| Method for identifying active anti-apoptosis compounds -> Monitor Keywords |
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Method for identifying active anti-apoptosis compoundsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) DoaiMethod for identifying active anti-apoptosis compounds description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060172921, Method for identifying active anti-apoptosis compounds. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This is a divisional of U.S. application Ser. No. 10/129,295, which is the U.S. national phase of international application PCT/EP00/1 0806 filed Nov. 2, 2000, the entire respective disclosures of which are hereby incorporated by reference. [0002] The invention relates to a method for identifying substances with antiapoptotic activity and to the substances identified thereby. The invention further relates to pharmaceutical preparations which comprise such substances, and to their use for the treatment of vascular disorders. [0003] Arteriosclerosis ("arterial calcification") is the most important and commonest pathological change in the arteries. It is associated with a change in the vessel content and with lesions of the endothelium (of the endothelial cells) and, caused thereby, metabolic and cellular reactions of the vessel wall. Disturbances of arterial blood flow are the commonest cause of death in industrialized countries (about 50%). The basis thereof in most cases is arteriosclerosis. [0004] The efficacy of agents currently employed against coronary heart disease is based essentially on reducing the myocardial oxygen consumption and adjusting the reduced coronary blood flow. These agents additionally bring about dilation of the coronary arteries. On the other hand, it is scarcely possible by medication to increase the coronary blood flow in cases of arteriosclerotic coronary stenosis because the diseased vessels can no longer be dilated. All medicaments act only in a very late stage of the disorder. No agents are yet known to be able to treat the cause of the disorder directly or to be suitable for early diagnosis. In the absence of an effective possibility of early diagnosis, most patients are treated only after a mild cardial infarction. In the advanced stage, usually a surgical operation (e.g. bypass) is the only option left. [0005] The inner walls of all blood vessels are lined by endothelial cells. They are involved in the regulation of various physiological processes such as, for example, the regulation of blood pressure and the degeneration and regeneration of vessels. A large number of pathological situations is associated with dysfumction of endothelial cells, for example the focal development of arteriosclerosis. [0006] Apoptosis (synonym: programmed cell death) is an irreversible process and cannot be stopped. Accordingly, an apoptotic cell inevitably dies. [0007] European patent application EP-A-0 903 149 describes a method for identifying apoptosis-inducing substances in immune cells. It showed that substances which bind to the integrin-associated protein (IAP or CD 47) on the surface of immune cells may have the ability to induce apoptosis. The mechanism of action was not described. [0008] It has already been proposed that IAP is involved in the formation of a specific calcium channel (Schwartz, M. A. et al., The Journal of Biological Chemistry, 268:27, 19931-19934). A role of this hypothetical calcium channel in the induction of apoptosis was not mentioned. [0009] Arteriosclerotic lesions form more frequently at bifurcations (forks) in the vascular system than in unbranched regions of the blood vessels. It has already been possible to observe apoptotic endothelial cells in the region of these lesions. It is suggested that apoptotic endothelial cells are involved in the development of arteriosclerosis [Asakura, T., Karino, T., Circulation Research, 66, 1045-1066 (1990)]. [0010] No agents are currently known for the prevention or therapeutic treatment of the occurrence of apoptosis in endothelial cells of the vascular system. [0011] The present invention is therefore based on the object of providing a method with which it is possible to find substances which inhibit apoptosis in endothelial cells. This method is simple to operate and reliable and cost-effective to carry out. The substances identified in this way are to be employed as ingredients of pharmaceutical preparations for the treatment of conditions in which an inhibition of apoptosis is indicated, in particular of vascular disorders, particularly preferably of arteriosclerosis. [0012] This object is achieved by a method for identifying apoptosis-inhibiting substances and substances with antiapoptotic activity, wherein cells which express both IAP and the integrin .alpha..sub.v.beta..sub.3 are cultivated, and/or the cells are caused to produce an apoptosis-inducing substance and/or a substance(s) which induces/induce apoptosis is/are added, a test substance is added and the apoptosis rate is measured. The invention likewise encompasses substances which can be identified by the claimed method, pharmaceutical preparations which comprise such a substance as active ingredient, and the use of this pharmaceutical preparation for the treatment of vascular disorders, in particular for the treatment of arteriosclerosis. The invention further encompasses the use of substances which have been identified with the aid of the method of the invention for treating vascular disorders, in particular for treating arteriosclerosis. [0013] The inventors have surprisingly shown that the simultaneous binding of thrombospondin-1 (TSP-1) to IAP and integrin .alpha..sub.v.beta..sub.3 induces apoptosis in endothelial cells. It has additionally been possible to show, surprisingly, that TSP-1 is produced by the endothelial cells themselves, and thus the apoptosis is self-induced or spontaneous. These investigations were carried out in conventional, static cell cultures. These are distinguished by the absence of any flows in the cell culture medium. However, it was unexpectedly possible to show that endothelial cells produce no TSP-1 in a perfusion culture, i.e. under conditions where the cells are confronted by a flowing cell culture medium, and apoptosis occurs to only a very small extent or not at all in this cell culture. [0014] Supplementation of fresh medium with TSP-1 causes an increase in spontaneous apoptosis in statically cultivated endothelial cells. This increase corresponds approximately to the effect of statically conditioned medium (i.e. medium which has previously been used for the cultivation of HUVEC in static culture) (table 1). This shows that statically conditioned medium has the ability to induce apoptosis via a mediator such as TSP-1. [0015] The term "conditioned" medium means herein a cell culture medium which has previously been used for cultivating other cells. This medium is distinguished by having dissolved in it soluble mediators, e.g. growth factors, hormones etc., which are produced by cells during their cultivation. [0016] It was possible to show by use of an anti-TSP-1 antibody which binds to and thus neutralizes TSP-1 that TSP-1 is the mediator of the apoptosis of endothelial cells. The effect of added TSP-1 can be suppressed, just like the effect of statically conditioned medium, by addition of a polyclonal antiserum against TSP-1 and by addition of a monoclonal anti-TSP-1 antibody (table 1). TABLE-US-00001 TABLE 1 Apoptosis Culture rate conditions Culture medium (24 h) (%) Static Fresh medium (a) 0.9 .+-. 0.1 Static Conditioned medium (b) 3.0 .+-. 0.2 Static Conditioned + anti-TSP 1 (c) 0.1 .+-. 0.1 Static Fresh + TSP 1 (e) 3.0 .+-. 0.4 Static Fresh + anti-TSP 1 (d) 0.2 .+-. 0.1 Static Fresh + TSP I + anti-TSP 1 (f) 0.2 .+-. 0.1 Static Fresh + mAb TSP 1 (g) 0.4 .+-. 0.1 [0017] The apoptosis rate (%) was determined as shown in example 6. The letters (a) to (g) relate to the investigations presented in example 10 and to the specific experimental conditions chosen therein (see example 10). [0018] It was additionally shown qualitatively by Western blot investigations that TSP-1 is secreted only by statically cultivated endothelial cells (FIG. 1). The secretion rates were determined for dynamic and static postconfluent cultures (FIG. 2). The results showed that induction of apoptosis can be controlled via the TSP-1 secretion rate. [0019] The term "static cell culture (conditions)" means here a cultivation of cells under conditions with which invariable, i.e. consistently directed, laminar flows do not occur in the cell culture medium surrounding the cells. The "static cell culture conditions" in this sense used herein thus include cell culture conditions under which turbulent or variable laminar flows, that is to say, for example, those with changing directions of flow or even with reversal of flow, occur. The term "dynamic cell culture (conditions)" means here cell culture conditions with which only consistently directed, laminar flow conditions prevail in the cell culture medium, i.e. cell culture conditions like those which can be achieved in the prior art for example with the aid of a so-called perfusion culture. It is clear that the conditions of idealized physical fluid mechanics are not attained either in the blood vessel system, i.e. in the in vivo situation, or under cell culture conditions. The flow conditions described herein are thus those which can approximately be attained, and are used, generally in cell culture experiments in the prior art. [0020] The shear stresses thus acting on the cultivated cells vary with the flow conditions. A consistently directed laminar flow results in a shear stress which is greater than 0.001 dyn/cm.sup.2 and whose vector sum is greater than under variable flow conditions with changing directions of flow. Static cell culture (conditions) are distinguished by a distinctly smaller (<0.001 dyn/cm.sup.2) or absolutely no stress. Dynamic cell cultures show shear stresses of >0.001 dyn/cm.sup.2 or a Reynolds number of >0.1. Turbulent flows may occur at Reynolds numbers of >200 (-1000) (depending on the geometry of the flow chamber) and, like static or variable flow conditions, no longer have a protective character in relation to induction of apoptosis. [0021] However, surprisingly, addition of TSP-1 to a perfusion culture generally has no effect on apoptosis. This shows that apoptosis depends not only on the occurrence of TSP-1 in the bloodstream but, on the contrary, also on the occurrence or accessibility of specific receptors on the surface of the cells. It was surprisingly possible to show in this connection that the integrin .alpha..sub.v.beta..sub.3 receptor is detectable on statically and dynamically cultivated cells, whereas the IAP receptor is expressed in detectable quantities only in static culture. [0022] It is known that TSP-1 binds to the integrin .alpha..sub.v.beta..sub.3. The binding of TSP-1 to the .alpha..sub.v.beta..sub.3 integrin is mediated by an RGD sequence motif. A potent agonist of RGD-mediated binding, a cyclic Arg-Gly-Asp-(D-Phe)-Val peptide (cyclic RGD peptide) (supplied by Merck), was therefore added to postconfluent endothelial cells. However, this unexpectedly had no effect on the apoptosis rate. This showed that binding of TSP-1 to the integrin .alpha..sub.v.beta..sub.3 at least does not on its own lead to apoptosis induction (table 2). TABLE-US-00002 TABLE 2 Culture medium (48 h) Apoptosis rate (%) Fresh medium (control) 1.5 .+-. 0.2 Conditioned medium 6.4 .+-. 0.5 Fresh + TSP1 6.9 .+-. 1.2 Fresh + active RGD 1.2 .+-. 0.5 Fresh + inactive RGD 1.0 .+-. 0.4 Fresh + CBD 2.0 .+-. 0.2 Fresh + CBD + active RGD 7.0 .+-. 0.6 Fresh + CBD + inactive RGD 1.7 .+-. 0.6 Fresh + CBD + active RGD + anti-TSP1 6.3 .+-. 1.3 [0023] The apoptosis rate was determined as shown in example 5. Cultivation in fresh medium serves as control and shows a spontaneous apoptosis rate in static cell culture. Conditioned medium has previously been incubated with endothelial cells for 48 to 72 hours, so that it contained the factors secreted by the endothelial cells. The apoptosis rate caused by the conditioned medium was at the same level as with fresh medium+TSP-1, which showed that one or more mediator(s) of apoptosis must have been present in this conditioned medium. The preparation of conditioned medium is explained in example 5. [0024] Another possible interaction of TSP-1 with a receptor on endothelial cells is the binding to IAP via the C-terminal cell binding domain (CBD). The C-terminal cell binding domain (CBD) is a TSP-1 domain which interacts specifically with IAP. A truncated TSP-1 which consists only of this C-terminal cell binding domain is marketed by Bachem as CBD peptide. Addition of the CBD peptide did not lead to an increase in the apoptosis rate (table 2). Simultaneous addition of the CBD peptide and the cyclic RGD peptide did, however, surprisingly lead to a marked increase in the apoptosis rate (table 2), which was at a similar level to the increase in the apoptosis rate by addition of TSP-1. It follows from this that only simultaneous binding to IAP and the integrin .alpha..sub.v.beta..sub.3 is effective for apoptosis. Continue reading about Method for identifying active anti-apoptosis compounds... Full patent description for Method for identifying active anti-apoptosis compounds Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for identifying active anti-apoptosis compounds patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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