Cyclosporin a conjugates and uses therefor

Details Cyclosporin a conjugates and uses therefor Cyclosporin a conjugates and uses therefor

1999-02-22

2001-11-13

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S002600, C514S014800, C514S012200, C435S007100, C530S317000, C530S326000, C530S327000

Reexamination Certificate

active

06316405

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed to conjugates of A&bgr;-binding peptides and cyclosporin analogs and conjugates of A&bgr;-binding peptides and FK506 Binding Protein inhibitors. The conjugates inhibit the aggregation and deposition of A&bgr; peptide into amyloid plaques and therefore find use in the prevention and treatment of disorders characterized by the formation of amyloid plaques.
DESCRIPTION OF THE PRIOR ART
Cyclosporin A (CsA) 1.1, marketed by Sandoz under the trademark “SANDIMMUNE,” currently is the drug of choice for preventing rejection of transplanted human organs. CsA is a highly lipophilic, cyclic undecapeptide, cyclo (-MeBmt
1
-Abu
2
Sar
3
-MeLeu
4
-Val
5
-MeLeu
6
-Ala
7
-(D)-Ala
8
-MeLeu
9
-MeLeu
10
—MeVal
11
-) (SEQ. ID. NO: 1), that contains 7 N-methyl amino acid residues and the novel amino acid (4R)-4-{(E)-2-butenyl}-N-methyl-(L)-threonine (abbreviated as MeBmt) in the 1-position. A number of synthetic routes are known in the art for solution-phase or solid-phase synthesis of CsA. See, for example, Rich et al. (1995), “Solid Phase Synthesis of Cyclosporin Peptides.”
J. Am. Chem. Soc
. 117:7279-7280; Wenger, R. M. (1984),
Helv. chim. Acta
67:502; and Wenger, R. M. (1985),
Angew. Chem. Int. Ed. Engl
. 24:77. CsA is depicted in structure 1.1:
CzA is produced by the fungus
Tolypocladium niveum
and was first isolated in 1976 by workers at Sandoz. In 1983, CsA was approved by the U.S. Food and Drug Administration for use as an immunosuppressant in the United States.
The structure of CsA has been confirmed by total synthesis, Wenger (1984),
Helv. Chim. Acta
, 67:502, and the conformations of CsA free in solution and bound to the protein cyclophilin have been solved by NMR and X-ray crystlography. Looslie et al. (1985),
Helv. Chim. Acta
, 68:682 and Mikol (1993),
J. Mol. Biol
., 234:1119, respectively.
Several modified cyclosporin derivatives are described in the prior art. A shorthand notation for designating cyclosporin analogs has developed in which any modified amino acids and their positions relative to unmodified CsA are listed. This makes for a very simple and unambiquous designation of cyclosporin analogs based upon their differences from natural CsA. For example, an analog of CsA possessing a serine residue in place of the normal valine as the fifth amino acid residue is designated (Ser
5
)-CsA. This conventional shall be consistently employed herein.
CsA analogs containing modified amino acids in the 1-position are reported by Rich et al. (1986),
J. Med. Chem
., 29:978. Strongly immunosuppressive, anti-inflammatory, and anti-parasitic CsA analogs are described in U.S. Pat. Nos. 4,384,996; 4,771,122; and 5,284,826, all assigned to Sandoz. Among the CsA analogs described in these patents are (AllylGly
2
)-CsA, ((D)-Ser
8
)-CsA, and (O-(2-hydroxyethyl)(D)Ser
8
) CsA.
In 1984, Handschumacher et al. reported the discovery of a CsA binding protein, named cyclophilin (Cyp), that binds CsA with a dissociation constant of approximately 20 nM. Handschumacher et al. (1984)
Sience
226:544. It was later shown that Cyp is homologous with peptidyl prolylisomerase (PPIase) a ubiquitous family of proteins found in a variety of cell types. See Takahashi (1989),
Nature
337:473 and Fischer et al. (1989)
Nature
337:476. Cyclophilins catalyze the cis-trans isomerization of Xaa-Pro bonds and are hypothesized to play a role in protein folding, although this functionality remains uncertain. See, for instance, Fischer (1994),
Angew. Chem. Int. Ed. Engl
. 33:1415 and Schmid (1993),
Ann. Rev. Biophys. Biomol. Struct
. 22:123.
The identification of Cyp as a PPIase suggested that CsA exerts its immunosuppressive effect by inhibiting the PPIase activity of Cyp, thereby causing improper folding of proteins which are crucial to the immune response Sigal et al. (1991),
J. Exp. Med
. 173:619. This hypothesis was originally strengthened by the discovery that the macrolide FK506, 1.2, has potent immunosuppressive activity and inhibits the PPIase activity of FK506 binding protein (FKBP). Siekerka et al. (1989),
Nature
341:755.
Further investigations, however, revealed several discrepancies regarding the inhibition of PPIase as a mechanism leading to immunosuppression. Foremost, the concentrations of CsA and FK506 required to ellicit immunosuppression are far lower than the concentrations of Cyp within a cell. Additionally, mutants of yeast and neurospora which lack the Cyp gene are resistant to cyclosporin but are still viable. See Agarwal et al. (1987),
Transplantation
42:627; Tropschung et al. (1989),
Nature
342:953; and Hayano et al. (1991),
Biochem
. 30:3041. Another observation at odds with the original hypothesis was that although CsA and FK506 exhibit very similar in vivo and in vitro effects, CsA does not bind to FKBP and FK506 does not bind to Cyp. Schreiber and Crabtree (1992),
Immunology Today
13:136. The PPIase inhibition hypothesis was further weakened with the discovery that several potent PPIase inhibitors do not cause immunosuppression. See, for example, Somers et al. (1991),
J. Am. Chem. Soc
. 113:8045.
In 1991, Liu et al. reported that the CsA-Cyp complex binds with high affinity to calcineurin, a calcium dependent serine/threonine phosphatase, Liu et al. (1991),
Cell
66:807; and Liu et al. (1992),
Biochem
. 31:3896. Calcineurin is thought to cleave a phosphate group from the nuclear factor of activated T-cells (NF-AT), allowing its translocation into the nucleus where it activates the gene for interleukin-2. See Schreiber and Crabtree (1992), supra. Inhibition of calcineurin is now generally accepted as the mechanism of immunosuppression by both CsA and FK506. See, for example, Ho et al. (1996),
Clin. Immun. and Immunopathology
, 80:S40. CsA binds to Cyp by an interaction between residues 9-10-11-1-2 of the CsA and an active site on Cyp residues. 9-10-11-1-2 of CsA are therefore referred to as the “binding domain.” Calcineurin is bound to CsA by an analogous interaction including residues 4-5-6-7-8 of CsA. These residues are therefore referred to as the “effector domain”:
Interestingly, both CsA and FK506 exhibit potent neurotrophic (nerve regeneration) effects, with FK506 showing significant neurotrophic activity at concentrations as low as 1 pM. Lyons et al. (1994),
Proc. Natl. Acad. Sci. U.S.A
. 91:3191; Steine et al. (1997),
Nature Medicine
3:421. The mechanism by which these compounds exert their neurotrophic activity is unclear, although Nerve Growth Factor (NGF) must be present for the activity to be present.
Chemical Induced Dimerization (CID) involves the dimerization of two biomolecules by a cell-permeable organic compound which “turns on” a specific biological event. (See, for example, Crabtrce and Schreiber (1996)
Trends in Biochemical Sciences
21:418.) The technique relies on the principle of induced proximity, where the process of bringing two parts of an activation system into close contact is sufficient to activate the biological process. Transcription factors, for example, are highly modular and the activation domain can be separated from the DNA binding domain. Attachment of each domain to a drug-binding protein allows the drug-dimer to bring the DNA binding and activation domains into close proximity by binding the fusion proteins, thereby turning on the gene expression. There are two important aspects CID that are attractive: the drug-dimer controlled gene expression can be “shut off” by addition of drug monomer, and the modular nature of the designed transcription factors means that each fusion protein/CID complex can be optimized for any given system, and that two or more biological events could be simultaneously controlled by using different fusion protein/CID complexes.
A significant advance in the CID technology was the use of orally-active rapamycin as the drug dimerizer. Rivera et al. (1990)
Nature Medicine
2:1028. Rapamycin can bind both FKBP and FRAP, a lipid kinase. FRAP could be further truncated to an 89-amino acid peptide, termed FRB, that retains binding to rapamycin. Add

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