TW201630928A - Use of short synthetic peptide for the treatment and/or prophylaxis of dry eye disease - Google Patents

Abstract

Disclosed herein are synthetic peptides and compositions comprising the same for the treatment and/or prophylaxis of an ocular disease, particularly, dry eye disease. Also disclosed herein are methods of treating and/or preventing an ocular disease by administering to a subject in need of such treatment a composition containing a synthetic peptide of the present disclosure.

Description

Short synthetic peptides and their use in the treatment and/or prevention of dry eye syndrome

The present invention is directed to the discovery of a short synthetic peptide, and its use for the treatment and/or prevention of ocular diseases.

Dry eye disease (DED) refers to various conditions of abnormal tear layer, which may be caused by damage to the stratum corneum. Symptoms of dry eye syndrome include sandy-gritty feeling, burning sensation, tingling sensation, and foreign body sensation, and patients experience more severe discomfort during the day. Patients with DED have different symptoms. Patients with more severe symptoms may experience persistent tingling of the eye, as well as epithelial lesions on the surface of the eye, damage to the sterile environment of the eyeball, or bacterial corneal ulceration. The only FDA-approved prescription drugs available today for the treatment of dry eye are corticosteroids and Restasis (0.05% cyclosporine). However, neither of the above drugs can effectively cure damaged corneal tissue, but it can significantly slow the symptoms of DED or heal damaged corneal tissue after prolonged treatment (about 4-6 months).

Therefore, there is a need in the art for an improved pharmaceutical and/or treatment. A method of treating and/or preventing DED.

SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

The present disclosure is directed to methods for developing novel compounds and/or for treating ocular diseases, particularly dry eye syndrome (DED).

A first aspect of the present disclosure provides a short synthetic peptide that can be used to treat DED. The short synthetic peptide is composed of 7 contiguous amino acid residues, the sequence of which is DLYRX ₁ X ₂ S (SEQ ID NO: 1), and the X ₁ and X ₂ are any amino acid residues, respectively. base.

According to other embodiments of the present disclosure, X ₁ and X ₂ in the synthetic peptide are valine and arginine, respectively, such as the amino acid sequence of SEQ ID NO: 2.

Further, in certain embodiments, X ₁ and X ₂ in the synthetic peptide are leucine and arginine, respectively, such as the amino acid sequence shown in SEQ ID NO: 3.

According to one embodiment, X ₁ and X ₂ in the synthetic peptide are glutamic acid and arginine, respectively, such as the amino acid sequence of SEQ ID NO: 4.

According to other embodiments, X ₁ and X ₂ in the synthetic peptide are glutamic acid and lysine, respectively, such as the amino acid sequence shown in SEQ ID NO: 5.

According to still another embodiment, the synthetic peptide of SEQ ID NO: 1 comprises at least one dextran amino acid residue. In some embodiments, the synthetic peptide of SEQ ID NO: 1 comprises at least two dextroamine amino acid residues.

A second aspect of the present disclosure is a method of treating an individual suffering from dry eye. The method comprises administering an effective amount of a synthetic peptide to the individual to alleviate symptoms associated with DED. The synthetic peptide consists of 7 contiguous amino acid residues having the sequence DLYRX ₁ X ₂ S (SEQ ID NO: 1), wherein X ₁ and X ₂ are each an amino acid residue, respectively.

According to other embodiments, X ₁ and X ₂ in the synthetic peptide are valine and arginine, respectively, such as the amino acid sequence shown in SEQ ID NO: 2.

According to some embodiments, X ₁ and X ₂ in the synthetic peptide are leucine and arginine, respectively, such as the amino acid sequence shown in SEQ ID NO: 3.

According to one embodiment, X ₁ and X ₂ in the synthetic peptide are glutamic acid and arginine, respectively, such as the amino acid sequence shown in SEQ ID NO: 4.

According to still another embodiment, X ₁ and X ₂ in the synthetic peptide are glutamic acid and lysine, respectively, such as the amino acid sequence shown in SEQ ID NO: 5.

According to other embodiments, the synthetic peptide of SEQ ID NO: 1 of the present disclosure comprises at least one dextran amino acid residue. In some embodiments, the synthetic peptide of SEQ ID NO: 1 comprises at least two dextrorotatory amino acid residues.

Method of the present disclosure in accordance with an optional embodiment Further comprising administering an effective amount of an agent to the individual to treat the dry eye, wherein the agent is selected from the group consisting of an anti-inflammatory agent, a calcineurin inhibitor, an antibiotic, and a nicotine B. A group consisting of a choline receptor agonist and an anti-lymphangiogenesis agent.

In certain embodiments, the anti-inflammatory agent is cyclosporine. The calcineurin is voclosporin. The antibiotic is selected from the group consisting of: amikacin, gentamycin, kanamycin, neomycin, netilmicin, streptomycin ( Streptomycin), tobramycin, teicoplanin, vancomycin, azithromycin, clarithromycin, dihithromycin, Erythromycin, roxithromycin, troleandomycin, amoxicillin, ampicillin, azlocillin, carbencilin, chlorine Sitting on cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin, piperacillin, ticarcillin (ticarcillin), bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin ), levofloxacin, lomefloxacin (lomefl) Oxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin (trovafloxacin), mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim, compound A group consisting of cotrimoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline. The nicotinic acetylcholine receptor agonist is pilocarpine, atropine, nicotine, epibatidine,洛beline or imidacloprid. The anti-lymphangiogenesis agent is a vascular endothelial growth factor C (VEGF-C) antibody, a VEGF-D antibody or a VEGF-3 antibody.

In all embodiments, the individual is a human.

A third aspect of the present disclosure is the use of a synthetic peptide for the preparation of a medicament for the treatment and/or prevention of dry eye disease (DED). The synthetic peptide is composed of 7 contiguous amino acid residues, the sequence of which is DLYRX ₁ X ₂ S (SEQ ID NO: 1), and the X ₁ and X ₂ are any amino acid residues, respectively. .

According to other embodiments, X ₁ and X ₂ in the synthetic peptide are valine and arginine, respectively, such as the amino acid sequence shown in SEQ ID NO: 2.

According to some embodiments, X ₁ and X ₂ in the synthetic peptide are leucine and arginine, respectively, such as the amino acid sequence shown in SEQ ID NO: 3.

According to one embodiment, X ₁ and X ₂ in the synthetic peptide are glutamic acid and arginine, respectively, such as the amino acid sequence shown in SEQ ID NO: 4.

According to still another embodiment, X ₁ and X ₂ in the synthetic peptide are glutamic acid and lysine, respectively, such as the amino acid sequence of SEQ ID NO: 5.

According to other embodiments, the synthetic peptide of SEQ ID NO: 1 of the present disclosure comprises at least one dextran amino acid residue. In some embodiments, the synthetic peptide of SEQ ID NO: 1 comprises at least two dextran amino acid residues.

The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. The description of the drawings is as follows: FIG. 1A is a view of a mouse corneal tissue according to an embodiment of the present disclosure. Photographs of light staining to show the preventive effect of the synthetic peptide of the present invention on desiccating stressed-mice; FIG. 1B is a bar graph of quantitative analysis of FIG. 1A; FIG. 2 is a map according to the present disclosure. The synthetic peptide of the present invention shown in the embodiment promotes the ability of dry stress mice to produce tear fluid; FIG. 3A is a photograph of fluorescent staining of corneal tissue according to an embodiment of the present disclosure to show that the synthetic peptide of the present invention is The therapeutic effect of the dry-pressure mice; the 3B is a bar graph shown in the quantitative analysis of FIG. 3A; and FIG. 4A is a photograph of the tissue examination of the mouse corneal section by H&E staining according to an embodiment of the present disclosure; Figure 4B is a bar graph for quantitative analysis of Figure 4A; Figure 5A is a photograph of conjunctival epithelium stained with PAS according to an embodiment of the present disclosure, wherein the arrow is referred to as goblet cells (pink; PAS staining) Positive); and Figure 5B is a bar graph of quantitative analysis of Figure 5A.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

Noun definition

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein.

The term "peptide" as used herein refers to a polymer composed of amino acid residues. The word "synthetic peptide" means that the peptide does not contain intact protein in nature. child. The reason why such a peptide is "synthetic" is that it is obtained by humans using technical means such as chemical synthesis, recombinant genetic techniques or segmentation of the entire antigen. In the present specification, any amino acid residue based on the position in a peptide is counted from the N-terminus of the peptide.

"Percent % amino acid sequence identity" as used herein with respect to the synthetic peptide sequence means the amino acid residue of the candidate synthetic peptide and the amino acid of a reference polypeptide The percentage of residues that are identical. When performing the above alignment, the candidate synthetic peptide can be side by side with the reference polypeptide, and a gap is introduced as necessary to form the highest sequence similarity of the two sequences, and when calculating the similarity, the Amino acid residues of conservative substitutions are taken into account. A variety of methods are available for the above-described side-by-side, such as publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art to which the present invention pertains may select appropriate parameters and calculation methods when performing side-by-side to obtain an optimal arrangement. In the present specification, sequence comparison between diamine acid sequences is carried out using the protein-protein BLAST analysis software Blastp provided by the National Center for Biotechnology Information (NCBI). Amino acid sequence similarity of a candidate amino acid sequence A compared to a reference amino acid sequence B (also referred to herein as sequence A and sequence B have a specific percentage (%) of amino acid sequence similarity Degree) is calculated as follows:

Where X is the use of Blastp software for sequences A and B. The number of identical amino acid residues obtained after alignment, and Y is the total number of amino acid residues in the shorter of the A and B sequences.

The term "treatment" refers to a prophylactic (eg, prophylactic), healing or palliative treatment whereby the desired pharmaceutical and/or physiological effect is achieved; for example, slowing or curing an individual Symptoms of dry eye syndrome. The above effects refer to the ability to partially or completely cure or prevent the symptoms of the disease in an individual. The "treatment" encompasses the prevention, treatment or alleviation of a mammalian disease, especially in humans. The treatment comprises: (1) preventing (eg, prophylactic use), treating or slowing down a disease or condition of a subject, wherein the individual may have a disease but has not yet been diagnosed; (2) inhibiting a disease (ie, reducing development) Risk of pathological changes associated with the disease, disorder, and/or condition; or (3) curing a disease (ie, reducing symptoms associated with the disease).

The term "administering" (administration) means providing the peptide or composition of the present invention to an individual. Such modes of delivery include, but are not limited to, intravenous, intramuscular, intraperitoneal, intraarterial, intracranial or subcutaneous delivery.

As used herein, the term "effective amount" means that the amount of a component is sufficient to induce a desired response or effect, i.e., to effectively treat the disease. For example, an agent (eg, a compound, a synthetic peptide, a nucleic acid encoding a therapeutic peptide) can be used to reduce, prevent, delay, or cure symptoms associated with dry eye. An effective amount of a drug does not necessarily cure a disease or condition, but delays, prevents or prevents the onset of disease or symptoms. Health, or delay the symptoms associated with the disease or symptoms. The therapeutically effective amount can be divided into one, two or more doses in a single administration dosage form and can be administered once, twice or more times over a specified period of time.

The term "mammal" encompasses all members of the Mammalia, including: humans, primates, livestock and farm animals. For example, the livestock or farm animals can be rabbits, pigs, sheep, and cattle. The mammals may also cover zoo or competition animals, pets, and rodents (eg, mice and rats). The term "non-human mammals" refers to all animals under the mammalian class except humans.

The term "subject" or "patient" is used interchangeably and refers to an animal (including human) that can accept the synthetic peptide and/or method of the present disclosure. Unless otherwise indicated, "individual" or "patient" generally includes males and females. Furthermore, the "individual" or "patient" encompasses an animal that has a good therapeutic effect from the therapeutic methods of the present disclosure. For example, the "individual" or "patient" includes, but is not limited to, human, rat, mouse, sylvestris, pig, monkey, pig, sheep, cow, horse, dog, cat, bird, and bird. In an example, the patient is a human.

The term "pharmaceutically acceptable carrier, excipient or stabilizer" means a carrier, agent or compound suitable for administration to the eye. The term "ophthalmic composition" refers to a composition that can be applied to the eye, or a device that can be used to treat contact with the eye (eg, a contact lens).

The term "dry eye syndrome (DED)" refers to conjunctival persistence Symptoms such as dryness and corneal opacity, and these symptoms are caused by decreased secretion of tears, excessive evaporation of tears, abnormal mucus production, or lipids in the tear layer. The causes of DED include, but are not limited to, exposure to dry environments (eg, aircraft and workplace), vitamin A deficiency, Sjögren's syndrome, rheumatoid arthritis and other rheumatoid diseases, compounds, heat Burns or drugs. The drug is atenolol, chlorpheniramine, hydrochlorothiazide, isotretinoin, ketorolac, ketotifen. , levocabastin, levofloxacin, oxybutynin and tolterodine.

Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0. 5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as indicated The effective number of bits is the value obtained by applying the general carry method.

In the absence of conflict with context, the singular noun used in this specification encompasses the plural of the noun.

2. Implementation

At least a portion of the present invention is based on the discovery of a short synthetic peptide that can be used to treat and/or prevent the development of a ocular disease; for example, dry eye syndrome (DED). Further, the present invention provides a method and composition for treating and/or preventing dry eye, wherein the composition comprises a newly discovered synthetic peptide.

2.1 The synthetic peptide of the present invention

The short synthetic peptide of the present disclosure consists of 7 contiguous amino acid residues having the sequence DLYRX ₁ X ₂ S (SEQ ID NO: 1), and the X ₁ and X ₂ are each an amine group, respectively. Acid residue.

In one embodiment, the X ₁ is leucine and the X ₂ is arginine.

In other embodiments, the X ₁ and X ₂ are valine and arginine, respectively.

In still other embodiments, the X ₁ and X ₂ are glutamic acid and arginine, respectively.

In still another embodiment, the X ₁ and X ₂ are glutamic acid and lysine, respectively.

In other optional embodiments, the synthetic peptide of SEQ ID NO: 1 comprises at least one dextran amino acid residue. In certain embodiments, the synthetic peptide of SEQ ID NO: 1 comprises a di-dextran amino acid residue.

The synthetic peptide of the present invention can be produced by any of the standard peptide synthesis methods described in the prior art. In one embodiment, the synthetic peptide of the present invention is a solid phase peptide synthesizer (ABI 433A peptide synthesizer; Applied Biosystems Inc.; Life Technologies Corp.; Foster City, CA, USA) and according to the operating manual. synthesis. The synthetic peptides of the present disclosure are 7-mer (X ₁ , X ₂ ) (SEQ ID NO: 1), 7-rner (SEQ ID NO: 2), 7-mer (V→L) (SEQ ID NO: 3), 7-mer (V→Q) (SEQ ID NO: 4) or 7-mer (VR→QK) (SEQ ID NO: 5), and each of the synthetic peptide sequences is shown in Table 1.

Those skilled in the art can modify the synthetic peptides by conventional methods (e.g., computer simulation programs), and the methods can predict the effect of peptide sequence configuration changes on activity. Furthermore, the synthetic peptide (eg, 7-mer) of the present invention can be "designed" or "modified" based on the disclosure herein; that is, based on teaching and testing, the modified synthesis is successful. Peptides to determine if the modified synthetic peptide has the desired function or configuration. Synthetic peptides of the invention (e.g., 7-mer) can be modified to alter the characteristics of the peptide without affecting its physiological activity. For example, the particular amino acid is altered and/or deleted without affecting the physiological activity shown in the present disclosure (ie, the efficacy of treating the DED). According to certain embodiments, the fifth and sixth amino acid residues (proline and arginine, respectively) on the 7-mer peptide sequence can be substituted with any amino acid without affecting its treatment. And / or the ability to prevent DED. Accordingly, the present invention encompasses derivatives which have an equivalent function to the preferred synthetic peptide (SEQ ID NO: 2), and include a peptide having an amino acid retention substitution.

According to other embodiments, the aspartic acid (D) at the N-terminus of SEQ ID NO: 2 and the serine acid (S) at the C-terminus are deleted, and the resulting modified peptide sequences are designated as 7-mer, respectively. - ΔD (LYRVRS; SEQ ID NO: 6) and 7-mer-ΔS (DLYRVR; SEQ ID NO: 7), and this dipeptide sequence does not have biological activity associated with DED. Therefore, aspartic acid (D) and C-terminal serine (S) located at the N-terminus of the sequence number 2 peptide sequence are important for maintaining the therapeutic peptide of the present invention for treating DED activity, the diamino acid residue It can only be substituted with a retaining amino acid residue.

Additionally, the synthetic peptides of the invention can be prepared using recombinant techniques. For example, a nucleic acid sequence encoding a peptide of the invention can be inserted into a performance vector using recombinant techniques, and wherein the nucleic acid sequence can be joined to a regulatory sequence to express the peptide of the invention in a host cell. The vector can then be sent to a suitable host cell to express the peptide of the invention. The expressed recombinant peptide can be purified by ammonium sulfate precipitation and column chromatography, and according to various embodiments of the present disclosure. The method shown is for activity testing.

The above nucleic acids or polynucleotides can be delivered using known biodegradable polymer microparticles or microcapsule delivery devices. Alternatively, the nucleic acid may be coated with a liposome by a conventional method to allow the nucleic acid to be absorbed by the host. The polynucleotides can be loaded separately or co-loaded into the delivery vehicle with other tissue specific antibodies. In addition, a molecular conjugate can be prepared which binds poly-L-lysine electrostatically or covalently by a plastid or other carrier. In addition, the present invention can also perform tissue specific calibration using known tissue-specific transcriptional regulatory elements. "Naked DNA (ie, no delivery vector)" is delivered to the intramuscular, intradermal or subcutaneous sites, and other methods are used for intracellular expression.

2.2 Composition for treating and/or preventing DED

The synthetic peptide of the present invention can be used to treat an individual suffering from dry eye syndrome or to prevent dry eye syndrome in an individual.

Another aspect of the present disclosure provides a pharmaceutical product for treating a DED comprising the synthetic peptide of the present invention. In the manufacture of the pharmaceutical product, an appropriate amount of the synthetic peptide of the present invention may be mixed with a pharmaceutically acceptable carrier, excipient or stabilizer to obtain a composition suitable for ocular administration, for example, a lyophilized composition. Formulation or an aqueous solution. In a specific embodiment, the synthetic peptide sequence is SEQ ID NO: 2-5 or a combination thereof.

The content of the peptide of the present invention in the composition varies depending on the peptide used. The peptide content in the composition is about 0.001% Up to 10% (% by weight); for example, about 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, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0%. In a preferred embodiment, the peptide is present in an amount of from about 0.001% to about 5% by weight; for example, about 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 Or 5.0%.

A wide variety of pharmaceutically acceptable carriers, excipients or stabilizers are known in the art to be suitable for use in the synthetic peptides of the present invention. For example, suitable buffers are phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (eg, octadecane) Octadecyldimethylbenzy ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol Alcohol), butyl alcohol, benzyl alcohol, alkyl parabens (eg methyl paraben or propyl paraben) , catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight peptide (less than about 10) Amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amines Base acids, such as: glycine, glutamic acid, Tianmen Guanamine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including: glucose mannose or dextrins; Chelating agents such as ethylenediaminotetraacetic acid (EDTA); sugars such as sucrose, mannitol, trehalose or sorbitol Salt-forming counter-ions such as sodium; metal complexes (eg, Zn-protein complexes) and/or nonionic surfactants such as polysorbates ( That is, TWEEN ^® ), poloxamers (ie, PLURONICS ^® ) or polyethylene glycol (PEG).

In a particular embodiment, the compositions provided herein can be formulated for topical application. Topically applied formula is usually liquid It has a physiologically acceptable pH and can usually be dispensed multiple times. Thus, in a specific embodiment, the composition is a topical ophthalmic composition comprising a therapeutically effective amount of a synthetic peptide (ie, 7-mer, 7-mer (V→L), 7-mer (V→Q) or 7-mer (VR→QK) and a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is a carrier suitable for topical application to the eye. Further, those having ordinary knowledge in the art have The various types of carriers are also suitable for use. Furthermore, the carrier is usually in the form of a liquid. Based on the convenience of preparation and convenience of the patient, it is usually preferred to formulate the aqueous solution for the formulation. Specifically, when the patient is infected with the eye, it is usually 1-2 drops of the composition are applied to the eye. However, the synthetic peptide of the present invention may also be used in combination with other types of compositions; for example, suspensions, viscous or semi-viscous gels or other solid or semi-solid Compositions. The peptides having relatively low water solubility can also be formulated as a suspension. The ophthalmic compositions of the present invention also comprise various other ingredients; for example, buffers, preservatives, solubilizers, and viscous constructs. .

In a preferred aspect, the composition of any of the embodiments of the present invention may comprise an effective amount of a buffer to maintain a pH between about 6 and 8; preferably, a pH of 7. The buffer may be a buffer known in the art; for example, acetate, ascorbate, borate, bicarbonate, carbonate, citrate (citrate) and phosphate buffers. In a preferred embodiment, the buffer comprises a borate. Those skilled in the art to which the present invention pertains can determine the effective amount of the buffer of the present invention by conventional conventional techniques. A buffer containing borate is exemplified, wherein the concentration of the borate is preferably about 0.6%.

In a preferred aspect, the composition of any of the embodiments of the present invention may comprise a tonicity agent. The addition of an isotonic agent to the ophthalmic composition can adjust the concentration of the dissolved material to the desired isotonic range. Isotonic agents can be isotonic agents known to those of ordinary skill in the eye, including, but not limited to, glycerin, mannitol, sorbitol, sodium chloride or Other electrolytes. In a preferred embodiment, the isotonic agent is sodium chloride.

Ophthalmic formulations are usually packaged in multiple doses. Therefore, preservatives are usually added to prevent microbial contamination during use. In certain instances, preservatives may be added even in single-use compositions. The "preservative" refers to a preservative conventionally known in the ophthalmology art. Preservatives are usually added to multi-use eye formulations to prevent bacterial contamination. The preservative includes, but is not limited to, benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol ( Phenylethyl alcohol), edetate disodium, sorbic acid, polyquaternium-1, stabilized oxychloro complexes (eg, Purite ^® )), phenylmercuric acetate, benzyl alcohol or other known agents. The concentration of the preservative in the composition is from about 0.001 to 1.0% (wt%).

In certain instances, a surfactant or other suitable cosolvent can be utilized in formulating with the compositions of the present invention. The "surfactant" and "cosolvent" are conventionally known reagents in the related art. Surfactants are used to aid in the dissolution of therapeutically active agents or other insoluble ingredients in the compositions. In a non-limiting example, the compound comprises polyethoxylated castor oils, polysorbates 20, 60, and 80; Pluronic ^® F-68, F-84, and P-103 (BASF Corp., Parsippany NJ, USA); cyclodextrins; or other known agents. Anionic, cationic, zwitterionic (zwitterionicc and amphoteric) and nonionic surfactants are suitable for use in the present invention. In order to achieve the object of the present invention, in a preferred embodiment, the surfactant may be a nonionic surfactant such as polysorbate, isopropyl acrylamide, ethanol, ethoxylate. (ethoxylates), ethylene glycol-propylene glycol block copolymers, fatty acids, alkylphenols or phospholipids. The concentration of the surfactant and co-solvent is from about 0.01 to 2% by weight.

To increase the efficiency of ocular absorption of the active ingredient, to reduce variations in the formulation process, to reduce the physical separation characteristics of a suspension formulation or emulsion formulation, and/or to improve other aspects of the ophthalmic formulation, the viscosity of the formulation can be adjusted to Larger than normal aqueous solution. The thickener comprises: polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl fiber Hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulos or other known agents. The concentration of the above agent is from about 0.01 to 2% by weight.

Another compound that can be added to the compositions of the present invention It is a chelating agent. The chelating agent is a compound capable of chelation with a metal. The addition of a chelating agent to the ophthalmic composition enhances the antiseptic effect. In a non-limiting embodiment, the chelating agents suitable for use in the present invention are edetate salts; for example, disodium edetate disodium, edetate calcium disodium , edetate sodium, edetate trisodium or edetate dipotassium.

In a specific embodiment, the synthetic peptide can be formulated into a topical application form (i.e., an ophthalmic composition) suitable for use in the eye. The topical formulation may be an eye drop, a gel, an ointment or an emulsion or the like.

The compositions of the invention may be topically applied to the eye. The dosage of the synthetic peptide of the present invention is from about 1 microgram/kg body weight to about 50 mg/kg body weight (e.g., 0.1-20 mg/kg body weight) as the severity of the patient's disease varies. The daily or weekly administration dose is from about 1 microgram/kg to about 20 mg/kg or more. For the purpose of any of the above topical administrations, the topical application amount is from about 0.01 to about 100 mg/kg body weight (mg/kg), administered once to several times a day (eg, daily administration of 4, 6, 8 or More times)

In one embodiment, the compositions provided herein comprise two or more synthetic peptides, wherein at least one of the synthetic peptides is a 7-mer or a derivative thereof (eg, 7-mer (V→L), 7- Mer (V→Q) or 7-mer (VR→QK), and it has the ability to treat or prevent DED. In this embodiment, the synthetic peptides can be formulated into different dosage forms depending on the mode of administration (eg, separate administration, simultaneous administration, or continuous administration).

The compositions provided by the present invention may be a set. can It is understood that the kit of the present invention is a product comprising a synthetic peptide of the present invention and/or other therapeutic compound which is packaged to form the composition to facilitate delivery, storage and simultaneous or Continuous application. Thus, the kits of the present invention may comprise one or more synthetic peptide suspensions, syringes and the like comprising the present invention to produce the peptides of the present invention in single or multiple dose forms. The kit further comprises a carrier for dissolving the synthetic peptide of the invention; for example, an aqueous medium, i.e., physiological saline, Ringer's solution, glucose, and sodium chloride. A water-soluble medium such as ethanol, polyethylene glycol, or propylethylene glycol. The water-insoluble carrier can also be applied to the present invention if necessary. The other items of the kit are a package for packaging the compositions of the present invention. Materials suitable for use in the package of the present invention may be glass, plastic (polyethylene, polypropylene, polycarbonate, etc.), bottles, cans, paper, bundles or the like.

The kit of the present invention may further comprise instructions describing the modes of administration of the various formulations of the invention (e.g., simultaneous administration, continuous administration, individual administration). Thus, the kit of the present invention may further comprise instructions for how to apply (e.g., simultaneous administration, continuous administration, individual administration) of the various components of the present invention. The instructions may be paper or an electronic medium that can be read, such as an electronic storage medium (disk, tape or the like), optical media (CD-ROM, DVD), and the like. The media may additionally or non-essentially add additional web pages to provide the above description information.

2.3 Methods of treating and/or preventing DED

As described above, the present invention can effectively prevent and/or treat DED.

Accordingly, the present invention relates to a method of preventing and/or treating a DED comprising topically administering a pharmaceutical or composition comprising a synthetic peptide of the present invention and a pharmaceutically acceptable carrier to a subject in need thereof, wherein The synthetic peptide is composed of 7 contiguous amino acid residues, the sequence of which is DLYRX ₁ X ₂ S (SEQ ID NO: 1), and wherein X ₁ and X ₂ are each an amino acid residue, respectively.

In a specific embodiment, the synthetic peptide is in the form of a composition wherein the composition comprises one or more synthetic peptides. In another specific embodiment, the synthetic peptide is a peptide sequence of any of SEQ ID NOs: 2-5, or a combination thereof.

In an optional embodiment, the method further comprises administering to the individual an effective amount of an agent to treat the DED. The agent is selected from the group consisting of an anti-inflammatory agent, a calcineurin inhibitor, an antibiotic, a nicotinic acetylcholine receptor agonist, and an anti-lymphangiogenic agent.

In certain embodiments, the anti-inflammatory agent is cyclosporine. The calcineurin is vancosin. The antibiotic is selected from the group consisting of: amikacin, gentamicin, kanalin, neomycin, netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin, Azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, doxycycline, amoxicillin, ampicillin, azlocillin, carbencillin, chlorhexidine, dichloroethylene Xilin, flucloxacillin, mezlocillin, nefcillin, penicillin, bepicillin, ticarcillin, subtilisin, kelimycin, polymycin B, cepsson, enoxacin, and alterne Sand star, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, Trovafloxacin, sulfamidamide, acesulfame, sulfamethoxazole, sulfamethoxazole, isosodium sulfonamide, trimiprin, cotrimoxazole, dimetridin, deoxytetracycline, minocycline In the group consisting of oxytetracycline and tetracycline. The nicotinic acetylcholine receptor agonist is a group consisting of pilocarpine, atropine, nicotine, echinolide, lobeline and imidacloprid. The anti-lymphangiogenesis agent may be a vascular endothelial growth factor C (VEGF-C) antibody, a VEGF-D antibody or a VEGF-3 antibody.

In all embodiments, the individual suitable for use in the methods of treatment of the invention is a human.

In the following, a plurality of experimental examples are set forth to illustrate certain aspects of the present invention, and the present invention is not limited by the scope of the present invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety.

Example

Materials and Methods

material

Carboxymethylcellulose sodium (CMC), periodic acid Schiff (PAS) and dexamethasone were purchased from Sigma-Aldrich (St. Louis, MO, USA). CMC (1% w/v) was dissolved in a balanced salt solution and used as a 7-mer carrier. All peptides were synthesized by GenScript (Piscataway, NJ, USA), in which the N-terminus and C-terminus of each peptide were acetylated and Amination modification to improve its stability followed by mass spectrometry (purity: >95%).

Experimental animal

Seven to eight week old C57BL/6 mice (about 18 to 25 grams per body weight) were used in this experiment. The experimental process was approved by the Ma Rong Memorial Hospital Review Committee and followed by the Animal Experimental Ophthalmology and Visual Research regulations set by the Academic Conference on Visual and Ophthalmology (ARVO).

Dry eye animal model

The mice were placed in a controlled environment chamber (CEC) and induced to produce dry eye in mice according to the experimental procedure disclosed by Barabino et al. (IVOS (2005) 46(8), 2766-2771). . Mice implanted with CEC were exposed to a relatively low humidity environment with relative humidity (RH) <25%; temperature 20 to 22 ° C; air flow 15 L/min; 12 hours daily. The mice in the control group were placed in a normal environment (RH > 50%; air flow was 0; temperature was 21-23 ° C) and passed the same period.

Corneal fluorescence staining

Experimental animals were injected intraperitoneally with a mixture (zoletil (6 mg/kg) and xylazine (3 mg/kg)) for anesthesia. Corneal damage was measured by local fluorescent staining (Fluor-I-Strip, Ayerst Laboratories, Philadelphia, PA). Corneal fluorescence staining was performed with a slit-lamp biomicroscope under cobalt blue light and imaged with a digital camera. The data were mean ± standard deviation (mean ± SD). According to standardized 5 components Table (0 points, the first level; 1-5 points, the first level; 6-15 points, The second level; 16-30 points, the third level; 30 points is the fourth level), the fluorescent staining points of the 1-mm central corneal area of each eye are graded. Furthermore, if there is a full-stained area in the staining result, an extra point is added, and if there are two or more full-filled areas, an additional two points are added (Li J et al., Mole Visions 2012, 18, 317). .

Determination of tear secretion

Tear production was determined by phenol red detection cotton (Zone-Quick; Oasis, Glendora, CA). The effectiveness of this mouse assay was performed in accordance with the procedures disclosed in the aforementioned Dursun et al. (IVOS (2002) 43, 632-638). The cotton thread was taken with a nickel clip and placed in the outer crotch portion of the mouse, and allowed to stand for 60 seconds. After the cotton thread is wetted by the tears, the wet area is turned into red, and the amount of tear secretion is expressed in mm.

Corneal epithelial histopathology

Corneal epithelial damage was assessed by the aforementioned method (Pflugfelder et al; 2005, Am J Pathol 166, 61-71). Briefly, the amount of apical cell exfoliation was calculated by two independent examiners, with a 20-gauge microscopy, calculating the cell volume under the microscope for complete field of view, and observing three different tissue sections.

Goblet cell PAS staining

After the experimental animals were euthanized, the eyes were surgically removed, fixed in 10% formaldehyde, embedded in paraffin, and sectioned (thickness: 5 μm). The sections were stained with PAS (Sigma-Aldrich) reagent to examine goblet cells in the upper and lower conjunctiva of the mice, and were observed under a microscope and photographed with a digital camera.

statistics

The results are expressed as mean ± standard error (SEM). Statistical analysis was performed by one-way analysis of variance (1-way ANOVA). P < 0.05 was considered significant.

Example 1 The synthetic peptide of the present invention can prevent dry eye induced dry eye syndrome

The mice were placed in CEC to induce dry eye syndrome (DED) according to the procedure described in Materials and Methods. On the first day of treatment, mice were placed in CEC, and the synthetic peptide of the present invention (ie, 7-mer; 100 μM), peptide carrier (1% CMC) or peptide derived from 7-mer was administered topically ( That is, 7-mer-ΔD, 7-mer-ΔS, 7-mer (V→L), and 7-mer (VR→QK); 100 μM ) to the mouse eye, application frequency: three times a day And continued for 14 days. 7-mer-ΔD peptide (LYRVRS; SEQ ID NO: 6) and 7-mer-ΔS peptide (DLYRVR; SEQ ID NO: 7) are N-terminal (D; aspartate) of 7-mer, respectively. And the C-terminal (S; serine) deleted the amino acid residue, and this dipeptide sequence was used as a negative control peptide. In addition, the single and two amino acid-substituted peptides in the 7-mer peptide sequence (ie, 7-mer (V→L) and 7-mer (VR→QK)) have therapeutic effects on dry eye. active.

The integrity of the corneal epithelium was assessed by fluorescent staining and the results are shown in Figures 1A and 1B. Figure 1A shows the results of treatment of dry eye mice with 1% CMC vector, 7-mer and their derivatives. Figure 1B is the result of quantitative analysis of Figure 1A.

As shown in Figure 1A, corneal epithelium treated with 1% CMC produced significant fluorescent staining. Therefore, the corneal epithelium treated with the carrier can be known, and the corneal epithelial damage induced by the dry pressure cannot be prevented. In contrast, mice treated with 7-mer showed a significantly lower fluorescence staining intensity than the vehicle group (3.8 ± 0.31 vs 1.3 ± 0.21), which showed that the mice treated with 7-mer had a relatively intact cornea. The 7-mer synthetic peptide is also shown to protect and/or prevent the development of DED in mice. Mice treated with 7-mer (ie, 7-mer (V→L) or 7-mer (VR→QK)) derivatives also had similar protective effects. Furthermore, mice treated with the 7-mer (ΔD) and 7-mer (ΔS) did not have any protective or preventive effects.

The amount of tear secretion from the experimental animals was measured to evaluate the protective effect of the 7-mer of the present invention and its derivatives on DED, and the results are shown in Fig. 2.

The amount of tear secretion from the mice placed in the CEC (period: 14 days) was examined. The results showed that the mice treated with the 1% CMC vehicle had a significant decrease in the amount of tear secretion (P < 0.05) compared to the healthy mice that were not placed in the CEC and did not receive any treatment. In contrast, mice treated with 7-mer, 7-mer (V→L) or 7-mer (VR→QK) maintained a higher amount of tear secretion (3.7±0.25) compared to the vehicle control group. Vs 5.1 ± 0.21, 5.0 ± 0.16 and 4.9 ± 0.15; Figure 2). Furthermore, 7-mer (ΔD) or 7-mer (ΔS) peptides have no relevant efficacy.

The results of this example demonstrate that the 7-mer synthetic peptide can prevent the development of dry stress induced DED in animals.

Example 2 The synthetic peptide of the present invention has the effect of treating dry stress-induced dry eye syndrome

The animals were placed in CEC for 14 days in accordance with the procedure shown in the "Treatment and Methods" described above to produce dry eye syndrome. After confirming the symptoms of DED by fluorescent staining (Fig. 3A; upper row; 14 days), the animals were removed from the CEC and placed in the general environment (Day 15), followed by administration of eye drops for 7 days (the first treatment) 3A; lower row; day 21; and 3B), wherein the eye drops contain 1% CMC, 1% dexamethasone (Dex), 7-mer (100 μ M) or 7-mer (△) S).

The quantitative results are shown in Figure 3B, and the results show that 0.1% dexamethasone and the control group peptide 7-mer (?S) are not effective in treating DED. In contrast, the 7-mer synthetic peptide of the present disclosure significantly reduced the fraction of the corneal fluorescence staining compared to the control group of mice (1% CMC) (3.6 ± 0.26 vs 1.5 ± 0.18; Figure 3B) ).

The mice treated with 1% CMC vector, 0.1% Dex and control group peptide 7-mer (△S) were stained with H&E, respectively, to identify the detached corneal surface apical cells in each specimen (Fig. 4A) ). There were no exfoliated cells in the corneal tissues of normal healthy mice (untreated) and 7-mer treated mice. The 7-mer synthetic peptide of the present disclosure significantly reduced detachment of apical cells compared to mice in the control group (1% CMC) or 7-mer (ΔS) treated groups (14 ± 1.06 and 13.8 ± 1.03) Vs2.1±0.37; Figure 4B).

Goblet cells are cells in the conjunctival epithelial layer that secrete mucin. The mucin is one of the constituents of the tear layer to place the cornea in an aqueous environment. The results of PAS staining showed that the number of goblet cells was significantly reduced in mice under dry conditions (14 days) and in dry-pressure mice administered with 1% CMC or 7-mer (ΔS) (Fig. 5A). ; lower row). The mice administered with the 7-mer synthetic peptide had the same amount of goblet cells as normal healthy mice (untreated) (Fig. 5A; upper row). Therefore, the 7-mer synthetic peptide of the present invention can be compared to the mouse administered with 1% CMC. Induction of goblet cell growth (7.5 ± 0.96 v.s. 2.7 ± 1.28; Figure 5B).

Based on the above experimental results, it was confirmed that the short synthetic peptide of the present disclosure has a novel function of treating and/or preventing dry eye.

Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

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Claims (14)

A synthetic peptide consisting of 7 contiguous amino acid residues having the sequence DLYRX ₁ X ₂ S (SEQ ID NO: 1), and X ₁ and X ₂ are each an amino acid residue, respectively. The synthetic peptide according to claim 1, wherein the X ₁ and X ₂ are leucine and arginine, respectively. The synthetic peptide of claim 1, wherein the X ₁ and X ₂ are valine and arginine, respectively. The synthetic peptide according to claim 1, wherein the X ₁ and X ₂ are glutamine and arginine, respectively. The synthetic peptide of claim 1, wherein the X ₁ and X ₂ are glutamic acid and lysine, respectively. The synthetic peptide according to claim 1, wherein at least one amino acid residue in the amino acid sequence of SEQ ID NO: 1 is a D-form amino acid. The synthetic peptide according to claim 6, wherein the at least diamino acid residue in the amino acid sequence of SEQ ID NO: 1 is d-amino acid. The use of a synthetic peptide for the preparation of a medicament for treating dry eye disease (DED), wherein the synthetic peptide consists of 7 consecutive amino acid residues, the sequence of which is DLYRX ₁ X ₂ S (sequence number: 1), and each of X ₁ and X ₂ is any amino acid residue. The use of claim 8, wherein the X ₁ and X ₂ are leucine and arginine, respectively. The use of claim 8, wherein the X ₁ and X ₂ are valine and arginine, respectively. The use of claim 8, wherein the X ₁ and X ₂ are glutamic acid and arginine, respectively. The use of claim 8, wherein the X ₁ and X ₂ are glutamic acid and lysine, respectively. The use of claim 8, wherein the at least one amino acid residue in the amino acid sequence of SEQ ID NO: 1 is d-amino acid. The use of claim 13, wherein the at least diamino acid residue in the amino acid sequence of SEQ ID NO: 1 is dextro-amino acid.