IL107066A - Pharmaceutical compositions containing diaminobenzenedisulfonic acid sodium salt oligomers for antagonizing inositol 1, 4, 5-triphosphate - Google Patents

Abstract

Oligomers of formula (I) have been demonstrated to be effective antagonists of inositol 1,4,5-triphosphate (IP3) by competitively vying with IP3 for binding sites. By competitively inhibiting the activity of IP3, the oligomers of this invention can modulate the release of intracellular calcium and elicit the resultant physiological effects. [WO9407507A1]

Description

107066/2 PHARMACEUTICAL COMPOSITIONS CONTAINING DIAMINO-BENZENEDISULFONIC ACID SODIUM SALTS OLIGOMERS FOR ANTAGONIZING INOSITOL 1 ,4,5-TRIPHOSPHATE , ,1 "pio ira 1213 niTJinn'? π",]ΐ9'?ιοιτ]Τ]αΊ]ιηκΊτ 4A ABSTRACT OF THE DISCLOSURE Oligomers of the formula have been demonstrated to be effective antagonists of inositol 1,4,5-trisphosphate (IP3) by competitively vying with IP3 for binding sites. By competitively inhibiting the activity of IP3, the oligomers of this invention can modulate the release of intracellular calcium and elicit the resultant physiological effects.

BACKGROUND OF THE INVENTION This application relates to a series of diamino benzenedisulfonic acid oligomers that have demonstrated an affinity for the receptor sites of inositol 1.4,5-triphosphate (IP3) and are, therefore useful in diminishing the bioactivity of IP3. especially with regard to its effect on the release of intracellular calcium ions.

DESCRIPTION OF THE PRIOR ART The diamino benzenedisulfonic acid oligomers demonstrating utility as IP3 antagonists according to this invention are described in detail in the European Patent Application published January 22, 1992 under Publication No. 0467185 AZ and IL98761. In that publication, the oligomers of the present invention were described as having utility in the diagnosis and/or treatment of AIDS and AIDS related complex.

SUMMARY OF THE INVENTION The invention herein disclosed relates to pharmaceutical compositions for inhibiting the activity of inositol 1 ,4, 5-triphosphate (IP3) by occupying the receptor sites specific to IP3 with a compound of the formula: -2- wherein n is a whole number selected from the range of 5 to 20 inclusive and the pharmaceutically acceptable salts thereof .

DETAILED DESCRIPTION OF THE INVENTION Inositol I, 4, 5-triphosphate (IP3) is a naturally occurring and active component of animal physiology. It is formed intracellularly upon the activation of cell-surface receptors linked to the enzyme phospholipase C. Once generated in sufficient quantities, IP3 acts to stimulate the release of calcium ions from storage organelles within the cell. In this role IP3 is characterized as a "second messenger". Depending upon the type of cell, the calcium released by IP3 functions to stimulate a variety of physiologic processes such as smooth muscle contraction, histamine secretion and the hyperpolarization of nerve cells. Any compound or agent that can promote or interfere with the function of IP3, will promote or interfere with the generation of calcium ions and thereby elicit predictable pharmacological effects.

The process by which IP3 releases calcium ions begins with the binding of IP3 to a specific receptor protein located on an intracellular calcium storage compartment located typically on the endoplasmic reticulum. This receptor protein has been cloned and has been shown to form a calcium "channel" with unique structural properties when bound to IP3. Therefore, when IP3 binds with its receptor, a calcium channel is opened causing the release of calcium stored in the cell's endoplasmic reticulum. In turn, the -3-released calcium will elicit the appropriate cellular response.

Heretofore, the only verified potent antagonist of the IP3 receptor was heparin, a complex glycosaminoglycan. The diamino benzenedisulfonic acid oligomers of this invention also appear to antagonize the effects of IP3 by competing for the receptor site. In most cases, these compounds are more effective than heparin and demonstrate fewer secondary effects. In addition to providing utility as laboratory "tools" in evaluating the therapeutic potential of other IP3 receptor antagonists, the oligomers of this invention would also be administered to modulate IP3-induced calcium release and have a salutary effect on any number of disorders that are caused or exacerbated by an inordinately productive IP3 second messenger pathway.

EXPERIMEN ALS Measurement of IP3 Binding Cerebella from male Sprague-Dawley rats (200 g) were homogenized in 30 volumes of ice-cold buffer A (50 mM Tris, 1 mM dithiothreitol, 1 mM EDTA, pH 7.7 with HC1) with a polytron (setting 9 for 10 seconds). The tissue is then washed twice by centrifugation (20,000 x g, 15 minutes; Sorvall 28-S., SS-34 rotor) and resuspended in 30 volumes of ice-cold buffer A.

For the binding assays, 1.5 ml eppendorf tubes containing 50 μΐ of test compound (made up as a lOx stock in water) or water, 50 μΐ [3H] IP3 (17Ci/mmol; Dupont-NEN; usually made as a 25 nM (lOx) stock solution in buffer), and 350 μΐ of buffer B (buffer A with pH adjusted to 8.4) on ice. Tubes for non-specific binding also contained 50 μΐ of non-radioactive IP3 (100 μΜ stock (lOx); final concentration 10 μΜ) , with an appropriate reduction in the volume of buffer B. Reactions were initiated by the addition of 50 μΐ tissue to make the final volume 500 μΐ, -4-followed by vortex mixing. Samples were incubated on ice for 10 minutes and then were centrifuged (14,000 x g) in a microfuge (Eppendorf model 5415) for 5 minutes followed by aspiration of the supernatant fraction. The tissue pellets were solubilized overnight in 100 yl of Protosol (Dupont-NEN) . After solubilization, 73 μΐ of glacial acetic acid were added to decrease chemiluminescence, and the mixture was transferred to scintillation vials. To these vials was added 7 ml of Ecoscint-A (National Diagnostics) and the radioactivity determined by liquid scintillation spectrophotometry.

Specific binding was defined as the difference between total binding (radioactivity in the absence of test compound and cold IP3) and non-specific binding (radioactivity in the absence of test compound but in the presence of cold IP3). This number was taken as 100% specific binding. Data points obtained with the test compounds were fit by a computer program (GraphPad-InPlot ) to determine their inhibitory potency. The inhibitory potencies of the test compounds were expressed as the concentration of compound that produces 50% inhibition of specific binding (the IC50 value).

The binding data are presented in Table 1 and demonstrate that compounds within the scope of the present invention effectively compete for [3H] IP3 binding sites in rat cerebellar membranes. The compound identified as DL 102,869 was the most potent competitor for binding with an IC50 of 50 nM, whereas low molecular weight heparin (5100 MW) had an IC50 of 74 nM. MDL 102,869 is the compound according to the claimed invention wherein n = 15.

The potency for binding also seems to correlate with the ability to antagonize IP3~induced calcium ion release. Thus, 1 and 3 μΜ of MDL 102,869 inhibited calcium ion release by 42 and 100%, respectively (see Fig. 1), whereas -5- 10 μ of heparin inhibited release by 90%. MDL 101,828, which had an IC50 binding of 104 nM, inhibited IP3~induced calcium ion release by 72% at 3 micro moles. MDL 101,828 is the compound according to the claimed invention wherein n = 9.

TABLE 1 The tracing of Fig. 1, dramatically illustrates the inhibition data set forth in the third column of Table 1 The y-axis represents the concentration of free calcium ions in arbitrary units. The tracing shows that two successive additions of 0.1 μΜ of IP3 stimulated similar amounts of calcium ion release from cerebellar microsomes. The addition of 1 μΜ of MDL 102,869 stimulated a small increase of calcium ion for unknown reasons. In the presence of 102,869, however, calcium ion release stimulated by 0.1 μΜ of IP3 was inhibited by 42%. This inhibition was overcome by the addition of 1 μΜ of IP3, consistent with competitive antagonism by MDL 102,869. 107066/2 -6-

Claims (3)

1.A pharmaceutical composition for inhibiting the activity of inositol 1 , 4, 5-trisphosphate by occupying a receptor site specific to inositol 1 ,4,5-trisphosphate containing an effective quantity of a compound of the formula wherein n is a whole number within the range of 5-20 and the pharmaceutically acceptable salts thereof.

2. he composition of claim 1 wherein the oligomer n is 9.

3. The composition of Claim 1 wherein the oligomer n is 15.