US20200308534A1 - Recombinant uricase enzyme - Google Patents

Recombinant uricase enzyme Download PDF

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US20200308534A1
US20200308534A1 US16/628,743 US201816628743A US2020308534A1 US 20200308534 A1 US20200308534 A1 US 20200308534A1 US 201816628743 A US201816628743 A US 201816628743A US 2020308534 A1 US2020308534 A1 US 2020308534A1
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uricase
utilis
recombinant mutant
substituted
subject
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Aditi R. Deshpande
Danica Grujic
Sridhar Govindarajan
Mark Welch
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ALLENA PHARMACEUTICALS Inc
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    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12N9/0048—Uricase (1.7.3.3)
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    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates generally to methods and compositions for treating diseases or disorders associated with an elevated amount of uric acid, and, more particularly, the invention relates to recombinant mutant Candida utilis uricases and methods using, and compositions containing, such uricases for treating diseases or disorders associated with an elevated amount of uric acid.
  • Uric acid is the final oxidation product of purine metabolism in humans and higher primates.
  • Uricase, or urate oxidase is an enzyme that degrades uric acid into allantoin and carbon dioxide. Due to mutational silencing, humans and higher primates lack a functional uricase gene. Therefore, unlike certain other mammals, humans have lost the capacity to metabolize uric acid by hepatic uricase due to mutational silencing of the enzyme. Although humans produce large quantities of uric acid, the majority of the uric acid is excreted in urine. Nevertheless, increased production and/or decreased excretion of uric acid can result in high levels of uric acid in blood (hyperuricemia) and urine (hyperuricosuria). Hyperuricemia and hyperuricosuria can result, for example, as in inflammatory arthritis due to urate deposits in joints and cutaneous tissue.
  • Gout is a condition that affects an estimated 8 million Americans and is characterized by recurring attacks of joint inflammation (arthritis).
  • the joint inflammation is precipitated by deposits of uric acid crystals in the joint fluid (synovial fluid) and joint lining (synovial lining).
  • Intense joint inflammation occurs as white blood cells engulf the uric acid crystals and release inflammatory chemicals, causing pain, heat, and redness of the joint tissues.
  • Chronic gout can additionally lead to decreased kidney function and kidney stones.
  • Functional uricase enzymes can be found in a wide range of organisms, including animals, plants, bacteria and fungi, and, as such, exogenous uricase has been used in the treatment of diseases or disorders associated with an elevated amount of uric acid.
  • Clinically approved uricases include Krystexxa® (pegloticase), which has been approved for the treatment of chronic refractory gout, and Elitek® (rasburicase), which has been approved for tumor lysis syndrome.
  • the invention is based, in part, upon the discovery of recombinant uricase enzymes that are active in humans and have greater stability and/or activity than naturally occurring enzymes.
  • the recombinant enzymes of the invention exhibit improved stability against proteolytic digestion by pancreatin (a collection of enzymes secreted by the pancreas) compared to naturally occurring versions of the enzyme.
  • the recombinant enzymes of the invention may have greater specific activity than a wild type uricase enzyme.
  • the recombinant enzymes described herein may be suitable for oral administration, and therefore potentially safer and more tolerable than the commercially available, injectable forms of uricase (e.g., Krystexxa® and Elitek®), because it is contemplated that the enzymes will remain active within the intestines and will not be absorbed through the intestinal wall.
  • uricase e.g., Krystexxa® and Elitek®
  • the invention provides a recombinant mutant Candida utilis uricase enzyme that comprises at least one (for example, one, two, three, four, five, six, seven or eight) mutation(s) at a position corresponding to wild type C.
  • utilis uricase of SEQ ID NO: 1 wherein the at least one mutation is selected from: (a) at position 180, isoleucine is substituted by valine or alanine (I180V or I180A), (b) at position 165, tyrosine is substituted by phenylalanine (Y165F), (c) at position 190, valine is substituted by glycine or alanine (V190G or V190A), (d) at position 51, glutamic acid is substituted by lysine (E51K), (e) at position 244, glutamine is substitute by lysine (Q244K), (f) at position 132, isoleucine is substituted by arginine or asparagine (I132R or I132N
  • the recombinant mutant C. utilis uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, V190A, E51K, Q244K, I132R, V97I, E92N, A87G, D142E, G44A, G128P, A236N, K208A, N213A, S140T, Y253Q, and A84S.
  • the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, I132R, V97I, E92N, A87G, D142E, and G44A.
  • the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, I132R, and G44A. In certain other embodiments, the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, E51K, I132R, and G44A. In certain other embodiments, the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, and I132R.
  • the invention provides a recombinant mutant C. utilis uricase enzyme comprising at least one (for example, one, two, three, four, five, or six) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 190, position 51, position 132, and position 44.
  • one or more mutations may be conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1
  • one or more mutations may be non-conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1.
  • the invention provides a recombinant mutant C. utilis uricase enzyme comprising at least one (for example, one, two, three, four, or five) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 51, position 132, and position 44.
  • one or more mutations may be conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1
  • one or more mutations may be non-conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1.
  • the invention provides a recombinant mutant C. utilis uricase comprising at least one (for example, one, two, three, four, or five) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 190, position 51, position 244, and position 132.
  • one or more mutations may be conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1
  • one or more mutations may be non-conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1.
  • the uricase comprises two, three, four, five, six, seven, or eight mutations.
  • the uricase comprises the following substitutions (i) I180V, Y165F, E51K, I132R, and G44A, (ii) I180A, Y165F, E51K, I132R, and G44A, (iii) I180V, Y165F, V190G, E51K, I132R, and G44A, (iv) I180A, Y165F, V190G, E51K, I132R, and G44A, (v) I180V and Y165F, or (vi) I180V, Y165F, V190G, E51K, Q244K, and I132R, either alone or in combination with other substitutions.
  • the invention provides a recombinant mutant C. utilis uricase enzyme comprising three substitutions listed in a given row of TABLE 1 hereinbelow. In certain embodiments, the invention provides a recombinant mutant C. utilis uricase enzyme comprising five substitutions listed in a given row of TABLE 2 hereinbelow.
  • the invention provides a recombinant mutant C. utilis uricase having a half-life of at least 35 minutes in the presence of pancreatin, e.g., a half-life of 35-200 minutes in the presence of pancreatin, for example, under the conditions set forth in Example 1.
  • any of the foregoing recombinant mutant Candida utilis uricases may, for example, have 5-50 fold, 10-40 fold, 10-30 fold, 20-40 fold, or 20-30 fold, higher stability in the presence of pancreatin, compared to the wild-type uricase.
  • the uricase may, for example, be more stable at a pH less than about 6.5 compared to the template (or reference) wild-type uricase.
  • any of the foregoing recombinant mutant Candida utilis uricases may, for example, be conjugated to a water soluble polymer, e.g., polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the uricase in any of the foregoing recombinant mutant C. utilis uricase enzymes, the uricase is isolated.
  • the invention provides an isolated nucleic acid comprising a nucleotide sequence encoding any one of the foregoing uricase enzymes.
  • the nucleotide sequence is codon optimized for expression in a host cell, e.g., an Escherichia coli cell.
  • the invention also provides an expression vector that comprises any one of the foregoing nucleotide sequences.
  • the invention provides host cells, e.g., Escherichia coli cells, comprising one or more of the foregoing expression vectors.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising any one of the foregoing recombinant mutant C. utilis uricase enzymes and at least one pharmaceutically acceptable carrier and/or an excipient.
  • the enzyme may be in a soluble form or in a crystal form.
  • the composition may comprise a pH increasing agent. It is contemplated that the pharmaceutical composition may, for example, be formulated as an oral dosage form or a parenteral dosage form. In certain embodiments, the composition is a formulated as a powder, granulate, pellet, micropellet, or a minitablet.
  • the composition is encapsulated in a capsule, e.g., a hydroxypropyl methylcellulose (HPMC) capsule, soft gelatin capsule, or a hard gelatin capsule, or the composition is formulated as a tablet dosage form.
  • a capsule e.g., a hydroxypropyl methylcellulose (HPMC) capsule, soft gelatin capsule, or a hard gelatin capsule
  • HPMC hydroxypropyl methylcellulose
  • the invention provides a method of treating a disease or disorder associated with an elevated amount of uric acid in a subject in need thereof.
  • the disease or disorder is associated with an elevated amount of uric acid in plasma or urine of the subject.
  • the method comprises administering to the subject an effective amount of any of the uricase enzymes or compositions described herein, to treat the disease or disorder in the subject.
  • the invention provides a method of treating hyperuricemia and/or hyperuricosuria in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of any of the uricase enzymes or compositions described herein, to treat the hyperuricemia and/or hyperuricosuria in the subject.
  • the invention provides a method of treating gout in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of any of the uricase enzymes or compositions described herein, to treat the gout in the subject.
  • the recombinant mutant C. utilis uricase is administered in combination with a xanthine oxidase inhibitor (e.g., allopurinol or febuxostat), a uricosuric (e.g., probenecid, benzbromarone, losartan or lesinurad), or a combination thereof.
  • a xanthine oxidase inhibitor e.g., allopurinol or febuxostat
  • a uricosuric e.g., probenecid, benzbromarone, losartan or lesinurad
  • FIG. 1A is a SDS-PAGE gel depicting pancreatin, wild-type C. utilis uricase (His-UO), and wild-type C. utilis uricase following a 90 minute incubation with pancreatin.
  • FIG. 1B is a line graph depicting wild-type C. utilis uricase activity as measured by loss of substrate uric acid concentration following incubation of wild-type C. utilis uricase with pancreatin for the indicated time points. Uric acid concentration is measured by absorbance at 298 nm.
  • FIG. 2A is a line graph depicting the activity of the indicated mutant C. utilis uricases in the presence of pancreatin. Data from two independent preparations are depicted for each uricase. Activity values are normalized to the activity in presence of pancreatin at time zero.
  • FIG. 2B is a line graph demonstrating the reproducibility across each preparation for the data depicted in FIG. 2A .
  • FIG. 3 is a line graph depicting the activity of the R2_V79, R2_15, R2_V16 and R2_Parent mutant C. utilis uricases following incubation with pancreatin for the indicated time-points. Activity values are normalized to the activity in presence of pancreatin at time zero.
  • FIG. 4 shows protein unfolding as determined by differential scanning fluorimetry (DSF) for wild-type C. utilis uricase and the indicated mutant C. utilis uricase enzymes.
  • FIG. 5 is an SDS-PAGE gel showing the R2_V17, R2_V4 and R2_V79 mutant C. utilis uricases following incubation with pancreatin for the indicated timepoints.
  • FIG. 6 is an SDS-PAGE gel showing the wild-type C. utilis uricase and R2_V17 mutant C. utilis uricase following incubation with pancreatin for the indicated timepoints.
  • FIG. 7 is a bar graph showing the pancreatin stability of the indicated mutant C. utilis uricases relative to wild-type.
  • R2 mutant C. utilis uricases described in Example 1 each containing five substitutions (right), and mutant C. utilis uricases described in Example 2, each containing a single substitution (left and middle), are depicted.
  • FIG. 8 is a waterfall chart showing the pancreatin stability of the mutant C. utilis uricases described in Example 2, each containing a single substitution, relative to wild-type. Enzymes are ordered relative to their effect on stability.
  • FIG. 9A is a bar graph showing the plasma urate levels (mg/dL) in Uricase knockout (UrOxKO) mice with severe hyperuricemia.
  • Mean (SEM) of pre-treatment plasma urate levels was measured in samples collected on day 7 after removal of maintenance dose of allopurinol
  • treatment plasma urate level was measured in samples collected on day 7 after administration of 50 mg/L of allopurinol, 150 mg/L of allopurinol, or 150 mg/day mutant C. utilis uricase, respectively
  • post-treatment plasma urate levels are shown.
  • FIG. 9B is a bar graph showing the urine uric acid levels (mg/dL) in UrOxKO mice with severe hyperuricosuria. Uric acid levels were measured in 24-hour urine samples collected during the last 3 days of pre-treatment and treatment periods, as indicated.
  • the invention is based, in part, upon the discovery of recombinant uricase enzymes that are active in humans and have greater stability and/or activity than naturally occurring enzymes.
  • the recombinant enzymes of the invention exhibit improved stability against proteolytic digestion by pancreatin (a collection of enzymes secreted by the pancreas) compared to naturally occurring versions of the enzyme.
  • the recombinant enzymes of the invention may have greater specific activity than a wild type uricase enzyme.
  • the recombinant enzymes described herein may be suitable for oral administration, and therefore potentially safer and more tolerable than the commercially available, injectable forms of uricase (e.g., Krystexxa® and Elitek®), because it is contemplated that the enzymes will remain active within the intestines and will not be absorbed through the intestinal wall because the size of the recombinant enzyme would preclude passive absorption, and no receptor has been identified for active transport of the enzyme from the intestine.
  • uricase e.g., Krystexxa® and Elitek®
  • Uric acid also known as urate
  • Uricase also known as urate oxidase or UrOx
  • UrOx degrades uric acid into allantoin by catalyzing the following reaction:
  • C. utilis uricase is a homo-tetrameric enzyme that does not require a metal atom or an organic co-factor for catalysis.
  • the amino acid sequence of wild type C. utilis uricase is as follows:
  • An exemplary nucleotide sequence encoding the wild type C. utilis uricase is as follows:
  • the invention provides a family of recombinant mutant Candida utilis uricase enzymes that, for example, are useful in treating disorders associated with elevated levels of uric acid in a subject, for example, disorders associated with elevated levels of uric acid in plasma of the subject.
  • the recombinant mutant C. utilis uricase enzymes described herein have higher stability compared to the wild-type C. utilis uricase, e.g., higher stability in the presence of pancreatin compared to the wild-type C. utilis uricase, and are therefore better suited for oral delivery and activity in the intestines than wild-type C. utilis uricase.
  • wild-type C. utilis uricase refers a C.
  • utilis uricase having the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof that can catalyze the oxidation of uric acid to 5-hydroxyisourate.
  • functional fragment is understood to be a protein fragment of wild type C. utilis uricase of SEQ ID NO: 1 that has at least 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the activity of wild type C. utilis uricase to catalyze the conversion of uric acid to 5-hydroxyisourate and/or allantoin.
  • the invention provides a recombinant mutant Candida utilis uricase enzyme that comprises at least one (for example, one, two, three, four, five, six, seven or eight) mutation(s) at a position corresponding to wild type C.
  • utilis uricase of SEQ ID NO: 1 wherein the at least one mutation is selected from: (a) at position 180, isoleucine is substituted by valine or alanine (I180V or I180A), (b) at position 165, tyrosine is substituted by phenylalanine (Y165F), (c) at position 190, valine is substituted by glycine or alanine (V190G or V190A), (d) at position 51, glutamic acid is substituted by lysine (E51K), (e) at position 244, glutamine is substitute by lysine (Q244K), (f) at position 132, isoleucine is substituted by arginine or asparagine (I132R or I132N
  • the recombinant mutant C. utilis uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, V190A, E51K, Q244K, I132R, V97I, E92N, A87G, D142E, G44A, G128P, A236N, K208A, N213A, S140T, Y253Q, and A84S.
  • the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, I132R, V97I, E92N, A87G, D142E, and G44A.
  • the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, I132R, and G44A. In certain other embodiments, the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, E51K, I132R, and G44A. In certain other embodiments, the uricase enzyme comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, and I132R.
  • the invention provides a recombinant mutant C. utilis uricase enzyme comprising at least one (for example, one, two, three, four, five, or six) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 190, position 51, position 132, and position 44.
  • one or more mutations may be conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1
  • one or more mutations may be non-conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1.
  • the invention provides a recombinant mutant C. utilis uricase enzyme comprising at least one (for example, one, two, three, four, or five) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 51, position 132, and position 44.
  • one or more mutations may be conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1
  • one or more mutations may be non-conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1.
  • conservative substitution refers to a substitution with a structurally similar amino acid.
  • conservative substitutions may include those within the following groups: Ser and Cys; Leu, Ile, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gln, Asn, Glu, Asp, and His.
  • Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution:p matrix (e.g., the PAM 250 matrix).
  • Non conservative substitutions are amino acid substitutions that are not conservative substitutions.
  • the invention provides a recombinant mutant C. utilis uricase comprising at least one (for example, one, two, three, four, five, or six) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 190, position 51, position 244, and position 132.
  • one or more mutations may be conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1
  • one or more mutations may be non-conservative substitutions relative to wild type C. utilis uricase of SEQ ID NO: 1.
  • the uricase comprises two, three, four, five, six, seven, or eight mutations.
  • the uricase comprises the following substitutions (i) I180V, Y165F, E51K, I132R, and G44A, (ii) I180A, Y165F, E51K, I132R, and G44A, (iii) I180V, Y165F, V190G, E51K, I132R, and G44A, (iv) I180A, Y165F, V190G, E51K, I132R, and G44A, (v) I180V and Y165F, or (vi) I180V, Y165F, V190G, E51K, Q244K, and I132R, either alone or in combination with other substitutions.
  • the invention provides a recombinant mutant C. utilis uricase enzyme comprising three substitutions listed in a given row of TABLE 1.
  • the invention provides a recombinant mutant C. utilis uricase comprising five substitutions listed in a given row of TABLE 2.
  • a recombinant mutant Candida utilis uricase disclosed herein may, for example, have higher specific activity than wild-type C. utilis uricase of SEQ ID NO.: 1.
  • a recombinant mutant C. utilis uricase may have from 5 to 50 fold higher specific activity than the wild-type C. utilis uricase.
  • the uricase has from about 5 to about 50, from about 5 to about 40, from about 5 to about 30, from about 5 to about 20, from about 5 to about 10, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, from about 20 to about 50, from about 20 to about 40, from about 20 to about 30, from about 30 to about 50, from about 30 to about 40, from about 40 to about 50, about 5, about 10, about 20, about 30, about 40, or about 50 fold higher specific activity than wild-type C. utilis uricase.
  • the recombinant mutant Candida utilis uricase disclosed herein may, for example, have higher stability, e.g., higher stability in the presence of pancreatin, compared to the wild-type C. utilis uricase.
  • a recombinant mutant C. utilis uricase may have from 5 to 50 fold higher stability in the presence of pancreatin compared to the wild-type C. utilis uricase.
  • the uricase has from about 5 to about 50, from about 5 to about 40, from about 5 to about 30, from about 5 to about 20, from about 5 to about 10, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, from about 20 to about 50, from about 20 to about 40, from about 20 to about 30, from about 30 to about 50, from about 30 to about 40, from about 40 to about 50, about 5, about 10, about 20, about 30, about 40, or about 50 fold higher stability in the presence of pancreatin compared to the wild-type C. utilis uricase.
  • the recombinant mutant Candida utilis uricase may, for example, have a half-life of at least 35 minutes in the presence of pancreatin.
  • the uricase has a half-life of at least from about 35 to about 200 minutes, from about 35 to about 175 minutes, from about 35 to about 150 minutes, from about 35 to about 125 minutes, from about 35 to about 100 minutes, from about 35 to about 75 minutes, from about 35 to about 50 minutes, from about 50 to about 200 minutes, from about 50 to about 175 minutes, from about 50 to about 150 minutes, from about 50 to about 125 minutes, from about 50 to about 100 minutes, from about 50 to about 75 minutes, from about 75 to about 200 minutes, from about 75 to about 175 minutes, from about 75 to about 150 minutes, from about 75 to about 125 minutes, from about 75 to about 100 minutes, from about 100 to about 200 minutes, from about 100 to about 175 minutes, from about 100 to about 150 minutes, from about 100 to about 125 minutes, from about 125 to about 200 minutes, from about 100 to about 175 minutes,
  • Uricase stability or half-life may be measured by any method known in the art, including absorption based assays or SDS-PAGE as described in Example 1. Uricase half-life in the presence of pancreatin will depend upon the experimental conditions in which the half-life is measured, including, e.g., the concentration of pancreatin. In certain embodiments, the half-life of a disclosed recombinant mutant Candida utilis uricase in the presence of pancreatin is measured in the presence of 20 ng/ ⁇ L or 80 ng/ ⁇ L pancreatin, e.g., pancreatin available from Sigma-Aldrich (Cat No. P7545).
  • a recombinant mutant Candida utilis uricase enzyme disclosed herein may, for example, have higher stability at a pH less than about 6.5 compared to the wild-type C. utilis uricase.
  • a recombinant mutant C. utilis uricase may have from 5 to 50 fold higher stability in the presence of pancreatin compared to the wild-type C. utilis uricase.
  • the uricase enzyme has from about 5 to about 50, from about 5 to about 40, from about 5 to about 30, from about 5 to about 20, from about 5 to about 10, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 20, from about 20 to about 50, from about 20 to about 40, from about 20 to about 30, from about 30 to about 50, from about 30 to about 40, from about 40 to about 50, about 5, about 10, about 20, about 30, about 40, or about 50 fold higher stability at a pH less than about 6.5 compared to the wild-type C. utilis uricase.
  • Uricase stability or half-life may be measured by any method known in the art, including absorption based assays or SDS-PAGE as described in Example 1.
  • the invention further provides a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, G44A, E51K, and I132R, e.g., a recombinant mutant C. utilis uricase comprising the following amino acid sequence, e.g., a recombinant mutant uricase referred to as R2_V17 herein:
  • the invention further provides a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, E51K, V97I, and A236N, e.g., a recombinant mutant C. utilis uricase comprising the following amino acid sequence, e.g., a recombinant mutant uricase referred to as R2_V4 herein:
  • the invention further provides a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, I132R, Q217L, and P285S, e.g., a recombinant mutant C. utilis uricase comprising the following amino acid sequence, e.g., a recombinant mutant uricase referred to as R2_V79 herein:
  • the invention further provides a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, E51K, V97I, and I196L, e.g., a recombinant mutant C. utilis uricase comprising the following amino acid sequence, e.g., a recombinant mutant uricase referred to as R2_V47 herein:
  • the invention further provides a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, E51K, D142E, and Q217L, e.g., a recombinant mutant C. utilis uricase comprising the following amino acid sequence, e.g., a recombinant mutant uricase referred to as R2_V39 herein:
  • the invention further provides a recombinant mutant C. utilis uricase that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a C. utilis uricase disclosed herein, and has at least 60% specific activity and/or 5 fold higher stability as wild type C. utilis uricase.
  • Sequence identity may be determined in various ways that are within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • BLAST Basic Local Alignment Search Tool
  • analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL.
  • the search parameters for histogram, descriptions, alignments, expect i.e., the statistical significance threshold for reporting matches against database sequences
  • cutoff, matrix and filter are at the default settings.
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated by reference).
  • a disclosed recombinant mutant C. utilis uricase may be modified, engineered or chemically conjugated.
  • a disclosed recombinant mutant C. utilis uricase can be conjugated to an effector agent using standard in vitro conjugation chemistries. If the effector agent is a polypeptide, the uricase enzyme can be chemically conjugated to the effector or joined to the effector as a fusion protein. Construction of fusion proteins is within ordinary skill in the art.
  • a disclosed recombinant mutant C. utilis uricase can be modified with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, or other tissues.
  • a disclosed recombinant mutant C. utilis uricase enzyme may be conjugated to a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide.
  • utilis uricase enzyme is conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone.
  • a water soluble polymer e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone.
  • polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof.
  • Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylates, carbomers, and branched or unbranched polysaccharides.
  • DNA molecules encoding a uricase enzyme can be chemically synthesized using the sequence information provided herein.
  • Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., expression control sequences, to produce conventional gene expression constructs encoding the desired uricase enzyme.
  • Nucleic acids encoding desired uricase enzymes can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the uricase enzyme.
  • Nucleic acids encoding recombinant mutant C. utilis uricases of the invention may be generated by mutating a nucleotide sequence encoding the wild type C. utilis uricase, e.g., SEQ ID NO: 7 disclosed herein, using methods known in the art. Furthermore, in certain embodiments, nucleic acids encoding recombinant mutant C. utilis uricases of the invention may be codon optimized for expression in a heterologous cell, e.g., an E. coli cell, using methods known in the art.
  • an exemplary nucleotide sequence encoding a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, G44A,
  • E51K, and I132R e.g., a recombinant mutant C. utilis uricase referred to as R2_V17 herein, is as follows:
  • An exemplary nucleotide sequence encoding a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, E51K, V97I, and A236N, e.g., a recombinant mutant C. utilis uricase referred to as R2_V4 herein, is as follows:
  • An exemplary nucleotide sequence encoding a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, I132R, Q217L, and P285S, e.g., a recombinant mutant C. utilis uricase referred to as R2_V79 herein, is as follows:
  • An exemplary nucleotide sequence encoding a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, E51K, V97I, and I196L, e.g., a recombinant mutant C. utilis uricase referred to as R2_V47 herein, is as follows:
  • An exemplary nucleotide sequence encoding a recombinant mutant C. utilis uricase that comprises the following substitutions: Y165F, I180V, E51K, D142E, and Q217L, e.g., a recombinant mutant C. utilis uricase referred to as R2_V39 herein, is as follows:
  • a gene is to be expressed in E. coli , it can be cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence.
  • a suitable bacterial promoter e.g., Trp or Tac
  • the expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication.
  • the refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art.
  • a uricase enzyme can be produced by growing (culturing) a host cell transfected with an expression vector encoding such uricase enzyme, under conditions that permit expression of the uricase enzyme. Following expression, the uricase enzyme can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine tags.
  • affinity tags such as glutathione-S-transferase (GST) and histidine tags.
  • a recombinant uricase enzyme described herein preferably is combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • the uricase enzymes can be formulated, or co-administered (either at the same time or sequentially), for example, by an enteral route (e.g., orally), with a pH increasing agent, for example, a protein pump inhibitor (PPI), to enhance the stability of the uricase enzyme, for example, in an acidic environment, for example, in the gastrointestinal tract.
  • a pH increasing agent for example, a protein pump inhibitor (PPI)
  • PPI protein pump inhibitor
  • Proton pump inhibitors are a group of drugs whose main action is pronounced and long-lasting reduction of gastric acid production.
  • Proton pump inhibitors act by blocking the hydrogen/potassium adenosine triphosphatase enzyme system (the H + /K + ATPase, or more commonly just gastric proton pump) of the gastric parietal cell.
  • the proton pump is the terminal stage in gastric acid secretion, being directly responsible for secreting H + ions into the gastric lumen, making it an ideal target for inhibiting acid secretion.
  • proton pump inhibitors examples include: Omeprazole (brand names: LOSEC®, PRILOSEC®, ZEGERID®); Lansoprazole (brand names: PREVACID®, ZOTON®, INHIBITOL®); Esomeprazole (brand names: NEXIUM®); and Pantoprazole (brand names: PROTONIX®, SOMAC®PANTOLOC®).
  • compositions containing a recombinant uricase enzyme disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration.
  • the pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form will depend upon the intended mode of administration and therapeutic application.
  • compositions preferably are formulated for administration enterally (for example, orally), such compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection).
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection.
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection.
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration.
  • Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • a pharmaceutical formulation that is sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
  • a disclosed composition comprises a polyionic reagent which may, e.g., coat the uricase (e.g., the composition comprises a polyionic coating).
  • exemplary polyionic reagents include PSS (poly(Sodium 4-styrenesulfonate), PAA (poly Acrylic acid sodium salt), PMG (poly(methylene-co-guanidine) hydrochloride), DS (dextran sulfate), PMA (poly(methyl acrylate)), or PVS (polyvinylsiloxane).
  • the recombinant uricase enzymes disclosed herein can be used to treat various diseases or disorders associated with an elevated amount of uric acid in a subject.
  • “elevated amount of uric acid in a subject” may refer to an elevated amount of uric acid in a body fluid (e.g., blood, plasma, serum, or urine), tissue and/or cell in a subject, relative to a subject without the disease or disorder.
  • uric acid concentrations between 2.4-6 mg/dL for females and 3.4-7.2 mg/dL for males are considered normal by the Clinical Mayo Reference laboratory.
  • the invention provides a method of treating a disease or disorder associated with an elevated amount of uric acid in a subject.
  • the disease or disorder is associated with an elevated amount of uric acid in plasma of the subject.
  • the method comprises administering to the subject an effective amount of a disclosed recombinant uricase, either alone or in a combination with another therapeutic agent to treat the disease or disorder in the subject.
  • effective amount refers to the amount of an active agent (e.g., a recombinant uricase of the present invention) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • the method comprises orally administering to the subject an effective amount of a disclosed recombinant uricase, either alone or in a combination with another therapeutic agent to treat the disease or disorder in the subject. It is contemplated that, in certain embodiments, the orally administered recombinant uricase may avoid passive absorption in the intestine due to its size, and if metabolized, the novel recombinant uricase of the present invention orally administered with food would be metabolized in a manner similar to that of any other ingested protein.
  • treat means the treatment of a disease in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state.
  • subject and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.
  • diseases or disorders associated with an elevated amount of uric acid include a metabolic disorder, e.g., metabolic syndrome, hyperuricemia, gout (e.g., gouty arthritis), Lesch-Nyhan syndrome, cardiovascular disease, diabetes, hypertension, renal disease, metabolic syndrome, uric acid nephrolithiasis (or kidney stones (see Wiederledge et al. (2011), Clin. Rev. Bone. Miner. Metab., 9(3-4):207-217 (“Uric acid nephrolithiasis is characteristically a manifestation of a systemic metabolic disorder. It has a prevalence of about 10% among all stone formers, the third most common type of kidney stone in the industrialized world.))), tumor lysis syndrome, and hyperuricosuria.
  • a metabolic disorder e.g., metabolic syndrome, hyperuricemia, gout (e.g., gouty arthritis), Lesch-Nyhan syndrome, cardiovascular disease, diabetes, hypertension, renal disease, metabolic syndrome, uric acid
  • the methods and compositions described herein can be used alone or in combination with other therapeutic agents and/or modalities.
  • administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.”
  • the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • a method or composition described herein is administered in combination with one or more additional therapies selected from a xanthine-oxidase inhibitor (e.g., allopurinol, TEI-6720 (2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid), febuxostat (2-[3-cyano-4-isobutoxyphenyl]-4-methylthiazole-5-carboxylic acid), oxypurinol, or pteridylaldehyde), a uricosuric (e.g., probenecid, lesinurad, sulfinpyrazone, sulfinpyrazone, or fenofibrate), ethylenediaminetetraacetic acid, acetazolamide, a potassium supplement, and any combination thereof.
  • a xanthine-oxidase inhibitor e.g., allopurinol, TEI-6720 (2-
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.
  • This example describes the design and testing of recombinant mutant Candida utilis uricases with improved pancreatin stability.
  • mutant C. utilis uricases were designed each with three amino acid substitutions relative to the wild-type sequence.
  • the mutant C. utilis uricases are indicated as R1_V1-R1_V95.
  • DNA fragments encoding the 95 mutant C. utilis uricases were cloned into a rhamanose pD861-NH expression vector (ATUM, Newark, Calif.) that encodes a N-terminal His-tag. All constructs were confirmed by sequencing. Following expression in Escherichia coli cells, each recombinant mutant C. utilis uricase enzyme was bound to a Ni-NTA column and eluted in a buffer containing 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 200 mM imidazole, and 50% (v/v) glycerol.
  • pancreatin Sigma-Aldrich Cat No. P7545; which converts at least 25 times its weight of potato starch into soluble carbohydrates in 5 minutes in water at 40° C., digests at least 25 times its weight of casein in 60 minutes at pH 7.5 at 40° C., and releases at least 2 microequivalents of acid per minute per mg pancreatin from olive oil at pH 9.0 at 37° C.
  • pancreatin stability 25 ng/ ⁇ L of uricase was incubated with 20 ng/ ⁇ L of pancreatin at 37° C. for up to 200 minutes.
  • the assay was performed in simulated intestinal fluid (SIF) buffer (50 mM potassium phosphate, pH 6.8) in 96 well plates. Following incubation with pancreatin for the indicated time points, enzymatic activity was monitored using an absorption based assay. Uric acid has a strong absorbance at 293 nm, and the enzymatic oxidation of uric acid to 5-hydroxyisourate by uricase results in a corresponding drop in 293 nm absorbance over time.
  • SIF simulated intestinal fluid
  • Results for C. utilis uricase mutants with the most improved pancreatin stability were confirmed over multiple protein preparations.
  • Representative data for wild type C. utilis uricase is depicted in FIG. 1
  • representative data for a subset of mutant C. utilis uricases is depicted in FIG. 2 .
  • TABLE 3 depicts the amino acid substitutions for the 95 recombinant mutant C. utilis uricases, as well as the specific activity ( ⁇ M/minute per 1.2 ng/ ⁇ l of uricase), pancreatin stability (half-life, minutes) and expression yield ( ⁇ g/ml) for each enzyme. “nd” indicates that activity and stability measurements were not determined due to insufficient expression yield.
  • TABLE 4 depicts the amino acid substitutions for the 95 mutant C. utilis uricases, as well as the specific activity ( ⁇ M/minute per 1.2 ng/ ⁇ l of uricase), pancreatin stability (half-life, minutes) and expression yield ( ⁇ g/ml) for each enzyme.
  • Pancreatin stability was assayed at 80 ng/ ⁇ L soluble pancreatin. “nd” indicates that activity and stability measurements were not determined due to insufficient expression yield.
  • pancreatin stability data for a subset of the mutant C. utilis uricases is depicted in FIG. 3 .
  • a subset of mutant C. utilis uricases were further tested for thermal stability by differential scanning fluorimetry (DSF).
  • DSF is a method to evaluate thermal stability by heating a protein in the presence of a fluorescent dye which will increase its fluorescence upon binding to the exposed hydrophobic interior of the protein after protein unfolding. Protein unfolding curves are depicted in FIG. 4 .
  • R2_V17 has the highest melting temperature among those tested, with a 5° C. increase relative to wild type uricase.
  • FIG. 5 shows the analysis of R2_V17, R2_V4, and R2_V79 C. utilis uricase enzymes by SDS-PAGE following incubation of 144 ng/ ⁇ L of uricase with 80 ng/ ⁇ L of pancreatin in SIF buffer at 37° C. for the indicated time points.
  • FIG. 6 shows the analysis of wild type and R2_V17 C. utilis uricase enzymes by SDS-PAGE following incubation of 100 ng/ ⁇ L of uricase with 320 ng/ ⁇ L of pancreatin in SIF buffer at 37° C. for the indicated time points.
  • the results from the SDS-PAGE analysis are consistent with the activity assay data.
  • the R2_V17, R2_V4 and R2_V79 mutants show increased stability in the presence of pancreatin relative to wild type.
  • This example describes the testing of individual substitutions included in the recombinant mutant Candida utilis uricases described in Example 1.
  • Example 1 Among the various substitutions included in the mutant Candida utilis uricases described in Example 1, a set of individual substitutions were selected for testing by protein modeling tools. In certain instances, conservative substitutions were tested along with the original substitution that was identified in Example 1. In total, 51 mutant C. utilis uricases, each with one amino acid substitution relative to the wild-type sequence, were designed and tested. The 51 mutant C. utilis uricases containing one amino acid substitution are indicated by the individual substitution in TABLE 5. The mutant C. utilis uricases were tested in a pancreatin stability assay along with a subset of the mutant C. utilis uricases described in Example 1. The subset of mutant C. utilis uricases described in Example 1 that were tested, containing five substitutions, are as set forth in TABLE 3. Results are summarized in TABLE 5, FIG. 7 , and FIG. 8 .
  • TABLE 5 depicts the amino acid substitutions for the mutant C. utilis uricases, as well as the specific activity ( ⁇ M/minute per 1.2 ⁇ M of uricase), pancreatin stability (half-life, minutes. ⁇ SEM), and expression yield ( ⁇ g/ml) for each enzyme.
  • Pancreatin stability was assayed at 40 ng/ ⁇ L soluble pancreatin. “nd” indicates that activity and stability measurements were not determined due to insufficient expression yield.
  • An expression vector comprising a codon-optimized nucleic acid sequence of SEQ ID NO: 13, which encodes a mutant Candida utilis uricase, was expressed in E. coli , and the expressed recombinant mutant uricase was isolated and purified.
  • ALLO allopurinol
  • ALLO allopurinol
  • the maintenance dose of 150 mg/L ALLO was removed.
  • the plasma urate levels were measured in plasma samples collected on day 7 after removal of the maintenance dose of ALLO, and urine uric acid levels were measured in 24-hour urine samples collected during the last 3 days of the pre-treatment period.
  • Plasma urate levels and urine uric acid levels were measured following the Liquick Cor-UA 30 plus protocol by Cormay, Poland (Liquick Cor-UA 30 plus, kit size 5 ⁇ 30 ml, Cat. No. 2-260.
  • mice treated with the recombinant mutant uricase (n 8) orally received approximately 62 mg/day (or 1,500 U/day) recombinant mutant uricase mixed with food (spray dried powder of 25% Uricase and 75% trehalose, mixed with 3.5 g food).
  • the plasma urate levels were measured in blood samples collected from the mice on day 7 of treatment with recombinant mutant uricase, ALLO 150 mg/L, and ALLO 50 mg/L, respectively, and urine uric acid levels were measured in 24-hour urine samples collected during the last 3 days of the treatment period.
  • plasma urate levels were measured in blood samples collected from the mice on day 7 after termination of treatment with recombinant mutant uricase, ALLO 150 mg/L, and ALLO 50 mg/L, respectively.
  • the assay for urine uric acid was performed according to the manufacturer's instructions (Liquick Cor-UA 30 plus protocol by Cormay, Poland (Liquick Cor-UA 30 plus, kit size 5 ⁇ 30 ml, Cat. No. 2-260)). For example, urine samples were diluted 1:4, 1:9, or 1:14 depending on groups of animals and the time of collection. To prevent precipitation of salts of uric acid, 1 drop of NaOH (500 g/L) was added to the collection tube before collection of a 24-hour specimen.
  • Plasma urate levels were also measured according to manufacturer's instructions (Liquick Cor-UA 30 plus protocol). Urate levels in the blood samples were measured without dilution or diluted 1:1 with double-distilled water (ddH 2 O).
  • hyperuricemia i.e., excess of uric acid in the blood
  • hyperuricosuria i.e., the presence of excessive amounts of uric acid in the urine
  • SEM standard of mean
  • digesta the semifluid mass into which food is converted by gastric secretion and which passes from the stomach into the small intestine
  • GIT gastrointestinal tract
  • Embodiments disclosed herein include embodiments P1 to P53, as provided in the numbered embodiments of the disclosure:
  • Embodiment P1 A recombinant mutant Candida utilis uricase comprising at least one (for example, one, two, three, four, five, six, seven or eight) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is selected from: (a) at position 180, isoleucine is substituted by valine or alanine (I180V or I180A), (b) at position 165, tyrosine is substituted by phenylalanine (Y165F), (c) at position 190, valine is substituted by glycine or alanine (V190G or V190A), (d) at position 51, glutamic acid is substituted by lysine (E51K), (e) at position 244, glutamine is substitute by lysine (Q244K), (f) at position 132, isoleucine is substituted by arginine or asparagine (I132R or I132N), (
  • Embodiment P2 The recombinant mutant C. utilis uricase of embodiment P1, wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, V190A, E51K, Q244K, I132R, V97I, E92N, A87G, D142E, G44A, G128P, A236N, K208A, N213A, S140T, Y253Q, and A84S.
  • Embodiment P3 The recombinant mutant C. utilis uricase of embodiment P1 or P2, wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, I132R, V97I, E92N, A87G, D142E, and G44A.
  • Embodiment P4 The recombinant mutant C. utilis uricase of any one of embodiments P1-P3, wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, I132R, and G44A.
  • Embodiment P5 The recombinant mutant C. utilis uricase of any one of embodiments P1-P4, wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, E51K, I132R, and G44A.
  • Embodiment P6 The recombinant mutant C. utilis uricase of any one of embodiments P1-P5, wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, and I132R.
  • Embodiment P7 A recombinant mutant Candida utilis uricase comprising at least one (for example, one, two, three, four, five, or six) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 190, position 51, position 132, and position 44.
  • Embodiment P8 A recombinant mutant Candida utilis uricase comprising at least one (for example, one, two, three, four, or five) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 51, position 132, and position 44.
  • Embodiment P9 A recombinant mutant Candida utilis uricase comprising at least one (for example, one, two, three, four, five, or six) mutation(s) at a position corresponding to wild type C. utilis uricase of SEQ ID NO: 1, wherein the at least one mutation is present at a position selected from position 180, position 165, position 190, position 51, position 244, and position 132.
  • Embodiment P10 The recombinant mutant C. utilis uricase of any one of embodiments P1-P9, wherein the uricase comprises two, three, four, five, six, seven, or eight mutations.
  • Embodiment P11 The recombinant mutant C. utilis uricase of any one of embodiments P1-P10, wherein the uricase comprises the following substitutions: I180V, Y165F, E51K, I132R, and G44A.
  • Embodiment P12 The recombinant mutant C. utilis uricase of any one of embodiments P1-P10, wherein the uricase comprises the following substitutions: I180A, Y165F, E51K, I132R, and G44A.
  • Embodiment P13 The recombinant mutant C. utilis uricase of any one of embodiments P1-P10, wherein the uricase comprises the following substitutions: I180V, Y165F, V190G, E51K, I132R, and G44A.
  • Embodiment P14 The recombinant mutant C. utilis uricase of any one of embodiments P1-P10, wherein the uricase comprises the following substitutions: I180A, Y165F, V190G, E51K, I132R, and G44A.
  • Embodiment P15 The recombinant mutant C. utilis uricase of any one of embodiments P1-P10, wherein the uricase comprises the following substitutions: I180V and Y165F.
  • Embodiment P16 The recombinant mutant C. utilis uricase of any one of embodiments P1-P10, wherein the uricase comprises the following substitutions: I180V, Y165F, V190G, E51K, Q244K, and I132R.
  • Embodiment P17 A recombinant mutant C. utilis uricase comprising a substitution listed in TABLE 1 or TABLE 2.
  • Embodiment P18 A recombinant mutant Candida utilis uricase having a half-life of at least 35 minutes in the presence of pancreatin.
  • Embodiment P19 The recombinant mutant C. utilis uricase of embodiment P17, wherein the half-life is 35-200 minutes in the presence of pancreatin.
  • Embodiment P20 The recombinant mutant C. utilis uricase of any one of embodiments P1-P19, wherein the uricase has 5-50 fold higher stability in the presence of pancreatin, compared to the wild-type uricase.
  • Embodiment P21 The recombinant mutant C. utilis uricase of embodiment P20, wherein the uricase has 20-30 fold higher stability in the presence of pancreatin, compared to the wild-type uricase.
  • Embodiment P22 The recombinant mutant C. utilis uricase of any one of embodiments P1-P21, wherein the uricase is isolated.
  • Embodiment P23 The recombinant mutant C. utilis uricase of any one of embodiments P1-P22, wherein the uricase is conjugated to a water soluble polymer.
  • Embodiment P24 The recombinant mutant C. utilis uricase of embodiment P23, wherein the uricase is conjugated to polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Embodiment P25 An expression vector comprising a nucleic acid sequence encoding the recombinant mutant C. utilis uricase of any one of embodiments P1-P24.
  • Embodiment P26 The expression vector of embodiment P25, wherein the nucleic acid sequence encoding the recombinant mutant uricase is codon optimized for expression in a heterologous cell.
  • Embodiment P27 The expression vector of embodiment P26, wherein the heterologous cell is Escherichia coli.
  • Embodiment P28 A cell comprising the expression vector of any one of embodiments P25-P27.
  • Embodiment P29 The cell of embodiment 28, wherein the cell is Escherichia coli.
  • Embodiment P30 A pharmaceutical composition comprising the recombinant mutant C. utilis uricase of any one of embodiments P1-P24.
  • Embodiment P31 The pharmaceutical composition of embodiment P30, further comprising a pharmaceutically acceptable carrier and/or an excipient.
  • Embodiment P32 The pharmaceutical composition of embodiment P30 or P31, wherein the composition is formulated as an oral dosage form or a parenteral dosage form.
  • Embodiment P33 The pharmaceutical composition of embodiment P32, wherein the composition is formulated as an oral dosage form.
  • Embodiment P34 The pharmaceutical composition of any one of embodiments P30-P33, wherein the composition is a formulated as a powder, granulate, pellet, micropellet, or a minitablet.
  • Embodiment P35 The pharmaceutical composition of any one of embodiments P30-P34, wherein the composition is encapsulated in a capsule or formulated as a tablet dosage form.
  • Embodiment P36 The pharmaceutical composition of embodiment P35, wherein the capsule is a hydroxypropyl methylcellulose (HPMC) capsule, soft gelatin capsule, or a hard gelatin capsule.
  • HPMC hydroxypropyl methylcellulose
  • Embodiment P37 The pharmaceutical composition of embodiment P32, wherein the composition is formulated as a parenteral dosage form.
  • Embodiment P38 The pharmaceutical composition of embodiment P37, wherein the composition is formulated as an intravenous dosage form.
  • Embodiment P39 A method of treating a disease or disorder associated with an elevated amount of uric acid in a subject in need thereof, the method comprising administering to the subject an effective amount of the recombinant mutant C. utilis uricase of any one of embodiments P1-P24, thereby treating the disease or disorder in the subject.
  • Embodiment P40 The method of embodiment P39, wherein the disease or disorder is associated with an elevated amount of uric acid in plasma of the subject.
  • Embodiment P41 A method of treating hyperuricemia in a subject in need thereof, the method comprising administering to the subject an effective amount of the recombinant mutant C. utilis uricase of any one of embodiments P1-P24, thereby treating hyperuricemia in the subject.
  • Embodiment P42 A method of treating gout in a subject in need thereof, the method comprising administering to the subject an effective amount of the recombinant mutant C. utilis uricase of any one of embodiments P1-P24, thereby to treat gout in the subject.
  • Embodiment P43 A method of treating hyperuricemia in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments P30-P38, thereby to treat hyperuricemia in the subject.
  • Embodiment P44 A method of treating gout in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments P30-P38, thereby to treat gout in the subject.
  • Embodiment P45 The method of any one of embodiments P39-P44, wherein the recombinant mutant C. utilis uricase is administered in combination with a xanthine oxidase inhibitor, a uricosuric, or a combination thereof.
  • Embodiment P46 The method of embodiment P45, wherein the xanthine oxidase inhibitor is selected from allopurinol and febuxostat.
  • Embodiment P47 The method of embodiment P45, wherein the uricosuric is selected from probenecid, benzbromarone, losartan and lesinurad.
  • Embodiment P48 A method of treating hyperuricosuria in a subject in need thereof, the method comprising administering to the subject an effective amount of the recombinant mutant C. utilis uricase of any one of embodiments P1-P24, thereby treating hyperuricosuria in the subject.
  • Embodiment P49 A method of treating hyperuricosuria in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments P30-P38, thereby to treat hyperuricosuria in the subject.
  • Embodiment P50 The method of embodiment P48 or P49, wherein the recombinant mutant C. utilis uricase is administered in combination with a xanthine oxidase inhibitor, a uricosuric, or a combination thereof.
  • Embodiment P51 The method of embodiment P48 or P49, wherein the recombinant mutant C. utilis uricase is administered subsequent to administration of a xanthine oxidase inhibitor, a uricosuric, or a combination thereof.
  • Embodiment P52 The method of embodiment P50 or P51, wherein the xanthine oxidase inhibitor is selected from allopurinol and febuxostat.
  • Embodiment P53 The method of embodiment P50 or P51, wherein the uricosuric is selected from probenecid, benzbromarone, losartan and lesinurad.

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