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Researchers Submit Patent Application, "Pyrazolyl Compounds for Use in Reversing Reactive Gliosis", for Approval

July 3, 2014



By a News Reporter-Staff News Editor at Gene Therapy Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors AL-Mohanna, Futwan (Riyadh, SA); Deniro, Michael (Riyadh, SA), filed on May 6, 2011, was made available online on June 19, 2014 (see also King Khalid Eye Specialist Hospital).

The patent's assignee is King Khalid Eye Specialist Hospital.

News editors obtained the following quote from the background information supplied by the inventors: "Diabetic retinopathy [DR] is a leading cause of visual disturbance in adults and is the leading cause of blindness in Americans between the ages of 20 and 74 years [48]. DR has been regarded as a retinal microvascular disease, which develops in two stages: an early, nonproliferative stage, and a later, proliferative stage. In the early non-proliferative stage, retinal vascular permeability can increase even before the appearance of clinical retinopathy [59]. Currently, this stage is diagnosed by dilation of retinal veins, retinal microaneurysmas, intraretinal microvascular abnormalities [which include intraretinal new vessels], areas of capillary nonperfusion, retinal hemorrhages, cotton wool spots [infarctions within the nerve fiber layer [NFL], edema, and exudates. All these signs indicate regional failure of the retinal microvascular circulation, which presumably results in ischemia. On the other hand, proliferative DR [PDR] is diagnosed based on the ischemia-induced formation of new blood vessels on the surface of the retina. New vessels can extend into the vitreous cavity of the eye and can hemorrhage into the vitreous, resulting in visual loss [7]. They also can cause tractional retinal detachments from the accompanying contractile fibrous tissue. Furthermore, during this stage, over-proliferation of capillary endothelial cells [EC] results in retinal neovascularisation [NV], abnormal formation of new vessels in the retina and in the vitreous, leading to PDR [68]. As a consequence retinal edema may ensue [15]. Retinal edema involves the breakdown of the blood-retinal barrier, with leakage of plasma from small blood vessels.

"Platelet-derived growth factor-B [PDGF-B] is secreted by endothelial cells. PDGF-B is both chemotactic and mitogenic to vascular endothelial cells in vitro [56] and may also have angiogenic effects in vivo [40]. Increased expression of PDGF-B in the retina causes severe proliferative retinopathy and retinal detachment similar to advanced stages of PDR [64]. PDGF-B promotes the recruitment, proliferation and survival of pericytes; recruits glial cells and retinal pigment epithelial [RPE] cells [9], which promotes scarring, a complication of ocular NV that is the major cause of permanent loss of vision.

"Nitric oxide [NO] is an important signaling pathway that mediates a variety of essential physiological processes, including neurotransmission, vasodilatation, and host cell defense [44]. NO is generated from L-arginine by the catalytic reaction of three different isoforms of nitric oxide synthase [NOS]. Neuronal and endothelial isoforms are constitutively expressed [cNOS] and upregulated by intracellular free calcium. At low concentrations NO regulates vessel tone; whereas at high concentrations NO mediates tissue damage [47]. In addition, NO directly contributes to tissue damage by combining with superoxide to form peroxynitrite, a highly reactive species that produces lipid peroxidation, mitochondrial and DNA damage. Moreover, inducible NOS [iNOS] is expressed at the transcriptional level by macrophages, neutrophils and a number of other cells in response to inflammatory stimuli such as LPS and cytokines [47]. iNOS is expressed in RPE, ciliary epithelial cells, Muller cells, retinal parenchyma, choroid vasculature and pericytes [45]. In addition, iNOS is independent of calcium, and generates large amounts of NO [nanomolar concentrations] over extended periods [hours to days]. It is believed that the relatively smaller amount of NO generated by eNOS and nNOS are involved in physiological functions, whereas the comparatively larger amount of NO generated by iNOS is involved in the pathophysiology of a number of inflammatory diseases [47]. Upregulation of iNOS level has been found in retinas of experimental diabetic rodents and human patients in most studies [19]. Furthermore, NO has been shown to have pro-angiogenic or anti-angiogenic effects depending upon the physiological or the pathological setting. In transgenic mice with increased expression of VEGF in photoreceptors, deficiency of any of the three isoforms caused a significant decrease in subretinal NV [10]. In mice with laser-induced rupture of Bruch's membrane, deficiency of iNOS or nNOS, but not eNOS, caused a significant decrease in choroidal NV. In the oxygen induced retinopathy [OIR] mouse model, deficiency of eNOS, but not iNOS or nNOS, caused a significant decrease in retinal NV and decreased expression of VEGF. These data suggest that NO contributes to both retinal and choroidal NV, but that different isoforms of NOS are involved. Several studies demonstrated that NO plays a critical role in VEGF-induced vascular hyperpermeability [41] and angiogenesis [49]. Furthermore, VEGF was shown to induce the expression of iNOS [39] and stimulate production of NO [72].

"The retina contains two types of macroglial cells. The most abundant are the Muller cells, which project from the retinal ganglion cell layer [GCL] to the photoreceptors, whereas the astrocytes, which originate in the optic nerve and migrate into the retina during development [71] reside as a single layer adjacent to the inner limiting membrane.

"Muller cells are the principal glial cells of the neural retina, and play a wealth of crucial roles in supporting neuronal function [4]. In response to virtually every pathological alteration of the ischemia, photic damage, retinal trauma, retinal detachment, glaucoma, DR, and age-related macular degeneration, Muller cells become reactivated [4]. Muller cells protect neurons after retinal injury, via release of neurotrophic factors and free radical scavengers, glutamate uptake, and facilitation of NV [4]. Hence, the close association between neurons and glia suggests that gene expression in these cell types is likely to be influenced by mutual interactions. Several lines of evidence indicate that glia influence the growth, migration and differentiation of neurons. Glial cells provide structural and metabolic support for retinal neurons and blood vessels, and the cells become reactive in certain injury states [35].

"It has been shown in animal studies that iNOS is localized at the GCL, at the inner nuclear layer [INL], and at the outer nuclear layer [ONL] in the retinas of diabetic rats. iNOS immunoreactivity was observed in the retina in other ischemic retinopathies as well. Retinal ischemia induced by common carotid artery occlusion in rats induced iNOS expression in Muller cells and retinal ganglion cells. In a murine model of OIR, iNOS mRNA was expressed in ischemic retina [16]: Ischemia has been shown to induce the expression of iNOS [62] and VEGF [46] in the retina. Yet, other studies demonstrated that VEGF induced the expression of iNOS in human EC [39] Recently, several studies demonstrated expression of VEGF receptors in retinal ganglion cells, INL, and Muller cells [51]. Induction of iNOS through VEGF stimulates production of NO from rabbit and human EC through activation of tyrosine kinases and an increase in intracellular calcium [72]. Several studies demonstrated that NO plays a critical role in VEGF-induced vascular hyperpermeability and angiogenesis. Previous studies demonstrated that an NOS inhibitor blocked VEGF-induced vascular hyperpermeability in all ocular and non-ocular tissues. Since the expression of iNOS protein was mainly localized at the level of the retinal vessels, it is possible that the overproduction of NO by the iNOS isoform contributes to blood-retinal barrier breakdown in the ischemic retinas of OIR mouse model. Since NOS inhibitors block the VEGF-induced proliferation, the mitogenic action of VEGF on EC is likely to be NO mediated [66]. In a murine model of OIR, iNOS expression was found to inhibit angiogenesis locally in the avascular retina mediated by a downregulation of VEGF R2 and to promote intravitreal NV [62]. iNOS inhibitors enhanced angiogenesis in the ischemic retina and inhibited pathological intravitreal NV. In addition, pathological intravitreal NV was significantly reduced in iNOS knockout mice [62]. In addition, data have demonstrated that that oxygen-induced retinal vaso-obliteration was significantly reduced by an NOS inhibitor, strongly supporting a putative role for NO in the retinal vaso-obliterative process [5].

"Several studies suggested that glial reactivity and altered glial metabolism are early pathological events in the retina during diabetes [58]. The most constant manifestation of reactivity is the increase in immunoreactivity for the intermediate filament protein glial fibrillary acidic protein [GFAP][43]. GFAP is mainly expressed in astrocytes for which it constitutes a selective marker. Previous reports have demonstrated that upregulation of astrocytic intermediate filaments is a crucial step and a hallmark of reactive gliosis [RG][54].

"RG is one of the pathophysiological features of retinal damage. RG includes morphological, biochemical, and physiological changes of Muller cells; these alterations vary with type and severity of insult. Under stress, Muller cells exhibit three crucial nonspecific gliotic responses, which are considered as 'hallmarks of glial cell activation', these are: [i] cellular hypertrophy due to alterations in intermediate filament [10], [ii] cellular proliferation [8], and [iii] the upregulation of the intermediate filament [IF] system [known also as nanofilament system] composed of GFAP, vimentin, nestin and synemin [32, 55]. There are other gliotic characteristics such; targeted cellular migration [69], changes in ion transport properties [57], and secretion of signaling molecules such as VEGF [1]. Like other glial cells of the CNS, Muller cells undergo RG following acute retinal injury or chronic neuronal stress [73]. The overexpression of GFAP is the most sensitive non-specific response to retinal disease and injury, and it may be considered as the hallmark of 'retinal stress', i.e. as a universal early cellular marker for retinal injury and Muller cell activation [4]. Furthermore, GFAP, which is located primarily to Muller cells, has specific immunoreactivities that occur in all retinal eccentricities. The vertebrate retina contains a specialized type of glia, the Muller glia, not found elsewhere in the CNS. Muller cell gliosis is characterized by proliferation [61], and changes in cell shape due to alterations in intermediate filament [60]. Successful inhibition of GFAP using antisense oligonucleotides has also been reported by several groups [71, 72, 40]. Ostensibly, gliosis is important for the protection and repair of retinal neurons, yet some pathologies such as DR may be exacerbated by RG properties [55, 1].

"In general, neuronal loss in the retina due to various eye and retinal diseases that are associated directly or indirectly with Reactive Gliosis. Such diseases include but are not limited to: retinopathy, ischemic retinopathy, hypertensive retinopathy, retinal neovascularization, macular degeneration [age-related macular degeneration (AMD)], Meckel syndrome, autosomal recessive inheritance diseases, Bardet-Biedl syndrome, retinal vessel occlusions [blockage of central retinal arteries and veins] [branch and central retinal vein occlusions] and retinal artery occlusion, cytomegalovirus (CMV) retinitis, diabetic retinopathy [retinopathy in diabetes], diabetic eye problems, epi-retinal membrane (or cellophane or macular pucker), flashin lights, eye floaters, flashes and posterior vitreous detachments, macular edema (CME), macular holes, macular translocation, cancers affecting the retina, melanoma, retinoblastoma (PDQ), retinal tear and retinal detachment, retinal detachment repair, proliferative vitreoretinopathy (PVR), photodynamic therapy (PDT), subretinal neovascular membranes and surgery (AMD, OHS, idiopathic, myopia, PXE, etc.), vitrectomy, Usher syndrome, retinoschisis, retinitis pigmentosa (RP), retinal tear, Bietti's crystalline dystrophy, choroideremia, retinopathy of prematurity (ROP), Behcet's disease, central serous choroidopathy, amaurosis fugax, Leber congenital amaurosis, juvenile retinoschisis, Refsum disease, neuropathy, ataxia, and retinitis pigmentosa, Leber congenital amaurosis, familial exudative vitreoretinopathy, and choroideremia.

"Few medicaments to treat reactive gliosis in the retinal or eye diseases are currently known. Thus, there is a strong need for novel effective substances and formulations for use as a medicament for the treatment, reversal or the attenuation of reactive gliosis and reactive gliosis associated with one of the aforementioned diseases.

"So far, a method for treating a neurodegenerative disease, comprising administrating a pyrazolyl compound like 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)-indazole or 1-benzyl-3-(5'-methoxymethyl-2'-furyl)-indazole has been previously described in US 2004/0077702. In contrast, reactive gliosis is an acute disease, while a neurodegenerative disease is a chronic disease. Reactive gliosis may lead to a neurodegenerative disease, however, this is not necessarily the case.

"The object of the invention is thus to provide an alternative substance for use as a medicament for the treatment, reversal or the attenuation of reactive gliosis and reactive gliosis associated with one of the aforementioned diseases."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "The object of the present invention is solved by the subject-matter as defined in the attached claims. The object of the invention is solved by a pyrazolyl compound comprising the chemical structure of formula I

"##STR00001##

"wherein Ar.sub.1 forms an aromatic or heteroaromatic ring, or is phenyl, wherein the aromatic or heteroaromatic ring is optionally substituted; Ar.sub.2 is selected from furyl, phenyl, alkyl, aryl, or heterocyclyl; wherein the alkyl, aryl, or heterocyclyl is optionally substituted; Ar.sub.3 is phenyl or substituted phenyl; n is 1 to 10; or any enantiomer, racemic form or mixture, prodrug, or analog of the substance, for use as a medicament for the treatment, reversal or the attenuation of reactive gliosis, and/or reactive gliosis directly or indirectly associated with eye diseases, diseases of the retina, or retinal disorders.

"In a preferred embodiment, the object of the invention is solved by a pyrazolyl compound comprising the chemical of structure formula I, wherein

"each of Ar.sub.1 and Ar.sub.3 is phenyl;

"Ar.sub.2 is furyl or phenyl;

"one of R.sub.1, and R.sub.2, is H, and the other is H, C.sub.1-C.sub.6 alkyl or halogen; or both R.sub.1 and R.sub.2 are H;

"one of R.sub.3 and R.sub.4 is H, and the other is CH.sub.2OH;

"each of R.sub.5 and R.sub.6 is H; n is 1 to 10

"or any enantiomer, racemic form or mixture, prodrug, or analog of the substance, for use as a medicament for the treatment, reversal or the attenuation of reactive gliosis and/or reactive gliosis directly or indirectly associated with eye diseases, diseases of the retina, or retinal disorders.

"In one embodiment, the compound is the compound as described above, wherein Ar.sub.2 is 5'-furyl. In a further embodiment, R.sub.3 is substituted at position 2 of furyl and R.sub.3 is CH.sub.2OH and R.sub.4 is H. In a further embodiment, R.sub.1 and R.sub.2, are substituted at positions 4 and 5 of phenyl and R.sub.1 is H, and R.sub.2 is CH.sub.3. In yet a further embodiment, R.sub.1 is H, and R.sub.2 is F.

"Preferably, the object is solved by the compound which is 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) and has the chemical structure of Formula II:

"##STR00002##

"or any enantiomer, racemic form or mixture, prodrug, or analog of the substance.

"In another preferred embodiment, the object is solved by the compound, which is 1-benzyl-3-(5-methyl-furan-2-yl)-1H-indazole, 1-benzyl-3-(5-methoxymethyl-furan-2-yl)-1H-indazole, or 1-benzyl-3-(5'-methoxymethyl-2'-furyl)-indazole, or any enantiomer, racemic form or mixture, prodrug, or analog of the substance.

"The compound of the invention may also be for use in reduction of PDGF-B, GFAP and iNOS expression and protein levels, in vivo and in vitro; reduction of filopodial length and number in endothelial tip cells; inhibition of ischemic pathologic neovascular response; promotion of the physiological retinal microvascular repair and intra-retinal revascularization [RV] of the avascular retina.

"In yet another preferred embodiment, the compound as described above is for use as a medicament for the treatment, reversal or the attenuation of gliosis, astrogliosis, reactive gliosis, preferably reactive gliosis, more preferably reactive gliosis that is directly or indirectly associated with retinal or eye diseases.

"In yet another preferred embodiment, the compound as described above is for use as a medicament for the treatment, reversal or the attenuation of reactive gliosis associated with retinopathy, ischemic retinopathy, hypertensive retinopathy, retinal neovascularization, macular degeneration [age-related macular degeneration (AMD)], Meckel syndrome, autosomal recessive inheritance diseases, Bardet-Biedl syndrome, retinal vessel occlusions [blockage of central retinal arteries and veins] [branch and central retinal vein occlusions] and retinal artery occlusion, cytomegalovirus (CMV) retinitis, diabetic retinopathy [retinopathy in diabetes], diabetic eye problems, epi-retinal membrane (or cellophane or macular pucker), flashin lights, eye floaters, flashes and posterior vitreous detachments, macular edema (CME), macular holes, macular translocation, cancers affecting the retina, melanoma, retinoblastoma (PDQ), retinal tear and retinal detachment, retinal detachment repair, proliferative Vitreoretinopathy (PVR), photodynamic therapy (PDT), subretinal neovascular membranes and surgery (AMD, OHS, idiopathic, myopia, PXE, etc.), vitrectomy, usher syndrome, retinoschisis, retinitis pigmentosa (RP), retinal tear, Bietti's crystalline dystrophy, choroideremia, retinopathy of prematurity (ROP), Behcet's disease, central serous choroidopathy, amaurosis fugax, Leber congenital amaurosis, juvenile retinoschisis, refsum disease, neuropathy, ataxia, retinitis pigmentosa, Leber congenital amaurosis, familial exudative vitreoretinopathy, and/or choroideremia.

"The object of the invention is also solved by a pharmaceutical composition comprising the compound as described above and a pharmaceutical acceptable carrier for use as a medicament for the treatment, reversal, or attenuation of an acute neuronal disease or disorder, preferably reactive gliosis, directly or indirectly associated with eye diseases, diseases of the retina, or retinal disorders. In a preferred embodiment, the acute disorder is reactive gliosis, preferably reactive gliosis associated with one of the aforementioned diseases.

"The object of the invention is also solved by a pharmaceutical composition for use in reduction of PDGF-B, GFAP and iNOS expression and protein levels, in vivo and in vitro; reduction of filopodial length and number in endothelial tip cells; inhibition of ischemic pathologic neovascular response; promotion of the physiological retinal microvascular repair and intra-retinal revascularization [RV] of the avascular retina.

"In yet another embodiment, the pharmaceutical composition as described above is for use as a medicament for the treatment, reversal, or attenuation of a disease or disorder that is reactive gliosis, reactive gliosis directly or indirectly associated with eye diseases, diseases of the retina, retinal disorders, preferably reactive gliosis associated with one of the aforementioned diseases, or reactive gliosis associated with retinopathy, ischemic retinopathy, hypertensive retinopathy, retinal neovascularization, macular degeneration [age-related macular degeneration (AMD)], Meckel syndrome, autosomal recessive inheritance diseases, Bardet-Biedl syndrome, retinal vessel occlusions [blockage of central retinal arteries and veins] [branch and central retinal vein occlusions] and retinal artery occlusion, cytomegalovirus (CMV) retinitis, diabetic retinopathy [retinopathy in diabetes], diabetic eye problems, epi-retinal membrane (or cellophane or macular pucker), flashin lights, eye floaters, flashes and posterior vitreous detachments, macular edema (CME), macular holes, macular translocation, cancers affecting the retina, melanoma, retinoblastoma (PDQ), retinal tear and retinal detachment, retinal detachment repair, proliferative vitreoretinopathy (PVR), photodynamic therapy (PDT), subretinal neovascular membranes and surgery (AMD, OHS, idiopathic, myopia, PXE, etc.), vitrectomy, Usher syndrome, retinoschisis, retinitis pigmentosa (RP), retinal tear, Bietti's crystalline dystrophy, choroideremia, retinopathy of prematurity (ROP), Behcet's disease, central serous choroidopathy, amaurosis fugax, Leber congenital amaurosis, juvenile retinoschisis, refsum disease, neuropathy, ataxia, retinitis pigmentosa, Leber congenital amaurosis, familial exudative vitreoretinopathy, and/or choroideremia.

"In another preferred embodiment, the dosage form of the pharmaceutical composition is a tablet, lozenge, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, solution or emulsion. In another preferred embodiment, the pharmaceutical composition is for oral, rectal, transmucosal, transdermal, intestinal, parenteral, intramuscular, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular, or subcutaneous administration.

"The object of the invention is further solved by a method for treating an acute disorder and the treatment, reversal or the attenuation of reactive gliosis and reactive gliosis associated with one of the aforementioned diseases, comprising administering to a subject in need thereof an effective amount of a therapeutic agent comprising a compound as described above or a pharmaceutical composition as described above. Preferably, the method comprises the administration of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole and the acute disease or disorder is one of the diseases or disorders as mentioned above, reactive gliosis, preferably reactive gliosis associated with one of the aforementioned diseases."

For additional information on this patent application, see: AL-Mohanna, Futwan; Deniro, Michael. Pyrazolyl Compounds for Use in Reversing Reactive Gliosis. Filed May 6, 2011 and posted June 19, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2169&p=44&f=G&l=50&d=PG01&S1=20140612.PD.&OS=PD/20140612&RS=PD/20140612

Keywords for this news article include: VEGF, Biotechnology, Edema, Myopia, Neurons, Surgery, Deafness, Diabetes, Ischemia, Oncology, Angiology, Blindness, Chemicals, Chemistry, Cytoplasm, Melanomas, Neonatology, Angiogenesis, Cytoskeleton, Ear Diseases, Nitric Oxide, Phototherapy, Choroideremia, Endocrinology, Ophthalmology, Retinoschisis, Brain Diseases.

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