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Title:  Treating retinal neuronal disorders by the application of insulin-like growth factors and analogs

United States Patent:  6,310,040

Inventors:  Bozyczko-Coyne; Donna (Norristown, PA); Neff; Nicola (Wallingford, PA); Lewis; Michael E. (West Chester, PA); Iqbal; Mohamed A. (Malvern, PA)

Assignee:  Cephalon, Inc. (West Chester, PA)

Appl. No.:  963329

Filed:  October 15, 1992

Abstract

Disclosed is a method for promoting retinal neuronal survival in a mammal, wherein the neuronal cells are at risk of dying. The method comprises administering to the mammal an effective dose of at least one of the following substances: IGF-I; a functional derivative of IGF-I; IGF-II; or a functional derivative of IGF-II.

SUMMARY OF THE INVENTION

In one aspect, the invention features a method of enhancing the survival of retinal neuronal cells at risk of dying in a mammal, by administering to the mammal an effective dose of at least one of the following substances: IGF-I; a functional derivative of IGF-I; IGF-II; or a functional derivative of IGF-II.

In preferred embodiments, where there is administered IGF-I, IGF-II, or a functional derivative of IGF-I or IGF-II, the method further comprises administering to the mammal an effective amount of a substance which produces an additive and/or synergistic effect. A combination of two or more of the substances, which act synergistically, can be administered to the mammal, or a combination of two or more of the substances, which act additively, can be administered to the mammal.

In other preferred embodiments, the retinal neuronal cells are photoreceptor cells, amacrine cells, horizontal cells, bipolar cells, or ganglion cells.

In yet other preferred embodiments, the method is used in a therapeutic context for the treatment of retinal neuronal tissues which are suffering from the effects of injury, aging and/or disease, wherein the term injury is a broad term which includes, but is not limited to, injury resulting in retinal degeneration, such as photodegeneration, trauma, axotomy, neurotoxic-excitatory degeneration or ischemic neuronal degeneration, and wherein the term disease is a broad term which includes, but is not limited to, any infectious or non-infectious disease such as inherited retinal dystrophy, diabetic retinopathy, Alzheimer's disease, infantile malignant osteopetrosis, ceroid-lipofuscosis or cholestasis.

In preferred embodiments, wherein a functional derivative of IGF-I is administered, IGF-I(4-70) (SEQ ID NO:2), also known as IGF-III or brain IGF, is the preferred IGF-I derivative. Where a functional derivative of IGF-II is administered, IGF-II(54-67) (SEQ ID NO:13) is the preferred IGF-II derivative. The substances can also be administered in conjunction with neurotransmitter enhancers and/or their derivatives.

IGF-I, IGF-II, or functional derivatives thereof administered in methods of the invention may be chemically modified in such a way as to increase its efficacy, e.g., by increasing the transport of these polypeptides across the blood-retina barrier, e.g., by modifications of the polypeptide that increase lipophilicity, alter glycosylation, or increase net positive charge.

The invention also features a composition comprising a solution containing IGF-I or IGF-II, or a functional derivative thereof, e.g., IGF-I(4-70) (SEQ ID NO:2), or IGF-II (54-67) (SEQ ID NO:13), with excipients for ophthalmic administration, contained within a chemically inert vessel which is closed at one end with a dropper or other device for the transfer of drops of the solution from the vessel to the eye of the recipient of the solution. The invention also features a composition comprising a solution containing IGF-I or IGF-II, or a functional derivative thereof, e.g., IGF-I(4-70) (SEQ ID NO:2), or IGF-II (54-67) (SEQ ID NO:13), with excipients for ophthalmic administration, contained within a chemically inert vessel, e.g., an implant, e.g., an implanted disk, which is implanted into a recipient for the transfer of the solution from the implant to the eye of the recipient.

The invention features a composition comprising an ointment containing IGF-I or IGF-II, or a functional derivative thereof, e.g., IGF-I(4-70) (SEQ ID NO:2), or IGF-II (54-67) (SEQ ID NO:13), with excipients for ophthalmic administration.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence CALLETYCATPAKSEC (SEQ ID NO:17, the amino acid sequence CTYCATPAKSEC (SEQ ID NO:57), the amino acid sequence CEPYCAPPAKSEC (SEQ ID NO:58), and the amino acid sequence CTYCAPAKSEC (SEQ ID NO:59), wherein the N-terminal cysteine is connected to the C-terminal cysteine by a covalent bond.

As a preferred embodiment to the method of the invention the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence CALLETDYCATPAKSEC (SEQ ID NO:47), the amino acid sequence CTDYCATPAKSEC (SEQ ID NO:48), and the amino acid sequence CTDYCAPAKSEC (SEQ ID NO:49), wherein the N-terminal cysteine is connected to the C-terminal cysteine by a covalent bond.

As a preferred embodiment to the method of the invention the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence CTYTAPAKSEC (SEQ ID NO:60), the amino acid sequence CALLETYATPAKSEC (SEQ ID NO:61), the amino acid sequence CRRLEMYCAPLKPAKSAC (SEQ ID NO:62), the amino acid sequence CGYGSSSRRAPQTC (SEQ ID NO:63), the amino acid sequence CYFNKPTGYGC (SEQ ID NO:64), the amino acid sequence CYFNKPTGYGSSSRRAPQTC (SEQ ID NO:65), and the amino acid sequence CKPTGYGSSSRC (SEQ ID NO:66), wherein the N-terminal cysteine is connected to the C-terminal cysteine by a covalent bond.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide selected from the group consisting of the amino acid sequence CDLRRLEMYC (SEQ ID NO:19), the amino acid sequence CCFRSCDLRRLEMYC (SEQ ID NO:20), the amino acid sequence CCFRSC (SEQ ID NO:22), and the amino acid sequence CFRSC (SEQ ID NO:23), wherein said peptide is cyclized by a covalent bond between two residues of said peptide.

As a preferred embodiment to the method of the invention the functional derivative is a substantially pure peptide selected from the group consisting of the amino acid sequence TYCATPAKSE (SEQ ID NO:68), and the amino acid sequence RRLEMYCAPLKPAKSA (SEQ ID NO:67). The residues flanking the amino acid sequence can be homologous to the naturally occurring sequence of IGF-I, or to the naturally occurring sequence of IGF-II.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure cyclized peptide consisting essentially of the amino acid sequences CGCELVDALQFVC (SEQ ID NO:18) and CCFRSCDLRRLEMYC (SEQ ID NO:20), wherein said cyclized peptide comprises at least one covalent bond between two residues of said cyclized peptide.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence CGCELVDALQFVC (SEQ ID NO:18), the amino acid sequence CDLRRLEMYCCPLKPAKSE (SEQ ID NO:21), the amino acid sequence CGPETLC (SEQ ID NO:26), the amino acid sequence CGYGSSSRRCPQTGIVDEC (SEQ ID NO:27), the amino acid sequence CGDRGFYFNKPTC (SEQ ID NO:28), the amino acid sequence CCPLKPAKSAC (SEQ ID NO:29), and the amino acid sequence CDLRRLEMYAPLKPAKSAC (SEQ ID NO:30), wherein the N-terminal cysteine is connected to the C-terminal cysteine by a covalent bond.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide selected from the group consisting of the amino acid sequence CGGELVDTLQFVC (SEQ ID NO:32), the amino acid sequence CCFRSCDDLALLETYC (SEQ ID NO:34), wherein said peptide is cyclized by a covalent bond between two residues of said peptide. Preferably, the residues flanking the amino acid sequence are homologous to the naturally occurring sequence of IGF-I, or to the naturally occurring sequence of IGF-II.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure cyclized peptide consisting essentially of the amino acid sequences CGGELVDTLQFVC (SEQ ID NO:32) and CCFRSCDLCLLETYC (SEQ ID NO:39), wherein said cyclized peptide comprises at least one covalent bond between two residues of said cyclized peptide.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence CDLCLLETYC (SEQ ID NO:33), the amino acid sequence CDLCLLETYCATPAKSE (SEQ ID NO:35), the amino acid sequence CCYRPSETLC (SEQ ID NO:40), CRPCSRVSRRSRGIVEEC (SEQ ID NO:41), CGDRGFYFSRPC (SEQ ID NO:42), CCTPAKSEC (SEQ ID NO:43), and CDLCLLETATPAKSEC (SEQ ID NO:44), wherein the N-terminal cysteine is connected to the C-terminal cysteine by a covalent bond.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence CATPAKSE (SEQ ID NO:53), YCAPAKSE (SEQ ID NO:54), YCAPA (SEQ ID NO:55), TYCAPA (SEQ ID NO:56), CAPAKSE (SEQ ID NO:24), EALLETYCATPAKSE (SEQ ID NO:36), and APSTCEYKA (SEQ ID NO:38).

As a preferred embodiment to the method of the invention the functional derivative is a substantially pure peptide selected from the group consisting of the amino acid sequence YFNKPTGYGSSSRRAPQT (SEQ ID NO:3), the amino acid sequence GYGSSSRRAPQT (SEQ ID NO:4), the amino acid sequence APLKPAKSA (SEQ ID NO:5), the amino acid sequence YFNKPTGYG (SEQ ID NO:6), the amino acid sequence SSSRRAPQT (SEQ ID NO:10), the amino acid sequence PTGYGSSSRRAPQT (SEQ ID NO:11), and the amino acid sequence KPTGYGSSSR (SEQ ID NO:12). Preferably, the residues flanking the amino acid sequence are homologous to the naturally occurring sequence of IGF-I, or to the naturally occurring sequence of IGF-II.

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence YFNKPTGYGSSSRRAPQT-NH2 (SEQ ID NO:7), the amino acid sequence SSSRRAPQT-NH2, the amino acid sequence GIVDECC(Acm)FRSCLDRRL-NH2 (SEQ ID NO:9), the amino acid sequence EPYCAPPAKSE (SEQ ID NO:69), the amino acid sequence TYCAPAKSE (SEQ ID NO:70), the amino acid sequence ALLETYSATPAKSE (SEQ ID NO:71), the amino acid sequence ETQCATPAKSE (SEQ ID NO:72), and the amino acid sequence GAELVDALQFYSGDRGFYFNKPTG (SEQ ID NO:73).

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide comprising a sequence selected from the group consisting of the amino acid sequence ALLETDYCATPAKSE (SEQ ID NO:45), the amino acid sequence TDYCATPAKSE (SEQ ID NO:46), and the amino acid sequence TDYCAPAKSE (SEQ ID NO:50).

As a preferred embodiment to the method of the invention, the functional derivative is a substantially pure peptide selected from the group consisting of the amino acid sequence ALLETYCATPAKSE (SEQ ID NO:13), the amino acid sequence TPAKSE (SEQ ID NO:14), and the amino acid sequence SRVSRRSR (SEQ ID NO:15).

As a additional embodiment, the functional derivative contains between 5 and 40 amino acids, preferably 6-25 amino acids. The functional derivative can be iodinated.

The functional derivative can also be a cyclic peptide, the cyclic peptide consisting essentially of 5-40 amino acid residues, or 6-25 amino acid residues. Preferably the cyclic peptide includes a fragment of the respective IGF-I, IGF-II, or IGF-III as at least a portion of its amino acid sequence. The cyclic peptide can include a disulfide bond between two cysteines of the peptide, the cysteines being located at either terminal or internal positions of the peptide. Alternatively or in addition to the disulfide bond, the cyclic peptide may include an amide bond between the amino and carboxyl ends of the peptide. Preferred cyclic peptides include, but are not limited to, those derived by cyclization, e.g., by disulfide bond formation or by amide bond formation.

As a preferred embodiment to the method of the invention, the functional derivative is a retro-inverso peptide, preferably a retro-inverso peptide that is homologous to IGF-I, or a fragment thereof, or a retro-inverso peptide that is homologous to IGF-II, or a fragment thereof. A "retro-inverso peptide", as used herein, refers to a peptide with a reversal of the direction of the peptide bond at at least one position, i.e., a reversal of the amino- and carboxy- termini with respect to the side chain of the amino acid. Retro-inverso peptides may contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids.

The functional derivative can also be a scrambled peptide. A "scrambled peptide", as used herein, is a peptide that contains the same residues of the naturally occurring peptide or a functional derivative thereof, but where the sequence of the residues has been rearranged.

With respect to any of the IGF-I or IGF-II peptides listed herein, most preferred are linear and cyclic peptides that contain at least one cysteine residue that is not involved in disulphide bond formation. In some cases where a naturally-occurring alanine has been changed to a cysteine, the invention embodies both the peptide containing the naturally-occurring alanine, which has at least partial activity, as well as the peptide containing the substituted cysteine, which has the preferred activity.

"Homologous" refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two polypeptide molecules is occupied by leucine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the amino acid sequences Leu-gly-val-ala-gly-pro and Leu-his-tyr-ala-gly-leu share 50% homology.

In addition to substantially full-length polypeptides, the invention also includes fragments of the IGF-I, IGF-II, or IGF-III polypeptides. As used herein, the term "fragment", as applied to a polypeptide, will ordinarily be at least about 5 contiguous amino acids, typically at least about 20 contiguous amino acids, usually at least about 40 contiguous amino acids, and preferably at least about 60 or more contiguous amino acids in length. Fragments of IGF I, II, or III can be generated by methods known to those skilled in the art.

In a final aspect, the invention includes a substantially pure peptide, the peptide comprising a sequence selected from the group consisting of the amino acid sequence ALLETYSATPAKSE (SEQ ID NO:71), the amino acid sequence ETQCATPAKSE (SEQ ID NO:72), and the amino acid sequence GAELVDALQFYSGDRGFYFNKPTG (SEQ ID NO:73). Any of the peptides of the invention may be iodinated.

The peptides described herein are provided as examples, and are not to be construed as limiting the range of peptides useful for the methods of the invention.

Survival of treated retinal neuronal cells denotes maintenance of the cell's viability to an extent greater than that of untreated controls. Since the preponderance of retinal neuronal cells are commonly believed to be incapable of cell division, the ability of an agent to promote survival of such cells may be measured by assays which reproducibly indicate relative numbers of cells, such as directly counting cells which stain as viable cells, or which possess other characteristics of viable neurons. The method and composition of the invention are useful for therapeutically treating a disorder of a human or other mammal characterized by death and/or dysfunction of retinal neuronal cells, including disorders attributable to a disease of aging of, or injury to, such retinal neuronal cells.

Claim 1 of 1 Claim

What is claimed is:

1. A method for promoting the survival of photoreceptors in a mammal, said photoreceptors being at risk of dying, said method comprising administering to said mammal an effective dose of Insulin-like Growth Factor-I.

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