Pharmacology/Drug Development

Despite the effort on developing new treatments, therapy for neuropathic pain is still a clinical challenge and combination therapy regimes of two or more drugs are often needed to improve efficacy. Accumulating evidence shows an altered expression and activity of histone acetylation enzymes in chronic pain conditions and restoration of these aberrant epigenetic modifications promotes pain-relieving activity. Recent studies showed a synergistic activity in neuropathic pain models by combination of histone deacetylases (HDACs) and bromodomain and extra-terminal domain (BET) inhibitors. On these premises, the present study investigated the pharmacological profile of new dual HDAC/BRD4 inhibitors, named SUM52 and SUM35, in the spared nerve injury (SNI) model in mice as innovative strategy to simultaneously inhibit HDACs and BETs. Intranasal administration of SUM52 and SUM35 attenuated thermal and mechanical hypersensitivity in the absence of locomotor side effects. Both dual inhibitors showed a preferential interaction with BRD4-BD2 domain, and SUM52 resulted the most active compound. SUM52 reduced microglia-mediated spinal neuroinflammation in spinal cord sections of SNI mice as showed by reduction of IBA1 immunostaining, inducible nitric oxide synthase (iNOS) expression, p65 nuclear factor-κB (NF-κB) and p38 MAPK over-phosphorylation. A robust decrease of the spinal proinflammatory cytokines content (IL-6, IL-1ß) was also observed after SUM52 treatment. Present results, showing the pain-relieving activity of HDAC/BRD4 dual inhibitors, indicate that the simultaneous modulation of BET and HDAC activity by a single molecule acting as multi-target agent might represent a promise for neuropathic pain relief.

Cannabinoids have been increasingly used to alleviate chronic pain; however, tolerance to the antinociceptive effects of cannabinoids, including delta-9-tetrahydrocannabinol (Δ -THC), may limit their therapeutic utility. Likewise, with more women than men now using medical cannabis for pain relief, it is imperative that we understand how sex may influence cannabinoid-mediated antinociception and subsequent tolerance. While studies in rats have consistently found female rats to be more sensitive to the acute antinociceptive effects of cannabinoids compared to male rats, work in our lab consistently finds the opposite finding that male mice are more sensitive to the acute antinociceptive effects of both Δ -THC and CP55,940 compared to female littermates. Studies in our lab have consistently utilized mice on a C57BL6/J (B6) background. Therefore, the purpose of the present study is to examine whether our observed sex-differences in Δ -THC-induced antinociception and tolerance are consistent across multiple mouse strains or are strain-dependent. Male and female B6 and DBA mice were first assessed for differences in acute Δ -THC-induced antinociception using the tail-flick assay across a range of doses of (0-100 mg/kg). After a significant washout period, these mice were subsequently assessed for sex-differences in antinociceptive tolerance development to 30 mg/kg Δ -THC following once-daily treatment for seven consecutive days. Consistent with our previous findings, male B6 mice were more sensitive to the acute antinociceptive effects of Δ -THC than female B6 mice. Male and female DBA, however, mice did not differ in their antinociceptive response to Δ -THC, suggesting that sex-differences in cannabinoid-induced antinociception in mice is likely strain-specific. These studies highlight the therapeutic potential of Δ -THC in pain management and underscore the importance of considering sex when evaluating their clinical utility.

Histone deacetylase 6 (HDAC6) is a Class IIb histone deacetylase, which is primarily localized to the cytoplasm and plays an important role in cell structure and dynamism, transcriptional repression, exocytosis and endocytocis. Preclinical evidence has suggested that HDAC6 inhibitors alleviate signs of chemotherapy-induced peripheral neuropathy (CIPN), such as mechanical allodynia. However, no group to our knowledge has investigated the mechanism of action of HDAC6 inhibitors in a severe nerve injury model, which has a different pathophysiology than milder models such as CIPN. In this study we use genetically modified mice and the tibial spared nerve injury (SNI) model to determine the impact of dorsal root ganglion (DRG) specific HDAC6 knockout in the induction and maintenance of sensory hypersensitivity. Downregulation of HDAC6 in the DRG was achieved by injection of the left sciatic nerve of adult male HDAC6 mice with AAV8-CMV-Cre-EGFP or AAV8-CMV-EGFP vectors. DRG-knockdown of HDAC6 prevented the development of mechanical allodynia after SNI. Using in situ hybridization (RNAScope), we also demonstrate that HDAC6 is upregulated at three weeks after the induction of nerve injury in L3-6 DRG neurons. Furthermore, treatment with the peripherally acting HDAC6 inhibitor ACY1215 (Regenacy Pharmaceuticals, Waltham, MA) after SNI leads to full recovery from mechanical allodynia. We are continuing this work by performing bulk RNA sequencing on L3-6 DRGs from mice who received 21 days of treatment with ACY1215 or vehicle starting at three weeks after SNI or Sham operation, in order to understand the transcriptomic events and upstream pathways associated with recovery from prolonged neuropathic states. Our findings highlight a promising therapeutic role of HDAC6 inhibitors for the prevention or recovery from sensory hypersensitivity behaviors associated with prolonged peripheral nerve injury. Our future work aims to define the mechanisms underlying HDAC6 action in the DRG and identify new pathways associated with recovery from sensory hypersensitivity to influence novel treatment strategies for pain.

Opioid analgesics are critical for acute and chronic pain management, but important side effects-including tolerance, constipation, respiratory depression, and abuse liability-limit their safety and utility. To provide patients with safer analgesic options, it is critically important to identify of new pharmacotherapeutic strategies to treat pain. Activation of µ-opioid receptors (MORs) in central and peripheral nociceptive pathways mediates opioid analgesia and their critical side effects. Antinociception can also be achieved via selective enhancement of GABAergic signaling at ionotropic GABA receptors. α2 and α3 subunit-containing GABA receptors (α2/α3GABA ), which are co-expressed with MORs in dorsal horn spinal pathways important to nociceptive transmission, can be selectively targeted with novel imidazodiazepine positive allosteric modulators (PAMs), such as MP-III-024, which produces antinociceptive effects with limited behavioral disruption. MP-III-024 co-administered with morphine produces synergistic antinociceptive and anti-hyperalgesic effects. In this study, we evaluated whether MP-III-024/morphine co-administration produces sub-additive or synergistic effects in behavioral tests sensitive to morphine side effects. Herein we report that co-administration of MP-III-024/morphine at a 0.94/1 ratio (synergistic in models of antinociception) produced sub-additive effects in morphine-induced hyperlocomotion and in measures of behavioral disruption in food-maintained operant responding. Ongoing studies are evaluating the effects of MP-III-024/morphine co-administration on tolerance in the hot plate test and in conditioned place preference. These experiments are the first comprehensive preclinical analyses of a dual MOR-α2/α3GABA pharmacotherapy strategy which may increase the therapeutic window between desirable opioid analgesic effects and side effects.

Chronic use of mu-opioid receptor (MOR) agonists, such as morphine, for pain management can lead to the rapid development of analgesic tolerance. In contrast, tolerance to morphine effects in the gastrointestinal tract develops at a different rate. This can limit the therapeutic utility of morphine as the undesired gastrointestinal effects can persist even in the absence of analgesia. It is unclear if the discrete rates of morphine tolerance are due to different underlying mechanisms. β-arrestin-2 is a multi-functional protein implicated in the mechanism of antinociceptive tolerance to opioids. We have recently shown that morphine tolerance in dorsal root ganglia nociceptive neurons is mediated via two distinct mechanisms that are dependent on the duration of drug exposure: a β-arrestin-2-dependent mechanism for short-term (15-18 hours) tolerance and a β-arrestin-2-independent mechanism for long-term (7 days) tolerance (Muchhala et al., 2021, European Journal of Pharmacology). In the gastrointestinal tract, myenteric plexus neurons regulate intestinal motility. However, it is not clear if morphine engages the same molecular mechanisms for tolerance development in myenteric plexus neurons and dorsal root ganglia neurons. Therefore, in the present study we investigated the role of β-arrestin-2 in the development of morphine tolerance in myenteric plexus neurons of the mouse ileum. Here, we used whole-cell patch clamp electrophysiology to examine morphine tolerance in individual ileum myenteric plexus neurons treated with 10 µM morphine for 15-18 hours or isolated from mice treated with morphine for 7 days. Acute 3 µM morphine significantly increased the threshold to fire action potentials in naïve myenteric plexus neurons. However, this effect was not observed in wild-type neurons exposed to morphine for 15 -18 hours or isolated from mice treated with morphine for 7 days, indicating the development of tolerance. Furthermore, genetic deletion of β-arrestin-2 did not prevent the development of morphine tolerance in ileum neurons. In contrast, Bisindolylmaleimide XI, a selective protein kinase C inhibitor, reversed tolerance in ileum myenteric plexus neurons exposed to morphine in vivo for 7 days. In these neurons acute 3 µM morphine significantly increased action potential threshold. Thus, unlike dorsal root ganglia neurons, morphine tolerance in myenteric plexus neurons does not utilize β-arrestin-2. These findings reveal a potential mechanism for the differences in the rates of tolerance to morphine and highlight the need to investigate tolerance mechanisms for mu-opioid analgesics in different tissues/neurons. These results also indicate that mu-opioid analgesics that preferentially signal through G-proteins over β-arrestin-2 i.e. biased agonists, such as Oliceridine, may induce tolerance in the ileum.

The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with pain therapeutics. In this study, we developed a mouse model of oxycodone misuse in order to gain insight into genes and molecular pathways in reward-related brain regions that are affected by prolonged exposure to oxycodone and subsequent withdrawal in the presence or absence of chronic neuropathic pain. RNA-sequencing (RNA-seq) and bioinformatic analyses revealed that oxycodone withdrawal alone triggers robust gene expression adaptations in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and ventral tegmental area (VTA), with numerous genes and pathways selectively affected by oxycodone withdrawal under peripheral nerve injury states. Our pathway analysis predicted that histone deacetylase 1 (HDAC1), an epigenetic modifier with a prominent role in striatal plasticity, is a top upstream regulator in opioid withdrawal in both the NAc and mPFC. Indeed, treatment with the novel HDAC1/2 inhibitor RCY1305 attenuated behavioral manifestations of oxycodone withdrawal, with the drug being more efficacious under states of neuropathic pain. Our studies also suggest that RCY1305 treated mice did not develop conditional place preference. Since RCY1305 displays antiallodynic actions with no rewarding effects in models of neuropathic pain, inhibition of HDAC1/2 may provide an avenue for chronic pain patients dependent on opioids to transition to non-opioid analgesics. Overall, our study highlights transcriptomic events in components of the reward circuitry associated with oxycodone withdrawal under pain-naïve and prolonged neuropathic pain states, thereby providing information on possible new targets for the treatment of physical dependence to opioids and transitioning individuals to non-opioid medications for chronic pain management.

Chronic neuropathic pain is a major health issue and an economic burden that affects large numbers of people. Patients suffering from chronic pain have a significantly lowered quality of life, and to date there are no effective treatments for neuropathic pain. The P2Y receptor (P2Y R) is a purinergic G-protein coupled receptor that binds nucleotide-sugars. P2RY14 is widely expressed in the body and is found in the immune system and nervous tissues. Few studies provide evidence that P2Y R is involved in pain conditions. In rat traumatic nerve injury-induced pain and post-surgical pain models, P2RY14 transcript levels were found to increase on the same side of the spinal cord as the nerve injury. In addition, P2Y R expression was elevated in an inflammatory pain model after a complete Freund's adjuvant injection in the rat trigeminal ganglia. Taken together, we hypothesize that the P2Y R plays a role in the development and maintenance of neuropathic pain. To test this, we used peripheral nerve injury model of neuropathic pain and tested several novel P2R R antagonists, which had varying potencies and bioavailabilities. Adult male ICR mice went through unilateral chronic constriction of the sciatic nerve, and on day 7 post injury they received a P2Y R antagonist intraperitoneally and the mechanical sensitivity in the hind paws was assessed. The antagonists rapidly (≤30 min) attenuated, some even reversed, mechanical sensitivity in the mice, with maximal effects observed typically within 1 h post-injection, in a dose-dependent manner. Overall, our findings provide evidence that the P2Y R is a potential therapeutic target for treating chronic pain, and its antagonists can be candidate drugs for pain treatment.