbH, Munchen, Germany) prior to cDNA synthesis with reverse transcriptase kit (Applied Biosystems, Carlsbad, CA, U.S.). cDNA samples were run in quantitative real time PCR (qRT-PCR). Target genes were identified with commercial available TaqMan probes: HTR2A (Hs01033524_m1 ), HTR2B

(Hs00168362_m1 ), HTR2C (Hs00968671_m1 ) and GAPDH (Hs02758991_g1 ) from Applied Biosystems. Gene expression levels were analysed by comparative Ct (cycle threshold) method, and related to endogenous control and total amount RNA. To identify cross contamination and genomic DNA, controls with RNA samples lacking reverse transcriptase and cDNA samples lacking DNA template were used. An initial analysis of four housekeeping genes was made to determine a stable endogenous control. Housekeeping genes GAPDH and 18S presented results indicative of stable gene expression and sufficient PCR efficiency, respectively (data not shown). Immunocvtochemistry (ICC)

HFL-1 cells, plated on glass chamber slides (Thermo Scientific) and cultured for 24h with or without fibrotic stimuli (TGF-βΙ 10ng/ml ± 5-HT 1 μΜ), were fixated in 4% buffered formaldehyde solution for 15 min in room temperature (RT). HFL-1 cells were labelled with anti-5HT₂A receptor antibody (^g/ml) (sc-166775, Santa Cruz, Dallas, TX, U.S.), anti-5-HT_2B receptor antibody (2.5Mg/ml) (AP01 188PU-N, Acris Antibodies GmbH, Herford, Germany) and anti-5-HT₂c receptor antibody (PA5-27164, Thermo Scientific) for 90 min at RT, followed by washing in Tris-buffered saline (TBS). Cells were incubated with fluorescent-conjugated secondary antibodies (A-21428, A-21235; diluted 1/200; Life Technologies, Carlsbad, CA, USA) in combination with DAPI, for 45 min at RT. For localization of antibody labelled receptors, comparable studies were made with intact and permeabilized cells (conditioned 5min with 0.2% Triton-X100). Negative controls were composed of isotype matched antibodies (X0903, X0931 ;

DakoCytomation, Glostrup, Denmark) and sole secondary antibody exposure. Imaging processing was performed with imaging systems: Nikon Eclipse 80i, Nikon DSQiMC (Tokyo, Japan) and Olympus DP80 (Olympus, Center Valley, P.A., U.S.), with imaging software: NIS-Elements BR 3.2 Ink, cellSens Dimension (Olympus, Center Valley, P.A., U.S.) and ImageJ 1 .45s (Wayne Rasband, NIH, Bethesda, MD, U.S.). The expression of intracellular a-SMA was examined in equal manner in antibody labelled

permeabilized HFL-1 cells, using anti-a-SMA antibody (dilution 1 :200) (C6198, Sigma).

SDS Page and Western Blot

HFL-1 cells, seeded in 6-well cell culture plate, were pretreated (1 h) with COMP1 or COMP2 (10μΜ) before addition of fibrotic stimuli (TGF-βΙ 10ng/ml ± 5-HT 1 μΜ or 10μΜ) or simultaneously treated with fibrotic stimuli and receptor antagonists. 24h post- treatment, cell lysate was collected with supplemented 1 % NP-40 (Sigma-Aldrich).

Whole cell lysate, prepared in reduced sample buffer, was electrophoresed in a

NuPAGE Bis-Tris Gel (Life Technologies) for 50 min at 200V constant. The size- separated protein samples were transferred to a PVDF membrane (Millipore,

Massachusetts, U.S.) by running the membrane at 30V constant for 16h at +4°C in transfer buffer (Invitrogen, Waltham, MA, U.S.) with 10% methanol. Optionally, the membrane was stained with 0.1 % Ponceau S acetic acid solution for verification of protein band transfer. Following unstaining using 1 % acetate, the membrane was blocked with 0.5% casein in TBS-Tween 0.5% (TBS-T) for 30 min at RT. The

membrane was incubated with anti-a-SMA antibody (diluted 1 :200,400,750) (ab5694, Abeam) along with anti-p-tubulin antibody (dilution 1 :30 00/40 000) (ab6046, Abeam) or anti-GAPDH antibody (sc-47724, Santa Cruz Dallas, Texas, U.S.) for 2h at +40°C. After washing in TBS-T, the membrane was incubated for 30 min at RT with secondary DyLight800-conjugated antibody (diluted 1 : 15 000) (5151 S, Cellular signalling

technologies, Danvers, MA, U.S.), followed by final washing steps. Core protein bands composed of a-SMA and endogenous controls (GAPDH and β-tubulin) were detected with imaging system Odyssey FC (LI-COR Inc., Lincoln, N.E., U.S.). The total content of α-SMA, quantified with Image Studio v.3.1 (LI-COR Inc.), was normalized to total protein content, measured in cell lysate with BCA protein assay kit (Thermo Scientific).

EXAMPLE 2: Lung fibroblasts expression of 5-HT₂ receptors

For the assessment of potential anti-fibrotic effects mediated through 5-HT2 receptors in vitro, we examined the expression of 5-HT₂B receptor in HFL-1 cells. Evaluations of four housekeeping genes were made to determine stable gene expression for the

endogenous control in qRT-PCR. The housekeeping gene 18S was found to have a stable gene expression in the phenotype transition from fibroblast to myofibroblast and therefore 18S was used throughout the study (data not shown). Lung fibroblasts (HFL-1 ) were shown to express the 5-HT₂B receptor at both mRNA levels (data not shown) and protein levels (Fig. 2A). The mRNA level of 5-HTR2B was studied at 6, 24 and 48 h post-treatment of fibrotic stimuli, which displayed equivalent expression patterns. The protein expression was sustained in TGF-βΙ + 5-HT treated fibroblasts which also displayed a distinct cellular phenotype with protruding cellular outgrowths and an overall larger cellular size in comparison to untreated HFL-1 cells (Fig. 2B). For verification of receptor location, cells were permeabilized with Triton-X100 which revealed a distinct expression pattern separated from the pattern seen from intact cells (Fig. 2C).

EXAMPLE 3: Regulation of alpha smooth muscle actin

The contractile protein a-SMA, a commonly used marker for myofibroblast, was quantified with Western Blot in HFL-1 cells treated with fibrotic stimuli and 5-HT₂B receptor antagonists. Exposure to TGF-βΙ induced a 1.8± 0.4 fold increase of a-SMA (Figure 3A). Interestingly, 5-HT at 10μΜ showed a possible additive effect on a-SMA production in cells co-exposed with TGF-βΙ (2.7 ± 0.9 fold increase, p=0.053) at 24h post-exposure. Treatment with COMP1 or COMP2 (10μΜ) significantly reduced the elevated production of a-SMA to protein levels comparable to those seen in untreated cells (Figure 3B). To further explore potential regulating effects on myofibroblast differentiation, a pretreatment with COMP1 and COMP2 prior to addition of fibrotic stimuli were examined (Figure 3B). No modification of a-SMA production was seen in HFL-1 cells exposed to receptor antagonist pretreatment (1 h) versus cells treated simultaneously with receptor antagonists. By immunocytochemistry, the expression of a-SMA was examined in HFL-1 cells cultured with and without fibrotic stimuli. Cells exposed to fibrotic stimuli displayed aligned stress fibres positive for a-SMA, supporting our previous findings seen at gene expression levels and quantitative protein levels, Figure 3C.

EXAMPLE 4: Production and regulation of proteoglycans

HFL-1 cells were cultured to confluence in a 6-well cell culture plate following three stepwise culture treatments: Step 1 ) 2h culture in supplemented DMEM (1 % Amfotericin + 0.5% Gentamicin + 1 % Glutamine) with 1 % FCIII. Step 2) 2h culture in with TGF-βΙ 10ng/ml ± 5-HT 1 μΜ and receptor antagonists in supplemented sulphate-poor medium (074-91083P, Invitrogen) containing 0.4% FCIII. Step 3) 22h culturing in 50 Ci/ml ³⁵S, following cell lysate and cell medium collection. Proteoglycans were extracted by column-based separation and size separated with gel electrophoresis according to previously described methodology [29]. Bands of ³⁵S-labeled proteoglycans (all and separate) were visualized with imaging system Molecular Imager FX. The relative proteoglycan quantity, analysed with Quantitative One 4.6, were evaluated in samples normalized to total protein content measured by BCA protein assay kit (Thermo

Scientific).

Exposure to TGF-βΙ ± 5-HT significantly increased the proteoglycan production seen 24h post-stimulation (Figure 4A). By antagonization of 5-HT₂B receptors with COMP1 or COMP2 (10μΜ), the total production of proteoglycans was reduced (Figure 4B). To further understand how 5-HT₂B receptors influence proteoglycan production, the regulation of individual proteoglycans was examined. Initial results showed that the production of decorin seems partly controlled by the 5-HT₂B receptor, with significantly reduced levels in COMP1 -treated fibroblasts (Figure 4C). EXAMPLE 5: Anti-fibrotic effects in the bleomycin-induced ling fibrosis model

C57/BI6 mice (female, 12.5 weeks old) were divided into seven mice per treatment group. Mice were s.c. injected three times a week with bleomycin (BLM, 50IE/animal) or saline solution for two weeks according to previous described methodology and housing conditions [30]. Compound-treated mice received daily p.o. administration of COMP1 or COMP2 solved in water-based Tween80 (Sigma Aldrich) solution (2.5(w/v) %) or 20% Solutol HS 15 (Sigma), in doses of 10mg/kg or 30mg/kg. All p.o. administrations were executed by the use of a 20 gauge silicone tipped feeding needle. Control animals received BLM or saline injections with p.o. administration of vehicle. The study was approved by the animal experimental committee of Malmo/Lund, Sweden (M103-14). Following euthanasia, lung lobes were extracted and fixated o/n in 4% buffered formaldehyde solution and paraffin embedded according to standard protocol.

IHC. De-paraffin ized and re-hydrated lung segments (4μηη) were stained with hematoxylin/eosin according to standard protocol. Tissue density was analysed with ImageJ in randomly taken microscopic images of the lung parenchyma and assessed as total amount positive stained tissue area per total area (%). 7-10 randomly taken images per lung segment were processed as previously described in ICC methodology. All microscopic examinations were performed under sample coded conditions until finalization of analysis.

The results showed that after two weeks of treatment, the lung tissue density was significantly increased (p=0.01 ) in BLM-treated mice in comparison to healthy control mice (Figure 5). BLM-treated mice with daily p.o. treatment of COMP1 or COMP2 (30mg/kg) displayed a significantly reduced tissue density with 86 ± 23.8% and 83 ± 15.4% (p=0.05), respectively.

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Claims

1 . A compound of general Formula I:

or a tautomer, diastereomer or enantiomer thereof, wherein n is 0, 1 , 2 or 3, wherein at least one of R1 , R2, R3, R4 and R5 is -S-R, wherein R is selected from alkyl having 1 to 5 carbon atoms, cycloalkyi having 3 to 6 carbon atoms, and aryl having 6 to 10 carbon atoms, and wherein the other R1 , R2, R3, R4 and R5 groups are the same or different and are selected from hydrogen, halogen, alkyl having 1 to 5 carbon atoms, alkoxy having 1 to 5 carbon atoms, hydroxy, cyano, nitro, trifluoroalkyl, amide, alkylamino having 2 to 6 carbon atoms, benzoyloxy, nitroxy, phenyl and sulpho; or a pharmacologically-acceptable salt thereof, for the treatment of fibrosis.

2. A compound as claimed in claim 1 , wherein R1 or R2 is -S-R.

3. A compound as claimed in claim 1 or claim 2, wherein R is alkyl, preferably methyl.

4. A compound as claimed in any one of the preceding claims, wherein at least one of R1 -R5 is halogen, preferably fluoro or chloro.

5. A compound as claimed in claim 4, wherein R1 or R2 is halogen.

6. A compound as claimed in any one of the preceding claims, wherein:

(i) R1 is -S-R and R2 is halogen; or

(ii) R1 is halogen and R2 is -S-R.

7. A compound as claimed in any one of the preceding claims, wherein at least one of R1 -R5 is alkoxy, preferably methoxy.

8. A compound as claimed in any one of the preceding claims, wherein n is 0 or 1 .

9. A compound as claimed in claim 1 , wherein the compound is selected from the group consisting of: N-(2-Methylthio-3-chlorobenzylideneamino)guanidine

N-(2-Chloro-4-Methoxy-3-methylthiobenzylideneamino)guanidine

N-(2-Chloro-3-Methoxy-4-methylthiobenzylideneamino)guanidine

N-(2-Chloro-3,4-dimethylthiobenzylideneamino)guanidine

N-(2-Methylthio-3-chlorophenylpropylideneamino)guanidine

N-(2-Chloro-4-Methoxy-3-methylthiophenylpropylideneamino)guanidine

N-(2-Chloro-3-Methoxy-4-methylthiophenylpropylideneamino)guanidine, and

N-(2-Chloro-3,4-dimethylthiophenylpropylideneamino)guanidine; or a pharmacologically-acceptable salt thereof.

10. A compound as claimed in claim 1 , wherein the compound is N-(2-methylthio-3- chlorobenzylideneamino)guanidine or a pharmacologically-acceptable salt thereof.

1 1 . A compound as claimed in claim 1 , wherein the compound is N-(2-chloro-4- methoxy-3-methylthiobenzylideneamino)guanidine or a pharmacologically-acceptable salt thereof.

12. A pharmaceutical composition comprising:

(i) a compound as defined in any one of the claims 1 to 1 1 ; and

(ii) one or more pharmaceutically-acceptable adjuvants, carriers or excipients, for use in the treatment of fibrosis.

13. Use of a compound as defined in any one of claims 1 to 1 1 in the manufacture of a medicament for the treatment of fibrosis.

14. A method of treating fibrosis comprising administering an effective amount of a compound as defined in any one of claims 1 to 1 1 to a patient in need thereof.

15. A compound as claimed in any one of claims 1 to 1 1 , a composition as claimed in claim 12, a use as claimed in claim 13 or a method as claimed in claim 14, wherein the fibrosis is pulmonary fibrosis.