Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "Liu et al, Blood, 2003, 101, 3014. Patent WO 02/24218 A1; US 2004/0038907 A1 The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), the new physiological."— Presentation transcript:

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

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

3 Leukemia, 1989, 4, 315 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

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

5 AraC : 30 mg/kg/inj s.c. 1x day, during 15d AcSDKP : 100 ng/inj i.p. 1x day, during 15d control AraC Arac + AcSDKP (LD 90) % mice survival (simult) (2h post) (6h post) (8h post) Ann NY Acad Sci,1991, 628, 126. 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, % CFU-S en S nat.synth. control AraC AraC + AcSDKP (100 ng/mouse)

6 AcSDKP (0.5  g/kg/day), i.v. infusion from -24h to +1h with regard to time of irradiation Ann Hematol, 1997, 74, 117. In vivo protective effect of AcSDKP against the myelotoxicity of total irradiation (300 cGy) + AcSDKP irradiated control Granulocytes / mm days after irradiation AcSDKP irradiated control % of survival 100

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

8 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 Clinical trials of AcSDKP “Goralatide” (Beaufour Laboratories - IPSEN Biotech)

9 Biosynthesis AcSDKP Receptor study Mechanism of action Catabolisme Analogues

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

11 T4T4 stimulation of angiogenesis increased in cancer tissues AcSDKP  role in angiogenesis? Question Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) BIOSYNTHESIS

12 AcSD * KP Angiotensin I-Converting Enzyme (ACE) AcSD + * KP * K + P Hg 2+ sensitive peptidase Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)CATABOLISM 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.

13 2468 placebo time (hours) captopril In vivo (humans) captopril (ACE inhibitor) AcSDKP in blood [nM] ACE ACE inhibitors  stimulation of angiogenesis AcSDKP  role in angiogenesis ? Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)CATABOLISM J Clin Invest, 1996, 97, 839.

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

15

16 AcSDKP is produced and secreted by endothelial cells

17 AcSDKP enhances the secretion of active form of matrix metalloproteinase (MMP-1) and stimulates migration of endothelial cells 123 control AcSDKP [M] MMP-1 content (ng/10 6 cells) + 84% ** * MMP-1 ** ** ** ** * 1 2 * 57 % control AcSDKP [M] migration score Migration

18 AcSDKP enhances endothelial cell proliferation

19 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 % increase in tube formation ** * FGF-2 AcSDKP [M] (1ng/ml)

20 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)

21 In vivo, AcSDKP stimulates the neovascularisation in the chicken embryo chorioallantoic membrane (CAM) control + AcSDKP embryos/dose 2 days of incubation *** *** *** ** pmol/cm 2 AcSDKP AcS D DKP % increase in vessels number

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

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

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

25 Angiogenesis Brain stroke Wound healing Limb arterial occlusive disease Cancer

26 Therapeutic effects of AcSDKP 1. Brain stroke * cortex sub-cortex brain saline + AcSDKP VOL T2 d8 / d2 AcSDKP reduces the extent of ischemic region in subcortical part of brain 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

27 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)

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

29 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) Therapeutic effects of AcSDKP Curative effect on the ischemic damages in brain and hindlimb Circulation, 2004, submitted Patent WO 02/24218 A1; US 2004/ A1

30 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 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 ( 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.

31 AcSDKP increases capillary density in rat hearts with myocardial infarction (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. Sham-MI saline AcSDKP MI + 75% Therapeutic effects of AcSDKP

32 Ex vivo, AcSDKP accelerates the process of wound healing of human skin explants injured with UVB irradiation (10J/cm 2 ) control + AcSDKP keratin 14 fibronectin Therapeutic effects of AcSDKP 3. Wound healing

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

34 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) Wound repair and regeneration, 2004 (submitted) Patent WO 02/24218 A1; US 2004/ A1 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

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

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

37 In vitro and in vivo evaluation of analogues’ angiogenic activity Analogues of AcSDKP coll. J. Thierry, ICSN  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  Analogues resistant to proteolysis Ac-N-Ser-Asp-Lys-Pro-OH  (CH 2 NH) Pro-NH 2 cyclic peptide

38 In vivo effect of AcSDKP analogues on neovascularisation in CAM [M] pmoles/cm 2 AcSDKP AcSDKP-NH 2 dimer head-head AcS D DKP Ac D Ser D Asp D Lys D Pro % increase in vascular density

39 Localisation of AcSDKP receptor on endothelial cell:  flow cytometry and confocal microscopy with: MAP ( Multiple Antigen Peptide) coumarin- SDKP labelled with fluorescein  autoradiography with [ 3 H]AcSDKP Lys-  Ala-OH Lys AcSDKP AcSDKP receptor exc. = 374 n m em. = 470 nm = 17x10 3  O O N CO - SDKP

40 Localization of AcSDKP receptor M MAP M MAP x AcSDKP Incubation of human dermal microvascular endothelial cells with MAP for 5 min at 37°C. Lys-  Ala-OH Lys AcSDKP MAP ex  em  Specific nuclear binding of AcSDKP

41 exc. = 374 nm em. = 470 nm = 17x10 3  O O N CO - Coum-SDKP SDKP Localization of AcSDKP receptor 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 Incubation of murine bone marrow microvascular endothelial cells with coum-AcSDKP for 5 min at 37°C.

42 Nuclear localisation of thymosin  4 (confocal section of fibroblasts NIH-3T3 following microinjection of labelled T  4) J Cell Sci, 2004, 117, 5333.

43 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…)

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

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

46 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 Role of AcSDKP in cancer angiogenesis ?

47 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 ** p < ** 6-fold increase

48 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 ** 0 day 4 day 6 day 7 day 8

49 Level of AcSDKP in bone marrow cells of leukemic mice time following transplantation of leukemic cells 0 day 4 day 6 day 7 day 8 NS SA9 ** AcSDKP (pmol/10 6 cells) control SA2 SA7 SA9 SA10 mice leukemic mice ** 6-fold increase

50 Concentration of AcSDKP in bone marrow cell supernatants of leukemic mice 1,0 2,0 AcSDKP (pmol/10 6 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 **

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

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

53 Concentration of AcSDKP in human plasma (coll. Prof. T. Robak) control AML CLM n = % AcSDKP (pmol/ml) + 61 % n = 33 n = 65 (acute myeloid (chronic lymphocytic leukemia) leukemia)

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

55 normal cancer Expression of AcSDKP in human ovarium tissue

56 cancer prostate normal prostate Expression of AcSDKP in human prostate

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

58 peripheric blood: tumor blood: AcSDKP [nM] N° of patient Thyroid B-cell lymphoma Thyroid lymphoma Thynoma invasivum Concentration of AcSDKP in plasma of patients with malignant tumors of thyroid (coll. Profs Kuzdak, Komorowski, Stepien)

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

60 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

61 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

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


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

Similar presentations


Ads by Google