U.S. patent application number 10/077475 was filed with the patent office on 2003-05-01 for matrices for drug delivery and methods for making and using the same.
Invention is credited to Babich, John W., Bonavia, Grant, Zubieta, Jon.
Application Number | 20030082238 10/077475 |
Document ID | / |
Family ID | 22386633 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082238 |
Kind Code |
A1 |
Babich, John W. ; et
al. |
May 1, 2003 |
Matrices for drug delivery and methods for making and using the
same
Abstract
In one aspect, biocompatible matrices such as sol-gels
encapsulating a reaction center may be administered to a subject
for conversion of prodrugs into biologically active agents. In
certain embodiments, the biocompatible matrices of the present
invention are sol-gels. In one embodiment, the enzyme L-amino acid
decarboxylase is encapsulated and implanted in the brain to convert
L-dopa to dopamine for treatment of Parkinson's disease.
Inventors: |
Babich, John W.; (North
Scituate, MA) ; Zubieta, Jon; (Syracuse, NY) ;
Bonavia, Grant; (Kensington, MD) |
Correspondence
Address: |
FOLEY, HOAG & ELIOT, LLP
PATENT GROUP
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Family ID: |
22386633 |
Appl. No.: |
10/077475 |
Filed: |
February 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10077475 |
Feb 15, 2002 |
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09503438 |
Feb 14, 2000 |
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6395299 |
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60119828 |
Feb 12, 1999 |
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Current U.S.
Class: |
424/491 ;
424/130.1; 424/94.1 |
Current CPC
Class: |
A61P 25/16 20180101;
A61K 9/0085 20130101; A61K 38/51 20130101; A61K 38/44 20130101;
A61K 47/6903 20170801; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 9/0024 20130101; G01N 33/5436 20130101; A61K 38/44
20130101; C12Y 401/01028 20130101; G01N 33/552 20130101; C12Q 1/00
20130101; A61K 38/51 20130101 |
Class at
Publication: |
424/491 ;
424/130.1; 424/94.1 |
International
Class: |
A61K 039/395; A61K
038/43; A61K 009/16; A61K 009/50 |
Claims
What is claimed is:
1. A method for producing a biologically active agent from a
prodrug, comprising: a. encapsulating a first cell-free reaction
center in a biocompatible matrix; and b. administering said
biocompatible matrix to a subject; wherein said biocompatible
matrix comprises an inorganic-based sol-gel matrix and wherein said
first reaction center converts a first prodrug into a first
biologically active agent in said subject.
2. The method of claim 1, wherein said biocompatible matrix
comprises a silica-based sol-gel matrix.
3. The method of claim 2, wherein said first reaction center
comprises one of the following: an enzyme, an antibody or a
catalytic antibody.
4. The method of claim 2, wherein said biocompatible matrix
encapsulates at least one additive.
5. The method of claim 2, wherein said first reaction center
comprises L-amino acid decarboxylase.
6. The method of claim 5, wherein said first prodrug comprises
L-dopa and said first biologically active agent comprises
dopamine.
7. The method of claim 2, wherein said first reaction center
comprises L-tyrosine decarboxylase.
8. The method of claim 7, wherein said first prodrug comprises
L-dopa and said first biologically active agent comprises
dopamine.
9. The method of claim 2, further comprising encapsulating a second
reaction center in said biocompatible matrix before administering
said biocompatible matrix to said subject.
10. The method of claim 9, wherein said first biologically active
agent produced by said first reaction center from said first
prodrug is a second prodrug for said second reaction center, and
wherein said second reaction center produces a second biologically
active agent that differs from said first biologically active
agent.
11. The method of claim 12, wherein said first reaction center
comprises tyrosine monooxygenase, and said second reaction center
is one of the following: L-amino acid decarboxylase or L-tyrosine
decarboxylase.
12. The method of claim 11, wherein said first prodrug comprises
tyrosine, said first biologically active agent and said second
prodrug comprises L-dopa, and said second biologically active agent
comprises dopamine.
13. The method of claims 4, 5, 6, 7, 8, 11 or 12, wherein
administering said biocompatible matrix comprises administering
said biocompatible matrix to a region of the brain of said
subject.
14. The method of claim 13, wherein said region of said brain of
said subject is one of the following: basal ganglia, substantia
nigra or striatum.
15. The method of claim 2, wherein said biocompatible matrix is
prepared from at least one type of oxysilane.
16. The method of claim 15, wherein said biocompatible matrix is
prepared from more than one type of oxysilane.
17. The method of claim 15, wherein said biocompatible matrix is
prepared from at least one type of inorganic oxide and at least one
type of oxysilane.
18. The method of claim 15 or 16, wherein said type of oxysilane
has at least one non-hydrolizable substituent.
19. The method of claim 2, wherein said biocompatible matrix
consists essentially of siloxane.
20. The method of claim 2, wherein said biocompatible matrix
comprises siloxane.
21. The method of claim 2, wherein administering said biocompatible
matrix comprises surgical implantation.
22. The method of claim 2, further comprising administering said
first prodrug to said subject.
23. The method of claim 2, wherein said first prodrug comprises an
exogenous prodrug.
24. The method of claim 2, wherein said first prodrug comprises an
endogenous prodrug.
25. The method of claim 2, wherein said first reaction center
comprises an enzyme or antibody that is xenogeneic to said
subject.
26. The method of claim 3, wherein the ratio of Km
(nonencapsulated) to Km (encapsulated) for said first reaction
center is greater than or equal to one.
27. The method of claim 3, wherein the ratio of Km
(nonencapsulated) to Km (encapsulated) for said first reaction
center is less than or equal to one.
28. The method of claim 2, wherein said first reaction center
comprises more than one weight percent of said biocompatible
matrix.
29. The method of claim 2, wherein said first reaction center
comprises less than one weight percent of said biocompatible
matrix.
30. The method of claim 29, wherein said first reaction center
comprises more than five weight percent of said biocompatible
matrix.
31. The method of claim 31, wherein said first reaction center
comprises more than ten weight percent of said biocompatible
matrix.
32. The method of claim 2, wherein said first reaction center is
attached to said biocompatible matrix.
33. The method of claim 2, wherein said biocompatible matrix is
immunoisolatory.
34. The method of claim 2, wherein administering said biocompatible
matrix comprises parenteral administration.
35. The method of claim 2, wherein administering said biocompatible
matrix comprises systemic administration.
36. The method of claim 2, wherein treatment of said subject by
said method results in long-term, stable production of said first
biologically active agent in said subject.
37. The method of claim 22, wherein said first prodrug is
administered to said subject on at least more than one
occasion.
38. The method of claim 2, wherein said first biologically active
agent is cytotoxic.
39. The method of claim 38, wherein said biocompatible matrix is
implanted in proximity to a neoplasm.
40. The method of claim 2, wherein said first reaction center does
not leach significantly from said biocompatible matrix.
41. The method of claim 2, wherein said biocompatible matrix
comprises a xero-gel.
42. The method of claim 15, wherein said oxysilane is one of the
following: TMOS or TEOS.
43. The method of claim 3, wherein said biocompatible matrix causes
prodrug activation.
44. The method of claim 2, wherein said first prodrug is a
deleterious agent to said subject and said-first biologically
active agent is less deleterious to said subject than said first
prodrug.
45. The method of claim 44, wherein said first prodrug is an agent
to which said subject is capable of becoming addicted, and wherein
said subject is less capable of becoming addicted to said first
biologically active agent.
46. The method of claim 45, wherein said first prodrug is one of
the following: ethanol or cocaine.
47. The method of claim 2, wherein said first prodrug is one of the
following: L-phenylalanine, noradrenalin, norepinephrine,
histadine, histamine, 1-methylhistamine, glutumate, GABA or
serine.
48. The method of claim 2, wherein said subject is human.
49. The method of claim 2, wherein said subject receives a
therapeutically effective amount of said biocompatible matrix and
said first prodrug.
50. The method of claim 23, wherein the ratio of the therapeutic
index of treatment using said first prodrug and said biocompatible
matrix over the therapeutic index of treatment using said first
prodrug alone is about five or more.
51. The method of claim 50, wherein the ratio of the therapeutic
index of treatment using said first prodrug and said biocompatible
matrix over the therapeutic index of treatment using said first
prodrug alone is about ten or more.
52. The method of claim 51, wherein the ratio of the therapeutic
index of treatment using said first prodrug and said biocompatible
matrix over the therapeutic index of treatment using said first
prodrug alone is at least about one hundred.
53. The method of claim 37, wherein the ratio of the therapeutic
index of treatment using said first prodrug and said biocompatible
matrix over the therapeutic index of treatment using the
biologically active agent of said first prodrug alone is at about
five or more.
54. The method of claim 53, wherein the ratio of the therapeutic
index of treatment using said first prodrug and said biocompatible
matrix over the therapeutic index of treatment using the
biologically active agent of said first prodrug alone is at about
ten or more.
55. The method of claim 51, wherein the ratio of the therapeutic
index of treatment using said first prodrug and said biocompatible
matrix over the therapeutic index of treatment using the
biologically active agent of said first prodrug alone is at least
about one hundred.
56. The method of claim 2, wherein said first biologically active
agent comprises a neutrophic factor.
57. The method of claim 2, wherein said first biologically active
agent comprises a type selected from the group consisting of
anti-angiogenesis factors, antiinfectives; antibiotics agents;
antiviral agents; analgesics; anorexics; antihelmintics;
antiarthritics; antiasthmatic agents; anticonvulsants;
antidepressants; antidiuretic agents; antidiarrheals;
antihistamines; antiinflammatory agents; antimigraine preparations;
antinauseants; antineoplastics; antiparkinsonism drugs;
antipruritics; antipsychotics; antipyretics, antispasmodics;
anticholinergics; sympathomimetics; xanthine derivatives;
cardiovascular preparations; calcium channel blockers;
beta-blockers; antiarrhythmics; antihypertensives; catecholamines;
diuretics; vasodilators; central nervous system stimulants; cough
preparations; cold preparations; decongestants; growth factors,
hormones; steroids,; corticosteroids; hypnotics;
immunosuppressives; muscle relaxants; parasympatholytics;
psychostimulants; sedatives; tranquilizers; proteins;
polysaccharides; glycoproteins; lipoproteins; interferons;
cytokines; chemotherapeutic agents; anti-neoplastics, antibiotics,
anti-virals, anti-fungals, anti-inflammatories, anticoagulants,
lymphokines, and antigenic materials.
58. The method of claim 3, wherein said first reaction center
comprises an enzyme that is a member of a class selected from the
group consisting of oxidoreductases; transferases; hydrolaaes;
isomerases; and ligases.
59. The method of claim 2, wherein said first reaction center
replaces, augments or supplements some endogenous biological
activity in said subject.
60. The method of claim 59, wherein said first reaction center
comprises an enzyme in which said subject is deficient.
61. The method of claim 60, wherein said first reaction center is
one of the following: glucocerebrosidase; .alpha.-1,4-glucosidase;
.alpha.-galactosidase; .alpha.-L-iduronidase; .beta.-glucuronidase;
aminolaevulinate dehydratase; bilirubin oxidase; catalase;
fibrinolysin; glutaminase; hemoglobin; heparinase; L-arginine
ureahydrolase (A1); arginase; liver microsomal enzymes;
phenylalanine ammonia lyase; streptokinase; superoxide dismutase;
terrilythin; tyrosinase; UDP glucuronyl transferase; urea cycle
enzymes; urease; uricase; or urokinase.
62. A method of toxicology testing, comprising: a. encapsulating at
least one reaction center in a silica-based sol-gel matrix; b.
interacting a compound with said matrix; and, c. evaluating for any
products of said compound resulting from conversion of said
compound by said reaction center, wherein production of any
cytotoxic or mutagenic products indicates that said compound may be
toxic to a subject upon administration.
63. The method of claim 62, wherein said reaction center comprises
an enzyme that is located in the liver of a mammal.
64. The method of claim 62, wherein said reaction center is an
enzyme prepared by recombinant methods.
65. The method of claim 62, wherein said reaction center is
cell-free.
66. The method of claim 63, wherein said mammal is one of the
following: a pig or a human.
67. A biocompatible matrix for treatment, comprising: a. a
inorganic-based sol-gel matrix that is biocompatible; and, b. a
first cell-free reaction center encapsulated in said matrix,
wherein said first reaction center, after administration of said
matrix to a subject, produces a therapeutically effective amount of
a first biologically active agent from a first prodrug in said
subject.
68. The biocompatible matrix of claim 67, wherein said
biocompatible matrix comprises a silica-based sol-gel matrix.
69. The biocompatible matrix of claim 67, wherein said first
reaction center comprises one of the following: an enzyme, an
antibody or a catalytic antibody.
70. The biocompatible matrix of claim 68, wherein said first
reaction center comprises one of the following: L-amino acid
decarboxylase or L-tyrosine decarboxylase.
71. The biocompatible matrix of claim 71, wherein said first
reaction center comprises L-amino acid decarboxylase, said first
prodrug comprises L-dopa, and said first biologically active agent
comprises dopamine.
72. The biocompatible matrix of claim 68, wherein said
biocompatible matrix further comprises a second reaction
center.
73. The biocompatible matrix of claim 72, wherein said first
biologically active agent produced by said first reaction center
from said first prodrug is a second prodrug for said second
reaction center, and wherein said second reaction center produces a
second biologically active agent that differs from said first
biologically active agent.
74. The biocompatible matrix of claim 73, wherein said first
reaction center comprises tyrosine monooxygenase, and said second
reaction center is one of the following: L-amino-acid decarboxylase
or L-tyrosine decarboxylase.
75. The biocompatible matrix of claims 70, 71 or 74, wherein
administering said biocompatible matrix comprises administering
said biocompatible matrix to a region of the brain of said
subject.
76. The biocompatible matrix of claim 75, wherein said region of
said brain of said subject is one of the following: basal ganglia,
substantia nigra or striatum.
77. The biocompatible matrix of claim 68, wherein said
biocompatible matrix is prepared from at least one type of
oxysilane.
78. The biocompatible matrix of claim 68, wherein said
biocompatible matrix is siloxane.
79. The biocompatible matrix of claim 77, wherein said type of
oxysilane has at least one non-hydrolizable substituent.
80. The biocompatible matrix of claim 68, wherein said first
prodrug is exogenous to said subject.
81. The biocompatible matrix of claim 67, wherein said first
prodrug is endogenous to said subject.
82. The biocompatible matrix of claim 68, wherein said first
reaction center comprises an enzyme that is xenogeneic to said
subject.
83. The biocompatible matrix of claim 69, wherein the ratio of Km
(nonencapsulated) to Km (encapsulated) for said first reaction
center is greater than or equal to one.
84. The biocompatible matrix of claim 67, wherein said first
reaction center comprises more than one weight percent of said
biocompatible matrix.
85. The biocompatible matrix of claim 82, wherein said xenogeneic
enzyme comprises more than five weight percent of said
biocompatible matrix.
86. The biocompatible matrix of claim 68, wherein said first
reaction center comprises more than ten weight percent of said
biocompatible matrix.
87. The biocompatible matrix of claim 68, wherein said
biocompatible matrix is immunoisolatory.
88. The biocompatible matrix of claim 68, wherein said
biocompatible matrix is capable of long-term, stable production of
said first biologically active agent in said subject.
89. The biocompatible matrix of claim 69, wherein said first
biologically active agent is cytotoxic.
90. The biocompatible matrix of claim 67, wherein said first
reaction center does not leach significantly from said
biocompatible matrix after administration.
91. The biocompatible matrix of claim 68, wherein said
biocompatible matrix comprises a xero-gel.
92. The biocompatible matrix of claim 77, wherein said oxysilane is
one of the following: TMOS or TEOS.
93. The biocompatible matrix of claim 68, wherein said first
prodrug is a deleterious agent to said subject and said first
biologically active agent is less deleterious to said subject than
said first prodrug.
94. The biocompatible matrix of claim 67, wherein said first
prodrug is an agent to which said subject is capable of becoming
addicted, and wherein said subject is less capable of becoming
addicted to said first biologically active agent.
95. The biocompatible matrix of claim 68, wherein said first
prodrug is one of the following: L-phenylalanine, noradrenalin,
norepinephrine, histadine, histamine, 1-methylhistamine, glutumate,
GABA or serine.
96. The biocompatible matrix of claim 80, wherein the ratio of the
therapeutic index of treatment using said first prodrug and said
first biocompatible matrix over the therapeutic index of treatment
using said first prodrug alone is about five or more.
97. The biocompatible matrix of claim 88, wherein the ratio of the
therapeutic index of treatment using said first prodrug and said
first biocompatible matrix over the therapeutic index of treatment
using said first prodrug alone is at least about one hundred.
98. The biocompatible matrix of claim 80, wherein the ratio of the
therapeutic index of treatment using said first prodrug and said
biocompatible matrix over the therapeutic index of treatment using
the biologically active agent of said first prodrug alone is at
least about ten or more.
99. The biocompatible matrix of claim 80, wherein said first
biologically active agent comprises a type selected from the group
consisting of anti-angiogenesis factors, antiinfectives;
antibiotics agents; antiviral agents; analgesics; anorexics;
antihelmintics; antiarthritics; antiasthmatic agents;
anticonvulsants; antidepressants; antidiuretic agents;
antidiarrheals; antihistamines; antiinflammatory agents;
antimigraine preparations; antinauseants; antineoplastics;
antiparkinsonism drugs; antipruritics; antipsychotics;
antipyretics, antispasmodics; anticholinergics; sympathomimetics;
xanthine derivatives; cardiovascular preparations; calcium channel
blockers; beta-blockers; antiarrhythmics; antihypertensives;
catecholamines; diuretics; vasodilators; central nervous system
stimulants; cough preparations; cold preparations; decongestants;
growth factors, hormones; steroids,; corticosteroids; hypnotics;
immunosuppressives; muscle relaxants; parasympatholytics;
psychostimulants; sedatives; tranquilizers; proteins;
polysaccharides; glycoproteins; lipoproteins; interferons;
cytokines; chemotherapeutic agents; anti-neoplastics, antibiotics,
anti-virals, anti-fungals, anti-inflammatories, anticoagulants,
lymphokines, and antigenic materials.
100. The biocompatible matrix of claim 68, wherein said first
reaction center comprises an enzyme that is member of a class of
one of the following: oxidoreductases; transferases; hydrolases;
isomerases; or ligases.
101. The biocompatible matrix of claim 67, wherein said first
reaction center replaces, augments or supplements some endogenous
biological activity in said subject.
102. The biocompatible matrix of claim 68, wherein said first
reaction center comprises an enzyme in which said subject is
deficient.
103. The biocompatible matrix of claim 102, wherein said first
reaction center is one of the following: glucocerebrosidase;
.alpha.-1,4-glucosidase; .alpha.-galactosidase;
.alpha.-L-iduronidase; .beta.-glucuronidase; aminolaevulinate
dehydratase; bilirubin oxidase; catalase; fibrinolysin;
glutaminase; hemoglobin; heparinase; L-arginine ureahydrolase (A1);
arginase; liver microsomal enzymes; phenylalanine ammonia lyase;
streptokinase; superoxide dismutase; terrilythin; tyrosinase; UDP
glucuronyl transferase; urea cycle enzymes; urease; uricase; or
urokinase.
104. A biologically active agent produced by a process comprising:
a. encapsulating a first cell-free reaction center in a
biocompatible matrix; and b, administering said biocompatible
matrix to a subject; wherein said biocompatible matrix comprises an
inorganic-based sol-gel matrix, and wherein said biologically
active agent is produced by said first reaction center from a first
prodrug in said subject.
105. The biologically active agent of claim 104, wherein said
biocompatible matrix comprises a silica-based sol-gel matrix.
106. The biologically active agent of claim 105, wherein said first
reaction center comprises one of the following: an enzyme, an
antibody or a catalytic antibody.
107. The biologically active agent of claim 106, wherein said first
reaction center is one of the following: L-amino acid
decarboxylase, L-tyrosine decarboxylase or tyrosine
monooxygenase,
108. The biologically active agent of claim 105, further comprising
encapsulating a second reaction center in said biocompatible matrix
before administering said biocompatible matrix to said subject.
109. The biologically active agent of claim 107, wherein
administering said biocompatible matrix comprises administering
said biocompatible matrix to one of the following regions of the
brain: basal ganglia, substantia nigra or striatum.
110. The biologically active agent of claim 105, further comprising
preparing said biocompatible matrix from at least one type of
oxysilane at substantially the same time as said encapsulating of
said first reaction center.
111. The biologically active agent of claim 105, wherein said
biocompatible matrix consists essentially of siloxane.
112. The biologically active agent of claim 105, wherein
administering said biocompatible matrix to a subject comprises
surgical implantation.
113. The biologically active agent of claim 110, further comprising
administering said first prodrug to said subject.
114. The biologically active agent of claim 105, wherein said first
prodrug comprises a prodrug exogenus to said subject.
115. The biologically active agent of claim 105, wherein said
process results in long-term, stable production of said
biologically active agent in said subject.
116. The biologically active agent of claim 114, wherein said first
prodrug is administered to said subject on at least more than one
occasion.
117. The biologically active agent of claim 105, wherein said first
prodrug is a deleterious agent to said subject and said first
biologically active agent is less deleterious to said subject than
said first prodrug.
118. The biologically active agent of claim 105, wherein said
subject is human.
119. The biologically active agent of claim 105, wherein said
biologically active agent comprises a type selected from the group
consisting of anti-angiogenesis factors, antiinfectives;
antibiotics agents; antiviral agents; analgesics; anorexics;
antihelmintics; antiarthritics; antiasthmatic agents;
anticonvulsants; antidepressants; antidiuretic agents;
antidiarrheals; antihistamines; antiinflammatory agents;
antimigraine preparations; antinauseants; antineoplastics;
antiparkinsonism drugs; antipruritics; antipsychotics;
antipyretics, antispasmodics; anticholinergics; sympathomimetics;
xanthine derivatives; cardiovascular preparations; calcium channel
blockers; beta-blockers; antiarrhythmics; antihypertensives;
catecholamines; diuretics; vasodilators; central nervous system
stimulants; cough preparations; cold preparations; decongestants;
growth factors, hormones; steroids,; corticosteroids; hypnotics;
immunosuppressives; muscle relaxants; parasympatholytics;
psychostimulants; sedatives; tranquilizers; proteins;
polysaccharides; glycoproteins; lipoproteins; interferons;
cytokines; chemotherapeutic agents; anti-neoplastics, antibiotics,
anti-virals, anti-fungals, anti-inflammatories, anticoagulants,
lymphokines, and antigenic materials.
120. The biologically active agent of claim 105, wherein said first
reaction center comprises an enzyme that is member of a class
selected from the group consisting of oxidoreductases;
transferases; hydrolases; isomerases; and ligases.
121. The biologically active agent of claim 111, wherein said first
reaction center comprises an enzyme in which said subject is
deficient.
122. A tissue assist device, comprising: a. a inorganic-based
sol-gel matrix that is biocompatible; and, b. a first reaction
center encapsulated in said matrix, wherein upon placing said
biocompatible matrix in contact with fluids of a subject, said
first reaction center converts a first prodrug into a first
biologically active agent, and wherein said first reaction center
provides a biological function characteristic of tissue of said
subject.
123. The device of claim 122, wherein said biocompatible matrix
comprises a silica-based sol-gel matrix.
124. The device of claim 123, wherein said tissue is an organ.
125. The device of claim 124, wherein said organ is a liver.
126. The device of claim 125, wherein said first reaction center is
one of the following: cytochrome P-450, hepatocytes or Kupffer
cells.
127. The device of claim 124, wherein said first prodrug is
endogenous to said subject and is more deleterious to said subject
than said first biologically active agent.
128. The device of claim 123, wherein said fluid is blood of said
subject.
129. The device of claim 125, wherein said first reaction center is
xenogeneic.
130. The device of claim 123, wherein said contact occurs
extracorporeal to said subject.
131. The device of claim 123, wherein said tissue of said subject
is deficient in converting said first prodrug into said first
biologically active agent.
132. A kit for treatment of a subject, comprising: a.
a-inorganic-based sol-gel matrix that is biocompatible; and, b. a
first cell-free reaction center encapsulated in said matrix,
wherein said first reaction center, after administration of said
matrix to a subject, produces a therapeutically effective amount of
a first biologically active agent from a first prodrug in said
subject.
133. The kit of claim 132, wherein said biocompatible matrix
comprises a silica-based sol-gel matrix.
134. The kit of claim 133, further comprising instructions for
treatment of said subject using said kit.
135. The kit of claim 133, further comprising one or more doses of
said first prodrug for administration to said subject.
136. The kit of claim 135, wherein said dose of said first prodrug
is formulated for controlled release of said first prodrug upon
administration to said subject.
137. A method of treatment of a subject, comprising: a. a step for
encapsulating a first cell-free reaction center in a biocompatible
matrix; and b. a step for administering said biocompatible matrix
to a subject; wherein said biocompatible matrix comprises a
silica-based sol-gel matrix, and wherein said first reaction center
converts a first prodrug into a first biologically active agent in
said subject.
138. The method of treatment of claim 137, further comprising a
step for administering said prodrug to said subject before, at the
same time or after said step for administering said biocompatible
matrix to said subject.
Description
1. RELATED APPLICATION INFORMATION
[0001] This Application claims the benefit of priority under 35
U.S.C. section 119(e) to Provisional Application No, 60/119,828,
filed Feb. 12, 1999.
2. INTRODUCTION
[0002] Many clinical conditions, deficiencies, and disease states
may be remedied or alleviated by providing to a patient beneficial
biologically active agents or removing from the patient deleterious
biologically active agents. In many cases, provision of beneficial
agents or removal of deleterious ones may restore or compensate for
the impairment or loss of function or homeostasis. An example of a
disease or deficiency state whose etiology includes loss of such an
agent include Parkinson's disease, in which dopamine production is
diminished. The impairment or loss of such agents may result in the
loss of additional metabolic functions.
[0003] Parkinson's disease, one of many motor system disorders,
results in symptoms such as tremor, bradykinesia, and impaired
balance. Keller in Handbook of Parkinson's Disease (Marcel-Dekker
Inc.: New York 1992). Parkinson's disease is both chronic and
progressive, and nearly 50,000 Americans are diagnosed with
Parkinson's disease each year. More than half a million Americans
are currently being treated for Parkinson's disease. Bennett et al.
Dis. Mon. 38:1 (1992).
[0004] A specific area of the brain known as the basal ganglia is
affected in Parkinson's disease. The basal ganglia plays a vital
role in voluntary movement control. A region of the basal ganglia
termed the substantia nigra is important in the synthesis of the
neurotransmitter dopamine. Deterioration of the dopamine producing
cells in the substantia nigra results in the characteristic
symptoms of Parkinson's disease. These symptoms are thought to be
due to a deficiency of dopamine in both the substantia nigra and
the striatum. Obeso et al., Advances in Neurology 74:143 (1997).
The striatum requires a balance of the neurotransmitters dopamine
and acetylcholine in order to control properly movement, balance,
and walking. The cause of the impairment or death of the cells
responsible for the production of dopamine in the substantia,
although currently unknown, has been attributed to a number of
factors, including oxidant stress, mitochondrial toxicity, and
autoimmunity. Olanow et al., in Neurodegenetaion and
Neuroprotection (Academic Press: San Diego 1996).
[0005] There are currently a number of methods being used for
treating Parkinson's disease, which can be grouped into two
categories, namely chemical and surgical methods. Yahr et al.
Advances in Neurology 60: 11-17 (1993). In chemical treatment
methods, the goal is to achieve a stasis between the
counterbalancing dopamine and acetylcholine neurotransmitters.
Jankovic et al., in Parkinson's Disease and Movement Disorders
115-568 (Williams and Wilkins: Baltimore). The correct balance of
the neurotransmitters produces a therapeutic effect in the
Parkinson's disease patient. At least three methods of
accomplishing or restoring a therapeutic balance are presently
possible. First, in the dopaminergic method, a balance may be
achieved by increasing deficient dopamine levels by using dopamine
precursors or by increasing levels of dopamine agonists in the
brain. Controlled release systems have been used to increase
dopamine levels. Becker et al. Brain Res. 508:60 (1990); Sabel
Advances in Neurology, 53:513-18 (1990). Second, monoamine oxidase
inhibitors (MAO) reduce the rate of doparnine breakdown catalyzed
by monoamine oxidase enzymes and thereby increase the dopamine
levels in the brain. Third, anticholinergics block the receptor
sites for acetylcholine in an attempt to compensate for low
dopamine levels.
[0006] Currently, there are at least two surgical methods being
utilized in Parkinson's therapy. Jankovic et al., supra. In
ablative surgeries, a small portion of the globus pallidus
(pallidotomy) or the thalamus (thalamotomy) is destroyed, which has
been shown to be effective in treating Parkinson's disease. In
tissue transplants, dopaminergic cells, such as fetal nigral
primordia and adrenal chromaffin cells, are grafted into the basal
ganglia region or striatum. Fetal dopaminergic neurons have been
observed to provide superior functional recovery in terms of both
magnitude and duration of effects. Kordower et al., in Therapeutic
Approaches To Parkinson's Disease 443-72 (Roller et al. eds.,
Mercer Dekker Inc.: New York (1990)). This is true for both rodent
and nonhuman primate models of Parkinson's disease as well as
clinical trials in Parkinson's disease patients. Bakay et al. Ann.
NY Acad. Sci. 495:623-40 (1987); Bankiewiez et al. Progress in
Brain Research 78:543-50 (1988); Freed et al. New England Journal
of Medicine 327:1549-55 (1992). In addition, such cells have been
encapsulated, Emerich et al. Neurosci. Behav. Rev. 16:437-47
(1992), and found to alleviate symptoms of Parkinson's disease in
rodents, Aebischer et al. Brain Res. 560:43 (1991); Lindner et al.
Exp. Neurol. 132:62-76 (1995); Subramanian et al. Cell Transplant
6:469-77 (1997).
[0007] Although both chemical and surgical methods help to decrease
the symptoms of Parkinson's disease, there are a number of areas
requiring improvement. With respect to chemical methods, delivery
to the striatal region of any biologically active agent, such as
dopamine, MAO inhibitors, or anticholinergics, is complicated, in
part, because of the presence of the blood-brain barrier, which may
result in low bioavailability of any such agents. As an
alternative, direct administration of dopamine into the central
nervous system may require the frequent and repeated use of
invasive procedures which compromise the integrity of the
blood-brain barrier. Those techniques require repeated infusions
into the brain, either through injections via cannulae, or from
pumps which must be replaced every time the reservoir is depleted.
Even with the careful use of sterile procedures, there is risk of
infection. It has been reported that even in intensive care units,
intracerebroventricular catheters used to monitor intracranial
pressure become infected with bacteria after about three days.
Saffran, Perspectives in Biology and Medicine 35:471-86 (1992). In
addition to the risk of infection, there seems to be some risk
associated with the infusion procedure. Infusions into the
ventricles have been reported to produce hydrocephalus, Saffran et
al. Brain Research 492:245-54 (1989), and continuous infusions of
solutions into the parenchyma is associated with necrosis.
[0008] Because of the fact that dopamine itself does not readily
cross the blood-brain barrier, many of the drug therapies utilize
the dopamine precursor L-dopa. Modern Pharmacology 108 (2d ed,
Craig et al. eds, 1986). Conversion of L-dopa to dopamine requires
the enzyme amino acid decarboxylase, which is found in the
substantia nigra of the brain. The progression of Parkinson's
disease and the need for larger doses of L-dopa in order to produce
therapeutic effects may be due to the loss of the enzyme required
for this conversion. This loss of therapeutic efficacy is known as
long-term L-dopa syndrome and occurs in 3 to 5 years in 50% of
Parkinson's disease patients being treated with L-dopa. Brannan et
al. Neurology 45:596 (1991).
[0009] Surgical tissue transplantation suffers from a number of
factors such as immunogenic complications, delayed improvement
results, and low tissue survival rates of around 10%. The use of
fetal tissue has formidable hurdles, including the failure to
reestablish the normal neural circuitry, high mortality and
morbidity associated with the transplant procedure, and the ethical
issue of human fetal tissue research. Aebischer et al. Transactions
of the ASME 113:178 (1991). Adrenal cells are generally only
implanted in patients less than 60 years of age, as the adrenal
gland of older patients may not contain sufficient
dopamine-secreting cells, which limits the usefulness of the
procedure as a treatment method because the disease most often
affects the elderly. With respect to encapsulation of dopamine
producing cells, questions remain concerning cell viability upon
encapsulation and their resulting durability and output. Lindner et
al. Cell Transplant 7:165-74 (1998).
[0010] Although the different therapies discussed above for
Parkinson's disease have met with some success, there remains a
need for additional treatment methods for the condition. In the
present invention, in part, novel methods of producing the
biologically active agent dopamine in the brain is contemplated. In
another aspect, the present invention contemplates treating
diseases or conditions by either producing or removing biologically
active agents in a patient.
3. SUMMARY OF THE INVENTION
[0011] In one aspect, the present invention contemplates matrices
encapsulating a reaction center, and methods of using the same.
[0012] In another aspect, the present invention is directed to
methods for producing a biologically active agent from a prodrug
involving encapsulating a cell-free reaction center in a
biocompatible matrix and administering the matrix to a subject,
wherein said reaction center converts a prodrug into a biologically
active agent in the subject. In one method of the present
invention, the matrices of the present invention are administered
to a subject for treatment of a disease or condition by production
or removal of a biologically active agent or agents.
[0013] In another aspect, the present invention involves methods of
enzyme replacement therapy for treating a subject involving
administering to the subject a reaction center which is
encapsulated in a biocompatible matrix, wherein said reaction
center replaces, augments, or supplements some activity in said
subject. The reaction center may be an enzyme in which a subject to
be treated is deficient, because of, for example, a disease or
condition or an inborn error of metabolism.
[0014] In another aspect, the present invention contemplates
methods for the extra-corporeal use of the subject matrices in, for
example, organ assist devices such as a liver assist device. In one
method of the present invention, the matrices of the present
invention are used ex vivo for treatment of a disease or condition
by production or removal of a biologically active agent or agents
from a patient.
[0015] In certain embodiments of the present invention, including
the foregoing aspects, the reaction center may be an enzyme, an
antibody, a catalytic antibody or other biological material. In
other embodiments, the matrix may be an inorganic-based sol-gel
matrix or a silica-based sol-gel matrix. More than one reaction
center may be encapsulated in a single matrix. In addition to any
encapsulated reaction center, the matrix may have encapsulated
additives. In one preferred embodiment, the reaction center may be
L-amino acid decarboxylase, the prodrug may be L-dopa and the
biologically active agent may be dopamine.
[0016] In still another aspect, the matrices of the present
invention, and methods of using the same, may be used in diagnostic
applications, such as in certain embodiments in which an imaging
agent is encapsulated therein.
[0017] In still another aspect, the matrices and compositions of
the present invention may be used in the manufacture of a
medicament for any number of uses, including for example treating
any disease or other treatable condition of a patient. In still
other aspects, the present invention is directed to a method for
formulating (either separately or together) matrices, prodrugs and
other materials and agents required for treatment in a
pharmaceutically acceptable carrier.
[0018] In another aspect, this invention contemplates a kit
including matrices of the present invention, and optionally
instructions for their use. For example, in one embodiment, such
kits include matrices and associated prodrug for treatment of a
patient. Such kits may have a variety of uses, including, for
example, imaging, diagnosis, therapy, vaccination, and other
applications.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1: Enzymatic activity assay for a matrix containing
.beta.-Glucosidase.
[0020] FIG. 2: Substrate and product spectra for penicillinase
assay.
[0021] FIGS. 3(a) and (b): Penicillinase activity assays showing
(a) multiple assays of a single matrix and (b) a single assay
performed on each of five matrices from one batch preparation.
[0022] FIG. 4: Change in absorbance at three hours as a function of
the enzyme concentration added to the matrix during
preparation.
[0023] FIG. 5: Yield of immobilized enzyme in
penicillinase-containing sol-gel matrices (observed activity was
calculated as the percentage of enzyme activity used in the
preparation of the matrices).
[0024] FIG. 6. Activity of crushed and whole matrices containing
penicillinase. FIGS. 6 and 7 both show data for five unique
matrices assayed one time each.
[0025] FIG. 7: Penicillinase activity in whole monoliths and
crushed matrices with points shown being the mean of five
measurements (error bars+/-one standard deviation).
[0026] FIGS. 8(a) and (b): (a) Activity of penicillinase-containing
matrices with varying surface areas, and (b) activity as a
percentage of the activity added in preparation. Surface areas
corresponding to the labeling in the graphs are: A=15.1 cm.sup.2,
B=39.2 cm.sup.2, C=71.3 cm.sup.2 and D=135 cm.sup.2.
[0027] FIG. 9: Tyrosine decarboxylase activity assay; elapsed time
since gel cast 19.5 h.
[0028] FIG. 10. Tyrosine decarboxylase activity assay of two
identical 16 day old matrices, A and B, with comparison to C (same
matrix composition aged 19 h, no cofactor present). Assay of A is
performed in the absence of pyridoxal-5-phosphate (cofactor) while
B is performed with cofactor present.
5. DETAILED DESCRIPTION OF THE INVENTION
[0029] 5.1. Definitions
[0030] For convenience, the meanings of certain terms employed in
the specification are provided below. The meanings for these terms,
as those of skill in the art would understand them, should be read
in light of the remainder of the specification for a full and
complete understanding of the scope of the invention.
[0031] The term "additives" refers to compounds, materials, and
compositions that may be included in a matrix along with a reaction
center. An additive may be encapsulated in or on a matrix or
attached to a matrix, either the interior or exterior, by some
interaction, including a covalent one or adhesion of the additive
to the matrix. Examples of additives include other molecules
necessary for the conversion mediated by the reaction center, solid
materials which serve as a framework for the matrix, etc.
[0032] The term "antibody" refers to a binding agent including a
whole antibody or a binding fragment thereof which is reactive with
a specific antigen. Antibodies can be fragmented using conventional
techniques and the fragments screened for utility in the same
manner as described above for whole antibodies. For example, F(ab)2
fragments can be generated by treating an antibody with pepsin. The
resulting F(ab)2 fragment can be treated to reduce disulfide
bridges to produce Fab fragments.
[0033] The term "biocompatible matrix" as used herein means that
the matrix, upon implantation in a subject, does not elicit a
detrimental response sufficient to result in the rejection of the
matrix or to render it inoperable, for example through degradation.
To determine whether any subject matrix is biocompatible, it may be
necessary to conduct a toxicity analysis. Such assays are well
known in the art. One non-limiting example of such an assay for
analyzing a composition of the present invention would be performed
with live carcinoma cells, such as GT3TKB tumor cells, in the
following manner: various amounts of subject matrices are placed in
96-well tissue culture plates and seeded with human gastric
carcinoma cells (GT3TKB) at 104/well density. The degraded products
are incubated with the GT3TKB cells for 48 hours. The results of
the assay may be plotted as % relative growth versus amount of
matrices in the tissue-culture well. In addition, matrices of the
present invention may also be evaluated by well-known in vivo
tests, such as subcutaneous implantations in rats to confirm that
they do not cause significant levels of irritation or inflammation
at the subcutaneous implantation sites.
[0034] The term "biologically active agent" as used herein means
any organic or inorganic agent that is biologically active, e.g.,
produces some biological affect in a subject.
[0035] The term "encapsulated reaction center" means a reaction
center that is contained within or on a matrix. For example, an
encapsulated reaction center may be immobilized somewhere in a
silica matrix; alternatively, it may be attached to the interior or
the surface of a matrix by some means other than physical
confinement, such as by covalent bonds or adhesion. Alternatively,
an encapsulated reaction center may be located on the surface of a
matrix.
[0036] The term "enzyme" refers to any polypeptide that converts a
prodrug into a biologically active agent. An enzyme may be isolated
from naturally occurring sources, or it may be prepared by
recombinant methods. An enzyme may be a fusion or chimeric protein
of a polypeptide that converts a prodrug and another polypeptide.
An enzyme may be a portion or a fragment of a full-length enzyme.
An enzyme may be substantially purified, or only partially
purified. Homologs, orthologs, and paralogs of an enzyme are also
enzymes. For purposes of the present invention, an enzyme is not a
catalytic antibody, a cell, or an organism.
[0037] "Homology" refers to sequence similarity between two
polypeptides or between two nucleic acid molecules. Homology may be
determined by comparing a position in each sequence which may be
aligned for purposes of comparison. When a position in the compared
sequence is occupied by the same base or amino acid, then the
molecules are homologous at that position. A degree of homology
between sequences is a function of the number of matching or
homologous positions shared by the sequences. An "unrelated" or
"non-homologous" sequence shares less than 40 percent identity,
though preferably less than 25 percent identity, with the sequence
to which it is being compared.
[0038] The term "immunoisolatory matrix" means that the matrix upon
administration to subject minimizes the deleterious effects of the
subject's immune system on the reaction center or other contents
contained within the matrix.
[0039] The term "long-term, stable production of biologically
active agent" as used herein means the continued production of a
biologically active agent at a level sufficient to maintain its
useful biological activity for periods greater than at least about
one month, more preferably about two months, four months, six
months, eight months, ten months, one year, one and a half years or
more.
[0040] The term "matrix" means any material in which a reaction
center has been encapsulated. For example, one type of matrix is a
silica-based sol-gel matrix. Another example of a matrix is an
inorganic-based sol-gel matrix. A matrix may have more than one
type of reaction center encapsulated.
[0041] The term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic
acid (RNA). The term should also be understood to include, as
equivalents, analogs of either RNA or DNA made from nucleotide
analogs, and, as applicable to the embodiment being described,
single (sense or antisense) and double-stranded
polynucleotides.
[0042] The phrases "parenteral administration" and "administered
parenterally" mean modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticulare, subcapsular, subarachnoid, intraspinal and
intrastemal injection and infusion.
[0043] A "patient" or "subject" to be treated by the present
invention can mean either a human or non-human animal.
[0044] The phrase "pharmaceutically acceptable" is employed to
refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0045] The phrase "pharmaceutically-acceptable carrier" means a
pharmaceutically-acceptable material, composition or vehicle, such
as a liquid or solid filler, diluent, excipient, solvent or
encapsulating material, involved in carrying or transporting a
prodrug, compound, material, or composition from one organ, or
portion of the body, to another organ, or portion of the body. Each
carrier must be "acceptable" in the sense of being compatible with
the other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0046] The term "prodrug" is intended to encompass compounds,
materials, and compositions which are converted by an encapsulated
reaction center into a biologically active agent. One means of
converting a prodrug to a biologically active agent is by an
enzyme-catalyzed reaction. A prodrug need not be biologically
inactive itself; instead, to be a prodrug, a compound need only
have some altered biological activity upon conversion by a reaction
center. A prodrug may be either endogenous or exogenous to a
subject. Also, many prodrugs produce more than one compounds upon
certain types of conversion, and the term "biologically active
agent" when used to refer the products of such a prodrug conversion
is intended to encompass all of those products.
[0047] The term "reaction center" means any material or compound
that may be encapsulated in a matrix and that converts or reacts a
prodrug into a biologically active agent or reacts with a
biologically active agent to, for example, degrade such agent. In
certain embodiments, the reaction center may be an enzyme, a
catalytic antibody, or a nonbiologically derived catalyst, such as
those commonly used for organic synthesis. In certain embodiments,
the reaction center may be prokaryotic or eukaryotic cells, such as
bacteria, yeast, or mammalian cells, including human cells, or
components thereof, such as organelles. In other embodiments of the
present invention, the reaction center is substantially pure, i.e.
95%, 96%, 97%, 98% or 99% pure and therefore essentially cell-free
or organism-free. Numerous examples of reaction centers are set
forth below.
[0048] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" mean the administration of a compound, drug or other
material other than directly into the central nervous system such
that it enters the patient's system and, thus, is subject to
metabolism and other like processes, for example, subcutaneous
administration.
[0049] The phrase "therapeutically effective amount" means that
amount of a prodrug, biologically active agent, compound, material,
or composition according to the present invention which is
effective for producing some desired therapeutic effect. Because in
certain embodiments of the present invention, a prodrug is
converted into a biologically active agent by an encapsulated
reaction center, it is necessary to consider this conversion in
determining what may be a "therapeutically effective amount" of a
prodrug. The amount can vary greatly according to the effectiveness
of a matrix, prodrug, or biologically active agent, the age,
weight, and response of the individual subject, as well as the
nature and severity of the subject's symptoms. Accordingly, there
is no upper or lower critical limitation upon the amount of the a
matrix, prodrug, or biologically active agent. The required
quantity to be employed of a matrix or prodrug in combination with
a matrix in the present invention may readily be determined by
those skilled in the art.
[0050] The terms "treating" or "method of treatment" (and
variations thereof) is intended to encompass curing as well as
ameliorating at least one symptom of a condition, deficiency, or
disease.
[0051] The term "ED.sub.50" means the dose of a drug, including,
for example, a matrix or a combination of a matrix and prodrug,
which produces 50% of a maximum response or effect. Alternatively,
the dose which produces a predetermined response in 50% of test
subjects or preparations.
[0052] The term "LD.sub.50" means the dose of a drug, including,
for example, a matrix or a combination of a matrix and prodrug,
which is lethal in 50% of test subjects.
[0053] The term "therapeutic index" refers to the therapeutic index
of a drug, including, for example, a matrix or a combination of a
matrix and prodrug, defined as LD.sub.50/ED.sub.50.
[0054] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
[0055] Herein, the term "aliphatic group" refers to a
straight-chain, branched-chain, or cyclic aliphatic hydrocarbon
group and includes saturated and unsaturated aliphatic groups, such
as an alkyl group, an alkenyl group, and an alkynyl group.
[0056] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In preferred embodiments, a straight chain or branched
chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.30 for straight chain, C.sub.3-C.sub.30 for branched
chain), and more preferably 20 or fewer. Likewise, preferred
cycloalkyls have from 3-10 carbon atoms in their ring structure,
and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0057] Moreover, the term "alkyl" (or "lower alkyl") as used
throughout the specification, examples, and claims is intended to
include both "unsubstituted alkyls" and "substituted alkyls", the
latter of which refers to alkyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. Such substituents can include, for example, a halogen, a
hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a thio
acetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphonate, a phosphinate, an amino, an amido, an amidine, an
imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a
sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a
heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
It will be understood by those skilled in the art that the moieties
substituted on the hydrocarbon chain can themselves be substituted,
if appropriate. For instance, the substituents of a substituted
alkyl may include substituted and unsubstituted forms of amino,
azido, imino, amido, phosphoryl (including phosphonate and
phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl
and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CF.sub.3, --CN and the like. Exemplary substituted alkyls are
described below. Cycloalkyls can be further substituted with
alkyls, alkenyls, alkoxys, alkylthios, alkylaminos,
carbonyl-substituted alkyls, --CF.sub.3, --CN, and the like.
[0058] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
[0059] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively.
[0060] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Throughout the
application, preferred alkyl groups are lower alkyls. In preferred
embodiments, a substituent designated herein as alkyl is a lower
alkyl
[0061] The term "aryl" as used herein includes 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics." The term
"aryl" refers to both substituted and unsubstituted aromatic rings.
The aromatic ring can be substituted at one or more ring positions
with such substituents as described above, for example, halogen,
azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,
alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or
heteroaromatic moieties, --CF.sub.3, --CN, or the like. The term
"aryl" also includes polycyclic ring systems having two or more
cyclic rings in which two or more carbons are common to two
adjoining rings (the rings are "fused rings") wherein at least one
of the rings is aromatic, e.g., the other cyclic rings can be
cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or
heterocyclyls.
[0062] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations. The abbreviations contained in said
list, and all abbreviations utilized by organic chemists of
ordinary skill in the art are hereby incorporated by reference.
[0063] The terms ortho, meta and para apply to 1,2-, 1,3- and
1,4-disubstituted benzenes, respectively. For example, the names
1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
[0064] The terms "heterocyclyl" or "heterocycle" refer to 4- to
10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathin, pyrrole, imidazole, pyrazole,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, quinoline, pteridine, carbazole, carboline,
phenanthridine, acridine, phenanthroline, phenazine, phenarsazine,
phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,
thiolane, oxazole, piperidine, piperazine, morpholine, lactones,
lactams such as azetidinones and pyrrolidinones, sultams, sultones,
and the like. The heterocyclic ring can be substituted at one or
more positions with such substituents as described above, as for
example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0065] The terms "polycyclyl" or "polycyclic group" refer to two or
more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings". Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,
ester, a heterocyclyl, an aromatic or heteroaromatic moiety,
--CF.sub.3, --CN, or the like.
[0066] The term "carbocycle", as used herein, refers to an aromatic
or non-aromatic ring in which each atom of the ring is carbon.
[0067] The phrase "fused ring " is art recognized and refers to a
cyclic moiety which can comprise from 4 to 8 atoms in its ring
structure, and can also be substituted or unsubstituted, (e.g.,
cycloalkyl, a cycloalkenyl, an aryl, or a heterocyclic ring) that
shares a pair of carbon atoms with another ring. To illustrate, the
fused ring system can be a benzodiazepine, a benzoazepine, a
pyrrolodiazepine, a pyrroloazepine, a furanodiazepine, a
furanoazepine, a thiophenodiazepine, a thiophenoazepine, an
imidazolodiazepine, an imidazoloazepine, an oxazolodiazepine, an
oxazoloazepine, a thiazolodiazepine, a thiazoloazepine, a
pyrazolodiazepine, a pyrazoloazepine, a pyrazinodiazepine, a
pyrazinoazepine, a pyridinodiazepine, a pyridinoazepine, a
pyrimidinodiazepine, or a pyrimidinoazepine.
[0068] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2--.
[0069] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
can be represented by the general formula: 1
[0070] wherein R.sub.9, R.sub.10 and R'.sub.10 each independently
represent a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.80, or R.sub.9 and R.sub.10 taken
together with the N atom to which they are attached complete a
heterocycle having from 4 to 8 atoms in the ring structure;
R.sub.80 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle or a polycycle; and m is zero or an integer in the
range of 1 to 8. In preferred embodiments, only one of R.sub.9 or
R.sub.10 can be a carbonyl, e.g., R.sub.9, R.sub.10 and the
nitrogen together do not form an imide. In even more preferred
embodiments, R.sub.9 and R.sub.10 (and optionally R'.sub.10) each
independently represent a hydrogen, an alkyl, an alkenyl, or
--(CH.sub.2).sub.m--R.sub.80. Thus, the term "alkylamine" as used
herein means an amine group, as defined above, having a substituted
or unsubstituted alkyl attached thereto, i.e., at least one of
R.sub.9 and R.sub.10 is an alkyl group.
[0071] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula: 2
[0072] wherein R.sub.9 is as defined above, and R'.sub.11
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.80, where m and R.sub.80 are as defined
above.
[0073] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula: 3
[0074] wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
[0075] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In preferred
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R.sub.80, wherein m and R.sub.80 are defined
above. Representative alkylthio groups include methylthio,
ethylthio, and the like.
[0076] The term "carbonyl" is art recognized and includes such
moieties as can be represented by the general formula: 4
[0077] wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.11 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.80 or a pharmaceutically acceptable salt,
R'.sub.11 represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.80, where m and R.sub.80 are as defined
above. Where X is an oxygen and R.sub.11 or R'.sub.11 is not
hydrogen, the formula represents an "ester". Where X is an oxygen,
and R.sub.11 is as defined above, the moiety is referred to herein
as a carboxyl group, and particularly when R.sub.11 is a hydrogen,
the formula represents a "carboxylic acid". Where X is an oxygen,
and R'.sub.11 is hydrogen, the formula represents a "formate". In
general, where the oxygen atom of the above formula is replaced by
sulfur, the formula represents a "thiolcarbonyl" group. Where X is
a sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thiolester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiolcarboxylic acid." Where X
is a sulfur and R.sub.11' is hydrogen, the formula represents a
"thioformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
[0078] The terms "alkoxyl" or "alkoxy" as used herein refers to an
alkyl group, as defined above, having an oxygen radical attached
thereto. Representative alkoxyl groups include methoxy, ethoxy,
propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons
covalently linked by an oxygen. Accordingly, the substituent of an
alkyl that renders that alkyl an ether is or resembles an alkoxyl,
such as can be represented by one of --O-alkyl, --O-alkenyl,
--O-alkynyl, --O--(CH.sub.2).sub.m--R.sub.80, where m and R.sub.80
are described above.
[0079] The terms "sulfoxido", as used herein, refers to a moiety
that can be represented by the general formula: 5
[0080] in which R'.sub.11 is as defined above, but is not
hydrogen.
[0081] A "sulfone", as used herein, refers to a moiety that can be
represented by the general formula: 6
[0082] in which R'.sub.11 is as defined above, but is not
hydrogen.
[0083] The term "sulfonamido" is art recognized and includes a
moiety that can be represented by the general formula: 7
[0084] in which R.sub.9 and R'.sub.11 are as defined above.
[0085] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by the general formula: 8
[0086] in which R.sub.9 and R.sub.10 are as defined above.
[0087] A "phosphoryl" can in general be represented by the formula:
9
[0088] wherein Q.sub.1 represented S or O, and R.sub.46 represents
hydrogen, a lower alkyl or an aryl. When used to substitute, e.g.,
an alkyl, the phosphoryl group of the phosphorylalkyl can be
represented by the general formula: 10
[0089] wherein Q.sub.1 represented S or O, and each R.sub.46
independently represents hydrogen, a lower alkyl or an aryl,
Q.sub.2 represents O, S or N. When Q.sub.1 is an S, the phosphoryl
moiety is a "phosphorothioate".
[0090] A "phosphoramidate" can be represented in the general
formula: 11
[0091] wherein R.sub.9 and R.sub.10 are as defined above, and
Q.sub.2 represents O, S or N.
[0092] A "phosphonamidate" can be represented in the general
formula: 12
[0093] wherein R.sub.9 and R.sub.10 are as defined above, and
Q.sub.2 represents O, S.
[0094] Analogous substitutions can be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0095] The definition of each expression, e.g. alkyl, m, n, etc.,
when it occurs more than once in any structure, is intended to be
independent of its definition elsewhere in the same structure.
[0096] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0097] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by derivitization with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts are formed with an appropriate
optically-active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0098] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof, wherein one or more
simple variations of substituents are made which do not adversely
affect the desired use of the compound.
[0099] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, hydrolysis,
etc.
[0100] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. The permissible substituents can be one or more and
the same or different for appropriate organic compounds. For
purposes of this invention, the heteroatoms such as nitrogen may
have hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0101] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover. Also for purposes of this invention, the term
"hydrocarbon" is contemplated to include all permissible compounds
having at least one hydrogen and one carbon atom. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic organic compounds which can be substituted or
unsubstituted.
[0102] By the terms "amino acid residue" and "peptide residue" is
meant an amino acid or peptide molecule without the --OH of its
carboxyl group (C-terminally linked) or the proton of its amino
group (N-terminally linked). In general the abbreviations used
herein for designating the amino acids and the protective groups
are based on recommendations of the IUPAC-IUB Commission on
Biochemical Nomenclature (see Biochemistry 11:1726-1732 (1972)).
For instance Met, Ile, Leu, Ala and Gly represent "residues" of
methionine, isoleucine, leucine, alanine and glycine, respectively.
By the residue is meant a radical derived from the corresponding
.alpha.-amino acid by eliminating the OH portion of the carboxyl
group and the H portion of the .alpha.-amino group. The term "amino
acid side chain" is that part of an amino acid exclusive of the
--CH(NH.sub.2)COOH portion, as defined by Kopple, Peptides and
Amino Acids 2, 33 W. A. Benjamin Inc., New York and Amsterdam,
1966; examples of such side chains of the common amino acids are
--CH.sub.2CH.sub.2SCH.s- ub.3 (the side chain of methionine),
--CH(CH.sub.3)--CH.sub.2CH.sub.3 (the side chain of isoleucine),
--CH.sub.2CH(CH.sub.3)2 (the side chain of leucine) or H-- (the
side chain of glycine).
[0103] For the most part, the amino acids used in the application
of this invention are those naturally occurring amino acids found
in proteins, or the naturally occurring anabolic or catabolic
products of such amino acids which contain amino and carboxyl
groups. Particularly suitable amino acid side chains include side
chains selected from those of the following amino acids: glycine,
alanine, valine, cysteine, leucine, isoleucine, serine, threonine,
methionine, glutamic acid, aspartic acid, glutamine, asparagine,
lysine, arginine, proline, histidine, phenylalanine, tyrosine, and
tryptophan. However, the term amino acid residue further includes
analogs, derivatives and congeners of any specific amino acid
referred to herein. For example, the present invention contemplates
the use of amino acid analogs wherein a side chain is lengthened or
shortened while still providing a carboxyl, amino or other reactive
precursor functional group for cyclization, as well as amino acid
analogs having variant side chains with appropriate functional
groups. For instance, such amino acid analogs include
.beta.-cyanoalanine, canavanine, djenkolic acid, norleucine,
3-phosphoserine, homoserine, dihydroxyphenylalanine,
5-hydroxytryptophan, 1-methylhistidine, or 3-methylhistidine. Other
naturally occurring amino acid metabolites or precursors having
side chains which are suitable herein will be recognized by those
skilled in the art and are included in the scope of the present
invention.
[0104] Also included are the D and L stereoisomers of such amino
acids when the structure of the amino acid admits of stereoisomeric
forms. The configuration of the amino acids and amino acid residues
herein are designated by the appropriate symbols D, L or DL,
furthermore when the configuration is not designated the amino acid
or residue can have the configuration D, L or DL. It will be noted
that the structure of some of the compounds of this invention
includes asymmetric carbon atoms. It is to be understood
accordingly that the isomers arising from such asymmetry are
included within the scope of this invention. Such isomers are
obtained in substantially pure form by classical separation
techniques and by sterically controlled synthesis. For the purposes
of the present invention, unless expressly noted to the contrary, a
named amino acid shall be construed to include both the D or L
stereoisomers, preferably the L stereoisomer.
[0105] The phrase "protecting group" as used herein means temporary
substituents which protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed. (Greene et al., Protective Groups in Organic Synthesis,
2.sup.nd ed.; Wiley: New York, 1991).
[0106] The phrase "N-terminal protecting group" or
"amino-protecting group" as used herein refers to various
amino-protecting groups which can be employed to protect the
N-terminus of an amino acid or peptide against undesirable
reactions during synthetic procedures. Examples of suitable groups
include acyl protecting groups such as, to illustrate, formyl,
dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl and
methoxysuccinyl; aromatic urethane protecting groups as, for
example, carbonylbenzyloxy (Cbz); and aliphatic urethane protecting
groups such as t-butyloxycarbonyl (Boc) or
9-Fluorenylmethoxycarbonyl (FMOC).
[0107] The phrase "C-terminal protecting group" or
"carboxyl-protecting group" as used herein refers to those groups
intended to protect a carboxylic acid group, such as the C-terminus
of an amino acid or peptide. Benzyl or other suitable esters or
ethers are illustrative of C-terminal protecting groups known in
the art.
[0108] 5.2. Uses
[0109] As a general introduction to the present invention, one way
to appreciate certain aspects of the invention is by considering
some of the different uses to which certain embodiments may be put.
The rendition of these uses is for illustrative purposes only, and
such categorization is not intended to limit the scope of the
present invention. Other uses may be readily apparent to those of
skill in the art, and other features of the present invention are
presented below and may be applicable to any of the following uses.
For all these uses, certain embodiments of the present invention
may be used in animal models for assaying new treatments, e.g., new
therapeutics or new treatment regimes.
[0110] 5.2.1. Prodrug Activation
[0111] In one aspect of the present invention, embodiments of the
present invention may be used as prodrug activators, that is, for
prodrug activation. When used in this fashion, the reaction center
encapsulated in a matrix reacts with a prodrug or prodrugs to
produce a biologically active agent or agents. The prodrug may be
exogenous to the subject, thereby requiring administration of the
prodrug, or the prodrug may be endogenous to the subject, in which
case administration of the prodrug to a subject may be used to add
to the prodrug present in the subject, but is not absolutely
necessary. For prodrug activation, one feature concerns matching
the prodrug of interest with the reaction center encapsulated so
that the reaction center may convert the prodrug into a
biologically active agent. In general, the therapeutic effect of a
matrix used for pending activation may vary greatly with its site
of administration.
[0112] One related example of prodrug activation using exogenous
sources involves ADEPT technology, or antibody directed enzymatic
prodrug therapy, whereby an enzyme that converts a prodrug into a
cytotoxic agent is attached to an antibody that is targeted to a
antigen of interest, often a surface cell receptor of a neoplastic
growth. See generally Denny et al. J. Pharm. Pharmacol. 50:387-94
(1998) and U.S. Pat. Nos. 6,015,556, 6,005,002, and 5,985,281.
After binding of the antibody-enzyme conjugate to the antigen,
prodrug is administered. The enzyme converts the exogenous prodrug
into the cytotoxic agent in the vicinity of the neoplastic growth.
In this fashion, ADEPT technology reduces toxicity to the subject
because the cytotoxic agent is only produced in the immediate
vicinity of the tumor. In gene-directed enzyme prodrug therapy
(GDEPT), the exogenous enzyme is generated selectively in the tumor
cells after delivery of a DNA construct containing the
corresponding gene. The present invention contemplates relying on
bystander effects and the like in the same fashion by
administration, e.g., implantation, of the matrix in the vicinity
of any neoplasm, whereupon administration of a prodrug causes
conversion of that prodrug into a biologically active agent
adjacent to the neoplasm. In contrast to ADEPT, the matrix may not
be cleared from the subject as is often observed for the
antibody-enzyme conjugate, so multiple administrations of prodrug
using the present invention may be feasible. The present invention
may also not result in nonspecific activation of the prodrug, which
may occur using ADEPT if the antibody-enzyme conjugated binds
indiscriminately or is not cleared by treatment with another
antibody prior to administration of the prodrug.
[0113] Another illustrative report of prodrug activation using a
exogenous source involved localized generation of 5-flurouracil
from 5-fluorocytosine by surgically implanting immobilized cytosine
deaminase adjacent to subcutaneous tumors in rats and injecting
intraperitomeally 5-fluorocytosine. Nishiyama et al., Cancer Res.
45:1753-61 (1985). In this example, the implanted enzyme was
encapsulated in a dialysis tube.
[0114] Embodiments of the present invention that may be grouped
under this category involve the production of dopamine by a matrix,
to which an example described below is directed. The biosynthesis
of dopamine involves a number of enzymatic steps. One traditional
treatment of Parkinson's disease uses an immediate precursor of
dopamine, L dopa. Although L dopa therapy is effective in reducing
Parkinson's symptoms, there is a lose of L dopa efficacy over time.
One possible means of overcoming such a loss of efficacy involves
increasing the enzymatic activity necessary to convert L dopa to
dopamine. One enzyme that may be used in this regard is aromatic
L-amino acid decarboxylase (AADC, E.C. 4.1.1.28). Cells stably
expressing AADC have been grafted into 6-hydroxy-dopamine
denervated rat striatum, and upon administration of L dopa, the
dopamine content was observed to increase. Kaddis et al. J.
Neurochem. 68:1520-26 (1997).
[0115] AADC catalzyes the irreversible decarboxylation reaction of
several aromatic L-amino acids, including L-dopa,
m-tyrosine,p-tyrosine, phenylalanine, 5-hydroxytrptophan, and
tryptophan. Hayashi et al. Biochemistry 32:812-18 (1993); Dominici
et al. Eur. J. Biochem. 169:209-13 (1987); Voltattorni et al.
Methods in Enzymology, 142:179-87 (1987); Sourkes, Methods in
Enzymology, 142:170-87 (1987); Lindstrom, Biochem Biophys. Acta
884:276-81 (1986); Jung Bioorganic Chem. 14:429-43 (1986);
Nishigaki Biochem. J. 252:331-35 (1988). AADC is a pyridoxal
phosphate dependent enzyme and is produced in the substantial
nigral cells. Nigrastriatal cell death and concomitant decrease in
AADC activity may result in decreased dopamine levels in the brain.
Encapsulation of AADC as the reaction center and appropriate
administration may allow for production of dopamine from L-dopa in
the nigrastriatal region for treatment of Parkinson's disease.
[0116] It is possible that, by using this embodiment or related
ones of the present invention, it may not be necessary to
administer L-dopa to produce a therapeutic effect. Encapsulated
enzymes may not require exogenous prodrug, e.g., administration of
L-dopa, because L-dopa levels that occur naturally in a subject may
be a sufficient source of dopamine upon conversion by the matrix.
As a result, side effects of L-dopa administration common in
present therapeutic treatments, including nausea, may be avoided.
L-dopa therapy presently requires simultaneous administration of a
peripheral decarboxylase inhibitor, such as carbidopa or
benserazide, to reduce decarboxylation of L-dopa to dopamine
outside the brain, which causes such nausea. Calne et al. New Eng.
J. Med. 329:1021-27 (1993).
[0117] Although AADC is the enzyme responsible for L-dopa
conversion to dopamine within the substantia nigra region of the
brain, at least one additional enzyme is capable of effecting this
conversion. L-Tyrosine decarboxylase (TD, E.C. 4.1.1.25) catalyzes
the removal of the carboxyl group from tyrosine to produce tyramine
and carbon dioxide. Pyridoxal 5'-phosphate is a necessary coenzyme.
Although TD has greater specificity for decarboxylation of
L-tyrosine to tyramine, TD also catalyzes decarboxylation of
L-dopa. Maraques et al. Plant Physiol, 88: 46-51 (1988).
Accordingly, TD may be encapsulated as the reaction center in a
matrix of the present invention as a treatment method for
Parkinson's disease.
[0118] In another approach to Parkinson's treatment using an
embodiment of the present invention, an encapsulated reaction
center may be used to produce a precursor of dopamine, such as
L-dopa. Such a matrix would be similar to traditional L-dopa
treatments, but in this case, L-dopa would be produced by the
matrix only in the location where the matrix was administered. In
this fashion, the matrix could be implanted so that L-dopa would be
produced only in the brain. Consequently, there would probably be
no side effects as reported for traditional L-dopa therapy.
Tyrosine is converted to L-dopa by the enzyme tyrosine
monooxygenase (TMO, E.C. 1.14.16.2). Encapsulation of TMO in a
matrix and administration to the brain should increase L-dopa
levels there. Tyrosine readily crosses the blood-brain barrier and
would result in very little systematic toxicity. Tyrosine may or
may not be necessary to administer to a subject to achieve the
desired therapeutic effect.
[0119] In another embodiment of the present invention, it is
possible to encapsulate both TMO and either AADC, TD, or both, in a
matrix. By doing so, it would be possible for a single matrix to
convert tyrosine to L-dopa, and L-dopa to dopamine. Because the
decarboxylating enzyme, either AADC or TD, would be in close
proximity to any L-dopa produced by the TMO, by virtue of both
enzyme being encapsulated in the same matrix, conversion to
dopamine readily proceed. Because tyrosine is less toxic than
L-dopa, it may be desirable to use a matrix to effect two
conversions, instead of just one. The present invention
contemplates encapsulating more than one reaction center per
matrix. Yamanka et al. J. Sol-Gel Sci. & Tech. 7:117-21
(1996).
[0120] In another embodiment, the enzyme monophenol monooxygenase
(MMO E.C. 1.14.18.1), or tyosinase, is encapsulated as the reaction
center. MMO is the key enzyme in melanin synthesis, catalyzing the
first two steps of the pathway: dehydroxylation of L-tyrosine to
L-dopa and oxidation of L-dopa to dopaquinone. The former reaction
is termed cresolase activity, and the later reaction is termed
catecholase activity. A number of assays have been used to measure
the tyrosinase hydroxylase and dopa oxidase activities, including
spectrophotometric, radiometric, HPLC and electrometric methods.
Winder, J. Biochem. Biophys. Methods 28:173-83 (1994); Vachtenheum
et al., Analytical Biochem. 146:405-10 (1985). MMOs from a number
of different sources are known. Kenji Adachi et al. Biochem.
Biophys. Res. Comm. 26 (1967); Seymour H. Pomerantz, Tyrosinases
(Hampster Melanoma) 620-626; Duckworth et al., J. Biol. Chem.
245:1613-25 (1970); Steiner et al., Analytical Biochem, 238:72-75
(1996). Oxidation of o-diphenols to benzoquinones is referred to as
catecholase activity. Although catecholase activity of MMO may
reduce the production of the desired therapeutic L-dopa product,
engineering of the sol-gel matrix may allow for increased
production of the diphenol product. For example, it has been
reported that administration of liposome-entrapped tyrosinase to
rat increases levels of L-dopa in rat plasma. Miranda et al. Gen.
Pharmacol. 24:1319-22 (1993). In the present invention, if the
matrix is administered in the brain, then the increase in L-dopa
would occur where it would have the greatest therapeutic
effect.
[0121] In another aspect of the present invention, modulation of
dopamine and related neurotransmitters may have use in treatment
for cocaine addiction. See U.S. Pat. No. 5,189,064. Chronic cocaine
users may experience dopamine deficiency, and dopamine
supplementation like that contemplated by the present invention may
reduce the feeling of dysphoria inadequate stimulation attributable
to depressed dopamine levels, which invites readministration of the
drug or recividism.
[0122] In another aspect, the present invention contemplates
applying the matrix-based technology to modulate the availability
of any compounds by augmenting the enzymes found in the biological
pathway for any such compounds. For example, tryptophan is
converted into 5-hydroxy-tryptophan by the enzyme tryptophan
hydroxylase with concomitant conversion of tertrahydrobiopterin to
dihydrobiopterin. AADC then converts 5-hydroxy-tryptophan to
serotonin, which is a neurotransmitter. Serotonin is found in the
gastrointestinal tract and in the brain, where it is synthesized
locally in the pineal gland. Monoamine oxidase converts serotonin
into 5-hydroxy-indoleacetaldehyde, which aldehyde dehydrogenase
metabolizes into 5-hydroxy-indoleacetic acid. The present invention
contemplate encapsulation any one or more of the enzymes in the
serotonin pathway either to produce, as was discussed for the
dopamine example discussed above, or to degrade serotonin in vivo
as clinically necessary to treat any disease or condition. Thus,
those of skill in the art may be able to use the present invention
to modulate any biological pathway using enzymes of such pathway as
reaction centers.
[0123] Some other possible enzymes, which may be useful in
neuropharmacology and which may be used as reaction centers in the
present invention, and the reaction that they catalyze, are listed
below:
1 Enzyme Chief Reactant Chief Product Choline acetyltransferase
acetyl CoA + choline CoA + o-acetylcholine Phenylalanine 4-
L-phenylalanine L-tyrosine monooxygenase Dopamine .beta.- dopamine
norepinephrine monooxygenase (noradenaline) Noradrenalin N-
noradrenalin adrenaline methyltransferase (epinephrine) Monoamine
oxidase norepinephrine 3,4-dihydroxy- phenylglycolaldehyde
Catecholamine-O-methyl norepinephrine 3-O- transferase (COMT)
methylnorepinephrine Histidine decarboxylase histidine histamine
Histamine histamine 1-methylhistamine methyltransferase Diamine
oxidase histamine 5-imidazole acetic acid Diamine oxidase
1-methylhistamine 1-methylimidazole acetic acid L-Glutamic acid-1-
glutamate .gamma.-aminobutyric acid decarboxylase (GABA)
GABA-.alpha.-oxoglutarate .gamma.-aminobutyric acid glutamate and
succinic transaminase (GABA) semialdehyde (plus
.alpha.-oxoglutarate) Serine hydroxymethylase L-serine glycine See
generally Enzymes (Dixon et al. eds; 3d ed. 1979).
[0124] In addition to these illustrative examples, the present
invention contemplates providing any biologically active agents to
treat a disease or condition. For example, in the nervous system,
chronic, low-level delivery of trophic factors is sufficient to
maintain the health of growth-factor dependent cell populations. In
chronic disorders such as Alzheimer's disease and Huntington's
disease, long-term delivery of one or more neurotrophic factors
such as NGF, BDNF, NT-3, NT-4/5, CNTF, GDNF and CDF/LIF may be
required to maintain neuronal viability. These growth factors
cannot be delivered through systemic administration as they are
unable to traverse the blood-brain barrier. Therefore, it may be
necessary to deliver such neurotrophic factors into the central
nervous system as a prodrug that is able to cross the blood-brain
barrier. The present invention contemplates preparing prodrugs of
such biologically active agents, and using an encapsulated reaction
center to convert the prodrug into such an agent in the central
nervous system. For example, CNTF is a potential therapeutic agent
for Huntington's disease. Emerich et al. Nature 386:395-99 (1997).
As discussed in more detail below, one of skill in the art could
prepare a prodrug for CNTF so that a specific encapsulated reaction
center would be capable of converting the prodrug to CNTF. As one
example of such a matching between a prodrug and a reaction center,
insulin may be obtained from recombinant proinsulin by reaction
with carboxypeptidase or trypsin. Markvicheva et al. App. Biochem.
Biotechnol. 61:75-84 (1996).
[0125] In certain embodiments of the present invention, an antibody
may be encapsulated as the reaction center and the matrix
administered to the subject. The matrix would be used to isolate
deleterious biologically active agents from the subject in the
matrix, as opposed to modifying as would a reaction center that
reacts with such a biologically active agent. After all the
antibodies have bound their corresponding hapten, the matrix may or
may not be removed from the subject.
[0126] In certain embodiments matrices of the present invention may
be used as prodrug activators in vivo after administration of the
matrix and, if necessary, any prodrug, to a subject. Alternatively,
the present invention contemplates using a matrix to convert a
prodrug into a biologically active agent ex vivo, whereupon the
biologically active agent is administered to a subject, as
described in more detail in U.S. Pat. No. 5,378,232.
[0127] 5.2.2. Enzyme Replacement, Augmentation, or
Supplementation
[0128] In other aspects of the present invention, a reaction center
is encapsulated in a matrix to replace or augment some lost
biological activity that is generally present in a healthy subject,
e.g., are without the disease or condition to be treated. Certain
embodiments of the production of dopamine described above are
examples of such replacement or augmentation. The encapsulated
reaction center may restore or augment vital metabolic functions,
such as the removal of toxins or harmful metabolites. Lost
biological activity might result from loss of activity generally,
or loss of activity in some location, e.g., a specific type of
tissue. The loss may be attributable to a genetic defect, for
example, an inborn error of metabolism, or may be caused by some
disease or condition. If loss of function is complete, then this
aspect of the invention may be referred to as enzyme replacement,
whereas if loss of function is only partial, then this aspect of
the invention may be referred to as enzyme augmentation. In other
embodiments, although there has been no loss of enzyme function,
enzyme augmentation may produce a desirable therapeutic effect. In
other embodiments, the enzyme encapsulated is new to the subject,
that is, there is supplementation. Through use of certain
embodiments of the present invention, homeostasis of particular
substances can be restored and maintained for extended periods of
time.
[0129] Based on conditions and diseases that those of skill in the
art know to result from loss of a particular enzymatic activity,
reaction centers that replace or augment lost or diminished
biological activity may be readily identified. Certain embodiments
of the present invention have advantages over more conventional
types of enzyme replacement therapy (ERT), which often rely on
administration of an enzyme whose activity is lost or diminished.
The matrices of the present invention for the most part may be
biocompatible and immunoisolatory with respect to any encapsulated
reaction center, whereas enzyme administration can result in
hypersensitivity and/or anaphylactic reaction during or immediately
after enzyme infusion. Brooks et al., Biochem. Biophys. Acta
1497:163-72 (1998). The development of antibodies to any enzyme
used in ERT may preclude its use in long-term therapeutic regimes
or following relapses. Likewise, the present invention may avoid
the need to dermitize an enzyme used in ERT with polyethylene
glycol (PEG). Goldberg et al. Biomedical Polymers 441-52 (Academic
Press 1980). Certain embodiments of the present invention, by
encapsulating an enzyme as the reaction center, prevent
degradation, and thereby may provide for prolonged treatment upon
administration of the matrix. In contrast, for ERT, multiple
infusions of the enzyme may be required for sustained therapy. Even
erythrocyte-entrapped enzymes may show only modest increases in
activity. See, for example, Thorpe et al. Pediatr. Res. 9:918-23
(1975).
[0130] By way of illustration, ERT has been used to treat Gaucher's
disease. See generally Morales, Ann. Pharmacother. 30:381-88
(1996). Gaucher's disease is caused by a genetic deficiency of the
enzyme glucocerebrosidase, and results in accumulation of
glucocerebrosidase within the reticuloedothelial system. Symptoms
include hepatosplenomegaly, bone marrow suppression, and bone
lesions. There are three subtypes, of which the most common, type
1, is non-neuronopathic. Types 2 and 3, which are neuronopathic,
may result in nerve cell destruction. Enzyme treatment first began
with a placentally derived form of glucocerebrosidase, Dale et al.
Proc. Natl. Acad. Sci. USA 73:4672-74 (1976), which has been
replaced with a recombinant version of the enzyme. The enzyme
treatment must be repeated every two weeks, and it has been
effective in reducing hepatosplenomegaly, improving anemia and
thrombocytopenia, and general health. Numerous studies have been
completed on ERT for Gaucher's disease. Magnaldi et al., Eur.
Radiol. 7:486-91 (1997); Ueda et al., Acta Paediatr. Jpn. 38:260-04
(1996); Lorberboym et al., J. Nucl. Med. 38:890-95 (1997); Charrow
et al., Arch Inter. Med. 158:1754-60 (1998). The present invention
contemplates encapsulation of this enzyme in a matrix and
administration to a subject suffering from Gaucher's disease for
treatment.
[0131] ERT has also been effected by using materials that act by
slow release. For example, L-asparaginase has been loaded into
nanoparticles composed of polymers that release the enzyme over a
few weeks, or loaded into erythrocytes, Updike et al. J. Lab. Clin.
Med. 101:679-91 (1983). The enzyme may be useful for treating
cancer, especially acute lymphocytic leukemia. By encapsulating
this enzyme as the reaction center in a matrix, the enzymatic
activity may be present for longer periods than that made possible
by using slow-release methods.
[0132] In another embodiment of the present invention, transport
proteins such as hemoglobin may be encapsulated to produce an
artificial red blood cell. In such an embodiment, the matrix must
be of appropriate morphology to travel throughout the vasculature
of a subject.
[0133] In addition to the examples already discussed, almost any
naturally occurring enzyme may be used in to augment or replace
enzymatic activity, and is therefore a candidate for this use. Some
other possible enzymes that may be used as reaction centers, and
the disease or condition that they may treat, are listed below.
(These enzymes may be used to treat other diseases and conditions
as well.)
2 Enzyme Disease or condition Reference .alpha.-1,4-Glucosidase
Type II glycogenosis (Pompe's disease) .alpha.-Galactosidase
Fabry's disease (heart and kidney failure due to ceramide
accumulation) .alpha.-L-iduronidase mucopolysaccharidosis Kakkis et
al. Biochem. Mol. type I. Med. 58:156-67 (1996)
.beta.-glucuronidase mucopolysaccharidosis type O'Connor et al. J.
Clin. VII Invest. 101:1394-400 (1998) Aminolaevulinate Lead
poisoning Bustos et al. Drug Des. Deliv. dehydratase 5:125-31
(1989) Bilirubin oxidase jaundice Catalase Acatalasemia
Fibrinolysin Thromboembolic occlusive vascular disease Glutaminase
(e.g., from Cancer Pseudomonas putrefaciens) Hemoglobin Respiratory
Heparinase (e.g., from Extracorporeal circulation Flavobacterium
heparinum) L-arginine ureahydrolase Hyperargininemia Wissmann et
al. Somot. Cell (Al), Arginase Mol. Genet. 22:489-98 (1996) Liver
microsomal enzymes Liver failure Brunner et al. Artif. Organs
(e.g., from rabbit liver) 3:27-30 (1979); U.S. Pat. No. 5,849,588
Phenylalanine ammonia lyase Phenylketonuria Bourget et al. Biochim
(e.g., from Rhodotorula Biophys Acta 883:432-48 glutinis) (1986)
Streptokinase (e.g., from Thromboembolic occlusive Streptococcus
sp.) vascular disease Superoxide dismutase (e.g., Inflammatory
diseases Ledwozyw Acta Vet Hung from bovine liver), catalase
thought to be mediated by 39:215-24 (1991); Turrnes et oxygen free
radicals, e.g., al. J. Clin. Invest. 73:87-95 bleomycin-induced
lung (1984) fibrosis Terrilythin Peritonitis Tyrosinase Liver
failure UDP Glucuronyl transferase Jaundice, liver disease (e.g.,
from rabbit liver) Urea cycle enzymes Liver failure Urease Renal
failure Uricase (e.g., from hog liver) Hyperuricemia due to gout
Urokinase (e.g., from human Thromboembolic occlusive urine)
vascular disease See generally Klein et al. TIBTECH July 1986,
179-86.
[0134] 5.2.3. Addiction Neutralization
[0135] In another aspect of the invention, the encapsulated
reaction center is chosen to degrade biologically active agents
that may result in addiction. Efforts to combat addiction, e.g.,
cocaine addiction, have included inducing anti-drug antibodies
specific to the drug. See generally U.S. Pat. No. 5,840,307. In
certain embodiments, the present invention may help to neutralize
the addiction, or in other words, cause addiction neutralization.
For example, investigators have encapsulated alcohol dehydrogenase
and/or acetaldehyde dehydrogeanse in human erthrocytes and reported
the continuous degradation of ethanol for up to seventy hours.
Lizano et al. Biochem. Biophys. Acta 1425:328-36 (1998). See also
U.S. Pat. No. 5,759,539. Encapsulation of either or both of these
enzymes in a matrix may allow for the complete metabolization of
ethanol upon administration of the matrix, which would thereby
combat addiction by neutralizing the addictive agent, ethanol.
[0136] In another example, catalytic antibodies have been elicited
that are capable of aiding hydrolysis of the cocaine molecule.
Landry et al. Science 259:1899-1901 (1993). See also U.S. Pat. No.
5,730,985. Encapsulation of such catalytic antibodies in a matrix
of the present invention and administration to a subject addicted
to cocain may allow for neutralization of any ingested cocaine. The
present invention, by encapsulating the catalytic antibody, may
avoid some of the drawbacks usually associated with passive
antibody therapy. In the same fashion, reaction centers targeted at
biologically active agents responsible for other types of addition
are known to those of skill in the art, and may be used in a
similar fashion.
[0137] 5.2.4. Mutagenic Assays
[0138] In another aspect of the invention, the present invention
contemplates using matrices as metabolic activating systems for
use, for example, as toxicology screens for cytotoxic and
pharmaceutical compounds in vivo. Such a use may reduce the need
for laboratory animals for toxicology testing. Numerous efforts
have been made to prepare human liver epithelian cell lines, and
liver cell and tissue culture systems for such uses. See, for
example, U.S. Pat. Nos. 5,849,588 and 5,759,765. In one report,
enzymes responsible for deactivation of many endogenous toxins have
been isolated and covalently bound onto a hemocompatible form of
agarose support. Brunner et al., Artif. Organs 3:27-30 (1979). In a
like manner, the present invention contemplates encapsulating such
enzymes in matrices of the present invention and evaluating
suspected mutagens. See generally Rueff et al Mutat. Res.
353:151-76 (1996). Enzymes usually found in the liver, such as
cytochrome P-450 for example, may be used in this or related
embodiments. Janig et al. Acta Biol. Med. Ger. 38:409-22
(1979).
[0139] 5.2.5. Tissue Assist Devices
[0140] In addition to the many methods and uses described herein in
which the subject matrices are administered for in vivo use, the
matrices of the present invention may be used ex vivo. Many of the
teachings herein described for in vivo use apply as well to ex vivo
use (and visa-versa).
[0141] In one aspect of the present invention, one example of an ex
vivo use is a tissue assist device, and in certain embodiments, an
organ assist device. In such a device, matrices encapsulating one
or more reaction centers could be used to replace, augment or
supplement the biological function of an organ or other tissue. It
is important to note that for this embodiment (and others described
herein in which the prodrug converted by the reaction center is
potentially deleterious to the subject being treated), it is not
always necessary that the prodrug be converted into the same
agent(s) that the enzymes and other catalysts usually present in
the tissue would have were it to react with the prodrug. In certain
embodiments of the subject invention, it is not the product of the
prodrug that is critical; instead it is the transformation of the
prodrug into a less toxic or otherwise undesirable compound(s) that
is the primary concern. For example, this principle applies to
certain of the embodiments used for addition neutralization
described above as well as certain tissue assist devices.
[0142] One example of such an assist device is particularly
well-suited to the subject matrices is an hepatic assist device.
Liver transplantation has become widely accepted as an effective
treatment for chronic and acute liver disease. One of the major
problems associated with the transplantation process, however, as
been the need for an effective means for providing temporary
support for patients awaiting an available donor organ.
Extracorporeal devices that are effective for liver support has
proven more elusive. See, for example, Takahashi et al., Digestive
Diseases and Sciences 36(9) (1991).
[0143] In certain embodiments of the present invention, one or more
enzymes generally localized in the liver of a patient could be
encapsulated in a subject matrix, and blood and other bodily fluids
of the patient could be passed through and over these matrices ex
vivo to augment the biological activity usually associated with the
liver. Functions of the liver that could be addressed by such liver
assist devices include, among others, carbohydrate, fat and protein
metabolism and detoxification of drugs, hormones and other
substances. At the point that the encapsulated reaction centers of
the matrices are exhausted or otherwise less efficient than
desired, they may be readily replaced by providing with new and
more efficient matrices.
[0144] A variety of reaction centers could be encapsulated for such
a liver assist device. Examples include cytochrome P-450, other
enzymes usually located in the liver, a less than highly purified
mixture of biologicals isolated from livers, transformed cells such
as those derived from hepatoblastoma cell lines (Sussman et al.,
Hepatology 16:60-65 (1992)), cultured or isolated hepatocytes (U.S.
Pat. No. 5,866,420; Rozga et al., Hepatology 17:258-65 (1993);
Rozga et al., Ann. Surg. 217:502-11 (1994)), cells from
hepatocarcinoma-derived cell lines (Richardson et al., J. Cell
Biol. 40:23647 (1969); Aden et al., Nature (London) 282:615-16
(1970)), Kupffer cells and other biologicals that are capable of
replacing, augmenting or supplementing the biological function of
the liver. For other examples of possible biologicals and liver
assist devices, see, for example, U.S. Pat. Nos. 6,008,049,
5,849,588, 5,290,684, 5,270,192, 5,043,260, 4,853,324, 3,734,851;
and WO 93/16171. Sources of suitable enzymes and biologicals for a
livers assist device for humans include, for example, porcine and
other mammals. In particular, use of certain biologicals, including
for example hepatocytes, has proved difficult because their
instability, and encapsulation of such biologicals in matrices of
the present invention may improve their stability.
[0145] A variety of bioreactor techniques known to those of skill
in the are could be used with such an assist device, including for
example, hollow fiber techniques, static maintenance reactor
systems, fluidized bed reactors, microporous membranes and
flat-bed, single-pass perfusion systems. See, for example, U.S.
Pat. Nos. 4,200,689, 5,081,035, 3,997,396; and WO 90/13639;
Halberstadt et al., Biotechnology and Bioengineering 43:740-46
(1994).
[0146] In addition to liver assist devices, other organs or
functions of a patient could be treated using matrices of the
present invention ex vivo.
[0147] 5.3. Matrices
[0148] The concept of encapsulating or immobilizing a reaction
center in or on a matrix of some kind is well precedented. For
example, significant efforts have been made to immobilize enzymes
on solid supports. Handbook of Enzyme Biotechnology (2d ed., ed.
Wiseman 1985). In these and other examples, encapsulation or
immobilization of the reaction center may impart desirable
characteristics on the reaction center.
[0149] A number of matrix chemistries that may be used in the
present invention have been used to immobilize enzymes or
biologically active agents. For instance, cells have been attached
to glass beads and implanted in rats. Cherksey et al. Neuroscience
75:657-64 (1996). Reaction centers may be immobilized on a type of
porous zirconia. Huckel et al., J. Biochem. Biophys Methods
31:165-79 (1996). Alternatively, reaction centers may be attached
to supports through silane coupling. Weetall, Appl. Biochem.
Biotechnol. 41:157-88 (1993). Biologics may be immobilized within a
composite fibre by using a gel formation of cellulose derivative
and metal alkoxide, e.g., titanium isopropoxide. Hatayama et al. J.
Sol-Gel Sci. & Tech. 7:13-17 (1996); Ohmori et al. J.
Biotechnol. 33:205-09 (1994). Poly(vinyl alcohol) synthetic polymer
foams may be used. Li et al. J. Biomater. Sci. Polm. Ed. 9:239-58
(1998). Other polymers known in the art may be used. See, for
example, U.S. Pat. Nos. 5,529,914 and 5,780,260; WO 93/16687. As
described in greater detail below, inorganic-based or silica-based
sol-gel matrices are contemplated by the present invention. Some
examples of suitable inorganic-based matrices include those
disclosed in the following references: Mazei et al., J. Materials
Chemistry 8:2095-101 (1998); Yoldas, J. Mater. Sci. 1098-92 (1986);
and Curran et al., Chemistry of Materials, 10:3156-66 (1998).
[0150] One feature of the matrix in certain embodiments of the
present invention is its ability to prevent leaching of any
encapsulated reaction center, at least to the extent necessary for
the intended use of the matrix. In certain embodiments of the
present invention, there may be negligible leaching. In others,
there may be some leaching, but usually only a small amount over
time. If leeching proses to be excessive, either the material to be
encapsulated, e.g., a reaction center or an additive, or the
sol-gel matrix may be modified to improve leaching characteristics,
e.g., reduce leaching. For example, to reduce leaching, the
reaction center may be derivatized to increase its size. For
example, an enzyme may be chemically modified to create derivatives
by forming covalent or aggregate conjugates with other chemical
moieties, such as glycosyl groups, lipids, phosphate, acetyl
groups, PEG, and the like. Covalent derivatives may be prepared by
linking the chemical moieties to functional groups on amino acid
sidechains of the protein or at the N-terminus or at the C-terminus
of the polypeptide. Alternatively, a fusion or chimeric polypeptide
retaining at least some of the activity of the enzyme may be used.
Alternatively, the reaction center of additive may be attached to
the sol-gel matrix in some fashion, e.g., by covalently
bonding.
[0151] The manner in which a reaction center is encapsulated in a
matrix, be it for example by physical entrapment, covalent
attachment, or some other physical attraction, may affect the
properties of such reaction center. For example, the micro
environment around any covalently attached reaction center may
differ from that encountered by the same reaction center
encapsulated during gelation of the sol-gel, and any difference may
affect the activity of the center. Thus, the present invention
contemplates adjusting encapsulation, if necessary, for each
intended use.
[0152] In preparing any matrix, the encapsulated material, e.g. the
reaction center and additives, must be robust enough to retain
their usefulness after being encapsulated. For example, many
biological materials may not be able to survive the high
temperatures and harsh conditions required to prepare some
inorganic materials. Consequently, such inorganic materials may not
be used with sensitive biolgicals. In the present invention,
matrices are matched with the reaction center(s) or additive(s) to
be encapsulated therein so as to retain sufficient activity of the
reaction center.
[0153] Another feature of the present invention is the ability of
the matrix to stabilize, in certain cases, the encapsulated
reaction center. For example, the present invention may protect
against degradation of any encapsulated biological material by
naturally occurring systems, such as proteases. The matrix may
protect against thermal denaturation of any encapsulated biological
materials. Finally, the matrix may even assist in the correct
re-folding of any denature polypeptide chain. Heichal-Segal et al.
Bio/Technology 13:798 (1995).
[0154] 5.3.1. Silica-based Sol-gel Matrices
[0155] In one aspect of the present invention, reactions centers
are encapsulated in silica-based sol-gel matrices. Silica-based
sol-gels have been applied to encapsulate a wide range of
materials, including biological materials, small organic molecules,
antibodies, antigens, and organic catalysts. See, for example,
Ellerby et al. Science 255:1113-15 (1992); Dave et al. Analytical
Chem. 66:1120-27 (1994); Avnir et al. Acc. Chem. Res., 28:328-34
(1995); Avnir et al. Chem. Mater., 6:1605-14 (1994) (listing
encapsulated purified enzymes and whole-cell extracts and whole
cells); Biochemical Aspects of Sol-Gel Science and Technology (eds.
Avnir et al. 1996); Shtelzer et al. Biotechnol. Appl. Biochem.
15:227-35 (1992). Biological materials encapsulated in
inorganic-based or silica-based sol-gel matrices have retained
significant activity for a substantial time period.
[0156] Inorganic-based sol-gels, and in particular, silica-based
sol-gels, have a variety of characteristics that are useful for
encapsulation of reaction centers and implantation in vivo. See
generally Dunn et al. Acta Mater. 46:737-41 (1998); Avnir et al.
Chem. Mater., 6:1605-14 (1994). Any or all of these features may or
may not be present in particular embodiments of the present
invention. Some such features include: stability to heat, light (no
photodegredation), and electrical current (no electrochemical
degradation); transparent in the visible region and into the UV-Vis
region; controllable surface area and porosity (average pore size
and pore size distribution); possibility of controlling
conductivity by appropriate use of other inorganic alkoxides during
preparation of the gel or addition of additives; capable of being
readily modified chemically; improved stability of any encapsulated
material, e.g., reaction center or additive, because of the rigid
matrix; little or no leaching of any encapsulated material; readily
manipulated in a variety of physical morphologies; and isolatory of
any encapsulated material from the surrounding environment, except
for any substance that is able to diffuse into the matrix. Many of
these features are explained in greater detail below.
[0157] One area of interest involves using doped sol-gels as
chemical sensors. As part of that effort, sol-gels have been used
to encapsulate enzymes and antibodies. Avnir, Acc. Chem. Res., 28:
328-334 (1995); Akbarian et al. J. Sol-Gel Sci & Tech.
8:1067-70 (1997). Immunosensors have been prepared using sol-gel
technology. Wang et al. Anal. Chem. 15:1171-75 (1998). For-example,
the enzyme glucose oxidase has been examined upon encapsulation in
a silica-based sol-gel matrix for use as a glucose sensing
material. Yamanaka et al. Chem. Mater. 4:495 (1992); Audebert et
al. Chem Mater. 5:911-13 (1993). Such sol-gel preparations have
been used as electrodes for electrochemical assays of glucose
concentrations. Sampath et al. J. Sol-Gel Sci. & Tech. 7:123-28
(1996).
[0158] Another area of interest of silica-based sol-gel technology
has been encapsulating enzymes for use as organic catalysts in a
variety of applications, including synthesis of chiral materials.
Jaeger et al. TIBTECH 16:396-403 (1998).
[0159] Silica-based sol-gel matrices have also been used as
controlled-release carriers of biologically active agents. See, for
example, U.S. Pat. No. 5,849,331 and WO 97/45367.
[0160] (a) Preparation
[0161] Modifications in well-known sol-gel processes permit the
incorporation of enzymes or other biologically derived reaction
centers in silica-based sol-gel matrices. See generally Avnir et
al. Chem Mater. 6:1605 (1994); U.S. Pat. Nos. 5,824,526, 5,650,311;
5,650,311; 5,371,018; 5,308,495; 5,300,564; 5,292,801.
[0162] Silica-based sol-gel matrices of the present invention may
be prepared in the sol-gel method by polymerization of a metal
alkoxide precursor. See generally Bruce Dunn et al. Chem. Mater.,
9:2280-91 (1997). The polymerization process is well documented and
known to proceed by the formation of colloidal silica particles. A
suspension of these particles is termed a sol. The synthesis
generally involves the use of metal alkoxides which may undergo
hydrolysis and condensation polymerization reactions. The
preparation process can ordinarily be divided into the following
steps: forming a solution, gelation, ageing, drying, and
densification. In the preparation of a silica-based matrix, one
starts with an appropriate alkoxide, for example,
Si(OC.sub.2H.sub.5).sub.4, tetraethyl orthosilicate or TEOS, or
Si(OCH.sub.3).sub.4, tetramethyl or thosilicate or TMOS, which is
mixed with water and a solvent, e.g., the alcohol of the alkoxide,
ethanol or methanol, to form a solution. A number of reactions
result, including hydrolysis, which leads to the formation of
silanol groups Si--OH, and condensation, which gives siloxane
Si--O--Si groups.
[0163] There are several parameters which influence the hydrolysis
and condensation polymerization reactions, including the
temperature, solution pH, particular alkoxide precursor and
solvent, and relative concentrations of the alkoxide precursor,
water, and solvent. Such parameters may be important to retaining
activity when the reaction center encapsulated is an enzyme or
other biological. For example, in encapsulating enzymes, greater
enzyme activity may been preserved by not adding any alcohol at the
start of polymerization. Another improvement may result from
buffering the reaction solution to some pH suitable for any
pH-sensitive materials to be encapsulated after the acid-catalyzed
hydrolysis of the oxysilanes. Ellerby et al. Science 255:1113-15
(1992); Rietti-Shati et al. J. Sol-Gel Sci. & Tech. 7:77-79
(1996).
[0164] Initial hydrolysis of the precursor alkoxide is catalyzed by
protons or hydroxide ions. It is possible to control the matrix
characteristics by controlling the rates of the individual steps by
which the matrix is condensed. Acidic catalysis tends to increase
the rate of hydrolysis and disfavors the condensation reactions
necessary to form the sol-gel, whereas base hydrolysis produces
rapid condensation. If the reaction center to be encapsulated is
not sensitive to pH conditions, the formation of the gel matrix can
be achieved fairly rapidly. However, in the case of reaction
centers or additives which may be sensitive to extreme pH
conditions, such as enzymes, the pH of the sol must be adjusted
prior to addition. Hence, preparation of the silica-based sol-gel
may involve buffering the sol before the addition of the reaction
center or other additives. For example, to retain the activity of
bacteriorhodopsin, the solution was buffered to pH 9 after addition
of the polypeptide. Weetall et al. Biochem Biophys. Acta
1142:211-13 (1993).
[0165] As the hydrolysis and condensation of polymerization
reactions continue, viscosity increases until the solution ceases
to flow. This sol-gel transition is irreversible, and at this stage
the one-phase liquid is transformed to a two-phase system. The gel
may consist of amorphous primary particles of variable size (5-10
nm or smaller) with an interstitial liquid phase. At this stage the
pores have yet to shrink and the liquid phase fills the pores.
After gelation, gels are generally subjected to an aging process
during which the gels are sealed and very little solvent loss or
shrinkage occurs. Condensation reactions continue, increasing the
degree of cross-linking in the network.
[0166] The drying process involves the removal of the liquid phase.
Ambient temperature evaporation may be employed, and there is
considerable weight loss and shrinkage. It is at this stage that
pore collapse may occur, deceasing pore size and thus decreasing
the solvent volume. The combination of these effects causes an
increase in the interaction between the reaction center and the
matrix. The final stage of the sol-gel process is that of
densification. It is at this point that the gel-to-glass conversion
occurs and the gel achieves the properties of the glass. Matrices
are found to contract to one eighth the pre-dried volume and are
termed "xero-gels." The drying process may affect the accessibility
of any encapsulated reaction center, and by adjusting such process,
the present invention contemplates another means of influencing the
activity of any encapsulated reaction center. Wamboldt et al. J.
Sol-Gel Sci & Tech 7:53-57 (1996).
[0167] (b) Composition and Characteristics
[0168] Any number of alkoxide precursors may be used in preparing
silica-based sol-gel matrices of the present invention. Those
silica-based sol-gel matrices prepared from oxysilanes other than
Si(OR.sup.1).sub.4 are known as organically modified silica
matrices, or Ormosil matrices. In preparing the matrices of the
present invention, for example, alkoxides of the form
Si(OR.sup.1).sub.4, R.sup.2Si(OR.sup.1).sub.3,
R.sup.2.sub.2Si(OR.sup.1).sub.2, R.sup.2.sub.3Si(OR') may be used,
in which each R.sup.1 is independently methyl, ethyl, or any
lower-weight alkyl (although the identity of R.sup.1 is usually the
same in any type of oxysilane), and R.sup.2 is independently any
alkyl, aryl, or other substituent that does not interfere
substantially with formation of the sol-gel, as discussed in more
detail below. A significant difference between R.sup.1 and R.sup.2
is that the R.sup.1 alkoxide the majority of R.sup.1 is hydrolyzed
during gelation, whereas the R.sup.2 substituent remains part of
the matrix. Because R.sup.2 is not hydrolized but remains in the
sol-gel matrix, the identity of R.sup.2 may have a significant
affect on the sol-gel matrix and any material encapsulated therein.
In contrast, for R.sup.1, much of which is hydrolized during
gelation, may not constitute a significant percentage of the
sol-gel matrix that results. Even so, the identity of R.sup.1 may
be important to the reaction, because HOR.sup.1, which is produced
upon hydrolysis of the oxysilane, may affect the formation of the
sol-gel and any material encapsulated therein. Accordingly, for
example, some biolgicals to be encapsulated may be stable to some
HOR.sup.1 and not others. In certain preferred embodiments, the
alkoxide used is Si(OR.sup.1).sub.4, in which R.sup.1 is methyl or
ethyl.
[0169] In certain embodiments, R.sup.2 may contain functional
groups. For example, aminopropyl, which has an amine functional
group, and mercaptopropyl, which has a thiol functional group, have
been used as R.sup.2 in preparing sol-gel matrices from
R.sup.2Si(OR.sup.1).sub.3 and mixtures of R.sup.2Si(OR.sup.1).sub.3
and Si(OR.sup.4. Collino et al. J. Sol-Gel Sci. & Tech 7:81-85
(1996); Venton et al. Biochim Biophys Acta 1250:117-25 (1995). Such
sol-gel matrices were used to prepare thin films. Almost any
chemical moiety may be used as R.sup.2 in the present invention as
long as any functional groups contained therein are not adverse to
formation of the sol-gel matrix. For those functional groups that
may not be compatible with the sol-gel chemistry, it may be
possible to protect them using standard protection technologies
know in the art of organic chemistry and then deprotect them after
preparation of the sol-gel matrix is complete.
[0170] Functional groups may be used to increase the stability or
reactivity of any encapsulated reaction center, especially when the
reaction center is a biologic. For example, functional groups
having hydrolizable functional groups, such a phenol or amine, may
affect the local pH, thereby improving reactivity or stability of
the encapsulated reaction center, or directly assist catalysis or
stabilize an enzyme. Alternatively, functional groups may affect
the characteristics of the surface of the matrix, which may affect
the biocompatability of the matrix. In addition, by incorporating
functional groups into the matrices of the present invention, the
exterior characteristics of the matrix may be altered by
derivitization. For example, chemical moieties, such as glycosyl
groups, lipids, phosphate, acetyl groups, PEG, and the like, could
be attached to the surface of the matrix though such functional
groups.
[0171] In other embodiments of the present invention, R.sup.2 may
incorporate chemistry that allows for covalent attachment of any
reaction center or additive directly to the sol-gel matrix. For
example, a reaction center or additive could be covalently attached
to the silicon of an oxysilane as R.sup.2 through a linker bound to
the silicon or other morganic. Such a modified silica alkoxide
could be reacted with nonsubstituted silica alkoxides to form the
sol-gel of interest. In another embodiment, the moiety covalently
attached to the silica alkoxide could be a biotin group, and the
reaction center or additive could be attached to avidin. The
biotin/avidin interaction would effectively attach the reaction
center or additive to the silica oxide framework of any sol-gel
matrix. An antibody/hapten pair could be used in the same
fashion.
[0172] For certain embodiments, the alkoxide used may be a single
silica alkoxide, or a mixture of silica alkoxides. Reetz et al. J.
Sol-Gel Sci. & Tech. 7:35-42 (1996). When using silica oxides
of structure R.sup.2Si(OR.sup.1).sub.3, R.sup.2.sub.2Si(OR.sup.2,
R.sup.2.sub.3Si(OR.sup.1), it may be necessary to react them with
sufficient Si(OR.sup.1).sub.4 to allow for adequate gelation and
formation of a physically robust matrix. For instance, the
oxysilanes substituted with non-hydrolizable substituents may
constitute 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,
90, or 100 percent of the oxysilane used in the preparation. If a
oxysilane has non-hydrolizable substituents, the percent of that
oxysilane may need to be less to ensure sufficient gelation.
[0173] Modification of the framework of a silica-based sol-gel by
variation of the precursor alkoxide presents the possibility of
tailoring the microenvironment of the encapsulated reaction center.
In this fashion, it is possible to, for example, maximize the
reactivity of any encapsulated reaction center. Increasing the
number of alkyl groups as well as increasing chain length of the
alkyl groups in the precursor material may produce increased matrix
hydrophobicity. Such hydrophobicity, for example, may be conducive
to stabilization of the reaction center. The local pH within the
sol-gel may also be affected by the chemical identity of the
matrix. In one report, the activity of an encapsulated enzyme
increased upon using a mixture of oxysilanes with various alkyl
substituents. Reetz et al. Angew. Chem. Int. Ed. Engl. 34:301-303
(1995). By varying the chemical identity and ratio of different
oxysilanes, the present invention contemplates customizing the
sol-gel matrix for each encapsulated reaction center, e.g., to
maximize catalytic activity. Other features of the matrix may
depend on the chemical identity of the oxysilane precursors, for
example wettability, which may affect the biocompatibility of any
matrix upon implantation. For example, cell adhesion and growth may
depend on the wettability of the matrix. Altankov et al. J. Biomed.
Mater. Res. 30:385-91 (1996); Altankov et al. J. Biomater. Sci.
Polym. Ed. 8:299-310 (1996).
[0174] Pore size is an important characteristic of any sol-gel
matrix, because it may affect what materials, e.g., prodrugs, may
diffuse in and out of the matrix, and the leachability of any
encapsulated reaction center(s) and/or additive(s). A number of
reports indicate that the pore size and/or shape may be varied by
adjusting the synthetic conditions by which any material is
encapsulated in a sol-gel. Dave et al. ACS Symp. Ser. 622:351
(1996). The present invention contemplates pore sizes ranging from
the angstrom level to the micron level.
[0175] In addition to preparing silica-based sol-gel matrices from
silica oxides, other oxides, including metal oxides, may be used to
encapsulate a reaction center in inorganic-based sol-gel matrices.
In one report, glucose oxidase was encapsulated within vanadium
pentaoxide, and the resulting sol-gel was used in electrochemical
studies. Glezer et al. J. Am. Chem. Soc., 115:2533-34 (1993). A
vanadium alkoxide has been co-condensed with TEOS, thereby
imparting the properties, including reactivity, of oxovanadium(V)
functional groups to the matrix. Stiegman et al. Chem. Mater.
5:1591-94 (1993). Oxysilines and other metal oxides may be combined
in any sol-gel matrix. Silica-based sol-gel matrices in which redox
active metal ions constitute part of the sol-gel framework may
prove useful in promoting reactions involving electron transfers
such as reductions and oxidations within the sol-gel itself. For
example, an electron source foreign to the matrix may transfer an
electron to a reaction center encapsulated in such a mixed metal
sol-gel by electron transfer through a pathway involving the metal
atoms in the framework of the sol-gel. Soghomonian et al. Chem.
Mater. 5:1595-97 (1993).
[0176] The local environment, or micro-environment, immediately
surrounding any encapsulated reaction center of the present
invention may play an important role in affecting the capability of
such reaction center to catalyze the conversion of prodrug to
biologically active agent. A number of studies have been completed
to better characterize the nature of the microenvironment around
any encapsulated material in a sol-gel. Samuel et al. Chem Mater.,
6:1457-61 (1994); Zheng et al. Anal Chem. 69:3940-49 (1997); Dave
et al. J. Sol-Gel Sci & Tech. 8:629-34 (1997); Avnir et al. J.
Phys. Chem. 88:5956-59 (1984). By adjusting any of the variables
delimited above, the properties of the silica-based sol-gel matrix
may be readily tailored by one of skill in the art to the reaction
center encapsulated.
[0177] 5.3.2. Other Features of the Matrix
[0178] The matrix may be immunoisolatory with respect to the
reaction center or other contents. Use of immunoisolatory matrices
allows the implantation of alkogenetic or xenogeneic reaction
centers and other additives, without a concomitant need to
immunosuppress the subject. Using immunoisolatory matrices, it is
possible to implant reactions centers that are foreign to the
subject, such as nonmamallian enzymes, provided that critical
substances necessary to the mediation of immunological attack are
excluded from the implant. These substances may comprise the
complement attack complex component Clq, or they may comprise
phagocytic or cytotoxic cells; the instant immunoisolatory matrix
protects against these harmful substances.
[0179] The present invention allows for coating or otherwise
modifying the exterior of the matrix. Such a coating may render the
matrix immunoisolatory. U.S. Pat. No. 5,676,943. For example, the
coating or other modification may confer protection of the reaction
center or other contents from the immune system of the host in whom
the matrix is implanted, by providing a physical barrier sufficient
to prevent detrimental immunological contact between the reaction
center and other additives and the host's immune system. The
thickness of a coating may vary, but it will always be sufficiently
thick to prevent direct contact between the reaction center and the
elements of the host's immune system. The thickness generally
ranges between 5 and 200 microns; thicknesses of 10 to 100 microns
are preferred, and thickness of 20 to 75 microns are particularly
preferred. Types of immunological attack which can be prevented or
minimized by the use of a coating or other modification include
attack by macrophages, neutrophils, cellular immune responses (e.g.
natural killer cells and antibody-dependent T cell-mediated
cytoloysis (ADCC), and humoral response (e.g., antibody-dependent
complement mediated cytolysis).
[0180] Various polymers and polymer blends can be used to
manufacture a coating, including polyacrylates (including acrylic
copolymers), polyvinylidenes, polyvinyl chloride copolymers,
polyurethanes, polystyrenes, polyamides, cellulose acetates,
cellulose nitrates, polysulfones, polyphosphazenes,
polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well
as derivatives, copolymers and mixtures thereof.
[0181] The solvents used in conjunction with the above-identified
polymers in forming the coating will depend upon the particular
polymer chosen. Suitable solvents include a wide variety of organic
solvents such as alcohols and ketones generally as well as
dimethylsulfoxide (DMSO), dimethylacetamide (DMA), and
dimethylformamide (DMF) and blends of these solvents as well.
[0182] The coating may also include a hydrophobic matrix such as an
ethylene vinyl acetate copolymer, or a hydrophilic matrix such as a
hydrogel. The coating may be post-production coated or treated with
an impermeable outer layer such as a polyurethane, ethylene vinyl
acetate, silicon, or alginate.
[0183] The polymeric solution for the coating may also include
various additives such as surfactants to enhance the formation of
porous channels and antioxidants to sequester oxides that are
formed during the coagulation process. Exemplary surfactants
include Triton-X 100 available from Sigma Chemical Corp. and
Pluronics P65, P32, and P18. Exemplary anti-oxidants include
vitamin C (ascorbic acid) and vitamin E.
[0184] In addition, anti-inflammatory agents can also be
incorporated into the coating to reduce immune response. Exemplary
anti-inflammatory agents include corticoids such as cortisone and
ACTH, dexamethasone, cortisol, interleukin-1 and its receptors and
agonists, and antibodies to TGF, interleukin-1, or
gamma-interferon. Alternatively, these materials may be added to
the implant after formation by a post-coating or spraying process.
For example, the implant could be immersed in a solution containing
an anti-inflammatory agent.
[0185] Post-coating procedures can also be used to provide a
protective barrier against immunogens and the like. For example,
after formation, the matrix may be coated (e.g., by immersion,
spraying or applying a flowing fluid, if applicable) with a surface
protecting material such as polyethylene oxide or polypropylene
oxide to inhibit protein interactions of the reaction centers of
the matrix with entities of the subject. Other protective coatings
include silicon and hydrogels such as alginates. Derivatives of
these coating materials, such as polyethylene oxide-polydimethyl
siloxane, may also be used.
[0186] The coating may be formed freely around the core without
chemical bonding, or alternatively, the coating may be directly
cross-linked to the material of the implant.
[0187] The present invention also allows for enclosing the matrix
in a membrane. The membrane may allow for passage of substances up
to a predetermined size but prevents the passage of larger
substances. More specifically, the surrounding or peripheral region
is produced in such a manner that it has pores or voids of a
predetermined range of size. As a result, the vehicle is
selectively permeable. The molecular weight cutoff (MWCO) selected
for a particular coating will be determined in part by the use
contemplated. Membranes useful in the instant invention are
ultrafiltration and microfiltration membranes.
[0188] If necessary, the present invention also allows for
modification or additions to be made to the matrix to support or
strengthen the matrix. U.S. Pat. No. 5,786,216. For example,
structural materials, such as a hollow tube or cylindrica support,
may be encapsulated in the matrix to improve its compression
strength, tensile strength, or other properties.
[0189] The present invention also allows for attachment of a tether
to the matrix to make implantation, placement, or recovery of the
matrix more facile. The present allow also contemplates altering
the surface of the matrix, for example by encapsulating a structure
that affects the surface of the matrix, as an aid in fixing the
matrix upon implantation.
[0190] 5.3.3. Additives
[0191] Additives may be encapsulated in the matrix in addition to
any reaction center or centers. Such additives may be used to alter
the properties of the matrix. Investigations show that the addition
of low concentrations of organic molecules to sol-gels has very
little effect on the network formation of the sol-gel Dunn et al.
Chem. Mater 9:2280-91 (1997).
[0192] For example, additives that may be used in the present
invention include sodium fluoride and polyethylene glycol. The use
of polyethylene glycol in place of any alcohol during condensation
of the sol-gel matrix may improve enzymatic activity of the
encapsulated enzyme. Likewise, sodium fluoride may be used and may
improve enzymatic activity of the encapsulated enzyme. Avnir et al.
Chem. Mater. 6:1605-14 (1994).
[0193] For example, additives may alter the porosity of the matrix.
Alternatively, additives may be used to provide necessary reactants
for any encapsulated reaction center. For example, coenzymes for a
reaction center may be encapsulated along with the enzyme itself
For AADC and TD, a required cofactor is pyridoxal phosphate.
Pyridoxal phosphate may be encapsulated directly during preparation
of the sol-gel matrix. Alternatively, pyridoxal phosphate itself
may be prepared in a slow release formulation, and the formulation
encapsulated. See generally U.S. Pat. No. 5,759,582.
[0194] Alternatively, additives may improve the physical
characteristics of the matrix, for example, for purposes of
handling and administration of the material. As with the reaction
center, additives may be physically encapsulated during the
synthesis of the sol-gel, or they may be covalently attached to the
matrix directly.
[0195] Another possible additive of the present invention allows
for ready detection of the matrix after administration. In this
fashion, the matrices of the present invention may also be used for
diagnostic purposes. Thus an X-ray contrast agent, such as a
poly-iodo aromatic compound, may be encapsulated in the matrix.
Matrices according to the invention may also contain paramagnetic,
superparamagnetic or ferromagnetic substances which are of use in
magnetic resonance imaging (MRI) diagnostics. Thus, submicron
particles of iron or a magnetic iron oxide may be encapsulated into
the matrix to provide ferromagnetic or superparamagnetic particles.
Alternatively, paramagnetic MRI contrast agents, which principally
comprise paramagnetic metal ions, such as gadolinium ions, ligated
by a chelating agent which prevents their release (and thus
substantially eliminates their toxicity), may be encapsulated.
Matrices of the present invention may also contain ultrasound
contrast agents such as heavy materials, e.g. barium sulphate or
iodinated compounds such as the X-ray contrast agents referred to
above, to provide ultrasound contrast media.
[0196] 5.3.4. Matrix Morphology
[0197] The matrices of the present invention may take any variety
of morphologies. As a practical matter, the form of the matrix may
initially be determined by the vessel in which the matrix is
synthesized. Such matrices may subsequently be processed in order
to produce matrices of desired morphology. The size of any matrix
may depend on its intended use, and the present invention
contemplates preparing matrices having dimensions of centimeters
(1, 10, 100, 1000 cm), millimeters (1, 10, 100 mm), micrometers (1,
10, 100), nanometers (1, 10, 100), and picometers (0.01, 0.01, 1,
10, 100 pm). Alternatively, the size of a matrix may be referred to
by mass, which the present invention contemplates may range
anywhere from 1000 gm to 1 ng.
[0198] The matrix may be any configuration appropriate for
providing sufficient activity of the encapsulated reaction center
necessary for its intended use. Possible morphologies include
cylindrical, rectangular, disk-shaped, patch-shaped, ovoid,
stellate, or spherical. For use in a subject, a matrix of the
present invention may provide, in at least one dimension,
sufficiently close proximity of any reaction centers to the
surrounding tissues of the subject, including the subject's
bloodstream, in order to make any biologically active agent
produced by the reaction center bioavailable.
[0199] If the matrix is to be retrieved after it is implanted,
configurations which tend to prevent migration of the matrix from
the site of implantation may be desirable; in contrast, if the
matrix is intended to migrate throughout a patient, other
morphologies, such as spherical capsules small enough to travel in
the recipient's blood vessels, may be desirable. The degree of
miniaturization of any matrix may affect mobility and localization
of a matrix in a subject. Certain shapes, such as rectangles,
disks, or cylinders may offer greater structural integrity.
[0200] The surface area of the matrix may be important for its use.
A greater surface area may result in a greater observed activity
with respect to any particular load of an encapsulated reaction
center. In particular, small bead or sphere shaped materials
ranging in size of radius from may be desirable because they have
increased surface area as compared to other morphologies. In order
to increase further the surface area of a matrix, a powder of the
matrix may be desirable. It may be desirable to enclose a matrix,
especially if the matrix is in powder form, in a capsule. See, for
example, U.S. Pat. Nos. 5,653,975; 5,773,286.
[0201] It may be possible to control the size of any matrix by
using the aqueous core of reverse cellular micellar droplets as
host reactors for preparation of the matrix, as reported by Jain et
al. J. Am. Chem. Soc. 120:11092-95 (1998) for a silica-based
sol-gel matrix. The matrix particles prepared in such a fashion may
be highly monodispersed and have a narrow size distribution. Other
hollow spheres may be used to prepare matrices of similar
dimensions. See for example Caruso et al. Science 282:1111-13
(1998), U.S. Pat. No. 5,770,416, Lu et al., Nature 398:223-26
(1999).
[0202] 5.4. Reaction Centers
[0203] A wide variety of compounds or materials may be used as the
reaction center in the present invention. In general, any compound
or material that converts a compound into a biologically active
agent may be encapsulated. Alternatively, in other embodiments, any
compound or material that degrades a biologically active agent may
be encapsulated. Alternatively, any compound or material exhibiting
reactivity of interest may be encapsulated. Many possible reaction
centers have already been described in setting forth some of the
uses of the present invention.
[0204] Possible types of reaction centers contemplated by the
present invention include, for example, enzymes, catalytic
antibodies, antibodies, and non-biologically derived catalysts.
Many reaction centers may be biologically derived. Numerous reports
describe encapsulating enzymes in sol-gels, and such teachings may
be of assistance in embodiments of the present inventions. Zink et
al. New J. Chem., 18:1109-15 (1994); Miller et al. J.
Non-Crystalline Solids. 202:279-89 (1996); Ji et al. J. Am. Chem.
Soc., 720: 222-23 (1998); Braun et al. J. Non-Crystalline Solids
147&148:739-43 (1992); Yamanaka et al. Chem. Mater. 4:497-500
(1992); Lin et al. J. Sol-Gel Sci. & Tech. 7:19-26 (1996).
Catalytic antibodies have been encapsulated in sol-gel matrices and
the resulting matrices used in either a batch-wise operation or in
a continuous flow apparatus for preparative scale organic
synthesis. Shabat et al. Chem. Mater. 9:2258-60 (1997). Optically
active polypeptides have been encapsulated with retention of
activity. For example, bacteriorhodopsin or mutated forms have been
encapsulated in sol-gel matrices, which are optically transparent.
Weetall et al. Biochem Biophys. Acta 1142:211-13 (1993); Wu et al.
Chem. Mater. 5:115-20 (1993). Phycobiliproteins have also been
encapsulated. Chen et al. J. Sol-Gel Sci. & Tech. 7:99-108
(1996). Antibodies against small organic antigens have been
encapsulated within the sol-gel. Bronshtein et al. Chem. Mater.
9:2632-39 (1997); Turniansky et al. J. Sol-Gel Sci. & Tech.
7:135-43 (1996). Wang et al. Chem. Mater. 65:2671-75 (1993).
Alternatively, antigens have been encapsulated. Roux et al. J.
Sol-Gel Sci. & Tech. 8:663-66 (1997); Livage et al. J. Sol-Gel
Sci. & Tech. 7:45-51 (1996). Biologics having novel magnetic
properties, such as ferritin have been encapsulated. Lan et al. J.
Sol-Gel Sci. & tech. 7:109-116 (1996). In certain embodiments,
the reaction center encapsulated need not be substantially purified
from its natural source. Bresslar et al. J. Sol-Gel Sci. &
Tech. 7:129-33 (1996).
[0205] Cells and organisms have been encapsulated in silica-based
sol-gel matrices. Peterson el al. P.S.E.B.M. 218:365-69 (1998).
Bacteria have been immobilized in sol-gel matrices to metabolize
herbicides for environmental clean-ups. Rietti-Shati et al. J.
Sol-Gel Sci. & Tech. 7:77-79 (1996). In using cells in a
subject, it may be important to accustom them to the implantation
site before implantation to improve their viability. Differences in
conditions such as glucose concentration, oxygen availability,
nutrient concentrations, in vitro and in vivo may have an adverse
affect on implantation of cells. See generally U.S. Pat. Nos.
5,550,050; 5,620,883.
[0206] In addition to biological reaction centers, a variety of
other materials have been encapsulated in silica-based sol-gels.
For example, organic fluorescent dyes and photochromic information
recording materials have been encapsulated. Avnir et al. J. Phys.
Chem. 88:5956-59 (1984); Avnir et al. Journal of Non-Crystalline
Solids, 74:395-406 (1985); Levy et al., Journal of Non-Crystalline
Solids, 113:137-45 (1989). It is also possible to entrap a reaction
center in a sol-gel for use in organic catalysis. For example,
lipases may be encapsulated in a sol-gel for use as a heterogeneous
biocatalyst. Reetz et al. Angew. Chem. Int. Ed. Engl. 34:301-03
(1995).
[0207] For those embodiments of the present invention that involve
administration of a sol-gel matrix to a subject, the reaction
center encapsulated therein need not be native to the subject. The
sol-gel matrix, as discussed above, may be immunoisolatory itself
or modified to make it so.
[0208] Any number of different types of reaction centers may be
encapsulated in a single matrix. By encapsulating more than type of
reaction center in a single matrix, certain embodiments of the
present invention may cause the conversion of one compound into a
second and then into a third, and so on. Yamanka et al. J. Sol-Gel
Sci. & Tech. 7:117-21 (1996); Chang et al. Artif Organs 3:38-41
(1979). Such a result may be advantageous if, for example, the
biological activity of the second compound is undesirable.
Alternatively, it may be the case that it is the third compound
that has a more valuable biological activity than the second.
Encapsulating more than one reaction center may increase the
activity of the second reaction center in any pathway. For
instance, the local concentration of reactants for the second
center may be increased because of the reactivity of the first
center. Fossel et al. Eur. J. Biochem. 30:165-71 (1987).
Alternatively, for example, if the first type of reaction center
catalyzes an oxidation or reduction, the second type of reaction
center could mediate electron transfer and thereby facilitate
greater catalytic activity. For instance, the electron-transfer
redox pair Cc:CcP complex has been encapsulated. Lin et al. J.
Sol-Gel Sci. & Tech. 7:19-26 (1996).
[0209] In addition to reaction of the reaction center with a single
other compound, e.g., a prodrug, the present invention also
contemplates the reaction of more than one material with the
encapsulated reaction center. For example, it has been shown that
NADP (nicotinamide adenine dinucleotide phosphate ester) may react
with the reaction center and another molecule. In one example,
D-glucose-6-phosphate was converted by
glucose-6phosphate-dehydrogenase to a oxidized byproduct with the
concomitant reduction of NADP.sup.+ to NADPH. Yamanaka et al., J.
Am. Chem. Soc. 117:9095-96 (1995). The types of reaction centers
which may be encapsulated in a sol-gel, as contemplated by the
present invention, thus includes those compounds or materials that
react with more than one reactant. For example, many enzymes
contemplated by the present invention for encapsulation require
coenzymes in addition to any substrate.
[0210] One important characteristic of any encapsulated reaction
center is the degree of loading of the matrix. For example, the
degree of loading may affect the reactivity of a reaction center in
the sol-gel matrix. It has been reported that the activity of
trypsin appeared to decrease with increased loading levels. Levels
of loading contemplated by the present invention include 0.001,
0.01, 0.1, 1, 3, 5, 10, 15, 20 weight percent reaction center(s)
and/or any additives to the matrix.
[0211] In certain embodiments, the present invention contemplates
using as a reaction center enzymes or other biological materials
that are isolated from, or otherwise substantially free of other
cellular proteins. The term "substantially free of other cellular
proteins" (also referred to herein as "contaminating proteins") or
"substantially pure or purified preparations" are defined as
encompassing preparations the reaction center of interest having
less than 20% (by dry weight) contaminating protein, and preferably
having less than 5% contaminating protein. The term "purified" as
used herein preferably means at least 80% by dry weight, more
preferably in the range of 95-99% by weight, and most preferably at
least 99.8% by weight, of biological macromolecules of the same
type present (but water, buffers, and other small molecules,
especially molecules having a molecular weight of less than 5000,
can be present). The term "pure" as used herein preferably has the
same numerical limits as "purified" immediately above. "Isolated"
and "purified" do not encompass either natural materials in their
native state or natural materials that have been separated into
components (e.g., in an acrylamide gel) but not obtained either as
pure (e.g. lacking contaminating proteins, or chromatography
reagents such as denaturing agents and polymers, e.g. acrylamide or
agarose) substances or solutions.
[0212] In certain embodiments, the present invention contemplates
using for the reaction center human homologs of any of the enzymes
or other biological materials described herein, as well as
orthologs and paralogs (homologs) in other species. The term
"ortholog" refers to proteins which are homologs via speciation,
e.g., closely related and assumed to have common descent based on
structural and functional considerations. Orthologous proteins
function as recognizably the same activity in different species.
The term "paralog" refers to genes or proteins which are homologs
via gene duplication, e.g., duplicated variants of a gene within a
genome. See also Fritch Syst Zool 19:99-113 (1970).
[0213] In certain embodiments, the present invention contemplates
homologs of any naturally occurring enzymes. Further, the present
invention contemplates modification of the structure of any enzyme
to enhance therapeutic or prophylactic efficacy, or stability
(e.g., ex vivo shelf life). Such modified peptides may be produced,
for instance, by amino acid substitution, deletion, or
addition.
[0214] For example, it is reasonable to expect that an isolated
replacement of a leucine with an isoleucine or valine, an aspartate
with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
(i.e. isosteric and/or isoelectric mutations) will not have a major
effect on the biological activity of the resulting molecule.
Conservative replacements are those that take place within a family
of amino acids that are related in their side chains. Genetically
encoded amino acids are can be divided into four families: (1)
acidic aspartate, glutamate; (2) basic lysine, arginine, histidine;
(3) nonpolar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes
classified jointly as aromatic amino acids. In similar fashion, the
amino acid repertoire can be grouped as (1) acidic=aspartate,
glutamate; (2) basic=lysine, arginine histidine, (3)
aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,
threonine, with serine and threonine optionally be grouped
separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,
tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)
sulfur-containing=cysteine and methionine. (see, for example,
Biochemistry (2nd ed., Stryer et al. eds. 1981). Whether a change
in the amino acid sequence of a peptide results in a functional
homolog to any naturally occurring enzyme (e.g., functional in the
sense that the resulting polypeptide mimics the wild-type form) can
be readily determined by assaying for activity. Polypeptides in
which more than one replacement has taken place can readily be
tested in the same manner.
[0215] This invention further contemplates a method for generating
sets of combinatorial mutants of any nucleic acid encoding for an
enzyme used as a reaction center, as well as truncation mutants,
and is especially useful for identifying potential variant
sequences (e.g. homologs) that are functional in modulating the
enzymatic activity of interest. The purpose of screening such
combinatorial libraries is, for example, to identify novel
polypeptides that may convert prodrugs to biologically active
agents. In certain embodiments, such novel polypeptides may convert
prodrugs that naturally occurring enzymes do not, which may
therefore allow a particular prodrug to be used in the present
invention that otherwise would not have been possible. In other
embodiments, the prodrug of interest is converted only by an
encapsulated, novel polypeptide. As a result, no biologically
active agent is produced in vivo except by the encapsulated
reaction center of the matrix. Such a specificity difference may
have value because prodrugs that become cyotoxic agents may have
reduced toxicity if they are stable in vivo. Site-directed
mutagenesis has been used in ADEPT to prepare mutants of
carboxypeptidase A, so that only the mutants and no naturally
occurring enzyme converts selected prodrugs into corresponding
cytotoxic agents. Smith et al. J. Biol. Chem. 272:15804-16 (1997).
Even a single amino acid change may be sufficient to affect the
specificity. Thus, combinatorially-derived homologs can be
generated to have an increased potency or different specificity
relative to a naturally occurring form of an enzyme or other
biological macromolecule.
[0216] In one aspect of this method, the amino acid sequences for a
population of homologs for any enzyme, or other related proteins,
are aligned, preferably to promote the highest homology possible.
Such a population of variants can include, for example, homologs
from one or more species. Amino acids which appear at each position
of the aligned sequences are selected to create a degenerate set of
combinatorial sequences. In a preferred embodiment, the variegated
library of variants is generated by combinatorial mutagenesis at
the nucleic acid level, and is encoded by a variegated gene
library. For instance, a mixture of synthetic oligonucleotides can
be enzymatically ligated into gene sequences such that the
degenerate set of potential sequences are expressible as individual
polypeptides, or alternatively, as a set of larger fusion proteins
containing the set of sequences therein.
[0217] There are many ways by which such libraries of potential
homologs can be generated from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
carried out in an automatic DNA synthesizer, and the synthetic
genes then ligated into an appropriate expression vector. The
purpose of a degenerate set of genes is to provide, in one mixture,
all of the sequences encoding the desired set of potential
sequences. The synthesis of degenerate oligonucleotides is well
known in the art. See, for example, Narang Tetrahedron 39:3 (1983);
Itakura et al. Recombinant DNA. Proc 3rd Cleveland Sympos.
Macromolecules273-89 (ed. A G Walton, Amsterdam: Elsevier 1981);
Itakura et al. Annu. Rev. Biochem. 53:323 (1984); Itakura et al.
Science 198:1056 (1984); Ike et al. Nucleic Acid Res. 11:477
(1983). Such techniques have been employed in the directed
evolution of other proteins. See, for example, Scott et al. Science
249:386-90 (1990); Roberts et al. PNAS 89:2429-33 (1992); Devlin et
al. Science 249:404-06 (1990); Cwirla et al. PNAS 87:6378-82
(1990); as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and
5,096,815.
[0218] Likewise, a library of coding sequence fragments can be
provided for an enzyme of interest as a reaction center in order to
generate a variegated population of fragments for screening and
subsequent selection of bioactive fragments. A variety of
techniques are known in the art for generating such libraries,
including chemical synthesis. In one embodiment, a library of
coding sequence fragments can be generated by (i) treating a double
stranded PCR fragment of an coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule;
(ii) denaturing the double stranded DNA; (iii) renaturing the DNA
to form double stranded DNA which can include sense/antisense pairs
from different nicked products; (iv) removing single stranded
portions from reformed duplexes by treatment with S1 nuclease; and
(v) ligating the resulting fragment library into an expression
vector. By this exemplary method, an expression library can be
derived which codes for N-terminal, C-terminal and internal
fragments of various sizes.
[0219] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations or truncation, and for screening cDNA libraries for gene
products having a certain property. Such techniques will be
generally adaptable for rapid screening of the gene libraries
generated by the combinatorial mutagenesis of homologs for any
enzyme of interest as a reaction center. The most widely used
techniques for screening large gene libraries typically comprises
cloning the gene library into replicable expression vectors,
transforming appropriate cells with the resulting library of
vectors, and expressing the combinatorial genes under conditions in
which detection of a desired activity facilitates relatively easy
isolation of the vector encoding the gene whose product was
detected.
[0220] 5.4.1. Enzymes used for ADEPT
[0221] Enzymes that have been used for ADEPT may generally be used
as the encapsulated reaction center in the present invention. Such
enzymes were originally chosen because they convert a prodrug into
a biologically active agent, and they are thereby of use in the
present invention as well. Some examples of such enzymes, and the
conversion that they catalyze, follow. Other examples may be found
in U.S. Pat. Nos. 5,714,148; 5,660,829, 5,587,161; and 5,405,990.
In the present invention, enzymes used in ADEPT need not
necessarily be used with the same prodrug as used in the ADEPT
application.
[0222] In one aspect of the invention, a variety of peptidases,
which cleave amide bonds, may be used as the reaction center.
Jungheim et al., Chem. Rev. 94:1553-66 (1994). In one embodiment,
carboxypeptidase G2 can be used as the reaction center with
prodrugs to cleave an amide bond. One biologically active agent is
nitrogen mustard which is an alkylating agent. Carboxypeptidase G2
cleaves an amide bond of a prodrug to give the free nitrogen
mustard and glutamic acid. Bagshawe, Br. J. Cancer 56:531 (1987);
Bagshawe et al. Br. J. Cancer 58:700 (1988). In another embodiment,
carboxypeptidase A from bovine pancrease can be used as the
reaction center with prodrugs to cleave an amide bond. In one
embodiment, the biologically active agent is methotrexate, which is
used for cancer chemotherapy, and the prodrugs are .alpha.-peptides
of methotrexate, e.g., Glu-.alpha.-L-Ala-methotrexate and
L-Glu-L-Phe-methotrexate. Vitols et al., Pteridines 3:125 (1992);
Kueffier et al., Biochem. 28:2288 (1989); Haenseler et al.,
Biochem. 31:891 (1992).
[0223] In another embodiment, penicillin V amidase from Fusarium
oxysporum can be used as the reaction center with prodrugs to
deacylate an N-acyl amine. In several embodiments, the biologically
active agent is doxorubicin or mephalan, which are anticancer
agents, and the prodrugs are N-acyl derivatives of doxorubicin or
mephalan. Kerr et al. Cancer Immun. Immunother. 31:202 (1990). In
another embodiment, penicillin G amidase can be used as the
reaction center with prodrugs to cleave phenylacetamides. In
several embodiments, the biologically active agent is doxorubicin
or mephalan, and the prodrugs are phenylacetamide derivatives of
doxorubicin or mephalan. Kerr et al. supra. In another embodiment,
the biologically active agent is palytoxin, which is a potent
cytotoxin, and the prodrug is
N-[(4'-hydroxyphenyl)acetyl]palytoxin. Because palytoxin asserts
its effect extracellularly, it may be able to overcome the
multidrug resistance phenotype.
[0224] In another embodiment, urokinase may be the reaction center.
Puromycin and doxorubicin have been produced using this enzyme. WO
91/09134. In another embodiment, a variety of .beta.-lactamases,
which cleave certain amide bonds, may be used as the reaction
center. In one embodiment, a biologically active agent is produced
by covalently attaching the agent to the C-3' position of
cephalosporin or a derivative of cephalosporin, whereupon
hydrolization of the prodrug by a .beta.-lactamase, e.g., P99
enzyme derived from Enterobacter cloacae 265A or enzyme derived
from B. cereus, produces the free agent. Biologically active agents
that have been covalently attached to cephalosporin or a derivative
of cephalosporin in this manner include: methotrexate;
5-fluorouracil, which is often used for the treatment of colon
cancer; LY233425, a potent analogue of the anticancer agent
vinblastine; desacetylvinblastine hydrazine, a potent vinca
alkaloid; nitrogen mustard alkylating agents; thioguanine;
doxorubicin; mitomycin C; and DACCP, a carboplatinum-based drug
that is a potent antitumor agent. Jungheim et al. Chem. Rev.
94:1553-66 (1994); Meyer et al. Antibody. Immunoconjugates.
Radiopharm. 3:66 (1990); Jungheim et al., Antibody,
Immunoconjugates. Radiopharm. 4:228 (1991); Shepard et al., Biomed.
Chem. Lett. 1:21 (1991); EP0382411A2; Alexander et al. Tetrahedron
Lett. 32:3269 (1991); EPo392745A2; Svensson et al. Bioconj. Chem.
3:176 (1992); Vrudhula et al. Bioconj. Chem. 4:334 (1993); Hudyma
et al. Biomed. Chem. Lett. 3:323 (1993); EP0484870A2; Junghein et
al. Heterocycles 35:33 (1993); Hanessian et al. Can J. Chem. 71:896
(1993).
[0225] In another aspect of the invention, alkaline phosphotase can
be used as the reaction center with prodrugs to remove phosphate
from organic phosphates. Biologically active agents that are
produced in this manner include etoposide, mitomycin-derived
agents, nitrogen mustard derived agents, and doxorubicin. Senter et
al. Proc. Nat. Acad. Sci. U.S.A. 85:4842 (1988); Senter et al.
Cancer Res. 49:5789 (1989); Senter, FASEB J. 4:188 (1990); Sahin et
al. Cancer Res. 50:6944 (1990).
[0226] In another aspect of the invention, glycosidases, which
cleave a glycosidic linkage may be used as the reaction center. In
one embodiment, .beta.-glucuronidase is used as the reaction center
to produce biologically active agents, including nitrogen mustard
derived agents, daunomycin, adriamycin, epirubicin. Roffier et al.
Biomed Pharmacol. 42:2062 (1991); Wang et al. Cancer Res. 52:4484
(1992); Deonarain et al. Br. J. Cancer 70:786-94 (1994). In another
embodiment, .beta.-glucosidase converts amygdalin into glucose,
benzaldehyde, and hydrogen cyanide, a toxic-species. In another
embodiment, .alpha.-galactosidase is used as the reaction center to
produce daunorubicin. Andrianomenjanahary et al. Biomed. Chem.
Lett. 2:1093 (1992).
[0227] In another aspect of the invention, cytosine deaminase,
which converts cytosine into uracil, may be used as the reaction
center. In one embodiment, cytosine deaminase isolated from Bakers'
yeast is used to produce the antitumor agent 5-fluroruracil from
5-fluorocytosine. Senter et al. Bioconj. Chem. 2:447 (1991). In
another aspect of the invention, nitroreductase, which requires the
presence of a cofactor such as NADH, may be used as the reaction
center. The enzyme has been used to produce
5-aziridin-4-hydroxyamino-2-nitrobenzamide from
5-aziridin-2,4-dinitroben- zamide. Knox et al. Cancer Metathesis
Rev. 12:195 (1993).
[0228] In another aspect of the invention, oxidases, which produce
reduced oxygen species, e.g., peroxide, superoxide, and hydroxyl
radicals, may be used as the reaction center. In one embodiment,
glucose oxidase and lactoperoxidase convert glucose and iodide into
hydrogen peroxide and toxic iodine species. Ito et al. Bone Marrow
Transplant. 6:395-98 (1990); Stanislawski (1989). In another
embodiment, xathine oxidase produces reduced oxygen species from
either xanthine or hypoxanthine. Dinota et al. Bone Marrow
Transplant. 6:31-36 (1990).
[0229] 5.4.2. Other Enzymes
[0230] In addition to the enzymes used for ADEPT, for which a
prodrug may have already been identified, other enzymes may be used
as the reaction center. In using any enzyme in the present
invention, it may be necessary to consider which compounds will be
converted by the enzyme. Which enzyme is most suitable for
encapsulation as the reaction center depends, in part, on the
expected use of any matrix.
[0231] In deciding which enzyme may be appropriate for any
application, the general classification of enzymes may be used in
identifying and considering different types of reactions that a
reaction center could possibly catalyze. These classes include: (i)
oxidoreductases (acting on the CH--OH group of donors; acting on
the aldehyde or oxo group of donors; acting on the CH--CH group of
donors; acting on the CH--NH(2) group of donors; acting on the
CH--NH group of donors; acting on NADH or NADPH; acting on other
nitrogenous compounds as donors; acting on a sulfur group of
donors; acting on a heme group of donors; acting on diphenols and
related substances as donors; acting on a peroxide as acceptor
(peroxidases); acting on hydrogen as donor; acting on single donors
with incorporation of molecular oxygen; acting on paired donors
with incorporation of molecular oxygen; acting on superoxide
radicals as acceptor; oxidizing metal ions; acting on --CH(2)
groups; acting on reduced ferredoxin as donor; acting on reduced
flavodoxin as donor; other oxidoreductases); (ii) transferases
(transferring one-carbon groups; transferring aldehyde or ketone
residues; acyltransferases; glycosyltransferases; transferring
alkyl or aryl groups, other than methyl groups; transferring
nitrogenous groups; transferring phosphorous-containing groups;
transferring sulfuir-containing groups; transferring
selenium-containing groups); (iii) hydrolases (acting on ester
bonds; glycosidases; acting on ether bonds; acting on peptide bonds
(peptide hydrolases); acting on carbon-nitrogen bonds, other than
peptide bonds; acting on acid anhydrides; acting on carbon-carbon
bonds; acting on halide bonds; acting on phosphorus-nitrogen bonds;
acting on sulfur-nitrogen bonds; acting on carbon-phosphorus bonds;
acting on sulfur-sulfir bonds); lyases (carbon-carbon lyases;
carbon-oxygen lyases; carbon-nitrogen lyases; carbon-sulfur lyases;
carbon-halide lyases; phosphorus-oxygen lyases; other lyases); (iv)
isomerases (racemases and epimerases; cis-trans-isomerases;
intramolecular oxidoreductases; intramolecular transferases
(mutases); intramolecular lyases; other isomerases); (v) ligases
(forming carbon-oxygen bonds; forming carbon-sulfur bonds; forming
carbon-nitrogen bonds; forming carbon-carbon bonds; forming
phosphoric ester bonds).
[0232] Illustrative examples of enzymes that may serve as reaction
centers in the present invention include, without limitation:
alcohol dehydrogenase (EC 1.1.1.1), homoserine dehydrogenase (EC
1.1.1.3), (R,R)-butanediol dehydrogenase (EC 1.1.1.4), glycerol
dehydrogenase (EC 1.1.1.6), glycerol-3-phosphate dehydrogenase
(NAD+) (EC 1.1.1.8), D-xylulose reductase (EC 1.1.1.9), L-xylulose
reductase (EC 1.1.1.10), L-iditol dehydrogenase (EC 1.1.1.14),
mannitol-1-phosphate dehydrogenase (EC 1.1.1.17), myo-inositol
2-dehydrogenase (EC 1.1.1.18), aldehyde reductase (EC 1.1.1.21),
quinate dehydrogenase (EC 1.1.1.24), shikimate dehydrogenase (EC
1.1.1.25), glyoxylate reductase (EC 1.1.1.26), L-lactate
dehydrogenase (EC 1.1.1.27), D-lactate dehydrogenase (EC 1.1.1.28),
glycerate dehydrogenase (EC 1.1.1.29), 3-hydroxybutyrate
dehydrogenase (EC 1.1.1.30), 3-hydroxyisobutyrate dehydrogenase (EC
1.1.1.31), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), malate
dehydrogenase (EC 1.1.1.37), malate dehydrogenase and aspartate
aminotransferase (EC 1.1.1.37 & 2.6.1.1), malate dehydrogenase
and citrate (si)-synthase (EC 1.1.1.37 and 4.1.3.7), malate
dehydrogenase (decarboxylating) (EC 1.1.1.39), malate dehydrogenase
(oxaloacetate-decarboxylating) (NADP+) (EC 1.1.1.40), isocitrate
dehydrogenase (NADP+) (EC 1.1.1.42), phosphogluconate dehydrogenase
(decarboxylating) (EC 1.1.1.44), glucose dehydrogenase (EC
1.1.1.47), galactose dehydrogenase (EC 1.1.1.48),
glucose-6-phosphate dehydrogenase (EC 1.1.1.49),
glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase (EC
1.1.1.49 & 3.1.1.31), 3-hydroxysteroid dehydrogenase (EC
1.1.1.50), 3(or 17)-hydroxysteroid dehydrogenase (EC 1.1.1.1.51),
lactaldehyde reductase (NADPH) (EC 1.1.1.55), ribitol dehydrogenase
(EC 1.1.1.56), 3-hydroxypropionate dehydrogenase (EC 1.1.1.59),
2-hydroxy-3-oxopropionate reductase (EC 1.1.1.160),
4-hydroxybutyrate dehydrogenase (EC 1.1.1.61), estradiol 1
7-dehydrogenase (EC 1.1.1.62), mannitol dehydrogenase (EC
1.1.1.67), gluconate 5-dehydrogenase (EC 1.1.1.69), glycerol
dehydrogenase (NADP+) (EC 1.1.1.72), glyoxylate reductase (NADP+)
(EC 1.1.1.79), aryl-alcohol dehydrogenase (EC 1.1.1.90),
phosphoglycerate dehydrogenase (EC 1.1.1.95), diiodophenylpyruvate
reductase (EC 1.1.1.96), 3-hydroxybenzyl-alcohol dehydrogenase (EC
1.1.1.97), 3-oxoacyl-[acyl-carrier-protein] reductase (EC
1.1.1.100), carnitine dehydrogenase (EC 1.1.1.108), indolelactate
dehydrogenase (EC 1.1.1.110), glucose dehydrogenase (NADP+) (EC
1.1.1.119), fructose 5-dehydrogenase (NADP+) (EC 1.1.1.124),
2-deoxy-D-gluconate dehydrogenase (EC 1.1.1.125), L-threonate
dehydrogenase (EC 1.1.1.129), sorbitol-6-phosphate dehydrogenase
(EC 1.1.1.140), 15-hydroxyprostaglandin dehydrogenase (NAD+) (EC
1.1.1.141), 21-hydroxysteroid dehydrogenase (NAD+) (EC 1.1.1.150),
sepiapterin reductase (EC 1.1.1.153), coniferyl-alcohol
dehydrogenase (EC 1.1.1.194), (R)-2-hydroxyglutarate dehydrogenase
(EC 1.1.1.a), sorbitol-6-phosphate dehydrogenase (NADP+) (EC
1.1.1.b), gluconate 2-dehydrogenase (EC 1.1.99.3), lactate-malate
transhydrogenase (EC 1.1.99.7), glucoside 3-dehydrogenase (EC
1.1.99.13), formate dehydrogenase (EC 1.2.1.2), acetaldehyde
dehydrogenase (acetylating) (EC 1.2.1.10), aspartate-semialdehyde
dehydrogenase (EC 1.2.1.11), glyceraldehyde-3-phosphate
dehydrogenase (EC 1.2.1.12), glyceraldehyde-3-phosphate
dehydrogenase and phosphoglycerate kinase (EC 1.2.1.12 &
2.7.2.3), glyoxylate dehydrogenase (acylating) (EC 1.2.1.17),
formate dehydrogenase (NADP+) (EC 1.2.1.43), succinate
dehydrogenase (EC 1.3.99.1), butyryl-CoA dehydrogenase (EC
1.3.99.2), dihydroorotate dehydrogenase (EC 1.3.99.11), alanine
dehydrogenase (EC 1.4.1.1), glutamate dehydrogenase (EC 1.4.1.2),
glutamate dehydrogenase (NAD(P)+) (EC 1.4.1.3), glutamate
dehydrogenase (NADP+) (EC 1.4.1.4), leucine dehydrogenase (EC
1.4.1.9), glycine dehydrogenase (EC 1.4.1.10),
L-erythro-3,5-diaminohexanoate dehydrogenase (EC 1.4.1.11),
2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12),
pyrroline-2-carboxylate reductase (EC 1.5.1.1),
pyrroline-5-carboxylate reductase (EC EC 1.5.1.2), dihydrofolate
reductase (EC EC 1.5.1.3), methylenetetrahydrofolate dehydrogenase
(NADP+) (EC 1.5.1.5), D-octopine dehydrogenase (EC 1.5.1.11),
methylenetetrahydrofolate dehydrogenase (NAD+) (EC 1.5.1.15),
alanopine dehydrogenase (EC 1.5.1.17), 1-piperidine-2-carboxylate
reductase (EC 1.5.1.21), NAD(P)+transhydrogenase (EC 1.6.1.1),
glutathione reductase (NAD(P)H) (EC 1.6.4.2), thioredoxin reductase
(NADPH) (EC 1.6.4.5), NADH dehydrogenase (EC 1.6.99.3),
5,10-methylenetetrahydrofolate reductase (FADH2) (EC 1.7.99.5),
dihydrolipoamide dehydrogenase (EC 1.8.1.4),
glutathione-CoA-glutathione transhydrogenase (EC 1.8.4.3),
cytochrome-c oxidase (EC EC 1.9.3.1), hydrogen dehydrogenase (EC
1.12.1.2), thetin-homocysteine S-methyltransferase (EC 2.1.1.3),
homocysteine S-methyltransferase (EC 2.1.1.10), thymidylate
synthase (EC 2.1.1.45), glycine hydroxymethyltransferase (EC
2.1.2.1), glycine formiminotransferase (EC 2.1.2.4), glutamate
formiminotransferase (EC 2.1.2.5), D-alanine
2-hydroxymethyltransferase (EC 2.1.2.7), aminomethyltransferase (EC
2.1.2.10), methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1),
ornithine carbamoyltransferase (EC 2.1.3.3), oxamate
carbamoyltransferase (EC 2.1.3.5), glycine amidinotransferase (EC
2.1.4.1), transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2),
imidazole N-acetyltransferase and phosphate acetyltransferase (EC
2.3.1.2 & 2.3.1.8), arylamine N-acetyltransferase (EC 2.3.1.5),
choline O-acetyltransferase (EC 2.3.1.6), camitine
O-acetyltransferase (EC 2.3.1.7), phosphate acetyltransferase (EC
2.3.1.8), phosphate acetyltransferase and formate
C-acetyltransferase (EC 2.3.1.8 & 2.3.1.54), phosphate
acetyltransferase and acetate kinase (EC 2.3.1.8 & 2.7.2.1),
acetyl-CoA C-acetyltransferase (EC 2.3.1.9), camitine
O-palmitoyltransferase (EC 2.3.1.21), glutamate N-acetyltransferase
(EC 2.3.1.35), [acyl-carrier-protein] S-acetyltransferase (EC
2.3.1.38), [acyl-carrier-protein] S-malonyltransferase (EC
2.3.1.39), formate C-acetyltransferase (EC 2.3.1.54), sucrose
phosphorylase (EC 2.4.1.7), maltose phosphorylase (EC 2.4.1.8),
levansucrase (EC 2.4.1.10), sucrose synthase (EC 2.4.1.13),
sucrose-phosphate synthase (EC 2.4.1.14), ,-trehalose-phosphate
synthase (UDP-forming) (EC 2.4.1.15), cellobiose phosphorylase (EC
2.4.1.20), laminaribiose phosphorylase (EC 2.4.1.31), ,-trehalose
phosphorylase (EC 2.4.1.64), galactinol-raffinose
galactosyltransferase (EC 2.4.1.67), sinapate 1-glucosyltransferase
(EC 2.4.1.120), purine-nucleoside phosphorylase (EC 2.4.2.1),
purine-nucleoside phosphorylase and pyrimidine-nucleoside
phosphorylase (EC 2.4.2.1 & 2.4.2.2), pyrimidine-nucleoside
phosphorylase (EC 2.4.2.2), uridine phosphorylase (EC 2.4.2.3),
nucleoside deoxyribosyltransferase (EC 2.4.2.6), adenine
phosphoribosyltransferase (EC 2.4.2.7), hypoxanthine
phosphoribosyltransferase (EC 2.4.2.8), orotate
phosphoribosyltransferase (EC 2.4.2.10), guanosine phosphorylase
(EC 2.4.2.15), thiamine pyridinylase (EC 2.5.1.2),
thiamin-phosphate pyrophosphorylase (EC 2.5.1.3), aspartate
transaminase (EC 2.6.1.1), malate dehydrogenase and aspartate
transaminase (EC 1.1.1.37 & 2.6.1.1), alanine transaminase (EC
2.6.1.2), histidinol-phosphate transaminase (EC 2.6.1.9),
ornithine-oxo-acid transaminase (EC 2.6.1.13), glutamine-pyruvate
aminotransaminase (EC 2.6.1.15), succinyldiaminopimelate
transaminase (EC 2.6.1.17), -alanine-pyruvate transaminase (EC
2.6.1.18), 4-aminobutyrate transaminase (EC 2.6.1.19), D-alanine
transaminase ( EC 2.6.1.21), pyridoxamine-pyruvate transaminase (EC
2.6.1.30), dTDP-4-amino-4,6-dideoxy-D-glucose transaminase (EC
2.6.1.33), glycine-oxaloacetate transaminase (EC 2.6.1.35),
2-aminoadipate transaminase (EC 2.6.1.39), serine-pyruvate
transaminase (EC 2.6.1.51), phosphoserine transaminase (EC
2.6.1.52), hexokinase (EC 2.7.1.1), galactokinase (EC 2.7.1.6),
6-phosphofructokinase (EC 2.7.1.11), NAD+kinase (EC 2.7.1.23),
dephospho-CoA kinase (EC 2.7.1.24), glycerol kinase (EC 2.7.1.30),
protein kinase (EC 2.7.1.37), pyruvate kinase (EC 2.7.1.40),
1-phosphatidylinositol kinase (EC 2.7.1.67), pyrophosphate-serine
phosphotransferase (EC 2.7.1.80),
pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC
2.7.1.90), acetate kinase (EC 2.7.2.1), carbamate kinase (EC
2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3),
glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate
kinase (EC 1.2.1.12 & 2.7.2.3), aspartate kinase (EC 2.7.2.4),
guanidinoacetate kinase (EC 2.7.3.1), creatine kinase (EC 2.7.3.2),
creatine kinase and myosin ATPase (EC 2.7.3.2 & 3.6.1.32),
arginine kinase (EC 2.7.3.3), taurocyamine kinase (EC 2.7.3.4),
lombricine kinase (EC 2.7.3.5), phosphomevalonate kinase (EC
2.7.4.2), adenylate kinase (EC 2.7.4.3), nucleoside-phosphate
kinase (EC 2.7.4.4), nucleoside-diphosphate kinase (EC 2.7.4.6),
guanylate kinase (EC 2.7.4.8), nucleoside-triphosphate-adenylate
kinase (EC 2.7.4.10), (deoxy)nucleoside-phosphate kinase (EC
2.7.4.13), cytidylate kinase (EC 2.7.4.14), ribose-phosphate
pyrophosphokinase (EC 2.7.6.1), nicotinamide-nucleotide
adenylyltransferase (EC 2.7.7.1), sulfate adenylyltransferase (EC
2.7.7.4), sulfate adenylyltransferase and inorganic pyrophosphatase
(EC 2.7.7.4 & 3.6.1.1), DNA-directed DNA polymerase (EC
2.7.7.7), UTP-glucose-1-phosphate uridylyltransferase (EC 2.7.7.9),
UDPglucose-hexose-1-phosphate uridylyltransferase (EC 2.7.7.12),
UDPglucose-hexose 1-phosphate uridylyltransferase and UDPglucose
(EC 2.7.7.12 & 5.1.3.2), mannose-1-phosphate
guanylyltransferase (EC 2.7.7.13), ethanolamine-phosphate
cytidylyltransferase (EC 2.7.7.14), choline-phosphate
cytidylyltransferase (EC 2.7.7.15), UDP-N-acetylglucosamine
pyrophosphorylase (EC 2.7.7.23), glucose-1-phosphate
thymidylyltransferase (EC 2.7.7.24), glucose-1-phosphate
adenylyltransferase (EC 2.7.7.27), glucose-1-phosphate
cytidylyltransferase (EC 2.7.7.33), glucose-1-phosphate
guanylyltransferase (EC 2.7.7.34), [glutamate-ammonia-ligase]
adenylyltransferase (EC 2.7.7.42), glucuronate-1-phosphate
uridylyltransferase (EC 2.7.7.44), pyruvate, orthophosphate
dikinase (EC 2.7.9.1), aryl sulfotransferase (EC 2.8.2.1),
3-oxoacid CoA-transferase (EC 2.8.3.5), acetate CoA-transferase (EC
2.8.3.8), triacylglycerol lipase (EC 3.1.1.3), acetylcholinesterase
(EC 3.1.1.7), retinyl-palmitate esterase (EC 3.1.1.21),
glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase (EC
1.1.1.49 & 3.1.1.31), alkaline phosphatase (EC 3.1.3.1), acid
phosphatase (EC 3.1.3.2), phosphoserine phosphatase (EC 3.1.3.3),
5'-nucleosidase (EC 3.1.3.5), fructose-biphosphatase (EC 3.1.3.11),
phosphodiesterase I (EC 3.1.4.1), 3',5'-cyclic-nucleotide
phosphodiesterase (EC 3.1.4.17), phosphohydrolase (unclassified)
(EC 3.1.4.a), ribonuclease T2 (EC 3.1.27.1), pancreatic
ribonuclease (EC 3.1.27.5), ribonuclease (unclassified) (EC
3.1.27.a), cyclomaltodextrin glucanotransferase and -amylase (EC
2.4.1.19 & 3.2.1.1), -amylase (EC 3.2.1.2), glucan
1,4--glucosidase (EC 3.2.1.3), oligo-1,6-glucosidase (EC 3.2.1.10),
-glucosidase (EC 3.2.1.20), -glucosidase (EC 3.2.1.21),
-galactosidase (EC 3.2.1.23), -mannosidase (EC 3.2.1.24),
-fructofuranosidase (EC 3.2.1.26), -dextrin endo-1,6-glucosidase
(EC 3.2.1.41), AMP nucleosidase (EC 3.2.2.4), NAD+nucleosidase (EC
3.2.2.5), NAD(P)+nucleosidase (EC 3.2.2.6), adenosine nucleosidase
(EC 3.2.2.7), adenosylhomocysteinase (EC 3.3.1.1), leucyl
aminopeptidase (EC 3.4.11.1), dipeptidyl-peptidase I(EC 3.4.14.1),
carboxypeptidase A (EC 3.4.17.1), gly-X carboxypeptidase (EC
3.4.17.4), -glu-X carboxypeptidase (EC 3.4.19.9), chymotrypsin (EC
3.4.21.1), trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), pepsin A
(EC 3.4.23.1), chymosin (EC 3.4.23.4), thermolysin (EC 3.4.24.27),
asparaginase (EC 3.5.1.1), glutaminase (EC 3.5.1.2), urease (EC
3.5.1.5), penicillin amidase (EC 3.5.1.11), aminoacylase (EC
3.5.1.14), pantothenase (EC 3.5.1.22), N-methyl-2-oxoglutaramate
hydrolase (EC 3.5.1.36), dihydroorotase (EC 3.5.2.3),
carboxymethylhydantoinase (EC 3.5.2.4), -lactamase (EC 3.5.2.6),
arginase (EC 3.5.3.1), allantoicase (EC 3.5.3.4), arginine
deiminase (EC 3.5.3.6), adenosine deaminase (EC 3.5.4.4), cytidine
deaminase (EC 3.5.4.5), AMP deaminase (EC 3.5.4.6),
methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), inorganic
pyrophosphatase (EC 3.6.1.1), sulfate adenylyltransferase and
inorganic pyrophosphatase (EC 2.7.7.4 & 3.6.1.1),
trimetaphosphatase (EC 3.6.1.2), nucleotide pyrophosphatase (EC
3.6.1.9), myosin ATPase (EC 3.6.1.32), creatine kinase and myosin
ATPase (EC 2.7.3.2 & 3.6.1.32), Ca2+-transporting ATPase (EC
3.6.1.38), chymotrypsin (EC 3.4.21.1), thermolysin (EC 3.4.24.4),
phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32),
phosphoenolpyruvate carboxykinase (diphosphate) (EC 4.1.1.38),
ribulose-biphosphate carboxylase (EC 4.1.1.39),
ketotetraose-phosphate aldolase (EC 4.1.2.2), deoxyribose-phosphate
aldolase (EC 4.1.2.4), fructose-biphosphate aldolase (EC 4.1.2.13),
fructose-biphosphate aldolase and triose-phosphate isomerase (EC
4.1.2.13 & 5.3.1.1), 2-dehydro-3-deoxyphosphogluconate aldolase
(EC 4.1.2.14), L-fuculose-phosphate aldolase (EC 4.1.2.17),
2-dehydro-3-deoxy-L-pentonat- e aldolase (EC 4.1.2.18),
rhamnulose-1-phosphate aldolase (EC 4.1.2.19),
2-dehydro-3-deoxy-6-phosphogalactonate aldolase (EC 4.1.2.21),
D-arabino-3-hexulose phosphate formaldehyde lyase (EC 4.1.2.a),
isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2),
N-acetylneuraminate lyase (EC 4.1.3.3), citrate (pro-3S)-lyase (EC
4.1.3.6), citrate (si)-synthase (EC 4.1.3.7), citrate (si)-synthase
and malate dehydrogenase (EC 4.1.3.7 & 1.1.1.37), ATP
citrate(pro-3S)-lyase (EC 4.1.3.8), 4-hydroxy-2-oxoglutarate
aldolase (EC 4.1.3.16), citramalate lyase (EC 4.1.3.22), malyl-CoA
lyase (EC 4.1.3.24), 2,3-dimethylmalate lyase (EC 4.1.3.32),
tryptophanase (EC 4.1.99.1), fumarate hydratase (EC 4.2.1.2),
aconitate hydratase (EC 4.2.1.3), 3-dehydroquinate dehydratase (EC
4.2.1.10), phosphopyruvate hydratase (EC 4.2.1.11), enoyl-CoA
hydratase (EC 4.2.1.17), tryptophan synthase (EC 4.2.1.20), maleate
hydratase (EC 4.2.1.31), (S)-2-methylmalate dehydratase (EC
4.2.1.34), (R)-2-methylmalate dehydratase (EC 4.2.1.35),
D-glutamate cyclase (EC 4.2.1.48), urocanate hydratase (EC
4.2.1.49), crotonoyl-[acyl-carrier-protein]hydratase (EC 4.2.1.58),
dimethylmaleate hydratase (EC 4.2.1.85), 3-hydroxybutyryl-CoA
dehyratase (EC 4.2.1.a), aspartate ammonia-lyase (EC 4.3.1.1),
methylaspartate ammonia-lyase (EC 4.3.1.2), histidine ammonia-lyase
(EC 4.3.1.3), phenylalanine ammonia-lyase (EC 4.3.1.5), -alanyl-CoA
ammonia lyase (EC 4.3.1.6), arginosuccinate lyase (EC 4.3.2.1),
adenylosuccinate lyase (EC 4.3.2.2), ureidoglycolate lyase (EC
4.3.2.3), lactoylglutathione lyase (EC 4.4.1.5), adenylate cyclase
(EC 4.6.1.1), alanine racemase (EC 5.1.1.1), glutamate racemase (EC
5.1.1.3), lysine racemase (EC 5.1.1.5), diaminopimelate epimerase
(EC 5.1.1.7), 4-hydroxyproline epimerase (EC 5.1.1.8), amino-acid
racemase (EC 5.1.1.10), ribulose-phosphate 3-epimerase (EC
5.1.3.1), UDPglucose 4-epimerase (EC 5.1.3.2), UDPglucose
4-epimerase and UDPglucose-hexose 1-phosphate (EC 5.1.3.2 &
2.7.7.12), L-ribulose-phosphate 4-epimerase (EC 5.1.3.4),
UDParabinose 4-epimerase (EC 5.1.3.5), UDPglucuronate 4-epimerase
(EC 5.1.3.6), N-acylglucosamine 2-epimerase (EC 5.1.3.8),
N-acylglucosamine-6-phosphate 2-epimerase (EC 5.1.3.9), CDPabequose
epimerase (EC 5.1.3.10), glucose-6-phosphate 1-epimerase (EC
5.1.3.15), GDP-D-mannose 3,5-epimerase (EC 5.1.3.18),
methylmalonyl-CoA epimerase (EC 5.1.99.1), retinal isomerase (EC
5.2.1.3), linoleate isomerase (EC 5.2.1.5), triose-phosphate
isomerase (EC 5.3.1.1), triose-phosphate isomerase and
fructose-bisphosphate aldolase (EC 5.3.1.1 & 4.1.2.13),
erythrose isomerase (EC 5.3.1.2), arabinose isomerase (EC 5.3.1.3),
L-arabinose isomerase (EC 5.3.1.4), xylose isomerase (EC 5.3.1.5),
ribose-5-phosphate isomerase (EC 5.3.1.6), mannose isomerase (EC
5.3.1.7), mannose-6-phosphate isomerase (EC 5.3.1.8),
glucose-6-phosphate isomerase (EC 5.3.1.9), glucosamine-6-phosphate
isomerase (EC 5.3.1.10), glucuronate isomerase (EC 5.3.1.12),
arabinose-5-phosphate isomerase (EC 5.3.1.13), L-rhamnose isomerase
(EC 5.3.1.14), D-lyxose ketol-isomerase (EC 5.3.1.15), ribose
isomerase (EC 5.3.1.20), L-mannose ketol-isomerase (EC 5.3.1.a),
phospho-3-hexuloisomerase (EC 5.3.1.b), phenylpyruvate tautomerase
(EC 5.3.2.1), oxaloacetate tautomerase (EC 5.3.2.2),
isopentenyl-diphosphate-- isomerase (EC 5.3.3.2), methylitaconate
-isomerase (EC 5.3.3.6), phosphoglycerate mutase (EC 5.4.2.1),
phosphoglucomutase (EC 5.4.2.2), phosphoacetylglucosamine mutase
(EC 5.4.2.3), -phosphoglucomutase (EC 5.4.2.6), phosphopentomutase
(EC 5.4.2.7), phosphomannomutase (EC 5.4.2.8), lysine
2,3-aminomutase (EC 5.4.3.2), D-omithine 4,5-aminomutase
(EC 5.4.3.5), methylaspartate mutase (EC 5.4.99.1),
methylmalonyl-CoA mutase (EC 5.4.99.2), 2-methyleneglutarate mutase
(EC 5.4.99.4), muconate cycloisomerase (EC 5.5.1.1),
tetrahydroxypteridine cycloisomerase (EC 5.5.1.3), chalcone
isomerase (EC 5.5.1.6), valine-tRNA ligase (EC 6.1.1.9),
acetate-CoA ligase (EC 6.2.1.1), butyrateCoA ligase (EC 6.2.1.2),
succinate-CoA ligase (GDP-forming) (EC 6.2.1.4), succinate-CoA
ligase (ADP forming) (EC 6.2.1.5), glutamate-ammonia ligase (EC
6.3.1.2), formate-tetrahydrofolate ligase (EC 6.3.4.3),
adenylosuccinate synthase (EC 6.3.4.4), arginosuccinate synthase
(EC 6.3.4.5), pyruvate carboxylase (EC 6.4.1.1), propanoyl-CoA
carboxylase (EC 6.4.1.3), hemoglobin, tyrosine hydroxylase,
prohormone convertase, bcl-2, dopa decarboxylase, and dopamine
beta-hydroxylase.
[0233] 5.4.3. Assay of Encapsulated Reaction Centers
[0234] Any number of methods are available to determine whether a
reaction center retains its ability to convert prodrug to
biologically active agent upon encapsulation. Those of skill in the
art will be able to modify, if necessary, any standard procedures
developed for assaying the reaction center in free solution for
assaying the encapsulated reaction center. For example, if the
matrix is transparent, as is true for silica-based sol-gel
matrices, then standard visible and UV-Vis techniques for solid
materials may be employed. Yamanka et al. J. Sol-Gel sci. &
tech. 7:117-21 (1996). If the reaction center is redox active
center, e.g., a transition metal, then other spectroscopies, such
as EPR, may be employed. Lin et al. J. Sol-Gel Sci. & Tech.
7:19-26 (1996). As discussed below for the prodrugs, assaying for
reaction center activity often involves measuring the reactants and
products, and solution techniques may be applicable. Alternatively,
coenzymes, cofactors, or other reactants involved in any reaction
center may be monitored as an assay for activity of any
encapsulated reaction center.
[0235] From such assays, it should be possible to determine the
reaction kinetics for the encapsulated reaction center. In general,
for enzymes, the reaction kinetics may correspond to the
Michaelis-Menten treatment. Zubay et al. Biochemistry 137-141
(1983). An apparent Michaelis constant (Km) may be determined for
the encapsulated reaction center. Dosoretz et al., J. Sol-Gel Sci.
& Tech. 7:7-11 (1996). The Km for the nonencapsulated reaction
center and the Km of the encapsulated reaction center may be
compared. In certain embodiments, the ratio of Km (nonencapsulated)
to Km (encapsulated) may be greater than one. Dosoretz et al.,
supra. In other embodiments, the ratio may be less than one. Venton
et al. Biochim Biophys Acta 1250:117-25 (1995). The present
invention contemplates ratios of 100, 10, 5, 1, 0.5, 0.1, 0.005,
0.01, and 0.001. Of course, for determining any of these ratios,
the conditions of the reaction should be kept as similar as
possible.
[0236] The encapsulation of reaction centers allows for the design
of novel assays for reaction center activity. For example, a second
reaction center may be encapsulated so as to help assay the
activity of a first encapsulated reaction center. Yamanka et al.
report encapsulating both oxalate oxidase and peroxidase. The
peroxidase converts two dye precursors into a detectable dye using
hydrogen peroxide, which is formed by oxalate oxidase from oxalate,
water, and dioxygen. Yamanka et al. J. Sol-Gel Sci. & Tech.
7:117-21 (1996). Hence, the peroxidase in this sol-gel matrix
assists in assaying the reaction kinetics of the oxalate oxidase.
Yamanka et al. report that this enzyme system is useful as a
diagnostic for the decreased secretion of oxalate in cases of
hyperglycinemia, hypoclycinuria, and hyperoxaluria. Ngo et al.
Anal. Biochem. 105:389 (1980).
[0237] 5.5. Prodrugs
[0238] 5.5.1. Prodrugs Contemplated by the Invention
[0239] A variety of materials or compounds may be employed as
prodrugs in the present invention. A number of such prodrugs have
been discussed elsewhere, including when considering possible
reaction centers and uses of the present invention. Any compound
that is biologically active may be used in the present invention as
a prodrug, as long as a suitable prodrug may be prepared that may
be converted by a reaction center into a biologically active
compound. The matrices of the present invention may be administered
by way of oral ingestion or implantation. If implantation is
desired, they can be implanted subcutaneously, constitute a part of
a prosthesis, or be inserted in a cavity of the human body.
Subcutaneous implantation using a syringe consists of injecting the
amtrices directly into subcutaneous tissue. Thus, the matrices of
the present invention can be suspended in a physiological buffer
and introduced via a syringe to the desired site. In certain cases,
a biologically active agent may itself be used as a prodrug in the
present invention if a reaction center modulates its biological
activity upon reaction.
[0240] A number of considerations may be weighed by those of skill
in the art in determining which prodrug is appropriate for any use
of the present invention. For example, it may be necessary to match
a prodrug with a reaction center that has a high activity for
conversion of the prodrug. Alternatively, in choosing a prodrug, it
may be important to consider where in a subject the resulting
matrix may be administered, e.g., the use of the prodrug L-dopa to
produce the biologically active agent doparnine in the striatum.
Another possible consideration may be the physical dimension of any
prodrug, for to operate as a prodrug, it may need to diffuse into
the matrix for conversion by the reaction center to a biologically
active agent. (Alternatively, the reaction center may be located on
the surface of the matrix, whereupon no diffusion is necessary for
conversion of the prodrug.) However, even antibodies have been
observed to diffuse into matrices of the present invention, so any
prodrug of at least that dimension may be used in the present
invention. As discussed in preparing the matrices of the present
invention, it may be necessary to ensure that the physical size of
the reaction center is greater than that of its counterpart prodrug
so as to prevent leaching. This criteria need not always apply,
however, because for example, the reaction center may be covalently
attached to the matrix, which may prevent any substantial leaching,
or alternatively, any leaching that may occur may be acceptable for
any use that the matrix is put.
[0241] Possible biologically active agents, which may be used as
prodrugs in the present invention after appropriate modification,
include without limitation, medicaments; vitamins; mineral
supplements; substances used for the treatment, prevention,
diagnosis, cure or mitigation of disease or illness; or substances
which affect the structure or function of the body.
[0242] Specific types of biologically active agents include,
without limitation: anti-angiogenesis factors, antiinfectives such
as antibiotics and antiviral agents; analgesics and analgesic
combinations; anorexics; antihelmintics; antiarthritics;
antiasthmatic agents; anticonvulsants; antidepressants;
antidiuretic agents; antidiarrheals; antihistamines;
antiinflammatory agents; antimigraine preparations; antinauseants;
antineoplastics; antiparkinsonism drugs; antipruritics;
antipsychotics; antipyretics, antispasmodics; anticholinergics;
sympathomimetics; xanthine derivatives; cardiovascular preparations
including calcium channel blockers and beta-blockers such as
pindolol and antiarrhythmics; antihypertensives; catecholamines;
diuretics; vasodilators including general coronary, peripheral and
cerebral; central nervous system stimulants; cough and cold
preparations, including decongestants; growth factors, hormones
such as estradiol and other steroids, including corticosteroids;
hypnotics; immunosuppressives; muscle relaxants;
parasympatholytics; psychostimulants; sedatives; and tranquilizers;
and naturally derived or genetically engineered proteins,
polysaccharides, glycoproteins, lipoproteins, interferons,
cytokines, chemotherapeutic agents and other anti-neoplastics,
antibiotics, anti-virals, anti-fungals, anti-inflammatories,
anticoagulants, lymphokines, or antigenic materials.
[0243] To illustrate further, other types of biologically active
agents that may be used as prodrugs upon appropriate modification
if necessary, including peptide, proteins or other biopolymers,
e.g., interferons, interleukins, tumor necrosis factor, nerve
growth factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), ciliary
neurotrophic factor (CNTF), glial cell line-derived neurotrophic
factor (GDNF), cholinergic differentiation factor/Leukemia
inhibitory factor (CDF/LIF), epidermal growth factor (EGF),
insulin-like growth factor (IGF), basic fibroblast growth factor
(bFGF), platelet-derived growth factor (PDGF), erythropoietin,
growth hormone, Substance-P, neurotensin, insulin, erythropoietin,
albumin, transferrin, and other protein biological response
modifiers.
[0244] Other examples of biologically active agents that may be
used as prodrugs in accordance with the present invention either
directly or after appropriate modification include acebutolol,
acetaminophen, acetohydoxamic acid, acetophenazine, acyclovir,
adrenocorticoids, allopurinol, alprazolam, aluminum hydroxide,
amantadine, ambenonium, amiloride, aminobenzoate potassium,
amobarbital, amoxicillin, amphetamine, ampicillin, androgens,
anesthetics, anticoagulants, anticonvulsants-dione type,
antithyroid medicine, appetite suppressants, aspirin, atenolol,
atropine, azatadine, bacampicillin, baclofen, beclomethasone,
belladonna, bendroflumethiazide, benzoyl peroxide, benzthiazide,
benztropine, betamethasone, betha nechol, biperiden, bisacodyl,
bromocriptine, bromodiphenhydramine, brompheniramine, buclizine,
bumetanide, busulfan, butabarbital, butaperazine, caffeine, calcium
carbonate, captopril, carbamazepine, carbenicillin, carbidopa &
levodopa, carbinoxamine inhibitors, carbonic anhydsase,
carisoprodol, carphenazine, cascara, cefaclor, cefadroxil,
cephalexin, cephradine, chlophedianol, chloral hydrate,
chlorambucil, chloramphenicol, chlordiazepoxide, chloroquine,
chlorothiazide, chlorotrianisene, chlorpheniramine,
6.times.chlorpromazine, chlorpropamide, chlorprothixene,
chlorthalidone, chlorzoxazone, cholestyramine, cimetidine,
cinoxacin, clemastine, clidinium, clindamycin, clofibrate,
clomiphere, clonidine, clorazepate, cloxacillin, colochicine,
coloestipol, conjugated estrogen, contraceptives, cortisone,
cromolyn, cyclacillin, cyclandelate, cyclizine, cyclobenzaprine,
cyclophosphamide, cyclothiazide, cycrimine, cyproheptadine,
danazol, danthron, dantrolene, dapsone, dextroamphetamine,
dexamethasone, dexchlorpheniramine, dextromethorphan, diazepan,
dicloxacillin, dicyclomine, diethylstilbestrol, diflunisal,
digitalis, diltiazen, dimenhydrinate, dimethindene,
diphenhydramine, diphenidol, diphenoxylate & atrophive,
diphenylopyraline, dipyradamole, disopyramide, disulfiram,
divalporex, docusate calcium, docusate potassium, docusate sodium,
doxyloamine, dronabinol ephedrine, epinephrine, ergoloidmesylates,
ergonovine, ergotamine, erythromycins, esterified estrogens,
estradiol, estrogen, estrone, estropipute, etharynic acid,
ethchlorvynol, ethinyl estradiol, ethopropazine, ethosaximide,
ethotoin, fenoprofen, ferrous fumarate, ferrous gluconate, ferrous
sulfate, flavoxate, flecainide, fluphenazine, fluprednisolone,
flurazepam, folic acid, furosemide, gemfibrozil, glipizide,
glyburide, glycopyrrolate, gold compounds, griseofuwin,
guaifenesin, guanabenz, guanadrel, guanethidine, halazepam,
haloperidol, hetacillin, hexobarbital, hydralazine,
hydrochlorothiazide, hydrocortisone (cortisol), hydroflunethiazide,
hydroxychloroquine, hydroxyzine, hyoscyamine, ibuprofen,
indapamide, indomethacin, insulin, iofoquinol, iron-polysaccharide,
isoetharine, isoniazid, isopropamide isoproterenol, isotretinoin,
isoxsuprine, kaolin & pectin, ketoconazole, lactulose,
levodopa, lincomycin liothyronine, liotrix, lithium, loperamide,
lorazepam, magnesium hydroxide, magnesium sulfate, magnesium
trisilicate, maprotiline, meclizine, meclofenamate,
medroxyproyesterone, melenamic acid, melphalan, mephenytoin,
mephobarbital, meprobamate, mercaptopurine, mesoridazine,
metaproterenol, metaxalone, methamphetamine, methaqualone,
metharbital, methenamine, methicillin, methocarbamol, methotrexate,
methsuximide, methyclothinzide, methylcellulos, methyldopa,
methylergonovine, methylphenidate, methylprednisolone,
methysergide, metoclopramide, metolazone, metoprolol,
metronidazole, minoxidil, mitotane, monamine oxidase inhibitors,
nadolol, nafcillin, nalidixic acid, naproxen, narcotic analgesics,
neomycin, neostigmine, niacin, nicotine, nifedipine, nitrates,
nitroftirantoin, nomifensine, norethindrone, norethindrone acetate,
norgestrel, nylidrin, nystatin, orphenadrine, oxacillin, oxazepam,
oxprenolol, oxyrnetazoline, oxyphenbutazone, pancrelipase,
pantothenic acid, papaverine, para-aminosalicylic acid,
paramethasone, paregoric, pemoline, penicillamine, penicillin,
penicillin-v, pentobarbital, perphenazine, phenacetin,
phenazopyridine, pheniramine, phenobarbital, phenolphthalein,
phenprocoumon, phensuximide, phenylbutazone, phenylephrine,
phenylpropanolamine, phenyl toloxamine, phenytoin, pilocarpine,
pindolol, piper acetazine, piroxicam, poloxamer, polycarbophil
calcium, polythiazide, potassium supplements, pruzepam, prazosin,
prednisolone, prednisone, primidone, probenecid, probucol,
procainamnide, procarbazine, prochlorperazine, procyclidine,
promazine, promethazine, propantheline, propranolol,
pseudoephedrine, psoralens, psyllium, pyridostigmine, pyrodoxine,
pyrilamine, pyrvinium, quinestrol, quinethazone, quinidine,
quinine, ranitidine, rauwolfia alkaloids, riboflavin, rifampin,
ritodrine, salicylates, scopolamine, secobarbital, senna,
sannosides a & b, simethicone, sodium bicarbonate, sodium
phosphate, sodium fluoride, spironolactone, sucrulfate,
sulfacytine, sulfamethoxazole, sulfasalazine, sulfinpyrazone,
sulfisoxazole, sulindac, talbutal, tamazepam, terbutaline,
terfenadine, terphinhydrate, teracyclines, thiabendazole, thiamine,
thioridazine, thiothixene, thyroblobulin, thyroid, thyroxine,
ticarcillin, timolol, tocainide, tolazamide, tolbutamide, tolmetin
trozodone, tretinoin, triamcinolone, trianterene, triazolam,
trichlormethiazide, tricyclic antidepressants, tridhexethyl,
trifluoperazine, triflupromazine, trihexyphenidyl, trimeprazine,
trimethobenzamine, trimethoprim, tripclennamine, triprolidine,
valproic acid, verapamil, vitamin A, vitamin B-12, vitamin C,
vitamin D, vitamin E, vitamin K, xanthine, parathyroid hormone,
enkephalins, and endorphins.
[0245] To illustrate further, antimetabolites may be used as
prodrugs upon appropriate modification if necessary, including
without limitation methotrexate, 5-fluorouracil, cytosine
arabinoside (ara-C), 5-azacytidine, 6-mercaptopurine,
6-thioguanine, and fludarabine phosphate. Antitumor antibiotics may
include but are not limited to doxorubicin, daunorubicin,
dactinomycin, bleomycin, mitomycin C, plicamycin, idarubicin, and
mitoxantrone. Vinca alkaloids and epipodophyllotoxins may include,
but are not limited to vincristine, vinblastine, vindesine,
etoposide, and teniposide. Nitrosoureas, including carmustine,
lomustine, semustine and streptozocin, may also be prodrugs, upon
appropriate modification if necessary. Hormonal therapeutics may
also be prodrugs, upon appropriate modification if necessary, such
as corticosteriods (cortisone acetate, hydrocortisone, prednisone,
prednisolone, methyl prednisolone and dexamethasone), estrogens,
(diethylstibesterol, estradiol, esterified estrogens, conjugated
estrogen, chlorotiasnene), progestins (medroxyprogesterone acetate,
hydroxy progesterone caproate, megestrol acetate), antiestrogens
(tamoxifen), aromastase inhibitors (aminoglutethimide), androgens
(testosterone propionate, methyltestosterone, fluoxymesterone,
testolactone), antiandrogens (flutamide), LHRH analogues
(leuprolide acetate), and endocrines for prostate cancer
(ketoconazole). Antitumor drugs that are radiation enhancers may
also be used as prodrugs, upon appropriate modification if
necessary. Examples of such biologically active agents include, for
example, the chemotherapeutic agents 5'-fluorouracil, mitomycin,
cisplatin and its derivatives, taxol, bleomycins, daunomycins, and
methamycins. Antibiotics may be used as prodrugs as well, upon
appropriate modification if necessary, and they are well known to
those of skill in the art, and include, for example, penicillins,
cephalosporins, tetracyclines, ampicillin, aureothicin, bacitracin,
chloramphenicol, cycloserine, erythromycin, gentamicin,
gramacidins, kanamycins, neomycins, streptomycins, tobramycin, and
vancomycin.
[0246] Other prodrugs, upon appropriate modification if necessary,
which may be used in the present invention include those presently
classified as investigational drugs, and can include, but are not
limited to alkylating agents such as Nimustine AZQ, BZQ,
cyclodisone, DADAG, CB10-227, CY233, DABIS maleate, EDMN,
Fotemustine, Hepsulfam, Hexamethylmelamine, Mafosamide, MDMS, PCNU,
Spiromustine, TA-077, TCNU and Temozolomide; antimetabolites, such
as acivicin, Azacytidine, 5-aza-deoxycytidine, A-TDA, Benzylidene
glucose, Carbetimer, CB3717, Deazaguanine mesylate, DODOX,
Doxifluridine, DUP-785, 10-EDAM, Fazarabine, Fludarabine, MZPES,
MMPR, PALA, PLAC, TCAR, TMQ, TNC-P and Piritrexim; antitumor
antibodies, such as AMPAS, BWA770U, BWA773U, BWA502U, Amonafide,
m-AMSA, CI-921, Datelliptium, Mitonafide, Piroxantrone,
Aclarubicin, Cytorhodin, Epirubicin, esorubicin, Idarubicin,
lodo-doxorubicin, Marcellomycin, Menaril, Morpholino
anthracyclines, Pirarubicin, and SM-5887; microtubule spindle
inhibitors, such as Amphethinile, Navelbine, and Taxol; the
alkyl-lysophospholipids, such as BM41-440, ET-18-OCH.sub.3, and
Hexacyclophosphocholine; metallic compounds, such as Gallium
Nitrate, CL286558, CL287110, Cycloplatam, DWA2114R, NK121,
Iproplatin, Oxaliplatin, Spiroplatin, Spirogermanium, and Titanium
compounds; and novel compounds such as, for example, Aphidoicolin
glycinate, Ambazone, BSO, Caracemide, DSG, Didemnin, B, DMFO,
Elsamicin, Espertatrucin, Flavone acetic acid, HMBA, HHT, ICRF-187,
Iododeoxyuridine, Ipomeanol, Liblomycin, Lonidamine, LY186641, MAP,
MTQ, Merabarone SK&F104864, Suramin, Tallysomycin, Teniposide,
THU and WR.sub.2721; and Toremifene, Trilosane, and
zindoxifene.
[0247] 5.5.2. Assays and Identification of Prodrugs
[0248] As a general matter, it will be clear to one of skill in the
art which prodrugs may be used with which reaction centers so as to
effect the any of the uses of the subject invention, e.g.,
producing a biologically active agent. Prodrugs that display
desired characteristics, e.g., certain kinetic profiles of
conversion of a prodrug by a reaction center to the corresponding
biologically active agent, may serve as lead compounds for the
discovery of more desirable prodrugs.
[0249] In general, there are a number of methods by which useful
prodrugs of any reaction center encapsulated in a sol gel may be
determined. For example, prodrugs may be individually prepared and
tested for production of the corresponding biologically active
agent upon interaction with the reaction center, whether
encapsulated or not.
[0250] In another embodiment of the present invention, the use of
prodrugs in this invention readily lends itself to the creation of
combinatorial libraries of compounds for screening prospective
prodrugs with any particular reaction center or group of reaction
centers to identify prodrugs of such reaction centers. For the
purposes of the present invention, the application of combinatorial
chemistry may be especially valuable because it may render
identification of a suitable prodrug of a biologically active agent
for use with a particular reaction center more facile. A
combinatorial library for the purposes of the present invention is
a mixture of chemically related compounds which may be screened
together for a desired property, e.g., conversion by a particular
reaction center or reaction centers to produce a biologically
active agent. Such libraries may be in solution or covalently
linked to a solid support. The preparation of many related
compounds as prospective prodrugs in a single reaction greatly
reduces and simplifies the number of screening processes which need
to be carried out. Screening for the appropriate reactivity of any
prospective prodrug may be done by conventional methods.
[0251] For purposes of this invention, diversity in a library may
be created at a variety of different levels. In general, for
instance, substrate aryl groups used in a combinatorial approach
can be diverse in terms of the core aryl moiety, e.g., a
variegation in terms of the ring structure, and/or can be varied
with respect to the other substituents. With respect to the subject
invention, for example, it is generally known that
carboxypeptidases hydrolyze amide bonds. Any biologically active
agent having an amine or carboxylic acid moiety may, in theory, be
derivatized in a combinatorial approach to form an amide with a
carboxylic acid or amine moiety, respectively. For example, a
peptidyl fragment having a varied number of amino acid residues
with a diverse identity could be coupled to a biologically active
agent of interest to give a library of prospective prodrugs,
whereupon conversion by reaction centers such as carboxypeptidases
of the prospective prodrugs in the library could be screened. In
this fashion, prospective prodrugs of a biologically active agent
could be prepared and screened for use with a particular reaction
center, or alternatively, a group of reaction centers that catalyze
a similar type of chemical conversion. As already noted above, the
reaction centers themselves may be prepared and screened by
combination methods as well. As will be clear to one of skill in
the art, in preparing any such library, some considerations to take
into account include the chemical conversion catalyzed by the
reaction center or centers of interest; in what fashion a
biologically active agent may be readily derivatized to provide
prodrugs so that the reaction center or centers of interest could
produce the biologically active agent from a prodrug; and the
specificity of the reaction center or centers of interest to a
variation in structure with respect to the reaction that it
normally catalyzes, e.g., the naturally occurring substrate for an
enzyme.
[0252] A variety of techniques are available in the art for
generating combinatorial libraries of small organic molecules. See
generally Blondelle et al. Trends Anal. Chem. 14:83 (1995); U.S.
Pat. Nos. 5,359,115, 5,362,899, 5,288,514, and 5,721,099; Chen et
al. JACS 116:2661 (1994; Kerr et al. JACS 115:252 (1993);
WO92/10092, WO93/09668, WO 94/08051, WO93/20242 and WO91/07087.
Accordingly, a variety of libraries on the order of about 16 to
1,000,000 or more diversomers can be synthesized and screened for a
particular activity or property.
[0253] In an exemplary embodiment, a library of substituted
diversomers can be synthesized using the subject reactions adapted
to the techniques described in WO 94/08051, e.g., being linked to a
polymer bead by a hydrolyzable or photolyzable group, e.g., located
at one of the positions of substrate. According to the technique
disclosed therein, the library is synthesized on a set of beads,
each bead including a set of tags identifying the particular
diversomer on that bead. In one embodiment, the beads can be
dispersed on the surface of a permeable membrane, and the
diversomers released from the beads by lysis of the bead linker.
The diversomer from each bead will diffuse across the membrane to
an assay zone, where it will interact with an assay for a reaction
center or centers. Detailed descriptions of a number of
combinatorial methodologies are provided below.
[0254] (a) Direct Characterization. A growing trend in the field of
combinatorial chemistry is to exploit the sensitivity of techniques
such as mass spectrometry (MS), e.g., which can be used to
characterize sub-femtomolar amounts of a compound, and to directly
determine the chemical constitution of a compound selected from a
combinatorial library. For instance, where the library is provided
on an insoluble support matrix, discrete populations of compounds
can be first released from the support and characterized by MS. In
other embodiments, as part of the MS sample preparation technique,
such MS techniques as MALDI can be used to release a compound from
the matrix, particularly where a labile bond is used originally to
tether the compound to the matrix. For instance, a bead selected
from a library can be irradiated in a MALDI step in order to
release the diversomer from the matrix, and ionize the diversomer
for MS analysis.
[0255] (b) Multipin Synthesis. The libraries of the subject method
can take the multipin library format. Briefly, Geysen and
co-workers, Geysen et al. PNAS 81:3998-4002 (1984), introduced a
method for generating compound libraries by a parallel synthesis on
polyacrylic acid-grated polyethylene pins arrayed in the microtitre
plate format. The Geysen technique can be used to synthesize and
screen thousands of compounds per week using the multipin method,
and the tethered compounds may be reused in many assays.
Appropriate linker moieties can also been appended to the pins so
that the compounds may be cleaved from the supports after synthesis
for assessment of purity and further evaluation. Compare Bray et
al. Tetrahedron Lett. 31:5811-14 (1990); Valerio et al. Anal
Biochem 197:168-77 (1991); Bray et al. Tetrahedron Lett. 32:6163-66
(1991).
[0256] (c) Divide-Couple-Recombine. In yet another embodiment, a
variegated library of compounds can be provided on a set of beads
utilizing the strategy of divide-couple-recombine. See, for
example, Houghten PNAS 82:5131-35 (1985); and U.S. Pat. Nos.
4,631,211; 5,440,016; 5,480,971. Briefly, as the name implies, at
each synthesis step where degeneracy is introduced into the
library, the beads are divided into separate groups equal to the
number of different substituents to be added at a particular
position in the library, the different substituents coupled in
separate reactions, and the beads recombined into one pool for the
next iteration.
[0257] In one embodiment, the divide-couple-recombine strategy can
be carried out using an analogous approach to the so-called "tea
bag" method first developed by Houghten, where compound synthesis
occurs on resin sealed inside porous polypropylene bags. Houghten
et al. PNAS 82:5131-35 (1986). Substituents are coupled to the
compound-bearing resins by placing the bags in appropriate reaction
solutions, while all common steps such as resin washing and
deprotection are performed simultaneously in one reaction vessel.
At the end of the synthesis, each bag contains a single
compound.
[0258] (d) Combinatorial Libraries by Light-Directed, Spatially
Addressable Parallel Chemical Synthesis. A scheme of combinatorial
synthesis in which the identity of a compound is given by its
locations on a synthesis substrate is termed a
spatially-addressable synthesis. In one embodiment, the
combinatorial process is carried out by controlling the addition of
a chemical reagent to specific locations on a solid support. Dower
et al. Annu Rep Med Chem 26:271-280 (1991); Fodor, Science 251:767
(1991); U.S. Pat. No. 5,143,854; Jacobs et al. Trends Biotechnol
12:19-26 (1994). The spatial resolution of photolithography affords
miniaturization. This technique can be carried out through the use
protection/deprotection reactions with photolabile protecting
groups.
[0259] The key points of this technology are illustrated in Gallop
et al. J Med Chem 37:1233-51 (1994). A synthesis substrate is
prepared for coupling through the covalent attachment of
photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers
or other photolabile linkers. Light is used to selectively activate
a specified region of the synthesis support for coupling. Removal
of the photolabile protecting groups by light (deprotection)
results in activation of selected areas. After activation, the
first of a set of amino acid analogs, each bearing a photolabile
protecting group on the amino terminus, is exposed to the entire
surface. Coupling only occurs in regions that were addressed by
light in the preceding step. The reaction is stopped, the plates
washed, and the substrate is again illuminated through a second
mask, activating a different region for reaction with a second
protected building block. The pattern of masks and the sequence of
reactants define the products and their locations. Since this
process utilizes photolithography techniques, the number of
compounds that can be synthesized is limited only by the number of
synthesis sites that can be addressed with appropriate resolution.
The position of each compound is precisely known; hence, its
interactions with other molecules can be directly assessed. With
respect to the above example, for example, a library of peptidyl
fragments could thereby be prepared, whereupon the biologically
active agent could be coupled in the final step to produce a
diverse library of prospective prodrugs when paired with reaction
centers that hydrolyze peptide bonds.
[0260] In a light-directed chemical synthesis, the products depend
on the pattern of illumination and on the order of addition of
reactants. By varying the lithographic patterns, many different
sets of test compounds can be synthesized simultaneously; this
characteristic leads to the generation of many different masking
strategies.
[0261] (e) Encoded Combinatorial Libraries. In yet another
embodiment, the subject method utilizes a compound library provided
with an encoded tagging system. A recent improvement in the
identification of active compounds from combinatorial libraries
employs chemical indexing systems using tags that uniquely encode
the reaction steps a given bead has undergone and, by inference,
the structure it carries. Conceptually, this approach mimics phage
display libraries, where activity derives from expressed peptides,
but the structures of the active peptides are deduced from the
corresponding genomic DNA sequence. The first encoding of synthetic
combinatorial libraries employed DNA as the code. A variety of
other forms of encoding have been reported, including encoding with
sequenceable bio-oligomers (e.g., oligonucleotides and peptides),
and binary encoding with additional non-sequenceable tags.
[0262] (1) Tagging with sequenceable bio-oligomers. The principle
of using oligonucleotides to encode combinatorial synthetic
libraries was described in 1992 Brenner et al. PNAS 89:5381-83
(1992), and an example of such a library appeared the following
year. Needles et al. PNAS 90:10700-04 (1993). A combinatorial
library of nominally 7.sub.7 (=823,543) peptides composed of all
combinations of Arg, Gln, Phe, Lys, Val, D-Val and Thr
(three-letter amino acid code), each of which was encoded by a
specific dinucleotide (TA, TC, CT, AT, TT, CA and AC,
respectively), was prepared by a series of alternating rounds of
peptide and oligonucleotide synthesis on solid support. In this
work, the amine linking functionality on the bead was specifically
differentiated toward peptide or oligonucleotide synthesis by
simultaneously preincubating the beads with reagents that generate
protected OH groups for oligonucleotide synthesis and protected
NH.sub.2 groups for peptide synthesis (here, in a ratio of 1:20).
When complete, the tags each consisted of 69-mers, 14 units of
which carried the code. The bead-bound library was incubated with a
fluorescently labeled antibody, and beads containing bound antibody
that fluoresced strongly were harvested by fluorescence-activated
cell sorting (FACS). The DNA tags were amplified by PCR and
sequenced, and the predicted peptides were synthesized. Following
such techniques, compound libraries can be derived for use in the
subject method, where the oligonucleotide sequence of the tag
identifies the sequential combinatorial reactions that a particular
bead underwent, and therefore provides the identity of the compound
on the bead.
[0263] The use of oligonucleotide tags permits exquisitely
sensitive tag analysis. Even so, the method requires careful choice
of orthogonal sets of protecting groups required for alternating
co-synthesis of the tag and the library member. Furthermore, the
chemical lability of the tag, particularly the phosphate and sugar
anomeric linkages, may limit the choice of reagents and conditions
that can be employed for the synthesis of non-oligomeric libraries.
In preferred embodiments, the libraries employ linkers permitting
selective detachment of the test compound library member for
assay.
[0264] Peptides have also been employed as tagging molecules for
combinatorial libraries. Two exemplary approaches are described in
the art, both of which employ branched linkers to solid phase upon
which coding and ligand strands are alternately elaborated. In the
first approach, Kerr et al. J Am Chem Soc 115:2529-31 (1993),
orthogonality in synthesis is achieved by employing acid-labile
protection for the coding strand and base-labile protection for the
compound strand.
[0265] In an alternative approach, Nikolaiev et al. Pept Res
6:161-70 (1993), branched linkers are employed so that the coding
unit and the test compound can both be attached to the same
functional group on the resin. In one embodiment, a cleavable
linker can be placed between the branch point and the bead so that
cleavage releases a molecule containing both code and the compound.
Ptek et al. Tetrahedron Lett 32:3891-94 (1991). In another
embodiment, the cleavable linker can be placed so that the test
compound can be selectively separated from the bead, leaving the
code behind. This last construct is particularly valuable because
it permits screening of the test compound without potential
interference of the coding groups. Examples in the art of
independent cleavage and sequencing of peptide library members and
their corresponding tags has confirmed that the tags can accurately
predict the peptide structure.
[0266] (2) Non-sequenceable Tagging: Binary Encoding. An
alternative form of encoding the test compound library employs a
set of non-sequencable electrophoric tagging molecules that are
used as a binary code. Ohlmeyer et al. PNAS 90:10922-26 (1993).
Exemplary tags are haloaromatic alkyl ethers that are detectable as
their trimethylsilyl ethers at less than femtomolar levels by
electron capture gas chromatography (ECGC). Variations in the
length of the alkyl chain, as well as the nature and position of
the aromatic halide substituents, permit the synthesis of at least
40 such tags, which in principle can encode 2.sup.40 (e.g., upwards
of 10.sup.12) different molecules. In the original report, Ohlmeyer
et al., supra, the tags were bound to about 1% of the available
amine groups of a peptide library via a photocleavable
o-nitrobenzyl linker. This approach is convenient when preparing
combinatorial libraries of peptide-like or other amine-containing
molecules. A more versatile system has, however, been developed
that permits encoding of essentially any combinatorial library.
Here, the compound would be attached to the solid support via the
photocleavable linker and the tag is attached through a catechol
ether linker via carbene insertion into the bead matrix. Nestler et
al. J Org Chem 59:4723-24 (1994). This orthogonal attachment
strategy permits the selective detachment of library members for
assay in solution and subsequent decoding by ECGC after oxidative
detachment of the tag sets.
[0267] Although several amide-linked libraries in the art employ
binary encoding with the electrophoric tags attached to amine
groups, attaching these tags directly to the bead matrix provides
far greater versatility in the structures that can be prepared in
encoded combinatorial libraries. Attached in this way, the tags and
their linker are nearly as unreactive as the bead matrix itself.
Two binary-encoded combinatorial libraries have been reported where
the electrophoric tags are attached directly to the solid phase,
Ohlmeyer et al. PNAS 92:6027-31 (1995), and provide guidance for
generating the subject compound library. Both libraries were
constructed using an orthogonal attachment strategy in which the
library member was linked to the solid support by a photolabile
linker and the tags were attached through a linker cleavable only
by vigorous oxidation. Because the library members can be
repetitively partially photoeluted from the solid support, library
members can be utilized in multiple assays. Successive photoelution
also permits a very high throughput iterative screening strategy:
first, multiple beads are placed in 96-well microtiter plates;
second, compounds are partially detached and transferred to assay
plates; third, a metal binding assay identifies the active wells;
fourth, the corresponding beads are rearrayed singly into new
microtiter plates; fifth, single active compounds are identified;
and sixth, the structures are decoded.
[0268] When prospective prodrugs are screened as libraries of
compounds, high throughput assays are desirable in order to
maximize the number of compounds surveyed in a given period of
time. The activity of the reaction center, e.g., enzymatic
activity, with any prospective prodrug may be determined by
monitoring either the disappearance of prodrug or the appearance of
the corresponding biologically active agent. Alternatively, the
reaction center activity may be determined by monitoring the
reduction or production or any reactants consumed or by-products
produced. Spectroscopic methods well-known to those of skill may be
used for such monitoring, or alternatively, any of the reactants or
products, e.g., the prodrug or the corresponding biologically
active agent, may be isolated and quantified.
[0269] In other embodiments, differential assays can be used to
identify prodrugs that react more readily with a encapsulated
reaction center than with any other naturally occurring enzyme, so
that any such prodrug are converted chiefly by any administered
encapsulated reaction center instead of by any naturally occurring
enzymes or catalysts. Such a feature may be desirable depending on
how the matrix is used.
[0270] 5.6. Administration
[0271] 5.6.1. Matrix Administration
[0272] Immobilized enzymes may be administered in a variety of
ways. See generally Ming et al. Methods for Therapeutic
Applications 46:676-699. The site of administration of the matrix
may affect its therapeutic effect depending on the reaction center
encapsulated therein. For example, the site of implantation of
encapsulated PC12 cells for treatment of Parkinson's disease
appears to affect the device output. Emerich et al. Cell
Transplant. 5:589-96 (1996).
[0273] A number of different implantation sites in a subject are
contemplated for the matrices of this invention. In particular, the
most preferred site is determined by the identity of the
encapsulated reaction center. Any site that results in a
therapeutic effect may be used. For example, for reaction centers
that produce biologically active agents that are cytotoxic, the
implants may be implanted near any neoplasm. ADEPT technology
relies on such proximity to deliver any cytotoxic agent essentially
directly to the tumor. In another instance, for matrices used to
treat Parkinson's disease by affecting dopamine levels in the
brain, implantation in the brain may be preferred. Other sites in
the brain for such matrices include the basal ganglia, the
substantia nigra, and the striatum.
[0274] The matrices of the present invention may be administered by
way of oral ingestion or implantation. If implantation is desired,
they can be implanted subcutaneously, constitute a part of a
prosthesis, or be inserted in a cavity of the human body.
Subcutaneous implantation using a syringe consists of injecting the
matrices directly into subcutaneous tissue. Thus, the matrices of
the present invention can be suspended in a physiological buffer
and introduced via a syringe to the desired site. Other sites
include the central nervous system, including the brain, spinal
cord, and aqueous and vitreous humors of the eye. Other sites in
the brain include the cerebral cortex, subthalamic nuclei and
nucleus Basalis of Maynert. Other sites include the cerebrospinal
fluid, the subarachnoid space, and the lateral ventricles. Other
sites includes the kidney subcapsular site, and intraperitoneal and
subcutaneous sites.
[0275] In other embodiments of the present invention, the matrices
of the present invention may be associated with a medical article
to be used as an implant. For example, matrices of the present
invention could be attached as thin films to such devices.
Alternatively, matrices of the present invention could be attached
as a capsule or incorporated into any medical device. Exemplary
structural medical articles include such implants as orthopedic
fixation devices, ventricular shunts, laminates for degradable
fabric, drug-carriers, burn dressings, coatings to be placed on
other implant devices, and the like.
[0276] For administration of matrices of the present invention, an
important feature may be whether the matrix is intended to stay in
place after administration or move in the subject. For example,
matrices administered to a subject may be transported and localized
in the lymphatic system as part of the subject immune response to
the foreign objects.
[0277] Once a matrix of the present invention is administered, it
may remain in at least partial contact with a biological fluid,
such as blood, internal organ secretions, mucus membranes,
cerebrospinal fluid, and the like.
[0278] The length of the period during which encapsulated reaction
center remains active enough so as to produce a therapeutic effect
may depend on a variety of features. Enzymes encapsulated in
silica-based sol-gel matrices have remained active for periods of
several months. The administration of any matrix of the present
invention may result in the long-term, stable production of a
biologically active agent.
[0279] 5.6.2. Formulations and Use of Matrices and Prodrugs
[0280] In addition to the general introduction, pharmaceutical
compositions for use in accordance with the present invention may
be formulated in a conventional manner using one or more
physiologically acceptable carriers or excipients. Thus, as
appropriate, matrices and any prodrug, including any
physiologically acceptable salts and solvates, may be formulated
for administration by, for example, injection, inhalation or
insufflation (either through the mouth or the nose) or oral,
buccal, parenteral or rectal administration. Appropriate
formulations may depend, in part, on the administration method used
and whether a prodrug or a matrix is being administered.
[0281] The matrices or prodrugs of the invention may be formulated
for a variety of loads of administration, including systemic and
topical or localized administration. Techniques and formulations
generally may be found in Remington's Pharmaceutical Sciences,
Meade Publishing Co., Easton, Pa. Intramuscular, intravenous,
intraperitoneal, and subcutaneous injection is possible. For
injection, the matrices or prodrugs of the invention can be
formulated in liquid solutions, preferably in physiologically
compatible buffers such as Hank's solution or Ringer's solution. In
addition, the prodrugs may be formulated in solid form and
redissolved or suspended immediately prior to use. Lyophilized
forms are also included.
[0282] For oral administration, the matrices or prodrugs may take
the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0283] Preparations for oral administration may be suitably
formulated to give controlled release of any prodrug. For buccal
administration the prodrugs may take the form of tablets or
lozenges formulated in conventional manner. For administration by
inhalation, the prodrugs for use according to the present invention
are conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0284] The prodrugs and/or matrices may be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the prodrug may be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0285] The prodrugs may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0286] In addition to the formulations described previously, the
prodrugs and matrices of the present invention may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the prodrugs may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt. Other suitable delivery
systems include microspheres which offer the possibility of local
noninvasive delivery of drugs over an extended period of time. This
technology utilizes microspheres of precapillary size which can be
injected via a coronary catheter into any selected part of the e.g.
heart or other organs without causing inflammation or ischemia.
Other methods of controlled release of the prodrugs and matrices of
the present invention are known to those of skill in the art.
[0287] Systemic administration may also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives. In addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the prodrugs of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art. A
wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0288] The prodrugs and/or matrices may, if desired, be presented
in a pack or dispenser device which may contain one or more unit
dosage forms containing the active ingredient. The pack may for
example comprise metal or plastic foil, such as a blister pack. The
pack or dispenser device may be accompanied by instructions for
administration.
[0289] The prodrugs may be employed in the present invention in
various forms, such as molecular complexes or pharmaceutically
acceptable salts. Representative examples of such salts are
succinate, hydrochloride, hydrobromide, sulfate, phosphate,
nitrate, borate, acetate, maleate, tartrate, salicylate, metal
salts (e.g., alkali or alkaline earth), ammonium or amine salts
(e.g., quaternary ammonium) and the like. Furthermore, derivatives
of the prodrugs such as esters, amides, and ethers which have
desirable retention and release characteristics but which are
readily hydrolyzed in vivo by physiological pH or enzymes can also
be employed.
[0290] 5.7. Treatment
[0291] The selected dosage level for the matrices and prodrugs, if
applicable, of the present invention will depend upon a variety of
factors including: the load of the reaction center within the
matrix; the activity of the reaction center, the activity of the
particular prodrug employed, or the ester, salt or amide thereof,
the route of administration; the time of administration, the rate
of excretion of the particular prodrug (and possibly matrix) being
employed, the duration of the treatment, other drugs, compounds
and/or materials used in combination with the particular matrix and
prodrug employed, the age, sex, weight, condition, general health
and prior medical history of the patient being treated, and like
factors well known in the medical arts.
[0292] Toxicity and therapeutic efficacy of the matrices of the
present invention may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50. (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. In certain embodiments,
those in which an exogenous prodrug is activated by the reaction
center encapsulated in a biocompatible matrix of the subject
invention, the efficacy of treatment using the subject invention
may be gauged by comparing any of the foregoing parameters
resulting from treatment using a prodrug alone (i.e., without the
subject matrix), the biologically active substance that results
from the prodrug alone, and treatment using the prodrug and a
subject matrix as disclosed herein. In certain embodiments,
treatments using the subject invention have ratios of about two (or
less), five, ten, one hundred, one thousand or even greater orders
of magnitude more favorable than treatment with the prodrug alone
or the biologically active substance that results from the prodrug
alone.
[0293] Because the matrix itself does not result in a therapeutic
effect without the involvement of some other compound, e.g., a
prodrug or naturally occurring metabolite, but also the
availability of other compounds that interact with the matrix may
affect any treatment regime. In general, matrices and the
biologically active agents that they produce which exhibit large
therapeutic indices are preferred. By targeting the matrix to a
particular region of a subject so as to localize the production of
the biologically active agent, the therapeutic efficacy may be
dramatically increased, and unwanted side effects may be minimized.
For example, by implanting the dopamine producing matrix in the
striatum, it may not be necessary to administer L-dopa with
carbidopa or benserazide, which is used to combat nausea resulting
from conversion of L-dopa to dopamine outside of the brain.
[0294] Because the matrix, upon administration, may be in place and
active for significant time periods, ant treatment regime may
involve multiple administrations of a prodrug so as to produce
biologically active agent.
[0295] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any matrix and/or prodrug used in the
present invention, the therapeutically effective dose can be
estimated initially from cell culture assays. For example, a dose
may be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma of a biologically active agent may be
measured, for example, by high performance liquid
chromatography.
Exemplifications
[0296] The present invention now being generally described, it may
be more readily understood by reference to the following examples
which are included merely for purposes of illustration of certain
aspects and embodiments of the present invention, and are not
intended to limit the invention in any way.
[0297] A. Reaction Center Encapsulation Studies
[0298] 1. Matrix Preparation
[0299] The general synthetic technique used for preparation of the
silica sol was addition of 21 mL of tetramethyl orthosilicate
(Aldrich, 99+%) and 5.08 mL of a 4 mM HCl solution to a
25.times.150 mm test tube equipped with a stirbar. The mixture is
stirred until homogeneous (approximately 15 minutes). The test tube
containing the sol is then transferred to an ice bath and allowed
to cool for 10 minutes. A 2 mL aliquot of sol is then transferred
to another chilled test tube in an ice bath and stirred. To this
sol, 1 mL of chilled buffer solution (appropriate to the enzyme to
be entrapped) is added, and stirred for ca. 10 s, followed by
addition of 1 mL of chilled, buffered solution containing the
desired enzyme. The sol is swirled briefly, and then pipetted into
a 4.5 mL polystyrene cuvette (cell culture dishes were also used
for surface area study matrices). The cuvette opening is sealed
with Parafilm following gel formation (cell culture dish covers
were used for surface area study matrices). The gel is then allowed
to age in the sealed container for a period of time ranging from 18
h to 50 d or more at temperatures ranging from 4.degree. C. to room
temperature. Selected samples were dried at ambient temperature
over a period of days to weeks by puncturing the Parafilm covering
the container opening. Other samples were assayed without
drying.
[0300] 2. Enzyme Encapsulation and Assays
[0301] a) b-Glucosidase
[0302] The entrapment of b-Glucosidase (from almonds, crude,
lyophilized powder, Sigma) was performed as outlined above, using
50 mM, pH 5.0 acetate buffer. The activity assay was performed
using 2.667 mL of a 10 mM solution of
para-nitrophenyl-b-D-glucopyranoside (Sigma, 99+%) in buffer and
37.33 mL acetate buffer in a 125 mL Erlenmeyer flask (40 mL total
solution volume). 2 mL aliquots of solution were removed for assay
and their UV-Vis spectra recorded.
[0303] b) Penicillinase
[0304] Entrapment of Penicillinase (Type I from Bacillus cereus,
lyophilized powder containing approx. 10% protein, Sigma) was
performed as outlined above, using 50 mM pH 6.5 phosphate buffer.
Penicillinase activity was determined using 100 mL of a 3 mM
solution of Penicillin G (Benzylpenicillin, sodium salt, Sigma) in
buffer. 2 mL aliquots of the reaction solution were removed for
assay and their UV-Vis spectra recorded.
[0305] c) Tyrosinase
[0306] Entrapment of Tyrosinase (from mushroom, Sigma) was
performed as outlined above, with 5 mM pH 6.5 phosphate buffer
solution. Tyrosinase activity assays were performed using 0.3 mM
L-tyrosine (Aldrich, 99+%) solution in buffer.
[0307] d) Tyrosine Decarboxylase
[0308] Entrapment of Tyrosine Decarboxylase (from Streptococcus
faecalis, Fluka) was performed using 50 mM pH 5.5 acetate buffer
and was accomplished by the method outlined above. The activity
assay was accomplished using a 50:50 mixture of 2.5 mM solution of
L-tyrosine (Aldrich, 99+%) in buffer and buffer. Total reaction
volume for this assay was either 40 mL (19.5 h data reported
herein) or 100 mL (all other data reported). 2 mL aliquots of
reaction mixture were removed from the reaction vessel for assay.
The method used for this assay was addition of 1 mL of a 1M K2CO3
solution to the 2 mL aliquot, followed by mixing. To this was added
2 drops of a solution of picrylsulfonic acid (5% w/v aquesous
solution of 2,4,6-trinitrobenzenesulfonic acid, Sigma). The mixture
was mixed well. 2 mL of toluene were added to this mixture, the
layers shaken well, and centrifuged. The toluene layer was removed
and its UV-Vis spectrum collected. Where applicable, a 0.1 mM
solution of pyridoxal-5-phosphate monohydrate (98%, Aldrich) in
buffer was substituted for the buffer solution in the assay
mixture. Assays performed in the presence of cofactor were carried
out in foil-covered reaction vessels due to the sensitivity of
pyridoxal-5-phosphate to light.
[0309] 3. Results of Enzyme Encapsulation and Assays
[0310] a) b-Glucosidase entrapment yielded active matrices which
were assayed using the synthetic substrate
para-nitrophenyl-b-D-glucopyranosid- e, shown below. Enzymatic
activity of matrix composites on the synthetic substrate results in
cleavage of the glucosidic bond producing a bathochromic shift in
the spectral band. This shift permits monitoring of the cleavage
process, as illustrated in FIG. 1. 13
[0311] The synthetic substrate
para-nitrophenyl-b-D-glucopyranoside.
[0312] b) Penicillinase entrapment provided active matrices which
were assayed using the sodium salt of the synthetic substrate
benzylpenicillin, shown below. Conversion of penicillin to
penicilloic acid via rupture of the .beta.-lactam ring may be
monitored spectrophotometrically, as shown in FIG. 2. 14
[0313] Substrate used for penicillinase activity assay, sodium
benzylpenicillin.
[0314] Reproducibility of the measurements done for penicillinase
was checked by performing activity assay multiple times for the
same matrix. As shown in FIG. 3(a), good agreement is observed for
multiple assays performed over six consecutive days. Likewise,
running multiple matrices from the same preparation to check
reproducibility of the matrix entrapment shows good agreement, as
seen in FIG. 3(b).
[0315] Loading studies utilizing penicillinase matrices were
performed in which the enzyme concentration was varied over a wide
range to determine the optimal enizyme concentration. The bar graph
shown in FIG. 4 shows the effects of varying enzyme concentration
on the activity of the matrix. The highest percentage of activity
observed as a function of enzyne entrapped within the matrix
(selected from the five compositions analyzed) occurs for the
lowest concentration of enzyme examined, as shown in FIG. 5.
[0316] Surface area effects were also examined utilizing
penicillinase matrices. Ten identical monoliths were prepared and
aged simultaneously. Five of these were assayed as whole monoliths
(cast in 4.5 mL cuvettes) while another five matrices were coarsely
crushed and then assayed. This qualitative examination of surface
area effects revealed that an increase in surface area does result
in an increase in the enzyme activity observed, as shown in FIGS. 6
and 7. It should be noted that there is no leaching of enzyme
observed from either the whole or crushed matrices, as determined
by soaking the matrices in buffer solution overnight and
subsequently checking the activity of the soak solution. The
reproducibility of the measurements for the assays shown in FIG. 6
is quite reasonable, with the larger deviation in the crushed
matrices attributable to the lack of control over particle size
when breaking up the samples. FIG. 7, showing the mean values for
each measurement with error bars, emphasizes the greater relative
activity of the crushed matrix samples.
[0317] The significant effect of changing surface area on the
observed enzyme activity prompted further investigation. Control
over total surface area was achieved by casting the sol containing
penicillinase into varying numbers of cell culture plate wells
(22.6 mm diameter). By varying the amount of sol cast into a given
well, the total 4 mL of material per matrix could be spread out
over a number of wells and the disks cast in these wells could be
recombined, after removal from the wells, for assay. Thus, the 4 mL
of sol that constitutes one matrix preparation could be cast into
one or multiple wells to generate samples with known, varying
surface areas. Surface area stated for a given matrix reflects the
initial surface area of the gel when freshly cast, and does not
attempt to correct for any shrinkage that occurred during aging.
FIG. 8(a) illustrates the difference in activity observed for
matrices of varying surface areas. FIG. 8(b) shows the activity as
a percentage of the penicillinase activity used in the preparation
of the matrices.
[0318] c) Following entrapment of tyrosinase, the bifunctional
activity of this enzyme was found to complicate spectrophotometric
assay of the matrix composite due to the variation in molar
extinction coefficient of the different species, and possible
retention within the matrix. Tyrosinase possesses both cresolase
(conversion of phenols to diphenols) and catecholase activity
(conversion of diphenols to the corresponding quinone), as shown
below. However, a qualitative analysis shows the conversion of the
natural substrate L-tyrosine to L-dopa, which then undergoes
dehydrogenation to give dopaquinone. Dopaquinone is unstable in
aqueous solutions and undergoes a Michealis rearrangement to form,
among other products, a number of melanin precursors which
eventually polymerize to produce pigments. This complicated
reaction may be followed qualitatively by monitoring a color change
within the matrix. Although the L-tyrosine solution is, itself,
colorless, the tyrosinase-containing matrices become noticeably
darkened within one hour of contact with the substrate solution,
suggesting formation of products with subsequent retention by the
matrix. A solution of L-dopa in the presence of tyrosinase,
likewise forms a gray-black precipitate. 15
[0319] d) Active Tyrosine Decarboxylase matrices were and assayed
using L-tyrosine as substrate. A complicating factor in the assay
of entrapped Tyrosine Decarboxylase was the unavailability of a
direct spectrophotometric method due to the equivalent molar
extinction coefficients of substrate and product. The observation
necessitated the development of an indirect assay provided by Phan
et. al., App. Biochem. Biotech. 8:127 (1983). The results of an
active Tyrosine Decarboxylase composite assay are shown in FIG.
9.
[0320] Longer aging times for Tyrosine Decarboxylase-containing
matrices resulted in matrices for which no enzyme activity was
observed without addition of cofactor. Addition of cofactor,
pyridoxal-5-phosphate monohydrate (0.05 mM), to the assay mixture
restored activity of the entrapped enzyme to varying degrees
depending on the aging of the monolith. FIG. 10 shows activity
assays for two 16 day old Tyrosine Decarboxylase-containing
matrices, one without cofactor present and one with cofactor, and
compares them to a matrix of the same composition assayed after
aging 19 h. The activity observed at 19 h without cofactor and at
16 d with cofactor present are nearly identical, whereas without
the presence of cofactor little, if any, activity is noted. For
matrices aged 50 d a significant portion of the activity is
retained in the presence in cofactor, although some loss of
activity is observed.
[0321] B. Matrix Optimization Studies
[0322] 1. Matrix Preparation
[0323] The general synthetic technique used for preparation of the
silica sol was addition of appropriate aliquots of the organically
substituted trimethoxysilane, tetramethyl orthosilicate and 4 mM
HCl solution to a 25.times.150 mm test tube equipped with a
stirbar. Total desired volume of sol was determined by the number
of matrices to be prepared. The RSi(OCH.sub.3).sub.3 and TMOS
reagents were combined in appropriate ratios to yield the desired
compositions.
3 Reagent Source Tetramethylorthosilicate (TMOS) Aldrich, 99+%
Methyltrimethoxysilane (MTMS) Aldrich, 98% Ethyltrimethoxysilane
(ETMS) Aldrich, 97+% Trimethoxypropylsilane (TMPS) Aldrich, 98%
iso-Butyltrimethoxysila- ne (i-BTMS) Aldrich, 97%
n-Butyltrimethoxysilane (n-BTMS) United Chemical Technologies,
95.3% Phenyltrimethoxysilane (PTMS) Aldrich, 97%
[0324] As with 100% TMOS matrices, the mixture is stirred until
homogeneous (approximately 15 minutes). The test tube containing
the sol is then transferred to an ice bath and allowed to cool for
10 minutes. A 2 mL aliquot of sol is then transferred to another
chilled test tube in an ice bath and stirred. To this sol, 1 mL of
chilled buffer solution (appropriate to the enzyme to be entrapped)
is added, and stirred for ca. 10 s, followed by addition of 1 mL of
chilled, buffered solution containing the desired enzyme. The sol
is swirled briefly, and then pipetted into a 4.5 mL polystyrene
cuvette (cell culture dishes were also used for surface area study
matrices). The cuvette opening is sealed with Parafilm following
gel formation (cell culture dish covers were used for surface area
study matrices). The gel is then allowed to age in the sealed
container for a period of time ranging from 14 to 50 days or more
at temperatures ranging from 4.degree. C. to room temperature.
[0325] 2. Enzyme Encapsulation and Assays
[0326] Entrapment of Penicillinase (Type I from Bacillus cereus,
lyophilized powder containing approx. 10% protein, Sigma) was
performed as outlined above, using 50 mM pH 6.5 phosphate buffer.
Penicillinase activity was determined using 100 mL of a 3 mM
solution of Penicillin G (Benzylpenicillin, sodium salt, Sigma) in
buffer. 2 mL aliquots of the reaction solution were removed for
assay and their UV-Vis spectra recorded.
[0327] 3. Results of Enzyme Encapsulation and Assays
[0328] Initial examination of which matrix compositions provided
matrices suitable for the purposes of this study excluded the
n-butyltrimethoxysilane composition, as well as some of the higher
ratios of other RSi(OCH.sub.3).sub.3 precursors, due to the failure
of these compositions to form a gel that was appropriate for our
intended uses. Compositions examined, and their reactivity relative
to 100% TMOS matrices are shown in Table 1.
4TABLE 1 Activity for given compositions relative to 100% TMOS.
Composition Relative Activity MTMS:TMOS 10% MTMS:90% TMOS 108% 20%
MTMS:80% TMOS 82% 30% MTMS:70% TMOS 92% 40% MTMS:60% TMOS 104% 50%
MTMS:50% TMOS 112%* ETMS:TMOS 10% ETMS:90% TMOS 99% 20% ETMS:80%
TMOS 93% 30% ETMS:70% TMOS 99% TMPS:TMOS 10% TMPS:90% TMOS 100% 20%
TMPS:80% TMOS 80% i-BTMS:TMOS 10% i-BTMS:90% TMOs 90% 20%
i-BTMS:80% TMOs 75% PTMS:TMOS 10% PTMS:90% TMOS 94% *Indicates a
composition in which slight enzyme leaching is observed at the time
of the assay. If insufficient aging is allowed, enzyme leaching is
observed for methyltrimethoxysilane composition with MTMS content
greater than 20%
[0329] In addition, it was observed that as the matrices age the
relative activity of the MTMS-containing matrices with respect to
100% TMOS drops. When matrices from the same preparation are
assayed after aging 104 days at 4.degree. C., the relative activity
observed is shown in Table 2.
5TABLE 2 Enzyme activity relative to 100% TMOS for varying
MTMS-containing matrices aged 104 days. Composition Relative
Activity MTMS:TMOS 10% MTMS:90% TMOS 85% 20% MTMS:80% TMOS 78% 30%
MTMS:70% TMOS 85% 40% MTMS:60% TMOS 85% 50% MTMS:50% TMOS 103%*
*Indicates a composition in which no enzyme leaching is observed at
the time of this assay, although leaching is observed for shorter
aging time.
[0330] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference and set forth in its entirety
herein. In case of conflict, the present application, including any
definitions herein, will control. In addition to the foregoing
materials, the practice of the present invention may employ in
part, unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature See, for example, Molecular Cloning a Laboratory Manual,
2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press: 1989); DNA Cloning Volumes I and II (D. N. Glover
ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S.
Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames &
S. J. Higgins eds. 1984); Transcription and Translation (B. D.
Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R.
I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And
Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors for Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods in Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods in Cell and Molecular Biology (Mayer
and Walker, eds., Academic Press, London, +987); Handbook of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986), all of
which references are hereby incorporated by reference to the same
extent as the other references specified herein.
[0331] The specification and examples should be considered
exemplary only with the true scope and spirit of the invention
suggested by the following claims.
* * * * *