Protocol 010

Zoledronic Acid Preclinical Profile
1
Bisphosphonates and the Bone Marrow
Microenvironment
Peter Croucher
University of Sheffield, UK
1

Zoledronic Acid Preclinical Profile
2
Myeloma Bone Disease - Targeting the
Interdependence Between Myeloma and Bone
Soluble Mediators
T cel s
Endo
Myeloma cells
Adhesion
RANKL
MIP1
BMSC
Others
Osteoclast
Precursor
Cytokines
Ob
Osteoclast
Growth
Factors
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Zoledronic Acid Preclinical Profile
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Targeting the Bone Microenvironment with
Bisphosphonates
ˇ Clodronate
ˇ Pamidronate
ˇ Ibandronate
ˇ Zoledronic Acid
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Zoledronic Acid Preclinical Profile
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Murine Models of Myeloma Bone Disease -
The Importance of Choice
Syngeneic Models
5T2MM / 5T33MM
Microenvironment
Dependent
5TGM1 model
SCID Mouse
ARH-77 / JJN-3 SCID models
SCID/Hu system
Microenvironment
Dependent
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Zoledronic Acid Preclinical Profile
5
Zoledronic Acid Prevents the Development of
Osteolysis in Mice Bearing 5T2MM Myeloma Cells
p<0.01
15.0
p<0.001 p<0.01
(%) 10.0
r
ea
5.0
Bone A
0
Control
5T2MM
5T2MM + Zol
p<0.001
15.0
)
p<0.005
10
p<0.05
(
%
m 10.0
5
a
s
t
P
5.0
teocl
No Lesions
*
*
*
Os
0
Con
5T2+
5T2+
5T2+
0
Con
5T2+
5T2+
5T2+
Veh
Zol (T) Zol (P)
Veh
Zol (T) Zol (P)
Croucher et al. JBMR 2003.
A series of studies have been conducted by Croucher and colleagues in the
5T2 MM murine myeloma model. In this model, mice injected with 5T2 MM
cells developed detectable paraprotein, characteristic osteolytic lesions on
plain radiograph, a decrease in cancellous bone volume, and an increase in
osteoclasts on the bone surface
Treatment with zoledronic acid (120ľg/kg) twice weekly by subcutaneous
injection, either from the time paraprotein was first detected approximately 8
weeks after tumor cell injection (TREAT) or at the time of tumor cell injection
(PREVENT) resulted in a significant reduction in the number of osteolytic
lesions and cancellous bone loss compared with vehicle controls
More importantly, zoledronic acid reduced levels of paraprotein by 31% with
a similar decrease in tumor area, suggesting a reduction in tumor burden
Reference
1. Croucher P, De Raeve H, Perry M, et al. Zoledronic acid prevents the development of
osteolytic bone disease and increases survival in a murine model of multiple myeloma. Bone.
2002;30(suppl):39S. Abstract B10.
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Zoledronic Acid Preclinical Profile
6
Bisphosphonate Treatment Decreases
Tumor Burden and Increases Survival
1.0
Median Survival
Con = 35 days (27-42 days)
0.75
(g/dl)
1.2
Zol = 47 days (34-60 days)
P<0.005
0.5
r
otein
1.0
0.25
i
v
al
Parap
0.8
u
rv
0
Zoledronic Acid
100
i
ve S
)
*P<0.05
0.6
%
u
lat
(
*
90
*
Control
0.4
rea
Cum
A
r 80
0.2
Control
70
Tumo
0
60
0
6 12 18 24 30 36 42 48 54
5T2MM
5T2MM
5T2MM
+Veh
+Zol (T)
+Zol (P)
Time (Days)
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Zoledronic Acid Preclinical Profile
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Zoledronic Acid Inhibits the Growth of Primary
Human Myeloma Cells in Nude Mice
Treatment,
Prevention,
100 ľg/kg/wk
3 x 100 ľg/kg/wk
500
Human Ig in
400
Control
mouse serum,
Zoledronic acid
ng/ml
300
Start
200
treatment
100
0
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14
Time after intraosseous tumor cell injection, weeks
Yaccoby et al. Br J Haematol. 2002.
Myeloma cells isolated from patients were injected into severe combined
immunodeficient-human (SCID-Hu) mice, and mice were treated with
zoledronic acid (100 ľg/kg/week) either from the time of tumor cell injection
(prevention) or after tumors were established in the bone (treatment)
In this experiment, serum levels of human immunoglobulin were significantly
reduced in mice treated with zoledronic acid in either the treatment or
prevention setting
When zoledronic acid was administered in the prevention setting, fewer
osteolytic lesions formed, and there was no detectable human
immunoglobulin even after 14 weeks
These results suggest that zoledronic acid significantly reduces growth of
established tumors and may prevent myeloma cells from establishing
osteolytic lesions by inhibiting tumor-induced osteolysis and perhaps also by
a direct anti-tumor effect
References
1. Yaccoby S, Pearse RN, Johnson CL, Barlogie B, Choi Y, Epstein J. Myeloma interacts with
the bone marrow microenvironment to induce osteoclastogenesis and is dependent on
osteoclast activity. Br J Haematol. 2002;116:278-290.
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Zoledronic Acid Preclinical Profile
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Mechanisms of Anti-Myeloma Activity
ˇ Inhibiting Bone Resorption
ˇ Blocking Tumour Cell Invasion and Adhesion
ˇ Inhibiting Proliferation and Promoting Apoptosis
ˇ Inhibiting Angiogenesis
ˇ Promoting T Cell Expansion
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Zoledronic Acid Preclinical Profile
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Biosphosphonates and Murine Models of
Myeloma Bone Disease
Model
Bone Effect Tumor Effect
Reference
Pamidronate 5T2MM
Positive
Survival
Radl et al
SCID/Hu Positive
Paraprotein
Yaccoby et al
Ibandronate
5TGM1
Positive
None
Dallas et al
ARH-77
Positive
None
Cruz et al
Zoledronate
5T2MM
Positive
Survival
Croucher et al
SCID/Hu Positive
Paraprotein
Yaccoby et al
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Zoledronic Acid Preclinical Profile
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Fc.OPG Prevents the Development of Myeloma
Bone Disease in the 5T2MM Murine Model
Control
Fc.OPG
Naive
5T2MM + vehicle
5T2MM + Fc.OPG
t 5
p<0.005
p<0.005
20
p<0.005
4
15
3
10
2
No of osteoclas 1
No of lesions 5
0
0
Naive
Control
Fc.OPG
Control
Fc.OPG
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Zoledronic Acid Preclinical Profile
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RANK.Fc Prevents the Development of Myeloma
Bone Disease in the 5T2MM Murine Model
p<0.001
p<0.001
10
8
Lytic
Lesions 6
(No)
4
2
Control
0
Control 5T2MM+ 5T2MM+
Veh
RANK.Fc
10.0
p<0.005
p<0.001
5T2MM+ Veh
Bone 7.5
Area 5.0
(%)
2.5
5T2MM+ RANK.Fc
0
Control 5T2MM+ 5T2MM+
Veh
RANK.Fc
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Zoledronic Acid Preclinical Profile
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The Effect of Fc.OPG on Tumour Burden In the
5T33MM Murine Model of Myeloma
3.5
(
g/dl)3.0
2.5
*
r
otein
2.0
1.0
Parap1.5
P<0.02
i
v
al
0.8
1.0
u
rv
0.6
Control
90
i
ve S
ells 85
0.4
Fc.OPG
C
*
u
lat
o
r 80
Cum0.2
75
0
% Tum
0
5 10 15 20 25 30 35 40
70
65
Days
Con 5T33MM+ 5T33MM+
* p<0.05
Veh
Fc.OPG
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Zoledronic Acid Preclinical Profile
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Targeting RANKL on Tumour Burden in Other Models
of Myeloma Bone Disease
ˇ RANK.Fc treatment reduces tumour burden in the 5TGM1
syngeneic model (Oyajobi et al 2000)
ˇ RANK.Fc decreases the serum
100
human Ig in the SCID/Hu model
(Pearce et al 2001)
1
1
ˇ
(
%)
NK.Fc
However, neither RANK.Fc or
NK.Fc
hIgG
hIgG
RA
OPG (lentiviral delivery)
hIg
RA
influenced serum Ig in the ARH-
77/SCID model (Pearce et al
2001, Doran et al 2002).
0
SCID/ARH77
SCID-hu
(Pearce et al 2001)
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Zoledronic Acid Preclinical Profile
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Conclusions
ˇ Bisphosphonates inhibit bone resorption in vivo
ˇ Treatment associated with anti-myeloma effect
ˇ Seen in microenvironment dependent models
ˇ Consistent with other resorption inhibitors
ˇ Suggests that the anti-myeloma effect is mediated
at least in part by inhibiting bone resorption
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Zoledronic Acid Preclinical Profile
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Acknowledgements
Claire Shipman
Mark Perry
Jenny Lippitt
Karin Vanderkerken (Brussels)
Bill Dougall (Amgen)
Colin Dunstan (Sydney)
Jonathan Green (Novartis)
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