Drug – bio-affecting and body treating compositions – Nonspecific immunoeffector – per se ; or nonspecific...
Reexamination Certificate
2000-05-01
2001-05-08
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Nonspecific immunoeffector, per se ; or nonspecific...
C424S280100, C514S011400, C514S016700, C514S018700
Reexamination Certificate
active
06228374
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to new peptides, a method for the preparation of said peptides and a pharmaceutical preparation containing said peptides. The peptides according to the present invention are excellent as immunomodulating agents.
1. Background of the Invention
There has been a longfelt need for new safe immunomodulatory agents in the treatment of many different diseases including malignant diseases, autoimmune diseases and asthma/allergy. Present immunomodulatory agents such as Cyclosporin A and steroids, are very potent immunosuppressive agents but also present severe side effects in a dose dependent manner. New immunomodulatory agents with higher specificity for the immune system, showing less side effects will be of great benefit in the treatment of diseases with a pathological immune response as an important component in the disease process.
2. Prior Art
Signalling between cells are to a major extent mediated by oligo- or polypeptide principles, including cytokines, neuropeptides and hormones. One possible way such as signal can be transmitted may involve oxidoreductase activity mediated by thiol-disulfide interaction of cysteine residues. This type of action can induce conformational changes of proteins which ultimately may result in a signal to the cell nuclei. Thus redox systems, based on oxidised or reduced cysteines, play important roles in initiating, maintaining and/or downregulating inflammatory responses. Redox systems that are characterized today are the thioredoxin (TR)/thioredoxin reductase (TRR) system (Holmgren et al, 1989, J. Biol. Chem., 264 13963) and similar systems like the glutaredoxin/glutathione reductase (Bushweller et al., 1992, Biochemistry, 31, 9288) and the protein disulfide isomerase (PDI) system (Noiva and Lennarz, 1992, J. Biol. Chem., 267, 3553). The TR/TRR system and related redox systems are potent regulators of different known immunological and inflammatory parameters, like IL-2R &agr;-chain expression (Espinoz-Adelgado et al, 1992, J. Immunol., 149, 2961), modulation of expression of IFN-&ggr; activity (Deiss and Kimchi, 1991, Science, 252, 117), differentiation and effector function of lymphocytes (Yodoi and Uchiyama, 1992, Immunol. Today 13, 405-411), regulation of eosinophil effector functions (Balcewics et al, 1991, J. Immunol., 147, 2170), activation of glucocorticoid receptor (Grippo et al, 1985, J. Biol. Chem. 260, 93-97) and modulation of immune response during pregnancy (Clarke et al, 1991, J. Reprod. Fert., 93, 525).
The active site of TR includes a sequence with a -Cys-Gly-Pro-Cys- motif. Selected virus proteins, e.g. gene products coded from X regions of human T-cell leukaemia viruses (Shimotohno et al, 1985, P.N.A.S. 82, 302-306) and human immunoregulatory proteins may have cysteine-containing sequences which are homologous to such a -Cys-Gly-Pro-Cys- motif. We have considered that these proteins may either express oxidoreductase activity or can be substrates for such an activity or possibily act as inhibitors of such an activity.
Previously peptides based on the cysteine-rich TR active site sequence mentioned above have been produced and shown to exhibit biological activities similar to the native protein. Another example of a cysteine-containing peptide with thioredoxin-like activity was obtained from hFSH-&bgr;-(81-95) (Grasso et al, 1991, Molecular and Cellular Endocrinology 78, 163).
Analogs of thymic humoral factor &ggr;2 (THF-&ggr;2) for use as immunomodulatory agents in pharmaceutical compositions are described in WO, A1, 9501182 (12.01.95). This document discloses two cyclic analogs: Leu-Glu-Cys-Gly-Pro-Cys-Phe-Leu (SEQ ID NO:34) and Leu-Cys-Ala-Gly-Pro-Cys-Phe-Leu (SEQ ID NO:35); which are excluded from the present invention. However, this document does not reveal the active importance of cysteine-containing sequences.
We have prepared peptides with cysteine-containing motifs, selected from virus structural protein e.g. retroviral transmembraneous protein p15E, and human proteins involved in regulation of inflammation, e.g., TGF-&bgr;. Peptides were then modified to get optimal immuno-regulatory properties.
Outline of the Invention
We have now surprisingly found a novel group of peptides which are excellent as immunomodulators. The peptides according to the present invention comprise 4-15 amino acids and can be described by the general formula (I):
A-X-Y-Cys-Z-B (I)
wherein
X is selected from Gly, Ala, Ile, Asp, Thr, Ser, Arg or Trp;
Y is selected from Pro, pipecolic acid (hereinafter called Pec) or Ile;
Z is selected from Ile, Phe, Pro, Ala, Tyr or Gly;
A is H, a protecting group, an amino acid in either L- or D-form with or without protected sidechain-functionality and/or N-terminal protection or an amino acid sequence with or without protected sidechain-functionalities and/or N-terminal protection;
B is OH, NH
2
, a protecting group, an amino acid in either L- or D-form with or without protected sidechain-functionality and ending with a C-terminal amide, a free carboxyl or a protecting group or an amino acid sequence with or without protected sidechain-functionalities and ending with a C-terminal amide, a free carboxyl or a protecting group; and
provided that the following sequences are excluded from the formula (I):
Leu-Glu-Cys-Gly-Pro-Cys-Phe-Leu (SEQ ID NO:34),
Leu-Cys-Ala-Gly-Pro-Cys-Phe-Leu (SEQ ID NO:35),
Tyr-Ile-Pro-Cys-Phe-Pro-Ser-Ser-Leu-Lys-Arg-Leu-Leu-Ile (SEQ ID NO:36),
Tyr-Ile-Pro-Cys-Phe-Pro-Ser-Leu-Lys-Arg-Leu-Ile (SEQ ID NO:37),
Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQ ID NO:38) and
Thr-Pro-Pro-Thr-Pro-Cys-Pro-Ser (SEQ ID NO:39).
The length of A and B can vary, as long as the criteria concerning length and possible amino acids or other substituents are fulfilled.
The amino acids according to the present invention can be both naturally occurring amino acids and non-naturally, synthetic amino acids or amino acid analogues.
Examples of protecting groups for A are a variety of carbamates and amides of which the following protecting groups are preferred: acetyl (Ac, 9-fluorenylmethyl carbamate (Fmoc), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), trityl (Trt), allyl carbamate (Alloc) and t-butyl carbamate (Boc).
Especially preferred protecting groups for A are acetyl (Ac), 9-fluorenylmethyl carbamate (Fmoc) and t-butyl carbamate (Boc).
Examples of protecting groups for B are a variety of esters such as C
1
-C
6
alkyl, allyl, adamantyl, benzyl, and t-butyl
Also within the scope of the present invention are homodimers according to the formulae (II), (III) and (IV)
i.e. homodimers of the peptides of the formula (I) according to the invention.
Also within the scope of the present invention are pharmaceutically acceptable salts of peptides of the formulae (I), (II), (III) and (IV).
Peptides of the formula (I) containing several cysteine residues may exist both in an oxidized and in a reduced form. The oxidized form may contain intramolecular disulfide bonds resulting in oxidized monomers or intermolecular disulfides resulting in both head to head and head to tail dimers of the peptides of formula (I).
Preferred peptides according to the present invention are peptides of the formulae (I), (II), (III) and (IV) wherein
X is Gly, Y is Pro and Z is Ile;
X is Gly, Y is Pro and Z is Gly;
X is Ala, Y is Pro and Z is Ala;
X is Ile, Y is Pro and Z is Tyr;
X is Ala, Y is Pro and Z is Ile;
X is Arg, Y is Pro and Z is Ile;
X is Ile, Y is Pro and Z is Ile;
X is Asp, Y is Pro and Z is Ile;
X is Trp, Y is Pro and Z is Ile;
X is Trp, Y is Pro and Z is Gly;
X is Gly, Y is Ile and Z is Ile;
X is Gly, Y is Pec and Z is Ile;
X is Thr, Y is Pro and Z is Tyr;
X is Thr, Y is Pec and Z is Phe;
X is Ala, Y is Pro and Z is Phe;
X is Ser, Y is Pro and Z is Phe;
X is Gly, Y is Pro and Z is Pro; or
X is Gly, Y is Pro and Z is Tyr;
wherein A and B can be varied as defined above; and
provided that the following sequence is excluded from the formulae (I), (II), (III) and (IV):
Ser-Gly-Pro-Cys-Pro-Lys-Asp-Gly-Gln-Pro-Ser (SEQ ID NO:38).
Preferred peptides according to the invention
Bergstrand Håkan
Eriksson Tomas
Karabelas Kostas
Lindvall Magnus
Sarnstrand Bengt
Astra Aktiebolag
Borin Michael
Fish & Richardson P.C.
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