Michael Klukinov

Nociceptive sensitization and BDNF up-regulation in a rat model of traumatic brain injury

Chronic pain after traumatic brain injury (TBI) is very common, but the mechanisms linking TBI to pain experienced in the periphery have not been described. In this set of studies we examined nociceptive sensitization and changes in spinal cord gene expression using the rat lateral fluid percussion model of mild TBI. We did not identify changes in thermal nociceptive thresholds in rats with mild TBI. However, mechanical allodynia in hind paws contralateral to TBI was significant and sustained. We also found that spinal cord levels of brain derived neurotrophic factor (BDNF) but not several other pain-related genes were up-regulated one week after injury. Our findings suggest that TBI-induced up-regulation of spinal BDNF levels might contribute to chronic TBI-related pain, and that the lateral fluid percussion model might be useful for exploring this relationship.

Oxytocin receptor: Expression in the trigeminal nociceptive system and potential role in the treatment of headache disorders.

Aims Our studies investigated the location of oxytocin receptors in the peripheral trigeminal sensory system and determined their role in trigeminal pain. Methods Oxytocin receptor expression and co-localization with calcitonin gene-related peptide was investigated in rat trigeminal ganglion using immunohistochemistry. Enzyme-linked immunosorbent assay was used to determine the effects of facial electrocutaneous stimulation and adjuvant-induced inflammation of the temporomandibular joint on oxytocin receptor expression in the trigeminal ganglion. Finally, the effects of oxytocin on capsaicin-induced calcitonin gene-related peptide release from dural nociceptors were investigated using isolated rat dura mater. Results Oxytocin receptor immunoreactivity was present in rat trigeminal neurons. The vast majority of oxytocin receptor immunoreactive neurons co-expressed calcitonin gene-related peptide. Both electrocutaneous stimulation and adjuvant-induced inflammation led to a rapid upregulation of oxytocin receptor protein expression in trigeminal ganglion neurons. Oxytocin significantly and dose-dependently decreased capsaicin-induced calcitonin gene-related peptide release from dural nociceptors. Conclusion Oxytocin receptor expression in calcitonin gene-related peptide containing trigeminal ganglion neurons, and the blockade of calcitonin gene-related peptide release from trigeminal dural afferents suggests that activation of these receptors may provide therapeutic benefit in patients with migraine and other primary headache disorders.

Single intrathecal administration of the transcription factor decoy AYX1 prevents acute and chronic pain after incisional, inflammatory, or neuropathic injury

Summary AYX1 is a DNA decoy designed to specifically inhibit, in the spinal cord and dorsal root ganglia network, the trauma‐induced transcription factor EGR1 responsible for long‐term neuronal hyper‐excitability. A single intrathecal administration of AYX1 around the time of surgery or trauma prevents acute and chronic pain in animal models of inflammatory, incisional, bone, and neuropathic pain. ABSTRACT The persistence of pain after surgery increases the recovery interval from surgery to a normal quality of life. AYX1 is a DNA‐decoy drug candidate designed to prevent post‐surgical pain following a single intrathecal injection. Tissue injury causes a transient activation of the transcription factor EGR1 in the dorsal root ganglia–dorsal horn network, which then triggers changes in gene expression that induce neuronal hypersensitivity. AYX1 is a potent, specific inhibitor of EGR1 activity that mimics the genomic EGR1‐binding sequence. Administered in the peri‐operative period, AYX1 dose dependently prevents mechanical hypersensitivity in models of acute incisional (plantar), inflammatory (CFA), and chronic neuropathic pain (SNI) in rats. Furthermore, in a knee surgery model evaluating functional measures of postoperative pain, AYX1 improved weight‐bearing incapacitance and spontaneous rearing compared to control. These data illustrate the potential clinical therapeutic benefits of AYX1 for preventing the transition of acute to chronic post‐surgical pain.

Pharmacology, pharmacokinetics, and metabolism of the DNA-decoy AYX1 for the prevention of acute and chronic post-surgical pain

Background AYX1 is an unmodified DNA-decoy designed to reduce acute post-surgical pain and its chronification with a single intrathecal dose at the time of surgery. AYX1 inhibits the transcription factor early growth response protein 1, which is transiently induced at the time of injury and triggers gene regulation in the dorsal root ganglia and spinal cord that leads to long-term sensitization and pain. This work characterizes the AYX1 dose-response profile in rats and the link to AYX1 pharmacokinetics and metabolism in the cerebrospinal fluid, dorsal root ganglia, and spinal cord. Results The effects of ascending dose-levels of AYX1 on mechanical hypersensitivity were measured in the spared nerve injury model of chronic pain and in a plantar incision model of acute post-surgical pain. AYX1 dose-response profile shows that efficacy rapidly increases from a minimum effective dose of ∼ 0.5 mg to a peak maximum effective dose of ∼ 1 mg. With further dose escalation, the efficacy paradoxically appears to decrease by ∼ 30% and then returns to full efficacy at the maximum feasible dose of ∼ 4 mg. The reduction of efficacy is associated to doses triggering a near-saturation of AYX1 metabolism by nucleases in the cerebrospinal fluid and a paradoxical reduction of AYX1 exposure during the period of early growth response protein 1 induction. This effect is overcome at higher doses that compensate for the effect of metabolism. Discussion AYX1 is a competitive antagonist of early growth response protein 1, which is consistent with the overall increased efficacy observed as dose-levels initially escalate. Chemically, AYX1 is unprotected against degradation by nucleases. The sensitivity to nucleases is reflected in a paradoxical reduction of efficacy in the dose-response curve. Conclusions These findings point to the importance of the nuclease environment of the cerebrospinal fluid to the research and development of AYX1 and other intrathecal nucleotide-based therapeutics.

Gene therapy for trigeminal pain in mice.

The aim of this study was to test the efficacy of a single direct injection of viral vector encoding for encephalin to induce a widespread expression of the transgene and potential analgesic effect in trigeminal behavioral pain models in mice. After direct injection of herpes simplex virus type 1 based vectors encoding for human preproenkephalin (SHPE) or the lacZ reporter gene (SHZ.1, control virus) into the trigeminal ganglia in mice, we performed an orofacial formalin test and assessed the cumulative nociceptive behavior at different time points after injection of the viral vectors. We observed an analgesic effect on nociceptive behavior that lasted up to 8 weeks after a single injection of SHPE into the trigeminal ganglia. Control virus-injected animals showed nociceptive behavior similar to naive mice. The analgesic effect of SHPE injection was reversed/attenuated by subcutaneous naloxone injections, a μ-opioid receptor antagonist. SHPE-injected mice also showed normalization in withdrawal latencies upon thermal noxious stimulation of inflamed ears after subdermal complete Freund’s adjuvant injection, indicating widespread expression of the transgene. Quantitative immunohistochemistry of trigeminal ganglia showed expression of human preproenkephalin after SHPE injection. Direct injection of viral vectors proved to be useful for exploring the distinct pathophysiology of the trigeminal system and could also be an interesting addition to the pain therapists’ armamentarium.

Oxytocin and Migraine Headache

This article reviews material presented at the 2016 Scottsdale Headache Symposium. This presentation provided scientific results and rationale for the use of intranasal oxytocin for the treatment of migraine headache. Results from preclinical experiments are reviewed, including in vitro experiments demonstrating that trigeminal ganglia neurons possess oxytocin receptors and are inhibited by oxytocin. Furthermore, most of these same neurons contain CGRP, the release of which is inhibited by oxytocin. Results are also presented which demonstrate that nasal oxytocin inhibits responses of trigeminal nucleus caudalis neurons to noxious stimulation using either noxious facial shock or nitroglycerin infusion. These studies led to testing the analgesic effect of intranasal oxytocin in episodic migraineurs—studies which did not meet their primary endpoint of pain relief at 2 h, but which were highly informative and led to additional rat studies wherein inflammation was found to dramatically upregulate the number of oxytocin receptors available on trigeminal neurons. This importance of inflammation was supported by a series of in vivo rat behavioral studies, which demonstrated a clear craniofacial analgesic effect when a pre‐existing inflammatory injury was present. The significance of inflammation was further solidified by a small single‐dose clinical study, which showed analgesic efficacy that was substantially stronger in chronic migraine patients that had not taken an anti‐inflammatory drug within 24 h of oxytocin dosing. A follow‐on open label study examining effects of one month of intranasal oxytocin dosing did show a reduction in pain, but a more impressive decrease in the frequency of headaches in both chronic and high frequency episodic migraineurs. This study led to a multicountry double blind, placebo controlled study studying whether, over 2 months of dosing, “as needed” dosing of intranasal oxytocin by chronic and high frequency migraineurs would reduce the frequency of their headaches compared to a 1‐month baseline period. This study failed to meet its primary endpoint, due to an extraordinarily high placebo rate in the country of most of the patients (Chile), but was also highly informative, showing strong results in other countries and strong post hoc indications of efficacy. The results provide a strong argument for further development of intranasal oxytocin for migraine prophylaxis.

Intrathecal Administration of AYX2 DNA Decoy Produces a Long-Term Pain Treatment in Rat Models of Chronic Pain by Inhibiting the KLF6, KLF9, and KLF15 Transcription Factors

Background Nociception is maintained by genome-wide regulation of transcription in the dorsal root ganglia—spinal cord network. Hence, transcription factors constitute a promising class of targets for breakthrough pharmacological interventions to treat chronic pain. DNA decoys are oligonucleotides and specific inhibitors of transcription factor activities. A methodological series of in vivo–in vitro screening cycles was performed with decoy/transcription factor couples to identify targets capable of producing a robust and long-lasting inhibition of established chronic pain. Decoys were injected intrathecally and their efficacy was tested in the spared nerve injury and chronic constriction injury models of chronic pain in rats using repetitive von Frey testing. Results Results demonstrated that a one-time administration of decoys binding to the Kruppel-like transcription factors (KLFs) 6, 9, and 15 produces a significant and weeks–month long reduction in mechanical hypersensitivity compared to controls. In the spared nerve injury model, decoy efficacy was correlated to its capacity to bind KLF15 and KLF9 at a specific ratio, while in the chronic constriction injury model, efficacy was correlated to the combined binding capacity to KLF6 and KLF9. AYX2, an 18-bp DNA decoy binding KLF6, KLF9, and KLF15, was optimized for clinical development, and it demonstrated significant efficacy in these models. Conclusions These data highlight KLF6, KLF9, and KLF15 as transcription factors required for the maintenance of chronic pain and illustrate the potential therapeutic benefits of AYX2 for the treatment of chronic pain.

Nasal application of HSV encoding human preproenkephalin blocks craniofacial pain in a rat model of traumatic brain injury

According to Centers for Disease Control and Prevention, each year, an estimated 1.7 million Americans sustain a traumatic brain injury (TBI), which frequently leads to chronic craniofacial pain. In this study we examine a gene therapy approach to the treatment of post-TBI craniofacial neuropathic pain using nasal application of a herpes simplex virus (HSV)-based vector expressing human proenkephalin (SHPE) to target the trigeminal ganglia. Mild TBI was induced in rats by the use of a modified fluid percussion model. Two days after mild TBI, following the development of facial mechanical allodynia, animals received either an intranasal application of vehicle or recombinant HSV encoding human preproenkephalin or lacZ reporter gene encoding control vector (SHZ.1). Compared with baseline response thresholds, mild TBI in SHZ.1 or vehicle-treated animals induced a robust craniofacial allodynia lasting at least 45 days. On the other hand, nasal SHPE application 2 days post-TBI attenuated facial allodynia, reaching significance by day 4–7 and maintaining this effect throughout the duration of the experiment. Immunohistochemical examination revealed strong expression of human proenkephalin in trigeminal ganglia of SHPE, but not SHZ.1-treated rats. This study demonstrates that intranasal administration of HSV-based gene vectors may be a viable, non-invasive means of treating chronic craniofacial pain, including post-TBI pain.

Intranasal Oxytocin Attenuates Reactive and Ongoing, Chronic Pain in a Model of Mild Traumatic Brain Injury

Approximately 1.7 million Americans sustain a traumatic brain injury (TBI) each year and chronic pain is a common complication.

Visualizing Nerve Injury in a Neuropathic Pain Model with [ 18 F]FTC-146 PET/MRI

The ability to locate nerve injury and ensuing neuroinflammation would have tremendous clinical value for improving both the diagnosis and subsequent management of patients suffering from pain, weakness, and other neurologic phenomena associated with peripheral nerve injury. Although several non-invasive techniques exist for assessing the clinical manifestations and morphological aspects of nerve injury, they often fail to provide accurate diagnoses due to limited specificity and/or sensitivity. Herein, we describe a new imaging strategy for visualizing a molecular biomarker of nerve injury/neuroinflammation, i.e., the sigma-1 receptor (S1R), in a rat model of nerve injury and neuropathic pain. The two-fold higher increase of S1Rs was shown in the injured compared to the uninjured nerve by Western blotting analyses. With our novel S1R-selective radioligand, [18F]FTC-146 (6-(3-[18F]fluoropropyl)-3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one), and positron emission tomography-magnetic resonance imaging (PET/MRI), we could accurately locate the site of nerve injury created in the rat model. We verified the accuracy of this technique by ex vivo autoradiography and immunostaining, which demonstrated a strong correlation between accumulation of [18F]FTC-146 and S1R staining. Finally, pain relief could also be achieved by blocking S1Rs in the neuroma with local administration of non-radioactive [19F]FTC-146. In summary, [18F]FTC-146 S1R PET/MR imaging has the potential to impact how we diagnose, manage and treat patients with nerve injury, and thus warrants further investigation.