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
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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
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ASO Mechanisms of Action
DeVos SL, Neurotherapeutics. 2013, 10(3):486-97
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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
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