Peptide composition

Abstract

Peptide compositions and methods for inhibiting angiogenesis, or pathological or abnormal blood vessel development, in a human or other animal subject are provided. The present invention provides a composition of matter comprising a peptide consisting of or including the amino acid sequence Arg-Ala-Glu.

Description

Technical Field

[0001] Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 62 / 521,984, filed on June 19, 2017, entitled "Peptide Compositions and Related Methods", the entire disclosure of which is hereby incorporated by reference in its entirety.

[0002] The present invention generally relates to the fields of biology and medicine, and more specifically, to peptide compositions and methods of using the same.

Background Art

[0003] In accordance with 37 CFR 1.71(e), this patent document contains material subject to copyright protection, and the owner of this patent document retains all copyrights whatsoever. Throughout this patent application, amino acids may be referred to interchangeably using the following names, three-letter codes, and one-letter codes:

[0004]

Table 1

[0005] Applicants have shown that the synthetic oligopeptide glycyl - arginyl - glycyl - cysteine (acid) - threonyl - proline inhibits a number of integrins and has significant anti - angiogenic, anti - inflammatory, neuroprotective and other effects (SEQ ID NO: 1)(Developed by (ALG-1001 or Luminate®, Allegro Ophthalmics, LLC). When administered to the eye, ALG-1001 causes vitreolysis and posterior vitreous detachment (PVD) and can be used for the treatment of eye diseases such as wet macular degeneration (WMD), dry macular degeneration (DMD), proliferative diabetic retinopathy (PDR), diabetic macular edema (DME), and vitreomacular traction (VMT). Further information regarding ALG-1001 and related compounds can be found in U.S. Patent No. 9,018,352 entitled "Peptide Compositions and Therapeutic Uses Thereof," No. 9,872,886 entitled "Compositions and Methods for Inhibiting Cellular Adhesion or Directing Diagnostic or Therapeutic Agents to RGD Binding Sites," and No. 9,896,480 entitled "Integrin Receptor Antagonists and Their Methods of Use," as well as U.S. Patent Application No. 15 / 874,814 entitled "Therapeutic and Neuroprotective Peptides" which is currently pending, and the entire disclosures of such patents and patent applications are hereby expressly incorporated by reference herein.)

[0006] The applicant conceived and synthesized the peptides listed in Table 1 below. [Table 2] As described below, Applicant has synthesized a number of additional novel peptides and conducted initial tests, many of which show a therapeutic effect in in vivo tests. [Summary of the Invention] [Means for Solving the Problems]

[0007] In accordance with the present invention, there are provided peptide compounds and methods for inhibiting angiogenesis of pathological or abnormal blood vessel development in a human or animal subject. According to one aspect of the present invention, a formula: Y-X-Z A composition of a substance comprising a peptide consisting of or containing an amino acid sequence having: Wherein: Y = R, H, K, Cys(acid), G, or D, X = G, A, Cys(acid), R, G, D, or E, Z = Cys(acid), G, C, R, D, N, or E, is provided. Such peptides have the amino acid sequences; R-G-Cys(acid), R-R-Cys, R-Cys ( acid)-G, Cys(acid)-R-G, Cys(acid)-G-R, R-G-D, R-G-Cys(acid) 、 H-G-Cys(acid), R-G-N, D-G-R, R-D-G, R-A-E, K-G-D, R-G-Cys(acid)-G-G-G-D-G (SEQ ID NO: 16) , cyclo-{R-G-Cys(acid)-F-N-Me-V} (SEQ ID NO: 4) , R-A-Cys(acid), R-G-C, K-G-D, Cys(acid)-R-G, Cys(acid)-G-R, cyclo-{R-G-D-D-F-NMe-V} (SEQ ID NO: 24) , H-G-Cys(acid), and may include or consist of their salts. Possible salts include, but are not limited to, acetate, trifluoroacetate (TFA), and hydrochloride. Such peptides are useful, at least, for inhibiting angiogenesis of pathological or abnormal blood vessel development in a human or animal subject.

[0008] Furthermore, in some embodiments, the composition of matter comprises, or consists of only, a peptide having the following general formula 1: Gly-X-Thr-Pro wherein X is selected from Arg-Ala-Cys(acid); Arg-Gly-Cys; Arg-Asp-Gly; Arg-Ala-Glu; Arg-Gly-Asn; Asp-Gly-Arg; Cys(acid)-Gly-Arg and Lys-Gly-Asp. Furthermore, in accordance with the present invention, the peptide of the present invention, or the synthetic oligopeptide glycyl-arginyl-glycyl-cysteic acid-threonyl-proline (SEQ ID NO: 1)It may be administered to a human or animal subject for the purpose of inhibiting angiogenesis of pathological or abnormal blood vessel development, in combination with taurine.

[0009] Furthermore, according to the present invention, there is provided a method for inhibiting angiogenesis of pathological or abnormal blood vessel development in a human or animal subject in need of inhibiting angiogenesis of pathological or abnormal blood vessel development, the method comprising the step of administering to such a subject a composition comprising a therapeutically effective amount of a peptide as summarized above. In some cases, such a method may be carried out for treating an eye disease or disorder in which angiogenesis or pathological or abnormal blood vessel development occurs. Such eye diseases or disorders include, but are not necessarily limited to, diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity (ROP), sickle cell retinopathy, retinal vein occlusion, ischemia-induced retinopathy, and certain eye inflammatory diseases.

[0010] Furthermore, according to the present invention, there is provided a method for inhibiting angiogenesis or pathological or abnormal blood vessel development at a location outside the eye of a human or animal subject. In some cases, such a method may be carried out for inhibiting the growth or metastasis of angiogenic tumors.

[0011] Still further aspects and details of the present invention will be understood by reading the detailed description and examples set forth below in this specification.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] The following detailed description and the accompanying drawings to which it refers are not necessarily intended to describe all examples or embodiments of the present invention, but are intended to describe some examples or embodiments. The embodiments described are considered to be illustrative in all respects and not limiting. The content of the detailed description of the present invention and the accompanying drawings do not limit the scope of the present invention in any way.

[0014] A number of diseases and disorders, including diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity (ROP), sickle cell retinopathy, retinal vein occlusion, ischemia-induced retinopathy, certain ocular inflammatory diseases, and the growth or metastasis of angiogenic tumors, are known to cause angiogenesis or the development of pathologic or abnormal blood vessels. Applicants have found a number of compounds that have been shown to be active in an anti-angiogenic mouse ROP model, as described below. Based on this, such compounds are potentially useful in the treatment of diseases and disorders known to cause angiogenesis or the development of pathologic or abnormal blood vessels, including but not limited to those described above.

[0015] Each test compound was prepared for injection in sterile water containing 0.08 mg / 100 μL of sodium chloride and 0.005 mg / 100 μL of trisodium citrate, the concentration of the peptide was 2.0 mg / 100 μL, and the pH = 2.7, and was dispensed into sterile vials by sterile filtration. The taurine test compound was obtained from Sigma Aldrich company, which was >99% pure, prepared by the same method as previously described, and had a concentration of 3.0 mg / 100 μL. 2.0 mg / 100 μL of R-G-Cys(acid) + 3.0 mg / 100 μL of taurine was prepared by the same method as above.

[0016] To screen test compounds for activity against hypoxia-induced retinal angiogenesis, a well-established model of retinopathy of prematurity (ROP) in mice was used. Litters of C57BI / 6 mice were placed in 75% oxygen on postnatal (P) day 7 and returned to room air on (P) day 12. The pups were randomly assigned to treatment groups of 4 - 10 animals per group. The pups were treated as follows. The treatment eye was treated by intravitreal injection of 1.0 microliter of a solution containing 20 micrograms of the test compound.

[0017] On postnatal day 17, 5 days after intravitreal injection, the animals were sacrificed, the retinas were flat-mounted, and the area of angiogenesis in each retina was measured by fluorescent dextran image analysis. The applicant 、R -G-cysteic acid (acid) as the oligopeptide glycyl-arginyl-glycyl-cysteic acid (acid)-threonyl-proline (SEQ ID NO: 1) (ALG-1001 or Luminate® , Allegro Ophthalmics, LLC) Active was identified as a motif. The trifluoroacetate (TFA) and acetate salts of the R-G-cysteic acid (acid) tripeptide (Test Compounds 1 and 2) were tested in both the ROP mouse model as described above, as well as in a mouse model of laser-induced choroidal neovascularization (the "CNV mouse model") generally described by Lambert, V et al., Laser-Induced Choroidal Neovascarization Model to Study Age Related Macular Degeneration in Mice, Nature Protocols, 8;2197 - 2211 (2013). Animals assigned to the "control" group were In this model known to be inactive Gly- Arg-Gly-Glu -Thr-Pro (SEQ ID NO: 26) ( Inactive control peptide) by intravitreal injection. Some of the experiments included an additional "positive control" group. Animals assigned to the "positive control" group were In this modelknown to be active Gly- Arg-Gly-Cys (acid) -Thr-Pro TFA (SEQ ID NO: 37) was treated by intravitreal injection of

[0018] The following table 2 summarizes the angiogenesis inhibitory effects of each test compound at the tested doses. In each case, data were obtained using the ROP mouse model, except for the two table entries specifically marked as "CNV". Only the table entries marked as "CNV" show data obtained from the CNV mouse model. The bar graphs showing the test results summarized in the table 2 are also provided herein as Figures 1 - 27. When shown in the figures, the tests were performed in a blinded fashion so that the identity or structure of each test compound was not known to the test operator.

[0019]

Table 3

[0020] For each of the test compounds, except for cyclic test compounds 12 and 13, the R - G - G - Cys (acid) - T - G motif of P (SEQ ID NO: 1) (ALG - 1001) was rearranged and / or substituted by three different amino acid motifs having the amino acid sequence defined by General Formula 2 (below). As a result, test compounds having the general formula defined by - G - ( 2 ) were obtained. The following ) test test compounds were obtained.

[0021]

Chemical formula

[0022] wherein Y = R*, H, K, Cys(acid), G, or D, X = G*, A, Cys(acid), R, G, D, or E, Z = Cys (acid) *, G, cysteine, R, D, N, or E.

[0023] * indicates a component of the GRGCys(acid)TP (SEQ ID NO: 1) (ALG - 1001) used as a positive control of R in the GCys(acid) binding motif. Based on the results of the ROP and CNV tests summarized above, the presence of arginine, alanine, and cysteic acid in the GRGCys(acid)TP peptide (SEQ ID NO: 1) (ALG - 1001 / Luminate), particularly the sequences R - G - Cys and R - A - Cys, play important roles in the inhibition of angiogenesis. Furthermore, in the presence of arginine, the substitution of cysteic acid with neutral amino acids showed a strong inhibitory effect in these experiments. Based on the initial data presented herein, a specific structure / activity relationship is suggested with respect to specific changes made to the R - G - cysteic acid active motif. For example, when the amino acid R (i.e., the Y component) of the R - G - cysteic acid binding motif is substituted with a basic or acidic amino acid, the anti - angiogenic effect of the peptide is reduced, while in the presence of arginine in the active motif, aspartic acid as the component Y seems to promote the anti - angiogenic effect of the peptide.

[0024] R - G - cysteic acid Active When the amino acid G (i.e., the X component) of the motif is substituted with a basic or acidic amino acid, the anti - angiogenic effect of the peptide decreases. However, in the presence of arginine (strong hydrogen bonding), two carbon - length spaces for hydrophobic interactions (alanine and aspartic acid) will not affect the anti - angiogenic effect of the peptide.

[0025] R - G - cysteic acid ActiveWhen the Cys(acid) (i.e., the Z component) of the motif is substituted with a neutral amino acid, the angiogenesis inhibitory activity of the peptide increases. On the other hand, substitution of the Z component with an acidic or basic amino acid causes a decrease in angiogenesis inhibitory activity.

[0026] All is the oligopeptide glycyl-arginyl-glycyl-cysteic acid(acid)-threonyl-proline (SEQ ID NO: 1) (ALG-1001 or Luminate® , Allegro Ophthalmics, LLC) has shown that the R-G-cysteic acid(acid) is important for the inhibition of angiogenesis. Also, the addition of 3 parts of taurine to 1 part of glycyl-arginyl-glycyl-cysteic acid(acid)-threonyl-proline (SEQ ID NO: 1) (ALG-1001) promotes angiogenesis inhibitory activity.

[0027] Although the present invention has been described above herein with reference to specific embodiments or implementations of the present invention, it should be understood that various additions, deletions, modifications, and alterations may be made to the described embodiments and implementations without departing from the intended spirit and scope of the present invention. Unless otherwise specified or unless it results in an embodiment or implementation that is inappropriate for its intended use, for example, any element, step, member, component, composition, reactant, part number, or portion of an embodiment or implementation may be incorporated into or used with another embodiment or implementation. Also, if the steps of a method or process are described or listed in a particular order, unless otherwise specified or unless it results in a method or process that is inappropriate for its intended use, the order of such steps may be changed. Further, unless otherwise noted, the elements, steps, members, components, compositions, reactants, part numbers, or portions of any invention or implementation described herein may optionally be present or may be utilized in the absence or substantial absence of any other element, step, member, component, composition, reactant, part number, or portion. All reasonable additions, deletions, modifications, and alterations are considered equivalents of the described embodiments and implementations and are included within the scope of the following claims. The technical idea that can be grasped from the above embodiments is described below as an appended note. [Appended Note 1] Formula: Y-X-Z A composition of matter comprising, or consisting of, a peptide having an amino acid sequence, wherein In the formula, Y = R, H, K, Cys(acid), G, or D, X = G, A, Cys(acid), R, G, D, or E, Z = Cys(acid), G, C, R, D, N, or E, Composition. [Appended Note 2] The composition according to Appended Note 1, wherein the amino acid sequence is R-G-Cys(acid). [Appended Note 3] The composition according to Appended Note 2, wherein the peptide comprises R-G-Cys(acid). [Appended Note 4] The composition according to Appended Note 2, wherein the peptide comprises R-G-Cys(acid). [Appended Note 5] The composition according to Appended Note 2, wherein the peptide comprises R-G-Cys(acid)-G-G-G-D-G. [Appended Note 6] The composition according to Appended Note 2, wherein the peptide comprises cyclo-{R-G-Cys(acid)-F-N-Me-V}. [Appended Note 7] The composition according to Appended Note 1, wherein the amino acid sequence is R-A-Cys(acid). [Appended Note 8] The composition according to Appended Note 1, wherein the amino acid sequence is R-G-cysteine. [Appended Note 9] The composition according to Appended Note 1, wherein the amino acid sequence is R-Cys(acid)-G. [Appended Note 10] The composition according to Supplementary Note 1, wherein the amino acid sequence is Cys(acid)-R-G. [Supplementary Note 11] The composition according to Supplementary Note 1, wherein the amino acid sequence is Cys(acid)-G-R. [Supplementary Note 12] The composition according to Supplementary Note 1, wherein the amino acid sequence is R-G-D. [Supplementary Note 13] The composition according to Supplementary Note 12, wherein the peptide comprises cyclo-{R-G-D-D-F-N-Me-V}. [Supplementary Note 14] The composition according to Supplementary Note 1, wherein the amino acid sequence is H-G-Cys(acid). [Supplementary Note 15] The composition according to Supplementary Note 1, wherein the amino acid sequence is R-G-N. [Supplementary Note 16] The composition according to Supplementary Note 1, wherein the amino acid sequence is D-G-R. [Supplementary Note 17] The composition according to Supplementary Note 1, wherein the amino acid sequence is R-D-G. [Supplementary Note 18] The composition according to Supplementary Note 1, wherein the amino acid sequence is R-A-E. [Supplementary Note 19] The composition according to Supplementary Note 1, wherein the amino acid sequence is K-G-D. [Supplementary Note 20] The composition according to any one of Supplementary Notes 1 to 19, wherein the peptide comprises a salt. [Supplementary Note 21] The composition according to Supplementary Note 20, wherein the salt is selected from the forms of trifluoroacetate, acetate, and hydrochloride. [Supplementary Note 22] The composition according to any one of Supplementary Notes 1 to 19, further comprising taurine. [Supplementary Note 23] A composition comprising G-R-G-Cys(acid)-T-P or a salt thereof in combination with taurine. [Supplementary Note 24] A method for inhibiting angiogenesis or the development of pathological or abnormal blood vessels in a human or animal subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition according to any one of Supplementary Notes 1 to 19. [Supplementary Note 25] The method according to Supplementary Note 24, wherein the composition further comprises taurine. [Supplementary Note 26] The method according to any one of Supplementary Notes 24 or 25, wherein the method is carried out to inhibit angiogenesis or the development of pathological or abnormal blood vessels in the eye. [Supplementary Note 27] The method according to Supplementary Note 26, wherein the method is carried out to treat a disease or disorder selected from diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity (ROP), sickle cell retinopathy, retinal vein occlusion, ischemia-induced retinopathy, and inflammatory diseases of the eye. [Supplementary Note 28] The method according to Supplementary Note 24, wherein the method is carried out to inhibit the angiogenesis development of pathological or abnormal blood vessels outside the eye. [Supplementary Note 29] The method according to appended claim 28, wherein the method is carried out to inhibit the growth or metastasis of an angiogenesis tumor.

Claims

1. A composition comprising a substance containing a peptide consisting only of the amino acid sequence Gly-Arg-Ala-Glu-Thr-Pro.

2. The composition according to claim 1, wherein the peptide is prepared as a solution for injection.

3. The composition according to claim 1 or 2, wherein the peptide is a salt.

4. The composition according to claim 3, wherein the peptide is a salt selected from the forms of trifluoroacetate, acetate, and hydrochloride.

5. A composition according to any one of claims 1 to 4, which is combined with taurine.

6. A composition according to any one of claims 1 to 5 for inhibiting angiogenesis or the development of pathological or abnormal blood vessels in a human or animal subject in need thereof, wherein a therapeutically effective amount of the composition is administered to the subject.

7. The composition according to claim 6, wherein the composition is administered to the eye of the subject to treat an eye disease or disorder of the subject in which angiogenesis or the development of pathological or abnormal blood vessels has occurred.

8. The eye disease or disorder according to claim 7, which is selected from diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity (ROP), sickle cell retinopathy, retinal vein occlusion, ischemia-induced retinopathy, and inflammatory eye diseases.

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