Andrea G. Hohmann - US grants

DESCRIPTION: (provided by applicant) Chronic pain affects one in three Americans. Socioeconomic costs attributed to inadequate pain management are estimated at $100 billion per year. The classic demonstration that analgesia can be elicited by environmental stressors provides evidence for endogenous pain inhibitory systems. Stress-induced analgesia is mediated in part by endogenous opioids. However, stress analgesia that is insensitive to blockade with opioid antagonists provides evidence for endogenous nonopioid analgesic systems. The mechanism underlying nonopioid stress-induced analgesia is completely unknown. Narrowing this gap is critical to harness the potential of endogenous nonopioid mechanisms for suppressing pain. The objective of this application is to identify the mechanism underlying nonopioid stress-induced analgesia. The central hypothesis is that environmental stressors release endocannabinoids -endogenous ligands for cannabinoid receptors- that suppress sensitivity to pain. The proposed research is expected to demonstrate that nonopioid stress-induced analgesia is: 1) blocked by a cannabinoid antagonist, 2) suppressed in rats rendered tolerant to cannabinoids, 3) attenuated by cannabinoid CB 1 antisense receptor knockdown, and 4) mediated by endogenous cannabinoids. The applicant is well-positioned to undertake this work because her research demonstrates that cannabinoids suppress pain neurotransmission and provides preliminary data that validates the central hypothesis. Completion of these aims is critical for establishing the biological significance of an endogenous cannabinoid transmitter system for suppressing pain. The proposed work is significant because the potential of nonopioid mechanisms for suppressing intractable pain cannot be realized until the underlying endogenous analgesic systems have been identify led. Pharmacological interventions that manipulate levels of endogenous cannabinoids by increasing synthesis and/or inhibiting inactivation represent novel targets for drug development. The development of effective pharmacotherapies for pain is likely to have a profound impact by driving down escalating health care costs and improving the quality of human life.

DESCRIPTION (provided by applicant): One in three Americans suffer from chronic pain. Available therapeutic interventions show limited efficacy and are plagued by adverse side effects (e g, altered mental status, sedation, nausea) that accompany systemic routes of drug administration Novel pharmacotherapies that specifically target the periphery therefore represent an alternative strategy for managing pain in the absence of unwanted central side-effects. Activation of a cannabinoid system- a nonopioid system that acts through a marijuana-like mechanism- in the periphery attenuates nociceptive responding The proposed research combines correlative behavioral and neurophysiological approaches to examine the functional consequences of peripheral cannabinoid actions on nociceptive transmission in vivo The use of subtype selective competitive antagonists and high affinity agonists provide the pharmacological tools required to study peripheral cannabinoid actions The proposed work uses a rat model of inflammation to test the hypothesis that a peripheral cannabinoid mechanism suppresses responses evoked by natural cutaneous stimulation in physiologically identified neurons of the spinothalamic tract Behavioral correlates for the electrophysiological studies will be established by assessing peripheral cannabinoid modulation of responsiveness to thermal and punctate mechanical stimulation under similar conditions The consequences of peripheral inflammation on axonal transport of cannabinoid receptors to peripheral nerve terminals is evaluated The development of effective pharmacotherapies for pain that are non-toxic, non-addicting and devoid of side-effects is likely to have a profound impact by improving quality of human life and reducing socioeconomic costs associated with inadequate pain management.

[unreadable] DESCRIPTION (provided by applicant): Environmental stressors transiently activate neural systems that inhibit pain responsiveness. This phenomenon, called stress-induced analgesia (SIA), engages both opioid and nonopioid mechanisms. We have recently reported that nonopioid SIA may be mediated by the mobilization of endogenous cannabinoid compounds, such as 2-arachidonoylglycerol (2-AG), in the midbrain periaqueductal gray (PAG) [Hohmann et al., Nature 435: 1108-1112, 2005]. Our results show, indeed, that nonopioid SIA is abolished by cannabinoid receptor antagonists, is associated with 2-AG accumulation in the PAG, and is enhanced by pharmacological agents that block 2-AG deactivation. However, the mechanisms governing 2-AG signaling in the PAG during SIA remain largely unknown. Our central hypothesis is that stress stimuli that result in nonopioid SIA activate phospholipase C (PLC) and diacylglycerol lipase (DGL) in select neurons of the PAG, causing the rapid mobilization of 2-AG. Newly released 2-AG engages local cannabinoid receptors to induce nonopioid SIA, and is subsequently deactivated through monoacylglycerol lipase (MGL)-mediated hydrolysis. Glutamatergic stimulation of group I metabotropic receptors (mGluR), which are positively coupled to PLC/DGL, may be responsible for initiating these events. We will test this hypothesis with a series of experiments, which are expected to demonstrate that: 1) foot shock stress elicits on-demand mobilization of 2-AG in the PAG by activating PLC/DGL; 2) selective inhibition of MGL activity in the PAG increases 2-AG accumulation in this structure and amplifies SIA; 3) group I mGluR activation in the PAG stimulates 2-AG mobilization through PLC/DGL to induce SIA; and 4) key components of the 2-AG signaling system (i.e. mGluRS, PLC, DGL, cannabinoid receptors, MGL) are expressed in select neurons of the PAG. The results of these studies will elucidate, for the first time, biochemical and physiological mechanisms governing 2-AG signaling in the brain and determine the role of this endocannabinoid mediator in pain modulation. Furthermore, the results will provide essential information on the mechanisms responsible for endocannabinoid deactivation in the brain, which may facilitate the development of novel analgesic and anti- stress medicines. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Environmental stressors transiently activate neural systems that inhibit pain responsiveness, thereby inducing a phenomenon known as stress-induced analgesia (SIA). We have recently reported that nonopioid SIA may be mediated by the mobilization of endogenous cannabinoid lipids, such as 2-arachidonoylglycerol (2-AG), in the midbrain periaqueductal gray (PAG) [Hohmann et al., Nature 435: 1108-1112, 2005]. However, the molecular mechanisms governing 2-AG signaling in the brain under physiological conditions remain unknown. Our central hypothesis is that stress stimuli that result in nonopioid SIA activate diacylglycerol lipase (DGL) in select neurons of the PAG, causing the rapid mobilization of 2-AG. Newly- released 2-AG engages local cannabinoid receptors to induce nonopioid SIA, and is then subsequently deactivated through monoacylglycerol lipase (MGL)-mediated hydrolysis. We will test this hypothesis by developing a novel, though high-risk, methodology that unites, for the first time, in vivo virally-mediated gene transfer with targeted lipidomic analyses. Our work will identify the functional consequences of virally- mediated RNA silencing of enzymes implicated in 2-AG formation (DGL) and deactivation (MGL) using three independent complementary approaches. We will use behavioral, neuroanatomical and mass spectrometric analyses (of related lipid mediators, their precursors and metabolites) to evaluate direct and indirect consequences of DGL and MGL silencing on lipid signaling pathways and SIA. Specifically, we will determine whether virally mediated silencing of the DGL and MGL genes within the PAG (i) influences 2-AG signaling and SIA; and (ii) causes distal changes in other endocannabinoid and non-cannabinoid lipid pathways. These studies are expected to demonstrate that 1) in vivo virally-mediated RNA silencing of DGL- beta - a DGL isoform recently shown to colocalize with phospholipase C, an enzyme implicated in formation of the 2-AG precursor diacylglycerol (DAG) - suppresses 2-AG formation in the PAG and SIA, and 2) in vivo virally-mediated RNA silencing of MGL stimulates 2-AG accumulation in the PAG and SIA. Validation of our in vivo gene transfer - targeted lipidomic approach should offer a critical advantage over existing methods which rely solely on neuroanatomical measurements of mRNA or protein. The results of these studies should elucidate, for the first time, the molecular, biochemical and physiological mechanisms governing 2-AG signaling in the brain and determine the role of this endocannabinoid mediator in pain modulation. Furthermore, the results will validate targeted lipidomics as a means to quantify changes in lipid signaling that occur downstream of targeted genetic manipulations. Successful validation of our technological approach and hypotheses may facilitate development of endocannabinoid-based pharmacological and gene therapies for pain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The present application "An Animal Model of Therapeutic Self-Medication for Neuropathic Pain" addresses the broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-DA-112: New Models and Measures in Preclinical Chronic Pain Research. Our long-term goal is to uncover mechanisms for suppressing pathological pain that lack drug abuse liability in humans. The objective of this application is to validate an animal model for assessing therapeutic self-medication of non-psychoactive analgesics. Our novel approach combines assessments of analgesic self-administration behavior with behavioral assessments of antinociception and dependence. Our central hypothesis is that animals will self-medicate with a nonpsychoactive cannabinoid analgesic to attenuate a neuropathic pain state. These studies are innovative because they exploit technological approaches commonly employed to study positive reinforcing effects of abused drugs and apply these methods to a novel context. Here, drug self-administration is used to study negative reinforcing properties of putative analgesics (i.e. ability to attenuate a neuropathic pain state) and dependence liability in the presence and absence of nerve injury. The investigators are well-positioned to conduct the proposed work because we have developed a new preclinical model which demonstrates that animals will self-administer a nonpsychoactive cannabinoid analgesic to attenuate a neuropathic pain state. We, consequently, believe that our self-medication model has broad translational value. We will complete experiments proposed under two Specific Aims: 1) To test the hypothesis that rats will self-administer a nonpsychoactive cannabinoid analgesic to attenuate a neuropathic pain state, 2) To test the hypothesis that a nonpsychoactive cannabinoid analgesic will produce minimal dependence (antagonist-precipitated withdrawal and conditioned place aversion), relative to the opiate analgesic morphine. Completion of this project is expected to validate a novel preclinical model for assessing both analgesic efficacy and drug abuse liability. The development of effective pharmacotherapies for pain with minimal abuse liability is expected to drive down health care costs and alleviate suffering in patients. PUBLIC HEALTH RELEVANCE: Pain is the number one reason Americans access the health care system. Socioeconomic costs of inadequate treatment for pain are estimated at $100 billion annually. The proposed work will develop and validate a rat model for therapeutic self-medication of analgesics. Development of safe and effective drugs that lack abuse potential is necessary to drive down health care costs and improve quality of life in patients.

DESCRIPTION (provided by applicant): The present application Protein-protein interaction inhibitors as novel analgesics addresses the critical need for efficacious analgesics lacking adverse side-effects. The NMDA receptor is involved in the maintenance of chronic pain and other pathological neuronal diseases. Dr. Lai, a co-principal investigator for this project, first showed that the small molecule inhibitor IC87201 disrupts the functional protein-protein interaction involving PDZ domains (neuronal nitric oxide synthase, nNOS and postsynaptic density protein 95, PSD95) required for NMDA receptor signaling. IC87201 also attenuated hyperalgesia in a rat model of neuropathic pain produced by traumatic nerve injury. However, whether nNOS-PSD95 inhibitors suppress nociceptive processing remains unknown. The objective of this application is to validate disruptors of nNOS-PSD95 protein-protein interactions as broad spectrum analgesics that suppress nociceptive processing using animal models of evoked and spontaneous pain. Persistent pain associated with central nervous system sensitization will be produced using inflammatory and toxic neuropathic insults. IC87201, one of the first small molecule protein-protein interaction disruptors, shows efficacy in several preclinical pain models. These findings support our hypothesis that disruption of signal compartmentalization represents an innovative approach to develop novel analgesics with fewer side-effects. The investigators, with extensive combined experience in drug discovery (Lai) and development of novel pain models (Hohmann) are well-positioned to conduct the proposed work. We will conduct experiments proposed under two Specific Aims: (1) To evaluate antinociceptive efficacy of small molecule inhibitors of nNOS-PSD95 on both inflammation-evoked behavioral hypersensitivities and neuronal activation; (2) To assess the efficacy of small molecule inhibitors of nNOS-PSD95 in suppressing spontaneous neuropathic pain using a conditioned place preference approach. Completion of this project is expected to validate the use of nNOS-PSD95 inhibitors as a new class of broad-spectrum analgesics. These studies are expected to validate the disruption of signal compartmentation as an innovative and feasible approach to drug development. The development of effective pharmacotherapies with novel chemical structures that possess limited side-effect profiles and minimal abuse liability is expected to drive down health care costs and alleviate suffering in patients.

Project Summary Excessive glutamate signaling through N-methyl-D-aspartate receptors (NMDARs) is implicated in altered forms of neuronal plasticity associated with opioid reward and dependence. The present Cutting Edge Basic Research Award proposes to functionally decouple signaling complexes downstream of NMDAR activation to eliminate aberrant NMDAR-dependent nitric oxide signaling and circumvent the development of opioid reward. In the United States, unintentional deaths due to prescription drug overdoses have more than tripled since 1990, more than deaths attributed to cocaine and heroin combined. The increase in unintentional drug overdose death rates has mainly been driven by increased use of opioid analgesics. Inadequate treatment for pain, exacerbated by incomplete analgesic efficacy and narcotic abuse liability, contributes to escalating drug use, resulting in socioeconomic costs estimated at $600 billion annually. Improving the safety, efficacy, side effect profile and abuse liability of opioid analgesics thus remains an urgent medical need. Excessive NMDAR stimulation triggers a signaling cascade involving activation of the enzyme neuronal nitric oxide synthase (nNOS), which catalyzes formation of the signaling molecule nitric oxide (NO), which promotes addiction- related behaviors. Inhibition of aberrant glutamatergic hyperexcitability and inhibition of nNOS reduces addiction-related behaviors in preclinical studies. However, the therapeutic potential of NMDA receptor antagonists and NOS inhibitors are limited by severe side effects. We propose to functionally decouple NMDARs from nNOS signaling to circumvent opioid reward without unwanted side effects of global NMDAR antagonists or nonselective NOS catalytic inhibitors. We propose to accomplish this objective by selectively disrupting the protein-protein interface between nNOS and postsynaptic density 95kDA, a scaffolding protein which tethers nNOS to NMDARs. Aim 1 will test the hypothesis that disruption of PSD95-nNOS protein-protein interactions will suppress morphine-induced reward using conditioned place preference and drug self- administration approaches. Aim 2 will test the hypothesis that disruption of PSD95-nNOS interactions will attenuate opioid-induced dopamine dynamics in the nucleus accumbens shell, a key component of the reward circuit. Disruption of protein-protein interactions was once considered an impossible target for drug development. Completion of these high risk, high impact studies will establish the feasibility of disrupting the PSD95-nNOS interface to eliminate opioid reward, while retaining therapeutic efficacy, bypassing unwanted side effects of both NMDAR antagonists and NOS catalytic inhibitors. Validation of our hypotheses will provide a strong rationale for undertaking lead optimization of PSD95-nNOS inhibitors for advancement toward clinical studies in opioid addiction, filling a major gap in an area of unmet therapeutic need.

? DESCRIPTION (provided by applicant): Chemotheraphy-induced peripheral neuropathy (CIPN) limits life saving anti-cancer treatment, can be permanent and negatively impacts quality of life. It is thus important to dissect the critical signaling pathways involved in development an maintenance of CIPN and identify therapeutic strategies to prevent or treat CIPN. The NMDA receptor (NMDAR) signaling complex plays a key role in central sensitization of chronic pain. While NMDAR antagonists are efficacious in decreasing pain sensitization, they have limited therapeutic uses because they disrupt normal physiological processes (e.g. motor function, memory and cognition). The NMDAR signaling complex consists of many protein partners including the scaffold postsynaptic density 95 kDA (PSD95) protein, the neuronal enzyme nitric oxide synthase (nNOS) and its adaptor protein NOS1AP. Disruption of specific steps downstream of NMDAR activation offers the opportunity to decrease pain sensitization while avoiding some of the broader side effects associated with upstream receptor blockade. Our preliminary studies suggest that the interface between nNOS and NOS1AP represents a previously unrecognized candidate target for the development of new analgesics for CIPN. Aim 1 will determine whether disruption of nNOS interactions with its upstream or downstream protein partners bias NMDAR signaling in a functionally selective manner using biochemical and cell based assays. Aim 2 will evaluate the therapeutic potential of disrupting the nNOS-NOS1AP protein-protein interface for suppressing neuropathic pain induced by the chemotherapeutic agent paclitaxel and correlate antinociceptive efficacy of intrathecally administered agents with disruption of the nNOS-NOS1AP complex in lumbar spinal cord of paclitaxel-treated rats. Aim 3 will identify signaling pathways downstream of NOS1AP that underly the ability of nNOS-NOS1AP inhibitors to attenuate paclitaxel-induced neuropathic pain. In this project, the mechanism by which peptide and small molecule protein-protein interaction inhibitors to selectively block NMDAR-induced hypersensitivity in a paclitaxel-induced neuropathic pain model will be elucidated. If confirmed, a new generation of modulators of pathological pain could be developed by targeting this interaction.

? DESCRIPTION (provided by applicant): Cancer chemotherapy frequently causes a painful peripheral neuropathy that is dose-limiting and can be irreversible. Gabapentin is clinically used to treat diverse forms of neuropathic pain. However, the mechanism(s) responsible for its antinociceptive effects remain poorly understood. Unpublished work from our groups suggests that gabapentin suppresses neuropathic pain induced by the chemotherapeutic agent paclitaxel in rodents through interactions with CB2 cannabinoid receptors. At the cellular level, gabapentin selectively increases ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) to recruit ?-arrestin to CB2 receptors. These observations suggest a previously unrecognized interaction between CB2 receptors, ?- arrestin/ERK1/2 signaling and gabapentin-induced antinociception. We postulate that gabapentin analgesic efficacy is due (at least in part) to a CB2-specific mechanism that involves increased ?-arrestin signaling in microglia or neurons. We will thoroughly test this hypothesis by completing three Specific Aims: Aim 1 will characterize the impact of gabapentin on CB2 receptor signaling using transfected cells lines, cell lines natively expressing CB2 receptors and primary cultures of CB2-expressing cells. In addition, potential allosteric interactions between gabapentin and CB2 will be probed. Aim 2 will use conditional deletion of CB2 from neurons, microglia, and astrocytes to determine which cell type(s) express the CB2 receptors mediating gabapentin antinociception during the development and maintenance phases of paclitaxel neuropathy. Since CB2 agonists efficaciously relieve paclitaxel-induced allodynia and hyperalgesia, this approach will also be used to determine the cell type(s) mediating antinociception elicited by direct acting CB2-agonists. Aim 3 will extend the findings of the first two aims to determine if gabapentin efficacy is also CB2-mediated in other nerve injury and inflammatory pain models. The relevant cell type(s) will be determined using conditional deletion of CB2 as warranted and as described in the second specific aim. This aim will also investigate the mechanism of direct-acting CB2 agonists in these pain models using the above conditional deletion approach. Our research team combines expertise in (1) CB2 receptor binding, signaling, trafficking, and regulation, (2) cannabinoid pharmacology and antinociceptive mechanisms, and (3) mouse preclinical models of pain. Our studies suggest a highly novel and previously unrecognized intersection between CB2 receptors, endocannabinoids, and arrestin signaling that underlies the therapeutic efficacy of gabapentin. Understanding the cross-talk between these pathways is critical both for elucidating the mechanism of action of gabapentin to exploit and optimize its therapeutic efficacy and for identifying novel therapeutic targets for drug development that lack unwanted side effects of conventional treatments. Lastly, our studies will also identify the cellular targets of CB2 agonist as they relieve a variety of pathological pain states.

Despite significant limitations, opioids remain a mainstay for the treatment of severe acute and chronic pain. Two clinically significant limitations that accompany the long-term therapeutic use of opioids are: (1) The development of tolerance (requiring escalating doses of opioid to maintain the desired therapeutic benefit or leading to diminished benefit with constant dose) and (2) Physical dependence (withdrawal symptoms on cessation of opioid use, which are very unpleasant, and seeking their avoidance may increase the risk for opioid addiction). Currently, there are no practical approaches for decreasing opioid tolerance or suppressing the development of physical dependence. However, in exploring potential interactions between CB2 cannabinoid receptor (CB2R) and mu opioid receptor (MOR) signaling, we made the exciting discovery that certain CB2 agonists effectively prevented the development of tolerance to opioid-induced anti-allodynic efficacy in a murine neuropathic pain model, while also blunting the physical dependence that accompanies chronic morphine exposure. These findings, if they can be translated to humans, hold the promise to significantly improve the clinical use of opioids. On the path to translating these findings, we will complete three specific aims to better understand how CB2 and mu opioid receptor agonists interact to suppress opioid tolerance and dependence: Aim 1: Identify the CB2-expressing cells engaged by LY28282360 to prevent opioid tolerance. We will delineate the cell types responsible for the ability of LY2828360 to block development of morphine tolerance and identify site of action using both pharmacological manipulations and a conditional deletion approach. Separate studies will target primary afferent nociceptors vs. microglia. Aim 2: Define the conditions under which CB2 agonists suppress opioid-induced physical dependence. We will delineate the cell types responsible for the effects of LY2828360 on opioid dependence, as measured using naloxone precipitated opioid withdrawal, using both pharmacological manipulations and a conditional deletion approach. Separate studies will target primary afferent nociceptors vs. microglia. Aim 3: Mechanistic characterization of CB2R/MOR interaction. We will characterize CB2 ligands for their G protein/arrestin signaling bias as well as their kinetics of G protein activation. We will also determine if the slow activation of G protein signaling by LY2828360 and related CB2 agonists is due to the kinetics of receptor binding. Finally, if the results of Aims 1 or 2 suggest that MOR and CB2R are interacting in the same cell, we will characterize the differences in CB2R/MOR crosstalk between slowly and rapidly signaling CB2 agonists. Completion of these aims will fully characterize interactions between CB2 and opioid receptors in preclinical and cell-based models. These studies will help define the clinical settings where CB2 agonists may be useful in countering two major limitations on the use of opioids in treating chronic pain.

This proposal requests support for the second renewal of a highly successful, integrative pre-doctoral training program in the neuroscience of drug abuse at Indiana University Bloomington. Despite substantial advances in understanding drug addiction within specific levels of analysis (e.g., behavioral, clinical, and molecular), the problem of drug abuse will not be solved by focusing on a single level of analysis. If the next generation of researchers is to make meaningful progress, they must be well-rounded scientists with an appreciation that drug abuse is a multi-faceted problem, while possessing the flexibility to respond to and incorporate rapidly evolving technologies that will enable them to understand mechanisms and develop treatments for drug abuse. To prepare trainees for success in the next decade and beyond, our program emphasizes a team-driven, inter- disciplinary approach based on the translational model. Our program is successful because it brings together 12 core faculty members who are committed to integrative training and have a long history of collaboration on questions integral to drug abuse research. They include senior and junior investigators, molecular neurobiologists, cognitive neuroscientists, epidemiologists, and clinical scientists. They come from several departments in the College of Arts and Sciences and the School of Public Health, and all have joint appointments in the campus-wide Program in Neuroscience. Working together in state-of-the-art facilities, this group has access to a pool of highly talented trainees motivated to pursue careers in drug abuse research. Our training program develops trainees by emphasizing three key components: integrative course work, translational research training, and professional skills development. Course work covers basic neuro- and psychopharmacology, provides an integrative view of biobehavioral processes in substance use disorders, and brings a translational perspective to theoretical and empirical knowledge. Research is guided by a mentor in molecular, systems, cognitive, or clinical neuroscience closely integrated with a co-mentor representing a complimentary level of analysis. This integrative approach is reinforced through discussion groups, attendance at colloquia, and participation at national meetings. Instruction in ethical scientific behavior includes formal course work and campus workshops as well as specialized instruction led by a core faculty member who has many years of experience leading seminars on ethical issues unique to substance use research. Trainees also learn to develop skills in grant writing, manuscript preparation, teaching, and community outreach and organize an annual program retreat with outside experts in drug abuse. In short, our program relies on a combination of course work and research training aimed at integrating and translating bench and bedside approaches to produce scientists well prepared for productive and transformative careers in drug abuse research.

ABSTRACT Chemotherapy-induced peripheral neuropathy (CIPN) is an often long-lasting neurological condition that arises frequently in cancer patients who receive broadly used chemotherapies such as taxanes. CIPN causes abnormal pain and other symptoms that can limit chemotherapy dosage and significantly impact quality of life for years. There are no drugs that prevent CIPN or treat it well. Duloxetine is the only clinically proven efficacious pain- reducing agent, though it can cause significant side effects and its efficacy limited to a subset of patients. Opioids are used off-label, but also carry serious side effects. Thus, there is a critical unmet need for drugs that safely and effectively treat or prevent CIPN. Peripheral Cannabinoid 2 receptors (CB2) are a promising target for CIPN treatment. CB2 is constitutively expressed on inflammatory immune cells and induced in peripheral neurons in neuropathic conditions. Its activation has powerful neuroprotective, anti-inflammatory, and analgesic effects. Rodent models of CIPN show that small molecule CB2 agonists alleviate neuropathic pain behavior, and when administered prophylactically suppress CIPN both during dosing and for 100 days after. Several companies have developed small molecule CB2 agonists that, unfortunately, are rapidly cleared, penetrate the blood-brain barrier and/or have off-target effects (notably cognitive ones) mediated by the CB1 receptor. Abalone Bio used its proprietary Functional Antibody Selection Technology (FAST) to isolate a selective CB2-activating nanobody (VHH), which we converted into a VHH-Fc fusion lead antibody, ABt140, for in vivo studies. Phase I SBIR results show that ABt140 rapidly and durably reversed allodynia in mice with CIPN. In this Phase II SBIR project, Abalone Bio will first improve ABt140?s immunogenicity and manufacturability by rational engineering, and then the FAST platform will be used to increase its potency to select durable agonists from millions of computationally- designed variants. We will select 1 lead and at least 2 alternates using in vitro and in vivo assays. Using the paclitaxel CIPN mouse model, we will assess the lead?s ability to prevent CIPN development, reduce in nerve damage and inflammation, show no impairment of motor skills, and not affect chemotherapy?s anti-tumor efficacy. Finally, we will determine the lead antibody?s half-life and tissue distribution in mice, and using blood markers and organs appearance and weight, we will assess its toxicity at high doses. Successful completion of these aims will de-risk the project sufficiently to advance Abalone?s antibody drug to IND-enabling studies and eventually first in human trials for painful CIPN. Abalone?s drug may also have broad utility for other neuropathic pains and inflammatory conditions.