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My Defence presentation of Msc

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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.

Health & MedicineTechnology
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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
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
Heparin on cancer Immune Modulation Anticoagulation Metastasis Courtesy: Sungwoo Chung
Heparin on cancer Immune Modulation Angiogenesis Anticoagulation Metastasis Courtesy: Sungwoo Chung
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
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
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
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
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
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
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
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
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.
Blood Pressure
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 !"#$%"& !"#$%&#$$ '()*(+
Change In Blood Pressure At Different Doses of HT10 $("# !"##$%&'())*'(%45%6*"78"(9%:;'#21:%0#)()% %!"##$%3'())*'(%45%677%.-89-%:12-"(%;#)(% &!"# * $'"# * /012304# !"##$%&'())*'(%% &'"# $"56786# !"##$%&'())*'(%% $&"# +,-../% %(56786# +,-../% &&"# $%"# ("56786# %"# $$"# $""56786# $"# $""# !"# !"# "# &# '# !# &"# &!# 01.(%+.12/% )*+#,# -*+#$(# -*+#%.# 0#)12-%31.(% ()*+,)-# ./&"#0&""123425# p value <0.01*
Change In Blood Pressure At Different Doses of HT10 $("# !"##$%&'())*'(%45%6*"78"(9%:;'#21:%0#)()% %!"##$%3'())*'(%45%677%.-89-%:12-"(%;#)(% &!"# * $'"# * /012304# !"##$%&'())*'(%% &'"# $"56786# !"##$%&'())*'(%% $&"# +,-../% %(56786# +,-../% &&"# $%"# ("56786# %"# $$"# $""56786# $"# $""# !"# !"# "# &# '# !# &"# &!# 01.(%+.12/% )*+#,# -*+#$(# -*+#%.# 0#)12-%31.(% ()*+,)-# ./&"#0&""123425# p value <0.01* No Significant Increase in Blood Pressure was observed up to 10 mg/kg dose after Chronic study
In vitro work plan HUVEC cells !"#$%"&'(%&)"'*+ "$%,#+-' ./'0"'1&-20"+-#3%, #+-' 4")%,)5'6#,0':; <='2+-,%#-#-8'8" 4")%,)5'6#,0' +>7'+"'?4@A' 70)-+('")5'*") )'$)5#%'2+-,% #-#-8'7+1#,#9)' 2+-,"+(' !"#$%&'()* !"#$%&'()*
In-vitro: PGI2 Secretion Effect of Blood Parameter, PGI2, after Binding Reversibility of HT10 with VEGF treating HUVEC with HT10 in secreting Blood Parameter, PGI2 (#$" !"*$ (" !")$ !#'" !"($ !"#$%&'(&%% )*+,-./% !"'$ !#&" !"&$ !#%" !"%$ !#$" !"#$ !" !$ !"($ #$ #"($ %$ !" 0.5 (" 2 $" 6 )" 8 %" 18 *" 24 &" + 01-2%)34/% !"#$%&'()% +,-./,0$ 1234$ 5-6575.,/$ ,-./0-1" 2345" 67(!8!#!(9:" 6.#!8!"!#93:90$ 6.#!8!"#93:90$ 6.#!8#93:90$ 67(!8!#(9:" ;:<=>.9:" ?.@ABA/-0" 6.#!8#!93:90$
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 (#$" !"*$ (" !")$ !#'" !"($ !"#$%&'(&%% )*+,-./% !"'$ !#&" !"&$ !#%" !"%$ !#$" !"#$ !" !$ !"($ #$ #"($ %$ !" 0.5 (" 2 $" 6 )" 8 %" 18 *" 24 &" + 01-2%)34/% !"#$%&'()% +,-./,0$ 1234$ 5-6575.,/$ ,-./0-1" 2345" 67(!8!#!(9:" 6.#!8!"!#93:90$ 6.#!8!"#93:90$ 6.#!8#93:90$ 67(!8!#(9:" ;:<=>.9:" ?.@ABA/-0" 6.#!8#!93:90$ The Reversible Binding of HT10 and VEGF would have beneficial effect in reversing the change in BP.
Effect on kidney function
Proteinuria Urine collection after over night inhabitation Bradford test
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
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
Histology: Kidney (2 WEEKS) X400 X400 Control HT10- 25 mg/kg X400 X400 HT10-10 mg/kg HT10- 50 mg/kg
Effect on liver function
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
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
liver histology X400 X400 Control HT10- 25 mg/kg X400 X400 HT10-10 mg/kg HT10- 50 mg/kg
Hematology
Platelet count 3-4+5-5+"()6*+"78*/-5"9)75" '#!!" '!!!" !"#$%"%$&'()*+,& &!!" Minimum platelet count for rat %!!" $!!" * * * p<0.001 #!!" * * !" ()*+,)-" '!./01/" #2./01/" 2!./01/" '!!./01/" -./*0& 1%'(2%30&!#4%3*&(5&!"#$%"%$&#6%3&78*9"%&#/:8*87$3#;(*& 1200 1050 !"#$%"%$&'()*$&+,-)".& 900 750 Minimum platelet count for rat 600 control 450 10mg/kg 300 50mg/kg 150 0 0 1 2 3 6 7 /#0&
Platelet behavior at splited dose Platelet behavior after split doses’ administration *+,-.+/" 01/2-"3+456'!"789:8" '#!!" '!!!" !"#$%"%$&'()*"+& &!!" Minimum platelet count for rat %!!" $!!" #!!" !" !" (" '!" '(" #!" #(" )!" ,-.%&'/0+&
HT10 uptake by platelet HT10 uptake by Platelet ,-./01"23"45'!" '!!" &!" !"#$%"%$&'($#)%&& *+,*&-./01"2& %!" $!" #!" !" !"()" '"()" '!"()" #*"()" *!"()" +*"()" '!!"()" 3,*.4#$%5&6789&*+,*&-./01"2& Platelet concentration: 200-250 K/ul Detection method: Chemosensor
!"#$%"%$&'()*$&#/%0&12%%34&(5&678-38&9(4%& '#!!" '!!!" !"#$%"%$&'()*$&+,-)".& &!!" %!!" $!!" #!!" !" ()*+,)-" ./'!0'12342" Platelet damage test by NAP-2 release #$!!" (!" #!!!" '!" !"#"$%"&'(&)*+,-&./0& )!!" &!" )*+,-&& 1234#& (!!" %!" &!!" $!" $!!" #!" !" !" !"*+,-." '!"*+,-." #!!"*+,-." $'!"*+,-." '!!"*+,-." /012$"3453" 6785+9":5";9.98<9"
Chronic safety evaluation At Therapeutic DOSE
I mg/kg dose safety evaluation !"#$%&'()*#+'$(&'(+"&'(#,(")$-(),$%"(.*/01/(2#-%( !"##$%&'())*'(#+%',-)%,+-('%.%/((0)%-'(,-1(2-% )2*&'&-$")$&#'( 150 140 #!!" 130 +!" !"##$%&'())*'(%+,,-./% *!" 120 )!" !"#$%#&'()* 110 (!" 100 '!" 90 &!" %!" 80 $!" 70 #!" 60 !" Control ,-./0-1" #23453" HT10-1mg/kg ,/."012",-."30456"78"#9:;<:"27=6" +!" *!" )!" (!" '!" !"#$% >71?@74" &!" %!" A.#!B#9:;<:" $!" #!" !" ,-." ,/."
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.
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.
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
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
Future direction • Platelet uptake by HT10 • More depth mechanism study of the toxicities • Toxicokinetics of HT10
Acknowledgement
Acknowledgement
Thank you for listening

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My Defence presentation of Msc

  • 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.
  • 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 !"#$%"& !"#$%&#$$ '()*(+
  • 16. Change In Blood Pressure At Different Doses of HT10 $("# !"##$%&'())*'(%45%6*"78"(9%:;'#21:%0#)()% %!"##$%3'())*'(%45%677%.-89-%:12-"(%;#)(% &!"# * $'"# * /012304# !"##$%&'())*'(%% &'"# $"56786# !"##$%&'())*'(%% $&"# +,-../% %(56786# +,-../% &&"# $%"# ("56786# %"# $$"# $""56786# $"# $""# !"# !"# "# &# '# !# &"# &!# 01.(%+.12/% )*+#,# -*+#$(# -*+#%.# 0#)12-%31.(% ()*+,)-# ./&"#0&""123425# p value <0.01*
  • 17. Change In Blood Pressure At Different Doses of HT10 $("# !"##$%&'())*'(%45%6*"78"(9%:;'#21:%0#)()% %!"##$%3'())*'(%45%677%.-89-%:12-"(%;#)(% &!"# * $'"# * /012304# !"##$%&'())*'(%% &'"# $"56786# !"##$%&'())*'(%% $&"# +,-../% %(56786# +,-../% &&"# $%"# ("56786# %"# $$"# $""56786# $"# $""# !"# !"# "# &# '# !# &"# &!# 01.(%+.12/% )*+#,# -*+#$(# -*+#%.# 0#)12-%31.(% ()*+,)-# ./&"#0&""123425# 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 !"#$%"&'(%&)"'*+ "$%,#+-' ./'0"'1&-20"+-#3%, #+-' 4")%,)5'6#,0':; <='2+-,%#-#-8'8" 4")%,)5'6#,0' +>7'+"'?4@A' 70)-+('")5'*") )'$)5#%'2+-,% #-#-8'7+1#,#9)' 2+-,"+(' !"#$%&'()* !"#$%&'()*
  • 19. In-vitro: PGI2 Secretion Effect of Blood Parameter, PGI2, after Binding Reversibility of HT10 with VEGF treating HUVEC with HT10 in secreting Blood Parameter, PGI2 (#$" !"*$ (" !")$ !#'" !"($ !"#$%&'(&%% )*+,-./% !"'$ !#&" !"&$ !#%" !"%$ !#$" !"#$ !" !$ !"($ #$ #"($ %$ !" 0.5 (" 2 $" 6 )" 8 %" 18 *" 24 &" + 01-2%)34/% !"#$%&'()% +,-./,0$ 1234$ 5-6575.,/$ ,-./0-1" 2345" 67(!8!#!(9:" 6.#!8!"!#93:90$ 6.#!8!"#93:90$ 6.#!8#93:90$ 67(!8!#(9:" ;:<=>.9:" ?.@ABA/-0" 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 (#$" !"*$ (" !")$ !#'" !"($ !"#$%&'(&%% )*+,-./% !"'$ !#&" !"&$ !#%" !"%$ !#$" !"#$ !" !$ !"($ #$ #"($ %$ !" 0.5 (" 2 $" 6 )" 8 %" 18 *" 24 &" + 01-2%)34/% !"#$%&'()% +,-./,0$ 1234$ 5-6575.,/$ ,-./0-1" 2345" 67(!8!#!(9:" 6.#!8!"!#93:90$ 6.#!8!"#93:90$ 6.#!8#93:90$ 67(!8!#(9:" ;:<=>.9:" ?.@ABA/-0" 6.#!8#!93:90$ 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
  • 31. Platelet count 3-4+5-5+"()6*+"78*/-5"9)75" '#!!" '!!!" !"#$%"%$&'()*+,& &!!" Minimum platelet count for rat %!!" $!!" * * * p<0.001 #!!" * * !" ()*+,)-" '!./01/" #2./01/" 2!./01/" '!!./01/" -./*0& 1%'(2%30&!#4%3*&(5&!"#$%"%$&#6%3&78*9"%&#/:8*87$3#;(*& 1200 1050 !"#$%"%$&'()*$&+,-)".& 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" '#!!" '!!!" !"#$%"%$&'()*"+& &!!" Minimum platelet count for rat %!!" $!!" #!!" !" !" (" '!" '(" #!" #(" )!" ,-.%&'/0+&
  • 33. HT10 uptake by platelet HT10 uptake by Platelet ,-./01"23"45'!" '!!" &!" !"#$%"%$&'($#)%&& *+,*&-./01"2& %!" $!" #!" !" !"()" '"()" '!"()" #*"()" *!"()" +*"()" '!!"()" 3,*.4#$%5&6789&*+,*&-./01"2& Platelet concentration: 200-250 K/ul Detection method: Chemosensor
  • 34. !"#$%"%$&'()*$&#/%0&12%%34&(5&678-38&9(4%& '#!!" '!!!" !"#$%"%$&'()*$&+,-)".& &!!" %!!" $!!" #!!" !" ()*+,)-" ./'!0'12342" Platelet damage test by NAP-2 release #$!!" (!" #!!!" '!" !"#"$%"&'(&)*+,-&./0& )!!" &!" )*+,-&& 1234#& (!!" %!" &!!" $!" $!!" #!" !" !" !"*+,-." '!"*+,-." #!!"*+,-." $'!"*+,-." '!!"*+,-." /012$"3453" 6785+9":5";9.98<9"
  • 35. Chronic safety evaluation At Therapeutic DOSE
  • 36. I mg/kg dose safety evaluation !"#$%&'()*#+'$(&'(+"&'(#,(")$-(),$%"(.*/01/(2#-%( !"##$%&'())*'(#+%',-)%,+-('%.%/((0)%-'(,-1(2-% )2*&'&-$")$&#'( 150 140 #!!" 130 +!" !"##$%&'())*'(%+,,-./% *!" 120 )!" !"#$%#&'()* 110 (!" 100 '!" 90 &!" %!" 80 $!" 70 #!" 60 !" Control ,-./0-1" #23453" HT10-1mg/kg ,/."012",-."30456"78"#9:;<:"27=6" +!" *!" )!" (!" '!" !"#$% >71?@74" &!" %!" A.#!B#9:;<:" $!" #!" !" ,-." ,/."
  • 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
  • 44. Thank you for listening