Compositions comprising PEDF-derived oligopeptides for treatment of neurotrophic keratitis disease

Abstract

The present invention relates to a method for treating neurotrophic keratitis (NK) comprising administering to a subject in need thereof a composition comprising a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-7. The pharmaceutical composition contains about 10 [mu] M to 200 [mu] M 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 peptide (PDSP) and its use in treating and / or improving neurotrophic keratitis. Background Art

[0002] Neurotrophic keratitis (NK) is a rare corneal degenerative disease caused by damage to the trigeminal nerve, resulting in loss of corneal sensitivity, corneal epithelial breakdown, and poor corneal healing. NK was originally described as "neuroparalytic keratitis" by Magendie in 1824, who hypothesized the presence of trophic nerve fibers in the trigeminal nerve that regulate tissue metabolism. Subsequently, studies have demonstrated that the trigeminal nerve provides corneal sensation and supplies trophic factors to the cornea, playing a key role in maintaining corneal anatomical integrity and ocular surface function. Damage to the trigeminal innervation of the cornea can lead to morphological and metabolic epithelial disorders and the development of recurrent or persistent epithelial defects. Damage to the corneal sensory innervation also leads to a decrease in protective reflexes and trophic neuromodulators, which are critical for the vitality, metabolism, and wound healing of ocular surface tissues.

[0003] A variety of ocular and systemic diseases, including herpetic keratitis, chemical burns to the eye, corneal surgery, diabetes, multiple sclerosis, and neurosurgery, may cause NK by disrupting trigeminal innervation. As mentioned above, loss or reduction of corneal innervation results in a reduction in the aqueous phase of the tear film, which in turn leads to a reduction in neurotransmitters / trophic factors that impair or inhibit the epithelial healing ability (impaired mitosis and migration). The combination of a deficient tear film and impaired epithelial healing ability makes affected individuals more susceptible to persistent epithelial defects, corneal ulcers, and perforations.

[0004] Early diagnosis, treatment, and careful monitoring of patients with neurotrophic keratitis are essential to achieve epithelial healing and prevent progression of corneal damage in patients with NK. Treatment of NK is based on clinical severity, and the goal of treatment is to arrest the progression of corneal damage and promote epithelial healing. To assess severity, the clinical stage of NK is evaluated, which ranges from corneal epithelial changes (stage 1) to persistent epithelial defects (stage 2) and ulcers (stage 3), which may progress to corneal perforation.

[0005] Clinical treatment of NK depends on the clinical stage and can be roughly divided into medical therapy, nonsurgical intervention, and surgical treatment. Surgical treatment is reserved for refractory cases. Although several medical and surgical treatments have been proposed, no therapy can restore corneal sensitivity, so the treatment of NK remains difficult and challenging. Only one topical treatment, nerve growth factor (NGF), is approved by the U.S. Food and Drug Administration (FDA) for the treatment of NK disease. Summary of the invention

[0006] One aspect of the present invention relates to a pharmaceutical composition for treating neurotrophic keratitis (NK) in a subject. According to one embodiment of the present invention, the pharmaceutical composition for treating NK comprises a peptide having an amino acid sequence selected from SEQ ID NO: 1-7.

[0007] One aspect of the present invention relates to a method of treating neurotrophic keratitis (NK) in a subject. According to one embodiment of the present invention, the method of treating NK comprises administering to a subject in need thereof a composition comprising a peptide having an amino acid sequence selected from SEQ ID NO: 1-7.

[0008] Other aspects of the invention will be explained using the following description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0011] Figure 3A and Figure 3B The therapeutic effects of BRM424 on corneal fluorescein staining score (FL score) and corneal surface image are shown respectively.

[0012] Figure 4A and Figure 4B The therapeutic effect of BRM424 on corneal sensitivity in the Von Frey test was shown. Figure 4A Contact force results for nylon sizes of Von Frey filaments triggering the corneal reflex are shown. Figure 4B Results are shown for the contact force that triggers the corneal reflex in grams.

[0013] Figure 5A The therapeutic effect of BRM424 was shown in the tear secretion test (Schirmer Test). Figure 5B The therapeutic effect of BRM424 on tear film breakup time (TFBUT) was shown. DETAILED DESCRIPTION

[0014] Human pigment epithelium derived factor (PEDF) is a secretory protein containing 418 amino acids with a molecular weight of about 50 kDa. PEDF is a multifunctional protein with many biological functions (see U.S. Patent Application Publication No. 2010 / 0047212). Different peptide regions of PEDF were found to be responsible for different functions. For example, a 34-mer fragment (residues 44 to 77 of PEDF) has been identified to have anti-angiogenic activity, while a 44-mer fragment (residues 78 to 121 of PEDF) has been identified 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 No. 9,051,547 and U.S. Patent No. 9617311 disclose that PEDF fragments (residues 93-121 of PEDF) with a length of 20-39 amino acids can promote stem cell proliferation and wound healing. These PEDF-derived short peptides are referred to as PDSP in this application. The PDSP used in the present invention is listed in Table 1 below:

[0016] Table 1

[0017]

[0018] Embodiments of the present invention relate to PDSP and its use in preventing and / or treating neurotrophic keratitis (NK). NK is a rare degenerative eye disease with reduced corneal sensitivity, spontaneous destruction of corneal epithelial cells, and impaired healing. In severe cases, NK can cause corneal ulcers, melting and perforation, resulting in impaired vision in patients. In preclinical studies, it has been found that all PDSPs listed in Table 1 can effectively prevent and / or treat NK. The following specific examples will use the preclinical study results of 29-mer (SEQ ID NO: 3, hereinafter referred to as "BRM424") to illustrate the embodiments of the present invention. Those skilled in the art will understand that the results of BRM424 are for illustration only and are not intended to limit the scope of the present invention.

[0019] Example

[0020] The NK model was established in Chinchilla rabbits, and NK was induced by chemically destroying the ophthalmic nerve branch (V1) of the trigeminal nerve. In brief, the animals were anesthetized. By careful blunt dissection, an opening was formed by pulling the fascia between the lateral rectus muscle and the inferior rectus muscle at the lower edge of the rabbit's eye. The trigeminal nerve was carefully separated from the surrounding fascia, and the ophthalmic nerve branch (V1) of the trigeminal nerve was isolated and injured with sodium hydroxide solution. V1 injury was performed on the unilateral eye of each animal 5 days before treatment.

[0021] Figure 1The study schedule including 28 days of treatment is shown. Before treatment on day 1, a total of 16 animals were randomly divided into two groups according to the area of ​​corneal ulcers, and these animals received BRM424 eye drops (OS containing 0.03% BRM424; TA-1 group) or vehicle (TA-2 group) in the operated eye, three times a day (TID, eye drops) for 28 days. During the study, the corneal ulcer area, corneal fluorescein staining, corneal sensitivity, tear secretion test (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.

[0022] Corneal ulcer area was measured based on images taken during slit-lamp examination with cobalt blue light. Figure 2 The results of corneal ulcer area measurement are shown. On day 0, the corneal ulcer areas of the TA-1 and TA-2 groups were 25.98 ± 1.88 mm, respectively. 2 and 27.65±2.66mm 2 By the 8th day, the corneal ulcer area in the TA-1 group and the TA-2 group was reduced to 7.80±1.45mm 2 and 13.30±2.36mm 2 The reduction in corneal ulcer area in the TA-1 group on the 8th, 15th, and 29th days was greater than that in the TA-2 group (p<0.05), indicating that the TA-1 group was more effective and was superior to the TA-2 group in promoting corneal epithelial healing.

[0023] Table 2 summarizes the results of corneal ulcer area.

[0024] Table 2. Corneal ulcer area (mm 2 , )

[0025]

[0026] NS, not significant

[0027] Corneal damage can be assessed by corneal fluorescein staining. Figure 3A The results of corneal fluorescein staining (FL score) are shown. Figure 3B The staining images are shown. The average corneal fluorescein sodium staining score of the TA-1 and TA-2 groups on day 0 was 3.62±0.18, which dropped to below 3 on day 8 (TA-1=1.38±0.18, TA-2=2.36±0.42), indicating a rapid onset of effect. Significant differences were observed between the TA-1 and TA-2 groups on days 8 and 15 (P<0.05). The fluorescein staining score of the TA-1 group reached 1 on day 29.

[0028] Table 3 summarizes the results of corneal ulcer area.

[0029] Table 3. Fluorescein staining scores ( )

[0030]

[0031]

[0032] NS, not significant

[0033] Central corneal sensitivity was monitored using Von Frey filaments. Various forces of a calibrated Von Frey filament (0.008-300 g) were applied to the center of the corneal surface of stationary animals. The mechanical threshold response was determined by evaluating the therapeutic blink response evoked by the same calibrated Von Frey filament twice out of three times. Figure 4A and Figure 4B The results of the corneal sensitivity test are shown. Before dosing (day 0), all animals had no corneal reflex even when the thickest nylon thread (force 0.008-300g) was used. This result is consistent with the fact that the corneal sensory nerves are damaged.

[0034] like Figure 4B As shown, after 29 days of treatment, corneal sensation in both groups recovered slowly, but the TA-1 group recovered faster and improved better than the TA-2 group. Improved corneal sensation indicates the recovery of functional corneal sensory nerves. These results suggest that BRM424 can accelerate the recovery of corneal sensory nerves and therefore can be used to treat neurotrophic keratitis.

[0035] Table 4 summarizes the results of the corneal sensitivity tests.

[0036] Table 4. Corneal sensitivity

[0037]

[0038] Figure 5A The results of the tear secretion (Schirmer) test for tear production are shown. After 29 days of topical administration, there was no significant difference in tear volume between the TA-1 group and the TA-2 group. However, the tear volume of the TA-1 group on day 29 was about 2 times higher than that on day 0. The above tear secretion test results are summarized in Table 5.

[0039] Table 5. Tear secretion test (mm, )

[0040]

[0041] Figure 5BThe results of the tear film breakup time study are shown and are summarized in Table 6. After 29 days of topical administration, there was no significant difference in tear film breakup time (TFBUT) between the TA-1 and TA-2 groups (see Table 6). Figure 5B ).

[0042] Table 6. TFBUT (second, )

[0043]

[0044] The above results clearly demonstrate the efficacy of the PDSP of the present invention in treating neurotrophic keratitis (NK). Embodiments of the present invention relate to methods for preventing and / or treating NK in a subject. The subject of an embodiment of the present invention may be a human or an animal. The method of one embodiment of the present invention may include administering a pharmaceutical composition to a subject in need of preventing or treating NK, the pharmaceutical composition comprising a peptide selected from any PDSP listed in Table 1. According to an embodiment of the present invention, the composition may comprise a peptide of the present invention or a salt of the peptide, and a pharmaceutically acceptable carrier or excipient, such as distilled water, saline, oil or gel.

[0045] The compositions of the present invention can be formulated in any suitable dosage form, such as solutions, ointments, suspensions, gels or emulsions, which can be formulated in any suitable concentration, such as 10-200 μM. Those skilled in the art will be able to formulate these drugs in appropriate concentrations to provide effective doses without creative effort. These dosage forms can be formulated for topical application to the eye or other suitable routes of administration (e.g., oral or injection).

[0046] A limited number of embodiments have been used to illustrate the implementation of the present invention. Those skilled in the art will appreciate that other modifications and variations 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 treating neurotrophic keratitis (NK), comprising: a peptide having an amino acid sequence selected from SEQ ID NO: 1-7.

2. The pharmaceutical composition according to claim 1, characterized in that The peptide has the amino acid sequence shown in SEQ ID NO:

3.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that The pharmaceutical composition comprises about 10-200 μM of the peptide.

4. The method according to any one of claims 1 to 3, characterized in that The pharmaceutical composition further comprises one or more other active ingredients.

5. A method for treating neurotrophic keratitis (NK), comprising: A pharmaceutical composition comprising a peptide having an amino acid sequence selected from SEQ ID NOs: 1-7 is administered to a subject in need thereof.

6. The method according to claim 5, characterized in that The peptide has the amino acid sequence shown in SEQ ID NO:

3.

7. The method according to claim 5 or 6, characterized in that: The pharmaceutical composition comprises about 10-200 μM of the peptide.

8. The method according to any one of claims 5 to 7, characterized in that: The pharmaceutical composition further comprises one or more other active ingredients.

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