Cyclosporin fermentation process

Details Cyclosporin fermentation process Cyclosporin fermentation process

1999-09-09

2001-07-03

Wessendorf, T. D. (Department: 1627)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S254100, C435S252000, C435S244000, C435S243000, C435S911000

Reexamination Certificate

active

06255100

ABSTRACT:

BACKGROUND
The present invention relates to novel cyclosporins, their use as pharmaceuticals and pharmaceutical compositions comprising them, as well as to processes for their production.
The cyclosporins comprise a class of structurally distinctive, cyclic, poly-N-methylated undecapeptides, commonly possessing pharmacological, in particular immunosuppressive, anti-inflammatory and/or antiparasitic activity. The first of the cyclosporins to be isolated was the naturally occurring fungal metabolite Ciclosporin or Cyclosporine, also known as cyclosporin A and commercially available under the Registered Trademark SANDIMMUNR or SANDIMMUNER. Ciclosporin is the cyclosporin of formula A.
where MeBmt represents the N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)threonyl residue of formula B
in which —x—y— is —CH═CH— (trans).
Since the original discovery of Ciclosporin, a wide variety of naturally occurring cyclosporins have been isolated and identified and many further non-natural cyclosporins have been prepared by total- or semi-synthetic means or by the application of modified culture techniques. The class comprised by the cyclosporins is thus now substantial and includes, for example, the naturally occurring cyclosporins A through Z [cf. Traber et al; 1, Helv. Chim. Acta, 60, 1247-1255 (1977); Traber et al; 2, Helv. Chim. Acta, 65, 1655-1667 (1982); Kobel et al, Europ. J. Applied Microbiology and Biotechnology, 14, 273-240 1982); and von Wartburg et al, Progress in Allergy, 38, 28-45, 1986)], as well as various non-natural cyclosporin derivatives and artificial or synthetic cyclosporins including dihydro-cyclosporins [in which the moiety —x—y— of the MeBmt residue (formula B above) is saturated to give —x—y—═—CH
2
—CH
2
—]; derivatised cyclosporins (e.g. in which the 3′-O-atom of the MeBmt residue is acylated or a further substituent is introduced at the &agr;-carbon atom of the sarcosyl residue at the 3-position); cyclosporins in which the MeBmt residue is present in isomeric form (e.g. in which the configuration across positions 6′ and 7′ of the MeBmt residue is cis rather than trans); and cyclosporins in which variant amino acids are incorporated at specific positions within the peptide sequence, e.g. employing the total synthetic method for the production of cyclosporins developed by R. Wenger—see e.g. Traber et al. 1, Traber et al, 2 and Kobel et al., loc. cit.; U.S. Pat. Nos. 4,108,985, 4,220,641, 4,288,431, 4,554,351, 4,396,542 and 4,798,823 European Patent Publications Nos. 34,567A, 56,782A, 300,784A, 300,785A and 414,632A; International Patent Publication No WO 86/02080 and UK Patent Publications Nos. 2,206,119 and 2,207,678; Wenger 1, Transpl. Proc., 15 Suppl. 1:2230 (1983); Wenger 2., Angew. Chem. Int. Ed. 24 77 (1985) and Wenger 3., Progress in the Chemistry of Organic Natural Products, 50, 123 (1986).
The class comprised by the cyclosporins is thus now very large indeed and includes, for example, [Thr]
2
-, [Val]
2
-, [Nva]
2
- and [Nva]
2
-[Nva]
5
-Ciclosporin (also known as cyclosporins C, D, G and M respectively), [3-O-acetyl-MeBmt]
1
-Ciclosporin (also known as cyclosporin A acetate), [Dihydro-MeBmt]
1
-[Val]
2
-Ciclosporin (also known as dihydro-cyclosporin D), [(D)Ser]
8
-Ciclosporin, [MeIle]
1
-Ciclosporin, [(D)MeVal]
11
-Ciclosporin (also known as cyclosporin H), [MeAla]
6
-Ciclosporin, [(D)Pro]
3
-Ciclosporin and so on.
In accordance with conventional nomenclature for cyclosporins, these are defined throughout the present specification and claims by reference to the structure of Ciclosporin (i.e. Cyclosporin A). This is done by first indicating those residues in the molecule which differ from those present in Ciclosporin and then applying the term “Ciclosporin” to characterise the remaining residues which are identical to those present in Ciclosporin. At the same time the prefix “dihydro” is employed to designate cyclosporins wherein the MeBmt residue is hydrogenated (dihydro-MeBmt) i.e. where —x—y— in formula B is —CH
2
—CH
2
—. Thus [Thr]
2
-Ciclosporine is the cyclosporin having the sequence shown in Formula A but in which &agr;Abu at the 2-position is replaced by Thr, and [Dihydro-MeBmt]
1
-[Val]
2
-Ciclosporin is the cyclosporin having the sequence shown in Formula A but in which the MeBmt residue at position 1 is hydrogenated and &Abu at the 2-position is replaced by Val.
In addition, amino acid residues referred to by abbreviation, e.g. Ala, MeVal, aAbu etc. are, in accordance with conventional practice, to be understood as having the (L)-configuration unless otherwise indicated, e.g. as in the case of “(D)Ala”. Residue abbreviations preceded by “Me” as in the case of “MeLeu”, represent a-N-methylated residues. Individual residues of the cyclosporin molecule are numbered, as in the art, clockwise and starting with the residue MeBmt or dihydro-MeBmt in position 1. The same numerical sequence is employed throughout the present specification and claims.
It is now well established that Ciclosporin acts by interfering with the process of T cell activation by blocking transcription initiation of IL-2, although the precise mechanism has not yet been elucidated. Ciclosporin has been shown to form a complex with a 17 kD cytosolic protein (cyclophilin) that occurs in many cell types and has been shown to be identical to peptidyl-prolyl cis-trans isomerase, an enzyme involved in protein folding. Up to now, however, it has not been clear whether binding to cyclophilin is directly correlated with immunosuppressive activity in cyclosporins, or indeed whether cyclophilin binding is itself a sufficient criterion for immunosuppressive activity.
SUMMARY OF THE INVENTION
It has now been found that there are cyclosporins which bind strongly to cyclophilin, but are not at all immunosuppressive. It therefore follows that binding to cyclophilin is a necessary, but not a sufficient, criterion for immunosuppressant activity.
The present invention provides cyclosporins which are active against HIV-1 replication.
DETAILED DESCRIPTION
Human immune deficiency virus (HIV) infects preferentially T-helper (T4) lymphocytes, although it replicates also in various other cell types, especially those of the monocytic lineage. It causes a slowly progressing disease of the immune system characterised by a gradual T4-cell destruction, named AIDS. Other immunological abnormalities of AIDS are increase of cytotoxic/suppressor (T8) lymphocytes, a defect in the antigen presentation/recognition process and polyclonal activation of B-cells. The mechanism of T4-cell destruction is still not clear. Relatively few T4-cells seem to be infected, thus, a direct cytopathic effect caused by the virus may not be the only reason for T4-cell depletion. It has been hypothesised that T4-cell destruction could be amplified by an autoimmune process triggered by HIV-producing or HIV-protein-coated T4-cells. This continuous antigenic stimulation may lead to a state of permanent activation of T4-cells which would enhance HIV-replication in these cells and expand T-cytotoxic clones. Uninfected T4-cells may be rendered antigenic by binding exogenous viral gp120 to their CD4 molecules and would thus be a target of a T-cytotoxic response.
U.S. Pat. No. 4,814,323 discloses that Ciclosporin has activity against AIDS, and that in general “cyclosporins known as immunosuppressors” may be useful in this indication. There is no suggestion that non-immunosuppressive cyclosporins might be expected to have this property.
Surprisingly, it has now been found that cyclosporins which bind to cyclophilin, but are not immunosuppressive, exhibit an inhibitory effect upon HIV-1 replication.
A cyclosporin is considered as binding to cyclophilin if it binds to human recombinant cyclophilin at least one fifth as well as does Ciclosporin in the competitive ELISA test described by Quesniaux in
Eur. J. Immunol.
1987 17 1359-1365. In this

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