WO2013065017A2

WO2013065017A2 - Platelet rich plasma (prp) and prominin-1 derived peptide stimulate lymphangiogenesis

Info

Publication number: WO2013065017A2
Application number: PCT/IB2012/056110
Authority: WO
Inventors: Paolo ERBA
Original assignee: Centre Hospitalier Universitaire Vaudois (C.H.U.V.)
Priority date: 2011-11-03
Filing date: 2012-11-02
Publication date: 2013-05-10
Also published as: WO2013065017A3

Abstract

The present invention relates to an isolated prom-1 peptide derived from a prominin-, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of 5 said prominin-1 peptide, as well as to isolated Platelet Rich Plasma (PRP), or a combination of PRP and said prominin-1 peptide for use in the treatment and/or prevention of lymphedema, lymphatic fistula, lymphorrea or lymphocele/seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

Description

Platelet Rich Plasma (PRP) and Prominin-1 derived Peptide stimulate

lymphangiogenesis

FIELD OF THE INVENTION

The present invention relates to an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, as well as to isolated Platelet Rich Plasma (PRP), or a combination of PRP and said prominin-1 peptide for use in the treatment and/or prevention of

lymphedema, lymphatic fistula, lymphorrea or lymphocele/seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

BACKGROUND OF THE INVENTION

Lymphatics remove the net fluid efflux that accumulates in the interstitial space. In normal conditions the venous system reabsorbs 90% of the fluid in the extracellular space and the remaining fluid is transported to the blood by the lymphatic system as lymph.

Lymphatic vessels also serve to remove macromolecules (eg. Proteins) from the interstitial space. Impaired removal of these macromolecules leads to an increased colloid osmotic pressure and though to an increased fluid accumulation in the interstitial space (1).

Lymphedema is the clinical manifestation of a lymph transport dysfunction and is

characterized by a chronic accumulation of protein-rich fluid, local inflammation, adipose tissue hypertrophy and fibrosis. The affected patients present with an important and debilitating swelling of the hidden extremity which usually leads to reduced mobility and function. Lymphedema is associated with a higher risk of severe life threatening soft tissue infections and may lead to an asymmetric charge and consequent deformation of the spine and bony skeleton.

Lymphedema can be classified, according to its aetiology, into:

1) Primary lymphedema, which is caused by congenital abnormality. The most known primary lymphedema is the Milroy disease and it is characterized by an inactivation-mutation of the Vascular Endothelial Growth Factor Receptor type 3 (VEGFR-3) (2, 3). 2) Secondary lymphedema, which is the consequence of a lymphatic system disruption due to a disease or a iatrogenic practice. The most frequent secondary lymphedema is the one which occurs after cancer treatment and more precisely following lymph node dissection and radiotherapy. These very common cancer therapies cause an interruption/disruption of the lymphatic circulation and lead to a fibrosis of the operated/irradiated tissues (4-13). After mastectomy lymphedema occurs in 24 to 49% of the cases (4, 14).

Because of the epidemic characteristics of cancer, more and more patients will suffer in the future of lymphedema. Unfortunately this pathology is often under diagnosed and because no effective treatment is available patients are forced to live with this uncomfortable condition for their entire life.

Fibrosis is a key component of lymphedema and has been shown to impair lymphatic regeneration, lymphatic endothelial cell proliferation and migration, interfere with tubule formation and impair lymphatic function (15-17). One of the mechanisms through which fibrosis acts is by impeding the interaction of Vascular endothelial growth factor-C (VEGF-C) with its receptors VEGFR-2 and VEGFR-3 (16, 18, 19).

Through the activation of VEGFR-3, VEGF-C improves secondary lymphedema by promoting lymphangiogenesis, stimulating the recanalization of injured lymphatic vessels, restoring the lymph flow and ameliorating the pump activity of the collecting lymphatics (20, 21, 22). Through the activation of VEGFR-2, VEGF-C improves lymphangiogenesis and more precisely improves the organization into functional lymphatic vessels (19).

Macrophages are known to be an important source of cytokines and growth factors (23, 24, 25). Macrophages isolated from diabetic mice have shown reduced inflammatory cytokines (like IL-Ιβ and tumor necrosis factor-β) secretions (26). There is some evidence that macrophages could express lymphatic endothelium markers (as LYVE-1, podoplanin and Prox-1) and that they could contribute to lymphatic vessels formation (27-29). Diabetes is characterized by a reduced number of macrophages and a reduced expression of VEGFR-3, VEGF-C and VEGF-A (24, 25).

Unfortunately there are no effective treatments for lymphedema. Currently, there are two available therapies:

1) Conservative therapies: - Decongestive lymphatic therapy (DLT) includes massage or manual lymphatic drainage, skin cares, compression bandaging and exercises. DLT have shown generally positive results. Some randomized controlled studies have shown as much as 40% to 60% mean decrease in excess volume (30-32). Other studies have however shown only minimal improvement (33) and a significant patient-to-patient variability (34).

- Multilayer inelastic bandaging (35), controlled compression therapy and external sequential pneumatic compression devices (36) have been described to reduce excess fluid accumulation by as much as 31% to 46% (37, 38).

- Numerous pharmacologic agents have been tested for the treatment of lymphedema. Most of them target to break down protein accumulation in the edematous tissues but present with unwilling effects such as hepatotoxicity (39).

The problem of conservative therapies is that they need to be done regularly (average once a week) lifelong and are therefore associated with high costs and life quality impairment.

2) Surgical therapies (36):

- Debulking procedures consist in the aggressive removal of all tissues (skin and

subcutaneous tissues) up to the muscles of the hidden extremity which is then covered by skin grafts. In spite of a reduced frequency of infections and increased walking capacity this therapy is highly aggressive and invasive and should be reserved for few selected cases (40, 41).

- Microsurgical interventions consist in 6-8 hours long surgical interventions to perform lymphatico -venous anastomoses (42-44); lymphatic grafts to connect gaps in the lymphatic system (45, 46) or lymph nodes transplants (47, 48). Microsurgical skills, long operations with high morbidity are needed. Results are variable and depend on surgeon's experience and lymphedema's stage (49).

- Liposuction is used only in certain circumstances where lymphedema has turned into adipose tissue hypertrophy and where compression device therapy is no longer effective. After treatment patients however still need to daily wear compression garments (50).

The problem of surgical therapies is that they are either too invasive, or associated with a significant morbidity, or require highly specific skills, long operations and that results are variable and often not satisfactory on the long term. Ideally, new therapies that are not invasive, that can improve the lymphatic circulation without heavy secondary effects, are needed.

Other pathological conditions related to the lymphatic system are lymphatic fistula, lymphorrea or lymphocele /seroma formation. These conditions are characterized by an escape and/or accumulation of lymph from ruptured, torn, or cut lymphatic vessels. The duration of such a condition depends from the regenerative ability of lymphatic vessels and lymphatic microcirculation. If the latter is delayed, insufficient, or altered, then lymphatic fistula, lymphorrea or lymphocele /seroma formation persist. Persistance of lymphatic fistula, lymphorrea or lymphocele /seroma formation leads prolonged hospitalization times, increased risk of infection, delayed healing, and/or other complications. These pathological conditions related to the lymphatic system and the associated complications could be avoided if new therapies that stimulate the regeneration of the lymphatic circulation are available. At the moment no effective treatment for such conditions is available.

SUMMARY OF THE INVENTION

This object has been achieved by providing an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, for use in the treatment and/or prevention of a disorder or disease related to the lymphatic system.

A further object of the present invention is to provide a pharmaceutical composition for the treatment and/or prevention of a disorder or a disease related to the lymphatic system comprising an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, of any one of the preceding claims and a pharmaceutically acceptable carrier.

Furthermore, the invention provides a method of promoting lymphangiogenesis in a tissue in need thereof, the method comprising contacting said tissue with a pharmaceutical composition of the invention. The invention further provides a method of treatment and/or prevention of a disorder or disease related to the lymphatic system comprising administering to a subject in need thereof a pharmaceutical composition of the invention. Also provided is a method of promoting regeneration of lymphatic vessels in a tissue in need thereof, the method comprising contacting said tissue with a pharmaceutical composition of the invention 1.

An isolated Platelet Rich Plasma (PRP) for use in the treatment and/or prevention of lymphedema, lymphatic fistula, lymphorrea, lymphocele/ seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds is also provided.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Lymphedema mouse tail model. A circular 2mm wound is made at 1cm from the base of mice tail. SEQ ID No. 8, PRP or Saline are injected in the wound. The wound site is harvested for immunhistochemistry at day 14. Lymphangiogenesis and angiogenesis are studied.

Figure 2: A) Lymphatic micro vessel densities were determined and expressed as a percentage of the total surface area in both wild type and diabetic mice in tail cross sections stained with Lyve-1. PRP achieved a significantly increased number of new lymphatic vessel in wild type mice. SEQ ID No. 8 (or P) achieved a significantly increased number of new lymphatic vessels in both wild type and diabetic mice. B) Representative figures of Lyve-1 stained tail cross sections showing dramatically increased lymphatic capillaries in SEQ ID No. 8 (or P) treated lymphedema. PRP treated lymphedema shows a moderately increased number of lymphatic vessels. Figure 3: Wound epithelialization from the proximal wound margin and wound size at day 14 in P, PRP and Saline treated wild type mice. Wounds show a significantly increased epithelialization and healing in PRP and SEQ ID No. 8 (or P) treated wounds. Figure 4: Micro vessel densities were determined and expressed as a percentage of the total surface area in tail cross sections stained with anti-CD-31. PRP achieved a non-significantly increased angiogenesis. SEQ ID No. 8 (or P) achieved significantly increased angiogenesis as previously described.

Figure 5: Proliferation/survival study of primary human intestinal microvascular lymphatic endothelial cells seeded onto fibronectin-coated 96-well plates in EBM2 medium without serum. Each condition was analyzed in 6 wells as followed : 20 ng/ml VEGF-C, 340 mg/ml amino acid sequence DRVQRQTTTVVA (SEQ ID. No. 8) with or without 20 ng/ml VEGF- C, and 2% PRP. After 48 hours, the number of viable cells was quantified using Cell Titer Blue kit (Promega) according to the manufacturer's instructions. After 48 hours, SEQ ID No. 8, the combination of amino acid sequence DRVQRQTTTVVA (SEQ ID. No. 8) and VEGF- C as well as PRP achieved superior cell proliferation and survival compared to VEGF-C positive control. The increase in proliferation achieved with amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) and the combination of amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) with VEGF-C was higher than with VEGF-C alone. BSA values are from different set of experiments and are provided only for indication purposes.

DESCRIPTION OF THE INVENTION

Surprisingly, the inventors of the present invention have shown that an isolated prom- 1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide can increase the lymphatic endothelial cell proliferation, promote lymphatic regeneration, promote epithelialisation and thus wound healing. Accordingly, the present invention relates to an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, for use in the treatment and/or prevention of a disorder or disease related to the lymphatic system. Usually, the disorder or disease related to the lymphatic system is selected from the group comprising lymphedema, lymphatic fistula, lymphorrea or lymphocele /seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

The term "comprise" or "comprising" is generally used in the sense of include/including, that is to say permitting the presence of one or more features or components. Additionally, the term "comprising" also encompasses the term "consisting".

As used herein, the term "prom-1 peptide" refers to a peptide derived from a prominin-1. Prom-1 peptide is a recently described peptide (WO2010/014616) which in several animal models proved to have a powerful pro-angiogenic effect when combined with VEGF. The mechanism of action seems to be an improved binding of VEGF with different cells (eg. vascular endothelial cells, melanoma cells, etc.) mediated by the said peptide. According to our knowledge, the effect of such described peptide on lymphatic endothelial cells or on VEGF receptors related to the lymphatic system have not been described so far.

Preferably, the prom-1 peptide is derived from human prominin-1 (Genbank Accession No. : NM 006017.1 ; NP 006008.1; AF027208.1 ; SEP ID NO: 1 :

MALVLGSLLL LGLCGNSFSG GQPSSTDAPK AWNYELPATN YETQDSHKAG PIGILFELVH IFLYVVQPRD FPEDTLRKFL QKAYESKIDY DKPETVILGL KIVYYEAGII LCCVLGLLFI ILMPLVGYFF CMCRCCNKCG GEMHQRQKEN GPFLR CFAI SLLVICIIIS IGIFYGFVAN HQVRTRIKRS RKLADSNFKD LRTLLNETPE QIKYILAQYN TTKDKAFTDL NSINSVLGGG ILDRLRPNII PVLDEIKSMA TAIKETKEAL ENMNSTLKSL HQQSTQLSSS LTSVKTSLRS SLNDPLCLVH PSSETCNSIR LSLSQLNSNP ELRQLPPVDA ELDNVNNVLR TDLDGLVQQG YQSLNDIPDR VQRQTTTVVA GIKRVLNSIG SDIDNVTQRL PIQDILSAFS VYVNNTESYI HRNLPTLEEY DSYWWLGGLV ICSLLTLIVI FYYLGLLCGV CGYDRHATPT TRGCVSNTGGVFLMVGVGLS FLFCWILMII VVLTFVFGAN VEKLICEPYT SKELFRVLDT PYLLNEDWEYYLSGKLFNKS KMKLTFEQVY SDCKKNRGTY GTLHLQNSFN ISEHLNINEH TGSISSELES LKVNLNIFLL GAAGRKNLQD FAACGIDRMN YDSYLAQTGK SPAGVNLLSF AYDLEAKANS LPPGNLRNSL KRDAQTIKTI HQQRVLPIEQ SLSTLYQSVK ILQRTGNGLL ERVTRILASL DFAQNFITNN TSSVIIEETK KYGRTIIGYF EHYLQWIEFS ISEKVASCKP VATALDTAVD VFLCSYIIDP LNLFWFGIGK ATVFLLPALI FAVKLAKYYR RMDSEDVYDD VETIPMKNME NGNNGYHKDH VYGIHNPVMT SPSQH and share at least 80%, at least 85%, at least 90%>, at least 95%>, or at least 99% sequence identity with SEQ ID NO: 1 . The human prominin-1 is a penta span transmembrane glycoprotein (5-TMD) expressed in stem cells, primarily on the apical membrane of epithelial cells, and is a marker of hematopoietic stem cells. It belongs to a molecular family of 5- transmembrane domain (TMD) proteins, pfam prominin. This "family" includes members from several different species including human, mouse, rat, fly, zebrafish and nematode worms. The 5 -TMD structure includes an extracellular N-terminus, two short intracellular loops, two large extracellular loops and an intracellular C-terminus.

Preferably, in the present invention the extracellular domain of the human prominin- 1 is selected from the group comprising

MALVLGSLLLLGLCGNSFSGGQPSSTDAPKAWNYELPATNYETQDSHKAGPIGILFEL VHIFL YVVQPRDFPEDTLR FLQKAYESKIDYDKPETVILGLKIVYYE (SEQ. ID. No. 2),

NHQVRTRIKRSR LADSNFKDLRTLLNETPEQIKYILAQYNTTKDKAFTDLNSINSVL GGGILDRLRPNIIPVLDEIKSMATAIKETKEALENMNSTLKSLHQQSTQLSSSLTSVKT SLRSSLNDPLCLVHPSSETCNSIRLSLSQLNSNPELRQLPPVDAELDNVNNVLRTDLDG LVQQGYQSLNDIPDRVQRQTTTVVAGIKRVLNSIGSDIDNVTQRLPIQDILS AFS VYVN NTESYIHRNLPTLEEYDS Y (SEQ. ID. No. 3), and TFVFGANVEKLICEPYTSKELFRVLDTPYLLNEDWEYYLSGKLFNKSKMKLTFEQVY SDCKKNRGTYGTLHLQNSFNISEHLNINEHTGSISSELESLKVNLNIFLLGAAGRKNLQ DFAACGIDRMNYDSYLAQTGKSPAGVNLLSFAYDLEAKANSLPPGNLRNSLKRDAQ TIKTIHQQRVLPIEQSLSTLYQSVKILQRTGNGLLERVTRILASLDFAQNFITNNTSSVII EETKKYGRTIIGYFEHYLQWIEFSISEKVASCKPVATALDTAVDVFLCSYIIDP (SEQ. ID. No. 4).

Prom-1 was initially shown to be expressed on primitive hematopoietic stem and progenitor cells and on retinoblastoma cells. However, prom-1 has since been shown to be expressed on hemangioblasts, and neural stem cells as well as on developing epithelia. The prom-1 positive fractions of human bone marrow, cord blood and peripheral blood efficiently engraft in xenotransplantation models, and contain the majority of the granulocyte/macrophage precursors, NOD/SCID repopulating cells and CD34 + dendritic cell precursors.

Phenotypically, prom-1 positive cells in blood and marrow are CD34 bright, with CD34 dim CD71 bright cells being negative for prom-1 expression. Prom-1 is also found in extracellular membrane particles in body fluids.

The present invention also includes the use of a conservative amino acid substitution variant of said prominin-1 peptide as well as of a fragment of said prominin-1 peptide.

The term "conservative amino acid substitution" refers to a peptide having an amino acid sequence that differ to some extent from a native sequence peptide, that is an amino acid sequence that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly

II. Polar, positively charged residues: His, Arg, Lys

III. Polar, negatively charged residues: and their amides: Asp, Asn, GIu, Gin

IV. Large, aromatic residues: Phe, Tyr, Trp

V. Large, aliphatic, nonpolar residues: Met, Leu, He, Val, Cys.

The term "fragment" refers to any peptide or polypeptide having an amino acid residue sequence shorter than that of a full-length polypeptide whose amino acid residue sequence is described herein. An isolated peptide of prom- 1 is shortened or truncated compared to its parent full-length prom-1. The polypeptide can have N-terminus or C-terminus truncations and/or also internal deletions. Examples of prom-1 fragments include fragments consisting of amino acids.

These sequence can be used as long as they exhibit the same biological properties as the native sequence from which they derive. Preferably, the peptide fragments of prom-1 described herein are derived from the extracellular domain set forth in

SEP. ID. No. 2

MALVLGSLLLLGLCGNSFSGGQPSSTDAPKAWNYELPATNYETQDSHKAGPIGILFEL VHIFL YVVQPRDFPEDTLRKFLQKAYESKIDYDKPETVILGLKIVYYE,

SEP. ID. No. 3

NHQVRTRIKRSRKLADSNFKDLRTLLNETPEQIKYILAQYNTTKDKAFTDLNSINSVL GGGILDRLRPNIIPVLDEIKSMATAIKETKEALENMNSTLKSLHQQSTQLSSSLTSVKT SLRSSLNDPLCLVHPSSETCNSIRLSLSQLNSNPELRQLPPVDAELDNVNNVLRTDLDG LVQQGYQSLNDIPDRVQRQTTTVVAGIKRVLNSIGSDIDNVTQRLPIQDILSAFSVYVN NTESYIHRNLPTLEEYDS Y, or SEP. ID. No. 4

TFVFGANVEKLICEPYTSKELFRVLDTPYLLNEDWEYYLSGKLFNKSKMKLTFEQVY SDCKKNRGTYGTLHLQNSFNISEHLNINEHTGSISSELESLKVNLNIFLLGAAGRKNLQ DFAACGIDRMNYDSYLAQTGKSPAGVNLLSFAYDLEAKANSLPPGNLRNSLKRDAQ TIKTIHQQRVLPIEQSLSTLYQSVKILQRTGNGLLERVTRILASLDFAQNFITNNTSSVII EETKKYGRTIIGYFEHYLQWIEFSISEKVASCKPVATALDTAVDVFLCS YIIDP

For example, the peptide fragments of prom-1 derived from the extracellular domain can be selected among the group comprising LCGNSFSGGQPS (SEQ ID. NP. 5); PNIIPVLDEIKS (SEQ. ID. No. 6); LCGVCGYDRHAT (SEQ ID. No. 7); ITNNTSSVIIEE (SEQ ID. No. 8); DRVQRQTTTVVA (SEQ ID. No. 9); and CSFAYDLEAKANSLPPGNLRN (SEQ ID.

No.10).

Also envisioned is a peptide of the invention having at least 80% identity with said prom-1 peptide derived from a prominin-1, said conservative amino acid substitution variant, or said fragment thereof.

Alternatively, said isolated prom-1 peptide derived from a prominin-1, conservative amino acid substitution variant of said prominin-1 peptide, or fragment of said prominin-1 peptide, is conjugated to a polymer for the purpose of improving serum half and pharmacokinetics in vivo. Generally, the polymer is selected from the group comprising a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co-glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene- vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly( vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefm, or a blend or copolymer thereof. Since an inherent problem with native peptides (in L-form) is degradation by natural proteases, said isolated prom-1 peptide derived from a prominin-1, conservative amino acid substitution variant of said prominin-1 peptide, or fragment of said prominin-1 peptide of the invention may be prepared to comprise at least one amino acid in the D-form and/or in a retro -inverso isomer form.. Protecting a peptide from natural proteolysis should therefore increase the effectiveness of the specific heterobivalent or heteromultivalent compound. A higher biological activity is predicted for the retro-inverso containing peptide when compared to the non-retro -inverso containing analog owing to protection from degradation by native proteinases. Furthermore they have been shown to exhibit an increased stability and lower immunogenicity (Sela and Zisman, 1997).

By "retro-inverso isomer" is meant an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted; thus, there can be no end-group complementarity.

This invention also relates to a pharmaceutical composition for the treatment and/or prevention of a disorder or a disease related to the lymphatic system comprising an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, of the invention and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers which facilitates processing of the active compounds into preparation which can be used pharmaceutically are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

The pharmaceutical composition of the invention is for systemic or topical applications or for injection. For example, administration of such a composition may be various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, buccal routes or via an implanted device, and may also be delivered by peristaltic means.

The pharmaceutical composition comprising an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, as described herein, as an active agent may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support. In addition the matrix may be comprised of a biopolymer. The bioabsorbable matrix can be an autologous tissue or a product derived from an autologous tissue (eg. adipose tissue and its derivates, dura mater, etc.)

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON

DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. The appropriate dosage of pharmaceutical compositions will depend upon the type of tissue and the associated medical conditions to be treated, the severity and course of the medical conditions, whether the pharmaceutical compositions are administered for preventative or therapeutic purposes, previous therapy, the patient's clinical history and response to the compositions and the discretion of the attending physician. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the condition being treated and should be decided according to the judgment of the practitioner and each subject's circumstances in view of, e.g., published clinical studies. Suitable effective dosage amounts for topical administration of the peptide compositions described herein range from about 10 micrograms to about 5 grams applied or administered about every 4 hours, although they are typically about 500 mg or less per every 4 hours. In one embodiment the effective dosage for topical administration is about 0.01 mg, 0.5 mg, about 1 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 g, about 1.2 g, about 1.4 g, about 1.6 g, about 1.8 g, about 2.0 g, about 2.2 g, about 2.4 g, about 2.6 g, about 2.8 g, about 3.0 g, about 3.2 g, about 3.4 g, about 3.6 g, about 3.8 g, about 4.0 g, about 4.2 g, about 4.4 g, about 4.6 g, about 4.8 g, or about 5.0 g, every 4 hours. Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The effective dosage amounts described herein refer to total amounts administered.

For systemic administration, the dosage ranges are typically from O.OOlmg/kg body weight to 5 g/kg body weight. In some embodiments, the dosage range is from 0.001 mg/kg body weight to lg/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight, from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001 mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg body weight to 0.1 mg/kg body weight, from 0.001 mg/kg body weight to 0.005 mg/kg body weight. Alternatively, in some embodiments the dosage range is from 0.1 g/kg body weight to 5 g/kg body weight, from 0.5 g/kg body weight to 5 g/kg body weight, from 1 g/kg body weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5 g/kg body weight, from 2 g/kg body weight to 5 g/kg body weight, from 2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight to 5 g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body weight, from 4 g/kg body weight to 5 g/kg body weight, from 4.5 g/kg body weight to 5 g/kg body weight, from 4.8 g/kg body weight to 5 g/kg body weight. In one embodiment, the dose range is from 5μg/kg body weight to 3(^g/kg body weight. Alternatively, the dose range will be titrated to maintain serum levels between 5μg/mL and 3(^g/mL.

Administration of the doses recited above can be repeated for a limited period of time. In some embodiments, the doses are given once a day, or multiple times a day, for example but not limited to three times a day. In a preferred embodiment, the doses recited above are administered daily for several weeks or months. The duration of treatment depends upon the subject's clinical progress and responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.

The pharmaceutical composition to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.

This pharmaceutical composition can also be used to decrease/stop lymphatic secretions in surgical wounds. Alternatively, this pharmaceutical composition may further comprise and additional active ingredient such as an isolated Platelet Rich Plasma (PRP) and/or at least one growth factor.

As used herein, Platelet Rich Plasma (PRP) refers to the part of blood plasma which after centrifugation presents enriched platelets concentration. PRP contains several growth factors and cytokines known to affect tissue regeneration. Among these the most important are platelet-derived growth factor, transforming growth factor beta, fibroblast growth factor, insulin- like growth factor 1, insulin- like growth factor 2, vascular endothelial growth factor, epidermal growth factor, Interleukin 8, keratinocyte growth factor, connective tissue growth factor to cite a few. The effect of PRP on lymphangiogenesis is not known. The only available manuscript which describes the effect of PRP on lymphatic tissues studied the survival rate and viability of avascular lymph node grafts without keening on studying lymphangiogenesis (51).

Also encompassed in the present invention is a method of promoting lymphangiogenesis in a tissue in need thereof, the method comprising contacting said tissue with a pharmaceutical composition of the invention.

Further encompassed is a method of treatment and/or prevention of lymphedema,

lymphatic fistula, lymphorrea, lymphocele/ seroma formation or lymph secretions in a surgical wound, the method comprising contacting said tissue with a pharmaceutical composition of the invention. Alternatively, this method of treatment and/or prevention can be practiced in combination with a conservative or surgical therapy for lymphedema, lymphatic fistula, lymphorrea, lymphocele /seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds. Preferably, the conservative or surgical therapy is selected among the group comprising decongestive lymphatic therapy (DLT), multilayer inelastic bandaging, controlled compression therapies, external sequential pneumatic compression devices, pharmacologic agents, debulking procedures, lymphatico- venous anastomoses, lymphatic grafts, lymph nodes transplants, liposuction, application of glues, application of haemostatic agents, negative pressure wound therapies.

Alternatively also, this method of treatment and/or prevention can be practiced alone or in combination with an isolated Platelet Rich Plasma (PRP), at least one growth factor, stem cell therapies or injections of autologous adipose tissue (lipofilling). Usually, the at least one growth factor is selected from the group comprising VEGF,

Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO),

Transforming growth factor alpha(TGF-a), Transforming growth factor beta(TGF-P), Tumor necrosis factor-alpha(TNF-a), Wnt Signaling Pathway, placental growth factor (P1GF), Foetal Bovine Somatotrophin (FBS), Interleukins (IL) (eg. IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7).

Another further aspect of the present invention concerns a method of promoting regeneration of lymphatic vessels in a tissue in need thereof, the method comprising contacting said tissue with a pharmaceutical composition of the invention. A further aspect of the present invention concerns an agent able to block or inactivate an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide as described supra, or able to bind and though impede any mediated activity of the same peptide with the aim to treat and/or prevent lymphatic tumour spread or metastasis. Preferably, said agent being an antibody.

The present invention relates also to an isolated Platelet Rich Plasma (PRP) for use in the treatment and/or prevention of lymphedema, lymphatic fistula, lymphorrea, lymphocele/ seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

Alternatively, the isolated Platelet Rich Plasma (PRP) can be in combination with an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide of the invention for use in the treatment and/or prevention of lymphedema, lymphatic fistula, lymphorrea, lymphocele/ seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

Preferably, said PRP is obtained from blood taken from the same person, other persons, and other species or from expired blood samples.

Further uses of the isolated prom-1 peptide derived from a prominin-1, conservative amino acid substitution variant of said prominin-1 peptide, or fragment of said prominin-1 peptide or Platelet Rich Plasma (PRP) of the invention are envisioned in cancer therapies or in surgeries that are associated with lymphatic wound secretions or in wounds that do not heal because of lymphorrhea.

Cancer is an epidemic pathology whose treatment often includes lymph node dissection and radiotherapy to prevent further tumour spread. Following these therapies the lymphatic circulation is disrupted and lymphedema may occur. As an example, in breast cancer lymphedema occurs in 24-49% of the patients having had mastectomy with lymph node dissection and 4-28% in patients having had lumpectomy. This means that about 20% of breast cancer patients (corresponding to 2.5% of all women) will suffer of lymphedema.

Most cancer spread through the lymphatic system. Rapidity of tumour spread is related to the amount of lymphatic vessels growing into the tumour and connecting the tumour with the lymphatic circulation and will determine duration of patient's survival. Methods avoiding or slowing down lymph vessel growth would be able to slow down tumour spread. If an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide stimulates lymphatic tissue generation and antibody against said an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide with lymphogenic receptors (VEGFR3) and though inhibit lymphatic tissue regeneration. Antibodies against an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide could therefore be used to slow down tumour spread and allow patients to live longer. Most surgeries require drains to evacuate wound secretions. Drains are an entry point for infections and for this reason patients usually can not leave the hospital with a drain in order to avoid possible complications. Because blood coagulates relatively soon, these secretions mostly consist of lymph. The application of i) PRP, ii) an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide of the invention, or iii) or PRP in combination with an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide in wound cavities to help restoring the disrupted lymphatic capillaries could help reducing/stopping lymph secretions and drains could be either avoided or taken earlier away. Some wounds do not heal because of lymphorrhea which impedes wound margins to remain in contact because of constant lymph flow. The administration of i) PRP, ii) an isolated prom- 1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide of the invention, or iii) or PRP in combination with an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide could help restore lymphatic circulation and decrease/stop lymph secretions / lymph flow into a wound cavity.

EXAMPLES

Example 1 Methods

Adult homozygous genetically diabetic 10 week-old, Lep/r - db/db male mice (strain

C57BL/KsJ-Lepr^db, Jackson Laboratory, Bar Harbor, ME) (n=6 per group) and adult male wild-type C57B16 mice (Jackson Laboratory, Bar Harbor, ME) (n=6 per group) were included in the study. Mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care certified facility under an approved experimental protocol. Under inhalation anaesthesia (Isoflurane 2%) and disinfection of the tail with alcohol patches, a circumferential 2-mm-wide piece of skin located 1 cm distal of the tail base was removed including the deeper draining lymphatics running alongside the major blood vessels.

Depending on the groups, either Platelet Rich Plasma (PRP), a 12MER peptide derived from Prominin-1 (P) of SEQ ID No. 8 or Saline, were topically injected to the wound (Figure 1).

Wounds were then kept covered by a Tegarderm 3M Dressing. Animals were sacrificed at day 14, whereby the proximal half of the wound and underlying tissue was excised, formalin fixed and embedded in paraffin.

Study Groups

The following substances were topically injected in both wild type and db/db mice:

1) Platelet Rich Plasma (PRP) was prepared from human fresh blood according to previously described protocols (115). PRP consists of a mixture of growth factors including VEGF. 2) a 12-mer peptide (P) consisting of the following amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) was synthetized by GenScript.

3) Results of lymphangiogenesis induced by PRP and amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) were compared to those of the control group where Saline was applied.

Immunohistochemistry

Lymphatic vessels were identified by immunohistochemistry with the use of rabbit primary antibodies against murine LYVE-1 (Santa Cruz, Heidelberg, Germany, 65647). Analysis of wound bed angiogenesis (CD31) was performed by immunohistochemistry with CD31 (Vector Laboratories Inc., Burlingame, CA). Microvessel densities were determined and expressed as a percentage of the total surface area. Micrographs of representatitve areas were choosen and evaluated with a Weibel grid. Wound Healing Analysis

Wounds were photographed immediately after wounding and at harvest time. Digital photographs captured at the end of the experiment were compared with initial photographs. Wound closure was calculated as a percentage of the original wound and measured by planimetric analysis (Image J, NIH, Bethesda, MD).

Statistics

For comparison of two groups, a two-tailed T-test was used. For greater than two groups, oneway analysis of variance (ANOVA) and ad hoc Fischer's LSD tests was used to determine the significant differences between treatment groups if the ANOVA was found to be significant. All statistical analyses were performed using WinStat (R. Fitch Software, Lehigh Valley, PA). A p-value less than 0.05 was considered statistically significant.

Results

Lymphatic regeneration measured as the percentage of the total analyzed section area was more than doubled in lymphedema treated by amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) and nearly doubled in lymphedema treated by

PRP (Figure 2). Platelet Rich Plasma and amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) achieved significantly increased lymphangiogenesis in wild type mice. SEQ ID No. 8 achieved significantly increased lymphangiogenesis in both wild type and db/db animals (Figure 2). Very significant results were achieved by amino acid sequence DRVQRQTTTVVA (SEQ ID. No. 8) in diabetic animals where the increase of lymphangiogenesis was nearly 5 times the value of the control group.

In accordance with previous studies PRP and amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) were associated with improved healing rates (Figure 3) and improved angiogenesis (Figure 4). Example 2

Lymphatic Endothelial Cell Proliferation Experiments

Methods

Primary human intestinal microvascular lymphatic endothelial cells were isolated from human jejunum as described (Norrmen et al., 2010). After enzymatic digestion of the jejunum mucosa, cells were seeded onto 5 μ§/ι 1 fibronectin-coated culture dishes and subjected to serial negative selection by immunomagnetic purification (DynaBeads, Invitrogen) using anti- CD44 antibodies (Southern Biothech) to deplete fibroblasts, and a final positive selection using anti-CD31 antibodies (Invitrogen) to select endothelial cells. The lineage-specific differentiation was confirmed by immunostaining for beta-catenin and the lymphatic marker Proxl . Cells were cultured in endothelial cell basal medium (EBM2, Lonza) supplemented with EBM2 supplements and 2% serum. Cells were used for experiment at passages 7-11. For proliferation/survival assay, confluent LECs (5 x 10³) were seeded onto fibronectin- coated 96-well plates in EBM2 medium without serum. Each condition was analyzed in 6 wells as followed : 20 ng/ml VEGF-C, 340 mg/ml amino acid sequence

DRVQRQTTTVVA (SEQ ID. No. 8) with or without 20 ng/ml VEGF-C, and 2% PRP. After 48 hours, the number of viable cells was quantified using Cell Titer Blue kit (Promega) according to the manufacturer's instructions. Results

After 48 hours, SEQ ID No. 8, the combination of amino acid sequence DRVQRQTTTVVA (SEQ ID. No. 8) and VEGF-C as well as PRP achieved superior cell proliferation and survival compared to VEGF-C positive control. The increase in proliferation achieved with amino acid sequence DRVQRQTTTVVA (SEQ ID. No. 8) and the combination of amino acid sequence DRVQRQTTTVVA (SEQ ID. No. 8) with VEGF-C was higher than with VEGF-C alone. BSA values are from different set of experiments and are provided only for indication purposes (Figure 5). REFERENCES

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Claims

1. An isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, for use in the treatment and/or prevention of a disorder or disease related to the lymphatic system.

2. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of claim 1 , wherein the disorder or disease related to the lymphatic system is selected from the group comprising a lymphedema, lymphatic fistula, lymphorrea or lymphocele /seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

3. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of claim 1 or 2, wherein the prominin-1 is human prominin-1 as set forth in SEQ ID No. 1.

4. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of any one of the preceding claims, wherein said isolated prom-1 peptide is derived from the extracellular domain of the human prominin-1.

5. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of any one of the preceding claims, wherein the extracellular domain of the human prominin-1 is selected from the group comprising

MALVLGSLLLLGLCGNSFSGGQPSSTDAPKAWNYELPATNYETQDSHKAGPIGILFEL VHIFL YVVQPRDFPEDTLRKFLQKAYESKIDYDKPETVILGLKIVYYE (SEQ. ID. No. 2),

NHQVRTRIKRSRKLADSNFKDLRTLLNETPEQIKYILAQYNTTKDKAFTDLNSINSVL GGGILDRLRPNIIPVLDEIKSMATAIKETKEALENMNSTLKSLHQQSTQLSSSLTSVKT SLRSSLNDPLCLVHPSSETCNSIRLSLSQLNSNPELRQLPPVDAELDNVNNVLRTDLDG LVQQGYQSLNDIPDRVQRQTTTVVAGIKRVLNSIGSDIDNVTQRLPIQDILSAFSVYVN NTESYIHRNLPTLEEYDS Y (SEQ. ID. No. 3), and

TFVFGANVEKLICEPYTSKELFRVLDTPYLLNEDWEYYLSGKLFNKSKMKLTFEQVY SDCKKNRGTYGTLHLQNSFNISEHLNINEHTGSISSELESLKVNLNIFLLGAAGRKNLQ DFAACGIDRMNYDSYLAQTGKSPAGVNLLSFAYDLEAKANSLPPGNLRNSLKRDAQ TIKTIHQQRVLPIEQSLSTLYQSVKILQRTGNGLLERVTRILASLDFAQNFITNNTSSVII EETKKYGRTIIGYFEHYLQWIEFSISEKVASCKPVATALDTAVDVFLCSYIIDP (SEQ. ID. No. 4).

6. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of any one of the preceding claims, wherein said fragment of the prominin-1 peptide is selected from the group comprising

LCGNSFSGGQPS (SEQ. ID. NO. 5),

PNIIPVLDEIKS (SEQ. ID. No. 6),

LCGVCGYDRHAT (SEQ. ID. No. 7),

ITNNTSSVIIEE (SEQ. ID. No. 8),

DRVQRQTTTVVA (SEQ. ID. No. 9),

and CSFAYDLEAKANSLPPGNLRN (SEQ. ID. No. 10).

7. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of any one of the preceding claims, wherein said isolated prom-1 peptide derived from a prominin-1, conservative amino acid substitution variant of said prominin-1 peptide, or fragment of said prominin-1 peptide, is conjugated to a polymer.

8. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of claim 7, wherein the polymer is selected from the group comprising a polyethylene glycol (PEG), a polyether, a poly(lactide), a poly(glycolide), a poly(lactide-co-glycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acid-co-glycolic acid), a polyanhydride, a polyorthoester, a polycarbonate, a polyetherester, a polycaprolactone, a polyesteramide, a polyester, a polyacrylate, a polymer of ethylene- vinyl acetate or another acyl substituted cellulose acetate, a polyurethane, a polyamide, a polystyrene, a silicone based polymer, a polyolefm, a polyvinyl chloride, a polyvinyl fluoride, a fluoropolymer, a polypropylene, a polyethylene, a cellulosic, a starch, a naturally occurring polymer, a poly( vinyl imidazole), a polyacetal, a polysulfone, a chlorosulphonate polyolefm, or a blend or copolymer thereof.

9. The isolated prom-1 peptide derived from a prominin-1, the conservative amino acid substitution variant of said prominin-1 peptide, or the fragment of said prominin-1 peptide of any one of the preceding claims, wherein said isolated prom-1 peptide derived from a prominin-1, conservative amino acid substitution variant of said prominin-1 peptide, or fragment of said prominin-1 peptide, comprises at least one amino acid in the D-form and/or in a retro-inverso isomer form.

10. A pharmaceutical composition for the treatment and/or prevention of a disorder or a disease related to the lymphatic system comprising an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, of any one of the preceding claims and a

pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, for systemic or topical applications or for injection.

12. A method of promoting lymphangiogenesis in a tissue in need thereof, the method comprising contacting said tissue with a pharmaceutical composition of claim 10 or 11.

13. A method of treatment and/or prevention of a disorder or disease related to the lymphatic system comprising administering to a subject in need thereof a pharmaceutical composition of claim 10 or 11.

14. The method of treatment and/or prevention of claim 13, wherein the disorder or the disease related to the lymphatic system is selected from the group comprising a lymphedema, lymphatic fistula, lymphorrea or lymphocele /seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

15. The method of treatment and/or prevention of claim 13 or 14 in combination with a conservative or surgical therapy for lymphedema, lymphatic fistula, lymphorrea, lymphocele /seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

16. The method of treatment and/or prevention of any one of claim 13 to 15, wherein the pharmaceutical composition is administered in combination with an isolated Platelet Rich Plasma (PRP).

17. The method of treatment and/or prevention of any one of claim 13 to 16, wherein the pharmaceutical composition is administered in combination with at least one growth factor.

18. The method of treatment and/or prevention of claim 17, wherein the at least one growth factor is selected from the group comprising VEGF, Adrenomedullin (AM),

Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain- derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF),

Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration- stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-a), Transforming growth factor beta(TGF-P), Tumor_necrosis_factor-alpha(TNF- a), Wnt Signaling Pathway, placental growth factor (P1GF), Foetal Bovine Somatotrophin (FBS), Interleukins (IL).

19. A method of promoting regeneration of lymphatic vessels in a tissue in need thereof, the method comprising contacting said tissue with a pharmaceutical composition of claim 10 or 1 1.

20. An isolated Platelet Rich Plasma (PRP) for use in the treatment and/or prevention of lymphedema, lymphatic fistula, lymphorrea, lymphocele/ seroma formation, any chronic wound not healing because of lymphorrhea or lymph secretions in surgical wounds.

21. The isolated Platelet Rich Plasma (PRP) of claim 20, wherein said PRP is obtained from blood taken from the same person, other persons, other species or from expired blood samples.

22. The isolated Platelet Rich Plasma (PRP) of claim 20 or 21, in combination with an isolated prom-1 peptide derived from a prominin-1, a conservative amino acid substitution variant of said prominin-1 peptide, or a fragment of said prominin-1 peptide, of any one of the preceding claims.