U.S. patent application number 11/714449 was filed with the patent office on 2007-07-19 for systems and methods related to degradation of uremic toxins.
This patent application is currently assigned to Brown University. Invention is credited to Jan Markus Bruder, Michael J. Lysaght, Jill A. O'Loughlin.
Application Number | 20070166285 11/714449 |
Document ID | / |
Family ID | 34634447 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166285 |
Kind Code |
A1 |
O'Loughlin; Jill A. ; et
al. |
July 19, 2007 |
Systems and methods related to degradation of uremic toxins
Abstract
The present invention generally relates to the treatment of
uremic toxins in vivo using uremic toxin-treating enzymes, and/or
cells capable of producing uremic toxin-treating enzymes or
otherwise reacting with uremic toxins. Non-limiting examples of
cases where the treatment of uremic toxins is desired include renal
disease or dysfunction, gout, subjects receiving chemotherapy, or
the like. In one aspect, the treatment includes an oral delivery
composition able to reduce the blood concentration of one or more
non-protein nitrogen compounds in vivo. The composition, in some
cases, may comprise one, two, or more uremic toxin-treating
enzymes, such as urease, uricase or creatininase. The oral delivery
composition may be able to deliver the uremic toxin-treating
enzymes, substantially undigested, to the intestines, where the
enzymes can interact with uremic toxins transported to the
intestines from the bloodstream. In another aspect, the treatment
includes an oral delivery composition comprising a cell able to
reduce the concentration of one or more uremic toxins in vivo. In
some cases, the cell may be designed to overexpress one, two, or
more uremic toxin-treating enzymes, such as urease, uricase or
creatininase, for example, by transfecting the cell with a
corresponding gene. In some embodiments, a species able to react
with or otherwise sequester by-products of the uremic
toxin-treating enzyme reactions may be included with the oral
delivery composition. For example, if the by-product is ammonium,
the species may be a sorbent able to adsorb ammonium, an enzyme
able to react with the ammonium, or the like.
Inventors: |
O'Loughlin; Jill A.;
(Rochester, NY) ; Bruder; Jan Markus; (Altena,
DE) ; Lysaght; Michael J.; (East Greenwich,
RI) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Brown University
Providence
RI
|
Family ID: |
34634447 |
Appl. No.: |
11/714449 |
Filed: |
March 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10731877 |
Dec 9, 2003 |
7198785 |
|
|
11714449 |
Mar 6, 2007 |
|
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Current U.S.
Class: |
424/93.2 ;
424/451; 424/94.6 |
Current CPC
Class: |
Y02A 50/473 20180101;
A61K 38/50 20130101; C12Y 107/03003 20130101; Y02A 50/30 20180101;
A61K 38/53 20130101; A61K 38/44 20130101; C12Y 305/01005 20130101;
A61K 9/4891 20130101; C12Y 603/01002 20130101; A61P 19/06 20180101;
C12Y 305/0201 20130101; A61K 9/4866 20130101; A61K 38/53 20130101;
A61K 2300/00 20130101; A61K 38/44 20130101; A61K 2300/00 20130101;
A61K 38/50 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/093.2 ;
424/094.6; 424/451 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 38/46 20060101 A61K038/46; A61K 9/48 20060101
A61K009/48 |
Claims
1-98. (canceled)
99. A method, comprising: administering, to a subject, an oral
delivery composition comprising a capsule, wherein the capsule
comprises an enteric coating comprising a polymer, and the capsule
contains isolated urease and an ammonium uptake species in an
effective amount to treat a disease characterized by elevated
levels of at least one non-protein nitrogen compound.
100. The method of claim 99, wherein the polymer comprises
cellulose acetate.
101. The method of claim 99, wherein the capsule, when ingested by
the subject, does not substantially release the isolated urease
externally of the capsule.
102. The method of claim 99, wherein the capsule does not
substantially impede mass transport of urea therethrough.
103. The method of claim 99, wherein the ammonium uptake species is
able to adsorb ammonium.
104. The method of claim 103, wherein the ammonium uptake species
comprises a sorbent.
105. The method of claim 104, wherein the sorbent comprises
zirconium phosphate.
106. The method of claim 104, wherein the sorbent comprises at
least one of carbon and oxystarch.
107. The method of claim 99, wherein the ammonium uptake species
comprises at least one enzyme able to react with ammonium.
108. The method of claim 99, wherein the subject is susceptible to
or exhibits symptoms of a disease characterized by elevated levels
of at least one non-protein nitrogen compound.
109. The method of claim 108, wherein the subject is susceptible to
or exhibits symptoms of a disease characterized by elevated levels
of more than one non-protein nitrogen compounds.
110. The method of claim 99, wherein the subject has end stage
renal disease.
111. The method of claim 99, wherein the subject has renal
dysfunction.
112. The method of claim 99, wherein the subject has gout.
113. The method of claim 99, wherein the subject has been treated
with chemotherapy.
114. The method of claim 99, further comprising administering
dialysis to the subject.
115. An article, comprising: an oral delivery composition
comprising a capsule comprising isolated urease and an ammonium
uptake species, wherein the capsule comprises an enteric coating
comprising a polymer.
116. The article of claim 115, wherein the polymer comprises
cellulose acetate.
117. The article of claim 115, wherein the ammonium uptake species
comprises a sorbent.
118. The article of claim 115, wherein the sorbent comprises
zirconium phosphate.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/731,877, filed Dec. 9, 2003, entitled
"Systems and Methods Related to Degradation of Uremic Toxins," by
O'Loughlin, et al., which application is incorporated herein by
reference in its entirety.
FIELD OF INVENTION
[0002] The present invention generally relates to the treatment of
disorders associated with uremic toxins in vivo and, in particular,
to the treatment of disorders associated with uremic toxins in vivo
using uremic toxin-treating enzymes, and/or cells capable of
[0003] The producing uremic toxin-treating enzymes or otherwise
reacting with uremic toxins.
DISCUSSION OF RELATED ART
[0004] The principle excretory function of the kidney is to
maintain fluid balance and excrete waste metabolites. Typical rates
of fluid and solute removal (per 24 hours) are .about.1.5 L of
water, .about.20 g of urea, .about.5 g of electrolytes, and
.about.6 g of other metabolites, such as uric acid or creatinine.
During renal dysfunction or failure, e.g., in end stage renal
disease patients, waste metabolites normally excreted in the urine
are instead retained in the blood and body tissues, leading to a
pathological state commonly known as uremia or uremic toxicity.
[0005] Urea is the predominant nitrogen waste product of dietary
protein catabolism. Uric acid is a product of nucleic acid
degradation. Creatinine typically results from muscular protein
breakdown. These components are normally eliminated in the urine
via the kidneys. These components are also commonly used as markers
to monitor kidney dialysis and other similar treatments. Although
these waste metabolites are relatively nontoxic when acting alone,
they are part of a more complex uremic toxicity syndrome, in which
toxicity may result from the combined effects of these metabolites.
Patients
[0006] There are currently .about.325,000 dialysis patients in the
United States and .about.1.3 million patients worldwide, with a
cost of about $70,000 per patient/year, which translates to an
estimated overall cost for dialysis care of about $80 billion (2003
figures). The patient population has an annual growth rate of 7%.
Since the early 1970's, the full cost of dialysis treatment in the
United States has been paid for by Medicare, regardless of patient
age or need. Nevertheless, the U.S. has quite a high mortality
rate: .about.50% of patients die within 3 years. A recent study
compared the expected remaining lifetime for patients with selected
diseases versus controls (i.e., free of disease) for the U.S.
resident population in 1990. Study participants (aged 45 to 54)
free of disease had an expected remaining lifetime of thirty years,
compared to ten years for colon cancer patients and seven years for
end stage renal disease patients. In another age bracket that was
surveyed (aged 55 to 64), study participants free of disease had an
expected remaining lifetime of twenty-two years, compared to ten
and five years remaining for colon cancer and end stage renal
disease patients respectively. In addition, .about.10% of patients
electively withdraw from dialysis treatment and accept a form of
suicide.
[0007] Dialysis treatments often interfere with normal activities
of daily living, since it typically is required three times a week,
for three to five hours per session. Blood access is usually by
percutaneous needle puncture. Secondary medical complications of
uremia often arise, most commonly hypotension, leading to nausea,
and cramps, and the like. These complications may be resolved by a
kidney transplant, although the transplant recipient must still
endure daily immunosuppressant treatment. Transplantation as a
treatment for renal failure is scarce, as only .about.14,000 donor
organs (in the U.S.) are available each year, and there are
currently over 80,000 patients on the waiting list (2003
figures).
[0008] There is thus a need for improvement in the treatment of
uremic toxins in vivo.
SUMMARY OF INVENTION
[0009] The present invention generally relates to the treatment of
disorders associated with uremic toxins in vivo using uremic
toxin-treating enzymes, and/or cells capable of producing uremic
toxin-treating enzymes or otherwise reacting with uremic toxins.
The subject matter of this invention involves, in some cases,
interrelated products, alternative Medicare, regardless of patient
age or need. Nevertheless, the U.S. has quite a high mortality
rate: .about.50% of patients die within 3 years. A recent study
compared the expected remaining lifetime for patients with selected
diseases versus controls (i.e., free of disease) for the U.S.
resident population in 1990. Study participants (aged 45 to 54)
free of disease had an expected remaining lifetime of thirty years,
compared to ten years for colon cancer patients and seven years for
end stage renal disease patients. In another age bracket that was
surveyed (aged 55 to 64), study participants free of disease had an
expected remaining lifetime of twenty-two years, compared to ten
and five years remaining for colon cancer and end stage renal
disease patients respectively. In addition, .about.10% of patients
electively withdraw from dialysis treatment and accept a form of
suicide.
[0010] Dialysis treatments often interfere with normal activities
of daily living, since it typically is required three times a week,
for three to five hours per session. Blood access is usually by
percutaneous needle puncture. Secondary medical complications of
uremia often arise, most commonly hypotension, leading to nausea,
and cramps, and the like. These complications may be resolved by a
kidney transplant, although the transplant recipient must still
endure daily immunosuppressant treatment. Transplantation as a
treatment for renal failure is scarce, as only .about.14,000 donor
organs (in the U.S.) are available each year, and there are
currently over 80,000 patients on the waiting list (2003
figures).
[0011] There is thus a need for improvement in the treatment of
uremic toxins in vivo.
SUMMARY OF INVENTION
[0012] The present invention generally relates to the treatment of
disorders associated with uremic toxins in vivo using uremic
toxin-treating enzymes, and/or cells capable of producing uremic
toxin-treating enzymes or otherwise reacting with uremic toxins.
The subject matter of this invention involves, in some cases,
interrelated products, alternative solutions to a particular
problem, and/or a plurality of different uses of one or more
systems and/or articles.
[0013] In one aspect, the present invention is an article. The
article, in one set of embodiments, includes an oral delivery
composition. In one embodiment, the oral delivery composition
includes at least one of isolated uricase and isolated
creatininase. In another embodiment, the oral delivery composition
includes at least one cell transfected with at least one of a
uricase gene and a creatininase gene. In yet another embodiment,
the oral delivery composition includes at least one cell designed
to overexpress at least one of uricase and creatininase. The oral
delivery composition includes, in still another embodiment, at
least one cell transfected with at least one of a urease gene, a
uricase gene, and a creatininase gene, where the at least one cell
is not E. coli. In yet another embodiment, the oral delivery
composition includes at least one cell able to reduce a blood
concentration of at least one non-protein nitrogen compound in a
subject when the oral delivery composition is ingested by the
subject, where the at least one cell is not E. coli. In still
another embodiment, the oral delivery composition includes at least
two isolated uremic enzymes. In some cases, the oral delivery
composition may include a capsule. The capsule, in some
embodiments, may include one or more of the above-described
compositions.
[0014] In another aspect, the present invention defines a method.
In one set of embodiments, the method includes administering, to a
subject, an oral delivery composition comprising at least one of
uricase and creatininase. The method, in another set of
embodiments, includes administering, to a subject, an oral delivery
composition comprising at least one cell transfected with at least
one of a uricase gene and a creatininase gene. The method, in yet
another set of embodiments, includes administering, to a subject,
an oral delivery composition comprising at least one cell designed
to overexpress at least one of uricase and creatininase. In still
another set of embodiments, the method includes administering, to a
subject, an oral delivery composition comprising at least one cell
transfected with at least one of a urease gene, a uricase gene, and
a creatininase gene, where the at least one cell is not E. coli.
The method, in yet another set of embodiments, includes
administering, to a subject, an oral delivery composition
comprising at least one cell able to reduce a blood concentration
of at least one non-protein nitrogen compound in the subject when
the oral delivery composition is ingested by the subject, where the
at least one cell is not E. coli. In another set of embodiments,
the method includes administering at least one of isolated uricase
and isolated creatininase to an intestine of a subject. The method
includes, in still another set of embodiments, administering, to a
subject, an oral delivery composition comprising at least two
isolated uremic enzymes.
[0015] In another aspect, the present invention is directed to a
method of making one or more of the embodiments described herein,
for example, an oral delivery capsule. In yet another aspect, the
present invention is directed to a method of using one or more of
the embodiments described herein, for example, an oral delivery
capsule. In still another aspect, the present invention is directed
to a method of promoting one or more of the embodiments described
herein, for example, an oral delivery capsule.
[0016] Other advantages and novel features of the invention will
become apparent from the following detailed description of the
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control.
BRIEF DESCRIPTION OF DRAWINGS
[0017] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For the
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0018] FIG. 1 illustrates certain enzymatic reactions of the
invention;
[0019] FIGS. 2A-2C are photocopies of photographs of alginate
microspheres and systems used to produce them, in accordance with
one embodiment of the invention;
[0020] FIG. 3 illustrates the reduction in uremic toxins in 24
hours in an embodiment of the invention, compared to typical levels
of reduction observed using clinical hemodialysis;
[0021] FIG. 4 illustrates the degradation of uremic toxins by
encapsulated enzymes, according to one embodiment of the
invention;
[0022] FIGS. 5A-5B illustrate urease degradation kinetics of an
embodiment of the invention;
[0023] FIGS. 6A-6D illustrate the oral passage of an embodiment of
the invention through a rat model;
[0024] FIG. 7 illustrates the effect of sorbent on urea
degradation, in accordance with one embodiment of the
invention;
[0025] FIG. 8 illustrates a comparison of the effectiveness of one
embodiment of the invention, as compared to the typical performance
of clinical hemodialysis;
[0026] FIGS. 9A-9C illustrates the degradation in vitro of urea,
uric acid, and creatinine using an embodiment of the invention;
and
[0027] FIG. 10 illustrates the in vivo delivery of an embodiment of
the invention.
DETAILED DESCRIPTION
[0028] The present invention generally relates to the treatment of
disorders associated with uremic toxins in vivo using uremic
toxin-treating enzymes, and/or cells capable of producing uremic
toxin-treating enzymes or otherwise reacting with uremic toxins to
reduce or eliminate the toxic activity of the uremic toxins.
Non-limiting examples of disorders associated with uremic toxins
include renal disease or dysfunction, gout, subjects receiving
chemotherapy, or the like. In one aspect, the treatment includes an
oral delivery composition able to reduce the blood concentration of
one or more non-protein nitrogen compounds in vivo. The
composition, in some cases, may comprise one, two, or more uremic
toxin-treating enzymes, such as urease, uricase or creatininase.
The oral delivery composition may be able to deliver the uremic
toxin-treating enzymes, substantially undigested, to the
intestines, where the enzymes can interact with uremic toxins
transported to the intestines from the bloodstream. In another
aspect, the treatment includes an oral delivery composition
comprising a cell able to reduce the concentration of one or more
uremic toxins in vivo. In some cases, the cell may be designed to
overexpress one, two, or more uremic toxin-treating enzymes, such
as urease, uricase or creatininase, for example, by transfecting
the cell with a corresponding gene. In some embodiments, a species
able to react with or otherwise sequester by-products of the uremic
toxin-treating enzyme reactions may be included with the oral
delivery composition. For example, if the by-product is ammonium,
the species may be a sorbent able to adsorb ammonium, an enzyme
able to react with the ammonium, or the like.
[0029] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0030] As used herein, "or" is understood to mean inclusively or,
i.e., the inclusion of at least one, but including more than one,
of a number or list of elements. Only terms clearly indicated to
the contrary, such as "exclusively or" or "exactly one of," will
refer to the inclusion of exactly one element of a number or list
of elements.
[0031] The term "patient" or "subject" as used herein includes
mammals such as humans, as well as non-human mammals such as
non-human primates, cows, horses, pigs, sheep, goats, dogs, cats,
rabbits, or rodents such as mice or rats.
[0032] As used herein, a "uremic toxin" is given its ordinary
meaning as used in the art, e.g., one or more compounds containing
nitrogen produced by the body as waste products, e.g., from the
breakdown of proteins, nucleic acids, or the like. Typically,
uremic toxins are not proteins. Non-limiting examples of uremic
toxins include urea, uric acid, creatinine, and beta-2
(.beta..sub.2) microglobulin (e.g., see FIG. 1). In healthy
individuals, uremic toxins are usually excreted from the body
through the urine. However, in certain individuals, uremic toxins
are not removed from the body at a sufficiently fast rate, leading
to uremic toxicity, i.e., a disease or condition characterized by
elevated levels of at least one uremic toxin with respect to
physiologically normal levels of the uremic toxin. Non-limiting
examples of such diseases include renal disease or dysfunction,
impaired or partial kidney function, gout, subjects receiving
chemotherapy, or the like. Subjects receiving chemotherapy or other
treatments may experience significant amounts of necrosis of cell
populations, which can cause the releases of purines which are
metabolized to uric acid. "Renal disease" includes early renal
disease states (i.e., the kidneys do not perform at physiologically
normal levels, but are able to process and remove some uremic
toxins from the bloodstream), as well as end stage renal disease
("ESRD"), where the kidneys are substantially nonfunctional.
Certain uremic toxins are transported between the bloodstream and
the intestine, for example, urea, uric acid, or creatinine. As used
herein, "transport" refers to any process in which a substance is
moved from one location to another, for example, through diffusion
(passive transport), facilitated diffusion, convection, transport
proteins or other active transport systems, etc.
[0033] One aspect of the present invention involves delivering one,
two, or more uremic toxin-treating enzymes to a subject, typically
to the intestines. Preferably, the enzymes are delivered in a
substantially undigested state. In some cases, one or more of the
enzymes are isolated (e.g., as described below). As used herein, a
"uremic toxin-treating enzyme," or a "uremic enzyme," is an enzyme
able to react with a uremic toxin as a substrate, for example, the
uremic toxic-treating enzyme may be an enzyme able to react with
urea as a substrate, with uric acid as a substrate, or with
creatinine as a substrate. Uremic enzymes can be determined in
vitro, for example, by allowing the enzyme to react with a uremic
toxin in solution and measuring a decrease in the concentration of
the uremic toxin. Examples of uremic toxin-treating enzymes
include, but are not limited to, ureases (which reacts with urea),
uricases (which reacts with uric acid), or creatininases (which
reacts with creatinine). FIG. 1 illustrates enzymatic reactions
that typically occur with these enzymes. In some cases, each enzyme
independently may originate from a different species (i.e.,
heterologous). In some cases, the enzyme is commercially available,
for example, isolated and purified from other sources. A specific
non-limiting example of a urease is urease from Canavalia
ensiformis, having a sequence SEQ ID NO.: 1 (GenBank Accession
number URJB GI:418642). A specific non-limiting example of a
uricase is uricase from Schizosaccharomyces pombe, having a
sequence SEQ ID NO.: 2 (GenBank Accession number T40869
GI:7493586). A specific non-limiting example of a creatininase is
creatininase from Arthrobacter sp., having a sequence SEQ ID NO.: 3
(GenBank Accession number BAA25929.1 GI:3116224). Those of ordinary
skill in the art will know of other suitable uremic toxin-treating
enzymes. Additionally, minor changes to such enzymes (for example,
through chemical changes or modifications, such as the addition of
reporting groups, linkage to a physical surface, changes or
substitutions in bases in the amino acid sequence of the enzyme,
etc.) that do not alter the ability of the enzyme to recognize and
react with its substrate are also included herein as uremic
toxin-treating enzymes. For example, a urease, uricase, or
creatininase may be covalently bound to a surface, for instance, in
a microarray or an ELISA.
[0034] One or more uremic toxin-treating enzyme described herein
may be isolated in certain cases. An "isolated" molecule (e.g., an
enzyme), as used herein, is a molecule that is substantially pure
and is free of other substances with which it is ordinarily found
in nature or in vivo systems to an extent practical and appropriate
for its intended use. For instance, the molecular species may be
sufficiently pure or sufficiently free from substances such as
biological constituents with which it is normally found in vivo so
as to be useful in, for example, producing pharmaceutical
preparations, or sequencing if the molecular species is a nucleic
acid, peptide, enzyme, or polysaccharide. Because an isolated
molecular species of the invention may be admixed with a
pharmaceutically-acceptable carrier in a pharmaceutical
preparation, and/or other physiologically-active agents (e.g., as
described below), the molecular species may comprise only a small
percentage by weight of the preparation. The molecular species is
nonetheless substantially pure in that it has been substantially
separated from the substances with which it may be associated in
living systems. As examples, a uremic toxin-treating enzyme, such
as urease, uricase, and/or creatininase may be associated with
other molecules, such as a pharmaceutically acceptable carrier, a
sorbent, a capsule (e.g., comprising alginate), etc.
[0035] Any suitable system or method may be used to orally deliver
the uremic enzyme in a substantially undigested state. As used
herein, an "oral delivery composition" is a composition that is
designed to be delivered orally to a subject, i.e., the composition
has been formulated in such a way that it is designed to be taken
orally by a subject in a therapeutically effective amount without
substantially adverse effects. For example, an enzyme may be
delivered to a subject in an oral delivery composition that is a
capsule, a sustained release pill, a controlled release
formulation, a liposome, etc. As used herein, a "substantially
undigested" enzyme (or other such substance) is an enzyme (or other
substance) that enters the gastrointestinal system of a subject,
and is not degraded or digested by the gastrointestinal system
until at least reaching the site of delivery (e.g., the
intestines), and/or is partially degraded or digested, but such
that a therapeutically effective amount of the enzyme or other
substance is able to reach the site of delivery. Degradation or
digestion by the gastrointestinal system of the enzyme (or other
substance) can occur, for example, through the action of pH,
gastric acid, mechanical action, hydrolysis, digestive enzymes such
as pepsin, trypsin, chymotrypsin, etc. In some cases, the enzyme is
not degraded or digested by the gastrointestinal system and is
excreted substantially intact.
[0036] In some cases, an enzyme is included in a formulation that
is not substantially susceptible to degradation or digestion by the
gastrointestinal system, i.e., a formulation that is able to
deliver the enzyme to the site of delivery in a substantially
undigested state. For example, the enzyme may be encapsulated in a
formulation that resists degradation or digestion, the enzyme may
be included in a formulation that is surrounded by a coating at
least partially resistant to degradation or digestion, or the like.
In certain instances, the formulation may be at least partially
susceptible to degradation or digestion, but over time scales
greater than the time it takes for the formulation to pass through
the gastrointestinal system; thus, the formulation is still able to
deliver the enzyme to the site of delivery in a substantially
undigested state, even though some degradation or digestion of the
enzyme may occur. As used herein, "substantially undigested state"
refers to a level of degradation or digestion of the enzyme that
does not impede the ability of the enzyme to recognize and react
with its substrate. In some cases, it is preferred that the
formulation be designed so as to not substantially release the
uremic toxin-treating enzyme externally of the capsule, i.e., into
the gastrointestinal system. That is, the formulation may be
designed such that any release of the uremic toxin-treating enzyme
externally of the capsule does not prevent the uremic
toxin-treating enzyme remaining within the capsule from being able
to react with uremic toxins found in the gastrointestinal system at
a therapeutically effective rate.
[0037] In certain embodiments, the formulation may be chosen or
designed to allow sufficient mass transport of uremic toxins into
the formulation to occur such that the enzyme is able to react with
uremic toxins found in the gastrointestinal system at a
therapeutically effective rate. As used herein, "mass transport" is
given its ordinary meaning as used in the art, i.e., the physical
movement of a substance from location to another, using processes
such as diffusion, convection, osmosis, etc. In some cases, the
formulation can be designed such that it does not substantially
impede mass transport of the uremic toxin into the formulation,
i.e., where "substantially impede" refers to the ability of the
uremic toxin to reach the site of the uremic toxin-treating enzyme
without being significantly rate-limited, for example, such that
the reaction of the uremic enzyme occurs over a time scale
comparable to the time scale of mass transport of the uremic toxin
from external the formulation to the enzyme. For example, a capsule
and/or an enteric coating may allow diffusion to occur therethrough
to a uremic enzyme at rates similar to rates of reaction of free
enzyme to the uremic toxin.
[0038] In one set of embodiments, a formulation of the invention,
such as a capsule, may contain one uremic toxin-treating enzyme, or
more than one uremic toxin-treating enzyme (i.e., a "combination"
formulation). More than one type of formulation may be given to a
subject. For example, a subject may be given a first capsule
containing a first enzyme and a second capsule containing a second
enzyme, e.g., serially or simultaneously.
[0039] As one example, a formulation of the invention may contain
one, two, or more uremic toxin-treating enzymes encapsulated within
a capsule. The capsule may comprise alginate or other suitable
polymers or materials able to at least partially resist degradation
or digestion in the gastrointestinal system. Alginates are salts of
alginic acid, a carbohydrate biopolymer that can be extracted from
brown algae or other sources. Typically, alginates include monomers
such as mannuronic acid or guluronic acid, although other monomers
may be included as well. Other examples of suitable materials
include lactic acid, glycolic acid, lysine, and hydroxyapatite, as
well as mixtures or copolymers of these and/or other materials,
e.g., poly(lactic-co-glycolic acid).
[0040] The capsules may be produced by any suitable technique known
to those of ordinary skill in the art. For example, one or more
uremic enzymes may be placed within a pre-polymeric solution that,
upon solidification or polymerization, forms a capsule embedding
the enzymes. Other compounds, such as sorbents, stabilizers,
buffers, or the like may also be included within the capsule, e.g.,
as further described below. In some cases, post-processing steps
may also be performed, for example, forming an enteric coating
around the capsule.
[0041] As used herein, an "enteric coating" is given its ordinary
meaning as used in the art, i.e., a coating that at least is
partially resistant to degradation or digestion within the
gastrointestinal system or at least a portion thereof, such as
within the stomach. Those of ordinary skill in the art will know of
suitable materials useful for enteric coatings. Non-limiting
examples include enteric polymers such as cellulose acetate
phthalate, cellulose acetate succinate, methylcellulose phthalate,
ethylhydroxycellulose phthalate, polyvinylacetatephthalate,
polyvinylbutyrate acetate, vinyl acetate-maleic anhydride
copolymer, styrene-maleic mono-ester copolymer, methyl
acrylate-methacrylic acid copolymer, methacrylate-methacrylic
acid-octyl acrylate copolymer, etc. These may be used either alone
or in combination, or together with other polymers than those
mentioned above. The enteric coating may also include insoluble
substances which are neither decomposed nor solubilized in living
bodies, such as alkyl cellulose derivatives such as ethyl
cellulose, crosslinked polymers such as styrene-divinylbenzene
copolymer, polysaccharides having hydroxyl groups such as dextran,
cellulose derivatives which are treated with bifunctional
crosslinking agents such as epichlorohydrin, dichlorohydrin, a
diepoxybutane, etc. The enteric coating may also include starch
and/or dextrin.
[0042] In some embodiments, a formulation of the invention may
include a species able to react with or otherwise sequester
(isolate) one or more by-products of the uremic toxin-treating
enzyme reactions. For example, in cases where ammonia (NH.sub.3) is
produced as a by-product, the species may react with or sequester
ammonia, and that species may be referred to as an "ammonium uptake
species." In some instances, the species may sequester the
by-product through physical means, such as through physisorption
mechanisms, i.e., by use of a sorbent such as zirconium phosphate,
carbon, or oxystarch. In other instances, an enzyme able to react
with a by-product may be used to react the by-products, for
example, into a metabolically neutral form, into a form that can is
useful (e.g., an amino acid), into a form that is non-toxic, etc.
As an example, in cases where ammonia is produced as a by-product,
an enzyme able to react with ammonia may be used, such as glutamine
synthetase. As yet another example, a cell able to react with one
or more by-products of the reaction may be included in the
formulation. Also, combinations of such techniques may also be used
in some cases, for example, a sorbent and an enzyme may be included
in the capsule or other formulation.
[0043] Other active compounds may be added to the formulation as
well, for example, other cells, enzymes, chemicals, drugs,
reporting agents, etc. For example, bacteria and/or enzymes
targeted toward other molecules elevated in uremia, such as beta-2
(.beta.2) microglobulin may be identified and added to the
formulation. In another embodiment, bacteria capable of recycling
ammonia into amino acid precursors may be used, which may, in some
cases, counteract the malnutrition which often accompanies renal
failure.
[0044] Another aspect of the invention involves using cells
designed to overexpress a uremic toxin-treating enzyme. Such cells
may be delivered to a subject in an oral delivery composition, such
as those previously described (for example, encapsulated),
optionally in combination with other cells, enzymes such as uremic
toxin-treating enzymes, sorbents, or other species able to react
with or otherwise sequester one or more reaction by-products, etc.
As used herein, a cell that is "designed" to overexpress a uremic
enzyme is intentionally chosen or selected to "overexpress" the
uremic enzyme, i.e., to express the uremic enzyme at expression
levels significantly greater than the expression level of the
enzyme for that cell type (which can include zero or negligible
expression levels). For example, such a cell may be artificially
selected through natural selection processes to overexpress the
uremic toxin-treating enzyme, the cells may be stimulated (e.g.,
with a hormone to overexpress the uremic toxin-treating enzyme, the
cell may be transfected to overexpress the uremic toxin-treating
enzyme, or the like.
[0045] The cell may be any cell able to overexpress the uremic
enzyme at levels that are therapeutically effective. For example,
the cell may be a bacterium or a mammalian cell. Bacteria may be
advantages in some cases. For example, bacteria can grow and expand
during their passage through the gastrointestinal system, thus
increasing the effectiveness of this form of treatment. In some
cases, the bacteria can metabolize some of the breakdown products
from the enzymatic reaction, e.g., preventing their resorption. In
certain cases, the bacteria efficiency (e.g. in terms of
weight/degradation power) may be higher than that of isolated
enzymes or encapsulated enzymes. Bacteria can also be relatively
easy to grow quickly in large amounts, and are often less expensive
than enzymes. Some bacteria can also metabolize uremic toxins
intracellularly, such that the uremic enzymes stay well-protected
from the environment of the gastrointestinal system. In one
embodiment, the bacteria is not E. coli.
[0046] In some cases, the cell may be transfected, e.g., with one,
two, or more genes for urease, uricase, creatininase, or other
uremic toxin-treating enzymes. Those of ordinary skill in the art
will know of suitable ways of transfecting cells. For example, some
techniques for transformation (micro-injection, electroporation,
calcium phosphate method, etc.) are described in Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
N.Y. (1989).
[0047] In one embodiment, a gene for urease, uricase, creatininase,
or other uremic toxin-treating enzymes may be transfected into a
cell using a DNA vector. The vector may be a vector in which the
gene is functionally linked to one or more control sequences which
allows expression of the corresponding enzymes. These include
plasmids which can be replicated and/or expressed in prokaryotes or
bacteria such as E. coli and/or in eukaryotic systems such as
yeasts or mammalian cell lines.
[0048] Expression in prokaryotes may be carried out using
techniques known in the art. The gene may be expressed as fusion
proteins or as intact, native proteins. In some cases, fusion
proteins may be produced in large quantities. The fusion proteins
are generally more stable than the native polypeptide and are easy
to purify. The expression of these fusion proteins can be
controlled by normal host DNA sequences.
[0049] Producing intact native polypeptides using bacteria such as
E. coli may require, in some cases, a strong, regulatable promoter
and an effective ribosome binding site. Promoters which may be used
for this purpose include, but are not limited to, the temperature
sensitive bacteriophage .lamda.P.sub.L-promoter, the tac-promoter
inducible with IPTG or the T7-promoter. Numerous plasmids with
suitable promoter structures and efficient ribosome binding sites
have been described, such as for example pKC30 (.lamda.P.sub.L;
Shimatake and Rosenberg, Nature, 292:128 (1981), pKK173-3 (tac,
Amann and Brosius, Gene, 40:183 (1985)) or pET-3 (T7-promoter
(Studier and Moffat, J Mol. Biol., 189:113 (1986)). A number of
other suitable vector systems for expressing the DNA according to
the invention in bacteria are known from the prior art and are
described, for example, in Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, N.Y. (1989).
[0050] Suitable bacterial strains which are specifically tailored
to a particular expression vector are known to those skilled in the
art (Sambrook, et al., Molecular Cloning. A Laboratory Manual, Cold
Spring Harbor Press, N.Y. (1989)). The experimental performance of
the cloning experiments, the expression of the polypeptides in
bacteria and the working up and purification of the polypeptides
are known.
[0051] In addition to prokaryotes, eukaryotic microorganisms such
as yeast may also be used in some cases. For expression in yeast,
the plasmid YRp7 (Stinchcomb et al. Nature, 282:39 (1979); Kingsman
et al., Gene 7:141 (1979); Tschumper et al., Gene, 10:157 (1980))
and the plasmid YEp13 (Bwach et al., Gene, 8:121-133 (1979)) are
used, for example. The plasmid YRp7 contains the TRP1-gene which
provides a selection marker for a yeast mutant (e.g., ATCC No.
44076) which is incapable of growing in tryptophan-free medium. The
presence of the TRP1 defect as a characteristic of the yeast strain
used then constitutes an effective aid to detecting transformation
when cultivation is carried out without tryptophan. The same is
true with the plasmid YEp13, which contains the yeast gene LEU-2,
which can be used to complete a LEU-2-minus mutant.
[0052] Other suitable marker genes for yeast include, for example,
the URA3- and HIS3-gene. Preferably, yeast hybrid vectors also
contain a replication start and a marker gene for a bacterial host,
so that the construction and cloning of the hybrid vectors and
their precursors can be carried out in a bacterial host. Other
expression control sequences suitable for expression in yeast
include, for example, those of PHO3- or PHO5-gene.
[0053] Other suitable promoter sequences for yeast vectors contain
the 5'-flanking region of the genes of ADH I (Ammerer, Methods of
Enzymology, 101: 192-210 (1983)), 3-phosphoglycerate kinase
(Hitzeman et al., J Biol. Chem., 255:2073 (1980)) or other
glycolytic enzymes (Kawaski and Fraenkel, Biochem. Biophys. Res.
Comm., 108:1107-1112 (1982)) such as enolase,
glycerinaldehyde-3-phosphate-dehydrogenase, hexokinase,
pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, phosphoglucose-isomerase and glucokinase. When
constructing suitable expression plasmids, the termination
sequences associated with these genes may also be inserted in the
expression vector at the 3'-end of the sequence to be expressed, in
order to enable polyadenylation and termination of the mRNA.
[0054] Generally, any vector which contains a yeast-compatible
promoter and origin replication and termination sequences is
suitable. Thus, hybrid vectors which contain sequences homologous
to the yeast 2 .mu.plasmid DNA may also be used. Such hybrid
vectors are incorporated by recombination within the cells of
existing 2 .mu.-plasmids or replicate autonomously.
[0055] The genetic constructs may generally contain one or more
suitable regulatory elements (such as one or more suitable
promoters, enhancers, terminators, etc.), 3'- or 5'-UTR sequences,
leader sequences, selection markers, expression markers/reporter
genes, and/or elements that may facilitate or increase (the
efficiency of) transformation. These and other suitable elements
for such genetic constructs will be clear to those of ordinary
skill in the art, and may, for instance, depend upon the type of
construct used, the intended host cell or host organism; the manner
in which the nucleotide sequences of the invention of interest are
to be expressed (e.g. via constitutive, transient or inducible
expression); and/or the transformation technique to be used.
[0056] In some cases, one or more elements may be "operably linked"
to the above-described genes and/or to each other, by which is
generally meant that they are in a functional relationship with
each other. For instance, a promoter is considered "operably
linked" to a coding sequence if said promoter is able to initiate
or otherwise control/regulate the transcription and/or the
expression of a coding sequence (in which said coding sequence
should be understood as being "under the control of" said
promoter). Generally, when two nucleotide sequences are operably
linked, they will be in the same orientation and usually also in
the same reading frame. They will usually also be essentially
contiguous, although this may also not be required. In some cases,
the optional further elements of the genetic construct(s) used in
the invention may be such that they are capable of providing their
intended biological function in the intended host cell or host
organism. For instance, a promoter, enhancer or terminator may be
"operable" in the intended host cell or host organism, by which is
meant that (for example) the promoter should be capable of
initiating or otherwise controlling/regulating the transcription
and/or the expression of a nucleotide sequence (e.g. a coding
sequence) to which it is operably linked (as defined above). Such a
promoter may be a constitutive promoter or an inducible promoter,
and may also be such that it (only) provides for expression in a
specific stage of development of the host cell or host organism,
and/or such that it (only) provides for expression in a specific
cell, tissue, organ or part of a multicellular host organism.
[0057] A selection marker may be chosen such that it allows (e.g.,
under appropriate selection conditions) host cells and/or host
organisms that have been (successfully) transformed with the
nucleotide sequence of the invention to be distinguished from host
cells/organisms that have not been (successfully) transformed. Some
preferred, but non-limiting examples of such markers are genes that
provide resistance against antibiotics (such as kanamycin or
ampicillin), genes that provide for temperature resistance, or
genes that allow the host cell or host organism to be maintained in
the absence of certain factors, compounds and/or (food) components
in the medium that are essential for survival of the
non-transformed cells or organisms.
[0058] A leader sequence may be chosen such that, in the intended
host cell or host organism, it allows for the desired
post-translational modifications, and/or such that it directs the
transcribed mRNA to a desired part or organelle of a cell. A leader
sequence may also allow for secretion of the expression product
from said cell. As such, the leader sequence may be any pro-, pre-,
or prepro-sequence operable in the host cell or host organism.
[0059] An expression marker or reporter gene may be chosen such
that, in the host cell or host organism, it allows for detection of
the expression of (a gene or nucleotide sequence present on) the
genetic construct. An expression marker may optionally also allow
for the localization of the expressed product, e.g. in a specific
part or organelle of a cell and/or in (a) specific cell(s),
tissue(s), organ(s) or part(s) of a multicellular organism. Such
reporter genes may also be expressed as a protein fusion with the
amino acid sequence of the invention. Some preferred, but
non-limiting examples include fluorescent proteins such as GFP. The
genetic constructs of the invention may generally be provided by
suitably linking the nucleotide sequence(s) of the invention to the
one or more further elements described above, for example using the
techniques described in the general handbooks such as Sambrook et
al., mentioned above.
[0060] Often, the genetic constructs will be obtained by inserting
a nucleotide sequence in a suitable (expression) vector known per
se. Some preferred, but non-limiting examples of suitable
expression vectors include: vectors for expression in bacterial
cells: pET vectors (Novagen) and pQE vectors (Qiagen); and vectors
for expression in yeast or other fungal cells: pYES2 (Invitrogen)
and Pichia expression vectors (Invitrogen).
[0061] The nucleotide sequences and/or genetic constructs may be
used to transform a host cell. The host cell may be any suitable
(prokaryotic or eukaryotic) cell or cell line, for example: a
bacterial strain, including, but not limited to, E. coli, Bacillus.
Streptomyces and Pseudomonas; and a yeast cell, including, but not
limited to, Kluyveromyces or Saccharomyces.
[0062] In one aspect, a formulation of the invention may be used to
control uremic toxins within the subject at an acceptable level.
The formulation may be used to treat a subject having or at risk
for uremic toxicity, as previously described. In some cases, the
formulation may used independently. For example, a formulation of
the invention may be given to a subject in lieu of dialysis, or
before a subject has reached a state where dialysis is required.
For instance, in a subject having or at risk for renal failure, a
formulation of the invention may be given to the subject to control
uremic toxin levels within the subject, to reduce the need for
dialysis or other forms of treatment, etc. As another example, in a
subject being treated using chemotherapy, a formulation of the
invention may be given to the subject to control uremic toxin
levels within the subject, for example, to prevent or at least
control uremic toxicity symptoms, or to supplement normal kidney
function. In other cases, the formulation may be used in
combination with other treatments or strategies for controlling
uremic toxins, such as dialysis, e.g., to supplement and/or enhance
such treatments. For example, the formulation may be given
simultaneously with dialysis, before and/or after dialysis,
interspersed with dialysis, etc. For instance, on days where no
dialysis is performed, a subject may be given a formulation of the
invention, once a day, twice a day, once every other day, or at any
other suitable frequency, for example, three times a week, four
times a week, or five times a week. As a specific example, in a
subject where dialysis is to be performed three times a week, a
formulation of the invention may be given to the subject on the
four days of the week where no dialysis is performed.
[0063] As one example, a formulation of the invention may be used
in a subject as a replacement of dialysis (e.g., kidney dialysis),
or as a supplement to dialysis. Those of ordinary skill in the art
will be able to identify suitable types of dialysis. For example,
in kidney dialysis, blood is typically pumped from a subject
through a semiporous membrane that allows urea and salt transport
across the membrane to occur, but does not allow passage of red
blood cells, white blood cells, and other important blood
components therethrough. Examples of dialysis techniques include
hemodialysis and peritoneal dialysis, for instance, continuous
ambulatory peritoneal dialysis ("CAPD"). Dialysis can be performed,
for example, using external machines or portable devices. By
supplying the subject with the compositions of the invention, the
time between dialysis treatments may be extended in some cases. For
instance, the subject may be able to prolong the time between
dialysis treatments to at least three days, at least four days, at
least five days, at least seven days, or at least ten days or more
in some cases.
[0064] As another example, a formulation of the invention may be
used in combination with other small-molecule drugs, and/or other
enzymes such as urate oxidase. As yet another example, a
formulation of the invention may be used in combination with
treatments that allow inhibition of uric acid synthesis, increased
uric acid excretion, and/or enzymatic degradation. For instance,
for the treatment of gout, a form of inflammatory arthritis in
which urate deposits are common in and around the joints and
characterizable by elevated levels of uric acid in the blood, the
most often used drugs include allopurinol and probenecid.
Allopurinol can interfere with uric acid synthesis by inhibiting
xanthine oxidase, an enzyme which is required in the formation of
uric acid, and probenecid can increase uric acid excretion by
inhibiting the reabsorption of urate in the renal tubules.
Rasburicase, a form of recombinant urate oxidase cloned from
Aspergillus flavus fungi, is an example of a treatment for
chemotherapy-induced hyperuricaemia. This enzyme, which may be
given by intravenous injection, can degrade the uric acid via
conversion of uric acid to allantoin, which is 5-10 times more
soluble than uric acid.
[0065] Another aspect of the present invention provides a method of
orally administering any of the above-described formulations to a
subject. After oral delivery, the formulation may stay within the
gastrointestinal system until being eliminated by the subject,
typically after roughly twenty-four hours after administration. The
formulation may be active during part or all of its transit through
the gastrointestinal system, for example, within the large and/or
small intestine.
[0066] When administered, the formulations of the invention are
applied in a therapeutically effective, pharmaceutically acceptable
amount as a pharmaceutically acceptable formulation. As used
herein, the term "pharmaceutically acceptable" is given its
ordinary meaning. Pharmaceutically acceptable formulations are
generally compatible with other materials of the formulation and
are not generally deleterious to the subject. Any of the
formulations of the present invention may be administered to the
subject in a therapeutically effective dose. A "therapeutically
effective" or an "effective" as used herein means that amount
necessary to at least partially decrease the concentrations of one
or more uremic toxins within the bloodstream of the subject. When
administered to a subject, effective amounts will depend on the
particular condition being treated and the desired outcome. A
therapeutically effective dose may be determined by those of
ordinary skill in the art, for instance, employing factors such as
those further described below and using no more than routine
experimentation.
[0067] In administering the formulations of the invention to a
subject, dosing amounts, dosing schedules, routes of
administration, and the like may be selected so as to affect known
activities of these formulations. Dosages may be estimated based on
the results of experimental models, optionally in combination with
the results of assays of formulations of the present invention.
Dosage may be adjusted appropriately to achieve desired drug
levels, local or systemic, depending upon the mode of
administration. The doses may be given in one or several
administrations per day. In the event that the response of a
particular subject is insufficient at such doses, even higher doses
may be employed to the extent that subject tolerance permits.
Multiple doses per day are also contemplated in some cases.
[0068] The dose of the formulations to the subject may be such that
a therapeutically effective amount of the active compound (enzyme
and/or cell, etc.) reaches the intestines of the subject. The
dosage may be given in some cases at the maximum amount while
avoiding or minimizing any potentially detrimental side effects
within the subject. The dosage of the formulation that is actually
administered is dependent upon factors such as the final
concentration desired, the efficacy of the formulation, the
longevity of the formulation within the subject, the timing of
administration, the effect of concurrent treatments (e.g., as in a
cocktail, or in conjunction with dialysis), etc. The dose delivered
may also depend on conditions associated with the subject, and can
vary from subject to subject in some cases. For example, the age,
sex, weight, size, environment, physical conditions, or current
state of health of the subject may also influence the dose
required. Variations in dosing may occur between different
individuals or even within the same individual on different days.
It may be preferred that a maximum dose be used, that is, the
highest safe dose according to sound medical judgment. Preferably,
the dosage form is such that it does not substantially
deleteriously affect the subject.
[0069] Formulations suitable for oral administration may be
presented as discrete units such as hard or soft capsules, pills,
cachettes, tablets, troches, or lozenges. Other oral formulations
suitable for use with the invention include solutions or
suspensions in aqueous or non-aqueous liquids such as a syrup, an
elixir, or an emulsion. In another set of embodiments, the
formulation may be used to fortify a food or a beverage.
[0070] In certain embodiments of the invention, a formulation can
be combined with a suitable pharmaceutically acceptable carrier,
for example, as incorporated into a liposome, incorporated into a
polymer release system, or suspended in a liquid, e.g., in a
dissolved form or a colloidal form. In general, pharmaceutically
acceptable carriers suitable for use in the invention are
well-known to those of ordinary skill in the art. As used herein, a
"pharmaceutically acceptable carrier" refers to a non-toxic
material that does not significantly interfere with the
effectiveness of the biological activity of the active compound(s)
to be administered (e.g., enzymes, cells, etc.), but is used as a
formulation ingredient, for example, to stabilize or protect the
active compound(s) within the formulation before use. The term
"carrier" denotes an organic or inorganic ingredient, which may be
natural or synthetic, with which one or more active compounds of
the invention are combined to facilitate the application of the
formulation. The carrier may be co-mingled or otherwise mixed with
one or more enzymes and/or cells, and with each other, in a manner
such that there is no interaction which would substantially impair
the desired pharmaceutical efficacy. The carrier may be either
soluble or insoluble, depending on the application. Examples of
well-known carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylase, natural and modified
cellulose, polyacrylamide, agarose and magnetite. The nature of the
carrier can be either soluble or insoluble. Those skilled in the
art will know of other suitable carriers, or will be able to
ascertain such, using only routine experimentation.
[0071] In some embodiments, the formulations of the invention
include pharmaceutically acceptable carriers with formulation
ingredients such as salts, carriers, buffering agents, emulsifiers,
diluents, excipients, chelating agents, fillers, drying agents,
antioxidants, antimicrobials, preservatives, binding agents,
bulking agents, silicas, solubilizers, or stabilizers that may be
used with the active compound. For example, if the formulation is a
liquid, the carrier may be a solvent, partial solvent, or
non-solvent, and may be aqueous or organically based. Examples of
suitable formulation ingredients include diluents such as calcium
carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or
sodium phosphate; granulating and disintegrating agents such as
corn starch or alginic acid; binding agents such as starch, gelatin
or acacia; lubricating agents such as magnesium stearate, stearic
acid, or talc; time-delay materials such as glycerol monostearate
or glycerol distearate; suspending agents such as sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone; dispersing or wetting agents such as lecithin
or other naturally-occurring phosphatides; thickening agents such
as cetyl alcohol or beeswax; buffering agents such as acetic acid
and salts thereof, citric acid and salts thereof, boric acid and
salts thereof, or phosphoric acid and salts thereof, or
preservatives such as benzalkonium chloride, chlorobutanol,
parabens, or thimerosal. Suitable carrier concentrations can be
determined by those of ordinary skill in the art, using no more
than routine experimentation. Those of ordinary skill in the art
will know of other suitable formulation ingredients, or will be
able to ascertain such, using only routine experimentation.
[0072] Preparations include sterile aqueous or nonaqueous
solutions, suspensions and emulsions Aqueous carriers include
water, alcoholic/aqueous solutions, or emulsions or suspensions,
including saline and buffered media. Preservatives and other
additives may also be present such as, for example, antimicrobials,
antioxidants, chelating agents and inert gases and the like. Those
of skill in the art can readily determine the various parameters
for preparing and formulating the formulations of the invention
without resort to undue experimentation.
[0073] In some embodiments, the present invention includes the step
of bringing a formulation of the invention into association or
contact with a suitable carrier, which may constitute one or more
accessory ingredients. The final formulations may be prepared by
any suitable technique, for example, by uniformly and intimately
bringing the formulation into association with a liquid carrier, a
finely divided solid carrier or both, optionally with one or more
formulation ingredients as previously described, and then, if
necessary, shaping the product.
[0074] In some embodiments, the formulations of the present
invention may be present as a pharmaceutically acceptable salt. The
term "pharmaceutically acceptable salts" includes salts of the
formulation, prepared in combination with, for example, acids or
bases, depending on the particular compounds found within the
formulation and the treatment modality desired. Pharmaceutically
acceptable salts can be prepared as alkaline metal salts, such as
lithium, sodium, or potassium salts; or as alkaline earth salts,
such as beryllium, magnesium or calcium salts. Examples of suitable
bases that may be used to form salts include ammonium, or mineral
bases such as sodium hydroxide, lithium hydroxide, potassium
hydroxide, calcium hydroxide, magnesium hydroxide, and the like.
Examples of suitable acids that may be used to form salts include
inorganic or mineral acids such as hydrochloric, hydrobromic,
hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, phosphorous acids and the like. Other
suitable acids include organic acids, for example, acetic,
propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic,
fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,
citric, tartaric, methanesulfonic, glucuronic, galacturonic,
salicylic, formic, naphthalene-2-sulfonic, and the like. Still
other suitable acids include amino acids such as arginate,
aspartate, glutamate, and the like.
[0075] The present invention also provides any of the
above-mentioned formulations useful for the treatment of uremic
toxins in a subject, optionally including instructions for use of
the formulation for such treatments. That is, the kit can include a
description of use of the formulation for participation in any
biological or chemical mechanism disclosed herein associated with
uremic toxicity. The kit can include a description of use of the
formulations as discussed herein. The kit can also include
instructions for use of a combination of two or more formulations
of the invention. Instructions also may be provided for
administering the drug by any suitable technique, as described
above. A "kit," as used herein, defines a package including any one
or a combination of the formulations of the invention, and/or
homologs, analogs, derivatives, enantiomers and functionally
equivalent formulations thereof, and may also include instructions
of any form that are provided in connection with the formulation in
a manner such that a clinical professional will clearly recognize
that the instructions are to be associated with the specific
formulation, for example, as described above. The kits described
herein may also contain, in some cases, one or more containers,
which can contain formulations such as those described above. The
kits also may contain instructions for mixing, diluting, and/or
administrating the formulation. The kits also can include other
containers with one or more solvents, surfactants, preservative
and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose)
as well as containers for mixing, diluting or administering the
formulation to the subject.
[0076] The formulations of the kit may be provided as any suitable
form, for example, as liquid solutions or as dried powders. When
the formulation provided is a dry powder, the formulation may be
reconstituted by the addition of a suitable solvent, which may also
be provided. In embodiments where liquid forms of the formulation
are used, the liquid form may be concentrated or ready to use. The
solvent will depend on the formulation and the mode of use or
administration. Suitable solvents for drug formulations are well
known and are available in the literature.
[0077] The kit, in one set of embodiments, may comprise a carrier
that is compartmentalized to receive in close confinement one or
more container means such as vials, tubes, and the like, each of
the compartments comprising one of the separate elements to be used
in the method. For example, one of the compartments may comprise a
positive control for an assay. Additionally, the kit may include
containers for other components of the formulations, for example,
buffers useful in the assay.
[0078] The invention also involves, in some embodiments, the
promotion of the treatment of uremic toxins in a subject according
to any of the techniques and formulations described herein. As used
herein, "promoted" includes all methods of doing business
including, but not limited to, methods of selling, advertising,
assigning, licensing, contracting, instructing, educating,
researching, importing, exporting, negotiating, financing, loaning,
trading, vending, reselling, distributing, replacing, or the like
that can be associated with the methods and formulations of the
invention, e.g., as discussed herein. Promoting may also include,
in some cases, seeking approval from a government agency to sell a
formulation of the invention for medicinal purposes. Methods of
promotion can be performed by any party including, but not limited
to, businesses (public or private), contractual or sub-contractual
agencies, educational institutions such as colleges and
universities, research institutions, hospitals or other clinical
institutions, governmental agencies, etc. Promotional activities
may include instructions or communications of any form (e.g.,
written, oral, and/or electronic communications, such as, but not
limited to, e-mail, telephonic, facsimile, Internet, Web-based,
etc.) that are clearly associated with the invention. As used
herein, "instructions" can define a component of instructional
utility (e.g., directions, guides, warnings, labels, notes, FAQs
("frequently asked questions"), etc., and typically involve written
instructions on or associated with the formulation and/or with the
packaging of the formulation, for example, use or administration of
the formulation, e.g., in the treatment of uremic toxins in a
subject. Instructions can also include instructional communications
in any form (e.g., oral, electronic, digital, optical, visual,
etc.), provided in any manner such that a user will clearly
recognize that the instructions are to be associated with the
formulation, e.g., as discussed herein.
[0079] The following examples are intended to illustrate certain
aspects of certain embodiments of the present invention, but do not
exemplify the full scope of the invention.
EXAMPLE 1
[0080] A uremic test solution containing metabolites at levels
comparable to those found in the blood of ESRD patients was
prepared. The solution included urea (100 mg/dl) (Fisher
Scientific), uric acid (10 mg/dl) (Sigma), and creatinine (10
mg/dl) (Fluka), dissolved in saline. The enzymatic reduction of
these metabolites was measured using spectrophotometric assay
(using kits from Sigma), at 535 nm, 686 nm, and 555 nm
respectively. All experiments were conducted at 37.degree. C. The
solutions were maintained in an orbital shaker, and samples were
stored in a -80.degree. C. freezer between collection and
measurement, except for the final time point.
[0081] To evaluate the capacity of unencapsulated enzymes to
degrade the uremic toxins in the test solution, an
enzyme-containing solution was prepared. The enzyme-containing
solution included 800 units (50 mg) of urease from jack beans
(solid powder), 10 units (0.6 mg) of uricase from Arthrobacter
globiformis (aqueous solution) and 40 units (0.14 mg) of
creatininase (aqueous solution) from Flavobacterium (Sigma). The
enzyme-containing solution was added to the test solution, and
changes in the metabolite concentration were monitored by
spectrophotometric assay. Samples were taken every 4 hours from 0
hours to 12 hours, and again at 24 hours. These experiments were
performed in triplicate. Dose-response trials were also conducted,
in which the concentrations of the enzymes in solution were reduced
by 10-fold and 100-fold and evaluated under the same conditions.
These experiments were performed in duplicate.
[0082] To evaluate the effectiveness of encapsulated enzymes,
identical amounts of enzyme were utilized as in the unencapsulated
trials (i.e., 800 units of urease, 10 units of uricase, and 40
units of creatininase), but the enzymes were mixed with 5 ml of
1.8% alginate solution (low viscosity alginic acid, Sigma);
therefore, these in vitro experiments maintained a similar
substrate-to-enzyme ratio for both unencapsulated and encapsulated
enzymes. 5 ml of a mixture of alginate and enzymes was extruded
through a 300 micrometer nozzle into a 1.4% calcium chloride bath
(Sigma) to produce capsules containing encapsulated enzymes. All
three enzymes were incorporated into each alginate capsule, thus
forming a combination capsule, using an automated vibrational (f
=1500 Hz) encapsulating method (Inotech). Alginate beads that were
substantially spherical were produced (see FIG. 2C), which measured
roughly 0.6 mm in diameter. The capsules were then added to 100 ml
of uremic test solution, with an estimated dilutional effect of 5%.
As in the unencapsulated trials, samples from the test solution
were taken in duplicate at intermediate points every 4 hours from 0
hours to 12 hours, and finally at 24 hours. Each sample was tested
for metabolite concentration by assay. This procedure was also
repeated in triplicate, measuring metabolite concentration at 0
hours and 24 hours. In addition, as a control, in one set of
experiments, empty alginate capsules were exposed to the uremic
test solution.
[0083] Results of these experiments showed that each enzyme
effectively lowered its respective toxin in the uremic test
solution, as shown in Table 1. In addition, the control (empty)
capsules had the expected dilutional effect, based on the volume of
alginate added to test solution, but did not otherwise show a
detectable alteration in any of the uremic toxins in the test
solution. Table 1 summarizes these results after 24 hours. The rate
of metabolite degradation by each of the encapsulated enzymes to
its respective toxin is shown in FIG. 4. This figure depicts the
effectiveness of metabolite degradation by a system in which all
three enzymes were present in capsules. FIG. 5A is based on
Michaelis-Menten kinetics and shows the substrate concentration of
urea versus the reaction velocity (change in concentration of urea
with respect to time) for the action of unencapsulated and
encapsulated urease on urea. The reaction velocity, V, was
calculated as the change in the substrate concentration (urea in
this case) over time, and plotted versus the substrate
concentration, S, averaged over the measurement interval. The
maximum velocity, V.sub.max, and the Michaelis constant, K.sub.m,
for the enzyme (urease in this case) was then determined. In
addition, the Michaelis-Menten equation was reformatted as a linear
Lineweaver-Burk double reciprocal plot of 1/V vs. 1/S in FIG. 5B,
where the expression 1/V.sub.o was calculated as
(K.sub.m/(V.sub.maxS) (1/V.sub.max). TABLE-US-00001 TABLE 1 Urea
Uric acid Creatinine Experiment Units of enzyme 800 10 40 #1 per
100 ml of test solution Number of trials 5 5 5 Percent 95 .+-. 1
>99 .+-. 0.2 59 .+-. 2 degradation in 24 hours Experiment Units
of enzyme 80 1 4 #2 per 100 ml of test solution Number of trials 2
1 2 Percent 13 .+-. 9 97 46 .+-. 13 degradation in 24 hours
Experiment Units of enzyme 8 0.1 0.4 #3 per 100 ml of test solution
Number of trials 2 1 2 Percent 9 .+-. 3 40 19 .+-. 7 degradation in
24 hours
[0084] Table 1 shows that these experiments reproducibly
demonstrated degradations of 95%, >99%, and 59%, respectively,
for urea, uric acid, and creatinine, as observed over 24 hours. The
degradation rates greatly decreased with dilution of enzyme
quantity. For example, with a 100-fold reduced dose of enzyme, the
amount of degradation was lowered to only 9%, 40%, and 19%,
respectively, for urea, uric acid, and creatinine. In further
experiments (data not shown), 5 ml of the encapsulated enzymes
effectively degraded 97% of the urea, nearly 100% of the uric acid,
and 70% of the creatinine within 24 hours in 100 ml of test
solution. Thus, these results show that these uremic enzymes, in
unencapsulated and encapsulated form, are able to efficiently
degrade metabolites. Additionally, no substantial difference in
efficacy between the unencapsulated and encapsulated enzyme was
found.
[0085] FIG. 5B is a Lineweaver-Burk plot of the reciprocal of
substrate concentration (urea in this case) versus the reciprocal
of reaction velocity, as derived from the Michaelis-Menten
equation. This figure demonstrates that the enzymatic behavior of
urease that was observed was consistent with theory. This figure
also shows that the observed urea degradation rates were nearly
identical with both the encapsulated and unencapsulated enzymes,
suggesting that the encapsulated enzymes are fully active within
the capsules, and that mass transfer across the capsules is not a
limiting factor.
[0086] These results may have important implications for a
supplemental therapy, involving a treatment format employing an
oral delivery composition (i.e., encapsulated) of enzymes for
metabolite degradation. A typical uremic patient has a total body
water volume of about 400 times what was employed in this example.
Thus, if 400 times more enzyme were to be delivered, the quantity
of enzymes would be approximately 2 g of urease, 0.2 g of uricase,
and 0.6 g of creatininase, or just under 3 g of enzymes in total
per day, a reasonable amount. Oral delivery composition of enzymes
can thus serve as a supplement for an existing therapy, such as
hemodialysis, or may be used independently, for example to delay
the starting point of dialysis treatment for patients with at least
some residual renal function and/or to prolong the time interval
between dialysis sessions for current patients.
EXAMPLE 2
[0087] In this example, certain experiments, alginate capsules
containing 30% barium sulfate (Mallinckrodt) by weight were
fabricated for delivery into rats in an in vivo system. These
capsules were otherwise identical to the capsules described
previously in Example 1. Barium sulfate was added for X-ray
visualization. Four Sprague-Dawley CD male rats weighing 250-300 g
(Charles River Laboratories) were fed .about.1 ml of the capsules
mixed with maple syrup by oral gavage. Full body X-rays were taken
at 0.5 hours, 2.5 hours, 4.5 hours, 6.5 hours, 10.5 hours, and 24
hours. The rats were briefly sedated for the X-ray photos with
isoflurane gas, and were allowed free access to food and water
throughout.
[0088] X-ray photographs of a rat, as shown in FIG. 6, show a
widespread dispersion of barium sulfate capsules throughout the
digestive system of the rat and a measurable residence time of at
least 12 hours in the GI tract. The X-ray photographs are from one
rat, but are representative of the four rats X-rayed after oral
delivery of the capsules.
[0089] A chemically-induced acute model of chronic renal failure
was used in this in vivo study. Several other methods of inducing
acute renal failure were investigated in this study including
nephrectomy and the injection of mercuric chloride (data not
shown). The chemically-induced renal failure model, based on
intramuscular injection of glycerin, was chosen for this example
due to its improved reliability and lack of technique dependence,
compared to the other methods. Glycerol injection causes local
tissue necrosis and the release of many soluble agents, and these
agents accumulate in the kidney, leading to kidney failure. Capsule
residence times were determined based on X-rays taken following
oral administration of the barium-alginate capsules to the rats. As
illustrated in FIG. 6, the initial bolus of capsules was
well-separated in the digestive system of the rats, and the
distribution of capsules reaches its maximum dispersion of up to
about 6.5 hours after delivery. The capsules were still prominent
at about 10.5 hours after gavage, but were located towards the
caudal part of the gut. At 24 hours, no traces of the capsules
could be seen. During the period the capsules were present in the
intestine, they were able to adsorb and degrade toxins, including
urea, uric acid, and creatinine. It was also shown that the
capsules were able to pass through the digestive system without
being substantially digested, i.e., the capsules were able to
"escape" digestion.
EXAMPLE 3
[0090] In this example, following overnight water deprivation with
free access to food, acute renal failure (ARF) was induced in
cohorts of Sprague-Dawley CD male rats weighing 250-300 g (Charles
River Laboratories) by intramuscular injection of hypertonic (50%)
glycerol solution (glycerin, Mallinckrodt) at a dose of 10 ml/kg
body weight, using methods similar to those described in Example 2.
Plasma urea, uric acid, and creatinine levels were measured at time
0 hours, 1 hour, 3 hours, 5 hours, and 24 hours after injection, in
both the lesioned rats and unlesioned control rats. Experiments
were conducted on groups consisting of four rats, where three of
the four received the injection, with one serving as a control.
Blood samples were collected from the tail and placed in
heparinized centrifuge tubes. The rats were briefly sedated during
sample collection with isoflurane gas. After centrifugation, the
samples were stored in the refrigerator and analyzed by assay after
the final time point.
[0091] Lesioned rats and controls were fed capsules containing all
three enzymes as described in the previous examples, in three
cohorts of four rats each. All cohorts received identical glycerol
injections. The first cohort received only the lesion, but no
capsules. Encapsulated enzymes were delivered to the second cohort,
and an oral sorbent (3 g/rat of ion exchange resin AG 50W-X8,
Bio-Rad) was administered in conjunction with the capsules to the
third cohort. In all instances of oral delivery, each rat received
roughly 100 mg of encapsulated enzymes.
[0092] Each cohort of four rats was deprived of water overnight
prior to glycerol injection. The capsules were administered
immediately after glycerol injection. For oral delivery of the
capsules, the rats were anesthetized using isoflurane gas, and
microcapsules suspended in maple syrup were fed through an
orogastric tube. The rats were subsequently allowed free access to
food and water. The rats were again sedated for blood collection
purposes taken via tail bleeding at time 0 hours, 1 hour, 3 hours,
5 hours, and 24 hours. The samples were collected in heparinized
tubes, centrifuged, and the plasma analyzed to determine urea, uric
acid, and creatinine concentrations by standard spectrophotometric
assay. At the completion of the study, the rats were sacrificed in
a CO.sub.2 chamber. All animal experiments were conducted according
to institutionally approved written protocols. The results are
reported as mean.+-.standard error (SEM). Statistical significance
was evaluated using Student's t test.
[0093] The results of these in vivo experiments are summarized in
Table 2 and plotted for urea in FIG. 7. These experiments showed
that acute renal failure was induced in the rats via intramuscular
glycerol injection at a dose of 10 ml/kg. Over a period of 24
hours, measured concentrations rose by eightfold for urea,
fifteen-fold for uric acid, and threefold for creatinine. Thus, as
demonstrated in Table 2, the encapsulated enzyme therapy
significantly decreased the magnitude of these metabolites within
the bloodstreams of the rats. TABLE-US-00002 TABLE 2 Solute
concentration 24 hours after lesion (mg/dl) Treatment N Urea Uric
acid Creatinine Unlesioned 4 35 .+-. 2 0.2 .+-. 0.3 1.4 .+-. 0.1
controls Lesion only 4 269 .+-. 9 3.3 .+-. 1 4.8 .+-. 0.9 Lesion +
4 163 .+-. 35 0.8 .+-. 0.4 2.6 .+-. 0.2 encapsulated enzymes Lesion
+ 4 85 .+-. 20 0.8 .+-. 0.3 3.5 .+-. 0.7 encapsulated enzymes + ion
exchange
[0094] Administration of the encapsulated enzymes along with an ion
exchange resin sorbent decreased the severity of azotemia (i.e.,
the effect of elevated or toxic levels of urea and other uremic
toxins) considerably. As shown in FIG. 7, rats receiving
encapsulated enzyme therapy plus a sorbent displayed lower urea
levels than those receiving only encapsulated enzymes, i.e., a 70%
vs. a 40% decrease in urea concentration relative to lesioned but
untreated cohort. The sorbent is believed to be beneficial to the
rate of urea degradation, not for uric acid and creatinine, since
ammonia is not formed from their degradation (see reactions in FIG.
1).
[0095] The results in FIG. 7 show that addition of an ion exchange
resin, which serves as a sorbent able to remove high concentrations
of ammonia in the GI tract, enhances the efficiency of urea
removal. Urease degrades urea into ammonia and carbon dioxide and
the ammonia needs to be removed, since it could potentially diffuse
from the intestine to the liver and be converted back to urea.
However, the resin is not required for degradation of uric acid and
creatinine (as shown by the data in Table 2). Alternative
approaches to ammonia uptake include metabolism to amino acids via
glutamine synthetase, and/or the use of a sorbent, such as carbon,
oxystarch, and zirconium phosphate.
[0096] FIG. 8 shows a comparison of the amount of major uremic
toxins remaining after one hemodialysis session, with encapsulated
enzyme therapy in vitro (Example 1) and in vivo. This figure is
useful as an assessment of what an embodiment of invention can
remove in 24 hours verses clinically-accepted hemodialysis. On
average, after a typical hemodialysis treatment, a subject retains
about 35% of the predialysis urea; the corresponding numbers for
uric acid and creatinine are about 60% and about 65%. In contrast,
for the experiments described in Examples 1 and 3, the
corresponding percentage retentions (relative to controls) were 3%,
0%, and 30%, respectively, for an in vitro, and, the percentage
retention (relative to controls) was 60%, 25%, and 53% for an in
vivo system.
EXAMPLE 4
[0097] In this example, microencapsulated enzymes and/or bacteria
were orally administered which take up and degrade urea, uric acid,
and creatinine while passing through the intestine. The targeted
solutes are generated throughout the body, especially the liver,
and diffuse into the capsules where they are degraded upon
conversion of: 1) urea into ammonia and carbon dioxide, 2) uric
acid into allantoin and hydrogen peroxide and 3) creatinine and
water into creatine (see FIG. 1). The degradation products are
excreted as waste.
[0098] This therapy may be useful in conjunction with hemodialysis
to decrease the frequency and duration of treatments, and/or to
provide improved outcomes for existing therapeutic regimens. In
some cases, this treatment may allow a postponement of the
initiation of dialysis in patients with early stage renal
failure.
[0099] In this example, the development and characterization of a
microcapsule containing a combination of both cells and enzymes is
described, for instance, when a. genetically modified cell
expressing creatinine is not readily available. In some cases, the
method of removing of non-protein nitrogen compounds can be
combined with the removal of other solutes, such as beta-2
(.beta.2) microglobulin for which bacterial degradation may not be
practical.
[0100] Enzymes purchased from a commercial source may be less
complicated in terms of biocompatibility, immune reactions, and/or
overall safety concerns. They may require much less effort in
storage, packaging, and transportation and are likely to need less
time to obtain FDA approval. However, bacteria can grow and expand
during their passage through the gut and the bacteria may be able
to metabolize some of the breakdown products from the enzymatic
reaction in certain cases. Bacteria are readily available, since
there are easy to grow quickly in large amounts, and less expensive
than enzymes. Furthermore, bacteria may metabolize uremic compounds
intracellularly, such that the metabolizing enzymes are protected
from the external environment.
[0101] Genetically modified E. coli DH 5 cells expressing urease
and E. Coli JM109 cells expressing uricase were prepared using
techniques known to those of ordinary skill in the art.
Luria-Bertani ("LB") growth medium was used for cell cultivation
with a composition of 10 g/L sodium chloride (Sigma), 10 g/L
tryptone, and 5 g/L yeast (Difco). The cell concentration was
obtained by measurement of optical density at 600 nm according to
the formula 1 Optical Density unit (O.D.) =1.times.10.sup.6 cells.
Urease was obtained from jack beans, uricase from Arthrobacter
globiformis and creatininase from Flavobacterium (all from
Sigma).
[0102] A test solution consisting of metabolites at levels
comparable to those found in the blood of kidney failure patients
was prepared with urea (100 mg/dl) (Fisher Scientific), uric acid
(20 mg/dl) (Sigma), and creatinine (10 mg/dl) (Fluka). Throughout,
the reduction of the metabolites by the enzymes was measured by
spectrophotometric assay (using kits from Sigma, 535, 686, and
555). All experiments were conducted at 37.degree. C., solutions
were maintained in an orbital shaker, and samples were stored in a
-80.degree. C. freezer between collection and measurement, except
for the final time point.
[0103] For the kinetic experiments, 80 ml of each cell type were
grown in LB medium along with ampicillin (1 mg/dl) overnight in a
37.degree. C. orbital shaker. Just prior to capsule fabrication,
the cell density was measured and the cells were centrifuged to
form a pellet. Cells and enzyme were suspended in 5 ml of 1.8%
alginate solution (low viscosity alginic acid, Sigma), loaded into
a syringe and then extruded through a 300 micron diameter
vibrational nozzle (frequency =1500 Hz), and formed into solid
capsules upon contact with a 1.4% solution of calcium chloride
(Sigma). This protocol, described in more detail in the previous
examples, resulted in capsules with .about.600 microns diameter.
Each capsule contained known concentrations of the two bacteria
types as well as the enzyme creatininase.
[0104] For in vitro studies, 5 ml of alginate capsules
(approximately 50,000 capsules) containing 35 million urease cells,
20 million uricase cells and 40 units (0.14 mg) of creatininase
were added to 100 ml of the test solution. 1 ml samples from the
solution were taken at intermediate points every 4 hours from 0
hours to 12 hours and finally at 24 hours, and monitored for
changes in metabolite concentration by assay. For dose response
studies, the study was repeated but the quantities of cells were
reduced to 40% and 10% of the amounts used in the initial
experiments and the enzyme concentration was reduced by 10- and
100-fold.
[0105] For in vivo studies, three cohorts of rats received
identical intramuscular glycerol injection at a dose of 10 ml/kg
bodyweight of 50% solution to produce acute renal failure. The
first group of four rats, received only the lesion but no capsules,
thus serving as a measure of average solute concentration in this
form of acute renal failure. The second group of two rats received
an identical lesion, followed by administration of 2 ml of capsules
(Table 3, Formulation A) containing a mixture of cells and enzymes,
co-administered with cation exchange resin AG 50W-X8 (Bio-Rad). The
third group of two rats received the lesion, followed by
administration of 2 ml of capsules (Table 3, Formulation B)
containing a mixture of cells and creatininase. TABLE-US-00003
TABLE 3 Quantity included per 2 ml dose (.about.20,000 capsules)
Formulation A Formulation B # of cells containing urease gene 65
million 65 million # of cells containing uricase gene 45 million 45
million Urease (units) 1600 -- Uricase (units) 20 -- Creatininase
(units) 60 60 Ion exchange resin AG 50W-X8 3 g --
[0106] Except for the contents of the capsules, the overall
procedure was similar to the procedure described in Example 3. Each
cohort of rats was deprived of water overnight prior to glycerol
injection. Capsules were administered immediately after glycerol
injection. For oral delivery of the capsules, the rats were
anesthetized using isoflurane gas and microcapsules suspended in
maple syrup and fed through an orogastric tube. Rats were
subsequently allowed free access to food and water. Rats were again
sedated for blood collection. Small samples of 0.3 ml were
collected from the tail at 0 hours, 1 hour, 3 hours, 5 hours and 24
hours for urea analysis. Larger samples of 0.7 ml were collected at
0 hours and 5 hours for determination of, uric acid, and creatinine
concentration. All samples were collected in heparinized tubes,
centrifuged, and the plasma analyzed by standard spectrophotometric
methods. At the completion of the study, rats were sacrificed in a
CO.sub.2 chamber. All animal experiments were conducted according
to institutionally-approved written protocols.
[0107] FIG. 9 demonstrates the rate of in vitro degradation of urea
(FIG. 9A), uric acid (FIG. 9B), and creatinine (FIG. 9C) by
encapsulated cells and enzyme exposed to the test solution for 24
hours. The concentration of the test solution is plotted versus
time at varying capsule dosages. The graph demonstrates
effectiveness of metabolite degradation by a system in which cells
and enzymes were co-encapsulated. 5 ml of these capsules completely
cleared >99% of the urea, 100% of the uric acid and 58% of the
creatinine from 100 ml of a challenge solution formulated to model
the concentration of these solutes in a hemodialysis patient.
[0108] In FIGS. 9A-9C, the standard dose, represented as diamonds,
includes 5 ml of capsules (.about.50,000) containing an initial
concentration of 55 million cells (35 million E. coli DH5 and 20
million E. coli JM109 cells) and 40 units of creatininase (0.14 mg)
added to 100 ml of test solution. Squares and triangles,
respectively, represent 40% and 10% fewer cells and enzyme
encapsulated in the same volume of alginate. These data are based
upon single determinations. These figures demonstrate a clear dose
response effect, with degradation rates decreasing with the
quantity of cells and enzymes provided. The degradation rates
appeared to decrease with dilution of cell and enzyme quantity.
[0109] In the in vivo trials with Wistar rats, orally administered
capsules decreased the severity of azotemia, hyperuricemia and
elevated creatinine following chemical induction of acute renal
failure. FIG. 10 contains a bar graph plotting the ratio of solute
concentration at different time intervals in animals receiving
active capsules to unlesioned controls for two different capsule
formulations. Both capsule formulations, described in Table 3, were
highly effective for uric acid and creatinine degradation.
[0110] In FIG. 10, circulating urea, uric acid, and creatinine
concentrations were each measured at 24 hours post lesion and
capsule delivery for urea and 5 hours after for uric acid and
creatinine. The results are reported as the ratio of concentration
in treated rats to that in control rats. Two trials were performed:
one with Formulation A (Table 3), in which the capsules contained
cells and the enzymes urease, uricase, and creatininase as well as
an ion exchange resin and the second with Formulation B (Table 3),
in which the capsules contained only cells and creatininase. It was
found that delivery of Formulation A generally decreases the
severity of the hyperuricemic condition, lowers elevated creatinine
after 5 hours, and reduces azotemia after 24 hours. The results
after administration of Formulation B were similar for uric acid
and creatinine at 5 hours and showed no effect on azotemia at 24
hours. The graph represents data from eight rats: four controls,
and a total of four, two each in the two study groups.
[0111] These data thus show that in vivo, metabolite concentrations
were lowered from the elevated levels following induction of acute
renal failure. In addition, it was found that a cation exchange
resin enhanced urea removal, while having no impact upon uric acid
or creatinine removal. This is likely because free (unadsorbed)
ammonia could diffuse from the intestine to the liver where it
would be converted back to urea by a complex enzymatic pathway.
However, the sorbent did not affect uric acid or creatinine
degradation in this experiment, since ammonia was not a by-product
of the enzymatic degradation of these compounds.
[0112] These data show that a combination of modified cells and
enzymes, co-encapsulated in a single capsule and shown capable of
efficiently degrading the most abundant uremic toxins (urea, uric
acid, and creatinine) in vitro and lessening the elevation of the
concentration of metabolites in vivo. This therapy format takes
advantage of the intestinal tract as an effective route for the
degradation of uremic waste. No apparent interference or blockage
among the cells and enzymes was identified.
[0113] In the combination capsule described in this example, cells
and enzymes displayed no synergistic or antagonistic effects.
Creatininase was found to operate effectively in both the presence
of enzymes and cells; the normal rise in creatinine following
induction of renal failure was reduced by .about.30%-45%. This
method may thus be useful for the delivery of agents for which
genetically modified cells are not available or difficult to
create.
[0114] While several embodiments of the invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations or modifications is deemed to be within the
scope of the present invention. More generally, those skilled in
the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and/or claimed. The present invention is directed to each
individual feature, system, material and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials and/or methods, if such features,
systems, articles, materials and/or methods are not mutually
inconsistent, is included within the scope of the present
invention.
[0115] All definitions as used herein are solely for the purposes
of this disclosure. These definitions should not necessarily be
imputed to other commonly-owned patents and/or patent applications,
whether related or unrelated to this disclosure. The definitions,
as used herein, should be understood to control over dictionary
definitions, definitions in documents incorporated by reference,
and/or ordinary meanings of the defined terms.
[0116] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one act, the order of the acts of the method is not
necessarily limited to the order in which the acts of the method
are recited.
[0117] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," and the like are to
be understood to be open-ended, i.e., to mean including but not
limited to. Only the transitional phrases "consisting of" and
"consisting essentially of" shall be closed or semi-closed
transitional phrases, respectively, as set forth in the United
States Patent Office Manual of Patent Examining Procedures, Section
2111.03.
Sequence CWU 1
1
3 1 840 PRT Canavalia ensiformis 1 Met Lys Leu Ser Pro Arg Glu Val
Glu Lys Leu Gly Leu His Asn Ala 1 5 10 15 Gly Tyr Leu Ala Gln Lys
Arg Leu Ala Arg Gly Val Arg Leu Asn Tyr 20 25 30 Thr Glu Ala Val
Ala Leu Ile Ala Ser Gln Ile Met Glu Tyr Ala Arg 35 40 45 Asp Gly
Glu Lys Thr Val Ala Gln Leu Met Cys Leu Gly Gln His Leu 50 55 60
Leu Gly Arg Arg Gln Val Leu Pro Ala Val Pro His Leu Leu Asn Ala 65
70 75 80 Val Gln Val Glu Ala Thr Phe Pro Asp Gly Thr Lys Leu Val
Thr Val 85 90 95 His Asp Pro Ile Ser Arg Glu Asn Gly Glu Leu Gln
Glu Ala Leu Phe 100 105 110 Gly Ser Leu Leu Pro Val Pro Ser Leu Asp
Lys Phe Ala Glu Thr Lys 115 120 125 Glu Asp Asn Arg Ile Pro Gly Glu
Ile Leu Cys Glu Asp Glu Cys Leu 130 135 140 Thr Leu Asn Ile Gly Arg
Lys Ala Val Ile Leu Lys Val Thr Ser Lys 145 150 155 160 Gly Asp Arg
Pro Ile Gln Val Gly Ser His Tyr His Phe Ile Glu Val 165 170 175 Asn
Pro Tyr Leu Thr Phe Asp Arg Arg Lys Ala Tyr Gly Met Arg Leu 180 185
190 Asn Ile Ala Ala Gly Thr Ala Val Arg Phe Glu Pro Gly Asp Cys Lys
195 200 205 Ser Val Thr Leu Val Ser Ile Glu Gly Asn Lys Val Ile Arg
Gly Gly 210 215 220 Asn Ala Ile Ala Asp Gly Pro Val Asn Glu Thr Asn
Leu Glu Ala Ala 225 230 235 240 Met His Ala Val Arg Ser Lys Gly Phe
Gly His Glu Glu Glu Lys Asp 245 250 255 Ala Ser Glu Gly Phe Thr Lys
Glu Asp Pro Asn Cys Pro Phe Asn Thr 260 265 270 Phe Ile His Arg Lys
Glu Tyr Ala Asn Lys Tyr Gly Pro Thr Thr Gly 275 280 285 Asp Lys Ile
Arg Leu Gly Asp Thr Asn Leu Leu Ala Glu Ile Glu Lys 290 295 300 Asp
Tyr Ala Leu Tyr Gly Asp Glu Cys Val Phe Gly Gly Gly Lys Val 305 310
315 320 Ile Arg Asp Gly Met Gly Gln Ser Cys Gly His Pro Pro Ala Ile
Ser 325 330 335 Leu Asp Thr Val Ile Thr Asn Ala Val Ile Ile Asp Tyr
Thr Gly Ile 340 345 350 Ile Lys Ala Asp Ile Gly Ile Lys Asp Gly Leu
Ile Ala Ser Ile Gly 355 360 365 Lys Ala Gly Asn Pro Asp Ile Met Asn
Gly Val Phe Ser Asn Met Ile 370 375 380 Ile Gly Ala Asn Thr Glu Val
Ile Ala Gly Glu Gly Leu Ile Val Thr 385 390 395 400 Ala Gly Ala Ile
Asp Cys His Val His Tyr Ile Cys Pro Gln Leu Val 405 410 415 Tyr Glu
Ala Ile Ser Ser Gly Ile Thr Thr Leu Val Gly Gly Gly Thr 420 425 430
Gly Pro Ala Ala Gly Thr Arg Ala Thr Thr Cys Thr Pro Ser Pro Thr 435
440 445 Gln Met Arg Leu Met Leu Gln Ser Thr Asp Asp Leu Pro Leu Asn
Phe 450 455 460 Gly Phe Thr Gly Lys Gly Ser Ser Ser Lys Pro Asp Glu
Leu His Glu 465 470 475 480 Ile Ile Lys Ala Gly Ala Met Gly Leu Lys
Leu His Glu Asp Trp Gly 485 490 495 Ser Thr Pro Ala Ala Ile Asp Asn
Cys Leu Thr Ile Ala Glu His His 500 505 510 Asp Ile Gln Ile Asn Ile
His Thr Asp Thr Leu Asn Glu Ala Gly Phe 515 520 525 Val Glu His Ser
Ile Ala Ala Phe Lys Gly Arg Thr Ile His Thr Tyr 530 535 540 His Ser
Glu Gly Ala Gly Gly Gly His Ala Pro Asp Ile Ile Lys Val 545 550 555
560 Cys Gly Ile Lys Asn Val Leu Pro Ser Ser Thr Asn Pro Thr Arg Pro
565 570 575 Leu Thr Ser Asn Thr Ile Asp Glu His Leu Asp Met Leu Met
Val Cys 580 585 590 His His Leu Asp Arg Glu Ile Pro Glu Asp Leu Ala
Phe Ala His Ser 595 600 605 Arg Ile Arg Lys Lys Thr Ile Ala Ala Glu
Asp Val Leu Asn Asp Ile 610 615 620 Gly Ala Ile Ser Ile Ile Ser Ser
Asp Ser Gln Ala Met Gly Arg Val 625 630 635 640 Gly Glu Val Ile Ser
Arg Thr Trp Gln Thr Ala Asp Lys Met Lys Ala 645 650 655 Gln Thr Gly
Pro Leu Lys Cys Asp Ser Ser Asp Asn Asp Asn Phe Arg 660 665 670 Ile
Arg Arg Tyr Ile Ala Lys Tyr Thr Ile Asn Pro Ala Ile Ala Asn 675 680
685 Gly Phe Ser Gln Tyr Val Gly Ser Val Glu Val Gly Lys Leu Ala Asp
690 695 700 Leu Val Met Trp Lys Pro Ser Phe Phe Gly Thr Lys Pro Glu
Met Val 705 710 715 720 Ile Lys Gly Gly Met Val Ala Trp Ala Asp Ile
Gly Asp Pro Asn Ala 725 730 735 Ser Ile Pro Thr Pro Glu Pro Val Lys
Met Arg Pro Met Tyr Gly Thr 740 745 750 Leu Gly Lys Ala Gly Gly Ala
Leu Ser Ile Ala Phe Val Ser Lys Ala 755 760 765 Ala Leu Asp Gln Arg
Val Asn Val Leu Tyr Gly Leu Asn Lys Arg Val 770 775 780 Glu Ala Val
Ser Asn Val Arg Lys Leu Thr Lys Leu Asp Met Lys Leu 785 790 795 800
Asn Asp Ala Leu Pro Glu Ile Thr Val Asp Pro Glu Ser Tyr Thr Val 805
810 815 Lys Ala Asp Gly Lys Leu Leu Cys Val Ser Glu Ala Thr Thr Val
Pro 820 825 830 Leu Ser Arg Asn Tyr Phe Leu Phe 835 840 2 296 PRT
Schizosaccharomyces pombe 2 Met Ser Glu Thr Thr Tyr Val Lys Gln Cys
Ala Tyr Gly Lys Thr Leu 1 5 10 15 Val Arg Phe Met Lys Lys Asp Ile
Cys Pro Lys Thr Lys Thr His Thr 20 25 30 Val Tyr Glu Met Asp Val
Gln Ser Leu Leu Thr Gly Glu Leu Glu Glu 35 40 45 Ser Tyr Thr Lys
Ala Asp Asn Ser Ile Val Val Pro Thr Asp Thr Gln 50 55 60 Lys Asn
Thr Ile Tyr Val Phe Ala Lys Asn Asn Asp Val Ser Val Pro 65 70 75 80
Glu Val Phe Ala Ala Lys Leu Ala Lys His Phe Val Asp Lys Tyr Lys 85
90 95 His Ile His Gly Ala Ala Leu Asp Ile Thr Ile Thr Pro Trp Thr
Arg 100 105 110 Met Glu Val Gln Gly Lys Pro His Ser His Ser Phe Ile
Arg Asn Pro 115 120 125 Gly Glu Thr Arg Lys Thr His Val Val Phe Ser
Glu Gly Lys Gly Phe 130 135 140 Asp Val Val Ser Ser Leu Lys Asp Val
Leu Val Leu Lys Ser Thr Gly 145 150 155 160 Ser Gly Phe Thr Asn Phe
His Lys Cys Glu Phe Thr Thr Leu Pro Glu 165 170 175 Val Thr Asp Arg
Ile Phe Ser Thr Ser Ile Asp Cys Asn Tyr Thr Phe 180 185 190 Lys His
Phe Asp Thr Phe Glu Glu Leu Ala Gly Phe Asp Phe Asn Ser 195 200 205
Ile Tyr Glu Lys Val Lys Glu Ile Thr Leu Glu Thr Phe Ala Leu Asp 210
215 220 Asp Ser Glu Ser Val Gln Ala Thr Met Tyr Lys Met Ala Asp Thr
Ile 225 230 235 240 Ile Asn Thr Tyr Pro Ala Ile Asn Glu Val Tyr Tyr
Ala Leu Pro Asn 245 250 255 Lys His Tyr Phe Glu Ile Asn Leu Ala Pro
Phe Asn Ile Asp Asn Leu 260 265 270 Gly Ser Asn Cys Ser Leu Tyr Gln
Pro Gln Ala Tyr Pro Ser Gly Tyr 275 280 285 Ile Thr Cys Thr Val Ala
Arg Lys 290 295 3 258 PRT Arthrobacter sp. 3 Met Lys His Leu Ile
Ser Asn Met Thr Trp Asn Glu Tyr Gln Asp Lys 1 5 10 15 Val Asp Lys
Gly Phe Leu Ile Leu Pro Val Gly Ser Thr Glu Gln His 20 25 30 Gly
Pro His Leu Pro Leu Gly Val Asp Ala Val Ile Ser Thr Gln Phe 35 40
45 Ser Leu Ala Ile Ala Arg Glu Leu Asn Ala Ala Val Ala Pro Val Leu
50 55 60 Ser Tyr Gly Tyr Lys Ser Leu Pro Ala Ser Gly Gly Gly Pro
Met Phe 65 70 75 80 Pro Gly Thr Ile Asp Leu Lys Gly Ser Thr Leu Thr
Ser Leu Val Tyr 85 90 95 Asp Leu Leu Glu Glu Phe Ile Ala Asp Gly
Trp Lys Lys Ile Leu Ile 100 105 110 Phe Ser Ala His Phe Glu Asn Glu
Ala Phe Leu Ser Glu Ala Cys Asp 115 120 125 Leu Leu Leu Arg Asn Gln
Lys Glu Glu Phe Pro Lys Val Leu Ile Cys 130 135 140 Asn Trp Trp Asp
Asn Leu Ser Ala Glu Thr Met Ser Lys Val Phe Asp 145 150 155 160 Glu
Val Arg Phe Pro Gly Trp Ala Leu Glu His Ala Ala Ile Ser Glu 165 170
175 Thr Ser Leu Met Met His Phe Ser Pro Glu Leu Val Lys Glu Asp Leu
180 185 190 Ile Thr Asp Glu Gly Val Asn Asn Pro Pro Thr Tyr Gln Ser
Phe Pro 195 200 205 Pro Ser Lys Thr Leu Ile Pro Ala Ser Gly Cys Leu
His Ser Ala Tyr 210 215 220 Ser Ser Ser Ala Glu Lys Gly Lys Leu Ile
Ala Leu Asp Ala Thr Lys 225 230 235 240 Asn Ile Val Ser Phe Leu Ile
Lys Glu Phe Ser Leu Glu Met Val Pro 245 250 255 Ile Glu
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