Post on 27-Jan-2021
1 © 2020 Alnylam Pharmaceuticals, Inc.
September 27, 2020
The ADME of siRNA GalNAc Conjugates Chris MacLauchlin, PhD
2
• GalNAc Absorption/PK• GalNAc Distribution• GalNAc Metabolism• GalNAc Excretion• GalNAc DDI profiles• GalNAc PK/PD insights
Presentation Outline
3
Innovation in siRNA to Target Tissues
Large MW ~16KNegatively charged (Na+ salt)Highly polarAqueous sol >200mg/ml
4
GalNAc-siRNA for Targeted Delivery to Liver
Asialoglycoprotein Receptor (ASGPR)• Highly expressed on hepatocytes• High capacity receptor• Conserved across species
Hepatocyte specific ligand• Trivalent GalNAc conjugated to sense strand• High affinity and specificity to ASGPR
HN
O
O
O
O
O
O
HN
HN
HN
HN
NH
NH
O
OHOH
HOAcNH
O
O
O
OHOH
HOAcNH
O
O
O
OHOH
HOAcNH
O
O
N
OH
OO
O
P
O
OO
Carito et alACVP Meeting 2016
RISC=RNA-Induced Silencing Complex
5
In Vivo Fate of GalNAc-siRNA after SC Administration
SC injection site
Systemic Circulation Liver
KidneyMetabolism
Excretion
BiliaryExcretion
Metabolism
ASGPR
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Plasma PK parameter
Rat Monkey Human
Cmax1[(µg/mL)/(mg/kg)]) 0.130 0.286 0.284
AUClast1 [(hr*µg/mL)/(mg/kg)]) 0.373 1.31 2.95
Tmax (hr) 1.0 1.8 4.1
T1/2 (hr) 0.9 1.9 4.8
• Low plasma exposure with short half-life due to targeted rapid liver uptake• Similar plasma PK conserved across species, scaled by allometry
Pharmacokinetics: Transient Plasma Exposure Due to Rapid Liver Uptake
Plasma Exposure Considerably Greater in the Absence of Liver-Targeted Delivery
1 Dose Normalized. Data from vutrisiran
Plasma PK Parameters Across Species
0 1 2 3 4 5
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
P la s m a C o n c e n tra t io n (n g /m L )
T im e p o s t-d o s e (h o u rs )
siR
NA
Pla
sm
a C
on
c (
ng
/mL
) T ri-G a lN A c
N o G a lN A c
No liver uptake
With liver uptake
SC dosing of 2 mg/kg tool compounds (siRNA with and without GalNAc) to rats
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Rat and Monkey PK after IV infusion and SC
Rat Monkey
SC administration results in a slow absorption from the SC injection site and facilitates efficient hepatic drug uptake without saturation of the ASGPR
LLQ in plasma: 0.01 µg/ml LLQ in plasma: 0.01 µg/ml
8
Liver PK in Rat and Monkey Shows Preferential Distribution
Dramatic difference in plasma PK vs. liver PK
0 5 0 0 1 0 0 0 1 5 0 0
0 . 0 0 1
0 . 0 1
0 . 1
1
1 0
1 0 0
1 0 0 0
T i m e ( h )
Co
nc
en
tra
tio
n (µ
g/m
L o
r g
)
L i v e r
P l a s m a
R a t
0 5 0 0 1 0 0 0 1 5 0 0
0 . 0 1
0 . 1
1
1 0
1 0 0
1 0 0 0
T i m e ( h )
Co
nc
en
tra
tio
n (µ
g/m
L o
r g
)
L i v e r
P l a s m a
M o n k e y
t1/2~3-8h
t1/2~120h
t1/2~5h
t1/2~150h
Rat Monkey
9
Rat Radiolabeled ADME Study Conducted withThree GalNAc-siRNA Conjugates
F
3H
2’F-3H-adenosine
H
Conjugate-A
Conjugate-B
Conjugate-C
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Rat Radiolabeled ADME Study Design
N (sex) Sample Collection Purpose
IntactRat
4 (M) Blood, Urine, Feces, Carcasses
Urinary/Fecal Excretion, Mass-Balance, Met ID
IntactRat
12 (M) Blood, Carcasses for QWBA
Tissue-Distribution, PK, Met ID
BDCRat
6 (M) Blood, Urine, Bile, Feces, Carcasses
Biliary Excretion, Met ID
QWBA=Quantitative whole-body autoradiographyBDC=Bile-duct cannulated (7 days only)
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Radiolabeled ADME Study Conducted withThree Conjugates in Intact Rats
Conjugate-A Conjugate-B Conjugate-C
% of Dose Excreted 81 76 82
% of Dose in Carcass 3 3 1
Total % of dose Recovered 84 79 83
Conjugate-A Conjugate-B Conjugate-C
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Biodistribution Studies Confirm On-Target DeliveryConjugate-A (40µm thick)
4hr0.5hr 2hr 96hr 336hr 1344hr
• Rapid distribution to the liver with tmax 4-6hr• Few other organs show significant levels of radioactivity.
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How Do GalNAc-siRNAs Get Metabolized?
3' & 5'-Exonuclease• End products are mononucleotidesEndonuclease• Cleaves internally
Localization• Exo- and endonucleases are ubiquitously distributed in both plasma and tissuesSpecies difference• Much higher cross species similarity compared to conventional drug metabolizing
enzymes
5’ 3’
5’ Exo- 3’ Exo-
Endo-
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5′
5′
Metabolites from 3ʹ -end antisense strand are typically active with comparable potency
Sense Strand
Anti-sense strand
14
M15: AS(N-1)3’ (pl, urine, bile)M11 (bile)M14 (urine, bile)M13 (bile)
M10 (bile)
M8 (bile)
5’ 3’Antisense Strand
• M15: AS(N-1)3’ metabolite is the major metabolite in all matrices
Metabolite ProfilesMetabolism of 3H-labeled ( ) siRNA in rats
15
GalNAc moieties are quickly cleaved after uptake into hepatocytes The linkers are metabolized by hydrolysis of the amide bonds
Metabolism: Linker and Ligand
HN
O
O
O
O
O
O
HN
HN
HN
HN
NH
NH
O
OHOH
HOAcNH
O
O
O
OHOH
HOAcNH
O
O
O
OHOH
HOAcNH
O
O
N
OH
OO
O
P
O
OO
15
Sense Strand Metabolism
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Species Conjugate-A Conjugate-B Conjugate-A
Rat 15-22%(58% total radioactivity)11%
(53% total radioactivity)9%
(68% total radioactivity)
Monkey 1% 19% 11-24%
Human 6 – 32% 3 – 17% 10 - 25%
• Renal elimination across compounds is low (
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Biliary Excretion
Conjugate-A Conjugate-B Conjugate-C% of Radioactivity Dose Recovered
Bile Feces Bile Feces Bile FecesIntact Rat 14 15 7BDC Rat 27 24 17
• Total radioactivity recovered in feces (intact rat) was lower than the total radioactivity excreted in bile (BDC rat), suggesting reabsorption of radioactivity via enterohepatic recycling
• Mostly full length and AS(N-1)3’ metabolite found in the bile
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Biliary Excretion (Conjugate-A)
• Classical double peak observed (enterohepatic recycling)• No Conjugate-A or AS(N-1)3’ metabolite detected in plasma• Full length and AS(N-1)3’ metabolite are further cleaved in the GI tract to
shortmers, and get reabsorbed
AS(N-1)3’Conjugate-A
Retention Time (min)
Rad
ioac
tivity
(CPM
) Shortmers
0 2 0 0 4 0 0 6 0 0 8 0 0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
ng/m
l equ
ivin
Pla
sma
Double Peak
Time (hr)
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siRNA
Lysopaint
Merge
GalNAc-siRNAs Co-localize With Lysosomes
• Free uptake (image taken 4 hours post-dose)• Lysosomes (Lysopaint)• siRNA(AD-326763, 10 nM)• Knockdown not seen until 6 hours
Mouse Hepatocytes (Live cells)
Brown, Nucleic Acids Research, 2020: 1-18.
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DDI Unlikely
DDI Possible
DDI Strategy at Alnylam
• Mechanistically siRNA DDIs seem unlikely
• Given the limited amount of data and unknown regulatory expectations a traditional approach to DDI evaluation was taken
• Retrospective analysis of the data has allowed a shift in amount of DDI data needed (data has been published: Ramsden D et al., DMD 2019 )
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Drug-Drug Interaction Potential: Summary
Major CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5) Major transporters (P-gp, BCRP, BSEP, MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2)
Ramsden D et al., DMD 2019
GalNAc-siRNAs are not:• Substrates of major CYPs or
transporters• Inducers of drug metabolizing
enzymes• Likely to cause clinically meaningful
inhibition of CYPs or transporters
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0 7 1 4 2 1 2 8 3 5 4 2
0 .1
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
Fit
us
iran
Co
nce
ntr
atio
n(n
g/m
L o
r n
g/g
)P
lasma A
T P
rotein
(% o
f Ba
se
line)
T im e p o s t-d o s e (D a y )
F itu s ira n in P la s m a (n g /m L )
F itu s ira n in L iv e r (n g /g )
F itu s ira n in R IS C (n g /g )
A T P ro te in (% o f B a s e lin e )
siR
NA
Con
cent
ratio
n (n
g/m
L or
ng/
g)PD
(biomarker in plasm
a) (%
of Baseline)
Time post-dose (Day)
Plasma PK
Liver PK
RISC PK
PD
Plasma PK, Liver PK, RISC PK and PD in Mice
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Cross Platform PK/PD Insights
0 3 6 9 1 2
0
2 0
4 0
6 0
8 0
D o s e ( m g / k g )
AU
Cla
st
(h*µ
g/m
L)
R2
= 0 . 9 0 1
(8 Conjugates)
0 2 4 6 8 1 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
D o s e ( m g / k g )
AU
Cla
st
(h*µ
g/g
)
R a t L i v e r
R2
= 0 . 8 2 7
0 2 4 6 8 1 0
0
3 0 0 0 0
6 0 0 0 0
9 0 0 0 0
1 2 0 0 0 0
D o s e ( m g / k g )
AU
Cla
st
(h*µ
g/g
)
M o n k e y L i v e r
R2
= 0 . 7 6 3
Human Plasma Exposure
Monkey and rat liver exposure is similar across platform Human plasma exposure is similar across platform
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ADME Summary of GalNAc-siRNAs
SC injection site
Circulation
Near completeabsorption
Predominantly delivered to liver
• ASGPR-mediated hepatocyte uptake
• Ligand deglycosylation• Intracellular trafficking
and release
RISC loading
Functional RISC
Degraded mRNA
mRNA cleavage
Biliary Excretion
Fecal Excretion
Renal Excretion
Metabolites only
Metabolites only
Intracellular: Trafficking and RISC LoadingHepatocyte UptakeSC Injection
Catalytic
Circulation
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AcknowledgmentsJing-Tao WuSaeho ChongYuanxin XuDiane RamsdenJing LiJu LiuXiumin LiuRobin McDougallJeff KurzXuemei ZhangChris TranValerie ClausenKirk Brown
Alnylam Early Development Group
Alnylam RNAi Platform Team
Chris BrownMartin MaierIvan ZlatevVarun GoelPete Smith
26 © 2020 Alnylam Pharmaceuticals, Inc.
To those who say “impossible, impractical, unrealistic,” we say:
CHALLENGE ACCEPTED
cmaclauchlin@alnylam.com
The ADME of siRNA GalNAc Conjugates Presentation OutlineInnovation in siRNA to Target TissuesGalNAc-siRNA for Targeted Delivery to LiverIn Vivo Fate of GalNAc-siRNA after SC AdministrationSlide Number 6Rat and Monkey PK after IV infusion and SCLiver PK in Rat and Monkey Shows Preferential DistributionSlide Number 9Slide Number 10Slide Number 11Conjugate-A (40µm thick)How Do GalNAc-siRNAs Get Metabolized? Slide Number 14Slide Number 15Slide Number 16Biliary ExcretionBiliary Excretion (Conjugate-A)GalNAc-siRNAs Co-localize With LysosomesDDI Strategy at AlnylamSlide Number 21Slide Number 22Cross Platform PK/PD InsightsADME Summary of GalNAc-siRNAsSlide Number 25Slide Number 26