U.S. patent application number 09/777324 was filed with the patent office on 2001-07-12 for prodrugs with enhanced penetration into cells.
This patent application is currently assigned to D-Pharm LTD.. Invention is credited to Kozak, Alexander.
Application Number | 20010007865 09/777324 |
Document ID | / |
Family ID | 23906125 |
Filed Date | 2001-07-12 |
United States Patent
Application |
20010007865 |
Kind Code |
A1 |
Kozak, Alexander |
July 12, 2001 |
Prodrugs with enhanced penetration into cells
Abstract
The invention relates to a pharmaceutically acceptable prodrug
which is a covalent conjugate of a pharmacologically active
compound and an intracellular transporting adjuvant, characterized
by the presence of a covalent bond which is scission-sensitive to
intracellular enzyme activity. The prodrug may be used in a
technique for treating a condition or disease in a mammal related
to supranormal intracellular enzyme activity, whereby on
administering it to a human having such condition or disease, the
bond is broken in response to such activity, and the
pharmacologically active compound is activated selectively within
cells having such supranormal intracellular enzyme activity.
Inventors: |
Kozak, Alexander; (Rehovat,
IL) |
Correspondence
Address: |
DAVIDSON, DAVIDSON & KAPPEL, LLC
485 SEVENTH AVENUE, 14TH FLOOR
NEW YORK
NY
10018
US
|
Assignee: |
D-Pharm LTD.
Kiryat Weizmann Science Park, Building 16 P.O. Box 2313
Rehovot
IL
76123
|
Family ID: |
23906125 |
Appl. No.: |
09/777324 |
Filed: |
February 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09777324 |
Feb 6, 2001 |
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08479959 |
Jun 7, 1995 |
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08479959 |
Jun 7, 1995 |
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08481243 |
Aug 21, 1995 |
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5985854 |
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08481243 |
Aug 21, 1995 |
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PCT/GB94/00669 |
Mar 30, 1994 |
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Current U.S.
Class: |
514/143 ;
514/162; 514/227.5; 514/252.11; 514/78 |
Current CPC
Class: |
A61K 47/542 20170801;
A61K 31/352 20130101; C07F 9/10 20130101; Y10S 514/826 20130101;
A61K 47/544 20170801 |
Class at
Publication: |
514/143 ;
514/227.5; 514/252.11; 514/78; 514/162 |
International
Class: |
A61K 031/685; A61K
031/66; A61K 031/54; A61K 031/497 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 1993 |
IL |
105244 |
Claims
What is claimed is:
1. A pharmaceutically acceptable prodrug comprising a
pharmacologically active compound covalently bonded to an
intracellular transporting adjuvant, said pharmacologically active
compound being effective to treat a disease or disorder related to
supranormal enzyme activity, said covalent bond being cleaved in
the presence of supranormal intracellular enzyme activity and said
prodrug being pharmacologically inactive prior to cleavage.
2. A prodrug according to claim 1, wherein said pharmacologically
active compound is a pharmacologically active carboxylic acid and
said intracellular transporting adjuvant comprises at least one
pharmaceutically acceptable alcohol which is selected from the
group consisting of glycerol, C.sub.3-20 fatty acid monoglycerides,
C.sub.3-20 fatty acid diglycerides, hydroxy-C.sub.2-6-alkyl esters
of C.sub.3-20 fatty acids, hydroxy-C.sub.2-6-alkyl esters of
lysophosphatidic acids, lyso-plasmalogens, lysophospho-lipids,
lysophosphatidic acid amides, glycerophosphoric acids,
lyso-phophatidalet~hanolamine, lysophosphatidyl-ethanolamine,
N-mono-(C.sub.1-4)-alkyl and N,N-di-(C.sub.1-4)-alkyl and
quaternary derivatives of the amines thereof.
3. A prodrug according to claim 2, wherein said pharmacologically
active carboxylic acid is selected from branched-chain aliphatic
carboxylic acids, salicylic acids, steroidal carboxylic acids,
monoheterocyclic carboxylic acids and polyheterocyclic carboxylic
acids.
4. A prodrug according to claim 1 wherein said intracellular
transporting adjuvant comprises a carboxylic acid compound of the
formula: (HOOC--CH.sub.2--).sub.2--N--A--N--(--CH.sub.2COOH).sub.2
where A is a linking radical selected from the group consisting of
an aliphatic, aromatic and heterocyclic organic radical comprising
from 2-8 carbon atoms is interrupted by n non-adjacent oxygen
atoms, wherein n is a number selected from the group consisting of
0, 2, 3 and 4, so that said carboxylic acid compound is an ester
when n ranges from 2 to 4, and said carboxylic acid compound is
covalently linked to a pharmaceutically acceptable alcohol
containing from 3 to 32 carbon atoms and from 1-3 hydroxyl
radicals; and pharmaceutically acceptable salts thereof; provided
that, when said carboxylic acid compound is an ester, the nitrogens
flanking A are not linked to any of said n non-adjacent oxygen
atoms.
5. A prodrug according to claim 4, wherein said pharmaceutically
acceptable alcohol is a C.sub.7-32 secondary monohydric
alcohol.
6. A prodrug according to claim 4, wherein said pharmaceutically
acceptable alcohol contains 3 to 6 carbon atoms and 1-3 hydroxyl
radicals.
7. An ester according to claim 4, wherein said linking radical A is
selected from the group consisting of --(CH.sub.2CH.sub.2).sub.m--,
where m is a number ranging from 1 to 4, and
--CR.dbd.CR--O--CH.sub.2CH.sub.2--- O--CR'.dbd.CR'--, wherein each
pair of radicals R--R and R'--R', together with the attached
--C.dbd.C-- moiety, complete an aromatic or heterocyclic ring
containing from 5 to 6 ring atoms, the ring completed by R--R being
the same as or different from the ring completed by R'--R'; wherein
p carbons of (CH.sub.2CH.sub.2).sub.m-- are each replaced by oxygen
and p is a number selected from the group consisting of 0, 2, 3 and
4.
8. An ester according to claim 4, wherein said linking radical A is
selected from the group consisting of --CH.sub.2CH.sub.2-- and
--CHFCH.sub.2--O--CH.sub.2CH.sub.2--O--CH.sub.2CH.sub.2--.
9. An ester according to claim 4, wherein said linking radical is
--CR.dbd.CR--O--CH.sub.2CH.sub.2--O--CR'.dbd.CR', where each of the
pairs of radicals R--R and R'--R', together with the attached
--C.dbd.C-- moiety, complete an aromatic or heterocyclic ring which
is selected from the group consisting of furan, thiophene, pyrrole,
pyrazole, imidazole, 1,2,3-triazole, oxazole, isoxazole,
1,2,3oxadiazole, 1,2,5-oxadiazole, thiazole, isothiazole,
1,2,3-thiadiazole, 1,2,5-thiadiazole, benzene, pyridine,
pyridazine, pyrimidine, pyrazine, 1,2,3triazine, 1,2,4-triazine,
and 1,2-, 1,3- and 1,4-oxazines and -thiazines, the ring completed
by R--R being the same as or different from the ring completed by
R'--R'.
10. An ester according to claim 9, wherein the linking radical A is
--CR.dbd.CR--O--CH.sub.2CH.sub.2--O--CR'.dbd.CR'--, where each of
the pairs of radicals R--R and R'--R', together with the attached
--C.dbd.C-- moiety, completes the same or different rings selected
from unsubstituted and substituted benzene rings, in which
substituted benzene rings contain from 1 to 4 substituents selected
from the group consisting of C.sub.1-3-alkyl, C.sub.1-3-alkoxy,
fluorine, chlorine, bromine, iodine and CF.sub.3, or a single
divalent substituent which is --O--(CH.sub.2).sub.n--O-- and n is a
number ranging from 1 to 3.
11. An ester according to claim 4, wherein said ester is a
chelating agent selected from the group consisting of
ethylene-1,2-diamine-N,N,N',N'-tetr- aacetic acid,
ethylene-1,2-diol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraac- etic
acid and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid.
12. An ester according to claim 7, wherein said pharmaceutically
acceptable alcohol contains from 3 to 6 carbon atoms and from 1 to
3 hydroxyl radicals.
13. An ester according to claim 11, wherein said pharmaceutically
acceptable alcohol comprises at least one member selected from the
group consisting of glycerol, C.sub.3-20 fatty acid monoglycerides,
C.sub.3-20 fatty acid diglycerides, hydroxy-C.sub.1-6-alkyl esters
of C.sub.3-20 fatty acids, hydroxy-C.sub.2-6-alkyl esters of
lysophosphatidic acids, lyso-plasmalogens, lysophospholipids,
lysophosphatidic acid amides, glycerophosphoric acids,
lyso-phophatidalethanolamine, lyso-phosphatidylethanolamine
N-mono-(C1-4)-alkyl and N,N-di-(C.sub.1-4)-alkyl and quaternary
derivatives of the amines thereof.
14. An ester according to claim 4, which is selected from the
mono-, di-, tri- and tetra-esters of
1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraac- etic acid with
heptanoyl-sn-3-glycerophosphoryl-choline or
octanoyl-sn-3-glycerophosphoryl-choline.
15. A prodrug according to claim 1, wherein the pharmacologically
active compound is a protein kinase inhibitor.
16. A prodrug according to claim 15, which is an ester of a protein
kinase inhibitor carboxylic acid covalently bonded with a
pharmaceutically acceptable alcohol selected from the group
consisting of glycerol, C.sub.3-20 fatty acid monoglycerides,
C.sub.3-20 fatty acid diglycerides, hydroxy-C.sub.2-6-alkyl esters
of C.sub.3-20 fatty acids, hydroxy-C.sub.2-6-alkyl esters of
lyso-phosphatidic acids, lysoplasmalogens, lysophospholipids,
lyso-phosphatidic acid amides, glycerophosphoric acids,
lysophophatidal-ethanolamine, lysophosphatidyl-ethanolamine,
N-moino-(C.sub.1-4)-alkyl and N,N-di-(C.sub.1-4)-alkyl and
quaternary derivatives of the amines thereof.
17. A prodrug according to claim 16, wherein the protein kinase
inhibitor is protein kinase inhibitor K252b from Nocardi.opsis
sp.
18. A prodrug according to claim 15, wherein the protein kinase
inhibitor contains an amine group with a replaceable N-linked
hydrogen atom, and the prodrug is an amide thereof with a
phosphoric acid derivative selected from the group consisting of
glycerophosphoric acids, O-acylglycerophosphoric acids, etherified
glycerophosphoric acids, and monoacylated monoetherified
glycerophosphoric acids.
19. A prodrug according to claim 18, wherein the protein kinase
inhibitor is isoquinoline-5-sulfonamide ad which is N-substituted
by a radical selected from the group consisting of an acyclic
aminoalkyl and a heterocyclic aminoalkyl radical.
20. A prodrug according to claim 19, wherein said aminoalkyl
radical is selected from the group consisting of
NHCH.sub.2CH.sub.2NHCH.sub.3 and 2-methylpiperazin-1-yl.
21. A prodrug according to claim 15, wherein the protein kinase
inhibitor contains at least one phenolic hydroxy group, and the
prodrug is an ester thereof with a phosphoric acid derivative
selected from the group consisting of glycerophosphoric acids,
O-acyl-glycerophosphoric acids, etherified glycerophosphoric acids,
and monoacylated monoetherified glycerophosphoric acids.
22. A prodrug according to claim 21, wherein the protein kinase
inhibitor is 4',5,7-trihydroxyisoflavonie.
23. A prodrug according to claim 1, wherein the pharmacologically
active compound is an antiepileptic compound.
24. The prodrug according to claim 23 wherein said antiepileptic
compound is valproic acid or a pharmaceutically acceptable
derivative thereof.
25. The prodrug according to claim 24 wherein said antiepileptic
compound is covalently bonded with a pharmaceutically acceptable
alcohol selected from the group consisting of glycerol, C.sub.3-20
fatty acid monoglycerides, C.sub.3-20 fatty acid diglycerides,
hydroxy-C.sub.2-6-alkyl esters of C.sub.3-20 fatty acids,
hydroxy-C.sub.2-6-alkyl esters of lyso-phosphatidic acids,
lysoplasmalogens, lysophospholipids, lyso-phosphatidic acid amides,
glycerophosphoric acids, lysophophatidal-ethanolamine,
lysophosphatidyl-ethanolamine, N-mono-(C.sub.1-4)-alkyl and
N,N-di-(C.sub.1-4)-alkyl and quaternary derivatives of the amines
thereof.
26. The proclrug according to claim 24 wherein
26. The prodrug according to claim 24 wherein said antiepileptic
compound is covalently bonded with
1-heptanoyl-sn-glycero-3-phosphorylcholine.
27. A method for treating a disease or disorder in a mammal
comprising administering to a mammal having a disease or disorder
related to supranormal intracellular enzyme activity, an amount a
pharmaceutically acceptable prodrug effective to treat the disease
or disorder, said prodrug comprising a pharmacologically active
compound covalently bonded to an intracellular transporting
adjuvant, said prodrug being cell membrane permeable and said
covalent bond being cleaved in the presence of supranormal enzyme
activity and cleavage of said covalent bond results in selective
intracellular accumulation of therapeutic amounts of the
pharmacologically active compound within cells having supranormal
intracellular enzyme activity.
28. The method according to claim 27, wherein said
pharmacologically active compound is a carboxylic acid and said
intracellular transporting adjuvant comprises at least one
pharmaceutically acceptable alcohol which is selected from the
group consisting of glycerol, C.sub.3-20 fatty acid monoglycerides,
C.sub.3-20 fatty acid diglycerides, hydroxy-C.sub.2-6-alkyl esters
of C.sub.3-20 fatty acids, hydroxy-C.sub.2-6-alkyl esters of
lysophosphatidic acids, lyso-plasmalogens, lyso-phospholipids,
lysophosphatidic acid amides, glycerophosphoric acids,
lysophophatidal-thanolamine, lysophosphatidyl-ethanolamine,
N-mono-(C.sub.1-4)-alkyl and N,N-di-(C.sub.1-4)-alkyl and
quaternary derivatives of the amines thereof.
29. The method according to claim 28, wherein said
pharmacologically active carboxylic acid is selected from the group
consisting of branched-chain aliphatic carboxylic acids, salicylic
acids, steroidal carboxylic acids, monoheterocyclic carboxylic
acids and polyheterocyclic carboxylic acids.
30. The method according to claim 27, wherein said
pharmacologically active compound is a calcium chelating agent.
31. The method according to claim 30, wherein said intracellular
transporting adjuvant comprises a carboxylic acid compound of the
formula: (HOOC--CH.sub.2--).sub.2--N--A--N--(--CH.sub.2COOH).sub.2
where A is a linking radical selected from the group consisting of
an aliphatic, aromatic and heterocyclic organic radical comprising
from 2-8 carbon atoms interrupted by n non-adjacent oxygen atoms,
wherein n is a number selected from the group consisting of 0, 2, 3
and 4, so that said carboxylic acid compound is an ester when n
ranges from 2 to 4, and said carboxylic acid compound is covalently
linked to a pharmaceutically acceptable alcohol containing from 3
to 32 carbon atoms and from 1-3 hydroxyl radicals; and
pharmaceutically acceptable salts thereof; provided that, when said
carboxylic acid compound is an ester, the nitrogens flanking A are
not linked to any of said n non-adjacent oxygen atoms.
32. The method according to claim 31, wherein said pharmaceutically
acceptable alcohol is a C.sub.7-32 secondary monohydric
alcohol.
33. The method according to claim 31 wherein said pharmaceutically
acceptable alcohol contains from 3 to 6 carbon atoms and from 1 to
3 hydroxyl radicals.
34. The method according to claim 31 wherein said linking radical A
is selected from the group consisting of
--(CH.sub.2CH.sub.2).sub.m--, where m is a number ranging from 1 to
4, and --CR.dbd.CR--O--CH.sub.2CH.sub.2--- O--CR'=CR'--, wherein
each pair of radicals R--R and R'--R', together with the
attached--C.dbd.C-- moiety, complete an aromatic or heterocyclic
ring containing from 5 to 6 ring atoms, the ring completed by R--R
being the same as or different from the ring completed by R'--R';
wherein p carbons of --(CH.sub.2CH.sub.2).sub.m-- are each replaced
by oxygen and p is a number selected from the group consisting of
0, 2, 3 and 4.
35. The method according to claim 31 wherein said linking radical A
is selected from the group consisting of --CH2CH2-- and
--CH2CH2--O--CH2CH2--O--CH2CH2--.
36. The method according to claim 31 wherein said linking radical
is --CR.dbd.CR--O--CH.sub.2CH.sub.2--O--CR'.dbd.CR'--, where each
of the pairs of radicals R--R and R'--R', together with the
attached --C.dbd.C-- moiety, complete an aromatic or heterocyclic
ring which is selected from the group consisting of furan,
thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, oxazole,
isoxazole, 1,2,3-oxadiazole, 1,2,5-oxadiazole, thiazole,
isothiazole, 1,2,3-thiadiazole, 1,2,5-thiadiazole, benzene,
pyridine, pyridazi.ne, pyrimidine, pyrazine, 1,2,3-triazine,
1,2,4-triazine, and 1,2-, 1,3- and 1,4-oxazines and -thiazines, the
ring completed by R--R being the same as or different from the ring
completed by R'--R'.
37. The method according to claim 36, wherein the linking radical A
is --CR.dbd.CR--O--CH.sub.2CH.sub.2--O--CR'.dbd.CR'--, where each
of the pairs of radicals R--R and R'--R', together with the
attached --C.dbd.C-- moiety, completes the same or different rings
selected from unsubstituted and substituted benzene rings, in which
substituted benzene rings contain from 1-4 substituents selected
from the group consisting of C.sub.1-3-alkyl, C.sub.1-3-alkoxy,
fluorine, chlorine, bromine, iodine and CF.sub.3, or a single
divalent substituent which is --O--(CH.sub.2).sub.n--O-- and n is a
number from 1 to 3.
38. The method according to claim 30 wherein said chelating agent
is selected from the group consisting of
ethylene-1,2-diamine-N,N,N',N'-tetr- aacetic acid,
ethylene-1,2-diol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraac- etic
acid and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid.
39. The method according to claim 31 wherein said pharmaceutically
acceptable alcohol contains from 3 to 6 carbon atoms and from 1 to
3 hydroxyl radicals.
40. The method according to claim 31, wherein said pharmaceutically
acceptable alcohol comprises at least one member of the group
consisting of glycerol, C.sub.3-20 fatty acid monoglycerides,
C.sub.3-20 fatty acid diglycerides, hydroxyy-C.sub.2-6-alkyl esters
of C.sub.3-20 fatty acids, hydroxy-C.sub.2-6-alkyl esters of
lysophosphatidic acids, lyso-plasmalogens, lysophospholipids,
lysophosphatidic acid amides, glycerophosphoric acids,
lysophophatidalethanolamine, lysophosphatidyl-ethanolamine,
N-mono-(C.sub.1-4)-alkyl and N,N-di-(C.sub.1-4)-alkyl and
quaternary derivatives of the amines thereof.
41. The method according to claim 31, wherein said ester is a
chelating agent selected from the group consisting of
ethylene-1,2-diamine-N,N,N',N- '-tetraacetic acid,
ethylene-1,2-diol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic
acid and 1,2-bis-(2aminophenoxy)ethane-N,N,N- ',N'-tetraacetic
acid.
42. The method according to claim 27, wherein said
pharmacologically active compound is a protein kinase
inhibitor.
43. The method according to claim 42, wherein said prodrug is an
ester of a protein kinase inhibitor carboxylic acid covalently
bonded with a pharmaceutically acceptable alcohol selected from the
group consisting of glycerol, C.sub.3-20 fatty acid monoglycerides,
C.sub.3-20 fatty acid diglycerides, hydroxy-C.sub.2-6-alkyl esters
of C.sub.3-20 fatty acids, hydroxy-C.sub.2-6-alkyl esters of
lyso-phosphatidic acids, lysoplasmalogens, lysophospholipids,
lyso-phosphatidic acid amides, glycerophosphoric acids,
lysophophatidal-ethanolamine, lysophosphatidyl- ethanolamine,
N-mono-(C.sub.1-4)-alkyl and N,N-di-(C.sub.1-4)-alkyl and
quaternary derivatives of the amines thereof.
44. The method according to claim 43, wherein the protein kinase
inhibitor is protein kinase inhibitor K252b from Nocardiopsis
sp.
45. The method according to claim 42, wherein the protein kinase
inhibitor contains an amine group with a replaceable N-linked
hydrogen atom, and the prodrug is an amide thereof with a
phosphoric acid derivative selected from the group consisting of
glycerophosphoric acids, O-acylglycerophosphoric acids, etherified
glycerophosphoric acids, and monoacylated monoetherified
glycerophosphoric acids.
46. The method according to claim 42, wherein the protein kinase
inhibitor is isoquinoline-5-sulfonamide which is N-substituted by
an acyclic or heterocyclic aminoalkyl radical.
47. The method according to claim 46, wherein said aminoalkyl
radical is selected from the group consisting of
NHCH.sub.2CH.sub.2NHCH.sub.3 and 2-methylpiperazin-1-yl.
48. The method according to claim 42, wherein the protein kinase
inhibitor contains at least one phenolic hydroxy group, and the
prodrug is an ester thereof with a phosphoric acid derivative
selected from the group consisting of glycerophosphoric acids,
O-acyl-glycerophosphoric acids, etherified glycerophosphoric acids,
and monoacylated monoetherified glycerophosphoric acids.
49. The method according to claim 42, wherein the protein kinase
inhibitor is 4',5,7-trihydroxyisoflavone.
50. The method according to claim 27, wherein the pharmacologically
active compound is an antiepileptic compound.
51. The method according to claim 50 wherein said antiepileptic
compound is vaiproic acid or a pharmaceutically acceptable
derivative thereof.
52. The method according to claim 50 wherein said antiepileptic
compound is covalently bonded with a pharmaceutically acceptable
alcohol selected from the group consisting of glycerol, C.sub.3-20
fatty acid monoglycerides, C.sub.3-20 fatty acid diglycerides,
hydroxy-C.sub.2-6-alkyl esters of C.sub.3-20 fatty acids,
hydroxy-C.sub.2-6-alkyl esters of lyso-phosphatidic acids,
lysoplasmalogens, lysophospholipids, lyso-phosphatidic acid amides,
glycerophosphoric acids, lysophophatidal-ethanolamine,
lysophosphatidyl-ethanolamine, N-mono-(C.sub.1-4)-alkyl and
N,N-di-(C.sub.1-4)-alkyl and quaternary derivatives of the amines
thereof.
53. The method according to claim 50 wherein said antiepileptic
compound is covalently bonded with
1heptanoyl-sn-glycero-3-phosphorylcholine.
54. The method according to claim 27, wherein the prodrug is
administered by a route selected from the group consisting of
intramuscular injection, intravenous injection, infusion into a
body cavity, cerebrospinal injection, localized infiltration of a
target tissue, buccal absorption and aerosol inhalation in an
amount effective to treat said disease or disorder.
55. The method according to claim 27, wherein said disease or
disorder is selected from the group consisting of localized tissue
ischemia, stroke, epilepsy, asthma and allergy.
Description
[0001] This application is a continuation-in-part of U.S. Ser. No.
______, filed on ______ as a U.S. national stage application of
PCT/GB94/000669, the disclosures of which are incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a technique for treating a
condition or disease in a mammal, including humans, related to
supranormal intracellular enzyme activity, and to a prodrug useful
in treating such a condition or disease.
BACKGROUND OF THE INVENTION
[0003] Many of the most prevalent diseases in humans including
ischemia, stroke, epilepsy, asthma and allergy are all believed to
be related to the phenomenon of cell hyperexcitation, a term used
herein to denote supranormal intracellular enzyme activity. Certain
pharmacological strategies are therefore aimed at inhibiting this
detrimental degradative activity.
[0004] In contrast to such known strategies which are aimed at
suppressing this dlegradative activity, it would be advantageous to
be able to selectively target diseased cells characterized by
enzyme hyperactivity, so as to introduce a pharmacologically active
molecule in the form of a prodrug into the cell, whereby such
hyperactivity would act on the prodrug, so that the
pharmacologically active molecule accumulates in the diseased cells
rather than in the healthy cells.
[0005] Different types of intracellular enzyme systems are known to
be significantly elevated in pathological conditions, and may be
used to achieve preferential release of the active drug compound
within the diseased cells. Candidate enzymes that could be utilized
to activate the prodrugs according to the present invention include
lipases, proteases or glycosidases. By way of example, in many
diseases cell membranes are broken down due to abnormal
intracellular lipase activity.
[0006] The use of prodrugs to impart desired characteristics such
as increased bioavailability or increased site- specificity on
known drugs is a recognized concept in the state of the art of
pharmaceutical development. The use of various lipids in the
preparation of particular types of prodrugs is also known in the
background art. In none of those instances are the prodrugs
characterized in that they achieve preferential accumulation of the
drug within the diseased cells of the organ, by activation with
intracellular lipases. Rather, they provide for the drug to be
transported to a specific site, or to be released within a specific
organ.
[0007] This approach is exemplified in the case of the phospholipid
prodrugs of salicylates and non-steroidal anti-inflammatory drugs
disclosed in WO 91/16920 which, taken orally, protect the gastric
mucosa and release the active principle in the gut.
[0008] In other examples of phospholipid prodrugs, formulation of
the prodrugs into liposomes or other micellar structures is the
feature that enables their preferential uptake, for instance by
macrophages or by liver cells as in the case of the phospholipid
conjugates of antiviral drugs disclosed in WO 90/00555 and WO
93/00910.
[0009] Generally, viral infection is not associated with
supranormal phospholipase activity and antiviral phospholipid
conjugates do not teach or suggest activation of the drug
preferentially in the diseased cells, or in the infected cells as
in the case of the phospholipid conjugates of antiviral nucleotides
and anti-sense oligonucleotides, such as those disclosed in WO
90/00555, in WO 90/10448 and in NTIS Technical Notes, no. 9, page
630, Springfield, Va., US, 1984.
[0010] In other instances specific types of polar lipids are used
to target the prodrugs to intracellular organelles as in the case
of the antiviral and antineoplastic nucleosides disclosed in U.S.
Pat. No. 5,149,794. Additional types of lipids have also been used
in specific types of prodrugs such as EP A-325160 which discloses
glycerin esters of ACE inhibitors, which form micelles absorbed
from the intestine into the lymphatic system, thereby bypassing the
liver and having increased access to the central nervous system,
for use in the treatment of hypertension and cognitive dysfunction.
The ACE inhibitors undergo enzymatic cleavage and exert their
therapeutic effects extracellularly.
[0011] Other types of lipophilic carriers that facilitate
intracellular transport are known in the art, as in CH A-679856
which discloses the use of salicyloyl-carnitine for the treatment
of pain, and in WO 89/05358 which discloses modified
oligonucleotide antisense drugs, transported into cells by
attachment of apolar groups such as phenyl or naphthyl groups.
[0012] Different classes of pharmacologically active molecules can
be administered as prodrugs according to the principles of the
present invention. Candidates include anti-inflammatory drugs,
anti-epileptic drugs, protease inhibitors, and anti-tumor drugs. A
non-limiting example of such pharmacologically active molecules is
a calcium chelating agent, which would have many advantages over
drugs presently used for the treatment of calcium associated
disorders.
[0013] Intracellular calcium is an important determinant for cell
death, irrespective of the initial insult sustained by the cell. It
may be involved in cell death in lymphocyte and killer cell
mediated damage of target cells, in organ damage during
transplantation, and in other types of tissue damage including
ischemic insults. Calcium channel blockers or cell membrane
permeable forms of calcium chelators have been suggested to protect
against tissue injury or to decrease tissue damage. Thus, it will
be apparent that the present invention has potential use (in the
embodiment employing a calcium chelator ) in relation to these
circumstances
[0014] The cell damage occurring in ischemia may be secondary to
the influx and/for intracellular release of Ca.sup.2+ions (Choi,
Trends Neurosci., 1988, 11, 465-469; Siesjo and Smith,
Arzneimittelforschung, 1991, 41, 288-292). Similarly, calcium
influx appears to play an important role in the genesis of
epileptic seizures. Although a significant portion of intracellular
calcium arrives from intracellular stores, current research
suggests that calcium entry blockers may have anticonvulsant
activity (see e.g. Meyer, 1989, Brain Res. Rev. 14, 227-243).
[0015] Drugs which are currently or potentially useful for
treatment of calcium associated disorders include: (1) calcium
channel blockers, (2) drugs affecting calcium balance by
modification of intracellular calcium storage sites, and (3)
intracellular calcium chelating agents. Calcium channel blockers
used in clinical practice are represented by Verapamil, Nifedipine
and Diltiazem. The major toxicities associated with the use of such
compounds involve excessive vasodilation, negative inotropy,
depression of the sinus nodal rate, and atrial ventricular (A-V)
nodal conduction disturbances. Drugs affecting calcium mobilization
and/or sequestration, like calcium channel blockers, exhibit rather
narrow specificity.
[0016] Though the use of calcium chelators for reducing injury to
mammalian cells is disclosed in WO 94/08573, there are no
intracellular calcium chelating agents suitable for clinical
requirements. Existing cell membrane permeable calcium chelators
include acetoxymethyl esters such as EGTA-AM (ethylene-1,2-diol bis
2-aminoethyl ether N,N,N',N',tetra-acetic acid acetoxymethyl ester)
EDTA-AM (ethylene-1,2-diamine tetra-acetic acid acetoxymethyl
ester), and BAPTA-AM (1,2-bis 2-aminophenoxy
ethan-N,N,N',N'-tetra-acetic acid acetoxymethyl ester). These known
complex molecules, are digested by ubiquitous esterases, thus
causing activation of the chelator in the intracellular space in a
manner which is random and uncontrolled, being unrelated to cell
activity.
[0017] It will also be self-evident that a similar concept can be
applied to the treatment of conditions or diseases other than those
related to the intracellular level of Ca.sup.2+ions. By way of
example, if the active entity incorporated in the prodrug molecule
is a protein kinase inhibitor, after administration of the prodrug
the inhibitor would be accumulated in a cell exhibiting abnormal
proliferation, thus providing potentially an important tool for use
in antitumor therapy.
SUMMARY OF THE INVENTION
[0018] In accordance with one object of the invention, there are
provided prodrugs which selectively undergo activation to release
pharmacologically active compounds in hyperactivated cells. In
accordance with another object of the invention, the
pharmacologically active compound is released from the prodrug in
response to enzyme activity in the targeted cells. In accordance
with yet another object of the invention, the pharmacologically
active compound, selectively accumulated in a cell characterized by
a relatively raised level of enzyme activity therein, is trapped in
the cell and therefore exhibits an enhanced desired activity
therein
[0019] The present invention accordingly provides in one aspect, a
prodrug which is a covalent conjugate of a pharmacologically active
compound and an intracellular transporting adjuvant, characterized
by the presence of a covalent bond which is scission-sensitive to
intracellular enzyme activity.
[0020] In another aspect, the present invention provides a
technique for treating a condition or disease in a mammal,
including a human, related to supranormal intracellular enzyme
activity, which comprises administering to a mammal having such
condition or disease, a pharmaceutically acceptable cell membrane
permeable prodrug, the prodrug being a covalent conjugate of a
pharmacologically active compound and an intracellular transporting
adjuvant, characterized by the presence of a covalent bond which is
scission- sensitive to intracellular enzyme activity, such that the
bond is broken in response to such activity, a whereby the
pharmacologically active compound accumulates selectively within
cells having supranormal intracellular enzyme activity, or in their
immediate environment. In one particular aspect, the technique or
method is used to treat, e.g., a human patient.
[0021] In yet another aspect, the invention provides pharmaceutical
compounds for treating a condition or disease in a mammal related
to supranormal intracellular enzyme activity, by selectively
accumulating a pharmacologically active compound within cells
having such activity, comprising a pharmaceutically acceptable cell
membrane permeable prodrug, which is a covalent conjugate of the
pharmacologically active compound and an intracellular transporting
adjuvant, and is characterized by the presence of a covalent bond
which is scission-sensitive to intracellular enzyme activity, such
that the bond is broken in response to such activity. In one
particular aspect, the pharmaceutical compounds are used to treat,
e.g., a human patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 presents the proportion of cells with elevated
intracellular calcium levels in lymphocytes from a healthy
individual and an asthmatic patient, and the effects of Prodrug 1
on these clacium levels, in comparison to treatment with BAPTA,
before (Panel A) or after (Panel B) IgE stimulation;
[0023] FIG. 2 compares cumulative mortality, with elapsed time (in
hours), in a rat model of permanent cerebral ischemia in the
presence (square) or absence (circle) of DP16.
[0024] FIG. 3 illustrates the dose-response curve for protection
afforded by DP16 against generalized epileptic seizures induced by
pilocarpine;
[0025] FIG. 4 illustrates the dose-response curve for protection
afforded by DP16 against pilocarpine induced fatal epileptic
events;
[0026] FIG. 5 illustrates the dose-response curve for protection
afforded by DPl6 in a metrazol minimum seizures test;
[0027] FIG. 6 illustrates results of experiments in
hypoxiareperfusion cardiopathology. The upper panel (1) shows an
EKG of a heart during cardiac perfusion, the middle panel (2) shows
an EKG of a heart during low flow perfusion, with and without 1
.mu.g/L DP16 treatment and the lower panel (3) shows an EKG of a
heart after reperfusion with and without 1 .mu.g/L DP16
treatment.
[0028] FIG. 7 presents the superior protection of DP16 compared to
BAPTA-AM in hypoxia-reperfusion induced cardiopathology;
[0029] FIG. 8 presents the dose response curve of TVA compared to
valproic acid itself.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Regulated Activation of Prodrugs by Hyperactive
Intracellular Enzymes
[0031] According to the present invention, compounds are provided
which are cell permeable prodrugs, comprising a pharmacologically
active compound covalently bound to a lipophilic moiety which
facilitates intracellular transport of the prodrug. As used herein
and in the claims the term prodrug denotes a molecule which is
incapable of exerting the pharmacological activity of the active
compound. The active compound will exert its therapeutic effects
after it is released from the prodrugs of the invention by the
action of intracellular enzymes. The covalent bond of these
prodrugs are scission sensitive to enzymes that are hyperactive in
the cells that are affected, thereby providing selective activation
of the pharmacological compound in the diseased cells.
[0032] In certain preferred embodiments, the pharmacologically
active molecule may be et cell impermeable drug. In these
embodiments wherein the pharmacological compound is a cell
impermeable-drug, the compound will be selectively accumulated in
the affected cells.
[0033] In other preferred embodiments, the pharmacological agents
that are incorporated into the prodrugs of the invention, are
themselves cell permeable molecules. In these embodiments the
regulated activation of the active compound is achieved in those
cells that require treatment, thereby significantly improving the
therapeutic index of the pharmacological agent.
[0034] Different types of intracellular enzyme systems that are
significantly elevated in pathological conditions may be used
according to the present invention, to achieve the preferential
release of the active drug compound within the diseased cells.
Suitable enzymes that are to be utilized according to the present
invention to activate the prodrugs include but are not limited to
lipases, proteases or glycosidases. Members of these classes of
enzymes are known to be elevated in a variety of diseases and
disorders.
[0035] In currently preferred embodiments, the enzymes that
activate the prodrugs are intracellular lipases. In most preferred
embodiments the covalent bond of the prodrug is scission sensitive
to phospholipases, a nion limitin exmle of which are the
phospholipases A2.
[0036] Distinction among the various phospholipases is based in
part on their substrate specificity as well as their tissue
localization, regulation and physicochemical attributes. The
different specificities of these classes of phospholipases can
serve as the basis of designing prodrugs which undergo specific
activation, as suitable for the pathology to be treated.
[0037] The cleavage sites of the various phospholipases are herein
depicted schematically in the following scheme. 1
[0038] Prodrugs designed as substrates for phospholipase C (PLC)
will much more useful for treatment of chronic excitatory disorders
such as epilepsy. In this type of disorder PLC is involved in the
earliest events of hyperactivation (preceding the physiological
attack), while PLA.sub.2 activation coincides with epileptic
seizures.
[0039] Prodrug activation by PLC could be most preferred for
targeting of antiepileptic drugs. Whereas prodrug activation by
Phospholipase D (PLD) could by appropriate for targeting of
antitumor drugs. In such prodrugs the P--O bond constituting the
bond between the drug and the phospholipid would be
scission-sensitive to enzyme PLD, thus releasing the antitumor
agents intracellularly, and accumulating these inhibitors in cells
having a supranormal level of PLD.
[0040] Phospholipases A.sub.2 are a family of esterases that
hydrolyze the sn-2 ester bonds in phosphoglyceride molecules
releasing a free fatty acid and a lysophospholipid. Classification
of the members of this family of enzymes is based on certain
structural features and/or their localization in different cells
and tissues. In principle, these enzymes are more active on
aggregated phospholipid substrates compared with monomeric soluble
substrates.
[0041] Phospholipid conjugates of drugs that will be cleaved by
Phospholipases A.sub.2 have previously been disclosed either a) to
enhance penetration into cells; b) to enable formulation of drugs
in liposomes; or c) as a form of "enterocoating" that prevents
exposure of the gastric mucosa to the drug.
[0042] None of the previously disclosed uses of phospholipid-drug
conjugates is an essential feature of the present methods of using
these prodrugs, inasmuch as a) the present invention is effective
even with drugs that are already capable of penetrating cells, as
in the example of antiepileptic drugs;
[0043] b) it is not desirable according to the current invention to
formulate the prodrugs into liposomes since this achieves
preferential distribution to specific organs (e.g., the liver) or
to specific cell types(e.g., macrophages) rather than to diseased
cells within an organ or cell population;
[0044] c) the prodrugs according to the present invention are
intended for parenteral administration in order to prevent their
premature digestion by phospholipases in the digestive tract.
[0045] The prodrugs according to the present invention are
contemplated to be useful in the treatment of patients in both
human and veterinary medical practice. The prodrugs can be
administered to a patient in need thereof by any of the
conventional parenteral routes of administration, as may be
appropriate for use in conjunction with the selective activation
afforded by the prodrugs according to the invention for the disease
or condition to be treated. These routes include, but are not
limited to, intravenous (i.v.) injection, intramuscular (i.m.)
injection, subcutaneous (s.c.) injection, infusion into a body
cavity, cerebrospinal injection, localized infiltration into a
target tissue, buccal absorption, and aerosol inhalation, in an
amount effective to treat the disease or disorder. Formulations of
the compounds of the present invention into pharmaceutical
compositions suitable for the chosen route of administration may
include any physiologically acceptable solutions, suspensions,
emulsions, microemulsions, micellar dispersions, or the like, with
any pharmaceutically acceptable excipients, as are known in the
art. In addition, formulations may include various encapsulations
or depots designed to achieve sustained release of the prodrug, as
in those circumstances where a chronic disorder is to be
treated.
[0046] According to one preferred embodiment of the present
invention, protease inhibitors are provided which comprise a
peptide or peptide analog which is a potent protease inhibitor,
covalently bound to a phospholipid. These prodrugs are cell
permeable molecules which are scission sensitive to abnormally
hyperactivated phospholipases. Preferred protease inhibitors may
inclue peptides, peptide analogs, or peptidomimetics.
[0047] A non-limiting example of such protease inhibitors are
inhibitors of the neutral calcium-activated protease Calpain.
Excessive activation of calpain may play a major role in a variety
of disorders, including cerebral ischemia, muscular dystrophy and
platelet aggregation (for review see Wang and Yuen, TIPS 15,
412-419, 1994). However, there are at present no selective and cell
permeable calpain inhibitors. The improvement according to the
present invention may be achieved with any of the known peptide or
peptide analogs that are known calpain inhibitors, such as those
reviewed by Wang and Yuen (ibid).
[0048] Within the scope of the present invention, additional
embodiments are provided wherein the covalent bond of the prodrug,
comprising said protease inhibitor, is scission sensitive to
hyperactive intracellular proteases. Such further embodiments have
a scission sensitive peptide bond between the protease inhibitor
and a lipophilic carrier, thereby releasing the inhibitor in those
cells that possess hyperactive protease activity. The use of
lipophilic carriers to facilitate transport of peptide analogs
across lipophilic barriers such as the blood brain barrier has been
disclosed for instance in International patent application
PCT/US93/09057. However, it is neither taught nor suggested in such
disclosures that lipid conjugates may be utilized to achieve
intracellular activation of a peptide drug.
[0049] In yet another embodiment, activation of the prodrugs is
regulated by enzymes which are intracellular glycosidases, a
non-limiting example-of which is heparanase. Interaction of
circulating cells of the immune system, as well as platelets, with
the subendothelial extracellular matrix is associated with
degradation of heparan sulfate by the specific endoglycosidase,
heparanase. This enzyme is released from intracellular compartments
in response to activation signals, implicating its involvement in
inflammation and immunity. In contrast, various tumor cells express
heparanase in a constitutive manner, in correlation with their
metastatic potential. This enzyme is a suitable candidate for
achieving regulated activation of antitumor drugs, or of drugs that
modulate the immune response.
[0050] Prodrugs Activated by Phospholipases
[0051] The pharmacologically active compound may be by way of
example a pharmacologically active carboxylic acid, when the
adjuvant may comprise for example at least one pharmaceutically
acceptable alcohol which is selected from glycerol, C.sub.3-20
fatty acid monoglycerides, C.sub.3-20 fatty acid diglycerides,
hydroxy-C.sub.2-6-alkyl esters of C.sub.3-20 fatty acids,
hydroxy-C.sub.2-6-alkyl esters of lysophosphatidic acids,
lyso-plasmalogens, lysophospholipids, lysophosphatidic acid amides,
glycerophosphoric acids, lysophophatidal-ethanolamine,
lyso-phosphatidylethanolamine and N-mono- and
N,N-di-(C.sub.1-4)-alkyl and quaternary derivatives of the amines
thereof.
[0052] Exemplary of pharmacologically active carboxylic acids are
branched-chain aliphatic carboxylic acids (e.g. valproic acid),
salicylic acids (e.g. acetylsalicylic acid), steroidal carboxylic
acids (e.g. lysergic and isolysergic acids), monoheterocyclic
carboxylic acids (e.g. nicotinic acid) and polyheterocyclic
carboxylic acids (e.g. penicillins and cephalosporins). While
pharmacologically active carboxylic acids are particularly
described herein, as exemplary of the active compounds which may be
conjugated with an intracellular transporting adjuvant, the
invention is not limited thereto. Thus, by way of further example,
it is entirely within the concept of the present invention to
conjugate therapeutically active nucleic acid (including RNA and
DNA) or fragments thereof with an intracellular transporting
adjuvant.
[0053] In a preferred embodiment, the prodrug according to the
invention comprises a conjugate of a calcium chelating agent and a
lipid, and may thus be of potential use for treating diseases or
conditions which are related to an unduly high level of
intracellular Ca.sup.2+ions.
[0054] In a most preferred embodiment, the prodrug contains at
least one covalent. bond between the pharmacologically active
compound and the intracellular transporting adjuvant, which
covalent bond is scission-sensitive to intracellular enzyme
activity, with the consequence that the greater part of the prodrug
molecules will move freely in and out of normal cells without
scission of such bond, whereas in the cells possessing the
supranormal enzyme activity only, the scission- sensitive bond in a
high proportion of prodrug molecules entering the cells will break.
In those embodiments where the pharmacologically active compound is
cell membrane impermeable the drug released from the prodrug will
accumulate intracellularly, within the abnormal cells possessing
supranormal enzyme activity.
[0055] Persons skilled in the art will appreciate in what manner
the concept of the invention may be applied to conditions and
diseases which are not necessarily related to an intracellular
excess of calcium ions, so that in such other cases, the prodrug
will incorporate an active compound which is not a calcium chelator
but which will possess other desired pharmacological activity.
[0056] The prodrug which comprises a calcium chelating agent is,
e.g., a partially or totally esterified carboxylic acid, which is
an ester of:
[0057] (a) a pharmaceutically acceptable chelating agent for
calcium having the formula
(HOOC--CH.sub.2--).sub.2--N--A--N--(--CH.sub.2COOH).su- b.2 where A
is saturated or unsaturated, aliphatic, aromatic or heterocyclic
linking radical containing, in a direct chain link between the two
depicted nitrogen atoms, 2-8 carbon atoms in a continuous chain
which may be interrupted by 2-4 oxygen atoms, provided that the
chain members directly connected to the two depicted nitrogen atoms
are not oxygen atoms, with
[0058] (b) a C.sub.3-32 pharmaceutically acceptable alcohol
containing 1-3 OH radicals (e.g. such a C.sub.3-6 alcohol, or e.g.
a C.sub.7-32 secondary monohydric alcohol);
[0059] and salts with alkali metals of the partially esterified
carboxylic acids, as well as acid addition salts of such of the
esterified carboxylic acids as contain one or more potentially
salt-forming nitrogen atoms.
[0060] The choice of the preferred alcohol that is appropriate for
any given prodrug is dependent on the intended therapeutic use of
the conjugate. Thus alcohols below C.sub.10 exhibit very low
substrate specificity, whereas alcohols above C.sub.12 or C.sub.14
are very good substrates for the phospholipases and will therefore
be readily activated. Regulated activation will best be achieved by
the intermediate length alcohols such as C.sub.2-C.sub.10, and
these will be preferred for the treatment of persistent or chronic
disease states or disorders.
[0061] In contradistinction, in certain disease states that require
the rapid release of the active agent the most preferred alcohols
will be the longer chain alcohols.
[0062] This is most suitable for conditions involving acute onset
pathology such as in the treatment of epilepsy with the prodrugs of
the invention. Further, in the case where there are relatively
minimal differences in intracellular enzymatic activity between
normal and diseased or disordered cells, relatively shorter chain
alcohols may be selected.
[0063] The ester of choice may be one in which the linking radical
A is a member selected from the group consisting of
--(CH.sub.2CH.sub.2).sub.m-- where m=1-4, in which 2-4 of the
carbon atoms not attached to nitrogen may be replaced by oxygen
atoms, and --CR.dbd.CR--O--CH.sub.2CH.sub.2--O-- -CR'.dbd.CR'--,
where each of the pairs of radicals R--R and R'--R', together with
the attached --C.dbd.C-- moiety, complete an aromatic or
heterocyclic ring containing 5 or 6 ring atoms, the ring completed
by R--R being the same as or different from the ring completed by
R'--R'.
[0064] In particular embodiments, the linking radical A may be,
e.g., selected from --CH.sub.2CH.sub.2-- and
--CH.sub.2CH.sub.2--O--CH.sub.2CH.- sub.2--O-- CH.sub.2CH.sub.2--;
or it may be e.g. --CR.dbd.CR--O--CH.sub.2C-
H.sub.2--O--CR'.dbd.CR'--, where each of the pairs of radicals R--R
and R'--R', together with the attached --C.dbd.C-- moiety, complete
an aromatic or heterocyclic ring which is selected from the group
consisting of furan, thiophene, pyrrole, pyrazole, imidazole,
1,2,3-triazole, oxazole, isoxazole, 1,2,3-oxadiazole,
1,2,5-oxadiazole, thiazole, isothiazole, 1,2,3-thiadiazole,
1,2,5-thiadiazole, benzene, pyridine, pyridazine, pyrimidine,
pyrazine, 1,2,3-triazine, 1,2,4-triazine, and 1,2-, 1,3- and
1,4-oxazines and thiazines, the ring completed by R--R being the
same as or different from the ring completed by R'--R'. In a
particularly preferred embodiment, the linking radical A is
--CR.dbd.CR-- O--CH.sub.2CH.sub.2--O--CR'.dbd.CR'--, where each of
the pairs of radicals R--R and R'--R', together with the attached
--C.dbd.C-- moiety, completes the same or different rings selected
from unsubstituted and substituted benzene rings, in which
substituted benzene rings contain 1-4 substituents selected from
the group consisting of C.sub.1-3alkyl, C.sub.1-3-alkoxy, F, Cl,
Br, I and CF.sub.3, or a single divalent substituent which is
--O--(CH.sub.2).sub.nO-- and n=1-3.
[0065] It is presently preferred that the calcium chelating agent
incorporated in the prodrug is selected from
ethylene-1,2-diamine-N,N,N',- N'-tetra-acetic acid,
ethylene-1,2-diol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic
acid and 1,2-bis-(2-aminophenoxy)ethane-N,N,- N',N'-tetraacetic
acid.
[0066] As mentioned above, C.sub.3-32, e.g. C.sub.3-6, alcohol
referred to above contains 1-3 OH radicals. When 2 OH radicals are
present, one of them may be esterified or otherwise derivatized,
and when 3 OH radicals are present, either 1 or 2 of the OH
radicals may be esterified or otherwise derivatized. Any carbon
atoms in the esterifying or otherwise derivatizing group(s) are not
counted for the purpose of the e.g. 3 to 6 carbon atoms which may
be contained in the pharmaceutically acceptable alcohols.
[0067] Thus, these alcohols may comprise, e.g., at least one member
of the group consisting of glycerol, C.sub.3-20 fatty acid
monoglycerides, C.sub.3-20 fatty acid diglycerides,
hydroxy-C.sub.2-6-alkyl esters of C.sub.3-20 fatty acids,
hydroxy-C.sub.2-6-alkyl esters of lysophosphatidic acids,
lysoplasmalogens, lysophospholipids, lysophosphatidic acid amides,
glycerophosphoric acids, lysophophatidalethanolamine,
lysophosphatidylethanolamine and the N-mono-C.sub.1-4-alkyl,
N,N-di-C.sub.1-4-alkyl and quaternary ammonium derivatives of such
of the foregoing as are amines. An example of a C.sub.7-32
secondary alcohol is 1-myristylmyristyl alcohol.
[0068] The person skilled in the art will appreciate that the
prodrug of the present invention can be tailored in such a manner
that the desired pharmacologically active entity is released by
action of the specific enzyme known to be the source of enzyme
hyperactivity in the condition or disease being treated. For
example, A membrane-associated calcium-independent
plasmalogen-selective PLA.sub.2 activity has been found to increase
over 400% during two minutes of global ischemia (P<0.01), was
greater than 10-fold (near to the maximum) after only five minutes
of ischemia, and remained activated throughout the entire ischemic
interval examined (up to 60 minutes), see Ford et al, J. Clin.
Invest., 1991, 88(1): 331-5. These facts suggest attaching the
pharmacological active entity to the 2-position in a
glycerophosphoric acid derivative, and that use of a
lysoplasmalogen may possibly be more effective as the intracellular
transporting adjuvant, to which the active entity is attached
covalently, than a lysophospholipid.
[0069] Many events (e.g. cytotoxic chemicals, physical stimuli and
infective agents) causing damage of the cell membrane can trigger a
cascade leading ultimately to a condition which mimics ischemic
damage(Robbins et al, Pathological Basis for Disease, 1984, p. 10,
W. B. Sanders Co.). The present invention will potentially be of
use for protecting cells in these circumstances, by introduction of
a calcium chelator intracellularly.
[0070] In this connection, it is noted that the antitumor drug
Adriamycin, which has been reported to inhibit Na--Ca exchange and
to overload the sarcoplasm with calcium, could induce contractile
heart failure; this would be consistent with the hypothesis that
calcium overload, in absence of ischemia, can leave behind
long-lasting contractile dysfunction (Kusuoka et al, J. Cardiovasc.
Pharmacol., 1991, 18(3): 437-44).
[0071] As indicated above, the concept of the present invention is
not restricted to the treatment of conditions or diseases related
to the intracellular level of Ca.sup.2+ions, so that the materials
used in practicing the invention are not restricted to calcium
chelators. Thus for example, the pharmacologically active compound
may be e.g. an antiepileptic compound such as valproic acid.
[0072] In this connection, it is contemplated that application of
the present invention in this embodiment would enable a much lower
effective dose of valproic acid to be used than is otherwise the
case, thus potentially substantially reducing the occurrence of
undesired side-effects. In principle, any of the range of alcohols,
and examples thereof, mentioned above in connection with
esterification of calcium chelators may also be applied to the
esterification of valproic acid in accordance with the concept of
the present invention.
[0073] In a non-limiting embodiment, valproic acid may be
esterified with, e.g.,
1-heptanoyl-sn-glycero-3-phosphorylcholine.
[0074] In another particular embodiment, the pharmacologically
active compound incorporated in the prodrug of the invention is a
protein kinase inhibitor. Where the protein kinase inhibitor is a
carboxylic acid, the prodrug may be e.g. an ester thereof with a
pharmaceutically acceptable alcohol such as glycerol, C.sub.3-20
fatty acid monoglycerides, C.sub.3-20 fatty acid diglycerides,
hydroxy-C.sub.2-6-alkyl esters of C.sub.3-20 fatty acids,
hydroxy-C.sub.2-6-alkyl esters of lysophosphatidic acids,
lysoplasmalogens, lysophospholipids, lysophosphatidic acid amides,
glycerophosphoric acids, lysophophatidalethanolamine,
lysophosphatidylethanolamine and N-mono- and
N,N-di-(C.sub.1-4)-alkyl and quaternary derivatives of the amines
thereof. Such a carboxylic acid is e.g. protein kinase inhibitor
K252b from Nocardiopsis sp.
[0075] Where the protein kinase inhibitor contains an amine group
with a replaceable N-linked hydrogen atom, the prodrug may be e.g.
an amide thereof with a phosphoric acid derivative selected from
glycerophosphoric acids, O-acylated or etherified glycerophosphoric
acids, and monoacylated monoetherified glycerophosphoric acids.
Such protein inhibitors are e.g. isoquinoline-5-sulfonamide
N-substituted by an acyclic or heterocyclic aminoalkyl radical such
as NHCH.sub.2CH.sub.2NHCH.sub.3 and 2-methylpiperazin-1-yl. Where
the protein kinase inhibitor contains at least one phenolic hydroxy
group, the prodrug may be e.g. an ester thereof with a phosphoric
acid derivative selected from glycerophosphoric acids, O-acylated
glycerophosphoric acids, etherified glycerophosphoric acids, and
monoacylated monoetherified glycerophosphoric acids. Such a protein
kinase inhibitor is e.g. 4',5,7-trihydroxyisoflavone.
[0076] In another particular embodiment, the pharmacologically
active compound incorporated in the prodrug of the invention is an
antitumor agent. The ordinary artisan will understand that the
principle of the invention can be applied to any suitable antitumor
agent by linking such an agent to an intracellular transporting
adjuvant as described above, to which the pharmacologically active
compound is attached covalently. The linkage is selected so that
supranormal intracellular enzyme activity characteristic of target
cells (e.g., tumor cells) will cleave the intracellular
transporting adjuvant from the pharmaceutically active compound. In
a particular aspect,the antitumor agent is, for example, a folic
acid agonist such as a 4-amino analog of folic acid. A
representative member of this class of compounds is methotrexate.
Methotrexate and related compounds are known to the art as
effective antitumor agents that have also been used in the
treatment of psoriasis and in the modulation of cell mediated
immunity. Impaired transport of methotrexate into target cells is
believed to be one mechanism for the development of tumor
resistance to that drug (Goodman and Gilman's, THE PHARMACOLOGICAL
BASIS OF THERAPEUTICS, 8Th Ed., 1990, Pergamon Press, hereby
incorporated by reference in its entirety). Thus, methotrexate
linked to a cell membrane permeable adjuvant cleavable by
supranormal intracellular enzyme associated with a diseased or
disordered target cell will enhance the specificity and
effectiveness of such treatment of tumor cells by antitumor drugs,
such as, e.g, methotrexate or other folic acid antagonists. Prodrug
derivatives of methotrexate are also contemplated to be used to
treat any of the other aformentioned conditions treatable by
methotrexate
[0077] When selecting the intracellular transporting adjuvant for
the purposes of the present invention, the skilled person will of
course take into consideration the necessity for avoiding such
adjuvants, e.g. certain 1,2-diacylglycerols, which are activators
of protein kinase C (see Lapetina et al, J. Biol. Chem., 1985, 260:
1358 and Boynton et al, Biochem. Biophys. Res. Comm., 1983, 115:
383), or intracellular transporting adjuvant which are likely to
give rise to undesirable products such as these in the cell. In
addition, the artisan will appreciate that the selected linker to
the intracellular transporting adjuvant should be selected to avoid
interaction with desired pharmacological activity and to avoid
rapid, nonspecific intracellular degradation after specific
cleavage.
[0078] The following examples are to be construed in a
non-limitating fashion and represent certain preferred embodiments
of the invention. One skilled in the art can readily devise many
variations and modifications of the principles disclosed herein
without departing from the scope of the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0079] The following examples illustrate various aspects of the
present invention and are not to be construed to limit the claims
in any manner whatsoever
EXAMPLES
Example 1
[0080] Preparation of Esters of
Heptanoyl-sn-3-glycero-phosporylcholine (Prodrug-1 and
Prodrug-2).
[0081] Introduction
[0082] "Prodrug-1" is the name used herein to denote a 1:1 ester of
1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)
with the choline derivative
ROCH.sub.2-CH(OH)-CH.sub.2O-(PO.sub.2)-OCH.sub.2N.-
sup.+(CH.sub.3).sub.2, wherein R is heptanoyl. BAPTA is a calcium
chelator, to which the human cell membrane is normally impermeable,
whereas the cell membrane is permeable to prodrug-1, which is not a
calcium chelator per se. The carboxylic ester links in prodrug-1
are digestible by PLA.sub.2, so that activated cells such as IgE
lymphocytes should exhibit a selective intracellular accumulation
of BAPTA, compared to the unactivated cells, with the result that
the [Ca.sup.2+], level in the activated cells should be reduced
when compared with unactivated cells. "Prodrug-2" is the 1:2 ester
of BAPTA with the depicted choline derivative.
[0083] Procedure
[0084] (a) Diheptanoyl-L-.alpha.-lecithin
[0085] In a dry 3-neck 500 ml flask equipped with oil-sealed
stirrer, CaCl.sub.2 tube and dropping funnel, were placed 100 ml of
5 mm diameter glass beads and 11.0 g (0.01 mole) of CdCl.sub.2
adduct of synthetic L-.alpha.-glycero-phosphorylcholine. The flask
was immersed in an ice-water bath, and to the rapidly-stirred
mixture there was added a thin stream of 29.7 g (0.2 mole) freshly
prepared heptanoyl chloride dissolved in 60 ml chloroform, followed
by 11 ml (0.14 mole) anhydrous pyridine dissolved in 100 ml
chloroform(anhydrous, alcohol-free). After 30 minutes, the bath
temperature was raised to 25.degree. C. and stirring continued for
2 hours. The reaction mixture was poured through a filter-less
Buchner, the glass beads washed with 3.times.50 ml chloroform and
the combined filtrates clarified by centrifugation. The supernatant
was concentrated under reduced pressure, the residue kept for
several hours at 0.1 mmHg vacuum and bath temperature 30-35.degree.
C. to remove most excess pyridine, and was then stirred with 500 ml
anhydrous acetone for 10 minutes, and centrifuged. The precipitate
was treated similarly with 2.times.100 ml anhydrous acetone and
2.times.100 ml anhydrous ether.
[0086] The residual solid material was dried under reduced pressure
and freed of the last traces of cadmium chloride and pyridine
hydrochloride, by dissolving in 200 ml of a 5:4:1 by volume mixture
of chloroform/methanol/water, and passing the solution through a
120 cm long.times.2.5 cm diameter column containing an equivolume
mixture of Amberlites IR-45 and IRC-50. The column was washed with
500 ml of the same chloroform/methanol/water mixture, the combined
effluents were concentrated to dryness under reduced pressure from
a bath at 40-45.degree. C., and the residue dried at 0.1 mm vacuum
and 45.degree. C. The crude product was purified by precipitation
from a solution in 50 ml chloroform, with 150 ml acetone,
centrifugation and recrystallization of the precipitate, 2.3 g
(47.6t) from chloroform and ether. (Di-octanoyl-L-.alpha.-lecithin
can be prepared similarly.)
[0087] (b) 1-Heptanoyl-sn-3-glycerophosphorylcholine.
[0088] A solution of the product of part (a) (1.2 mmol) in a
mixture of ether (196 ml) and methanol (12 ml) was stirred
vigorously in presence of (HOCH.sub.2).sub.3C--NH.sub.2.HCl (50 ml
of 0.1M, pH 8.,7) containing CaCl.sub.2 (0.72 mM) and 5 mg of crude
rattle snake venom (Crotalus adamanteus) as a source of
phospholipase A.sub.2, at 37.degree. C. for 3 hours. The reaction
was monitored by TLC (70:25:4 by volume chloroform/methanol/water).
After completion of reaction, the organic layer was separated, and
the aqueous layer was washed with ether and then lyophilized. The
residue was extracted with 2:1 by volume chloroform/methanol and
centrifuged. On evaporation of the clear supernatant, the title
product was obtained in 90% yield. Thin layer chromatography using
70:25:4 by volume chloroform/methanol/water showed that it was free
from starting material and heptanoic acid. Any fatty acid in the
product can however be remove by crystallization from
ethanol-ether.
[0089] Note: this is a general method for scission of the
glycerol-2-ester bond. (Octanoyl-sn-3-glycerophosphoryl-choline can
be prepared similarly.)
[0090] (c) Prodrug-1 and Prodrug-2
[0091] A solution of the product of part (b) (0.5 g, 1.04 mmol) in
chloroform (15 ml, freshly distilled over P.sub.2O.sub.5) was added
to a solution of BAPTA (0.495 g, 1.03 mmol for the monoester
Prodrug-1, or 0.248 g, 0.51 mmol for the diester Prodrug-2),
N,N'-dicyclohexyl-carbodii- mide (0.214 g, 1.03 mmol) and
4-dimethylaminopyridine (0.025 g, 0.202 mmol) and HCONMe.sub.2 (20
ml, freshly distilled over CaH.sub.2) under a nitrogen atmosphere,
and the mixture was stirred at room temperature for two days. The
reaction was monitored by TLC (65:35:5 by volume
chloroform/methanol/water).
[0092] The precipitate was removed by filtration, the filtrate was
concentrated by evaporation in vacuo at 35.degree. C. and the
residue was dissolved in 2:1:2 by volume chloroform/isopropanol/
water). The organic layer was separated, dried (Na.sub.2SO.sub.4)
and then passed through a 20 cm long x 1.8 cm diameter column of
silicic acid (Bio-Sil-ILk). The column was thoroughly washed with
chloroform until free from BAPTA (TLC) and then eluted with a
gradient of chloroform/methanol (1:1 by volume) to pure methanol,
the elution being monitored by TLC.
[0093] The eluted fractions were combined and concentrated by
evaporation. The desired title product (i.e. Prodrug-1 or
Prodrug-2, depending on the number of molar equivalents of BAPTA
used) was crystallized from ether and dried in vacuo over
P.sub.2O.sub.5 at 30.degree. C.: yield 0.3 g (30%).
[0094] It will be apparent that the corresponding triester or
tetraester may be obtained by varying appropriately number of molar
equivalents of BAPTA. (The analogous octanoyl esters are prepared
similarly.)
Example 2
[0095] Application of Prodrug-1 for Reduction of the Intracellular
Calcium Level in Hyperactivated Cells.
[0096] Method
[0097] Intracellular free [Ca.sup.2+].sub.i content was monitored
by flow cytometry using the Ca.sup.2+-sensitive dye fluo-3/AM
(Molecular Probe Inc., OR)(Minta, Kao and Tsien, 1989, J. Biol.
Chem. 264:8171-8178). Cells obtained from donor blood and those
from the blood of an asthmatic patient were further washed twice in
DMEM and resuspended to a concentration of 10.sup.7 cells/ml.
Fluo-3/AM (1 mM) was prepared in DMSO augmented with the nonionic
surfactant Pluronic F-127 (Wyandotte Corp., MI). Aliquots of
fluo-3/AM stock solution were added to cell suspensions in
DMEM/HEPES at a final concentration of 3 .mu.M (loading buffer).
Loading was allowed to proceed for 30 min. at 37.degree. C. and
continued for 1 hour at 23.degree. C. with gentle agitation. Cells
were then adjusted to desired concentrations using fresh
DMEM/HEPES, supplemented with 2% horse serum. Autofluorescence was
eliminated by setting the threshold sensitivity above the levels
obtained in absence of dye. Fluorescence intensity data was
collected from 5000 single cells and values were expressed as
arbitrary fluorescence units. Prodrug-1 (1 mM) was prepared in DMSO
and added when appropriate at a final concentration of 3 .mu.M to
the cells for 5 min. prior to calcium treatment.
[0098] Results
[0099] Lymphocytes from donor blood and from the blood of an
asthmatic patient were exposed to prodrug-1. Accumulation of the
liberated BAPTA chelator within the cell was estimated by
measurement of [Ca.sup.2+].sub.i, by flow cytometry using fluo-3/AM
as described above. The results are presented in FIG. 1, in which
the [Ca.sup.2+] levels are shown as follows:
[0100] Panel A presents a comparison between the lymphocytes
isolated from a healthy domor and those of the asthmatic patient,
in terms of the proportion of cells having high intracellular free
calcium.
[0101] Panel B presents a comparison of the same cell populations
after stimulation with IgE. As shown in panel B, the prodrug also
provides protection against high intracellular calcium in IgE
stimulated cells.
[0102] It was found that lymphocytes from an asthmatic patient have
a dual partition according to the [Ca.sup.2+], level. About 50% of
the cells exhibit a high [Ca.sup.2+] level indicating cell
hyperactivation (panel A), while the second part of the population
is similar to the normal one. In the case where the cells have been
treated with prodrLg-1, the population of hyperactivated cells is
back to normal, while the population of non-activated cells remains
unchanged. These data demonstrate that prodrug-1 provides selective
accumulation of the chelator within activated, but not in
non-activated cells BAPTA itself, which is a cell impermeable
molecule is ineffective in reducing the intracellular calcium
levels, in either stimulated or untreated cells
Example 3
[0103] Prodrugs of Potential Application in the Treating
Tumors.
[0104] Introduction
[0105] In this Example, there are presented a number of
illustrative embodiments of the present invention in which a
prodrug comprises a protein kinase inhibitor. After administration
of the prodrug, the inhibitor would be accumulated in a cell
exhibiting abnormal proliferation, thus providing potentially an
important tool for use in antitumor therapy.
[0106] (i) The compound QSO.sub.2{circumflex over ( )}where
Q=5-isoquinolyl and N{circumflex over (
)}=NHCH.sub.2CH.sub.2NHCH.sub.3, is a selective inhibitor of
cAMP-dependent protein kinase: Hidaka et al, Biochemistry, 1984,
23: 5036, and Tash et al, J. Cell Biol., 1986, 103: 649. Similarly,
the compound QSO.sub.2 where Q=5isoquinolyl and
=2-methylpiperazin-1-yl, is a potent inhibitor of cyclic nucleotide
dependent protein kinase and protein kinase C: Hidaka et al, 10c
cit, and Kikuchi et al, Nucl. Acid Res., 1988, 16: 10171. These
compounds can be covalently conjugated to an intracellular
transporting adjuvant by methods known to persons of the art, e.g.
illustratively: 2 3
[0107] In scheme (b), R is an aliphatic hydrocarbon group such as
is found in plasmalogens (or it may be inserted in a conventional
synthetic procedure) and A is an aliphatic acyl radical, e.g.
lauroyl, myristoyl, palmitoyl, stearyl and oleyl.
[0108] The compound QSO.sub.2N{circumflex over ( )} where
Q=5-isoquinolyl and N{circumflex over ( )}=2-methylpiperazin-1-yl,
may be attached in a similar manner by means of the piperazine
N.sup.4 atom.
[0109] It would be expected that the P--N bond in prodrugs (A) and
(B) depicted above would be scission-sensitive to enzyme PLD, thus
releasing the described protein kinase inhibitors intracellularly,
and accumulating these inhibitors in cells having a supranormal
level of PLD.
[0110] (ii) 4',5,7-trihydroxyflavone is an inhibitor of tyrosine
specific protein kinase: Akiyama et al, J. Biol. Chem., 1987, 262:
5592. This compound can be conjugated to an intracellular
transporting adjuvant by methods (a) and (b) described in part (i),
above. The illustrative conjugates would have structures (C) &
(D): 4
[0111] where R' and A have the meanings given above and Q' is the
residue of 4',5,7-trihydroxyisoflavone from which one phenolic
hydrogen atom has been removed and which is thus attached to the
rest of the molecule by an O atom forming a P--Obond. It would be
expected that this P--O bond in p:rodrugs (C) and (D) depicted
above would be scission-sensitive to enzyme PLD, thus releasing the
described protein kinase inhibitors intracellularly, and
accumulating these inhibitors in cells having a supranormal level
of PLD.
[0112] (iii) Protein kinase inhibitor K252b from Nocardiopsis sp.
is a carboxylic acid believed to have the following formula: 5
[0113] This compound can be conjugated to an intracellular
transporting adjuvant, e.g., by the method described in Example 1,
above. Exemplary conjugates are esters of the carboxylic function
in the above formula, with e.g.
heptanoyl-sn-3-glycerophosphoryl-choline or
octanoyl-sn-3-glycerophosphoryl-choline.
Example 4
[0114] Preparation and Biological Properties of DP16.
[0115] 4.1) Preparation of DP16
[0116] "DP16" denotes herein to denote a 1:1 ester of BAPTA with
the phosphorylcholine derivative
ROCH.sub.2--CH(OH)--CH.sub.2O--(PO.sub.2)--O-
CH.sub.2N.sup.+(CH.sub.3).sub.2, where R is hexadecanoyl. DP16 was
prepared according to the method described in Example 1.
[0117] 4.2) DP16 Testing in Models of Brain Ischemia
[0118] a) Permanent Ischemia Model in Rats:
[0119] Bilateral ligation of the common carotid arteries is the
simplest and most direct approach for inducing permanent partial
ischemia. In the rats there is almost 64% mortality 24 h later. The
causes of mortality are largely brain swelling (edema) and focal
lesions (infarcts). Permanent partial global is achieved by
isolation of the common carotid artery through an incision on the
ventral surface of the neck. The salivary glands are moved
laterally and the carotid sheath exposed. Both the vagus and
sympathetic nerves are separated from the common carotid artery,
which is then permanently ligated. Sprague-Dawley rats (250-300 g)
were anesthetized with halothane or by intramuscular injection of
0.1 ml Ketamine (0.1 g/ml, Parke Davis UK) and 0.1 ml Rompun (2%,
Bayer, FRG) per 300 g body weight. DP16 was administered
intraperitoneally (i.p., 0.001-0.1 mg/kg) when appropriate
following the artery ligation. Every experimental and control group
included 14 male rats. Statistical analysis was performed according
to t-test criteria.
[0120] b) Embolic Stroke:
[0121] Sprague-Dawley rats (300 g) are anesthetized with halothane.
The right common carotid artery is exposed and the external carotid
and pterygopalatine arteries are ligated with No. 0 silk thread.
The common carotid artery is cannulated with a plastic tube
previously filled with heparinized saline. The cannula is then
injected (0.5 ml gas-tight Hamilton syringe) with a suspension of
polystyrene spheres, followed by a flush of 0.5 ml saline. The
common carotid artery is then permanently ligated. The polystyrene
15 .mu.m spheres are prepared in 0.05% Tween-80 in normal saline
followed by 5 min. of full power sonication. A 100 .mu.l aliquot is
taken and immediately transferred to the syringe.
[0122] c) Ischemic Fetal Brain Model:
[0123] Sprague-Dawley pregnant rats were used at 20 days gestation.
Animals were anesthetized by intramuscular injection of 0.1 ml
Ketamine (0.1 g/ml, Parke Davis, UK) and 0.1 ml Rompun (2%, Bayer,
FRG) per 300 g body weight. An abdominal incision was performed and
the two uterine horns were exposed and kept moist throughout the
surgery. Intracerebral injection of 1-2mCi/2 ml
[.sup.3H]arachidonic acid (Na+, 240 mCi/mmol from New England
Nuclear, Boston, Mass.) and/or 1.5 mCi/2 ml [.sup.14C]palmitic acid
(Na+, 819 mCi/mmol from Amersham, Searle, UK) in isotonic salt
solution containing NaHCO.sub.3 (1.32 g%), into the embryos was
performed through the uterine wall into the fontanellae. Custom
made syringes (33 gauge, 0.375" length from Hamilton, Reno, Nev.)
were used to reduce brain edema. After injection fetuses were
returned to the abdominal cavity for maintenance at physiological
temperature. After 1h they were subjected to blood flow restriction
for 20 min. (restriction session) by clamping the blood vessels in
the placenta manifold. Whenever desired, circulation was restored
for 30 min. by removal of the clamps (reperfusion session). At all
times both restricted and sham-operated fetuses were maintained in
the abdominal cavity before surgical delivery. After delivery
through a transverse cut in the uterus, viable fetuses with no
apparent edema were killed without delay and excised fetal brains
were immediately homogenized in suitable organic solvents for
further treatment.
[0124] d) Fetal Cerebral Hemispheres Model:
[0125] Rat fetuses were removed from the uterine horns in a viable
state and their cerebral hemispheres were dissected within 15 sec
after decapitation. The cerebral hemispheres freed of blood and
meninges were separated and each (50.+-.2.5 mg) was placed in a
well of a 24-well Falcon culture dish. Tissue was quickly washed
twice in cold Dulbecco's Modified Eagle Medium (DMEM, Grand Island
Biol. Co) and then incubated at 37.degree. C. in 0.6-1.2 ml DMEM
flushed with oxygen and supplemented with various additives.
Aliquots of incubation medium (0.1 ml) were taken for eicosanoid
determination by a radioimmunoasay (RIA) technique. After
acidification with 5 ml formic acid, 0.1 ml of isopropanol and 0.5
ml diethylether were added. After mixing and low speed
centrifugation (2500.times.g, 5 min.) the organic layer was
collected and dried under a stream of nitrogen. The resulting
residue was dissolved in 0.1 ml sodium phosphate buffer pH 7,4,
containing 0.1% bovine serum albumin. Samples were incubated
overnight at 4.degree. C. with the appropriate polyclonal
antiserum, and .sup.3H-labeled tracer (4000 cpm/tube) in a 24 final
volume of 0.3 ml. Unbound material was precipitated with 0.3 ml
dextran-coated charcoal (Pharmacia, Sweden). After centrifugation
at 4.degree. C. aliquots of the supernatant (0.4 ml) were
transferred to vials and after addition of scintillation liquid
samples were counted in a Packard Tricarb scintillation counter.
[.sup.3H]Arachidonic acid (240 Ci/mmol) (New England Nuclear,
Boston, Mass.) dissolved in isotonic NaHCO.sub.3 (1.32% w/v) was
injected through the uterine wall and the fontanellae into the
embryonic brain. After injection fetuses were returned to the
abdominal cavity for maintenance under physiological conditions.
After 1 h, fetuses were delivered and immediately sacrificed.
Cerebral hemispheres were rapidly excised for subsequent ex vivo
incubation or for lipid extraction.
[0126] e) Results
[0127] Bilateral Permanent Cerebral Ischemia causes progressive
loss of experimental animals up-to 6-7 days after surgery. As
illustrated in FIG. 2, DP16 decreases post-ischemic mortality by
250%, compared with control using non-protected rats (p<0.01).
These data demonstrate the potential ability of DP16 to treat
otherwise fatal ischemic conditions.
[0128] f) Heart Ischemia-Langendorff Perfused Heart Model:
[0129] White rats were sacrificed by cervical dislocation and their
hearts were rapidly removed and reperfused at 60 mmHg with modified
Krebs-Henselleit buffer utilizing a Langendorff perfused heart
model. Hearts were perfused for 10-min. preequlibration interval
and were subsequently rendered either global ischemic (zero flow)
or continuously perfused for the indicated time. Perfusion were
terminated by rapid excision of ventricular tissue and directly
submersion into cold homogenization buffer (10 mM imidazole, 10 mM
KCl, 0.25 M sucrose [grade 1], pH 7.8) Both the activation of
phospholipase A2 and its reversibility during reperfusion were
temporally correlated to alterations in myocytic anaerobic
metabolism and electron microscopic analyses.
[0130] g) Ventricular Fibrillation Model by Coronary Occlusion:
[0131] Dogs (11.6-20.7 kg) were anesthetized and connected to
instrumentation to measure left circumflex coronary blood flow,
left ventricular pressure, and ventricular electrogram. The left
anterior descending artery was ligated and an anterior wall
myocardial infarction was then produced. All leads to the
cardiovascular instrumentation were tunneled under the skin to exit
on the back of the animal's neck. Appropriate medicine was given to
minimize postoperative pain and prevent inflammation. The ischemia
test was performed after 3-4 weeks.
[0132] 4,3) DP 16 Testing in Treatment of Epileptic Disorders:
[0133] a) Pilocarpine Based Model of Experimental Epilepsy:
[0134] Acetylcholine, acetylcholinesterase inhibitors and
acetylcholine analogues are effective epileptogenic agents when
applied intracerebrally or systematically (see ref. in Leite et
al., Neurosci. & Biobeh. Rev., 1990, 14:511-17). It was
demonstrated in different species that systemic administration of
muscarinic cholinergic agonists produced electroencephalographic
and behavioral limbic seizure accompanied by widespread brain
damage resembling topographically that produced by kainic acid and
folates and are frequently observed in autopsied human epileptics.
Systemic injections of the pilocarpine, a potent muscarinic
cholinergic agonist, are capable of producing a sequence of
behavioral alterations including stirring spells, facial
automatisms and motor limbic seizures, that develop over 1-2 hours
and build progressively into limbic status and following by general
status epilepticus.
[0135] b) Results
[0136] Immediately following injection of pilocarpine, akinesia,
ataxic lurching, facial automatism and heart tremor dominated the
animals' behavior. Further development of epileptic events is
dose--dependent. Administration of pilocarpine in doses of 300-350
mg/kg causes appearance of limbic seizures with rearing, forelimb
clonus, salivation, intense masticatory jaw movements and falling.
Motor limbic seizures commenced after 20-30 min., recurred every
2-8 min and lead to status epilepticus. Increase of the dose of
pilocarpine up-to 400mg/kg abolished limbic seizures and after
15-25 min of initial behavioral alterations causes fatal general
tonic--clonic convulsions. We consider this dose as the
LD.sub.100.
[0137] Administration of DP16 prior to pilocarpine prevented death
in the animals and decreased epileptiform manifestations. As shown
in FIG. 3, DP16 protected animals in a dose dependent fashion
against generalized epileptic events induced by pilocarpine. As
shown in FIG. 4, DPIL6 exhibits dose dependent therapeutic effects
at doses in the range 10.sup.-8 to 10.sup.-5 mg/kg, and decreased
the severity of the attacks as well, with a significant reduction
in fatal seizures. For this particular model of epilepsy
(pilocarpine 400 mg/kg; rats) the estimated therapeutic index (ET)
of DP16 is 0.5 mg/kg/5.times.10.sup.-7 mg/kg=1.times.10.sup.6. The
data obtained suggest that DP16 is an extremely promising prodrug
for the treatment of epileptic disorders.
[0138] c) Antiepileptic Effects of DP16:
[0139] Metrazol Minimal Seizures Test.
[0140] Testing of DP16 as a possible antiepileptic drug was
performed on 3-4 week old male BALB/c mice (18-27 g). Animals were
maintained on an adequate diet and allowed free access to food and
water except briefly during the experimental period. Animals were
separately housed for one hour in transparent plastic cages before
treatment and during the experimental period. Drugs were dissolved
in normal saline with injection volume adjusted to 0.01 ml/g of
body weight. DP16 was administered i.p., in doses ranging from 0.1
to 300 .mu.g/kg: (0.1 .mu.g/kg: n=10, 5 .mu.g/kg: n=10, 25
.mu.g/kg: n=20, 75 .mu.g/kg: n=20, 150 .mu.g/kg: n=20, and 300
.mu.g/kg: n=10 animals respectively). Control animals received
injections i.p.--of normal saline. DP16 or saline administration
followed in 30 minutes by Metrazol (50 .mu.g/kg, s.c.).
Subsequently epileptic signs were observed for the next 30 minutes.
Absence or relative delay of myoclonic jerks (MJ) in the
experimental group was considered as indication of possible
antiepileptic activity. Data were subjected to chi-square analysis
with the computer statistic package "StatViewII".
[0141] d) Results and Conclusions:
[0142] Metrazol in a dosage of 50 .mu.g/kg, s.c. caused myoclonic
jerks (MJ) in all of control mice with a latent period of 1011 min
(n=11). The effect of DP16 on the appearance of minimal metrazol
induced seizures is shown in FIG. 5. The doses are presented in
this figure in terms of mg/kg of the active pharmacological
component of the drug, i.e. BAPTA.
[0143] Mice treated with 0.1 .mu.g/kg DP16 showed the same response
to metrazol as control (untreated) animals. DP16 in doses ranging
from 5 to 300 .mu.g/kg exhibited a significant protective effect
(p<0.001). The results of the test suggest a significant
dose-dependent antiepileptic effect of DP16 on the metrazol induced
seizures.
[0144] 4.4) Investigation of cardioprotective effect of DP16:
[0145] a) Ex-vivo Rat Heart Low-Flow--Reperfusion Model.
[0146] Method and Results
[0147] The following experiments demonstrate the protective effects
of DP16 in models of cardiac diseases. Low-flow Reperfusion
Langendorff's heart (Meely and Rovetto, 1975, METHODS IN
ENZYMOLOGY, v39:43-60) is an established ex vivo model of a human
ischemic heart. A severe decrease in perfusion pressure (PP) below
20 mm Hg (low-fl.ow period) causes sinus bradycardia culminating by
stable AV block ("AVB"; 10 out of 11 hearts) frequently followed by
ventricular arythmia. Restoration of perfusion pressure causes
paraxysmal tachyarrhythmia followed by irreversable ventricular
fibrillation (VF).
[0148] The experiments were preformed ex vivo on 39 rat hearts.
Heart electrical activity and perfusion pressure were stable
following 15 min., each. Perfusion buffer was supplemented with
DP16 (1.0 .mu.g/l) following the stable AV block during low flow
perfusion and during the Reperfusion period.
[0149] Treatment of Cardiac Ischemia--Reperfusion with DP16.
[0150] The experimental protocol documented by FIG. 16 included
peroiods of Normal Coronary Flow (FIG. 6, NF, panel 1) followed by
Low-Flow (LF) and then by Normal flow-reperfusion (NF-Rp) (panels 2
and 3, respectively).
[0151] The experiments were performed by addition of DP16 (0.5-500
.mu.g/l) to the perfusion buffer after AV block establishment. In
11 out of 16 experiments DP16 (1.0 .mu.g/l) to the perfusion buffer
caused complete restoration of AV synchronism and in the additional
5 cases it resulted in a decrease dlevel of AVB and prevented
ventricular fibrillation (FIG. 6). Moreover, DP16 showed notable
cardioprotective effects during the reperfusion period. Full
restoration of the sinum rhythm was observed in 11 out of 16
experiments.
[0152] Conclusion
[0153] Evaluation of the cardioprotective effect of DP16 in the Low
Flow-Reperfusion model as compared to treatment with parent
compound BAPTA and to cell permeable BAPTA derivative, BAPTA-AM
(supplied by Molectular Probes) and shown to have much better
efficiency in resolving atrio-ventricular blockade and preventing
ventricular fibrillation as indicated by FIG. 7.
[0154] b) DP16 prevents isoproterenol induced myocardial damage
[0155] Method and Results
[0156] Administration of the potent .beta.-adrenoreceptor agonist
isoproterenol (ISO) is commonly accepted model of experimental
myocardial pathology. The cardioprotective effect of DP16 was
tested on 82 Sprague-Dawley female rats weighing 250-350 g.
Myocardial damage was induced in rats by two consecutive injections
of ISO (85 .mu.g/kg, s.c.). When appropriate, the injections of ISO
were followed in 30 and 180 minutes by DP16 (0.01 .mu.g/kg, i.p.).
The effect of DP16 was estimated by ECG analysis and determination
of serum glutamate-oxaloacetate transaminase (SGOT) and
lactatdehydrogenase (LDH) activity. Mortality of control rats after
ISO intervention was 17.1.+-.5.9% (7 out of 41). The surviving
animals exhibited striking hyperacute deviation ST-segment in lead
1 and 2 ECG. Pathological signs on ECG were aggravated during the
experimental period. In 48 hours after the second ISO injection all
treated animals displayed pathological displacement of ST-segment.
Administration of DP16 decreased mortality in 2 cases (2 out of
30). Animals receiving DP16 exhibited significantly (p<0.05)
fewer alterations in the ECG. Pathological displacement of the
ST-segment was found only on 28 and 40% of ECG (in 24 and in 48
hours following ISO respectively). Biochemical determination
demonstrated a 1.7-1.9 fold increase if SGOT and LDH in ISO treated
control rats (p<0.05). Treatment with DP16 substantially
decreased the percentage of experimental animals exhibiting
abnormal level of SGOT and LDH activity.
[0157] Conclusions
[0158] The data above suggest a significant cardioprotective effect
of DP16 in an in vivo model of myocardial pathology.
[0159] c) Pilocarpine and Cardiotoxicity.
[0160] Two types of death were found in rats treated with
pilocarpine: first death due to fatal convulsions and second,
retarded death not immediately due to epileptic events. We
attempted to understand the actual reason of retarded death of rats
after pilocarpine-induced convulsions. Under macroscopic autopsy of
these animals signs of cardiopulmonary damages were seen: lung
edema and hemorrhages, dilated and in same cases deformed hearts.
Dyeing of hearts with 0.1% Trypan blue in surviving animals
revealed spotted picture of myocardia with areas of intensive dye
absorption, i.e., damaged parts, and pale areas, i.e., infarctions.
Thus, we can consider that after pilocarpine administration, there
developed heart damage, which we term
post-pilocarpine--seizure-car- diopathy (PSCP). Studies of PSCP in
relation to DP16 evaluation were performed in vivo and in vitro
with rats which survived after convulsive and sub-convulsive doses
of Pilocarpine.
[0161] d) Post Seizure Cardiopathy (PSCP) Model:
[0162] Adult (2-3 months) male Sprague-Dawley rats were used for
all experiments. They were fed with standard briquette chow with
water ad libitum and were maintained in standard plastic cages (4-5
individuals in each cage) under natural illumination. A
pilocarpine-scopolamine epileptic status model (pilocarpine) was
performed as described earlier. In a group of 23 rats, pilocarpine
was administered i.p. in different doses which ranged from 100 to
400 mg/kg body weight (B/W) for different periods of time; a second
group of 17 rats was treated with DP16 prior to pilocarpine
administration, wherein the DP16 was injected for 30 min before
pilocarpine in the next dose range and its effect was investigated
in the ensuing periods.
[0163] In vivo ECG (Birtcher-Cardio-Tracer, model 375, USA) in
three standard leads were recorded under ketamine anesthesia (3.3
mg/kg Imalgene 100, Rhone Merieux, France and 7 mg/kg Rompun, Bayer
Leverkusen, Germany, i.m.). ECG recordings were made in the period
before pilocarpine injections (control), 24h after pilocarpine
administration (acute period) and after relative stabilization of
cardiac function, on the 3-14th day after pilocarpine
administration. Part of the ECG recordings were made under nembutal
anesthesia (35 mg/kg, i.p.) in the period before establishing
Langendorff's perfusion isolated heart preparation.
Perfusion-Hypoxia-Reperfusion isolated heart model (PHR) was
performed with the conventional Langendorff technique
(non-recirculating perfusion system) adjusted to 37.degree. C. in
two modifications: 1. under constant Perfusion Pressure (PP)--60 mm
Hg; or 2. under constant flow, established after the first 10-15
min perfusion with PP as above, by adjusting flow with help of
peristaltic pump (Ismlatec SA, Laboratoriumstechnic,
Switzerland).
[0164] In the case of constant PP the volume of effluent flow was
measured on electron balance (Precisa 1000C-3000D,
Switzerland).
[0165] In case of constant flow, established at the control period,
flow did not change during subsequent experimental periods and PP
was recorded frequently. After 30 min of the control period,
perfusion was stopped for 30 min and subsequent reperfusion period
lasted 30 min. Direct ECG were recorded from ventricular apex (lead
1), auriculum (lead 2) and in-between (lead 3). The coronary
vessells perfusion resistance (CVPR) was calculated in arbitrary
units as follows: PP/flow/heart weight. Following the protocol
above, hearts were subjected to perfusion with the dye Trypan blue
(0.1%), in order to evaluate cellular damage and infarction.
[0166] e) Results and Discussion
[0167] ECG results in vivo demonstrated distinct ECG changes after
pilocarpine injections in an acute stage of PSCP: statistically
significant depressions of R- peak were noted under leads 1 and 2
(47% & 16% of control one respectively). DP16 treatment of PSCP
50 normalized electrical activity at the acute stage in 5 out of 7
treated rats. It is known that the amplitude of ECG events are
partly connected with the intensity of correspondent physiological
processes. Thus, the pilocarpine-induced change of R-wave and its
normalization by DP16 may reflect the ability of DP16 to cure
ventricular weakness, at least under PSCP. Control rats display
relative normalization of R-wave in 3-14 days after pilocarpine.
However, R normalization apparently was correlated with drastically
increased S-wave depth under lead 3 (36%) and lead 2 (61% ). The
last was not statistically significant in view of large
variability. Increase of S-wave depth reflected damage typical of
myocardial ischemia and possibly suggests infarction in Pilocarpine
treated control animals. As during the acute stage of PSCP in the
phase of stabilization, DP16 prevents the appearance of ECG
alterations noted in control rats. The difference between animals
protected with DP16 and those not protected, is statistically
significant (p<0.01). In this period of PSCP there is marked
elevation of Heart Rate in both control Pilocarpine, and in DP16
treated animals. Such tachycardia possibly is connected with
hemodynamic insufficiency, which is characteristic for infarction
20 pathophysiology . Thus, in vivo ECG investigation during long-
term period after Pilocarpine injections revealed definite
alteration of cardiac functions (PSCP), which in some animals may
be cured by DP16-treatment.
[0168] f) Langendorff's Heart Model.
[0169] In the first 30 min of control, isolated Langendorff's
hearts CVPR steadily increased and this elevation is statistically
significant after 20 min. In all hearts, perfused after pilocarpine
administration, initial perfusion flow was larger then in control,
and subsequent CVPR significantly decreased. This decrease of
coronary vessel tone was possibly connected with intracardial
noradrenaline deficiency or paralysis, evoked by hypoxia.
[0170] Treatment of rats with DP16 prior to pilocarpine application
prevents damage of CVPR regulation in both the initial and final
periods of perfusion, thus providing evidence relating to the
ability of DPl6 to normalize coronary vessels function under
hypoxic conditions. Cessation of perfusion for 30 min and
subsequent reperfusion is characterized by the well-known broad
class of cardiac damage events, which we classified with an
arbitrary scale. Control hearts from non-treated control rats
generally were restored after cessation of perfusion with distinct
range of alterations (e.g., impaired myocardial excitability,
conductivity and contractility). Mean point of recovery in control
group is 6.3.+-.0.6 (n=7). Hearts from pilocarpine-treated rats on
different stages of PSCP demonstrated an increase of the spectrum
and severity of pathological events, as the mean point of recovery
was just 3.3.+-.0.8, n=7, p<0.05. Recovery was frequently
accompanied by ventricular fibrillation. Some of the hearts were
not restored completely or restored atrial activity only. DP16
treatment prior to pilocarpine administrations increased ability of
damaged hearts to restore after reperfusion cessation: the mean
point was 6.4.+-.0.6 (n=9). In this group of rats there was an
increased incidence of cases of complete recovery. Thus, DP16
treatment of pilocarpine-induced heart damage (PSCP) produced a
definite improvement in cardiac function.
[0171] 4.5) General Conclusions.
[0172] The prodrug denoted DP16 exhibited significant therapeutic
and protective effects in experimental models of stroke and
ischemia as well as in models of epilepsy, comparable with using
the corresponding drug in conventional form in an amount which is
105-106 times the amount when used in the form of the prodrug of
the invention.
Example 5
[0173] Preparation of Prodrug-3.
[0174] "Prodrug-3" is the name used herein to denote a 1:1 ester of
1,2-bis-(2-aminophenoxy)elhane-N,N,N',N'-tetraacetic acid (BAPTA)
with 1-myristylmyristyl alcohol and is prepared as follows. A
solution of BAPTA (0.!) g, 1.05 mmol) in dimethylformamide (25 ml,
freshly distilled over CaH.sub.2), 1-myristylmyristyl alcohol
(0.451 g, 1.1 mmol), N,N'-dicyclohexylcarbodiimide (0.216 g, 1.1
mmol) and 4-dimethylaminopyridine (0.025 g, 0.202 mmol) were
stirred together for two days at room temperature under argon, in a
50 ml flask equipped with a magnetic stirrer. After two hours,
N,N'-dicyclohexylurea began to precipitate. The reaction was
monitored by TLC (90:10 v/v chlorof orm:methanol); R.sub.f of the
product=0.62. The precipitate was removed by filtration and the
filtrate was concentrated at 35.degree. C. in vacuum. The residue
was extracted with 25 ml of a 2:1:2 v/v mixture of
chloroform:isopropanol:water. The organic layer was separated,
washed with 1% aq. NaCl solution and dried over Na.sub.2SO.sub.4;
it was then evaporated and the residue was passed through a
160.times.30 mm column of Kieselgel 60 (230-400 mesh ASTM), the
desired product being eluted with a 90:10 v/v chloroform:methanol
mixture. The 1-myristylmyristyl alcohol was prepared according to
the method of Molotkovski, V. G. and Bergelson, L. D.
(Biologicheska Chimia, 1982, 8(9): 1256-1262). The
BAPTA-1-myristylmyristyl alcohol ester link in Prodrug-3 is
susceptible to digestion by esterases.
Example 6
[0175] Preparation and Biological Properties of TVA16.
[0176] "TVA16" is the name used herein to denote a 1:1 ester of
valproic acid with the phosphorylcholine derivative
[0177]
ROCH.sub.2--CH(OH)--CH.sub.2O--(PO.sub.2)--OCH.sub.2N(CH.sub.3).sub-
.2, where R is hexadecanoyl, and was prepared as follows. A
solution of 1-hexadecanoyl-sn-glycero-3-phosphorylcholine (1.04
mmol) in chloroform (25 ml, freshly distilled over P.sub.2O.sub.5),
valproic acid (0.159 g, 1.1 mmol), N,N'-dicyclohexylcarbodiimide
(0.216 g, 1.1 mmol) and 4-dimethylaminopyridine (0.025 g, 0.202
mmol) were stirred together for two days at room temperature under
argon, in a 50 ml flask equipped with a magnetic stirrer and glass
beads (10 g, 5 mm diameter). After two hours, N,N'-dicyclohexylurea
began to precipitate. The reaction was monitored by TLC (65:25:4
v/v chloroform:methanol:water); R.sub.f of the product=0.41. The
precipitate and glass beads were removed by filtration and the
filtrate was concentrated at 35.degree. C. in vacuum. The residue
was extracted with 25 ml of a 2:1:2 v/v mixture of
chloroform:isopropanol:water. The organic layer was separated, 10
washed with 1% aq. NaCl solution and dried over Na.sub.2SO.sub.4;
it was then evaporated and the residue was passed through a
160.times.30 mm column of Kieselgel 60 (230-400 mesh ASTM), the
desired product being eluted with a 65:25:4 v/v
chloroform:methanol:water mixture; R.sub.f=0.4.
[0178] A test sample of TVA16 was administered i.p. (0.01 to 100
mg/kg) to a group of three mice, one hour before an s.c. dose of
metrazol (80 mg/kg). An effective dose was the amount which
prevented convulsions (scored 2 points per animal) and/or death 20
-=(scored :L point per animal) in the subsequent 30 minutes. On
this basis, the ED.sub.100 could be calculated and is compared to
known anticonvulsants in the following table.
1TABLE 1 Anticonvulsant activity of known drugs and TVA16
ED.sub.100 ED.sub.100 Compound (mg/kg) Compound (mg/kg)
chlordiazepoxide 25 muscimol (i.p.) 2.5 diazepam 2.5 nifedipine
>100 diphenylhydantoin >100 nimodipine >300 flunarizine
>300 phenobarbital 50 glutethimide 150 sodium valproate 500
meprobamate 200 verapamil >100 MK-801 0.5 TVA16 20
[0179] From the above data it may be seen that TVA16 has
significant anticonvulsant activity and appears to be more than
500.times. as potent as sodium valproate.
[0180] FIG. 8 presents the dose response curves of valproic acid
itself, in comparison to TVA, which clearly shows the improvement
obtained with the prodrug according to the invention. The doses of
each of the two drugs are calculated on the basis of mg of valproic
acid administered per kg body weight of the animal.
Conclusion
[0181] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the claims. Various
publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
* * * * *