SBIR-STTR Award

Development of Anti-OLAM Aptamers as Novel Analgesics The management of pain remains a major health care problem due to an incomplete understanding of pain mechanisms. TRPV1, a prominent member of the transient receptor potential (TRP) family of ligand-gated ion channel, detects noxious chemical and physical stimuli in peripheral tissues. Both pharmacological and gene deletion studies have demonstrated a pivotal role for TRPV1 in inflammatory heat hyperalgesia and other pain conditions. Oxidized linoleic acid metabolites (OLAMs) have been recently demonstrated to comprise a novel family of endogenous TRPV1 agonists that contributes to acute and inflammatory pain conditions. Therefore, compounds that block the OLAM system are likely to constitute a novel family of analgesics. In direct support of this prediction, preliminary data provided herein demonstrate that injection of antibodies against two of the major OLAMs, 9-HODE and 13-HODE, produce significant analgesia in two pain models. Although these data provide evidence for proof-of-concept, the clinical development of rabbit polyclonal antibodies is not feasible due to adverse effects related to immunogenicity. Accordingly, Operational Technologies Corporation (OpTech) proposes to use combinatorial aptamer chemistry to discover DNA aptamers that specifically bind to these OLAMs and neutralize their pain-producing activities. This would permit replacing antibodies with more specific, less expensive and perhaps higher affinity DNA aptamers. In Phase 1, OpTech expects to complete two overall Specific Aims. Aim 1 will develop, clone, and sequence several highly specific DNA aptamers that bind 9-hydroxydecadienoic acid (HODE) and 13-HODE without binding to the precursor lipid, linoleic acid (Fig 1A). Aim 2 will evaluate the analgesic activity of the aptamers using several in vivo preclinical rat models of pain. In Phase 2, OpTech will refine, optimize, and begin commercialization of its anti-OLAM aptamer compounds. The Phase 2 optimization process will include 3-D modeling of putative aptamer binding pocket interactions with the OLAMs. Based on 3-D modeling findings, OpTech anticipates adding modified bases having various functional groups (e.g., primary amines, methyl, thiol groups, etc.) that are now commercially available for addition to oligonucleotides at the point of chemical synthesis. The effects of these additional functional groups on aptamer-OLAM binding affinity are expected to better emulate amino acid side chains and will be studied by ELISA-like plate assays and surface plasmon resonance (SPR). The highest affinity and most specific unmodified or modified anti-OLAM aptamers will move into animal studies. The most effective aptamers in animal pain studies will be modified for longer in vivo lifetimes (3'-cholesterol addition and inclusion in liposomes, PEGylation, etc.) or time-released formulation and enter the FDA approval pipeline.

Public Health Relevance:
Development of Anti-OLAM Aptamers as Novel Analgesics Millions of patients suffer from pain and many available analgesic drugs ("pain killers") suffer from either incomplete analgesia or unacceptable side-effects. The proposal will develop a novel class of analgesics that work by blocking the endogenous capsaicin-like substances that are released during tissue injury.

Public Health Relevance Statement:
Development of Anti-OLAM Aptamers as Novel Analgesics Millions of patients suffer from pain and many available analgesic drugs ("pain killers") suffer from either incomplete analgesia or unacceptable side-effects. The proposal will develop a novel class of analgesics that work by blocking the endogenous capsaicin-like substances that are released during tissue injury.

NIH Spending Category:
Biotechnology; Neurosciences; Pain Conditions - Chronic; Pain Research

Project Terms:
3-Dimensional; Absence of pain sensation; Acids; Acute inflammatory pain; Adverse effects; Afferent Neurons; Affinity; Agonist; American; Amines; Amino Acids; Analgesics; animal pain; Animals; Antibodies; aptamer; Arthritis; base; Binding (Molecular Function); Biological Assay; Capsaicin; chemical synthesis; Chemicals; Chemistry; Cholesterol; chronic pain; Clinical; Clinical Trials; combinatorial; commercialization; Coronary heart disease; cost; Data; Development; Diabetes Mellitus; Diagnosis; DNA; Drug Formulations; Enzyme-Linked Immunosorbent Assay; Evolution; Family; Figs - dietary; Foundations; functional group; Gated Ion Channel; Gene Deletion; Healthcare; Heating; Hyperalgesia; immunogenicity; in vivo; Incidence; Inflammatory; inhibiting antibody; Injection of therapeutic agent; injured; Injury; Ligands; Linoleic Acids; Lipids; Liposomes; Malignant Neoplasms; Mechanics; Medical; member; Modeling; Nociceptors; novel; Nucleic Acids; Oligonucleotides; Oryctolagus cuniculus; Pain; Pain management; Patients; pegaptanib; Peripheral; Pharmaceutical Preparations; Phase; Physiological; polyclonal antibody; pre-clinical; Process; process optimization; Quality of life; Rattus; receptor; Reporting; Role; Side; Small Business Innovation Research Grant; Sodium; Stimulus; Sulfhydryl Compounds; Surface Plasmon Resonance; System; Technology; Testing; Time; Tissues; TRPV1 gene; Work

Development of Anti-OLAM Aptamers as Novel Analgesics (Phase 2) The management of pain remains a major health care problem due to an incomplete understanding of pain mechanisms. TRPV1, a prominent member of the transient receptor potential (TRP) family of ligand-gated ion channels, detects noxious chemical and physical stimuli in peripheral tissues. Both pharmacological and gene deletion studies have demonstrated a pivotal role for TRPV1 in inflammatory heat hyperalgesia and other pain conditions. Oxidized linoleic acid metabolites (OLAMs) have been recently demonstrated to comprise a novel family of endogenous TRPV1 agonists that contribute to acute and inflammatory pain conditions. Therefore, compounds that block the OLAM system are likely to constitute a novel family of analgesics. In direct support of this prediction, Phase 1 data provided herein demonstrate that injection of high affinity DNA aptamers developed against two of the major OLAMs: 9-HODE and 13-HODE, produced significant analgesia in patch- clamp and rat behavioral models of heat pain. Although these data provide evidence for proof-of-concept, the aptamers must be developed into heavier conjugates in order to avoid rapid clearance by the kidneys and other major organs. Accordingly, Operational Technologies Corporation (OpTech) proposes to continue developing its successful high affinity anti-OLAM aptamers from Phase 1 into long-lived aptamer conjugates in vivo which specifically bind to 9-HODE and 13-HODE and neutralize their pain-producing activities for extended periods of time. This would permit replacing anti-HODE polyclonal antibodies with more specific, less expensive and higher affinity DNA aptamers. In Phase 2, OpTech expects to complete several specific aims targeted toward enhancing pharmacokinetics (PK) by addition of ibuprofen to the 3' end (ref. 80), thereby enabling association with serum albumin to add weight and retard renal and hepatic clearance. Alternative, but proven, methods to slow in vivo clearance and protect aptamers in vivo including 3'-polyethylene glycol (PEG) and covalent 3' rat albumin attachment will also be investigated. Moreover, 3-dimensional molecular models of aptamer-HODE interactions will be generated by the Southwest Research Institute (SwRI) to enable potential molecular engineering of improvements to aptamer affinity (already exhibiting low nM KD values) and specificity, if possible, using modified or unnatural deoxynucleotides. In addition to in vitro patch clamp studies in the presence and absence of various doses of the aptamer conjugates, rats will be studied for behavioral changes before and after injection and exposure to 43oC noxious heat with the various aptamer-3'-conjugates. If relatively long- term (on the order of hours) PK is observed and heat/burn analgesia can again be demonstrated, OpTech will seek to file an IND or pre-IND application with the FDA.

Public Health Relevance Statement:


Public Health Relevance:
Development of Anti-OLAM Aptamers as Novel Analgesics (Phase 2) Millions of patients suffer from pain and many available analgesic drugs ("pain killers") suffer from either incomplete analgesia or unacceptable side-effects. This Phase 2 SBIR proposal will further develop a novel class of long-lived aptamer conjugate analgesics that work by blocking the endogenous capsaicin-like substances that are released during tissue injury, thereby specifically treating heat/burn pain, cancer pain, and possibly counteracting some types of shock which are mediated by OLAMs (oxidized linoleic acid metabolites) including 9- and 13- hydroxyoctadecadienoic acid ("HODEs").

Project Terms:
13-hydroxyoctadecadienoic acid; 3-Dimensional; 9-hydroxy-10,12-octadecadienoic acid; Absence of pain sensation; Acute inflammatory pain; addiction; Adverse effects; Afferent Neurons; Affinity; Agonist; Albumins; American; Analgesics; Animals; Antibodies; aptamer; Arthritis; base; Behavioral Model; Binding (Molecular Function); Burn injury; Burning Pain; cancer pain; Capsaicin; Chemicals; chronic pain; Clinical; Coronary heart disease; cost; Data; design; Development; Diabetes Mellitus; Diagnosis; DNA; Dose; Drug Kinetics; Engineering; Evolution; Exhibits; Exposure to; Family; FDA approved; Foundations; Gated Ion Channel; Gene Deletion; Healthcare; Heart; Heating; Hepatic; Hour; Hyperalgesia; Ibuprofen; immunogenic; immunogenicity; In Vitro; in vivo; Incidence; Inflammatory; inhibiting antibody; Injectable; Injection of therapeutic agent; injured; Injury; Investigational Drugs; Investigational New Drug Application; Kidney; Life; Ligands; Linoleic Acids; Liver; Malignant Neoplasms; Mechanics; Mediating; Medical; member; Metabolite Interaction; Methods; Modeling; Molecular; molecular modeling; Molecular Models; Nociceptors; novel; Nucleic Acids; Nucleotides; Opioid; Organ; Oryctolagus cuniculus; Pain; pain inhibition; Pain management; patch clamp; Patients; pegaptanib; Peripheral; Pharmaceutical Preparations; Phase; Physiological; polyclonal antibody; Polyethylene Glycols; Process; Proteins; public health relevance; Quality of life; Rattus; receptor; Reporting; Research Institute; Role; Serum Albumin; Shock; Small Business Innovation Research Grant; Sodium; Specificity; Stimulus; stroke; System; Technology; Testing; Time; Tissues; TRPV1 gene; Ulcer; Weight; Work