Advances in understanding migraine pathophysiology: a bench to bedside review of research insights and therapeutics.

The individual and global burden of migraine is of such significance that there are accelerated efforts to develop new therapies. New migraine therapeutics are needed to address the current deficiencies that exist in the efficacy and adherence rate of approved anti-migraine medications. The recent discovery of the calcitonin gene related peptide as an add-on to the role of serotonin has markedly increased the range of new treatment options for acute and chronic migraine. Despite this, tackling the complexity of migraine disorders requires a complete understanding of its pathophysiology. Preclinical animal models can shed light on disease-related pathophysiology, including migraine. Indeed, the use of animal models has been instrumental in developing many therapeutics. However, an animal model is limited by the predictive and face validity of that model, and this extends to preclinical migraine models. In this review, a summary of the current understanding of the pathophysiology of migraine is given from both a preclinical and clinical perspective, and an emphasis is placed on the animal models of migraine. We will discuss the strengths and pitfalls of common preclinical migraine models as well as experimental research areas to explore further.

Characterization of a Nonselective Opioid Receptor Functional Antagonist: Implications for Development as a Novel Opioid Dependence Medication.

Opioids represent the most extensive category of abused substances in the United States, and the number of fatalities caused by these drugs exceeds those associated with all other drug overdoses combined. The administration of naltrexone, a potent pan-opioid receptor antagonist, to an individual dependent on opioids can trigger opioid withdrawal and induce severe side effects. There is a pressing demand for opioid antagonists free of opioid withdrawal effects. In our laboratory, we have identified a compound with affinity to mu, delta, and kappa opioid receptors in the range of 150-250 nM. This blood-brain barrier (BBB)-permeant compound was metabolically stable in vitro and in vivo. Our in vivo work demonstrated that 1-10 mg/kg intraperitoneal administration of our compound produces moderate efficacy in antagonizing morphine-induced antiallodynia effects in the chemotherapy-induced peripheral neuropathy (CIPN) model. The treatment was well-tolerated and did not cause behavioral changes. We have observed a fast elimination rate of this metabolically stable molecule. Furthermore, no organ toxicity was observed during the chronic administration of the compound over a 14-day period. Overall, we report a novel functional opioid antagonist holds promise for developing an opioid withdrawal therapeutic.

Cannabidiol and mitragynine exhibit differential interactive effects in the attenuation of paclitaxel-induced mechanical allodynia, acute antinociception, and schedule-controlled responding in mice.

BACKGROUND: For many chemotherapy patients peripheral neuropathy is a debilitating side effect. Mitragyna speciosa (kratom) contains the alkaloid mitragynine (MG), which produces analgesia in multiple preclinical pain models. In humans, anecdotal reports suggest cannabidiol (CBD) may enhance kratom-related analgesia. We examined the interactive activity of MG and CBD in a mouse chemotherapy-induced peripheral neuropathy (CIPN) model. We also examined MG + CBD in acute antinociception and schedule-controlled responding assays, as well as examined underlying receptor mechanisms. METHODS: Male and female C57BL/6J mice received a cycle of intraperitoneal (ip) paclitaxel injections (cumulative dose 32 mg/kg). The von Frey assay was utilized to assess CIPN allodynia. In paclitaxel-naïve mice, schedule-controlled responding for food was conducted under a fixed ratio (FR)-10, and hot plate antinociception was examined. RESULTS: MG dose-relatedly attenuated CIPN allodynia (ED50 102.96 mg/kg, ip), reduced schedule-controlled responding (ED50 46.04 mg/kg, ip), and produced antinociception (ED50 68.83 mg/kg, ip). CBD attenuated allodynia (ED50 85.14 mg/kg, ip) but did not decrease schedule-controlled responding or produce antinociception. Isobolographic analysis revealed 1:1, 3:1 MG + CBD mixture ratios additively attenuated CIPN allodynia. All combinations decreased schedule-controlled responding and produced antinociception. WAY-100635 (serotonin 5-HT1A receptor antagonist) pretreatment (0.01 mg/kg, ip) antagonized CBD anti-allodynia. Naltrexone (pan opioid receptor antagonist) pretreatment (0.032 mg/kg, ip) antagonized MG anti-allodynia and acute antinociception but produced no change in MG-induced decreased schedule-controlled behavior. Yohimbine (α2 receptor antagonist) pretreatment (3.2 mg/kg, ip) antagonized MG anti-allodynia and produced no change in MG-induced acute antinociception or decreased schedule-controlled behavior. CONCLUSIONS: Although more optimization is needed, these data suggest CBD combined with MG may be useful as a novel CIPN therapeutic.

Medicinal Cannabis and Central Nervous System Disorders.

Cannabinoids, including those found in cannabis, have shown promise as potential therapeutics for numerous health issues, including pathological pain and diseases that produce an impact on neurological processing and function. Thus, cannabis use for medicinal purposes has become accepted by a growing majority. However, clinical trials yielding satisfactory endpoints and unequivocal proof that medicinal cannabis should be considered a frontline therapeutic for most examined central nervous system indications remains largely elusive. Although cannabis contains over 100 + compounds, most preclinical and clinical research with well-controlled dosing and delivery methods utilize the various formulations of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), the two most abundant compounds in cannabis. These controlled dosing and delivery methods are in stark contrast to most clinical studies using whole plant cannabis products, as few clinical studies using whole plant cannabis profile the exact composition, including percentages of all compounds present within the studied product. This review will examine both preclinical and clinical evidence that supports or refutes the therapeutic utility of medicinal cannabis for the treatment of pathological pain, neurodegeneration, substance use disorders, as well as anxiety-related disorders. We will predominately focus on purified THC and CBD, as well as other compounds isolated from cannabis for the aforementioned reasons but will also include discussion over those studies where whole plant cannabis has been used. In this review we also consider the current challenges associated with the advancement of medicinal cannabis and its derived potential therapeutics into clinical applications.

Novel roles of phentolamine in protecting axon myelination, muscle atrophy, and functional recovery following nerve injury.

Incomplete functional recovery after peripheral nerve injury (PNI) often results in devastating physical disabilities in human patients. Despite improved progress in surgical and non-surgical approaches, achieving complete functional recovery following PNI remains a challenge. This study demonstrates that phentolamine may hold a significant promise in treating nerve injuries and denervation induced muscle atrophy following PNI. In a sciatic nerve crush injury mouse model, we found that phentolamine treatment enhanced motor and functional recovery, protected axon myelination, and attenuated injury-induced muscle atrophy in mice at 14 days post-injury (dpi) compared to saline treatment. In the soleus of phentolamine treated animals, we observed the downregulation of phosphorylated signal transducer and activator of transcription factor 3 (p-STAT3) as well as muscle atrophy-related genes Myogenin, muscle ring finger 1 (MuRF-1), and Forkhead box O proteins (FoxO1, FoxO3). Our results show that both nerve and muscle recovery are integral components of phentolamine treatment-induced global functional recovery in mice at 14 dpi. Moreover, phentolamine treatment improved locomotor functional recovery in the mice after spinal cord crush (SCC) injury. The fact that phentolamine is an FDA approved non-selective alpha-adrenergic blocker, clinically prescribed for oral anesthesia reversal, hypertension, and erectile dysfunction makes this drug a promising candidate for repurposing in restoring behavioral recovery following PNI and SCC injuries, axonal neuropathy, and muscle wasting disorders.

Evaluation of the terpenes ß-caryophyllene, α-terpineol, and γ-terpinene in the mouse chronic constriction injury model of neuropathic pain: possible cannabinoid receptor involvement.

Pain is one of the most common reasons to seek medical attention, and chronic pain is a worldwide epidemic. Anecdotal reports suggest cannabis may be an effective analgesic. As cannabis contains the terpenes α-terpineol, ß-caryophyllene, and γ-terpinene, we hypothesized these terpenes would produce analgesia in a mouse model of neuropathic pain. We used the chronic constriction injury of the sciatic nerve mouse model, which produces mechanical allodynia, assessed via the von Frey assay, as well as thermal hyperalgesia assessed via the hotplate assay. Compounds were further assessed in tests of locomotor activity, hypothermia, and acute antinociception. Each terpene produced dose-related reversal of mechanical allodynia and thermal hyperalgesia. Thermal hyperalgesia displayed higher sensitivity to the effects of each terpene than mechanical allodynia, and the rank order potency of the terpenes was α-terpineol > ß-caryophyllene > γ-terpinene. To examine the involvement of cannabinoid receptors, further tests were conducted in mice lacking either functional cannabinoid type 1 receptors (CB1R (-/-)) or cannabinoid type 2 receptors (CB2R (-/-)). Compared to wild type mice, CB1R (-/-) mice treated with α-terpineol displayed a 2.91-fold decrease in potency to reverse mechanical allodynia; in CB2R (-/-) mice, the potency of α-terpineol was decreased 11.73-fold. The potency of ß-caryophyllene to reverse mechanical allodynia decreased 1.80-fold in CB2R (-/-) mice. Each terpene produced a subset of effects in tests of locomotor activity, hypothermia, and acute antinociception. These findings suggest α-terpineol, ß-caryophyllene, and γ-terpinene may have differential cannabinoid receptor activity and a pharmacological profile that may yield new efficacious analgesics.

DT2216-a Bcl-xL-specific degrader is highly active against Bcl-xL-dependent T cell lymphomas.

BACKGROUND: Patients with advanced T cell lymphomas (TCLs) have limited therapeutic options and poor outcomes in part because their TCLs evade apoptosis through upregulation of anti-apoptotic Bcl-2 proteins. Subsets of TCL cell lines, patient-derived xenografts (PDXs), and primary patient samples depend on Bcl-xL for survival. However, small molecule Bcl-xL inhibitors such as ABT263 have failed during clinical development due to on-target and dose-limiting thrombocytopenia. METHODS: We have developed DT2216, a proteolysis targeting chimera (PROTAC) targeting Bcl-xL for degradation via Von Hippel-Lindau (VHL) E3 ligase, and shown that it has better anti-tumor activity but is less toxic to platelets compared to ABT263. Here, we examined the therapeutic potential of DT2216 for TCLs via testing its anti-TCL activity in vitro using MTS assay, immunoblotting, and flow cytometry and anti-TCL activity in vivo using TCL cell xenograft and PDX model in mice. RESULTS: The results showed that DT2216 selectively killed various Bcl-xL-dependent TCL cells including MyLa cells in vitro. In vivo, DT2216 alone was highly effective against MyLa TCL xenografts in mice without causing significant thrombocytopenia or other toxicity. Furthermore, DT2216 combined with ABT199 (a selective Bcl-2 inhibitor) synergistically reduced disease burden and improved survival in a TCL PDX mouse model dependent on both Bcl-2 and Bcl-xL. CONCLUSIONS: These findings support the clinical testing of DT2216 in patients with Bcl-xL-dependent TCLs, both as a single agent and in rational combinations.

Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity.

Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors.

Inhibition of USP7 activity selectively eliminates senescent cells in part via restoration of p53 activity.

The accumulation of senescent cells (SnCs) is a causal factor of various age-related diseases as well as some of the side effects of chemotherapy. Pharmacological elimination of SnCs (senolysis) has the potential to be developed into novel therapeutic strategies to treat these diseases and pathological conditions. Here we show that ubiquitin-specific peptidase 7 (USP7) is a novel target for senolysis because inhibition of USP7 with an inhibitor or genetic depletion of USP7 by RNA interference induces apoptosis selectively in SnCs. The senolytic activity of USP7 inhibitors is likely attributable in part to the promotion of the human homolog of mouse double minute 2 (MDM2) ubiquitination and degradation by the ubiquitin-proteasome system. This degradation increases the levels of p53, which in turn induces the pro-apoptotic proteins PUMA, NOXA, and FAS and inhibits the interaction of BCL-XL and BAK to selectively induce apoptosis in SnCs. Further, we show that treatment with a USP7 inhibitor can effectively eliminate SnCs and suppress the senescence-associated secretory phenotype (SASP) induced by doxorubicin in mice. These findings suggest that small molecule USP7 inhibitors are novel senolytics that can be exploited to reduce chemotherapy-induced toxicities and treat age-related diseases.