U.S. patent application number 10/635951 was filed with the patent office on 2005-10-13 for method of treating inflammatory, tissue repair and infectious conditions.
This patent application is currently assigned to PERSTORP AB. Invention is credited to Siren, Matti.
Application Number | 20050227946 10/635951 |
Document ID | / |
Family ID | 35457895 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050227946 |
Kind Code |
A1 |
Siren, Matti |
October 13, 2005 |
METHOD OF TREATING INFLAMMATORY, TISSUE REPAIR AND INFECTIOUS
CONDITIONS
Abstract
The present invention relates to a method of treating
inflammatory, tissue repair and infectious conditions in a mammal
suffering therefrom which comprises administering to a mammal in
need thereof an effective amount of a cyclic compound selected from
the group consisting of cyclopentane, cyclohexane, cycloheptane,
monosaccharide. disaccharide, trisaccharide, tetrasaccharide,
piperidine, tetrahydrothiopyran, 5-oxotetrahydrothiopyra- n,
5,5-dioxotetrahydrothiopyran, tetrahydroselenopyran,
tetrahydrofuran, pyrrolidine, tetrahydrothiophene,
5-oxotetrahydrothiophene, 5,5-dioxotetrahydrothiophene,
tetrahydroselenophene, benzene, cumene, mesitylene, naphthalene and
phenanthrene, in which said cyclic compound is substituted by at
least three vicinal phosphorus containing radicals of the formula:
1
Inventors: |
Siren, Matti;
(Fin-Helsingfors, FI) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
SUITE 300
GARDEN CITY
NY
11530
US
|
Assignee: |
PERSTORP AB
PERSTORP
SE
|
Family ID: |
35457895 |
Appl. No.: |
10/635951 |
Filed: |
August 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10635951 |
Aug 7, 2003 |
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09876637 |
Jun 7, 2001 |
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6632797 |
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09876637 |
Jun 7, 2001 |
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09202908 |
Oct 12, 1999 |
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09202908 |
Oct 12, 1999 |
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PCT/SE97/01115 |
Oct 12, 1997 |
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Current U.S.
Class: |
514/79 ;
514/102 |
Current CPC
Class: |
A61K 31/675
20130101 |
Class at
Publication: |
514/079 ;
514/102 |
International
Class: |
A61K 031/675 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 1996 |
SE |
9602463-3 |
Claims
1-17. (canceled)
18. A method of treating inflammatory conditions comprising
administering to a mammal in need thereof an effective amount of a
cyclic compound selected from the group consisting of cyclopentane,
cyclohexane, cycloheptane, monosaccharide, disaccharide,
trisaccharide, tetrasaccharide, piperidine, tetrahydrothiopyran,
5-oxotetrahydrothiopyra- n, 5,5-dioxotetrahydrothiopyran,
tetrahydroselenopyran, tetrahydrofuran, pyrrolidine,
tetrahydrothiophene, 5-oxotetrahydrothiophene,
5,5-dioxotetrahydrothiophene, tetrahydroselenophene, benzene,
cumene, mesitylene, naphthalene and phenanthrene, in which said
cyclic compound is substituted by at least three vicinal phosphorus
containing radicals of the formula: 5wherein V.sup.1 to V.sup.4 are
independently Y.sup.8.sub.m6T.sub.o3U; T.sub.01 to T.sub.03 are
independently (CH.sub.2).sub.n, CH.dbd.CH, or
CH.sub.2CH.dbd.CHCH.sub.2; o1 to o3 are independently 0 or 1; n is
0 to 4; U is R.sup.1Y.sup.9m.sub.7,
CY.sup.10Y.sup.11R.sup.2,SY.sup.12Y.sup.13Y.sup.14R.sup.3,PY.sup.15Y.sup.-
16Y.sup.17R.sup.4R.sup.5,
Y.sup.18PY.sup.19Y.sup.20Y.sup.21R.sup.6R.sup.7, CH.sub.2NO.sub.2,
NHSO.sub.2R.sup.8, or NHCY.sup.22Y.sup.23R.sup.9; m1 to m7 are
independently 0 or 1; Y.sup.1 to Y.sup.23 are independently
NR.sup.10, NOR.sup.11, O, or S; and where R.sup.1 to R.sup.11 are
independently i) hydrogen; ii) a straight or branched saturated or
unsaturated alkyl group, containing 1-22 carbon atoms; iii) a
saturated, unsaturated aromatic or non-aromatic homo- or
heterocyclic group containing 3-22 carbon atoms and 0-5 heteroatoms
selected from the group consisting of nitrogen, oxygen and sulfur;
iv) a straight or branched saturated or unsaturated alkyl group
containing 1-22 carbon atoms substituted with a saturated or
unsaturated aromatic or non-aromatic homo- or heterocyclic group
containing 3-22 carbon and 0-5 heteroatoms selected from the group
consisting of nitrogen, oxygen, and sulfur; v) an aromatic or
non-aromatic homo- or heterocyclic group containing 3-22 carbon and
0-5 heteroatoms selected from the group consisting of nitrogen,
oxygen, and sulfur which aromatic or non-aromatic homo- or
heterocyclic group is substituted with a straight or branched
saturated or unsaturated group containing 1-22 carbon atoms;
whereby said groups in ii-v are unsubstituted or are substituted by
1-6 of the following groups hydroxy, alkoxy, aryloxy, acyloxy,
carboxy, alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyl,
aryloxycarbonyloxy, carbamoyl, fluoro, chloro, bromo, azido, cyano,
oxo, oxa, amino, imino, alkylamino, arylamino, acylamino, arylazo,
nitro, alkylthio or alkylsulfonyl.
19. The method according to claim 18 wherein the inflammatory
conditions is rheumatoid arthritis.
20. The method according to claim 18 or 19 wherein the
phosphorus-containing radicals have the following formula: 6wherein
V.sup.1 and V.sup.2 are OH, (CH.sub.2).sub.pOH, COOH, CONH.sub.2,
CONOH, (CH.sub.2).sub.pCOOH, (CH.sub.2).sub.pCONH.sub.2,
(CH.sub.2).sub.pCONOH, (CH.sub.2).sub.pSO.sub.3H,
(CH.sub.2).sub.pSO.sub.3NH.sub.2, (CH.sub.2).sub.pNO.sub.2,
(CH.sub.2).sub.pPO.sub.3H.sub.2, O(CH.sub.2).sub.pOH,
O(CH.sub.2).sub.pCOOH, O(CH.sub.2).sub.pCONH.sub.2,
O(CH.sub.2).sub.pCONOH, O(CH.sub.2).sub.pSO.sub.3H,
O(CH.sub.2).sub.pSO.sub.3NH.sub.2, O(CH.sub.2).sub.pNO.sub.2,
O(CH.sub.2).sub.pPO.sub.3H.sub.2 or CF.sub.2COOH; and p is 1 to
4.
21. The method according to claim 18 or 19 wherein the
phosphorus-containing radicals are phosphate groups.
22. The method according to claim 18 or 19 wherein the cyclic
compound is a monosaccharide.
23. The method according to claim 22 wherein the monosaccharide is
D/L-ribose, D/L-arabinose, D/L-xylose, D/L-lyxose, D/L-allose,
D/L-altrose, D/L-glucose, D/L-mannose, D/L-gulose, D/L-idose,
D/L-galactose, D/L-talose, D/L-ribulose, D/L-xylulose, D/L-psicose,
D/L-sorbose, D/L-tagatose, or D/L-fructose.
24. The method according to claim 22 wherein the monosaccharide is
substituted with three phosphorus-containing radicals having the
following formula: 7wherein V.sup.1 and V.sup.2 are OH,
(CH.sub.2).sub.pOH, COOH, CONH.sub.2, CONOH, (CH.sub.2).sub.pCOOH,
(CH.sub.2).sub.pCONH.sub.2, (CH.sub.2).sub.pCONOH,
(CH.sub.2).sub.pSO.sub.3H, (CH.sub.2).sub.pSO.sub.3NH.sub.2,
(CH.sub.2).sub.pNO.sub.2, (CH.sub.2).sub.pPO.sub.3H.sub.2,
O(CH.sub.2).sub.pOH, O(CH.sub.2).sub.pCOOH,
O(CH.sub.2).sub.pCONH.sub.2, O(CH.sub.2).sub.pCONOH,
O(CH.sub.2).sub.pSO.sub.3H, O(CH.sub.2).sub.pSO.sub.3NH.sub.2,
O(CH.sub.2).sub.pNO.sub.2, O(CH.sub.2).sub.pPO.sub.3H.sub.2 or
CF.sub.2COOH; and p is 1 to4.
25. The method according to claim 24 wherein the phosphorous
containing radicals are phosphate groups.
26. The method according to claim 18 or 19 wherein the cyclic
compound administered to the mammal is selected from the group
consisting of mannose-2,3,4-trisphosphate,
rhamnose-2,3,4-trisphosphate, galactose-2,3,4-trisphosphate,
methyl-6-O-butyl-.alpha.-D-mannopyranoside- -2,3,4-trisphosphate,
1,5-anhydro-D-arabinitol-2,3,4-trisphosphate,
fructose-2,3,4-trisphosphate,
1,2-O-ethylene-.beta.-D-fructopyranoside-2,- 3,4-trisphosphate,
cyclohexane-1,2,3-triol trisphosphate,
1,5-dideoxy-1,5-iminoarabinitol-2,3,4-trisphosphate,
altrose-2,3,4-trisphosphate, or
methyl-6-O-butyl-.alpha.-D-altropyranosid- e
2,3,4-trisphosphate.
27. The method according to claim 18 or 19 wherein the compound is
administered by parenteral or non-parenteral administration.
28. The method according to claim 18 or 19 wherein the effective
amount ranges from about 0.1 to about 100 mg per kg body weight of
the mammal.
29. A method of treating tissue repair conditions comprising
administering to a mammal in need thereof an effective amount of a
cyclic compound selected from the group consisting of cyclopentane,
cyclohexane, cycloheptane, inositol, monosaccharide, disaccharide,
trisaccharide, tetrasaccharide, piperidine, tetrahydrothiopyran,
5-oxotetrahydrothiopyra- n, 5,5-dioxotetrahydrothiopyran,
tetrahydroselenopyran, tetrahydrofuran, pyrrolidine,
tetrahydrothiophene, 5-oxotetrahydrothiophene,
5,5-dioxotetrahydrothiophene, tetrahydroselenophene, benzene,
cumene, mesitylene, naphthalene and phenanthrene, in which said
cyclic compound is substituted by at least three vicinal phosphorus
containing radicals of the formula: 8wherein V.sup.1 to V.sup.4 are
independently Y.sup.8.sub.m6T.sub.o3U; T.sub.01 to T.sub.03 are
independently (CH.sub.2).sub.n, CH.dbd.CH, or
CH.sub.2CH.dbd.CHCH.sub.2; o1 to o3 are independently 0 or 1; n is
0 to 4; U is R.sup.1Y.sup.9m.sub.7,
CY.sup.10Y.sup.11R.sup.2,SY.sup.12Y.sup.13Y.sup.14R.sup.3,PY.sup.15Y.sup.-
16Y.sup.17R.sup.4R.sup.5,
Y.sup.18PY.sup.19Y.sup.20Y.sup.21R.sup.6R.sup.7, CH.sub.2NO.sub.2,
NHSO.sub.2R.sup.8, or NHCY.sup.22Y.sup.23R.sup.9; m1 to m7 are
independently 0 or 1; Y.sup.1 to Y.sup.23 are independently
NR.sup.10, NOR.sup.11, O, or S; and where R.sup.1 to R.sup.11 are
independently i) hydrogen; ii) a straight or branched saturated or
unsaturated alkyl group containing 1-22 carbon atoms; iii) a
saturated, unsaturated aromatic or non-aromatic homo- or
heterocyclic group containing 3-22 carbon atoms and 0-5 heteroatoms
selected from the group consisting of nitrogen, oxygen and sulfur;
iv) a straight or branched saturated or unsaturated alkyl group
containing 1-22 carbon atoms substituted with a saturated or
unsaturated aromatic or non-aromatic homo- or heterocyclic group
containing 3-22 carbon and 0-5 heteroatoms selected from the group
consisting of nitrogen, oxygen, and sulfur; v) an aromatic or
non-aromatic homo- or heterocyclic group containing 3-22 carbon and
0-5 heteroatoms selected from the group consisting of nitrogen,
oxygen, and sulfur which aromatic or non-aromatic homo- or
heterocyclic group is substituted with a straight or branched
saturated or unsaturated group containing 1-22 carbon atoms;
whereby said groups in ii-v are unsubstituted or are substituted by
1-6 of the following groups hydroxy, alkoxy, aryloxy, acyloxy,
carboxy, alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyl,
aryloxycarbonyloxy, carbamoyl, fluoro, chloro, bromo, azido, cyano,
oxo, oxa, amino, imino, alkylamino, arylamino, acylamino, arylazo,
nitro, alkylthio or alkylsulfonyl.
30. A method according to claim 29 wherein the tissue repair
condition is wound healing, matrix formation, collagen synthesis or
scar formation.
31. The method according to claim 29 or 30 wherein the
phosphorus-containing radicals have the following formula: 9wherein
V.sup.1 and V.sup.2 are OH, (CH.sub.2).sub.pOH, COOH, CONH.sub.2,
CONOH, (CH.sub.2).sub.pCOOH, (CH.sub.2).sub.pCONH.sub.2,
(CH.sub.2).sub.pCONOH, (CH.sub.2).sub.pSO.sub.3H,
(CH.sub.2).sub.pSO.sub.3NH.sub.2, (CH.sub.2).sub.pNO.sub.2,
(CH.sub.2).sub.pPO.sub.3H.sub.2, O(CH.sub.2).sub.pOH,
O(CH.sub.2).sub.pCOOH, O(CH.sub.2).sub.pCONH.sub.2,
O(CH.sub.2).sub.pCONOH, O(CH.sub.2).sub.pSO.sub.3H,
O(CH.sub.2).sub.pSO.sub.3NH.sub.2, O(CH.sub.2).sub.pNO.sub.2,
O(CH.sub.2).sub.pPO.sub.3H.sub.2 or CF.sub.2COOH; and p is 1 to
4.
32. The method according to claim 29 or 30 wherein the
phosphorus-containing radicals are phosphate groups.
33. The method according to claim 29 or 30 wherein the cyclic
compound is a monosaccharide.
34. The method according to claim 33 wherein the monosaccharide is
D/L-ribose, D/L-arabinose, D/L-xylose, D/L-lyxose, D/L-allose,
D/L-altrose, D/L-glucose, D/L-mannose, D/L-gulose, D/L-idose,
D/L-galactose, D/L-talose, D/L-ribulose, D/L-xylulose, D/L-psicose,
D/L-sorbose, D/L-tagatose, or D/L-fructose.
35. The method according to claim 33 wherein the monosaccharide is
substituted with three phosphorus-containing radicals having the
following formula: 10wherein V.sup.1 and V.sup.2 are OH,
(CH.sub.2).sub.pOH, COOH, CONH.sub.2, CONOH, (CH.sub.2).sub.pCOOH,
(CH.sub.2).sub.pCONH.sub.2, (CH.sub.2).sub.pCONOH,
(CH.sub.2).sub.pSO.sub.3H, (CH.sub.2).sub.pSO.sub.3NH.sub.2,
(CH.sub.2).sub.pNO.sub.2, (CH.sub.2).sub.pPO.sub.3H.sub.2,
O(CH.sub.2).sub.pOH, O(CH.sub.2).sub.pCOOH,
O(CH.sub.2).sub.pCONH.sub.2, O(CH.sub.2).sub.pCONOH,
O(CH.sub.2).sub.pSO.sub.3H, O(CH.sub.2).sub.pSO.sub.3NH.sub.2,
O(CH.sub.2).sub.pNO.sub.2, O(CH.sub.2).sub.pPO.sub.3H.sub.2 or
CF.sub.2COOH; and p is 1 to 4.
36. The method according to claim 35 wherein the phosphorous
containing radicals is a phosphate group.
37. The method according to claim 29 or 30 wherein the cyclic
compound is inositol.
38. The method according to claim 37 wherein the inositol is
alloinositol, cisinositol, ipiinositol, D/L-chiroinositol,
scylloinositol, myoinositol, mycoinositol or neoinositol.
39. The method according to claim 38 wherein the inositol is
substituted with three phosphorus-containing radicals having the
following formula: 11wherein V.sup.1 and V.sup.2 are OH,
(CH.sub.2).sub.pOH, COOH, CONH.sub.2, CONOH, (CH.sub.2).sub.pCOOH,
(CH.sub.2).sub.pCONH.sub.2, (CH.sub.2).sub.pCONOH,
(CH.sub.2).sub.pSO.sub.3H, (CH.sub.2).sub.pSO.sub.- 3NH.sub.2,
(CH.sub.2).sub.pNO.sub.2, (CH.sub.2).sub.pPO.sub.3H.sub.2,
O(CH.sub.2).sub.pOH, O(CH.sub.2).sub.pCOOH,
O(CH.sub.2).sub.pCONH.sub.2, O(CH.sub.2).sub.pCONOH,
O(CH.sub.2).sub.pSO.sub.3H, O(CH.sub.2).sub.pSO.sub.3NH.sub.2,
O(CH.sub.2).sub.pNO.sub.2, O(CH.sub.2).sub.pPO.sub.3H.sub.2 or
CF.sub.2COOH; and p is 1 to 4.
40. The method according to claim 35 wherein the phosphorous
containing radicals are phosphate groups.
41. The method according to claim 29 or 30 wherein the cyclic
compound administered to the mammal is selected from the group
consisting of myoinositol-1,2,6-trisphosphate,
mannose-2,3,4-trisphosphate, rhamnose-2,3,4-trisphosphate,
galactose-2,3,4-trisphosphate,
methyl-6-O-butyl-.alpha.-D-mannopyranoside-2,3,4-trisphosphate,
1,5-anhydro-D-arabinitol-2,3,4-trisphosphate,
fructose-2,3,4-trisphosphat- e,
1,2-O-ethylene-.beta.-D-fructopyranoside-2,3,4-trisphosphate,
cyclohexane-1,2,3-triol trisphosphate,
1,5-dideoxy-1,5-iminoarabinitol-2,- 3,4,-trisphosphate,
altrose-2,3,4-trisphosphate, or
methyl-6-O-butyl-.alpha.-D-altropyranoside 2,3,4-trisphosphate.
42. The method according to claim 29 or 30 wherein the compound is
administered by parenteral or non-parenteral administration.
43. The method according to claim 29 or 20 wherein the effective
amount ranges from about 0.1 to about 100 mg per kg body weight of
the mammal.
44. A method of treating infectious conditions comprising
administering to a mammal in need thereof an effective amount of a
cyclic compound selected from the group consisting of cyclopentane,
cyclohexane, cycloheptane, inositol, monosaccharide, disaccharide,
trisaccharide, tetrasaccharide, piperidine, tetrahydrothiopyran,
5-oxotetrahydrothiopyra- n, 5,5-dioxotetrahydrothiopyran,
tetrahydroselenopyran, tetrahydrofuran, pyrrolidine,
tetrahydrothiophene, 5-oxotetrahydrothiophene,
5,5-dioxotetrahydrothiophene, tetrahydroselenophene, benzene,
cumene, mesitylene, naphthalene and phenanthrene, in which said
cyclic compound is substituted by at least three vicinal phosphorus
containing radicals of the formula: 12wherein V.sup.1 to V.sup.4
are independently Y.sup.8.sub.m6T.sub.o3U; T.sub.01 to T.sub.03 are
independently (CH.sub.2).sub.n, CH.dbd.CH, or
CH.sub.2CH.dbd.CHCH.sub.2; o1 to o3 are independently 0 or 1; n is
0 to 4; U is R.sup.1Y.sup.9.sub.m7,
CY.sup.10Y.sup.11R.sup.2,SY.sup.12Y.sup.13Y.sup.14R.sup.3,PY.sup.15Y.sup.-
16Y.sup.17R.sup.4R.sup.5,
Y.sup.18PY.sup.19Y.sup.20Y.sup.21R.sup.6R.sup.7, CH .sub.2NO.sub.2,
NHSO.sub.2R.sup.8, or NHCY.sup.22Y.sup.23R.sup.9; m1 to m7 are
independently 0 or 1; Y.sup.1 to Y.sup.23 are independently
NR.sup.10, NOR.sup.11, O, or S; and where R.sup.1 to R.sup.11 are
independently i) hydrogen; ii) a straight or branched saturated or
unsaturated alkyl group containing 1-22 carbon atoms; iii) a
saturated, unsaturated aromatic or non-aromatic homo- or
heterocyclic group containing 3-22 carbon atoms and 0-5 heteroatoms
selected from the group consisting of nitrogen, oxygen and sulfur;
iv) a straight or branched saturated or unsaturated alkyl group
containing 1-22 carbon atoms substituted with a saturated or
unsaturated aromatic or non-aromatic homo- or heterocyclic group
containing 3-22 carbon and 0-5 heteroatoms selected from the group
consisting of nitrogen, oxygen, and sulfur; v) an aromatic or
non-aromatic homo- or heterocyclic group containing 3-22 carbon and
0-5 heteroatoms selected from the group consisting of nitrogen,
oxygen, and sulfur which aromatic or non-aromatic homo- or
heterocyclic group is substituted with a straight or branched
saturated or unsaturated group containing 1-22 carbon atoms;
whereby said groups in ii-v are unsubstituted or are substituted by
1-6 of the following groups hydroxy, alkoxy, aryloxy, acyloxy,
carboxy, alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyl,
aryloxycarbonyloxy, carbamoyl, fluoro, chloro, bromo, azido, cyano,
oxo, oxa, amino, imino, alkylamino, arylamino, acylamino, arylazo,
nitro, alkylthio or alkylsulfonyl.
45. A method according to claim 44 wherein the infectious condition
is trypanosomiasis.
46. The method according to claim 44 or 45 wherein the
phosphorus-containing radicals have the following formula:
13wherein V.sup.1 and V.sup.2 are OH, (CH.sub.2).sub.pOH, COOH,
CONH.sub.2, CONOH, (CH.sub.2).sub.pCOOH,
(CH.sub.2).sub.pCONH.sub.2, (CH.sub.2).sub.pCONOH,
(CH.sub.2).sub.pSO.sub.3H, (CH.sub.2).sub.pSO.sub.3NH.sub.2,
(CH.sub.2).sub.pNO.sub.2, (CH.sub.2).sub.pPO.sub.3H.sub.2,
O(CH.sub.2).sub.pOH, O(CH.sub.2).sub.pCOOH,
O(CH.sub.2).sub.pCONH.sub.2, O(CH.sub.2).sub.pCONOH,
O(CH.sub.2).sub.pSO.sub.3H, O(CH.sub.2).sub.pSO.sub.3NH.sub.2,
O(CH.sub.2).sub.pNO.sub.2, O(CH.sub.2).sub.pPO.sub.3H.sub.2 or
CF.sub.2COOH; and p is 1 to 4.
47. The method according to claim 44 or 45 wherein the
phosphorus-containing radicals are phosphate groups.
48. The method according to claim 44 or 45 wherein the cyclic
compound is a monosaccharide.
49. The method according to claim 48 wherein the monosaccharide is
D/L-ribose, D/L-arabinose, D/L-xylose, D/L-lyxose, D/L-allose,
D/L-altrose, D/L-glucose, D/L-mannose, D/L-gulose, D/L-idose,
D/L-galactose, D/L-talose, D/L-ribulose, D/L-xylulose, D/L-psicose,
D/L-sorbose, D/L-tagatose, or D/L-fructose.
50. The method according to claim 48 wherein the monosaccharide is
substituted with three phosphorus-containing radicals having the
following formula: 14wherein V.sup.1 and V.sup.2 are OH,
(CH.sub.2).sub.pOH, COOH, CONH.sub.2, CONOH, (CH.sub.2).sub.pCOOH,
(CH.sub.2).sub.pCONH.sub.2, (CH.sub.2).sub.pCONOH,
(CH.sub.2).sub.pSO.sub.3H, (CH.sub.2).sub.pSO.sub.3NH.sub.2,
(CH.sub.2).sub.pNO.sub.2, (CH.sub.2).sub.pPO.sub.3H.sub.2,
O(CH.sub.2).sub.pOH, O(CH.sub.2).sub.pCOOH,
O(CH.sub.2).sub.pCONH.sub.2, O(CH.sub.2).sub.pCONOH,
O(CH.sub.2).sub.pSO.sub.3H, O(CH.sub.2).sub.pSO.sub.3NH.sub.2,
O(CH.sub.2).sub.pNO.sub.2, O(CH.sub.2).sub.pPO.sub.3H.sub.2 or
CF.sub.2COOH; and p is 1 to4.
51. The method according to claim 50 wherein the phosphorous
containing radicals are phosphate groups.
52. The method according to claims 44 or 45 wherein the cyclic
compound is inositol.
53. The method according to claim 52 wherein the inositol is
alloinositol, cisinositol, ipiinositol, D/L-chiroinositol,
scylloinositol, myoinositol, mycoinositol or neoinositol.
54. The method according to claim 53 wherein the inositol is
substituted with three phosphorus-containing radicals having the
following formula: 15wherein V.sup.1 and V.sup.2 are OH,
(CH.sub.2).sub.pOH, COOH, CONH.sub.2, CONOH, (CH.sub.2).sub.pCOOH,
(CH.sub.2).sub.pCONH.sub.2, (CH.sub.2).sub.pCONOH,
(CH.sub.2).sub.pSO.sub.3H, (CH.sub.2).sub.pSO.sub.- 3NH.sub.2,
(CH.sub.2).sub.pNO.sub.2, (CH.sub.2).sub.pPO.sub.3H.sub.2,
O(CH.sub.2).sub.pOH, O(CH.sub.2).sub.pCOOH,
O(CH.sub.2).sub.pCONH.sub.2, O(CH.sub.2).sub.pCONOH,
O(CH.sub.2).sub.pSO.sub.3H, O(CH.sub.2).sub.pSO.sub.3NH.sub.2,
O(CH.sub.2).sub.pNO.sub.2, O(CH.sub.2).sub.pPO.sub.3H.sub.2 or
CF.sub.2COOH; and p is 1 to4.
55. The method according to claim 54 wherein the phosphorous
containing radicals is a phosphate group.
56. The method according to claim 44 or 45 wherein the cyclic
compound administered to the mammal is selected from the group
consisting of myoinositol-1,2,6-trisphosphate,
mannose-2,3,4-trisphosphate, rhamnose-2,3,4-trisphosphate,
galactose-2,3,4-trisphosphate,
methyl-6-O-butyl-.alpha.-D-mannopyranoside-2,3,4-trisphosphate,
1,5-anhydro-D-arabinitol-2,3,4-trisphosphate,
fructose-2,3,4-trisphosphat- e,
1,2-O-ethylene-.beta.-D-fructopyranoside-2,3,4-trisphosphate,
cyclohexane-1,2,3-triol trisphosphate,
1,5-dideoxy-1,5-iminoarabinitol-2,- 3,4-trisphosphate,
altrose-2,3,4-trisphosphate, or methyl-6-O-butyl-.alpha-
.-D-altropyranoside 2,3,4-trisphosphate.
57. The method according to claim 44 or 45 wherein the compound is
administered by parenteral or non-parenteral administration.
58. The method according to claim 44 or 45 wherein the growth
factor modulating effective amount ranges from about 0.1 to about
100 mg per kg body weight of the mammal.
Description
[0001] Growth factors comprise a family of polypeptides with a
manyfold of properties regulating for example cell proliferation
and cell metabolism. As being multi-functional molecules, they may
stimulate or inhibit cell proliferation as well as affect cell
function depending on the type of the target cells and the presence
of other signal peptides. The family of polypeptides include for
example platelet derived growth factor (PDGF), epidermal growth
factor (EGF), transforming growth factors (TGF-, TGF-.beta.),
insulin like growth factors (IGF-1, IGF-2), fibroblast growth
factors (a FGF, b FGF), vascular endothelial growth factor (VEGF),
nerve growth factor (NGF) and bone morphogenetic proteins
(BMP).
[0002] The activities of these growth factors are considered to be
casual components of several conditions such as cardiovascular
conditions, for example diabetes, inflammatory conditions for
example rheumatoid arthritis, analgetic conditions, viral
conditions, carcinogenic conditions, several eye diseases and wound
healing.
[0003] Angiogenesis is under normal conditions under stringent
control but in many pathological conditions e.g. solid tumours,
rheumatoid arthritis, diabetic retinopathy and artherosclerosis
unregulated vessel proliferation occurs. The angiogenetic process
is controlled by a balance between many positive and negative
regulating signals where growth factors such as TGF, FGF and VEGF
play a dominant role.
[0004] Under normal conditions in the body growth factor activity
is to a large extent regulated by the interactions with heparin and
heparan sulfate. The interaction between these polyanionic
glycosaminoglycans and growth factors is thought to be of
functional significance serving as storage depots for growth
factors and protecting them from various degradative processes
(Vlodavsky, I., Fuks, Z., Ishai-Michaeli, R., Bashkin, P., Levi,
E., Korner, G., Bar-Shavit, R., and Klagsbrun, M. (1991) J. Cell
Biochem. 45, 167-176).
[0005] Under some circumstances the administration of growth
factors as therapeutic agents has been utilized. However the
limited stability of this type of molecules reduces their activity
before reaching the target in the body.
[0006] Under abnormal conditions when growth factor regulation is
required to a larger extent than in the normal situation, the
administration of heparin and derivatives of heparin has been
considered. However the administration of these type of compounds
renders side-effects like anticoagulant activity which limits their
possible usage.
[0007] Other sulphonated compounds like suramin, a polysulphonated
napthylurea, has been shown to have activity in the treatment of
adrenocortical carbinoma but the limitation also for this type of
compounds is the narrow margin between the dose required to achieve
anti-tumor activity and the dose leading to the onset of toxic side
effects.
[0008] According to the present invention the use of a compound
containing a high density, negatively charged domain of vicinally
oriented radicals for the preparing of a medicament providing a
growth factor modulating activity in mammals including man is
described.
[0009] In preferred embodiments of the invention the negatively
charged domain comprises, at least three vicinal
phosphorus-containing radicals.
[0010] The invention also relates to the use of a compound wherein
the phosphorus-containing radicals have the following formula:
2
[0011] wherein.
[0012] V.sup.1 to V.sup.4 are Y.sup.8.sub.m6 T.sub.o3 U
[0013] T.sub.o1 to T.sub.o3 are (CH.sub.2).sub.n, CHCH, or
CH.sub.2CHCHCH.sub.2;
[0014] o1 to o3 are 0 or 1;
[0015] n is 0 to 4;
[0016] U is R.sup.1Y.sup.9.sub.m7, CY.sup.10Y.sup.11R.sup.2,
SY.sup.12Y.sup.13Y.sup.14R.sup.3,
PY.sup.15Y.sup.16Y.sup.17R.sup.4R.sup.5- ,
[0017] Y.sup.18PY.sup.19Y.sup.20Y.sup.21R.sup.6R.sup.7,
CH.sub.2NO.sub.2, NHSO.sub.2R.sup.8, or
NHCY.sup.22Y.sup.23R.sup.9;
[0018] m1 to m7 are 0 to 1;
[0019] Y.sup.1 to Y.sup.23 are NR.sup.10, NOR.sup.11, O, or S;
[0020] and where R.sup.1 to R.sup.11 are
[0021] i) hydrogen
[0022] ii) a straight or branched saturated or unsaturated alkyl
residue containing 1-22 carbon atoms
[0023] iii) a saturated or unsaturated aromatic or non-aromatic
homo- or heterocyclic residue containing 3-22 carbon atoms and 0-5
heteroatoms consisting of nitrogen, oxygen or sulfur
[0024] iv) a straight or branched saturated or unsaturated alkyl
residue containing 1-22 carbon atoms substituted with a saturated
or unsaturated aromatic or non-aromatic homo- or heterocyclic
containing 3-22 carbon and 0-5 heteroatoms consisting of nitrogen,
oxygen or sulfur
[0025] v) an aromatic or non-aromatic homo- or heterocyclic residue
containing 3-22 carbon and 0-5 heteroatoms consisting of nitrogen,
oxygen or sulfur substituted with a straight or branched saturated
or unsaturated alkyl residue containing 1-22 carbon atoms in the
said groups ii-v the residues and/or the substituents the thereof
being substituted with 0-6 of the following groups: hydroxy,
alkoxy, aryloxy, acyloxy, carboxy, alkoxycarbonyl,
alkoxycarbonyloxy, aryloxycarbonyl, carbamoyl, fluoro, chloro,
bromo, azido, cyano, oxo, oxa, amino, imino, alkylamino, aryl
amino, acylarmino, arylazo, nitro, alkylthio or alkylsulfonyl.
[0026] The streight or branched saturated or unsaturated alkyl
residue in groups i-v above can be exemplified by methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, eicosyl, heneicosyl, doeicosyl, isopropyl,
isobutyl, isopentyl, isohexyl, isoheptyl, isooctyl, isononyl,
isodecyl, isodoecosyl, 2-butyl, 2-pentyl, 2-hexyl, 2-heptyl,
2-octyl, 2-nonyl, 2-decyl, 2-doeicosyl, 2-methylbutyl,
2-methylpentyl, 2-methylhexyl, 2-methylheptyl, 2-methyloctyl,
2-methylnonyl, 2-methyldecyl, 2-methyleicosyl, 2-ethylbutyl,
2-ethylpentyl, 2-ethylhexyl, 2-ethylheptyl, 2-ethyloctyl,
2-ethylnonyl, 2-ethyldecyl, 2-ethyleicosyl, tertbutyl, ethenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,
decenyl undecenyl, dodecenyl, tridecenyl, tetradecenyl,
pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl,
eicosenyl, heneicosenyl, doeicosenyl, butadienyl, pentadienyl,
hexadienyl, heptadienyl, octadienyl, nonadienyl, decadienyl,
doeicodienyl, ethynyl, propynyl, doeicosynyl.
[0027] The saturated or unsaturated aromatic or non-aromatic homo-
or heterocyclic residue in groups i-v above can be exemplified by
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cycllododecyl,
cyclotridecyl, cyclotetradecyl, cyclopentadecyl, cyclohexadecyl,
cycloheptadecyl, cyclooctadecyl, cyclononadecyl, cycloeicosyl,
cycloheneicosyl, cyclodoeicosyl, adamantyl, cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,
cyclooctenyl, cyclpnbnenyl, cyclodecenyl. phenyl, biphenyl,
naphthyl, hydroxyphenyl, aminophenyl, mercaptophenyl, fluorophenyl,
chlorophenyl, azidophenyl, cyanophenyl, carboxyphenyt,
alkoxyphenyl, acyloxyphenyl, acylphenyl, oxiranyl, thiiranyl,
faziridinyl, oxetanyl, thietanyl, azetidinyl, tetrahydrofuranyl,
tetrahydrothiophenyl, pyrrolidinyl, tetrahydropyranyl,
tetrahydrothiopyranyl, piperidinyl, quinuclidinyl, dioxanyl,
dithanyl, trioxanyl, furyl, pyrrolyl, thienyl, pyridyl, quinolyl,
benzofuryl, indolyl, benzothienyl, oxazolyl, imidazolyl, thiazolyl,
pyridazinyl, pyrimidyl, pyrazinyl, purinyl, or a carbohydrate.
[0028] Substituents may be selected from the group of: hydroxy,
alkoxy, aryloxy, acyloxy, carboxy, alkoxycarbonyl,
alkoxycarbonyloxy, aryloxycarbonyl, aryloxycarbonyloxy, carbamoyl,
fluoro, chloro, bromo, azido, cyano, oxo, oxa, amino, imino,
alkylamino, arylamino, acylamino, nitro, alkylthio,
alkylsulfonyl.
[0029] Furthermore the invention relates to the use a compound
wherein the phosphorus-containing radicals have the following
formula. 3
[0030] wherein V.sup.1 and V.sup.2 are the same or different and
are OH, (CH.sub.2).sub.pOH, COOH, CONH.sub.2, CONOH,
[0031] (CH.sub.2).sub.pCOOH, (CH.sub.2).sub.pCONH.sub.2,
(CH.sub.2).sub.pCONOH, (CH.sub.2).sub.pSO.sub.3H,
[0032] (CH.sub.2).sub.pSO.sub.3NH.sub.2, (CH.sub.2).sub.pNO.sub.2,
(CH.sub.2).sub.pPO.sub.3H.sub.2, O(CH.sub.2).sub.pOH,
[0033] O(CH.sub.2).sub.pCOOH, O(CH.sub.2).sub.pCONH.sub.2,
O(CH.sub.2).sub.pCONOH, O(CH.sub.2).sub.pSO.sub.3H,
[0034] O(CH.sub.2).sub.pSO.sub.3NH.sub.2,
O(CH.sub.2).sub.pPO.sub.3H.sub.2 or CF.sub.2COOH
[0035] p is 1 to 4
[0036] In this embodiment of the invention the
phphohorus-containing radicals are phosphonates or phospates or
derivatives thereof.
[0037] In one embodiment of the invention the backbone to the high
density negatively charged region of vicinally oriented radicals is
a cyclic moiety.
[0038] The cyclic moiety comprises a saturated or unsaturated
aromatic or non-aromatic homo- or heterocyclic moiety. When the
moiety is heterocyclic the heteroatoms are selected from the group
of oxygen, nitrogen, sulfur or selenium.
[0039] Preferably the cyclic moiety comprises 4 to 24 atoms, most
preferably 5 to 18 atoms. The cyclic moiety is for example selected
from the group of cyclopentane, cyclohexane, cycloheptane,
cyclooctane, inositol, monosacharide, disacharide, trisacharide,
tetrasacharide, piperidin, tetrahydrothiopyran,
5-oxotetrahydrothiopyran, 5,5-dioxotetrahydrothiopyran,
tetrahydroselenopyran, tetrahydrofuran, pyrrolidine,
tetrahydrothiophene, 5-oxotetrahydrothiophene,
5,5-dioxotetrahydrothiophene, tetrahydroselenophene, benzene,
cumene, meritylene, naphtalene and phenantrene. When the cyclic
moiety is an inositol it could be selected from the group of
alloinositol, cisinositol, epiinositol, D/L-chiroinositol,
scylloinositol, myomositol, mucoinositol and neoinositol.
[0040] In one preferred embodiment of the invention the compounds
are phosphates, phosphonates or phosphinates of cyclohexane such as
1, 2, 3.beta.-cyclohexane-1,2,3-trioltrisphosphate.
[0041] In other preferred embodiments of this type of the invention
the compounds are phosphates, phosphonates or phosphinates of
inositol. Preferably the number of phosphate-, phosphonate- or
phosphinate radicals per inositol moiety is at least three. The
remaining hydroxyl groups on the inositol moiety may be derivatized
in the form of ethers or esters.
[0042] In one preferred embodiment the compound is
myo-inositol-1,2,6-tris- phosphate or
myo-inositol-1,2,3-trisphosphate.
[0043] In one most preferred embodiment the compounds are selected
from the group of D-myo-inositol-1,2,6-trisphosphate,
D-myo-inositol-1,2,6-tri- s(carboxymethylphosphate),
D-myo-inositol-1,2,6-tris(carboxymethylphosphon- ate),
D-myo-inositol-1,2,6-tris(hydroxymethylphosphonate),
D-3,4,5-tri-O-methyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-hexanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-butanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-pentanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-isobutanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-propanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-(6-hydroxy-4-oxa)hexanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-3-(ethylsulphonyl)propanoyl-myo-inositol-1,2,6-trisphosphat-
e,
D-3,4,5-tri-O-3-hydroxypropanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-(6-hydroxy)-hexanoyl-myo-inositol-1,2,6-trisphosphate,
D-5-O-hexanoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-phenylcarb- amoyl-myo-inositol-1,2,6-trisphosphate,
D-3,4,5-tri-O-propanoyl-myo-inosit-
ol-1,2,6-tris(carboxymethylphosphate),
D-3,4,5-tri-O-butanoyl-myo-inositol-
-1,2,6-tris(carboxymethylphosphate),
D-3,4,5-tri-O-isobutanoyl-myo-inosito-
l-1,2,6-tris(carboxymethylphosphate),
D-3,4,5-tri-O-pentanoyl-myo-inositol-
-1,2,6-tris(carboxymethylphosphate),
D-3,4,5-tri-O-hexanoyl-myo-inositol-1-
,2,6-tris(carboxymethylphosphate),
D-3,4,5-tri-O-propanoyl-myo-inositol-1,-
2,6-tris(carboxyrethylphosphonate),
D-3,4,5-tri-O-butanoyl-myo-inositol-1,-
2,6-tris(carboxymethylphosphonate),
D-3,4,5-tri-O-iso-butanoyl-myo-inositl-
-1,2,6-tris(carboxymethylphosphonate),
D-3,4,5-tri-O-pentanoyl-myo-inosito-
l-1,2,6-tris(carboxymethylphosphonate),
D-3,4,5-tri-O-hexanoyl-myo-inosito-
l-1,2,6-tris(carboxymethylphosphonate),
D-3,4,5-tri-O-propanoyl-myo-inosit-
ol-1,2,6-tris(hydroxymethylphosphonate),
D-3,4,5-tri-O-butanoyl-myo-inosit-
ol-1,2,6-tris(hydroxymethylphosphonate),
D-3,4,5-tri-O-isobutanoyl-myo-ino-
sitol-1,2,6-tris(hydroxymethylphosphonate),
D-3,4,5-tri-O-pentanoyl-myo-in-
ositol-1,2,6-tris(hydroxymethylphosphonate),
D-3,4,5-tri-O-hexanoyl-myo-in-
ositol-1,2,6-tris(hydroxymethylphosphonate).
[0044] When the cyclic moiety is a sacharide it could be selected
from the group of D/L-ribose, D/L-arabinose, D/L-xylose,
D/L-lyxose, D/L-allose, D/L-altrose, D/L-glucose, D/L-mannose,
D/L-gulose, D/L-idose, D/L-galactose, D/L-talose, D/L-ribulose,
D/L-xylulose, D/L-psicose, D/L-sorbose, D/L-tagatose and
D/L-fructose or derivatives thereof. In preferred embodiments of
this type of the invention the compounds are phosphates,
phosphonates or phosphinates of sacharides. Preferably the number
of phosphate-, phosphonate- or phosphinate radicals per sacharide
unit is at least three. The remaining hydroxyl groups on the
sacharide moiety may be derivatized in the form of ethers or
esters. In many instances the ether form is desired as this type of
radical propongs the stability and half-life in vivo as the
susceptibility to enzymatic degradation is reduced. In one
preferred embodiment of this type of the invention the compound is
selected from the group of mannose-2,3,4-trisphosphate,
galactose-2,3,4-trisphosphate, fructose-2,3,4-trisphosphate,
altrose-2,3,4-trisphosphate and rhamnose-2,3,4-trisphosphate. In
one most preferred embodiment the, compound is selected from the
group of R.sub.1-6-O-R.sub.2-.alpha.-D-mann-
opyranoside-2,3,4-trisphosphate,
R.sub.1-6-O-R.sub.2-.alpha.-D-galacto-pyr-
anoside-2,3,4-trisphosphate,
R.sub.1-6-O-R.sub.2-.alpha.-D-altropyranoside- -2,3,4-trisphosphate
and R.sub.1-6-O-R.sub.2-.beta.-D-fructopyranoside-2,3-
,4-trisphosphate where R.sub.1 and R.sub.2 independently are as
defined above and preferably are methyl, ethyl, propyl, butyl,
pentyl or hexyl. Most preferred compounds in this type of the
invention are
methyl-6-O-butyl-.alpha.-D-mannopyranoside-2,3,4-trisphosphate,
methyl-6-O-butyl-.alpha.-D-galactopyranoside-2,3,4-trisphosphate,
methyl-6-O-butyl-.alpha.-D-glycopyranoside-2,3,4-trisphosphate,
methyl-6-O-butyl-.alpha.-D-altropyranoside-2,3,4-trisphosphate,
methyl-6-O-butyl-.beta.-D-fructopyranoside-2,3,4-trisphosphate,
1,5-anhydro-D-arabinitol-2,3,4-trisphosphate,
1,5-anhydroxylitol-2,3,4-tr- isphosphate,
1,2-O-ethylene-.beta.-D-fructopyranoside-2,3,4-trisphosphate,
methyl-.alpha.-D-rhamnopyranoside-2,3,4-trisphosphate,
methyl-.alpha.-D-mannopyranoside-2,3,4-triphosphate,
metyl-6-O-butyl-.alpha.-D-mannopyranoside-2,3,4-tris-(carboxymethylphosph-
ate),
methyl-6-O-butyl-.alpha.-D-mannopyranoside-2,3,4-tris-(carboxymethyl-
phosphonate),
methyl-6-O-butyl-.alpha.-D-mannopycanoside-2,3,4-tris(hydrox-
ymethylphosphonate),
methyl-6-O-butyl-.alpha.-D-galactopyranoside-2,3,4-tr-
is(carboxymethylphosphate),
methyl-6-O-butyl-.alpha.-D-galactopyranoside-2-
,3,4-tris(carboxymethylphosphonate),
methyl-6-O-butyl-.alpha.-D-galactopyr-
anoside-2,3,4-tris(hydrbxymethylphosphonate),
methyl-6-O-butyl-.alpha.-D-g-
lucopyranoside-2,3,4-tris(carboxymethylphosphate),
methyl-6-O-butyl-.alpha-
.-D-glucopyranoside-2,3,4-tris(carboxymethylphosphonate),
methyl-6-O-butyl-.alpha.-D-glucopyranoside-2,3,4-tris(hydroxymethylphosph-
onate),
methyl-6-O-butyl-.alpha.-D-altropyranoside-2,3,4-tris-(carboxymeth-
ylphosphoate),
methyl-6-O-butyl-.alpha.-D-altropyranoside-2,3,4-tris-(carb-
oxymethylphosphonate),
methyl-6-O-butyl-.alpha.-D-altropyranoside-2,3,4-tr-
is-(hydroxymethylphosphonate),
methyl-6-O-butyl-.beta.-D-fructopyranoside--
2,3,4-tris-(carboxymethylphosphate),
methyl-6-O-butyl-.beta.-D-fructopyran-
oside-2,3,4-tris-(carboxymethylphosphonate),
methyl-6-O-butyl-.beta.-D-fru-
ctopyranoside-2,3,4-tris-(hydroxymethylphosphonate).
[0045] In other preferred embodiments of the invention the
compounds are phosphates, phosphonates or phosphnates of
heterocyclic moieties such as
1,5-dideoxy-1,5-iminoarabinitol-2,3,4-trisphosphate,
1,5-dideoxy-1,5-iminoarabinitol-2,3,4-tris-(carboxymethylphosphate),
1,5-dideoxy-1,5-iminoarabinitol-2,3,4-tris(carboxymethylphosphonate),
1,5-dideoxy-1,5-iminoarabinitol-2,3,4-tris(hydroxymethylphosphonate),
1,5-dideoxy-1,5-imino-N-(2-phenylethyl)arabinitol-2,3,4-trisphosphate,
1,5-dideoxy-1,4-imino-N-(2-phenylethyl)arabinitol-2,3,4-tris(carboxymethy-
lphosphate),
1,5-dideoxy-1,5-imino-N-(2-phenylethyl)arabinitol-2,3,4-tris--
(carboxymethylphosphonate)
1,5-dideoxy-1,5-imino-N-(2-phenylethyl)arabinit-
ol-2,3,4-tris(hydroxymethylphosphonate).
[0046] The growth factor modulating activity of the described
compounds are expressed on at least four levels. One level is a
type of interaction with growth factors such as hepatin-binding
growth factors and/or the specific receptors of these growth
factors. The compounds according to the invention interact with
domains comprising high basicity on the growth factors. This type
of interaction is considered to be an important factor in
connection with growth factors such as basic fibroblast growth
factor, acidic fibroblast growth factor, platelet-derived growth
factor, vascular endothelial growth factor and hepatocyte growth
factor.
[0047] The interactions are considered to be especially important
in connection with growth factors from the fibroblast growth family
such as different types of acidic and basic fibroblast growth
factors. Processes which are regulated by the interaction are e.g.
dimerization and interactions with receptors and ligands.
[0048] Another level of interaction is a more specific interaction
with the internalisation process of a group of growth factors. Many
growth factors are exerting their effect after internalisation to
the intracellular domain. The pathway includes an
interaction/binding of the growth factor to the external part of a
transmembrane receptor. Binding is followed by endocytosis i.e. the
formation of a vesicle which is transferred over the plasma
membrane and released into the internal part of the cell. This
process of internalisation is controlled by specific assembly
proteins. One specific assembly protein, AP-2 is considered to be
important in the internalisation process of this group of growth
factors. Within the scope of the invention it is considered that
the described compounds interact with the external receptor and an
ion-channel connected to AP-2. The binding of this group of growth
factors to their receptor is effected by interaction with receptor
bound small monophosphorylated sugars such as mannose-phosphate.
When the binding to the monophosphorylated sugar is reversed by the
existence of compounds according to the invention, the binding of
the growth factor is retarded or does not happen at all.
[0049] The other interaction on this level is the binding of the
compounds according to the invention to an ion-channel, most often
a potassium-channel belonging to AP-2. Such a binding retards or in
some case inhibit the internalisation process and thus retards or
inhibits the consequences of growth factors entering the internal
part of the cell and the subsequent metabolic events. To the group
of growth factors functioning according to the described pathway
belong for example epidermal growth factor, transforming growth
factor, insulin growth factor and nerve growth factor.
[0050] A third level of interaction includes a very specific
interaction between the compounds according to the invention and a
specific group of growth factors. The negatively charged domain of
the compounds according to the invention interacts with domains in
the growth factor, characterized by the existence of basic amino
acids such as arginine, lysine and histidine structurally arranged
in a way that renders condensed binding sites on a small area. The
binding area is typically not exceeding 500 square Angstrom and
requires a specific stereochemistry of the binding compound.
Accordingly the preferred compounds have two equatorial and one
axial equatorial negatively charged groups attached to a cyclic
moiety. Within this group of growth factors transforming growth
factor .beta. is mentioned as one example. The interaction is for
example expressed by three lysine moieties, lysine 25, lysine 31
and lysine 37 and three phosphate radicals in a way that the
distance between the radicals is less than 10 Angstrom.
[0051] The interaction between the growth factors and the compounds
according to the invention is characterized by a binding constant,
K.sub.D. Typically, the K.sub.D-value is less than 100 .mu.M.
Preferably the binding constant is less than 60 .mu.M and most
preferably less than 30 .mu.M.
[0052] The interaction is also described as a consequence of the
acid constants, pK.sub.A1 of the compounds according to the
invention. Typically pK.sub.A1 is in the range of 7 to 9 and for
some compounds in the range of 7 to 12.
[0053] A fourth level of interaction is expressed by a receptor
interaction on the cell surface which transfer the signal from for
example a specific growth factor via a signalling cascade to the
internal compartments of the cell. The compounds bind to the
receptor on the cell surface characterized by a IC.sub.50-value
less than 50 .mu.M and preferably less than 5 .mu.M. The binding is
depending on the concentration, either allosteric or
hyperbolic.
[0054] The effects described renders the growth factor modulating
activity which is characteristic to the use of the compounds
according to the invention.
[0055] According to the invention the compounds are most often
present in a salt form or in a form where only a few of the
negative charges are protonated. The salt can contain one or more
cations in different combinations. Examples of cations are sodium
and potassium ions.
[0056] The pharmaceutical composition according to the invention
may be administered orally, topically, parentally, rectally or by
inhalation spray in dosage forms or formulations comprising
conventional, nontoxic pharmaceutically acceptable carriers,
adjuvants and vehicles.
[0057] The pharmaceutical composition for oral use can be present
in different forms such as capsules, granules, tablets, troches,
lozenges, aqueous suspensions, dispensible powders, emulsions,
syrups or elixirs. When the composition is present in liquid form
capsules are preferably utilized. At the use of granules, these
preferably have a size of 0.15-2 mm. Either the granules can
consist of the pharmaceutical composition per se or of the
composition and suitable fillers. When the pharmaceutical
composition is used in a tablet form, the tablets can have a weight
of 50-1500 mg, preferably 50-800 mg and most preferably 100-500
mg.
[0058] Formulations for oral use include tablets which contain the
active ingredient in a mixture with nontoxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents, such as calcium carbonate, sodium chloride, lactose,
calcium phosphate or sodium phosphate; granulating and
disintegrating agents, for example, potato starch, or alginic acid,
binding agents, for example, starch, gelatin or acacia, and
lubricating agents, for example, magnesium stearate, stearic acid
or talc. The tablets may be uncoated or they may be coated by known
techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate may be employed.
[0059] Formulations for oral use may also be presented as hard
gelatin capsules wherein the activ ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example,
peanut oil, liquid paraffin, or olive oil.
[0060] For the parentaral application of this invention, typical
dosage forms include intravenous, intramuscular, intraperitoneal
formulations.
[0061] For the rectal application of the composition of this
invention, typical dosage forms include suppositories, rectal
gelatin capsules (solutions and suspensions), and enemas or
micro-enemas (solutions and suspensions). Thus, in a typical
suppository formulation, any one of the compounds of this invention
is combined with any pharmaceutically acceptable suppository base
such as cocoa butter, esterified fatty acids, glycerinated gelatin,
and various water-soluble or dispersible bases like polyethylene
glycols and polyoxyethylene sorbitan fatty acid ester. Various
additives like salicylates or surfactant materials may be
incorporated.
[0062] For topical use, creams, ointments, gels, solutions or the
like containing the compostions are employed according to methods
recognized in the art.
[0063] Naturally, the therapeutic dosage range for the compounds of
the present invention will vary with the size and needs of the
patient and the particular condition or disease symptom being
treated.
[0064] The administration of the pharmaceutical composition
according to the invention can be in a combined dosage form or in
separate dosage forms.
[0065] For administration to human patients appropriate dosages can
routinely be determined by those skilled in this art by extension
of the results obtained in animals at various dosages. The
preferred dosage for humans falls within the range of 0.1 to 100 mg
compound per day and kg bodyweight, especially 0.1 to 50. mg/day/kg
bodyweight.
[0066] The administration of the compounds according to the
invention to mammals are considered to be benefical within the
following conditions:
[0067] Bone disorders such as heterotopic ossification,
osteoporosis, osteoarthritis, osteomalacia, Paget's disease
[0068] Inflammatory conditions such as rheumatoid arthritis
[0069] Analgesic conditions such as hyperalgesia
[0070] Tumour conditions such as glioma, prostate cancer and other
conditions where inhibition of angiogenes is crucial
[0071] Wound healing or tissue repair such as matrix formation,
collagen synthesis and scarr formation
[0072] Viral conditions such as HIV
[0073] Cardiovascular conditions such as atherosclerosis
[0074] Eye diseases
[0075] Infectious conditions such as trypanosomiasis
[0076] According to the present invention a process of modulating
growth factor activity by using a compound containing a high
density, negatively charged domain of vicinally oriented radicals
is described.
[0077] In preferred embodiments of the invention the negatively
charged domain comprises at least three vicinal
phosphorus-containing radicals.
[0078] The invention also relates to the use of a compound wherein
the phosphorus-containing radicals have the following formula:
4
[0079] as described above.
[0080] The invention will be further, explained with the following
embodiment examples however without limiting it thereto.
[0081] Example 1 and 2 describe the interaction between growth
factors and D-myo-inositol-1,2,6-trisphosphate (IP.sub.3). In
example 3 and 4 the growth factor modulating effect, of IP.sub.3 in
tumor models are illustrated while example 5 describes the
counteraction of heterotopic bone formation by IP.sub.3.
[0082] Example 6 shows the inhibitory effect of
D-3,4,5-tri-O-hexanoyl-myo- -inositol-1,2,6-trisphosphate
(PP10-202) on HIV-infection.
[0083] Example 7 describes a growth factor modulating effect of
IP.sub.3 in oedema conditions while example 8, 9 and 10 illustrate
the growth factor modulating effect of
1,2-O-ethylene-.beta.-D-fructopyranoside 2,3,4-trisphosphate (PP
35-405), 1,5-dideoxy-1,5-imino-N-(2-phenylethyl)a-
rabinitol-2,3,4-trisphosphate (PP 35-508) and
myo-inositol-1,2,6-tris(carb- oxymethylphosphonate) (PP 40-402),
respectively in inflammatory conditions.
[0084] In example 11 the growth factor modulating effect of
IP.sub.3 in eye opening of mice is examined.
[0085] The effect of the application of IP.sub.3 on scarring is
shown in example 12. Example 13 describes the interaction between
assembly protein-2 (AP-2) and IP.sub.3. Example 14 illustrates the
binding of compounds accordings to the invention to spermine, While
example 15 shows the analgetic effect of
methyl-.alpha.-D-rhamnopyranoside-2,3,4-trisphosp- hate (PP
35-406). Example 16 describes the effect of IP.sub.3 on PI
3'-kinase. The effect of IP.sub.3 against bone tumours is described
in Example 17. Example 18 illustrates the inhibitory effect of
IP.sub.3 against b FGF-inducted damages, while Example 19 describes
the interactions between IP.sub.3 and different fibroblast growth
factors in computer modelling experiments.
EXAMPLE 1
[0086] Nerve growth factors (NGFA from Vipera lebetina Venom and
NGFB from Mouse Submaxillary glands) (0.31 nmol and 0.08 nmol
respectively) were mixed with 2.7 nmol of radioactive
D-myo-inositol-1,2,6-trisphosphate (IP.sub.3) (specific activity 17
Ci/mmol) and diluted with a buffer containing 50 mM Hepes and 1 mM
EDTA, pH 7.4 to a final volume off 1 ml. The mixture was incubated
for 1 hour at 4.degree. C.
[0087] The interaction between NGF and IP.sub.3 was determined by
fractioning the incubate on a Sephadex G-25 column (Pharmacia
Biotech., Sweden) (volume 9.1 ml, bed height 5 cm). This type of
column fractionate substances according to molecular weight.
Substances with molecular weight exceeding 1500 Will not penetrate
the microspheres of the column and will accordingly elute quite
soon after being loaded to the column, while smaller molecules will
appear later as they partly or totally penetrate the microspheres
depending on the molecular weight. Thus a mixture of a polypeptide
such as NGF (Mw.gtoreq.1500) and a substance with Mw 500, such as
IP.sub.3 will elute in two separated peaks if there is no
interaction between the substances.
[0088] The incubate was loaded on the column and was eluted with 50
mM Hepes in fractions of 0.4 ml each. Each fraction was determined
for radioactivity, indicating the occurrence of IP.sub.3 and with
UV (280 nm) indicating the occurrence of NGF. Maximum radioactivity
and UV absorbance was measured in the same fraction, number 10,
indicating that the two components eluted together.
[0089] To further determine the interaction, the fractions with
radioactivity and UV-absorbance were loaded on a Heparin-Sepharose
CL-6B column (Pharmacia Biotech, Sweden) (volume 25 ml, height 20
cm). As heparin is known to strongly interact with growth factors
such as NGF this type of column is able to bind NGF while other
substances such as IP.sub.3 will be eluted. Elution was performed
with 50 mM Hepes, pH 7.4 and each fraction of 1 ml was collected
for determination of radioactivity and UV absorbance. A peak with
radioactivity but without UV absorption was loaded on a similar
Sephadex G-25 column as described above and was eluted with the
same type of buffer. Maximum radioactivity was obtained in fraction
number 13 i.e. in this case the substance with radioactivity was
more retained on the column.
[0090] The experiment shows that there exists a strong interaction
between NGF and IP.sub.3. When eluted from a Sephadex G-25 column
they elute together, demonstrating the interaction. After binding
NGF to a Heparin-Sepharose column, IP.sub.3 without NGF elutes at a
different place after passage through a Sephadex G-25 column
showing that this type of column is capable of differing between
the NGF-IP.sub.3 complex and pure IP.sub.3.
EXAMPLE 2
[0091] Fibroblast growth factor (FGF) (0.027 nmol) was mixed with
2.7 nmol of radioactive D-myo-inositol-1,2,6-trisphosphate
(IP.sub.3) specific activity 17 Ci/mmol).and diluted with a buffer
containing 50 MM Hepes and 1 mM EDTA, pH 7.4,to a final volume of 1
ml. The mixture was incubated for 1 hour at 4.degree. C.
[0092] The interaction between FGF and IP3 was determined by
fractioning the incubate on a Sephadex. G-25 column (Pharmacia
Biotech., Sweden) (volume 9.1 ml, bed height 5 cm). This type of
column fractionate substances according to molecular weight.
Substances with molecular weight exceeding 1500 will not penetrate
the microspheres of the column and will accordingly elute quite
soon after being loaded to the column, while smaller molecules will
appear later as they partly, or totally penetrate the microspheres
depending on the molecular weight. Thus a mixture of a polypeptide
such as FGF (Mw.gtoreq.1500) and a substance with Mw 500, such as
IP.sub.3 will elute in two separated peaks if there is no
interaction between the substances.
[0093] The incubate was loaded on the column and was uluted with 50
mM Hepes in fractions of 0.4 ml each. Each fraction was determined
for radioactivity, indicating the occurrence of IP3 and with UV
(280 nm) indicating the occurrence of FGF. Maximum radioactivity
and UV absorbance was measured in the same fraction, number 10,
indicating that the two components eluted together.
[0094] To further determine the interaction the fractions with
radioactivity and UV absorbance were loaded on a Heparin-Sepharose
CL-6B column (Pharmacia Biotech, Sweden) (volume 25 ml, height 20
cm). As heparin is known to strongly interact with growth factors
such as FGF this type of column is able to bind FGF while other
substances such as IP.sub.3, will be eluted. Elution was performed
with 50 mM Hepes, pH 7.4 and each fraction of 1 ml was collected
for determination of radioactivity and UV absorbance. A peak with
radioactivity but without UV absorption was loaded on a similar
Sephadex G-25 column as described above and was eluted with the
same type of buffer. Maximum radioactivity was obtained in fraction
number 13 i.e. in this case the substance with radioactivity was
more retained on the column.
[0095] The experiment shows that there exists a strong interaction
between FGF and IP.sub.3. When eluted from a Sephadex G-25 column
they elute together, demonstrating the interaction. After binding
FGF to a Heparin-Sepharose column IP.sub.3 without FGF elutes at a
different place after passage through a Sephadex G-25 column
showing that this type of column is capable of differing between
the FGF-IP.sub.3 complex and pure IP.sub.3.
EXAMPLE 3
[0096] The aim of this experiment was to investigate the effect of
IP.sub.3 in a model ressembling a clinical situations in injured
tissue where fibroblast growth factors mediate autocrine and
paracrine cell growth phases.
[0097] Fibroblast growth factor acidic (aFGF) from Sigma (USA), was
diluted in a buffer, RPMI-1640. (Life Technologies, UK). 25 .mu.g
of aFGF was diluted in 125 .mu.l buffer and 50 .mu.l was
transferred to a small Eppendorf tube 150 .mu.l of Matrigel.RTM.
basement membrane matrix from Becton and Dickinson (UK) was added
to the tube and the content was thoroughly mixed for 60 minutes.
All equipment was sterilized by irradiation or autoclaving
(115.degree. C. in 10 minutes) before use and all procedures were
performed at 7.degree. C. The mixture was kept in an icebath before
the animal experiment.
[0098] Ten female, specific pathogen free BALBc nude mice with a
weight of 18-22 g were maintained in autoclaved type III makrolon
cages with filter tops in horizontal air flow cabinets in a barrier
unit equipped with HEPA filters. Autoclaved bedding, irradiated
feed and sterile filtered water was used. Temperature and humidity
was kept constant at 22.+-.2.degree. C., 65.+-.5 RH.
[0099] When fibroblast growth factor (FGF) and Matrigepl.RTM. is
mixed and inovulated to nude mice, developments of a swelling
process ressembling tumor begin to develop.
[0100] A portion of the mixture of Matrigel.RTM. and a FGF was
inoculated subcutaneously by a 23 G needle in the right flank of
the mice. Two days before all mice were surgically prepared with
osmotic minipumps (Alzet, model 2002). To five mice,
D-myo-inositol-1,2,6-trisphosphate (IP.sub.3) was administered (pup
volume 226 .mu.l, duration 14 days, concentration 0.4 g/ml).
[0101] To another group of five mice saline was administered as a
control group.
[0102] The volume of the formed matrix was measured during 9 days
and the results are shown in the following table.
1 Treatment IP.sub.3 Control Day volume (mm.sup.3) volume
(mm.sup.3) 1 150 150 4 125 125 5 90 125 6 50 130 7 50 140 8 70 130
9 70 120
[0103] The results show a marked reduction in the aFGF-induced
swelling process when the animals were treated with IP.sub.3. The
administration of IP.sub.3 in vivo counteracts produced and
released a FGF which indicates clinically favourable effects of the
compound as a FGF-induced processes are basic phenomenons in
different phases of many disceases.
EXAMPLE 4
[0104] In a procedure similar to example 3 fibroblast grouth factor
basic (bFGF) was used.
[0105] D-myo-inositol-1,2,6-trisphosphate. (IP.sub.3) was
administered to five mice while another group of five mice,
recieving saline, were a control group.
[0106] The volumes of the formed matrix were measured during 9 days
and the results are shown in the followng table:
2 Treatment IP.sub.3 Control Day volume (mm.sup.3) volume
(mm.sup.3) 1 180 220 3 120 140 5 110 150 7 120 170 8 110 200 9 120
200
[0107] The treatment with IP.sub.3 reduces the volume of the tumor
compared to control in a marked way demonstrating the
down-regulatory effect of IP.sub.3 on bFGF-induced injuries.
Histological examinations of the animals revealed anti-angiogenic
effects in the group where IP.sub.3 was administered.
EXAMPLE 5
[0108] Using the rabbit heterotopic bone (HB) development model,
the aim of this experiment was to investigate the effect of
D-myo-inositol-1,2,6-phosphate (IP.sub.3) against heterotopic bone
formation. Heterotopic bone formation is caused by a local growth
and formation of bone. One essential component in this process is
the activation of growth factors.
[0109] The right hind limb of adult rabbits (weight about 4.0 kg)
was immobilized with the knee in extension by means of a plastic
splint and an elastic bandage, so that the Vastus intermedius
profundus (VIP) muscle was in a shortened position and the hip
joint could be moved freely.
[0110] According to the standard model the rabbits were treated by
removing the splint daily for 2-3 minutes for forcible and rapid
manipulation of the knee.
[0111] IP.sub.3 was administered via subcutaneously operated
osmotic minipumps to three rabbits. The dose level was 1
mg/kg/hour, and the time of application period was 14-21 days. The
control group, another three rabbits received NaCl-solution by
osmotic minipumps.
[0112] Anteposterior and lateral radiographs were made of the hind
limbs of all rabbits weekly throughout the duration of the
experiment which lasted 42 days.
[0113] At the end of the experiment the rabbits were killed with an
overdose of Mebunat and the left and right femoral bones of each
rabbit were dissected and boiled for macroscopic examination.
[0114] The amount and localization of HB formation in the rabbits
after the experimental period are shown in Table 1.
3TABLE 1 Treatment and final average amount of heterotopic bone in
different groups Application of Amount Group of animals Treatment
IP.sub.3 of HB Control Imm 0 +++ Control Imm 0 ++++ Control Imm 0
+++ Experim Imm 14 days + Experim Imm 14 days 0 Experim Imm 21 days
0 HB = heterotopic bone Imm = immobilization ++++ = very marked
amount of HB +++ = marked amount of HB ++ = between slight and
marked amount of HB + = slight amount of HB 0 = none amount of
HB
[0115] The final HB formation in the control group developed in the
same way and to the same extent as has been previously found in
corresponding manipulation experiments.
[0116] The animals which received IP.sub.3 subcutaneously were
found to have drastically less ectopic bone in the femur than the
control animals.
[0117] The results obtained in the experiment indicate that the
occurrence, the amount and the localization of HB formation in the
rabbits could be blocked by the administration of IP.sub.3.
EXAMPLE 6
[0118] Growth factor activity is considered to be involved in the
process of viral infections. Viral isolates from HIV-infected
patients were used in order to induce an infection by adding
peripheral blood mononuclear cells (PBMC) to a medium containing
different concentrations of the sodium salt of
D-3,4,5-tri-O-hexanoyl-myo-inositol-1,2,6-trisphosphate (PP10-202).
25 CCID.sub.50 (50% cell culture infections dose) of PBMC from
HIV-infected patients were incubated with four different
concentrations of PP10-202; 0.0625 mg/ml, 0.125 mg/ml, 0.250 mg/ml
and 0.500 mg/ml. Another preparation, without any PP10-202 served
as a control. The growth medium consisted of 10% fetal calf serum 2
.mu.M glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycin and
20 .mu.g/ml gentamicin. The concentration of cells were 2.times.10
per ml. All preparations were incubated for 1 hour at 37.degree.
C.
[0119] PBMCs from healthy donors were then added to the
preparation. Before addition, these cells were stimulated for three
days with phytohemaggclutinin (PHA). 0.5.times.10.sup.6.
PHA-stimulated PBMCs were added to each preparation, followed by
incubation for 3 hours at 37.degree. C. After extensive washing the
cells were resuspended in growth medium supplemented with 10 IU/ml
of interleukin-2 and seeded in quadruplicates of 100.000 cells in a
96-well microtite plate before further cultivation for 7 days. The
HIV-antigen production was assayed at the seventh day using an
ELISA-technique. The obtained values were normalized and are
summarized in the following table:
4 Concentration of PP10-202 mg/ml HIV-infection (%) 0 100 0.0625 75
0.125 41 0.250 33 0.500 20
[0120] The results show that PP10-202 possess a strong effect to
counteract the viral infection.
EXAMPLE 7
[0121] Vascular permeability factor (VPF) promotes extravasation
and oedema formation. Evans Blue was injected intravenously to 15
guinea pigs. This dye binds to plasma proteins and thereby provides
a marker for vascular permeability to macromolecules.
[0122] One group of animals, 7 guinea pigs received an intravenous
injection of 5 mg/kg D-myo-inositol-1,2,6-trisphosphate (IP.sub.3)
while the other group of animals, 8 guinea pigs served as a control
group.
[0123] 50 mg of VPF was injected to each animal and the
distribution of Evans Blue was measured.
[0124] After 60 minutes, the control group had an average area
covered with Evans Blue of 62 mm.sup.2 while the group which
recieved IP.sub.3 had an average area covered with Evans Blue of 15
mm.sup.2.
[0125] The results show that the treatment with IP.sub.3 markedly
reduces the permeability caused by VPF. It has been shown that
antibodies directed to VPF demonstrates a similar effect indicating
that IP.sub.3 has a regulating effect on VPF.
EXAMPLE 8
[0126] Growth factors are implicated to participate in inflammatory
conditions. Carrageenan (0.1 ml, 1%) was injected intraplantarly in
one hind paw of two groups of mice, five animals in each group.
[0127] An intravenous injection of 64 mg/kg of
1,2-O-ethylene-.beta.-D-fru- ctopyranoside-2,3,4-trisphosphate
(PP35-405) was administered immediately before the injection of
carrageenan.
[0128] Injection of carrageenan induces inflammation which is
measured by comparing the increase in paw volume using a
plethysmometer.
[0129] After 3.5 hrs a comparison shows that the inflammation was
retarded by 51% in the group recieving PP35-405. Thus the
administration of PP35-405 significantly counteract the
inflammatory conditions.
EXAMPLE 9
[0130] In a procedure similar to the one described in Example 8, an
intravenous injection of
1,5-dideoxy-1,5-imino-N-(2-phenylethyl)arabinito-
l-2,3,4-trisphosphate (PP.sub.35-508) was administered.
[0131] 3,5 hrs after the injection of carrageenan, the inflammation
was retarded with 27% in the group recieving PP35-508. Thus the
administration of PP35-508 markedly retard the inflammatory
condition.
EXAMPLE 10
[0132] In a procedure similar to the one described in Example 8, an
intravenous injection of myo-inositol-1,2,6-tris(carboxymethyl
phosphonate) (PP40-402) was administered. 3.5 hrs after the
injection of carrageenan the inflammation was retarded with 32% in
the group recieving PP40-402. Thus the administration of PP40-402
markedly reduces the inflammatory condition.
EXAMPLE 11
[0133] Growth factors are involved in processes occuring in newborn
mammals. The biological process of eye opening is one example where
specific growth factors affect the timing and completeness of the
event.
[0134] Three groups of mice, 40 animals in each group were used.
One group recieved daily injections of
D-myo-inositol-1,2,6-trisphosphate, sodium salt (IP.sub.3) from day
0 to day 7 (192 .mu.g/g/day) while the second group recieved saline
under a similar regime. The third group served as a control without
any treatment.
[0135] Eye opening was significantly delayed in the group recieving
IP.sub.3. As a medium value, eyes were opened after 13.6 and 13.5
days respectively in the groups serving as a blank control,
respectively. The medium value for the group recieving IP.sub.3 was
15.3 days, i.e. nearly two days delay in eye opening. These results
demonstrate the growth factor modulating actiity of IP.sub.3.
EXAMPLE 12
[0136] In order to investigate the effect of
D-myo-inositol-1,2,6-trisphos- phate (IP.sub.3) on the process of
scarr formation on skin, scratches to the epidermis of the back of
a human hand was performed with a knife. To two of the scratches a
solution comprising IP.sub.3 was applied topically while two of the
scratches served as controls. Occular investigation was performed
every day over a period of 10 days.
[0137] It was observed that the scratches treated with IP.sub.3
formed scarr in a delayed manner compared to the control and that
the formation of the scars occurred in a more smooth way. The
existing scarrs after the IP.sub.3-treated scratches were
considered to have a nicer appearance. Thus the administration of
IP.sub.3 modulates the growth factor activity involved in the wound
healing process in a favourable way.
EXAMPLE 13
[0138] The binding of D-myo-inositol-1,2,6-trisphosphate sodium
salt (IP.sub.3) to bovine brain clathrin assembly protein-2 (AP-2)
was studied. The binding assay was done in a total volume of 100
.mu.l buffer containing 25 nM Na HEPES pH 7.1, 1 nM EDTA, 1 nM DTT,
5 mg/ml IgG, 10 nM tritiated inositoltetraphosphate and 1.46 .mu.g
of AP-2. The concentrations of IP.sub.3 ranged from 0.04 to 30
.mu.M. Incubation was carried out on ice for a period of ten
minutes and then the protein was co-precipitated with IgG using 5%,
(w/w) polyethylene glycol and pelleted by centrifugation at 95000
rpm for 10 minutes. The supernatants were aspirated, the pellets
were wasted with 0.2 ml water and resuspended in 0.2 ml water and
transferred to scintillation vials. 5.0 ml of cyto scint fluid was
added to each vial and the radioactivity was measured. IP.sub.3 was
found to compete with inositoltetraphosphate for binding. The
IC.sub.50 was determined to be 0.60.+-.0.1 .mu.M.
[0139] The observed binding affinity is well within the
concentration of IP.sub.3 when administered to animals in different
pharmacological models and supports the property of IP.sub.3 to
modulate the activity, of certain growth factors via retarding or
inhibiting the internalisation process.
EXAMPLE 14
[0140] The interaction between a polyamine, spermine and different
triphosphorylated compounds were investigated in order to determin
the binding constants. Spermine is a basic polyamine comprising
domains of similar basicity to basic domains in growth factors.
Spermine and the different phosphorylated compounds forms different
complexes and the 1:1:5 complex was used as the complex for
determination of binding constants.
[0141] The following binding constants, log K.sub.s was determined
in 0.1 M N-tetramethyl-bromide at 25.degree. C.
5 Compound log K.sub.s D-myo-inositol-1,2,6-trisphosphate 5.16
D-3,4,5-tri-O-hexanoyl-my- o-inositol- 4.20 1,2,6-trisphosphate,
PP10-202 D-3,4,5-tri-O-phenylcarbamoyl-myo- 4.71
inositol-1,2,6-trisphosph- ate, PP11-201
Methyl-6-O-butyl-.alpha.-D-mannopyranoside 5.36
2,3,4-trisphosphate, PP35-402 Trimethylolpropane trisphosphate,
<1 PP50-202
[0142] The results show that the cyclic moieties have binding
constants, log K.sub.s, higher than 4 indicating a strong binding,
while the linear compound PP50-202 does not bind.
EXAMPLE 15
[0143] Two groups of mice, 10 animals per group, were used in order
to investigate the analgetic effect of methyl
.alpha.-D-rhamnopyranoside-2,3- ,4-trisphosphate (PP 35-406). The
control group was given an intravenous dose of saline while the
other group was given a dose of 64 mg/kg of the sodium salt of PP
35-406. Three minutes after intravenous dosing each rat received an
intraperitoneal injection of 1 ml of a 1% (w/w) solution of acetic
acid. Directly after that procedure each animal was placed into
individual observation chambers and the numbers of writhes elicited
during the subsequent 10-minute period were recorded. After the
observation period the animals were killed by, cervical
dislocation. The number of writhes during the observation period is
an, expression of the pain experienced by the animal. Percent
protection was calculated as follows. 10.times. ((number of writhes
in control group--number of writhes in treated animals)/(number of
writhes in control group).
[0144] The protection determined for PP 35-406 was 19%
demonstrating a reduction in pain when the compound is
administered.
EXAMPLE 16
[0145] The enzyme phosphatidyl inositol 3' kinase (PI 3'-kinase) is
implicated in a number of growth factor Induced responses such as
mitogenesis, chemotaxis, membrane ruffling and the balance between
osteoclasts and, osteoblasts. Furthermore it is known that
inactivation of PI 3' kinase leads to increased oedema formation
expressed as a decrease of interstitial fluid pressure (P.sub.IF)
in animal models.
[0146] An inhibitor of PI 3' kinase such as wortmannin renders a
decrease of P.sub.IF as a consequence of inactivation of PI 3'
kinase.
[0147] The measurements were performed on female Wistar Moller rats
(200-250 g), anesthetized with pentobarbital (50 mg/kg i.p.).
[0148] P.sub.IF was measured on the dorsal side of the hind paw
with sharpened glass capillarier (tip diameter 2-7 .mu.m) filled
with 0.5 M NaCl coloured with Evans Blue and connected to a
serve-controlled counter pressure system. The punctures were
performed through intact skin using a stereomicroscope. The
pipette-tip was located 0.3-0.5 mm below the skin. Pressure
measurements were made 2.5-3.0 mm from the center of injection. The
normal transcapillary pressure gradient is about 0.5 mm Hg.
[0149] Three sets of experiments were performed with six animals in
each set.
[0150] In the first set of experiment the vehicle for wortmannin,
DMSO diluted in saline, 5 .mu.l, was injected subdermally using a
chromatography syringe. In this control group the P.sub.IF stayed
constant at ca -0.7 mm Hg over the entire determination, on period
of 100 minutes. In the second set of experiments, wortmannin 2.3 M
in DMSO/saline 51 .mu.l was injected. The measurement of P.sub.IF
showed a decrease to -2.5 mm Hg after 30 minutes. This value stayed
constant over the subsequent determination period.
[0151] In the third set of experiments, the animals were pretreated
with an intravenous injection of D-myo-inositol-1,2,6-trisphosphate
(IP.sub.3) 5 .mu.g in 0.1 ml before the administration of
wortmannin, 2.3 M in DMSO/saline.
[0152] The pretreatment with IP.sub.3 inhibited the effect of
wortmannin which resulted in an abolished decrease in P.sub.IF.
[0153] The results demonstrate an interaction of IP.sub.3 with the
PI 3' kinase pathway i.e. a pathway implicated in growth factor
induced responses.
EXAMPLE 17
[0154] In an experimental model of prostate tumours the effect of
D-myo-inositol-1,2,6-trisphosphate (IP.sub.3) was assessed. The
injection of a specific cell-line Wish tumour cells, induces
proliferation of osteoblasts on the calvaria of nude mice leading
to bone tumours.
[0155] Ten mice were divided into two groups, one group receiving
an infusion of IP.sub.3 for 14 days while the other group, serving
as a control group, received saline for 14 days.
[0156] IP.sub.3 and saline respectively were administered with
osmotic minipumps (Alzet.sup.R). with a dosage of 120
mg/kg/day.
[0157] Wish cells (American Type Culture Collection.sup.R, ATCC (R)
F-12784 25 CCL WITH.times.01166) were used for inocculation. Cells
were grown on MEM supplemented with 15% FCS, 2% glutamine and
streptomycin-penicillin/garamycin. Cells were grown as a monolayer
and trypsinised and split in the ratio 1:3,to 1:4. All together 360
million cells were harvested and suspended in 7 ml for a final
concentration of approximately 10 million cells per 0.2 ml.
[0158] The osmotic pumps with IP.sub.3 and saline respectively were
implanted on the same day as the inocculation of Wish-cells were
performed.
[0159] A 27 gauge needle on a 1 ml syringe was used for the
injections of Wish-cells. The injection was done under the skin of
the calvaria scratching the periosteum.
[0160] At day 13 all animals were given an intraperiotoneal
injection of demeclocycline (Sigma Chemicals Co. St Louis). All
animals were investigated regarding the occurrence of and size of
tumours. The size of tumours were measured according to a scale
from
[0161] + which represents small and non-severe tumours to
[0162] ++++ which represents large and severe tumours.
[0163] The results are shown in the following table:
6 Animal no Treatment Severeness of tumours 1 IP.sub.3 ++ 2 -"- ++
3 -"- +++ 4 -"- ++++ 5 Saline ++++ 6 -"- ++++ 7 -"- ++++ 8 -"- ++++
9 -"- ++++ 10 -"- ++++
[0164] The animals receiving IP.sub.3 showed a lower degree of size
and severeness of the tumours which demonstrates that the
administration of IP.sub.3 counteracts tumour formation in a bone
tumour model.
EXAMPLE 18
[0165] Injection of basic fibroblast growth factor (b FGF)
stimulates osteoblastic activity and leads to bone formation. In an
experiment in mice the effect of D-myo-inositol-1,2,6-trisphosphate
(IP.sub.3) to counteract the growth factor induced process was
assessed.
[0166] Once group with five mice recieved an infusion of IP.sub.3
for 14 days while another group of five mices, serving as a control
group, recieved saline for 14 days.
[0167] IP.sub.3 and saline respectively were administered with
osmotic minipumps (Alzet.sup.R) with a dosage of 120 mg/kg/day.
[0168] The osmotic minipumps were implanted on day 1.
[0169] To the calvaria of each animal, 10 .mu.l of b FGF
(concentration 50 .mu.g/ml) was injected four times daily of the
period of day 2 to day 4.
[0170] After the experimental period of 14 days each animal was
investigated macroscopically and the histological preparations were
further examined by conventional routines.
[0171] Macroscopically visible bone formation was measured
according to scale from
[0172] 0 which represents no bone formation to
[0173] ++++ which represents severe bone formations
[0174] The results are shown in the following table:
7 Animal no Treatment Severeness of bone formation 1 IP.sub.3 0 2
-"- ++ 3 -"- 0 4 -"- 0 5 -"- 0 6 Saline ++++ 7 -"- ++++ 8 -"- ++++
9 -"- ++++ 10 -"- ++++
[0175] The results show that IP.sub.3 has a strong counteractive
effect against b FGF-induced bone formation. Furthermore the
histological examination showed clear differences between the group
expressed as structure and thickness of the calvaria. The
preparation show that IP.sub.3 has anti-angiogenic properties.
EXAMPLE 19
[0176] The interaction between D-myo-inositol-1,2,6-trisphosphate
(IP.sub.3) and several growth factors has been examined by the
utilization of computer modelling programs.
[0177] Especially the interaction between IP.sub.3 and the
fibrobast growth factor family reveals the pattern that the two
axial and one equatorial phosphate group interacts with regions
consisting of basic amino acids such as lysine, arginine and
histidine. These domain are essential for the dimerization of
growth factors but also for the interaction between growth factors
and their cellular receptors.
[0178] In the interaction between IP.sub.3 and acid Fibroblast
Growth Factor (a FGF) specific binding is observed with lysine 112,
lysine 118 and arginine 122 in such a manner that the distance is
approximately 2 .ANG. indicating strong and firm interactions. The
interaction is shown in FIG. 1.
[0179] Furthermore IP.sub.3 interacts in a very specific way with
basic Fibroblast Growth Factor (b FGF). The different phosphate
groups of IP.sub.3 interacts with arginine 120; distance 1.94
.ANG.; with lysine 125; distance 2,40 .ANG., with lysine 135;
distance 2.37 .ANG. and with aspargine 27; distance 2,68 .ANG.. The
interaction is further described in FIG. 2. A specific interaction
is observed to the site in b FGF where the dimerization process is
initiated. To this site IP.sub.3 interacts with arginine 72;
distance 2,26 .ANG., with arginine 81; distance 2.36 .ANG. and
arginine 39; distance 1.45 .ANG.. This interaction is further
described in FIG. 3.
[0180] One of the essential elements of the interaction is the fact
that the three phosphate groups of IP.sub.3 stereochemically is in
a form where two moities are axial and one equatorial. As a
consequence IP.sub.3 can be regarded as a model substance where the
same type of interactions can be foreseen with other
stereochemically similar compounds according to the invention.
* * * * *