liu et al, blood, 2003, 101, 3014. patent wo 02/24218 a1; us 2004/0038907 a1 the tetrapeptide...

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 regulator of angiogenesis

CO2H

NNH

HN

OCO2H

O

NH2

O

NH

HOO

• 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

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)

Proc Natl Acad Sci USA, 1989, 86, 779

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

% o

f C

FU

-S in

S-p

has

e50

anti-AcSDKP

Go S S

Go Go

S

S

Hematopoieticstem cell

Chemo- or radiothera

py

chemo- or radiothera

py

+ proliferation inhibitor

Bone marrow aplasia

Cancer cell

Cell survival

(immunodeficiency, hemorrhages…)

cell proliferation

Bone marrow toxicity => major limiting factor in the treatment of cancer

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

(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, 779.

40

20

% CFU-S en S

nat. synth.

control AraC AraC + AcSDKP

(100 ng/mouse)

AcSDKP (0.5 g/kg/day), i.v. infusionfrom -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 controlG

ran

ulo

cyte

s / m

m3

10 20 30 40 50days after irradiation

10000 + AcSDKP

irradiatedcontrol

% o

f su

rviv

al

100

->> anticancer drugs :- AraC (cytosine arabinoside)- CTX (cyclophosphamide)- 5-FU (5-fluorouracil)- doxorubicine->> irradiation

->> anticancer drugs :- AraC- AZT (azidothymidine)- AstaZ (mafosfamide)->> phototherapy->> hyperthermy

In vivo In vitro

Myeloprotective effect of AcSDKP

AcSDKP => new myeloprotective agent

Clinical application:in vivo: radio- and chemotherapyin vitro: bone marrow purging before autologous grafting

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)

Biosynthesis

AcSDKP

Receptor studyMechanism of action

Catabolisme

Analogues

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)BIOSYNTHESIS

Thymosin 4 (T4) = metabolic precursor of AcSDKP ?

- intracellular co-localisation of T4 and AcSDKP- inhibits proliferation of hematopoietic stem cells-[3H]T4 + bone marrow cells => [3H]AcSDKP

AcSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES

43 AA

AcSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES?

AcSDKPDMAEIEKFD-prolylendopeptidase

AcSDKP

T4

FEBS Lett, 1990, 274, 30.Cell Prolif, 1996, 29, 437.

T4

stimulation of angiogenesis

increased in cancer tissues

AcSDKP role in angiogenesis?

Question

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)BIOSYNTHESIS

AcSD*KPAngiotensin I-Converting Enzyme (ACE)

AcSD + *KP

*K + P

Hg2+ sensitive peptidase

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)CATABOLISMCATABOLISM

AcSDKP = physiological substrate of N-active site of ACE

C-active siteACE

Ang I -> Ang IIbradykinin -> inactive form

AcSDKP -> AcSD + KP

N-active site

Biochem J, 1993, 296, 373.J Biol Chem, 1995, 270, 3656.

2 4 6 8

placebo0

3

6

9

1 2

1 5

time (hours)

captopril

In vivo (humans)

captopril(ACE inhibitor)

AcS

DK

P i

n b

loo

d [

nM

]

ACE

ACE inhibitors stimulation of angiogenesis

AcSDKP role in angiogenesis ?

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP)CATABOLISMCATABOLISM

J Clin Invest, 1996, 97, 839.

EC receptor binding

EC activation (MMPs production)

EC proliferation

EC migration

ECM remodeling

Tube formation

Loop formation

Vascular stabilisation

Angiogenesis QuickTime™ et un décompresseur

GIF sont requis pour visualisercette image.

Angiogenesis QuickTime™ et un décompresseur

GIF sont requis pour visualisercette image.

AcSDKP is produced and secreted by endothelial cells

.

0

0.5

1.0

1.5

2.0

2.5

3.0

0

1

2

3

4

5

time of culture (days)0 1 2 3 4 7 8 10

cell n

um

be

r x 1

06

AcS

DK

P [

pm

ol/

10

6 c

ells

]

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]

MM

P-1

co

nte

nt

(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]

mig

rati

on

sc o

r e

Migration

AcSDKP enhances endothelial cell proliferation

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

% in

crea

se in

tu

be

form

atio

n

**

**

**

****

*

**

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/dose2 days of incubation

***

***

***

30

20

10

0.002 0.02 0.2 2 200 2

**

pmol/cm2

AcSDKPAcSDDKP

% i

nc

rea

se

in

ve

ss

els

nu

mb

er

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

In vivo, AcSDKP stimulates corneal neovascularisation

control tumor 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 andcontinued for 10 days.

+200%

(9L-gliosarcoma cell spheroid)

Am J Physiol, 2004, 287, 2099.

In vivo, AcSDKP induces blood vessel invasion

Matrigel alone or containing AcSDKP injected s.c. in rats. Histological analysis

of Matrigel plugsperformed at day 7 %

in

crea

se

in v

esse

ls n

um

ber

100

200

AcSDKP [M]

* *

*

010-9 10-7 10-5

AcSDKP stimulates vascular infiltration into Matrigel plugs implanted subcutaneously in rats

+ control

+ AcSDKP

QuickTime™ et un décompresseurGIF sont requis pour visualiser

cette image.

Angiogenesis

Brain stroke

Wound healing

Limb arterial occlusive disease

Cancer

Therapeutic effects of AcSDKP1. Brain stroke

*

cortex sub-cortex brain

0.5

1.5

1.0

+saline+AcSDKP

VO

L T

2 d

8 / d

2AcSDKP reduces the extent of

ischemic region in subcortical part of brain

Focal cerebral ischemia inducedexperimentally 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

Therapeutic effects of AcSDKP2. 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)

Isch

N.Isch

AcSDKPcontrol

AcSDKP administration significantly increasesthe angiogenic score in the ischemic limb

2. Hindlimb ischemia

An

gio

gra

ph

ic s

core

(Isc

h./N

.Isch

.)

0.5

1

1.5

**

control AcSDKP

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 AcSDKPCurative effect on the ischemic damages in brain and hindlimb

Circulation, 2004, submittedPatent WO 02/24218 A1; US 2004/0038907 A1

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-SDKPon 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.

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

Ex vivo, AcSDKP accelerates the process of wound healing

of human skin explants injured with UVB irradiation (10J/cm2)

control + AcSDKP

keratin 14

fibronectin

Therapeutic effects of AcSDKP3. Wound healing

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 AcSDKP4. Postinjury tissue repair in surgical skin flap with ischemia

QuickTime™ et un décompresseurTIFF (LZW) sont requis pour visualiser

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Therapeutic effects of AcSDKPCurative 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 2004/0038907 A1

These findings identify AcSDKP as a new tissue-repair agents andsuggest its potential clinical use for the management of skin woundsand as adjuvant to healing in plastic and reconstutive surgery

Structure-activity relationship study

Conception of new molecules with biological efficacy higher than that displayed by AcSDKP

A I M

short sequence

stereoisomers:

modification of AA lateral chains

modification of N- terminus

Ac Ser-DAsp-Lys-Pro-OH, AcDSer-DAsp-DLys-DPro-

OH

Analogues of AcSDKP

Ac-Ser-Asp-Lys-Pro-OH

Succinyl

Homo-Seror Ala Glu

Arg or Ornithine

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

(CH2NH)Pro-NH2

cyclic peptide

In vivo effect of AcSDKP analogues on neovascularisation in CAM

10-10 10-9 10-8 10-7 10-5 [M]0.002 0.02 0.2 2 200 pmoles/cm2

AcSDKP

AcSDKP-NH2 dimer head-head

AcSDDKPAcDSerDAspDLysDPro

% i

ncr

eas

e in

vas

cul a

r d

ensi

ty

1 2 3 4 5

10

20

30

Localisation of AcSDKP receptor on endothelial cell:

flow cytometry and confocal microscopy with:

MAP (Multiple Antigen Peptide)

coumarin- SDKP labelled with fluorescein

autoradiography with [3H]AcSDKP

Lys-Ala-OH

Lys

Lys

Lys

Lys

Lys

Lys

AcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP receptor

ex c . = 3 74 n m

e m . = 470 n m

= 1 7x1 03

O ON

C O -SDKP

Localization of AcSDKP receptor

10-9M MAP 10-9M MAP + 1000x AcSDKP

Incubation of human dermal microvascular endothelial cells with MAP for 5 min at 37°C.

Lys-Ala-OH

Lys

Lys

Lys

Lys

Lys

Lys

AcSDKPAcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP

AcSDKP

MAP

ex

em

Specific nuclear bindingof AcSDKP

exc. = 374 nm

em. = 470 nm

= 17x103

O ON

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.

Nuclear localisation of thymosin 4(confocal section of fibroblasts NIH-3T3 following microinjection of labelled T4)

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…)

AcSDKP

Links between biological effects of AcSDKP

Stimulation of angiogenesis

Hematoprotection duringradio- and chemotherapy

Enhancement of bone marrow graft

???

Role of AcSDKP in cancer angiogenesis ?

is there a correlation betweenlevels of endogenous AcSDKP

and the developement of neoplastic diseases ?

QuickTime™ et un décompresseurPhoto - JPEG sont requis pour visualiser

cette image.

AcSDKP

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 revealsignificantly 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)

AcS

DK

P (

pm

ol/m

l)

control SA2 SA7 SA9 SA10 mice leukemic mice

0

10

20

** **

**

**p < 0.001**6-fold increase

Concentration of AcSDKP in plasma of SA9 miceincreases with the progression of disease

time following transplantation of leukemic cells

AcS

DK

P (

pm

ol/m

l)

0 day 4 day 6 day 7 day 80

2

4

6

8

10

12

** **

**

**


Level of AcSDKP in bone marrow cells of leukemic mice

0

1

2

time following transplantation of leukemic cells


NS

SA9

**

**

**

0

1

2

3

4

AcS

DK

P (

pm

ol/1

06 cel

ls)


**

** **

**

6-fold increase

Concentration of AcSDKPin bone marrow cell supernatants of leukemic mice

1,0

2,0

AcS

DK

P (

pm

ol/1

06 cel

ls)


NS

****

**

0,5

1,0

1,5

0 day 4 day 6 day 7 day 8time following transplantation

of leukemic cells

SA9

**

**

**

**

NBM

SA9 BM

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

QuickTime™ et un décompresseurTIFF (non compressé) sont requis pour visualiser

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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 0

1

2

3

4

5

6

n = 34

+ 203 %A

cSD

KP

(p

mol

/ml)

+ 61 %

n = 33 n = 65

(acute myeloid (chronic lymphocytic leukemia) leukemia)


cette image.


cette image.

Expression of AcSDKP in human leukemic (CLM) bone marrow

chronic lymphocytic leukemia

normal cancer

Expression of AcSDKP in human ovarium tissue

cancer prostate

normal prostate

Expression of AcSDKP in human prostate


cette image.


cette image.

(coll. Profs Kuzdak, Komorowski, Stepien)

normal lobus papillary thyroid cancer lobus

Expression of AcSDKP in human thyroid

0

2

4

6

8

10

12

peripheric blood: 1.59 + 0.45tumor blood: 4.34 + 2.98

AcS

DK

P [

nM

]

8 11 12 13 15 23 24 25 26 27 29 30N° of patient

Thy

roid

B-c

ell l

ymph

oma

Thy

roid

lym

phom

aThy

nom

a in

vasi

vum

Concentration of AcSDKP in plasma of patients with malignant tumors of thyroid(coll. Profs Kuzdak, Komorowski, Stepien)

Ac S

DK

P [

nM

]

2

4

6

8

10

12

peripheric bloodtumor 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 increasen = 18


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 preparationBr 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

AcSDKP

Links between biological effects of AcSDKP

Stimulation of angiogenesis

Hematoprotection duringradio- and chemotherapy

Enhancement of bone marrow graft

???

liu et al, blood, 2003, 101, 3014. patent wo 02/24218 a1; us 2004/0038907 a1 the tetrapeptide...

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