U.S. patent application number 16/445922 was filed with the patent office on 2020-04-30 for crystallized oxalate decarboxylase and methods of use.
The applicant listed for this patent is Allena Pharmaceuticals, Inc. Invention is credited to Teresa G. Cachero, Danica Grujic, Margaret Ellen McGrath, Reena J. Patel, Aftab Rashid, Bhami C. Shenoy, John Shin, Lekai Zhang.
Application Number | 20200129599 16/445922 |
Document ID | / |
Family ID | 39721749 |
Filed Date | 2020-04-30 |
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United States Patent
Application |
20200129599 |
Kind Code |
A1 |
Shenoy; Bhami C. ; et
al. |
April 30, 2020 |
CRYSTALLIZED OXALATE DECARBOXYLASE AND METHODS OF USE
Abstract
Oxalate decarboxylase crystals, including stabilized crystals,
such as cross-linked crystals of oxalate decarboxylase, are
disclosed. Methods to treat a disorder associated with elevated
oxalate concentration using oxalate decarboxylase crystals are also
disclosed. Additionally disclosed are methods of producing protein
crystals.
Inventors: |
Shenoy; Bhami C.; (South
Grafton, MA) ; Cachero; Teresa G.; (Hingham, MA)
; Shin; John; (Burlington, MA) ; Zhang; Lekai;
(Watertown, MA) ; Rashid; Aftab; (Natick, MA)
; Grujic; Danica; (Boston, MA) ; Patel; Reena
J.; (Woburn, MA) ; McGrath; Margaret Ellen;
(Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allena Pharmaceuticals, Inc |
Newton |
MA |
US |
|
|
Family ID: |
39721749 |
Appl. No.: |
16/445922 |
Filed: |
June 19, 2019 |
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15790556 |
Oct 23, 2017 |
10369203 |
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16445922 |
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14843751 |
Sep 2, 2015 |
9821041 |
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14283794 |
May 21, 2014 |
9155785 |
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14843751 |
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13372274 |
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8741284 |
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60854540 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0034 20130101;
A61P 7/00 20180101; C07K 2299/00 20130101; A61M 1/16 20130101; A61P
43/00 20180101; A61M 2205/75 20130101; A61P 13/04 20180101; A61P
13/12 20180101; G01N 2333/988 20130101; A61K 38/00 20130101; A61P
15/00 20180101; A61P 13/02 20180101; A61P 9/10 20180101; A61P 1/14
20180101; A61K 38/51 20130101; A61P 9/00 20180101; A61P 1/00
20180101; A61P 1/18 20180101; C12Q 1/527 20130101; A61M 2202/0413
20130101; A61M 1/3687 20130101; A61P 1/04 20180101; A61P 7/08
20180101; C12Y 401/01002 20130101; A61P 1/16 20180101; A61P 13/00
20180101; C12N 9/88 20130101; A61P 1/12 20180101; A61P 19/00
20180101; A61P 29/00 20180101 |
International
Class: |
A61K 38/51 20060101
A61K038/51; A61M 1/36 20060101 A61M001/36; A61M 1/16 20060101
A61M001/16; A61K 9/00 20060101 A61K009/00; C12Q 1/527 20060101
C12Q001/527; C12N 9/88 20060101 C12N009/88 |
Claims
1. A pharmaceutical composition comprising an oxalate decarboxylase
crystal.
2.-33. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. application Ser.
No. 60/834,933, filed on Aug. 2,2006 and U.S. application Ser. No.
60/854,540, filed on Oct. 26, 2006. the contents of which are
hereby incorporated by reference in their entireties.
BACKGROUND
[0002] Oxalic acid is a dicarboxylic acid of the formula
HO.sub.2C--CO.sub.2H. Oxalic acid exists primarily as oxalate in
biological organisms, which is the salt form of oxalic acid.
Oxalate is found in foods, such as, e.g., spinach, rhubarb,
strawberries, cranberries, nuts, cocoa, chocolate, peanut butter,
sorghum, and tea. Oxalate is also a metabolic end product in humans
and other mammals. It is excreted by the kidneys into the urine.
When combined with calcium, oxalic acid produces an insoluble
product, calcium oxalate, which is the most prevalent chemical
compound found in kidney stones. Because mammals do not synthesize
enzymes that degrade oxalate, oxalate levels in an individual are
normally held in check by excretion and low absorption of dietary
oxalate. Elevated concentrations of oxalate are associated with a
variety of pathologies, such as primary hyperoxaluria, enteric
hyperoxaluria, and idiopathic hyperoxaluria. Leumann et al.,
Nephrol. Dial. Transplant. 14:2556-2558 (1999) and Earnest, Adv.
Internal Medicine 24:407-427 (1979). Increased oxalate can be
caused by consuming too much oxalate from foods, by hyperabsorption
of oxalate from the intestinal tract, and by abnormalities of
oxalate production. Hyperabsorption of oxalate in the colon and
small intestine can be associated with intestinal diseases,
including hyperabsorption caused by diseases of bile acid and fat
malabsorption; ileal resection; and, for example, by steatorrhea
due to celiac disease, exocrine pancreatic insufficiency,
intestinal disease, and liver disease.
[0003] Hyperoxaluria, or increased urinary oxalate excretion, is
associated with a number of health problems related to the deposit
of calcium oxalate in the kidney tissue (nephrocalcinosis) or
urinary tract (e.g., kidney stones, urolithiasis, and
nephrolithiasis). Calcium oxalate may also be deposited in, e.g.,
the eyes, blood vessels, joints, bones, muscles, heart and other
major organs, causing damage to the same. See. e.g., Leumann et
al., J. Am. Soc. Nephrol. 12:1986 1993 (2001) and Monico et al.,
Kidney International 62:392 400 (2002). The effects of increased
oxalate levels can appear in a variety of tissues. For example,
deposits in small blood vessels cause painful skin ulcers that do
not heal, deposits in bone marrow cause anemia, deposits in bone
tissue cause fractures or affect growth in children, and calcium
oxalate deposits in the heart cause abnormalities of heart rhythm
or poor heart function.
[0004] Existing methods to treat elevated oxalate levels are not
always effective and intensive dialysis and organ transplantation
may be required in many patients with primary hyperoxaluria.
Existing therapies for various hyperoxalurias include high-dose
pyridoxine, orthophosphate, magnesium, iron, aluminum, potassium
citrate, cholestyramine, and glycosaminoglycan treatment, as well
as regimes for adjusting diet and fluid intake, for dialysis, and
for surgical intervention, such as renal and liver transplantation.
These therapies (e.g., low-oxalate or low-fat diet, pyridoxine,
adequate calcium, and increased fluids), are only partially
effective and they may have undesirable adverse side effects, such
as the gastrointestinal effects of orthophosphate, magnesium, or
cholestyramine supplementation and the risks of dialysis and
surgery. Accordingly, methods that safely remove oxalate from the
body are needed. Moreover, methods that degrade oxalate to reduce
oxalate levels in a biological sample are advantageous over a
therapy, for example, that solely blocks absorption or increased
clearance of oxalate.
SUMMARY
[0005] The invention relates to crystals of oxalate decarboxylase
("OXDC") and cross-linked forms thereof ("CLEC") and their uses to
treat oxalate-associated disorders, e.g., hyperoxaluria. In one
embodiment, crystalline oxalate decarboxylase can be administered
to a mammal, e.g., orally or directly to the stomach, to reduce
oxalate levels and/or to reduce damage caused by calcium oxalate
deposits in the mammal. Additionally disclosed are methods of
producing protein crystals from cell extracts. Compositions, e.g.,
pharmaceutical compositions, including the crystals of oxalate
decarboxylase ("OXDC") and cross-linked forms thereof ("CLEC") are
also disclosed.
[0006] In one aspect, the invention provides cross-linked oxalate
decarboxylase crystals. The cross-linking agent can be
multifunctional, and in certain embodiments, the agent is a
bifunctional agent, such as glutaraldehyde. In certain embodiments,
the oxalate decarboxylase crystals are cross-linked with
glataraldahyde at a concentration that does not substantially alter
enzyme activity, e.g., at a concentration of at least about 0.02%
(w/v), in embodiments, the level of cross-linking of the oxalate
decarboxylase crystal is equivalent to that produced by treatment
with 0.02% (w/v) glutaraldehyde. The level of cross-linking can be
determined by methods known in the art or disclosed herein, e.g.,
determining the level of protein leaching, e.g., as disclosed in
Examples 10-11.
[0007] The invention farther provides oxalate decarboxylate
crystals, e.g., oxalate decarboxylate crystals that have a higher
activity, e.g., at least about 100%, 200%, 300%, 400% or 500%,
compared to the soluble oxalate decarboxylate.
[0008] The invention further provides a stabilized, e.g.,
cross-linked, oxalate decarboxylase crystal, wherein said
stabilized crystal retains an activity and/or stability, in acidic
conditions at least 2-, 3-fold higher than the activity and/or
stability retained by a soluble oxalate decarboxylase in similar
acidic conditions (e.g., an acidic pH of about 2 to 3). In
embodiments, the stabilized oxalate decarboxylase crystal is at
least 200%, 300%, 400% more active and/or stable than a soluble
oxalate decarboxylase in acidic conditions.
[0009] The invention farther provides a stabilized, e.g.,
cross-linked, oxalate decarboxylase crystal, wherein said
stabilized crystal retains an activity and/or stability, in the
presence of a protease, at least 2-, 3-fold higher than the
activity and/or stability retained by a soluble oxalate
decarboxylase in similar conditions. In embodiments, the stabilized
oxalate decarboxylase crystal is at least 200%, 300%, 400% more
active and/or stable than a soluble oxalate decarboxylase in the
presence of a protease. The protease can be chosen from one or more
of, pepsin, chymotrypsin or pancreatin. In embodiments, the
activity of the stabilized or soluble oxalate decarboxylase is
measured after exposing the stabilized crystal or soluble oxalate
decarboxylase to acidic conditions and/or a protease for a
predetermined length of time, e.g., at least one, two, three, four
or five hours as described in the Examples herein).
[0010] In a related aspect, the invention features a cross-linked,
oxalate decarboxylase crystal which is substantially active and
stable in variable pH conditions (e.g., about pH 2.5 or 3 to 7.5 or
8.5), and/or in the presence of a protease, e.g., a protease can be
chosen from one or more of, e.g., pepsin, chymotrypsin or
pancreatin. In embodiments, the cross-linked crystal retains an
activity at least 2-, 3-fold higher than the activity retained by a
soluble oxalate decarboxylase in acidic conditions (e.g., an acidic
pH of about 2 to 3) and in the presence of a protease, as described
herein. In other embodiments, the stabilized oxalate decarboxylase
crystal is at least 200%, 300%, 400% more stable than a soluble
oxalate decarboxylase in acidic conditions (e.g., an acidic pH of
about 2 to 3) and in the presence of a protease, as described
herein.
[0011] Compositions, e.g., pharmaceutical compositions, that
include the crystals and/or the cross-linked oxalate decarboxylase
crystals as described herein are also within the scope of the
invention.
[0012] In some embodiments, the crystals include oxalate
decarboxylase having a sequence identical or substantially
identical to an oxalate decarboxylase sequence found in a natural
source, such as a plant, bacterium and fungus, in particular from
Bacillus subtilis, Collybia velulipes or Flammulina velutipes,
Aspergillus niger, Pseudomonas sp. Synechocystis sp. Streptococcus
mutans, Trametes hirsute, Sclerotinia sclerotiorum, T. versicolor,
Postia placenta, Myrothecium verrucaria, Agaricus bisporus,
Methylobactetium extorquens, Pseudomonas oxalaticus, Rafstonia
eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibactenum
flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.
oxalativorans, Varlovorax paradoxus, Xanthobacter autotrophicus,
Aspergillus sp., Penicillium sp., and Mucor species. In other
embodiments, the oxalate decarboxylase is recombinantly
produced.
[0013] In one aspect, the invention provides a method of reducing
oxalate concentration in a subject by administering a composition,
e.g., a pharmaceutical composition, that includes oxalate
decarboxylase crystals, e.g., cross-linked oxalate decarboxylase
crystals, as disclosed herein. In one embodiment, the oxalate
decarboxylase crystals are stabilized by a cross-linking agent,
such as glutaraldchyde. Administration of the composition can cause
a reduction of oxalate concentration by at least 10%. at least 20%,
at least 30%, or at least 40% or more. In some embodiments, the
composition is administered orally or via an extracorporeal device.
In one embodiment, the extracorporeal device is a catheter, e.g., a
catheter coated with oxalate decarboxylase crystals. In other
embodiments, the composition is administered as a suspension, dry
powder, capsule, or tablet. In one embodiment, the method of
reducing oxalate concentration in a mammal includes a step of
assaying the oxalate concentration in a biological sample of the
mammal, such as a urine, blood, plasma, or serum sample.
[0014] In another aspect, the invention provides a method of
treating, prevolting, and/or slowing the progression of a disorder
associated with elevated oxalate concentrations in a mammal by
administering oxalate decarboxylase crystals and/or stabilized,
e.g., cross-linked, oxalate decarboxylase crystals to the mammal.
In one embodiment, the disorder associated with elevated oxalate
concentration is a kidney disorder, joint disorder, eye disorder,
liver disorder, gastrointestinal disorder, or pancreatic disorder,
in certain embodiments, the disorder is primary hyperoxaluria,
enteric hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol
poisoning, cystic fibrosis, inflammatory bowel disease,
urolithiasis, nephrolithiasis, chronic kidney disease,
hemodialysis, and gastrointestinal bypass.
[0015] In another aspect, the invention provides a composition,
e.g., a pharmaceutical composition, that includes oxalate
decarboxylase crystals, e.g., cross-linked oxalate decarboxylase
crystals (e.g., the crystals and/or cross-linked crystals, as
disclosed herein).
[0016] In yet another aspect, the invention provides a method of
treating a mammal by administering an effective amount of a
pharmaceutical composition that includes oxalate decarboxylase
crystals, e.g., cross-linked oxalate decarboxylase crystals (e.g.,
the crystals and/or cross-linked crystals, as disclosed
herein).
[0017] In another aspect, the invention provides methods of
producing protein crystals, e.g., enzyme crystals (e.g., oxalate
decarboxylase crystals), that include: providing a preparation of
cell extract or pellets/precipitate/solution containing the
protein, and crystallizing the protein from the preparations. In
embodiments, the methods include one or more of: culturing a
prokaryotic host cell culture expressing the protein; obtaining, a
preparation of pellets or extracts containing desired. protein;
solubilizing the preparation of pellets; allowing, protein crystals
to form, and/or further stabilizing the crystals by cross-linking.
Typically, the protein is expressed recombinantly. In embodiments,
the pellet preparation includes inclusion bodies.
[0018] In embodiments, the solubilization step includes adding to
the preparation of pellets one or more of: a solution comprising a
mild denaturant concentration (e.g., urea or guanidine
hydrochloride at a concentration, e.g., about 1 M to about 3M); a
solution comprising a high salt concentration, e.g., a salt chosen
from one or more of sodium chloride, potassium chloride, calcium
chloride, or other salts at a concentration of, e.g., about 0.3 to
about 0.8 M; a solution comprising a mild denaturant concentration
under basic conditions, e.g., at a pH of about 9 to about 12; or a
solution comprising a high denaturant concentration, e.g., about 4
M to about 8 M urea or guanidine hydrochloride.
[0019] In embodiments, the purifying step includes removing debris
from the pellets, e.g., by separating, e.g., by one or more
spinning or centrifugation steps, the solubilized preparation of
pellets and/or collecting the supernatant. The purifying step may,
optionally, further include passing the solubilized preparation of
pellets through ion exchange chromatography, and/or filtering the
solubilized preparation.
[0020] In embodiments, the crystallizing step includes
concentrating the purified protein, thereby forming crystallized
protein. In embodiments, the crystallized protein is obtained from
the supernatant collected after the separation step, e.g., after
one or more spinning or centrifugation steps. The crystallization
step can additionally include contacting the crystallized protein
with a cross-linking agent, e.g., across-linking agent disclosed
herein (e.g., glutaraldehyde). The concentration of crosslinking
agent used can be in the range of 0.01% to 20% w/v; typically,
0.02% to 10% w/v; more typically, 0.02%, 0.5% or 1% w/v.
[0021] In embodiments, the yield of the protein in the pellet
preparation is at least about 50%, 60%, 70%, 80% of the specific
protein found in the cell preparation from which the pellet
preparation is obtained. In other embodiments, the yield of the
solubilized protein is at least about 90%, 95% or higher of that
found in the pellet preparation. In yet other embodiments, the
yield of the crystallized protein is at least about 50%, 60%, 70%,
80% of that found in the pellet preparation.
[0022] The invention further provides protein crystals, e.g.,
enzyme crystals (e.g., oxalate decarboxylase crystals) produced by
the methods disclosed herein.
[0023] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description
below.
DESCRIPTION OF DRAWINGS
[0024] FIG. 1 is a graph showing the pH activity profile of soluble
oxalate decarboxylase ("Soluble"), oxalate decarboxylase crystals
("Crystal"), and cross-linked oxalate decarboxylase crystals
("CLEC").
[0025] FIG. 2 is a bar graph depicting urinary oxalate levels in
Sprague Dawley rats after administration of OXDC-CLEC. Each bar
represents a mean .+-.SE (standard error). The asterisks indicate a
significant difference between the control group and three
treatment groups at p<0.05 at the time point, using a two tailed
Student t-test. FIG. 3 is a bar graph depicting the effect of
OXDC-CLEC on reduction of urinary oxalate levels in ethylene
glycol-challenged AGT1 knock-out (KO) mice. Each bar represents the
mean SE. Asterisks indicate a significant difference between the
control group and three treatment groups at p<0.05 at the time
point, calculated using a two tailed Student's t-test.
[0026] FIG. 4 is a bar graph depicting the effect of OXDC-CLEC on
creatinine clearance in ethylene glycol-challenged AGT1 KO mice.
Each bar represents the mean .+-.SE. Asterisks indicate a
significant difference between the control group and 80 mg
treatment group at p<0.05 by a two tailed Student's t-test.
[0027] FIGS. 5A-5C are images showing the prevention of calcium
oxalate deposits on kidney parenchyma following treatment with
OXDC-CLEC. FIG. 5A is a section of kidney parenchyma from EG
challenged mice treated with 80 mg OXDC-CLEC. FIGS. 5B and 5C arc
section of kidney parenchyma from the control group. The section if
FIG. 5B demonstrates moderate nephrocalcinosis and the section in
FIG. 5C demonstrates severe nephrocalcinosis. The dark patches are
calcium oxalate deposits (example indicated by a white arrow), and
the light patches are areas with interstitial fibrosis (examples
indicated by gray arrows).
[0028] FIG. 6 is a Kaplan-Meier survival plot comparing the
survival times of EG-challenged mice treated with three different
doses of OXDC-CLEC or a vehicle control.
[0029] FIG. 7 is a graph depicting the stability of soluble oxalate
decarboxylase ("Sol"), oxalate decarboxylase crystals ("XTAL"), and
cross-linked oxalate decarboxylase crystals ("CLEC") at low pH at
the time intervals indicated.
[0030] FIG. 8 is a graph depicting the stability of soluble oxalate
decarboxylase ("Sol"), oxalate decarboxylase crystals ("XTAL"), and
cross-linked oxalate decarboxylase crystals ("CLEC") in the
presence of pepsin at pH 3.0 at the time intervals indicated.
[0031] FIG. 9 is a graph depicting the stability of soluble oxalate
decarboxylase ("Sol"), oxalate decarboxylase crystals ("XTAL") and
cross-linked oxalate decarboxylase crystals ("CLEC") in the
presence of chymotrypsin at pH 7.5 at the time intervals
indicated.
[0032] FIG. 10 is a graph depicting the stability of soluble
oxalate decarboxylase ("Sol"), oxalate decarboxylase crystals
("XTAL"), and cross-linked oxalate decarboxylase crystals ("CLEC")
in the presence of simulated intestinal juice with pancreatin at pH
6.8 at the time intervals indicated.
DETAILED DESCRIPTION
[0033] The present invention is based, in part, on the discovery
that administering crystals of oxalate decarboxylase (OXDC) can
reduce the symptoms of hyperoxaluria in a mammal. Methods of
administering OXDC crystals to treat various oxalate-related
disorders are described herein. Additionally, OXDC crystals and
cross-linked crystals (CLECs) are provided, as are compositions
comprising and using the same. Additionally disclosed are methods
of producing large quantities of protein crystals from cell
extracts of a prokaryotic host cell.
[0034] Definitions. In order that the present invention may be more
readily understood, certain terms are first defined. Additional
definitions are set forth throughout the detailed description.
[0035] As used herein, a "biological sample" is biological material
collected from cells, tissues, organs, or organisms, for example,
to detect an analyte. Exemplary biological samples include a fluid,
cell, or tissue sample. Biological fluids include, for example,
serum, blood, plasma, saliva, urine, or sweat. Cell or tissue
samples include biopsy, tissue, cell suspension, or other specimens
and samples, such as clinical samples.
[0036] A "crystal" is one form of the solid state of matter,
comprising atoms arranged in a pattern that repeats periodically in
three dimensions (see, e.g., Barret, Structure of Metals, 2.sup.nd
ed., McGraw-Hill, New York (1952)). A crystal form of a
polypeptide, for example, is distinct from a second form the
amorphous solid state. Crystals display characteristic features
including shape, lattice structure, percent solvent, and optical
properties, such as, e.g., refractive index.
[0037] An "extracorporeal device" is a structure that is not within
the body for bringing a body fluid in contact with OXDC crystals in
the treatment of an individual. Preferably, an extracorporeal
device is a device used for dialysis, including kidney dialysis, a
device for continuous arteriovenous hemofiltration, an
extracorporeal membrane oxygenator, or other device used to filter
waste products from the bloodstream. Similarly, components of
devices to filter waste products are encompassed by the term,
including a tube, a porous material, or a membrane, for example. In
particular, an extracorporeal device may be a dialysis device. It
may also be a membrane of a dialysis device.
[0038] A "functional fragment" of OXDC is a portion of an OXDC
polypeptide that retains one or more biological activities of OXDC,
such as the ability to catalyze the decarboxylation of oxalate. As
used herein, a functional fragment may comprise terminal
truncations from one or both termini, unless otherwise specified.
For example, a functional fragment may have 1, 2, 4, 5, 6, 8, 10,
12, 15, or 20 or more residues omitted from the amino and/or
carboxyl terminus of an OXDC polypeptide. Preferably, the
truncations are not more than 20 amino acids from one or both
termini. A functional fragment may optionally be linked to one or
more heterologous sequences.
[0039] The term "individual" or "subject" refers to any mammal,
including but not limited to, any animal classified as such,
including humans, non human primates, primates, baboons,
chimpanzees, monkeys, rodents (e.g., mice, rats), rabbits, cats,
dogs, horses, cows, sheep, goats, pigs, etc.
[0040] The term "isolated" refers to a molecule that is
substantially free of its natural environment. For instance, an
isolated protein is substantially free of cellular material or
other proteins from the cell or tissue source from which it is
derived. The term refers to preparations where the isolated protein
is sufficiently pure to be administered as a therapeutic
composition, or at least 70% to 80% (w/w) pure, more preferably, at
least 80% 90% (w/w) pure, even more preferably, 90 to 95% pure;
and, most preferably, at least 95%, 96%, 97%, 98%, 99%, 99.5%,
99.8% or 100% (w/w) pure.
[0041] As used herein, the term "about" refers to up to 10% of the
value qualified by this term. For example, about 50 mM refers to 50
mM.+-.mM; about 4% refers to 4.+-.0.4%.
[0042] As used herein, "oxalate-associated disorder" refers to a
disease or disorder associated with pathologic levels of oxalic
acid or oxalate, including, but not limited to hyperoxaluria,
primarily hyperoxaluria, enteric hyperoxaluria, idiopathic
hyperoxaluria, ethylene glycol (oxalate) poisoning, idiopathic
urinary stone disease, renal failure (including progressive,
chronic, or end-stage renal failure), steatorrhoea, malabsorption,
ileal disease, vulvodynia, cardiac conductance disorders,
inflammatory bowel disease, cystic fibrosis, exocrine pancreatic
insufficiency, Crohn's disease, ulcerative colitis,
nephrocalcinosis, urolithiasis, and nephrolithiasis. Such
conditions and disorders may optionally be acute or chronic.
Oxalate-associated disorders associated with kidneys, bone, liver,
gastrointestinal tract, and pancreas are known in the art. Further,
it is well known that calcium oxalate can deposit in a wide variety
of tissues including, but not limited to, the eyes, blood vessels,
joints, bones, muscles, heart, and other major organs leading to a
number of oxalate-associated disorders.
[0043] "Oxalic acid" exists predominantly in its salt form, oxalate
(as salts of the corresponding conjugate base), at the pH of urine
and intestinal fluid (pK.sub.a11.23, pK.sub.a2=4.19). Earnest, Adv
Internal Medicine 24:407 427 (1979), The terms "oxalic acid" and
"oxalate" are used interchangeably throughout this disclosure.
Oxalate salts comprising lithium, sodium, potassium, and iron (II)
are soluble, but calcium oxalate is typically very poorly soluble
in water (for example, dissolving only to about 0.58 mg/100 ml at
18.degree. C. Earnest, Adv. Internal Medicine 24:407 427 (1979)).
Oxalic acid from food is also referred to as dietary oxalate.
Oxalate that is produced by metabolic processes is referred to as
endogenous oxalate. Circulating oxalate is the oxalate present in a
circulating body fluid, such as blood.
[0044] The terms "therapeutically effective dose," or
"therapeutically effective amount," refer to that amount of a
compound that results in prevention, delay of onset of symptoms, or
amelioration of symptoms of an oxalate-related condition, including
hyperoxaluria, such as primary hyperoxaluria or enteric
hyperoxaluria. A therapeutically effective amount will, for
example, be sufficient to treat, prevent, reduce the severity,
delay the onset, and/or reduce the risk of occurrence of one or
more symptoms of a disorder associated with elevated oxalate
concentrations. The effective amount can be determined by methods
well known in the art and as described in subsequent sections of
this description.
[0045] The terms "treatment," "therapeutic method," and their
cognates refer to treatment of an existing disorder and/or
prophylactic/preventative measures. Those in need of treatment may
include individuals already having a particular medical disorder,
as well as those at risk or having, or who may ultimately acquire
the disorder. The need for treatment is assessed, for example, by
the presence of one or more risk factors associated with the
development of a disorder, the presence or progression of a
disorder, or likely receptiveness to treatment of a subject having
the disorder. Treatment may include slowing or reversing the
progression of a disorder.
[0046] Oxalate decarboxylase. As used herein, oxalate decarboxylase
(OXDC) (EC 4.1.1.2) refers to an oxalate carboxy-lyase enzyme.
Oxalate decarboxylases are a group of enzymes known in the art
capable of catalyzing the molecular oxygen (O.sub.2) independent
oxidation of oxalate to carbon dioxide and formate according to the
following reaction:
HO.sub.2C--CO.sub.2H.fwdarw.1 CO.sub.2+HCOOH
[0047] Isoforms of oxalate decarboxylase, and glycoforms of those
isoforms, are included within this definition. OXDC from plants,
bacteria and fungi are encompassed by the term, including the true
oxalate decarboxylases from bacteria and fungi, such as Bacillus
subtilis, Collybia velutipes or Flammulina velutipes, Aspergillus
niger, Pseudomonas sp., Synechocystis sp., Streptococcus mutans,
Trametes hirsute, Selerotinia sclerotiorum, T. versicolor, Postia
placenta, Mywrothecium verrucaria, Agaricus bisporus,
Methylobacterium extorquens, Pseudomonas oxalaficus, Kalstonia
eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium
flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.
oxalativorans. Variovorax paradoxus, Xanthobacter autotrophicus,
Aspergillus sp., Penicillium sp., and Mucor species. Optionally,
the OXDC will be additionally dependent on coenzyme A, such as OXDC
from organisms in the intestinal tract. In certain circumstances,
OXDC is a soluble hexameric protein. Oxalate decarboxylases are
produced by higher plants, bacteria, and fungi and have oxalate
carboxy-lyase enzymatic activity. Oxalate decarboxylases include
those produced by Bacillus subtilis, Collybia velutipes or
Flammulina velutipes, Aspergillus niger, Pseudomonas sp.,
Synechocystis sp., Streptococcus mutans, Trametes hirsute,
Sclerotinia sclerotiorum, T. versicolor, Postia placenta,
Myrothecium verrucaria, Agaricus bisporus, Methylobacterium
extorquens, Pseudomonas oxalaticus, Rahtonia eutropha, Cupriavidus
oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus
oxalaticus, Vibrio oxalaticus, A. oxalativorans, Variovorax
paradoxus, Xanthobacter autotrophicus, Aspergillus sp., Penicillium
sp., and Mucor species are generally identified as cupin type
OXDCs. The cupin-like proteins are a large class of proteins
sharing certain structural features. OXDCs, such as G-OXDCs from
Collybia sp., are active as, for example, hexameric
glycoproteins.
[0048] Oxalate decarboxylases used to prepare the crystals, and
which are used in methods described herein, may be isolated, for
example, from a natural source, or may be derived from a natural
source. As used herein, the term "derived from" means having an
amino acid or nucleic acid sequence that naturally occurs in the
source. For example, oxalate decarboxylase derived from Bacillus
subtilis will comprise a primary sequence of a Bacillus subtilis
oxalate decarboxylase protein, or will be encoded by a nucleic acid
comprising a sequence found in Bacillus subtilis that encodes an
oxalate decarboxylase or a degenerate thereof. A protein or nucleic
acid derived from a source encompasses molecules that are isolated
from the source, recombinantly produced, and/or chemically
synthesized or modified. The crystals provided herein may be formed
from polypeptides comprising amino acid sequences of OXDC, or a
functional fragment of OXDC that retains oxalate degrading
activity. Preferably, the OXDC retains at least one functional
characteristic of a naturally occurring OXDC, e.g., retains one or
more of the ability to catalyze degradation of oxalate, ability to
multimerize, and/or manganese requirement.
[0049] Isolated Oxalate Decarboxylase. Oxalate decarboxylases have
been previously isolated and are thus available from many sources,
including Bacillus subtilis, Collybia velutipes or Flammulina
velutipes, Aspergillus niger, Pseudomonas sp., Synechocystis sp.,
Streptococcus mutans, Trametes hirsute, Sclerotinia sclerotiorum,
T. versicolor, Postia placenta, Myrothecium verrucaria, Agaricus
bisporus, Methylobacterium extorquens, Pseudomonas oxalaticus,
Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp.,
Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio
oxalaticus, A. oxalativorans, Variovorax paradoxus, Xanthobacter
autotrophicus, Aspergillus sp., Penicillium sp., and Mucor species.
OXDC may also be purchased from commercial purveyors, such as,
e.g.. Sigma. Methods to isolate OXDC from a natural source are
previously described, for example, in the following references:
Tanner et al., The Journal of Biological Chemistry. 47:43627-43634.
(2001); Dashek, W. V. and Micales. J. A., Methods in plant
biochemistry and molecular biology. Boca Raton, Fla.: CRC Press.
5:49-71. (1907); Magro et al., FEMS Microbiology Letters. 49:
49-52. (1988); Anand et al., Biochemistry. 41: 7659-7669. (2002);
and Tanner and Bornemann, S. Journal of Bacteriology. 182;
5271-5273 (2000). These isolated oxalate decarboxylases may be used
to form the crystals and methods described herein.
[0050] Recombinant Oxalate Decarboxylase. Alternatively,
recombinant OXDCs may be used to form the crystals and methods
provided herein. In some instances, recombinant OXDCs encompass or
arc encoded by sequences from a naturally occurring OXDC sequence.
Further, OXDCs comprising an amino acid sequence that is homologous
or substantially identical to a naturally occurring sequence are
herein described. Also, OXDCs encoded by a nucleic acid that is
homologous or substantially identical to a naturally occurring
OXDC-encoding nucleic acid are provided and may be crystallized
and/or administered as described herein.
[0051] Polypeptides referred to herein as "recombinant" are
polypeptides which have been produced by recombinant DNA
methodology, including those that are generated by procedures which
rely upon a method of artificial recombination, such as the
polymerase chain reaction (PCR) and/or cloning into a vector using
restriction enzymes. "Recombinant" polypeptides arc also
polypeptides having altered expression, such as a naturally
occurring polypeptide with recombinantly modified expression in a
cell, such as a host cell.
[0052] In one embodiment, OXDC is recombinantly produced from a
nucleic acid that is homologous to a Bacillus subtilis or Coltybia
velutipes OXDC nucleic acid sequence, and sometimes it is modified,
e.g., to increase or optimize recombinant production in a
heterologous host. An example of such a modified sequence is
provided in SEQ ID NO: 1 (nucleic acid), which includes the nucleic
acid sequence of the open reading frame of Collybia velutipes OXDC,
for expression in Candida boidinii. The OXDC sequence has been
modified to reduce its GC. content, is linked to an .alpha. Mating
Factor secretion signal sequence, and is flanked by engineered
restriction endonuclease cleavage sites. In another embodiment,
OXDC is recombinantly produced from SEQ ID NO:2, or the unmodified
Bacillus subtilis OXDC nucleic acid sequence that is available at
GenBank Accession No: Z99120. The amino acid sequence encoded by
SEQ ID NO:2 is provided as SEQ ID NO:3.
[0053] OXDC polypeptides useful for forming OXDC crystals may be
expressed in a host cell, such as a host cell comprising a nucleic
acid construct that includes a coding sequence for an OXDC
polypeptide or a functional fragment thereof. A suitable host cell
for expression of OXDC may be yeast, bacteria, fungus, insect,
plant, or mammalian cell, for example, or transgenic plants,
transgenic animals or a cell-free system. Preferably, a host cell
is capable of glycosylating the OXDC polypeptide if necessary,
capable of disulfide linkages, capable of secreting the OXDC and/or
capable of supporting multimerization of OXDC polypeptides.
Preferred host cells include, but are not limited to E. coli
(including E. coli Origami B and E. coli BL21), Pichia pastoris,
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Bacillus
subtilis, Aspergillus, Sf9 cells. Chinese hamster ovary (CHO), 293
cells (human embryonic kidney), and other human cells. Also
transgenic plants, transgenic animals including pig, cow, goat,
horse, chicken, and rabbit are suitable hosts for production of
OXDC.
[0054] For recombinant production of OXDC, a host or host cell
should comprise a construct in the form of a plasmid, vector,
phagemid, or transcription or expression cassette that comprises at
least one nucleic acid encoding an OXDC or a functional fragment
thereof. A variety of constructs are available, including
constructs which are maintained in single copy or multiple copy, or
which become integrated into the host cell chromosome. Many
recombinant expression systems, components, and reagents for
recombinant expression are commercially available, for example from
Invitrogen Corporation (Carlsbad, Calif.); U.S. Biological
(Swampscott, Mass.); BD Biosciences Pharmingen (San Diego, Calif.);
Novagen (Madison, Wis.); Stratagene (La Jolla, Calif.); and
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).
(Braunschweig, Germany).
[0055] Recombinant expression of OXDC is optionally controlled by a
heterologous promoter, including a constitutive and/or inducible
promoter. Promoters such as, e.g., T7, the alcohol oxidase (AOX)
promoter, the dihydroxy-acetone synthase (DAS) promoters, the GaI
1,10 promoter, the phosphoglycerate kinase promoter, the
glyceraldehyde-3-phosphate dehydrogenase promoter, alcohol
dehydrogenase promoter, copper metallothioncin (CUP1) promoter,
acid phosphatase promoter, CMV and promoters polyhedrin are also
appropriate. The particular promoter is selected based on the host
or host cell. In addition, promoters that are inducible by
methanol, copper sulfate, galactose, by low phosphate, by alcohol,
e.g., ethanol, for example, may also be used und are well known in
the art.
[0056] A nucleic acid that encodes OXDC may optionally comprise
heterologous sequences. For example, a secretion sequence is
included at the N-terminus of an OXDC polypeptide in some
embodiments. Signal sequences such as those from .alpha. Mating
Factor, BGL2, yeast acid phosphatase (PHO), xylanase. alpha
amylase, from other yeast secreted proteins, and secretion signal
peptides derived from other species that are capable of directing
secretion from the host cell may be useful. Similarly other
heterologous sequences such as linkers (e.g., comprising a cleavage
or restriction endonuclease site) and one or more expression
control elements, an enhancer, a terminator, a leader sequence, and
one or more translation signals are within the scope of this
description. These sequences may optionally be included in a
construct and/or linked to the nucleic acid that encodes OXDC.
Unless otherwise specified, "linked" sequences can be directly or
indirectly associated with one another.
[0057] Similarly, an epitope or affinity tag such as Histidine, HA
(hemagglutinin peptide), maltose binding protein, AviTag.RTM.,
FLAG, or glutatthione-S-transferase may be optionally linked to the
OXDC polypeptide. A tag may be optionally cleavable from the OXDC
after it is produced or purified. A skilled artisan can readily
select appropriate heterologous sequences, for example, match host
cell, construct, promoter, and/or secretion signal sequence.
[0058] OXDC homologs or variants differ from an OXDC reference
Sequence by one or more residues. Structurally similar amino acids
can be substituted for some of the specified amino acids, for
example. Structurally similar amino acids include: (I, L and V); (F
and Y); (K and R); (Q and N), (D and E); and (G and A). Deletion,
addition, or substitution of amino acids is also encompassed by the
OXDC homologs described herein. Such homologs and variants include
(i) polymorphic variants and natural or artificial mutants, (ii)
Modified polypeptides in which one or more residues is modified,
and (iii) mutants comprising one or more modified residues.
[0059] OXDC polypeptide or nucleic acid is "homologous" (or is a
"homolog") if it is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%,
90%, 95%, 97%, 98%, 99%, or 100% identical to a reference sequence.
If the homolog is not identical to the reference sequence, it is a
"variant." A homolog is "substantially identical" to a reference
OXDC sequence if the nucleotide or amino acid sequence of the
homolog differs from the reference sequence (e.g. , truncation,
deletion, substitution, or addition) by no more than 1, 2, 3, 4, 5,
8, 10, 20, or 50 residues, and retains (or encodes a polypeptide
that retains) the ability to catalyze the degradation of oxalate.
Fragments of all oxalate decarboxylase may be homologs, including
variants and/or substantially identical sequences. By way of
example, homologs may be derived from various sources of OXDC, or
they may be derived from or related to a reference sequence by
truncation, deletion, substitution, or addition mutation. Percent
identity between two nucleotide or amino acid sequences may be
determined by standard alignment algorithms such as, for example,
Basic Local Alignment Tool (BLAST) described in Altschul et al.,J.
Mol. Biol., 215:403 410 (1990), the algorithm of Needleman et al.,
J. Mol. Biol., 48: 444 453 (1970), or the algorithm of Meyers et
al., Compute. Appl. Biosci. 4: 11 17 (1988). Such algorithms are
incorporated into the BLASTN, BLASTP and "BLAST 2 Sequences"
programs (reviewed in McGinnis and Madden, Nucleic. Acids Res.
32:W20-W25, 2004). When utilizing such programs, the default
parameters can be used. For example, for nucleotide sequences the
following settings can be used for "BLAST 2 Sequences": program
BLASTN, reward for match 2, penalty for mismatch 2, open gap and
extension gap penalties 5 and 2 respectively, gap x_dropoff 50,
expect 10, word size 11, filter ON. For amino acid sequences the
following settings can be used tor "BLAST 2 Sequences": program
BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11
and 1 respectively, gap x_dropoff 50, expect 10, word size 3,
filter ON. The amino acid and nucleic acid sequences for OXDCs that
are appropriate to form the crystals described herein, may include
homologous, variant, or substantially identical sequences.
Purification of Oxalate Decarboxylase. Oxalate decarboxylase
proteins or polypeptides may be purified from the source, such as a
natural or recombinant source, prior to crystallization. A
polypeptide that is referred to herein as "isolated" is a
polypeptide that is substantially free of its natural environment,
such as proteins, lipids, and/or nucleic acids of their source of
origin (e.g., cells, tissue (i.e., plant tissue), or fluid or
medium (in the case of a secreted polypeptide)). Isolated
polypeptides include those obtained by methods described herein or
other suitable methods, and include polypeptides that are
substantially pure or essentially pure, and polypeptides produced
by chemical synthesis, by recombinant production, or by
combinations of biological and chemical methods. Optionally, an
isolated protein has undergone further processing after its
production, such as by purification steps.
[0060] Purification may comprise buffer exchange and
chromatographic steps. Optionally, a concentration step may be
used, e.g., by dialysis, chromatofocusing chromatography, and/or
associated with buffer exchange. In certain instances, cation or
anion exchange chromatography is used for purification, including
Q-sepharose, DEAE sepharose, DE52, sulfopropyl Sepharose
chromatography or a CM52 or similar cation exchange column. Buffer
exchange optionally precedes chromatographic separation, and may be
performed by tangential flow filtration such as diafiltration. In
certain preparations, OXDC is at least 70%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% pure.
[0061] Purification in gram-scale runs is appropriate to prepare
OXDC, and procedures are optimized for efficient, inexpensive,
manufacturing-scale OXDC purification. For example, purification of
at least 0, 5, 1, 2, 5, 10, 20, 50, 100, 500, or 1000 grams or more
of OXDC in a purification procedure is provided. In one exemplary
procedure, tangential flow filtration of starting samples of at
least 10L, 50L, 100L, 500L, 1000L or more is provided, allowing
buffer exchange and precipitation of contaminant proteins. A single
Q-sepharose column is optionally used for purification of OXDC.
[0062] Crystallization of purified OXDC may also remove
contaminants, for example to further purify OXDC preparations. For
example, OXDC cystallized as described in Examples 2-6, has reduced
levels of low molecular weight contaminants, as compared to soluble
purified OXDC. In some aspects, contaminants having a measured mass
(by matrix assisted laser desorption ionization mass spectroscopy
(MALDI-MS)) of 0-10 KDa, 1-10 KDa, 0.5-5 KDa, or 2-5 KDa are
selectively excluded from the crystal form. For example,
contaminants having measured masses of approximately 2.5, 3.0, 3.7,
3.8, 4.0. 4.2, or 5.0 KDa can be substantially removed by
crystallization. Purification by crystallization may also be done
using, e.g. crude oxalate decarboxylase containing fermentation
media.
[0063] Crystallization of Oxalate Decarboxylase. Oxalate
decarboxylase crystals can be prepared using an (OXDC polypeptide,
such as a hexamer, as described above (see Anand et al.,
Biochemistry 41:7659-7669 (2002)). Vapor diffusion (such as, e.g.,
hanging drop and sitting drop methods), and batch methods of
crystallization, for example, can be used. Oxalate decarboxylase
crystals may be grown by controlled crystallization of the protein
out of an aqueous solution or an aqueous solution that includes
organic solvents. Conditions to be controlled include the rate of
evaporation of solvent, the presence of appropriate co-solutes and
buffers, pH, and temperature, for example.
[0064] For therapeutic administration, such as to treat a condition
or disorder related to oxalate levels, a variety of OXDC crystal
sizes are appropriate. In certain embodiments, crystals of less
than about 500 .mu.m average dimension are administered. Oxalate
decarboxylase crystals with an average, maximal, or minimal
dimension (for example) that is about 0.01, 0.1, 1, 5, 10, 25, 50,
100, 200, 300, 400, 500, or 1000 .mu.m in length are also provided.
Microcrystalline showers are also suitable.
[0065] Ranges are appropriate and would be apparent to the skilled
artisan. For example, the protein crystals may have a longest
dimension between about 0.01 .mu.m and about 500 .mu.m,
alternatively, between 0.1 .mu.m and about 50 .mu.m, in a
particular embodiment, the longest dimension ranges from about 0.1
.mu.m to about 10 .mu.m. Crystals may also have a shape chosen from
spheres, needles, rods, plates, such as hexagons and squares,
rhomboids, cubes, bipyramids and prisms. In illustrative
embodiments, the crystals are cubes having a longest dimension of
less than 5 .mu.m.
[0066] In general, crystals are produced by combining the protein
to be crystallized with an appropriate aqueous solvent or aqueous
solvent containing appropriate crystallization agents, such as
salts or organic solvents. The solvent is combined with the protein
and optionally subjected to agitation at a temperature determined
experimentally to be appropriate for the induction of
crystallization and acceptable for the maintenance of protein
activity and stability. The solvent can optionally include
co-solutes, such as monovalent or divalent cations, co-factors or
chaotropes, as well as buffer species to control pH. The need for
co-solutes and their concentrations are determined experimentally
to facilitate crystallization. In an industrial scale process, the
controlled precipitation leading to crystallization can be carried
out by the combination of protein, precipitant, co-solutes and,
optionally, buffers in a batch process, for example. Alternative
laboratory crystallization methods and conditions, such as dialysis
or vapor diffusion, can be adopted (McPherson, et al., Methods
Enzymol. 114:112-20 (1985) and Gilliland, Crystal Growth 90:51-59
(1998)). Occasionally, incompatibility between the cross-linking
agent and the crystallization medium might require changing the
buffers (solvent) prior to cross-linking.
[0067] As set forth in the Examples, oxalate decarboxylase
crystallizes under a number of conditions, including a wide pH
range (e.g.. pH 3.5 to 8.0). A precipitant such as a polyethylene
glycol (such as e.g., PEG 200, PEG 400, PEG 600, PEG 1000, PEG
2000, PEG 3000, PEG 8000 [See examples 7 and 8]) or an organic
cosolvent such as 2-methyl-2,4-pentanediol (MPD) is included in
some embodiments as described. Common salts that may be used
include sodium chloride, potassium chloride, ammonia sulfate, zinc
acetate, etc.
[0068] Oxalate decarboxylase may be at a concentration of, e.g., at
least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100
mg/ml. or more in a crystallization broth. The efficiency or yield
of a crystallization reaction is at least 50%, 60%, 70%, 80%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In one
embodiment, crystals of oxalate decarboxylase are grown or produced
by a batch process by mixing a solution of oxalate decarboxylase
with an appropriate buffer. In certain embodiments, the buffer is
100 mM Tris-HCl buffer, pH 8.0 and 100 mM NaCl with 2 mM
cysteine-HCl.
[0069] Crystallization from cells or cell extract. Crystals may be
prepared directly from cells or crude cell extracts. In one
embodiment, bacteria cells expressing oxalate decarboxylase are
harvested. Cells are resuspended with or without DNase and
homogenized. A salt solution is added to the cell lysis to reach a
salt concentration of about 0.3 M. 0.4 M, 0.5 M, 0.6 M or more. The
salt added can be a sodium salt, a potassium salt, a calcium salt,
or other salts. Proteins may be optionally extracted from the cell
mixture by removing cell debris. In one embodiment, homogenized
cell mixture is centrifuged, leaving proteins in the supernatant
solution. Crystals are generated by reducing salt concentration of
the cell mixture or protein solution. In one embodiment, salt is
removed through dialysis to maintain protein concentration. To
increase crystal yield, protein solution may be concentrated before
salt concentration of the solution is reduced. Crystals may be
generated at a solution with a pH of about 6, 7, or 8.
[0070] Crystals may be prepared from a protein precipitate or
pellet. In one embodiment, cells expressing desired proteins are
harvested and oxalate decarboxylase protein is collected in a
precipitate or pellet. Pellet or precipitate containing oxalate
decarboxylase protein is solubilized in a salt solution. Crystals
are formed by reducing salt concentration in the protein solution.
For increased crystal yields, the salt concentration in the
solubilized protein solution is at least about 0.3 M, 0.4 M, 0.5 M
or more before it is reduced to produce crystals.
[0071] Crystals may also be prepared from a protein solution. In
one embodiment, an oxalate decarboxylase protein solution is
concentrated in a salt solution, and crystals are formed when the
salt concentration in the solution is reduced. For increased
crystal yields, the salt concentration is at least about 0.3 M, 0.4
M. 0.5 M or more before it is reduced to produce crystals.
[0072] Stabilized crystals. Once oxalate decarboxylase crystals
have been grown in a suitable medium they can be optionally
stabilized, such as by cross-linking. Cross-linking results in
stabilization of the crystal lattice by introducing covalent links
between the constituent protein molecules of the crystal. This
makes possible transfer of the protein into an alternate
environment that might otherwise be incompatible with the existence
of the crystal lattice or even with the existence of intact
protein. Oxalate decarboxylase crystals may be cross-linked
through, e.g., lysine groups, thiol (sulthydryl) groups, and
carbohydrate moieties. Cross-linked crystals are also referred to
herein as "OXDC-CLEC," "CLEC-OXDC," or "CLEC."
[0073] A cross-linked crystal may alter the enzymatic stability
(e.g., pH, temperature, mechanical and/or chemical stability), the
pH profile of OXDC activity, the solubility, the uniformity of
crystal size or volume, the rate of release of enzyme from the
crystal, and/or the pore size and shape between individual enzyme
molecules in the underlying crystal lattice.
[0074] Advantageously, cross-linking, or stabilizing according to
the present invention carried out in such a way that the crystals
comprise an OXDC that shows at least 60%, 80%, 100%, 150%, 200%,
250%, 300% or more of the activity as compared to soluble OXDC.
Stability may be increased by at least 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 150%, 200%, 250%, 300% or more as compared to
soluble OXDC. Stability can be measured under conditions of
storage, such as pH stability, temperature stability, stability
against gut proteases, dissolution stability, and as in vivo
biological stability, for example.
[0075] In some embodiments, cross-linking slows the dissolution of
the OXDC polypeptides in the crystal into solution, effectively
immobilizing the protein, molecules into microcrystalline
particles. Upon exposure to a trigger in the environment
surrounding the cross-linked protein crystals, such as under
conditions of use rather than storage, the protein molecules slowly
dissolve, releasing active OXDC polypeptide and/or increasing OXDC
activity. The rate of dissolution is controlled, for example, by
one or more of the following factors: the degree of cross-linking,
the length of time of exposure of protein crystals to the
cross-linking agent, the rate of addition of cross-linking agent to
the protein crystals, the nature of the cross-linker, the chain
length of the cross-linker, pH, temperature, presence of
sulfahydryl reagents like cysteine, glutathione, the surface area
of the cross-linked protein crystals, the size of the cross-linked
protein crystals, and the shape of the cross-linked protein
crystals.
[0076] Cross-linking can be achieved using one or a combination of
a wide variety of cross-linking agents, including a multifunctional
agent, at the same time (in parallel) or in sequence. Upon exposure
to a trigger in the surrounding environment, or over a given period
of time, the cross-links between protein crystals cross-linked with
such multifunctional cross-linking agents lessen or weaken, leading
to protein dissolution or release of activity. Alternatively, the
cross-links may break at the point of attachment, leading to
protein dissolution or release of activity. See U.S. Pat. Nos.
5,976,529 and 6,140,475.
[0077] In some embodiments, the cross-linking agent is a
multifunctional cross-linking agent having at least 2, 3, 4, 5, or
more active moieties. In various embodiments, the agent may be
chosen from glutaraldehyde, succinaldehyde, octanedialdehyde,
glyoxal, dithiobis(succinimidylpropionate), 3,3'
dithiobis(sulfosuccinimidylpropionate), dimethyl
3,3'-dithiobispropionimidate-HCl,
N-succinimidyl-3-(2-pyridyldithio)propionate, hexamethylenediamine,
diaminooctane, ethylenediamine, succinc anhydride, phenylglutaric
anhydride, salicylaldehyde, acetimidate, formalin, acrolein,
succinic semialdehyde, butyraldehyde, dodecylaldehyde,
glyceraldehyde, and trans-oct-2-enal.
[0078] Additional multifunctional cross-linking agents include halo
triazines, e.g., cyanuric chloride; halo-pyrimidines, e.g.,
2,4,6-trichloro/bromo pyrimidine; anhydrides or halides of
aliphatic or aromatic mono- or di-carboxylic acids, e.g., maleic
anhydride, (meth)acryloyl chloride, chloroacetyl chloride;
N-methylol compounds, e.g., N-methylol chloro-acetamide;
di-isocyanates or di-isothiocyanates, e.g.,
phenylene-1,4-di-isocyanate and aziridines. Other cross-linking
agents include epoxides, such as, for example, di-epoxides,
tri-epoxides and tetra-epoxides. In one embodiment, the
cross-linking agent is glutaraldehyde, a bifunctional agent, and
glutaraldehyde is used alone or in sequence with an epoxide. Other
cross-linking reagents (see, for example, the 1996 catalog of the
Pierce Chemical Company) may also be used, at the same time (in
parallel) or in sequence with reversible cross-linking agents, such
as those described below.
[0079] According to an alternate embodiment of this invention,
cross-linking may be carried out using reversible cross-linking
agents, in parallel or in sequence. The resulting cross-linked
protein crystals are characterized by a reactive multi-functional
linker, into which a trigger is incorporated as a separate group.
The reactive functionality is involved in linking together reactive
amino acid side chains in a protein and the trigger consists of a
bond that can be broken by altering one or more conditions in the
surrounding environment (e.g., pH, presence of reducing agent,
temperature, or thermodynamic water activity).
[0080] The cross-linking agent may be homofunctional or
heterofunctional. The reactive functionality (or moiety) may, e.g.,
be chosen from one of the following functional groups (where R, R',
R'' and R''' may be alkyl, aryl or hydrogen groups):
[0081] I. Reactive acyl donors, such as, e.g.: carboxylate esters
RCOOR', amides RCONHR', Acyl azides RCON.sub.3, carbodiimides
R--N.dbd.C.dbd.N--R', N hydroxyimide esters, RCO--O--NR',
imidoesters R--C.dbd.NH2' (OR'), anhydrides RCO--O--COR',
carbonates RO--CO--O--R', urethanes RNHCONHR', acid halides RCOHal
(where HaI=a halogen), acyl hydrazides RCONNR'R'', and O
acylisources RCO--O--C.dbd.NR'(--NR''R''')
[0082] II. Reactive carbonyl groups, such as, e.g.: aldehydes RCHO
and ketones RCOR', acetals RCO(H.sub.2)R', and ketals RR'CO2 R'R''
(Reactive carbonyl containing functional groups known to those well
skilled in the art of protein immobilization and cross-linking are
described in the literature (Pierce Catalog and Handbook, Pierce
Chemical Company, Rockford, Ill. (1994); S. S. Wong, Chemistry of
Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.
(1991));
[0083] III. Alkyl or aryl donors, such as, e.g.: alkyl or aryl
halides R-Hal, azides R--N, sulfate esters RSO.sub.3 R', phosphate
esters RPO(OR'.sub.3), alkyloxonium salts R.sub.3 O+, sulfonium
R.sub.3S+, nitrate esters RONO.sub.2, Michael acceptors
RCR'.dbd.CR'''COR'', aryl fluorides ArF, isonitriles RN+=C--,
haloamines R.sub.2 N-Hal, alkenes, and alkynes;
[0084] IV. Sulfur containing groups, such as, e.g.: disulfides
RSSR', sulfhydryls RSH, and epoxides R.sub.2 C_.sup.OCR'.sub.2;
and
[0085] V. Salts, such as, e.g.: alkyl or aryl ammonium salts
R.sub.4 N+, carboxylate RCOO--, sulfate ROSO.sub.3--, phosphate
ROPO.sub.3'', and amines R.sub.3 N.
[0086] Reversible cross-linking agents, for example, comprise a
trigger. A trigger includes an alkyl, aryl, or other chain with
activating group that can react with the protein to be
cross-linked. Those reactive groups can be any variety of groups
such as those susceptible to nuclcophilic, free radical or
electrophilic displacement including halides, aldehydes,
carbonates, urethanes, xanthanes, and epoxides among others. For
example, reactive groups may be labile to acid, base, fluoride,
enzyme, reduction, oxidation, thiol, metal, photolysis, radical, or
heat.
[0087] Additional examples of reversible cross-linking agents are
described in T. W. Green, Protective Groups in Organic Synthesis,
John Wiley & Sons (Eds.) (1981). Any variety of strategies used
for reversible protecting groups can be, incorporated into a
cross-linker suitable for producing cross-linked protein crystals
capable of reversible, controlled solubilization. Various
approaches are listed, in Waldmann's review of this subject, in
Angewante Chemie Inl. Ed. Engl., 35:2056 (1996).
[0088] Other types of reversible cross-linking agents are disulfide
bond-containing cross-linkers. The trigger breaking cross-links
formed by such cross-linking agents is the addition of reducing
agent, such as cysteine, to the environment of the cross-linked
protein crystals. Exemplary disulfide cross-linking agents are
described in the Pierce Catalog and Handbook (1994-1995). Examples
of such cross-linkers and methods are disclosed in U.S. Pat. No.
6,541,606, relevant portions of which are incorporated by
reference.
[0089] In addition, cross-linking agents which cross-link between
carbohydrate moieties or between a carbohydrate, moiety and an
amino acid may also be used.
[0090] The concentration of the cross-linking agent may be from
about 0.01% to 20%, about 0.02% to 10%, or about 0.05% to 5% w/v in
solution. Typically, the crosslinking agent is about 0.5% or about
1% w/v. For example, the concentration of the cross-linking agent
may be, e.g., about 0.0.1%, 0.02%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%,
0.5%, 1%, 2%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%
w/v in solution [see Table 2 in the examples]. It may be necessary
to exchange buffers prior to cross-linking, Crystals, including
CLECs, may be optionally lyophilized or otherwise formulated.
[0091] The crystals, including the cross-linked crystals described
herein are useful in the methods of treatment and methods to reduce
oxalate levels described herein. The OXDC crystals are also useful
in methods relating to industrial processes (e.g., synthesis,
processing, bioremediation, disinfection, sterilization), and
methods to treat plants, such as plant fungal infections, for
example as reviewed in, e.g., Svedruzic el al., Arch. Biochem.
Biophys. 433:176-192 (2005). Such non-therapeutic applications for
soluble or amorphous OXDC are described, for example, in U.S. Pat.
Nos. 5,866,778; 6,218,134; 6,229,065; 6,235,530; and 6,503,507. The
crystals described herein can be applied to these uses, based on
one or more properties of the stabilized OXDC crystals described
above, such as increased stability of the oxalate decarboxylase
enzyme.
[0092] Drying of Crystals of Oxalate Decarboxylase. Crystals of
oxalate decarboxylase are dried by removal of water, organic
solvent or liquid polymer by means including drying with N.sub.2,
air or inert gases, vacuum oven drying, lyophilization, washing
with a volatile organic solvent followed by evaporation of the
solvent, evaporation in a fume hood, tray drying, fluid bed drying,
spray drying, vacuum drying, or roller drying. Typically, drying is
achieved when the crystals become a free flowing powder. Drying may
be carried out by passing a stream of gas over wet crystals. The
gas may be selected from the group consisting of: nitrogen, argon,
helium, carbon dioxide, air or combinations thereof.
[0093] In principle, dried crystals can be prepared by
lyophilization. However, this technique involves rapid cooling of
the material and can be applied only to freeze stable products. In
one embodiment, the aqueous solution containing a crystalline
oxalate decarboxylase is first frozen to between -40 and
-50.degree. C., followed by removal of the under vacuum.
[0094] Production of crystals of oxalate decarboxylase, or
formulations or compositions comprising such crystals. In one
aspect, crystals of oxalate decarboxylase, or formulations or
compositions comprising such crystals are disclosed. Such
compositions can be prepared according to the following
process:
[0095] First, the oxalate decarboxylase is crystallized. Next,
excipients or ingredients selected from sugars, sugar alcohols,
viscosity increasing agents, wetting or solubilizing agents, buffer
salts, emulsifying agents, antimicrobial agents, antioxidants, and
coating agents are added directly to the mother liquor.
Alternatively, the mother liquor is removed, after which the
crystals are suspended in an excipient solution for a minimum of 1
hour to a maximum of 24 hours. The excipient concentration is
typically between about 0.01 and about 10% (w/w). The ingredient
concentration is between about 0.01 and about 90% (w/w). The
crystal concentration is between about 0.01 and about 99%
(w/w).
[0096] The mother liquor is then removed from the crystal slurry
either by filtration or by centrifugation. Subsequently, the
crystals are washed optionally with solutions of about 50 to 100%
(w/w) of one or more organic solvents such as, for example,
ethanol, methanol, isopropanol or ethyl acetate, either at room
temperature or at temperatures between about -20.degree. C. to
about 25.degree. C.
[0097] The crystals are then dried either by passing a stream of
nitrogen, air, or inert gas over them. Alternatively; the crystals
are dried by air drying, spray drying, lyophilization or vacuum
drying. The drying is carried out for a minimum of about 1 hour to
a maximum of about 72 hours after washing, until the moisture
content of the final product is below about 10% by weight, most
preferably below about 5% by weight. Finally, micromizing (reducing
the size) of the crystals can be performed if necessary.
[0098] According to one embodiment of this invention, when
preparing crystals of oxalate decarboxylase, or formulations or
compositions comprising such crystals, enhancers, such as
surfactants, are not added during crystallization. Excipients or
ingredients are added to the mother liquor after crystallization,
at a concentration of between about 1 and about 10% (w/w),
alternatively at a concentration of between about 0.1 and about 25%
(w/w), alternatively at a concentration of between about 0.1 and
about 50% (w/W). The excipient or ingredient is incubated with the
crystals in the mother liquor for about 0.1 to about 3 hrs,
alternatively the incubation is carried out for about 0.1 to about
12 hrs, alternatively the incubation is carried out for about 0.1
to about 24 hrs.
[0099] In another embodiment of this invention, the ingredient or
excipient is dissolved in a solution other than the mother liquor,
and the crystals are removed from the mother liquor and suspended
in the excipient or ingredient solution. The ingredient or
excipient concentrations and the incubation times are the same as
those described above.
[0100] Another advantage of the present invention is that crystals
of oxalate decarboxylase, or formulations thereof, that are
encapsulated within polymeric carriers to form compositions
comprising microspheres can be dried by lyophilization.
Lyophilization, or freeze-drying allows water to be separated from
the composition. The oxalate decarboxylase crystal composition is
first frozen and then placed in a high vacuum. In a vacuum, the
crystalline water sublimes, leaving the oxalate decarboxylase
crystal composition behind, containing only the tightly bound
water. Such processing further stabilizes the composition and
allows for easier storage and transportation at typically
encountered ambient temperatures.
[0101] Spray drying allows water to be separated from the crystal
preparation. It is highly suited for the continuous production of
dry solids in either powder, granulate or agglomerate form from
liquid feedstocks as solutions, emulsions, and pumpable
suspensions. Spray drying involves the atomization of a liquid
feedstock into a spray of droplets and contacting the droplets with
hot air in a drying chamber. The sprays are produced by either
rotary tea (wheel) or nozzle atomizers. Evaporation of moisture
from the droplets and formation of dry particles proceed under
controlled temperature and airflow conditions. Relatively high
temperatures are needed for spray drying operations. However, heat
damage to products is generally only slight, because of an
evaporative cooling effect during the critical drying period and
because the subsequent time of exposure to high temperatures of the
dry material may be very short. Powder is discharged continuously
from the drying chamber. Operating conditions and dryer design are
selected according to the drying characteristics of the product and
the powder specification. Spray drying is an ideal process where
the end product must comply with precise quality standards
regarding particle size distribution, residual moisture content,
bulk density and particle shape.
[0102] Compositions. OXDC crystals, including cross-linked crystals
are provided as a composition, such as a pharmaceutical composition
(see, e.g., U.S. Pat. No. 6,541,606, describing formulations and
compositions of protein crystals). Pharmaceutical compositions
comprising OXDC crystals include the OXDC crystal with one or more
ingredients or excipients, including, but not limited to sugars and
biocompatible polymers. Examples of excipients are described in
Handbook of Pharmaceutical Excipients, published jointly by the
American Pharmaceutical Association and the Pharmaceutical Society
of Great Britain, and further examples are set forth below.
[0103] The OXDC enzyme may be administered as a crystal in a
composition as any of a variety of physiologically acceptable salt
forms, and/or with an acceptable pharmaceutical carrier and/or
additive as part of a pharmaceutical composition. Physiologically
acceptable salt forms and standard pharmaceutical formulation
techniques and excipients are well known to persons skilled in the
art (see, e.g., Physician's Desk Reference (PDR) 2003, 57th ed..
Medical Economics Company, 2002; an Remington: The Science and
Practice of Pharmacy, eds. Gennado et al. 20th ed, Lippincott,
Williams & Wilkins, 2000). For the purposes of this
application, "formulations" include "crystal formulations."
[0104] Oxalate decarboxylase useful in the methods of this
invention may be combined with an excipient. According to this
invention, an "excipient" acts as a filler or a combination of
fillers used in pharmaceutical compositions. Exemplary ingredients
and excipients for use in the compositions are set forth as
follows.
[0105] Biocompatiblc polymers. Biocompatible polymers are polymers
that are non-antigenic (when not used as an adjuvant),
non-carcinogenic, non-toxic and which are not otherwise inherently
incompatible with living organisms may be used in the OXDC crystal
compositions described herein. Examples include: poly (acrylic
acid), poly (cyanoacrylates), poly (amino acids), poly
(anhydrides), poly (depsipeptide), poly (esters) such as poly
(lactic acid) or PLA, poly (lactic-co-glycolic acid) or PLGA, poly
(.beta.-hydroxybutryate), poly (caprolactone) and poly (dioxanone);
poly (ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly
[(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol),
poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether
copolymers, pluronic polyols, albumin, alginate, cellulose and
cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid,
oligosaccharides, glycaminoglycans, sulfated polysaccharides,
blends and copolymers thereof.
[0106] Biodegradable polymers, i.e., polymers that degrade by
hydrolysis or solubilization may be included in OXDC crystal
compositions. Degradation can be heterogenous (occurring primarily
at the particle surface), or homogenous (degrading evenly
throughout the polymer matrix).
[0107] Ingredients such as one or more excipients or pharmaceutical
ingredients or excipients may be included in OXDC crystal
compositions. An ingredient may be an inert or active
ingredient.
[0108] Methods of Treating Oxalate-Associated Disorders with OXDC
Crystals. The methods of the invention comprise administering an
oxalate decarboxylase, e.g., crystals of OXDC or cross-linked forms
thereof, to a mammalian subject to treat, prevent, or reduce the
risk of occurrence of a condition associated with elevated levels
of oxalate. The elevated levels of oxalate may be detected, e.g.,
in a biological sample from the subject, such as a body fluid,
including urine, blood, serum, or plasma. In certain embodiments,
urinary oxalate levels are detected. The crystals and/or the
compositions comprising crystals may be administered in the methods
described herein.
[0109] In some embodiments, methods for treading hyperoxaluria in
individuals with primary hyperoxaluria, enteric hyperoxaluria,
hyperoxaluria caused by surgical intervention, idiopathic
hyperosaluria, oxalosis are provided. In other instances, elevated
oxalate-related disorders of the kidneys, bone, liver
gastrointestinal tract and pancreas are amenable to treatment with
the methods disclosed herein. Further disorders or diseases treated
by the methods provided herein include, but are not limited to
ethylene glycol (oxalate) poisoning, idiopathic urinary stone
disease, renal failure (including progressive, chronic, or
end-stage renal failure), steatorrhoea, malabsorption, ileal
disease, vulvodynia, cardiac conductance disorders, inflammatory
bowel disease, cystic fibrosis, exocrine pancreatic insufficiency,
Crohn's disease, ulcerative colitis, nephrocalcinosis,
osteoporosis, urolithiasis, and nephrolithiasis. Such conditions
and disorders may optionally be acute or chronic.
[0110] The methods of the invention may reduce oxalate levels in a
subject by at least 10%, 20%, 30%, 40%, 50%, 60% 70%, 80% 90%, 95%,
or more as compared to levels in an untreated or control subject,
to some embodiments, reduction is measured by comparing the oxalate
level in a subject before and after administration of OXDC. In some
embodiments, the invention provides a method of treating or
ameliorating an oxalate-associated condition or disorder, to allow
one or more symptoms of the condition or disorder to improve by at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In
certain embodiments the methods reduce levels of endogenous oxalate
and/or adsorption of dietary oxalate.
[0111] In some embodiments, methods for treating individuals having
a genotype associated with high oxalate levels are provided, such
as individuals homozygous or heterozygous for a mutation that
reduces activity of, e.g., alanine:glyoxalate aminotransferase,
glyoxylate reductase/hydroxypyruvate reductase, hepatic glycolate
oxidase, another enzyme involved in oxalate metabolism or
associated with hyperoxaluria. In other embodiments, methods for
treating individuals having reduced or lacking Oxalobacter
formigenes enteric colonization are provided.
[0112] The disclosed methods include, administering therapeutically
effective amounts of oxalate decarboxylase to a mammalian subject
at risk for, susceptible to, or afflicted with a condition
associated with elevated levels of oxalate. The populations treated
by the methods of the invention include, but are not limited to,
subjects suffering from, or at risk for developing an
oxalate-associated disorder such as, e.g., primary hyperoxaluria or
enteric hyperoxaluria.
[0113] Subjects treated according to the methods of the invention
include but are not limited to mammals, including humans, non human
primates, primates, baboons, chimpanzees, monkeys, rodents (e.g.,
mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs,
etc.
[0114] Indications, Symptoms, and Disease Indicators. Many methods
are available to assess development or profession of an
oxalate-associated disorder or a condition associated with elevated
oxalate levels. Such disorders include, but are not limited to, any
condition, disease, or disorder as defined above. Development or
progression of an oxalate-associated disorder may be assessed by
measurement of urinary oxalate, plasma oxalate, measurement of
kidney or liver function, or detection of calcium oxalate deposits,
for example.
[0115] A condition, disease, or disorder may be identified by
detecting or measuring oxalate concentrations, for example, in a
urine sample or other biological sample or fluid. An early symptom
of hyperoxaluria is typically kidney stones, which may be
associated with severe or sudden abdominal or flank pain, blood in
the urine, frequent urges to urinate, pain when urinating, or fever
and chills. Kidney stones may be symptomatic or asymptomatic, and
may be visualized, for example by imaging the abdomen by x-ray,
ultrasound, or computerized tomography (CT) scan. If hyperoxaluria
is not controlled, the kidneys are damaged and kidney function is
impaired. Kidneys may even fail. Kidney failure (and poor kidney
function) may be identified by a decrease in, or lacking urine
output (glomerular filtration rate), general ill, feeling,
tiredness, and marked fatigue, nausea, vomiting, anemia, anchor
failure to develop and grow normally in young children. Calcium
oxalate deposits in other tissues and organs may also be detected
by methods including direct visualization (e.g. in the eyes),
x-ray, ultrasound, CT, echocardiogram, or biopsy (e.g., bone,
liver, or kidney).
[0116] Kidney and liver function, as well as oxalate
concentrations, may also be assessed using art-recognized direct
and indirect assays. The chemical content or urine, blood or other
biological sample may also be tested by well known techniques. For
example, oxalate, glycolate, and glycerate levels may be measured.
Assays for liver and kidney function are well known, such as, for
example, the analysis of liver tissue for enzyme deficiencies and
the analysis of kidney tissue for oxalate, deposits. Samples may
also be tested for DNA changes known to cause primary
hyperoxaluria.
[0117] Other indications for treatment include, but are not limited
to, the presence of one or more risk factors, including those
discussed previously and in the following sections. A subject at
risk for developing or susceptible to a condition, disease, or
disorder or a subject who may be particularly receptive to
treatment with oxalate decarboxylase may be identified by
ascertaining the presence or absence of one or more such risk
factors, diagnostic, or prognostic indicators. Similarly, an
individual at risk for developing an oxalate-related disorder may
be identified by analysts of one or more genetic or phenotypic
markers.
[0118] The methods disclosed are useful in subjects with urinary
oxalate levels of at least 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,
250, 260, 270, 280, 290, 300, ,310, 320, 330, 340, 350, 360, 370,
380, 390, or 400 mg of oxalate per 24 hour period, or more. In
certain embodiments, the oxalate level is associated with one or
more symptoms or pathologies. Oxalate levels may be measured in, a
biological sample, such as a body fluid including blood, serum,
plasma, or urine. Optionally, oxalate is normalized to a standard
protein or substance, such as creatinine in urine. In some
embodiments, the claimed methods include administration of oxalate
decarboxylase to reduce circulating oxalate levels in a subject to
undetectable levels, or to less than 1%, 2%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, or 80% of the subject's oxalate levels prior to
treatment, within 1, 3, 5, 7, 9, 12, or 15 days.
[0119] Hyperoxaluria in humans can be characterized urinary oxalate
excretion of greater than 40 mg (approximately 440 .mu.mol) or 30
mg per day. Exemplary clinical cutoff levels are 43 mg/day
(approximately 475 .mu.mol) for men and 32 mg/day (approximately
350 .mu.mol) for women, for example. Hyperoxaluria can also be
defined as urinary oxalate excretion greater than 30 mg per day per
gram of urinary creatinine. Persons with mild hyperoxaluria may
excrete at least 30-60 (342-684 .mu.mol) or 40-60 (456-684 .mu.mol)
mg of oxalate per day. Persons with enteric hyperoxaluria may
excrete at least 80 mg of urinary oxalate per day (912 .mu.mol),
and persons with primary hyperoxaluria may excrete at least 200 mg
per day (2280 .mu.mol), for example. Borowski A. E., Langman C B.
Hyperoxaluria and Oxalosis: Current Therapy and Future directions.
Exp Opinion Phrama (2006, in press).
[0120] Administration of OXPC Crystals and Compositions Thereof
[0121] Administration of oxalate decarboxylase in accordance with
the methods of the invention is not limited to any particular
delivery system and includes administration via the upper
gastointestinal tract, e.g., the mouth (for example in capsules,
suspension, tablets, or with food), or the stomach, or upper
intestine (for example by tube or injection) to reduce oxalate
levels in an individual. In certain cases, the OXDC is administered
to reduce endogenous oxalate levels and/or concentrations. OXDC may
also be provided by an extracorporeal device, such as a dialysis
apparatus, a catheter, or a structure or device that contacts a
biological sample from an individual.
[0122] Administration to an individual may occur in a single dose
or in repeat administrations, and in any of a variety of
physiologically acceptable forms, and/or with an acceptable
pharmaceutical carrier and/or additive as part of a pharmaceutical
composition (described earlier). In the disclosed methods, oxalate
decarboxylase may be administered alone, concurrently or
consecutively over overlapping or nonoverlapping intervals with one
or more additional biologically active agents, such as, e.g.,
pyridoxine (vitamin B-6), orthophosphate, magnesium,
glycosaminoglycans, calcium, iron, aluminum, magnesium, potassium
citrate, cholestyramine, organic marine hydrocolloid, plant juice,
such as, e.g., banana stem juice or beet juice, or L-cysteine.
Biologically active agents that reduce oxalate levels or that
increase the activity or availability of OXDC are provided. In
sequential administration, the oxalate decarboxylase and the
additional agent or agents may be administered in any order. In
some embodiments, the length of an overlapping interval may be more
than 2, 4, 6, 12, 24, or 48 weeks or more.
[0123] The oxalate decarboxylase may be administered as the sole
active compound or in combination with another active compound or
composition. Unless otherwise indicated, the oxalate decarboxylase
is administered as a dose of approximately from 10 .mu.g/kg to 25
mg/kg or 100 mg/kg, depending on the severity of the symptoms and
the progression of the disease. The appropriate therapeutically
effective dose of OXDC is selected by a treating clinician and
would range approximately from 10 .mu.g/kg to 20 mg/kg, from 10
.mu.g/ka to 10 mg/kg, from 10 .mu.g/kg to 1 mg/kg, from 10 .mu.g/kg
to 100 .mu.g/kg, from 100 .mu.g/kg to 1 mg/kg, from 100 .mu.g/kg to
10 mg/kg, from 500 .mu.g/kg to 5 mg/kg, from 500 .mu.g/kg to 20
mg/kg, from 1 mg/kg to 5 mg/kg, from 1 mg/kg to 25 mg/kg, from 5
mg/kg to 100 mg/kg, from 5 mg/kg to 50 mg/kg, from 5 mg/kg to 25
mg/kg, and from 10 mg/kg to 25 mg/kg. Additionally, specific
dosages indicated in the Examples or in the Physician's Desk
Reference (PDR) 2003, 57th ed,, Medical Economics Company, 2002,
may be used.
[0124] The oxalate deearboxylase crystal of the present invention
may be administered through an extracorporeal device or catheter,
such as for delivery of oxalate decarboxylase to a patient.
Catheters, for example, urinary catheters, may be coated with
Compositions containing oxalate decarboxylase crystals.
[0125] The following Qxamples provide illustrative embodiments of
the invention. One of ordinary skill in the art will recognize the
numerous modifications and variations that may be performed without
altering the spirit or scope of the present invention. Such
modifications and variations are encompassed within the scope of
the invention. The Examples do not in any way limit the
invention.
EXAMPLES
Example 1. Fermentation and Purification of Oxalate
Decarboxylase
[0126] Oxalate decarboxylase (OXDC) from Bacillus subtilis (B.
subtilis) is a 261 kDa homohexameric protein that consists of six
identical monomers. Each monomer contains 385 amino acids, with a
calculated molecular weight of .about.43-44 kDa and an isoelectric
point of 5.2. The OXDC gene, formerly known as yvrK, was PCR
amplified using B. subtilis genomic DNA as template.
[0127] The amplified OXDC gene was first cloned into the pCRII
vector (Invitrogen, Carlsbad, Calif.) and then subcloned and
expressed from pET-11a expression vector using E. coli BL21 (DE3)
pLysS cells. Since gene expression in the pET-11a vectors is under
control of the T7 promoter, OXDC expression was regulated by
induction with IPTG (isopropyl-beta-D-thiogalactopyranoside).
[0128] Fermentation was used to achieve high expression levels of
recombinant OXDC in E. coli. Expression was performed in 800 L
(liter) fermentation media containing casein hydrolysate (USB
Corporation, Cleveland. Ohio) or soy peptone, yeast extract (USB
Corporation), NaCl (Fisher Scientific), PPG 2000 anti-foam (PPG),
KOH (Mallinckrodt Baker, Inc., Phillipsburg, N.J.), and ampicillin
(USB Corporation). Since OXDC is a manganese-dependent enzyme, 5 mM
MnCl.sub.2.4H.sub.2O (Mallinckrodt Baker, Inc.) was included in the
fermentation medium. Expression of OXDC was induced by addition of
0.4 mM IPTG (Lab Scientific). The cells expressing OXDC were grown
either in shake flasks or in a fermenter. By this method, OXDC was
expressed mainly in pellet preparations,
[0129] A glycerol stock of BL21 cells transformed with pET11a-OXDC
was used for fermentation. For 800 L fermentation, a pre-seed
culture was prepared with 2.times.250 ml flasks, each containing 50
ml of medium (LB plus 100 .mu.g/ml of ampicillin). 2.times.0.5 ml
of inoculum was used. The cultures were incubated at 35.degree. C.,
250 rpm for 6 hours. This culture was then transferred to 6.times.2
L flasks each containing 1 L of media (LB plus 100 .mu.g/ml of
ampicillin). In each flask, 10 ml of starter culture was added.
These cultures were incubated at 35.degree. C., 250 rpm for 12
hours. 6 L of this culture was transferred to the 800 L fermenter
containing the appropriate media as described above. The culture
was grown at 37.degree. C., pH 7.0, shaking at 100 rpm with
dissolved oxygen around 30-40 until the OD.sub.600 reached about
0.3 (this process took approximately 2-3 hours). Expression of OXDC
was induced with 0.4 mM of IPTG plus 5 mM MnCl.sub.2.4H.sub.2O. The
culture was induced for 4 hours at 37.degree. C., 100 rpm. The
cells were then harvested and frozen for later use. The OD.sub.600
at the time of harvesting was about 5.5-8.0.
[0130] Cells were resuspended in a ratio of 1 kg cell paste per 4 L
buffer containing 50 mM Tris pH 8, 100 mM NaCl, and in the presence
or absence of 15-25 U/ml DNase. Cell suspension was blended (50-60
rpm) overnight at 4.degree. C. The cell suspension was passed
through a pre-cooled homogenizer three times on ice. The efficiency
of cell lysis was checked under the microscope and unbroken cell
suspension was used as a control. Cells were centrifuged at 4,000
rpm for 40 min in 1 L bottles at 4.degree. C. Supernatant and
pellet were saved for characterization by SDS-PAGE. The expression
of OXDC was analyzed by SDS-PAGE. The majority of the OXDC was
found in the pellet, which included inclusion bodies and other
precipitates. The pellet was harvested by centrifugation at 4000
rpm for 40 min and was used immediately or frozen at -70.degree. C.
for later use.
[0131] From an 800 L fermentation reaction, we obtained between
5,000 and 5,500 g of cells, which yielded between 2,800 and 3,000 g
of wet weight pellets.
Example 2. Crystallization of Oxalate Decarboxylase from
Solubilized Pellets with Mild Denaturant Concentration Followed by
Anion Exchange Chromatography
[0132] OXDC pellets stored frozen at -20.degree. C. were used to
prepare crystals of OXDC.
[0133] In this procedure, pellets were solubilized under mild
conditions of denaturant concentration and pH. The solubilized
protein was then refolded using an anion exchange matrix
column.
[0134] The pellets were solubilized in 2 M Urea, 100 mM Tris pH
10.0, 10 mM DTT and 100 mM NaCl (1:10 w/v). The solution was
stirred at room temperature (RT) for 2 h, and then the solution was
centrifuged at 15 K. for 30 min at 4.degree. C. The supernatant was
carefully decanted and saved. The pellet was carefully weighed and
saved separately.
[0135] The solubilized pellets in supernatant were added dropwise,
at a flow rate of 10 ml/min, to 10 vol. of a solution consisting of
2 M Urea, 100 mM Tris pH 8.0, 1 mM DTT, and 1 mM MnCl.sub.2, with
constant and gentle stirring. After complete addition of the
supernatant the protein solution was incubated at RT for 1 h. After
incubation, the protein solution was made ready for anion exchange
chromatography by centrifuging at 15 K for 30 min at 4.degree. C.
to remove any possible precipitation.
[0136] An anion exchange chromatography column was prepared by
packing Q sepharose matrix in glass column. The column was attached
to an FPLC and equilibrated by washing with 10 column volumes (CV)
of a solution of 0.5 M Urea, 100 mM Tris pH 8.0, 1 mM DTT, and 1 mM
MnCl.sub.2. The flow-rate was maintained at 6 ml/min. Solubilized
pellet was loaded onto the column. Protein load was 8-10 mg/ml of
matrix. After loading the sample, the column was washed with at
least 10 CV of 100 mM Tris pH 8.0, 1 mM DTT and 1 mM MnCl2 at a
flow rate of 6 ml/min. This step removed urea from the bound
protein sample and allowed the protein to refold to its native
conformation.
[0137] The protein was eluted from the column with 1 M NaCl, 100 mM
Tris pH 8.0, and 1 mM DTT by either a step gradient or by step
elution. The protein elution was monitored at 280 nm and fractions
of 10 ml were collected. The peak fractions were pooled together
and tested by SDS-PAGE and enzyme activity.
[0138] The peak fractions containing OXDC were concentrated to 15
mg/ml by stirring cells using a 10,000 MWCO membrane, and dialyzing
against 10 vol. of 100 mM Tris pH 8.0, 100 mM NaCl, and 1 mM DTT.
Two changes of buffer were made at one hour intervals and after a
third change of buffer, the dialysis continued overnight for
maximum crystal recovery. Protein crystallized in dialysis bags was
recovered by centrifuging the sample at 2K, for 15 min at 4.degree.
C. After dialysis, about 70% of the refolded OXDC was crystallized.
The crystals were cubic in shape and of uniform size. The crystals
showed an activity of .about.44 units.
Example 3. Crystallization of Oxalate Decarboxylase by Solubilizing
Pellets Using High Denaturant Concentration Followed by Anion
Exchange Chromatography
[0139] By this method, pellets were solubilized in 5 M urea, 50 mM
Tris pH 8.6, 100 mM NaCl, 10 mM DTT (1:5 w/v). The solution was
stirred at RT for 2 h, and then the solution was centrifuged at 15K
for 30 min at 4.degree. C. The supernatant was carefully decanted
and saved. The pellet was weighed and saved separately.
[0140] Anion exchange chromatography column was prepared by packing
Q sepharose matrix in glass column. The column was attached to an
FPLC and equilibrated by washing with 3 column volumes (CV) of the
4 M Urea, 100 mM Tris pH 8.6 and 10 mM DTT. The column was further
washed with 7 CV in 100 mM NaCl, 50 mM Tris pH 8, 1 mM MnCl.sub.2,
10 mM DTT and eluted in a single step with 3 CV of 0.5 M NaCl, 50
mM Tris pH 8.0, 1 mM DTT, 1 mM MnCl.sub.2.
[0141] Appropriate fractions were collected and the protein was
identified by SDS-PAGE and activity assay. The soluble protein was
concentrated in the presence of high salt (0.5 M NaCl). A total
volume of 450 ml was eluted with 0.5 M salt and concentrated to 45
ml with pellicon filtration into 100 mM NaCl, 50 mM Tris pH 8.0, 1
mM DTT. After protein concentration, dilution was performed in
order to reduce the salt concentration from 0.5 M to 0.1 M NaCl. At
this point, crystals of OXDC begun to form. In this example, the
volume was brought to 210 mL in 100 mM NaCl, 50 mM Tris pH 8.0, 1
mM DTT equivalent. Formed crystals were spun and recovered in 100
mM NaCl, and 50 mM Tris pH 8.0, in the presence or absence of 1 mM
DTT.
Example 4. Crystallization of Oxalate Decarboxylase by Solubilizing
Pellets Using High pH and Mild Denaturant Concentration Followed by
Concentration on Hollow Fiber
[0142] Pellets Containing Inclusion Bodies and Other Precipitates
(3.93 kg) were solubilized in 9.5 L of 50 mM Tris pH 12, 500 mM
NaCl, 2 M urea, 10 mM DTT for 2 hours at room temperature. Final
volume was 12 L. pH 9.9. The sample was spun at 7000 rpm for 45
minutes and supernatant was recovered. The total volume after
spinning was 11.1 L, pH 9.9. The sample was concentrated on hollow
fiber to 5 L for an hour and the volume was then slowly brought up
to 20 L with 50 mM Tris pH 8.0. 500 mM NaCl, 2 M urea, 10 mM DTT.
This process was done for an hour. The final concentration of urea
at this point is estimated around 0.5 M urea. The volume was
concentrated to 6 L in hollow fiber for 2.5 h. The sample was
diluted to 24 L with 50 mM Iris pH 8.0, 500 mM NaCl, 1 mM DTT, 1 mM
MnCl, 200 mM L-Arginine for 30 min. At this point the concentration
of urea was estimated to be 125 mM. Another round of concentration
was done to a final volume of 5 L. The sample was diluted to 18 L
with 50 mM Tris pH 8.0, 500 mM NaCl, 1 mM DTF, 1 mM MnCl.sub.2 and
concentrated again to 6.5 L. The pH was at this time 8.1. The
sample was spun 45 minutes at 7000 rpm and the final pellet was
saved for analysis. After centrifugation, a dilution step with 50
mM Tris pH 8.0, 1 mM DTT, was performed to obtain crystals. The
dilution was to 30 L using a peristaltic pump, with mixing, at room
temperature. The flow rate of dilution was estimated to be 50
ml/min. This dilution took about 9 hours. Crystals were harvested
by centrifugation and the supernatant was saved for analysis. The
crystals were washed three times with 50 mM Tris, 100 mM NaCl pH 8
and resuspended in 50 mM Tris, 100 mM NaCl pH 8. The crystals were
stored at 4.degree. C.
Example 5. Crystallization of Oxalate Decarboxylase from Cell
Extracts Using High Salt
[0143] (1) Crystallization by Solubilizing Protein-Containing
Pellets Using High Salt Concentration Followed by Concentration and
Dilution
[0144] Frozen pellets (465 g) of Example 1 were solubilized in 2.3
L 100 mM Tris, 1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours
at room temperature, forming soluble oxalate decarboxylase. The
sample was spun at 7000 rpm for 45 minutes and supernatant was
recovered. The final volume was 2.15 L and the protein
concentration measured was 24.14 mg/ml. The sample was concentrated
to 550 ml by tangential flow filtration (10 kD Pall) for an hour,
and then diluted to 2,750 L with 100 mM Tris pH 8.0, 1 mM
L-cysteine HCl over 30 minutes at a flow rate of 73 ml/min and
stirring for an hour at room temperature. Crystals were allowed to
form overnight in the cold room. Crystals were harvested by
centrifugation, and the supernatant was saved for analysis. The
crystals were washed three times with 100 mM Tris. 100 mM NaCl pH 8
and then resuspended in 100 mM Tris, 100 mM NaCl pH 8. The crystals
were stored at 4.degree. C. Recombinant B. subtilis is OXDC
purified from E.coli expression medium exhibits a specific activity
of about 50-60 U/mg under standard assay conditions (see Example
15).
[0145] (2) Crystallization by Solubilizing Pellets Using High Salt
Concentration Followed by Concentration and Dialysis
[0146] Frozen pellets (510 g) of Example 1 were solubilized in 3 L
100 mM Tris, 1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours
at room temperature. The sample was spun at 7000 rpm for 30 minutes
and supernatant was recovered. The sample was concentrated to 500
ml by tangential flow filtration (10 kD Pall) for an hour, and then
dialyzed against 100 mM Tris pH 8.0 with stirring. Crystals were
allowed to form overnight in the cold room. Crystals were harvested
by centrifugation, and the supernatant was saved for analysis. The
crystals were washed three times with 100 mM Tris, pH 8.0 and then
resuspended in 100 mM Tris pH 8.0. The crystals were stored at
4.degree. C. The crystal yield was 60%.
[0147] (3) Crystallization by Solubilizing Pellets Using High Salt
Concentration Followed by Concentration and Dialysis
[0148] Frozen pellets (510 g) of Example 1 were solubilized in 3 L
100 mM Tris, 1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours
at room temperature. The sample was spun at 7000 rpm for 30 minutes
and supernatant was recovered. The sample was concentrated to 500
ml by tangential flow filtration 10 kD Pall) for an hour, and then
dialyzed against 100 mM Tris pH 7.5 with stirring. Crystals were
allowed to form overnight in the cold room. Crystals were harvested
by centrifugation, and the supernatant was saved for analysis. The
crystals were washed three times with 100 mM Tris, pH 7.5 and then
resuspended in 100 mM Iris pH 7.5. The crystals were stored at
4.degree. C. The crystal yield was 67%.
[0149] (4) Crystallization by Solubilizing Pellets Using High Salt
Concentration Followed by Concentration and Dialysis
[0150] Frozen pellets (510 g) of Example 1 were solubilized in 3 L
100 mM Tris, 1 mM HCL, 0.5 M NaCl, pH 8.0, for 2 hours at room
temperature. The sample was spun at 7000 rpm for 30 minutes and
supernatant was recovered. The sample was concentrated to 500 ml by
tangential flow filtration (10 kD Pall) for an hour, and then
dialyzed against 100 mM Tris pH 7.0 with stirring. Crystals were
allowed to form overnight in the cold room. Crystals were harvested
by centrifugation, and the supernatant was saved for analysis. The
crystals were washed three times with 100 mM Tris, pH 7.0 and then
resuspended in 100 mM Tris pH 7.0. The crystals were stored at
4.degree. C. The crystal yield was about 80%.
[0151] (5) Crystallization by Solubilizing Pellets Using High Salt
Concentration Followed by Concentration and Dialysis
[0152] Frozen pellets (510 g) of Example 1 were solubilized in 3 L
100 mM Tris, 1 mM HCL, 0.5 M NaCl, pH 8.0. for 2 hours at room
temperature. The sample was spun at 7000 rpm for 30 minutes and
supernatant was recovered. The sample was concentrated to 500 ml by
tangential flow filtration (10 kD Pall) for an hour, and then
dialyzed against 100 mM. sodium citrate buffer pH 6.5 with
stirring. Crystals were allowed to form overnight in the cold room.
Crystals were harvested by centrifugation, and the supernatant was
saved for analysis. The crystals were washed three times with 100
mM sodium citrate buffer, pH 6.5 and then resuspended in 100 mM
sodium citrate buffer pH 6.5. The crystals were stored at
-4.degree. C. The crystal yield was about 70%,
[0153] (6) Crystallization by Solubilizing Pellets Using High Salt
Concentration Followed by Concentration and Dialysis
[0154] Frozen pellets (510 g) of Example 1 were solubilized in 3 L
100 mM Tris, 1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours
at room temperature. The sample was spun at 7000 rpm for 30 minutes
and supernatant was recovered. The sample was concentrated to 500
ml by tangential flow filtration (10 kD Pall) for an hour, and then
dialyzed against 100 mM sodium citrate butter pH 6.0 with stirring.
Crystals were allowed to form overnight in the cold room. Crystals
were harvested by centrifugation, and the supernatant was saved for
analysis. The crystals were washed three times with 100 mM sodium
citrate buffer, pH 6.0 and then resuspended in 100 mM sodium
citrate butter pH 6.0. The crystals w ere stored at 4.degree. C.
The crystal yield was about 60%.
[0155] (7) Crystallization of OXDC from Cell Paste After
Homogenization and Solubilization
[0156] Cells were resuspended in a ratio of 1 kg cell paste per 3 L
buffer containing 100 mM Tris pH 7.5, 500 mM NaCl, 5 mM cysteine,
and 1 mM manganese chloride. Cell suspension was blended (50-60
rpm) overnight at 4.degree. C. The cell suspension was passed
through a pre-cooled homogenizer two times on ice. The efficiency
of cell lysis was checked under the microscope and unbroken cell
suspension was used as a control. The suspension was made up to 10
L and mixed with overhead stirrer for 3 hrs at room temperature.
The crude extract was then centrifuged at 7,000 rpm for 30 min at
4.degree. C. and supernatant was recovered. Supernatant and pellet
were saved for characterization by SDS-PAGE. The expression of OXDC
was analyzed by SDS-PAGE. The majority of the OXDC was found in the
supernatant. The final volume was 10 L and the protein
concentration measured was 34 mg/ml. The sample was concentrated to
3.5 L by tangential flow filtration (10 kD Pall) for an hour, and
then diluted using crystallization buffer (100 mM Tris. 100 mM NaCl
pH 7.5) with stirring for an hour at room temperature. Crystals
were harvested by centrifugation, and the supernatant was saved for
analysis. The crystals were washed three times with 100 mM Tris,
100 mM NaCl pH 7.5 and then resuspended in 100 mM Tris and 100 mM
NaCl, pH 7.5. The crystals were stored at 4.degree. C.
Example 6. Crystallization of OXDC from Soluble Protein
[0157] OXDC was expressed in E. coli in a shaking flask. The cells
were lysed by microfluidizer with 25 mM Tris-HCl buffer, pH 8.0,
100 mM NaCl containing 25 U/ml of DNase I. The cell lysate was
incubated at room temperature for one hour to allow OXDC crystals
to form. The crystals were spun down and reconstituted in 100 mM
Tris, 100 mM NaCl pH 8.
Example 7. Crystallization of OXDC by Vapor Diffusion
[0158] Hanging drop crystallization trials were performed using
commercially available sparse matrix crystallization kits: Crystal
Screen (Hampton Research; Aliso Viejo, Calif.), Crystal Screen 2
(Hampton Research), Wizard I (Emerald Biosystems; Bainbridge
Island, Wash.), Wizard II (Emerald Biosystems), Cryo I (Emerald
Biosystems), and Cryo II (Emerald Biosystems).
[0159] 600 .mu.l of reagent was placed in each well. 3 .mu.l of
reagent was dispensed onto a glass microscope coverslip and 3 .mu.l
of OXDC was dispensed into the reagent drop with minimal mixing. Up
to five more drops were made from this 6 .mu.l reagent and OXDC
drop. As the drops were minimally mixed, each of the subsequent
(smaller) drops had a different and unknown ratio of protein to
reagent, thereby increasing the likelihood of obtaining crystals in
a short period of time. The hanging drops were examined for
crystals under a microscope after overnight incubation at room
temperature. A large number of crystallization conditions were
obtained, as shown in Table 1.
TABLE-US-00001 TABLE 1 Crystallization conditions for OXDC in
hanging drops..sup.a Description Precipitant of Crystals 40% (v/v)
Ethanol, 0.1M phosphate citrate, needles pH 4.20, 5% (w/v) PEG 1000
20% (w/v) PEG-3000, 0.1M HEPES, pH 7.50, rods 0.2M NaCl 20% (w/v)
PEG-3000, 0.1M acetate pH 4.5 Rods, plates 20% (w/v) PEG 8000, 0.1M
phosphate-citrate, plates pH 4.20, 0.2M NaCl 10% (w/v) PEG 8000,
0.1M Cacodylate, pH 6.50, rods 0.2M Magnesium acetate 9% (w/v PEG
8000, 0.1M Cacodylate, pH 6.50, needles 0.2M Calcium acetate 40%
(v/v) PEG 400, 0.1M Na/phosphate, needles pH 6.20, 0.2M NaCl 10%
(v/v) PEG 8000, 0.1M Na/phosphate, rods pH 6.20, 0.2M NaCl 20%
(w/v) PEG 2000 MME, 0.1M Tris, pH 7.0 plates .sup.aOXDC
concentration was determined by Bradford assay to be about 1.7
mg/mL.
Example 8. Crystallization of OXDC by Microbatch
[0160] Oxalate decarboxylase could be crystallized by the
microbatch method from a number of crystallization conditions:
[0161] (i) 10 .mu.l of purified OXDC at a concentration of 23.46
mg/ml was mixed with 10 .mu.of 16% PEG 8000. Crystallization
occurred immediately in 2-5 seconds. Big crystals formed with some
precipitates.
[0162] ii) 10 .mu.l of purified OXDC at a concentration of 23.46
mg/ml was mixed with 10 .mu.l of 20% PEG 8000. Crystallization
occurred immediately in 2-5 seconds. Big crystals formed with no
precipitates.
[0163] (iii) 10 .mu.l of purified OXDC at a concentration of 23.46
mg/ml was mixed with 10 .mu.l of 24% PRO 8000. Crystallization
occurred immediately in 2-5 seconds. Smaller cube shaped crystals
formed.
[0164] (iv) 10 .mu.l of purified OXDC at a concentration of 23.46
mg/ml was mixed with 10 .mu.l of 28% PRO 8000. Crystallization
occurred immediately in 2-5 seconds. Very small cube shaped
crystals formed. There was no precipitation.
[0165] (v) 8 .mu.l of purified OXDC at a concentration of 23.46
mg/ml was mixed with 12 .mu.l of 24% PEG 8000. Crystallization
occurred immediately in 2-5 seconds. Very small cube shaped
crystals formed. There was no precipitation.
[0166] (vi) 9 .mu.l of purified OXDC at a concentration of 23.46
mg/ml was mixed with 11 .mu.l of 24% PEG 8000. Crystallization
occurred immediately in 2-5 seconds. Small cube shaped crystals
formed. There was no precipitation.
[0167] (vii) 10 .mu.l of purified oxalate decarboxylase at a
concentration of 23.46 mg/ml was mixed with 10 .mu.l of 24% PEG
8000. Crystallization occurred immediately in 2-5 seconds. Small
cube shaped crystals formed. There was no precipitation.
[0168] (viii) 11 .mu.l of purified oxalate decarboxylase at a
concentration of 23.46 mg/ml was mixed with 9 .mu.l of 24% PEG
8000. Crystallization occurred immediately in 2-5 seconds. Cube
shaped crystals formed. There was no precipitation.
[0169] (ix) 12 .mu.l of purified OXDC at a concentration of 23.46
mg/ml was mixed with 8 .mu.l of 24% PEG 8000. Crystallization
occurred immediately in 2-5 seconds. Cube shaped crystals formed.
There was no precipitation.
Example 9. Activities of Soluble OXDC and OXDC Crystals
[0170] Soluble OXDC was collected as described in Example 5 after
the pellets were solubilized, spun and the supernatant recovered.
OXDC crystals were collected as described in Example 5 after the
crystals were harvested and washed. Activities of soluble OXDC and
OXDC crystals were measured according to Example 15. In one
experiment, the activity of soluble OXDC was 12 Units/mg, and the
activity of OXDC crystal was 35 Units/mg.
[0171] Soluble OXDC is collected as described in any of Examples
2-5 and OXDC crystals are harvested as described in any of Examples
2-8. Activities of soluble and crystal OXDC are measured according
to Example 15. Activities of OXDC crystals can be at least about
100%, 200%, 300%, 400% or 500% of the activities of soluble
OXDC.
Example 10. Cross-Linking of Oxalate Decarboxylase Crystals with
Glutaraldehyde.
[0172] Oxalate decarboxylase crystals prepared according to any of
the Examples 2-8 were cross-linked using glutaraldehyde. After
crystallization, OXDC crystals were concentrated to 20-30 mg/ml.
0.8 ml of 25% glutaraldchyde was added to 20 ml of crystals to make
a solution of 1% glutaraldehyde, and crystals were tumbled for 18
hours at room temperature. Cross-linked crystals were washed five
times with 100 mM Tris, pH 7.00 and resuspended in 10 mM Tris, pH
7.00.
[0173] Specific activities of crystalline OXDC and cross-linked
OXDC (referred to as OXDC-CLEC) were compared (six trials) and it
was shown that cross-linked oxalate decarboxylase crystals retain
more than 30% to more than 50% of the original activity of the
crystalline protein, in various preparations.
[0174] To test the effects of varying concentrations of
glutaraldehyde on enzyme activity, 1 ml aliquots of OXDC crystals
(60 mg/ml) were crosslinked with different concentrations of
glutaraldehyde (from 0.05% to 2%, final concentration) at pH 8.0 at
25.degree. C. for 18 hrs. The crosslinking was terminated by
separation of the crosslinked crystals by centrifugation at 2000
rpm in an Eppendorf tube and then resuspension of the crosslinked
crystals in 1 ml of 100 mM Tris.HCl pH 7.0. The cross-linked OXDC
(OXDC-CLEC) was then washed five times with 100 mM Tris HCl buffer,
pH 7.5, followed by three washes with 10 mM Tris HCl buffer, pH 7.5
(see results in Table 2. below).
Example 11. pH Controlled Solubility of Crosslinked Oxalate
Decarboxylase Crystals
[0175] Solubility of various crosslinked oxalate decaroboxylase
crystals was examined following a decrease in pH from 7.5 to 3.0.
The cross-linked crystals were incubated at 1 mg/ml in 50 mM
glycine-HCl (pH 3.0). Aliquots were removed after 5 hour incubation
at 37.degree. C. with stirring. Soluble protein concentration was
measured at OD.sub.280 nm after separation of the undissolved
crosslinked crystals by centrifugation at 2000 rpm and filtration
of the supernatant through 0.22 .mu.m filter. The results are
described in Table 2 below.
TABLE-US-00002 TABLE 2 Crosslinking of OXDC Crystals with Different
Percentages of Glytaraldehyde and pH-Controlled Solubility of
OXDC-CLEC Sample % Glutaraldehyde % Protein Leaching OxDc-CLEC-1
0.005 100.0 OxDc -CLEC-2 0.010 100.0 OxDc -CLEC-3 0.050 2.2 OxDc
-CLEC-4 0.075 0.0 OxDc -CLEC-5 0.100 0.0 OxDc -CLEC-6 0.200 0.0
OxDc -CLEC-7 1.000 0.0
[0176] These results indicate that a substantially stable
glutaraldehyde OXDC crosslinked crystal is formed in the presence
of at least about 0.05% (final concentration) glutaraldehyde.
[0177] Oxalate decarboxylase crystals, prepared as described in
Examples 2-8, were crosslinked by addition of glutaraldehyde
(Sigma). A 1 ml aliquot of OXDC crystals (30-40 mg/ml) was
crosslinked with 1% glutaraldehyde (final concentration) at pH 8.0
at 25.degree. C. for 18 hrs. The crosslinking was terminated by
separation of the crosslinked crystals by centrifugation at 2000
rpm in an Eppendorf tube and then resuspension of the crosslinked
crystals in 1 ml of 100 mM Tris HCl, pH 7.0. The OXDC-CLEC was then
washed five times with 100 mM Tris-HCl buffer, pH 7.0, followed by
three washes with 10 mM Tris-HCl buffer, pH 7.0. The pH activity
profile of OXDC-CLEC was assayed by measuring the activity of the
crystals as described in Example 15 using various buffers and pHs:
50 mM glycine-HCl buffer at pH 2.0 and 3.0; 50 mM succinate buffer
at pHs 4.0, 5.0, and 6.0; and 50 mM Tris buffer at pH 7.0. The
activity level at each pH was performed twice and the average
activity was calculated. The results, shown in FIG 1, indicate that
OXDC-CLEC is most active than its soluble counterpart between pH
3.5 and 6.0. The uncrosslinked crystals showed much higher
activity, ranging from about 50% to about 200%, 300% or 400% more,
than the soluble form of oxalate decarboxylase at different pH.
Example 13. Oxalate Decarboxylase Therapy in Animal Model for
Enteric Hyperoxaluria
[0178] Rat Model for Enteric Hyperoxaluria, Dose Range Study: Male
Sprague Dawley (SD) rats fed a diet high in oxalate constitute a
suitable animal system for the study of enteric hyperoxaluria. In
this study, administration of 1.1% dietary potassium oxalate
resulted in a 5- to 10-fold increase in urinary oxalate.
[0179] Twenty Sprague Dawley (SD) rats less than 35 days old and
weighing 100-120 grams were randomly divided into a control group
and experimental groups (five rats per group). Rats were acclimated
for 7 days to individual metabolic cages (LabProducts, Inc.;
Seaford, Del.) prior to treatment. During this period, rats were
provided ad libitum with supplemented acidified water and fed a
synthetic diet having 1.1% potassium oxalate and a low (0.5%)
concentration of calcium (Research Diets TD89222PWD: Harlan Teklad;
Madison, Wis.). Rats were maintained on this diet for the duration
of the treatment.
[0180] Following the acclimation period, three different doses of
recombinant oxalate decarboxylase, formulated as cross-linked
crystals with 1% glutaraldehyde (see, e.g., Example 9), were
administered to the test rats for 4 consecutive weeks. The crystals
were administered orally as a freeze/dried food enzyme mixture (5,
25 and 80 mg of OXDC-CLEC slurry in 10 mM Tris HCl pH 7.0, each
separately mixed with 15 g food and freeze dried; each morning,
food containers were re-filled with .about.20 g of food/enzyme
mixture). Prior to the treatment, rats were randomly divided
between a control group and experimental group based on their basal
urinary oxalate.
[0181] Analysis of urine samples: 24 hour urinary samples were
collected in metabolic cages over acid (250 .mu.l of 6N
hydrochloride acid was mixed with urine sample collected during 24
h) in order to minimize the spontaneous breakdown of urinary
ascorbic acid to oxalate. Samples were stored at -70T until further
analysis. Daily diuresis and multiple 24 (hour) urine samples were
collected for oxalate and creatinine measurements. Assays for
oxalate and creatinine are described in Example 15. Urinary
excretion of oxalate and creatinine were expressed as .mu.mol of
oxalate and creatinine detected in 24 h urine samples. All data
were analyzed statistically using the Student's t-test.
[0182] As shown in FIG. 2, oral administration of OXDC-CLEC to SD
rats with chronic hyperoxaluria resulted in a sustained reduction
of urinary oxalate from day 4 of the treatment. Maximal continuous
reduction between 25-40% was recorded in the highest dose group (80
mg (OXDC-CLEC). The lower doses of 5 mg and 25 mg OXDC-CLEC
produced smaller reduction in urinary oxalate (up to 30 in the 25
mg group and 20% in the 5 mg group, respectively). The doses of 25
mg and 80 mg produced significant is reduction during, all tested
days (except day 21 for 25 mg group), while the lowest dose of 5 mg
had a minimal effect which was not significant. This result reveals
a dose dependant effect of OXDC-CLEC therapy.
Example 14: Oral Oxalate Decarboxylase therapy in animal model for
primary Hyperoxaluria
[0183] Mouse Model For Type 1 Primary Hyperoxaluria: AGT1 knockout
mice lack the liver peroxisomal enzyme alanine:glyoxylate
aminotransferase, a deficiency that causes primary hyperoxaluria,
Type 1. Chimeric mice were bred to homozygosity in (C57B16 and
129/sv background strains. All homozygous Agxt mice showed mild
hyperoxaluria 1-2 mmol/L), 5-10 fold urinary oxalate elevation over
normal values, as compared to wildtype (0.2 mmol/L). It was also
found that 30-50% of males, and 0% of females developed mild
nephrocalcinosas and calcium oxalate calculi in the urinary tract
later in life (4-7 months of age). Interestingly, when the mutation
was analyzed in a homogeneous (C57B16 strain, hyperoxaluria was not
associated with development of urinary stones in either sex;
underscoring the phenotypic variability typically observed in this
disease.
[0184] A total of 44 male mice (strain ATG1 KO/129sv, developed by
Dr, Salido, La Laguna Tenerife, Spain) were used in these
experiments. Mice were randomly divided between a control group and
three experimental groups. Mice weighed 20-25 grams and were less
than 6 months of age.
[0185] The AGT1 KO (129sv) mice were challenged with ethylene
glycol (EG) to provoke severe hyperoxaluria and the formation to
calcium oxalate deposits in the kidney parenchyma. EG is a common
alcohol that is metabolized in the liver to oxalate. Usually, after
2-6 weeks of EG challenge, AGT1 KO mice in the 129/sv background
show signs of impaired kidney function as determined by (i) the
variable excretion of oxalate in the urine, (ii) decreased
creatinine clearance, and (iii) nephrocalcinosis that ultimately
leads to renal failure and death.
[0186] Mice were acclimated for 7 days prior to treatment to
individual metabolic cages (Teeniplast USA Inc. Exton, Pa., USA),
and were fed standard breeder diet (17% proteins, 11% fat, 53.5%
carbohydrate) containing less than 0.02-0.08% oxalate and
approximately 0.5-0.9% calcium. After the acclimation period, mice
were divided into four groups; three treatment groups were fed
oxalate decarboxylase-CLEC mixed with food, while mice in the
matched control group received the same diet without addition of
the test article. Drinking water supplemented with 0.7% EG was
provided to all mice ad libitum from the first day of treatment
until the end of the study. After several days of challenge, mice
excrete approximately 3-6 mmol/L oxalate in their urine per day,
which is approximately 10-20 fold more than wild type
(unchallenged) mice.
[0187] Administration of OXDC-CLEC Enzyme; Dose Range Study: A
total of 44 male mice from strain AGT1 KO/129sv were used in a dose
study of OXDC-CLEC. The mice weighed 20-25 grams and were less than
6 months of age. The mice were challenged with EG, and then were
randomly divided between a control group and an experimental group.
The efficacy of three different doses of recombinant oxalate
decarboxyl formulated as cross-linked crystals (1% glutaraldehyde;
see Example 9), was monitored over four consecutive weeks. The term
"OXDC-CLEC as used in this Example refers to recombinant oxalate
decarboxylase, formulated as cross-linked crystals (1%
glutaraldehyde, as described in Example 9. OXDC-CLEC was orally
administered as a freeze/dried food enzyme mixture at nominal doses
of 5, 25 and 80 mg/day. An adequate amount of enzyme slurry in 10
mM Tris-HCl buffer (pH 7.0) was mixed with 3.5 g food and freeze
dried. Each morning, food containers were re-filled with .about.7 g
of the food/enzyme mixture.
[0188] Assessment of the efficacy of OXDC-CLEC: The efficacy of the
enzyme therapy was monitored by urinary oxalate reduction,
prevention of calcium oxalate deposition in kidney parenchyma, and
survival. At the end of the study, surviving mice were sacrificed
and blood samples taken for creatinine measurement.
[0189] Analysis of urine samples: 24 h urinary samples were
collected in metabolic cages over acid (50 .mu.l of 6 N
hydrochloride acid per 3-4 ml of urine) in order to minimize the
spontaneous breakdown of urinary ascorbic acid to oxalate. Urine
samples were stored at -20.degree. C. until further analysis. Daily
diuresis and multiple 24 h urine samples were collected and
analyzed for oxalate and creatinine levels. Assays for oxalate and
creatinine are described in Example 15. Urinary excretion of
oxalate and creatinine was expressed as .mu.mol of oxalate or
creatinine excreted in 24 h urine sample (mL). Data were analyzed
statistically using Student's t-test.
[0190] Analysis of blood samples: At the end of the study, mice
were sacrificed and serum samples were collected. For scrum
creatinine measurement, a slightly modified version of the Jaffe
reaction method (see, e.g., the Creatinine Microassay Plate Kit
from Oxford Medical Research. Inc.; Slot, Scand J. Clin. Lab.
Invest. 17:381, 1965; and Heinegard D, Clin. Chim. Acta 43:305,
1973) was used. 80 .mu.l of undiluted serum samples was mixed with
800 .mu.l of picric alkaline in the cuvettes and incubated for 30
minutes at room temperature. Color development was measured
spectrophotometrically at 510 nm; 33.3 .mu.l of 60% acetic acid was
then added to quench the unspecific reaction. Samples were
thoroughly mixed and after 5 minutes incubation at room temperature
were read again at 510 nm. Final absorbance is present as a
difference of two readings. Serial dilutions of 1 mM creatinine
solution was used for a standard curve.
[0191] Kidney function was monitored indirectly by measuring
creatinine clearance. Creatinine clearance is expressed as
excretion rate of creatinine (U.sub.er.times.V), where U.sub.er
represents the concentration of creatinine (.mu.mol/L) in a urine
sample, divided by plasma creatinine (P.sub.er). This is
represented as:
C.sub.er=(U.sub.er.times.V)/P.sub.er=mL/h
[0192] The safety parameters monitored during the study were
mortality, food and water intake, and body weight. Mortality checks
and cage side clinical observations were performed once daily
throughout the study. Food intake was measured daily and water
intake was recorded weekly. Body weights of all animals were
recorded at the beginning of the study and at the end of the
study.
[0193] As shown in FIG. 3, oral administration of OXDC-CLEC to
EG-challenged AGT1 KO (129sv) mice resulted in significant
reduction of urinary oxalate levels from day 4 of the treatment
until the end of the study when compared with matched untreated
control mice. A reduction of between 30 and 50% was observed in all
three treatment groups, with the maximal reduction observed in the
highest dose group (80 mg of OXDC-CLEC). Lower closes of 25 mg and
5 mg of OXDC-CLEC produced reduction in urine oxalate up to
35%.
[0194] The results are analyzed by unpaired two tail Student's
t-test. At the beginning of the study, each dosage group had n=11
mice, but several mice died during the course of the study due to
the ethylene glycol challenge. The results presented include only
mice that were alive at the particular day of urine oxalate
measurements. The bell shaped curve of urinary oxalate excretion in
the control group is best explained by the observation that initial
elevation of urinary oxalate leads to nephrocalcinosis, reduced
renal filtering function, and consequent lowered excretion rate of
oxalate over time, and eventual death in the worst case.
[0195] Assessment of the renal function by creatinine clearance
measurement. At the end of the study, all animals that survived 4
weeks of EG challenge were sacrificed and blood was collected to
measure plasma creatinine and creatinine clearance. All 11 mice in
tire 80 mg dosage group survived the 4 weeks of EG challenge; 8 of
11 mice in the 25 mg OXCD-CLEC dosage group survived; 8 of 11 mice
in the 5 mg OXCD-CLEC dosage group survived; and 7 of 11 mice in
the control group survived. For serum creatinine measurement, the
slightly modified Jaffe reaction method described above was used
(see, e.g., the Creatinine Microassay Plate Kit from Oxford Medical
Research, Inc.; Slot, Scand J. Clin. Lab. Invent. 17:381, 1965; and
Heinegard. Clin. Chim. Acta 42:305, 1973).
[0196] The efficacy of orally administered OXDC-CLEC on kidney
function was assessed by measuring creatinine clearance. Creatinine
clearance in the mice that survived the entire one month study
period is shown in FIG. 4. When compared with the control group,
creatinine clearance was significantly higher in surviving mice
that received 80 mg of OXDC-CLEC (p<0.05).
[0197] All mice (11/11) in the 80 mg OXDC-CLEC treatment group
survived the 4 week EG challenge regimen, while only 7 mice (7/11)
in the control group survived the regimen. The kidney filtration
rate, assayed by creatinine clearance, was also significantly lower
than in the 80 mg dosage group (FIG. 4).
[0198] Kidney histopathology analysis: Mouse kidneys were routinely
processed for paraffin embedding and positioned in order to obtain
complete cross sections of the kidneys. Each kidney was cut in 12
serial sections at 4 .mu.m per kidney and stained with either
hemotoxylin and eosin for routine histological examination, or by
specific Yasue metal substitution histochemical method to detect
the presence of calcium oxalate crystals in the renal tissue.
Slides were examined under the microscope using 20.times.
magnification and examiner scored sections under 4-category scale,
applying the same criteria to each of the anatomic areas in the
kidney (cortex, medulla and papilla). The scoring was (i) none (no
oxalate crystals in any field); (ii) minimal (1-5 crystals in any
field); (iii) moderate (6-10 crystals in any field); and (iv)
severe (all fields with multiple collections of crystals).
[0199] Representative images of kidney tissue from both treatment
and control animals are shown in FIGS. 5A-5C. Yasue-positive
calcium oxalate crystals were visible in the kidney parenchyma at
20.times. magnification. All mice treated with 80 mg of OXDC-CLEC
treatment group had normal healthy kidney with no traces of calcium
oxalate deposits (FIG. 5A). Moderate nephrocalcinosis (FIG. 5B) and
severe nephrocalcinosis (FIG. 5C were observed in the control group
and in some mice from low dose treatment groups. The white arrows
indicate calcium oxalate deposits and the gray arrows in FIG. 5C
indicate large area with interstitial fibrosis.
[0200] Histological examination of the kidneys using the specific
Yasue metal substitution method showed deposits primarily in the
cortex and medullar part of the kidneys. In the case of severe
nephrocalcinosis (FIG. 5C), calcium oxalate deposits were randomly
distributed in the kidneys. Signs of fibrosis and inflammation were
also visible and the morphology of glomeruli was changed with the
occasionally formation of calcium oxalate deposits within the
glomeruli. All mice (11/11) from the 80 mg dosage group had normal
morphology of the kidneys upon necropsy with no traces of calcium
oxalate deposition either in the kidney or in the urinary bladder.
In contrast, 100% (11/11) of mice from untreated control group had
calcium oxalate deposits. In the low treatment groups (25 mg or 5
mg of OXDC-CLEC), 63% (7/11) of mice had calcium oxalate deposits
in the kidneys. These results demonstrate a positive, dose
dependent effect of the oral therapy with OXDC-CLEC in the
EG-challenged AGT1 KO mice on the reduction of hyperoxaluria and
the prevention of calcium oxalate crystal deposition in the
kidneys. A summary of the histopathological analysis from all 4
groups of mice is presented in Table 3.
TABLE-US-00003 TABLE 3 Severity of Nephrocalcinosis and Number of
Mice Affected in Treated and Control Group Following Oral OXDC-CLEC
Treatment. NUMBER OF MICE WITH NEPHROCALCINOSIS CALCIUM OXALATE
DOSE MOD- DEPOSITS GROUPS* SEVERE ERATE MINIMAL NONE (%) CONT 4 (4
died) -- 7 -- 100 n = 11 80 mg -- -- -- 11 0 n = 11 25 mg 3 (3
died) 2 2 4 63 n = 11 5 mg 3 (3 died) -- 4 4 63 n = 11
[0201] The efficacy of the oral OXDC-CLEC treatment on the
frequency of urinary stone formation in control mice and mice from
three different treatment groups was also evaluated. Two main types
of calculi were found in the urinary bladder from EG AGT1 KO mice:
calcium oxalate monohydrate stones and calcium oxalate dihydrate
stones. Grossly visible bladder stones were present in 36% (4/11)
of the mice in the control group and in 19% (2/11) of the mice in
the two lower treatment groups. No bladder stones were observed in
the 80 mg high dose OXDC-CLEC group. The X-ray diffraction analysis
showed that stones with whitish, rough budding surface are mostly
composed of calcium oxalate monohydrate, while relatively large
stones with sharp crystal angles correspond to calcium oxalate
dehydrate.
[0202] Survival rate analysis by Kaplan-Meier estimator. The effect
of OXDC-CLEC treatment on survival rate of mice challenged with
ethylene glycol was analyzed using the Kaplan-Meier method where
survival of subjects that died in the certain time point is divided
by the number of subjects who were still in the study at the time.
This method graphically illustrates the difference between the
groups in the study (FIG. 6). Often statistical programs such as
Kaleida graph and STATS are used for calculations.
[0203] Oral treatment with OXDC-CLEC increased the survival rate of
EG-challenged AGT1 KO mice as compared to that of the matched
controls. All mice (11/11) from the 80 mg OXDC-CLEC treatment group
survived the 30 day study period without signs of sickness, while 4
of 11 mice from the control group had severe nephrocalcinosis with
development of urinary stones.
[0204] Since OXDC-CLEC is intended for oral administration, the
potential for adverse reactions of the crosslinked crystals on
gastrointestinal (GI) tissues was evaluated. The digestive tracts
of treated EG-challenged AGT1 KO mice were examined macroscopically
and histological analysis with a hemotoxylin-eosin stain was
performed on different parts of the GI tract including the stomach
(corpus and antrum) and small bowel (jejunum and ileum). The
evaluation confirmed that four weeks of oral treatment with
OXDC-CLEC was well-tolerated and did not cause structural or
morphological changes in the GI tract. Similar results were
observed (or the large bowel.
[0205] Summary of the dose range study in EG-challenged AGT1 KO
mice orally treated with oxalate decarboxylase-CLEC. Oral treatment
with OXDC-CLEC in a primary hyperoxaluria animal model was safe and
efficacious. In summary, four weeks of oral treatment with
OXDC-CLEC reduced urinary oxalate by 30-50%. Significant and
sustained reduction was recorded with each of the three doses of
the test article evaluated. Four weeks of oral treatment prevented
calcium oxalate deposition in the kidney parenchyma at the highest
treatment dose. Four weeks of oral treatment increased the survival
rate at the two lower doses and prevented animal mortality at the
highest dose studied. Finally, the 4 week treatment regimen, did
not produce macroscopic or microscopic changes in the GI tract.
Example 15. Assays
[0206] Protein Concentration Determination: The concentration of
oxalate decarboxylase was determined by measuring absorbance at 280
nm. The absorbance of 1.36 optical density (OD) was considered as 1
mg/ml.
[0207] OXDC: Activity Assay: The modified Sigma Aldrich protocol
(Enzymatic Assay of Oxalate Decarboxylase EC 4.1.1.2) was used to
measure the activity of soluble oxalate decarboxylase, oxalate
decarboxylase crystals, and cross-linked oxalate decarboxylase
crystals (OXDC-CLEC). This is an indirect two-step activity assay;
in the first reaction OXDC converts the substrate oxalate to
formate and carbon dioxide. In the second reaction, formate
hydrogen is stochiometrically transferred to NAD to form NADH by
formate dehydrogenase. The ensuing concentration of NADH is
quantified spectrophotomerically at 340 nm. Unit (u) of enzymatic
activity is defined as follows: one Unit of oxalate decarboxylase
will form 1.0 .mu.mol of oxalate to formate and carbon dioxide per
minute at pH 5 and 37.degree. C.
[0208] Assay samples were normalized at a concentration of
0.007-0.02 and 0.009-0.03 mg/mL for OXDC crystals and OXDC-CLEC,
respectively, in 5 mM potassium phosphate buffer pH 7.0 with 1 mM
DTT (Sigma). Protein concentrations were determined by absorbance
at 280 nm. Formate dehydrogenase (FDH) at 40 U/mL was prepared in
cold diH.sub.2O prior to use and kept on ice. All other reagents
were kept at room temperature. Reagents were added to 2 ml
microtubes with stir bar in the following order: 300 .mu.L of 100
mM potassium phosphate and 200 .mu.L of potassium oxalate pH 4.0
were mixed and warmed for 5 min in a water bath at 37.degree. C.,
then 100 .mu.L, of 5 mM potassium phosphate with 1 mM DTT was added
to the blank vial and 100 .mu.L of diluted oxalate decarboxylase
was added to all other vials. After 2 minutes, the reaction was
stopped with 130 mM potassium phosphate dibasic. In the second
reaction, 25 .mu.L of NAD solution was added to 100 .mu.L of FDH
solution, and the incubation was continued for another 20 min. All
samples were then centrifuged for 1 min at 16,100 rpm. Reaction
mixtures were then transferred to 1.5 mL UV cuvettes, and
absorbance at 340 nm was determined and recorded using a Shimadzu
BioSpec (Shimadzu Scientific Instruments, Columbia, Md.).
[0209] Enzyme Specific Activity was Calculated as Follows:
Units / mL OXDC = [ Abs .times. Total volume ( 1.725 mL ) .times.
dil . factor ] ( Ext . NADH = 6.22 ) ( Vol . OXDC = 0.1 mL ) (
assay time = 2 / 5 min ) ##EQU00001##
[0210] In a specific experiment, cross-linked crystals of oxalate
decarboxylase (OXDC-CLEC) (diamond shaped crystals cross-linked
with 1% glutaraldehyde by tumbling overnight; see Example 9) were
compared to OXDC crystals. OXDC-CLEC retained 50% of the activity
of the corresponding crystalline OXDC preparation.
[0211] Oxalate determination by colorimetric method: Oxalate
colorimetric kit for quantitative determination of oxalate in the
urine were purchased from Trinity Biotech USA (St. Louis, Mo.) or
Greiner Diagnostic AG (Dennliweg 9, Switzerland). The urine samples
were diluted and treated according to the manufacturer's
instruction. The assay comprises two enzymatic reactions: (a)
oxalate is oxidized to carbon dioxide and hydrogen peroxide by
oxalate oxidase, and (b) the hydrogen peroxide thus formed reacts
with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and
3-(dimethylamino)benzoic acid (DMAB) in the presence of peroxidase
to yield an indamine dye which can be detected by absorbance at 590
nm. The intensity of the color produced is directly proportional to
the concentration of oxalate in the sample. Urine oxalate values
are calculated from standard curve.
[0212] Creatinine determination by colorimetric method: Creatinine
colorimetric kits for the quantitative determination of creatinine
in the urine were purchased from Quidel Corporation (San Diego,
Calif.: METRA Creatinine Assay kit) or Randox Laboratories (Antrim,
United Kingdom). The assay is based on the principle that
creatinine reacts with picric acid in alkaline solution to form a
product that has an absorbance at 492 nm. The amount of complex
formed is directly proportional to the creatinine concentration. 24
h rat urine samples collected from single metabolic cages were
diluted 15 fold with double distilled water. 20 .mu.l of diluted
urine sample was mixed with 20 .mu.l picric acid/sodium hydroxide
(1:1). Absorbance at 492 nm was measured after incubating for 2
minutes incubation at room temperature. Urinary creatinine values
were calculated from standard curve.
Example 16. OXDC Therapy for the Treatment of Oxalate-Associated
Disorders in Humans
[0213] Humans in need of treatment or prevention of an
oxalate-associated disorder such as hyperoxaluria can be treated by
oral administration of cross-linked oxalate decarboxylase crystals.
The oxalate decarboxylase crystals are administered at an
approximate dose of 10 .mu.g/kg to 25 mg/kg; 1 mg/kg to 25 mg/kg,
or 5 mg/kg to 100 mg/kg, as determined by a treating clinician and
depending on the severity of the symptoms and the progression of
the disease. The oxalate decarboxylase crosslinked crystals are
administered 1, 2, 3, 4, or 5 times daily, or are administered less
frequently, such as once or twice a week. This oral administration
of OXDC-CLEC results in a decrease in urinary oxalate levels of at
least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or
more.
Example 17. Recombinant Production of Oxalate Decarboxylase
[0214] In Human Embryonic Kidney (HEK293) cells: DMA encoding OXDC
(e.g., SEQ ID NO: 1 or 2) is cloned into a suitable expression
vector. After sequence confirmation, the vector can be linearized
and transformation of the linearized vector to pre-seeded HEK293
cells may be carried out using Lipofectamine.TM. 2000 Transfection
Reagent in a 6 cm diameter dish. The transfection reaction is
cultured overnight in appropriate medium, and then transformants
are selected in medium supplemented with 0.5 g/L of neomycin.
Stably transfected HEK293 cell clones are identified after growth
in neomycin-containing medium for up to 3 weeks. The clones are
then isolated and propagated, and used for OXDC expression.
[0215] In Chinese Hamster Ovary (CHO) cells: DNA encoding OXDC gene
is cloned into a suitable expression vector. Cultured CHO lec
3.2.8.1 cells are then detached by trypsin digestion and harvested
by centrifugation. The cells are then suspended in Electroporation
phosphate buffered saline butter (EPBS) to a final concentration of
.about.1.times.10.sup.7/ml, and transformed with the linearized
vector by electroporation. After overnight culture, the medium is
exchanged with medium supplemented with 0.5 g/L neomycin.
Successive changes of medium are performed to screen for stably
transfected CHO cell clones. Once the stably transfected cell
clones are established and propagated, the cells are used for OXDC
expression.
[0216] In Pichia Pastoris: DNA encoding the OXDC gene is cloned
into a suitable expression vector. After sequence confirmation, the
vector can be linearized then transformed into a Pichia Pastoris
host cell (see, Whittaker et al. J. Biol. Inorg, Chem. 7:136-145,
2002). Transformants are selected with Zeocin, expanded in buffered
glycerol complex medium (BMGY), and induced with methanol. OXDC may
then be isolated from the culture medium.
[0217] In Saccharomyces cerevisiae: The synthetic OXDC, gene can be
cloned into a suitable expression vector containing, for example,
the Gal1 promoter (pGal) and terminator sequences for expression.
After sequence confirmation, the expression vector is transformed
into the competent Saccharomyces cerevisiae W303-1A by
electroporation. The transformants are screened and propagated
before use for OXDC expression.
[0218] In insect cells: DNA encoding OXDC may be cloned into a
suitable expression vector, such as, e.g., a baculovirus system.
After sequence confirmation, the vector may be transformed into
competent DH10Bac E. coli cells, and E. coli cells containing the
recombinant bacmid can be screened and verified. The recombinant
bacmid DNA is isolated and used to transfect insect Sf9 cells using
reagents such as Cellfectin.TM. reagent (Invitrogen, Carlsbad,
Calif.). The recombinant baculovirus particles can then be
isolated, propagated, and titered before use to infect Sf9 cells
for OXDC expression. In E. coli: DNA encoding OXDC is cloned into a
suitable E. coli expression vector. After sequence confirmation,
the vector is transformed into competent E. coli BL21 or if
necessary E. coli Origami B (DE3), which allows the formation of
disulfide bonds in the recombinant protein expressed in this
strain. The transformants are screened by growing the transformants
on nutrient plates containing antibiotics and verified by colony
HCR using OXDC gene specific primers. The transformants are then
cultured in the liquid medium and induced with
isopropyl-beta-D-thiogalactopyranoside (IPTG) for OXDC
expression.
Example 18. Sequences
[0219] Collybia velutipes sequence (SEQ ID NO:1) to express in
Candida boidinii. Two Not I sequences are underlined; the ATA
triplet in bold is a spacer codon; the double-underlined sequence
is alpha mating factor sequence optimized for C. Boidinii; the OXDC
coding sequence is depicted in lower-case letters.
TABLE-US-00004 1 ##STR00001## 50
TTATTTGCTGCTTCTTCTGCTTTAGCTGCTCCAGTTAATACTACTACTGA 100
AGATGAAACTGCTCAAATTCCAGCTGAAGCTGTTATTGGTTATTCTGATT 150
TAGAAGGTGATTTTGATGTTGCTGTTTTACCATTTTCTAATTCTACTAAT 200
AATGGTTTATTATTTATTAATACTACTATTGCTTCTATTGCTGCTAAAGA 250
AGAAGGTGTTTCTTTAGAAAAAAGAGAAGCTGAAGCTatgtttaataatt 300
ttcaaagattattaactgttattttattatctggttttactgctggtgtt 350
ccattagettctactactactggtactggtactgctactggtacttctac 400
tgctgctgaaccatctgctactgttccatttgcttctactgatccaaatc 450
cagttttatggaatgaaacttctgatccagctttagttaaaccagaaaga 500
aatcaattaggtgctactattcaaggtccagataatttaccaattgattt 550
acaaaatccagatttattagctccaccaactactgatcatggttttgttg 600
gtaatgctaaatggccattttctttttctaaacaaagattacaaactggt 650
ggttgggctagacaacaaaatgaagttgttttaccattagctactaattt 700
agcttgtactaatatgagattagaagctggtgctattagagaattacatt 750
ggcataaaaatgctgaatgggcttatgttttaaaagggtctactcaaatt 800
tctgctgttgataatgaagggagaaattatatttctactgttggtccagg 850
tgatttatggtattttccaccaggcattccacattctttacaagctactg 900
ctgatgatccagaaggttctgaatttattttagtttttgattctggtgct 950
tttaatgatgatggtacttttttattaactgattggttatctcatgttcc 1000
aatggaagttattttaaaaaattttagagctaaaaatccagctgcttggt 1050
ctcatattccagctcaacaattatatatttttccatctgaaccaccagct 1100
gataatcaaccagatccagtttctccacaagggactgttccattaccata 1150
ttcttttaatttttcttctgttgaaccaactcaatattctggtgggactg 1200
ctaaaattgctgattctactacttttaatatttctgttgctattgctgtt 1250
gctgaagttactgttgaaccaggtgctttaagagaattacattggcatcc 1300
aactgaagatgaatggactttttttatttctggtaatgctagagttacta 1350
tttttgctgctcaatctgttgcttctacttttgattatcaaggtggtgat 1400
attgcttatgttccagcttctatgggtcattatgttgaaaatattggtaa 1450
tactactttaacttatttagaagtttttaatactgatagatttgctgatg 1500
tttctttatctcaatggttagctttaactccaccatctgttgttcaagct 1550
catttaaatttagatgatgaaactttagctgaattaaaacaatttgctac 1600
taaagctactgttgttggtccagttaattaaGCGGCCGCtaaactat 1646
Bacillus subtilis sequence (SEQ ID NO:2): The underlined "G" at
position 705 indicates an A.fwdarw.G base substitution. This base
substitution does not alter the amino acid sequence.
TABLE-US-00005 1 ATGAAAAAACAAAATGACATTCCGCAGCCAATTAGAGGAGACAAAGGAG
50 CAACGGTAAAAATCCCGCGCAATATTGAAAGAGACCGGCAAAACCCTGAT 100
ATGCTCGTTCCGCCTGAAACCGATCATGGCACCGTCAGCAATATGAAGTT 150
TTCATTCTCTGATACTCATAACCGATTAGAAAAAGGCGGATATGCCCGGG 200
AAGTGACAGTACGTGAATTGCCGATTTCAGAAAACCTTGCATCCGTAAAT 250
ATGCGGCTGAAGCCAGGCGCGATTCGCGAGCTTCACTGGCATAAAGAAGC 300
TGAATGGGCTTATATGATTTACGGAAGTGCAAGAGTCACAATTGTAGATG 350
AAAAAGGGCGCAGCTTTATTGACGATGTAGGTGAAGGAGACCTTTGGTAC 400
TTCCCGTCAGGCCTGCCGCACTCCATCCAAGCGCTGGAGGAGGGAGCTGA 450
GTTCCTGCTCGTGTTTGACGATGGATCATTCTCTGAAAACAGCACGTTCC 500
AGCTGACAGATTGGCTGGCCCACACTCCAAAAGAAGTCATTGCTGCGAAC 550
TTCGGCGTGACAAAAGAAGAGATTTCCAATTTGCCTGGCAAAGAAAAATA 600
TATATTTGAAAACCAACTTCCTGGCAGTTTAAAAGATGATATTGTGGAAG 650
GGCCGAATGGCGAAGTGCCTTATCCATTTACTTACCGCCTTCTTGAACAA 700
GAGCCGATCGAATCTGAGGGAGGAAAAGTATACATTGCAGATTCGACAAA 750
CTTCAAAGTGTCTAAAACCATCGCATCAGCGCTCGTAACAGTAGAACCCG 800
GCGCCATGAGAGAACTGCACTGGCACCCGAATACCCACGAATGGCAATAC 850
TACATCTCCGGTAAAGCTAGAATGACCGTTTTTGCATCTGACGGCCATGC 900
CAGAACGTTTAATTACCAAGCCGGTGATGTCGGATATGTACCATTTGCAA 950
TGGGTCATTACGTTGAAAACATCGGGGATGAACCGCTTGTCTTTTTAGAA 1000
ATCTTCAAAGACGACCATTATGCTGATGTATCTTTAAACCAATGGCTTGC 1050
CATGCTTCCTGAAACATTTGTTCAAGCGCACCTTGACTTGGGCAAAGACT 1100
TTACTGATGTGCTTTCAAAAGAAAAGCACCCAGTAGTGAAAAAGAAATGC 1150 AGTAAATAA
1158
[0220] The translated oxalate decarboxylase protein from Bacillus
subtilis sequence is shown below (Swiss-Prot: O34714) (SEQ ID
NO:3).
TABLE-US-00006 1 MKKQNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMK
50 FSFSDTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKE 100
AEWAYMIYGSARVTIVDEKGRSFIDDVGEGDLWYFPSGLPHSIQALEEGA 150
EFLLVFDDGSFSENSTFQLTDWLAHTPKEVIAANFGVTKEEISNLPGKEK 200
YIFENQLPGSLKDDIVEGPNGEVPYPFTYRLLEQEPIESEGGKVYIADST 250
NFKVSKTIASALVTVEPGAMRELHWHPNTHEWQYYISGKARMTVFASDGH 300
ARTFNYQAGDVGYVPFAMGHYVENIGDEPLVFLEIFKDDHYADVSLNQWL 350
AMLPETFVQAHLDLGKDFTDVLSKEKHPVVKKKCSK 385
Example 19. Stability of Soluble OXDC Crystalline OXDC and
OXPC-CLEC at Low pH 3.0
[0221] Soluble OXDC, 2.5 mg/mL (Example 5), crystalline OXDC, 5.0
mg/mL (Example 5) and OXDC-CLEC, 5.0 mg/mL (Example 10) were taken
in sodium citrate buffer pH 3.0, 1 mL each in Eppendorf tubes and
incubated for 5 h at 37.degree. C. Samples were taken at 0, 2 and 5
hrs for measurement of stability of the enzyme. The activities were
determined as in Example 15. Results showing the stability of OXDC
and OXDC-CLEC at pH 3.0 after 0.2. and 5 hrs are shown in FIG 7.
OXDC-CLEC retained about 100% activity after 5h of incubation at pH
3.0, while soluble OXDC within first 2 h lost about 51% activity
and after 5 h had only about 40% activity remained when compared
with original. The crystalline OXDC was more stable than the
soluble OXDC by retaining about 68% activity in 1 hr and about 67%
activity in 2 hr.
Example 20. Stability of Soluble OXDC, Crystalline OXDC, and
OXDC-CLEC Crystals in the Presence of Pensin
[0222] Soluble OXDC, 1.0 mg/mL (Example 5), crystalline OXDC, 10.0
mg/mL (Example 5) and OXDC-CLEC, 1.0 mg/mL (Example 10) were taken
in sodium citrate buffer pH 3.0, 1 mL each in Eppendorf tubes and
incubated with pepsin (pepsin stock was made at a concentration of
1 mg/mL in 25 mM Tris-HCL buffer, pH 7.5) at a ratio of 50:1
between OXDC to pepsin for 5 h at 37.degree. C. Samples were taken
at 0, 2 and 5 hrs for measurement of stability of the enzyme. The
activities were determined as in Example 15. Results showing the
stability of soluble OXDC, crystalline OXDC and OXDC-CLEC in the
presence of pepsin after 0, 2, and 5 hrs are depicted in FIG.
8.
[0223] As shown in FIG. 8, superiority of the OXDC-CLEC formulation
versus soluble OXDC was revealed at both low pH and in the presence
of pepsin. OXDC-CLEC sample retained about 60% activity after 5 h
incubation at 37.degree. C., while majority of soluble OXDC and
uncrosslinked crystalline OXDC were degraded after 2 h by pepsin
and only about 20% activity remained after 5 h.
Example 21. Stability of Soluble OXDC, Crystalline OXDC, and
OXDC-CLEC in the Presence of Chymotrypsin
[0224] Soluble OXDC, 1.0 mg/mL (Example 5), crystalline OXDC, 10.0
mg/mL (Example 5) and OXDC-CLEC, 1.0 mg/mL (Example 10) were taken
in 25 mM Tris-HCl buffer at pH 7.5, 1 mL each in Eppendorf tubes
and incubated with chymotrypsin (chymotrypsin stock was made at a
concentration of 1 mg/mL in 25 mM Tris-HCl buffer, pH 7.5, freshly
prepared) at a ratio of 50:1 between OXDC to chymotrypsin for 5 h
at 37.degree. C. Samples were taken at 0, 2 and 5 hours for
measurement of stability of the enzyme. The activities were
determined as in Example 15. Results showing the stability of
soluble OXDC, crystalline OXDC and OXDC-CLEC in the presence of
chymptrypsin at 0, 2 and 5 hours are shown in FIG 9. Results from
FIG. 9 show that OXDC-CLEC and uncrosslinked crystalline OXDC were
stable and resistant to proteolytic cleavage by chymotrypsin after
5 hours of incubation at 37.degree. C. Analysis showed that
OXDC-CLEC and uncrosslinked crystalline OXDC enzyme retained about
100% activity when compared to its original values in 5 hours. At
the same time, soluble protein lost about 50% activity after 2
hours exposure to chymotrypsin and 5 hours later all soluble OXDC
was degraded, with 0% activity remained.
Example 22. Stability of Soluble OXDC, crystalline OXDC, and
OXDC-CLEC in Soluble OXDC, 5.0 mg/mL (Example 5), crystalline OXDC,
10.0 mg/mL
[0225] (Example 5) and OXDC-CLEC, 10.0 mg/mL (Example 10) were
taken in simulated intestinal juice (simulated intestinal juice
made according to USB recommendation. 6.8 gm of monobasic potassium
phosphate was dissolved in 250 ml of water, mixed with 77 ml 0.2 N
of sodium hydroxide and 500 ml of deionized water; then, 10 gm of
pancreatin was added and pH was adjusted with either 0.2N
hydrochloric acid or 0.2 N sodium hydroxide to a pH of 6.8; water
was added to 1L), 1 mL each, in Eppendorf tubes and incubated for 2
h at 37.degree. C. Samples were taken at 0, 1 and 2 hrs for
measurement of stability of the enzyme. The activities were
determined as in Example 15. The results are shown in FIG. 10.
[0226] The results, shown in FIG. 10 indicate that OXDC-CLEC is
stable in simulated intestinal juice with pancreatin with about
100% activity preserved. In contrast, the majority of soluble OXDC
was degraded by pancreatin (mix of lipase, amylase and protease)
within 1 hour and only between about 26%-28% activity remained
after 1 h and 2 h incubation, respectively. The uncrosslinked
crystalline OXDC was much more stable than its soluble form in the
presence of pancreatin where it lost only about 76% and about 49%
activities after 1 and 2 hr incubation, respectively.
[0227] In addition, OXDC-CLEC protects the enzyme from cleavage
from proteases, such as pepsin and chymotrypsin. Stability of
OXDC-CLEC can be at least about 100%, 200%, 300%, 400% or more of
that of the stability of soluble OXDC in the same conditions.
OXDC-CLEC maintains its activities at least about 2, 3, or 4 folds
higher than soluble OXDC maintaining its activities under the same
conditions. Thus, compared to soluble OXDC, OXDC-CLEC is both
active and stable in the severe conditions of the gut, ranging from
acidic pH of about 2.5 or 3 to pH of about 7.5 or 8.5 and
containing various proteases.
[0228] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
311646DNAArtificial SequenceSynthetically generated oligonucleotide
1ataagaatgc ggccgcataa tgagatttcc atctattttt actgctgttt tatttgctgc
60ttcttctgct ttagctgctc cagttaatac tactactgaa gatgaaactg ctcaaattcc
120agctgaagct gttattggtt attctgattt agaaggtgat tttgatgttg
ctgttttacc 180attttctaat tctactaata atggtttatt atttattaat
actactattg cttctattgc 240tgctaaagaa gaaggtgttt ctttagaaaa
aagagaagct gaagctatgt ttaataattt 300tcaaagatta ttaactgtta
ttttattatc tggttttact gctggtgttc cattagcttc 360tactactact
ggtactggta ctgctactgg tacttctact gctgctgaac catctgctac
420tgttccattt gcttctactg atccaaatcc agttttatgg aatgaaactt
ctgatccagc 480tttagttaaa ccagaaagaa atcaattagg tgctactatt
caaggtccag ataatttacc 540aattgattta caaaatccag atttattagc
tccaccaact actgatcatg gttttgttgg 600taatgctaaa tggccatttt
ctttttctaa acaaagatta caaactggtg gttgggctag 660acaacaaaat
gaagttgttt taccattagc tactaattta gcttgtacta atatgagatt
720agaagctggt gctattagag aattacattg gcataaaaat gctgaatggg
cttatgtttt 780aaaagggtct actcaaattt ctgctgttga taatgaaggg
agaaattata tttctactgt 840tggtccaggt gatttatggt attttccacc
aggtattcca cattctttac aagctactgc 900tgatgatcca gaaggttctg
aatttatttt agtttttgat tctggtgctt ttaatgatga 960tggtactttt
ttattaactg attggttatc tcatgttcca atggaagtta ttttaaaaaa
1020ttttagagct aaaaatccag ctgcttggtc tcatattcca gctcaacaat
tatatatttt 1080tccatctgaa ccaccagctg ataatcaacc agatccagtt
tctccacaag ggactgttcc 1140attaccatat tcttttaatt tttcttctgt
tgaaccaact caatattctg gtgggactgc 1200taaaattgct gattctacta
cttttaatat ttctgttgct attgctgttg ctgaagttac 1260tgttgaacca
ggtgctttaa gagaattaca ttggcatcca actgaagatg aatggacttt
1320ttttatttct ggtaatgcta gagttactat ttttgctgct caatctgttg
cttctacttt 1380tgattatcaa ggtggtgata ttgcttatgt tccagcttct
atgggtcatt atgttgaaaa 1440tattggtaat actactttaa cttatttaga
agtttttaat actgatagat ttgctgatgt 1500ttctttatct caatggttag
ctttaactcc accatctgtt gttcaagctc atttaaattt 1560agatgatgaa
actttagctg aattaaaaca atttgctact aaagctactg ttgttggtcc
1620agttaattaa gcggccgcta aactat 164621158DNABacillus subtilis
2atgaaaaaac aaaatgacat tccgcagcca attagaggag acaaaggagc aacggtaaaa
60atcccgcgca atattgaaag agaccggcaa aaccctgata tgctcgttcc gcctgaaacc
120gatcatggca ccgtcagcaa tatgaagttt tcattctctg atactcataa
ccgattagaa 180aaaggcggat atgcccggga agtgacagta cgtgaattgc
cgatttcaga aaaccttgca 240tccgtaaata tgcggctgaa gccaggcgcg
attcgcgagc ttcactggca taaagaagct 300gaatgggctt atatgattta
cggaagtgca agagtcacaa ttgtagatga aaaagggcgc 360agctttattg
acgatgtagg tgaaggagac ctttggtact tcccgtcagg cctgccgcac
420tccatccaag cgctggagga gggagctgag ttcctgctcg tgtttgacga
tggatcattc 480tctgaaaaca gcacgttcca gctgacagat tggctggccc
acactccaaa agaagtcatt 540gctgcgaact tcggcgtgac aaaagaagag
atttccaatt tgcctggcaa agaaaaatat 600atatttgaaa accaacttcc
tggcagttta aaagatgata ttgtggaagg gccgaatggc 660gaagtgcctt
atccatttac ttaccgcctt cttgaacaag agccgatcga atctgaggga
720ggaaaagtat acattgcaga ttcgacaaac ttcaaagtgt ctaaaaccat
cgcatcagcg 780ctcgtaacag tagaacccgg cgccatgaga gaactgcact
ggcacccgaa tacccacgaa 840tggcaatact acatctccgg taaagctaga
atgaccgttt ttgcatctga cggccatgcc 900agaacgttta attaccaagc
cggtgatgtc ggatatgtac catttgcaat gggtcattac 960gttgaaaaca
tcggggatga accgcttgtc tttttagaaa tcttcaaaga cgaccattat
1020gctgatgtat ctttaaacca atggcttgcc atgcttcctg aaacatttgt
tcaagcgcac 1080cttgacttgg gcaaagactt tactgatgtg ctttcaaaag
aaaagcaccc agtagtgaaa 1140aagaaatgca gtaaataa 11583385PRTBacillus
subtilis 3Met Lys Lys Gln Asn Asp Ile Pro Gln Pro Ile Arg Gly Asp
Lys Gly1 5 10 15Ala Thr Val Lys Ile Pro Arg Asn Ile Glu Arg Asp Arg
Gln Asn Pro 20 25 30Asp Met Leu Val Pro Pro Glu Thr Asp His Gly Thr
Val Ser Asn Met 35 40 45Lys Phe Ser Phe Ser Asp Thr His Asn Arg Leu
Glu Lys Gly Gly Tyr 50 55 60Ala Arg Glu Val Thr Val Arg Glu Leu Pro
Ile Ser Glu Asn Leu Ala65 70 75 80Ser Val Asn Met Arg Leu Lys Pro
Gly Ala Ile Arg Glu Leu His Trp 85 90 95His Lys Glu Ala Glu Trp Ala
Tyr Met Ile Tyr Gly Ser Ala Arg Val 100 105 110Thr Ile Val Asp Glu
Lys Gly Arg Ser Phe Ile Asp Asp Val Gly Glu 115 120 125Gly Asp Leu
Trp Tyr Phe Pro Ser Gly Leu Pro His Ser Ile Gln Ala 130 135 140Leu
Glu Glu Gly Ala Glu Phe Leu Leu Val Phe Asp Asp Gly Ser Phe145 150
155 160Ser Glu Asn Ser Thr Phe Gln Leu Thr Asp Trp Leu Ala His Thr
Pro 165 170 175Lys Glu Val Ile Ala Ala Asn Phe Gly Val Thr Lys Glu
Glu Ile Ser 180 185 190Asn Leu Pro Gly Lys Glu Lys Tyr Ile Phe Glu
Asn Gln Leu Pro Gly 195 200 205Ser Leu Lys Asp Asp Ile Val Glu Gly
Pro Asn Gly Glu Val Pro Tyr 210 215 220Pro Phe Thr Tyr Arg Leu Leu
Glu Gln Glu Pro Ile Glu Ser Glu Gly225 230 235 240Gly Lys Val Tyr
Ile Ala Asp Ser Thr Asn Phe Lys Val Ser Lys Thr 245 250 255Ile Ala
Ser Ala Leu Val Thr Val Glu Pro Gly Ala Met Arg Glu Leu 260 265
270His Trp His Pro Asn Thr His Glu Trp Gln Tyr Tyr Ile Ser Gly Lys
275 280 285Ala Arg Met Thr Val Phe Ala Ser Asp Gly His Ala Arg Thr
Phe Asn 290 295 300Tyr Gln Ala Gly Asp Val Gly Tyr Val Pro Phe Ala
Met Gly His Tyr305 310 315 320Val Glu Asn Ile Gly Asp Glu Pro Leu
Val Phe Leu Glu Ile Phe Lys 325 330 335Asp Asp His Tyr Ala Asp Val
Ser Leu Asn Gln Trp Leu Ala Met Leu 340 345 350Pro Glu Thr Phe Val
Gln Ala His Leu Asp Leu Gly Lys Asp Phe Thr 355 360 365Asp Val Leu
Ser Lys Glu Lys His Pro Val Val Lys Lys Lys Cys Ser 370 375
380Lys385
* * * * *