Method for inhibiting cytokine production by cells

Details Method for inhibiting cytokine production by cells Method for inhibiting cytokine production by cells

2000-10-13

2002-06-18

Lankford, Jr., Leon B. (Department: 1651)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Azoxy doai

C514S150000, C514S151000, C514S579000

Reexamination Certificate

active

06407081

ABSTRACT:

This application is a U.S. national phase of PCT/BE98/00183, filed Nov. 25, 1998, which claims priority of Belgium Application Serial No. 9700949, filed Nov. 26, 1997.
The present invention relates to a process for in vitro inhibition of the production of cytokines by cells, in particular animal or human cells, and of the secretion thereof.
The object of the present invention is to provide a process for in vitro inhibition of the production of cytokines which does not jeopardise said cells. Advantageously, this process will allow inhibition of the production and secretion of substances promoting the appearance of immunoallergic phenomena in these cells.
This object is achieved according to the invention by a process as described above comprising application onto said cells of at least one azo derivative of the formula
in which A represents a carboxyl group or a group
B represents a carboxyl group or a group
R
1
, R
2
, R
3
and R
4
are identical or different and each mutually independently represent a hydrogen or halogen atom, an optionally substituted aliphatic or aromatic hydrocarbon residue or a hydroxy group, R
1
and R
2
possibly being joined together to form a heterocyclic nucleus with the nitrogen atom adjacent thereto, and R
3
and R
4
possibly being joined together to form a heterocyclic nucleus with the nitrogen atom adjacent thereto, X
1
and X
2
are identical or different and each mutually independently represent an oxygen atom or a group NR
5
, in which R
5
is a hydrogen or halogen atom, an optionally substituted aliphatic or aromatic hydrocarbon residue or a nitro group, and in which, when two groups NR
5
are simultaneously present, each R
5
may be identical to or different from the other, together with the salts, esters and isomers thereof.
Various of these azo derivatives are compounds which are known in particular for the antiviral activity thereof, especially against viruses of the retrovirus group, in particular the AIDS virus (c.f. WO-A-9116054 and WO-A-9107876, together with U.S. Pat. No. 5,585,367).
More particularly in relation to 1,1′-azobisdimethylformamide, which is also known as diamide, many researchers have studied the activation phenomenon of the intracellular transcription factor NF-kappaB and have demonstrated that diamide blocks the function of certain enzymes in the activation cascade for this factor.
Other authors have demonstrated that at certain concentrations diamide induces apoptosis in certain cell lines.
Still other authors have demonstrated that diamide has an effect on certain membrane receptors.
However, none of these studies reveals any inhibitory action of diamide on the production of cytokines by the cells under observation. It may even be noted that C. Mendez et al. in “Oxidants augment endotoxin-induced activation of alveolar macrophages”, SHOGK, volume 6, no. 3, pp. 157-163, 1996, observe an insignificant increase in the production of tumour necrosis factor at a dose of 1 mmol, i.e. the reverse effect to that repeatedly observed on various cytokines according to the present invention.
It is moreover known that the azo derivatives according to the invention exhibit extremely low intrinsic toxicity towards the human body or healthy cells which are treated.
According to one embodiment of the invention, the process comprises in vitro inhibition of the production and secretion of interleukins by cells. Interleukins which may in particular be mentioned are interleukins IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 and IL-15, with inhibition of IL-2 and IL-5 most particularly being intended.
According to another embodiment of the invention, the process comprises in vitro inhibition of the production and secretion of interferon-&ggr; (IFN-&ggr;) by cells. According to still another embodiment, the process comprises inhibition of the production and secretion of tumour necrosis factor &agr; (TNF-&agr;).
According to an advantageous embodiment of the invention, in the above-stated general formula, R
1
to R
5
each represent an aliphatic or aromatic hydrocarbon residue comprising 1 to 6 carbon atoms. The heterocycles optionally formed in the general formula may contain, apart from one nitrogen atom, at least one other heteroatom, for example oxygen. The heterocycle is, for example, pentagonal to octagonal, preferably being hexagonal. The azo derivative according to the invention may be selected from among the group comprising azobisformamidine derivatives, such as 1,1′-azobisform-amidine, 1,1-azobisnitroformamidine, 2,2′-azobismethyl-formamidine, 1,1′-azobisfluoroformamidine, 1-monochloro-azobisformamidine, azobis [chloroformamidine], azobisformamide derivatives, such as 1,1′-azobisformamide and dimethylazobisformamide, 1,1′-(azodicarbonyl)-dipiperidine, 1,1′-(azodicarbonyl)dimorpholine, azodihydroxamic acid and the salts thereof, azodicarboxylic acid and the salts thereof.
The process advantageously comprises application of the azo derivative onto the cells at a dose which does not induce apoptosis thereof. A micromolar concentration of approx. 0.4 to approx. 200, preferably of 2 to 20, advantageously of 2 to 10, may be considered.
It this regard, 1,1′-azobisdimethylformamide, also known as diamide, may under certain circumstances exhibit the disadvantage of bringing about cellular apoptosis at certain excessively high concentrations, while providing a relatively short half-life of only a few minutes.
According to one embodiment of the invention, at least one of the stated azo derivatives is applied onto cells isolated from macroorganisms or cells of microorganisms, for example originating from cell cultures. The treated cells may also be those from an organ or multicellular tissue taken from the body of a human or an animal, such as for example the cells from a sample of blood or lymph.
According to one embodiment of the invention, at least one of the stated azo derivatives is applied, for example, onto cytokine producing cells isolated from human blood. These cells may be extracted in an overall manner, in which case overall effects will be observed on the various types of lymphocytes, on cells having antigens and on macrophages in co-cultures. A more advanced stage of the investigation may consist in measuring the production of cytokines by highly purified cell lines, for example CD4 lymphocytes.
The cell lines concerned may also be treated by administering azo derivatives to living organisms, including human beings, and measuring the various groups of cytokines in the biological fluids taken from the body of a human or an animal before, during and/or after treatment, and by measuring the production capacity for the lymphokines under consideration after extraction of the cells present in these fluids.
It is also possible to consider treating grafts or cellular tissues, prior to the transplantation thereof into the body of a human or an animal, with an azo derivative according to the invention.
It is also possible to provide the use of pharmaceutical preparations based on azo derivatives according to the invention to treat patients against graft rejection.
Cytokines are produced in the body by different cell reservoirs.
It is known, for example, that lymphocytes specialise specifically in the production of cytokines and that, depending upon the type of cytokine produced, a distinction is drawn between Th1 and Th2 CD4 lymphocytes.
Cytokines are also produced not only by CD8 lymphocytes (IL-4 and IL-5), but also by mast cells and eosinophils and, during uterine implantation, by the ectodermal cells of the trophoblast.
Cytokines are produced by these various cell reservoirs during the appearance of various inflammatory and allergic reactions in general and during the phenomenon of graft rejection. The following is a non-exhaustive list of pathological conditions which may inter alia be mentioned: asthma, atopic dermatitis, allergic seasonal rhinitis, psoriasis, pemphigus, thyroiditis, myasthenia gravis, rheumatoid arthritis, IgA nephropathy, scleroderma, lupus erythematosus

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