Purpose (the aim of the study): Despite different clinical phenotypes have been identified, the osteoarthritis (OA) pathogenesis remains unknown and no disease-modifying agent has been approved so far. In the inflammatory OA phenotype, characterized by marked synovitis and pain, an important role is played by tyrosine kinases. Inhibition of Src, Fyn and Lck, members of the Src family kinases (SFKs), and VEGFR2 reduced synovial inflammation, cartilage degradation, bone remodeling and chronic pain in preclinical models of OA. Moreover, blocking FGFR1 and DDR2 reduced cartilage degradation in OA models. Finally, inhibition of TrkB resulted in a reduction of chronic OA pain. The simultaneous inhibition of these specific kinases can provide a valid therapeutic strategy to treat OA patients with an inflammatory phenotype.

New graft copolymers were prepared by reaction of poly (vinyl alcohol) (PVA) with mono-imidazolide or bis-imidazolide derivatives of ferulic acid (FA) with the formation of ester bonds. The obtained graft copolymers, thanks to the crosslinking capability of FA, formed in water strong gels as verified by rheological analyses. The resulting hydrogels were characterized to evaluate their applicability as wound dressing. In this perspective, their capability to absorb and retain a large amount of fluid without dissolving was verified by swelling kinetics and Moisture Vapour Transmission Rate measurements. Their stability towards mechanical solicitations was assessed by quantifying elasticity, compliance, stress-relaxation, and adhesivity properties. The analyses pointed out that hydrogel PVA-FA2-3 obtained by feruloylation of PVA with bis-imidazole derivative of ferulic acid using an acylation agent/polymer molar ratio 0.03/1 resulted the best candidate for the foreseen application.

The COVID-19 pandemic and the need for additional safe, effective, and affordable vaccines gave new impetus into development of vaccine genetic platforms. Here we report the findings from the phase 1, first-in-human, dose-escalation study of COVID-eVax, a DNA vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Sixty-eight healthy adults received two doses of 0.5, 1, or 2 mg 28 days apart, or a single 2-mg dose, via intramuscular injection followed by electroporation, and they were monitored for 6 months. All participants completed the primary safety and immunogenicity assessments after 8 weeks. COVID-eVax was well tolerated, with mainly mild to moderate solicited adverse events (tenderness, pain, bruising, headache, and malaise/fatigue), less frequent after the second dose, and it induced an immune response (binding antibodies and/or T cells) at all prime-boost doses tested in up to 90% of the volunteers at the highest dose. However, the vaccine did not induce neutralizing antibodies, while particularly relevant was the T cell-mediated immunity, with a robust Th1 response. This T cell-skewed immunological response adds significant information to the DNA vaccine platform and should be assessed in further studies for its protective capacity and potential usefulness also in other therapeutic areas, such as oncology.

CR4056 is an imidazoline-2 receptor ligand having potent analgesic activity and synergistic effect with opioids. Very recently it has been found that CR4056 can revert the cognitive impairment in animal models of Alzheimer's disease (AD). Since several lines of evidence highlight the importance of NMDAR modulators in nociceptive signaling and in AD progression, we considered as important to investigate the effects of CR4056 on NMDAR activity. In primary culture of cortical neurons, application of NMDA and glycine elicits a current that is decreased in a dose-dependent fashion by CR4056 (IC50 5.3 ± 0.1 µM). CR4056 antagonism is reversible, not competitive and voltage-independent and it is not blocked by pertussis toxin. CR4056 interacts with the co-agonist glycine site in a competitive way, indeed high glycine concentrations diminish its effect. Fibroblasts expressing different recombinant NMDA receptors are differently modulated by CR4056: the potency and the efficacy of the compound are higher in GluN1- GluN2B than in GluN1-GluN2A containing receptors. In lamina II neurons of spinal cord slices, single stimulation of afferent fibers evokes an NMDA-mediated current that is inhibited by 10 µM CR4056. Repetitive stimulation of the dorsal root at high frequency and high intensity produces a firing activity that is significatively depressed by CR4056. Taken together, our results broad the understanding of the molecular mechanisms of CR4056 analgesic activity, involving the modulation of NMDAR activity. Therefore, we propose that the analgesic action of CR4056 and the neuroprotective effects in AD models may be mediated also by NMDAR inhibition.

Recent evidence suggests that I2-imidazoline ligands have neuroprotective properties in animal models of neurodegeneration, such as Alzheimer's disease (AD). We recently demonstrated that the I2-ligand BU224 reversed memory impairments in AD transgenic mice and this effect was not because of reductions in amyloid-β (Aβ) deposition. In this study, our aim was to determine the therapeutic potential of the powerful analgesic I2-imidazoline ligand CR4056 in the 5xFAD model of AD, since this ligand has been proven to be safely tolerated in humans. Sub-chronic oral administration of CR4056 (30 mg/kg for 10 days) led to an improvement in recognition memory in 6-month-old 5xFAD mice, but not in wild-type littermates, without affecting Aβ levels or deposition. Our results also revealed a change in the profile of microglia by CR4056, resulting in a suppression of pro-inflammatory activated microglia, but increased the density of astrocytes and the expression of ApoE, which is mainly produced by these glial cells. In addition, CR4056 restored fibrinogen extravasation, affecting the distribution of markers of astrocytic end feet in blood vessels. Therefore, these results suggest that CR4056 protects against Aβ-mediated neuroinflammation and vascular damage, and offers therapeutic potential at any stage of AD.

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.

COVID-19 is a rapidly spreading disease, posing a huge hazard to global health. The plasmid vaccine pTK1A-TPA-SpikeA (named COVID-eVax) encodes the severe acute respiratory syndrome coronavirus 2 S protein receptor-binding domain, developed for intramuscular injection followed by electroporation (EP). The aim of this study was to assess the systemic toxicity and local tolerance of COVID-eVax delivered intramuscularly followed by EP in Sprague Dawley (SD) rats. The animals were killed 2 days and 4 weeks after the last injection (30-day and 57-day, respectively). No mortality was observed, and no signs of toxicity were evident, including injection site reactions. A lasting and specific immune response was observed in all treated animals, confirming the relevance of the rat as a toxicological model for this vaccine. Histopathological evaluation revealed muscle fiber necrosis associated with subchronic inflammation at the injection sites (at the 30-day time point), with a clear trend for recovery at the 57-day time point, which is expected following EP, and considered a desirable effect to mount the immune response against the target antigen. In conclusion, the intramuscular EP-assisted DNA vaccine, COVID-eVax showed an excellent safety profile in SD rats under these experimental conditions and supports its further development for use in humans.

The COVID-19 pandemic caused by the β-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already 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, a technology previously utilized for cancer vaccines. 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 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 significantly 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 in Italy.

Abstract BACKGROUND: Immune checkpoint inhibitors (ICIs) have marked therapeutic effects in many cancer patients. This is not the case for the large majority (~85%) of colorectal cancers (CRC), i.e. those classified as microsatellite stable (MSS) tumors. Cancers that do not respond to immunotherapies, such as this predominant CRC form, have a T cell non-infiltrated phenotype and are known as “cold” tumours. They are poorly immunogenic, as opposed to ICI-responsive “hot” tumours that are associated with high T cell infiltration. A typical immunosuppressive mechanism used by tumors is the production of prostaglandin E2 (PGE2), which binds four EP receptor subtypes on tumor and immune cells to promote tumor survival. The EP4 receptor plays a major role in PGE2-induced immunosuppression. CR6086 is a clinical stage EP4 receptor antagonist acting as a targeted immunomodulator. This study tested the hypothesis that the EP4 antagonist CR6086 could turn cold into hot tumors, favoring the response to ICI therapy. METHODS: We investigated the efficacy of combining CR6086 with an ICI (i.e. anti-mouse PD-1 (CD279), clone RMP1-14 [mAb]) in a syngeneic model of CRC resistant to ICI therapy. CT26 colon carcinoma cells (1 × 106) were inoculated subcutaneously in the right flank of female BALB/c mice. Treatments started on day 7 post-inoculation, when all tumors were within the target volume range of 50-100 mm3. CR6086 (30 mg/kg) was administered orally once daily for 14 days (QDx14); anti-PD-1 mAb (150 μg/mouse) was administered intraperitoneally on days 7, 10, 14 and 17. RESULTS: The combination of CR6086 and anti-PD-1 significantly decreased tumor growth vs vehicle-treated mice (mean tumor volume: 721±166 mm3 vs 1457±219 mm3; P<0.05, Two-way RM ANOVA followed by Dunnett's test, n=11/group), as measured after 14 days of treatment. The anti-PD-1 alone did not affect tumor progression, similarly to CR6086 monotherapy. RT-PCR analysis on tumor tissues showed that combined treatment increased (≥2-fold change) the gene expression of key factors for lymphocyte recruitment and activation. They include specific chemokines (CCL4, CCL5, CXCL10) responsible for increased T cell and dendritic cell infiltrate; CD8α, a major indicator for the presence of cytotoxic T lymphocytes; the antigen processing marker H2-Eb1s; INFγ, a T cell activation factor; TNFα, a cytokine produced by M1 macrophages and involved in tumor cell death; PD-L1, a prognostic biomarker with predictive value for the response of patients under anti-PD-1 therapy. These findings on gene expression are well reflected at the cellular level, because combined treatment increased general and T cell infiltrate, as assessed by haematoxylin/eosin staining and CD3+ immunohistochemistry. Conversely, the combination of CR6086 and anti-PD-1 reduced the gene expression of MMP-9, a matrix metalloproteinase involved in tumor growth, invasion, metastasis, extracellular matrix remodeling, and angiogenesis. CONCLUSION: These results suggest that the EP4 receptor antagonist CR6086 may turn a cold MSS murine colorectal cancer, non-responsive to ICI treatment, into a hot tumor responsive to ICIs. Citation Format: Gianfranco Caselli, Flora Ferrari, Tiziana Piepoli, Adriana Grotti, Rosanna Cavagnoli, Giuseppe M. Montagna, Pierpaolo Romanelli, Albino Bonazzi, Marco Lanza, Camilla Recordati, Lucio C. Rovati. Combination of the EP4 antagonist CR6086 and anti-PD-1 monoclonal antibody inhibits tumor growth in a microsatellite stable colorectal cancer in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2208.

Background and Purpose Prolonged use of opioids causes analgesic tolerance and adverse effects including constipation and dependence. Compounds targeting imidazoline I 2 receptors are known to potentiate opioid analgesia in rodents. We investigated whether combination with the I 2 receptor ligand CR4056 could improve efficacy and safety of morphine and explored the mechanisms of the CR4056–opioid interaction. Experimental Approach We used the complete Freund's adjuvant (CFA) model in rats to study the effects of treatments on hyperalgesia, morphine tolerance and microglia activation as measured by immunofluorescence. Opioid‐induced adverse effects were assessed in rodent models of morphine‐induced constipation, sedation (open field, sedation rating scale, and rotarod), physical dependence (naloxone‐induced withdrawal), and abuse (conditioned place preference‐associated reward). Chemiluminescence assays tested CR4056 as allosteric modulator of μ‐opioid receptors. Key Results CR4056 (ED 50 = 4.88 mg·kg −1 ) and morphine (ED 50 = 2.07 mg·kg −1 ) synergized in reducing CFA‐induced hyperalgesia (ED 50 = 0.52 mg·kg −1 ; 1:1 combination). Consistently, low doses of CR4056 (1 mg·kg −1 ) spared one third of the cumulative morphine dose administered during 4 days and prevented/reversed the development of tolerance to morphine anti‐hyperalgesia. These opioid‐sparing effects were associated with decreased activation of microglia, independent of CR4056 interactions on μ‐opioid receptors. Importantly, the low doses of CR4056 and morphine that synergize in analgesia did not induce constipation, sedation, physical dependence, or place preference. Conclusion and Implications We showed selective synergism between CR4056 and morphine as analgesics. Their combination showed an improved safety and abuse liability profile over morphine alone. CR4056 could be developed as an opioid‐sparing drug in multimodal analgesia.

Abstract BACKGROUND: Currently there is no cure for glioblastoma (GBM), the most malignant primary brain cancer. Glioblastoma etiopathogenesis involves mutations and alterations of key signaling pathways including tyrosine kinases (TKs). Notably, aberrant activation of receptor tyrosine kinases (RTKs) such as EGFR (amplified in 40% of glioblastoma cases) and KDR represents one of the driving forces for cellular proliferation and angiogenesis. The ability to cross the blood brain barrier (BBB) and appropriate pharmacodynamics (target selectivity) and pharmacokinetics (brain penetration) are critical issues for the generation of potential drug candidates against glioblastoma. CR13626 is a novel brain penetrant small molecule able to inhibit different TKs relevant to the development of this cancer. The present study describes the in vitro and in vivo properties of CR13626 and its antitumor activity in a mouse model of glioblastoma. METHODS: We identified the TKs targeted by CR13626 against a panel of 173 kinase enzymes. The effects of CR13626 on cellular proliferation were assessed in different 2D human GBM cell lines (U87MG, U373, U87MG vIII) by ViaCount, and in U87MG 3D spheroids by CellTiter-Glo 3D viability assay. The antitumor activity in vivo was determined in an orthotopic mouse model of GBM based on the injection of U87MG-Luciferase cells in nude mice. Animals were orally treated with CR13626 (50 mg/kg/daily) for 10 days, starting on day 9 post-implantation. Measurement of bioluminescence (BLI) at day 19 (end of dosing) and during follow-up (days 26-33) allowed the monitoring of tumor progression. CR13626 pharmacokinetics and brain exposure were assessed by LC/MS/MS in plasma and brain homogenate tissues of CD1 and tumor-bearing nude mice. RESULTS: CR13626 potently inhibited tyrosine kinases relevant to GBM development, with IC50 values in the nanomolar range: FYN (69 nM), YES (3.6 nM), KDR (82 nM) and EGFR (6 nM). CR13626 reduced the growth of different human glioblastoma cell lines with GI50 values in the range 1-3 µM. In vivo, CR13626 treatment led to a time-dependent reduction of tumor growth, reaching 60% on the last BLI evaluation 33 days post-implantation (i.e. 15 days after the end of dosing). A 25% significant increase in the median survival of animals compared to the vehicle group was also observed. The antitumor effects of CR13626 were in line with the exposure of tumor-bearing mice to the compound. Pharmacokinetic studies in CD1 mice showed good oral bioavailability (72%) and brain penetration (brain/plasma ratio of 1.4) for CR13626. CONCLUSION: The ability of CR13626 to cross the BBB without being a substrate of efflux transporters that mediate tumor resistance, to simultaneously inhibit the activity of different TKs involved in GBM development, and to reduce tumor growth eventually leading to an increased survival of animals, warrant its further development as a drug candidate in glioblastoma. Citation Format: Chiara Galimberti, Tiziana Piepoli, Giuseppe M. Montagna, Silvia Zerbi, Ornella Letari, Roberto Artusi, Milena Colovic, Stefano Persiani, Gianfranco Caselli, Lucio C. Rovati. Efficacy of CR13626, a novel oral brain penetrant multi-kinase inhibitor, in a mouse model of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4219.

Abstract BACKGROUND Glioblastoma multiforme (GBM) is the most malignant primary brain cancer. Several mutations and alterations of key cellular pathways including tyrosine kinases (TKs) are involved in GBM etiopathogenesis. Currently there is no cure for GBM. Tumour heterogeneity and the presence of the blood brain barrier (BBB), with efflux transporters, are some of the causes of failure of novel therapeutic agents. Thus, appropriate target selectivity and pharmacokinetics (including brain penetration) are critical issues for the generation of potential drug candidates. The main in-vitro and in-vivo properties and antitumour activity of CR13626, a novel brain penetrant TK inhibitor, are presented. MATERIAL AND METHODS CR13626 inhibitory activity against a panel of 173 kinases was assessed. The effect on cellular proliferation was verified in different 2D human glioblastoma cell lines (U87MG, U373, U87MG vIII) and in a U87MG 3D spheroid model by ViaCount assays. The in-vivo antitumour activity was determined in a mouse model of glioblastoma based on the orthotopic injection of U87MG-Luciferase GBM cells in nude mice: oral treatment started on day 9 post-implantation and continued for 10 days (50 mg/kg/daily). Tumour progression was evaluated through the measurement of bioluminescence (BLI) at the end of dosing (day 19) and during follow-up (day 26–33). Survival was also monitored. The pharmacokinetics and brain exposure of CR13626 were assessed by LC/MS/MS in plasma and brain homogenate tissues of CD1 and tumour-bearing nude mice. RESULTS CR13626 potently inhibited FYN, YES, KDR and EGFR kinases, relevant for GBM development, with IC50 values of 69 nM, 3.6 nM, 82 nM and 6 nM, respectively. The compound reduced the proliferation of different human glioblastoma cell lines (GI50 1–3 µM). In CD1 mice, CR13626 had a good oral bioavailability (72%) and brain penetration (brain/plasma ratio of 1.4). In-vivo BLI analysis indicated a time-dependent reduction of tumour growth, reaching 60% on the last BLI evaluation 33 days post-implantation (i.e. 15 days after the end of dosing). Tumour growth inhibition translated into an increase of 25% of the median survival time of animals treated with CR13626 compared to the vehicle group (p<0.05). The observed antitumour effects agreed with the exposure of tumour-bearing mice to CR13626, which was above the TKs in-vitro IC50 values. CONCLUSION The combined abilities of CR13626 to inhibit the activity of TKs involved in GBM development, to cross the BBB, and to reduce tumour growth in-vivo leading to increased survival, warrant its further development as a drug candidate in GBM.

Water-soluble MBHA derivatives were found to self-assemble in a water environment to generate aggregates showing core-shell architectures. The aggregates appeared to be capable of working as nanoreactors performing a multi-functionalization of poly-histidine fragments, which after an initial interaction with the solvated oligo(ethylene glycol) shell reach the reactive core.

The reactivity of functional nanoreactors was evaluated in CRB0137 as a model protein to develop a new methodology for the site-specific PEGylation of proteins bearing poly-histidine tags.

Background and PurposeCR4056 is a first‐in‐class imidazoline‐2 (I2) receptor ligand characterized by potent analgesic activity in different experimental animal models of pain. In a recent phase II clinical trial, CR4056 effectively reduced pain in patients with knee osteoarthritis. In the present study, we investigated the effects of CR4056 on PKCε translocation in vitro and on PKCε activation in vivo in dorsal root ganglia (DRG) neurons.Experimental ApproachEffects of CR4056 on bradykinin‐induced PKCε translocation were studied in rat sensory neurons by immunocytochemistry. PKCε activation was investigated by immunohistochemistry analysis of DRG from complete Freund's adjuvant‐treated animals developing local hyperalgesia. The analgesic activity of CR4056 was tested on the same animals.Key ResultsCR4056 inhibited PKCε translocation with very rapid and long‐lasting activity. CR4056 decreased hyperalgesia and phospho‐PKCε immunoreactivity in the DRG neurons innervating the inflamed paw. The effect of CR4056 on PKCε translocation was blocked by pertussis toxin, implying that the intracellular pathways involved Gi proteins. The inhibition of PKCε translocation by CR4056 was independent of the α2‐adrenoeceptor and, surprisingly, was also independent of idazoxan‐sensitive I2 binding sites. The I2 agonist 2BFI had no effect alone but potentiated the activity of low concentrations of CR4056.Conclusions and ImplicationsOur results demonstrate that CR4056 shares the ability to inhibit PKCε translocation with other analgesics. Whether the inhibition of PKCε involves binding to specific subtype(s) of I2 receptors should be further investigated. If so, this would be a new mode of action of a highly specific I2 receptor ligand.

A small series of Morita-Baylis-Hillman adduct (MBHA) derivatives was synthesized and made to react with imidazole, N-acetylhistidine, and N-acetylhexahistidine as models of poly-histidine derivatives. Intriguingly, the reaction of MBHA derivatives 1a and b with imidazole in acetonitrile-phosphate buffered saline (PBS) gave the imidazolium salt biadducts 3a and b as the main reaction products. These results were confirmed by experiments performed with N-acetylhistidine and 1b and suggested the possible occurrence of these structures in the products of poly-histidine labeling with MBHA derivatives 1a and b. These compounds were then transformed into the corresponding water-soluble derivatives 1c-e by introducing oligo(ethylene glycol) chains and their reactivity was evaluated in preliminary experiments with imidazole and then with N-acetylhexahistidine in PBS. The structure of polymeric materials Ac-His-6-MBHA-1d and Ac-His-6-MBHA-1e obtained using ten-fold excesses of compounds 1d and e was investigated using mass spectrometry, NMR spectroscopy, and photophysical studies, which suggested the presence of biadduct residues in both polymeric materials. These results provide the basis for the preparation of fishbone-like polymer brushes, the characterization of their properties, and the exploration of their potential applications in different fields of science such as in vivo fluorogenic labeling, fluorescence microscopy, protein PEGylation, up to the production of smart materials and biosensors.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.