Myelin regulatory factor is a target of individual and interactive effects of HIV-1 Tat and morphine in the striatum and pre-frontal cortex.

HIV-associated neurocognitive disorders (HAND) remain pervasive even with increased efficacy/use of antiretroviral therapies. Opioid use/abuse among HIV + individuals is documented to exacerbate CNS deficits. White matter (WM) alterations, including myelin pallor, and volume/structural alterations detected by diffusion tensor imaging are common observations in HIV + individuals, and studies in non-human primates suggest that WM may harbor virus. Using transgenic mice that express the HIV-1 Tat protein, we examined in vivo effects of 2-6 weeks of Tat and morphine exposure on WM using genomic and biochemical methods. RNA sequencing of striatal tissue at 2 weeks revealed robust changes in mRNAs associated with oligodendrocyte precursor populations and myelin integrity, including those for transferrin, the atypical oligodendrocyte marker N-myc downstream regulated 1 (Ndrg1), and myelin regulatory factor (Myrf/Mrf), an oligodendrocyte-specific transcription factor with a significant role in oligodendrocyte differentiation/maturation. Western blots conducted after 6-weeks exposure in 3 brain regions (striatum, corpus callosum, pre-frontal cortex) revealed regional differences in the effect of Tat and morphine on Myrf levels, and on levels of myelin basic protein (MBP), whose transcription is regulated by Myrf. Responses included individual and interactive effects. Although baseline and post-treatment levels of Myrf and MBP differed between brain regions, post-treatment MBP levels in striatum and pre-frontal cortex were compatible with changes in Myrf activity. Additionally, the Myrf regulatory ubiquitin ligase Fbxw7 was identified as a novel target in our model. These results suggest that Myrf and Fbxw7 contribute to altered myelin gene regulation in HIV.

Kratom Alkaloids, Natural and Semi-Synthetic, Show Less Physical Dependence and Ameliorate Opioid Withdrawal.

Chronic administration of opioids produces physical dependence and opioid-induced hyperalgesia. Users claim the Thai traditional tea "kratom" and component alkaloid mitragynine ameliorate opioid withdrawal without increased sensitivity to pain. Testing these claims, we assessed the combined kratom alkaloid extract (KAE) and two individual alkaloids, mitragynine (MG) and the analog mitragynine pseudoindoxyl (MP), evaluating their ability to produce physical dependence and induce hyperalgesia after chronic administration, and as treatments for withdrawal in morphine-dependent subjects. C57BL/6J mice (n = 10/drug) were administered repeated saline, or graded, escalating doses of morphine (intraperitoneal; i.p.), kratom alkaloid extract (orally, p.o.), mitragynine (p.o.), or MP (subcutaneously, s.c.) for 5 days. Mice treated chronically with morphine, KAE, or mitragynine demonstrated significant drug-induced hyperalgesia by day 5 in a 48 °C warm-water tail-withdrawal test. Mice were then administered naloxone (10 mg/kg, s.c.) and tested for opioid withdrawal signs. Kratom alkaloid extract and the two individual alkaloids demonstrated significantly fewer naloxone-precipitated withdrawal signs than morphine-treated mice. Additional C57BL/6J mice made physically dependent on morphine were then used to test the therapeutic potential of combined KAE, mitragynine, or MP given twice daily over the next 3 days at either a fixed dose or in graded, tapering descending doses. When administered naloxone, mice treated with KAE, mitragynine, or MP under either regimen demonstrated significantly fewer signs of precipitated withdrawal than control mice that continued to receive morphine. In conclusion, while retaining some liabilities, kratom, mitragynine, and mitragynine pseudoindoxyl produced significantly less physical dependence and ameliorated precipitated withdrawal in morphine-dependent animals, suggesting some clinical value.

Expression of Human Immunodeficiency Virus Transactivator of Transcription (HIV-Tat1-86) Protein Alters Nociceptive Processing that is Sensitive to Anti-Oxidant and Anti-Inflammatory Interventions.

Despite the success of combined antiretroviral therapy (cART) in reducing viral load, a substantial portion of Human Immunodeficiency Virus (HIV)+ patients report chronic pain. The exact mechanism underlying this co-morbidity even with undetectable viral load remains unknown, but the transactivator of transcription (HIV-Tat) protein is of particular interest. Functional HIV-Tat protein is observed even in cerebrospinal fluid of patients who have an undetectable viral load. It is hypothesized that Tat protein exposure is sufficient to induce neuropathic pain-like manifestations via both activation of microglia and generation of oxidative stress. iTat mice conditionally expressed Tat(1-86) protein in the central nervous system upon daily administration of doxycycline (100 mg/kg/d, i.p., up to 14 days). The effect of HIV-Tat protein exposure on the well-being of the animal was assessed using sucrose-evoked grooming and acute nesting behavior for pain-depressed behaviors, and the development of hyperalgesia assessed with warm-water tail-withdrawal and von Frey assays for thermal hyperalgesia and mechanical allodynia, respectively. Tissue harvested at select time points was used to assess ex vivo alterations in oxidative stress, astrocytosis and microgliosis, and blood-brain barrier integrity with assays utilizing fluorescence-based indicators. Tat protein induced mild thermal hyperalgesia but robust mechanical allodynia starting after 4 days of exposure, reaching a nadir after 7 days. Changes in nociceptive processing were associated with reduced sucrose-evoked grooming behavior without altering acute nesting behavior, and in spinal cord dysregulated free radical generation as measured by DCF fluorescence intensity, altered immunohistochemical expression of the gliotic markers, Iba-1 and GFAP, and increased permeability of the blood-brain barrier to the small molecule fluorescent tracer, sodium fluorescein, in a time-dependent manner. Pretreatment with the anti-inflammatory, indomethacin (1 mg/kg/d, i.p.), the antioxidant, methylsulfonylmethane (100 mg/kg/d i.p.), or the immunomodulatory agent, dimethylfumarate (100 mg/kg/d p.o.) thirty minutes prior to daily injections of doxycycline (100 mg/kg/d i.p.) over 7 days significantly attenuated the development of Tat-induced mechanical allodynia. Collectively, the data suggests that even acute exposure to HIV-1 Tat protein at pathologically relevant levels is sufficient to produce select neurophysiological and behavioral manifestations of chronic pain consistent with that reported by HIV-positive patients.

Controlling opioid receptor functional selectivity by targeting distinct subpockets of the orthosteric site.

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gi-partial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism.

Multifunctional opioid receptor agonism and antagonism by a novel macrocyclic tetrapeptide prevents reinstatement of morphine-seeking behaviour.

BACKGROUND AND PURPOSE: The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) is a multifunctional µ-opioid receptor and κ-opioid receptor agonist and κ-opioid receptor antagonist that produces antinociception and prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference (CPP). We hypothesized that an analogue of CJ-15,208, cyclo[Pro-Sar-Phe-d-Phe], would demonstrate multifunctional µ-opioid receptor and κ-opioid receptor ligand activity, producing potent antinociception with fewer liabilities than selective µ-opioid receptor agonists, while preventing both drug- and stress-induced reinstatement of morphine-induced CPP. EXPERIMENTAL APPROACH: The opioid receptor agonist and antagonist activity of cyclo[Pro-Sar-Phe-d-Phe] was characterized after i.c.v. and i.p. administration to C57BL/6J or transgenic opioid receptor "knockout" mice using the 55°C warm-water tail-withdrawal assay. Liabilities of locomotor coordination, respiration and spontaneous ambulation, and direct rewarding or aversive properties were assessed. Finally, the ability of cyclo[Pro-Sar-Phe-d-Phe] to block morphine- and stress-induced reinstatement of extinguished CPP was determined. KEY RESULTS: cyclo[Pro-Sar-Phe-d-Phe] demonstrated dose-dependent, short-lasting antinociception, with an ED50 (and 95% confidence interval) of 0.15 (0.05-0.21) nmol i.c.v. and 1.91 (0.40-3.54) mg·kg-1 i.p., mediated by µ- and κ-opioid receptors. The macrocyclic tetrapeptide also demonstrated potent dose-dependent κ-opioid receptor antagonist-like activity at 2.5, but not at 4.5, h after administration. cyclo[Pro-Sar-Phe-d-Phe] displayed reduced liabiities compared with morphine, attributed to its additional activity at κ-receptors. Pretreatment with cyclo[Pro-Sar-Phe-d-Phe] prevented stress- and drug-induced reinstatement of extinguished morphine-place preference responses in a time-dependent manner. CONCLUSIONS AND IMPLICATIONS: These data suggest that cyclo[Pro-Sar-Phe-d-Phe] is a promising lead compound for both the treatment of pain with reduced sideeffects and preventing both drug- and stress-induced relapse in morphine-abstinent subjects.

Lyophilized Kratom Tea as a Therapeutic Option for Opioid Dependence.

BACKGROUND: Made as a tea, the Thai traditional drug "kratom" reportedly possesses pharmacological actions that include both a coca-like stimulant effect and opium-like depressant effect. Kratom has been used as a substitute for opium in physically-dependent subjects. The objective of this study was to evaluate the antinociception, somatic and physical dependence produced by kratom tea, and then assess if the tea ameliorated withdrawal in opioid physically-dependent subjects. METHODS: Lyophilized kratom tea (LKT) was evaluated in C57BL/6J and opioid receptor knockout mice after oral administration. Antinociceptive activity was measured in the 55 °C warm-water tail-withdrawal assay. Potential locomotor impairment, respiratory depression and locomotor hyperlocomotion, and place preference induced by oral LKT were assessed in the rotarod, Comprehensive Lab Animal Monitoring System, and conditioned place preference assays, respectively. Naloxone-precipitated withdrawal was used to determine potential physical dependence in mice repeatedly treated with saline or escalating doses of morphine or LKT, and LKT amelioration of morphine withdrawal. Data were analyzed using one- and two-way ANOVA. RESULTS: Oral administration of LKT resulted in dose-dependent antinociception (≥1 g/kg, p.o.) absent in mice lacking the mu-opioid receptor (MOR) and reduced in mice lacking the kappa-opioid receptor. These doses of LKT did not alter coordinated locomotion or induce conditioned place preference, and only briefly reduced respiration. Repeated administration of LKT did not produce physical dependence, but significantly decreased naloxone-precipitated withdrawal in morphine dependent mice. CONCLUSIONS: The present study confirms the MOR agonist activity and therapeutic effect of LKT for the treatment of pain and opioid physical dependence.