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 (IP ₃ ) and are, therefore useful in diminishing the bioactivity of IP ₃ , 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 IP ₃ antagonists according to this invention are described in detail in the European Patent Application published January 22, 1992 under Publication No. 0467185 A2. 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 a method of inhibiting the activity of inositol 1,4,5-triphosphate (IP ₃ ) by occupying the receptor sites specific to IP ₃ with a compound of the formula:

wherein n is a whole number selected from the range of 5 to , _fl 20 inclusive and the pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION Inositol 1,4,5-triphosphate (IP ₃ ) is a naturally

15 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, IP ₃ acts to stimulate the release of calcium ions from storage organelles within

20 the cell. In this role IP ₃ is characterized as a "second messenger". Depending upon the type of cell, the calcium released by IP ₃ functions to stimulate a variety of physiologic processes such as smooth muscle contraction, histamine secretion and the hyperpolarization of nerve

25 cells. Any compound or agent that can promote or interfere with the function of IP ₃ , will promote or interfere with the generation of calcium ions and thereby elicit predictable pharmacological effects.

30 The process by which IP ₃ releases calcium ions begins with the binding of IP ₃ 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

35 a calcium "channel" with unique structural properties when bound to IP ₃ . Therefore, when IP ₃ binds with its receptor, a calcium channel is opened causing the release of calcium stored in the cell's endoplasmic reticulum. In turn, the

released calcium will elicit the appropriate cellular response.

Heretofore, the only verified potent antagonist of the IP ₃ receptor vas heparin, a complex glycosaminoglycan. The diamino benzenedisulfonic acid oligomers of this invention also appear to antagonize the effects of IP ₃ 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 IP ₃ -induced calcium release and have a salutary effect on any number of disorders that are caused or exacerbated by an inordinately productive IP ₃ second messenger pathway.

EXPERIMENTALS 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 μl of test compound (made up as a lOx stock in water) or water, 50 μl [3H] IP ₃ (17Ci/mmol; Dupont-NEN; usually made as a 25 nM (lOx) stock solution in buffer), and 350 μl of buffer B (buffer A with pH adjusted to 8.4) on ice. Tubes for non-specific binding also contained 50 μl of non-radioactive IP ₃ (100 μM stock (lOx); final concentration 10 μM) , with an appropriate reduction in the volume of buffer B. Reactions were initiated by the addition of 50 μl tissue to make the final volume 500 μl.

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 μl of Protosol (Dupont- NEN). After solubilization, 73 μl 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 IP ₃ ) and non-specific binding

(radioactivity in the absence of test compound but in the presence of cold IP ₃ ). 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 IC ₅₀ value).

The binding data are presented in Table 1 and demonstrate that compounds within the scope of the present invention effectively compete for [3H] IP ₃ binding sites in rat cerebellar membranes. The compound identified as MDL 102,869 was the most potent competitor for binding with an IC ₅₀ of 50 nM, whereas low molecular weight heparin (5100 MW) had an IC ₅₀ 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 IP ₃ ~induced calcium ion release. Thus, 1 and 3 μM of MDL 102,869 inhibited calcium ion release by 42 and 100%, respectively (see Fig. 1), whereas

10 μM of heparin inhibited release by 90%. MDL 101,828, which had an IC ₅₀ binding of 104 nM, inhibited IP ₃ ~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 IP ₃ 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 μM of IP ₃ stimulated similar amounts of calcium ion release from cerebellar microsomes. The addition of 1 μM 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 μM of IP ₃ was inhibited by 42%. This inhibition was overcome by the addition of 1 μM of IP ₃ , consistent with competitive antagonism by MDL 102,869.

WHAT IS CLAIMED IS:

1. A method of inhibiting the activity of inositol 1,4,5-trisphosphate by occupying a receptor site specific to inositol 1,4,5-trisphosphate with a compound of the formula:

wherein n is a whole number within the range of 5-20 and the pharmaceutically acceptable salts thereof.

2. The oligomer of claim 1 wherein n is 9.

3. The oligomer of claim 1 wherein n is 15.