Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1994-04-26
1996-08-06
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 4, 530351, 424 851, 4241951, C12Q 102, C07K 1452, C07K 14525, A61K 3578
Patent
active
055433006
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method and composition for assaying certain bioactive substances and determining their immunological activity with respect to their ability to induce production of cytokines. Cytokines, such as interferons (IFNs) and tumor necrosis factors-(TNFs), are hormone-like proteins which play an important role in virtually all immunological reactions, as well as in the regulatory process responsible for the maintenance of homeostasis. The production of such cytokines can be induced by certain substances which, on account of their bioactive and immunomodulating activity, are useful in the therapy of immunodeficiencies and related diseases.
There is, of course, a substantial need of methods for properly and easily assaying the immunological activity of such bioactive substances. It is the object of the present invention to provide such a method.
The method according to the present invention allows one to determine whether a tested substance is able to induce production of different cytokines and permits a quick and efficient check of the properties of certain substances, namely their immunological activity, at each stage of production, separation, purification and formulation into final compositions. The present invention provides also a composition and kits suitable for carrying out said method.
Tests used until now to determine immunological activity are numerous but difficult to carry out. When a new therapeutical composition having an immunological effect is clinically tested, its immunological effectiveness is evaluated by monitoring the numerous, well known and analytically determinable features of immunological systems employing standard methods. Once the activity is proven, there emerges the necessity for representative single tests enabling a quick check of the status of a patient in terms of his or her immunological response.
In clinical studies, evaluating IFN serum levels may be difficult, unprecise and misleading. Interferons are often produced locally in various tissues, they are short-lived, their action is paracrine (in the immediate vicinity only) and they are strongly bound to cellular receptors and carrier proteins.
On the other hand, technological processes for manufacturing, purification and/or separation of immunologically active substances such as in particular, extracts from raw peat are usually multi-step processes, and there is always the need for establishing a reliable and fast test enabling a thorough control of the production. Moreover, since the substances are frequently of a complex nature, as it is the case with peat extracts, it is important to have a similar test for determining the activity of individual fractions of such substances. It is also necessary to find out a microscale testing method, since the products to be analysed are very costly.
The following four basic findings allowed the inventors to solve the above described problems.
1. Some immunoactive peat extracts induce the production of cytokines, such as interferons and tumor necrosis factors, in in-vitro cultures of peripheral blood leukocytes, the induction being dependent on the dose.
2. Interferon-.beta. (and TNF-.alpha.) is produced in significantly higher quantities by BALB/c mice resident peritoneal cells (RPC) when treated with immunoactive peat extracts as compared to a spontaneous release of these cytokines in non-treated samples.
3. Certain peat extracts, when tested in-vitro as mentioned above show a synergistic effect in combination with known immunomodulators, such as organoselenium compounds, the p-chlorophenylamide of 3-methyl-5-benzoylamino-isothiazol-4-carboxylic acid and indomethacin. This allows the use of much smaller quantities of immunologically active substances to show their activity.
4. PBL of healthy volunteers treated with TTP (a certain peat-derived product) administered orally at a dosage of 5 mg/day, tested for induction of certain cytokines, IFN-.alpha., IFN-.gamma. and TNF-.alpha., lose the ability to respond to induction of the cytokines with TTP soluti
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Blach-Olszewska Zofia
Inglot Anna
Degen Nancy J.
Fleisher Mindy
Torf Establishment
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