Dr. Gabriel López-Berestein- Simposio Internacional 'Terapias oncológicas avanzadas'

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