Use of 2-phenyl-6-(1H-imidazol-1-yl)quinazoline for treating neurodegenerative diseases, preferably Alzheimer's disease

Abstract

The present invention relates to a compound of formula 2-phenyl-6-(1H-imidazol-1-yl)quinazoline or a pharmaceutically acceptable salt thereof for use in the treatment of a neurodegenerative disease selected from the group consisting of Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion diseases, HIV-associated dementia, and any form of cognitive impairment associated with neurodegeneration, preferably Alzheimer's disease. [Drawings] None

Description

Technical Field

[0001] The present invention provides 2-phenyl-6-(1H-imidazol-1-yl)quinazoline for treating neurodegenerative diseases, preferably Alzheimer's disease.

Background Art

[0002] Neurodegenerative diseases (NDs) are a cause of increasing mortality and morbidity worldwide, particularly in the elderly. NDs include Alzheimer's disease, Parkinson's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion diseases, etc., and particularly those with a high prevalence and a diffuse pathological condition are focused on, especially the similarities and differences between these syndromes.

[0003] Due to their prevalence, complex biochemistry, and pathology, neurodegenerative diseases pose a major challenge to basic science and clinical medicine. Therefore, treatments related to new mechanisms are a typical example of unmet medical needs.

[0004] Alzheimer's disease (AD) is the most common type of dementia associated with progressive cognitive decline and memory loss. It is the sixth leading cause of death in the United States and is estimated to afflict 14 million people in this country by 2050.

[0005] Current treatments for AD provide little benefit for symptoms, and the last approval by the US Food and Drug Administration was in 2003. Since then, more than 400 clinical trials on AD therapeutics have been registered, and the failure rate in the trials for which results have been reported is almost 100%. It is frustrating that there has been no progress in the treatment and prevention of AD targeting the β-amyloid protein and paired helical filament tau protein that accumulate in the brains of AD patients, and new pathways, targets, and a change in perspective are needed to obtain new effective disease-modifying drugs.

[0006] AD is characterized by distinct tissue changes associated with the accumulation of extracellular amyloid-β (Aβ) peptides derived from the cleavage of amyloid precursor protein (APP), and intracellular deposition of hyperphosphorylated tau. Since Aβ and tau aggregates are neurotoxic and induce neurodegenerative processes in the brain, it is suggested that Aβ and tau are central to the etiology of AD. Furthermore, many genes that influence the risk of AD are expressed in microglia (Zhang B, Cell, 2013), and microglia and astrocytes change their activation phenotypes during aging and disease stages. These findings raise the possibility that innate immune activation contributes actively to the etiology of AD. In the presence of harmful stimuli, including misfolded proteins such as amyloid β, microglial cells mount an acute immune response in the brain. If the response does not subside, the physiological and beneficial functions are transformed by chronic activation of microglia and recruitment of astrocytes.

[0007] Imidazole (imidazoline) / guanidine compounds elicit central and peripheral actions through interaction with non-adrenergic receptor sites, so-called imidazoline receptors (Escriba P, Ann. N. Y. Acad. Sci., 1999). This receptor is classified into two major types. I1 binding site (BS) subtype: Identified by drugs such as clonidine and involved in blood pressure regulation. I2 binding site: First identified by idazoxan (a mixed I2BS and α2-adrenergic receptor ligand) and characterized by selective ligands lacking I1-IBS and α2-adrenergic receptor affinity (e.g., 2-BFI, BU224). Furthermore, I3BS was identified as a typical imidazoline subtype present in pancreatic β-cells, involved in insulin secretion, and recognized by efaroxan.

[0008] One of the endogenous ligands of the imidazoline receptor is agmatine, which is an amine intermediate in polyamine biosynthesis. Agmatine is widely distributed in the body and probably acts as a neurotransmitter and / or neurotransmission regulator in the brain. In addition to the imidazoline receptor, agmatine also binds to other target receptors such as the α2-adrenergic receptor, N-methyl-D-aspartic acid (NMDA) receptor, and serotonin receptor with low affinity and exerts physical effects.

[0009] I2BS is widely distributed and expressed on neurons in the brain, but mainly on glial cells localized on the outer membrane of mitochondria (Ruggiero DA, Brain Res., 1998).

[0010] Both the neuroprotective and anti-inflammatory effects of imidazoline drugs have been reported (Regunathan S, Ann. N. Y. Acad. Sci., 1999), but the pharmacology of I2BS remains unclear. The I2 receptor is a group of binding sites present on various proteins, and its nature and biological significance remain unclear (Escriba P, Ann. N. Y. Acad. Sci., 1999).

[0011] Some experimental evidence collected over the past 30 years indicates that I2 ligands may exert at least partial neuroprotective effects through different mechanisms and in different models of neurodegeneration.

[0012] Chronic imidazoline drug treatment has long been established to increase GFAP expression in astrocytes (Olmos G, Br. J. Pharmacol., 1994).

[0013] In astrocytes and macrophages, idazoxan can inhibit inducible NOS (iNOS) activity, thereby reducing the level of NO-mediated neurotoxicity (Feinstein D, Mol. Pharmacol., 1999).

[0014] BU224 (selective I2 ligand) has been demonstrated to downregulate apoptosis-promoting factors in the rat cerebral cortex and, by itself, may mediate neuroprotective effects by inhibiting important components of standard apoptotic signaling in the brain (Garau C J, Psychopharmacol., 2013).

[0015] 2-BFI (selective I2 ligand) shows neuroprotective effects in both in vitro and in vivo models of ischemic stroke. In vitro, 2-BFI prevents lipid peroxidation and mitochondrial apoptosis in astrocyte oxygen-glucose deprivation (Tian J, J.Neurosci. Res., 2018). On the other hand, in brain injury induced by middle cerebral artery occlusion, a rat model of transient cerebral ischemia, 2-BFI induces Bcl-2 expression (a gene that plays an important role in neuron survival during cerebral ischemia) (Han Z, Brain Res., 2010), protects blood-brain barrier integrity, reduces matrix metalloproteinase 9 (MMP-9) expression, and upregulates tight junction protein and collagen IV (Zhang ZJ, Stroke Cerebrovasc. Dis., 2018).

[0016] I2 receptor ligands bind to the NMDA receptor with low affinity and regulate its activity non-competitively and reversibly, similar to memantine, which reduces NMDA-mediated glutamate toxicity in vitro and in vivo (Jiang SX, Eur. J. Pharmacol., 2010).

[0017] I2 imidazoline drugs inhibit Aβ-induced neuronal toxicity by reducing Erk1 / 2 activation (Montolio M, J. Med. Chem., 2012).

[0018] Chronic treatment with 2-BFI attenuates experimental autoimmune encephalomyelitis (a mouse model of multiple sclerosis) by restoring B-CK and CaATPase enzyme activities and maintaining calcium-dependent calpain activation at basal levels (Wang P, Biochem. Biophys. Res. Commun., 2011), reduces the levels of the pro-inflammatory cytokines IL-17A and IFN-γ, and increases the level of the anti-inflammatory cytokine IL-10 (Zhu YB, Neurochem. Res., 2015).

[0019] In an AD model (induced by injection of Aβ1-42 into the rat hippocampus), 2-BFI improves learning and memory abilities, reduces oxidative stress, downregulates the release of inflammatory factors, and inhibits neuronal apoptosis (Tian JS, J. Integr. Neurosci., 2017).

[0020] BU224 is partially neuroprotective against kainic acid-induced excitotoxic signaling (Keller BJ, Psychopharmacol., 2016).

[0021] Acute treatment with BU224 increases the hippocampal p-FADD / FADD ratio (an indicator of cell survival) and decreases the cleavage of p35 to neurotoxic p25 (Abas S, ACS Chem. Neurosci.,2017).

[0022] Treatment with agmatine, an endogenous imidazoline receptor ligand, significantly improves cognitive function and restores memory impairment induced in diabetic rats (Bhutada P, Prog. Neuro-Psychopharmacology Biol. Psychiatry 2012).

[0023] On the other hand, an increase in I2R density using radioligand binding assays has been observed during aging and in the AD brain, probably due to their location in astrocytes (Garcia-Sevilla, Neurosci. Lett., 1998).

[0024] Protein kinase C (PKC) is a phospholipid-dependent family of serine / threonine protein kinases that constitute an extensive signaling network in the brain. Molecular cloning studies have revealed 12 PKC isozymes, which are divided into three subgroups: (1) classical PKC, (2) novel PKC, and (3) atypical PKC. PKC isoforms play important roles in various cognitive functions, including learning and memory. In particular, PKCε, which is classified as a novel PKC (not requiring calcium for its activation), is a phorbol ester / diacylglycerol (DAG)-sensitive and calcium-independent serine / threonine kinase. It is an important regulator of various signaling events, and thus, the need for it to be present at several intracellular locations is fulfilled by the translocation of the kinase by isozyme-specific chauffeur proteins. Abnormal translocation of the kinase can misdirect the output of signaling and is therefore harmful to cell physiology. Due to this complex biology, its role in neurodegenerative diseases is still under debate. Some papers have suggested that inhibition of PKCε activation provides overall protection against neurodegenerative diseases (e.g., for Alzheimer's disease, Zara S, Brain Research 2011; for ischemia-induced neurodegeneration, Kumar V, J Neuro Res 2019), while most past papers have pointed out a protective role of PKCε activation in these diseases. Based on this concept, for example, in US Patent Application Publication No. 2008 / 0004332 and US Patent Application Publication No. 2016 / 0025704, there has been a proposal of an activator (bryostatin-1) of this kinase as a clinical candidate for Alzheimer's disease. In the former, it is stated that an inhibitor of PKC in peripheral tissues may be associated with a PKC activator in order to merely attenuate the possible side effects induced by activating PKC in peripheral tissues (where peripheral tissues mean tissues other than the brain).Unfortunately, in May 2017, in the second-phase proof of concept study, it was reported that this approach using PKCε bryostatin-1 did not meet the primary evaluation item that measured the improvement of the Severe Impairment Battery (SIB) score versus placebo. Summary of the Invention Means for Solving the Problems

[0025] The inventors have found that a compound of formula 2-phenyl-6-(1H-imidazol-1-yl)quinazoline (also referred to as CR4056) can be used for the treatment of neurodegenerative diseases, preferably Alzheimer's disease.

[0026] This molecule combines 1) strong activity against the I2 binding site, 2) long-term inhibition of the translocation of PKCε to the cell membrane in neurons, and 3) outstanding ability to cross the blood-brain barrier. The combination of these three characteristics results in surprising effectiveness of CR4056 in models of memory impairment and Alzheimer's disease. This is a particularly innovative result since the prior art explicitly excludes the use of brain-penetrant PKC inhibitors in the treatment of neurodegenerative diseases.

[0027] Accordingly, in a first aspect, the present invention relates to a compound of formula 2-phenyl-6-(1H-imidazol-1-yl)quinazoline or a pharmaceutically acceptable salt thereof for use in the treatment of neurodegenerative diseases, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-associated dementia.

[0028] In the present invention, when referring to a neurodegenerative disease, it is intended to be a disease selected from the group of chronic, progressive disorders characterized by the gradual loss of neurons in distinct regions of the central nervous system (CNS).

[0029] The neurodegenerative diseases according to the present invention are diseases selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease, and HIV-related dementia, preferably Alzheimer's disease.

[0030] Although the mechanism underlying the progressive nature of such neurodegenerative diseases remains unclear, a timely and well-controlled inflammatory response is essential for the integrity and proper function of the CNS.

[0031] The compound 2-phenyl-6-(1H-imidazol-1-yl)quinazoline (also named as CR4056) is a first-in-class imidazoline-2 receptor ligand characterized by potent analgesic activity in various animal models of inflammatory, neurogenic, neuropathic, postoperative, fibromyalgia-like, and osteoarthritis pain (WO2008014822 A1, WO2009152868 A1, Ferrari F, JPAINI, 2011; Lanza M, B. J. of Pharmacol, 2014).

[0032] Furthermore, CR4056 is endowed with the ability to inhibit the translocation of PKCε to the cell membrane of primary neurons induced by inflammatory stimuli, which persists for a long time (but is still reversible). This long-lasting activity is characteristic of CR4056 and is not shared by other anti-inflammatory or analgesic compounds. However, this activity is idazoxan (a prototype I2 receptor antagonist) resistant and thus appears to be independent of the classical pathway induced by I2 ligands. Without being bound by any theory, the inventors believe that the inhibition of the translocation of PKCε to the cell membrane in neurons induced by inflammatory stimuli can be an unexpectedly excellent target for controlling neurodegeneration, and have found that CR4056 is the best candidate for doing so.

[0033] Finally, CR4056 has the unique ability to cross the blood-brain barrier and target the central nervous system: The presence of CR4056 in the brain was examined in rats 1 hour after a single oral administration at a dose of 30 mg / kg suspended in 0.5% Methocel. The average level of CR4056 in the brain was 32272 ng / g. The average brain / plasma ratio was 11.8, suggesting a significant ability of CR4056 to concentrate in the brain.

[0034] The inventors tested CR4056 in in vitro and in vivo models (transgenic or pharmacological) for neurodegenerative diseases, particularly Alzheimer's disease, and surprisingly found that CR4056 significantly reduced microglial activation, that it contributed to neuroprotection, that it significantly improved cognitive ability and could reverse memory impairment in both transgenic and pharmacological models of Alzheimer's.

[0035] From a safety perspective, even at supra-pharmacological concentrations and considering the specific affinity of CR4056 for the brain, CR4056 showed no central adverse effects in rats. In particular, no neurobehavioral changes (evaluated by the Irwin test) and no impairment of motor coordination and spontaneous movement (evaluated by the rotarod test and open field test) were observed. These findings indicate that CR4056 could be a safe medicine for treating neurodegenerative diseases, particularly Alzheimer's disease.

Brief Description of the Drawings

[0036]

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[0037] The inventors have found that the compound 2-phenyl-6-(1H-imidazol-1-yl)quinazoline (also referred to as CR4056) can be used for treating neurodegenerative diseases, preferably Alzheimer's disease.

[0038] Accordingly, in a first aspect, the present invention relates to a compound of formula 2-phenyl-6-(1H-imidazol-1-yl)quinazoline or a pharmaceutically acceptable salt thereof for use in the treatment of neurodegenerative diseases, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-associated dementia.

[0039] In the present invention, when referring to a neurodegenerative disease, it is intended to mean a disease selected from the group of chronic, progressive disorders characterized by the gradual loss of neurons in distinct regions of the central nervous system (CNS).

[0040] The neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-related dementia, preferably Alzheimer's disease.

[0041] The mechanism underlying the progressive nature of such neurodegenerative diseases is still unclear, but a timely and well-controlled inflammatory response is essential for the integrity and proper function of the CNS.

[0042] The present invention further provides a method for treating or preventing the onset of a neurodegenerative disease in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition of the present invention, thereby treating or reducing the risk of developing a neurodegenerative disease.

[0043] According to the present invention, the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-related dementia, preferably Alzheimer's disease.

[0044] The compound 2-phenyl-6-(1H-imidazol-1-yl)quinazoline can be used as the free base or in the form of a salt. Preferably, the pharmaceutically acceptable salts are salts selected from hydrochloride, hydrobromide, hydrogen sulfate and sulfate, maleate, fumarate, oxalate, methanesulfonate, succinate, ascorbate, tartrate, acetate, salicylate, citrate, aspartate, ethylenediaminetetraacetate, benzoate and glutamate. Further examples of pharmaceutically acceptable salts are reported in S.M. Berge et al, J.Pharm Sci. 1977, 66,2.

[0045] The compound 2-phenyl-6-(1H-imidazol-1-yl)quinazoline can also be in a crystalline polymorphic form or a hydrate form, as described in EP2438058A.

[0046] In a second aspect, the present invention relates to a pharmacological composition for use in the treatment of neurodegenerative diseases, comprising the compound 2-phenyl-6-(1H-imidazol-1-yl)quinazoline or a pharmaceutically acceptable salt thereof and a carrier, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-associated dementia.

[0047] Preferably, the composition of the present invention is used for treating Alzheimer's disease.

[0048] The composition for use can also contain pharmaceutically acceptable excipients and can be administered in a pharmaceutical form suitable for the desired route of administration.

[0049] Pharmaceutically acceptable additives can be excipients, ligands, dispersants, colorants, wetting agents commonly used in the preparation of tablets, capsules, pills, solutions, suspensions, emulsions for oral administration. Injectable solutions are also intended for parenteral administration including subcutaneous, intraspinal and transdermal administration.

[0050] The pharmaceutical composition according to the present invention is preferably for intravenous, oral, transdermal, intrathecal, intranasal, intraperitoneal or intramuscular administration.

[0051] The pharmaceutical composition according to the present invention can be used alone, or in combination with one or more further drugs, or can comprise them. These drugs can be drugs known for the treatment of neurodegenerative diseases, preferably Alzheimer's disease.

[0052] A composition for use in the treatment of neurodegenerative diseases contains 2-phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) or a pharmaceutically acceptable salt thereof in an amount of 15 to 250 mg per unit dosage form, and can provide a daily intake of 15 to 500 mg.

[0053] The present invention also relates to a combination preparation for simultaneous, sequential or separate use in the treatment of neurodegenerative diseases, comprising CR4056 or a pharmaceutically acceptable salt thereof and at least one of an NMDA (N-methyl-D-aspartic acid) receptor antagonist and / or an acetylcholinesterase inhibitor, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-associated dementia, preferably Alzheimer's disease.

[0054] CR4056 is administered at the dosages indicated above, optionally in combination with drugs belonging to the class of drugs currently approved for treating the cognitive symptoms of Alzheimer's disease, namely NMDA (N-methyl D-aspartic acid) receptor antagonists and acetylcholinesterase inhibitors.

[0055] In particular, the NMDA receptor antagonist is memantine, and the acetylcholinesterase inhibitor is selected from donepezil, rivastigmine and galantamine.

[0056] The administration of memantine (Namenda (registered trademark) or Ebixa (registered trademark)), donepezil (Aricept (registered trademark)), rivastigmine (Exelon (registered trademark)), galantamine (Razadyne (registered trademark) or Reminyl (registered trademark)) complies with the recommendations of the manufacturers, the Namenda label information (2007), the Aricept label information, the Exelon label information (2006), and the Razadyne label information (2008), respectively.

[0057] Next, the present invention will be described with reference to examples by using in vitro and in vivo models (transgenic and non-transgenic) for neurodegenerative diseases.

Examples

[0058] Experimental section Example 1: In vitro effect of 2-phenyl-6-(1H-imidazol-1-yl)quinazoline on the expression of inflammatory genes Method A model of astrocytes, the human glioblastoma astrocytoma cell line U373 MG (Uppsala), was used. Adherent cells were grown in DMEM medium supplemented with 10% FBS at 37 °C under CO2. 72 hours after plating, cells were treated with 2-phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) (10 μM) prepared according to EP2066653 for 1 hour, and then stimulated with the pro-inflammatory cytokine IL-1β (2 ng / mL) for an additional 6 hours and 24 hours.

[0059] At the end of the incubation period, total RNA was obtained and reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). The expression levels of COX-2, IL-1β, IL-6 and TNFα were evaluated by RT-PCR analysis using specific TaqMan assays and the 18S Pre-Developed TaqMan® assay (Thermo Fisher Scientific) as an endogenous control on an Applied Biosystems 7500 Fast Real-Time PCR System. Data analysis with 18S amplification values normalized was performed according to the Thermo Fisher Scientific specific instructions for gene expression relative quantification. All individual data were the result of at least three different measurements for each sample.

[0060] Results 2-Phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) decreased COX2 and IL-1β gene expression after 6 hours of incubation, with inhibitory effects of 45% on COX2 expression and 20% on IL-1β gene expression. At this point, the gene expression of IL-6 and TNFα did not appear to be regulated by CR4056 yet.

[0061] After 24 hours of stimulation, CR4056 decreased the gene expression of all analyzed inflammatory markers as reported in the following table, with inhibitory effects of 48% on COX2 expression, 29% on IL-1β expression, 39% on IL-6 expression, and 52% on TNFα gene expression.

[0062]

Table 1

[0063] Conclusion The above-reported results showed that CR4056 has a regulatory effect on the production of inflammatory cytokines in the astrocytoma cell line. This effect may contribute to a neuroprotective effect.

[0064] Example 2: In Vitro Effect of 2-Phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) on the Translocation of PKCε to the Cell Membrane in Cultured Neurons Method The rat dorsal root ganglia (DRGs) were obtained from freshly dissected vertebrae after careful removal of the nerve trunk and connective tissue. The larger ganglia, which were then cut into 2 - 4 smaller fragments, were incubated for 1 hour at 37°C in 0.125% collagenase (Worthington, Freehold, NJ) dissolved in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin and 1% L - glutamine (Euroclone, Milan, Italy). After enzymatic digestion, the ganglia were mechanically dissociated and the neurons were plated in a Petri dish containing wells with glass bottom coverslips (pre - coated with 10 μg / mL poly - L - lysine and 20 μg / mL laminin, Sigma - Aldrich, Milan, Italy) at a density such that the neurons formed a monolayer covering approximately 30% of the coverslip surface. The cells were incubated in DMEM as described above for 2 - 3 days, and 1.5 μg / ml of cytosine 1 - d - arabinofuranoside (ARA - C, Sigma - Aldrich) was added to slow down the proliferation of non - neuronal cells, and 100 ng / ml of nerve growth factor (NGF, Sigma - Aldrich) was added to increase the expression of receptors linked to cell health and PKCε translocation upon stimulation. Activation of membrane receptors coupled to the phospholipase C pathway results in the translocation of PKCε from the cytoplasm to the cell membrane. To study PKCε behavior, a well - established technique was used (Vellani V, Neuroscience, 2006). This technique involves rapid (30 seconds) activation of PKCε by inflammatory mediators such as bradykinin (BK) or prokineticin 2 (PK2), followed by fixation with 4% paraformaldehyde and 4% sucrose in phosphate - buffered saline (PBS, 50% dilution), staining for PKCε, and quantification of the number of neurons in which translocation is observed. CR4056 was applied to the culture medium 10 minutes prior to or simultaneously with the stimulation. After fixation, the cells were permeabilized with 0.2% Triton X - 100 (Sigma - Aldrich, Milan, Italy) and exposed overnight to a rabbit polyclonal antibody highly specific for PKCε.After sufficient rinsing, in dark cells, PKCε was visualized with a secondary antibody (1:200 diluted Alexa Fluor 488 goat anti-rabbit, Thermo Fisher Scientific, Monza, Italy) applied at room temperature for 2 - 4 hours. Neurons showing PKCε translocation were observed using a confocal microscope (Leica SP2, Leica, Switzerland) by measuring the fluorescence intensity along a line drawn through the cytoplasm and membrane, avoiding the nucleus. Neurons with a fluorescence intensity at the cell membrane across the whole cell more than 1.5 times the average cytoplasmic intensity were considered positive.

[0065] Results CR4056 inhibited PKCε translocation obtained with either BK or PK2 in a dose-dependent manner with IC 50 values of 0.20 and 0.17 μM, respectively. When CR4056 was applied for 10 minutes, the dose-response curve approached saturation at approximately 10 μM. This concentration was tested at different time intervals to examine the kinetics of the CR4056 effect. Prolonging (up to 24 hours) or shortening (10 seconds) the pre-incubation time did not change the extent of the effect. Next, the time required to wash out the effect of this drug was analyzed. In Figures 1A and B, PKCε translocation is reported at different time points after CR4056 (10 μM): first, immediately after a 10-minute application, and then after repeated washing (washout) with a large volume of culture medium (DMEM + 10% FBS, 37 °C) expected to remove any trace amounts of CR4056 from the extracellular environment. The effect of CR4056 remained unchanged until 1 hour after washout, after which it slowly decreased and completely reversed at 3 - 4 hours.

[0066] Next, the sensitivity of the PKCε translocation assay to idazoxan was tested. Idazoxan was pre-applied at high concentrations (10 and 100 μM) for 10 minutes towards 1 μM CR4056 (a concentration that induces a block less than the maximum amount of PKCε translocation). Neither concentration of idazoxan had any effect.

[0067] Conclusion From the above-reported results, CR4056 efficiently blocked the translocation of PKCε in neurons challenged with inflammatory stimuli. The CR4056 effect showed rapid onset but was removed very slowly from the cells. The pathway by which CR4056 acts was shown to be idazoxan-resistant, demonstrating the involvement of the non-classical I2 receptor. The mechanism of the specific anti-inflammatory action of CR4056 in such neurons, in addition to the established I2-mediated action, may contribute to the overall efficacy of this agent in neurodegenerative processes and cognitive impairment.

[0068] Example 3: In Vivo Effect of 2-Phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) on Microglial Activity in a CFA Inflammation Model Methods Activation of microglial cells was evaluated by immunofluorescence staining, and the expression of ionized calcium-binding adapter molecule 1 (Iba-1) in the ipsilateral L5 spinal cord in a complete Freund's adjuvant (CFA) model was measured.

[0069] Unilateral inflammation was induced by injecting 100 μL of CFA (1 mg / mL diluted 1:1 with saline) into the plantar surface of the right hind paw of rats.

[0070] CR4056 (6 mg / kg, oral) was administered 72 hours after CFA. Ninety minutes later, the animals were deeply anesthetized with an overdose of urethane (1.5 g / kg-1, intraperitoneal), and then perfused transcardially with 250 mL of 0.9% saline containing 1% heparin (5000 UI / mL-1), followed by 500 mL of 10% formalin (i.e., 4% paraformaldehyde, Bio-Optica Spa, Milano, Italy). The L5 segment of the spinal cord was harvested, fixed overnight at 4 °C, and embedded in paraffin blocks for sectioning. Transverse sections of the spinal cord were sliced with a fully automated rotary microtome at a thickness of 5 μm, mounted on slides coated with poly-L-lysine, and then processed for immunofluorescence. Antigen retrieval was performed at 90 °C for 20 minutes using 10 mM citrate buffer (pH 6.0). The sections were blocked with 10% normal horse serum in PBS containing 0.3% Triton-X for 90 minutes at room temperature and then incubated overnight at 4 °C with rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) primary polyclonal antibody (1:350; Wako Chemicals, Neuss, Germany, #019-19741). For secondary detection, the sections were incubated with Alexa-Fluor 488 donkey anti-rabbit secondary antibody (1:400; Thermo Fisher Scientific, Waltham, MA, USA #A-21206) for 1 hour at room temperature. The slides were mounted with FluoroShield mounting medium containing 4’,6-diamidino-2-phenylindole or DAPI (Sigma-Aldrich, Milano, Italy, #F6057) to counterstain the nuclei. Spinal cord sections were visualized with an Invitrogen EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). The contralateral site of each section was identified with a small section of the ventral horn. Laminae I-III of the dorsal horn were identified with reference to the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1982).For each section, representative images of both the ipsilateral and contralateral dorsal horns (laminae I–III) of the L5 spinal cord were captured at 20× magnification using a consistent exposure time across all sections and then analyzed. Iba1-positive microglial cells showing an amoeboid / activated state (i.e., clearly swollen cell bodies with reduced processes) were manually counted. The results are presented as the percentage of the ipsilateral / contralateral ratio of the number of Iba1-positive microglial cells showing an activated state. For each animal (n = 5 per group), six non-consecutive sections were analyzed and the results were averaged.

[0071] Results The CFA-induced arthritis rat model is a paradigm of chronic inflammatory pain. Intraplantar injection of CFA results in increased sensitivity to noxious heat and increased sensitivity to mechanical tactile stimuli in peripheral tissue injury, and induces a significant increase in the ratio of Iba1-positive, morphologically activated microglial cells, i.e., microglial activation, in laminae I–III of the ipsilateral versus contralateral dorsal horn of the L5 spinal cord.

[0072] CR4056, by single administration (6 mg / kg, oral), completely restored the basal state of microglia pre-activated by CFA treatment (Figure 2).

[0073] Conclusion During the course of several diseases, microglial cells lose their homeostatic molecular characteristics and functions, become chronically inflamed, and come to induce harmful effects. This is evident not only in neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, but also in aging and autism spectrum disorder (Butovsky O, Nature Rev Neuroscience 2018, Henstridge CM, Frontiers in Cellular Neuroscience, 2019; Wes PD, Glia 2016; Salter, MW, Nat. Med., 2017) and chronic pain (Malcangio M, Pain, 2016).

[0074] In this animal model, CR4056 significantly reduced microglial activation as highlighted by the decrease in Iba1 - positive cells, and thus contributed to neuroprotection.

[0075] Example 4: In - vivo effect of 2 - phenyl - 6-(1H - imidazol - 1 - yl) quinazoline (CR4056) on scopolamine - induced memory impairment model: Passive avoidance Method Scopolamine, a non - selective muscarinic receptor antagonist, blocks the activity of muscarinic acetylcholine receptors and simultaneously induces transient cognitive amnesia and electrophysiological changes, which are similar to those observed in Alzheimer's disease. Therefore, scopolamine administration may be considered as a psychopharmacological model of Alzheimer's disease (Lenz RA, Psychopharmacology (Berl), 2012).

[0076] Passive avoidance is a behavioral model of memory impairment.

[0077] The apparatus consisted of a two - compartment box with a bright and a dark compartment separated by a door. With the door between the two compartments closed, a rat was placed at the center of the bright compartment. After 4 seconds, the door was opened, and the latency, i.e., the time it took for the rat to place all four paws into the dark compartment, was recorded.

[0078] When the rat had completely moved into the dark compartment, the door was closed and a mild foot shock was immediately delivered through the grid floor. Thus, during the initial stage, the animal learned that moving into the dark compartment had a negative outcome.

[0079] Forty - eight hours after training, the rat was placed in the bright compartment and the same procedure as in training was followed, except that no shock was applied to the grid floor. Memory performance was positively correlated with the latency to exit the bright compartment.

[0080] To induce memory impairment, scopolamine (1 mg / kg, subcutaneous) was administered 30 minutes before the learning trial.

[0081] CR4056 (10 mg / kg, twice daily) was orally administered immediately after the learning trials to avoid the possibility of bias due to its analgesic effect.

[0082] Results Figure 3 shows that on the test day, sham animals (not treated with scopolamine) had a significantly increased latency to enter the light compartment compared to training. No increase in latency was observed in animals treated with scopolamine.

[0083] CR4056 10 mg / kg (twice daily) counteracted the memory impairment induced by scopolamine (as shown by the increased escape latency).

[0084] The escape latency in sham animals treated with CR4056 did not differ from that of its vehicle (methylcellulose, MC).

[0085] Conclusion CR4056 was able to reverse memory impairment in pharmacological models of Alzheimer's disease and dementia.

[0086] Example 5: Effect of 2-Phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) on a Scopolamine-Induced Memory Impairment Behavioral Model: Morris Water Maze Methods The Morris water maze test was used to evaluate distinct cognitive functions in two stages. First, acquisition and spatial localization of a hidden platform, and subsequently, processing, integration, retention, and retrieval of the acquired information to successfully locate the position of the platform to escape the water.

[0087] Place navigation required the rat to learn to swim from an arbitrary starting position to a hidden escape platform, thereby acquiring a long-term memory of the spatial location of the platform. The animal was placed in various quadrants of the pool, and the elapsed time and the distance traveled to reach the hidden platform were recorded. Various objects were placed in the laboratory so that the animal could use these visual cues as a means of navigating the maze. After repeated entries into the maze, the animal became increasingly efficient at locating the platform and thus escaped the water by learning the location of the platform relative to distal visual cues.

[0088] To induce memory impairment, scopolamine (1 mg / kg, subcutaneous) was administered 30 minutes before the test and CR4056 (20 mg / kg, oral) was administered 60 minutes before the test. Four trials were conducted over four days, and the reported final latency was the average for the entire day.

[0089] Results CR4056 at 20 mg / kg restored the memory impairment induced by scopolamine. As reported in Figure 4, rats treated with scopolamine required more time to locate the hidden platform compared to the control group. Co-administration of CR4056 and scopolamine significantly reduced the time required to reach the platform for animals treated with scopolamine alone.

[0090] Conclusions CR4056 treatment restored the impairment of memory ability that characterized the animal model of dementia obtained with scopolamine treatment.

[0091] Example 6: Effect of 2-Phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) in a Scopolamine-Induced Memory Impairment Model: Novel Object Recognition Test Methods Using a novel object recognition test, cognitive function was evaluated based on the ability of mice to recognize various objects proposed in the arena test. During the training phase, a pair of identical objects was placed in the cage test. Twenty-four hours after the first stage, during the test phase, one of the two objects was replaced with a new, different object. The exploration time of each object was recorded during the training and test phases. A typical approach of the mice was similar exploration of the objects during the training phase and preference for the new object during the test phase. Administration of a substance that can deteriorate cognitive ability (i.e., scopolamine) made it impossible to remember the old object and led to exploration of the two different objects in the same way.

[0092] Scopolamine 1 mg / kg was administered intraperitoneally 20 minutes before training, and 6 mg / kg and 20 mg / kg of CR4056 were administered orally 40 minutes before training.

[0093] Results 6 mg / kg and 20 mg / kg of CR4056 increased the exploration time (preference) for the new object during the test phase. Mice treated with scopolamine alone did not show preference for the new object (Figure 5).

[0094] Conclusion CR4056 dose-dependently improved mouse memory performance in this pharmacological model of scopolamine-induced dementia, which supported the previous results obtained in rats.

[0095] Example 7: Effect of 2-Phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) in a Transgenic 5XFAD Mouse Model of Alzheimer's Disease: Novel Object Recognition Test Methods Transgenic 5XFAD mice overexpressing human amyloid precursor protein (APP695) with Swedish (K670N / M671L), Florida (I716V) and London (V717I) familial Alzheimer's disease (FAD) mutations, and human presenilin 1 (PS1) with M146L and L286V FAD mutations were used as Alzheimer's disease models.

[0096] Six-month-old female transgenic 5XFAD mice and wild-type controls (WT) were treated orally (gavage) once daily for 10 days with 30 mg / kg of CR4056 or vehicle (n = 4 WT / vehicle, 8 WT / CR4056, 2 5XFAD / vehicle, and 3 5XFAD / CR4056). Following repeated treatment, hippocampal-dependent memory was tested using a novel object recognition task. The object recognition test measures working and spatial memory. In the training phase, animals were placed in an arena with an object constructed from large plastic bricks. In the test phase, one object was replaced with a novel object. The animals were returned to the arena and allowed to explore. Object exploration was observed and recorded. The mice should recognize that a novel object has been placed and should therefore explore this object for a longer period of time.

[0097] Results On the test day, the 5XFAD vehicle group showed cognitive impairment, while 5XFAD animals treated with CR4056 showed significant improvement in working and spatial memory (Figure 6).

[0098] Conclusions 5XFAD mice are a model of Alzheimer's disease characterized by intraneuronal Aβ aggregation, neurodegeneration, neuronal loss, and memory impairment associated with glial activation (Oakley H, The journal of Neuroscience, 2006; Mirzaei N, Glia 2006).

[0099] In this transgenic model, CR4056 significantly improved memory ability, consistent with the results obtained in a pharmacological model of scopolamine-induced dementia.

[0100] Example 8: Brain Penetration of 2-Phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) Method CR4056 was measured in rat plasma and rat brain and plasma using an LC / MS / MS method. Male Sprague Dawley rats (Harlan) were orally treated with a CR 4056 suspension (Methocel 0.5%) at a dose of 30 mg / kg. Oral administration was performed by gastric gavage at 5 ml / kg per group of 3 animals. The compound was detected in plasma and brain 1 hour after treatment.

[0101] Each rat brain was weighed and cut in half. Each cerebral hemisphere was weighed and homogenized in two different ways. One was fluidization by an Ultra turrax tube drive (IKA), and an aliquot of buffer solution (10 mM ammonium formate pH 3.5) was added three times the weight of the hemisphere. Then, 30 μl of the extraction solution was added to 0.25 ml of methanol in a 1.5 ml Eppendorf tube, vortex mixed, centrifuged at 13,500 rpm for 5 minutes at 4°C, and the supernatant was transferred to a 2 mL round 96 deep well plate (Axygen) and injected into the LC / MS / MS system. The second was cryogenic grinding by a Mikro Dismembrator (Sartorius). Each cerebral hemisphere was weighed, placed in a 20 mL Teflon container with steel balls (7.85 g / mL diameter 10 mm), and frozen by immersion in liquid nitrogen for 5 minutes. The deeply frozen brain was ground at 2500 rpm for 30 seconds using a Mikro Dismembrator, and the freeze / mill cycle was repeated twice. A portion of the powder was accurately weighed (about 10 mg) in a 1.5 ml Eppendorf tube, then 0.25 ml of methanol was added. After vortex kneading and centrifuging at 13,500 rpm for 5 minutes at 4°C, the supernatant was placed in a 2 mL round 96 deep well plate (Axygen) and injected into the LC / MS / MS system. Both methods used for homogenization gave similar results indicating that the extraction efficiency was similar for both methods.

[0102] Result Individual plasma and brain levels and brain-to-plasma ratios after oral administration of CR4056 suspension (30 mg / kg) to male Sprague Dawley rats in a fasting state are shown in the following table (rats were sacrificed 60 minutes after PO administration).

[0103] [Table 2]

[0104] This experiment showed that, as described above, CR4056 effectively entered the brain and had a direct effect on the central nervous system (CNS).

Claims

1. A medicament for the treatment of neurodegenerative diseases, comprising 2-phenyl-6-(1H-imidazol-1-yl)quinazoline or a pharmaceutically acceptable salt thereof, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-related dementia.

2. The medicament according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

3. The pharmaceutically acceptable salt of 2-phenyl-6-(1H-imidazol-1-yl)quinazoline is a salt selected from the group consisting of hydrochloride, hydrobromide, hydrogen sulfate and sulfate, maleate, fumarate, oxalate, methanesulfonate, succinate, ascorbate, tartrate, acetate, salicylate, citrate, aspartate, ethylenediaminetetraacetate, benzoate and glutamate, the medicament according to claim 1 or 2.

4. A pharmacological composition for the treatment of neurodegenerative diseases, comprising 2-phenyl-6-(1H-imidazol-1-yl)quinazoline or a pharmaceutically acceptable salt thereof and a carrier, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease and HIV-related dementia.

5. The composition according to claim 4, wherein the neurodegenerative disease is Alzheimer's disease.

6. The composition according to claim 4 or 5, wherein 2-phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) or a pharmaceutically acceptable salt is in an amount of 15 to 250 mg per unit dosage form, resulting in a daily intake of 15 to 500 mg.

7. The composition according to any one of claims 4 to 6, wherein the composition comprises at least one additional drug.

8. A combination pharmaceutical preparation for simultaneous, sequential, or separate use in the treatment of neurodegenerative diseases, comprising 2-phenyl-6-(1H-imidazol-1-yl)quinazoline (CR4056) or a pharmaceutically acceptable salt thereof and at least one of an NMDA (N-methyl-D-aspartic acid) receptor antagonist and / or an acetylcholinesterase inhibitor, wherein the neurodegenerative disease is a disease selected from the group consisting of Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion disease, and HIV-associated dementia.

9. The combination pharmaceutical preparation according to claim 8, wherein the neurodegenerative disease is Alzheimer's disease.

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