Upload
ledien
View
235
Download
5
Embed Size (px)
Citation preview
1
Preclinical Development of ALN-AS1, an
Investigational RNAi Therapeutic for the
Treatment of the Hepatic Porphyrias
Amy Chan, Abigail Liebow, Makiko Yasuda, Scott Barros, Tim Racie, Martin Maier, Satya Kuchimanchi, Don Foster, Stuart Milstein, Klaus Charisse, Muthiah Manoharan, Robert Desnick, Rachel Meyers, Kevin Fitzgerald, Amy Simon, and William Querbes
International Conference of Poprhyrins and Porphyrias
15 September 2015
2
Therapeutic Approach to Harnessing RNAi
dsRNA dicer
Cleavage
Natural Process of RNAi
Synthetic siRNA
mRNA
mRNA degradation
Strand separation
Complementary pairing
Cleavage
(A)n
(A)n
Targeted Gene
Silencing
RISC
3
Receptor Targeted siRNA Conjugates for Delivery to Hepatocytes
Lead Selection & Optimization Subcutaneous Delivery
siRNA sequence » Specificity » Crossreactivity » Potency
Introduce chemical modifications for
“drug-like” properties
Small Scale Gene walks In vitro assays
Chemistry, Manufacturing and Controls
Medium Scale In vivo biology
Large Scale GMP Production Clinical trials
Selection
Optimization
GalNac-siRNA
Conjugates
siRNA chemistry » Stability » Immuno-silence » Potency
ASGPR Receptor » Highly expressed
on hepatocytes » Rapidly recycles » Conserved across
species
4
Clinical Experience with GalNac-siRNA Conjugates
GalNAc Conjugate Platform
• Currently utilized in 6 clinical stage ALNY
programs
• Over 250 patients and >1500 doses
administered
• Generally well tolerated
Human Proof of Concept
Ex: TTR Lowering in TTR Amyloidosis Program
Human Clinical Efficacy
Ex: Reduced Bleed Rate in Hemophilia Program
Gilmore, ACC, March 2015, Sorenson, ISTH, June 2015
Alnylam Development Pipeline
5
ALN-AS1 Therapeutic Hypothesis
Brennan et al. Nature 1979, Hermes-Lima et al. Biochem Biophys Acta 1991, Dar et al., Hepatobiliary Pancreat Dis
Int.; 9:93-6 (2010), Dowman et al., Ann Intern Med; 154:571-2 (2011)
Targeting Liver ALAS1 to Lower Heme Intermediate Production
ALN-AS1
Role of Liver in Disease
• Liver and domino transplant
experience
◦ ALA/PBG and symptoms
improve or come on rapidly
after transplant
• ALN-AS1 potential to treat
all acute hepatic porphyrias
including AIP, VP and HCP
6
ALN-AS1 Preclinical Development Overview
Lead ID Disease
Model POC NHP
Evaluation
Drug Safety and
Metabolism
Phase 1 Testing
• Identify ALAS1 target sequence and optimized chemistry
• Liver ALAS1 knockdown in rodents
• Impact of ALN-AS1 on ALA/PBG
• Define ALAS1 and ALA/PBG relationship
• Assay Development
• ALAS1 lowering kinetics
• Dose and dose regimen exploration
• Tolerability of ALN-AS1 in different species
• PK/PD relationships
• Evaluate any potential on target risks
Key
Data
7
ALN-AS1 Multidose Prophylaxis in Rat AIP Model
ALAS-GalNAc 3, 1, 0.3mg/kg (QWx4)
Phenobarb
D18 PBGD
LNP
D0 D7 D14 D21 D25 (sac)
ALAS1 mRNA
0.0
1.0
2.0
3.0
4.0
4.5
PBS ALN-AS1
3mg/kg 1mg/kg 0.3mg/kg
- + + + +
+ + + + +
rAL
AS
1/r
GA
PD
H,
Re
lative
to
pb
s=
1
PBGD LNP
siRNA
PB
ALA/PBG
0
2
4
6
8
10
PBS 3 mg/kg 1 mg/kg 0.3 mg/kg
- + + + +
+ + + + +
Urinary
ALA
or
PB
G
(m
mol/m
ol c
reatinin
ine)
PB
S=
1
-
PBGD LNP
ALN AS1
PB
PBG ALA
8
ALN-AS1 Tissue Distribution in Rats
Exposure is the highest in the liver, followed by kidney; remaining tissues have
<2% of delivered dose deposited
Single Dose 10mg/kg 4 hours
0
1
10
100
1000
AL
N-A
S1 (
µg
/g)
Less than 2% of total exposure combined
~12X lower tissue concentration kidney vs. liver
9
Circulating Extracellular RNA Detection (cERD)
Monitoring RNAi Activity in Liver
• mRNA or 5’RACE product in tissue
• Circulating secreted protein
Detection of Circulating ALAS1 mRNA
• Exosomes are shed into bodily fluids from many
different cell types and contain mRNA and miRNA
derived from tissue of origin
• Exosomes can be used to monitor ALAS1 mRNA
levels after ALN-AS1 dosing in serum/urine without
need for biopsy
Method for Circulating ALAS1 mRNA Detection
0
20
40
60
80
100
120
PBS 1.25 2.5 5.0
ALAS-GalNAc
(mg/kg) N
orm
ali
ze
d A
LA
S-1
by cERD
by liver biopsy
AS1 Transcript
QDx5, EOD, d15 (not DC)
Sehgal et al. RNA 2012
10
Use of cERD Assay to Monitor ALN-AS1
Pharmacology in NHPs
QW-weekly
11
Liver ALN-AS1 Concentrations and ALAS1
mRNA Knockdown in NHP
Single Dose 10mg/kg Multiple Dose 2.5mg/kg
• ALN-AS1 liver drugs levels correlate with extent and duration
of ALAS1 mRNA reduction
Weekly x8
12
0
100
200
300
400
500
600
700
NH
V 1
NH
V 2
NH
V 3
NH
V 4
11304
11313
11305
11309
11311
11319
11337
11513
11320
12049
Re
lati
ve
AL
AS
1 m
RN
A L
eve
ls (
% N
HV
)
Serum Urine
cERD Assay Demonstrates ALAS1 Upregulation
in Porphyria Patients vs. Healthy Volunteers
• ~3-fold upregulation of ALAS1 mRNA in AIP patients (n=10) vs. NHV (n=4)
• Good correlation of ALAS1 mRNA in urine and serum
• Chart review suggests trend of ALAS1 upregulation correlating with worse disease
severity
AIP Patients Normal Healthy
Volunteers
13
Day to Day Variability in ALAS1 Levels with
cERD Assay in AIP Patient Samples
Human cERD Proof of Concept
• Minimal day to day variability
• Potential clinical utility
◦ ALAS1 changes during attacks
◦ Compare patient populations
◦ Monitoring PD with ALN-AS1 treatment
• Exploratory use in our EXPLORE natural
history study and in Phase 1
0
50
100
150
200
250
300
350
113
04-
Colle
ction 1
113
04-
Colle
ction 2
113
13-
Colle
ction 1
113
13-
Co
llectio
n 2
Rela
tive
AS
1 m
RN
A L
eve
ls (
% N
HV
)
Donor ID
Serum Urine
AIP Patient 1 AIP Patient 2
14
ALN-AS1 Nonclinical Safety and Metabolism
Safety margins of >300x (rat) and >1500x (NHP), based on respective
no adverse effect levels (NOAELs) and clinical starting doses (mg/kg)
CTA-Enabling Studies for Start of Phase I Clinical Trial
Exploratory Studies Cytokine screening in vitro (hWBA) and in vivo (mouse) No acute pro-inflammatory
effects
Dose range-finding toxicity studies in mouse, rat, NHP
(repeat dose, 3-5 weeks in duration)
Establish dose levels for GLP
studies
GLP-Compliant
Safety Studies
Safety pharmacology (cardiovascular and respiratory) in
telemetered NHPs
NOAEL ≥150 mg/kg
Genetic toxicology in vitro (Ames, chromosomal
aberrations in hPBL) and in vivo (rat BM micronucleus)
All negative up to limit doses
General Toxicology studies in rat and NHP
• 13 week studies + 13 week recovery
• Toxicokinetics and CNS evaluation (NHP)
Rat NOAEL ≥ 30 mg/kg
NHP NOAEL ≥ 150 mg/kg
Absorption,
Distribution,
Metabolism,
Excretion (ADME)
Studies
PK/PD in rat, NHP
Toxicokinetics included in all Tox studies (plasma; liver and kidney in rodents)
Metabolic profiling in rat, NHP (plasma, liver)
Plasma protein binding (mouse, rat, NHP, human)
In vitro, ex vivo CYP studies
15
ALN-AS1 Impact on P450s No Signs of Heme Deficiency in Preclinical Studies
Study/Species Doses CYPs Results
In vitro direct CYP
inhibition, human liver
microsomes
0.01-
10,000
ug/mL
1A2, 2B6,
2C8, 2C9,
2C19, 2D6,
3A4/5
No inhibition of
any isoform
In vitro CYP induction,
primary human
hepatocytes
0.1, 1, 10,
100 ug/mL
1A2, 2B6,
3A4
No changes in
CYP levels or
enzyme activity
Ex vivo CYP Activity
after ALN-AS1 dosing,
rat liver microsomes
30, 100,
300 mg/kg,
weekly x5
1A, 2B, 2D,
2E, 3A
No biologically
significant
changes
Ex vivo CYP Activity
after ALN-AS1 dosing,
NHP liver microsomes
30, 100,
300 mg/kg,
weekly x5
1A, 2B, 3A,
2D, 2E
No biologically
significant
changes
In vivo DDI with CYP
substrate probes
following ALN-AS1
dosing, NHP
5 mg/kg,
daily x5
1A, 2D, 2E,
3A
No reduction in
metabolism of
any CYP
substrate probes
No significant reduction
in P450 function or signs
of heme deficiency
detected with ALAS1
lowering
3A4,5 29%
2C8,9,18
18% 1A2 13%
2E1 7%
2D6 2%
2B6 1%
2A6 4%
Other 26%
~75% of total human liver CYPs
evaluated in these studies
16
Conclusions and Next Steps for ALN-AS1
Conclusions
• ALN-AS1 is a subcutaneous administered RNAi therapeutic targeting ALAS1 in development for
the treatment of the hepatic porphyrias
◦ Potent silencing of liver ALAS1 mRNA and reduction in ALA/PBG in preclinical models
◦ Robust safety profile supporting the initiation of Phase 1
• The cERD assay is a non invasive method for monitoring circulating ALAS1 mRNA
◦ ALAS1 mRNA upregulated in serum/urine from AIP patients consistent with role in disease
pathophysiology
◦ Potential utility of the assay to understand ALAS1 levels during attacks, in different patient
populations, and following ALN-AS1 dosing in clinical trials
ALN-AS1 Clinical Development Plan
• EXPLORE natural history study (ongoing)
◦ Non interventional study in AIP patients with recurrent attacks
• Phase 1 Study (ongoing)
◦ Part A/B- Single ascending dose (SAD) and multiple ascending dose (MAD) study in AIP
asymptomatic high excreter patients (ASHE)
◦ Part C- Multiple dose study in AIP patients with multiple recurrent attacks
• Pivotal study in patients with recurrent attacks
17
Acknowledgements
ALN-AS1 Core Team
• Bill Querbes (PL)
• Tanya Sengupta (PM)
• Amy Simon
• Craig Penz
• Amy Chan
• Scott Barros
• Lauri Binne
• Jason Costigan
• Satya Kuchimanchi
• Anshul Gupta
• Rena Denoncourt
• Nate Taneja
• Jon O’Shea
• Sarfraz Shaikh
• Mano Manoharan
• Rajeev Kallanthottathil
• Jeff Rollins
• Lubo Nechev
• John Frenz
• Jolly Bhatia
• Patrick Igwenagu
• Husain Attarwala
• Renta Hutabarat
• Ju Liu
• Krishna Aluri
• Chris Tran
• Yongli Gu
• Minggeng Gao
• Qianfan Wang
• Xuemei Zhang
• Mary Carioto
• Julia Hettinger
• Garvin Warner
• Mike Placke
• Matt Algarin
• Dave Mosher
• Svetlana Shulga Morskaya
• Meghan Collins
• Tim Mooney
EXPLORE Investigators
• Karl Anderson
• Herb Bonkovsky
• Montgomery Bissel
• John Phillips
• Charles Parker
• Manisha Balwani
• Joseph Bloomer
• Pauline Harper
• Eliane Sardh
• David Rees
• Mike Badminton
• Penelope Stein
• Raili Kauppinen
• Ulrich Stölzel
• Jorge Frank
• Elisabeth Minder
• Jean Charles Deybach
• Laurent Gouya
• Pavel Martesek
• Janne Langendonk
• Sverre Sandberg
• Delia D’Avola
• Maria Ivanova
• Paulo Ventura
• Maria Capellini
• Jerzy Wingyga
• Peter Meissner
• Joanne Marsden
ALN-AS1 Advisors and Extended Team
• Abigail Liebow
• Tim Racie
• Brian Bettencourt
• Kirsten McCarthy
• Siddharth Jain
• Don Foster
• Stu Milstein
• Rick Duncan
• Jared Gollob
• Martin Maier
• Klaus Charisse
• Matthias Kretschmer
• Lauren Melton
• Akshay Vaishnaw
• Jeff Cehelsky
• Kevin Fitzgerald
• Rachel Meyers
• Dave Watkins
• Oved Amitay
• Sara Nochur
• Carol Pitcher Towner
• Donna Mackey
• Ligang Zhang
• Lubomir Tchangov
• Karen Dobson
• Jeff Kopacz
• Jennifer Pena
• Kelley Hanson
Preclinical
Collaborators
• Robert Desnick
• Makiko Yasuda
• John Phillips
APF
• Desiree Lyon
• Jessica Hungate
• Natalia Sturza
APC Coordinator
• Hetanshi Naik
Phase 1 Investigators
• Eliane Sardh
• Nabil AL-Tawil
• Pauline Harper
• David Rees
• Penelope Stein
• Manisha Balwani
• Karl Anderson
• Monty Bissell
• Joseph Bloomer