DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100… · DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100, A CASE IN POINT Erik DE CLERCQ Rega Institute for Medical Research,

DRUG DISCOVERY AND DEVELOPMENT:THE BICYCLAM AMD3100, A CASE IN

POINT

Erik DE CLERCQRega Institute for Medical Research, K.U.Leuven

B-3000 Leuven, Belgium

LAV Replication, tested by reverse transcriptase (RT) activity in supernatantsof T cell cultures, during HPA 23 treatment of a patient with AIDS

HPA-23: (NH4)18(NaW21Sb9O86)17

0

10

20

30RT activity

I x 103 (cp

m)

HPA 23 HPA 23

May 83 July Aug Sep Oct Nov Dec Jan 84 Nov 84

NSNS

NS

The two periods of treatment were from July 27 to August 20, 1983, and from October 17 to December 8, 1983.Cumulative doses were, respectively, 890 mg and 1980 mg. HPA 23 doses were slightly increased from 1 mg/kg to 3.3 mg/kg. Solid columns show RT activity (NS = no virus detectable).

Rozenbaum et al., Lancet i: 450-451 (1985)

De Clercq, Clin. Microbiol. Rev. 8, 200-239 (1995)

JM1493H4SiW12O40

De Clercq, Metal-Based Drugs 4, 173-192 (1997)

JM1590K13[Ce(SiW11O39)2].26H20

De Clercq, Clin. Microbiol. Rev. 8, 200-239 (1995)

JM2820

Anti-HIV activity of polyoxometalates

> 156> 5003.2JM2820: [Me3NH]8[Si2W18Nb6O77]

> 263> 5001.9JM2815: K5[SiW11(C5H5)TiO39]

> 178> 5002.8JM2766: K6[BW11Ga(H2O)O39]

266 2931.1JM1809: K8HP2W15V3O62.34H2O

666 4660.7JM1596: K10[P2W18Zn4(H2O)2O68].20H2O

1130 3390.3JM1591: K12H2P2W12O48.24H2O

328 2300.7JM1590: K13[Ce(SiW11O39)2].26H2O

467 6541.4JM1583: K5[BW12O40]

Selectivityindex

CC50(µg/ml)

EC50(µg/ml)

Compound

De Clercq, Metal-Based Drugs 4, 173-192 (1997)

Anti-HIV activity of metalloporphyrins

25> 180 > 90

23> 90> 90

0.9 0.5

1

H2FeNi

Selectivityindex

CC50(µg/ml)

EC50(µg/ml)

MR

COOH

Song et al., Antiviral Chem. Chemother. 8, 85-97 (1997)

N

M

N

N N

R

RR

R

Cyclam Metal-cyclam complex

NH

HNNH

HN N

NN

NM

M

4H

Anti-HIV activity of JM1657 in MT-4 cells

1248 150399JM1498

319 1.01 0.144JM1657

HIV-2(ROD)HIV-1(IIIB)

CC50 (µM)EC50 (µM)StructureCompound

NH

HNNH

HN

H H

HN

HNNH

NH

NH

HNNH

HN

De Clercq et al., Proc. Natl. Acad. Sci. USA 89: 5286-5290 (1992)


> 622 1.00 0.248

HIV-2(ROD)HIV-1(IIIB)

CC50 (µM)EC50 (µM)Structure

De Clercq et al., Proc. Natl. Acad. Sci. USA 89: 5286-5290 (1992)

HN

HNNH

NNH

HNNH

N


0.005 > 100,000

Selectivityindex

> 500

CC50(µg/ml)

EC50(µg/ml)

HIV-1(IIIB)

Structure

De Clercq et al., Antimicrob. Agents Chemother. 38: 668-674 (1994)

HN

HNNH

NNH

HNNH

N

8 HBr 2 H2O

Anti-HIV activity of bicyclam JM2987

> 100> 100MOLT-4MND-GB1

> 100> 100MOLT-4AGM-3

> 100> 100MT-4MAC-251SIV

> 125,000> 500 0.004MT-4EHO

> 71,400> 500 0.007MT-4RODHIV-2

> 167,000> 500 0.003MT-4HE

> 500,000> 500 0.001MT-4RF

> 100,000> 500 0.005MT-4IIIBHIV-1

Selectivityindex

CC50(µg/ml)

EC50(µg/ml)

Cell lineStrainVirus

De Clercq et al., Antimicrob. Agents Chemother. 38: 668-674 (1994)

HN

HNNH

NNH

HNNH

N

JM3100AMD3100

1,1’-[1,4-phenylene-bis(methylene)]-bis(1,4,8,11-tetra-azacyclotetradecane) octahydrochloride dihydrate

8 HCl 2 H2O

Mutations detected in gp120 of bicyclam-resistant NL4-3viruses:

N269Y R272T S274R Q278H I288V N293H A297T ∆∆∆∆FNSTWP385L Q410E S433P V457I

De Vreese et al., Antimicrob. Agents Chemother. 41, 2616-2620 (1997)

Passage Fold resistance to Mutations detected

no JM2763 JM3100 N269Y R272T S274R Q278H I288V N293H A297T ∆FNSTW P385L Q410E S433P V457I

JM2763 resistant 16 2 3 0 0 + + 0 0 0 0 0 0 0 0

25 200 11 0 0 + + + 0 0 + 0 0 0 0

28 200 6 0 0 + + + 0 + + + 0 0 0

JM3100 resistant 28 450 7 0 0 + + + 0 + 0 + + 0 0

0 0 + + + 0 + + + + + 0

42 > 740 60 0 + + + + + + + + + + +

60 > 740 200 + + + + + + + + + + + +

Mechanism of action of bicyclams:interaction with monoclonal antibody binding to CXCR4

.

isotype 1 2 G 5 AM D3100 25 µµµµg/m l

AM D3100 5 µµµµg/m l AM D3100 1 µµµµg/m l

0 .5 % 8 9 % 1 %

1 % 2 % 4 7 %

M FI 3 M FI 23 M FI 4

M FI 4 M FI 4 M FI 10

CXCR-4 EXPRESSIO N

R

AM D3100 0.2 µµµµg/m l

REL

ATI

VE C

ELL

NU

MB

ER

Schols et al., Antiviral Res. 35: 147-156 (1997)

Anti-HIV activity profile of AMD3100 correlated withcoreceptor use

4> 1,000> 25,000CCR5 (CCR2b, CCR3)HIV-1 JR-FL10> 1,000> 25,000CCR5 (CCR2b, CCR3)HIV-1 ADA5> 1,000> 25,000CCR5HIV-1 SF-16225> 1,000> 25,000CCR5HIV-1 BaL

M-tropic> 1,000557CXCR4HIV-2 ROD> 1,0001003CXCR4HIV-1 NL4-3> 1,000505CXCR4HIV-1 RF> 1,000202CXCR4HIV-1 IIIB

T-tropicRANTESSDF-1ααααAMD3100

EC50 (ng/ml)Coreceptor usedStrain

Schols et al., J. Exp. Med. 186: 1383-1388 (1997)

-1000

-500

0

500

1000

1500

2000

0 50 100 150 200 250 300

Control AMD3100 200 ng/mlAMD3100 40 ng/ml AMD3100 8 ng/ml

Concentration-dependent inhibition of SDF-1-inducedCa2+ flux in Sup-T1 cells by AMD3100

Time (seconds)

Fluo

resc

ent c

hang

e (c

ount

s)

De Clercq, Int. J. Antimicrob. Agents 18: 309-328 (2001)

0

20

40

60

80

100

120

0 1000 100 10 1 0.1

Concentration of AMD3100 (ng/ml)

% o

f inh

ibiti

on—■■■■ — Binding of CXCR4 mAb—■■■■ — SDF-1-induced Ca2+ flux—●●●● — HIV-1 NL4.3 replication

Correlation between inhibitory effects of AMD3100 on HIV-1 replication, CXCR4 mAb binding and SDF-1-mediated signal transduction

De Clercq, Mol. Pharmacol. 57: 833-839 (2000)

Hatse et al., FEBS Lett. 527, 255-262 (2002)

-1000

0

1000

2000

3000

4000

5000

6000

0 50 100 150 200 250

Control5000 ng/ml1000 ng/ml200 ng/ml40 ng/ml

U87.CD4.CXCR4

Concentration-dependent inhibition of SDF-1/CXCR4-mediatedintracellular calcium flux by AMD3100 in U87.CD4.CXCR4 cells

Time (seconds)

Fluo

resc

ence

cha

nge

(cou

nts)


-500

500

1500

2500

3500

0 50 100 150


PBMCs

Time (seconds)

Fluo

resc

ence

cha

nge

(cou

nts)

Concentration-dependent inhibition of SDF-1/CXCR4-mediatedintracellular calcium flux by AMD3100 in freshly isolated human PBMCs


-500

0

500

1000

1500

2000

2500

3000

3500

0 50 100 150


HSB-2

Time (seconds)

Fluo

resc

ence

cha

nge

(cou

nts)

Concentration-dependent inhibition of SDF-1/CXCR4-mediatedintracellular calcium flux by AMD3100 in human T-lymphoid HSB-2 cells


-1000

0

1000

2000

3000

4000

5000

6000

7000

0 50 100 150


L1210

Time (seconds)

Fluo

resc

ence

cha

nge

(cou

nts)

Concentration-dependent inhibition of SDF-1/CXCR4-mediated intracellular calcium flux by AMD3100 in murine B-lymphoblastic leukemia L1210 cells


-500

500

1500

2500

0 50 100 150 200 250 300-500

1500

3500

5500

0 50 100 150 200 250 300-1000

0

1000

2000

3000

0 50 100 150 200

-500

500

1500

2500

0 50 100 150 200 250 300-500

500

1500

2500

3500

4500

0 50 100 150 200 250 300-500

0

500

1000

1500

2000

0 50 100 150 200 250 300

Time (seconds)

Fluo

resc

ence

cha

nge

(cou

nts)

Lack of inhibitory effect of AMD3100 (25 µg/ml) on chemokine-inducedsignaling mediated by CXCR1, CXCR2, CXCR3, CCR1, CCR2 and CCR3

CXCR1 CXCR2 CXCR3

CCR1 CCR2 CCR3


-500

1500

3500

5500

7500

9500

0 50 100 150 200 250 300-500

1500

3500

5500

0 50 100 150 200 250 300-500

0

500

1000

1500

2000

2500

3000

0 50 100 150 200

-1000

0

1000

2000

3000

4000

0 50 100 150 200 250 300-200

0

200

400

600

800

1000

1200

0 50 100 150 200 250 300-1000

0

1000

2000

3000

4000

0 50 100 150 200

Time (seconds)

Fluo

resc

ence

cha

nge

(cou

nts)

Lack of inhibitory effect of AMD3100 (25 µg/ml) on chemokine-inducedsignaling mediated by CCR4, CCR5, CCR6, CCR7, CCR8 and CCR9

CCR4 CCR5 CCR6

CCR7 CCR8 CCR9

AMD3100/CXCR4 Interactions

Hatse et al., Mol. Pharmacol. 60: 164-173 (2001)

Esté et al., Mol. Pharmacol. 60: 67-73 (1999)

Correlation of the anti-HIV-1 (NL4-3) activity and inhibitory effect of different bicyclam analogues ( ) on binding of CXCR4 mAb (12G5) !!!! : SDF-1αααα


Correlation of the anti-HIV-1 (NL4-3) activity and inhibitory effect of different bicyclam analogues ( ) on SDF-1αααα-dependent intracellular Ca2+ flux

ANTI-HIV-1 ACTIVITY, INHIBITION OF ANTI-CXCR4 mAbBINDING AND INHIBITION OF SDF-1-INDUCED [Ca2+]i

FLUX BY DIFFERENT METAL COMPLEXES OF AMD3100

Compound IC50 (ng/ml)

Inhibition of HIV-1

replication

Inhibition of anti-CXCR4 mAb binding

Inhibition of SDF-1-induced

[Ca2+]i flux AMD3479 (Zn) 8 1 3 AMD3462 (Ni) 8 16 2 AMD3469 (Cu) 48 200 50 AMD3461 (Co) 740 500 600 AMD3158 (Pd) 68,620 12,500 70,000

AMD3100 9 10 5

(average ∼ 5.6 years(average ∼ 5.6 years


Esté et al., J. Virol. 73: 5577-5585 (1999)

Coreceptor use of clinical HIV strains after AMD3100 treatment

′′′′ U87-CD4-CXCR4≤≤≤≤ U87-CD4-CCR5

Antiviral activity of AMD3100 in SCID-hu Thy/Livmice*

0.00373 ± 1510

0.010122 ± 333

0.028161 ± 461

0.61280 ± 480.3

---394 ± 1070

p value( treatedversus untreated)

p24(pg/million cells)

Dose(mg/kg/day)

Datema et al., Antimicrob. Agents Chemother. 40: 750-754 (1996)

*Infected with CXCR4-using virus (clinical isolate HIV-1 EW)

Figure 2. AMD-3100 Single Dose Pharmacokinetics following 15-minuteIntravenous Infusion (median and range). Inset: natural log scale.

Hours0 2 4 6 8 10 12 14

AMD-

3100

(ng/

ml)

0

100

200

300

400

500

600

80 µg/kg 40 µg/kg 20 µg/kg10 µg/kg

Hours

0 2 4 6 8 10 12 14

Ln A

MD-

3100

(ng/

ml)

e2

e3

e4

e5

e6

Hendrix et al., Antimicrob. Agents Chemother. 44: 1667-1673 (2000)

Single-dose AMD3100 pharmacokinetics following 15-min. intravenousinfusion (median and range)

AM

D31

00 (n

g/m

l)

Hours0 2 4 6 8 10 12 14 16 18 20 22 24

AM

D-3

100

(ng/

ml)

-100

0

100

200

300

400

500

600

80 µg/kg AM D40 µg/kg AM D20 µg/kg AM D10 µg/kg AM D

WB

C R

atio

(B

asel

ine

Ref

eren

ce)

1

2

3W BCW BCW BCW BC

Hendrix et al., Antimicrob. Agents Chemother. 44: 1667-1673 (2000)

WBC ratio versus time compared to AMD3100 concentration versustime following single-dose intravenous AMD3100 administration

AM

D31

00 (n

g/m

l)

Patients whose CXCR4-using HIV variants were less than 10% at entry

09.8Yes401-33

08.6Yes401-35

1.2*2.4No2.51-4*

0.052.0No203-26

01.5Yes53-11

01.0Yes52-10

01.0Yes52-9

00.8Yes403-32

00.5Yes201-31

00.3Yes202-28

00.2Yes806-73

% X4 day 11% X4 day 1Dual to R5 switchDay 1 to day 11

Dose (µg/kg/hr)Patient

*Treated for 9 days, day 9 sample tested.

Schols et al., Abstracts of the 9th Conference on Retroviruses and Opportunistic Infections, Seattle, WA, USA, 24-28 February 2002, p. 53, no. 2

Phase I/II clinical study with AMD3100 in HIV-infected individuals

Patients whose CXCR4-using HIV variants were 25-100% at entry

100100vRNA reduction0.89 log10

1601-40

26.094.0No101-2143.8*47.3No404-72*32.041.0No2.51-218.431.7No406-71

22.825.0No406-70

% X4 day 11% X4 day 1Dual to R5 switchDay 1 to day 11

Dose(µg/kg/hr)

Patient

*Treated for 2 days, sample from day 4 of study.


Phase I/II clinical study with AMD3100 in HIV-infected individuals

AMD3100-2001 Patient 1-40

-4 1

6

11

18

39

2.2

2.7

3.2

3.7

4.2

-5 2 9 16 23 30 37

Study Day

Log

Vira

l RN

A


Patient 1-40

Mobilization of CD34+ cells into peripheral blood:AMD3100 dose response (mean ±±±± SEM)

Data (from G. Calandra et al.) presented at International Society of Hematology Meeting, Quebec, 5-9 July 2002

Time (hours)

-!!!!- 40 µg/kg (n=3)-""""- 80 µg/kg (n=10)-ππππ- 160 µg/kg (n=5)-σσσσ- 240 µg/kg (n=5)

Abs

olut

e nu

mbe

r of C

D34

+ cel

ls in

pe

riphe

ral b

lood

(cel

ls p

er µ

L)

+ AMD3100

Peripheral blood CD34+ cell response followingadministration of G-CSF ±±±± AMD3100

(n = 6 per group; mean ±±±± SEM)

Data (from G. Calandra et al.) presented at International Society of Hematology Meeting, Quebec, 5-9 July 2002

On days 1 to 4:G-CSF 10 µg/kg/day

On day 5:—!!!!— G-CSF (10 µg/kg)--- O--- AMD3100 (160 µg/kg)—ππππ— G-CSF + AMD3100

Time (hours)

Abs

olut

e nu

mbe

r of C

D34

+ cel

ls in

pe

riphe

ral b

lood

(cel

ls p

er µ

L)

Mobilization and collection of CD34+ cellswith AMD3100 and G-CSF

• Target (range: 2-5 x 106/kg) CD34+ celltransplantation dose: 4.0 x 106/kg

• G-CSF (10 µg/kg) x 5 days: 3.73 x 106/kg• AMD3100 (240 µg/kg on day 5): 3.02 x 106/kg• G-CSF (10 µg/kg) x 5 days + AMD3100 (160

µg/kg on day 5): 9.88 x 106/kg

Liles et al., presented at the American Society of Hematology Meeting, 6-10 December 2002, Philadelphia, PA

For AMD3100 to be of relevant clinical utility, itwould also have to mobilize self-renewing stemcells.

AMD3100 proved to be a potent mobilizer ofCompetitive Repopulating Long Term Marrowself-renewing Stem Cells (CRLTMSCs) in mice.

Broxmeyer et al., presented at the American Society of Hematology Meeting, 6-10 December 2002, Philadelphia, PA

AMD3100 augments incorporation of bonemarrow (BM)-derived endothelial progenitorcells (EPCs) into sites of neovascularizationafter mycocardial infarction (MI) by mobilizingEPCs from BM to peripheral blood.

AMD3100 might provide a novel strategy forpreserving cardiac function in patients withacute MI.

Iwakura et al., presented at the American Society of Hematology Meeting, 6-10 December 2002, Philadelphia, PA

0 5 10 15 20 25 30 35 40 45 50 55

Days after immunization

0

1

2

3

4

5

6

7

TreatmentTreatment

Dis

ease

sco

re (m

ean ± ±±±

SEM

)

Matthys et al., J. Immunol. 167: 4686-4692 (2001)

!!!! AMD3100#### Control

Inhibition of collagen-induced arthritis by treatment with AMD3100

Mice were immunized with chicken collagen type II (CII) in CFA on day 0 and implanted on day 22 with osmotic minipumps delivering AMD3100 at a rate of 600 µg/day during a period of 14 days. Clinical symptoms of arthritis started to appear on day 22

-5 0 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

T im e (s e c o nd s )

Co n tro lAMD3 1 0 0 0 .0 4 µg /m l

AMD3 1 0 0 0 .2 µg /m lAMD3 1 0 0 1 µg /m l

AMD3 1 0 0 5 µg /m l

-5 0 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

T im e (s e c o nd s )

Co n tro lAMD3 1 0 0 0 .0 4 µg /m l

AMD3 1 0 0 0 .2 µg /m lAMD3 1 0 0 1 µg /m l

AMD3 1 0 0 5 µg /m l


Inhibition by AMD3100 of SDF-1-elicited intracellular Ca2+ flux inMac-1+ cells harvested from the spleens of mice on day 18

postimmunization with CII in CFA

0 5 1 0 1 5 2 0 2 5 3 0 3 5

P e rc e nta g e o f m ig ra te d c e lls

SDF-1 AMD3100(µg/ml) (µg/ml)

0 0

0.5 0

0.1 0

0.1 25

0.1 5

0.1 1

0.1 0.2

0 5 1 0 1 5 2 0 2 5 3 0 3 5



0 0

0.5 0

0.1 0

0.1 25

0.1 5

0.1 1

0.1 0.2

0 5 1 0 1 5 2 0 2 5 3 0 3 5



0 0

0.5 0

0.1 0

0.1 25

0.1 5

0.1 1

0.1 0.2


Inhibition by AMD3100 of SDF-1-elicited chemotaxis ofMac-1+ cells harvested from the spleens of mice on day 18

postimmunization with CII in CFA

Percentage of migrated cells

Expression of chemokine receptors in human tumor cellsQuantitative RT-PCR analyses of all known chemokine receptors in seven

human breast cancer cell lines

Müller et al., Nature 410: 50-56 (2001)

0

500

1,000

1,500

fg p

er 1

00 n

g cD

NA

Müller et al., Nature 410: 50-56 (2001)

Effect of CXCR4-neutralization on tumor metastasis in mice

a. Lung colony formation after i.v. injection of MDA-MB-231 cells.b. Spontaneous lung metastasis after orthotopic injection of MDA-MB-231 cells.

a b

AMD3100 inhibits stromal derived factor-1(SDF-1)-dependent migration and proliferationof Acute Lymphoblastic Leukemia (ALL) cells inbone marrow.

AMD3100 may be useful for the treatment ofALL.

Juarez et al., presented at the American Society of Hematology Meeting, 6-10 December 2002, Philadelphia, PA

The CXCR4-CXCL12 (= SDF-1) circuitry iscrucial for the migration of follicular non-Hodgkin’s lymphoma (NHL) cells.

AMD3100 strongly inhibits NHL cell migrationacross endothelial and stromal cell layers.AMD3100 enhances NHL cell apoptosis andinhibits proliferation of NHL cells.

Paul et al., presented at the American Society of Hematology Meeting, 6-10 December 2002, Philadelphia, PA

Milestones in the development of the bicyclamAMD3100

• Isolation of the bicyclam JM1657 as impurity in acommercial cyclam preparation

• Anti-HIV activity of JM2763 (with an aliphatic bridgebetween the two cyclam rings)

• Anti-HIV activity of AMD3100, alias JM3100 (with anaromatic bridge between the two cyclam rings)

• Implication of the viral envelope gp120 as the (indirect)target of action

• Identification as CXCR4 as the direct target of action

Milestones in the development of the bicyclamAMD3100

• Phase I clinical studies reveal elevation of WBCcounts (following intravenous administration ofAMD3100)

• Phase I/II clinical trials reveal proof of principle:AMD3100 causes a reduction of X4 HIV load inhumans

• AMD3100 pursued clinically for mobilization ofhematopoietic progenitor cells

• AMD3100 shown to be effective in a mouse model forarthritis

• AMD3100 predicted to be effective against CXCR4-dependent tumor progression and metastasis

Anti-arthritisactivity

Anti-cancer activity

Stem cellmobilization

Anti-HIV activityin patients

Increased whiteblood cell counts

Direct target: CXCR4

Indirect target: gp120

Identification ofclinical drug candidate

Anti-HIV activityin cell culture

Impurity

P. Matthys

G. Calandra

D.C. Dale

K. VermeireK. Princen T. Schwartz

S. HatseC.W. Hendrix

J.P. Moore J. Esté

K. De VreeseN. Yamamoto

G. Werner B. Rosenwirth R. Datema

D. Schols

R. Skerlj G. Bridger G. Henson

M. Abrams D. Picker

M. Perutz

Documents

DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100… · DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100, A CASE IN POINT Erik DE CLERCQ Rega Institute for Medical Research,