Early Drug Development: Making it HappenEarly Drug Development: Making it Happen
Gabriel Lopez-Berestein, M.D., Professor of Medicine and Cancer Gabriel Lopez-Berestein, M.D., Professor of Medicine and Cancer Biology, Department of Experimental Therapeutics, M D Anderson Biology, Department of Experimental Therapeutics, M D Anderson Cancer CenterCancer Center
Professor, Department of NanoMedicine and Bioengineering, Professor, Department of NanoMedicine and Bioengineering, UTHealthUTHealth
Gabriel Lopez-Berestein, M.D., Professor of Medicine and Cancer Gabriel Lopez-Berestein, M.D., Professor of Medicine and Cancer Biology, Department of Experimental Therapeutics, M D Anderson Biology, Department of Experimental Therapeutics, M D Anderson Cancer CenterCancer Center
Professor, Department of NanoMedicine and Bioengineering, Professor, Department of NanoMedicine and Bioengineering, UTHealthUTHealth
Fundacion Areces Symposium
Madrid October 15, 2014
The New Drug Development Process:The New Drug Development Process:Steps from Test Tube to New Drug Application ReviewSteps from Test Tube to New Drug Application Review
http: www.fda.gov.cder.handbook.develop.htm
Nucleus
siRNA
dsRNA
Liposomal siRNAViral vectors
Translation
miRNA
mRNA cleavage
Dicer RISC
Translational inhibition
miRNP
**
Chemically-modified siRNA
RNA InterferenceRNA Interference
OBJECTIVEOBJECTIVE OBJECTIVEOBJECTIVE
Develop a systemic nanoliposomal
delivery system for siRNA
HYPOTHESISHYPOTHESISHYPOTHESISHYPOTHESIS
Tumoral in vivo siRNA delivery can be improved by using neutral liposomal delivery
Downregulation of an ovarian cancer relevant target protein with siRNA reduces tumor growth in an orthotopic mouse model of ovarian cancer
Tumoral in vivo siRNA delivery can be improved by using neutral liposomal delivery
Downregulation of an ovarian cancer relevant target protein with siRNA reduces tumor growth in an orthotopic mouse model of ovarian cancer
IN VIVO IN VIVO SIRNA DELIVERYSIRNA DELIVERYIN VIVO IN VIVO SIRNA DELIVERYSIRNA DELIVERY
Successful siRNA delivery in vivo requires methods that are clinically limitedIntratumoral
Intrathecal
Intraocular
Viral vectors
Nanoliposomes as an alternative
Successful siRNA delivery in vivo requires methods that are clinically limitedIntratumoral
Intrathecal
Intraocular
Viral vectors
Nanoliposomes as an alternative
Phospholipid structurePhospholipid structure
Effect of temperature on the Effect of temperature on the packing of the phospholipidspacking of the phospholipids
Unsaturated and Saturated Unsaturated and Saturated PhospholipidsPhospholipids
1,2-Dioleyl-sn-Glycero-3-Phosphocholine (DOPC)
Lamellar vs Hexagonal PhaseLamellar vs Hexagonal Phase
Liposomes incorporate siRNALiposomes incorporate siRNA
All pictures 100XArturo Chavez-Reyes
siRNADOPC
Tween-20Tert-butanol
siRNA:DOPC1:10
Lyophilized Reconstitutedwith sterile saline
Electron Microscopy of DOPC NanoparticlesElectron Microscopy of DOPC Nanoparticles
Average size = 60 nanometersAverage size = 60 nanometers
Normal vs Tumor VasculatureNormal vs Tumor Vasculature
Untagged siRNA / DOPC Alexa555-siRNA / DOPC
siRNA/DOPC is delivered deeply siRNA/DOPC is delivered deeply into tumorinto tumor
Red: siRNABlue: Nuclei
siRNA in DOPC is not primarily siRNA in DOPC is not primarily scavenged by macrophagesscavenged by macrophages
Green: Macrophages: f4/80 Red: siRNABlue: Nuclei
IHC: CD31
H&E Immunofluorescence
Confocal Confocal MicroscopyMicroscopy
Bottom of slideTop of slide
Green: Macrophages Red: siRNABlue: Nuclei
siRNA/DOPC compared to delivery siRNA/DOPC compared to delivery ““nakednaked”” or in cationic liposomes* or in cationic liposomes*
Green: CD31
“Naked” siRNA *DOTAP-encapsulated siRNA
siRNA delivery in DOPC is not siRNA delivery in DOPC is not limited to vascularitylimited to vascularity
Green: CD31 Blue: NucleiRed: siRNA
siRNA uptake by other organssiRNA uptake by other organs
LIVER
KIDNEY
LUNG
H&E siRNA-Alexa 555 Untagged siRNA
Potential benefits of targeting EpHA2Potential benefits of targeting EpHA2
Advanced ovarian cancer is largely incurable
Advantages:
High-specificity
EphA2 is not expressed in most normal adult tissues
Harnessing a naturally occurring mechanism
Nanoliposomal-siRNA should be well-tolerated
EphA2/eckEphA2/eck
Developmental role
in neuronal migration
Low expression in adults
Overexpressed in 76% of ovarian cancerPredictive of a poor outcome
Valid target for systemic downregulation
EphA2
EphA2
Thaker ………Sood, Clin Ca Res 2004
Landen …….Sood, Abstract #1702, AACR 2005
EphA2 Expression and Clinical EphA2 Expression and Clinical Parameters in Ovarian CancerParameters in Ovarian Cancer
Low EphA2
EphA2 Overexpression
EphA2 Variable Overexpression PStage Low 38.4% High 83.3% 0.001Grade Low 50.0% High 80.6% 0.02Histology Serous 73.6% Other 80.8% 0.48
P=0.004
Univariate Analysis
Thaker…….Sood, Clin Cancer Res, 2004
Multivariate analysisVariable Decreased survival
Residual disease p<0.04
EphA2 Overexpression p<0.01
Grade NS
Stage NS
EphA2 Expression and Clinical EphA2 Expression and Clinical Parameters in Ovarian CancerParameters in Ovarian Cancer
Thaker…….Sood, Clin Cancer Res, 2004
Anti-human EphA2 siRNA Anti-human EphA2 siRNA downregulates EphA2 downregulates EphA2 in vitroin vitro
EphA2
-actin
No txNo tx
siRNA
No tx
siRNA
Non-sile
ncing
EphA2-targeted
48 hrs 2 days72 hrs 2 days
4 days
6 days
2 days
EphA2-targeted siRNA/DOPC EphA2-targeted siRNA/DOPC downregulates EphA2 downregulates EphA2 in vivoin vivo
control siRNA / DOPC EphA2 siRNA / DOPCEphA2 siRNA-naked
Therapy SchemaTherapy Schema
* SKOV3ip1 or HeyA8
Group 1: Empty liposomes
Group 2: Nonspecific siRNA/DOPC
Group 3: EphA2-targeting siRNA/DOPC
Group 4: Paclitaxel plus Nonspecific siRNA/DOPC
Group 5: Paclitaxel plus EphA2-targeting siRNA/DOPC
Week:
Intraperitoneal cell* injection:
siRNA (150g/kg) tx:
paclitaxel (100g) tx:
sacrifice:
1 2 3 4
SKOV3ip1 tumor weight after SKOV3ip1 tumor weight after EphA2-targeting siRNA therapyEphA2-targeting siRNA therapy
Empty
Lip
osom
es
Non-sile
ncing s
iRNA/D
OPC
EphA2 si
RNA/DOPC
Paclit
axel
+Non-s
ilenci
ng
siRNA/D
OPC
Paclit
axel
+
EphA2 si
RNA/DOPC
Tu
mo
r W
eig
ht
(g)
p=0.020
p=0.57
p<0.001
Mean tumor weights Individual weights
HeyA8 tumor weight after HeyA8 tumor weight after EphA2-targeting siRNA therapyEphA2-targeting siRNA therapy
Tu
mo
r W
eig
ht
(g)
p=0.155
p=0.036
p<0.003
Mean tumor weights Individual weights
Empty
Lip
osom
es
Non-sile
ncing s
iRNA/D
OPC
EphA2 si
RNA/DOPC
Paclit
axel
+Non-s
ilenci
ng
siRNA/D
OPC
Paclit
axel
+
EphA2 si
RNA/DOPC
Efficacy of IP delivery of Efficacy of IP delivery of EphA2-siRNA/DOPC: SKOV3ip1EphA2-siRNA/DOPC: SKOV3ip1
Mean tumor weights Individual weights
cntrl EphA2 cntrl
Tu
mo
r W
eig
ht
(g)
EphA2cntrlsiRNA:
delivery: IV IP IP IV IV
paclitaxel: NO + + + +Landen……..Sood, AACR, 2006
cntrl EphA2 cntrl EphA2cntrlsiRNA:
delivery: IV IP IP IV IV
paclitaxel: NO + + + +
Efficacy of IP delivery of Efficacy of IP delivery of EphA2-siRNA/DOPC: HeyA8EphA2-siRNA/DOPC: HeyA8
Mean tumor weights Individual weights
Tu
mo
r W
eig
ht
(g)
Landen……..Sood, AACR, 2006
SIRNA EPHA2 ANIMAL TOXICOLOGYSIRNA EPHA2 ANIMAL TOXICOLOGY
Mouse Study:
•Dual phase study (Acute: 24 hours, Delayed: 28 days)
•Single exposure
Control and 5 dose levels
5 animals per group, both male and female gender
•Observations:
No morbidity or mortality observed
•No dose-related alterations in group means for hematologic or non-hematologic parameters
•No gross or histological organ dysfunction
NOAEL > 225 g/kg
Mouse Study:
•Dual phase study (Acute: 24 hours, Delayed: 28 days)
•Single exposure
Control and 5 dose levels
5 animals per group, both male and female gender
•Observations:
No morbidity or mortality observed
•No dose-related alterations in group means for hematologic or non-hematologic parameters
•No gross or histological organ dysfunction
NOAEL > 225 g/kg
SIRNA EPHA2: ANIMAL TOXICOLOGYSIRNA EPHA2: ANIMAL TOXICOLOGY
Rhesus Monkey
•EphA2-Rhesus has 100% homology to EphA2-human
Toxicology Protocol:
•Methods:
10 animals (4 Rx-males, 4 Rx-females, 2 controls),
2 dose-levels (500 g/m2, 750 g/m2)
IV twice weekly x 4 weeks
•In Life: Hematology, clinical chemistry, urinalysis, coagulation, bone marrow evaluation revealed no test-article effects
•Anatomic Pathology: Necropsy – no test article effects; Histopathology: mild immunologic effects observed in both controls and test animals – ascribed as unrelated to test article
Rhesus Monkey
•EphA2-Rhesus has 100% homology to EphA2-human
Toxicology Protocol:
•Methods:
10 animals (4 Rx-males, 4 Rx-females, 2 controls),
2 dose-levels (500 g/m2, 750 g/m2)
IV twice weekly x 4 weeks
•In Life: Hematology, clinical chemistry, urinalysis, coagulation, bone marrow evaluation revealed no test-article effects
•Anatomic Pathology: Necropsy – no test article effects; Histopathology: mild immunologic effects observed in both controls and test animals – ascribed as unrelated to test article
Effects on immune parameters
Dicer and Drosha – Validation StudiesDicer and Drosha – Validation StudiesDicer and Drosha – Validation StudiesDicer and Drosha – Validation Studies
Ovarian cancer
New Engl J Med, 2008
Functional Impact of Low DicerFunctional Impact of Low Dicer
New Engl J Med, 2008
Clinical Trial DesignClinical Trial Design IND 072924Clinical Trial DesignClinical Trial Design IND 072924
Clinical ProtocolClinical ProtocolIND IND 072924
Clinical ProtocolClinical ProtocolIND IND 072924
Title: Therapeutic EphA2 Gene Targeting using Neutral Liposomal Small Interfering RNA Delivery: A Phase I Clinical Trial
Investigators:
PI: Robert L. Coleman, M.D.
Radiology: Vikas Kundra, M.D., Ph.D.
.
Title: Therapeutic EphA2 Gene Targeting using Neutral Liposomal Small Interfering RNA Delivery: A Phase I Clinical Trial
Investigators:
PI: Robert L. Coleman, M.D.
Radiology: Vikas Kundra, M.D., Ph.D.
.
SiRNA EphA2: Phase I StudySiRNA EphA2: Phase I StudySiRNA EphA2: Phase I StudySiRNA EphA2: Phase I Study
Primary Objectives: To determine the safety and tolerability of IV
siRNA-DOPC-EphA2 in patients with advanced solid tumors
To determine the maximum tolerated dose or optimal biological dose
To determine the target efficacy in escalating doses
Primary Objectives: To determine the safety and tolerability of IV
siRNA-DOPC-EphA2 in patients with advanced solid tumors
To determine the maximum tolerated dose or optimal biological dose
To determine the target efficacy in escalating doses
Bridge Study OCT 2014 Bridge Study OCT 2014
Secondary Objectives:
To determine the pharmacokinetic profile of IV siRNA-DOPC-EphA2 (twice weekly)
To evaluate the impact of therapy on by non-invasive imaging (DCE-MRI, DW-MRI FDG-PET)
To evaluate the impact of therapy on surrogate biomarkers (CF-DNA, CTCs, VEGFplasma)
SiRNA EphA2: Phase I StudySiRNA EphA2: Phase I StudySiRNA EphA2: Phase I StudySiRNA EphA2: Phase I Study
Eligibility: Solid tumors - recurrent or
considered incurable with standard therapy
Bi-dimensionally measurable disease (>2 cm)
Amenable to biopsy EphA2 overexpression
(IHC)
Eligibility: Solid tumors - recurrent or
considered incurable with standard therapy
Bi-dimensionally measurable disease (>2 cm)
Amenable to biopsy EphA2 overexpression
(IHC)
DoseDose MulMult.t.
FrequencyFrequency
450 µg/m450 µg/m22 -- 2/weekly2/weekly
900 µg/m900 µg/m22 100100%%
2/weekly2/weekly
1800 µg/m1800 µg/m22 100100%%
2/weekly2/weekly
3600 µg/m3600 µg/m22 100100%%
2/weekly2/weekly
7200 µg/m7200 µg/m22 100100%%
2/weekly2/weekly
SIRNA EPHA2 Murine Bridge StudySIRNA EPHA2 Murine Bridge Study
AcknowledgementsAcknowledgements
Anil K. Sood Alan G. Brady
Robert Coleman Beth. K. Chaffee
Cristian Rodriguez-Aguayo Hee-Dong Han
Mangala Selanere Kirstin Barnhart
Chip Landen
Arturo Chavez
Rahul Mitra
Wallace Baze
Chris R. Abee
Anil K. Sood Alan G. Brady
Robert Coleman Beth. K. Chaffee
Cristian Rodriguez-Aguayo Hee-Dong Han
Mangala Selanere Kirstin Barnhart
Chip Landen
Arturo Chavez
Rahul Mitra
Wallace Baze
Chris R. Abee