AU670962B2 - Method for antagonizing inositol 1,4,5-triphosphate - Google Patents

Description

if OPI DATE 26/04/94 APPLN. ID 48417/93 AOJP DATE 14/07/94 PCT NUMBER PCT/US93/08168 1111111111111111 11111 ilill AU9348417 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 A61K 31/795 (11) International Publication Number: Al (43) International Publication Date: WO 94/07507 14 April 1994(14.04.94) (21) International Application Number: (22) International Filing Date: 3 Priority data: 07/952,393 28 Septer PCT/US93/08168 30-August 1993 (30.08.93) nber 1992 (28.09.92) US (81) Designated States: AU, CA, FI, HU, JP, KR, NO, NZ, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

Published With international search report.

With amended claims.

67O9b~ (71) Applicant: MERRELL DOW PHARMACEUTICALS INC. [US/US]; 2110 East Galbraith Road, P.O. Box 156300, Cincinnati, OH 45215-6300 (US).

(72) Inventor: STAUDERMAN, Kenneth, A. 1343 Meier Avenue, Cincinnati, OH 45208 (US).

(74) Agent: SAYLES, Michael, Marion Merrell Dow Inc., 2110 East Galbraith Road, P.O. Box 156300, Cincinnati, OH 45215-6300 (US).

(54)Title: METHOD FOR ANTAGONIZING INOSITOL 1,4,5-TRIPHOSPHATE

O

H3C N SO3-Na+ N3-NaO H3-Na+

S

0 3 Na

'N

4 CH3 n (57) Abstract Oligomers of formula have been demonstrated to be effective antagonists of inositol 1,4,5-triphosphate (IP 3 by competitively vying with IP 3 for binding sites. By competitively inhibiting the activity of IP 3 the oligomers of this invention can modulate the release of intracellular calcium and elicit the resultant physiological effects.

L-I ii c i L i rl IA WO 94/07507 PCT/US93/08168 -1- METHOD FOR ANTAGONIZING INOSITOL 1,4,5-TRISPHOSPHATE 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 1,4,5triphosphate (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 public; ion, 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 j 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: L ,I; WO 94/07507 PCT/US93/08168 -2-

S

0 3-Na 0 o SH3C N N N CH 3 SO3-Na 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 1,4,5-triphosphate (IP 3 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, IP 3 acts to stimulate the release of calcium ions from storage organelles within 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 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.

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 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 WO 94/07507 PCT/US93/08168 -3released calcium will elicit the appropriate cellular response.

Heretofore, the only verified potent antagonist of the IP3 receptor was heparin, a complex glycosaminoglycan. The diamino benzenecisulfonic 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 IP 3 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 Ul of test compound (made up as a 10x stock in water) or water, 50 ul [3H] IP 3 (17Ci/mmol; Dupont-NEN; usually made as a 25 nM (10x) stock solution in buffer), and 350 p. of buffer B (buffer A with pH adjusted to 8.4) on ice. Tubes for non-specific binding also contained l of non-radioactive IP 3 (100 pM stock (10x); final concentration 10 UM), with an appropriate reduction in the volume of buffer B. Reactions were initiated by the addition of 50 4l tissue to make the final volume 500 l, WO 94/07507 PCT/US93/08168 -4followed 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 il of Protosol (Dupont- NEN). After solubilization, 73 ul 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 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] IP3 binding sites in rat cerebellar membranes. The compound identified as MDL 102,869 was the most potent competitor for binding with an

IC

5 s of 50 nM, whereas low molecular weight heparin (5100 MW) had an ICo 5 of 74 nM. MDL 102,869 is the compound according to the claimed invention wherein n The potency for binding also seems to correlate with the ability to antagonize IP3-induced calcium ion release.

Thus, 1 and 3 pM of MDL 102,869 inhibited calcium ion release by 42 and 100%, respectively (see Fig. whereas J 1 1 i 'r 1 1 11

V

I

WO 94/07507 PCT/US93/08168 UM of heparin inhibited release by 90%. MDL 101,828, which had an IC 50 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 Compound Binding Inhibition of Ins(1,4,5)P 3

IC

50 (nM) Induced Ca2+-release 0 Heparin (low MW) 74 90% at 10 uM MDL 101,828 104 42% at 3 uM MDL 102,869 50 42% at 1 uM 100% at 3 pM The tracing of Fig. 1, dramatically illustrates the IP 3 inhibition data set forth in the third column of Table 1.

j j N -CH 3

H

n The y-axis represents the concentration of free calcium ions in arbitrary units. The tracing shows that two successive additions of 0.1 uM of IP 3 stimulated similar amounts of calcium ion release from cerebellar microsomes.

The addition of 1 pM 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 uM of IP 3 was inhibited by 42%. This inhibition was overcome by the addition of 1 1M of IP 3 consistent with competitive antagonism by MDL 102,869.

consistent with competitive antagonism by MDL 102,869.

Claims (3)

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: H 3 C HC- H SO3-Na+ O H -3N H S 0

3-Na N CH 3 H r LI C i tilt II I C I( C. CI wherein n is a whole number within the range of 5-20 and the pharmaceutically acceptable salts thereof.. The method of claim 1 wherein n is 9. 3. The method of claim 1 wherein n is 15. 1 1 it: I I, INTERNATIONAL SEARCH REPORT Inte i nal Application No PCT/US 93/08168 A. CLASSIFICATION OF SUBJECT MATER IPC 5 A61K31/795 According to Internatonal Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) IPC 5 A61K Documentation searched other than minimum documentation to the extent that such documents are includl mn the fields searched Elecronic data base consulted during the international search (name of data base and, where practical, search terms used) ;i C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. Y EP,A,O 467 185 (THE DOW CHEMICAL COMPANY) 1 22 January 1992 cited in the application X see abstract; claims; example 3 2,3 Y THE BIOCHEMICAL JOURNAL 1 vol. 267, no. 2 1990 pages 297 302 F. O'ROURKE ET AL. 'THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR BINDING SITES OF PLATELET MEMBRANES' see the whole document SFurther documents are listed in the continuation of box C. Patent family mnbers are listed in annex. Special categories of cited documents: T later document published after the international filing date A' document defining the general state of the art which isor priority date and not in confiet with the applicton but d euedntoebeiof pargcerar levance a ich ntcited to understand the principle or theory underlying the consdered to be of paracular relevance mventon E" earlier document but published on or after the international X document of particular relevance; the claimed invention fling date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken alone which is cited to establish the publicaton date of another Y document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but m the art. later than the pnority date claimed document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report 14 December 1993

23.' '1. Name and mailing address of the ISA Authorized officr European Patent Office, P.B. 5818 Patentaan 2 NL 2280 HV Rijswijk Tel. (+31-70) 340-2040, Tx. 31 651 epo nl, ff Fa (+31-70) 340-3016 Hoff, P Form PCT/ISA/210 (second sheet) (July 1992) page 1 of 2 I V INTERNATIONL SEARCH REPORT I Intet. ral Apiplication No PCT/US 93/08168 C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category' Citation Of document, with indication, where appropriate, of the relevant paaages Relevant to claim No. A FEBS LETTERSI vol. 252, nu. 1,2 1989 pages 105 108 M.A. TONES ET AL. 'THE EFFECT OF HEPARIN ON THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN RAT LIVER MICROSOMES' see the whole document A THE JOURNAL OF BIOLOGICAL CHEMISTRY1 vol. 263, no. 23 1988 pages 11075 11079 T.K. GHOSH ET AL. 'COMPETITIVE, REVERSIBLE, AND POTENT ANTAGONISM OF INOSITOL 1,4, CALCIUM RELEASE BY HEPARIN' see the whole document A GB,A,781 479 (CIBA LIMITED) 21 August 1957 1-3 see page 2, line 8 line 30; claims 1,2,9-12,22; example Form PCT/ISA/21 0 (continuation of second sheet) (July 1992) page 2 of 2 IN'NJ7WATICI1 cr ADf'! .D D1D Int. -ional application No. PCT/US 93/08168 a nrjstj in is I~vrlr oL.s r %n A Box I Observations where certain claims were found unsearchable (Continuation of item I of first sheet) This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: Although claim 1 is directed to a method of treatment of the human/animal body the search has been carried out and based on the alleged effects of th e compound/composition. 2. M Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: 1. D As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. O As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. D As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 4. D No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest O The additional search fees were accompanied by the applicant's protest D No protest accompanied the payment of additional search fees. I Form PCT/ISA/210 (continuation of first sheet (July 1992) FomPTIS/20(cniutino irtset I)(ul 92 '1- I INTERNATIONAL SEARCH REPORT Information on patent family members Intar. ,nal Application No IPCT/US 93/08168 Patent document I Publication IPatent family I Publication cited in search report I date I member(s) I date EP-A-0467 185 22-01-92 AU-B- 635850 0 1-04-93 AU-A- 8024291 09-01-92 AU-A- 8286791 04-02-92 CA-A- 2046491 10-01-92 CN-A- 1058959 26-02-92 EP-A- 0538373 28-04-93 JP-A- 4226521 17-08-92 WO-A- 9200749 23-01-92 GB-A-781479 NONE I L. Form PCT/ISA/210 (patent family annex) (July 1992)