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Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP): A Physiological Regulator of Angiogenesis

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) is a tetrapeptide that regulates angiogenesis and inhibits hematopoietic stem cell proliferation. It has shown protective effects against bone marrow toxicity caused by chemotherapy and radiation. AcSDKP is currently being studied for its myeloprotective effects in cancer patients undergoing monochemotherapy. This peptide is also a potential target for developing anticancer drugs and has shown to stimulate migration and tube formation in endothelial cells, promoting angiogenesis.

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Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP): A Physiological Regulator of Angiogenesis

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  1. 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

  2. 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

  3. 50 anti-AcSDKP % of CFU-S in S-phase control AcSDKP + antiAcSDKP (incubated overnight) complex = AcSDKP is a physiological regulator of hematopoiesis (in vivo administration of anti-AcSDKP antibodies triggers hematopoietic stem cell into S-phase) Leukemia, 1989, 4, 315

  4. 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 (immunodeficiency, hemorrhages…) + proliferation inhibitor chemo- or radiotherapy Cell survival Go Go

  5. % mice survival % CFU-S en S 100 40 ( s i m u l t ) (2h post) (6h post) 50 20 (8h post) nat. synth. AraC : 30 mg/kg/inj s.c. 1x day, during 15d control AraCArac + AcSDKP (LD 90) control AraC AraC + AcSDKP AcSDKP : 100 ng/inj i.p. 1x day, during 15d (100 ng/mouse) Proc Nat Acad Sci USA, 1989 , 86, 779. 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)

  6. 10000 + AcSDKP 100 + AcSDKP % of survival Granulocytes / mm3 irradiated control irradiated control 10 20 30 40 50 days after irradiation 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 Ann Hematol, 1997, 74, 117.

  7. AcSDKP => new myeloprotective agent Clinical application: in vivo:radio- and chemotherapy in vitro: bone marrow purging before autologous grafting 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

  8. 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

  9. Catabolisme Biosynthesis AcSDKP Mechanism of action Receptor study Analogues

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

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

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

  13. In vivo (humans) 1 5 1 2 captopril AcSDKP in blood [nM] 9 6 3 placebo 0 2 4 6 8 time (hours) captopril (ACE inhibitor) ACE inhibitors  stimulation of angiogenesis ACE 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. Angiogenesis

  16. AcSDKP is produced and secreted by endothelial cells

  17. MMP-1 Migration + 84% 80 ** 57 % 2 60 migration score * * * * * * * MMP-1 content (ng/106 cells) * * * 40 * 1 20 1 2 3 control10-11 10-9 AcSDKP [M] control10-13 10-11 10-9 10-7 10-5 AcSDKP [M] AcSDKP enhances the secretion of active form of matrix metalloproteinase (MMP-1) and stimulates migration of endothelial cells

  18. AcSDKP enhances endothelial cell proliferation

  19. 80 ** 60 ** ** ** * 40 % increase in tube formation ** ** 20 10-15 10-13 10-11 10-9 10-7 10-5 FGF-2 AcSDKP [M] (1ng/ml) 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

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

  21. 30 AcSDKP AcSDDKP * * * * * * 20 % increase in vessels number * * * * * 10 0.0020.020.2 2 200 2 pmol/cm2 control + AcSDKP 30- 40 embryos/dose 2 days of incubation In vivo, AcSDKP stimulates the neovascularisation in the chicken embryo chorioallantoic membrane (CAM)

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

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

  24. 200 * * * % increase in vessels number 100 0 10-9 10-7 10-5 + AcSDKP AcSDKP [M] AcSDKP stimulates vascular infiltration into Matrigel plugs implanted subcutaneously in rats + control 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

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

  26. 1.5 +saline +AcSDKP Focal cerebral ischemia induced experimentally in the rat by occlusion of the middle cerebral artery (90 min) 1.0 VOL T2 d8 / d2 * 0.5 AcSDKP infusion (osmotic mini-pumps) 7 days The volumes of the injured ischemic areas were monitored using magnetic resonance imaging (MRI) cortex sub-cortex brain AcSDKP reduces the extent of ischemic region in subcortical part of brain Therapeutic effects of AcSDKP 1. Brain stroke

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

  29. 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

  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 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.

  31. 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.

  32. 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

  33. 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

  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 inrats -> 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

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

  36. Homo-Ser orAla ArgorOrnithine Glu Succinyl Ac-Ser-Asp-Lys-Pro-OH Analogues of AcSDKP  shortsequence  modification of N- terminus  modification of AA lateral chains stereoisomers: Ac Ser-DAsp-Lys-Pro-OH, AcDSer-DAsp-DLys-DPro-OH

  37. 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

  38. 1 2 3 4 5 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

  39. l m = 3 7 4 n e x c . l e m . = 4 7 0 n m 3 e = 1 7 x 1 0 N O O SDKP - C O AcSDKP receptor Localisation of AcSDKP receptor on endothelial cell:  autoradiography with [3H]AcSDKP  flow cytometry and confocal microscopy with: MAP(MultipleAntigenPeptide)coumarin- SDKPlabelled with fluorescein AcSDKP Lys AcSDKP Lys AcSDKP Lys AcSDKP Lys-bAla-OH AcSDKP Lys AcSDKP Lys AcSDKP Lys AcSDKP

  40. MAP AcSDKP Lys AcSDKP Lys AcSDKP Lys AcSDKP Lys-bAla-OH AcSDKP Lys AcSDKP Lys AcSDKP ex  lem  Lys AcSDKP Specific nuclear binding of AcSDKP 10-9M MAP 10-9M MAP + 1000x AcSDKP Localization of AcSDKP receptor Incubation of human dermal microvascular endothelial cells with MAP for 5 min at 37°C.

  41. Coum-SDKP N O O l = 3 7 4 n m e x c . l e m . = 4 7 0 n m 3 e = 1 7 x 1 0 SDKP - C O Localization of AcSDKP receptor 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

  42. Nuclear localisation of thymosin b4 (confocal section of fibroblasts NIH-3T3 following microinjection of labelled Tb4) 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. ??? ? Links between biological effects of AcSDKP Hematoprotection during radio- and chemotherapy Stimulation of angiogenesis AcSDKP ? Enhancement of bone marrow graft

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

  46. 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

  47. 20 p < 0.001 ** 6-fold increase ** ** 10 AcSDKP (pmol/ml) ** ** 0 control SA2 SA7 SA9 SA10 mice leukemic mice Increased level of AcSDKP in plasma of leukemic mice SA2, SA7, SA9, SA10 => post-irradiation AML (different proportion of blast cells)

  48. 12 10 ** 8 6 ** AcSDKP (pmol/ml) ** 4 ** 2 0 0 day 4 day 6 day 7 day 8 0 day 4 day 6 day 7 day 8 time following transplantation of leukemic cells Concentration of AcSDKP in plasma of SA9 mice increases with the progression of disease

  49. 4 3 6-fold increase 2 ** ** 2 SA9 ** ** ** 1 AcSDKP (pmol/106 cells) 1 ** 0 ** NS control SA2 SA7 SA9 SA10 mice leukemic mice 0 day 4 day 6 day 7 day 8 0 time following transplantation of leukemic cells Level of AcSDKP in bone marrow cells of leukemic mice

  50. 2,0 ** SA9 1,5 ** 1,0 ** ** 1,0 ** AcSDKP (pmol/106 cells) ** 0,5 NS ** 0 day 4 day 6 day 7 day 8 control SA2 SA7 SA9 SA10 mice leukemic mice time following transplantation of leukemic cells Concentration of AcSDKP in bone marrow cell supernatants of leukemic mice

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