Preclinical safety assessment of aptamer therapeutics
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Preclinical Safety Assessment of Aptamer Therapeutics. Scott A. Barros, PhD, DABT Sr. Scientist, Toxicology. What is an Aptamer?. apto: “to fit” mer: “smallest unit of repeating structure”.

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Preclinical safety assessment of aptamer therapeutics l.jpg

Preclinical Safety Assessment of Aptamer Therapeutics

Scott A. Barros, PhD, DABT

Sr. Scientist, Toxicology


What is an aptamer l.jpg
What is an Aptamer?

apto:“to fit”

mer:“smallest unit of repeating structure”

Aptamers are single stranded folded oligonucleotides that bind to molecular (protein) targets with high affinity and specificity


Aptamer structure l.jpg

Nature Structural Biology, 7(1):53-57

Aptamer Structure

  • Unique tertiary structures allow aptamers to fold into stable scaffolds for carrying out molecular recognition

  • van der Waals, hydrogen bonding, and electrostatic interactions drive high affinity target binding

  • Designed to block protein-protein interactions

  • Share properties of both small molecules and biologics

  • SELEX (Systematic Evolution of Ligands by Exponential Enrichment)

    • Tuerk and Gold (1990) Science 249, p505-510


Medicinal chemistry process l.jpg

optimized lead

early lead

single

substitutions,

nucleotide B, etc

single

substitutions,

nucleotide A

composites

composites

1

2

3

N

S

O

O

B

B

B

P

P

P

O

O

O

-O

-O

H3C

O

O

O

beneficial

tolerated

OMe

O

O

O

H

OCH3

P=O  P-Me

2’-OMe  2’-deoxy

P=O  P=S

2’-deoxy  2’-OMe

Medicinal Chemistry Process

  • Proprietary processes

  • Multiple chemistries employed

  • Increased plasma stability

  • Increased affinity

  • Increased potency


Considerations in safety assessment of aptamers l.jpg
Considerations in Safety Assessment of Aptamers

In general, aptamers have toxicological properties similar to other oligonucleotide therapeutics, but with a few modality-specific considerations:

  • The diversity and combinations of chemical compositions employed distinguish aptamers from other oligonucleotide therapeutic modalities

    • 15-40 mer, with variety of stabilizing 2’ ribose modifications and 3’-idT

    • Often with large molecular weight PEG conjugate

  • Species restriction and pharmacological activity

    • Species restriction is often observed; similar to mAbs

    • Two species toxicology testing, typically rat (off-target species) and monkey (on-target species)

  • Our goal is to keep aptamer at the site of action in the plasma and interstitial tissue compartments, outside of cells

    • Plasma concentration and plasma exposure is more of a focal point than tissue concentrations

  • Dose regimens vary widely depending on the aptamer compositions and the intended use

    • IV bolus, infusion, repeated bolus, SC bolus, etc.


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Discovery Toxicology

Purpose of Discovery Toxicology:

  • To detect potential development-limiting toxicological liabilities as early as possible and avoid or engineer them out

    Discovery Toxicology for Aptamers:

  • Thus far, the general toxicological properties of aptamer therapeutics have been mostly predictable, class-based, and with good safety margins for the intended uses

    • Therefore, we do not consider in vivo discovery toxicology important since we would only expect to find the predictable outcomes (discussed later)

  • But, we do not fully understand what attributes modulate the occurrence or potency of the known class-based effects (not yet fully predictable)

    • Therefore, we screen in vitro for oligo class-based toxicities during lead optimization in order to detect early and engineer if necessary

  • These in vitro screening assays include:

    • Anti-coagulation – Polyanion effect, sequence independent, influenced by composition

    • Complement activation – Polyanion effect, sequence independent, influenced by composition

    • Immune Stimulation – Sequence dependent, influenced by composition (TLRs)


In vitro complement activation l.jpg
In vitro Complement Activation

Assay method:

  • Add aptamer or control oligo to human serum or blood anti-coagulated with direct thrombin inhibitor

  • Incubate 37°C, 30 min

  • Quench complement reaction with EDTA

  • Assay for generation of C3a or C5a split products

Oligonucleotides, especially phosphorothioate oligos, can stimulate complement activation via Factor H or other mechanisms


In vitro anticoagulation l.jpg
In vitro Anticoagulation

Assay method:

  • Add aptamer or control oligo to citrated human plasma

  • Add aPTT reagent and calcium, and measure time to clot

Oligonucleotides, especially phosphorothiate oligos, inhibit coagulation, likely via intrinsic tenase complex (factors IXa and VIIIa, phospholipids, calcium)


In vitro immune stimulation screens l.jpg

IL-6 release from PBMCs

2000

1800

1600

1400

ARCxxx

1200

ARCyyy

pg/mL IL-6

1000

ARCzzz

800

CpG-B

600

400

200

0

1

10

100

1000

nM ODN

In vitro Immune Stimulation Screens

  • Cytokine release and proliferation assays measure TLR 3,7/8,9 activation

  • CpG oligonucleotides and transfected immunostimulatory RNAs induce PBMC/mouse splenocytes to produce IL-6 and interferon alpha

  • Class A and C type CpGs induce PBMCs and mouse splenocytes to proliferate

ARCxxx


Secondary pharmacology l.jpg
Secondary Pharmacology

  • “Off-target” protein interactions with ASOs have been referred to as “aptamer effects”

  • All oligonucleotides can have relatively low affinity interactions with unintended target proteins (polyanion effects)

  • This is to be distinguished from a therapeutic aptamer which has been selected and optimized for high potency interactions with a target protein

  • These “off-target” effects can manifest as secondary pharmacology, at some concentration

  • How do we test for secondary pharmacology?

    • Directed specificity testing depending on the target protein

    • Discovery in vitro toxicology screens (C’ activation, anti-coagulation, immune stimulation)

    • Receptor/enzyme panel screens

    • In vivo safety pharmacology and general toxicology


Safety pharmacology l.jpg
Safety Pharmacology

  • We adhere to the principles of ICH S7a

    • CNS:

      • Standard CNS study in rats

    • CV

      • hERG patch clamp

      • Telemetered cynomolgus monkey in vivo study

    • Respiratory:

      • Respiratory endpoints incorporated into cynomolgus monkey CV study

  • We have seen no significant test article related effects in these studies to date


Genetic toxicology l.jpg
Genetic Toxicology

  • We have conducted standard ICH battery of genetic toxicity studies

    • Ames assay

    • Human HPBL chromosomal aberrations

    • In vivo micronucleus (rat)

  • We have tested the final development compound in these assays (e.g., PEGylated) using standard practice for dose selections

  • All results have been negative for genotoxic effects


General toxicology principles l.jpg
General Toxicology - Principles

  • Species selection:

    • We conduct two species general toxicology testing

    • Rodents often non-pharmacologically responsive “off-target” species

    • Monkeys generally pharmacologically responsive “on-target and off-target species”

  • Route and regimens appropriate for the intended clinical use

    • Can vary widely (IV bolus, infusion, SC bolus; continuous, daily, weekly, etc)

    • Have successfully used single-dose toxicology to support single dose in man

    • Repeated-dose designs may mimic those for mAbs when PEGylated aptamer has long half-life (e.g., twice weekly dosing, etc)

  • Dose selection

    • Clinical equivalent (low), max feasible or chosen multiple of human (high), and log mean (mid), based on plasma exposure multiples

    • Clinically-relevant dose range is generally similar to what is seen with mAbs

    • We generally express dose on basis of aptamer mass, exclusive of PEG; PEG doses are generally 3-4X aptamer doses


Typical findings in general toxicology studies l.jpg
Typical Findings in General Toxicology Studies

  • Exaggerated pharmacology

    • Expected based on target biology

  • Anticoagulation

    • Generally a modest effect with good safety margins

  • C’ activation

    • Rarely seen and only at very high concentrations with aptamers tested to date

  • Bone marrow suppression

    • Seen in repeated-dose toxicity studies, modest effect with good safety margins

  • Hemodilution (PEGylated oligos only)

    • Osmotic properties of PEG at high plasma concentrations

  • Basophilic granulation and/or vacuolization

    • Mononuclear phagocytes and kidney tubule epithelial cells

    • Presence of drug-related material in these specific cells


Exaggerated pharmacology l.jpg
Exaggerated pharmacology

Cynomolgus Monkey

No spontaneous bleeding despite <3% vWF activity and prolonged cutaneous bleeding times, even at 25X projected human effective dose


Anticoagulation l.jpg
Anticoagulation

Cynomolgus Monkey

Concentration-dependent prolongation of aPTT


Complement activation dose rate concentration dependent l.jpg

Henry, JPET 1997, 281:810-816

Complement Activation Dose-, Rate-, Concentration-Dependent

Cynomolgus Monkeys

Threshold for Bb elevation:

~50 µg P=S ASO/mL, ~300 µg DNA aptamer/mL


Bone marrow suppression l.jpg
Bone marrow suppression

Sprague-Dawley Rat; Subcutaneous bolus, 3x/week for two weeks

Lower hemoglobin and reticulocyte counts after 14-day repeated-dose in rats


Hemodilution peg associated plasma volume expansion l.jpg
Hemodilution; PEG-Associated Plasma Volume Expansion

Cynomolgus Monkey

Other parameters comparably affected included:

alb, glob, ALT, LD, ALP, GGT, chol, trig, RBC, Hgb, Hct, retic, WBC, neut, lymph, plat

PEG doses and concentrations are 4X oligo


Basophilic granulation and or vacuolization mostly in mononuclear phagocytes l.jpg
Basophilic granulation and/or vacuolization, mostly in mononuclear phagocytes

Liver; Kupffer cell vacuolization

Spleen; PAMS vacuolization

Kidney; Basophilic granules in proximal tubulular epithelium

  • Presence of test article-related material in cells has not been associated with apparent adverse effects on those cells or tissues.

  • Therefore, we have not considered this finding alone to be an adverse effect.


Additional toxicology testing l.jpg
Additional Toxicology Testing

  • We plan to do standard ICH-guided testing for reproductive toxicology, chronic toxicology and carcinogenicity, when appropriate

  • We desire to test in at least 1 pharmacologically active species whenever possible

  • We do not propose to use surrogate molecules in toxicology testing (surrogate molecules would always differ appreciably in sequence, composition, potency, specificity, etc.)


Conclusions l.jpg
Conclusions

  • Aptamers share many “class- based” properties with other oligonucleotides

  • But aptamers also differ appreciably from other oligonucleotides in both MOA and chemical compositions

  • We have developed a customized toxicology testing strategy for aptamers

  • The toxicities we have seen are class-based, as seen with other oligonucleotides or with other PEGylated macromolecules

  • The aptamers tested to date have shown good safety margins between efficacious dose and concentrations and NOAELs in toxicology studies



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