Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics

Definitions

• Glaucoma represents a heterogeneous group of chronically progressive optic neuropathies. It is the leading cause of irreversible blindness worldwide, affecting around 64 million people globally. This figure is projected to increase to 80 million in 2020 and 112 million by 2040 (THAM, Y.C., LI, X., WONG, T.Y., QUIGLEY, H.A., AUNG, T deliberately CHENG, C.Y. Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthal molog v.121 (11 ) : 2081-2090, 2014; ALIANCY, J., STAMER, W.D., WIROSTKO, B. A Review of Nitric Oxide for the Treatment of Glaucomatous
• Glaucoma is generically described as a slow degeneration of retinal ganglion cells (RGC) followed by axon loss. It manifests as a progressive thinning of the retinal nerve fiber layer, and optic nerve head cupping, events which functionally result in a characteristic pattern of visual field loss.
• Optic nerve head cupping is the enlargement of the central portion of the optic disc, which is called the “cup”, a whitish portion without nerve fibers which is usually quite small in comparison with the entire optic disc.
• the diameter of the cup in relation to the total diameter of the optic disc is called the cup-to-disc ratio, a measurement used to assess the progression of glaucoma.
• the conventional outflow system comprises the trabecular meshwork (TM), the juxtacanalicular tissue (JCT), and the Schlemm’s canal (SC) in humans, non-human primates, and rats (MORRISON, J.C., FRAUNFELDER, F.W., MILNE, S.T., MOORE, C.G. Limbal microvasculature of the rat eye. Invest Ophthalmol Vis Sci. 36: 751- 756, 1995; MORRISON, J.C., CEPURNA, W.O., JOHNSON, E.C. Modeling glaucoma in rats by sclerosing aqueous outflow pathways to elevate intraocular pressure, EXP Eve Res.
• TM trabecular meshwork
• JCT juxtacanalicular tissue
• SC Schlemm’s canal
• POAG primary open-angle glaucoma
• the initial target aims, for example, at a 20% to
• Topic prostaglandin analogs such as latanoprost, travoprost, tafluprost, unoprostone, bimatoprost, are the first therapeutic option. These drugs act mainly by improving the AH drainage through the uveoscleral, unconventional, pathway. Although less effective, second-line agents are generally used when there is intolerance or contraindication to prostaglandin analogs.
• Topic alternative drug classes comprise b-Adrenergic and a- Adrenergic blockers, carbonic anhydrase inhibitors and cholinergic agonists.
• the drugs that act by this pathway tend potential to be more potent than the drugs that act thought unconventional outflow, or at least could have a complementary action.
• a drug that could act causing blood flow increase to the optic nerve could directly prevent or delay the optic nerve damage (i.e . neuroprotection).
• the convenient topical administration preferably with a low frequency of treatment, is also a valuable nice-to-have feature. Considerable efforts have been made in this direction, but with very limited success (WEINREB, R.N., AUNG, T deliberately MEDEIROS, F.A. The pathophysiology and treatment of glaucoma: a review. JAMA.
• Nitric Oxide (NO) has gained a lot of attention recently as a potential new target for the treatment of glaucoma.
• the biologic effects of NO could simultaneously mediate increased AH drainage through the conventional outflow and protect the optical nerve from further injury (CAVET, M.E., VITTITOW, J.L, IMPAGNATIELLO, F hinder ONGINI, E awkward BASTIA, E.
• NO signaling pathway has a role in the ocular homeostasis, regulating AH drainage and, therefore, IOP.
• the capacity to form NO is found in the anterior ocular tissues.
• nNOS neuronal nitric oxide synthase
• eNOS endothelial NOS
• TM ciliary muscle
• SC retinal vasculature
• inducible NOS INOS is not constitutively expressed in the eye at physiological conditions, only expressed only after stimulation in macrophages located in the stroma and in the ciliary process, and in astrocytes (WAREHAM, L.K., BUYS, E.S., SAPPINGTON, R.M.
• TM cells are known to be highly contractile in nature, analogous to vascular smooth muscle cells (VSMC), in which the role of NO-cGMP signaling in endothelium-dependent relaxation is well understood (CAVET, M.E., VITTITOW, J.L., IMPAGNATIELLO, F tolerate ONGINI, E awkward BASTIA, E. Nitric oxide (NO): an emerging target for the treatment of glaucoma. Invest Ophthalmol Vis Sci. 55(8): 5005-5015, 2014).
• VSMC vascular smooth muscle cells
• the optic nerve head - the site of glaucomatous axonal injury - is supplied by the posterior ciliary artery circulation and retinal circulation.
• Posterior ciliary artery the main source of blood supply, branching off the ophthalmic artery, divides later into a number of short posterior ciliary arteries that enter the globe around the optic nerve, and contribute to the perfusion of the anterior optic nerve head.
• the surface nerve fiber layer of the retina is fed by arteriolar branches from the central retinal artery.
• endogenous NO is essential for maintaining basal blood flow.
• Nitrovasodilators could be considered as a new class of ocular hypotensive agents, considering that NO mediates a multitude of diverse ocular effects and maintenance of IOP. NO donors have been shown to mediate IOP-lowering effects in both preclinical models and clinical studies, primarily through cell volume and contractility changes in the conventional outflow tissues. A NO-donating associated with a prostaglandin F receptor agonist, latanoprostene bunod, was more effective than the reference compound, latanoprost, in lowering IOP.
• a NO-based therapy that enhances optic nerve and retinal vascular NO signaling may have the potential to exert beneficial effects on injured RGC (TSAI, J.C., GRAY, M.J., CAVALLERANO, T. Nitric oxide in glaucoma: what clinicians needs to know. Candeo Clinical/Science Communications. LLC, 2017).
• NAION non-arteritic ischemic optic neuropathy
• Therapeutic peptides have shown great promise as novel therapeutics in the treatment of ocular diseases. These molecules offer several advantages such as high potency, low unspecific binding, less toxicity, and minimization of drug-drug interaction. However, factors such as physical and chemical degradation, short in vivo half-lives, clearance by the mononuclear phagocytes (MPS) of the reticulum endothelial system (RES), risk of immunogenicity, and failure to permeate cell membranes pose high challenges to topic ocular administration of peptides.
• MPS mononuclear phagocytes
• RES reticulum endothelial system
• Patent US 9,279,004 discloses a peptide with 19 amino acids (PnTx(19)) and molecular weight of 2,485.85 Da, built from the toxin PnTx2-6.
• the natural toxin causes priapism in male patients bitten by the spider Phoneutria nigriventer.
• the peptide PnTx(19), also called PnPP-19 is a non-naturally occurring molecule, engineered from noncontiguous domains of the natural toxin.
• the disclosures reveal that PnPP- 19 is capable of enhancing the erectile function, as demonstrated by an improved relaxation of isolated strips of murine penile corpus cavernosum ex vivo.
• PnPP19 a synthetic and nontoxic peptide designed from a Phoneutria nigriventer Toxin, potentiates erectile function via NO/cGMP. J Urol: 194(5): 1481-90. 2015).
• PnPP-19 was claimed as a potential candidate for the treatment of erectile dysfunction, with a potential application in patients that are resistant to the therapy based on phosphodiesterase 5 inhibitors (PDE5i).
• the present specification describes methods of treatment and pharmaceutical compositions comprising a NOS-enhancer peptide that can simultaneously improve the conventional outflow of AH and directly prevent the progression of optic nerve degeneration. Therefore, the methods and compositions described herein are useful for treating and/or preventing eye diseases related to intraocular hypertension and/or optic nerve degeneration, such as PCAG, POAG, NTG, and elevated IOP.
• the present invention unexpectedly shows that when topically administered as eye drops, the synthetic peptide PnPP-19 is capable of penetrating the eye, reducing the IOP in animals with healthy eyes and also glaucomatous untreated eyes, and protecting against ischemic injury to the retina and optic nerve. Accordingly, the embodiments of this description include the following:
• a method of reducing the intraocular pressure comprising topically administering to an eye an effective amount of PnPP-19.
• a method of treating or preventing ischemic optic neuropathy comprising topically administering to an eye an effective amount of PnPP-
• - PnPP-19 also has neuroprotective effects, as vision preservation, reduction of histological damage, protection of retinal cells against ischemic injury, in either prevention or therapeutic mode of treatment, confirmed in an animal model of retinal ischemia.
• FIG. 1 Figure 1 - HET-CAM's Photographs after 5 minutes exposure to (A) 0.1 M NaOH (positive control); (B) NaCI 0.9% (negative control); (C-F) PnPP-19, at concentrations that vary from 40 mg of peptide in 20 mL (0.2%) to 320 mg of peptide in 20 mL (1 .6%).
• Figure 5 - PnPP-19 reduces IOP in normotensive rats.
• Figure 6 - PnPP-19 reduces IOP in rats with glaucoma.
• Figure 7 - PnPP-19 preserves the number of RGCs. There was a smaller number of RGCs in the retinas of glaucomatous animals compared to healthy rats. Glaucomatous animals treated with PnPP-19 (80 mg/eye, 0.4%) have an RGC count higher than untreated glaucomatous rats and was not statistically different compared to healthy rat. Asterisks represent statistical difference in relation to the healthy animals: **p ⁇ 0.01 , one way ANOVA.
• Figure 8 - PnPP-19 permeates the cornea and reaches the retina. Comparison between Control (saline) and the treatment group using PnPP-19 (80 mg/eye, 0.4%). The images represent the fluorescence intensity (color green) from the cornea (A), vitreous body (B) and retina (C). The charts on the right represent the fluorescence intensity from the cornea and retina. Eyes were removed 3 hours after application of one drop (20 mI). Fluorescence microscopy performed using APOTOME.2 ZEISS, 10X objective, the bar is 100 mm in length. FITC was excited at 490 nm and the emission was detected at 526nm.
• Black arrows denote areas of vacuolization and pyknotic nuclei.
• A ischemic/untreated;
• B ischemic/PnPP-19 pre- treatment;
• C healthy.
• the method of treatment of the present comprises the administration of PnPP-19 to a patient in need thereof.
• PnPP-19 relates to the polypeptide having a sequence of SEQ ID NO 1 : Gly Glu Arg Arg Gin Tyr Phe Trp He Ala Trp Tyr Lys Leu Ala Asn Ser Lys Lys, which is optionally N-terminal acetylated and/or C-terminal amidated.
• therapeutic activity refers to a demonstrated or potential biological activity whose effect is consistent with a desirable therapeutic outcome in humans, or to desired effects in nonhuman mammals or in other species or organisms.
• a given therapeutic peptide may have one or more therapeutic activities, however, the term “therapeutic activities' as used herein may refer to a single therapeutic activity or multiple therapeutic activities.
• “Therapeutic activity” includes the ability to induce the desired response and may be measured in vivo or in vitro. For example, a desirable effect may be assayed in cell culture, isolated tissues, animal models, clinical evaluation, EC 50 assays, IC 50 assays, or dose-response curves.
• therapeutic activity includes preventive or curative treatment of a disease, disorder, or condition. Treatment of a disease, disorder or condition can include improvement of a disease, disorder or condition by any amount, including the elimination of a disease, disorder or condition.
• the "similarity" between two sequences is determined by comparing the sequences of amino acids of the polypeptides when aligned in order to maximize the superposition, minimizing the gaps of the sequence, followed by an accounting of identical residues between the sequences.
• the percentage of identity of two sequences of amino acids or nucleic acids can be determined by visual inspection and/or mathematical calculation, commonly done for longer sequences comparing the information of the sequence using a computer program. Examples of programs that can be used by a person skilled in the art for comparison of sequences of peptides and nucleic acids are the BLAST (BLASTP) and BLASTN, freely available on the website of the National Library of Medicine http://www.ncbi.nlm.nih.gov/BLAST.
• a peptide fragment is “derived from” an original peptide if there is a sequence of amino acids that is identical or homologous to the sequence of amino acids of the original peptide or polypeptide.
• the said fragment may be produced by synthetic methods ⁇ e.g. solid-state peptide synthesis, recombinant DNA expression in a modified cell and enzymatic degradation in vitro) or natural degradation of an original peptide.
• the said fragment results from a process occurred in a living organism (i.e . an isolated cell or a tissue in vitro, or an animal, for instance, but not limited to, a human being, in vivo), therefore, a product of the metabolism of the said organism.
• metabolite The said product or products from the metabolic degradation of an original peptide (or drugs in general) is called metabolite.
• Such metabolites may or may not have a biological effect. When such metabolites still have a biological activity that resembles the original peptide, they are considered active metabolites. Therefore, those skilled in the art could promptly capture that fragments of a therapeutic peptide, produced either synthetically or naturally, may exhibit lower, equal or higher lOP-lowering activity than the original peptide.
• a composition comprises, in a pharmaceutically acceptable medium, an effective amount of a polypeptide having a sequence of SEQ ID NO 1 , which is optionally N-terminally acetylated and/or C-terminally amidated.
• the polypeptide is referred to herein as PnPP-19.
• the polypeptide is in the form of a pharmaceutically acceptable salt.
• the polypeptide is acetylated, e.g. in some embodiments, using acetyl group into the N- terminus (peptide glycine (G)).
• the polypeptide is amidated, e.g.
• a method comprises, administering an effective amount of PnPP-19 a pharmaceutical composition noted herein. In some embodiments, the method is for reducing the IOP. In some embodiments, the method if for treating or preventing ischemic optic neuropathy, such as glaucoma, including normal-tension glaucoma; age-related macular degeneration; or diabetic neuropathy.
• ischemic optic neuropathy such as glaucoma, including normal-tension glaucoma; age-related macular degeneration; or diabetic neuropathy.
• Myopia is a significant risk factor for glaucoma, especially in those of Asian descent (MCMONNIES, C.W. Glaucoma history and risk factors. J Ootom. 10(2): 71-78, 2017).
• MMONNIES C.W. Glaucoma history and risk factors. J Ootom. 10(2): 71-78, 2017.
• myocilin mutations in advanced POAG and of copy number variations of TBK1 in NTG illustrating the contribution of genetics for glaucoma risk prediction (SOUZEAU, E., BURDON, K.P., DUBOWSKY, A. Higher prevalence of myocilin mutations in advanced glaucoma in comparison with less advanced disease in an Australian Disease Registry.
• a patient who has progressed to definite glaucoma may have sufficient visual field loss to complain of impaired night driving, near vision, reading speed, or outdoor mobility.
• an eye examination will diagnosis glaucoma if one of the following conditions are present: (i) consistently elevated IOP, (ii) suspicious-appearing optic nerve (such as abnormal nerve fiber layer on optical coherence tomography (OCT) or disc hemorrhage), or (ill) abnormal visual field.
• suspicious-appearing optic nerve such as abnormal nerve fiber layer on optical coherence tomography (OCT) or disc hemorrhage
• OCT optical coherence tomography
• ill abnormal visual field.
• nNOS is expressed in the ciliary non-pigmented epithelium
• PnPP-19 as an INOS enhancer, is capable to lower IOP up to 24h with one daily administration, and the reduction of IOP is sustained during this period, without great variation in IOP, a desirable effect to avoid vision loss.
• NO is produced locally in the targeted cells and therefore avoiding lack of effect due to low NO levels or side effects due to NO action outside the target.
• PnPP-19 demonstrates to be non-irritant and to not cause corneal or retinal damage in preclinical studies.
• Late-stage AMD can present in two forms: a “dry”, atrophic form of AMD, characterized by macular drop out of RPE and photoreceptors, termed geographic atrophy, and “wet” neovascular form of AMD, characterized by the invasion of RPE and/or the retina by abnormal blood vessels, therefore neovascular, or exudative AMD, since this presentation involves choroid neovascularization (RICKMAN, C.B., FARSIU, S., TOTH, C.A., KLINGEBORN, M. Dry Age-Related Macular Degeneration: Mechanisms, Therapeutic Targets, and Imaging. Invest Ophthalmol Vis Sci. 54(14): ORSF68-ORSF8O, 2013).
• the disease also has an important neurodegenerative component, that includes neural apoptosis of ganglion, amacrine, and Muller cells, as well as inflammatory glial activation, and altered glutamate metabolism (WANG, W. and LO, A.C.Y. Diabetic Retinopathy: Pathophysiology and treatments. Int J Mol Sci. 19(6): 1816, 2018; BARBER, A.J. A new view of diabetic retinopathy: A neurodegenerative disease of the eye. Progress in Neuro-DSvchoDharmacoloQv & Biological Psychiatry. 27(2): 283-290, 2003; LYNCH, S. K., ABRAMOFF, M.D. Diabetic Retinopathy is a neurodegenerative disorder. Vision Research. 139: 101-107, 2017).
• NAION is caused by the the ischemia of the anterior protion of the optic nerve, particularly the lamina cribrosa, the same localion as glaucoma.
• the ischemia of the head of the optic nerve should be associated with a "disc at risk", some anormality that increase the risk of nerve damage, as anatomical anormalities, optic-nerve drusen and papilledema.
• Hipertension, diabetes, hypercholesterolemia, stroke, ischemic heart disease, tobacco use, systemic aterosclerosis and hypercoagulability are some diseases related with NAION (BIOUSSE, V. and NEWMAN, N.J. Ischemic Optic Neuropathies. N Engl J Med, 372:2428-2436, 2015).
• NAION is a vasodilator and therefore acting against ischemia; NO can down regulate the NMDA receptor (the glutamate biding receptor), and therefore reduce the excitoxicity; NO can act as a scavenger, consuming free radicals, and tehrefore reducing the oxidative stress.
• PnPP-19 can rech the retina, the local of the ischemia of the head of the optic-nerve, and increase the local levels of NO. Therefore, PnPP-19 has the potential to be the first drug useful for NAION patients.
• the peptides of the present description can be prepared by any methodologies known by those skilled in the art, including recombinant and non-recombinant methods. Synthetic pathways (nonrecombinant) include, without limitation, the chemical synthesis of the peptide in solid phase, the chemical synthesis of the peptide in liquid phase and biocatalyzed synthesis. In a preferred embodiment, the peptides are obtained by chemical synthesis, in the liquid or solid phase, using manual, automated or semi-automated systems.
• SPPS Solid-phase peptide synthesis
• the free terminal amine of the amino acid bound to the support reacts with the terminal carboxy portion of the subsequent amino acid.
• the terminal amine of the dipeptide is then deprotected and the process is repeated until the polypeptide is completed. Whenever adequate, the starting amino acids can also have protections in the side chains.
• an exemplificative protocol includes: construction of the nucleic acid that encodes the peptide of interest; cloning of the said nucleic acid in an expression vector; transformation of a host cell (cells, vegetable, bacteria, such as Escherichia coli, yeasts, such as Saccharomyces cerevisiae, or mammal cells, such as Chines Hamster Ovary Cells) with the said vector; expression of the nucleic acid to produce the peptide of interest.
• a host cell cells, vegetable, bacteria, such as Escherichia coli, yeasts, such as Saccharomyces cerevisiae, or mammal cells, such as Chines Hamster Ovary Cells
• a topically applied drug that has to be delivered into the anterior eye chamber has to cross the cornea, a tri-layer tissue composed by an outer epithelium, with tight junctions connecting the most superficial cells, a central connective tissue, made of highly organized collagen, and an endothelium, mainly involved in the maintenance of the correct corneal hydration.
• the permeability of the cornea is low and the diffusion of drugs, particularly hydrophilic and of high molecular weight, such as peptides, is very difficult (PESCINA, S., OSTACOLO, C., GOMEZ-MONTERREY, I.M., SALA, M severely BERTAMINO, A., SONVICO, F compromise PADULA, C., SANTI, P mil BIANCHERA, A., NICOLI, S. Cell penetrating peptides in ocular drug delivery: State of the art. J Control Release. 2018 Aug 28;284:84-102).
• peptides related to those demonstrated herein include analogues and/or derivatives that retain some or all of the therapeutic activity of the original peptides.
• the term “analogue” indicates variants obtained by substitutions, deletions or additions of amino acids to the peptides described herein; while “derivative” indicates variants containing chemical modifications on the primary sequence of the peptides described herein and/or their analogues. In certain aspects, such variants may evidence improvements in at least one of the therapeutic activities of the peptides.
• the peptides of the present description may be comprised of L-amino acids, D-amino acids or a combination of both in any ratio.
• Another embodiment includes prodrugs or drug precursors that are chemically or enzymatically converted into any of the active peptides before, after or during the administration to a patient in need thereof.
• Such compounds may include among others esters, N-alkyl, phosphates or conjugates of amino acids (ARNAB, D.E., Application of Peptide-Based Prodrug Chemistry in Drug Development; Springer. New York Heidelberg Dordrecht London. 2013), more lipophilic peptides (CACCETTA, Rchromat BLANCHFIELD, J.T., HARRISON, J cache TOTH, I., BENSON, H.A.E.
• Another embodiment also includes any cyclic peptide able to be converted into the linear active peptide. It further includes chemical modification with bioconjugates or macromolecules such as glycosylation or pegylation (HUTTUNEN, K.M., RAUNIO, H Rule RAUTIO, J. Prodrugs— from Serendipity to Rational Design. Pharmacol Rev. 63:750- 771 , 2011).
• bioconjugates or macromolecules such as glycosylation or pegylation (HUTTUNEN, K.M., RAUNIO, H Rule RAUTIO, J. Prodrugs— from Serendipity to Rational Design. Pharmacol Rev. 63:750- 771 , 2011).
• Another embodiment includes a peptidomimetic approach using any of the active peptides as a support to project active structures based on bioesters of groups of amino acids (VAGNER, J., QU, H. and HRUBY, V.J. Peptidomimetics, a synthetic tool of Drug Discovery. Curr Opin Chem Biol. 12(3): 292-296. 2008).
• Desirable amino acid conservatives substitutions can be determined by those skilled in the art using routine methodologies. Natural amino acids may be classified in terms of the side chains properties of the as: nonpolar (glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), methionine (Met)); uncharged polar (cysteine (Cys), serine (Ser), threonine (Thr), proline (Pro), asparagine (Asn), glutamine (Gin); acid (aspartic acid (Asp), glutamic acid (Glu)); basic (histidine (His), lysine (Lys), arginine (Arg)); and aromatic (tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe)).
• nonpolar glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), me
• hydrophilicity The average hydrophilicity of a polypeptide, determined by the hydrophilicity of the adjacent amino acids, is correlated with the biological properties of the compound.
• the natural amino acids have the following hydrophilicity values: Arg (+3.0); Lys (+3.0); Asp (+3.0 ⁇ 1); Glu (+3.0 ⁇ 1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5 ⁇ 1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).
• Cationic CPPs have highly positive net charges at physiological pH, primarily from arginine (Arg) and lysine (Lys) residues.
• Hydrophobic CPPs predominantly contain non-polar amino acids, resulting in a low net charge. This family of peptides could translocate across lipid membranes in an energy-independent manner.
• the class of hydrophobic CPPs includes but is not limited to gH 625, CPP-C, PFVYLI, Pep-7, and SG3 (PESCINA, S acrylic OSTACOLO, C Montgomery GOMEZ-MONTERREY, I.M., SALA, M strictly BERTAMINO, A., SONVICO, F compromise PADULA, C., SANTI, P legislative BIANCHERA, A., NICOLI, S. Cell penetrating peptides in ocular drug delivery: State of the art. J Control Release. 2018 Aug 28;284:84-102).
• the above-mentioned linkers of amino acids, fusion peptides, and the permeation-enhancing sequences may have 5 to 40 additional amino acids and can be connected to the NO-inducer peptide by means of linking moieties.
• moieties may be an atom or a collection of atoms optionally used to link a therapeutic peptide to another therapeutic peptide.
• the connector molecule may consist of a sequence of amino acids designed for proteolytic cleavage in order to allow the release of the biologically active portion in an appropriate environment.
• the smooth muscle tone modulating peptides described here may be fused to peptides designed to improve pharmacological properties (pharmacokinetic and/or pharmacodynamic) and or physicochemical properties.
• the NOS-inducer peptide can contain chemical modifications with one or more methyl or another small alkyl group in one or more positions of the peptide chain.
• examples of such groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, etc.
• the NOS-inducer peptide modification results from the attachment of one or more glycosidic moieties to the peptide sequence.
• the cited derivatives may be obtained by the attachment of one or more monosaccharides, disaccharides or trisaccharides to the peptide sequence at any position.
• the scope of some embodiments of the present invention also includes variants that have a therapeutic activity higher than 100%, 110%, 125%, 150%, 200% or more than 300%, or yet that evidence a 100 or 100 times higher activity, and any range derivable therefrom, in comparison with the non- modified peptides.
• the NOS-enhancer peptide described in some embodiments of the present invention may also be covalently conjugated to hydrosoluble polymers, either directly or by means of a spacer group.
• Examples of peptide-polymer conjugates inserted in the scope of some embodiments of this invention include: conjugates containing a hydrosoluble polymer coupled to the peptide in a detachable or stable manner, particularly coupled to the N-terminal portion; conjugates containing a hydrosoluble polymer coupled to the peptide in a detachable or stable manner, particularly coupled to the C-terminal portion; conjugates containing a hydrosoluble polymer coupled to the peptide in a detachable or a stable manner, particularly coupled to an amino acid located internally in the peptide chain; conjugates containing more than one hydrosoluble polymer coupled to the peptide in a detachable or a stable manner, coupled to the peptide in distinct regions such as, for example, to the N-terminal portion and to the side chain of an amino acid located internally
• the above-contemplated polymer is hydrophilic, non-peptidic, biocompatible and non-immunogenic.
• a substance is deemed biocompatible if the beneficial effects associated with the administration thereof to living organisms, either alone or combined with another substance (for example, a biologically active ingredient such as a therapeutic peptide), overcomes any deleterious effect that is clinically observable.
• a substance is deemed non-immunogenic if the intended use of the substance in vivo does not produce an undesirable immunological response (for example, the formation of antibodies) or, if an immunological response is triggered, such event is not deemed clinically significant or important.
• hydrosoluble polymers are not limited to a particular architecture and may have linear of no-linear structures, such as branched, bifurcated, multi-branched (for example, PEGs coupled to a polyol core), or dendritic (densely branched structure with several terminal groups).
• Methods for the conjugation of polymers to peptides are described in the prior art, as well as the adequate reagents, which may be selected among alkylating or acylating agents (see HARRIS, J. M. and ZALIPSKY, S., Polyethylene glycol), Chemistry and Biological Applications. ACS. Washington, 1997; VERONESE, F., and HARRIS, J.M. Peptide and Protein PEGylation.
• benzalkonium chloride decrease whenever the pH of the formulation falls below 5.0; they are incompatible with anionic therapeutic agents and also with non-ionic hydrophilic polymers used for viscosity.
• Cationic preservatives as 0.1% w/v disodium edetate (disodium EDTA), commonly used in ocular solutions, can be included in ocular formulations in which benzalkonium chloride is used to enhance its antimicrobial activity by chelating divalent cations in the outer membrane of the bacterial cell.
• Parabens Mixtures of methyl and propyl esters of para- hydroxybenzoic acid are used in ophthalmic formulations (typically at a combined concentration of 0.2% w/w).
• Chlorobutanol and phenylethylalcohol are examples of organic alcohols. Chlorobutanol can be used at a concentration of 0.5% w/v. Hydrolysis of chlorobutanol occurs under alkaline condition, and HCI is liberated as a by-product (the rate of reaction increases with increasing temperature, e.g. during autoclaving). The use of chlorobutanol is reserved for acidic ophthalmic preparations, Chlorobutanol is volatile and may be lost from the solution if stored in polyolefin containers, due to partitioning.
• the release rate may be varied using methods well known in the art including (I) variation of thickness of composition of the coating, alteration of the quantity of manner of addition of plasticizer on a coating, (iii) inclusion of additional ingredients, such as agents that modify the release, (iv) alteration of the composition, particle size or format of particle of the matrix and (v) provision of one or more passages through the coating.
• the amount of modulator contained within a sustained release formulation depends, for example, from the method of administration (for example, the location of the implant), the rate and duration of release that is expected and the nature of the condition to be treated or prevented.
• the matrix material which may or not have a controlled release function, is generally any material that supports the active ingredient(s).
• Electroretinography showed the maintenance of the curve shape of a and b wave in all treatment groups, compared to day 0 (control) (data not shown), demonstrating the absence of retinal nerve damage at any tested dose of PnPP-19.
• IOP reduction after a single dose application of PnPP-19 was: 19.08 ⁇ 2.29 mmHg, after 2 hours of treatment, compared to control, 23.25 ⁇ 2.06 mmHg; 18.20 ⁇ 2 mmHg, after 4 hours of treatment, compared to control, 22.95 ⁇ 4.35 mmHg; 17.16 ⁇ 2.13 mmHg, after 5 hours of treatment, compared to the control 22.5 ⁇ 2.13 mmHg.
• D % of IOP reduction
• PnPP-19 reduced IOP by 40, 36 and 45% after 2, 4 and 5 hours of administration, respectively. This reduction was maintained for up to 6 hours (Figure 5).
• the outer nuclear layer exhibits a decreased cell number, and greater edema and cell disorganization compared with control retinas.
• PnPP-19 preserves the number of RGCs: glaucomatous animals treated with PnPP-19 have an RGC count higher than untreated glaucomatous rats and was not statistically different compared to healthy rat (66.6 ⁇ 12.5 cells vs. 93.3 ⁇ 34.6 cells, respectively, p ⁇ 0.01) (Fig 7).
• FITC PnPP-19 was applied, compared to vehicle, in the cornea, vitreous body and retina. This data demonstrated that labeled PnPP-19, in a simple saline solution, permeates from the cornea to retinal epithelium within 3 hours after application.
• Retinal ischemia is very common in many ocular disorders such as age-related macular degeneration (AMD), diabetic retinopathy, retinal vascular occlusion, or glaucoma. Retinal ischemia induces irreversible morphological and functional changes that may result in blindness.
• AMD age-related macular degeneration
• l/R ischemia/reperfusion
• Other changes include optic nerve damage, neuronal degeneration, a tissue dissolution, structural distortion, and increased microglial activation.
• IOP Glutamate Release in Experimental Ischaemia of the Retina: An Approach Using Microdialysis. J Neurochem. 59(1):358-63, 1992).
• IOP was elevated by cannulating the eye anterior chamber, with a sterile 27-gauge needle attached to a manometer/pump connected to an air reservoir (HUGHES, W.F. Quantitation of ischemic damage in the rat retina. Exo Eve Res. 53(5): 573-82, 1991) elevating the IOP to 155mmHg for 40 min evoking ischemia (indicated by whitening of the eye fundus as blood flow is interrupted).
• the IOP was allowed to return to normal levels for 45 min (reperfusion period, during which the fundus color returns to normal).
• the left retina of each animal was subjected to the experimental condition, ischemia and/or reperfusion, while the right retina served as a nonischemic control.
• ERG electroretinogram
• ISCEV International Society for Clinical Electrophysiology
• ERG was performed 0 and 72 hours after the PnPP-19 administration (80 mg/eye).
• ERGs were recorded using an Espion e2 electrophysiology system and a Ganzfeld LED stimulator (ColorDomeTM desktop Ganzfeld, Diagnosys LLC, Littleton, MA). All ERGs were recorded after 12 h of dark adaptation.
• the pupils were dilated using one drop of 0.5% tropicamide (Mydriacyl; Alcon, Sao Paulo, Brazil) and the animals were anesthetized by intramuscular injection (ketamine hydrochloride 90 mg/kg and xylazine hydrochloride 10.0 mg/kg) before the recording of ERG.
• the eyes were topically anesthetized with 0.5% proxy metacaine hydrochloride (Anestalcon; Alcon, Sao Paulo, Brazil) immediately before the ERG recordings.
• Bipolar contact lenses electrode were placed on both corneas and a needle electrode was inserted into the back. Impedance was set to less than 5 kW at 25 Hz in each electrode.
• the dark-adapted (scotopic) ERG protocol was recorded according to a modified ISCEV protocol and presented in the following sequence: rod (0.01 cd.s/m2) and combined response (3 cd.s/m2) with 30s interstimulus interval (ISI), with a duration of 4 ms.
• the thicknesses of the entire retina (between the inner limiting membrane and the pigment epithelium), the inner nuclear layer (INL) and the outer nuclear layer (ONL) were measured.
• the measurements (400X) were made 0.5 mm dorsal and ventral from the optic disc.
• the number of cells in the ganglion cell layer (GCL) was calculated using the linear cell density (cells per 200 mm). For each eye, three measurements at adjacent locations in each hemisphere were made. The mean of three or more eyes was recorded as the representative value for each group.
• the GCL showed lower cell density, and increases in vacuolization, number of pyknotic nuclei (black arrows), and cellular disorganization; the INL also had fewer cells and had more pyknotic nuclei and cytoplasmic vacuoles; and there were also fewer cells in the ONL, in comparison with healthy and ischemic/PnPP-19 post-treatment retinas ( Figure 9A-C).
• the overall thickness of the retina of ischemic/ PnPP-19 post-treatment group was similar to the healthy group (174.66 ⁇ 19.66mm versus 175.31 ⁇ 14.22mm); on the other hand, in the ischemic/untreated group, it was reduced around 30% (121.08 ⁇ 21.38mm) compared to healthy and to ischemic/PnPP-19 treatment groups (p: ⁇ 0.001 for both comparisons).
• the thickness of the INL and ONL of ischemic/PnPP-19 treatment group was similar to the healthy group (INL - 31.82 ⁇ 3.14mm versus 31.16 ⁇ 3.80mm), (ONL - 47.39 ⁇ 1.93 mm versus 44.98 ⁇ 9.47 mm); on the other hand, in the ischemic/untreated group the thickness of the INL and ONL were reduced by 20% and 28%, respectively, compared to healthy group (p ⁇ 0.05 and p: ⁇ 0.001 , respectively) and 24% and 30% compared to ischemic/PnPP-19 treatment group (p: ⁇ 0.05 and p: ⁇ 0.001 , respectively).
• the GCL number was similar between ischemic/PnPP-19 treatment and healthy groups (31 .75 ⁇ 3.5 versus 30.0 ⁇ 5.5 cells per 200 mm) (Table 4).
• the GCL density was reduced by 35% and 31% in the ischemic/untreated group compared with ischemic/PnPP-19 treatment and healthy groups, respectively (p: ⁇ 0.001 for both) ( Figure 9). This results demonstrated that PnPP-19 reduces the histological damage caused by retinal ischemia and avoid the loss of RGC.
• Table 4 The thickness of the retinal layers and GCL cell count at 7 days after Isquemia and treatment (PnPP-19 post-treatment).
• the overall thickness of the retina in the ischemic/untreated group was reduced around 21% compared to the healthy group (140.68 ⁇ 13.37 versus 179.47 ⁇ 12.42 mm, p ⁇ 0.001); the ischemic/PnPP-19 pre-treatment group presented with 11% higher thickness compared to ischemic/untreated group (157.12 ⁇ 8.43 mm versus 140.68 ⁇ 13.37 mm, p ⁇ 0.05), and reduced around 17% compared to the healthy group, although not statistically significant (157.12 ⁇ 8.43 mm versus 179.47 ⁇ 12.42 mm) (table 5).
• the thickness of the INL of the ischemic/untreated group was reduced by 22% compared to the healthy group (24.83 ⁇ 4.08 versus 31.91 ⁇ 3.94 mm, p ⁇ 0.05).
• the thickness of the ONL although presented with a reduction of 10%, was not statistically different from the healthy group (39.77 ⁇ 7.09 versus 44.40 ⁇ 3.70 mm).
• the thickness of the INL and ONL of ischemic/PnPP-19 pre-treatment group was not statistically different neither from the healthy group nor from the ischemic/untreated group (table 3).
• Values are (means ⁇ SD), n33. Values compared between groups by oneway AN OVA with Dunnett’s post-test, a: compared with the healthy group; b: compared with the treat PnPP-19 group p ⁇ 0.05; aa or bb p ⁇ 0.001.
• Untreated high lOP-induced ischemia injury without treatment;
• Treatment PnPP-19 high lOP-induced ischemia injury with PnPP-19 treatment.
• NO level was indirectly determined by measuring the concentration of nitrite using the Griess methodology.
• the detection limit of the assay was ⁇ 1.5 mMin distilled water.
• the total amount of protein found in the eye tissue was estimated by NanoDrop 2000 Spectrophotometer (Thermo Scientific Madison, Wl) and the nitrite release was normalized per mg of protein.
• PnPP-19 stimulate an increase in nitrite production in healthy rat's eye tissues, compared to vehicle (48.70 +/- 1.19 vs 31.01 +/- 0.38 of nitrite nmol/mg of protein) ( Figure 13).
• the tolerability was analyed thought a daily tolerability questionnaire. Four events were reported: one patient had puritis in the eye, mild and of very short duration, soon after the PnPP-19 was instillated in just one day; Two patients related eye burning, mild and of very short duration, soon after the PnPP-19 (in one patient) and vehicle (in the other) was instillated in just one day. One patient reported mild headache, that last minutes. The investigator considered that none of those adverse events were related to PnPP-19.
• IOP was measured with a non-contact tonometer before and 1 ,2,4 6 and 24 hours after the instillations of PnPP-19 or vehicle.
• the IOP of the 3rd day of treatment was statistically lower than the IOP of the first day ( Figure 14).

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