This document summarizes a toxicity study of a low molecular weight heparin-taurocholate (LMWH-TCA) derivative. LMWH was chemically modified with taurocholate to create the derivative in order to reduce its anticoagulant effects while maintaining its anti-angiogenic properties. The derivative was shown to inhibit blood vessel formation in vitro and suppress tumor growth in vivo more effectively than unmodified LMWH at the same dosage. The LMWH-TCA derivative shows potential as a safer alternative to heparin for anti-angiogenic cancer therapy.

1. Toxicity study of a potent
angiogenesis inhibitor: a Low
molecular weight Heparin-
taurocholate derivative
Farzana Alam
MSc Candidate
Lab of cell and macromocular delivery
Physical Pharmacy GRoup
College of Pharmacy
Seoul National university

2. Heparin
Preparation of Tissues
Heparin
extraction
- Oxidation
- Deaminative degradation
- Chemical !-elimination
- Enzymatic !-elimination
LMWH
Degradation
(4,500 ~ 6,000 Da)
UFH (12,000 Da)
Courtesy: Sungwoo Chung

3. Heparin on cancer
Immune
Modulation
Anticoagulation
Metastasis
Courtesy: Sungwoo Chung

4. Heparin on cancer
Immune
Modulation
Angiogenesis
Anticoagulation
Metastasis
Courtesy: Sungwoo Chung

5. Controversy and limitation
of heparin
! Experimental studies have observed both inhibitory and stimulatory eff
ects of UFH on cancer progression
- Heparin inhibited tumor metastasis (Invasion Metastasis, 1994)
- UFH stimulates angiogenesis systemically (Haemstasis, 1993)
! High dose of heparin in clinical application is strictly limited due to stro
ng anticoagulant activity
Side effects : Bleeding, Heparin-induced thrombocytopenia, osteoporosis, etc.
! Strategy to overcome limitations of heparin
Chemically modified heparin derivatives with non-anticoagulant activity
!
Minimize incidences of heparin side effects and improve therapeutic effects
Courtesy: Sungwoo Chung

6. Library of LMWH derivatives
HEPARIN
Naphthalenetrisulfonate
Low Molecular Weight Heparin (LMWH)
Highly sulfated Heparin (
HHS)
Glucose
Deoxycholic acid Taurocholic acid
HEPARIN
HEPARIN
LMWH-(DOCA)n
LMWH-Glucose
cRGDyK
Cholesteryl Chloroformate
Chol-LMWH-TCA
HEPARIN
HEPARIN
cRGDyK-LMWH-TCA
Courtesy: Sungwoo Chung

7. Library of LMWH derivatives
HEPARIN
Naphthalenetrisulfonate
Low Molecular Weight Heparin (LMWH)
Highly sulfated Heparin (
HHS)
Glucose
Deoxycholic acid Taurocholic acid
HEPARIN
HEPARIN HEPARIN
LMWH-(DOCA)n
LMWH-TCA LMWH-Glucose
cRGDyK
Cholesteryl Chloroformate
Chol-LMWH-TCA
HEPARIN
HEPARIN
cRGDyK-LMWH-TCA
Courtesy: Sungwoo Chung

8. Nanostructured heparin-
Taurocholate derivative
HT10
3D structure
(TCA-LMWH)
HEPARIN
30000
CD spectrum
Molar ellipticity [deg cm2 dmol-1]
20000
10000
0
-10000
-20000
LMWH HT1
-30000
HT2 HT3
1 Heparin -40000 HT5 HT10
+ 8 TCA -50000
200 220 240 260
wavelength (nm)
Courtesy: Sungwoo Chung Ref: Essac Lee, Int J Cancer, 124,2009

9. Some results of its efficacy
Control (PBS)
Ref: Essac Lee, Int J Cancer, 124,2009
Control (VEGF)
VEGF + HT10
4000 3500
Control Control
VEGF + LMWH 3500 LMWH (5mg/kg, once/3days)
3000 LMWH (0.5mg/kg, everyday)
Tumor Volume (mm3)
HT2 (5mg/kg, once/3days) HT10 (0.5mg/kg, everyday)
Tumor Volume (mm3)
3000
HT10 (5mg/kg, once/3days) 2500 HT10 (1mg/kg, everyday)
2500 Avastin (5mg/kg, once/4days)
Matrigel plug assay
HT10 (5mg/kg, everyday)
2000
HT10 (5mg/kg, once/3days)
2000
1500
120 1500
1000 1000
500 500
100
0 0
Tube Branches (%)
0 5 10 15 0 5 10 15
Day Day
80
4000 31
Control Control
3500 HT10 (1mg/kg, everyday) 30 HT10 (1mg/kg, everyday)
HT10 (1mg/kg, once/2days)
60 HT10 (1mg/kg, once/2days)
Tumor Volume (mm3)
3000 29 HT10 (1mg/kg, once/3days)
Body Weight (g)
HT10 (1mg/kg, once/3days)
2500 28 HT10 (1mg/kg, once/week)
HT10 (1mg/kg, once/week)
2000 27
40
1500 26
1000 25
20 500 24
0 23
0 5 10 15 0 5 10 15
0 Day Day
control LMWH LMWH LMWH HT 10µg/ HT 100µg/ HT 250µg/
10µg/mL 100µg/mL 250µg/mL mL mL mL
Tumor inhibition test
Tubular formation assay
Courtesy: Sungwoo Chung

10. Some results of its efficacy
Control (PBS)
Ref: Essac Lee, Int J Cancer, 124,2009
Control (VEGF)
VEGF + HT10
4000 3500
Control Control
VEGF + LMWH 3500 LMWH (5mg/kg, once/3days)
3000 LMWH (0.5mg/kg, everyday)
Tumor Volume (mm3)
HT2 (5mg/kg, once/3days) HT10 (0.5mg/kg, everyday)
Tumor Volume (mm3)
3000
HT10 (5mg/kg, once/3days) 2500 HT10 (1mg/kg, everyday)
2500 Avastin (5mg/kg, once/4days)
Matrigel plug assay
HT10 (5mg/kg, everyday)
2000
HT10 (5mg/kg, once/3days)
2000
1500
120 1500
1000 1000
500 500
100
0 0
Tube Branches (%)
0 5 10 15 0 5 10 15
Day Day
80
4000 31
Control Control
3500 HT10 (1mg/kg, everyday) 30 HT10 (1mg/kg, everyday)
HT10 (1mg/kg, once/2days)
60 HT10 (1mg/kg, once/2days)
Tumor Volume (mm3)
3000 29 HT10 (1mg/kg, once/3days)
Body Weight (g)
HT10 (1mg/kg, once/3days)
2500 28 HT10 (1mg/kg, once/week)
HT10 (1mg/kg, once/week)
2000 27
40
1500 26
1000 25
20 500 24
0 23
0 5 10 15 0 5 10 15
0 Day Day
control LMWH LMWH LMWH HT 10µg/ HT 100µg/ HT 250µg/
10µg/mL 100µg/mL 250µg/mL mL mL mL
Tumor inhibition test
Tubular formation assay
Therapeutic dose: 1mg/kg - once per two days up to 5mg/kg
Courtesy: Sungwoo Chung

11. Angiogenesis and mechanism of HT10
Binding LMWH-
HT10-VEGF
affinity VEGF
KD(M) (3.21 ± (1.860 ±
0.04)x10-7 0.10)10-5
HT10
Ref: Essac Lee, Int J Cancer, 124,2009

12. Adverse Effects of VEGF Blocking
(Clinical Outcomes)
Toxicities Possible mechanism
Bleeding, disturbed •Platelet dysfunction;
wound healing •Decreased expression of endothelial TF
Thrombotic events •Endothelial cell apoptosis;
•Lack of endothelial cell renewal leading to exposure of the ECM to the circulating
blood (results in platelet activation);
•Increased TF expression;
•Reduced TM and NO; direct
•Platelet activation
Hypertension •Decreased NO and/or PGI2 production;
•Inappropriateness of the density of vessels (arterioles and
capillaries);
•Vascular stiffness;
•Disturbed endothelin function
Proteinuria and oedema •Podocyte dysfunction owing to VEGF blockade;
•Hypertension

13. goal of my study
• Find out the Clinically Applied Safety Dose of
HT10
• No Observed Adverse Effect Level (NOEAL) value
of HT10
• The Possible Mechanism of any Observed Adverse
Effects at High Doses.

14. Blood Pressure

15. Work Plan
Dose:
Single dose- 100 mg/kg (highest dose)
1week- 10 mg/kg, 25 mg/kg, 50 mg/kg and 100 mg/kg
2 weeks- 10 mg/kg, 25 mg/kg, 50 mg/kg and 100 mg/kg
4weeks- 10 mg/kg, 25 mg/kg and 1 mg/kg,(chronic safety measurement)
Surgery and catheterization
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16. Change In Blood Pressure At
Different Doses of HT10
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p value <0.01*

17. Change In Blood Pressure At
Different Doses of HT10
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p value <0.01*
No Significant Increase in Blood Pressure was observed up to 10 mg/kg dose after Chronic study

18. In vitro work plan
HUVEC cells
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19. In-vitro: PGI2 Secretion
Effect of Blood Parameter, PGI2, after Binding Reversibility of HT10 with VEGF
treating HUVEC with HT10 in secreting Blood Parameter, PGI2
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6.#!8#!93:90$

20. In-vitro: PGI2 Secretion
Effect of Blood Parameter, PGI2, after Binding Reversibility of HT10 with VEGF
treating HUVEC with HT10 in secreting Blood Parameter, PGI2
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The Reversible Binding of HT10 and VEGF would have beneficial effect in reversing the change in BP.

21. Effect on kidney
function

22. Proteinuria
Urine collection after
over night inhabitation
Bradford
test

23. Proteinuria
80
**
70 ***
60
*p<0.05, **p<0.01
50
**
Protein Amount
(ug/ml/24 hr)
control
40 10mg
25mg
30 50mg
* 100mg
20
10
0
0 5 10 15 20 25 30 35
Days

24. Proteinuria
80
**
70 ***
60
*p<0.05, **p<0.01
50
**
Protein Amount
(ug/ml/24 hr)
control
40 10mg
25mg
30 50mg
* 100mg
20
10
0
0 5 10 15 20 25 30 35
Days
Increase in Proteinuria level was not observed up to 25 mg/kg dose at Chronic study

25. Histology: Kidney (2 WEEKS)
X400 X400
Control HT10- 25 mg/kg
X400 X400
HT10-10 mg/kg HT10- 50 mg/kg

26. Effect on liver
function

27. Alt level
ALT after single dose treatment
ALT after multiple doses
1600
160
1400
140
**
1200
120
ALT(IU/L)
ALT (IU/L)
1000
100 control
800
80 10mg/kg
600
60
400 25mg/kg
40
200 50mg/kg
20
0
0 100mg/kg
control 10mg/kg 25mg/kg 50mg/kg 100mg/kg
7 14 28
Group
Day
** p<0.001

28. ASt level
AST after multiple doses
AST after single dose treatment 800 ** control
1000
700
900 10mg/kg
800 600
25mg/kg
AST (IU/L)
700
AST( IU/L)
500
600 50mg/kg
500 400
100mg/kg
400 300
300
200
200
100 100
0 0
control 10mg/kg 25mg/kg 50mg/kg 100mg/kg 7 14 28
Group Day

29. liver histology
X400 X400
Control HT10- 25 mg/kg
X400 X400
HT10-10 mg/kg HT10- 50 mg/kg

30. Hematology

31. Platelet count
3-4+5-5+"()6*+"78*/-5"9)75"
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'!!!"
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&!!" Minimum platelet count for rat
%!!"
$!!" *
* * p<0.001
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1200
1050
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900
750 Minimum platelet count for rat
600 control
450 10mg/kg
300 50mg/kg
150
0
0 1 2 3 6 7
/#0&

32. Platelet behavior at splited dose
Platelet behavior after split doses’ administration
*+,-.+/"
01/2-"3+456'!"789:8"
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33. HT10 uptake by platelet
HT10 uptake by Platelet ,-./01"23"45'!"
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Platelet concentration: 200-250 K/ul Detection method: Chemosensor

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35. Chronic safety
evaluation At
Therapeutic DOSE

36. I mg/kg dose safety evaluation
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37. Conclusion
The Effect of HT10 in Blood Pressure at High & Chronic dose, would to
be Reversible as observed in In-vitro model.
No Change in Protein amount in urine was observed up to 25 mg/kg
dose, even after 1 month continuous dosing.
Liver and kidney Toxicity was only observed at very High Doses.
In antigen-antibody experiment, HT10 does not show any positive effect
Increase in Bleeding Time might be associated with Increase in
Platelet Uptake of HT10 as observed both in-vivo & in-vitro.
The Maximum dose of HT10 at which Platelet uptake is minimum, must be
determined.

38. Conclusion
The Effect of HT10 in Blood Pressure at High & Chronic dose, would to
be Reversible as observed in In-vitro model.
No Change in Protein amount in urine was observed up to 25 mg/kg
dose, even after 1 month continuous dosing.
Liver and kidney Toxicity was only observed at very High Doses.
In antigen-antibody experiment, HT10 does not show any positive effect
Increase in Bleeding Time might be associated with Increase in
Platelet Uptake of HT10 as observed both in-vivo & in-vitro.
The Maximum dose of HT10 at which Platelet uptake is minimum, must be
determined.
The NOEAL value of HT10 is 10 mg/kg.

39. Interpretation
mg/kg Dose-scale Therapeutic and toxic level
100 Major toxic dose
50 Minor toxic dose
25 Tolerable toxic dose
10 NOAEL Value
5
Therapeutic dose
1

40. Solution for toxic effects
• Treatment duration is not more than 3 months
• Administration time is not regular; 2-3 days dose
interval
• In case of high doses- dose can be splited
• Chemical modification; e.g- very low molecular
wight heparin

41. Future direction
• Platelet uptake by HT10
• More depth mechanism study of the toxicities
• Toxicokinetics of HT10

42. Acknowledgement

43. Acknowledgement

44. Thank you for listening