The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), the new physiological regulator of angiogenesis Liu et al, Blood, 2003, 101, 3014. Patent WO 02/24218 A1; US 2004/0038907 A1

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) purified from bone marrow (10 - 50 µg/kg tissue) present in the blood at concentrations of 10-9 M structure determined by AA analysis, 1D and 2D 1H-RMN, FAB-MS, MS-MS inhibitor of hematopoietic stem cell proliferation Proc Natl Acad Sci USA, 1989, 86, 779

AcSDKP is a physiological regulator of hematopoiesis (in vivo administration of anti-AcSDKP antibodies triggers hematopoietic stem cell into S-phase) AcSDKP + antiAcSDKP (incubated overnight) complex = control % of CFU-S in S-phase 50 anti-AcSDKP Leukemia, 1989, 4, 315

Chemo- or radiotherapy chemo- or radiotherapy Bone marrow toxicity => major limiting factor in the treatment of cancer S S Cancer cell Hematopoietic stem cell Chemo- or radiotherapy cell proliferation Bone marrow aplasia Go S S + proliferation inhibitor (immunodeficiency, hemorrhages…) chemo- or radiotherapy Cell survival Go Go

In vivo, AcSDKP inhibits hematopoietic stem cell (CFU-S) proliferation and increases the survival of mice given lethal doses of chemotherapy (AraC) Proc Nat Acad Sci USA, 1989 , 86, 779. 40 20 % CFU-S en S nat. synth. control AraC AraC + AcSDKP (100 ng/mouse) AraC : 30 mg/kg/inj s.c. 1x day, during 15d AcSDKP : 100 ng/inj i.p. 1x day, during 15d 100 50 control AraC Arac + AcSDKP (LD 90) % mice survival ( s i m u l t ) (2h post) (6h post) (8h post) Ann NY Acad Sci, 1991 , 628, 126.

In vivo protective effect of AcSDKP against the myelotoxicity of total irradiation (300 cGy) AcSDKP (0.5 mg/kg/day), i.v. infusion from -24h to +1h with regard to time of irradiation + AcSDKP irradiated control % of survival 100 + AcSDKP irradiated control Granulocytes / mm3 10 20 30 40 50 days after irradiation 10000 Ann Hematol, 1997, 74, 117.

Myeloprotective effect of AcSDKP In vivo In vitro ->> anticancer drugs : AraC (cytosine arabinoside) CTX (cyclophosphamide) 5-FU (5-fluorouracil) doxorubicine ->> irradiation ->> anticancer drugs : AraC AZT (azidothymidine) AstaZ (mafosfamide) ->> phototherapy ->> hyperthermy AcSDKP => new myeloprotective agent Clinical application: in vivo: radio- and chemotherapy in vitro: bone marrow purging before autologous grafting

Clinical trials of AcSDKP “Goralatide” (Beaufour Laboratories - IPSEN Biotech) Phase I: devoided of toxicity (continuous infusion), excellent tolerance Phase II: myeloprotective effect of AcSDKP in cancer patients undergoing monochemotherapy (AraC or ifosphamide) - decrease of neutropenia - decrease of leucopenia

AcSDKP Biosynthesis Catabolisme Mechanism of action Receptor study Analogues

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) BIOSYNTHESIS Thymosin b4 (Tb4) = metabolic precursor of AcSDKP ? - intracellular co-localisation of T4 and AcSDKP - inhibits proliferation of hematopoietic stem cells [3H]Tb4 + bone marrow cells => [3H]AcSDKP AcSDKP DMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES 43 AA AcSDKP DMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES ? DMAEIEKFD a-prolylendopeptidase T b 4 FEBS Lett, 1990, 274, 30. Cell Prolif, 1996, 29, 437.

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) BIOSYNTHESIS Tb4 stimulation of angiogenesis increased in cancer tissues AcSDKP  role in angiogenesis? Question

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) CATABOLISM AcSD * KP Angiotensin I-Converting Enzyme (ACE) AcSD + K + P Hg2+ sensitive peptidase AcSDKP = physiological substrate of N-active site of ACE C-active site ACE Ang I -> Ang II bradykinin -> inactive form AcSDKP -> AcSD + KP N-active site Biochem J, 1993, 296, 373. J Biol Chem, 1995, 270, 3656.

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) CATABOLISM 2 4 6 8 placebo 3 9 1 5 time (hours) captopril In vivo (humans) (ACE inhibitor) AcSDKP in blood [nM] J Clin Invest, 1996, 97, 839. ACE ACE inhibitors  stimulation of angiogenesis AcSDKP  role in angiogenesis ?

Angiogenesis EC receptor binding EC activation (MMPs production) EC proliferation EC migration ECM remodeling Tube formation Loop formation Vascular stabilisation

Angiogenesis

AcSDKP is produced and secreted by endothelial cells

AcSDKP enhances the secretion of active form of matrix metalloproteinase (MMP-1) and stimulates migration of endothelial cells 1 2 3 control 10-11 10-9 AcSDKP [M] MMP-1 content (ng/106 cells) + 84% ** * 20 40 60 80 MMP-1 * 1 2 57 % control 10-13 10-11 10-9 10-7 10-5 AcSDKP [M] migration score Migration

AcSDKP enhances endothelial cell proliferation

Assay of tube formation on Matrigel measures both the migration AcSDKP stimulates formation of vascular capillaries by endothelial cells Assay of tube formation on Matrigel measures both the migration and differentiation of endothelial cells into capillary-like structures 80 20 40 60 % increase in tube formation ** * 10-15 10-13 10-11 10-9 10-7 10-5 FGF-2 AcSDKP [M] (1ng/ml)

In vitro, AcSDKP promotes an angiogenic response of endothelial cells AcSDKP Receptor binding EC activation EC proliferation Directional migration ECM remodeling Tube formation Loop formation Vascular stabilisation (increase in the secreted active form of MMP-1)

In vivo, AcSDKP stimulates the neovascularisation in the chicken embryo chorioallantoic membrane (CAM) control + AcSDKP 30- 40 embryos/dose 2 days of incubation * 30 20 10 0.002 0.02 0.2 2 200 2 pmol/cm2 AcSDKP AcSDDKP % increase in vessels number

In vivo, AcSDKP stimulates the neovascularisation in abdominal muscle in the rat Day 7 AcSDKP i.m. (5 µg/kg/injection) during 5 days Day 28 control treated with AcSDKP

In vivo, AcSDKP stimulates corneal neovascularisation (9L-gliosarcoma cell spheroid) control tumor spheroid tumor spheroid + AcSDKP control spheroid spheroid + AcSDKP Administration of AcSDKP (800 mg/kg/day) via osmotic mini-pumps was started on the day of operation and continued for 10 days. +200% Am J Physiol, 2004, 287, 2099.

In vivo, AcSDKP induces blood vessel invasion AcSDKP stimulates vascular infiltration into Matrigel plugs implanted subcutaneously in rats + control + AcSDKP % increase in vessels number 100 200 AcSDKP [M] * 10-9 10-7 10-5 Matrigel alone or containing AcSDKP injected s.c. in rats. Histological analysis of Matrigel plugs performed at day 7

Limb arterial occlusive Angiogenesis Brain stroke Wound healing Limb arterial occlusive disease Cancer

Therapeutic effects of AcSDKP 1. Brain stroke Focal cerebral ischemia induced experimentally in the rat by occlusion of the middle cerebral artery (90 min) The volumes of the injured ischemic areas were monitored using magnetic resonance imaging (MRI) AcSDKP infusion (osmotic mini-pumps) 7 days * cortex sub-cortex brain 0.5 1.5 1.0 +saline +AcSDKP VOL T2 d8 / d2 AcSDKP reduces the extent of ischemic region in subcortical part of brain

Therapeutic effects of AcSDKP 2. Hindlimb ischemia Limb ischemia, in patients with arterial occlusive disease in the leg, is a major health problem surgically induced ischemia in mice (femoral artery occlusion) AcSDKP infusion (osmotic mini-pumps) 3 weeks - high-definition iliac microangiography - assessment of capillary density (immunohistochemistry) - laser Doppler perfusion imaging (DPI) (extent of blood flow = functional evidence for changes in vascularisation)

2. Hindlimb ischemia AcSDKP administration significantly increases the angiogenic score in the ischemic limb Isch N.Isch AcSDKP control Angiographic score (Isch./N.Isch.) 0.5 1 1.5 ** control AcSDKP

Therapeutic effects of AcSDKP Curative effect on the ischemic damages in brain and hindlimb AcSDKP induces revascularisation in brain and limb ischemic tissues and ameliorates the outcome of ischemic disease as shown by improvement of blood flow measured by Doppler (limb ischemia) Circulation, 2004, submitted Patent WO 02/24218 A1; US 2004/0038907 A1

density was studied in rats with MI given either vehicle or Ac-SDKP. Objective: N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a natural inhibitor of pluripotent hematopoietic stem cell proliferation, has been suggested as capable of promoting an angiogenic response. We studied whether Ac-SDKP 1) stimulates endothelial cell proliferation, migration and tube formation; 2) enhances angiogenic response in the rat cornea following implantation of a tumor spheroid; and 3) increases capillary density in rat hearts with myocardial infarction (MI). Methods and Results: 1) In vitro: an immortal BALB/c mouse aortic endothelial 22106 cell line was used to determine the effects of Ac-SDKP on endothelial cell proliferation and migration and tube formation. 2) In vivo: : a 9L-gliosarcoma cell spheroid (250-300 µm in diameter) was implanted in the rat cornea and vehicle or Ac-SDKP (800 µg/kg/day) infused i.p. via osmotic mini-pump. 3) Myocardial capillary density was studied in rats with MI given either vehicle or Ac-SDKP. We found that Ac-SDKP: 1) stimulated endothelial cell proliferation and migration and tube formation in a dose-dependent manner; 2) enhanced corneal neovascularization; and 3) increased myocardial capillary density. Conclusions: Endothelial cell proliferation and angiogenesis stimulated by AcSDKP could be beneficial in cardiovascular diseases such as hypertension and MI. Furthermore, since AcSDKP is mainly cleaved by ACE, it may partially mediate the cardioprotective effect of ACE inhibitors.

Therapeutic effects of AcSDKP AcSDKP increases capillary density in rat hearts with myocardial infarction (MI) + 75% Sham-MI saline AcSDKP MI MI was induced by ligation of left anterior descending coronary artery. AcSDKP was infused ip via osmotic mini-pumps 7 days before MI and continuing for 4 months.

Therapeutic effects of AcSDKP 3. Wound healing Ex vivo, AcSDKP accelerates the process of wound healing of human skin explants injured with UVB irradiation (10J/cm2) keratin 14 fibronectin control + AcSDKP

Therapeutic effects of AcSDKP 4. Postinjury tissue repair in surgical skin flap with ischemia control + AcSDKP control + AcSDKP AcSDKP injected s.c. for 5 days following operation. Evaluation of skin flap survival and skin flap vascularisation at day 7 In vivo, AcSDKP rescues the impaired vascularisation of ischemic experimental skin flaps and reduces their necrosis

Therapeutic effects of AcSDKP Curative effect on the skin injury AcSDKP actively participates in the repair of cutaneous damages: -> enhanced healing of injured explants of human skin -> accelerated wound healing of experimentally induced pseudo-ulcers in rats -> improvement of the viability of ischemic skin flaps in rats (promoting of post-operative angiogenesis) These findings identify AcSDKP as a new tissue-repair agents and suggest its potential clinical use for the management of skin wounds and as adjuvant to healing in plastic and reconstutive surgery Wound repair and regeneration, 2004 (submitted) Patent WO 02/24218 A1; US 2004/0038907 A1

Structure-activity relationship study A I M Conception of new molecules with biological efficacy higher than that displayed by AcSDKP

Ac-Ser-Asp-Lys-Pro-OH Analogues of AcSDKP  short sequence  modification of N- terminus  modification of AA lateral chains Ac-Ser-Asp-Lys-Pro-OH Succinyl Homo-Ser or Ala Glu Arg or Ornithine  stereoisomers: Ac Ser-DAsp-Lys-Pro-OH, AcDSer-DAsp-DLys-DPro-OH

Ac-N-Ser-Asp-Lys-Pro-OH Analogues of AcSDKP coll. J. Thierry, ICSN  Analogues resistant to proteolysis (CH2NH) Pro-NH2 Ac-N-Ser-Asp-Lys-Pro-OH cyclic peptide  Dimers (tail-head or head-head): Ac-Ser-Asp-Lys-Pro-Ser-Asp-Lys-Pro-OH HO-Pro-Lys-Asp-Ser-Succinyl-Ser-Asp-Lys-Pro-OH In vitro and in vivo evaluation of analogues’ angiogenic activity

In vivo effect of AcSDKP analogues on neovascularisation in CAM 30 AcSDKP AcSDKP-NH2 dimer head-head AcDSerDAspDLysDPro AcSDDKP 20 % increase in vascular density 10 10-10 10-9 10-8 10-7 10-5 [M] 0.002 0.02 0.2 2 200 pmoles/cm2 1 2 3 4 5

AcSDKP receptor Localisation of AcSDKP receptor on endothelial cell: O  autoradiography with [3H]AcSDKP  flow cytometry and confocal microscopy with: MAP (Multiple Antigen Peptide) coumarin- SDKP labelled with fluorescein l e x c . = 3 7 4 n m 1 O N C - SDKP AcSDKP Lys AcSDKP Lys AcSDKP Lys AcSDKP Pour localiser le site de fixation du tétrapeptide sur la cellule endothéliale , nous allons marquer ces molécules à l’aide de sondes marquées au tritium, biotinylées ou en fin fluorescentes… Et notamment nous allons préparer cette molécule : le MAP en anglais,un peptide multi antigenique qui permet d’augmenter la concentration locale du peptide et donc de pouvoir mieux visualiser les zones où le peptide se fixe Lys-bAla-OH AcSDKP Lys AcSDKP Lys AcSDKP Lys AcSDKP

Localization of AcSDKP receptor Lys-bAla-OH Lys AcSDKP MAP ex  lem  10-9M MAP Incubation of human dermal microvascular endothelial cells with MAP for 5 min at 37°C. 10-9M MAP + 1000x AcSDKP Pour localiser le site de fixation du tétrapeptide sur la cellule endothéliale , nous allons marquer ces molécules à l’aide de sondes marquées au tritium, biotinylées ou en fin fluorescentes… Et notamment nous allons préparer cette molécule : le MAP en anglais,un peptide multi antigenique qui permet d’augmenter la concentration locale du peptide et donc de pouvoir mieux visualiser les zones où le peptide se fixe Specific nuclear binding of AcSDKP

Localization of AcSDKP receptor x c . = 3 7 4 n m 1 O N C - Coum-SDKP SDKP Incubation of murine bone marrow microvascular endothelial cells with coum-AcSDKP for 5 min at 37°C. Project: to characterise the AcSDKP receptor and then to design new ligands offering an improved affinity for this receptor and consequently an improved angiogenic efficacy

Nuclear localisation of thymosin b4 (confocal section of fibroblasts NIH-3T3 following microinjection of labelled Tb4) J Cell Sci, 2004, 117, 5333.

Objectives To provide a basic understanding of the mechanisms involved in angiogenesis Developement of new drugs which will stimulate deficient angiogenesis (e.g. in hindlimb, cerebral and cardiac ischemia, wound healing, skin graft…)

? ? Links between biological effects of AcSDKP ??? Hematoprotection during radio- and chemotherapy Stimulation of angiogenesis AcSDKP ? Enhancement of bone marrow graft

Role of AcSDKP in cancer angiogenesis ? is there a correlation between levels of endogenous AcSDKP and the developement of neoplastic diseases ?

Role of AcSDKP in cancer angiogenesis ? Angiogenesis is essential not only for solid tumor growth but also plays a crucial role in hematological malignancies Hematological disorders (leukemia, lymphoma, myeloma, myeloplastic syndromes…) were shown to be angiogenesis-dependent and reveal significantly increased neovascularity in the bone marrow

Increased level of AcSDKP in plasma of leukemic mice SA2, SA7, SA9, SA10 => post-irradiation AML (different proportion of blast cells) AcSDKP (pmol/ml) control SA2 SA7 SA9 SA10 mice leukemic mice 10 20 ** p < 0.001 6-fold increase

Concentration of AcSDKP in plasma of SA9 mice increases with the progression of disease time following transplantation of leukemic cells AcSDKP (pmol/ml) 0 day 4 day 6 day 7 day 8 2 4 6 8 10 12 ** 0 day 4 day 6 day 7 day 8

time following transplantation Level of AcSDKP in bone marrow cells of leukemic mice 1 2 3 4 AcSDKP (pmol/106 cells) control SA2 SA7 SA9 SA10 mice leukemic mice ** 6-fold increase 1 2 time following transplantation of leukemic cells 0 day 4 day 6 day 7 day 8 NS SA9 **

Concentration of AcSDKP in bone marrow cell supernatants of leukemic mice 1,0 2,0 AcSDKP (pmol/106 cells) control SA2 SA7 SA9 SA10 mice leukemic mice NS ** 0,5 1,0 1,5 0 day 4 day 6 day 7 day 8 time following transplantation of leukemic cells SA9 **

in mouse normal and leukemic (SA9) bone marrow Expression of AcSDKP in mouse normal and leukemic (SA9) bone marrow SA9 BM NBM

in mouse normal and leukemic (SA2) spleen cells Expression of AcSDKP in mouse normal and leukemic (SA2) spleen cells normal spleen SA2 spleen

Concentration of AcSDKP in human plasma (coll. Prof. T. Robak) control AML CLM 1 2 3 4 5 6 n = 34 + 203 % AcSDKP (pmol/ml) + 61 % n = 33 n = 65 (acute myeloid (chronic lymphocytic leukemia) leukemia)

in human leukemic (CLM) bone marrow Expression of AcSDKP in human leukemic (CLM) bone marrow chronic lymphocytic leukemia

Expression of AcSDKP in human ovarium tissue normal cancer

Expression of AcSDKP in human prostate normal prostate cancer prostate

(coll. Profs Kuzdak, Komorowski, Stepien) Expression of AcSDKP in human thyroid (coll. Profs Kuzdak, Komorowski, Stepien) normal lobus papillary thyroid cancer lobus

Concentration of AcSDKP in plasma of patients with malignant tumors of thyroid (coll. Profs Kuzdak, Komorowski, Stepien) 2 4 6 8 10 12 peripheric blood: 1.59 + 0.45 tumor blood: 4.34 + 2.98 Thynoma invasivum AcSDKP [nM] Thyroid B-cell lymphoma Thyroid lymphoma 8 11 12 13 15 23 24 25 26 27 29 30 N° of patient

Concentration of AcSDKP in plasma of patients with malignant tumors of thyroid (coll. Profs Kuzdak, Komorowski, Stepien) 2 4 6 8 10 12 5 fold 6 fold peripheric blood tumor blood AcSDKP [nM] 3 fold 2 fold increase n = 18 papillary thyroid thyroid thyroid thynoma cancer lymphoma B-cell lymphoma invasivum

Role of AcSDKP in cancer angiogenesis ? AcSDKP, a potent stimulator of angiogenesis in vivo, may contribute to leukemia and solid tumor angiogenesis. This finding permit to hypothesise that AcSDKP expression may be critical factor for leukemia and solid tumor expansion Leukemia & Lymphoma, in preparation Br J Cancer, in preparation

Objectives To provide a basic understanding of the mechanisms involved in angiogenesis Developement of new drugs which will stimulate deficient angiogenesis (e.g. in hindlimb, cerebral and cardiac ischemia, wound healing, skin graft…) or to inhibit it in the case of cancer

? ? Links between biological effects of AcSDKP ??? Hematoprotection during radio- and chemotherapy Stimulation of angiogenesis AcSDKP ? Enhancement of bone marrow graft