US2020308534A1PendingUtilityA1

Recombinant uricase enzyme

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Assignee: ALLENA PHARMACEUTICALS INCPriority: Jul 7, 2017Filed: Jul 6, 2018Published: Oct 1, 2020
Est. expiryJul 7, 2037(~11 yrs left)· nominal 20-yr term from priority
Inventors:Aditi R. DeshpandeDanica GrujicSridhar GovindarajanMark Welch
C12N 1/20A61K 31/4196A61K 31/195A61K 45/06C12N 9/0048A61K 31/426C12N 1/16A61K 38/44A61P 7/00A61K 9/0019A61K 31/4178A61K 31/519C12Y 107/03003Y02A50/30
51
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Claims

Abstract

Disclosed are recombinant mutant Candida utilis uricase enzymes with improved pancreatin stability and/or activity, compositions containing such uricase enzymes, which can be used, among other things, to treat diseases or disorders associated with an elevated amount of uric acid, including, for example, hyperuricemia, hyperuricosuria, and gout.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . 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), (g) at position 97, valine is substituted by isoleucine (V97I), (h) at position 92, glutamic acid is substituted by asparagine (E92N), (i) at position 87, alanine is substituted by glycine (A87G), (j) at position 142, aspartic acid is substituted by glutamic acid (D142E), (k) at position 44, glycine is substituted by alanine (G44A), (1) at position 128, glycine is substituted by proline (G128P), (m) at position 236, alanine is substituted by asparagine (A236N), (n) at position 208, lysine is substituted by alanine (K208A), (o) at position 213, asparagine is substituted by alanine (N213A), (p) at position 140, serine is substituted by threonine (5140T), (q) at position 253, tyrosine is substituted by glutamine (Y253Q), (r) at position 84, alanine is substituted by serine (A84S), (s) at position 47, threonine is substituted by glutamic acid (T47E), (t) at position 95, serine is substituted by proline (595P), (u) at position 103, lysine is substituted by threonine (K103T), (v) at position 134, aspartic acid is substituted by glutamic acid (D134E), (w) at position 136, tyrosine is substituted by arginine (Y136R), (x) at position 196, isoleucine is substituted by leucine (I196L), (y) at position 224, threonine is substituted by aspartic acid (T224D), (z) at position 285, proline is substituted by serine (P285S), and (aa) at position 296, valine is substituted by alanine (V296A). 
     
     
         2 . The recombinant mutant  C. utilis  uricase of  claim 1 , 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. 
     
     
         3 . The recombinant mutant  C. utilis  uricase of  claim 1  or  2 , wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, I132R, V97I, E92N, A87G, D142E, and G44A. 
     
     
         4 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 3 , wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, I132R, and G44A. 
     
     
         5 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 4 , wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, E51K, I132R, and G44A. 
     
     
         6 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 5 , wherein the uricase comprises at least one mutation selected from: I180V, I180A, Y165F, V190G, E51K, Q244K, and I132R. 
     
     
         7 . 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. 
     
     
         8 . 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. 
     
     
         9 . 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. 
     
     
         10 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 9 , wherein the uricase comprises two, three, four, five, six, seven, or eight mutations. 
     
     
         11 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 10 , wherein the uricase comprises the following substitutions: I180V, Y165F, E51K, I132R, and G44A. 
     
     
         12 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 10 , wherein the uricase comprises the following substitutions: I180A, Y165F, E51K, I132R, and G44A. 
     
     
         13 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 10 , wherein the uricase comprises the following substitutions: I180V, Y165F, V190G, E51K, I132R, and G44A. 
     
     
         14 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 10 , wherein the uricase comprises the following substitutions: I180A, Y165F, V190G, E51K, I132R, and G44A. 
     
     
         15 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 10 , wherein the uricase comprises the following substitutions: I180V and Y165F. 
     
     
         16 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 10 , wherein the uricase comprises the following substitutions: I180V, Y165F, V190G, E51K, Q244K, and I132R. 
     
     
         17 . A recombinant mutant  C. utilis  uricase comprising a substitution listed in TABLE 1 or TABLE 2. 
     
     
         18 . A recombinant mutant  Candida utilis  uricase having a half-life of at least 35 minutes in the presence of pancreatin. 
     
     
         19 . The recombinant mutant  C. utilis  uricase of  claim 17 , wherein the half-life is 35-200 minutes in the presence of pancreatin. 
     
     
         20 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 19 , wherein the uricase has 5-50 fold higher stability in the presence of pancreatin, compared to the wild-type uricase. 
     
     
         21 . The recombinant mutant  C. utilis  uricase of  claim 20 , wherein the uricase has 20-30 fold higher stability in the presence of pancreatin, compared to the wild-type uricase. 
     
     
         22 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 21 , wherein the uricase is isolated. 
     
     
         23 . The recombinant mutant  C. utilis  uricase of any one of  claims 1 - 22 , wherein the uricase is conjugated to a water soluble polymer. 
     
     
         24 . The recombinant mutant  C. utilis  uricase of  claim 23 , wherein the uricase is conjugated to polyethylene glycol (PEG). 
     
     
         25 . An expression vector comprising a nucleic acid sequence encoding the recombinant mutant  C. utilis  uricase of any one of  claims 1 - 24 . 
     
     
         26 . The expression vector of  claim 25 , wherein the nucleic acid sequence encoding the recombinant mutant uricase is codon optimized for expression in a heterologous cell. 
     
     
         27 . The expression vector of  claim 26 , wherein the heterologous cell is  Escherichia coli.    
     
     
         28 . A cell comprising the expression vector of any one of  claims 25 - 27 . 
     
     
         29 . The cell of  claim 28 , wherein the cell is  Escherichia coli.    
     
     
         30 . A pharmaceutical composition comprising the recombinant mutant  C. utilis  uricase of any one of  claims 1 - 24 . 
     
     
         31 . The pharmaceutical composition of  claim 30 , further comprising a pharmaceutically acceptable carrier and/or an excipient. 
     
     
         32 . The pharmaceutical composition of  claim 30  or  31 , wherein the composition is formulated as an oral dosage form or a parenteral dosage form. 
     
     
         33 . The pharmaceutical composition of  claim 32 , wherein the composition is formulated as an oral dosage form. 
     
     
         34 . The pharmaceutical composition of any one of  claims 30 - 33 , wherein the composition is a formulated as a powder, granulate, pellet, micropellet, or a minitablet. 
     
     
         35 . The pharmaceutical composition of any one of  claims 30 - 34 , wherein the composition is encapsulated in a capsule or formulated as a tablet dosage form. 
     
     
         36 . The pharmaceutical composition of  claim 35 , wherein the capsule is a hydroxypropyl methylcellulose (HPMC) capsule, soft gelatin capsule, or a hard gelatin capsule. 
     
     
         37 . The pharmaceutical composition of  claim 32 , wherein the composition is formulated as a parenteral dosage form. 
     
     
         38 . The pharmaceutical composition of  claim 37 , wherein the composition is formulated as an intravenous dosage form. 
     
     
         39 . 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  claims 1 - 24 , thereby treating the disease or disorder in the subject. 
     
     
         40 . The method of  claim 39 , wherein the disease or disorder is associated with an elevated amount of uric acid in plasma of the subject. 
     
     
         41 . 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  claims 1 - 24 , thereby treating hyperuricemia in the subject. 
     
     
         42 . 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  claims 1 - 24 , thereby to treat gout in the subject. 
     
     
         43 . 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  claims 30 - 38 , thereby to treat hyperuricemia in the subject. 
     
     
         44 . 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  claims 30 - 38 , thereby to treat gout in the subject. 
     
     
         45 . The method of any one of  claims 39 - 44 , wherein the recombinant mutant  C. utilis  uricase is administered in combination with a xanthine oxidase inhibitor, a uricosuric, or a combination thereof. 
     
     
         46 . The method of  claim 45 , wherein the xanthine oxidase inhibitor is selected from allopurinol and febuxostat. 
     
     
         47 . The method of  claim 45 , wherein the uricosuric is selected from probenecid, benzbromarone, losartan and lesinurad. 
     
     
         48 . 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  claims 1 - 24 , thereby treating hyperuricosuria in the subject. 
     
     
         49 . 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  claims 30 - 38 , thereby to treat hyperuricosuria in the subject. 
     
     
         50 . The method of  claim 48  or  49 , wherein the recombinant mutant  C. utilis  uricase is administered in combination with a xanthine oxidase inhibitor, a uricosuric, or a combination thereof. 
     
     
         51 . The method of  claim 48  or  49 , wherein the recombinant mutant  C. utilis  uricase is administered subsequent to administration of a xanthine oxidase inhibitor, a uricosuric, or a combination thereof. 
     
     
         52 . The method of  claim 50  or  51 , wherein the xanthine oxidase inhibitor is selected from allopurinol and febuxostat. 
     
     
         53 . The method of  claim 50  or  51 , wherein the uricosuric is selected from probenecid, benzbromarone, losartan and lesinurad.

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