U.S. patent application number 10/137049 was filed with the patent office on 2002-09-05 for materials and processes for controlled release of thioamide moiety-containing therapeutic agents by linking to thiol-containing polymers.
Invention is credited to Qiu, Bo, Stein, Stanley, Zhang, Guobao.
Application Number | 20020122785 10/137049 |
Document ID | / |
Family ID | 26842739 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020122785 |
Kind Code |
A1 |
Stein, Stanley ; et
al. |
September 5, 2002 |
Materials and processes for controlled release of thioamide
moiety-containing therapeutic agents by linking to thiol-containing
polymers
Abstract
This invention pertains to disulfide-linked conjugates of
therapeutic agents containing at least one thioamide group with
polymer comprising at least one thiol group, so as to provide a
controlled release pharmaceutical composition for administration to
animals for the prophylaxis or treatment of various conditions or
diseases. The therapeutic agent conjugate may comprise an inactive
or weakly active prodrug form which may be converted into the
original therapeutic compound by the natural action of reducing
agents in vivo. The composition may comprise a mixture of polymers
each with a different thioamide-containing agent attached, or a
polymer conjugated with a mixture of thioamide-containing agents.
Modified properties of the therapeutic compound potentially
provided by the polymer itself, as well as by other compounds also
appended to the polymer, include but are not limited to greater
water solubility, longer in-vivo half-life (due to larger size of
the conjugate), slower release from a sustained-release depot (due
to larger size of the conjugate), better oral bioavailability and
tissue-specific targeting.
Inventors: |
Stein, Stanley; (East
Brunswick, NJ) ; Zhang, Guobao; (Piscataway, NJ)
; Qiu, Bo; (New Brunswick, NJ) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
|
Family ID: |
26842739 |
Appl. No.: |
10/137049 |
Filed: |
May 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10137049 |
May 1, 2002 |
|
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09621109 |
Jul 21, 2000 |
|
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60145177 |
Jul 22, 1999 |
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Current U.S.
Class: |
424/78.27 |
Current CPC
Class: |
A61K 47/60 20170801;
Y10S 424/16 20130101 |
Class at
Publication: |
424/78.27 |
International
Class: |
A61K 031/74 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising a disulfide-linked
conjugate of at least one therapeutic agent comprising prior to
conjugation a thioamide moiety, and at least one polymer comprising
prior to conjugation at least one thiol group.
2. The composition of claim 1 wherein said polymer comprising prior
to conjugation at least one thiol group is a conjugate of a polymer
comprising at least one functional group and a bifunctional
compound comprising at least one functional group and at least one
thiol group, said at least one functional group of said polymer
linked to at least one said functional group of said bifunctional
compound.
3. The composition of claim 1 wherein said polymer comprising prior
to conjugation at least one thiol group has a polymer backbone
selected from the group consisting of polyethylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol,
N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone,
poly-1,3-dioxolane, poly-1,3,6-trioxane, polypropylene oxide,
copolymers of ethylene/maleic anhydride copolymer,
polylactide/polyglycolide copolymers, polyaminoacids, copolymer of
polyethylene glycol and an amino acid, and polypropylene
oxide/ethylene oxide copolymers.
4. The composition of claim 1 wherein said polymer comprising prior
to conjugation at least one thiol group is a branched polymer or a
dendrimer.
5. The composition of claim 3 wherein said polymer backbone is a
polyethylene glycol polymer.
6. The composition of claim 5 wherein said polyethylene glycol
polymer has a molecular weight of from about 300 to about 30,000
Da.
7. The composition of claim 6 wherein said polyethylene glycol
polymer has a molecular weight of from about 600 to about 5,000
Da.
8. The composition of claim 1 wherein said polymer comprising prior
to conjugation at least one thiol group has a polymer backbone
selected from the group consisting of a polyethylene
glycol-thiomalic acid copolymer, a polyethylene glycol-cysteamine
conjugate, a polyethylene glycol-1-amino-2-methyl-2-propanethiol
conjugate, and a polyethylene glycol-lysine conjugate wherein free
carboxy groups on said lysine are derivatized to form thiol
groups.
9. The composition of claim 2 wherein said polymer comprising at
least one functional group is selected from the group consisting of
.alpha.,.omega.-dihydroxy-polyethylene glycol;
.alpha.,.omega.-dicarboxy-- polyethylene glycol; and
.alpha.,.omega.-diamino-polyethylene glycol.
10. The composition of claim 1 wherein said polymer comprising
prior to conjugation at least one thiol group has from one to about
ten thiol groups per polymer.
11. The composition of claim 10 wherein said polymer comprising
prior to conjugation at least one thiol group has from one to about
three thiol groups per polymer.
12. The composition of claim 1 wherein said therapeutic agent is
selected from the group consisting of UC781; R82150; HBY097; S2720;
thiouridine; UC38, trovirdine and
2',3'-dideoxy-3'-fluoro-4-thiothymidine.
13. The composition of claim 1 wherein said therapeutic agent is
derivatized to comprise a thioamide moiety.
14. The composition of claim 1 wherein said polymer additionally
comprises a functional group.
15. The composition of claim 14 wherein said additional functional
group is derivatized with a compound selected from the group
consisting of an antibody, a cell uptake enhancer, and a tissue
targeting agent.
16. The composition of claim 1 wherein the thiol group on said
polymer comprising at least one thiol group is sterically
hindered.
17. The composition of claim 16 wherein said sterically hindered
thiol group decreases the susceptibility of said conjugate to
cleavage under reducing conditions.
18. The composition of claim 1 comprising a second therapeutic
agent.
19. The composition of claim 18 comprising a third therapeutic
agent.
20. The composition of claim 1 wherein said therapeutic agent is
released in vivo under reducing conditions.
21. The composition of claim 1 wherein the in-vivo half life of
said therapeutic agent in said composition is increased compared
with that of the therapeutic agent alone in vivo.
22. The composition of claim 1 wherein said therapeutic agent is
therapeutically inactive or weakly active in said composition.
23. The composition of claim 1 wherein the water solubility of said
therapeutic agent is increased in said composition compared to its
inherent water solubility.
24. A method for preparing a composition comprising a
disulfide-linked conjugate of at least one therapeutic agent
comprising prior to conjugation a thioamide moiety, and at least
one polymer comprising prior to conjugation at least one thiol
group, comprising the steps of: a) providing said at least one
therapeutic agent comprising a thioamide moiety; b) providing said
at least one polymer comprising a thiol group; c) reacting said at
least one therapeutic agent comprising a thioamide moiety under
oxidizing conditions to form at least one disulfide cross-linked
homodimer of said at least one therapeutic agent comprising a
thioamide moiety; d) reacting said at least one disulfide-linked
homodimer with said at least one polymer comprising a thiol group,
under conditions in which a disulfide exchange reaction occurs, to
form a disulfide-linked disulfide-linked conjugate of at least one
therapeutic agent comprising prior to conjugation a thioamide
moiety, and at least one polymer comprising prior to conjugation at
least one thiol group; and e) isolating said at least one
disulfide-linked conjugate of at least one therapeutic agent
comprising prior to conjugation a thioamide moiety, and at least
one polymer comprising prior to conjugation at least one thiol
group.
25. The method of claim 24 wherein said polymer comprising at least
one thiol group is a conjugate of a polymer comprising at least one
functional group and a bifunctional compound comprising at least
one functional group and at least one thiol group, said at least
one functional group of said polymer linked to at least one said
functional group of said bifunctional compound.
26. The method of claim 24 wherein said polymer comprising a thiol
group is a branched polymer or a dendrimer.
27. The method of claim 24 wherein said polymer comprising a thiol
group has a polymer backbone selected from the group consisting of
polyethylene glycol, carboxymethylcellulose, dextran, polyvinyl
alcohol, N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone,
poly-1,3-dioxolane, poly-1,3,6-trioxane, polypropylene oxide,
copolymers of ethylene/maleic anhydride copolymer,
polylactide/polyglycolide copolymers, polyaminoacids, copolymer of
polyethylene glycol and an amino acid, and polypropylene
oxide/ethylene oxide copolymers.
28. The-method of claim 27 wherein said polymer backbone is a
polyethylene glycol polymer.
29. The method of claim 28 wherein said polyethylene glycol polymer
has a molecular weight of from about 300 to about 30,000 Da.
30. The method of claim 29 wherein said polyethylene glycol polymer
has a molecular weight of from about 600 to about 5,000 Da.
31. The method of claim 24 wherein said polymer comprising at least
one thiol group is selected from the group consisting of a
polyethylene glycol/thiomalic acid conjugate, a polyethylene
glycol/cysteamine conjugate, and a polyethylene
glycol/1-amino-2-methyl-2-propanethiol conjugate.
32. The method of claim 25 wherein said polymer comprising at least
one functional group is selected from the group consisting of
.alpha.,.omega.-diamino-polyethylene glycol;
.alpha.,.omega.-dihydroxy-po- lyethylene glycol; and
.alpha.,.omega.-dicarboxy-polyethylene glycol.
33. The method of claim 24 wherein said polymer comprising at least
one thiol group has from one to about ten thiol groups per
polymer.
34. The method of claim 33 wherein said polymer comprising at least
one thiol group has from one to about three thiol groups per
polymer.
35. The method of claim 24 wherein said therapeutic agent is
selected from the group consisting of UC781; R82150; HBY097; S2720;
thiouridine; UC-38, trovirdine and
2',3'-dideoxy-3'-fluoro-4-thiothymidine.
36 The method of claim 24 wherein said therapeutic agent is
derivatized to comprise a thioamide moiety.
37. The method of claim 24 wherein said oxidizing conditions
comprises reaction in the presence of an oxidizing agent selected
from the group consisting of molecular oxygen, hydrogen peroxide,
and molecular iodine.
38. The method of claim 24 wherein the reaction conditions of step
(d) comprise a degassed nonaqueous solvent.
39. The method of claim 38 wherein said solvent is a 1:1 mixture of
dimethylformamide and dichloromethane.
40. The method of claim 24 wherein said polymer additionally
comprises a functional group.
41. The method of claim 40 wherein said additional functional group
is derivatized with a compound selected from the group consisting
of an antibody, a cell uptake enhancer or a tissue targeting
agent.
42. The method of claim 24 wherein the thiol group on said polymer
comprising a thiol group is sterically hindered.
43. The method of claim 24 wherein said composition comprises a
second therapeutic agent.
44. The method of claim 43 wherein said composition comprises a
third therapeutic agent.
45. The method of claim 24 wherein said therapeutic agent is
released in vivo under reducing conditions.
46. The method of claim 24 wherein the in-vivo half life of said
therapeutic agent in said composition is increased compared with
that of the therapeutic agent alone in vivo.
47. The method of claim 24 wherein said therapeutic agent is
therapeutically inactive or weakly active in said composition.
48. The method of claim 24 wherein the water solubility of said
therapeutic agent is increased in said composition compared to its
inherent water solubility.
49. The method of claim 24 wherein said disulfide-linked agent
polymer conjugate is entrapped in a matrix providing a controlled
release depot.
50. A method for preparing a composition comprising a
disulfide-linked conjugate of at least one therapeutic agent
comprising prior to conjugation a thioamide moiety, and at least
one polymer comprising prior to conjugation at least one thiol
group, comprising the steps of: a) providing said at least one
therapeutic agent comprising a thioamide moiety; b) providing a
bifunctional thiol-containing compound, said compound comprising at
least one functional group other than said thiol group; c)
providing a polymer with a functional group; d) reacting said at
least one therapeutic agent comprising a thioamide moiety under
oxidizing conditions to form at least one disulfide cross-linked
homodimer of said at least one therapeutic agent comprising prior
to said reacting a thioamide moiety; e) reacting said at least one
disulfide-linked homodimer with said bifunctional thiol-containing
compound under conditions in which a disulfide exchange reaction
occurs to form a disulfide-linked heterodimer of said at least one
therapeutic agent comprising prior to said reacting a thioamide
moiety and said bifunctional thiol-containing compound; f) reacting
said disulfide-linked heterodimer with said polymer with a
functional group to form a covalent conjugate thereof comprising a
therapeutic agent comprising prior to said reacting a thioamide
moiety and said polymer comprising a thiol group; and g) isolating
said at least one disulfide-linked conjugate of said at least one
therapeutic agent comprising a thioamide moiety, and said at least
one polymer comprising a thiol group.
51. A method for the controlled release in an animal of at least
one therapeutic agent comprising prior to conjugation a thioamide
moiety comprising administering to said animal a composition
comprising at least one pharmaceutical composition of claim 1.
52. The method of claim 51 wherein said composition comprises a
second therapeutic agent.
53. The method of claim 52 wherein said composition comprises a
third therapeutic agent.
54. The method of claim 50 wherein said therapeutic agent is
released in vivo under reducing conditions.
55. The method of claim 50 wherein the in-vivo half life of said
therapeutic agent in said animal is increased compared with that of
the therapeutic agent alone in said animal.
56. The method of claim 50 wherein said therapeutic agent is
therapeutically inactive or weakly active in said composition.
57. A method for the controlled release in an animal of a
therapeutic agent within a preselected body compartment comprising
administering to said animal the composition of claim 1, said
composition additionally comprising a targeting agent for targeting
said composition to said compartment.
58. The method of claim 57 wherein said targeting agent is selected
from the group consisting of an antibody, a cell uptake enhancer,
and a tissue targeting agent.
59. A method for the controlled release in an animal of at least
one therapeutic agent comprising prior to conjugation a thioamide
moiety comprising administering to said animal a composition
comprising at least one pharmaceutical composition of claim 2.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to provisional application
serial No. 60/145,177, filed Jul. 22, 1999, and is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention pertains to disulfide-linked conjugates of
therapeutic agents containing at least one thioamide group with
thiol-containing polymers, so as to provide a controlled release
pharmaceutical composition for administration to animals for the
prophylaxis or treatment of various conditions or diseases.
BACKGROUND OF THE INVENTION
[0003] Derivatives of polymers such as polyethylene glycol (PEG)
containing thiol (--SH) groups may be used as a controlled release
carrier for therapeutic agents with thiol groups, by administering
the polymer to which the therapeutic agent is linked by a disulfide
bridge. Reduction of the disulfide group by endogenous reducing
agents results in the release of the therapeutic agent (U.S. patent
application Ser. No. 09/320,609; Huang et al.[10]; Woghiren et
al.[19]). Furthermore, the therapeutic agent linked to the polymer
may be in an inactive, or prodrug form, which when released becomes
active. The inclusion of various targeting agents which also have
been conjugated to the same polymer to target the therapeutic agent
to particular sites within the body or to enhance cellular uptake
have been described.
[0004] Appended PEG chains may provide the favorable pharmacologic
properties of protection of the underlying protein from immune
surveillance and proteolytic enzymes, in addition to a lower rate
of clearance from the bloodstream (Davis et al., 1981).
Furthermore, based on the properties provided by the PEG portion of
the conjugate (Davis et al., 1981), conjugates of therapeutic
agents as prodrugs with polymers provides certain advantages such
as reduction in possible toxicity, since biological activity of a
large bolus of that drug would not appear immediately upon
administration to the patient. Thus, the biological activity might
be present at a relatively constant, therapeutic level in the
bloodstream over an extended time period due to two opposing
actions, the conversion of inactive prodrug to active drug and the
clearance of active drug from the body.
[0005] While therapeutic agents which have a thiol group, or may be
derivatized to have one without loss of activity, are suitable for
the above process, numerous other compounds without such groups
cannot be bound to thiol-containing polymers following standard
procedures to produce a controlled release composition. This is
particularly true for compounds with a thioamide group. It is
toward the development of a controlled release delivery system for
therapeutic agents with thioamide groups that the present
application is directed.
[0006] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
SUMMARY OF THE INVENTION
[0007] In its broadest aspect, the present invention is directed to
a pharmaceutical composition which is a disulfide-linked conjugate
between at least one therapeutic agent comprising prior to
conjugation a thioamide moiety, and at least one polymer comprising
prior to conjugation at least one thiol group. The polymeric
portion of the polymer which comprises prior to conjugation at
least one thiol group may be a homopolymer or a copolymer, and may
be by way of non-limiting example, polyethylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol,
N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone,
poly-1,3-dioxolane, poly-1,3,6-trioxane, polypropylene oxide,
copolymers of ethylene/maleic anhydride copolymer,
polylactide/polyglycolide copolymers, polyaminoacids, copolymer of
polyethylene glycol and an amino acid, or polypropylene
oxide/ethylene oxide copolymers. The polymer may also be a branched
polymer or a dendrimer, i.e., a multi-branched polymer.
[0008] In a preferred embodiment, the polymer is a polyethylene
glycol polymer (PEG), for example, of a molecular weight of from
about 300 to about 30,000 Da, and preferably, from about 600 to
about 5,000 Da. The PEG has functional groups or may be derivatized
to bear functional groups to which a compound providing a free
thiol group may be attached. The polymer comprising at least one
thiol group may have from one to about ten thiol groups per
polymer; preferably from one to about three thiol groups per
polymer. The thiol group on the polymer may be sterically
hindered.
[0009] The polymer comprising at least one thiol group may be
prepared from, for example, .alpha.,.omega.-diamino-polyethylene
glycol and thiomalic acid; .alpha.,.omega.-dihydroxy-polyethylene
glycol and thiomalic acid; .alpha.,.omega.-dicarboxy-polyethylene
glycol and cysteamine; .alpha.,.omega.-dicarboxy-poly(ethylene
glycol) and 1-amino-2-methyl-2-propanethiol; or
.alpha.,.omega.-dicarboxy-PEG subunits and lysine, wherein carboxy
groups on the lysines are derivatized to form thiol groups. The
selection of the thiol compound providing the disulfide link to the
thioamide-containing compounds and the covalent link to the polymer
may be selected from a number of compounds containing a thiol group
and a reactive group which may be attached to a polymer.
[0010] The therapeutic agent comprising prior to conjugation a
thioamide moiety may be an agent that contains such a thioamide
group in its active form, or a therapeutic agent which is modified
to contain a thioamide group. For example, therapeutic agents with
thioamide groups include UC781; R82150; HBY097; troviridine; S2720;
UC38 and 2',3'-dideoxy-3'-fluoro-4-thiothymidine. However, the
invention is not so limiting. Furthermore, other compounds with
thioamide-like groups of similar reactivity to thioamide-containing
compounds as described herein are likewise suitable as compositions
as described herein. Such compounds include but are not limited to
thioureas and thiourethans.
[0011] In a further aspect of the invention, the polymer may
additionally have a functional group, which may be derivatized with
a compound such as but not limited to a cell uptake enhancer or a
tissue targeting agent.
[0012] The composition of the present invention may include a
second therapeutic agent, or a second and a third therapeutic
agent. This may be achieved by preparing polymers conjugated to
each therapeutic agent separately, and then mixing these polymers
to provide a composition with more than one therapeutic agent. In
another embodiment, a single polymer to which at least two thiol
groups is attached may be derivatized with a mixture of therapeutic
agents. The relative amounts of the different agents conjugated to
the polymer may be selected to correspond with the therapeutic
effectiveness of each compound. The therapeutic agents conjugated
to the polymer of the invention are released in vivo under reducing
conditions. The in-vivo half life of the therapeutic agent in the
composition may be increased compared with that of the therapeutic
agent alone in vivo. Furthermore, the therapeutic agent may be
therapeutically inactive or weakly active in the composition. The
water solubility of the therapeutic agent may be increased in said
composition compared to its inherent water solubility.
[0013] In another broad aspect, the invention is directed to a
pharmaceutical composition which is a disulfide-linked conjugate
between at least one therapeutic agent comprising prior to
conjugation a thioamide moiety, a bifunctional compound comprising
prior to conjugation at least one thiol group, and at least one
polymer attached to one or more of the bifunctional compounds. The
polymer may be a homopolymer or a copolymer, and may be by way of
non-limiting example, poly(ethylene glycol),
carboxymethylcellulose, dextran, polyvinyl alcohol,
N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone,
poly-1,3-dioxolane, poly-1,3,6-trioxane, polypropylene oxide,
copolymers of ethylene/maleic anhydride copolymer,
polylactide/polyglycolide copolymers, polyaminoacids, copolymer of
polyethylene glycol and an amino acid, or polypropylene
oxide/ethylene oxide copolymers. The polymer may also be a branched
polymer or a dendrimer, i.e., a multi-branched polymer. For linkage
to the bifunctional compound, the polymer may have one or more
similar or different functional groups such as an amino or carboxy
group, which may be cross-linked to the functional group on the
bifunctional compound with a cross-linking agent, or the polymer
may be an activated polymer, such as but not limited to
polyethylene glycol bis(imidazoyl carbonyl), which is capable of
reacting with an amino group. The bifunctional compound comprising
prior to conjugation at least one thiol group may for example
comprise a thiol group and an amino group, such as but not limited
to cysteamine or 1-amino-2-methyl-2-propan- ethiol.
[0014] In another broad aspect of the present invention, methods
for preparing a composition comprising a disulfide-linked conjugate
of at least one therapeutic agent comprising prior to conjugation a
thioamide moiety with at least one polymer comprising prior to
conjugation at least one thiol group are described. The conjugate
may be prepared by following the steps of: (A) providing at least
one therapeutic agent comprising a thioamide moiety or modified to
have a thioamide moiety; (B) providing at least one polymer
comprising at least one thiol group; (C) reacting the at least one
therapeutic agent comprising a thioamide moiety under oxidizing
conditions to form a disulfide cross-linked dimer of the
therapeutic agent comprising a thioamide moiety; (D) reacting the
disulfide-linked dimer with the polymer comprising at least one
thiol group, under conditions in which a disulfide exchange
reaction occurs to form a disulfide-linked conjugate between the
therapeutic agent comprising a thioamide moiety, and the at least
one polymer comprising at least one thiol group; and (E) isolating
the disulfide-linked conjugate.
[0015] The thiol-containing polymer may have a thiol-containing
moiety thereon, which may be prepared by any of a number of
methods. By way of non-limiting example, a compound with a thiol
group and another functional group, for example, an amino group,
may be covalently coupled to a polymer with carboxylic acid
moieties, for example, 1-amino-2-methyl-2-propanethiol or
cysteamine may be conjugated to a PEG polymer with carboxylic acid
moieties, using a carbodiimide reagent. By way of another example,
thiol-containing compounds containing a carboxylic acid moiety such
as thiomalic acid may be conjugated to a PEG bearing amino moieties
using a carbodiimide. In the previous examples, the thiol group of
1-amino-2-methyl-2-propanethiol is sterically hindered, while that
of cysteamine is less so.
[0016] The polymer comprising at least one thiol group may be
prepared from, for example, .alpha.,.omega.-diamino-poly(ethylene
glycol) and thiomalic acid; .alpha.,.omega.-dihydroxy-poly(ethylene
glycol) and thiomalic acid; .alpha.,.omega.-dicarboxy-poly(ethylene
glycol) and cysteamine; .alpha.,.omega.-dicarboxy-poly(ethylene
glycol) and 1-amino-2-methyl-2-propanethiol; or
.alpha.,.omega.-dicarboxy-PEG subunits and lysine, wherein carboxy
groups on the lysine are derivatized to form thiol groups. The
selection of the thiol compound providing the disulfide link to the
thioamide-containing compounds and the covalent link to the polymer
may be selected from a number of compounds containing a thiol group
and a reactive group which may be attached to a polymer.
[0017] In a further aspect of the invention, methods for preparing
a composition comprising a disulfide-linked conjugate between at
least one therapeutic agent comprising prior to conjugation a
thioamide moiety, a bifunctional compound comprising prior to
conjugation at least one thiol group, and at least one polymer
attached to one or more bifunctional compounds are described. In
one embodiment, a disulfide exchange-produced heterodimer is
prepared between the thioamide-containing compound and a
bifunctional compound comprising at least one thiol group and an
amino group, thus forming a disulfide-linked conjugate comprising
the therapeutic agent and the functional (amino) group.
Subsequently, the functional group of the heterodimer is covalently
linked to the polymer, for example, using a cross-linking reagent
to cross-link the functional group of the bifunctional compound and
a functional group on the polymer, or by use of an activated
polymer capable of reacting directly with the functional group on
the bifunctional compound. In yet a further embodiment, the
bifunctional group comprising a thiol group is first conjugated to
the polymer, for example by any of the foregoing methods, leaving
at least one free thiol group, and subsequently, a homodimer of the
oxidized therapeutic agent is reacted under disulfide exchange
conditions with the polymer to produce the desired conjugate. In
this fashion, additional control over the selection of sterically
hindered thiol groups is provided to tailor the release
characteristics of the therapeutic agent to the particular
condition to be treated or prevented, and the target organ, tissue
or cells.
[0018] The polymer portion of the polymer comprising at least one
thiol group and the bifunctional compound are as described
hereinabove.
[0019] The therapeutic agents in the present invention have a
thioamide group, whether present in or introduced synthetically
into the agent. For example, the agent may be UC781; R82150;
HBY097; troviridine; S2720; thiouridine; UC38 and
2',3'-dideoxy-3'-fluoro-4-thiothymidine. Many other therapeutic
agents, for various uses, are embraced herein. Furthermore, a
therapeutic agent may be prepared or chemically modified to provide
a therapeutically active analog having a thioamide group.
[0020] The oxidizing conditions to form the dimer of the
thioamide-containing therapeutic agent comprises reaction in the
presence of an oxidizing agent which may include, but is not
limited to, molecular oxygen, hydrogen peroxide, and molecular
iodine. The subsequent disulfide exchange reaction may be carried
out under conditions which promote the reaction, for example, in a
degassed nonaqueous solvent, such as a 1:1 mixture of
dimethylformamide and dichloromethane. The invention is not so
limiting to these conditions and any suitable conditions may be
employed to achieve the preparation of the desired conjugate.
[0021] Furthermore, any therapeutic agent having a thioamide-like
group that may be oxidized to form a dimer and then attachable via
a disulfide exchange reaction to a thiol-containing polymer via a
disulfide bond is suitable for use herein. Such moieties such as
but not limited to thioureas and thiourethans are included
herein.
[0022] The polymer comprising at least one thiol group may
additionally have a functional group, such as an amino or carboxyl
group, and by way of non-limiting examples, the additional
functional group is optionally derivatized with a cell uptake
enhancer or a tissue targeting agent. The polymer may also be a
branched polymer or a dendrimer, i.e., a multi-branched
polymer.
[0023] A pharmaceutical composition of the present invention
prepared as described above may have a second therapeutic agent or
a second and third therapeutic agent. The preparation step may
include a mixture of disulfide-linked dimers of more than one
agent, which during the disulfide exchange reaction become
conjugated to the polymer. The relative efficiency of disulfide
exchange and/or the relative concentration for each dimer may be
used to provide conditions such that the resulting polymer has the
therapeutic agents present at the desired ratio. In another
embodiment, polymers conjugated separately to particular
therapeutic agents following the process described above are mixed
at the desired ratio before administration to the patient.
[0024] The therapeutic agent is of the composition released in vivo
under reducing conditions. The in-vivo half life of said
therapeutic agent in the composition may be increased compared with
that of the therapeutic agent alone in vivo. Furthermore, the
therapeutic agent may be therapeutically inactive or weakly active
in the composition. The water solubility of the therapeutic agent
may be increased in said composition compared to its inherent water
solubility. In a further embodiment, the disulfide-linked agent
polymer conjugate may be entrapped in a matrix providing a
controlled release depot.
[0025] In another aspect of the present invention, a method is
provided for the controlled release in an animal of at least one
therapeutic agent having a thioamide moiety comprising
administering to the animal a composition comprising a
pharmaceutical composition as described hereinabove. The
composition may comprise a second therapeutic agent, or a second
and a third therapeutic agent. The therapeutic agent may be
released in vivo under reducing conditions.
[0026] The in-vivo half life of said therapeutic agent in the
animal is increased compared with that of the therapeutic agent
alone in said animal. The therapeutic agent may be therapeutically
inactive or weakly active in said composition.
[0027] In another aspect of the present invention, a method is
provided for the controlled release in an animal of a therapeutic
agent within a preselected body compartment comprising
administering to said animal a composition comprising the
pharmaceutical composition as described above, wherein the
pharmaceutical composition additionally comprises a targeting agent
for targeting said composition to said compartment. Non-limiting
examples of targeting agents include an antibody, a cell uptake
enhancer, or a tissue targeting agent.
[0028] These and other aspects of the present invention will be
better appreciated by reference to the following drawing and
Detailed Description.
BRIEF DESCRIPTION OF THE DRAWING
[0029] FIG. 1 depicts the reactions for the synthesis of a
conjugate between a PEG modified with thiol groups and UC781
(referred to as PEG-S-S-UC781), and for the regeneration of the
original active drug, UC781. Side reactions giving sulfur
elimination are also shown.
[0030] FIG. 2 shows thin layer chromatography demonstrating
conversion of UC781 into disulfide-linked homodimer. Detection was
by UV shadowing.
[0031] FIG. 3 is a thin layer chromatogram demonstrating release of
the drug, UC781, by reductive cleavage of the prodrug,
PEG-S-S-UC781. Detection was by UV shadowing.
[0032] FIG. 4 depicts .sup.1H NMR in deuterated dimethylsulfoxide
of original UC781. The peak at about 3.7 ppm is due to water, and
is not from the drug compound.
[0033] FIG. 5 shows .sup.1H NMR in deuterated dimethylsulfoxide of
UC781 regenerated from its prodrug conjugate, PEG-S-S-UC781. All
peaks are the same as in FIG. 4. The peak at about 3.7 ppm is due
to water, and is not from the drug compound.
[0034] FIG. 6 shows the results of TLC of 4-thiouridine
reactions.
[0035] FIG. 7 depicts the kinetics following intravenous injection
of PEG-S-S-UC781 in rabbits. Results from 2 separate rabbits are
shown.
[0036] FIG. 8 depicts kinetics following intramuscular injection of
PEG-S-S-UC781 in mice. Results from 2 separate mice are shown.
[0037] FIG. 9 shows dose-response graphs for reverse transcriptase
inhibition assay. The main panel is a replot of the linear range
data taken from the inset panel.
DETAILED DESCRIPTION OF THE INVENTION
[0038] This invention is directed to disulfide-linked conjugates of
therapeutic agents comprising at least one thioamide group with a
polymer comprising at least one thiol group, so as to provide a
controlled release pharmaceutical composition for administration to
animals for the prophylaxis or treatment of various conditions or
diseases. The therapeutic agent conjugate may comprise an inactive
or weakly active prodrug form which may be converted into the
original therapeutic compound by the natural action of reducing
agents in vivo. The composition may comprise a mixture of polymers
each with a different thioamide-containing agent attached, or a
polymer conjugated with a mixture of thioamide-containing agents.
Modified properties of the therapeutic compound potentially
provided by the polymer itself, as well as by other compounds also
appended to the polymer, include but are not limited to greater
water solubility, longer in-vivo half-life (due to larger size of
the conjugate), slower release from a sustained-release depot (due
to larger size of the conjugate), better oral bioavailability and
tissue-specific targeting.
[0039] The compositions of the present invention comprise
disulfide-linked conjugates between a polymer comprising prior to
conjugation at least one thiol group and therapeutic agents
containing a thioamide moiety prior to conjugation, or a polymer
attached to a bifunctional compound which is disulfide-linked with
a therapeutic agent comprising prior to conjugation a thioamide
group. These conjugates have a general structure
R.sup.1--N.dbd.C(R.sup.2)--S--S-polymer or
polymer-F.sub.2--F.sub.1--X--S- --S--C(R.sup.2).dbd.N--R.sup.1. As
will be described in more detail below, the thiol group of the
polymer of bifunctional compound, respectively, may be derived from
a compound covalently attached to the polymer or compound,
providing the thiol group and optionally a means for sterically
hindering the thiol group to provide particular characteristics of
the composition, such as susceptibility to reducing agents and
consequent release rate. As used herein, the phrase "therapeutic
agent comprising prior to conjugation a thioamide moiety," refers
to a conjugate in which a reactant is a compound having a thioamide
moiety (--NH--C.dbd.S); however, this moiety is present in the form
of a --N.dbd.C--S-- moiety in the conjugate. Moreover, the phrase
"polymer comprising prior to conjugation at least one thiol group"
may refer both to a polymer derivatized to comprise thiol group(s),
or to a conjugate of a polymer comprising a functional group and a
bifunctional compound as described herein, in both instances
providing a polymer subsequently conjugatable to a thioamide
compound by the methods herein to form the final disulfide-linked
thioamide compound as described herein.
[0040] The present invention extends to therapeutically useful
compounds having at least one thioamide group (--NH--C.dbd.S). By
way of non-limiting example, compounds with thioamide groups
include several reverse transcriptase inhibitor (RTI) compounds
useful for HIV/AIDS therapy or prophylaxis, as well as those with
moieties of similar reactivity, including: UC781; R82150; HBY097;
troviridine; S2720; UC38 and
2',3'-dideoxy-3'-fluoro-4-thiothymidine, and are applicable to the
preparation of the compositions of the present invention.
Furthermore, other compounds with thioamide-like groups of similar
reactivity to thioamide-containing compounds as described herein as
likewise suitable for the preparation of a composition as described
herein. Such compounds include but are not limited to thioureas
(e.g., R82150 and trovirdine) and thiourethans (e.g., UC38). The
term thioamide used herein embraces thioamides as well as the
related structures mentioned above, as well as their relatives.
Model therapeutic compounds with a thioamide moiety, such as
thiouridine, is also embraced herein.
[0041] UC781, chemically known as
N-[4-chloro-3-(3-methyl-2-butenyloxy)phe-
nyl]-2-methyl-3-furancarbothioamide, was described by Borkow et al.
(18). R82150, chemically known as
(+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-
-2-butenyl)-imidazo[4,5,1jk][1,4]-benzodiazepin-2(1H)-thione, was
described by Pauwels et al., 1990. Troviridine, or LY-300046, is
N-[2-(2-pyridylethyl)-N'-[2-(5-bromopyridyl)]thiourea. HBY 097,
known chemically as
(S)-7-methoxy-3,4-dihydro-2-[(methylthio)methyl]-3-thioxo-2-
(1H)-quinoxalinecarboxylic acid, isopropyl ester, was described by
Kleim et al., 1997. UC-38 is
4-chloro-3-(isopropoxycarbonyl)phenyl-carbamothioi- c acid,
O-isopropyl ester. 3'-F-4-thio-ddT, an abbreviation for
2',3'-dideoxy-3'-fluoro-4-thiothymidine, was described by Matthes
et al., 1989.
[0042] Examples of suitable subunit polymers comprising at least
one thiol group include both homopolymers or copolymers. By way of
non-limiting example, suitable polymers, which may have
modifications to attach thiol group(s), include poly(ethylene
glycol) [also known as polyethylene glycol or PEG, polyethylene
oxide or PEO], carboxymethylcellulose, dextran, polyvinyl alcohol,
N-(2-hydroxypropyl)methacrylamide, polyvinyl pyrrolidone,
poly-1,3-dioxolane, poly-1,3,6-trioxane, polypropylene oxide,
copolymers of ethylene/maleic anhydride copolymer,
polylactide/polyglycolide copolymers, polyaminoacids, copolymer of
polyethylene glycol and an amino acid, or polypropylene
oxide/ethylene oxide copolymers. By way of illustration, described
in more detail below with regard to the methods for preparing the
compositions, such polymers are then derivatized or further
polymerized to introduce thiol groups; chemical modification of the
polymer may be necessary as a step prior to the further
derivatization to incorporate thiol groups. For example, a
copolymer of the present invention may be derived from a
poly(ethylene glycol) (PEG) derivative, for example,
.alpha.,.omega.-dihydroxy-PEG, .alpha.,.omega.-dicarboxy-PEG or
.alpha.,.omega.-diamino-PEG, but other derivatives are embraced
herein. The polymer comprising thiol groups may be, for example,
.alpha.,.omega.-diamino-poly(ethylene glycol) and thiomalic acid;
.alpha.,.omega.-dihydroxy-poly(ethylene glycol) and thiomalic acid;
.alpha.,.omega.-dicarboxy-poly(ethylene glycol) and cysteamine;
.alpha.,.omega.-dicarboxy-poly(ethylene glycol) and
1-amino-2-methyl-2-propanethiol; or a copolymer of
.alpha.,.omega.-dicarboxy-PEG subunits and lysine wherein the free
carboxy groups on said lysine are derivatized to form thiol groups.
These polymers are only examples of possible choices, as the
skilled artisan will be aware of numerous alternatives. As will be
noted below, the selection of the polymer, or combinations thereof,
will be guided by the desired properties of the final product,
particularly the duration of release of the therapeutic agent and
the release kinetics. As will also be noted below, a product of the
invention may comprise more than one polymer component in order to
provide two or more different release characteristics. Of course,
more than one therapeutic agent may be included.
[0043] The polymer is preferably PEG. Thiol-PEG (MW=5 kDa) may be
purchased from Fluka, but thiol-PEG of different molecular weights
or with multiple thiol-attachment sites may be used. A combination
of 2 or more drugs may be appended on the multivalent PEG in a
preselected ratio. Furthermore, moieties with other functions, such
as cell uptake enhancement or tissue-selective targeting, may be
appended to the multivalent PEG. Such cell uptake enhancement
compounds have been described, for example, in U.S. patent
application Ser. No. 09/320,609. Although PEG has been used for the
Examples given below, substitution of PEG by another biocompatible
thiol-containing polymer is within the scope and spirit of this
Invention.
[0044] In one particular embodiment, a copolymer of the present
invention is derived from a poly(ethylene glycol) (PEG) derivative,
for example, .alpha.,.omega.-dihydroxy-PEG,
.alpha.,.omega.-dicarboxy-PEG or .alpha.,.omega.-diamino-PEG, but
other derivatives are embraced herein. Examples of such polymers
with particular molecular weights include
.alpha.,.omega.-dihydroxy-PEG.sub.3,400;
.alpha.,.omega.-dihydroxy-PEG.su- b.1000;
.alpha.,.omega.-diamino-PEG.sub.3,400; and .alpha.,.omega.-diamino-
-PEG.sub.1,000 PEG is known to be a particularly nontoxic polymer.
One commercially-available polymer with a thiol group useful for
the practice of the present invention is
O-(2-mercaptoethyl)-O'-methyl polyethylene glycol 5000 (mPEG-SH;
5000 Da).
[0045] In the embodiment wherein a bifunctional compound is
provided in the conjugate which is disulfide-linked to the
thioamide-containing agent as well cross-linked to the polymer,
several examples of conjugates between polymers and bifunctional
agents comprising at least one thiol group are described above,
such as cysteamine, thiomalic acid, etc., conjugated to PEG, for
example using a homobifunctional or heterobifunctional
cross-linking agent. In an alternate embodiment, an activate
polymer capable of directly reacting with the functional group on
the bifunctional compound may be employed, such as, in the example
of a bifunctional compound comprising a thiol group and an amino
group, the activated polymer polyethylene glycol bis(imidazolyl
carbonyl), from Sigma Chemical Co., will react with amino groups to
form the conjugate.
[0046] In an example of the preparation of a therapeutic agent of
the invention, a dimer of UC781 may be prepared by oxidation with
iodine. Conversion to the dimer by oxidation may be monitoring by
thin layer chromatography (TLC), to indicate the nearly complete
conversion as compared to the original UC781 monomer. The dimer is
subsequently purified. Then, mPEG-SH as described above and the
UC781 dimer are reacted to form PEG-S-S-UC781, and the product
purified therefrom.
[0047] In another strategy, the UC781 homodimer is prepared as
described above, but then converted by disulfide exchange into a
heterodimer with another bifunctional thiol compound, for example,
with 1-amino-2-methyl-2-propanethiol hydrochloride or cysteamine,
both of which also contain an amino group. This UC781 heterodimer
comprising an amino group is then appended to a polymer, such as
.alpha.,.omega.-dicarboxy-PEG by amide bond formation, using the
amino group on the heterodimer, by use, for example, of a
carbodiimide. As noted above, direct cross-linking of the amino
group to the polymer may be achieved with an activated polymer.
[0048] The foregoing examples are not meant to be limiting and
other reactants and processes are embraced herein for preparing the
compounds described herein.
[0049] In another example, a copolymer of
.alpha.,.omega.-dicarboxy-PEG subunits and lysine may be prepared,
and subsequently the free carboxy groups on the lysine are
derivatized to form thiol groups. These examples are provided by
way of illustration only and such methods for adding a thiol group
to a polymer are known to one skilled in the art.
[0050] The polymer comprising at least one thiol group may have
from one to about ten thiol groups per polymer; from one to about
three thiol groups per polymer is preferred. The polymer comprising
at least one thiol group may have a molecular weight of from about
300 to about 30,000 k Da, preferably from about 600 to about 5,000
Da.
[0051] The thiol group on the thiol-containing polymer may be
sterically hindered. For example, when an intermediate bifunctional
compound, such as the amino- and thiol-containing compound in the
second method above, is used between the thioamide-containing
compound and the polymer, a sterically hindered thiol group such as
is present in the compound 1-amino-2-methyl-2-propanethiol
decreases the releasability of the thioamide-containing compound in
the presence of reducing agents such as glutathione. If a compound
with a less-sterically-hindered thiol group such as cysteamine is
used, the conjugate is more easily reduced by reducing agents. By
judicious selection of the intermediate thiol/amine-containing
compound and the degree of steric hindrance of the thiol group, a
range of release over 2-3 logs may be selected. Thus, the desired
release characteristics for a particular compound for a particular
target organ or tissue, as well as other kinetic parameters, may be
built into the composition of the present invention by following
the guidance herein.
[0052] The present invention is also directed to methods for the
preparation of the compositions described hereinabove. The
compositions are prepared from a polymer comprising at least one
thiol group, depicted as polymer-SH, and a thioamide-containing
compound, depicted with the structural formula
R.sup.1--NH--C(R.sup.2).dbd.S. The methods described herein provide
for the preparation of the conjugate, which has the structural
formula R.sup.1--N.dbd.C(R.sup.2)--S--S-polymer. The following
steps are carried out, in schematic form, and will be described in
more detail below:
[0053] 1. Oxidation:
R.sup.1--NH--C(R.sup.2).dbd.S+R.sup.1--NH--C(R.sup.2).dbd.S.fwdarw.R.sup.1-
--N.dbd.C(R.sup.2)--S--S--C(R.sup.2).dbd.N--R.sup.1
[0054] 2. Disulfide Exchange:
R.sup.1--N.dbd.C(R.sup.2)--S--S--C(R.sup.2).dbd.N--R.sup.1+polymer-SH.fwda-
rw.polymer-S--S--C(R.sup.2).dbd.N--R'+S.dbd.C(R.sup.2)--NH--R.sup.1
[0055] In vivo, under reducing conditions, a reducing agent, such
as glutathione, depicted as R--SH, reduces the polymer to release
the therapeutic agent:
Polymer-S--S--C(R.sup.2).dbd.N--R.sup.1+R--SH.fwdarw.R.sup.1--NH--C(R.sup.-
2).dbd.S+polymer-S--S--R.
[0056] The conjugate may derived from the two reactants described
above, but as it will be shown below, the two reactants cannot
simply be reacted under oxidizing conditions to form the disulfide
bond. The preparation of the conjugate is carried out by first
oxidizing the thioamide-containing agent to form homodimers, and
then performing a disulfide exchange reaction between the dimer and
the polymer comprising at least one thiol group, forming the
conjugate. The details of this reaction will be elaborated upon
below.
[0057] As noted above, a direct-oxidation of the thioamide compound
with the thiol-containing polymer does not achieve the preparation
of the conjugate of the present invention. A disulfide exchange
reaction is performed between the thiol-containing polymer or
thiol-containing compound and dimers prepared by the oxidation of
the thioamide-containing agent. Various other methods of
preparation are embraced within the present invention to achieve
the preparation of the desired product; these will be described
further below. In one example, the following steps are carried out
with the reactants. First, the therapeutic agent comprising a
thioamide moiety is reacted under oxidizing conditions to form
disulfide cross-linked dimers of the therapeutic agent. The
oxidizing conditions entail the reaction in the presence of an
oxidizing agent, for example, by molecular oxygen, hydrogen
peroxide, or molecular iodine. Other oxidizing conditions for
forming dimers of the thioamide-containing agent are embraced
herein; the skilled artisan will be aware of other suitable agents
and conditions for preparing the dimer.
[0058] After preparation of the dimer, a disulfide exchange
reaction is performed in the presence of the polymer comprising at
least one thiol group, to form the desired conjugate. The
conditions for performing this reaction are also known by the
skilled artisan. For example, the reaction between the dimer and
the polymer may be performed in a degassed nonaqueous solvent, such
as a 1:1 mixture of dimethylformamide and dichloromethane. However,
the reaction conditions for the preparation of the product
described here is not so limiting and may be practiced by any one
of a number of suitable conditions.
[0059] It is important that the reaction conditions employed to
permit the disulfide exchange reaction to proceed does not result
in a rearrangement reaction in which an atom of sulfur is
eliminated from the disulfide linkage, thereby forming a
thioether-linked dimer (Schaeffer et al., 1967; Zabicky, 1970).
Such an undesirable reaction can be caused by the inappropriate
application of heat, or the carrying out of the reaction at a low
pH, for example, around pH 4. The elimination of sulfur will
produce a product in which the thioamide-containing compound is
conjugated to the polymer through a single sulfur atom, and the
conjugate is not reducible to yield the free therapeutic agent and
polymer.
[0060] By way of example of the aforementioned procedure, dimers of
UC781 are prepared by oxidation in the presence of iodine. Next,
the UC781 dimers are mixed with a thiol-containing polymer,
O-(2-mercaptoethyl)-O'-- methyl polyethylene glycol 5000 under
conditions favoring disulfide exchange. The product is subsequently
purified.
[0061] The releasability of the therapeutic agent from the polymer
by reaction with reducing agents is easily demonstrated, as shown
below in Example 1.
[0062] In another embodiment for the preparation of the
compositions herein, the thioamide-containing therapeutic agent may
be oxidized as described above to form disulfide-linked homodimers
of the agent. The homodimers are then, by disulfide exchange,
reacted with a thiol-containing compound with a functional group
(depicted as F.sub.1--X--SH) to produce heterodimers, and
subsequently, the heterodimers are linked to a polymer through a
functional group on the polymer. The polymer with a functional
group is depicted as F.sub.2-polymer. The reactions are depicted as
follows, and described in more detail below.
[0063] 1. Oxidation:
R.sup.1--NH--C(R.sup.2).dbd.S+R.sup.1--NH--C(R.sup.2).dbd.S.fwdarw.R.sup.1-
--N.dbd.C(R.sup.2)--S--S--C(R.sup.2).dbd.N--R.sup.1
[0064] 2. Disulfide Exchange with Thiol-Containing Compound.:
R.sup.1--N.dbd.C(R.sup.2)--S--S--C(R.sup.2).dbd.N--R.sup.1+F.sub.1--X--SH.-
fwdarw.F.sub.1--X--S--S--C(R.sup.2).dbd.N--R.sup.1+S.dbd.C(R.sup.2)--N--R.-
sup.1
[0065] 3. Conjugation of Functional Groups:
F.sub.1--X--S--S--C(R.sup.2).dbd.N--R.sup.1+F.sub.2-polymer.fwdarw.polymer-
-F.sub.2--F.sub.1--X--S--S--C(R.sup.2).dbd.N--R.sup.1
[0066] The preparation of the homodimers under oxidizing conditions
are as described above. Subsequently, the homodimers may be reacted
in the presence of a thiol-containing bifunctional compound such
as, by way of non-limiting example, cysteamine or
1-amino-2-methyl-2-propanethiol, under disulfide exchange
conditions, forming heterodimers of the therapeutic agent and the
bifunctional thiol-containing compound. After purification of the
desired heterodimers, they may be conjugated to a polymer by taking
advantage of functional groups on both the polymer and the
bifunctional thiol-containing compound. For example, the
aforementioned thiol-containing compounds have amino groups, which
may be coupled to carboxy groups on the polymer, for example,
dicarboxy-PEG, using a carbodiimide reaction. Other cross-linking
agents, including homobifunctional and heterobifunctional agents,
may be used to achieve the desired product. The final product must
be sensitive to reducing conditions in order that the
thioamide-containing therapeutic agent is released from the
disulfide link under the appropriate reducing conditions. The
skilled artisan will be aware of the criteria needed for the
selection of the appropriate reaction scheme and conditions, to
increase yield and ensure stability of the reactants and product.
The process may be performed with a reduced number of steps
depending on the reactivity of the reactants and the tolerable
yield and ease in purification of the desired intermediates or
products from byproducts.
[0067] For example, UC781 homodimers are prepared as described in
Example 1 below. Subsequently, 1-amino-2-methyl-2-propanethiol
hydrochloride is added under conditions to favor disulfide
exchange, and the heterodimer product is purified using a silica
gel column. The heterodimers are then reacted with polyethylene
glycol bis(imidazolyl carbonyl) to directly form the final
conjugate. The product is then purified.
[0068] As mentioned above, the composition of the present invention
may comprise a plurality of different therapeutic agents attached
to a single type of thiol-containing polymer with at least two
thiol groups, or may be a mixture of different polymers of at least
one thiol group each containing a different agent. These variations
allow controlled delivery of multiple agents at a predetermined
ratio.
[0069] In a further embodiment of the present invention, the
pharmaceutical compositions comprising at least one thioamide agent
may also be derivatized with a functional group, such as an amino
or carboxyl group. These functional groups may optionally serve as
sites for attachment of other compounds or agents, such as a
targeting agent for targeting said composition to said compartment.
Such compounds as antibodies, cell uptake enhancers, and tissue
targeting agents may be employed.
[0070] As will be seen in the examples below, administration of the
polymer comprising UC781 of the invention to mice or rabbits
results in release of UC781 in vivo, demonstrating the in-vivo
reductive cleavage of the polymer as expected. Moreover, further
experiments on blood samples from animals administered the polymer
comprising UC781 demonstrate reverse transcriptase activity,
confirming biological activity of the compound released in
vivo.
[0071] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed, however, as limiting
the broad scope of the invention.
EXAMPLE 1
Synthesis and Analysis of a Reversible Conjugate,
PEG-S-S-UC781.
[0072] I. Preparation of UC781 dimer. UC781 (MW 335 Da, 165 mg, 0.5
mmol) was dissolved in 10 ml of ethyl ether and cooled to 0.degree.
C., and 87 .mu.L (0.5 mmol) of diisopropylethyl amine (DIEA) was
added to this solution. Then 63.5 mg iodine (0.25 mmol) was
dissolved in 10 ml of ethyl ether and cooled to 0.degree. C. The
iodine solution was added to the UC781 solution dropwise while
stirring in an ice bath. The reaction was allowed to go for 4
hours. On thin layer chromatography (TLC) (ethyl acetate/hexane:
20/80), the reaction mixture gave a new spot (Rf=0.6) with nearly
complete conversion (FIG. 2), compared to the original UC781
monomer (Rf=0.4). The salt, diisopropylethyl ammonium iodide,
precipitated and was filtered through paper. The product was taken
to dryness and used without further purification.
[0073] II. Preparation of PEG-S-S-UC781.
O-(2-mercaptoethyl)-O'-methyl polyethylene glycol 5000 (mPEG-SH;
5000 Da) was from Fluka. MPEG-SH (50 mg, 0.01 mmol) and 20 mg of
UC781 dimer were dissolved in a degassed, mixed solvent of 1 ml
dimethylformamide and 1 ml of dichloromethane (DCM). The reaction
mixture was kept under argon overnight. TLC (ethyl acetate/hexane:
20/80) showed a spot remaining at the origin, indicating the
formation of the PEG-S-S-UC781 product, since MPEG-SH cannot
otherwise be seen by UV shadowing and since any PEG compound would
be expected to remain at the origin. The DCM was then evaporated
and the product was recovered by ether precipitation of the
reaction mixture in 10 ml of cold ether. The product was washed
four times with 5 ml of cold ether, and TLC showed only one spot
remaining at the origin (FIG. 3), indicating complete removal of
unreacted UC781 monomer and dimer. Spectroscopic analysis of the
conjugate showed a broad band of absorption in the UV, similar to
that of the original UC781.
[0074] III. Release of UC781 from mPEG-S-S-UC781. In an organic
phase reaction, the PEG-S-S-UC781 conjugate was dissolved in 0.5 ml
of DCM. Then, 0.5 ml of 6 mM dithiothreitol (DTT) in DCM was added
to the conjugate solution. After 2 hours, TLC showed a new spot at
an Rf of 0.4, indicating that UC781 had been cleaved from the
polymer (FIG. 3). The cleaved UC781 was purified by flash silica
gel column and .sup.1H NMR (FIGS. 4 and 5) showed the released
product to be the same as the original UC781.
[0075] In an aqueous phase reaction, the PEG-S-S-UC781 conjugate
was dissolved in 0.2 ml of phosphate-buffered saline (PBS), pH 7.4,
and 0.2 ml of 6 mM glutathione was added to this solution. After 2
hours at room temperature, the reaction mixture was taken to
dryness by speed vacuum. Then the dry residue was dissolved in DCM
and TLC showed a spot with the same Rf of 0.4 as did the original
UC781. The conclusion from these release studies is that the
original drug, UC781, can be regenerated from its prodrug form,
PEG-S-S-UC781, by reductive cleavage, including under conditions
that might be found in a living cell.
EXAMPLE 2
Conjugate Synthesis by Means of an Intermediate bifunctional Thiol
Compound
[0076] In an alternate strategy to that described in Example 1
above, the UC781 homodimer is prepared as in Example 1, but then
converted by disulfide exchange into a heterodimer with another
bifunctional thiol compound, cysteamine, which also contains an
amino group. This UC781 heterodimer is then appended to PEG by
reaction of the amino group on the heterodimer with polyethylene
glycol bis(imidazolyl carbonyl). By selecting the structure of the
moiety next to the thiol group, the glutathione-induced release
rate can be provided over a range of 2-3 orders of magnitude.
[0077] Synthesis of
NH.sub.2--CH.sub.2--C(CH.sub.3).sub.2--S--S--UC781: 133 mg of UC781
dimer (MW 670, 0.2 mmol) prepared as described in Example 1 was
dissolved in 3 ml of degassed methylene chloride (degassed by
bubbling with helium). Subsequently, 14 mg of
1-amino-2-methyl-2-propanet- hiol hydrochloride (MW 141.6, 0.1
mmol) was added to the solution. The reaction was kept under argon
for 2 days. Thin Layer Chromatography (TLC) showed that all the
1-amino-2-methyl-2-propanethiol hydrochloride had reacted.
[0078] Coupling of
NH.sub.2--CH.sub.2--C(CH.sub.3).sub.2--S--S-UC781 to dicarboxy-PEG:
The purified NH.sub.2--CH.sub.2--C(CH.sub.3).sub.2--S--S-U- C781
was reacted with polyethylene glycol bis(imidazolyl carbonyl)
(Sigma Chemical Co.) having a polymer average molecular weight of
3,350 Daltons. The product was purified by ether precipitation.
[0079] In an alternate procedure to the above, cysteamine may be
used in place of 1-amino-2-methyl-2-propanethiol, which produces a
UC781 conjugate (PEG-CO--NH--CH.sub.2--CH.sub.2--S--S-UC781) with
less sterically hindered thiol groups and therefore with a
.about.100-fold faster release under reducing conditions.
EXAMPLE 3
Synthesis of PEG-S-S-4-thiouridine
[0080] 4-Thiouridine (MW 260, 8 mg, 0.031 mmol) was dissolved in 3
ml of acetonitrile and kept on ice. DIEA (5.3 .mu.l, 0.031 mmol)
was added to the solution. Then 2.9 mg of iodine was dissolved in 3
ml ice cold acetonitrile and added to the flask dropwise while
stirring. The reaction was kept on ice. TLC (10% methanol/90% DCM)
showed that the reaction was completed after 2 hours. The reaction
mixture was dried by speed vacuum. Then 4 ml of water and 6 ml of
acetonitrile, degassed by helium bubbling, were added to dissolve
the salt. Then thiol-PEG (MW 5000, 30 mg, 0.006 mmol) was added to
the reaction mixture and kept under argon at room temperature
overnight. The reaction mixture was dried by speed vacuum. The
product was extracted into DCM.
[0081] The formation of the 4-thiouridine homodimer by iodine
oxidation and the disulfide exchange reaction to give the
PEG-S-S-4-thiouridine conjugate proceeded similarly to the UC781
reactions. On TLC analysis, the thiouridine dimer migrated more
slowly than the monomer (FIG. 6, lanes 1 and 2), but returned to
the monomer position upon treatment with the reducing agent DTT
(FIG. 6, lane 3). One notable difference from UC781 is that
thiouridine is water-soluble, so water was used to dissolve the
homodimer. As a result, the DIEA-iodide salt also dissolved, but
its presence did not interfere with the subsequent disulfide
exchange reaction to make the PEG-S-S-4-thiouridine conjugate (FIG.
6, lane 5).
[0082] As with PEG-S-S-UC781, the original 4-thiouridine could be
reductively released from its disulfide conjugate by exposure to
DTT (FIG. 6, lane 6). Lane 7 shows DTT alone.
EXAMPLE 4
HPLC Analysis and In-Vivo Release
[0083] UC781 was analyzed using an HPLC reverse phase column, PRP-1
(Hamilton, Reno, Nev.), under the following condition: mobile phase
A: 20% acetonitrile, mobile phase B: 90% acetonitrile, flow rate: 1
ml/min. Gradient: 0-2 min, 100% A; 2-20 min, linear gradient from
100% A to 100% B; 20-28 min, 100% B; 28-30 min, linear gradient
from 100% B to 100% A; 30-35 min, 100% A. Under these experimental
conditions, the retention time for UC781 is 24.2 min. The
wavelength for detection of UC781 is 290 nm.
[0084] Since 1 mole of UC781 can be appended to 1 mole of PEG-SH,
the maximum drug content of this conjugate is 335/5335=6.3% (w/w).
Quantitation of drug content was done by measuring the amount of
UC781 released from the PEG-S-S-UC781 conjugate. An aliquot (500
mg) of PEG-S-S-UC781 was treated with 30 mM DTT in acetonitrile for
2 hours. The treated sample was analyzed by RP-HPLC. The amount of
UC781 released, based on the standard curve, was 13 .parallel.g.
Since all of the disulfide-linked UC781 should be releasable by
this procedure, the UC781 content in the conjugate is 2.6%. The
discrepancy from the theoretical maximum of 6.3% most likely
results from a large portion of the thiol-PEG molecules being
otherwise oxidized, such as to PEG-S-S-PEG. Since this conjugate is
readily soluble in water (PBS) at 100 mg/ml, the now-soluble drug
concentration is about 3 mg/ml. Thus, imparting water solubility to
an otherwise insoluble drug was achieved by making this prodrug
form. This improvement in solubility by carrying out the method of
the invention may be likewise achieved with other insoluble
agents.
[0085] To each 1 ml sample of EDTA-treated rabbit blood, 2 ml of
acetonitrile was added, mixed and kept on ice for 10 min. The
mixture was centrifuged at 1000.times. g for 10 min. The
supernatant was collected and an aliquot (300 .mu.l) was injected
into the reverse phase column. To evaluate the accuracy of UC781
analysis in blood samples, UC781 (10 .mu.g) in 10 ml of
dimethylsulfoxide (DMSO) was added to blood, mixed by vortexing and
then kept at room temperature for 10 min. Similarly, 500 .mu.g of
PEG-S-S-UC781 in 50 .mu.l of water was spiked into blood. After
centrifugation, 400 .mu.l of the supernatant was analyzed, either
without or with DTT pretreatment (3 mM for 2 hours at room
temperature).
[0086] Quantitation of UC781 and PEG-S-S-UC781 in extracted blood
samples was by reverse phase HPLC with monitoring at 292 nm. UC781
elutes at 24.2 minutes. Sample analysis without prior reduction by
DTT should give the concentration of free UC781 since the
conjugated form does not give a peak at 24.2 minutes. Indeed, it
has been observed that PEG conjugates in general tend to aggregate
at the top of reverse phase HPLC columns. However, prior reduction
with DTT should give the total concentration of UC781 since the
conjugated UC781 is converted into the free form prior to HPLC. The
dose-response curve for UC781 was linear from 0.1 .mu.g to 10
.mu.g. Recovery through the extraction procedure was determined by
spiking a known amount of either free or conjugated UC781 into
EDTA-treated rabbit blood prior to acetonitrile extraction. The
recovery of UC781 was found to be 113%. The recovery of UC781 from
spiked PEG-S-S-UC781 was found to be 107% with DTT treatment and
106% without DTT treatment, indicating that the disulfide bond in
PEG-S-S-UC781 is readily cleaved during exposure to whole blood.
That these recovery percentages are a little higher than 100% may
be because UC781 is partially excluded from erythrocytes.
[0087] An in-vivo experiment was done in duplicate in rabbits
(about 3.6 kg, New Zealand white, female), performed according to
protocol #I99-054 approved by the IACUC of UMDNJ-Robert Wood
Johnson Medical School. In detail, 55 mg of PEG-S-S-UC781
(equivalent to 1.5 mg of UC781) was dissolved in 1 ml of water,
diluted into 5 ml of U.S.P. saline and injected intravenously into
the marginal ear vein. Blood samples were drawn from the auricular
artery of the opposite ear prior to and at predetermined time
points after injection. Immediately after the blood was drawn, 1 ml
was mixed with 2 ml of acetonitrile, vortexed, kept on ice for 10
min and then centrifuged at 1000.times. g for 10 min at 4.degree.
C. The supernatant was collected and analyzed by reverse phase
HPLC, as described above. Blood levels were determined with time by
HPLC with and without the DTT reductive cleavage step (FIG. 7). At
every time point, the concentration of free UC781 was found to be
the same in both the DTT-treated and the untreated samples,
indicating, as anticipated, that UC781 had been rapidly released
from its PEG carrier in vivo.
[0088] Estimating a total blood volume of 200 ml for the rabbits,
then 52% and 70% of the total injected dose of PEG-S-S-UC781 is
present in blood at the earliest time points, 5 min and 3 min,
respectively in separate experiments. The half-life of UC781 in the
bloodstream was found to be just several minutes (FIG. 7). This
data is consistent with the results given by Conover et al (6), in
which the bioreversible ester-linked conjugate of PEG with the
anticancer drug, camptothecin, had a blood t.sub.1/2a of less than
5 min. When plasma was prepared by centrifugation of a duplicate
sample of EDTA-treated blood, the UC781 concentration was found to
be about 10% higher than in the matched sample of whole blood, in
agreement with the spiking experiment described above.
[0089] An in-vivo experiment was done twice in mice (about 25 g,
Sprague-Dawley), performed according to protocol #100-001, approved
by the IACUC. PEG-S-S-UC781. Twenty mg, equivalent to 0.5 mg of
UC781, was dissolved in 1 ml of PBS and injected into the leg
muscle. At each time point, a mouse was euthanized, blood was
collected and the injected muscle was removed. The blood samples
were treated with 2 volumes of acetonitrile as described above. The
tissue samples were homogenized with 1 ml of PBS and then treated
with 2 volumes of acetonitrile, as above. The samples were then
analyzed by reverse phase HPLC. As with the intravenous experiment,
essentially the same values were obtained for UC781 whether or not
the samples were pretreated with DTT. Recovery and extraction
efficiency from the muscle was tested by injection into the excised
muscle from a freshly euthanized mouse. This "zero" time point
resulted in 40% recovery of the injected dose. As shown on FIG. 8,
the half-time of diffusion of the drug from the muscle, assuming no
metabolism in muscle, was about 10 minutes. Although there were
relatively high levels of UC781 in the tissue samples even after 1
hour (more than 10%), blood samples were below the detection limit
of the HPLC methods. This finding suggests that in mice the rate of
elimination from the bloodstream is faster than the rate of
diffusion from the injected muscle into the bloodstream.
EXAMPLE 5
Reverse Transcriptase (RT) Inhibition Assay
[0090] UC781 standard solutions were prepared in DMSO. The stock
solution of HIV-RT (13.5 Units/.mu.l) was diluted to 25
mUnits/.mu.l in buffer comprising 50 mM Tris-HCl, pH 8.0, 1 mM DTT,
0.01% bovine serum albumin (BSA). Each assay tube contained 40
.mu.l of a solution comprising 50 mM Tris-HCl, pH 8.0, 4 mM DTT,
12.4 nM MgCl.sub.2, 50 mM KCl, 0.01% BSA, 10 .mu.g/ml poly(A), 0.01
mM dTTP and 10 mCi [.alpha.-.sup.32P]dTTP, 2 .mu.l of diluted RT
and 3 .mu.l of sample dissolved in DMSO. Samples were incubated at
37.degree. C. for 30 minutes. The polymerization reaction was
stopped by adding 0.15 mg/ml of salmon sperm DNA and chilling on
ice. An aliquot (20 .mu.l) of each reaction mixture was spotted
onto square stamped GF/C filter paper (12.5 cm in diameter)
presoaked with 10% trichloroacetic acid (TCA). After the spots were
dry, the paper was rinsed with 50 ml of ice cold 10% TCA twice, 50
ml of ice cold water three times and then once with 25 ml of ice
cold 95% ethanol in a Buchner funnel. The paper was dried, cut into
squares and each square was counted in 4 ml of LSC cocktail
(National Diagnostics).
[0091] The reverse transcriptase inhibition (RT) assay was used to
confirm that the compound released in blood is truly active UC781.
The peak fraction at 24.2 minutes was collected from the HPLC
analysis of a sample of blood collected at the 5 minute time point
from the intravenous injection of PEG-S-S-UC781. As a control,
authentic UC781 was run on the HPLC column and the peak was
collected at 24.2 minutes. As shown in FIG. 9, the RT assay is
linear with inhibitor concentration in the range of 5 nM to 50 nM
UC781. An aliquot of each HPLC fraction, providing a predicted
concentration of 30 nM in the RT assay, was analyzed. The RT assay
gave results of 26 nM and 27 nM for the blood and standard samples,
respectively, indicating that the recovered compound is fully
active.
[0092] The present invention is not to be limited in scope by the
specific embodiments describe herein. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
[0093] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
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* * * * *