Los días 15 y 16 de octubre de 2014, la Fundación Ramón Areces y la Real Academia Nacional de Farmacia, en colaboración con la Fundación de la Innovación Bankinter, reunieron en Madrid a algunos de los mayores expertos mundiales en nuevas terapias contra el cáncer. El Simposio Internacional, coordinado por la profesora y académica María José Alonso, analizó el momento actual de la lucha contra esta enfermedad. También fue un punto de encuentro para científicos de los más innovadores institutos de investigación en oncología, quienes debatieron sobre tres grandes temas: la Medicina Personalizada contra el cáncer, los nanomedicamentos en la terapia del cáncer y las terapias basadas en la inmunomodulación.

1. Early Drug Development: Making it Happen
Fundacion Areces Symposium
Madrid October 15, 2014
Gabriel Lopez-Berestein, M.D., PPrrooffeessssoorr ooff MMeeddiicciinnee aanndd CCaanncceerr
BBiioollooggyy,, DDeeppaarrttmmeenntt ooff EExxppeerriimmeennttaall TThheerraappeeuuttiiccss,, MM DD AAnnddeerrssoonn
CCaanncceerr CCeenntteerr
PPrrooffeessssoorr,, DDeeppaarrttmmeenntt ooff NNaannooMMeeddiicciinnee aanndd BBiiooeennggiinneeeerriinngg,,
UUTTHHeeaalltthh

2. The New DDrruugg DDeevveellooppmmeenntt PPrroocceessss::
SStteeppss ffrroomm TTeesstt TTuubbee ttoo NNeeww DDrruugg AApppplliiccaattiioonn RReevviieeww
http: www.fda.gov.cder.handbook.develop.htm

3. Nucleus
RRNNAA IInntteerrffeerreennccee
siRNA
dsRNA
Liposomal siRNA
Viral vectors
Translation
miRNA
mRNA cleavage
Chemically-modified
siRNA
*
*
Dicer RISC
miRNP
Translational inhibition

4. OOBBJJEECCTTIIVVEE
 Develop a systemic nanoliposomal
delivery system for siRNA

5. HHYYPPOOTTHHEESSIISS
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

6. IINN VVIIVVOO SSIIRRNNAA DDEELLIIVVEERRYY
Successful siRNA delivery in vivo requires methods that
are clinically limited
Intratumoral
Intrathecal
Intraocular
Viral vectors
Nanoliposomes as an alternative

7. PPhhoosspphhoolliippiidd ssttrruuccttuurree

8. Effect ooff tteemmppeerraattuurree oonn tthhee
ppaacckkiinngg ooff tthhee pphhoosspphhoolliippiiddss

9. UUnnssaattuurraatteedd aanndd SSaattuurraatteedd
PPhhoosspphhoolliippiiddss

10. 1,2-Dioleyl-sn-Glycero-3-Phosphocholine
(DOPC)

11. LLaammeellllaarr vvss HHeexxaaggoonnaall PPhhaassee

12. LLiippoossoommeess iinnccoorrppoorraattee ssiiRRNNAA
All pictures 100X
Arturo Chavez-Reyes
siRNA
DOPC
Tween-20
Tert-butanol
siRNA:DOPC
1:10
Lyophilized Reconstituted
with sterile saline

13. Electron Microscopy ooff DDOOPPCC NNaannooppaarrttiicclleess
AAvveerraaggee ssiizzee == 6600 nnaannoommeetteerrss

15. NNoorrmmaall vvss TTuummoorr VVaassccuullaattuurree

16. ssiiRRNNAA//DDOOPPCC iiss ddeelliivveerreedd ddeeeeppllyy
iinnttoo ttuummoorr
Untagged siRNA / DOPC Alexa555-siRNA / DOPC
Red: siRNA
Blue: Nuclei

17. ssiiRRNNAA iinn DDOOPPCC iiss nnoott pprriimmaarriillyy
ssccaavveennggeedd bbyy mmaaccrroopphhaaggeess
H&E Immunofluorescence
Green: Macrophages: f4/80
Red: siRNA
Blue: Nuclei
IHC: CD31

18. CCoonnffooccaall
MMiiccrroossccooppyy
Top of slide Bottom of slide
Green: Macrophages
Red: siRNA
Blue: Nuclei

19. ssiiRRNNAA//DDOOPPCC ccoommppaarreedd ttoo ddeelliivveerryy
““nnaakkeedd”” oorr iinn ccaattiioonniicc lliippoossoommeess**
“Naked” siRNA *DOTAP-encapsulated siRNA
Green: CD31

20. ssiiRRNNAA ddeelliivveerryy iinn DDOOPPCC iiss nnoott
lliimmiitteedd ttoo vvaassccuullaarriittyy
Green: CD31
Blue: Nuclei
Red: siRNA

21. siRNA uuppttaakkee bbyy ootthheerr oorrggaannss
LIVER
KIDNEY
LUNG
H&E siRNA-Alexa 555 Untagged siRNA

22. PPootteennttiiaall bbeenneeffiittss ooff ttaarrggeettiinngg EEppHHAA22
 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

23. EEpphhAA22//eecckk
Developmental role
in neuronal migration
Low expression in adults
Overexpressed in 76% of ovarian cancer
Predictive of a poor outcome
Valid target for systemic downregulation
EphA2
EphA2
Thaker ………Sood, Clin Ca Res 2004
Landen …….Sood, Abstract #1702, AACR 2005

24. EEpphhAA22 EExxpprreessssiioonn aanndd CClliinniiccaall
PPaarraammeetteerrss iinn OOvvaarriiaann CCaanncceerr
Low EphA2
EphA2 Overexpression
EphA2
Variable Overexpression P
Stage
Low 38.4%
High 83.3% 0.001
Grade
Low 50.0%
High 80.6% 0.02
Histology
Serous 73.6%
Other 80.8% 0.48
P=0.004
Univariate Analysis
Thaker…….Sood, Clin Cancer Res, 2004

25. EEpphhAA22 EExxpprreessssiioonn aanndd CClliinniiccaall
PPaarraammeetteerrss iinn OOvvaarriiaann CCaanncceerr
Multivariate analysis
Variable Decreased survival
Residual disease p<0.04
EphA2 Overexpression p<0.01
Grade NS
Stage NS
Thaker…….Sood, Clin Cancer Res, 2004

26. AAnnttii--hhuummaann EEpphhAA22 ssiiRRNNAA
ddoowwnnrreegguullaatteess EEpphhAA22 iinn vviittrroo
EphA2
b-actin
No tx
siRNA
Non-silencing
No tx
No tx
siRNA
EphA2-targeted
48 hrs
2 days
72 hrs
4 days
2 days
6 days
2 days

27. EEpphhAA22--ttaarrggeetteedd ssiiRRNNAA//DDOOPPCC
ddoowwnnrreegguullaatteess EEpphhAA22 iinn vviivvoo
control siRNA / DOPC EphA2 siRNA-naked EphA2 siRNA / DOPC

28. TThheerraappyy SScchheemmaa
1 2 3 4
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
* SKOV3ip1 or HeyA8
Week:
Intraperitoneal cell* injection:
siRNA (150mg/kg) tx:
paclitaxel (100mg) tx:
sacrifice:

29. SKOV3ip1 ttuummoorr wweeiigghhtt aafftteerr
EEpphhAA22--ttaarrggeettiinngg ssiiRRNNAA tthheerraappyy
Mean tumor weights Individual weights
Empty Liposomes
Non-silencing siRNA/DOPC
EphA2 siRNA/DOPC
Paclitaxel +Non-silencing
Paclitaxel +
EphA2 siRNA/DOPC
siRNA/DOPC
Tumor Weight (g)
p=0.020
p=0.57
p<0.001

30. HeyA8 ttuummoorr wweeiigghhtt aafftteerr
EEpphhAA22--ttaarrggeettiinngg ssiiRRNNAA tthheerraappyy
Tumor Weight (g)
Mean tumor weights Individual weights
p=0.036
p=0.155
p<0.003
Empty Liposomes
Non-silencing siRNA/DOPC
EphA2 siRNA/DOPC
Paclitaxel +Non-silencing
Paclitaxel +
EphA2 siRNA/DOPC
siRNA/DOPC

31. Efficacy ooff IIPP ddeelliivveerryy ooff
EEpphhAA22--ssiiRRNNAA//DDOOPPCC:: SSKKOOVV33iipp11
0.87
0.21
0.04
0.13
0.04
1.5
1.0
0.5
0.0
Mean tumor weights Individual weights
cntrl EphA2 cntrl
Tumor Weight (g)
siRNA: cntrl EphA2
delivery: IV IP IP IV IV
paclitaxel: NO + + + +
Landen……..Sood, AACR, 2006

32. EEffffiiccaaccyy ooff IIPP ddeelliivveerryy ooff
EEpphhAA22--ssiiRRNNAA//DDOOPPCC:: HHeeyyAA88
Mean tumor weights Individual weights
2.16
0.66
0.34 0.42
0.23
4.0
3.0
2.0
1.0
0.0
Tumor Weight (g)
siRNA: cntrl cntrl EphA2 cntrl EphA2
delivery: IV IP IP IV IV
paclitaxel: NO + + + +
Landen……..Sood, AACR, 2006

33. SIRNA 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 mg/kg

34. SIRNA 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 mg/m2, 750 mg/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

35. Effects on immune parameters

36. DDiicceerr aanndd DDrroosshhaa –– VVaalliiddaattiioonn SSttuuddiieess
Ovarian cancer
New Engl J Med, 2008

37. FFuunnccttiioonnaall IImmppaacctt ooff LLooww DDiicceerr
New Engl J Med, 2008

38. CClliinniiccaall TTrriiaall DDeessiiggnn
IND 072924

39. CClliinniiccaall PPrroottooccooll
IINNDD 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.
.

40. SSiiRRNNAA EEpphhAA22:: PPhhaassee II SSttuuddyy
 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

41. 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)

42. SSiiRRNNAA EEpphhAA22:: PPhhaassee II SSttuuddyy
 Eligibility:
 Solid tumors - recurrent or
considered incurable with
standard therapy
 Bi-dimensionally
measurable disease (>2
cm)
 Amenable to biopsy
 EphA2 overexpression
(IHC)
DDoossee MMuull
tt.. FFrreeqquueennccyy
445500 μμgg//mm22 -- 22//wweeeekkllyy
990000 μμgg//mm22 110000
%% 22//wweeeekkllyy
11880000 μμgg//mm22 110000
%% 22//wweeeekkllyy
33660000 μμgg//mm22 110000
%% 22//wweeeekkllyy
77220000 μμgg//mm22 110000
%% 22//wweeeekkllyy

43. SIRNA EPHA2
Murine Bridge Study
•Three groups of mice, 5 males, 5 males in each
•Study Design Assess toxicology when EPHARNA was
administered twice weekly intravenously
• Conclusion: All mice administered the test article
completed the study with no clinical findings.

44. Acknowledgements
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