DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100, A CASE IN - - PowerPoint PPT Presentation

DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100, A CASE IN POINT Erik DE CLERCQ

Rega Institute for Medical Research, K.U.Leuven B-3000 Leuven, Belgium

LAV Replication, tested by reverse transcriptase (RT) activity in supernatants

• f T cell cultures, during HPA 23 treatment of a patient with AIDS

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

1 0 2 0 3 0 R T act i vi t y I x 1 0 3 (cp m ) HPA 23 HPA 23 May 83 July Aug Sep Oct Nov Dec Jan 84 Nov 84 NS NS 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)

JM1493

H4SiW12O40

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

JM1590

K13[Ce(SiW11O39)2].26H20

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

JM2820

Anti-HIV activity of polyoxometalates

> 156 > 500 3.2 JM2820: [Me3NH]8[Si2W18Nb6O77] > 263 > 500 1.9 JM2815: K5[SiW11(C5H5)TiO39] > 178 > 500 2.8 JM2766: K6[BW11Ga(H2O)O39] 266 293 1.1 JM1809: K8HP2W15V3O62.34H2O 666 466 0.7 JM1596: K10[P2W18Zn4(H2O)2O68].20H2O 1130 339 0.3 JM1591: K12H2P2W12O48.24H2O 328 230 0.7 JM1590: K13[Ce(SiW11O39)2].26H2O 467 654 1.4 JM1583: K5[BW12O40] Selectivity index 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 H2 Fe Ni Selectivity index CC50 (µg/ml) EC50 (µg/ml) M R

COOH

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

N M N N N R R R R

Cyclam Metal-cyclam complex

NH HN NH HN N N N N M

M 4H

Anti-HIV activity of JM1657 in MT-4 cells

1248 150 399 JM1498 319 1.01 0.144 JM1657 HIV-2(ROD) HIV-1(IIIB) CC50 (µM) EC50 (µM) Structure Compound

NH HN NH HN H H HN HN NH NH NH HN NH HN

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

Anti-HIV activity of JM2763 in MT-4 cells

> 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 HN NH N NH HN NH N

Anti-HIV activity of JM2987 in MT-4 cells

0.005 > 100,000 Selectivity index > 500 CC50 (µg/ml) EC50 (µg/ml) HIV-1(IIIB) Structure

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

HN HN NH N NH HN NH N

8 HBr 2 H2O

Anti-HIV activity of bicyclam JM2987

> 100 > 100 MOLT-4 MND-GB1 > 100 > 100 MOLT-4 AGM-3 > 100 > 100 MT-4 MAC-251 SIV > 125,000 > 500 0.004 MT-4 EHO > 71,400 > 500 0.007 MT-4 ROD HIV-2 > 167,000 > 500 0.003 MT-4 HE > 500,000 > 500 0.001 MT-4 RF > 100,000 > 500 0.005 MT-4 IIIB HIV-1 Selectivity index CC50 (µg/ml) EC50 (µg/ml) Cell line Strain Virus

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

HN HN NH N NH HN NH N JM3100 AMD3100 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 NL4-3 viruses: N269Y R272T S274R Q278H I288V N293H A297T ∆ ∆ ∆ ∆FNSTW P385L 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

+ +

25 200 11

+ + + +

28 200 6

+ + + + + + JM3100 resistant

28 450 7

+ + + + + + + + + + + + + +

42 > 740 60

+ + + + + + + + + + +

60 > 740 200

+ + + + + + + + + + + +

Mechanism of action of bicyclams:

interaction with monoclonal antibody binding to CXCR4

i sotype 1 2 G 5 A M D3100 25 µ µ µ µg/m l A M D3100 5 µ µ µ µg/m l AM D3100 1 µ µ µ µg/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 EXPRESSI O N R

A M D3100 0. 2 µ µ µ µg/m l

R E L A T I V E C E L L N U M B E R

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

Anti-HIV activity profile of AMD3100 correlated with coreceptor use

4 > 1,000 > 25,000 CCR5 (CCR2b, CCR3) HIV-1 JR-FL 10 > 1,000 > 25,000 CCR5 (CCR2b, CCR3) HIV-1 ADA 5 > 1,000 > 25,000 CCR5 HIV-1 SF-162 25 > 1,000 > 25,000 CCR5 HIV-1 BaL M-tropic > 1,000 55 7 CXCR4 HIV-2 ROD > 1,000 100 3 CXCR4 HIV-1 NL4-3 > 1,000 50 5 CXCR4 HIV-1 RF > 1,000 20 2 CXCR4 HIV-1 IIIB T-tropic RANTES SDF-1α α α α AMD3100 EC50 (ng/ml) Coreceptor used Strain Schols et al., J. Exp. Med. 186: 1383-1388 (1997)

• 1000
• 500

500 1000 1500 2000 50 100 150 200 250 300

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

Concentration-dependent inhibition of SDF-1-induced Ca2+ flux in Sup-T1 cells by AMD3100

Time (seconds) Fluorescent change (counts)

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

20 40 60 80 100 120 1000 100 10 1 0.1 Concentration of AMD3100 (ng/ml) % of inhibition

—■ ■ ■ ■ — 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

1000 2000 3000 4000 5000 6000 50 100 150 200 250 Control 5000 ng/ml 1000 ng/ml 200 ng/ml 40 ng/ml

U87.CD4.CXCR4

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

Time (seconds) Fluorescence change (counts)

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

• 500

500 1500 2500 3500 50 100 150 Control 1000 ng/ml 100 ng/ml 10 ng/ml 1 ng/ml

PBMCs

Time (seconds) Fluorescence change (counts)

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

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

• 500

500 1000 1500 2000 2500 3000 3500 50 100 150 Control 1000 ng/ml 200 ng/ml 40 ng/ml 8 ng/ml

HSB-2

Time (seconds) Fluorescence change (counts)

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

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

• 1000

1000 2000 3000 4000 5000 6000 7000 50 100 150 Control 1000 ng/ml 200 ng/ml 40 ng/ml 8 ng/ml

L1210

Time (seconds) Fluorescence change (counts)

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

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

• 500

500 1500 2500 50 100 150 200 250 300

• 500

1500 3500 5500 50 100 150 200 250 300

• 1000

1000 2000 3000 50 100 150 200

• 500

500 1500 2500 50 100 150 200 250 300

• 500

500 1500 2500 3500 4500 50 100 150 200 250 300

• 500

500 1000 1500 2000 50 100 150 200 250 300

Time (seconds) Fluorescence change (counts)

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

CXCR1 CXCR2 CXCR3 CCR1 CCR2 CCR3

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

• 500

1500 3500 5500 7500 9500 50 100 150 200 250 300

• 500

1500 3500 5500 50 100 150 200 250 300

• 500

500 1000 1500 2000 2500 3000 50 100 150 200

• 1000

1000 2000 3000 4000 50 100 150 200 250 300

• 200

200 400 600 800 1000 1200 50 100 150 200 250 300

• 1000

1000 2000 3000 4000 50 100 150 200

Time (seconds) Fluorescence change (counts)

Lack of inhibitory effect of AMD3100 (25 µg/ml) on chemokine-induced signaling 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-1α α α α

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 SDF-1α α α α-dependent intracellular Ca2+ flux

ANTI-HIV-1 ACTIVITY, INHIBITION OF ANTI-CXCR4 mAb BINDING 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., Mol. Pharmacol. 60: 67-73 (1999)

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/Liv mice*

0.003 73 ± 15 10 0.010 122 ± 33 3 0.028 161 ± 46 1 0.61 280 ± 48 0.3

• 394 ± 107

p value( treated versus 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-minute Intravenous Infusion (median and range). Inset: natural log scale.

Hours

2 4 6 8 10 12 14

AMD-3100 (ng/ml)

100 200 300 400 500 600 80 µg/kg 40 µg/kg 20 µg/kg 10 µg/kg

Hours

2 4 6 8 10 12 14

Ln AMD-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. intravenous infusion (median and range)

AMD3100 (ng/ml)

Hours

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

AMD-3100 (ng/ml)

• 100

100 200 300 400 500 600

80 µg/kg AMD 40 µg/kg AMD 20 µg/kg AMD 10 µg/kg AMD W BC Ratio (Baseline Reference)

1 2 3

W BC W BC W BC W BC

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

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

AMD3100 (ng/ml)

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

9.8 Yes 40 1-33 8.6 Yes 40 1-35 1.2* 2.4 No 2.5 1-4* 0.05 2.0 No 20 3-26 1.5 Yes 5 3-11 1.0 Yes 5 2-10 1.0 Yes 5 2-9 0.8 Yes 40 3-32 0.5 Yes 20 1-31 0.3 Yes 20 2-28 0.2 Yes 80 6-73 % X4 day 11 % X4 day 1 Dual to R5 switch Day 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

100 100 vRNA reduction 0.89 log10 160 1-40 26.0 94.0 No 10 1-21 43.8* 47.3 No 40 4-72* 32.0 41.0 No 2.5 1-2 18.4 31.7 No 40 6-71 22.8 25.0 No 40 6-70 % X4 day 11 % X4 day 1 Dual to R5 switch Day 1 to day 11 Dose (µg/kg/hr) Patient

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

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

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 Viral RNA

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

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)

• π

π π π- 160 µg/kg (n=5)

• σ

σ σ σ- 240 µg/kg (n=5)

Absolute number of CD34+ cells in peripheral blood (cells per µL)

+ AMD3100

Peripheral blood CD34+ cell response following administration 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)

—π π π π— G-CSF + AMD3100

Time (hours) Absolute number of CD34+ cells in peripheral blood (cells per µL)

Mobilization and collection of CD34+ cells with AMD3100 and G-CSF

• Target (range: 2-5 x 106/kg) CD34+ cell

transplantation 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, it would also have to mobilize self-renewing stem cells. AMD3100 proved to be a potent mobilizer of Competitive Repopulating Long Term Marrow self-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 bone marrow (BM)-derived endothelial progenitor cells (EPCs) into sites of neovascularization after mycocardial infarction (MI) by mobilizing EPCs from BM to peripheral blood. AMD3100 might provide a novel strategy for preserving cardiac function in patients with acute MI.

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

5 10 15 20 25 30 35 40 45 50 55

Days after immunization

1 2 3 4 5 6 7

Treatment Treatment Disease score (mean ± ± ± ± 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

• n 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

5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

Time (s e c o nd s )

Control AMD3100 0.04 µg/ml AMD3100 0.2 µg/ml AMD3100 1 µg/ml AMD3100 5 µg/ml

• 5 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 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

Time (s e c o nd s )

Control AMD3100 0.04 µg/ml AMD3100 0.2 µg/ml AMD3100 1 µg/ml AMD3100 5 µg/ml

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

Inhibition by AMD3100 of SDF-1-elicited intracellular Ca2+ flux in Mac-1+ cells harvested from the spleens of mice on day 18 postimmunization with CII in CFA

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

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

SDF-1 AMD3100 (µg/ml) (µg/ml) 0.5 0.1 0.1 25 0.1 5 0.1 1 0.1 0.2

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

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

SDF-1 AMD3100 (µg/ml) (µg/ml) 0.5 0.1 0.1 25 0.1 5 0.1 1 0.1 0.2

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

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

SDF-1 AMD3100 (µg/ml) (µg/ml) 0.5 0.1 0.1 25 0.1 5 0.1 1 0.1 0.2

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

Inhibition by AMD3100 of SDF-1-elicited chemotaxis of Mac-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 cells Quantitative RT-PCR analyses of all known chemokine receptors in seven human breast cancer cell lines

Müller et al., Nature 410: 50-56 (2001) 500 1,000 1,500 fg per 100 ng cDNA

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 proliferation

• f Acute Lymphoblastic Leukemia (ALL) cells in

bone marrow. AMD3100 may be useful for the treatment of ALL.

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

The CXCR4-CXCL12 (= SDF-1) circuitry is crucial for the migration of follicular non- Hodgkin’s lymphoma (NHL) cells. AMD3100 strongly inhibits NHL cell migration across endothelial and stromal cell layers. AMD3100 enhances NHL cell apoptosis and inhibits 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 bicyclam AMD3100

• Isolation of the bicyclam JM1657 as impurity in a

commercial cyclam preparation

• Anti-HIV activity of JM2763 (with an aliphatic bridge

between the two cyclam rings)

• Anti-HIV activity of AMD3100, alias JM3100 (with an

aromatic 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 bicyclam AMD3100

• Phase I clinical studies reveal elevation of WBC

counts (following intravenous administration of AMD3100)

• Phase I/II clinical trials reveal proof of principle:

AMD3100 causes a reduction of X4 HIV load in humans

• AMD3100 pursued clinically for mobilization of

hematopoietic progenitor cells

• AMD3100 shown to be effective in a mouse model for

arthritis

• AMD3100 predicted to be effective against CXCR4-

dependent tumor progression and metastasis

Anti-arthritis activity Anti-cancer activity Stem cell mobilization Anti-HIV activity in patients Increased white blood cell counts Direct target: CXCR4 Indirect target: gp120 Identification of clinical drug candidate Anti-HIV activity in cell culture Impurity

• P. Matthys
• G. Calandra

D.C. Dale

• K. Vermeire
• K. Princen
• T. Schwartz
• S. Hatse

C.W. Hendrix J.P. Moore

• J. Esté
• K. De Vreese
• N. Yamamoto
• G. Werner
• B. Rosenwirth
• R. Datema
• D. Schols
• R. Skerlj
• G. Bridger
• G. Henson
• M. Abrams
• D. Picker
• M. Perutz