Non-Imuunosuppressive cyclosporins and their use in the...

Details Non-Imuunosuppressive cyclosporins and their use in the... Non-Imuunosuppressive cyclosporins and their use in the...

1999-02-22

2001-08-07

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C424S278100

Reexamination Certificate

active

06270957

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed to non-immunosuppressive cyclosporin analogs and conjugates thereof which possess anti-HIV activity. The compounds and compositions containing the same are useful in the prevention and treatment of HIV infection in humans.
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
-)a (SEQ. ID. NO: 1), that contains 7 N-methyl amino acid residues and the novel amino acid (4R)-4-{(E)-2-butenyl}-4-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:
CsA 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 crystallography. 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 unambiguous 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 approxinately 20 nM. Handschumacher et al. (1984)
Science
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. Signal 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 “effective domain”:
In the late 1980's, CsA was reported to exhibit anti-HIV activity. See, for instance, Wainberg et al. (1988),
Blood,
72:1904; Karpas et al. (1992),
Proc. Natl. Acad. Sci. USA,
89:8351; and Bell et al. (1993),
Proc. Natl. Acad. Sci. USA,
90:1411. Although it originially seemed counterintuitive to use an immunosuppressant suppressant to treat a viral infection that compromises the immune system, the anti-HIV activity of CsA was at first attributed to the inhibition of T-cell activation. However this hypotheses was disproved when non-immunosuppressive CsA analogs were also found to have anti-HIV activity. Bartz et al. (1995),
Proc. Natl. Acad. Sci. USA,
92:5381; and Rosenwirth et al. (1994),
Antimicrobial Agents and Chemotherapy,
38:1763.
Regarding HIV and its replication in human T-cells, HIV protease is an aspartic protease that cleaves the immature viral protein gag-pol into mature structural proteins and enzymes. HIV protease is an essential enzyme in the replication of the HIV virus. Katz et al. (1994),
Annual Rev. Biochem.,
63:133. A decade of intense research has produced four FDA-approved HIV protease inhibitors, saquinivar 1.9, ritonavir 1.10, indinivar 1.11, and nelfinivar 1.12; and one compound currently in phase III trials, VX-478 1.13. The structure of these compounds are shown below:
Unfortunately, initial reports of clinical success by treatment with a single protease inhibitor have been tempered by the rapid onset of resistance. Molla et al. (1996),
Nature Medicine,
2:760. Due to the poor fidelity of reverse transcriptase, the enzyme that produces double-stranded DNA from the viral RNA, a large number of genetic mutations of the virus are produced. The low fidelity of the reverse transcriptase reaction is compounded by the massive turnover of viral particles during the HIV life cycle. Ho et al. (1996),
Science,
271:1582, have calculated that the decay half-life of virions is on the order of 0.24 days and that for infected cells the decay half-life is 1.5 days. These numbers indicate that every six hours approximately one-half of the c

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