CR13626: a novel oral brain penetrant tyrosine kinase inhibitor that reduces tumor growth and prolongs survival in a mouse model of glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant primary brain cancer. Despite aggressive treatments currently there is no cure for GBM. Many challenges should be considered for the development of new therapeutical agents for glioblastoma, including appropriate target selectivity and pharmacokinetics. Several mutations and alterations of key cellular pathways including tyrosine kinases (TKs) are involved in malignant transformation and tumor progression. Thus, the targeting of multiple pathways and the development of innovative combination drug regimens is expected to yield improved therapies. Moreover, the abilities to cross the blood-brain barrier (BBB) reaching effective concentrations in brain and to remain into this tissue avoiding the effects of efflux transporters are also critical issues in the development of new therapeutics for GBM. CR13626 is a novel brain penetrant small molecule able to potently inhibit in vitro the activity of EGFR, VEGFR2 (aka KDR), Fyn, Yes, Lck, HGK (aka MAP4K4) and RET kinases relevant for GBM development. CR13626 shows good oral bioavailability (72%) and relevant brain penetration (brain/plasma ratio of 1.4). In an orthotopic xenograft glioblastoma mouse model, oral treatment with CR13626 results in a time-dependent reduction of tumor growth, leading to a significant increase of animal survival. The unique properties of CR13626 warrant its further investigation as a potential new drug candidate in glioblastoma.

CR4056, a powerful analgesic imidazoline-2 receptor ligand, inhibits the inflammation-induced PKCε phosphorylation and membrane translocation in sensory neurons.

BACKGROUND AND PURPOSE: CR4056 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 APPROACH: Effects 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 RESULTS: CR4056 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 IMPLICATIONS: Our 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.

Pharmacological characterisation of CR6086, a potent prostaglandin E2 receptor 4 antagonist, as a new potential disease-modifying anti-rheumatic drug.

BACKGROUND: Prostaglandin E2 (PGE2) acts via its EP4 receptor as a cytokine amplifier (e.g., interleukin [IL]-6) and induces the differentiation and expansion of inflammatory T-helper (Th) lymphocytes. These mechanisms play a key role in the onset and progression of rheumatoid arthritis (RA). We present the pharmacological characterisation of CR6086, a novel EP4 receptor antagonist, and provide evidence for its potential as a disease-modifying anti-rheumatic drug (DMARD). METHODS: CR6086 affinity and pharmacodynamics were studied in EP4-expressing HEK293 cells by radioligand binding and cyclic adenosine monophosphate (cAMP) production, respectively. In immune cells, IL-6 and vascular endothelial growth factor (VEGF) expression were analysed by RT-PCR, and IL-23 and IL-17 release were measured by enzyme-linked immunosorbent assay (ELISA). In collagen-induced arthritis (CIA) models, rats or mice were immunised with bovine collagen type II. Drugs were administered orally (etanercept and methotrexate intraperitoneally) starting at disease onset. Arthritis progression was evaluated by oedema, clinical score and histopathology. Anti-collagen II immunoglobulin G antibodies were measured by ELISA. RESULTS: CR6086 showed selectivity and high affinity for the human EP4 receptor (Ki = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE2-stimulated cAMP production. In models of human immune cells in culture, CR6086 reduced key cytokine players of RA (IL-6 and VEGF expression in macrophages, IL-23 release from dendritic cells, IL-17 release from Th17 cells). In the CIA model of RA in rats and mice, CR6086 significantly improved all features of arthritis: severity, histology, inflammation and pain. In rats, CR6086 was better than the selective cyclooxygenase-2 inhibitor rofecoxib and at least as effective as the Janus kinase inhibitor tofacitinib. In mice, CR6086 and the biologic DMARD etanercept were highly effective, whereas the non-steroidal anti-inflammatory drug naproxen was ineffective. Importantly, in a study of CR6086/methotrexate, combined treatment greatly improved the effect of a fully immunosuppressive dose of methotrexate. CONCLUSIONS: CR6086 is a novel, potent EP4 antagonist showing favourable immunomodulatory properties, striking DMARD effects in rodents, and anti-inflammatory activity targeted to immune-mediated inflammatory diseases and distinct from the general effects of cyclooxygenase inhibitors. These results support the clinical development of CR6086, both as a stand-alone DMARD and as a combination therapy with methotrexate. The proof-of-concept trial in patients with RA is ongoing.

Synthesis and structure-activity relationship studies in serotonin 5-HT(1A) receptor agonists based on fused pyrrolidone scaffolds.

A new class of serotonin 5-HT1A receptor ligands related to NAN-190, buspirone and aripiprazole has been designed using our potent 5-HT3 receptor ligands as templates. The designed pyrrolidone derivatives 10a-n were prepared by means of the straightforward chemistry consisting in the reaction of the appropriate γ-haloester derivatives with the suitable arylpiperazinylalkylamines. The nanomolar 5-HT1A receptor affinity and the agonist-like profile shown by fused pyrrolidone derivatives 10k,m stimulated the rationalization of the interaction with an homology model of the 5-HT1A receptor and the evaluation of their selectivity profiles and the pharmacokinetic properties. Interestingly, the results of the profiling assays suggested for close congeners 10k,m a significantly divergent binding pattern with compound 10m showing an appreciable selectivity for 5-HT1AR.

Discovery, synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists.

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.

CR4056, a new analgesic I2 ligand, is highly effective against bortezomib-induced painful neuropathy in rats.

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.

Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain.

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.

Efficacy of CR3294, a new benzamidine derivative, in the prevention of 5-fluorouracil-induced gastrointestinal mucositis and diarrhea in mice.

PURPOSE: Gastrointestinal mucositis, commonly associated with diarrhea, is a dose-limiting toxicity of chemotherapy. The new benzamidine derivative CR3294 reduces tissue damage in animal models of intestinal inflammation. Thus, we tested whether CR3294 had the potential to prevent chemotherapy-induced mucositis. METHODS: In tests on isolated cells, reactive oxygen species (ROS) formation and cytokine release were measured by chemiluminescence and immunoassays, respectively. In studies in vivo, BDF1 mice were given oral CR3294 (2.5-20 mg/kg) for 3 days before receiving 5-fluorouracil. Intestinal crypt survival, cell apoptosis and proliferation, and diarrhea score were assessed. Additionally, nude mice bearing tumor xenografts were treated with CR3294 and/or 5-fluorouracil, and tumor growth was monitored. RESULTS: CR3294 significantly inhibited cytokine release from stimulated leukocytes at concentrations similar to the IC(50) (2.9 +/- 0.2 muM) for ROS production by these cells. Consistent with these molecular findings, CR3294 dose-dependently protected the intestinal mucosa against 5-fluorouracil-induced toxicity in a mouse model of mucositis. The number of surviving crypts per cross-section in mice receiving 20 mg/kg CR3294 was 2.8-fold that in vehicle-treated animals (18.1 +/- 1.9 vs. 6.5 +/- 0.9, P < 0.001). Moreover, CR3294 decreased the cumulative diarrhea score by 50%, reduced by nearly 70% the incidence of severe episodes, and increased by 3-fold the number of mice without diarrhea. CR3294 neither affected the growth of tumor xenografts nor protected tumors from the cytotoxic activity of 5-fluorouracil. CONCLUSIONS: This study demonstrates that CR3294 acts on key molecular targets to reduce the signs of mucositis and the occurrence of diarrhea in mice exposed to the chemotherapy drug 5-fluorouracil.

Induction of autophagic cell death by a novel molecule is increased by hypoxia.

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.