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/ A1

2. Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)
purified from bone marrow ( 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. 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

4. 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

5. 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.

6. 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 50
days after irradiation
10000
Ann Hematol, 1997, 74, 117.

7. 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

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. AcSDKP Biosynthesis Catabolisme Mechanism of action Receptor study
Analogues

10. 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.

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

12. 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.

13. 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 ?

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

15. Angiogenesis

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 1 2 3
control
AcSDKP [M]
MMP-1 content (ng/106 cells)
+ 84% ** * 20 40 60 80
MMP-1 * 1 2
57 %
control
AcSDKP [M]
migration score
Migration

18. AcSDKP enhances endothelial cell proliferation

19. 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 ** *
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 * 30 20 10
pmol/cm2
AcSDKP
AcSDDKP
% increase in vessels number

22. 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

23. 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.

24. 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] *
Matrigel alone or
containing AcSDKP
injected s.c. in rats.
Histological analysis
of Matrigel plugs
performed at day 7

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

26. 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

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. 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

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/ A1

30. 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 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. 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 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/ A1

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

36. 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

37. 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

38. In vivo effect of AcSDKP analogues on neovascularisation in CAM30
AcSDKP
AcSDKP-NH2
dimer head-head
AcDSerDAspDLysDPro
AcSDDKP 20
% increase in vascular density 10
[M]
pmoles/cm2 1 2 3 4 5

39. 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

40. 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 x 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

41. Localization of AcSDKP receptorx 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

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 ?
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. 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 SA SA SA9 SA10
mice leukemic mice 10 20 **
p < 0.001
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)
day day day 7 day 8 2 4 6 8 10 12 **
day day day 7 day 8

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

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

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

52. in mouse normal and leukemic (SA2) spleen cells
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 1 2 3 4 5 6
n = 34
+ 203 %
AcSDKP (pmol/ml)
+ 61 %
n = 33
n = 65
(acute myeloid (chronic lymphocytic
leukemia) leukemia)

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

55. Expression of AcSDKP in human ovarium tissue
normal cancer

56. Expression of AcSDKP in human prostate
normal prostate
cancer prostate

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

58. 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:
tumor blood:
Thynoma invasivum
AcSDKP [nM]
Thyroid B-cell lymphoma
Thyroid lymphoma
N° of patient

59. 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

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