Pharmacology/Drug Development

The plasma of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene-Related Peptide (iCGRP) release as a measure of nociceptor activation and found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the mM range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We showed that also at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the μM range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an over-additive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of TRPV1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Voltage-gated sodium (Nav) channels respond to changes in the membrane potential of excitable cells through the concerted action of four voltage-sensor domains (VSDs). Subtype Nav1.7 plays an important role in the propagation of signals in pain-sensing neurons and is a target for the clinical development of novel analgesics. Certain inhibitory cystine knot (ICK) peptides produced by venomous animals potently modulate Nav1.7; however, the molecular mechanisms underlying their selective binding and activity remain elusive. This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of Nav1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-S2 and helix S3 of VSD2 in a chimeric channel system. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on Nav channels.

Developing potent non-opioid pain medications is an integral part of the battle to conquer both chronic pain and the current opioid crisis. Although most screening approaches use in vitro surrogate targets, in vivo screening of analgesic candidates is a necessary pre-clinical step in drug discovery. Here, we report the design of a new automated behavioral testing apparatus based on the principle of a thermal place preference test (TPPT). This new design can detect, quantify, and differentiate behavioral responses to cold stimuli between sham and chronic constriction injury (CCI) rodents with up to 12 animals tested simultaneously. At an optimized temperature pair of 12.5°C vs 30.0°C (± 0.5℃), the TPPT design has captured the antinociceptive effects of morphine and pregabalin on CCI rats in individual 10-min tests. Moreover, it can differentiate analgesic effects by morphine or pregabalin from anxiolytic effects by diazepam. The results, along with the relatively low cost to construct the apparatus and moderately high throughput, make our TPPT design applicable for behavioral studies of chronic pain in rodents and for high-throughput in vivo screening of the next generation of pain medications.

Paclitaxel-induced neuropathic pain (PINP) is refractory to currently used analgesics. Previous studies show a pivotal role of oxidative stress in PINP. Because the nuclear factor erythroid-2-related factor 2 (Nrf2) has been considered as the critical regulator of endogenous antioxidant defense, we here explored whether activation of Nrf2 could attenuate PINP. A rat model of PINP was established by intraperitoneal injection of paclitaxel (2 mg/kg) every other day with a final cumulative dose of 8 mg/kg. Hind paw withdrawal thresholds (PWTs) in response to von Frey filament stimuli were used to assess mechanical allodynia. We showed that a single dose of Nrf2 activator, oltipraz (10, 50, and 100 mg/kg), dose-dependently attenuated established mechanical allodynia, whereas repeated injection of oltipraz (100 mg· kg· d, i.p. from d 14 to d 18) almost abolished the mechanical allodynia in PINP rats. The antinociceptive effect of oltipraz was blocked by pre-injection of Nrf2 inhibitor trigonelline (20 mg/kg, i.p.). Early treatment with oltipraz (100 mg· kg· d, i.p. from d 0 to d 6) failed to prevent the development of the PINP, but delayed its onset. Western blot and immunofluorescence analysis revealed that the expression levels of Nrf2 and HO-1 were significantly upregulated in the spinal cord of PINP rats. Repeated injection of oltipraz caused further elevation of the expression levels of Nrf2 and HO-1 in the spinal cord of PINP rats, which was reversed by pre-injection of trigonelline. These results demonstrate that oltipraz ameliorates PINP via activating Nrf2/HO-1-signaling pathway in the spinal cord.

Normally, opioids function in a receptor-dependent manner. They bind to opioid receptors, activate or inhibit receptor activation, and subsequently modulate downstream signal transduction. However, the complex functions of opioids and the low expression of opioid receptors and their endogenous peptide agonists in neural stem cells (NSCs) suggest that some opioids may also modulate NSCs via a receptor-independent pathway. In the current study, two opioids, morphine and naloxone, are demonstrated to facilitate NSC proliferation via a receptor-independent and ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-dependent pathway. Morphine and naloxone penetrate cell membrane, bind to TET1 protein via three key residues (1,880-1,882), and subsequently result in facilitated proliferation of NSCs. In addition, the two opioids also inhibit the DNA demethylation ability of TET1. In summary, the current results connect opioids and DNA demethylation directly at least in NSCs and extend our understanding on both opioids and NSCs.