PEPTIDE COMPOSITIONS AND RELATED METHODS

Abstract

Peptide compositions and methods for inhibiting neovascularization or development of pathological or aberrant blood vessels in human or other animal subjects.

Claims

1. A composition of matter comprising a peptide which consists of or includes an amino acid sequence having the formula
YXZ wherein: Y=R, H, K, Cys(acid), G or D; X=G, A, Cys(acid), R, G, D or E; and Z=Cys(acid), G, C, R, D, N or E.

2. (canceled)

3. (canceled)

4. (canceled)

5. A composition according to claim 2 wherein the peptide comprises R-G-Cys(acid)-G-G-G-D-G.

6. A composition according to claim 2 wherein the peptide comprises Cyclo-{R-G-Cys(acid)-F-N-Me-V}.

7. A composition according to claim 1 wherein the amino acid sequence is R-A-Cys (acid).

8. A composition according to claim 1 wherein the amino acid sequence is R-G-Cysteine.

9. A composition according to claim 1 wherein the amino acid sequence is R-Cys(acid)-G.

10. A composition according to claim 1 wherein the amino acid sequence is Cys(acid)-R-G.

11. A composition according to claim 1 wherein the amino acid sequence is Cys(acid)-G-R.

12. A composition according to claim 1 wherein the amino acid sequence is R-G-D.

13. A composition according to claim 12 wherein the peptide comprises Cyclo-{R-G-D-D-F-NMe-V}.

14. A composition according to claim 1 wherein the amino acid sequence is H-G-Cys(acid).

15. A composition according to claim 1 wherein the amino acid sequence is R-G-N.

16. A composition according to claim 1 wherein the amino acid sequence is D-G-R.

17. A composition according to claim 1 wherein the amino acid sequence is R-D-G.

18. A composition according to claim 1 wherein the amino acid sequence is R-A-E.

19. A composition according to claim 1 wherein the amino acid sequence is K-G-D.

20. A composition according to any of claims 1 through 19 wherein the peptide comprises a salt.

21. A composition according to claim 20 wherein the salt is selected from trifluoroacetate, acetate and hydrochloride salt forms.

22. A composition according to any of claims 1 through 19 further comprising taurine.

23. A composition comprising G-R-G-Cys(acid)-T-P or a salt thereof in combination with Taurine.

24.-29. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either His-Gly-Cys(acid) (Test Compound No. 14) or Control Peptide (Arg-Gly-Glu).

[0018] FIG. 2 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Arg-Ala-Cys (Test Compound No. 3) or Control Peptide (Arg-Gly-Glu).

[0019] FIG. 3 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Test Compound No. 1/positive control), Arg-Ala-Asp (Test Compound No. 23) or Control Peptide (Arg-Gly-Glu).

[0020] FIG. 4 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Arg-Ala-Cys(Acid) (Test Compound No. 3) or Control Peptide (Arg-Gly-Glu).

[0021] FIG. 5 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Arg-Gly-Cys (Test Compound No. 4) or Control Peptide (Arg-Gly-Glu).

[0022] FIG. 6 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Arg-Gly-Cys(acid)TFA (Masked) (Test Compound No. 1) or Control Peptide (Arg-Gly-Glu).

[0023] FIG. 7 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Lys-Glys-Asp (Test Compound No. 20) or Control Peptide (Arg-Gly-Glu).

[0024] FIG. 8 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either His-Gly-Cys(Acid) (Test Compound No. 14) or Control Peptide (Arg-Gly-Glu).

[0025] FIG. 9 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Lys-Gly-Cys(acid) (Test Compound No. 6) or Control Peptide (Arg-Gly-Glu).

[0026] FIG. 10 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Arg-Cys(Acid)-Gly (Test Compound No. 5) or Control Peptide (Arg-Gly-Glu).

[0027] FIG. 11 is a bar graph of retinal neovascular area in CNV mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Arg-Gly-Cys(Acid) Acetate (Test Compound No. 2) or Control Peptide (Arg-Gly-Glu).

[0028] FIG. 12 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Arg-Gly-Cys(Acid) Acetate (Test Compound No. 2) or Control Peptide (Arg-Gly-Glu).

[0029] FIG. 13 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Asp-Gly-Arg (Test Compound No. 17) or Control Peptide (Arg-Gly-Glu).

[0030] FIG. 14 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Asp (Test Compound No. 15) or Control Peptide (Arg-Gly-Glu).

[0031] FIG. 15 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Cys(Acid)-Gly (Test Compound No. 18) or Control Peptide (Arg-Gly-Glu).

[0032] FIG. 16 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)-Gly-Gly-Asp-Gly (Test Compound No. 7) or Control Peptide (Arg-Gly-Glu).

[0033] FIG. 17 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Ala-Glu (Test Compound No. 19) or Control Peptide (Arg-Gly-Glu).

[0034] FIG. 18 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Gly-Cys(acid)-Arg (Test Compound No. 11) or Control Peptide (Arg-Gly-Glu).

[0035] FIG. 19 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Cys(Acid)-Ala-Arg (Test Compound No. 10) or Control Peptide (Arg-Gly-Glu).

[0036] FIG. 20 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Glu-Gly (Test Compound No. 22) or Control Peptide (Arg-Gly-Glu).

[0037] FIG. 21 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Cys(acid)-Arg-Gly (Test Compound No. 8) or Control Peptide (Arg-Gly-Glu).

[0038] FIG. 22 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Asn (Test Compound No. 16) or Control Peptide (Arg-Gly-Glu).

[0039] FIG. 23 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Cyclo-{R-G-D-D-F-NMe-V} (Test Compound No. 13) or Control Peptide (Arg-Gly-Glu).

[0040] FIG. 24 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Cyclo-{R-G-Cys(acid)-F-N-Me-V} (Test Compound No. 12) or Control Peptide (Arg-Gly-Glu).

[0041] FIG. 25 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Cys(Acid)-Gly-Arg (Test Compound No. 9) or Control Peptide (Arg-Gly-Glu).

[0042] FIG. 26 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either His-Gly-Cys(Acid) (Test Compound No. 14) or Control Peptide (Arg-Gly-Glu).

[0043] FIG. 27 is a bar graph of retinal neovascular area in ROP mouse eyes following treatment with either Arg-Gly-Cys(acid)TFA (Positive Control), Taurine (Test Compound No. 25), Arg-Gly-Cys(acid).TFA+Taurine (Test Compound No. 24) or Control Peptide (Arg-Gly-Glu).

DETAILED DESCRIPTION

[0044] The following detailed description and the accompanying drawings to which it refers are intended to describe some, but not necessarily all, examples or embodiments of the invention. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The contents of this detailed description and the accompanying drawings do not limit the scope of the invention in any way.

[0045] A number of diseases and disorders are known to cause neovascularization or development of pathological or aberrant blood vessels, including diabetic retinopathy, neovascular age-related macular degeneration, retinopathy of prematurity (ROP), sickle cell retinopathy, retinal vein occlusion, ischemia-induced retinopathy, certain inflammatory diseases of the eye and the growth or metastasis of a vascularized tumors. Applicant has discovered a number of compounds that are shown to be active in an anti-neovascularization mouse ROP model as described below. On this basis, such compounds are potentially useful in the treatment of diseases and disorders which are known to cause neovascularization or development of pathological or aberrant blood vessels, including but not limited to those diseases and disorders listed above.

[0046] Each test compound was prepared in sterile water for injection, containing 0.08 mg/100 L of sodium chloride and 0.005 mg/100 L of trisodium citrate, the concentration of the peptide was at a concentration of 2.0 mg/100 L and pH=2.7 and dispensed by sterile filtration into sterile vials. The Taurine test compound was obtained from Sigma Aldrich company, which was >99% pure, and prepared the same way as mentioned previously, having a concentration of 3.0 mg/100 L. The R-G-Cys(acid) at 2.0 mg/100 L+Taurine at 3.0 mg/100 L were prepared the same way as mentioned above

[0047] To screen the test compounds for activity against ischemia-induced retinal neovascularization, the well-established model of retinopathy of prematurity (ROP) in mice was used. Litters of C57BI/6 mice were placed in 75% oxygen at postnatal day (P) 7, returned to room air at day (P) 12. The Pups were randomly assigned to treatment groups of 4 to 10 animals per group. The pups were treated as follows: Treatment eyes were treated by intravitreal injection of 1.0 microliters of solution containing 20 micrograms of Test Compound.

[0048] On post-natal day 17, 5 days after intravitreal injection, the animals were sacrificed, the retinas were flat mounted and the area of neovascularization in each retina was determined by Fluorescein-dextran image analysis.

[0049] Applicant has identified the tripeptide R-G-Cysteic(Acid) as an integrin binding motif of the oligopeptide Glycinyl-Arginyl-Glycinyl-Cysteic(Acid)-Threonyl-Proline (ALG-1001 or Luminate, Allegro Ophthalmics, LLC). The trifluoroacetate (TFA) and acetate salts of the R-G-Cysteic(Acid) tripeptide (Test Compound Nos. 1 and 2) were tested in both the ROP Mouse Model as described above as well as in a mouse model of choroidal neovascularization induced by laser photocoagulation (CNV Mouse Model), as generally as described in 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 Control groups were treated by intravitreal injection of Arg-Gly-Glu (Control Peptide), which is known to be inactive. In some of the experiments, an additional Positive Control group was included. Animals assigned to a Positive Control group were treated by intravitreal injection of Arg-Gly-Cys(acid)TFA, which is known to be active.

[0050] The following Table 1 summarizes the neovascularization inhibiting effect of each Test Compound at the dose tested. In each instance, the data was obtained using the ROP Mouse Model, except for the two table entries specifically labeled CNV. Only those table entries labeled CNV show data obtained from the CNV Mouse Model. Bar graphs showing the test results summarized in Table 1 are also provided herewith as FIGS. 1 through 27. Where indicated in the figures, the tests were performed in a blinded manner such that the persons performing the testing did not know the identity or structure of each test compound.

TABLE-US-00002 TABLE 1 SUPPRESSION OF RETINAL NEOVASCULARIZATION IN MOUSE MODEL OF ROP (ISCHEMIC) RETINOPATHY Mean % Reduction of Retinal Neovascularization Test Compound Number Test Compound In ROP Model Activity At Dose Tested 1 R-G-Cys(acid)TFA-ROP 61 Active 1(CNV) R-G-Cys(acid)TFA-CNV 49 - FIG. 11 Active 2(CNV) R-G-Cys(acid)Acetate-CNV 56 - FIG. 11 Active 2 R-G-Cys(acid)Acetate-ROP 72 Active 3 R-A-Cys (acid)TFA 60 Active 4 R-G-CysteineTFA 66 Active 5 R-Cys(acid)-GTFA 33 Slightly Active 6 K-G-Cys (acid)TFA 0 Not Active 7 R-G-Cys(acid)-G-G-G-D-GTFA 62 Active 8 Cys(acid)-R-GTFA 21 Slightly Active 9 Cys(acid)-G-RTFA 63 Active 10 Cys(acid)-A-RTFA 0 Not Active 11 G-Cys(acid)-RTFA 0 Not Active 12 Cyclo-{R-G-Cys(acid)-F-N-Me-V} Acetate 57 Active 13 Cyclo-{R-G-D-D-F-NMe-V}TFA 75 Active 14 H-G-Cys(acid)TFA 28 Slightly Active 15 R-G-DTFA 37 Slightly Active 16 R-G-NTFA 64 Active 17 D-G-RTFA 56 Active 18 R-D-GTFA 44 Active 19 R-A-ETFA 63 Active 20 K-G-DTFA 40 Active 21 R-G-ETFA 0 Not Active 22 R-E-GTFA 0 Not Active 23 R-A-DTFA 0 Not Active 24 R-G-Cys(acid)TFA + Taurine 58 Active 25 Taurine 33 Slightly Active

[0051] In some of the Test Compounds, the amino acid sequence of the binding motif RGCys(acid) tripeptide in GRGCys(acid)TP (ALG-1001) was rearranged and/or replaced by other basic, acidic and neutral amino acids. Based on the results of the ROP and CNV testing summarized above, the result indicates that the presence of Arginine, Alanine and Cysteic Acid in the GRGCys(acid)TP peptide (ALG-1001/Luminate) plays an important role in the suppression of the neovascularization, notably the sequence of R-G-Cys and R-A-Cys. Furthermore, in the presence of arginine, replacement of Cysteic (Acid) by a neutral amino acid exhibited a strong suppressive effect in these experiments.

YXZGeneral Formula 1

[0052] Wherein: [0053] Y=R*, H, K, Cys(acid), G or D; [0054] X=G*, A, Cys(acid), R, G, D or E; and [0055] Z=Cys*, G, Cysteine, R, D, N or E. [0056] *indicates component of the RGCys(acid) binding motif of tripeptide in GRGCys(acid)TP (ALG-1001), which was used as a Positive Control.

[0057] Based on the initial data set forth herein, certain structure/activity relationships are suggested in relation to specific changes made to the R-G-Cysteic Acid binding motif. For example, when the amino acid R (i.e., the Y Component) of the R-G-Cysteic(Acid) binding motif is replaced by a basic amino acid or acidic amino acid, the peptide's anti-neovascularization effects diminish, whereas in the presence of arginine in the binding motif aspartic acid as Component Y appears to promote the peptide's anti-neovascularization effects.

[0058] When amino acid G (i.e., the X Component) of the R-G-Cysteic Acid binding motif is replaced by a basic or acidic amino acid, the peptide's anti-neovascularization effects decrease. However, in the presence of arginine (a strong hydrogen bonding), two carbon length-space for hydrophobic interaction (Alanine and Aspartic Acid) may not influence the peptide's anti-neovascularization effects.

[0059] When Cys(Acid) (i.e., the Z Component) of the R-G-Cysteic(Acid) binding motif is replaced by a neutral amino acid, the peptide's neovascularization inhibiting activity increases whereas replacement of the Z component by acidic or basic amino acids causes the neovascularization inhibiting activity to decrease.

[0060] All indications are that the R-G-Cysteic(Acid) of the oligopeptide Glycinyl-Arginyl-Glycinyl-Cysteic(Acid)-Threonyl-Proline (ALG-1001 or Luminate, Allegro Ophthalmics, LLC) is important for suppression of neovascularization. Also, addition of three parts taurine to one part of the Glycinyl-Arginyl-Glycinyl-Cysteic(Acid)-Threonyl-Proline (ALG-1001) improves the neovascularization suppressing activity.

[0061] It is to be appreciated that, although the invention has been described hereabove with reference to certain examples or embodiments of the invention, various additions, deletions, alterations and modifications may be made to those described examples and embodiments without departing from the intended spirit and scope of the invention. For example, any elements, steps, members, components, compositions, reactants, parts or portions of one embodiment or example may be incorporated into or used with another embodiment or example, unless otherwise specified or unless doing so would render that embodiment or example unsuitable for its intended use. Also, where the steps of a method or process have been described or listed in a particular order, the order of such steps may be changed unless otherwise specified or unless doing so would render the method or process unsuitable for its intended purpose. Additionally, the elements, steps, members, components, compositions, reactants, parts or portions of any invention or example described herein may optionally exist or be utilized in the absence or substantial absence of any other element, step, member, component, composition, reactant, part or portion unless otherwise noted. All reasonable additions, deletions, modifications and alterations are to be considered equivalents of the described examples and embodiments and are to be included within the scope of the following claims.