WO2025088052A1

WO2025088052A1 - Cyclic analogue of glycine-proline for use in the prevention and/or treatment of medical implant-related fibrosis

Info

Publication number: WO2025088052A1
Application number: PCT/EP2024/080110
Authority: WO
Inventors: Sara VLEMINCKX; Elien Monique GEVAERT
Original assignee: Rousselot BV Belgium
Current assignee: Rousselot BV Belgium
Priority date: 2023-10-26
Filing date: 2024-10-24
Publication date: 2025-05-01
Anticipated expiration: 2026-04-26
Also published as: BE1032058B1; BE1032058A1

Abstract

The present invention pertains to a cyclic analogue of glycine-proline for use in the prevention and/or treatment of fibrosis, for example skin fibrosis, hypertrophic scars, keloids, organ-specific fibrosis or implant-related fibrosis. The peptide of the invention can be administered orally, but also instance provided in a topical formulation or cosmetic composition. Moreover, the peptide of the invention can be provided as a coating on an implantable device and/or medical device, to prevent excess fibrosis and encapsulation thereof.

Description

CYCLIC ANALOGUE OF GLYCINE-PROLINE FOR USE IN THE PREVENTION AND/OR TREATMENT OF MEDICAL IMPLANT-RELATED FIBROSIS

TECHNICAL FIELD

The present invention pertains to the field of prevention and/or treatment of fibrosis and an active ingredient for use therein.

BACKGROUND OF THE INVENTION

Wound healing is a dynamic and multi-stage process necessary to reconstruct and regenerate the tissue after damage. The wound healing process can be classified into the four distinct phases:

(1) hemostasis, involving vasoconstriction, primary hemostasis, and secondary hemostasis, and that stops the bleeding;

(2) inflammatory phase, in which damaged and dead cells, along with pathogens or debris, are cleared out;

(3) proliferative phase, in which new tissue formation and angiogenesis occurs;

(4) remodelling phase, wherein appropriate tensile strength is achieved through reorganization, degradation, and resynthesis of the extracellular matrix (in particular collagens).

An abnormal or exuberant response in one or more of the wound healing phases can lead to an undesirable amount of pathological fibrous tissue formation, referred to as fibrosis or scar tissue formation. The activation of fibroblastic or highly contractile myofibroblasts is thought to be a major contributor to the accumulation of excessive and stiff scar tissue as seen in pathological fibrotic conditions. Although mild fibrosis to a certain extent can be part of the normal tissue replacement process, excessive fibrosis can lead to physical, along with significant psychological, emotional, and social problems. Several examples of the negative impact of excessive fibrosis are illustrated next:

First, fibrosis can manifest as an organ-specific condition. Organ-specific fibrosis can have different underlying reasons, but is often associated with inflammation and/or occurring as a symptom of underlying disease. Unchecked fibrosis can result in destruction of the architecture of the underlying organ and impairment of organ function. Organ-specific fibrotic disorders include, for example pulmonary fibrosis, pulmonary hypertension, asthma, chronic obstructive pulmonary disease, liver fibrosis, kidney fibrosis, nonalcoholic steatohepatitis (NASH), musculoskeletal fibrosis, atrial fibrosis, Dupuytren’s contracture, cellulite, endometriosis, keloid formation in the connective tissue and the like.

Second, fibrosis can occur after tissue manipulation resulting from surgery, i.e. in the form of post-operative fibrosis. For example, post-operative fibrosis is a typical problem seen following abdominal, neurological, spinal, vascular, thoracic or other types of surgery using both classical open and arthroscopic/laparoscopic procedures. Post-operative fibrosis can negatively influence the result of surgery and healing. For example, glaucoma surgery is a challenging procedure and not performed widespread clinically because of the severe complications that can arise from excessive fibrosis. Postoperative adhesions (PA) are examples of fibrotic tissues that are the most common driver of long-term morbidity after for instance abdominal, thoracic and pelvic surgery.

Third, fibrosis can occur around a medical implant and/or device, e.g. due to trauma, abnormal inflammation/infection, a foreign body response and/or implant/device (micro)movement. In that case, the fibrosis can also be referred to as implant-related fibrosis. For instance, breast implants and methods for breast reconstruction and augmentation are well known and have been used for a period of over twenty years. However, the formation of excess fibrosis remains an important issue associated with breast implants. The excessive fibrosis may lead to pathological tightening around the implant, also known as capsular contracture. Capsular contraction can lead to an implant that is misshapen, painful, hard and can attain an unnatural appearance and feel. Unwanted fibrosis around implants is also typically seen for implantable sensors, drug delivery devices, pacemakers, cardiovascular stents, and orthopedic implants, to name a few. The encapsulation of such implants by a fibrotic membrane can “shield off” the implant from the body, thus preventing adequate tissue integration and ultimately necessitating implant removal.

Fourth, skin-related fibrosis can occur as a result of acute injury or irritation to the skin, and which mostly can be classified as being either hypertrophic scars or keloids. The difference between hypertrophic scars and keloids lies in that hypertrophic scars typically develop within the original wound boundaries and are prone to regress over time, while keloids typically grow without limitation and rarely regressing. Hypertrophic scars or keloids frequently are the result of cuts, burns, acne scars, chickenpox scars, piercings, scratches, surgical cuts, tattoos, tattoo removal, insect bites, and vaccination sites, to name a few. The mechanisms of formation of hypertrophic scar and keloids are not yet completely understood, however it is believed that inflammation is involved in modulating collagen synthesis, since the degree of inflammation tend to positively correlate to final scar size.

Finally, fibrosis is a common symptom resulting from medical conditions such as fatty liver disease, chronic obstructive pulmonary disease, asthma, or systemic sclerosis.

There are several anti-fibrotic drugs known that can play a role in reducing excessive fibrosis. These include for lung fibrosis-approved drug Pirfenidone or pan-av integrin inhibitors for lung fibrosis. A limitation of these drugs is that they require approval from federal agencies and medical prescription, and are associated with several adverse effects. For example, Pirfenidone reduces fibrosis by downregulating the production of growth factors and procollagens I and II. However, Pirfenidone is also associated with gastrointestinal, skin, and hepatic side effects. Other, non-pharmaceutical, anti-fibrotic products including both traditional plant-based materials and protein, however, there currently is no strong evidence to support their efficacy. Overall, the currently-known anti-fibrotic products appear insufficiently effective particular with regard to limiting tissue contraction and activation and/or transdifferentiation in skin cells and/or stromal cells such as myofibroblasts.

In the case of medical implants, a known strategy is to coat (e.g. immobilise) their surface with anti-fibrotic molecules in an attempt to reduce excessive fibrosis. These include coatings based on anti-inflammatory drugs, anti-fibrotic drugs, anti-proliferative drugs, and/or pro- angiogenic drugs to prevent excessive cellular fibrosis encapsulation. For instance, US6110155A describes a medical catheter carrying the anti-inflammatory corticosteroid dexamethasone, as a means of preventing tissue fibrosis. However, anti-inflammatory agents are preferably avoided, in order not to negatively influence normal wound healing and to avoid common side effects of corticosteroids and other anti-inflammatory agents.

Overall, the anti-fibrotic products known to date are typically administered after fibrosis has already manifested, but their efficacy in the prevention of fibrosis is unknown. Furthermore, the current anti-fibrotic products do not seem to sufficiently target the increased cellular contraction, activation and/or transdifferentiation in skin cells and/or stromal cells such as myofibroblasts, prior to the onset of fibrosis.

There remains an unmet need for active ingredients that ameliorate fibrosis, in particular more effectively and/or safely. Foremost, there is a need for active ingredients that can prevent fibrosis, e.g. by reducing cellular contraction, activation and/or transdifferentiation in skin cells and/or stromal cells such as myofibroblasts, in particular as implant coatings with myofibroblast-suppressing and contraction-supressing features.

The present invention aims to provide one or more solutions to the existing limitations in ameliorating excessive fibrosis.

SUMMARY OF THE INVENTION

The present inventors found that peptides which are cyclic analogues of glycine-proline peptide are surprisingly effective in preventing and/or treating fibrosis, at least in part by preventing the contraction, activation, transdifferentiation and/or collagen (type I) overproduction in skin cells and/or stromal cells (e.g. fibroblasts and myofibroblasts).

The peptide of the invention was found to be more beneficial in the context of the current invention than other (related) cyclic or linear peptides, or mixtures thereof.

The present inventors found a dose-response effect of the peptide of the invention in ameliorating fibrosis. Hence, the current invention pertains also to the desirable amount of the peptide for preventing and/or treating fibrosis.

The peptide of the invention was found to be capable to prevent fibrosis in particular when provided as a coating on an implantable device. The present inventors show that the peptide of the invention is particularly suitable for preventing fibrosis (rather then only able to treat fibrosis after it has developed), making it particularly suitable as a preventive measure in a coating. It is advantageous to use the peptide of the invention in a coating, in contrast to the use of anti-inflammatory agents and immune suppressants known in the art.

The present findings by the inventors are surprising, because cyclic glycine-proline has been suggested in the art (J P2019206481 A) as a promoter of production of type I and type V collagen and therefore suggested as a suitable skin anti-aging agent. Considering that fibrosis is characterized by excess collagen synthesis (mostly collagen type I), this points to the direction that cyclic glycine-proline worsens fibrosis. However, the current inventors unexpectedly found that glycine-proline may allow for collagen synthesis, but at the same time inhibits excessive collagen synthesis as a hallmark of fibrosis. Without being bound by theory, providing for a balanced collagen synthesis may be in part a mechanism by which cyclic glycine-proline and analogues effectively prevent and/or treat fibrosis. In an aspect, the present invention pertains to a cyclic analogue of glycine-proline for use in the prevention and/or treatment of fibrosis.

In a preferred embodiment, the fibrosis herein is preferably one or more of skin fibrosis, cellulite, Dupuytren’s contracture, hypertrophic scars, keloids, atrial fibrosis, heart, fibrosis, liver fibrosis, pulmonary fibrosis, pancreatic fibrosis, kidney fibrosis, vascular fibrosis, heart fibrosis, organ-specific fibrosis, surgery-related fibrosis, medical implant-related fibrosis and adhesion formation.

The cyclic analogue of glycine-proline can be administered orally, such as in the form of a food supplement.

In an aspect, the present invention pertains to a topical formulation comprising the cyclic analogue of glycine-proline.

In an aspect, the present invention pertains to a cosmetic composition comprising the cyclic analogue of glycine-proline, e.g. suitable for non-therapeutic improvement of the skin appearance in fibrosis.

In an aspect, the present invention pertains to a substrate coated with a cyclic analogue of glycine-proline, wherein the substrate is preferably a medical device such as a medical implant.

DETAILLED DESCRIPTION OF THE INVENTION

The present invention pertains to a use of a cyclic analogue of glycine-proline for the prevention and/or treatment of fibrosis.

Cyclic analogue of glycine-proline

The term “cyclic analogue of glycine-proline” in the context of the current invention means a cyclic glycine-proline (CAS 3705-27-9, (8aS)-2,3,6,7,8,8a-Hexahydropyrrolo[1 ,2-a]pyrazine- 1 ,4-dione), or an analogue thereof, including cyclo-L-glycyl-L-2-allylproline, cyclic cyclopentyl- G-2-MeP, cyclic cyclohexyl-G-2-MeP and cyclic(tri(pro-glycine)). In the context of the current invention, the “cyclic analogue of glycine-proline” may be synthetic or natural and/or derived from a natural material. In addition or alternatively, the term “cyclic analogue of glycine- proline” can mean any cyclic form of peptide or analogue thereof, which is formed by the condensation of a glycine- and (hydroxy)proline-containing dipeptide or tripeptide, preferably a glycine- and (hydroxy)proline collagen dipeptide or a glycine- and (hydroxy)proline collagen tripeptide. The term “cyclic glycine-proline analogue” in the context of the current invention means an adapted form of cyclic glycine-proline, e.g. to improve the therapeutic potential of the peptide. For instance, cyclo-L-glycyl-L-2-allylproline is a synthetic analogue of cyclic glycine-proline obtainable by inserting an -allyl substituent at the C-8a position of the diketopiperazine skeleton in cyclic glycine-proline.

In an embodiment, the cyclic analogue of glycine-proline is an active ingredient.

In an embodiment, the cyclic analogue of glycine-proline is synthetic (i.e. produced by chemical synthesis). Chemical synthesis encompasses for example solid phase synthesis, chemoenzymatic synthesis, Merrifield synthesis and Bailey peptide synthesis.

In an embodiment, the cyclic analogue of glycine-proline is natural and/or derived from a natural material.

In an embodiment, the cyclic analogue of glycine proline is a compound according to Formula

I (Shown in Table 1), wherein

X¹ is selected from the group consisting of NR', O and S;

X² is selected from the group consisting of CH2, CR⁶R⁷, NR', O and S;

R¹, R², R³, R⁴ and R⁵ are independently selected from the group consisting of — H, — OR', — SR', — NR'R', — NO₂, — CN, — C(O)R', — C(O)OR', — C(O)NR'R', — C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, allenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl and substituted heteroarylalkyl;

R⁶ and R⁷ are each independently selected from hydrogen and alkyl; each R' is independently selected from the group consisting of — H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl; or R⁴ and R⁵ taken together are — CH2 — (CH2)n — CH2 — where n is an integer from 0-6; or R² and R³ taken together are — CH2 — (CH2)n — CH2 — where n is an integer from 0-6; wherein when R¹=methyl and R²=R³=R⁴=H then R⁵ benzyl and; when R¹=H, at least one of R² and R³ H.

II (shown in Table 1), wherein R¹ is selected from the group consisting of -H, -OR', -SR', - NR'R', -NO₂, -CN, -C(O)R', -C(O)OR', -C(O)NR'R', -C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, allenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl substituted heteroarylalkyl, and -CH2-(CH2)n- CH2-, where n is an integer from 0-6.

In an embodiment, the cyclic analogue of glycine proline is a compound according to Formula III (shown in Table 1), wherein R⁴ is independently selected from the group consisting of -H, - OR', -SR', -NR'R', -NO₂, -CN, -C(O)R', -C(O)OR', -C(O)NR'R', -C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl, alkynyl, allenyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl substituted heteroarylalkyl and -CH2- (CH2)n-CH2-, where n is an integer from 0-6.

In an embodiment, the cyclic analogue of glycine-proline is cyclic glycine-proline and/or a compound according to Formula IV (shown in Table 1).

In the context of the current invention, “cyclic glycine-proline” means the same and can be used interchangeably with “Cyclo(Gly-Pro)”, “Cyclo(prolylglycyl)”, “cyclic proline-glycine”, “Cyclo (Pro-Gly)”, “Cyclo(glycylprolyl)”. The cyclic glycine-proline encompasses synthetic cyclic glycine-proline. In addition or alternatively, the cyclic glycine-proline encompasses peptides obtained from the cyclization reaction of a linear peptide, preferably a linear dipeptide (e.g. Gly-Pro, Pro-Gly, Gly-Hyp or Hyp-Gly) or a linear tripeptide (e.g. Gly-Pro-Z, Pro-Gly-Z, Gly-Hyp-Z or Hyp-Gly-Z, wherein Z can be any amino acid).

In an embodiment, the cyclic analogue of glycine-proline is cyclo-L-glycyl-L-2-allylproline and/or a compound according to Formula V (shown in Table 1).

In the context of the current invention, “cyclo-L-glycyl-L-2-allylproline” means the same as and can be used interchangeably with “Cyclic G-2-AllylP” or “NNZ2591”. In an embodiment, the cyclo-L-glycyl-L-2-allylproline and/or compound according to formula V is a compound according to Formula I, wherein X¹ is NH; X² is CH2 ; R¹ is - CH2 CHCH2 ; and R² , R³ , R⁴ , and R⁵ are each hydrogen.

In an embodiment, the cyclic analogue of glycine-proline is Cyclic Cyclopentyl- G-2-MeP and/or a compound according to Formula VI (shown in Table 1).

In an embodiment, the Cyclic Cyclopentyl- G-2-MeP and/or compound according to formula VI is a compound of Formula I, wherein X¹ is NH; X² is CH2; R¹ is CH3 ; R² and R³ are each hydrogen; and R⁴ and R⁵ taken together are -CH2 -(CH2)2 -CH2

In an embodiment, the cyclic analogue of glycine-proline is Cyclic cyclohexyl-G-2-MeP and/or a compound according to Formula VII (shown in Table 1).

In an embodiment, the Cyclic cyclohexyl-G-2-MeP and/or compound according to formula VII is a compound of Formula I, wherein X¹ is NH; X² is CH2 ; R¹ is CH3; R² and R³ are each hydrogen; and R⁴ and R⁵ taken together are -CH2 -(CFhh -CH2

In an embodiment, the cyclic analogue of glycine-proline is glycyl-L-prolylglycyl-L- prolylglycyl- L-prolyl . The terms “glycyl-L-prolylglycyl-L- prolylglycyl-L-prolyl”, “cyclic(tri(Pro- Gly))” and “c(PG)3” mean the same and can be used interchangeably in the context of the current invention.

Table 1. Compounds according to formula l-VII

The compounds of the invention encompass cyclic analogues of glycine-proline that contain chiral (asymmetric) centers, or which as molecule as a whole may are chiral. The compounds of the present invention include individual stereoisomers (enantiomers and diastereoisomers) of the cyclic analogues of glycine-proline and mixtures of these. In the context of the current invention, the term “stereoisomer” means a compound with the same molecular formula and sequence of bonded atoms (i.e., atom connectivity), though differ in the three-dimensional orientations of their atoms in space. In the context of the current invention, the term “enantiomer” means two compounds that are stereoisomers in that they are non- superimposable mirror images of one another, which may be denoted with either (R)- or (S)- configuration.

The compounds of the invention encompass a pharmaceutically acceptable salt of a cyclic analogue of glycine-proline. In the context of the current invention, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable organic or inorganic salts. Acid addition pharmaceutically acceptable salts suitable in the context of the current invention include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1 ,l'-methylene-bis- (2-hydroxy-3-naphthoate)) salts. Base addition pharmaceutically acceptable salts suitable in the context of the current invention include ammonium salts, alkali metal salts (e.g. those of potassium and sodium), alkaline earth metal salts (e.g. those of calcium and magnesium), and salts with organic bases [e.g. dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine (for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl -propylamine), or a mono-, di- or trihydroxy lower alkylamine (for example mono-, di- or triethanolamine)]. Pharmaceutically acceptable salts may be formed for instance as embodiments of the compound of Formula I- VII, which contain a suitable acidic or basic group.

The compounds of the invention encompass a hydrate of the cyclic analogue of glycine- proline.

The cyclic analogue of glycine-proline or the pharmaceutically acceptable salt thereof, may be present in an amorphous form or in a crystalline form in the context of the current invention. The cyclic analogue of glycine-proline or the pharmaceutically acceptable salt thereof, may be in any polymorph form in the context of the current invention. Types of fibrosis

The term "fibrosis" in the context of the current invention means a condition in which there is abnormal production, accumulation, and/or deposition of extracellular matrix (proteins) such as by skin cells and/or stromal cells, in particular fibroblasts and/or myofibroblasts. For example, an “abnormal production” means that the production of extracellular matrix (proteins) exceeds the degradation rate. The term “fibrosis” encompasses an abnormal contraction of extracellular matrix (proteins). For example, an “abnormal contraction” means that the contraction is too high, so that the tissue is overly stiff and such that it is associated with negative functional and/or cosmetic effects. Typically, contraction occurs during the proliferative phase, during which a cell-rich granulation tissue is formed. The skilled person is aware that contraction is an important part of wound healing, as it enables wound closure. However, wound contraction in has both positive and negative effects. It is beneficial to wound healing by narrowing the wound margins, which leads to wound closure; however, excessive contraction causes formation of undesirable contracture and scarring, leading to cosmetic and functional problems (Li et al. J Tissue Viability. 2011 Nov; 20(4): 108-120).

There are different methods that are capable of measuring presence of fibrosis (and the inhibition thereof) in the context of the current invention, which encompass in vitro and in vivo methods.

Suitable in vitro models include the hydrogel contraction assay and/or the myofibroblast transformation assay as disclosed herein in the Examples.

A suitable in vivo model is the subcutaneous or intraperitoneal implantation model as disclosed by Farah et al. (Nat Mater. 2019 August; 18(8): 892-904), for instance to study efficacy of peptide-loaded and/or - coated implants.

Suitable models to study fibrosis include models that have been developed to asses the amount of foreign body response (FBR) in vitro or in vivo. For example, Sharifi et al. (Adv Healthc Mater. 2019 Feb;8(4):e1801425) have developed an in vitro FBR-on-a-chip model as a physiologically relevant in vitro setting which reproduces the innate immune cell interactions with implants. The FBR-on-a-chip model is a suitable model to study fibrosis in the context of the current invention.

The prevention and/or the treatment of the fibrosis can in the context of the current invention be of therapeutic or non-therapeutic, depending on the severity of fibrosis and/or the context. The professional medical practitioner is able to determine on a case-by-case basis whether the fibrosis is pathological or in a pathological subject, or non-pathological or in a healthy subject. Therapeutic use can for example mean a use in severe fibrosis and/or pathological situation and/or where there are symptoms of pain and suffering which may lead to (serious) health and psychological risks. Non-therapeutic use can for example mean a use fibrosis having a nature or severity such that there are no symptoms of pain and suffering and/or it naturally disappears over time (i.e. it is not chronic). A non-therapeutic use in particular encompasses a cosmetic use e.g. to improve the appearance of the skin affected by fibrosis, but in a non-pathological context and/or in absence of symptoms of pain and suffering.

In a preferred embodiment, the fibrosis is one or more selected from the group consisting of medical implant-related fibrosis, surgery-related fibrosis, adhesion formation including post- surgical adhesion formation, skin fibrosis, a hypertrophic scar, a keloid, liver fibrosis, pulmonary fibrosis, pancreatic fibrosis, kidney fibrosis, vascular fibrosis, endometriosis, atrial fibrosis and heart fibrosis.

In an preferred embodiment, the fibrosis is organ-specific fibrosis. The organ-specific fibrosis is preferably one or more selected from the group consisting of pulmonary fibrosis , liver fibrosis, pancreatic fibrosis, kidney fibrosis, musculoskeletal fibrosis, atrial fibrosis, heart fibrosis, Dupuytren’s contracture, cellulite, endometriosis and keloid formation in the connective tissue.

In a preferred embodiment, the fibrosis is a symptom in a condition selected from the group consisting of pulmonary hypertension, fatty liver disease, Dupuytren’s contracture, cellulite, chronic obstructive pulmonary disease, nonalcoholic steatohepatitis (NASH), asthma and systemic sclerosis.

In an embodiment, the fibrosis as disclosed herein is not the condition cystic fibrosis and/or the cyclic analogue of glycine-proline is not for the prevention and/or treatment of the condition cystic fibrosis. Cystic fibrosis is a genetic condition that typically causes damage and scarring in different organs and may necessitate treatment of thick mucus formation and infection. Nevertheless, the cyclic analogue of glycine-proline may prevent and/or treat the fibrosis as a symptom in cystic fibrosis (without targeting the disease itself). Hence, in an embodiment, the fibrosis is fibrosis in the condition cystic fibrosis.

In an embodiment, the fibrosis as disclosed herein is not the condition myelofibrosis and/or the cyclic analogue of glycine-proline is not for the prevention and/or treatment of the condition myelofibrosis. Myelofibrosis is a bone marrow blood cancer, wherein healthy marrow is typically replaced by scar tissue. Prevention and/or treatment of myelofibrosis may necessitate a therapy that targets the cause of these disease and/or the underlying myeloproliferative condition. Nevertheless, the cyclic analogue of glycine-proline may prevent and/or treat the fibrosis as a symptom in myelofibrosis (without targeting the disease itself). Hence, in an embodiment, the fibrosis is fibrosis in the condition myelofibrosis.

Mechanism(s) of action

Without being bound by theory, there may be different mechanisms that alone or in conjunction lead to the beneficial anti-fibrosis effects.

The present inventors found that a cyclic analogue of glycine-proline is surprisingly effective in preventing fibrosis, at least in part by preventing the contraction, activation and/or transdifferentiation in skin cells and/or stromal cells, particularly fibroblasts and/or myofibroblasts. In particular, it was found that the cyclic analogue of glycine-proline can ameliorate the (trans)differentiation of skin cells (e.g. fibroblasts) and/or stromal cells into myofibroblasts.

Moreover, the present inventors also found that the cyclic analogue of glycine-proline inhibits an overproduction of collagen and/or provides for a balanced collagen synthesis (e.g. in skin cells and/or stromal cells), foremost seen for collagen type I.

Excess collagen production is typically a hallmark in fibrosis. The cells responsible for collagen production in fibrosis are believed to be fibroblasts and myofibroblasts. For instance, lung fibrosis and other types of fibrosis typically favor a predominance of at least type I and/or type III collagen, but also collagen type V and other types of collagen can be overexpressed in fibrosis. There often additionally is an increase in other extracellular matrix proteins such as fibronectin, elastin, laminin, hyaluronan, chondroitin sulfate and/or heparin sulfate proteoglycans.

Without being bound by theory, providing for a balanced collagen synthesis may be in part a mechanism cyclic glycine-proline and analogues thereof are highly effective in ameliorating (in particular preventing) fibrosis, without counteracting the normal wound healing (by not completely bringing collagen synthesis to a halt).

In a preferred embodiment, the preventing and/or treating fibrosis is by ameliorating, preferably decreasing, cell activation. In a preferred embodiment, the preventing and/or treating fibrosis is by ameliorating, preferably decreasing, cell transdifferentiation. In a preferred embodiment, the preventing and/or treating fibrosis is by ameliorating, preferably decreasing, overproduction of collagen.

In an embodiment, the cyclic analogue of glycine-proline reduces or increases the migration of a cell, preferably a skin cell and/or a stromal cell. In an embodiment, the cyclic analogue of glycine-proline reduces or increases proliferation of a cell, preferably a skin cell and/or stromal cell. In an embodiment, the cyclic analogue of glycine-proline reduces or increases the (trans)differentiation of a cell, preferably a skin cell and/or a stromal cell. In an embodiment, the cyclic analogue of glycine-proline reduces or increases the activation of a cell, preferably a skin cell and/or a stromal cell. In an embodiment, the cyclic analogue of glycine-proline reduces or increases the contraction of extracellular matrix, a skin cell and/or a stromal cell. In an embodiment, the cyclic analogue of glycine-proline reduces or increases wound contraction.

In a preferred embodiment, the cyclic analogue of glycine-proline ameliorates, preferably reduces, the (trans)differentiation of a skin cell (e.g. fibroblast) and/or a stromal cell into a myofibroblast.

In an embodiment, a reduced contraction means a reduction in at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, most preferably at least 50%, e.g. compared to a negative control group (not receiving the cyclic analogue of glycine-proline). A reduction in contraction is preferably measured in the collagen hydrogel contraction assay as disclosed herein.

In an embodiment, a reduced differentiation and/or transdifferentiation means a reduction in at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, most preferably at least 50%, e.g. compared to a negative control group (not receiving the cyclic analogue of glycine-proline). A reduction in differentiation and/or transdifferentiation is preferably measured in the myofibroblast transformation assay as disclosed herein.

In an embodiment, the cyclic analogue of glycine-proline reduces or inhibits the overexpression of one or more extracellular matrix proteins, preferably one or more collagens, more preferably one or more of collagen type I, III and/or V (e.g. in a skin cell and/or stromal cell). In an embodiment, the cyclic analogue of glycine-proline promotes a balanced synthesis of one or more extracellular matrix proteins, preferably one or more collagens, more preferably one or more of collagen type 1, 111 and/or V (e.g. in a cell such as a skin cell and/or stromal cell).

The term “skin cell” in the context of the current invention means any type of cell that is predominantly found in the skin (particularly epidermis) any type of cell that provides a specialized function in the skin (particularly epidermis), and/or any type of cell that forms the skin (particularly epidermis), and encompasses keratinocytes, melanocytes, Langerhans cells, and Merkel cells. The term “stromal cell” in the context of the current invention means any multipotent or pluripotent cell capable of developing specifically into a distinct type of connective tissue cell. The stromal cell may be a fibroblast, myofibroblast, smooth muscle cell, pericyte, or mesenchymal stem/stromal cell. The term “myofibroblast” in the context of the current invention means a cell type that is in between a fibroblast and a smooth muscle cell in differentiation. The “myofibroblast” may be formed for instance by one or more of the following pathways: partial smooth muscle differentiation of a fibroblastic cell, activation of a stellate cell, loss of contractile phenotype of a smooth muscle cell, direct myofibroblastic differentiation of a progenitor cell resident in a stromal tissue, homing and recruitment of a circulating mesenchymal precursor which can directly differentiate as above or indirectly differentiate through the other cell types as intermediates and/or epithelial to mesenchymal transdifferentiation of an epithelial cell. In a preferred embodiment, the myofibroblast is a cell that is positive (e.g. shown by staining) for the markers intermediate filament vimentin, a- smooth muscle actin and for paladin.

Coatings, immobilization

The cyclic analogue of glycine-proline appears particularly suitable to prevent fibrosis around a medical implantable, e.g. to thereby prevent and/or treat medical implant-related fibrosis.

In an aspect, the present invention pertains to a substrate coated with the cyclic analogue of glycine-proline.

In an embodiment, the cyclic analogue of glycine-proline is provided as a coating on a substrate. The substate is preferably a medical medical device such as a medical implant. The medical device is preferably selected from the group consisting of a suture material, a drug-eluting bead, a cardiovascular implant, a stent, a catheter, a pacemaker, an electrical lead, an implantable cardiac defibrillator, a nerve stimulator, a degradable implant, an interocular lense, an implantable drug delivery device, an implantable pump, an intra-uterine device, a surgical mesh implant, a breast implant and a prosthesis. The term “medical implant” in the context of the current invention means a device or tissue that is placed inside or on the surface of the body. The medical implant can be prosthetics, intended to replace missing body parts. The medical implant can be intended to deliver medication (e.g. as a drug delivery device), monitor body functions, or provide support to organs and tissues. The term “medical implant” encompasses implants made from skin, bone or other body tissues. The “medical implant” in the context of the current invention can be placed permanently or it may be removed once no longer needed. For example, a stent or hip implant is typically intended to be permanent, but chemotherapy ports or screws to repair broken bones can be removed when they no longer needed. The term “medical device” in the context of the current invention means any instrument, machine, implant, intended to treat, cure, prevent, mitigate, diagnose disease, and includes any medical device classified as such by the Medical Device Regulation in the European Union and/or the U.S. Food and Drug Administration.

In an embodiment, the implant is a cardiac implant, preferably one or more selected from the list consisting of pacemaker, implantable cardioverter defibrillator, left ventricular assist device, artificial heart valve and transcatheter aortic valve replacement.

In an embodiment, the implant is a cochlear implant.

In an embodiment, the implant is a deep brain stimulation device.

In an embodiment, the implant is an artificial hip or artificial knee joint.

In an embodiment, the implant is a gastric band.

In an embodiment, the implant is a spinal cord stimulator and/or neuro-stimulator.

In an embodiment, the implant is an implantable sensor, such as one or more selected from the list consisting of glucose sensor, pressure sensor, oxygen sensor, neural sensor and pH sensor.

In various embodiments, the cyclic analogue of glycine-proline of the invention is immobilized on the substrate.

The term “immobilization” or “to immobilize” in the context of the current invention means that a molecule (e.g. peptide) is chemically linked to a substrate, wherein the linking may be directly (e.g. by direct chemical conjugation) or indirectly (e.g. through a linker moiety or following chemical functionalization of the substrate). The immobilization in the context of the current invention may be reversible or irreversible.

In an embodiment, the cyclic analogue of glycine-proline is not released from the substrate preferably following immobilization. The immobilization in the context of the current invention encompasses non-specific immobilization (i.e. leading to immobilized molecules oriented in a random fashion), such as non-covalent adsorption or covalent non-specific attachment. The immobilization in the context of the current invention encompasses specific immobilization (i.e. leading to uniform orientation of immobilized molecules). The “immobilization”, preferably the “specific immobilization”, may be achieved by using thiazolidine ring formation, Diels-Alder reaction, Staudinger ligation, a-oxo semicarbazone ligation, His-Ni-NTA, biotin binding to biotin-binding moiety (e.g. biotin binding to streptavidin, avidin, NeutrAvidin) and/or native chemical ligation. The immobilization in the context of the current invention may be by chemical attachment such as by one or more of a chemical bond, a covalent chemical bond, a non-covalent chemical bond (such as Van der Waals forces, hydrogen bonding, electrostatic interaction, hydrophobic interaction, hydrophilica interaction, and receptor-ligand interaction). The chemical attachment encompasses non-covalent physical adsorption.

The molecule or compound may directly immobilized to the substrate without requiring a linker. In addition or alternatively, one or more linker molecules may be provided as intermediate between the substrate and the molecule to be immobilized. The “linker” in the context of the current invention preferably means a chemical linker, providing one or more chemical bonds as disclosed herein. The linker can be one or more functional chemical moieties including but are not limited to, amino, hydroxyl, carboxyl, carboxylate, aldehyde, ester, ether (e.g. thio-ether), amide, amine, nitrile, vinyl, sulfide, sulfonyl, siloxanes, phosphoryl, oxo, thiol, or similar chemically reactive functional groups. In addition or alternatively, moieties that can be used as functional groups as part of the linker are maleimide, N-hydroxysuccinimide, sulfo-N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl (e.g., bromoacetyl, iodoacetyl), activated carboxyl, hydrazide, epoxy, aziridine, sulfonylchloride, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, vinylsulfone, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, biotin and avidin.

The immobilization may involve a click chemistry reaction, such as one or more of cycloaddition reactions, such as the 1 ,3-dipolar family, and hetero Diels-Alder reactions; nucleophilic ring-opening reactions (e.g., epoxides, aziridines, cyclic sulfates, and so forth); carbonyl chemistry, such as the formation of oxime ethers, hydrazones, and aromatic heterocycles; in addition to carbon-carbon multiple bonds, such as epoxidation and dihydroxylation and azide-phosphine coupling (Staudinger ligation). In an embodiment, the substrate is functionalized during or after immobilization of the molecule to be immobilized, e.g. such as when the substrate is considered chemically inert and/or lacks functional moieties for chemical conjugation. The term “functionalization” in the context of the current invention means to alter the physical and/chemical properties of a surface, preferably to provide functional groups allowing for improved immobilization of a molecule. In an embodiment, the functionalization involves silanization, which is a known method in the art (Somasundaram et al. J Biomed Mater Res B Appl Biomater. 2018 Nov;106(8):2901-2918). In an embodiment, the functionalization involves plasma treatment, which is a known method in the art (Akdogan et al. Mater Sci Eng C Mater Biol Appl. 2021 Dec;131 : 112474). In a preferred embodiment, the surface is functionalized with a polydopamine film, which is known method in the art (Messersmith et al. Science, 2007, 318, 426-430). A polydopamine film can deposit on a substrate via the oxidative selfpolymerization of dopamine at slightly basic pH onto many kinds of surfaces, and providing in part amine and hydroxyl functional groups for immobilization of molecules. The present inventors consider that polydopamine is able to form on virtually any type of substrate surface, including metals, metal oxides, ceramics, synthetic polymers and a wide range of other hydrophilic and hydrophobic materials, and is particular suitable for immobilization of the cyclic analogue of glycine of proline.

In various embodiments, the cyclic analogue of glycine-proline is provided in an amount of 0.00001 - 10 g, preferably 0.0001 - 1 g, more preferably 0.001 - 0.1 g, even more preferably 0.01 - 0.1 g, wherein the amount is the average amount per cm² surface area of the substrate.

In various embodiments, the cyclic analogue of glycine-proline is provided in a carrier material (i.e. carrier) on the substrate. The carrier material may for instance be a degradable material, allowing for more sustained and/or tuneable release of an embedded molecule. The carrier may for instance be one or more of a polymer film, microparticle, hydrogel, collagen gel, extracellular matrix gel, or mixtures thereof. The term “hydrogel” in the context of the current invention encompasses gels which contain water, but also all hydrophilic gels and gel composites, including those containing organic non-polymeric components in the absence of water. The term “gel” in the context of the current invention means a state of matter that is intermediate between solids and liquids, and which exists as a solvent inside a solid or semisolid three dimensional network. Example of carrier materials suitable in the context of the current invention include carbohydrates and polysaccharides such as starch, cellulose, dextran, methylcellulose, and hyaluronic acid, proteins or polypeptides such as albumin, fibrin collagen, and gelatin, or combinations thereof. The carrier material in the context of the current invention is preferably a biodegradable polymer, such as chitosan, heparin, chitosan-heparin complexes, copolymers formed from monomers of lactide, glycolide, dioxanone, and caprolactone, collagen, fibrin, and silk; poly- (orthoesters) and poly-(anhydrides), polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid [e.g. poly(lactic-co-glycolic acid, PLGA], polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels. The term “glycolide” in the context of the current invention encompasses polyglycolic acid. The term “lactide” in the context of the current invention encompasses L-lactide, D-lactide, blends thereof, and lactic acid polymers and copolymers.

In an embodiment, the biodegradable polymer is a polymer film assembled on the substrate through the sequential deposition of interacting polymers, known as layer-by-layer (LbL) deposition (Campbell et al. Polymers. 2020, 12(9), 1949).

In an embodiment, the carrier material is an inert material such as synthetic silicones, for example, Silastic, silicone rubber or other polymers.

In various embodiments, the substrate and/or carrier material may be fibrous, wherein the fibers can be for instance in the micron and/or nanometer diameter range such as 100 nm-50 pm, or 200 nm-10 pm, or 500 nm 5 pm or 1-2 pm. Said fibers may mimic the extracellular matrix, can influence cell behaviour, and have a high surface area-to-volume ratio that increases cell contact area. In an embodiment, the substrate and/or carrier is made up of fibers of a biodegradable polymer as disclosed herein, such as obtainable by electrospinning of a solution of said polymer (yielding electrospun fibers).

In various embodiments, the cyclic analogue of glycine-proline is provided in a carrier in an amount of for example 0.00001 - 25 wt.%, or 0.0001 - 10 wt.% or 0.005 - 1 wt.%, calculated on the weight of the carrier. Based on dose-response curves in vitro, the present inventors found that a preferred concentration for anti-fibrosis may be in the range of 0.0001 - 5 wt.%, preferably 0.001 - 1 wt.%, more preferably 0.01 - 0.1 wt.%, calculated on the weight of the carrier.

Preferably, the carrier is adapted and/or provides a release profile that lasts for at least 2 days, or 3 days, or 4 days, or 5 days, or 6 days, 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 12 weeks. Preferably, the carrier is adapted and/or provides a release profile that lasts for no more than 12 weeks, or 8 weeks, or 6 weeks, or 4 weeks, or 3 weeks, or 2 weeks, or 1 week.

The skilled person knows how to adapt the carrier to accommodate the appropriate release rate and/or release profile, e.g. by selecting the appropriate polymer of mixture thereof, the concentration of said polymer, the thickness of the coating.

In an embodiment, the cyclic analogue of glycine-proline is releasable from the substrate and/or the coating.

In an embodiment, the substrate and/or the coating is configured for release of the cyclic analogue of glycine-proline.

In an embodiment, the release is a sustained release. In an embodiment, the release is according to a sustained release profile.

As used herein the terms "sustained release" or "sustained release profile" mean a release of substance of compound (e.g. peptide) gradually over an extended period of time (e.g. a period of several days, weeks, or months). In an embodiment, the amount released over an initial period is similar to or less than the amount released over the same period after several days or weeks. Preferably, the sustained release profile lasts at least 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 12 weeks.

In an embodiment, the release exhibits a substantially linear rate of release. The term “linear rate of release” in the context of the current invention means that the substance or compound (e.g. peptide) is released at a rate that does not vary by more than about 20% over the desired period of time, preferably by not more than 10%.

In an embodiment, the cyclic analogue of glycine-proline is released for least 2 days, or 3 days, or 4 days, or 5 days, or 6 days, 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 12 weeks. In addition or alternatively, the cyclic analogue of glycine- proline is released for no more than 12 weeks, or 8 weeks, or 6 weeks, or 4 weeks, or 3 weeks, or 2 weeks, or 1 week.

In an embodiment, the cyclic analogue of glycine-proline is released in an amount of 0.0001 g - 10 g, or 0.001 g - 1 g, or 0.01 g - 0.1 g per day on average. The desired release rate, release profile and/or the amount released can vary depending on the bodily site and/or the application.

The “release” in the context of the current invention preferably means the in vivo release, i.e. following implantation. The in vivo release can be determined by implanting coated samples with known peptide content, harvesting samples at various time points, and then determining the amount of residual peptide (i.e. non-released peptide) in the samples. Based on these values, a release profile curve can be plotted. In addition or alternatively, the “release” can be determined in vitro, preferably under conditions that approximate or mimic the in vivo conditions (temperature, pH, aqueous medium, and/or presence of enzymes etc...) that may influence the release rate in vivo. A preferred in vitro protocol for measuring release involves incubating samples in phosphate buffered saline (PBS) at 37 °C, and collecting/refreshing the PBS with eluted peptide at different time points. The concentration of peptide in the released samples can be measured by any suitable method, such as HPLC. A suitable HPLC protocol for measuring cyclic glycine-proline involves using two eluents, A [0.1% (v/v) TFA/water] and B [0.1% (v/v) TFA/CAN], linearly increasing eluent B: 0% (0 - 15 min), 0% - 50% (15 - 45 min), 50% - 0% (45 - 50 min), and 0% (50 - 60 min). A suitable flow rate is 1.0 mL/min, and a suitable column temperature is 30°C. Elution can be monitored by absorbance at 214 nm. Concentrations are normalized against a calibration curve obtained by diluting stock solutions cyclic glycine-proline in PBS.

Formulations

In an embodiment, the cyclic analogue of glycine-proline may be provided in a food formulation, food supplement formulation, or pharmaceutical formulation, preferably a food supplement formulation. In an embodiment, the cyclic analogue of glycine-proline may be provided in a solid dosage form such as a capsule, a tablet, or a powder, preferably a powder.

In a preferred embodiment, the cyclic analogue of glycine-proline is administered orally.

The terms “administer” or “administration” in the context of the current invention encompass providing a compound, substance, or composition to a subject consuming it. The term “administering” includes self-administering.

The cyclic analogue of glycine-proline may be provided in a formulation such as a drinkable solution or suspension, drink such as beer, syrup, artificially-flavoured drink, carbonated beverage, (water-soluble) powdered mixture, (water-soluble) paste, (water-soluble) powder, (water-soluble) tablet, (water-soluble) pill, (water-soluble) dragee, (water-soluble) caplet, (water-soluble) sachet, or (water-soluble) capsule. In addition or alternatively, the cyclic analogue of glycine-proline as taught herein may be present in a functional food, e.g. a juice, shake, dairy drink, yoghurt, yoghurt drink, dessert, energy bar, nutritional bar, slimming bar, or confectionery such as gummies or center-filled gummies.

The unit dose and/or daily dose of cyclic analogue of glycine-proline may be at least 0.001, 0.05, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g, wherein the dose is the total dry weight amount of the peptide administered. In addition or alternatively, in different embodiments, the unit and/or daily dose of the peptide of the invention may be no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, or 0.01 g, wherein the dose is the total dry weight amount of the peptide administered.

In a preferred embodiment, the cyclic analogue of glycine-proline is administered at a unit and/or daily dose of 0.001 - 50 g, preferably 0.01 - 10 g, more preferably 0.1 - 1 g, wherein the dose is the total dry weight amount of the peptide administered.

The unit dose and/or daily dose of the cyclic analogue of glycine-proline may be at least 0.01, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g, wherein the dose is the total dry weight amount of the cyclic analogue of glycine-proline. In addition or alternatively, in different embodiments, the unit and/or daily dose of the cyclic analogue of glycine-proline may be no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1 g, wherein the dose is the total dry weight amount of the cyclic analogue of glycine-proline.

The term “daily dose” in the context of the current invention means the total dry weight amount administered to a subject per day. The term “unit dose” in the context of the current invention means the total dry weight amount administered to a subject in a single dose. The unit dose typically is in a pre-prepared form (e.g. prepacked dosage) ready for administration. The unit dose may for example (also) be identifiable from the product packaging or label. Examples of unit dosage forms are individual tablets, pre-filled sachets, individual capsules, bulk powders, and liquid solutions, emulsions or suspensions.

In an embodiment, the cyclic analogue of glycine-proline is administered every day or every other day. In an embodiment, the cyclic analogue of glycine-proline is administered at least once a day. In an embodiment, the dosage regimen comprises administering cyclic analogue of glycine-proline for at least 2 consecutive days, preferably for at least 4 consecutive days, more preferably for at least 7 consecutive days. In an embodiment, the cyclic analogue of glycine-proline is administered for at least 1 consecutive week, preferably for at least 2 consecutive weeks, more preferably for at least 4 consecutive weeks.

The terms “consecutive” or “consecutively” used in the context of administration means that the administrations follow one another in order without gaps in a given time period. For example, when the cyclic analogue of glycine-proline is “administered for 4 consecutive days”, this means that the cyclic analogue of glycine-proline is administered for at least once a day for 4 days in a row (e.g. on Monday, Tuesday, Wednesday, and Thursday of the same week), irrespective of the number of administrations per day or the total number of administrations. For example, when “the cyclic analogue of glycine-proline is administered for

4 consecutive weeks”, this means that the cyclic analogue of glycine-proline is administered for at least once a week for 4 weeks in a row, irrespective of the number of administrations per day, the number of administrations per week or the total number of administrations.

In a preferred embodiment, the cyclic analogue of glycine-proline is administered locally, such as parenterally, preferably subcutaneously and/or trans-dermally.

In an embodiment, the peptide and/or peptide composition is administered by injection.

In a preferred embodiment, the cyclic analogue of glycine-proline is provided in a topical formulation. The topical formulation is preferably selected from the group consisting of a wound dressing, a hydrogel, a gel, an ointment, a plaster, a skin substitute, a tissue adhesive, a tissue sealant, a hemostat, a spray and a bandage. In various embodiments, the current invention pertains to a topical formulation comprising the cyclic analogue of glycine-proline disclosed herein.

The cyclic analogue of glycine-proline may be provided in the topical formulation in an amount of for example 0.00001 - 25 wt.%, or 0.0001 - 10 wt.% or 0.005 - 1 wt.%, calculated on the weight of the topical formulation. Based on dose-response curves in vitro, the present inventors found that a preferred concentration for anti-fibrosis may be in the range of 0.0001 -

5 wt.%, preferably 0.001 - 1 wt.%, more preferably 0.01 - 0.1 wt.%, calculated on the weight of the topical formulation.

In various embodiments, the current embodiment pertains to a non-therapeutic, cosmetic use of the cyclic analogue of glycine-proline disclosed herein. The cosmetic use can for example be for non-therapeutically improving skin appearance, such as due to scars, wrinkles, dyscolorations of the skin, skin irritation, volume augmentation, baldness, after skin peeling, dermabrasion and medical needling. The cosmetic use can be one or more of topical, intralesional, intraepithelial, intra-epidermal, intra-cutaneous, and subcutaneous administration into or onto the skin. In a preferred embodiment, the administration is topical administration onto the skin, e.g. wounded skin.

In various embodiments, the current invention pertains to a cosmetic composition comprising the cyclic analogue of glycine-proline.

The cyclic analogue of glycine-proline can be in a cosmetic composition, and therefore preferably further include acceptable excipients, lubricants and additives. The cosmetic composition may be in any form suitable for local application, for example, a solution, a gel, a solid, a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water phase, an emulsion obtained by dispersing a water phase in an oil phase, a multi-emulsion, a suspension, a microemulsion, a microcapsule, microgranules, ionic (liposome) and non-ionic vesicle dispersants, a foam, and an aerosol or patch form further containing a compressed propellant. The cosmetic composition may be selected from the group consisting of a soaptype preparation, skin toner, nutrition lotion, essence, nutrition cream, massage cream, pack, gel, makeup base, foundation, powder, lipstick, patch, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, a cleanser, hair shampoo, hair conditioning, hair treatment, hair essence, hair lotion, scalp hair tonic, scalp essence, hair gel, hair spray, hair pack, body lotion, body cream, body oil and body essence.

Clauses

Herein, clauses are embodiments of the invention. Features of clauses (embodiments) herein can be combined.

Clause 1 : Cyclic analogue of glycine-proline for use in the prevention and/or treatment of fibrosis, wherein the cyclic analogue of glycine-proline is a compound according to formula I, wherein

X¹ is selected from the group consisting of NR', O and S;

X² is selected from the group consisting of CH2, CR⁶R⁷, NR', O and S;

R¹, R², R³, R⁴ and R⁵ are independently selected from the group consisting of — H, — OR', —SR', — NR'R', — NO₂, — CN, — C(O)R', — C(O)OR', — C(O)NR'R', — C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, allenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl and substituted heteroarylalkyl;

Clause 2: Cyclic analogue of glycine-proline for use according to clause 1 , wherein the cyclic analogue of glycine-proline is a compound according to formula II or III, wherein R¹ and R⁴ are independently selected from the group consisting of -H, - OR', -SR', -NR'R', -NO₂, -CN, -C(O)R', -C(O)OR', -C(O)NR'R', -C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, allenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, and -CH2-(CH2)n-CH2-, where n is an integer from 0-6; each R' is independently selected from the group consisting of -H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

Clause 3: Cyclic analogue of glycine-proline for use according to clause 1 or 2, wherein the cyclic analogue of glycine-proline is cyclic glycine-proline according to formula IV.

Clause 4: Cyclic analogue of glycine-proline for use according to clause 1 or 2, wherein the cyclic analogue of glycine-proline is cyclo-L-glycyl-L-2-allylproline according to formula V. Clause 5: Cyclic analogue of glycine-proline for use according to any one of clauses 1-4, wherein the cyclic analogue of glycine-proline is synthetic.

Clause 6: Cyclic analogue of glycine-proline for use according to any one of clauses 1-5, wherein the fibrosis is one or more selected from the group consisting of medical implant- related fibrosis, surgery-related fibrosis, organ-specific fibrosis, post-surgical adhesion formation, skin fibrosis, cellulite, Dupuytren’s contracture, a hypertrophic scar, a keloid, liver fibrosis, pulmonary fibrosis, pancreatic fibrosis, kidney fibrosis, vascular fibrosis, endometriosis, atrial fibrosis, and heart fibrosis.

Clause 7: Cyclic analogue of glycine-proline for use according to any one of clauses 1-6, wherein the fibrosis is a symptom in a condition selected from the group consisting of pulmonary hypertension, fatty liver disease, chronic obstructive pulmonary disease, nonalcoholic steatohepatitis (NASH), asthma and systemic sclerosis. Clause 8: Cyclic analogue of glycine-proline for use according to any one of clauses 1-7, wherein preventing and/or treating fibrosis is by ameliorating one or more of cell activation, cell transdifferentiation, cell contraction, and/or overproduction of collagen.

Clause 9: Cyclic analogue of glycine-proline for use according to clause 8, wherein the cell is a skin cell and/or a stromal cell.

Clause 10: Cyclic analogue of glycine-proline for use according to clause 9, wherein the cell is a myofibroblast.

Clause 11 : Cyclic analogue of glycine-proline for use according to any one of clauses 1-10, wherein the cyclic analogue of glycine-proline is administered orally.

Clause 12: Cyclic analogue of glycine-proline for use according to clause 11 , wherein the cyclic analogue of glycine-proline is administered at a daily dose of 0.01 - 10 g, preferably 0.1 - 1 g.

Clause 13: Cyclic analogue of glycine-proline for use according to any one clauses 1-10, wherein the cyclic analogue of glycine-proline is administered parenterally, preferably subcutaneously and/or trans-dermally.

Clause 14: Cyclic analogue of glycine-proline for use according to any one of clauses 1-10, wherein the cyclic analogue of glycine-proline is provided in a topical formulation.

Clause 15: Cyclic analogue for use according to clause 14, wherein the topical formulation is selected from the group consisting of a wound dressing, a hydrogel, a gel, an ointment, a plaster, a skin substitute, a tissue adhesive, a tissue sealant, a hemostat, a spray and a bandage.

Clause 16: Cyclic analogue of glycine-proline for use according to clauses 14 or 15, wherein the cyclic analogue of glycine-proline is provided in the topical formulation in an amount of 0.001 - 1 wt.%, preferably 0.01 - 0.1 wt.%, calculated on the weight of the topical formulation.

Clause 17: Cyclic analogue of glycine-proline for use according to any one of clauses 1-10, wherein the cyclic analogue of glycine-proline is provided as a coating on a substrate.

Clause 18: Cyclic analogue of glycine-proline for use according to clause 17, wherein the substrate is a medical device.

Clause 19: Cyclic analogue of glycine-proline for use according to clause 18, wherein the medical device is one or more selected from the group consisting of a suture material, a drugeluting bead, a cardiovascular implant, a stent, a catheter, a pacemaker, an electrical lead, an implantable cardiac defibrillator, a nerve stimulator, a degradable implant, an interocular lense, an implantable drug delivery device, an implantable pump, an intra-uterine device, a surgical mesh implant, a breast implant and a prosthesis.

Clause 20: Cyclic analogue of glycine-proline for use according to any one of clauses 17-19, wherein the cyclic analogue of glycine-proline is provided as a coating on a substrate in an amount of 0.0001 - 1 g, preferably 0.001 - 0.1 g, wherein the amount is the average amount per cm² surface area of the substrate.

Clause 21 : Topical formulation, comprising a cyclic analogue of glycine-proline, wherein the cyclic analogue of glycine-proline is as defined in any one of clauses 1-9. Clause 22: Topical formulation according to clause 21, wherein the topical formulation is selected from the group consisting of a wound dressing, a hydrogel, a gel, an ointment, a plaster, a skin substitute, a tissue adhesive, a tissue sealant, a hemostat, a spray and a bandage.

Clause 23: Topical formulation according to clauses 21 or 22, wherein the cyclic analogue of glycine-proline is provided in an amount of 0.001 - 1 wt.%, preferably 0.01 - 0.1 wt.%, calculated on the weight of the topical formulation.

Clause 24: Substrate coated with a cyclic analogue of glycine-proline, wherein the cyclic analogue of glycine-proline is as defined in any one of clauses 1-5.

Clause 25: Substrate according to clause 24, wherein the cyclic analogue of glycine-proline is immobilized onto the substrate.

Clause 26: Substrate according to clause 24 or 25, wherein the cyclic analogue of glycine- proline is provided in a carrier covering the substrate.

Clause 27: Substrate according to clause 26, wherein the carrier is a degradable carrier. Clause 28: Substrate according to clause 26 or 27, wherein the carrier is adapted for sustained release of the cyclic analogue of glycine proline.

Clause 29: Substrate according to any one of clauses 24-28, wherein the substrate is a medical device.

Clause 30: Substrate according to clause 29, wherein the medical device is one or more selected from the group consisting of a suturing material, a drug-eluting bead, a cardiovascular implant, a stent, a catheter, a pacemaker, an electrical lead, a implantable cardiac defibrillator, a nerve stimulator, a degradable implant, an interocular lense, an implantable drug delivery device, an implantable pump, an intra-uterine device, a surgical mesh implant, a breast implant and a prosthesis.

Clause 31 : Substrate according to any one of clauses 24-30, wherein the cyclic analogue of glycine-proline is provided in an amount of 0.0001 - 1 g, preferably 0.001 - 0.1 g, wherein the amount is the average amount per cm² surface area of the substrate.

General definitions

The terms ‘comprising’ or ‘to comprise’ and their conjugations are used in the context of the current invention in their non-limiting sense to indicate that items following the word are included, but items not specifically mentioned are not excluded. Reference to an element by the indefinite article ’a’ or ‘an’ does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article ‘a’ or ‘an’ thus usually means ‘at least one’.

In the context of the current invention, a level is considered “increased” or “decreased” when it is at least 1% (such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) higher or lower, respectively, than the corresponding level in a control or reference. In addition or alternatively, a level is considered increased or decreased when it is statistically significantly higher or lower, respectively, compared to a level in a control or reference (including compared to an earlier time point), irrespective of the size of the change. The term “to reduce” may in the context of the current invention be used interchangeably with the term “to decrease”.

In the context of the current invention, the terms “to prevent”, or “prevention” and their conjugations mean that an intervention keeps a condition (e.g. fibrosis) from happening. An intervention is herein also considered to be preventive when a condition is delayed, reduced in severity and/or reduced in incidence, even when the condition is not entirely kept from happening. In the context of the current invention, the terms “to prevent”, or “prevention” and their conjugations encompass the situation wherein a subject previously has experienced a condition, but an intervention keeps the condition from recurring. The “preventing”, or "prevention” can be defined by any delay, change in severity, and/or change in incidence, such as of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of change in between, as compared to a control or reference as measured by any standard technique.

In the context of the current invention, the terms “to treat” or “treatment” and their conjugations mean that an intervention reduces, decreases and/or ameliorates a condition (e.g. fibrosis) once the condition is already existing, and also includes the complete curing of a condition or disease. The terms “to treat” or “treatment” and their conjugations encompass any reduction in severity, incidence, and/or frequency of the condition, such as of at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, or any value in between, as compared to a control or reference as measured by any standard technique.

In the context of the current invention, the terms “to ameliorate” or “amelioration” and their conjugations mean an improvement, such as an improvement relative to a control or reference (population) not receiving the cyclic analogue of glycine-proline and/or an improvement compared to the situation prior to initiation of administration of the cyclic analogue of glycine-proline. The improvement can be an at least 1% improvement, such as at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% improvement, or 100% improvement, compared to a corresponding level in a control or reference and/or compared to a level prior to initiating administration of the cyclic analogue of glycine-proline. In addition or alternatively, a level can be considered to be improved when it is significantly improved based on an appropriate statistical test, e.g. compared to a level in a control or reference and/or compared to a level prior to initiating administration of the cyclic analogue of glycine-proline. The “amelioration” can mean either a decrease or an increase in a certain biological process, depending on whether said biological process is desirable or not. For example, it is considered that collagen overproduction is involved in development of fibrosis, hence is undesirable. Therefore an amelioration of collagen overproduction preferably means a decrease or inhibition in collagen overproduction in the context of the current invention.

FIGURE LEGENDS

Figure 1. Cyclic glycine proline reduces hydrogel contraction in a fibroblast contraction assay. Fibroblast contraction after 14 days of exposure to different concentrations of peptide D or TGF-pi. Each symbol represents an independent experiment and donor, n=3. At least 100 cells per hydrogel were counted. Repeated Measures One-way ANOVA with Dunnett multiple comparison test. *p<0,05; **p<0,01; ***p<0,005; ****p<0,001

Figure 2. Cyclic glycine proline reduces hydrogel contraction in an adipose stromal cell contraction assay. Adipose stromal cell contraction after 14 days of exposure to different concentrations of peptide D or TGF-pi. Each symbol represents an independent experiment and donor, n=3. At least 100 cells per hydrogel were counted. Repeated Measures One-way ANOVA with Dunnett multiple comparison test. *p<0,05; **p<0,01 ; ***p<0,005; ****p<0,001

Figure 3. Cyclic glycine proline reduces differentiation of fibroblasts into myofibroblasts. Myofibroblast differentiation in fibroblasts after 14 days of exposure to different concentrations of peptide D or TGF-pi . Each symbol represents an independent experiment and donor, n=3. At least 100 cells per hydrogel were counted. Repeated Measures One-way ANOVA with Dunnett multiple comparison test. *p<0,05; **p<0,01 ; ***p<0,005; ****p<0,001

Figure 4. Cyclic glycine proline reduces differentiation of adipose stromal cells into myofibroblasts. Myofibroblast differentiation in adipose stromal cells after 14 days of exposure to different concentrations of peptide D or TGF-pi. Each symbol represents an independent experiment and donor, n=3. At least 100 cells per hydrogel were counted. Repeated Measures One-way ANOVA with Dunnett multiple comparison test. *p<0,05; **p<0,01 ; ***p<0,005; ****p<0,001

EXAMPLES

Example 1

Example 1 shows the effect of cyclic glycine-proline (cGP) on the prevention of fibrosis in terms of contraction by connective tissue cells and myofibroblast differentiation.

Methods

Cyclic glycine-proline

“Peptide D” is a synthetic cGP, purchased at Bachem AG (Switzerland).

Fibroblast and Adipose stromal cell (A SC) isolation

Normal skin (including subcutaneous fat) was obtained from patients undergoing abdominal dermolipectomy or breast reduction surgery and collected anonymously. Fibroblasts and adipose stromal cells (ASCs) were isolated from skin and subcutaneous fat tissue respectively. Fat was removed from the dermis and epidermis, and cut into pieces. The epidermis was removed from the dermis after dispase incubation. Both dermis and adipose tissue were incubated separately in collagenase type II (Gibco, Invitrogen, Paisly, UK)/dispase II (Roche, Mannheim, Germany) solution for 2 h at 37 °C. Cells were passed through a 40 pm cell strainer and cultured in a 37 °C, 5% CO2 atmosphere in DMEM supplemented with 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 1% UltroserG (BioSepra SA, Cergy-Saint-Christophe, France). Cells from n=3 donors were used for experiments at passage 2-4.

Cell culture assay

Collagen I was isolated from rat tails and reconstituted in 0.1% acetic acid (4 mg/ml). Fibroblasts and ASCs were seeded in 4 mg/ml collagen 1 solution at 2x10⁵ cells/ml and 1 ml hydrogel/well was poured into 12 wells plates. Hydrogels were allowed to polymerize for 2 hours at 37 °C. Hydrogels were detached from the well surface to allow contraction and culture medium was added to the wells. Medium with peptides was filtered using a 0.2 pm filter and allowed to cool to 37 °C before adding to the cultures. Three times per week the culture medium (including peptides) was changed and pictures were taken using a Canon Powershot G12 camera during a total culture period of two weeks. Hydrogel surface was measured using Imaged software. Each experimental condition had an intra-experimental replicate. After two weeks of culture, the hydrogels were fixed in 4% paraformaldehyde and embedded in paraffin.

Myofibroblast staining

The presence of myofibroblasts was determined by immunohistochemical staining for alpha Smooth Muscle Actin (a-SMA). Hydrogels were fixed in 4% paraformaldehyde and embedded in paraffin. 5 pm sections were dehydrated, incubated 1 hour with mouse-anti-human a-SMA (1 :200, clone 1A4, M0851 , DAKO) followed a blocking step with Brightvision Gold for 15 minutes and BrightVision Poly-HRP-Anti Mouse/Rabbit IgG Ruby for 30 minutes. 3-Amino-9- ethylcarbazole was used as a substrate and slides were counterstained with hematoxylin.

The percentage of a-SMA positive cells was determined by manually counting at least 50 cells per hydrogel (counted on average 100 per hydrogel).

Results

Peptide D was titrated in order to identify the lowest concentration which resulted in maximum effect of the above mentioned parameters. The titration of peptide D showed a linear doseresponse curve with a maximum effect of 29.9% reduced contraction with fibroblasts, where the minimum concentration with maximum effect was 0.2 mg/ml (0.15-0.25 mg/ml) (Figure 1). The titration of peptide D showed a linear dose-response curve with a maximum effect of 24.3% reduced contraction with ASCs, where the minimum concentration with maximum effect was 0.4 mg/ml (0.3-0.5 mg/ml)(Figure 2).

At the end of the 14 days culture period, hydrogels were processed for paraffin embedding and the percentage of a-SMA positive cells determined by immunohistochemistry. In accordance with the contraction data, the percentage of a-SMA positive cells was significantly increased in TGF-pi stimulated hydrogels compared to unexposed control. A clear trend in reduced percentage of a-SMA positive cells was observed with increasing concentrations of peptide D, both for fibroblasts (Figure 3) and ASCs (Figure 4).

Overall, in fibroblast-populated hydrogels, exposure to peptide D resulted in only 10% contraction, as compared to 36% contraction in unexposed hydrogels. In ASC-populated hydrogels, contraction decreased from 31% in unexposed controls to 8% contraction upon exposure to peptide D. The minimum concentration to reach this maximum effect is estimated at 0.25 mg/ml for fibroblasts and 0.4 mg/ml for ASCs.

A comparison between cGP and non-cyclic glycine-proline peptide shows that cGP has highest anti-fibrotic properties and prevents fibrosis, indicating that cyclization plays a role in the anti-fibrotic properties.

An anti-fibrotic effect is also found for cyclo-L-glycyl- L-2-allylprol ine, indicating that different cyclic analogues of glycine-proline may have anti-fibrotic properties.

Overall, this shows that cyclic analogues of glycine-proline are more effective in preventing fibrosis than other peptides.

It was furthermore found that peptide D ameliorates the overproduction of collagen I in connective tissue cells such as fibroblasts. Peptide D also appears to ameliorate the production of collagen III and V in connective tissue cells such as fibroblasts.

It was found that peptide D can inhibit macrophage enhanced contraction of fibroblast populated hydrogels, suggesting anti-inflammatory properties of the peptide. It was also found that peptide D stimulates monocyte derived dendritic cell (moDC) maturation and stimulate macrophage skewing towards the inflammatory M1 phenotype rather than the fibrotic M2 phenotype.

Overall, without being bound by theory, blocking or reducing the overproduction of collagen(s) and/or modulating the immune response by cyclic GP may be mechanism of actions that (further) lead to the prevention of fibrosis.

The results show that cyclic glycine-proline is surprisingly effective in the prevention of fibrosis, at least in part by preventing the contraction and/or myofibroblast differentiation in connective tissue cells. There is a dose-dependent effect of cyclic glycine-proline in preventing fibrosis.

Example 2

Example 2 shows the effect of cGP on the prevention of fibrosis around an implantable medical device.

Methods Polydimethylsiloxane (PDMS) is a biomaterial which is regulatory approved for different medical devices. Subcutaneous implantation in mice of PDMS incorporating cGP was performed to test the anti-fibrotic activity of cGP as additive in a well-known medical device material, following the model as described by Farah et al. (Nat Mater. 2019 August; 18(8): 892-904).

Synthetic cGP is mixed into PDMS (-100 mg /cc PDMS) prior to curing into discs and discs are implanted into C57BL/6 mice. The discs are retrieved after 8 weeks. Masson’s trichrome and H&E staining is performed to assess general histological appearance, immune infiltrations, fibrotic encapsulation, and amount of fibrosis.

Result

Samples with cGP show reduced fibrotic (collagen) deposition compared to samples without cGP. A normal cellular infiltration (e.g. normal macrophage activity) was seen in samples with cGP, indicating that there was no excessive immunosuppression. There are no signs of toxicity due to cGP.

Claims

1. Cyclic analogue of glycine-proline for use in the prevention and/or treatment of medical implant-related fibrosis, wherein the cyclic analogue of glycine-proline is a compound according to formula II or III:

(II) (HI)

wherein R¹ and R⁴are independently selected from the group consisting of -H, -OR', - SR', -NR'R', -NO₂, -CN, -C(O)R', -C(O)OR', -C(O)NR'R', -C(NR')NR'R', trihalomethyl, halogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, allenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, and -CH2-(CH2)n-CH2-, where n is an integer from 0-6; each R' is independently selected from the group consisting of -H, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.

2. Cyclic analogue of glycine-proline for use according to claim 1, wherein the cyclic analogue of glycine-proline is cyclic glycine-proline according to formula IV: (IV)

3. Cyclic analogue of glycine-proline for use according to claim 1 , wherein the cyclic analogue of glycine-proline is cyclo-L-glycyl-L-2-allylproline according to formula V: (V)

4. Cyclic analogue for use according to any one of claims 1-3, wherein the cyclic analogue of glycine-proline device is provided as a coating on a substrate selected from the group consisting of a suture material, a drug-eluting bead, a cardiovascular implant, a stent, a catheter, a pacemaker, an electrical lead, an implantable cardiac defibrillator, a nerve stimulator, a degradable implant, an interocular lense, an implantable drug delivery device, an implantable pump, an intra-uterine device, a surgical mesh implant, a breast implant and a prosthesis.

5. Cyclic analogue of glycine-proline for use according to claim 4, wherein the cyclic analogue of glycine-proline is immobilized onto the substrate.

6. Cyclic analogue of glycine-proline for use according to claim 4, wherein the cyclic analogue of glycine-proline is provided in a carrier covering the substrate.

7. Cyclic analogue of glycine-proline for use according to claim 6, wherein the carrier is a degradable carrier.

8. Cyclic analogue of glycine-proline for use according to claim 6 or 7, wherein the carrier is adapted for sustained release of the cyclic analogue of glycine proline.

9. Cyclic analogue of glycine-proline for use according to any one of claims 4-8, wherein the cyclic analogue of glycine-proline is provided in an amount of 0.0001 - 1 g, preferably 0.001 - 0.1 g, wherein the amount is the average amount per cm² surface area of the substrate.

10. Cyclic analogue of glycine-proline for use according to any one of claims 1-4, wherein the cyclic analogue of glycine-proline is administered orally.

11. Cyclic analogue of glycine-proline for use according to claim 10, wherein the cyclic analogue of glycine-proline is administered at a daily dose of 0.01 - 10 g, preferably 0.1 - 1 g.

12. Substrate coated with a cyclic analogue of glycine-proline, wherein the cyclic analogue of glycine-proline is a compound according to formula II or III, wherein the substrate is one or more selected from the group consisting of a suturing material, a drug-eluting bead, a cardiovascular implant, a stent, a catheter, a pacemaker, an electrical lead, a implantable cardiac defibrillator, a nerve stimulator, a degradable implant, an interocular lense, an implantable drug delivery device, an implantable pump, an intra-uterine device, a surgical mesh implant, a breast implant and a prosthesis.

13. Substrate according to claim 12, wherein the cyclic analogue of glycine-proline is a compound according to formula IV.

14. Substrate according to claim 12 wherein the cyclic analogue of glycine-proline is a compound according to formula V.

15. Substrate according to any one of claims 12-14, wherein the cyclic analogue of glycine-proline is provided in a carrier covering the substrate.

16. Substrate according to claim 15, wherein the carrier is a degradable carrier.

17. Substrate according to claim 15 or 16, wherein the carrier is adapted for sustained release of the cyclic analogue of glycine proline.

18. Substrate according to any one of claims 12-17, wherein the cyclic analogue of glycine-proline is provided in an amount of 0.0001 - 1 g, preferably 0.001 - 0.1 g, wherein the amount is the average amount per cm² surface area of the substrate.