Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-04-06
2002-05-07
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S326000, C435S007200
Reexamination Certificate
active
06384193
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a system for regulating oestrogen synthesis, a method of regulating an oestrogen-producing enzyme, an assay for determining whether a compound is an agent capable of regulating oestrogen synthesis, a related kit, and the use of an agent in an oestrogen-dependent system.
BACKGROUND OF THE INVENTION
Oestrogens are the most potent mitogens known to stimulate the growth of breast tumours and in postmenopausal women most of the oestrogen required for tumour growth is formed in situ within the breast (Reed et al., 1989). Three main enzyme complexes are involved in oestrogen synthesis in breast tumours, i.e. aromatase, which converts androstenedione to oestrone; oestrone sulphatase (or E1-STS), which regulates the formation of oestrone from oestrone sulphate; and oestradiol dehydrogenase (or E2DH), which converts oestrone to the biologically active oestrogen, oestradiol.
In Singh et al.,(1995) we proposed that the interleukin-6 soluble receptor (or IL-6sR) may regulate the ability of IL-6 to stimulate oestrogen synthesis in breast cancer cells and breast tumours. Significant arbmatase activity was detectable in IL-6 stimulated fibroblasts derived from sub-cutaneous adipose tissue, but the combination of IL-6 sR plus IL-6 resulted in a marked 21-fold stimulation of aromatase activity. To examine the control of IL-6sR release, the effects of oestradiol, 4-hydroxytamoxifen (or 4-OHT), dexamethasone, TPA, TNF&agr; or IL-6 on this process was examined using MCF-7 breast cancer cells. Oestradiol, TNF&agr; and dexamethasone all markedly increased IL-6sR release. While 4-OHT had a small stimulatory effect on IL-6sR release, it blocked the ability of oestradiol to increase IL-6sR release. Significant concentrations of IL-6sR were also detected in conditioned medium collected from lymphocytes and macrophages and in cytosols prepared from normal and malignant breast tissues. These results indicate that IL-6sR may have a role in potentiating the effect of IL-6 on oestrogen synthesis in breast cancer cells.
Cytokine, including IL-6, act by binding to membrane spanning receptors. For IL-6, the receptor (or IL-6R) complex consists of a 80 kDa (gp80) ligand binding sub-unit and a 130 kDa (gp130) signal transducing protein. The small gp80 sub-unit binds IL-6 with low affinity and must associate with the larger gp130 protein in order for high affinity binding and signal transduction to occur. A 55 kDa soluble form of gp80 (i.e. IL-6sR) is also found in serum, but unlike other known soluble cytokine receptors, which antagonise the effects of their respective cytokines, IL-6sR enhances the response to IL-6 in some biological systems. The IL-6sR is formed by limited proteolysis (shedding), but little is known about the factors which regulate this process (Mullberg et al., 1993).
Without wishing to be bound by any theory, we believe that a IL-6-IL-6sR complex is formed which has an important role in regulating at least aromatase activity. The development of a polypeptide which blocks the ability of the IL-6-IL-6sR complex to interact with gp130 provides a novel and surprising way of inhibiting the activity of this enzyme, and will thus influence oestrogen production and oestrogen-dependent systems.
Grube and Cochrane (1994) identified a 16 amino acid peptide, based upon part of the IL-6R external domain, but for a completely different application namely for blocked stimulation of B9 cell mitogenesis.
Reference is also made to U.S. Pat. No. 5,210,075, International Patent application No. WO 95/11303, Purohit et al., Regulation of Aromatase and Sulphatase in Breast Tumor Cells, Journal of Endocrinology, vol. 250, September 1996, pp S65-S71, and Grube et al., “Identification of a Regulatory Domain of the Interleukin-6 Receptor”, Journal of Biological Chemistry, vol. 269, no. 32, 1994, pp. 20791-20797.
SUMMARY OF THE INVENTION
The present invention provides a system for regulating oestrogen synthesis, a method of regulating an oestrogen-producing enzyme, an assay for determining whether a compound is an agent capable of regulating oestrogen synthesis, a related, the use of an agent in an oestrogen-dependent system (e.g., a system that depends upon oestrogen), and other embodiments which are disclosed in or obvious from the following Detailed Description.
DETAILED DESCRIPTION
Thus according to one aspect of the present invention there is provided a system comprising a IL-6-IL-6sR complex and an agent that can block the interaction of the complex with gp130 to regulate oestrogen synthesis.
According to another aspect of the present invention there is provided a method of regulating an oestrogen-producing enzyme in a system comprising a member of the IL-6 superfamily, IL-6sR and gp130, the method comprising adding to the system an agent that can block the interaction of a IL-6-IL-6sR complex with gp130.
Preferred members of the IL-6 superfamily include IL-6, IL-11 and oncostatin M, with IL-6 being especially preferred. However, it will be appreciated that other members of the IL-6 superfamily, including those which may become available, may be useful in the present invention, given that members work by a similar mechanism to IL-6 itself.
For the avoidance of doubt we would mention that the terminology “IL-6-IL6sR” as used in connection with the present invention indicates a complex of IL-6sR with members of the IL-6 superfamily and not just with IL-6.
The present invention is specifically exemplified using aromatase activity. However, other oestrogen-producing enzymes, especially E2DH and E1-STS, are regulated by a similar mechanism and a skilled worker would expect these other enzymes to be regulated by the present invention.
The agent useful in the present invention and which blocks the interaction of a IL-6-IL-6sR complex with gp130 can be termed an “anti-oestrogen”. These may include compounds which are known to have an effect on breast cancer, such as 4-hydroxytamoxifen (or 4-OHT), but which have previously been implicated in a different system, compounds which are newly identified as having an anti-oestrogen effect and completely new compounds. Such a compound with a newly identified effect is the polypeptide whose sequence is shown in Seq. ID No. 1 and which has been designated “Arohib”. Novel compounds include variants, derivatives and fragments of Arohib which retain the ability to block the interaction of a IL-6-IL-6sR complex with gp130. The terms “variant”, “derivative” or “fragment” include any substitution of, variation of, modification of, replacement of, deletion of or addition of one or more amino acids from or to the sequence providing the resultant polypeptide is capable of behaving as an agent in accordance with the present invention. It is expected that the useful compounds will be peptides or polypeptides, but this may not necessarily be the case.
The term “anti-oestrogen” is a term of art, and its use in this description can be in accordance with its use in the art (see, e.g., Thomas et al., Hum Reprod, 1994, 9(11):1991-6; Singh et al., J Reprod Fertil, 1994, 100(2):367-74; Ribot et al., Ann Endochrinol (Paris), 1995, 56(6):603-8; Jarvinen et al., Scand J Immunol, 1996, 44(1):15-20: Jordan, Scientific American, Oct. 1998, 279(4):60-67 (“Designer Estrogens”; “Drugs that Prevent Breast Cancer”)).
As IL-6sR is produced in large amounts by malignant cells, the agent may preferentially inhibit breast oestrogen synthesis. This would be an advantage in long-term prevention of breast cancer, for example, preventing possible bone loss due to other types of inhibition.
Indeed, the present invention provides an assay for determining whether a compound is an agent capable of regulating oestrogen synthesis comprising adding the compound under test to a system comprising a member of the IL-6 superfamily, IL-6sR and gp130, and determining whether the compounds blocks the interaction of a IL-6-IL6sR complex with gp130, wherein if the compounds blocks the interaction, then the compound is an agent capable of regulating oestrogen synthesis.
The present inven
Purohit Atul
Reed Michael John
Singh Anita
Eyler Yvonne
Frommer & Lawrence & Haug LLP
Imperial College of Science Technology and Medicine
Kowalski Thomas J.
Murphy Joseph F.
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