1 / 23

Preclinical Safety Assessment of Aptamer Therapeutics

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”.

paul
Download Presentation

Preclinical Safety Assessment of Aptamer Therapeutics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Preclinical Safety Assessment of Aptamer Therapeutics Scott A. Barros, PhD, DABT Sr. Scientist, Toxicology

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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)

  7. 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

  8. 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)

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. Exaggerated pharmacology Cynomolgus Monkey No spontaneous bleeding despite <3% vWF activity and prolonged cutaneous bleeding times, even at 25X projected human effective dose

  16. Anticoagulation Cynomolgus Monkey Concentration-dependent prolongation of aPTT

  17. 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

  18. 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

  19. 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

  20. 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.

  21. 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.)

  22. 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

  23. The Archemix Gang

More Related