A PRECLINICAL Evaluation Of ZIO-201 (ISOPHOSPHORAMIDE Mustard (IPM)-lysine)

In Sarcoma.

E.Anders Kolb, Pooja Gidwani, Robert Peter Gale, Richard Gorlick

Isophosphoramide Mustard-lysine (Zio-201)

• IPM: active moiety of ifosfamide.

• Alkylator: crosslinks DNA via G:C base pairs irreversibly.

• Activity: variety of cancers.

• Unstable for clinical administration.

• Lysine: provides stability.

Metabolism of Oxazaphosphorines

Ifosfamide Versus Zio-201

Chloroacetaldehyde“fuzzy brain”

Acrolein“bladder toxicity”

IFOS

X XZio-201

Courtesy of ZiopharmCourtesy of Ziopharm

Advantages of Zio-201

• Effective

• No toxic metabolites (acrolein, CAA)

• Bypassing resistance mechanisms (ALDH over-expression)

Objectives

• To test the activity of Zio-201 in a broad variety of sarcomas in-vitro.

• To test the activity of Zio-201 in sarcoma tumor xenografts including cyclophosphamide resistant tumors.

0

0.5

1

0.01 0.1 1 10 100

Concentration (mcg/ml)

Via

bil

ity

(O

D-T

/OD

-C)

RD

RH30

0

0.5

1

0.01 0.1 1 10 100

Concentration (mcg/ml)

Via

bil

ity

(O

D-T

/OD

-C)

OS230

OS229

OS222

SaOS

0

0.5

1

0.01 0.1 1 10 100

Concentration (mcg/ml)

Via

bil

ity

(O

D-T

/OD

-C)

SYO1

HSSYII

0

0.5

1

0.01 0.1 1 10 100

Concentration (mcg/ml)

Via

bil

ity

(O

D-T

/OD

-C)

SKES1

SKPNDW

a. Rhabdomyosarcoma b. Ewing’s Sarcoma

c. Osteosarcoma d. Synovial Sarcoma

Zio-201 has Broad Activity in Sarcoma Lines In Vitro

0

0.5

1

0.01 0.1 1 10 100 1000

Concentration (mcg/ml)

Via

bil

ity

(T

-OD

/C-O

D)

Daily x 1

Daily x 3

RH30-Rhabdomyosarcoma

0

0.5

1

0.01 0.1 1 10 100 1000

Concentration (mcg/ml)

Via

bil

ity

(O

D-T

/OD

-C)

Daily x 1

Daily x 3

OS229-Osteosarcoma

In Vitro, in most lines evaluated, the IC50 of daily x1 and daily x3 dosing are comparable

In Vitro, in most lines evaluated, the IC50 of daily x1 and daily x3 dosing are comparable

Cell Line Histology Daily x 3 IC50 Daily x 1 IC50

SK-PN-DW Ewing’s Sarcoma 0.44 µg/ml 1.13 µg/ml

SK-ES-1 Ewing’s Sarcoma 0.31 µg/ml 1.01 µg/ml

RH30 Alveolar Rhabdomyosarcoma 0.85 µg/ml 0.86 µg/ml

RD Embryonal Rhabdomyosarcoma 1.02 µg/ml 0.99 µg/ml

SYO-1 Synovial Sarcoma 0.080 µg/ml 0.16 µg/ml

HSSY-II Synovial Sarcoma 0.45 µg/ml 0.45 µg/ml

SaOS Osteosarcoma 1.08 µg/ml 1.23 µg/ml

OS222 Osteosarcoma 1.21 µg/ml 7.0 µg/ml

OS229 Osteosarcoma 0.31 µg/ml 0.50 µg/ml

OS230 Osteosarcoma 1.09 µg/ml 1.50 µg/ml

0.1

1

10

0 7 14 21 28 35 42

Time (days)

Rel

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um

or

Vo

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e

Control175mg/kg daily x 1 days 1 and 21100mg/kg daily x 3 starting days 1 and 21

In the OS31 osteosarcoma tumor line, Zio-201 at the MTD for each dose schedule results in significant tumor growth

delay

p=0.09

Houghton PJ, Morton CL, Tucker C, Payne D, Favours E, Gorlick R, Kolb EA, Zhang W, Lock R, Carol H, Tajbakhsh M, Reynolds CP, Maris JM, Courtright J, Keir ST, Friedman HS, Stopford C, Zeidner J, Wu1 J, Liu T, Billups CA, Khan J, Ansher S, Zhang J, Smith MA. The Pediatric Preclinical Testing Program: Description of Models and Early Testing Results. Pediatric Blood and Cancer (in press)

Cyclophosphamide resistance in Osteosarcoma Lines

OS31 OS33

Aldehyde dehydrogenase 3A1 is differentially expressed in

cyclophosphamide-resistant lines compared to sensitive lines

Affy U133a Micorarray data courtesy of Peter Houghton, PI, Pediatric Preclinical Testing Program,St. Judes Children’s Research Hospital

OS

160

OS

1

OS

17O

S2

OS

29O

S31

OS

33O

S9

ALD3A1

0.1

1

10

0 7 14 21 28 35 42

Time (weeks)

Rel

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Control

100mg/kg daily x3

1 2 3 4 5 60

Zio-201 is active in the cyclophosphamide-resistant, ALD3A1-overexpressing OS31 line

Summary• Zio-201 has broad activity in sarcomas in-

vitro.

• In many cell lines, IC50 for daily x1 and daily x3 dosing are comparable.

• Significant responses are noted in OS 31 osteosarcoma tumor line in-vivo at both daily x1 and daily x3 dosing.

Summary

• ALD3A1 is overexpressed in cyclophosphamide resistant tumors (OS31, OS2) suggesting this as a potential mechanism of resistance.

• IPM-lysine is active in cyclophosphamide resistant OS31 line.