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WO2011054993A1 - Peptides pour le traitement de l'hypertension oculaire et/ou du glaucome - Google Patents

Peptides pour le traitement de l'hypertension oculaire et/ou du glaucome Download PDF

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Publication number
WO2011054993A1
WO2011054993A1 PCT/ES2010/070710 ES2010070710W WO2011054993A1 WO 2011054993 A1 WO2011054993 A1 WO 2011054993A1 ES 2010070710 W ES2010070710 W ES 2010070710W WO 2011054993 A1 WO2011054993 A1 WO 2011054993A1
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WO
WIPO (PCT)
Prior art keywords
peptide
peptide compound
amino acid
ala
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2010/070710
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English (en)
Spanish (es)
Inventor
Miguel MORALES FUCIÑOS
Xavier Gasull Casanova
Ángel ACEBES VINDEL
Alberto FERRÚS GAMERO
Jesús PINTOR JUST
Francesc Rabanal Anglada
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Consejo Superior de Investigaciones Cientificas CSIC
Universidad Complutense de Madrid
Universitat de Barcelona UB
Fundacion Rioja Salud
Institut d'Investigacions Biomèdiques August Pi i Sunyer
Original Assignee
Consejo Superior de Investigaciones Cientificas CSIC
Universidad Complutense de Madrid
Universitat de Barcelona UB
Fundacion Rioja Salud
Institut d'Investigacions Biomèdiques August Pi i Sunyer
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Consejo Superior de Investigaciones Cientificas CSIC, Universidad Complutense de Madrid, Universitat de Barcelona UB, Fundacion Rioja Salud, Institut d'Investigacions Biomèdiques August Pi i Sunyer filed Critical Consejo Superior de Investigaciones Cientificas CSIC
Publication of WO2011054993A1 publication Critical patent/WO2011054993A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is generally framed in the field of medicine and specifically in the field of ophthalmology.
  • the invention relates to compounds for the treatment of glaucoma and / or ocular hypertension.
  • IOP intraocular pressure
  • glaucoma Although the causes of glaucoma are not fully known, its symptoms, in most cases, include an elevated IOP that can be caused by overproduction or by an inappropriate elimination of aqueous humor.
  • the elevation of the IOP is associated with clinical manifestations characteristic of glaucomatous optic neuropathy.
  • Optic nerve dysfunction could be the result of changes in pressure at the level of the structure of the optic nerve head and / or a low blood supply in the head of the optic nerve and in the retina.
  • the absence of treatment or inadequate treatment of glaucoma can lead to significant loss of vision or total blindness.
  • various drugs are useful for the treatment of glaucoma. These include: miotics (e.g.
  • pilocarpine pilocarpine, carbacol, and acetylcholinesterase inhibitors
  • sympathomimetics eg epinephrine, dipivalylepinephrine and apraclonidine
  • beta-blockers eg betaxolol, levobunolol and timolol
  • carbonic anhydrase inhibitors eg dorzolamide hydrochloride, acetazolamide,
  • sympathomimetics decrease IOP by increasing evacuation flow of aqueous humor
  • beta-blockers and carbonic anhydrase inhibitors decrease IOP by reducing the formation of aqueous humor.
  • the four types of drugs have potentially serious side effects. Miotics such as pilocarpine can cause blurred vision and other side effects in vision, which leads to a sick person's discomfort or abandonment of treatment.
  • Carbonic anhydrase inhibitors can also have important effects.
  • At least one beta-blocker timolol
  • timolol has frequently been associated with important pulmonary side effects that are attributed to its effect on beta-2 receptors present in lung tissue.
  • prostaglandin analogues have proven useful in the treatment of glaucoma topically. These compounds decrease IOP by increasing the flow of evacuation of aqueous humor through the uveoscleral route.
  • the inventors provide a new approach for the treatment of glaucoma and ocular hypertension that involves the use of peptide compounds, or their pharmaceutically acceptable salts, selected from the group consisting of: (i) a peptide with the amino acid sequence of formula (I ):
  • X it is none, one or more amino acid residues spacers, and pTyr is a phosphorylated tyrosine; (ii) a variant of the peptide of formula (I) that has substitution, elimination and / or addition of amino acid residues along the amino acid sequence, and / or that has another addition of 1 to 10 amino acid residues in the N and / or C terminal ends of the sequence; and (iii) a peptide derivative of formula (I) having chemical modifications in amino acid residues.
  • the peptide compounds have at least 80% sequence identity with the peptide of SEQ ID NO: 1, and have the ability to activate phosphatidylinositol-3-kinase
  • the peptide compounds of the invention show important differences with commercial drugs normally used in the treatment of ocular hypertension.
  • the peptide with sequence SEQ ID NO: 1 has greater effects than any of the commercial drugs tested: IOP reduction of 49% versus 35.3% obtained with timolol
  • the application of this peptide implies a maximum reduction of the IOP greater and of lasting effect.
  • the peptide compounds of the invention do not act by stimulating a receptor (unlike carbacol), or activating a tyrosine kinase receptor (as opposed to platelet-derived growth factor or PDGF), or antagonizing a receptor (a unlike timolol), nor inhibiting an enzyme (unlike dorzolamide).
  • the peptide compounds of the invention act through the activation of Pi3K, recruiting the Pi3K complex to the membrane and inducing its activation. This represents a new pharmacological target not previously described for the control of ocular hypertension.
  • the present invention relates to peptide compounds with the amino acid sequence of formula (I), as well as variants or derivatives of said peptide.
  • the amino acids can be substituted by other amino acids selected from natural amino acids or from unnatural / modified amino acids.
  • the amino acid substitution may be conservative (i.e. substituted by other residues with similar physicochemical properties) or non-conservative (i.e. substituted by other residues with different physicochemical properties) but without implying a substantial alteration of the primary structure.
  • the peptide may have another addition of between 1 and 10 amino acid residues added at the N and / or C terminal ends of the sequence.
  • substitutions, deletions or additions are selected so that no affect peptide activity.
  • Particularly suitable are those amidated, acetylated, sulfated, phenylated, alkylated phosphorylated, glycosed, oxidized or modified with polyethylene glycol.
  • the modifications are selected so as not to affect the activity of the peptide.
  • Preferred modifications for the peptide of formula (I) are the amidation of the C-terminal end to prevent degradation thereof; the replacement of aspartic acid and glycine (positions 14 and 15 of formula (I)) to avoid spontaneous hydrolysis; and the substitution of methionine (formula (II)) with norleucine (formula (III)) to avoid spontaneous oxidation of methionine.
  • the peptide compound has one or both Met substituted by norleucine (Nle).
  • Derivatives and variants of the peptide of formula (I) have at least 80% identity in sequence with the peptide of SEQ ID NO: 1, and have the ability to activate Pi3K. This activity is preferably established in an assay where phosphorylation levels of Akt are measured. Akt or PKB (protein kinase B) is an effector of Pi3K found further down in the signaling cascade. In the description of particular embodiments a suitable test is described. The basal levels of phosphorylated Akt (p-Akt) are very low, so the activation of Akt and, consequently, the Pi3K activation means an increase of this basal level. The activation percentage was calculated as the p-Akt level after treatment. compared to the level of p-Akt under control conditions.
  • pTyr (formula (IV)) has been replaced by a birradical selected from the group consisting of 4-sulfophenylalanine (formula (V)), 4-carboxy-L-phenylalanine (formula (VI)) , N- (oxalyl) -4-aminophenylalanine (formula (VII)), N- (sulfo) -4-aminophenylalanine (formula (VIII)), 4- (sulfomethyl) -phenylalanine (formula (IX)), N- ( 2-sulfoethyl) -L- asparagine (formula (X)), N- (sulfomethyl) -L-asparagine, 4- (phosphon
  • the formulas indicate the anionic form of the amino acid residue.
  • the peptide compound has the amino acid sequence of formula (I) where (X) is none, one or more spacer amino acid residues, and pTyr is a phosphorylated tyrosine.
  • (X) is a glycine residue. More particularly, the peptide compound has the sequence SEQ ID NO: 1.
  • the peptide compound has an amino acid sequence selected from the group consisting of SEQ ID NO: 2-22.
  • the peptide compounds of the invention can be synthesized by any method analogous to those detailed herein, including, but not limited to, solid phase synthesis, liquid phase synthesis, protein expression by transformed cells, excision of a synthetic or semi-synthetic polypeptide, or a combination of these methods.
  • the amino acids that make up the sequence may have an L or D configuration. Those with a D configuration are more expensive, but more resistant to degradation by proteases.
  • Peptide compounds according to the present invention in the form of pharmaceutically and / or biologically acceptable salts such as sodium, potassium, calcium, magnesium salt or acid addition salts, can also be obtained by methods analogous to those described herein.
  • these latter salts include salts of inorganic acids (eg hydrochloric acid, sulfuric acid and phosphoric acid) and organic acids (eg acetic acid, propionic acid, citric acid, tartaric acid, malic acid and acid
  • Another aspect of the invention relates to the peptide compounds of the invention for use in medicine.
  • Another aspect of the invention relates to the use of the peptide compounds of the invention for the manufacture of a medicament for reducing intraocular pressure. They can also be used for the manufacture of a medicament for the treatment and / or prevention of ocular hypertension and / or glaucoma.
  • This aspect can also be expressed as referring to a method for the treatment and / or prevention of glaucoma and / or ocular hypertension in a mammal, including a human, where an effective amount of the peptide compound of the invention is administered to the affected eye. of said mammal or human.
  • the peptide compound of the invention could additionally be combined or linked to other sequences such as histidine sequences to allow purification of the peptide, or to antigenic sequences such as hematoglutinin, T7 or myc, to recognize the peptide compound.
  • compositions comprising a therapeutically effective amount of the peptide compound of the invention, together with sufficient amounts of excipients.
  • the excipients Pharmaceutically acceptable are appropriate for topical ophthalmic administration, that is, suitable for use in contact with eye tissues without inducing toxicity, irritation, incompatibility, instability, allergic response or the like.
  • the amount of peptide compound of the invention to be administered can be determined without too much experimentation by a person skilled in the art. Due to the direct transduction of the peptide compound after its corneal application, the effective amount necessary to have a therapeutic effect is less than with other antiglaucomatous drugs. In general, amounts between 0.1 and 1 ⁇ g ml, and preferably 0.5 ⁇ g ml, are used for topical administration.
  • compositions can be prepared in different dosage forms suitable for topical ophthalmic application, including solutions, suspensions, emulsions, gels, creams, ointments and sprays.
  • the peptide compound may be administered through other forms, such as direct injection into the anterior chamber of the eye, or by any type of slow-release system.
  • compositions of the present invention may comprise
  • compositions of the invention may comprise various ingredients as preservatives.
  • compositions may comprise the peptide compound as a single agent for the treatment of ocular hypertension and glaucoma, or combinations of several of these compounds, or combinations with other therapeutic agents.
  • FIG. 1 shows the effect on IOP after a single application of a 10 ⁇ drop of the peptide of SEQ ID NO: 1, at a concentration of 0.5 Mg / ⁇ .
  • t (h) means time in hours
  • pep means peptide
  • with means saline control.
  • FIG. 1 refers to the "Single application” section of the description of particular embodiments.
  • FIG. 2 shows the effect on IOP after applying 10 ng of platelet-derived growth factor (PDGF) in a final volume of 10 ⁇ .
  • IOP is represented as% of the control, "t (h)” means time in hours; “go” means vehicle.
  • FIG. 2 refers to the "Comparison of the Peptide with PDGF” section of the description of particular embodiments.
  • FIG. 3 shows a graph of IOP levels during a five-day treatment with the peptide of SEQ ID NO: 1.
  • the arrows indicate the time of the different applications, "t (h)” means time in hours; “pep” means peptide.
  • FIG. 3 refers to the "Chronic application” section of the
  • FIG. 4 shows the increase in p-Akt levels induced by the peptide of SEQ ID NO: 1.
  • the ability of the peptide to activate the Pi3K-Akt-GSK3 signaling pathway was quantified by measuring p-Akt levels in the Ser-473 position using a SH-SY5Y human glioma cell line. The cells were fasted in serum for 16 hours before the peptide was added. Quantification shows that p-Akt levels increased to a maximum after 60-120 minutes and remained constant during the time interval examined (6 hours), "t (min)" means time in minutes.
  • FIG. 4 refers to the "Quantification of Pi3K activity by Western Blot" section of the description of particular embodiments.
  • FIG. 4 refers to the "Quantification of Pi3K activity by Western Blot" section of the description of particular embodiments.
  • IOP network in mm Hg
  • the peptide with sequence: Tyr-Ala-Arg-Ala-Ala-Ala-Arg-GIn-Ala-Arg-Ala Gly-Ser-Asp-Gly-Gly-pTyr-Met-Asp-Met-Ser (SEQ ID NO: 1) will be called next "The Peptide". It was obtained by solid phase peptide synthesis. The Peptide was formulated in isotonic saline solution and used at a
  • the Peptide was applied unilaterally to the cornea at a fixed volume of 10 ⁇ .
  • the contralateral eye received the same volume of control solution (0.9% NaCI, vehicle). Since tonometry can cause some discomfort in rabbits, the corneas were anesthetized by applying 10 ⁇ of a 1: 10 (v: v) solution of oxybuprocaine / tetracaine (4 mg and 1 mg respectively, from Alcon Cus ⁇ , Barcelona, Spain) .
  • the ⁇ was measured once every 30 minutes during the first hour and then once every hour.
  • the instillation time was considered time 0.
  • the Peptide mimics the intracellular domain of the activated form of the PDGF receptor, theoretically, it should produce results similar to those of the PDGF application. For this purpose, changes in IOP levels were studied after a 10 ng corneal application of PDGF (in a final volume of 10 ⁇ ).
  • the application of PDGF only decreased IOP levels up to 20%, and the reduction only lasted 3-4 hours.
  • the peptide of the invention has a better range of effects, reduction of the maximum IOP and more lasting effects than those obtained after the application of PDGF. Chronic application
  • the Peptide's ability to activate Pi3K was monitored by measuring phosphorylation levels of the Akt protein, an effector of Pi3K.
  • Cultures of the human glioma cell line (SH-SY5Y) were treated in serum fasting, with a concentration of 50 g / ml of the Peptide.
  • P-Akt levels were quantified at 10, 30, 120, 180 and 360 minutes after application of the peptide.
  • the cells were grown in 12 mm diameter plates and washed twice with PBS (4 ° C). They were scraped and lysed with 300 ⁇ of lysis buffer solution containing: 62.5 mM Tris HCI, pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT and 0.01% bromophenol.
  • the objective of the study was to evaluate the reduction of IOP in an animal model of induced glaucoma.
  • the selected model was the cauterization of three episcleral veins that causes an extension of cell death equivalent to human glaucoma.
  • dexamethasone No surgery was applied to the right eyes (RE), which were used as a control.
  • IOP measurements were performed with a Tonopen ® XL contact tonometer at baseline (pretreatment) and at the times indicated after instillation of the compound.
  • the rats were kept awake (not anesthetized) and a single drop of local anesthetic was applied to the eye before measurement.
  • the person responsible for taking the measures was always the same person to avoid possible modifications in the measurement methodology and to avoid the stress of the rat.
  • the measurements were always taken at the same time of day. The first measure began at 9 am and the second after treatment began at 1 pm. The measurements were taken in the same order of the rats to keep the times between measurements.
  • Treatment methodology the LOPs of LE and RE were measured at 9 a.m. Immediately afterwards, a 10 ⁇ drop of saline solution was instilled in the ER and 10 ⁇ of the Peptide in the LE: Four hours after instillation, all rats for both eyes were measured in the same order. The next day, the LOPs were measured at 9 a.m. before applying the new drop to the rat. These measures, 24 hours after the first drops, serve to know if the effect of the treatment is maintained in the long term (24 hours). The treatment was applied for five consecutive days.
  • FIG. 5 shows that the application of the Peptide induced a significant reduction in IOP 4 hours after the instillation of the Peptide. The reduction in the level of IOP remained significantly below the control value during all the days studied.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Ophthalmology & Optometry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des composés peptidiques ayant la séquence aminoacide représentée par la formule (I): Tyr-Ala-Arg-Ala-Ala-Ala-Arg-Gln-Ala-Arg-Ala-(X)-Ser-Asp-Gly-Gly-pTyr-Met- Asp-Met-Ser (I), dans cette formule, (X) représente zéro, un ou plusieurs restes d'aminoacide espaceurs, et pTyr représente une tyrosine phosphorylée, et des variants et des dérivés de ceux-ci; ces composés sont utilisés en médecine pour réduire la pression intraoculaire. Les peptides ou les compositions pharmaceutiques les contenant sont utilisés pour traiter et/ou prévenir l'hypertension oculaire et/ou le glaucome.
PCT/ES2010/070710 2009-11-04 2010-11-04 Peptides pour le traitement de l'hypertension oculaire et/ou du glaucome Ceased WO2011054993A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200902164 2009-11-04
ESP200902164 2009-11-04

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WO2011054993A1 true WO2011054993A1 (fr) 2011-05-12

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008119850A1 (fr) * 2007-03-29 2008-10-09 Universidad De Barcelona Préparation topique pour le traitement de l'hypertension oculaire et du glaucome

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008119850A1 (fr) * 2007-03-29 2008-10-09 Universidad De Barcelona Préparation topique pour le traitement de l'hypertension oculaire et du glaucome

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANDRIEU-SOLER C. ET AL.: "Ocular gene therapy: A review of nonviral strategies.", MOLECULAR VISION., vol. 12, 30 October 2006 (2006-10-30), pages 1334 - 1347 *
HUANG Y. ET AL.: "Differential roles of phosphatidylinositol 3-kinase/akt pathway in retinal ganglion cell survival in rats with or without acute ocular hypertension.", NEUROSCIENCE., vol. 153, 2008, pages 214 - 225 *
MORALES M. ET AL.: "Hypotensive effect of profilin on rabbit intraocular pressure.", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 567, 22 April 2007 (2007-04-22), pages 145 - 148 *
SAKAI H. ET AL.: "Transduction of TAT fusion proteins into the human and bovine trabecular meshwork.", INVESTIGATIVE OPHTHALMOLOGY AND VISUAL SCIENCE., vol. 47, no. 10, October 2006 (2006-10-01), pages 4427 - 4434 *

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