Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Nitrogen containing other than solely as a nitrogen in an...
Reexamination Certificate
2000-06-29
2001-12-25
Weddington, Kevin E. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Nitrogen containing other than solely as a nitrogen in an...
C514S318000, C514S481000, C514S724000
Reexamination Certificate
active
06333359
ABSTRACT:
The present invention relates to the use of &bgr;2-adrenergic receptor (&bgr;2-AR) modulators for the treatment of pathologies associated with a deregulation of apoptosis.
The term “apoptosis” denotes a particular form of cell death which depends on a genetically programmed process. Apoptosis is a physiological mechanism which occurs normally during the development and life of an organism. It is, inter alia, involved in the morphological changes of organs during ontogenesis, the regulation of the number of cells in tissues, the removal of certain sub-populations of lymphocytes in the regulation of the immune system, and the removal of abnormal cells.
Certain diseases are associated with a deregulation of apoptosis. If this is abnormally increased, it leads to excessive cell death (for example in the case of hepatitis, cardiac ischemia, neurodegenerative diseases and AIDS). If, on the other hand, it is insufficient, it allows the survival and proliferation of undesirable cells (for example in the case of carcinogenesis, auto-immune diseases and certain viral infections).
Cell death brought about by apoptosis results from the activation of endogenous endonucleases which destroy the cellular DNA; various factors resulting in this activation have been described. However, the mechanisms governing the mode of action of these factors are still poorly understood.
Two proteins, TNF-&agr; and the Fas ligand [ITOH et al., Cell, 66, pp. 233-240, (1991); OEHM et al., J. Biol. Chem., 267, pp. 10709-10715, (1992)] play a particularly important role in triggering apoptosis processes in many cells. These proteins interact with their respective receptors, TNFR [or TNF-&agr;r] and Fas [or FasAg, CD95, APO-1], at the cell surface. It has been found that the expression of these proteins and/or of their membrane receptors is impaired in certain pathologies associated with a deregulation of apoptosis; for example, in hepatitis, an increase in the expression of TNF-&agr; and TNFR has been observed [SPENGLER et al., Cytokine, 8(11), pp. 864-872, (1996)] and likewise for the expression of the Fas receptors [HIRAMATSU et al., Hepatology, 19, pp. 1354-1350, (1994)]; [MOCHIZUKI et al., J. Hepatology, 24, pp. 1-7, (1996)], which is, in contrast, reduced in the case of hepatocarcinomas [HIGAKI et al., Am. J. Pathol., 149, pp. 429-437, (1996)].
Anti-Fas antibodies having an activity similar to that of FasL are used in experimental models of apoptosis. Injection of these antibodies into mice brings about a fulminant hepatitis due to a massive apoptosis of the hepatocytes, leading to the death of the mice within hours of the injection [OGASAWARA et al., Nature, 364, pp. 806-809, (1993); NAGATA, Prog. Mol. Subcell. Biol., 16, pp. 87-103, (1996)].
Various in vivo inhibitory factors of apoptosis have been demonstrated. It has been observed, for example, that transgenic mice expressing the human Bcl-2 protein [LACRONIQUE et al., Nature Medecine, 2, pp. 80-86, (1996); RODRIGUEZ et al., J. Exp. Med., 183, pp. 1031-1036, (1996)], the T antigen of SV40 [ROUQUET et al., Oncogene, 11, pp. 1061-1067, (1995)], and mice to which protease inhibitors of ICE (interleukin 1&bgr; converting enzyme) type had been administered [ROUQUET et al., Curr. Biol., 6, pp. 1192-1195, (1996); RODRIGUEZ et al., J. Exp. Med., 184, pp. 2067-2071, (1996)], or an immunomodulator, linomide [REDONDO et al., J. Clin. Invest., 98, pp. 1245-1252, (1996)], were protected against the hepatic apoptosis induced experimentally with anti-Fas antibodies.
Moreover, the team of the Inventors has previously obtained a transgenic mouse, named F28, which expresses functional human &bgr;2-adrenergic receptors (H&bgr;2-ARs) [ANDRE et al., Eur. J. Biochem., 241, pp. 417-424, (1996)]. In the F28 mouse, the H&bgr;2-ARs are expressed in the muscle, the heart, the brain, the lungs and, especially, in the liver, in which the level of expression is very high. The expression of the &bgr;2-ARs in the liver of the F28 mice is closer to that observed in man than to that observed in normal mice.
The Inventors have now observed that when anti-Fas antibodies are administered to transgenic F28 mice, these mice survive this administration, and that their liver shows few apoptotic foci, whereas in normal mice, anti-Fas antibodies administered under the same conditions bring about a massive apoptosis of the hepatocytes, and the death of the animals.
The Inventors have thus put forward the hypothesis of a relationship between the increase in the number of &bgr;2-ARs in the liver cells of F28 mice, and the resistance of these cells to apoptosis, and have investigated whether or not this resistance could effectively result in an increase in the hepatic &bgr;2-adrenergic activity.
Several types of &bgr;-adrenergic receptor are known, and have been detected in greater or lesser amounts in various tissues of the body. The &bgr;1-adrenergic receptors (&bgr;1-ARs) and/or &bgr;2-adrenergic receptors (&bgr;2-ARs) are present in many tissues such as, for example, heart, kidney, lung, liver, brain, adipose tissue, smooth muscle, striated muscle, the cells of the immune system, etc. The &bgr;3-adrenergic receptors (or &bgr;3-ARs), which are present in smaller number than the above receptors, have been detected in the colon, the heart, the kidneys, the lungs, the liver, the brain, muscle and in particular in adipose tissue. A fourth type of &bgr;-AR has been described recently in the human heart [KAUMANN and MOLENAAR, Naunyn-Schmiedeberg's Arch. Pharmacol., 355(6), pp. 667-681, (1997), KAUMANN and LYNHAM, Br. J. Pharmacol., 120(7), pp. 1187-1189, (1997)].
The &bgr;-adrenergic receptors intervene in stimulation of the cAMP signaling pathway. According to observations carried out in vitro on various types of cell, it appears that the activation of adenylate cyclase and/or the stimulation of the cyclic AMP (cAMP) signaling pathway have variable effects on apoptosis; for example, apoptosis is stimulated in the thymocytes [KIZAKI et al., Cytokine, 5, pp. 342-347, (1993); MENTZ et al., Eur. J. Immunol., 25, pp. 1798-1801, (1995)], in granular cells [AHARONI et al., Exp. Cell. Res., 218, pp. 271-282, (1995)] and in B cells [LOMO et al., J. Immunol., 154, pp. 1634-1643, (1995); BAIXERAS et al., Scand. J. Immunol., 43, pp. 406-412, (1996)], whereas it is inhibited in T-cell hybridomas [LEE et al., J. Immunol., 151, pp. 5208-5217, (1993); HOSHI et al., Int. Immunol., 6, pp. 1081-1089, (1994)], human neutrophils [ROSSI et al., Biochem. Biophys. Res. Commun., 217, pp. 892-899, (1995)], MCF-7 mammary carcinoma cells [BOE et al., Br. J. Cancer, 72, pp. 1151-1159, (1995)] and cells derived from the spinal cord [BERRIDGE et al., Exp. Hematol., 21, pp. 269-276, (1993)].
In order to determine the possible role of the hepatic &bgr;2-adrenergic activity in the resistance to apoptosis, the Inventors administered an agonist specific for the &bgr;2-ARs to normal mice, and found that they survived the injection of anti-Fas antibodies, and were protected against the hepatic apoptosis induced by these antibodies. They also found, on the other hand, that the administration to mice of a &bgr;2-AR antagonist inhibited the resistance to apoptosis of F28 mice, or that of normal mice treated with the &bgr;2-AR agonist.
These results thus show that &bgr;2-adrenergic regulation plays a major role in the physiological control of the apoptosis process in vivo.
A subject of the present invention is the use of a &bgr;2-adrenergic modulator to obtain a medicinal product for regulating cell apoptosis.
The &bgr;2-adrenergic activity modulators which can be used in accordance with the present invention comprise &bgr;-AR agonists and antagonists which are active on the &bgr;2-ARs; it is possible, for example, to use isoproterenol as an agonist, or propranolol as an antagonist.
Advantageously, modulators that are specific for
Andre Claudine
Briand Pascale
Guillet Jean-Gerard
Institut National de la Sante et de la Recherche Medicale (Inser
Morgan & Lewis & Bockius, LLP
Weddington Kevin E.
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