WO2022183360A1 - Peptides synthétiques courts et leurs utilisations pour le traitement de la sécheresse oculaire - Google Patents

Peptides synthétiques courts et leurs utilisations pour le traitement de la sécheresse oculaire Download PDF

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WO2022183360A1
WO2022183360A1 PCT/CN2021/078641 CN2021078641W WO2022183360A1 WO 2022183360 A1 WO2022183360 A1 WO 2022183360A1 CN 2021078641 W CN2021078641 W CN 2021078641W WO 2022183360 A1 WO2022183360 A1 WO 2022183360A1
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synthetic peptide
amino acid
ded
peptide
vtn
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PCT/CN2021/078641
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English (en)
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Yeou-Ping Tsao
Tsung-Chuan Ho
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Tsao Yeou Ping
Ho Tsung Chuan
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Priority to PCT/CN2021/078641 priority Critical patent/WO2022183360A1/fr
Publication of WO2022183360A1 publication Critical patent/WO2022183360A1/fr

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    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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 disclosure relates to the discovery of short synthetic peptides having antagonistic activity toward ⁇ v integrins, thus are useful in the treatment or prophylaxis of dry eye disease (DED) .
  • DED dry eye disease
  • Dry eye disease is a multifactorial disorder of the ocular surface characterized by symptoms of discomfort, visual disturbance, decreased tear quality, and chronic inflammation. Reduced tear secretion and/or increased evaporation of tear lead to tear film instability and tear hyperosmolarity that are associated with ocular surface inflammation and corneal epithelial injury in DED.
  • steroid anti-inflammation agents are commonly prescribed by most eye doctors. Although they are effective but the risk of causing glaucoma and cataract cannot be ignored.
  • Topical cyclosporine takes several months before its effect kick in and shows effectiveness in only 15%of the patients. Accordingly, there exist in the related art an unmet medical need for DED therapy.
  • Integrins are transmembrane receptors and present as ⁇ heterodimers to execute extensive functions by interacting with a range of extracellular ligands, such as vitronectin (VTN) .
  • VTN vitronectin
  • Pierschbacher and Ruoslahti reported that the Arg-Gly-Asp (RGD) is a critical sequence served as cell attachment domain within fibronectin. Subsequently, the RGD sequence was found within other ECM proteins including VTN, von Willebrand factor, osteopontin, and laminin to promote cell attachment and migration.
  • integrins including ⁇ v-RGD integrin ( ⁇ v ⁇ 1, ⁇ v ⁇ 3, ⁇ v ⁇ 5, ⁇ v ⁇ 6, ⁇ v ⁇ 8) , ⁇ 5 ⁇ 1, ⁇ 8 ⁇ 1, and the platelet receptor ⁇ IIb ⁇ 3 are identified as cell surface receptor recognizing the small RGD tripeptide. Integrins have been found that are involved in a variety of pathogenic mechanisms. For examples, integrin ⁇ v ⁇ 3 is up-regulated in the angiogenic vessels for regulation of endothelial cell survival, proliferation and vessel permeability. In addition, ⁇ v integrin was studied in various disease models.
  • ⁇ v ⁇ 3 integrin has been shown to be highly expressed in activated macrophages in the atherosclerotic plaque, however, its expression in quiescent macrophages in normal aorta is minimal.
  • depletion of ⁇ v integrin in hepatic stellate cells (HSCs) may protect mice from hepatic fibrosis, whereas individual depletion of ⁇ subunit has no such effect.
  • Inventors of the present disclosure unexpectedly identified that ⁇ v integrins are involved in the macrophages associated inflammation and pathogenesis of DED, thus have designed and synthesized RGD-base peptides that serve as antagonists of ⁇ v integrins. Accordingly, these novel synthesized RGD-base peptides of the present disclosure are potential candidates for the development of a medicament for the treatment of DED.
  • the present disclosure relates to novel synthetic peptides, pharmaceutical compositions comprising the same, and/or methods for treating dry eyes disease (DED) by use of these novel synthetic peptides.
  • DED dry eyes disease
  • the first aspect of the present disclosure aims at providing a short synthetic peptide capable of antagonizing ⁇ v integrins.
  • the synthetic peptide has the amino acid sequence of formula (I) ,
  • R is arginine
  • G is glycine
  • D is aspartic acid
  • X aa is a D-form amino acid selected from the group consisting of isoleucine (I) , and phenylalanine (F) .
  • the synthetic peptide of formula (I) may be in linear or head-to-tail cyclic form.
  • the synthetic peptide of formula (I) is in linear form, and the N-terminus of the amino acid sequence of the synthetic peptide of formula (I) is acetylated while the C-terminus is amidated.
  • the synthetic peptide of formula (I) has an amino acid sequence at least 80%identical to SEQ ID NO: 10.
  • the synthetic peptide of formula (I) is in head-to-tail cyclic form, and has an amino acid sequence at least 85%identical to SEQ ID NO: 16 or 18.
  • the second aspect of the present disclosure aims at providing a medicament and/or a pharmaceutic composition suitable for treating DED.
  • the medicament or pharmaceutic composition comprises, the synthetic peptide described above, and a pharmaceutically acceptable carrier.
  • the synthetic peptide is in linear form and has an amino acid sequence at least 85%identical to SEQ ID NO: 10.
  • the synthetic peptide is in cyclic form and has an amino acid sequence at least 80%identical to SEQ ID NO: 16 or 18.
  • the synthetic peptide may be encapsulated or embedded in a delivery vehicle before being formulated into a medicament and/or a pharmaceutically composition.
  • the delivery vehicle may be a liposome, a lysosome, a microcapsule, or a nanoparticle.
  • the medicament and/or pharmaceutically composition is in a form of liquid, gel, cream, or ointment.
  • the third aspect of the present disclosure is thus directed to a method of treating a subject suffering from DED.
  • the method comprises the step of, administering to the subject an effective amount of the medicament or pharmaceutical composition of the present disclosure described above to ameliorate or alleviate symptoms associated with DED.
  • the medicament or pharmaceutical composition of the present disclosure may be administered to the subject via oral, enteral, rectal, pulmonary (e.g., inhalation) , nasal, topical (including transdermal, buccal and sublingual) , intravesical, intravitreal, subconjunctival, intraperitoneal, vaginal, brain delivery (e.g., intracerebroventricular, and intracerebral) , CNS delivery (e.g., intrathccal, perispinal, and intra-spinal) or parenteral (e.g., subcutaneous, intramuscular, intravenous, and intradermal) , transmucosal administration or administration via an implant, or other delivery routes known in the art.
  • pulmonary e.g., inhalation
  • nasal including transdermal, buccal and sublingual
  • brain delivery e.g., intracerebroventricular, and intracerebral
  • the medicament or pharmaceutical composition of the present disclosure may be administered in the amount of 0.001-1,500 mg/Kg to the subject; preferably, in the amount of 0.1-1,200 mg/Kg; more preferably, in the amount of 15-500 mg/Kg.
  • the subject is a human.
  • FIG 1 Vitronectin (VTN) is overexpressed in the tear and conjunctival stroma in DED mice.
  • A Tear fluid and serum concentrations of VTN in DED mice were assessed using ELISA. Data (mean ⁇ SE) are obtained from 10 mice from day 0 through 3 weeks. *P ⁇ 0.001 versus day 0.
  • FIG 2 Expression of ⁇ v integrin in macrophages is induced in dry eye.
  • A Immunofluorescence staining of ⁇ v integrin (red) and F4/80 (green; a macrophage marker) in conjunctiva at day 12 after DED induction. Insets are the images before superimposition. Nuclear was counterstained by Hoechst 33258 (blue) . Star indicates conjunctival epithelium. Representative images are from six sections per mouse eye, with six mice per group. NS group: mice housed at non-stress environment.
  • FIG 3 The effect of RGDVF–based peptides on the VTN-induced TNF- ⁇ mRNA expression in THP-1 macrophages. 5 ⁇ 10 5 THP-1 monocytic cells were differentiated into macrophages by addition of 10 ng/ml PMA in complete medium for 2 days and then cells were serum-starved for additional 16 h.
  • THP-1 macrophages were treated with (A) 20 ⁇ M RGDVF–based peptide (composed of amino acids in the L-configuration) and (B) RGDVF–based peptide with one D-amino acid (lowercase letters) in fresh serum-free medium for 10 min, and then the VTN (10 ⁇ g/mL) was added to incubate for further 3 hours.
  • VTN 10 ⁇ g/mL
  • Real-time qPCR analysis was conducted to determine the TNF- ⁇ mRNA levels. Gapdh was used as a loading control. Data are representative of three independent experiments. *p ⁇ 0.00007 versus solvent-treated cells. a P ⁇ 0.03, b P ⁇ 0.03, and c P ⁇ 0.005 versus solvent/VTN-treated cells.
  • FIG 4 The respective effects of the present synthetic peptides and known ⁇ v ⁇ 3 integrin antagonists on VTN-induced TNF- ⁇ mRNA expression in THP-1 macrophages.
  • 5 ⁇ 10 5 THP-1 macrophages were serum-starved for 16 h and then treated with VTN (10 ⁇ g/mL) and 20 ⁇ M (A) or 5 ⁇ 20 ⁇ M (B) integrin inhibitor as indications in fresh serum-free medium for another 3 h.
  • Real-time qPCR analysis was conducted to determine the TNF- ⁇ mRNA levels. Gapdh was used as a loading control. The results were presented as the percent inhibition of VTN/solvent control (100%) .
  • Each bar represents mean ⁇ SE of three independent experiments. *p ⁇ 0.05 versus solvent/VTN-treated cells.
  • FIG 5 The present synthetic peptides may ameliorate corneal epithelial damage of dry eye.
  • A Schedule of dry eye mouse model: To induce DED, C57BL/6 mice were housed at a low humidity CEC and subcutaneous injection of scopolamine at day 3 and day 5. Subsequently, mice were topically treated with vehicle (BSS) , c (RGDfD) and c (RGDiD) eye drop at day 8 ⁇ 12 and day 15 ⁇ 19. The severity of corneal epithelial damage is examined by corneal fluorescein staining before (day 8) and after treatment (day 12 and 19) .
  • B Representative images of corneal fluorescein staining score.
  • FIG 6 The therapeutic effect ⁇ v ⁇ 3 integrin antagonists on corneal epithelial damage in experimental dry eye.
  • Error bars represent the SE.
  • FIG 7 PAS staining of the goblet cells in the conjunctival fornices of DED mice at day 19.
  • A The representative photographs of goblet cells (pink) show dry eyes treated with vehicle, c (RGDfD) , and c (RGDiD) eye drop.
  • FIG 8 Real-time PCR analysis evaluates the transcriptional level of TNF- ⁇ , IL-1 ⁇ , IL-6 and MMP-9 in conjunctiva of dry eye. Schedule of induction and treatment of dry eye in mice is same as FIG 5. Data are presented as mean ⁇ SE. P value versus vehicle-treated group as indications.
  • peptide denotes a polymer of amino acid residues.
  • synthetic peptide as used herein, it is meant a peptide which does not comprise an entire naturally occurring protein molecule.
  • the peptide is “synthetic” in that it may be produced by human intervention using such techniques as chemical synthesis, recombinant genetic techniques, or fragmentation of whole antigen or the like.
  • positions of any specified amino acid residues within a peptide are numbered starting from the N terminus of the peptide.
  • amino acids are not designated as either D-or L-amino acids, the amino acid is either an L-amino acid or could be either a D-or L-amino acid, unless the context requires a particular isomer. Further, the notation used herein for the polypeptide amino acid residues are those abbreviations commonly used in the art.
  • minor variations in the amino acid sequences of peptides are contemplated as being encompassed by the presently disclosed and claimed inventive concept (s) , providing that the variations in the amino acid sequence maintain at least 70%identity, such as at least 70%, 71%, 72%, 73%, 75%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%and 100%identity.
  • 70%identity such as at least 70%, 71%, 72%, 73%, 75%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%and 100%identity.
  • Percentage (%) amino acid sequence identity with respect to the synthetic peptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • sequence comparison between two amino acid sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by National Center for Biotechnology Information (NCBI) .
  • NCBI National Center for Biotechnology Information
  • the percentage amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B is calculated by the formula as follows:
  • X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of amino acid residues in A or B, whichever is shorter.
  • the present synthetic peptide may be modified specifically to alter a feature of the peptide unrelated to its physiological activity. For example, certain amino acids can be changed and/or deleted without affecting the physiological activity of the peptide in this study (i.e., its ability to antagonize ⁇ v integrins) . In particular, conservative amino acid replacements are contemplated. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the peptide derivative. Fragments or analogs of peptides can be readily prepared by those of ordinary skill in the art. Preferred amino-and carboxy-termini of fragments or analogs occur near boundaries of functional domains.
  • one amino acid residue of the present synthetic peptide is conservatively replaced by its D-form amino acid residue, for example, L-form phenylalanine (F) and L-form isoleucine (I) are respectively replaced by the corresponding D-form residues.
  • L-form phenylalanine (F) and L-form isoleucine (I) are respectively replaced by the corresponding D-form residues.
  • treatment as used herein are intended to mean obtaining a desired pharmacological and/or physiologic effect, e.g., antagonistic effect against ⁇ v integrin.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment includes preventative (e.g., prophylactic) , curative or palliative treatment of a disease in a mammal, particularly human; and includes: (1) preventative (e.g., prophylactic) , curative or palliative treatment of a disease or condition (e.g., retinal degeneration or tissue injury) from occurring in an individual who may be pre-disposed to the disease but has not yet been diagnosed as having it; (2) inhibiting a disease (e.g., by arresting its development) ; or (3) relieving a disease (e.g., reducing symptoms associated with the disease) .
  • preventative e.g., prophylactic
  • a disease or condition e.g., retinal degeneration or tissue injury
  • administering are used interchangeably herein to refer a mode of delivery, including, without limitation, intraveneously, intramuscularly, intraperitoneally, intraarterially, intracranially, or subcutaneously administering an agent (e.g., a compound or a composition) of the present invention.
  • agent e.g., a compound or a composition
  • the synthetic peptide of the present disclosure and/or its analogues are formulated into eye drops for direct application on the corneal surface.
  • the synthetic peptide of the present disclosure are formulated into skin ointments or lotions for direct application on the skin.
  • the synthetic peptide of the present disclosure are formulated into powders for mixed with suitable carrier (e.g., buffer solution) before use, such as intraveneous injection.
  • an effective amount refers to an amount effective, at dosages, and for periods of time necessary, to achieve the desired result with respect to the treatment of a disease.
  • an agent i.e., a compound, a synthetic peptide, or a nucleic acid encoding the present therapeutic peptide
  • An effective amount of an agent is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered or prevented, or the disease or condition symptoms are ameliorated.
  • the effective amount may be divided into one, two or more doses in a suitable form to be administered at one, two or more times throughout a designated time period.
  • subject or “patient” is used interchangeably herein and is intended to mean a mammal including the human species that is treatable by the synthetic peptide and/or method of the present invention.
  • mammal refers to all members of the class Mammalia, including humans, primates, domestic and farm animals, such as rabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals; and rodents, such as mouse and rat.
  • subject or “patient” intended to refer to both the male and female gender unless one gender is specifically indicated. Accordingly, the term “subject” or “patient” comprises any mammal which may benefit from the treatment method of the present disclosure.
  • Examples of a “subject” or “patient” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl.
  • the patient is a human.
  • the present disclosure is based, at least in part, on the discovery of short synthetic peptides that are capable of antagonizing ⁇ v integrins. Accordingly, this invention provides method and composition comprising the newly identified synthetic peptides for the treatment and/or prophylaxis of DED.
  • the short synthetic peptide of the present disclosure consists of the amino acid sequence set forth as formula (I) ,
  • R is arginine
  • G is glycine
  • D is aspartic acid
  • X aa is a D-form amino acid selected from the group consisting of isoleucine (I) , and phenylalanine (F) .
  • the synthetic peptide of formula (I) may be in linear or head-to-tail cyclic form.
  • the synthetic peptide of formula (I) is in linear form, and the N-terminus of the amino acid sequence of the synthetic peptide of formula (I) is acetylated while the C-terminus is amidated.
  • the synthetic peptide of formula (I) has the amino acid sequence of SEQ ID NO: 10.
  • the synthetic peptide of formula (I) is in head-to-tail cyclic form, and has the amino acid sequence of SEQ ID NO: 16 or 18.
  • the fourth amino acid residue of the synthetic peptide of formula (I) must be in D-form, or else the synthetic peptide will lose its antagonistic activity against ⁇ v integrin.
  • the present synthetic peptide may be synthesized in accordance with any standard peptide synthesis protocol in the art.
  • the linear peptide may be readily synthesized by conventional procedures for the formation of a peptide linkage between amino acids.
  • Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acid residue having its carboxyl group and other reactive groups protected and the free primary carboxyl group of another amino acid residue having its amino group or other reactive groups protected.
  • the peptides of the present disclosure may be synthesized by solid-phase synthesis and purified according to methods known in the art.
  • any of a number of well-known procedures utilizing a variety of resins and reagents may be used to prepare the peptides of the present disclosure.
  • the process for synthesizing the peptides may be carried out by a procedure whereby each amino acid residue in the desired sequence is added one at a time in succession to another amino acid residue or by a procedure whereby peptide fragments with the desired amino acid sequence are first synthesized conventionally and then condensed to provide the desired peptide.
  • the present peptides may be synthesized by use of a solid-phase peptide synthesizer (ABI433A peptide synthesizer, Applied Biosystems Inc., Life Technologies Corp., Foster City, CA, USA) in accordance with the manufacturer’s protocols.
  • a solid-phase peptide synthesizer ABSI433A peptide synthesizer, Applied Biosystems Inc., Life Technologies Corp., Foster City, CA, USA
  • the synthesis of peptides can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods.
  • cyclic peptide it may be first synthesized by procedures described above, the resulting peptide is then cyclized to yield a cyclic peptide of the present disclosure. Any method of cyclization may be employed. In some embodiments, the peptide can be cyclized prior to cleavage from the peptide resin. For cyclization in the presence of reactive side chain moieties, the side chains are protected, and the peptide suspended in a suitable solvent and a cyclic coupling agent added. Suitable solvents include, for example dimethyl formaldehyde (DMF) , dichloromethane (DCM) or 1-methyl-2-pyrrolidone (NMP) .
  • DMF dimethyl formaldehyde
  • DCM dichloromethane
  • NMP 1-methyl-2-pyrrolidone
  • Suitable cyclic coupling reagents include, for example, 2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate (TBTU) , 2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate (HBTU) , benzotriazole-1-yl-oxy-tris (dimethylamino) phosphoniumhexafiuorophosphate (BOP) , benzotriazole-1-yl-oxy-tris (pyrrolidino) phosphoniumhexafluorophosphate (PyBOP) , 2- (7-aza-1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyturonium tetratluoroborate (TATU) , 2- (2-oxo-1 (2H) -pyridyl) -1, 1, 3, 3-
  • Coupling is conventionally initiated by use of a suitable base, such as N, N-diisopropylethylatnine (DIPEA) , 2, 4, 6-trimethyl pyridine or N-methylmorpholine (NMM) .
  • DIPEA N, N-diisopropylethylatnine
  • NMM N-methylmorpholine
  • the cyclized peptides can then be cleaved from solid phase, using any suitable reagent, such as ethylamine in DCM or various combinations of agents, such as trifluoroacetic acid (TFA) , tri-isopropylsilane (TIS) , dimethoxybenezene (DMB) , water and the like.
  • TFA trifluoroacetic acid
  • TIS tri-isopropylsilane
  • DMB dimethoxybenezene
  • the resulting crude peptide is dried and remaining amino acid side chain protecting groups, if any, are cleaved using any suitable reagent, such as TFA in the presence of water, TIS, 2-mercaptopethane (ME) , and/or 1, 2-ethanedithiol (EDT) .
  • the final product may be precipitated by adding cold ether and collected by filtration.
  • Final purification is by reverse phase high performance liquid chromatography (RP-HPLC) , using a suitable column, such as a C18 column, or other methods of separation or purification, such as methods based on the size or charge of the peptide, may also be employed.
  • RP-HPLC reverse phase high performance liquid chromatography
  • the peptide can be characterized by any number of methods, such as high performance liquid chromatography (HPLC) , amino acid analysis, mass spectrometry, and the like.
  • HPLC high performance liquid chromatography
  • amino acid analysis amino acid analysis
  • mass spectrometry mass spectrometry
  • the present linear synthetic peptide may be modified at its N-terminus or C-terminus.
  • N-terminal modifications include, but are not limited to, N-glycated, N-alkylated, and N-acetylated amino acid.
  • a terminal modification can also include a pegylation.
  • An example of C-terminal modification is a C-terminal amidated amino acid.
  • one or more peptide bond may be replaced by a non-peptidyl linkage, the individual amino acid moieties may be modified through treatment with agents capable of reacting with selected side chains or terminal residues.
  • Various functional groups may also be added at various points of the synthetic peptide that are susceptible to chemical modification. Functional groups may be added to the termini of the peptide. In some embodiments, the function groups improve the activity of the peptide with regard to one or more characteristics, such as improving the stability, efficacy, or selectivity of the synthetic peptide; improving the penetration of the synthetic peptide across cellular membranes and/or tissue barrier; improving tissue localization; reducing toxicity or clearance; and improving resistance to expulsion by cellular pump and the like.
  • Non-limited examples of suitable functional groups are those that facilitate transport of a peptide attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the peptide, these functional groups may optionally and preferably be cleaved in vivo, either by hydrolysis or enzymatically, inside the cell.
  • Hydroxy protecting groups include esters, carbonates and carbamate protecting groups.
  • Amine protecting groups include alkoxy and aryloxy carbonyl groups.
  • Carboxylic acid protecting groups include aliphatic, benzylic and aryl esters.
  • a “peptidomimetic organic moiety” can optionally be substituted for amino acid residues in the present synthetic peptide both as conservative and as non-conservative substitutions.
  • the peptidomimetic organic moieties optionally and preferably have steric, electronic or configuration properties similar to the replaced amino acid and such peptidomimetics are used to replace amino acids in the essential positions, and are considered conservative substitutions.
  • Peptidomimetics may optionally be used to inhibit degradation of peptides by enzymatic or other degradative processes.
  • the peptidomimetics can optionally and preferably be produced by organic synthetic techniques.
  • Non-limiting examples of suitable petidomimetics include isosteres of amide bonds, 3-amino-2-propenidone-6-carboxylic acid, hydroxyl-1, 2, 3, 4-tetrahydro-isoquinoline-3-carboxylate, 1, 2, 3, 4-tetrahydro-isoquinoline-3-carboxylate, and histidine isoquinolone carboxylic acid.
  • any part of the synthetic peptide may optionally be chemically modified, such as by the addition of functional groups.
  • the modification may optionally be performed during the synthesis of the present peptide.
  • Non-limiting exemplary types of the modification include carboxymethylation, acylation, phosphorylation, glycosylation or fatty acylation.
  • Ether bonds can optionally be used to join the serine or threonine hydroxyl to the hydroxyl of a sugar.
  • Amide bonds can optionally be used to join the glutamate or aspartate carboxy groups to an amino group of a sugar.
  • Acetal and ketal bonds can also optionally be formed between amino acids and carbon hydrates.
  • the present synthetic peptides are suitable for treating a subject suffering from DED. Accordingly, a further aspect of the present disclosure is to provide a medicament and/or pharmaceutical composition comprising the present synthetic peptide for treating DED.
  • the medicament and/or pharmaceutical composition is manufactured by mixing suitable amount of the present synthetic peptide with a pharmaceutically acceptable carrier, excipient or stabilizer into a composition.
  • the synthetic peptide is selected from the group of peptides as described above, which include but are not limited to, RGDfD (SEQ ID NO: 10) , c (RGDiD) (SEQ ID NO: 16) , c (RGDfD) (SEQ ID NO: 18) , and a combination thereof.
  • the amount of the peptide present in the medicament or the pharmaceutical composition will depend on the peptide used.
  • the peptide typically will be present in the pharmaceutical composition in the amount from about 0.001%to about 10%by weight, such as 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2., 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,
  • the synthetic peptide may be encapsulated or embedded in a delivery vehicle before being formulated into a medicament and/or a pharmaceutically composition.
  • the delivery vehicle may be a liposome, a lysosome, a microcapsule, or a nanoparticle.
  • compositions for use with the synthetic peptides are well known in the relevant art, and include but are not limited to non-toxic inert solid, semi-solid, or liquid filler, diluent, encapsulating agent or formulation auxiliary.
  • Typical pharmaceutically acceptable carrier is water or physiological saline.
  • Examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch; cellulose and its derivatives such as carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; as well as other agents such as non-toxic lubricants (e.g., lauryl sulfate and magnesium stearate) , coloring agents, releasing agents, flavoring agents, preservatives and antioxidants.
  • Suitable routes of administration of the medicament or the pharmaceutical composition of the present invention are intravascular delivery (e.g., injection or infusion) , oral, enteral, rectal, pulmonary (e.g., inhalation) , nasal, topical (including transdermal, buccal and sublingual) , intravesical, intravitreal, intraperitoneal, vaginal, brain delivery (e.g., intracerebroventricular, and intracerebral) , CNS delivery (e.g., intrathccal, perispinal, and intra-spinal) or parenteral (e.g., subcutaneous, intramuscular, intravenous, and intradermal) , transmucosal administration or administration via an implant, or other delivery routes known in the art.
  • intravascular delivery e.g., injection or infusion
  • oral enteral
  • rectal pulmonary
  • pulmonary e.g., inhalation
  • nasal including transdermal, buccal and sublingual
  • composition suitable for oral administration may be formulated into discrete dosage units such as pills, tablets, lozenges or hard or soft capsules, or as a dispersible powder or granules, or as a solutions or suspension for example, aqueous or oily suspensions, emulsions, syrups, elixirs, or enteral formulas.
  • the pharmaceutical composition is an eye drop.
  • the composition may be presented in uni-dose or multi-dose containers, such as sealed vials or ampoules, and may be stored in a lyophilized condition requiring the addition of sterile liquid carrier (e.g., water or saline) prior to use.
  • sterile liquid carrier e.g., water or saline
  • composition suitable for parental administration may be formulated into aqueous or non-aqueous sterile injection by mixing or dispersing the present synthetic peptide with a sterile solvent, such as water, Ringer’s solution, saline, 1, 3-butanediol, alcohol and etc. .
  • a sterile solvent such as water, Ringer’s solution, saline, 1, 3-butanediol, alcohol and etc.
  • Alternatively, fixed oil, fatty acid or synthetic mono-or diglycerides may be used as the solvent.
  • the composition may be sterilized by filtering through a filter.
  • compositions of the invention are administered topically to the eye.
  • pharmaceutical composition is an ointment for skin use.
  • about 1 ⁇ g/kg to about 1,500 mg/kg (e.g., 0.01-1,200 mg/kg) of the present synthetic peptide is administered to the patient, such as 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760
  • a typical daily or weekly dosage might range from about 0.1 mg/Kg to about 1200 mg/kg or more, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
  • the daily dosage ranges from about 15 mg/Kg to 500 mg/Kg, such as 15, 16, 17, 18, 19, 20, , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270
  • composition suitable for pulmonary administration is formulated as find dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulisers or insufflators.
  • kits of the invention can contain one or more sealed ampoules respectively contain the synthetic peptides of the invention, and which can be prepared in a single dose or as multiple doses.
  • the kit can additionally contain a vehicle suitable for solubilizing the synthetic peptides such as aqueous media such as saline solution, Ringer's solution, dextrose and sodium chloride; water-soluble media such as alcohol, polyethylene glycol, propylethylene glycol (PEG) ; and water-insoluble vehicles if necessary.
  • a vehicle suitable for solubilizing the synthetic peptides such as aqueous media such as saline solution, Ringer's solution, dextrose and sodium chloride; water-soluble media such as alcohol, polyethylene glycol, propylethylene glycol (PEG) ; and water-insoluble vehicles if necessary.
  • a vehicle suitable for solubilizing the synthetic peptides such as aqueous media such as saline solution, Ringer's solution, dextrose and sodium chloride
  • water-soluble media such as alcohol, polyethylene glycol, propylethylene glycol (PEG)
  • water-insoluble vehicles if necessary.
  • the kit of the present disclosure can additionally contain instructions for the simultaneous, successive or separate administration of the different formulations present in the kit. Therefore, the kit of the present disclosure can further comprise instructions for the simultaneous, successive or separate administration of the different components.
  • Said instructions can be in the form of printed material or in the form of an electronic support which can store the instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like) , optical media (CD-ROM, DVD) and the like.
  • the media can additionally or alternatively contain Internet webpages providing said instructions.
  • the findings described in the present disclosure are useful for the prevention and/or treatment of a subject suffering from DED.
  • the present disclosure therefore relates to a method for the prevention and/or treatment of DED, which comprises administering to a subject in need thereof a medicament or a pharmaceutical composition described above, which comprises a synthetic peptide of formula (I) ,
  • R is arginine, G is glycine, D is aspartic acid, and X aa is a D-form amino acid selected from the group consisting of isoleucine (I) , and phenylalanine (F) ; and
  • the synthetic peptide of formula (I) may be in linear or head-to-tail cyclic form.
  • the synthetic peptide of formula (I) in linear form, then the N-terminus of the amino acid sequence of the synthetic peptide is acetylated and the C-terminus of the amino acid sequence is amidated.
  • the synthetic peptide of formula (I) has the amino acid sequence of SEQ ID NO: 10.
  • the synthetic peptide of formula (I) is in head-to-tail cyclic form, and has the amino acid sequence of SEQ ID NO: 16 or 18.
  • the medicament and/or pharmaceutical composition when administrated to the subject is capable of ameliorating or alleviating the symptoms associated with DED.
  • the synthetic peptide is selected from the group of peptides described above, which include and are not limited to, RGDfD (SEQ ID NO: 10) , c (RGDiD) (SEQ ID NO: 16) , c (RGDfD) (SEQ ID NO: 18) , and a combination thereof.
  • the present invention is related to a method for treating DED, which comprises administering to a subject in need thereof an effective amount of a medicament or a pharmaceutical composition of the present invention.
  • the method includes the step of, administering to a subject in need thereof about 1 ⁇ g/kg to about 1,500 mg/kg (e.g., 0.01-1,200 mg/kg) of a medicament or a pharmaceutical composition of the present disclosure to the patient, such as 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 7
  • a typical daily or weekly dosage might range from about 0.1 mg/Kg to about 1200 mg/kg or more, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
  • the daily dosage ranges from about 15 mg/Kg to 500 mg/Kg, such as 15, 16, 17, 18, 19, 20, , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270
  • the subject suitable for treatment is a human.
  • RPMI 1640 medium, fetal bovine serum (FBS) , antibiotic–antimicotic solutions, and trypsin were purchased from Invitrogen (Carlsbad, CA) .
  • Recombinant Human Vitronectin (140-09) was purchased from PeproTech (Rocky Hill, NJ, USA) .
  • Hoechst 33258 dye and all chemicals were from Sigma-Aldrich (St. Louis, MO, USA) .
  • Anti-integrin alpha V (ab179475) and anti-F4/80 antibody (ab6640) were obtained from Abcam (Cambridge, MA, USA) .
  • Anti-vitronectin antibody (GTX61399) was purchased from GeneTex (Taipei, Taiwan) .
  • c (RGDfK) ATN-161, SB273005, cilengitide and Tirofiban were from Selleckchem (Houston, TX, USA) .
  • the RGDVF-based linear peptides were modified for stability by acetylation at the NH 2 termini and amidation at the COOH termini.
  • the RGDVF-based linear and head-to tail cyclic peptides were synthesized and characterized by mass spectrometry (>90%purity) at GenScript (Piscataway, NJ, USA) .
  • the human THP-1 monocytic leukemia cell line was cultured in RPMI 1640 with 25 mM HEPES buffer (supplemented with 10%FBS, 2mM l-glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin) at 37 °C in 5%CO 2 .
  • Differentiation of THP-1 cells was induced by resuspending the cells in fresh 10%FBS medium containing 10 ng/ml PMA for 48 h to induce the conversion of THP-1 cells into mature cells functionally resembling macrophages.
  • VTN 10 ⁇ g/ml
  • integrin inhibitors 20 ⁇ M, unless differently specified
  • Inhibitors dissolved in DMSO were added to the cell culture (the final concentration of DMSO was less than 0.05%) .
  • mice Male; 8-week-old mice were housed in an animal room under temperature control (24–25 °C) and a 12: 12 h light-dark cycle. Standard laboratory chow and tap water were available ad libitum. Experimental procedures were approved by the Mackay Memorial Hospital Review Board (New Taipei City, Taiwan) (project code: MMH-A-S-107-41, date: 1 January 2019 –31 December 2021) and were performed in compliance with national animal welfare regulations (Council of Agriculture) . DED was induced by housing mice in a controlled environment chamber (CEC) and administering scopolamine for maximal ocular surface dryness.
  • CEC controlled environment chamber
  • CEC conditions were controlled to provide a relative humidity of ⁇ 25%, airflow of 10 L/min, and temperature of 24–25 °C for 24 hours a day.
  • Mice were exposed to the CEC for 12 days and scopolamine hydrobromide (0.375 mg dissolved in 0.15 ml PBS per mouse; Escopan, Taiwan) was injected subcutaneously into mice two times per day at day 3 and day 5. Untreated age-and sex-matched mice were served as control.
  • balanced salt solution BSS, Alcon
  • 30 ⁇ l of 100 ⁇ M peptide or vehicle was topically administered to eyes three times daily.
  • fluorescein 100 mg/ml; Alcon Laboratories, Inc., Texas, USA
  • was instilled via a micropipette into the inferior-lateral conjunctival sac 0.6 ⁇ l of 0.5%sodium fluorescein dissolved in 4.4 ⁇ l of PBS per mouse
  • fluorescein staining for 15 sec mouse eyes were washed one time by PBS and then examined using a slit lamp microscope under cobalt blue light.
  • Proteins of interest were detected using the appropriate IgG-horseradish peroxidase secondary antibody (Santa Cruz Biotechnology) and ECL reagent (Amersham Biosciences) .
  • X-ray films were scanned on the model GS-700 Imaging Densitometer (Bio-Rad) and analyzed using Labworks 4.0 software. For quantification, blots of at least three independent experiments were used.
  • PFA-fixed, paraffin-embedded dry eye specimens were deparaffinized in xylene and rehydrated in a graded series of ethanol concentrations.
  • the slides were blocked with 10%goat serum and 5%bovine serum albumin (BSA) in PBS containing 0.5%Triton X-100 (PBST) for 60 min and then incubated with primary antibody against VTN (1: 100 dilution) at 37°C for 3 h.
  • the slides were subsequently incubated with peroxidase-labeled goat immunoglobulin (1: 500 dilution) for 20 min and then incubated with chromogen substrate (3, 3 ⁇ -diaminobenzidine) for 2 min before counterstaining with hematoxylin.
  • Deparaffinized tissue sections or 4%PFA-fixed BMDMs were blocked with 10%goat serum and 5%BSA in PBST for 20 min. Staining was performed using primary antibodies against F4/80 (1: 100 dilution) at 37°C for 3 h. For staining of ⁇ v integrin (1: 100 dilution) , sections were stained at 4°C for overnight.
  • the slides were subsequently incubated with the appropriate fluorescent-labeled secondary antibodies (1: 500 dilution) at 37°C for 1 h and then counterstained with Hoechst 33258 for 6 min and viewed with an epifluorescence microscope (Zeiss Axioplan 2 imaging; Zeiss, Oberkochen, Germany) equipped with a charge-coupled device camera (Zeiss AxioCam HRm, Zeiss) and quantification was performed using Axiovert software (Zeiss AxioVision Release 4.8.2, Zeiss) .
  • the concentration of VTN in the tear fluid was determined by ELISA.
  • Balanced salt solution (BSS; Alcone) was instilled onto the inferior fornix of a mouse at weekly intervals.
  • the tear-washing fluids of the two eyes (each 5 ⁇ l; referred to here as tear specimens) in the same mouse were pooled together and stored at -80°C until ELISA was performed.
  • the pooled tear specimens were measured by a Mouse VTN (vitronectin) ELISA kit (Fine Test; EM0500) , according to the manufacturer's instructions.
  • Fresh serum samples were isolated by centrifuging tubes for 15 min at 2,000g and diluted 1: 100 in phosphate-buffered saline (PBS) . Serum samples were stored at -20°C until ELISA performed.
  • PBS phosphate-buffered saline
  • VTN vitronectin
  • VTN Soluble VTN is found at high concentrations in blood serum that is predominantly produced in the liver by hepatocytes. VTN is also expressed in various tissues as an extracellular matrix component. VTN has been observed to be induced in the capillary to facilitate the macrophage infiltration in the ischemic hindlimb. In human eye, VTN is synthesized locally at retinal pigment epithelium-choroidal complex involved in the pathogenesis of age-related macular degeneration (AMD) . However, little is known about the role of VTN in the development of dry eye disease (DED) .
  • AMD age-related macular degeneration
  • VTN protein concentrations in tear fluid assayed by VTN ELISA showed slightly raised after DED induction for 1 week and further increased nearly 2-fold after DED induction for 2 and 3 weeks, compared with those of the NS mice (230 ⁇ 22 and 229 ⁇ 18 versus 114 ⁇ 7 ng/m1, FIG 1, (A) ) .
  • VTN was found at high concentrations in blood serum, but was not elevated in sera of DED mice.
  • the ⁇ v integrins including ⁇ v ⁇ 1, ⁇ v ⁇ 3, ⁇ v ⁇ 5, ⁇ v ⁇ 6 and ⁇ v ⁇ 8 are cell surface receptors for VTN.
  • the expression of ⁇ v integrin in macrophages of DED animals was investigated. Macrophages represent a heterogeneous cell population (M1 and M2 or more) . It is found that the pro-inflammatory M1 macrophage in ocular surface of DED mice predominantly reside in conjunctiva rather than cornea after DED induction for 10 days (You et al., Arch Immunol Ther Exp. 2015, 63: 299-304) .
  • macrophages may represent a novel drug target for patients with DED.
  • qPCR analysis of iNOS (M1 pro-inflammatory macrophage marker) and ⁇ v integrin mRNA expressions in the conjunctiva isolated from dry eye revealed a 2.7 ⁇ 0.6-fold and 3.4 ⁇ 0.5-fold higher than NS group, respectively (FIG 2, (C) ) .
  • THP-1 macrophages were synthesized, and their efficacies against ⁇ v integrin were evaluated by the expression of pro-inflammatory cytokine TNF- ⁇ gene in THP-1 macrophages in accordance with procedures described in “Material and Methods” section. Briefly, THP-1 macrophages were treated with both the soluble VTN and the designated peptide (any of SEQ ID NOs: 1 to 18) for 3 h and the gene expression was monitored by real-time qPCR. Results are depicted in FIG 3.
  • soluble VTN induced TNF- ⁇ mRNA expression approximately 14.3-fold greater than that of the solvent-treated control cells.
  • RGDVF-based linear and cyclic peptides composed of amino acids in the L-configuration exhibited inhibitory effect on TNF- ⁇ mRNA expression (FIG 3, (A) ) .
  • the results suggest that these RGD peptides have no antagonistic effect on ⁇ v ⁇ 3 integrin, while manifesting certain agonistic effect similar to VTN.
  • RGDfD, c (RGDfD) and c (RGDiD) may serve as antagonist of ⁇ v integrins by impairing VTN-mediated TNF- ⁇ gene expression in THP-1 macrophages.
  • THP-1 macrophages were treated with various dose levels (5, 10, or 20 ⁇ M) of cilengitide, c (RGDfK) , RGDfD, c (RGDiD) , and c (RGDfD) to evaluate their inhibitory effects on VTN-induced TNF- ⁇ mRNA expression.
  • the results were presented as the percent inhibition of VTN/solvent control (set as 100%) .
  • the five ⁇ v ⁇ 3 integrin antagonists showed dose-dependent response against VTN-induced TNF- ⁇ gene expression, albeit RGDfD and c (RGDiD) to a lesser degree.
  • Example 3 c (RGDfD) and c (RGDiD) independently exhibited beneficial effect on the recovery of corneal epithelial damage in experimental dry eye
  • mice treated with c (RGDfD) or c (RGDiD) for 5 days (Day 12) showed a significant decrease in the fluorescein staining score, as compared to that of the vehicle group (FIG 5, (C) ; 2.1 ⁇ 0.2 and 2.4 ⁇ 0.3 versus 3.0 ⁇ 0.0; P 0.0003 and 0.04, respectively) .
  • the result in this example indicates that the DED mice exhibited markedly corneal epithelial damage, however, the pre-existing ocular surface damage in dry eye may be ameliorated by the treatment of eye drops that contain c (RGDfD) or c (RGDiD) peptides.
  • eye drops containing the present synthetic peptides (RGDfD or c (RGDfD) ) or any of four other well-known ⁇ v ⁇ 3 integrin antagonists including c (RGDfK) , ATN-161, SB273005, and cilengitide, were independently used to treat DED mice.
  • mice in all groups exhibited obvious injury in corneal epithelium, which was assayed by fluorescein staining (score upon 2) .
  • Eye drop treatment for 4 days (day 12) revealed that c (RGDfK) , ATN-161, SB273005, cilengitide, RGDfD and c (RGDfD) exhibited therapeutic ability on DED, whereas vehicle and Tirofiban had no such effect (Table 4, and FIG 6) .
  • c (RGDfD) displayed much more pronounced therapeutic ability on DED than other groups, even though c (RGDfD) , c (RGDfK) , and cilengitide independently manifested similar activity to suppress VTN-induced TNF- ⁇ mRNA expression in THP-1 macrophages.
  • Example 5 The present synthetic peptides prevent loss of goblet cells in conjunctiva during development of experimental DED
  • Goblet cells are mainly resided in the superficial epithelium of conjunctival fornix and are responsible for mucous tear production.
  • PAS staining of goblet cells in NS mice displayed a continuous homogeneous pattern at the conjunctival fornix (FIG 7, (A) ) .
  • the vehicle-treated DED mice exhibited markedly decrease of goblet cells as compared to that of NS, c (RGDfD) or c (RGDiD) groups (number/0.1 mm 2 : 5 versus 9, 8 and 7; FIG 7, (B) ) .
  • Example 6 The present synthetic peptides suppress the inflammatory responses in the experimental dry eye
  • TNF- ⁇ pro-inflammatory cytokines
  • IL-1 ⁇ pro-inflammatory cytokines
  • MMP matrix metalloproteinase
  • the mRNA levels of TNF- ⁇ , IL-1 ⁇ , IL-6 and MMP-9 in dry eyes were respectively monitored via real-time qPCR. It was found that the respective mRNA levels of TNF- ⁇ , IL-1 ⁇ , IL-6 and MMP-9 were significantly up-regulated by 1.6-, 1.7-, 2.3-and 29.6-fold, as compared to that of the NS group (FIG 8) . Further, treatment with c (RGDfD) and c (RGDiD) significantly attenuated the gene expression of TNF- ⁇ , IL-1 ⁇ , and IL-6 to near their respective baseline levels. In addition, c (RGDfD) and c (RGDiD) also significantly repressed the gene expression of MMP-9 by factors of approximately 1.33 and 1.65-fold, respectively, as compared to that of the vehicle-treated group.

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Abstract

L'invention concerne des peptides synthétiques et des compositions pharmaceutiques les comprenant pour le traitement de la sécheresse oculaire (DED). L'invention concerne également des procédés de traitement de la sécheresse oculaire, par l'administration à un sujet ayant besoin d'un tel traitement, d'une quantité efficace d'une composition pharmaceutique contenant le peptide synthétique.
PCT/CN2021/078641 2021-03-02 2021-03-02 Peptides synthétiques courts et leurs utilisations pour le traitement de la sécheresse oculaire WO2022183360A1 (fr)

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