Composition comprising a PEDF-derived short-chain peptide for the treatment of neurotrophic keratitis disease

Abstract

A method for treating neurotrophic keratitis (NK) includes administering to a subject in need thereof a composition comprising a peptide having an amino acid sequence selected from SEQ ID NOs: 1-7. The pharmaceutical composition comprises about 10-200 μM of PDSP. The pharmaceutical composition further comprises one or more other active ingredients.

Description

Technical Field

[0001] The present invention relates to PEDF-derived short-chain peptides (PDSP) and their use in the treatment and / or amelioration of neurotrophic keratitis diseases.

[0002] Neurotrophic keratitis (NK) is a rare degenerative disease of the cornea caused by damage to the trigeminal nerve, resulting in loss of corneal sensitivity, disruption of the corneal epithelium, and poor corneal healing. NK was first described as "neuroparalytic keratitis" by Magendie in 1824, and Magendie hypothesized that trophic nerve fibers exist in the trigeminal nerve that regulate tissue metabolism. Subsequently, it has been demonstrated that the trigeminal nerve provides corneal sensation, supplies trophic factors to the cornea, and plays an important role in maintaining the anatomical integrity of the cornea and the function of the ocular surface. Disorders of corneal trigeminal innervation cause morphological and metabolic epithelial disorders, leading to the development of recurrent or persistent epithelial defects. Also, disorders of corneal sensory innervation cause a decrease in both protective reflexes and trophic nerve regulatory factors that are essential for the viability, metabolism, and wound healing of the ocular surface tissue.

[0003] A wide range of ocular and systemic conditions, including herpes simplex keratitis, chemical burns of the eye, corneal surgery, diabetes, multiple sclerosis, and cranial neurosurgery, can cause NK by damaging trigeminal innervation. As described above, loss or reduction of corneal innervation leads to a decrease in the aqueous phase of the tear film, which in turn leads to a decrease in neurotransmitters / trophic factors that impair or inhibit epithelial healing ability (disorders of mitosis and migration). The combination of tear film deficiency and reduced epithelial healing ability makes affected individuals prone to persistent epithelial defects, corneal ulcers, and perforation.

[0004] Early diagnosis, treatment, and careful monitoring of neurotrophic keratitis patients are essential to achieve epithelial healing and prevent the progression of corneal damage in NK patients. The management of NK is based on clinical severity, and the goal of treatment is to halt the progression of corneal damage and promote epithelial healing. To assess severity, the clinical stage of NK is evaluated, ranging from changes in the corneal epithelium (stage 1) to persistent epithelial defects (stage 2) and ulceration (stage 3) that can progress to corneal perforation.

[0005] The clinical management of NK depends on the clinical stage and can be broadly classified into medical management, non-surgical interventions, and surgical management. Surgical treatment is only performed in refractory cases. Although several medical and surgical treatments have been proposed, there is currently no treatment method to restore corneal sensitivity, so NK remains difficult to treat. For the treatment of NK disease, local treatment using nerve growth factor (NGF) has been approved by the FDA.

Summary of the Invention

[0006] One aspect of the present invention relates to a pharmaceutical composition for treating neurotrophic keratitis (NK) in a subject. The pharmaceutical composition for treating NK according to one embodiment of the present invention comprises a peptide having an amino acid sequence selected from SEQ ID NOs: 1-7.

[0007] One aspect of the present invention relates to a method for treating neurotrophic keratitis (NK) in a subject. The method for treating NK according to one embodiment of the present invention comprises administering a composition comprising a peptide having an amino acid sequence selected from SEQ ID NOs: 1-7 to a subject in need thereof.

[0008] Other aspects of the present invention will become apparent from the following description and the accompanying drawings.

Brief Description of the Drawings

[0009] Figure 1 shows a schematic diagram of a treatment plan according to an embodiment of the present invention.

[0010] Figure 2 shows the therapeutic effect of PDSP (SEQ ID NO: 3; referred to as BRM424) on corneal ulcer area. The TA-1 group received BRM424 eye drops treatment, and the TA-2 group received solvent treatment.

[0011] Figures 3A and 3B show the therapeutic effects of BRM424 on corneal fluorescein staining score (FL score) and the image of the corneal surface, respectively.

[0012] Figures 4A and 4B show the therapeutic effects of BRM424 on corneal sensitivity in the von Frey test. Figure 4A shows the results of the contact force regarding the nylon size of the von Frey filament that induced the corneal reflex. Figure 4B shows the results of the contact force in gram units that induced the corneal reflex.

[0013] Figure 5A shows the therapeutic effect of BRM424 on the Schirmer test. Figure 5B shows the therapeutic effect of BRM424 on the tear film break-up time (TFBUT). Detailed Description of the Invention

[0014] Human pigment epithelium-derived factor (PEDF) is a secreted protein containing 418 amino acids and has a molecular weight of approximately 50 kDa. PEDF is a multifunctional protein with many biological functions. (See, for example, US Patent Application Publication No. 2010 / 0047212). Different peptide regions of PEDF are known to undertake different functions. For example, a 34-mer fragment (residues 44-77 of PEDF) has been confirmed to have anti-angiogenic activity, and a 44-mer fragment (residues 78-121 of PEDF) has been confirmed to have neurotrophic properties.

[0015] U.S. Patent Application Publication No. 2010 / 0047212 discloses that PEDF can promote the self-renewal of stem cells. U.S. Patent Nos. 9,051,547 and 9,617,311 disclose that fragments of PEDF that are 20 to 39 amino acids in length (residues 93 to 121 of PEDF) can promote the proliferation of stem cells and wound healing. These short-chain peptides derived from PEDF are referred to herein as PDSP. The PDSP used in the present invention is listed in Table 1 below. [Table 1]

[0016] Embodiments of the present invention relate to PDSP and its use in the prevention and / or treatment of neurotrophic keratitis (NK). NK is a rare degenerative eye disease accompanied by a decrease in corneal sensitivity, spontaneous destruction of corneal epithelial cells, and impaired healing. In severe cases, NK can cause corneal ulceration, melting, and perforation, which may lead to visual impairment in patients. Preclinical studies have shown that all of the PDSP listed in Table 1 are effective in the prevention and / or treatment of NK. In the following specific example, the results of preclinical studies of the 29-mer (SEQ ID NO: 3, hereinafter referred to as "BRM424") are used to illustrate embodiments of the present invention. Those skilled in the art will understand that the results of BRM424 are for illustrative purposes and do not limit the scope of the present invention.

[0017] The NK model is established in chinchilla rabbits by destroying the ophthalmic branch (V1) of the trigeminal nerve using a chemical method to induce NK. Briefly, the animals were anesthetized. Through careful blunt dissection, the fascia between the lateral rectus muscle and the inferior rectus muscle at the lower edge of the rabbit's eye was pulled to form an opening. The trigeminal nerve was carefully separated from the surrounding fascia, and the ophthalmic nerve (V1) of the trigeminal nerve was separated and damaged with a sodium hydroxide solution. The (V1) damage was performed on one eye of each animal for 5 days before treatment.

[0018] Figure 1 shows the research schedule including 28 days of treatment. Based on the corneal ulcer area on day 1 before treatment, a total of 16 animals were randomly assigned to two groups. The animals were administered either BRM424 eye drops (OS containing 0.03% BRM424; TA-1 group) or vehicle (TA-2 group) three times a day (TID, eye drops) to the operated eye for 28 days. During the research period, corneal ulcer area, corneal fluorescein staining, corneal sensitivity, Schirmer test, and tear film breakup time (TFBUT) were measured at baseline (day 0), day 4, day 8, day 15, day 22, and day 29.

[0019] The corneal ulcer area was measured from images taken during slit lamp examination using cobalt blue light. Figure 2 shows the results of corneal ulcer area measurement. The corneal ulcer area on day 0 was 25.98 ± 1.88 mm2 in the TA-1 group and 27.65 ± 2.66 mm2 in the TA-2 group. However, on day 8, it decreased to 7.80 ± 1.45 mm2 and 13.30 ± 2.36 mm2 in the TA-1 group and TA-2 group, respectively. The decrease in the corneal ulcer area in the TA-1 group was greater than that in the TA-2 group on day 8, day 15, and day 29 (p < 0.05), indicating that the TA-1 group had a faster onset of action and was superior to the TA-2 group in promoting corneal epithelial healing.

[0020] The results of the corneal ulcer area are summarized in Table 2.

Table 2

[0021] Corneal injury can be evaluated using corneal fluorescein staining. Figure 3A shows the results of corneal fluorescein staining (FL score), and Figure 3B shows the stained images. The average value of the corneal fluorescein sodium staining score on day 0 was 3.62 ± 0.18 in both the TA-1 group and the TA-2 group, and it decreased to less than 3 by day 8 (TA-1 = 1.38 ± 0.18, TA-2 = 2.36 ± 0.42), indicating rapid onset of action. A significant difference was observed between the TA-1 group and the TA-2 group on days 8 and 15 (P < 0.05). The fluorescein staining score of the TA-1 group reached 1 by day 29.

[0022] The staining results of the corneal area are summarized in Table 3.

Table 3

[0023] Corneal central sensitivity was monitored using a von Frey filament. Calibrated von Frey filaments of various forces (0.008 - 300 g) were applied to the center of the corneal surface of the immobilized animals. The mechanical threshold response was determined by evaluating the blink response of the treated eye induced by the same calibrated von Frey filament twice out of three times. Figures 4A and 4B show the results of the corneal sensitivity test. Before administration (day 0), no corneal reflex was observed in any of the animals even when using the coarsest nylon filament (force 0.008 - 300 g). This result is consistent with the fact that the corneal sensory nerves are damaged.

[0024] As shown in Figure 4B, after 29 days of treatment, corneal sensation recovered slowly in both groups, but corneal sensation in the TA-1 group recovered earlier and to a higher degree than in the TA-2 group. The improvement in corneal sensation indicates the recovery of functional corneal sensory nerves. These results indicate that BRM424 accelerates the recovery of corneal sensory nerves and can thus be used for the treatment of neurotrophic keratitis.

[0025] The results of the corneal sensitivity test are summarized in Table 4. [Table 4]

[0026] Figure 5A shows the results of the Schirmer test regarding tear production. There was no significant difference in the amount of tears between the TA-1 group and the TA-2 group 29 days after local administration. However, the amount of tears on the 29th day in the TA-1 group was approximately twice that on the 0th day. The results of the Schirmer test are summarized in Table 5. [Table 5]

[0027] Figure 5B shows the research results of the tear film break-up time, and these results are summarized in Table 6. There was no significant difference between the TA-1 group and the TA-2 group in the tear film break-up time (TFBUT) 29 days after local administration (see Figure 5B). [Table 6]

[0028] The above results clearly show that the PDSP of the present invention is effective in the treatment of neurotrophic keratitis (NK). Embodiments of the present invention relate to methods for preventing and / or treating NK in a subject. The subject according to the embodiments of the present invention can be a human or an animal. The method according to the embodiments of the present invention may include administering to a subject in need of prevention or treatment of NK a composition comprising a peptide selected from any of the PDSPs listed in Table 1. According to an example of the present invention, the composition may comprise the peptide of the present invention, or a salt of such a peptide, together with a pharmaceutically acceptable carrier or excipient such as distilled water, physiological saline, oil, or gel.

[0029] The pharmaceutical composition of the present invention can be formulated in any suitable dosage form such as a solution, ointment, suspension, gel, or emulsion, and can be formulated at any suitable concentration such as 10 to 200 μM. A person skilled in the art would be able to formulate these at appropriate concentrations to deliver an effective dose without inventive effort. These dosage forms can be formulated for topical application to the eye or for other suitable routes of administration (e.g., oral or injection).

[0030] The embodiments of the present invention have been described by way of a limited number of examples, but a person skilled in the art will understand that other modifications or changes are possible without departing from the scope of the present invention. Therefore, the scope of protection should be limited by the appended claims.

Claims

1. A pharmaceutical composition for the treatment of neurotrophic keratitis (NK), comprising a peptide having any amino acid sequence selected from SEQ ID NOs: 1 to 7.

2. A pharmaceutical composition according to claim 1, comprising a peptide having the amino acid sequence of SEQ ID NO:

3.

3. A pharmaceutical composition according to claim 1 or 2, comprising 10 to 200 μM of the peptide.

4. A pharmaceutical composition according to claim 1, further comprising one or more other active ingredients.

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