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

6

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

7

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-

14

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