Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Hemic or immune system
Reexamination Certificate
1997-08-01
2001-05-15
Wortman, Donna C. (Department: 1642)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Hemic or immune system
Reexamination Certificate
active
06231893
ABSTRACT:
This invention relates to a new immunosuppressive and tumour-suppressive factor obtained from mammalian bone marrow. This factor appears to belong to a previously undescribed class of immunosuppressive molecules.
BACKGROUND OF THE INVENTION
A number of negative regulators of immune responses, cellular proliferation and hematopoiesis have been described. Protein or peptide negative regulators include transforming growth factor-beta (1,2), leukemia inhibitory factor (3), macrophage inflammatory protein-1-alpha (4,5), Ac-Ser-Asp-Lys-Pro (6), and pGlu-Glu-Asp-Cys-Lys (7). Lipid and lipid-related negative regulators include the prostaglandins (8), the leukotrienes (9), and the glycosphingolipids (10).
Preparations derived from bone marrow cells and showing immunosuppressive activity have also been described (11 and PCT application number WO91/19506).
The new factor described herein is distinguishable from all of these previously described factors both by chemical properties and by its biological activity, as will be more fully described below.
SUMMARY OF THE INVENTION
In accordance with the present invention, a novel immunosuppressive material of molecular weight less than 1000 Da, produced by mammalian bone marrow (BM) cells, has been identified and its biological properties described.
The new bone marrow factor described herein has been named Reptimed, in view of its source and immunosuppressive activity. The name Reptimed has been derived from the following Latin words: reprimere (to suppress); temperate (to modulate); immunitas (freedom from disease); and medulla (the bone marrow). “Reptimed” as used herein means a bone marrow-derived factor having the chemical, physical and biological properties described herein.
Reptimed has been purified using solid-phase extraction, gel filtration, gradient reverse-phase high performance liquid chromatography (RP-HPLC), and isocratic RP-HPLC, as described in Example 1 and has molecular weight less than 1000 daltons. Ionspray mass spectrometry indicates a molecular weight of about 625 daltons when protonated, as described in Example 3.
Reptimed is fully soluble in water and methanol but insoluble in hexans or chloroform.
An alternate method or purification has also been used, employing solid-phase extraction, gel filtration, anion exchange fast phase liquid chromatography (FPLC), NH
2
-HPLC and reverse phase HPLC, as described in Example 3.
Reptimed can be distinguished from previously described, naturally occurring compounds with immunosuppressive or cancer-suppressive properties.
Reptimed is an effective immunosuppressor and has been shown to suppress T cell activation in response to a variety of stimulants.
Reptimed has also been demonstrated to have tumour-suppressing activity against mammalian tumours.
A further activity of Reptimed is its inhibition of the development of autoimmune responses.
Reptimed is highly conserved across species and can be obtained from bone marrow of the human, rat, mouse, cow, pig, and rabbit. Rat Reptimed has been most highly characterised to date but c18 cartridge-purified material has been isolated from bone marrow of all the above-noted species and in each case has ben shown to have the same biological activity in the WEHI-3 cell assay described in Example 1 (data not shown).
The biological activity of Reptimed is not species restricted. As seen in the examples herein, Reptimed obtained from rat bone marrow was active against human and mouse cell lines, indicating its cross-species effectiveness.
Reptimed has also been shown to interfere with early T cell signalling events involving activation of tyrosine kinases.
In accordance with one embodiment of the invention, a purified, water soluble immunosuppressive factor is provided characterised by a molecular weight less than 1000 daltons and movement as a single peak on reverse phase high performance liquid chromatography.
In accordance with a further embodiment of the invention, a factor is provided having the following biological activities:
(a) prevention or reduction of IL-2 production by activated T lymphocytes;
(b) prevention or reduction of TNF-&agr; production by activated macrophages;
(c) prevention or reduction of proliferation of activated T lymphocytes; and
(d) inhibition of mixed lymphocyte reaction.
In accordance with another embodiment of the invention, a factor is provided having the following further biological activity:
(a) inhibition of tumour growth in a mammal.
In accordance with a further embodiment of the invention, a factor is provided having the following further biological activity:
(a) inhibition of leukemia cell proliferation.
In accordance with a further embodiment of the invention, a factor is provided having the following further biological activity:
(a) inhibition of a graft versus host reaction in a mammal.
In accordance with a further embodiment of the invention, a method is provided for inhibiting T lymphocyte activation in a mammal comprising administering to the mammal an effective amount of the immunosuppressive factor described herein.
In accordance with a further embodiment of the invention, a pharmaceutical composition is provided for inhibiting T lymphocyte activation in a mammal comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier.
In accordance with a further embodiment of the invention, a composition is provided comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier for use to inhibit T lymphocyte activation in a mammal.
In accordance with a further embodiment of the invention, a method is provided for treating or preventing a disorder associated with undesirable or excessive T lymphocyte activation, in a mammal, comprising administering to the mammal an effective amount of the factor described herein.
In accordance with a further embodiment of the invention, there is provided a pharmaceutical composition for treating or preventing a disorder associated with undesirable or excessive T lymphocyte activation comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier.
In accordance with a further embodiment of the invention, composition is provided comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier for use to treat or prevent a disorder associated with undesirable or excessive T lymphocyte activation.
In accordance with a further embodiment of the invention, a method is provided for inhibiting TNF-&agr; production in a mammal comprising administering to the mammal an effective amount of the factor described herein.
In accordance with a further embodiment of the invention, a pharmaceutical composition is provided for inhibiting TNF-&agr; production in a mammal comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier.
In accordance with a further embodiment of the invention, a composition is provided comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier for use to inhibit TNF-&agr; production in a mammal.
In accordance with a further embodiment of the invention, a method is provided for treating or preventing a disorder associated with undesirable or excessive TNF-&agr; production, in a mammal, comprising administering to the mammal an effective amount of the factor described herein.
In accordance with a further embodiment of the invention, a method is provided for inhibiting growth of a tumour in a mammal comprising administering to the mammal an effective amount of the factor described herein.
In accordance with a further embodiment of the invention, a pharmaceutical composition is provided for inhibiting growth of a tumour in a mammal comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier.
In accordance with a further embodiment of the invention, a composition is provided comprising an effective amount of the factor described herein and a pharmaceutically acceptable carrier for
Brumback Brenda G.
Klarquist Sparkman Campbell & Leigh & Whinston, LLP
London Health Services Centre
Wortman Donna C.
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