WO2021186041A1

WO2021186041A1 - Josamycin for use in prevention and treatment of fibrosis

Info

Publication number: WO2021186041A1
Application number: PCT/EP2021/057106
Authority: WO
Inventors: Stephan STRUCKMANN; Thomas STAHNKE; Anselm Jünemann; Georg FUELLEN
Original assignee: Universität Rostock
Priority date: 2020-03-19
Filing date: 2021-03-19
Publication date: 2021-09-23
Also published as: EP4121062A1

Abstract

The invention relates to the use of josamycin or a derivative of josamycin, particularly a derivative selected from kitasamycin, spiramycin and midecamycin, in a method of treatment or prevention of fibrosis.

Description

Josamycin for use in prevention and treatment of fibrosis

The present invention relates to the use of josamycin and its derivatives in the prevention and treatment of fibrosis, particularly fibrosis of the eye, more particularly fibrosis occurring after glaucoma surgery.

Description

Fibrosis and scarring are involved in the pathogenesis or failure of treatment of virtually all the major blinding diseases. Glaucoma is the second leading cause of blindness in the world. By 2020, its prevalence is estimated to reach around 79.6 million people worldwide, with more than 11 million individuals suffering from bilateral blindness. Glaucoma filtration surgery is the mainstay of surgical treatment for medically uncontrolled glaucoma. Even with new surgical techniques, postoperative scarring remains a critical determinant of the long-term surgical outcome and reduction of intraocular pressure after drainage surgery. The antimetabolites, mitomycin C (MMC) and 5-fluorouracil (5-FU), have improved the surgical outcome of glaucoma filtration surgery, but lead to non-specific cytotoxicity and the risk of sight-threatening complications like tissue damage, ruptures, and infection. In addition, some patients still develop scarring and fail surgery despite antimetabolite therapy. There is thus a large unmet need to develop new anti-fibrotic therapeutics to prevent scarring and post-surgical fibrosis in the eye (Yu-Wai-Man and Khaw, Expert Review of Ophtalmology 2015, 65-76).

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to treat and prevent fibrosis in the eye. This objective is attained by the subject matter of the claims of the present specification.

Terms and definitions

The term josamycin in the context of the present specification relates to the known macrolide antibacterial drug identified by CAS No. 16846-24-5. Josamycin is abbreviated as JM.

The term kitasamycin in the context of the present specification relates to the known macrolide antibacterial drug identified by CAS No. 1392-21-8.

The term spiramycin in the context of the present specification relates to the known macrolide antibacterial drug identified by CAS No. 24916-50-5.

The term midecamycin in the context of the present specification relates to the known macrolide antibacterial drug identified by CAS No. 35457-80-8.

The term “derivative” in the context of the present specification relates to an pharmaceutical agent approved by a regulatory agency for pharmaceutical use in humans. The derivative of a parent compound has the same chemical structure as the parent but may have groups attached to it such as acetyl, formyl, methyl, or other moieties commonly used in modifying the pharmacological of a drug substance without altering its target specificity. One example of a derivative of midecamycin is diacetylmidecamycin.

A first aspect of the invention relates to a pharmaceutical compound selected from josamycin, and a derivative of josamycin for use in treatment or prevention of fibrosis.

In certain embodiments, the compound is selected from josamycin, kitasamycin, spiramycin and midecamycin.

In certain embodiments, the compound is selected from josamycin or kitasamycin,

An alternative of the first aspect of the invention relates to josamycin for use in treatment or prevention of fibrosis.

In certain embodiments, the fibrosis is ocular fibrosis (fibrosis occurring in the eye).

Fibrosis may occur as the result of, or associated with, a number of pathological contexts.

Fibrosis can occur subsequent to viral or bacterial infection of the eye or as a result of injury.

Another context in which fibrosis can occur is corneal opacification.

In certain embodiments, fibrosis is caused by, or occurs subsequent to, glaucoma surgery.

In certain embodiments, the compound is administered via ocular injection.

In certain embodiments, the compound is administered during or after glaucoma surgery.

Similarly, the invention relates to a method of treating fibrosis after glaucoma surgery in a patient in need thereof, comprising administering to the patient an effective dosage of the compound, and to a dosage form for the prevention or treatment of fibrosis after glaucoma surgery, comprising an effective dose of the compound according to one of the above aspects of the invention.

The skilled person is aware that any specifically mentioned drug may be present as a pharmaceutically acceptable salt of the drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

Dosage forms prepared for topical administration provide particular advantages in practicing the invention. The skilled artisan is aware of a broad range of possible recipes for providing topical formulations, as exemplified by the content of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011, ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2^nd Ed., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1^st Ed. CRC Press 1989; ISBN-13: 978- 0824781835). Intraocular dosage forms are particularly preferred.

As used herein, the term pharmaceutical composition refers to a compound as specified in the description or claims, particularly josamycin, kitasamycin, spiramycin and midecamycin, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

As used herein, the term treating or treatment of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow. Method of Manufacture and Method of Treatment according to the invention

The invention further encompasses, as an additional aspect, the use of a compound as specified in the description or claims, particularly josamycin, kitasamycin, spiramycin and midecamycin, or a pharmaceutically acceptable salt thereof, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of fibrosis, particularly after ocular surgery.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Brief description of the figures

Fig. 1 In-vitro induction of fibrosis in ocular cells by TGF-bI . The figure shows microscopic images of the immunofluorescence of primary human Tenon fibroblasts (hTF) treated with increasing TGF-bI concentrations in vitro (A) and quantification of TGF-bI induced fibrosis markers fibronectin (B) and alpha-SMA (smooth muscle actin) expression (C). Data are presented as mean ± SD.

Fig. 2 Relative viability (A) and relative proliferation (B) of hTFs in response to JM in vitro. hTFs were treated with JM in concentrations as indicated. CellQuanti- Blue-assay was carried out to quantify relative cell viability. Cell Proliferation ELISA, BrdU (chemiluminescence)-assay was carried out to quantify relative proliferation. Data are presented as mean ± SD. The results represent the means of three independent experiments. Levels of significance: *p£0.05;

**p£0.01 ; ***p£0.001; ****p£0.0001.

Fig. 3 In-vitro reduction of fibrosis marker in ocular cells by JM. The figure shows microscopic images of the immunofluorescence of hTF treated with increasing JM concentrations (A) and quantification of fibronectin expression (B). * indicates significances obtained by comparison of JM5 [5 pg JM /ml, 6*1 O ⁹ mol/ml], JM10 [10 pg JM /ml, 12*10 ^® mol/ml], JM25 [25 pg JM /ml, 30*10 ^® mol/ml] and JM50 [50 pg JM /ml, 60*1 O ⁹ mol/ml] to untreated cultures. Level of significances: *p£0.05; **p£0.01; ***p£0.001.

Fig. 4 In-vitro reduction of fibrosis marker in ocular cells by JM. Immunofluorescence of fibrosis markers a-SMA (green) and fibronectin (red) in hTF treated with JM75 [75pg/ml, 90*1 O ⁹ mol/ml], JM100 [100pg/ml, 120*10 ^® mol/ml] and JM150 [150pg/ml, 180*1 O ^® mol/ml] concentrations. Bar represents 10pm. Fig. 5 In-vitro reduction of TGF^1-induced fibrosis in ocular cells by JM.

Immunofluorescence of fibrosis markers a-SMA (green) and fibronectin (red) in hTF treated with TGF-bI [10ng/ml], TGF^1+JM75, TGF^1+JM100 and TGF^1+JM150 (A); and quantification of fibronectin (B) and a-SMA (C) expression after incubating with JM75, JM100, JM150, TGF-bI, TGF- b1+ M75, TGF^31+JM100 and TGF^31+JM150 in hTF. ^* indicates significances obtained by comparison of TGF-bI, JM75, JM100, JM150, TGF- b1+ M75, TGF^1+JM100 and TGF^1+JM150 to untreated cultures. # indicates significances obtained by comparison of TGF^1-treated cultures and cultures with combined treatment TGF^1+JM75, TGF^1+JM100 and TGF^1+JM50. Level of significances: *(#)p£0.05; **(##)p£0.01;

***(###)p£0.001.

Fig. 6 In-vitro reduction of TGF^1-induced fibrosis in ocular cells by JM. Western blot analysis of cell lysates of hTFs under different culture conditions, using antibodies against fibrosis markers fibronectin, collagen I, vimentin, collagen VI, a-SMA, b-actin; and b-tubulin as a loading control. kDa are marked on the left, as indicated (A). Quantification of Western blot data of fibronectin (B), a- SMA (C), Collagen I (D) and Collagen VI (E) for hTFs is provided. Each column represents the mean ± SD from three independent experiments. * indicates significances obtained by comparison of TGF-bI, JM25, JM75,

JM50, TGF^1+JM25, TGF- b1+ M75 and TGF- b1+ M150 to untreated cultures. # indicates significances obtained by comparison of TGF-bI -treated cultures and cultures with combined treatment TGF^1+JM25, TGF^1+JM75 and TGF^1+JM150. Level of significances: *(#)p£0.05; **(##)p£0.01;

***(###) p£0.001.

Fig. 7 In-vitro reduction of TGF^1-induced fibrosis in ocular cells by kitasamycin (KM).

Representation of the fibrotic cell culture model based on primary human tenon fibroblasts (hTF). The fluorescent labeling shows an increased expression rate of fibronectin and a-SMA by the application of the cytokine TGF-bI indicating a transformation of the cells to myofibroblasts. The parallel addition of KM can suppress this transformation partially (a), [10mM]) and completely at higher concentrations (b), [50mM]). Measuring bar in a) and b): 50pm. Examples

All experiments were conducted according to standard cell biology and analysis methods such as detailed for example in Harris, Graham and Rickwood, Wiley (ISBN 0470847581) or Helgason and Miller, Basic Cell Culture Protocols, Springer Protocols, ISBN 1493956523).

Example 1:

Primary human Tenon fibroblasts (hTF) were isolated. After Tenon fibroblasts proliferated to a confluent monolayer, cells were trypsinized and subcultured in 25 cm² cell culture flasks and after reaching a confluent layer in 25 cm² culture flasks, fibroblasts were trypsinized again and seeded in 75 cm² cell culture flasks. For immunofluorescence analysis, cells were seeded on 12 mm glass coverslips (PAA, Colbe, Germany) and cultured until 60%-70% confluence was reached. For all analyses fibroblasts of passage three to five were used. The fibroblastic phenotype was confirmed by immunohistochemistry using anti-vimentin antibody to verify the mesenchymal origin.

For stimulation- and inhibition experiments, human fibroblast subpopulations were starved for 24 h under serum-free conditions, followed by the application of increasing TGF-bI concentrations [5ng/ml, 10ng/ml, and 20ng/ml] for 48 hours.

The effective concentration of TGF-bI was determined by the induction of a-SMA expression in immunofluorescence experiments. Additionally, the protein fibronectin, a member of the extracellular matrix (ECM), was analyzed.

Cells were fixed with 3% paraformaldehyde (PFA) for 10 min, followed by incubation of the primary antibodies (directed against a-SMA and fibronectin) in a dilution of 1:100, respectively. After incubation cells were washed three times with PBS, followed by an incubation with the secondary antibodies for 45 min. Secondary antibodies were used at the following dilutions: donkey anti-mouse IgG (H+L)-Alexa Fluor 488 (1:50), or donkey anti-rabbit IgG (H+L)-Cy3 (1 :100). After incubation with secondary antibodies, cells were washed again three times with PBS and mounted. Fluorescent labeling was analyzed using a Nikon confocal fluorescence microscope equipped with a digital camera (Nikon Eclipse E400 with D-Eclipse C1 , Dusseldorf, Germany). All images depicted were from a single plane through fibroblast cell monolayers equipped with a 40x objective using the same settings.

After stimulation of hTF with TGF-bI, a concentration depending increase of a-SMA was observed, demonstrating fibroblast transformation into myofibroblasts (Fig. 1 A). Also, the expression of the ECM protein fibronectin increased. Software based analysis (ImageJ) of the fluorescence signal density confirmed these results (Figs. 1B,C).

In further experiments, the influence of the inhibitor josamycin (JM) at different concentrations was examined in hTF cultures. For cell culture experiments, a JM-stock solution (1mg of JM in 50mI of ethanol) was diluted with culture medium (0% FCS) to a concentration of 1mg in 1ml. Cytotoxicity-assays demonstrated that JM did not have an influence on cell viability even in highest concentrations (Fig. 2A) but reduced cell proliferation of hTF (Fig. 2B).

To analyze the inhibitory effect of JM on fibroblasts (hTF), cells were starved with 0% medium for 24h. Subsequently, the solution of JM was added in different concentrations for 24h into multi well plates, with 1ml of 0% medium.

After 24h of incubation, cells were fixed and primary antibodies directed against fibronectin were applied, followed by secondary antibody incubation. Analysis of cell behavior and protein expression was performed as before. The immunofluorescence is shown in Fig. 3A and the quantification of fibronectin expression in Fig. 3B. The incubation of hTFs in the presence of JM resulted in a concentration dependent decrease of fibronectin expression. The expression of vimentin was not affected. JM was tested also at a wider concentration range.

In further experiments, the influence of the combination of josamycin (JM) with TGF-b was examined. For cell culture experiments, JM-stock solution was diluted with culture medium (0% FCS) to a concentration of 1mg in 1ml. Fibroblasts (hTF) grown on glass coverslips were used for the experiments. As in previous experiments, cells were starved with 0% medium for 24h followed by the incubation with TGF-bI, JM, and the combination.

Coverslips with TΰRb1 were incubated for 48h. Total incubation of JM in both samples was 24h. After incubation, cells were fixed and primary antibodies directed against fibronectin and a-SMA were applied, followed by secondary antibody incubation. Analysis of cell behavior and protein expression was performed as in previous experiments.

After stimulation of hTF with 10ng/ml of TGF-bI concentration the inventors observed, similarly to the first experiment, increase of a-SMA and fibronectin expression. The incubation of hTFs with the inhibitor JM resulted in a decrease of fibronectin expression. Combination of TGF-bI and JM resulted in decrease in fibronectin and a-SMA, compared to “fibrotic” cells stimulated by TGF-bI alone. Higher concentrations of JM up to 150pg/ml were used to increase the inhibitory effect on fibrotic ECM expression in hTFs. These higher concentrations resulted in a more intense decrease of fibronectin and a-SMA expression in cells without (Fig. 4) and in cells with TGF-bI stimulation (Fig. 5). The combination of TGF-bI stimulation and application of JM resulted in a statistically significant decrease in both fibronectin and a-SMA expression, compared to cells stimulated by TGF-bI alone. Cell death or apoptotic nuclei were not observed. The quantification of fibronectin and a-SMA expression is shown in Figure 5B and Figure 5C. Cell lysates were also analyzed by immunoblotting in order to quantify and compare the amount of various ECM components. As loading control, b-tubulin was quantified, showing no notable differences in signal intensity, and about equal amounts of loaded proteins. In the immunoblot analyses, an increase in the amounts of fibronectin was observed, as well as of collagen I and a-SMA, in hTF cultures stimulated with TGF-bI when compared to untreated controls. When culturing hTFs with JM alone in increasing concentrations, a marked decrease in the respective proteins could be observed. Moreover, the combined application of TGF-bI and JM in increasing concentrations resulted in a concentration dependent decrease specifically of fibrosis markers a-SMA and collagen I expression (Fig. 6A). The cytoskeletal proteins vimentin and b-actin were not affected. Mitomycin C was also applied for comparison. Quantification of western blot analyses are shown in Figure 6B-E.

The incubation of hTFs with the macrolide KM also resulted in a decrease of fibronectin expression. Combination of TGF-bI and KM resulted in a dose dependent decrease in fibronectin and a-SMA expression, compared to “fibrotic” cells stimulated by TGF-bI alone. The results of 3 independent immunofluorescence experiments are demonstrated in Figure 7. This effect is comparable to the outcome of the macrolide JM.

Discussion

The aim of this study was to discover if a macrolide antibiotic can inhibit the process of fibrosis in ocular fibroblasts in vitro. Glaucoma surgery leads to tissue trauma by inducing inflammatory response. Activated mediators (VEGF-A, TGF-bI) promote migration of polymorphonuclear cells and macrophages, remove debris and prevent infection. TGF-b induces transdifferentiation of fibroblasts to myofibroblasts, expressing a-SMA and also promotes expression of ECM proteins (fibronectin, collagens). Persisting presence of myofibroblasts leads to excessive scar formation. Currently, MMC and 5-FU are the substances that are commonly used to prevent scarring after glaucoma (trabeculectomy) surgery. They proved to be efficient in many studies, but they can lead to hypotony with maculopathy, bleb infection, thin vascular blebs and endophthalmitis, which are potentially causing vision loss. In the literature one can find studies examining efficacy of Bevacizumab, an anti-VEGF drug, alone or together with MMC in treatment of post-surgical fibrosis. These experiments demonstrate that the combination of both compounds does not provide a higher benefit than using MMC alone. Also, Bevacizumab alone is not more efficient than using only MMC in inhibiting fibrosis after trabeculectomy. Pirfenidone was also recently demonstrated as a small molecule with potential to attenuate TGF-bI induced expression of fibronectin and a-SMA in ocular fibroblast cultures, without impairing cell viability.

The inventors found and examined Josamycin (JM), a 16-membered ring macrolide, by in-silico comparative gene expression-based studies as a potential anti-fibrotic small molecule. As an antibiotic JM was reported to be effective against S. pyogenes and bronchopulmonary infections. Macrolides were also examined in various studies, suggesting an impact on fibrotic processes. One of them was performed by Kanai et al. who examined the influence of two macrolides, roxithromycin (RXM) and JM on matrix metalloproteinase (MMP) production from nasal polyp fibroblasts in vitro. They observed that addition of RXM, but not JM, suppressed production of MMPs. Effects of RXM in inhibiting fibroblasts growth on nasal polyp fibroblasts were also confirmed in other studies. It was also confirmed that some of the 14-membered ring macrolides, including RXM, have an antiangiogenic and antitumor effect.

In other studies, the macrolide JM was identified to be an effective inhibitor of T-cell proliferation by inhibiting their synthesis of IL-2. IL-2 synthesis by myofibroblasts (activated fibroblasts) was already reported to be increased in patients suffering from post-radiation fibrosis. Dysregulation of IL-2 levels in T-lymphocytes was also observed in patients with cystic fibrosis suggesting that this cytokine plays a role in fibrotic processes.

The inventors surprisingly found the anti-fibrotic activity of JM in ocular fibroblasts, motivating its exploration as a candidate for inhibiting post-surgical fibrosis in trabeculectomy.

In the immunofluorescence experiments that the inventors conducted, they focussed on fibronectin and a-SMA, which are both synthesized by myofibroblasts. a-SMA is an indicator for myofibroblasts, whereas synthesis of fibronectin as a component of the ECM is increased in comparison to fibroblasts. It was already confirmed that TGF-b is expressed in primary human ocular fibroblasts subpopulations and that it plays a key role in wound healing and scarring process. Following incubation of hTF with TGF-bI, the inventors confirmed increase in a-SMA and fibronectin expression emphasizing fibroblast transformation into fibrotic active myofibroblasts.

In the next step the inventors examined the antifibrotic effect of JM, on “not fibrotic” hTF. They observed that with increasing concentrations of JM, up to 50pg/ml and later to 150pg/ml, expression of fibronectin was gradually decreasing (Fig. 3, 4). These findings demonstrated the suppressing effect of JM on synthesis of ECM components, in hTF not stimulated with TGF-bI Additionally, the inventors did not observe any expression of a-SMA which confirms that JM did not induce fibroblast transformation into a-SMA positive myofibroblasts.

In the next steps the inventors examined how increasing doses of JM affected the expression of fibronectin and a-SMA in TGF-bI stimulated hTF. They observed that both, fibronectin and a-SMA expression were decreasing with higher values of JM and expression was the lowest with concentration of 150pg/ml. This finding demonstrates that JM can be efficient in reducing fibrosis in “fibrotic” ocular fibroblasts in cell culture in vitro, stimulated by TGF-bI, by inhibiting fibrotic markers which are involved in scar formation processes in vivo.

To confirm these results, Western Blot analyses of hTF cell lysates, stimulated and not- stimulated with TGF-bI and incubated with the combination of TGF-bI and of JM in increasing concentrations were performed. The inventors observed a strong and significant reduction of a-SMA expression. Fibronectin and Collagen VI quantity was also affected and decreased with increasing concentrations of JM. The inventors did not observe notable differences in Collagen I expression. These findings confirm the immunofluorescence results that JM has a notable impact specifically on the TGF-bI induced process of fibrosis in vitro. Evaluation of another member of the macrolide family (KM) could already show a dose dependent antifibrotic effect in the primary human fibroblast cell culture model. This effect is comparable to the outcome of the macrolide JM.

It can be possible to apply JM in form of eye drops or by implants after glaucoma surgery. To conclude, JM is able to decrease synthesis of proteins relevant in fibrosis in hTF and also in activated myofibroblasts in vitro. Therefore, it constitutes a promising candidate for the treatment of fibrosis after glaucoma filtration surgery or drainage device implantation in vivo.

Claims

1. A pharmaceutical compound selected from josamycin, and a derivative of josamycin for use in treatment or prevention of fibrosis.

2. The compound for use according to claim 1, wherein the compound is selected from josamycin, kitasamycin, spiramycin and midecamycin.

3. The compound for use according to claim 1 or 2, wherein the compound is selected from josamycin or kitasamycin,

4. The compound josamycin for use in treatment or prevention of fibrosis.

5. The compound for use according to any one of claims 1 to 4, wherein the fibrosis is ocular fibrosis.

6. The compound for use according to any one of claims 1 to 5, wherein fibrosis is caused by, or occurs subsequent to, glaucoma surgery.

7. The compound for use according to any one of claims 5 or 6, wherein the compound is administered via ocular injection.

8. The compound for use according to any one of the above claims 6 or 7, wherein the compound is administered during or after glaucoma surgery.