Pharmacology/Drug Development

Eight P2YR antagonists, including three newly synthesized analogues, containing a naphthalene or phenyl-triazolyl scaffold were compared in a mouse model of chronic neuropathic pain (sciatic constriction). P2YR antagonists rapidly (≤30 min) reversed mechano-allodynia, with maximal effects typically within 1 h after injection. Two analogues (4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid and -acetyl analogue , 10 μmol/kg, i.p.) achieved complete pain reversal (100%) at 1 to 2 h, with relief evident up to 5 h for (41%). A reversed triazole analogue reached 87% maximal protection. Receptor affinity was determined using a fluorescent antagonist binding assay, indicating similar mouse and human P2YR affinity. The mP2YR affinity was only partially predictive of efficacy, suggesting the influence of pharmacokinetic factors. Thus P2YR is a potential therapeutic target for treating chronic pain.

The voltage-gated calcium channels CaV3.1-3.3 constitute the T-type subfamily, whose dysfunctions are associated with epilepsy, psychiatric disorders, and chronic pain. The unique properties of low voltage-activation, faster inactivation, and slower deactivation of these channels support their role in modulation of cellular excitability and low-threshold firing. Thus, selective T-type calcium channel antagonists are highly sought after. Here, we explored Ugi-azide multicomponent reaction (MCR) products to identify compounds targeting T-type calcium channel. Of the 46 compounds tested, an analog of benzimidazolonepiperidine – 5bk (1-{1-[(R)-{1-[(1S)-1-phenylethyl]-1H-1,2,3,4-tetrazol-5-yl}(thiophen-3-yl)methyl]piperidin-4-yl}-2,3-dihydro-1H-1,3-benzodiazol-2-one) modulated depolarization-induced calcium influx in rat sensory neurons. Modulation of T-type calcium channels by 5bk was further confirmed in whole-cell patch clamp assays in dorsal root ganglion (DRG) neurons, where pharmacological isolation of T-type currents led to a time- and concentration-dependent regulation with a low micromolar IC50. Lack of an acute effect of 5bk argues against a direct action on of T-type channels. Genetic knockdown revealed CaV3.2 to be the isoform preferentially modulated by 5bk. High voltage-gated calcium, as well as tetrodotoxin-sensitive and -resistant sodium, channels were unaffected by 5bk. 5bk inhibited spontaneous excitatory post synaptic currents and depolarization-evoked release of calcitonin gene-related peptide (CGRP) from lumbar spinal cord slices. Notably, 5bk did not bind human mu, delta, or kappa opioid receptors. 5bk reversed mechanical allodynia in rat models of HIV-associated neuropathy, chemotherapy-induced peripheral neuropathy (CIPN), and spinal nerve ligation (SNL)-induced neuropathy, without effects on locomotion or anxiety. Thus, 5bk represents a novel T-type modulator that could be used to develop non-addictive pain therapeutics.

Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS and NTS receptors and NT analogs provide stronger pain relief than morphine on a molecular basis. Here, we examined the analgesic/adverse effect profile of a new NT(8-13) derivative denoted JMV2009, in which the Pro residue was substituted by a silicon-containing unnatural amino acid silaproline. We first report the synthesis and in vitro characterization (receptor-binding affinity, functional activity and stability) of JMV2009. We next examined its analgesic activity in a battery of acute, tonic and chronic pain models. We finally evaluated its ability to induce adverse effects associated with chronic opioid use, such as constipation and analgesic tolerance or related to NTS activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS and NTS, improved proteolytic resistance as well as agonistic activities similar to NT, inducing sustained activation of p42/p44 MAPK and receptor internalization. Intrathecal injection of JMV2009 produced dose-dependent antinociceptive responses in the tail-flick test and almost completely abolished the nociceptive-related behaviors induced by chemical somatic and visceral noxious stimuli. Likewise, increasing doses of JMV2009 significantly reduced tactile allodynia and weight bearing deficits in nerve-injured rats. Importantly, repeated agonist treatment did not result in the development of analgesic tolerance. Furthermore, JMV2009 did not cause constipation and was ineffective in inducing hypothermia. These findings suggest that NT drugs can act as an effective opioid-free medication for the management of pain or can serve as adjuvant analgesics to reduce the opioid adverse effects.

Novel synthetic opioids are appearing in recreational drug markets worldwide as adulterants in heroin or ingredients in counterfeit analgesic medications. 3,4-Dichloro-N-[(1R,2R)-2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) is an example of a non-fentanyl synthetic opioid linked to overdose deaths. Here, we examined the pharmacodynamics and pharmacokinetics of U-47700 in rats. Male Sprague-Dawley rats were fitted with intravenous (i.v.) catheters and subcutaneous (s.c.) temperature transponders under ketamine/xylazine anesthesia. One week later, rats received s.c. injections of U-47700 HCl (0.3, 1.0 or 3.0 mg/kg) or saline, and blood samples (0.3 mL) were withdrawn via i.v. catheters at 15, 30, 60, 120, 240, 480 min post-injection. Pharmacodynamic effects were assessed at each blood withdrawal, and plasma was assayed for U-47700 and its metabolites by liquid chromatography tandem mass spectrometry. U-47700 induced dose-related increases in hot plate latency (ED = 0.5 mg/kg) and catalepsy (ED = 1.7 mg/kg), while the 3.0 mg/kg dose also caused hypothermia. Plasma levels of U-47700 rose linearly as dose increased, with maximal concentration (C) achieved by 15-38 min. C values for N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 were delayed but reached levels in the same range as the parent compound. Pharmacodynamic effects were correlated with plasma U-47700 and its N-desmethyl metabolite. Using radioligand binding assays, U-47700 displayed high affinity for μ-opioid receptors (Ki = 11.1 nM) whereas metabolites were more than 18-fold weaker. Our data reveal that U-47700 induces typical μ-opioid effects which are related to plasma concentrations of the parent compound. Given its high potency, U-47700 poses substantial risk to humans who are inadvertently exposed to the drug.

The goal of this research is the identification of new treatments for neuropathic pain. We characterized the GABAergic system of immortalized mouse and human microglia using electrophysiology and qRT-PCR. Cells from both species exhibited membrane current changes in response to γ-aminobutyric acid, with an EC50 of 260 nM and 1940 nM, respectively. Human microglia expressed high levels of the γ-aminobutyric acid type A receptor (GABAAR) α3 subunit, which can assemble with β1 and γ2/δ subunits to form functional GABAARs. Mouse microglia contained α2, α3 and α5, in addition to β1-3, γ1-2 and δ, mRNA, enabling a more diverse array of GABAARs than human microglia. Benzodiazepines are well-established modulators of GABAAR activity, prompting a screen of a library of diverse benzodiazepines in microglia for cellular effects. Several active compounds were identified by reduction of nitric oxide (NO) in interferon gamma and lipopolysaccharide activated microglia. However, further investigation with GABAAR antagonists flumazenil, picrotoxin, and bicuculline demonstrated that GABAARs were not linked to the NO response. A screen of 48 receptors identified the κ-opioid receptor and to a lesser extent the µ-opioid receptor as molecular targets, with opioid receptor antagonist norbinaltorphimine reversing benzodiazepine induced reduction of microglial NO. Functional assays identified the downregulation of inducible NO synthase as the mode of action of imidazodiazepines MP-IV-010 and GL-IV-03. Like other κ-opioid receptor agonists, GL-IV-03 reduced the agitation response in both phases of the formalin nociception test. However, unlike other κ-opioid receptor agonists, MP-IV-010 and GL-IV-03 did not impair sensorimotor coordination in mice. Thus, MP-IV-010 and GL-IV-03 represent a new class of non-sedative drug candidates for inflammatory pain.