Bispecific sigma-1 receptor antagonism and mu-opioid receptor partial agonism: WLB-73502, an analgesic with improved efficacy and safety profile compared to strong opioids.

Opioids are the most effective painkillers, but their benefit-risk balance often hinder their therapeutic use. WLB-73502 is a dual, bispecific compound that binds sigma-1 (S1R) and mu-opioid (MOR) receptors. WLB-73502 is an antagonist at the S1R. It behaved as a partial MOR agonist at the G-protein pathway and produced no/unsignificant ß-arrestin-2 recruitment, thus demonstrating low intrinsic efficacy on MOR at both signalling pathways. Despite its partial MOR agonism, WLB-73502 exerted full antinociceptive efficacy, with potency superior to morphine and similar to oxycodone against nociceptive, inflammatory and osteoarthritis pain, and superior to both morphine and oxycodone against neuropathic pain. WLB-73502 crosses the blood-brain barrier and binds brain S1R and MOR to an extent consistent with its antinociceptive effect. Contrary to morphine and oxycodone, tolerance to its antinociceptive effect did not develop after repeated 4-week administration. Also, contrary to opioid comparators, WLB-73502 did not inhibit gastrointestinal transit or respiratory function in rats at doses inducing full efficacy, and it was devoid of proemetic effect (retching and vomiting) in ferrets at potentially effective doses. WLB-73502 benefits from its bivalent S1R antagonist and partial MOR agonist nature to provide an improved antinociceptive and safety profile respect to strong opioid therapy.

Propionamide Derivatives as Dual µ-Opioid Receptor Agonists and σ1 Receptor Antagonists for the Treatment of Pain.

A new series of propionamide derivatives was developed as dual µ-opioid receptor agonists and σ1 receptor antagonists. Modification of a high-throughput screening hit originated a series of piperazinylcycloalkylmethyl propionamides, which were explored to overcome the challenge of achieving balanced dual activity and convenient drug-like properties. The lead compound identified, 18g, showed good analgesic effects in several animal models of both acute (paw pressure) and chronic (partial sciatic nerve ligation) pain, with reduced gastrointestinal effects in comparison with oxycodone.

Preliminary in vitro approach to evaluate the drug-drug interaction potential of EST73502, a dual µ-opioid receptor partial agonist and σ1 receptor antagonist.

The potential for drug-drug interactions (DDI) of EST73502 was preliminary explored in vitro. EST73502 is a new chemical entity intended for oral pain treatment with dual sigma-1 receptor (σ1R) antagonism and µ-opioid receptor (MOR) partial agonism, that presents a promising potent analgesic activity.Several enzymes were involved in EST73502 metabolism catalysing the formation of different metabolites, CYP3A4 and CYP2D6 being the main ones.Fraction unbound was determined due to its impact in interactions, a considerable proportion of EST73502 being available.EST73502 showed a low potential for CYP inhibition, except for CYP2D6 that showed time-dependent inhibition.No induction potential was found for CYP1A2 and 3A4, while CYP2B6 was induced at high concentration.EST73502 seemed to be a potential efflux transporter substrate (efflux ratio ≥ 2) but a negligible in vivo impact would be expected due to its high solubility and permeability in Caco-2 cells. P-gp inhibition was observed while no BCRP inhibition was detected.Preliminary in vitro interaction studies suggested that neither CYPs nor efflux transporters interactions would preclude further development of EST73502 to thoroughly assess the clinical relevance of these findings.

Preliminary in vitro assessment of pharmacokinetic drug-drug interactions of EST64401 and EST64514, two sigma-1 receptor antagonists.

EST64401 and EST64514 are two selective sigma-1 receptor ligands that showed a good profile in a lead optimization process for oral pain treatment. Their potential for pharmacokinetic-based drug-drug interactions was assessed to anticipate clinical interactions.Both compounds showed a low potential for CYP inhibition with percentages of inhibition <50% at 1 µM in recombinant human CYPs (CYP1A2, 2C9, 2C19, 2D6 and 3A4) and IC50 ≥75 µM for CYP3A4 and 2D6 in human liver microsomes.No CYP induction was observed for CYP1A2, 2B6 and 3A4 at concentrations ≤25 µM (EST64401) or ≤50 µM (EST64514) in human hepatocytes using as endpoints CYP activities and mRNA levels.More than one enzyme participated in compound metabolism. The main enzymes involved were CYP3A4 for EST64401 and CYP2D6 besides CYP3A4 for EST64514.Neither EST64401 nor EST64514 seemed to be substrates of P-gp or BCRP in Caco-2 cells (efflux ratio ≤2). Transporter inhibition was observed at concentrations ≥20 µM; EST64401 only inhibiting P-gp at higher concentrations (≥125 µM).Preliminary in vitro interaction studies suggest a similar profile for EST64401 and EST64514. Therefore, other properties will have to be considered for compound differentiation and selection for further development.

Discovery of EST73502, a Dual µ-Opioid Receptor Agonist and σ1 Receptor Antagonist Clinical Candidate for the Treatment of Pain.

The synthesis and pharmacological activity of a new series of 4-alkyl-1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the σ1 receptor (σ1R) and the µ-opioid receptor (MOR) are reported. A lead optimization program over the initial 4-aryl analogues provided 4-alkyl derivatives with the desired functionality and good selectivity and ADME profiles. Compound 14u (EST73502) showed MOR agonism and σ1R antagonism and a potent analgesic activity, comparable to the MOR agonist oxycodone in animal models of acute and chronic pain after single and repeated administration. Contrary to oxycodone, 14u produces analgesic activity with reduced opioid-induced relevant adverse events, like intestinal transit inhibition and naloxone-precipitated behavioral signs of opiate withdrawal. These results provide evidence that dual MOR agonism and σ1R antagonism may be a useful strategy for obtaining potent and safer analgesics and were the basis for the selection of 14u as a clinical candidate for the treatment of pain.

Preliminary in Vitro Assessment of the Potential of EST64454, a Sigma-1 Receptor Antagonist, for Pharmacokinetic Drug-Drug Interactions.

EST64454 is a selective sigma-1 receptor ligand intended for orally administered pain treatment that showed a promising profile in the lead optimization process. As part of the preliminary compound profiling, the potential for future drug-drug interactions was explored in vitro. Both direct and time-dependent CYP inhibition for CYP1A2, 2C9, 2C19, 2D6 and 3A4 was studied in human liver microsomes. EST64454 showed a low potential for CYP inhibition (IC50 between 100 and 1000 µM) and as time-dependent inhibitor (IC50 shift mainly around 1). CYP induction studies with HepaRG™ cells revealed no CYP induction at concentrations ≤50 µM, as shown by the CYP1A2, 3A4 and 2B6 activities measured. Reaction phenotyping was assessed after incubation with recombinant human enzymes. Although a very low metabolism was observed, several enzymes catalyzed the formation of metabolites, including CYP3A4, 2C19 and flavin monooxygenases (FMO) 1 and 3. EST64454 was not a P-glycoprotein (P-gp) substrate and was highly permeable in Caco-2 cells. P-gp inhibition was only observed at 200 µM, the highest concentration studied. Preliminary studies suggest that neither CYP nor P-gp interaction of EST64454 would be of any concern for further development. At later stages, the interaction kinetics and the clinical relevance of these findings will be thoroughly evaluated.

Tetrahydro-3-benzazepines with fluorinated side chains as NMDA and σ1 receptor antagonists: Synthesis, receptor affinity, selectivity and antiallodynic activity.

The class of tetrahydro-1H-3-benzazepines was systematically modified in 1-, 3- and 7-position. In particular, a F-atom was introduced in ß- or γ-position of the 4-phenylbutyl side chain in 3-position. Ligands with the F-atom in γ-position possess higher GluN2B affinity than analogs bearing the F-atom in ß-position. This effect was attributed to the reduced basicity of ß-fluoro amines. 3-Benzazepines with a benzylic OH moiety show moderate GluN2B affinity, but considerable selectivity over the σ2 receptor. However, removal of the benzylic OH moiety led to increased GluN2B affinity, but reduced GluN2B/σ2 selectivity. With respect to GluN2B affinity the phenol 17b with a γ-fluorophenylbutyl moiety in 3-position represents the most interesting fluorinated ligand (Ki(GluN2B) = 16 nM). Most of the synthesized ligands reveal either similar GluN2B and σ1 affinity or higher σ1 affinity than GluN2B affinity. The methyl ether 16b shows high σ1 affinity (Ki(σ1) = 6.6 nM) and high selectivity over a broad panel of receptors and transporters. The high antiallodynic activity in the mouse capsaicin assay proved the σ1 antagonistic activity of 16b.

Oleoyl-estrone is a precursor of an estrone-derived ponderostat signal.

Oleoyl-estrone (OE) is a powerful anti-obesity compound that decreases food intake, decreases insulin resistance and circulating cholesterol. OE stimulates a severe loss of body fat by decreasing adipose tissue lipid synthesis and maintaining lipolysis. Therefore, the body economy loses lipid energy because energy expenditure is maintained. This study analyses the discrepancy between OE effects and the distribution of labelled OE in plasma. Estrone radioimmunoassay of organic solvent plasma extracts of rats treated with OE showed the massive presence of acyl-estrone, but saponification did not release estrone, but containing similar unknown compound. Analysis of label distribution in plasma after oral gavages of (3)H-OE showed the presence of a more hydrophilic compound than OE or any estrogen as well as (3)H(2)O, formed from (3)H-OE in the acidic stomach medium. OE was not attached to a specific transporter in plasma. Through serum HPLC analysis we found W, a labelled derivative more hydrophilic than OE or estrone. The results were confirmed using (14)C-OE. HPLC-MS/MS studies showed that plasma OE levels were one order of magnitude lower than those of W. When liver cell cytosols from rats laden with (3)H-OE were incubated with nuclei from untreated rats, the OE-derived label (i.e., Ws) was found attached to nuclear DNA. Neither estradiol nor estrone interfered with its binding. W is a fairly hydrophilic compound of low molecular weight containing the estrone nucleus, but it is not an ester because saponification or esterases do not yield estrone as OE does. It is concluded that OE acts through its conversion to W, its active form; which binds to a nuclear receptor different from that of estrogen. The estimated W serum levels are proportional to the pharmacological OE effects in vivo. We postulate W as a new type of hormone that exerts the full range of in vivo effects thus far attributed to OE. The full identification of W is anticipated to open the way for the development of new OE-like anti-obesity drugs.

Avidin and streptavidin ligands based on the glycoluril bicyclic system.

Glycoluril derivatives with a carboxylic acid side chain have been synthesized and shown to bind to both avidin and streptavidin. Introduction of a valerate side chain in glycoluril led to an increased binding to both proteins only when the valerate group was bound to a N atom and with the proper stereochemistry [(+)-enantiomer]. On the other hand, introduction of the valerate side chain either on the bridgehead carbon or on the N atom with the opposite stereochemistry [(-)-enantiomer] led to a decrease in binding constant compared with unsubstituted glycoluril. Direct spectrophotometric competitive titration of each protein with a racemic ligand allowed measurement of the enantioselectivity of the ligand-protein complexation, together with the binding constant of the two enantiomers. In the case of the N-substituted glycoluril, the extension of the side chain by one methylene group, from valerate to caproate, led to an increase in the binding constant to both proteins. Docking studies using AutoDock 3.05 have been performed in order to predict the binding modes of these ligands to streptavidin. The effect of the stereochemistry and the position of the side chain on the binding constant to streptavidin is discussed in view of the predicted binding modes.