ADME and PK/PD Considerations for Antisense Oligonucleotide Therapeutics

Xiao Shelley Hu, PhD

Director, Head of DMPK and Clinical PharmacologyWave Life Sciences

June 12, 2019

2

Outlines• Introduction to antisense oligonucleotides (ASOs)

• ADME considerations

• PK/PD considerations

• Summary

3

History of oligonucleotide therapeutics

Backbone modifications• Introduce chiral centers• Generate mixtures

Sugar modifications Drug approvals (FDA)

Chiral Phosphorothioate Chiral Phosphorodiamidite Morpholino (PMO)

Mixtures of 2n molecules(n=No. of chiral centers)

SEQUENCE

STEREOCHEMISTRY

CHEMISTRY

Fomiversen

Pegaptanib

Mipomersen

Patisiran

Stereopure ASOs enter clinic

Wave Stereopure ASOs

Oka N, Wada T, Saigo K.JACS. 2002

Stec WJ, et al.J Am Chem Soc. 1989

~500,000 differentmolecules per dose

Tegsedi

EteplirsenNusinersen

4

ASO Mechanisms of Action

DeVos SL, Neurotherapeutics. 2013, 10(3):486-97

5

Wave backbone chemistryRp or Sp LinkerNucleotide

Uncontrolled Controlled

RpSp

Nucleotide

~0.5 million molecules 1 molecule

Traditional backbone chemistry

Stereorandom Stereopure

Wave chemistry controls nucleic acid backbone chirality

>500,000 permutations in every dose One defined and consistent profile

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Precedents: Overview of Preclinical ADME packages

In vitro In vivo

Transporter ProteinBinding

CYP inhibition/CYP induction

Hepatic/metabolic stability

Plasma PK QWBA Metabolite Profiling

Mass balance/Urinary Excretion

Eteplirsen √ √ √ √ √ √ √ √

Mipomersen √ √ √ √ √ √ √ √

Drisapersen No √ √ √ √ √ √ √

Nusinersen √ √ a √ √ √ No √ No

Pegaptanib(Macugen) No No No √ √ √ √ √

a Plasma and CSFQWBA: quantitative whole body autoradiography,

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ADME Considerations - Absorption• Considerations for bioanalytical method selection

– Sensitivity– Specificity– Pharmacologically relevant concentration– Understand the assay

• Duration– Sufficient to characterize elimination phase

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Commonly utilized platforms for quantitation of antisense oligonucleotides (ASO)

Technology Hybridization ELISA LC-MS (triple quad/HRMS)

Hybridization LC-FL

Main concept Binding Mass Binding

Sensitivity (LLOQ, ng/mL) High: ≤1 Moderate 10 High, ≤1

Specificity Moderate Highest High

Dynamic Range Low High High

Complexity Moderate High Moderate

Throughput LowModerately long

method development

Reasonable method development

LowModerately long

method development

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Relative selectivity of ASO-1 in Ligation HELISA

Depending on the 3’- or 5’-end liabilities, probes can be designed to increase selectivity

0 10 20 30 40 50 600

5000

10000

15000

20000

25000

30000

35000

Conc. of ASO (ng/mL)

RFU

Full length Oligomer3' N-1 mer3' N-2 mer5' N-1 mer

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Characterization of Full PK Profile

AUC0–last=area under the plasma concentration-time curve from time zero to the last quantifiable concentration; Cmax=maximum plasma concentration; EOI=end of infusion; t1/2=terminal half-life.

Characteristic, mean (SD)

Suvodirsen5 mg/kg

n=6

Cmax, μg/mL 40.7 (21.4)

AUC0-last, μg⋅h/mL 81.0 (43.6)

t1/2, h 20.7 (15.0)

• High sensitivity enables full characterization of PK Profiles

• Support adequate PK model: 3 compartment model

Wagner, MDA conference, Orlando, FL, 2019

0.1

1

10

100

1000

10000

100000

Time Postinfusion

Pla

sma

Con

cen

trat

ion

, n

g/m

L

Suvodirsen 0.5 mg/kg Suvodirsen 1 mg/kg Suvodirsen 2 mg/kg Suvodirsen 5 mg/kg

EOI 12 h 24 h 7 d

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ADME Considerations - Distribution• On-target and off-target concentrations are usually measured

• Selective high tissue uptake

• Using plasma concentration to predict tissue concentration

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ADME Considerations - Metabolism• Metabolized by endo-nuclease and exo-nuclease

• Metabolism in all tissues

• Metabolite: shortened metabolites

• May be pharmacologically active

• May interfere with the assay

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Characterization of Metabolism in AnimalsDrug Species Metabolites

Drisapersena Mouse, monkey 3’-shortened metabolites up to N-13

Eteplirsen In vitro mouse, rat, monkey, and human hepatic microsomes

Not significantly metabolized

Inotersen Mouse and monkey 6- to 12-mers

Mipomersena Monkey Cleavage at central 10-base; 7-15 bases in lengths

Nusinersen Monkey Plasma: 3’ N-1 (2%); tissue: 3’ N-1 (20%), 3’ N-2, N-3, N-4, 5’ N-1, N-2, N-3 (each <5%)

Patisiran In vitro S9 of mice, rats monkey and human

Comparable degradation by nuclease

bFDA Pharmacology review

• Patterns were consistent between animals and patients

• Regulatory metabolite guidance on small molecule metabolite applies

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ADME Considerations - Excretion• Parent, shortened metabolites, and modified monomers in urine were usually measured

using radio-labeled method or LC/MS over certain period of time (e.g. 24 hours)

• Parent and metabolites were recovered in urine

• Clearance pathway:– Urinary excretion– Shortened metabolites followed by urine excretion

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Characterization of Urine Excretion in AnimalsDrug Species Urine Recovery

Drisapersena CD-1 Mouse, MDX mouse 25 %(CD-1) and 12.4 % (MDX) within 24 h

Eteplirsenb Rat 60% as parent over 2 weeks

Inotersenb Mouse, rat 2-8% excreted intact in mice; 45% over ~2 weeks in rats, mostly as shortened metabolites

Mipomersenc Mouse, rat, monkey <10% over 24 h (parent + shortened metabolites)

Nusinersenb Not evaluated

Patisiranb Information unavailable

aEMA Kyndrisa Assessment reportbFDA Pharmacology reviewcGeary, Clin Pharmacokinet, 2015(54):133–146

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PK/PD Study to Support Drug Development

Kimko H and Pinheiro J. British Journal of Clinical Pharmacology. 2015

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Pharmacologically Active Dose Projection• MOA: mRNA knockdown

• Pharmacological model: transgenic mouse model

• PD markers: mRNA and protein

• Project pharmacologically active dose in mouse for various dosing regimen

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mRNA Knockdown model: PK/PD Model Structure

Absorption Elimination

Synthesis (Kin)

Elimination (Kout)

PK

mRNA

Inhibition(Imax, IC50)

Central

Periph

mRNA

Protein Degradation

Synthesis: proportional to (b*mRNAa)

Protein

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PK/PD Model for a Tool Molecule

• A PK/PD model was built and described the data adequately

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Simulation for Various Dosing Regimen in Mice

• Both 50 μg Q2M and 100 μg Q3M are sufficient to reduce protein level by ~50%

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Summary• Basic DMPK principles of small molecules are applicable to ASOs

• Special considerations should be taken into account for BA assay development and metabolite characterization

• Measurable PD markers for ASOs enable extensive application of PK/PD model to inform drug development

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Acknowledgement

• Jaya Goyal, PhD

• Hyun Gyang Jang, PhD

• Pallavi Lonkar, PhD

• Ravindra Kodihalli, PhD

• Susovan Mohapatra, PhD

• Michael Panzara, MD, MPH