SummaryThioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca 2 ϩ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.

Two decades of investigations have failed to unequivocally clarify the functions and the molecular nature of imidazoline-2 receptors (I2R). However, there is robust pharmacological evidence for the functional modulation of monoamino oxidase (MAO) and other important enzyme activities by I2 site ligands. Some compounds of this class proved to be active experimental tools in preventing both experimental pain and opioid tolerance and dependence. Unfortunately, even though these compounds bind with high potency to central I2 sites, they fail to represent a valid clinical opportunity due to their pharmacokinetic, selectivity or side-effects profile. This paper presents the preclinical profile of a novel I2 ligand (2-phenyl-6-(1H-imidazol-1yl) quinazoline; [CR4056]) that selectively inhibits the activity of human recombinant MAO-A in a concentration-dependent manner. A sub-chronic four day oral treatment of CR4056 increased norepinephrine (NE) tissue levels both in the rat cerebral cortex (63.1% ±4.2%; P < 0.05) and lumbar spinal cord (51.3% ± 6.7%; P < 0.05). In the complete Freund’s adjuvant (CFA) rat model of inflammatory pain, CR4056 was found to be orally active (ED50 = 5.8 mg/kg, by mouth [p.o.]). In the acute capsaicin model, CR4056 completely blocked mechanical hyperalgesia in the injured hind paw (ED50 = 4.1 mg/kg, p.o.; ED100 = 17.9 mg/kg, p.o.). This effect was dose-dependently antagonized by the non-selective imidazoline I2/α2 antagonist idazoxan. In rat models of neuropathic pain, oral administration of CR4056 significantly attenuated mechanical hyperalgesia and allodynia. In summary, the present study suggests a novel pharmacological opportunity for inflammatory and/or neuropathic pain treatment based on selective interaction with central imidazoline-2 receptors.

The lupus-like autoimmune syndrome of MRL͞Mp-Tnfrsf6 lpr (lpr) mice is characterized by progressive lymphadenopathy and autoantibody production, leading to early death from renal failure. Activation of T helper lymphocytes is one of the events in the pathogenesis of the disease in these mice and likely in human systemic lupus erythematosus. Among T helper lymphocytedependent cytokines, IFN-␥ plays a pivotal role in the abnormal cell activation and the fatal development of the lpr disease. IL-18, an inducer of IFN-␥ in T lymphocytes and natural killer cells, may contribute to the disease because cells from lpr mice are hypersensitive to IL-18 and express high levels of IL-18. To assess the contribution of IL-18 to the pathogenesis in the animal model, in vivo inhibition of IL-18 was attempted. Young lpr mice were vaccinated against autologous IL-18 by repeated administration of a cDNA coding for the murine IL-18 precursor. Vaccinated mice produced autoantibodies to murine IL-18 and exhibited a significant reduction in spontaneous lymphoproliferation and IFN-␥ production as well as less glomerulonephritis and renal damage. Moreover, mortality was significantly delayed in anti-IL-18-vaccinated mice. These studies support the concept that IL-18 plays a major role in the pathogenesis of the autoimmune syndrome of lpr mice and that a reduction in IL-18 activity could be a therapeutic strategy in autoimmune diseases.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

Our findings suggest that chondrocytes express a self-sufficient machinery for glutamate signaling, including a peripheral NMDA receptor with unique properties. This receptor may have a role in the inflammatory process associated with cartilage degradation, thus emerging as a potential pharmacological target in OA.

Although bortezomib (BTZ) is the frontline treatment for multiple myeloma, its clinical use is limited by the occurrence of painful peripheral neuropathy, whose treatment is still an unmet clinical need. Previous studies have shown chronic BTZ administration (0.20 mg/kg intravenously three times a week for 8 weeks) to female Wistar rats induced a peripheral neuropathy similar to that observed in humans. In this animal model of BTZ-induced neurotoxicity, the present authors evaluated the efficacy of CR4056, a novel I2 ligand endowed with a remarkable efficacy in several animal pain models. CR4056 was administered in a wide range of doses (0.6–60 mg/kg by gavage every day for 2–3 weeks) in comparison with buprenorphine (Bupre) (28.8 μg/kg subcutaneously every day for 2 weeks) and gabapentin (Gaba) (100 mg/kg by gavage every day for 3 weeks). Chronic administration of BTZ reduced nerve conduction velocity and induced allodynia. CR4056, Bupre, or Gaba did not affect the impaired nerve conduction velocity. Conversely, CR4056 dose-dependently reversed BTZ-induced allodynia (minimum effective dose 0.6 mg/kg). The optimal dose found, 6 mg/kg, provided a constant pain relief throughout the treatment period and without rebound after suspension, being effective when coadministered with BTZ, starting before or after allodynia was established, or when administered alone after BTZ cessation. A certain degree of tolerance was seen after 7 days of administration, but only at the highest doses (20 and 60 mg/kg). Bupre was effective only acutely, since tolerance was evident from the fourth day onwards. Gaba showed a significant activity only at the fourth day of treatment. CR4056, over the range of concentrations of 3–30 μM, was unable to hinder BTZ cytotoxicity on several tumor cell lines, which could indicate that this substance does not directly interfere with BTZ antitumor activity. Therefore, CR4056 could represent a new treatment option for BTZ-induced neuropathic pain.

Background and PurposeCR4056 is a novel imidazoline-2 (I2) ligand exhibiting potent analgesic activity in animal models of pain. In this study, we investigated the effects of CR4056 in a well-established model of postoperative pain where rats develop hyperalgesia in the injured hind paw.Experimental ApproachBy measuring paw withdrawal threshold to mechanical pressure, we studied the pharmacology of CR4056, potential sex differences in pain perception and response to treatment, and the pharmacodynamic interaction of CR4056 with morphine.Key ResultsOral CR4056 and subcutaneous morphine dose-dependently reversed the hyperalgesic response. Analgesic effects of CR4056 were completely suppressed by the non-selective imidazoline I2/α2-adrenoceptor antagonist idazoxan, were partially reduced (∼30%; P < 0.05) by the selective α2-adrenoceptor antagonist yohimbine, but were not influenced by the non-selective I1/α2-adrenoceptor antagonist efaroxan or by the μ opioid receptor antagonist naloxone. We found no differences in responses to CR4056 or morphine between male and female rats. However, females had a lower pain threshold than males, and needed lower doses of drugs to reach a significant analgesia. When CR4056 and morphine were combined, their median effective doses were lower than expected for additive effects, both in males and in females. Isobolographic analysis confirmed a synergism between CR4056 and morphine.Conclusions and ImplicationsCR4056 is a novel pharmacological agent under development for postoperative pain both as stand-alone treatment and in association with morphine. CR4056 has successfully completed Phase I studies for tolerability and pharmacokinetics in healthy volunteers, and is currently entering the first proof-of-concept study in patients.

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