Sustained delivery of angiopoetin-like 3 polypeptides

Abstract

Described herein are drug delivery systems for delivering biologically active agents comprising primary or secondary amines, or a ring nitrogen atom of an azaheteroaryl ring, pharmaceutically acceptable salts thereof, drug delivery reagents related thereto, pharmaceutical compositions comprising the drug delivery systems, and the use of the drug delivery systems as sustained release therapeutics.

What is claimed is: 
     
         1 . A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a drug delivery system or pharmaceutically acceptable salt thereof comprising D-R, that is represented by Formula (I), where D is an ANGPTL3 polypeptide comprising at least one primary amine; and
 R has the formula:   
       
         
           
           
               
               
           
         
         where the dashed line indicates attachment to the primary amine of D; 
         R 1  is hydrogen or C 1 -C 4 alkyl; 
         R 1a  is hydrogen or C 1 -C 4 alkyl, or CR 1 R 1a , taken in combination form a C 3 -C 6 cycloalk-1,1-diyl; 
         R 2  is independently selected at each occurrence from C 1 -C 4 alkyl or oxo, or two R 2  groups taken in combination with the carbon atom(s) to which they are attached form a fused C 3 -C 6  cycloalkyl or spiro C 3 -C 6 cycloalk-1,1-diyl group; 
         a is 0, 1, 2, 3 or 4; 
         R 3  is hydrogen or C 1 -C 4 alkyl; 
         R 3a  is hydrogen, C 1 -C 4 alkyl, or CR 3 R 3a , taken in combination form a C 3 -C 6 cycloalk-1,1-diyl; 
         Y is C(O)R 4 , C(O)OR 4 , C(O)NHR 4 , C(O)NR 5 R 6 , SiR 5 R 6 R 7 , or CR 12 R 12a OR 13 ; 
         R 12  is hydrogen or C 1 -C 4 alkyl; 
         R 12a  is hydrogen or C 1 -C 4 alkyl, or CR 12 R 12a , taken in combination form a C 3 -C 6 cycloalk-1,1-diyl; 
         R 13  is C 1 -C 4 alkyl; or 
         R 12  and R 13 , taken in combination with C(R 12a ) and O form a 5, 6, or 7-member cyclic ether; 
         R 4  is C 1 -C 8 alkyl or C 3 -C 7 cycloalkyl, wherein cycloalkyl is optionally substituted with 0, 1, or 2 independently selected C 1 -C 4 alkyl groups and wherein alkyl is optionally substituted by C 1 -C 4 alkoxy; 
         R 5  and R 6  are each independently selected from C 1 -C 4 alkyl and C 3 -C 6 cycloalkyl; 
         R 7  is C 1 -C 8 alkyl, C 3 -C 7 cycloalkyl, C 1 -C 8 alkoxy, C 3 -C 7 cycloalkyloxy, heterocycloalkyloxy, or —(OCHR 3 CH 2 ) b O—C 1 -C 4 alkyl, wherein the heterocycloalkyloxy is a 4 to 7 member saturated heterocyclic ring having one ring heteroatom selected from N, O, and S and optionally substituted with 0, 1, or 2 independently selected C 1 -C 4 alkyl groups; 
         b is an integer of from 1 to 10; 
         Z is, N-L-A, N-A, or NR 9 ; 
         L is an optionally substituted bivalent linker Q-[Sp-Q]h-Q; 
         Q is independently selected at each occurrence from a bond, O, C(O), N(H), N(C 1 -C 4 alkyl), C(O)NH, C(O)N(C 1 -C 4 alkyl), N(H)C(O), N(C 1 -C 4 alkyl)C(O), N(H)C(O)O, N(C 1 -C 4 alkyl)C(O)O, OC(O)N(H), OC(O)N(C 1 -C 4 alkyl), N(H)C(O)N(H), N(C 1 -C 4 alkyl)C(O)N(H), N(H)C(O)N(C 1 -C 4 alkyl), N(C 1 -C 4 alkyl)C(O)N(C 1 -C 4 alkyl), C(O)O, OC(O), OC(O)O, S, S(O) 2 , N(H)S(O) 2 , N(C 1 -C 4 alkyl)S(O) 2 , S(O) 2 N(H), S(O) 2 N(C 1 -C 4 alkyl), C 1 -C 2 alkyl-C(O)N(H), N(H)C(O)C 1 -C 2 alkyl, C 1 -C 2 alkyl-C(O)O, OC(O)C 1 -C 2 alkyl, 1,2,3-triazole, OP(O) 2 , P(O) 2 O, C 1 -C 4 alkyl-P(O) 2 —O, or O—P(O) 2 —C 1-4 alkyl; 
         Sp is independently selected at each occurrence from an optionally substituted C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, [W—O] g , C 1 -C 8 alkyl-[O—W] g , [O—W] g —O—C 1 -C 8 alkyl, C 1 -C 8 Calkyl-[O—W] g —O—C 1 -C 8 alkyl, or oligopeptide; 
         h is an integer of between 1 and 20; 
         g is a weighted average number of between about 2 and about 50; 
         R 9  is selected from hydrogen, C 1 -C 8  alkyl, C(O)—(CH 2 ) n -Q-A, C(O)C 1 -C 8  alkyl, or —C(O)(CH 2 ) q [O—W] g (NHC(O)) m (CH 2 ) q [O—W] p -Q-A, wherein the alkyl group is optionally substituted with 0 or 1 Q-A; 
         W is C 2 -C 4 alkyl-1,2-diyl in which a hydrogen, methyl, or ethyl side chain may be present on either backbone carbon atom; 
         A is hydrogen, C 1 -C 8 alkyl, C(O)C 1 -C 8 alkyl, C(O)N(H)C 1 -C 8 alkyl, C(O)OC 1 -C 8 alkyl, or R 11 ; and 
         R 11  is a carrier. 
       
     
     
         2 . The method of  claim 1 , wherein D is an ANGPTL3 polypeptide having at least 95% identity to any one of SEQ ID NOS: 1 or 3-45. 
     
     
         3 . The method of  claim 1 , wherein D is an ANGPTL3 polypeptide comprising amino acid residues 201-460; 207-460; 225-455; 225-455; 225-460; 225-460; 226-455; 226-455; 226-460; 226-460; 228-455; 228-455; 228-460; 228-460; 233-455; 233-455; 233-460; 233-460; 241-455; 241-455; 241-460; 241-460; 242-455; 242-455; 242-460; or 242-460, each in reference to SEQ ID NO:1. 
     
     
         4 . The method of  claim 1 , wherein D is an ANGPTL3 polypeptide comprising at least 95% identity to amino acid residues 242-460 in reference to SEQ ID NO:1 and a K423Q substitution. 
     
     
         5 . The method of  claim 1 , wherein R 1  is hydrogen or methyl, R 1a  is hydrogen or methyl, or CR 1 R 1a , taken in combination form a cyclopropan-1,1-diyl group. 
     
     
         6 . The method of  claim 1 , wherein the variable a is 0. 
     
     
         7 . The method of  claim 1 , wherein R 3  and R 3a  are each hydrogen. 
     
     
         8 . The method of  claim 1 , wherein
 Y is C(O)R 4  and R 4  is C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or C 1 -C 2 alkoxyC 1 -C 2 alkyl, or   Y is SiR 5 R 6 R 7 ;   wherein   R 5  and R 6  are each methyl, ethyl, propyl or isopropyl; and   R 7  is C 1 -C 4  alkyl, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, 2-ethoxyethoxy, 2-isopropoxy-ethoxy, tetrahydropyranyloxy, or —(OCHR 3 CH 2 ) b O—C 1 -C 4 alkyl where b is 2, 3, or 4.   
     
     
         9 . The method of  claim 8 , wherein Y is C(O)R 4  and R 4  is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl, or methoxymethyl. 
     
     
         10 . The method of  claim 1 , wherein Z is NR 9 ;
 R 8  and R 9  are each independently selected from hydrogen, C 1 -C 8  alkyl, C(O)—(CH 2 ) n -Q-A, C(O)C 1 -C 8  alkyl, or —C(O)(CH 2 ) q [O—W] g (NHC(O)) m (CH 2 ) q [O—W] p -Q-A, wherein the alkyl group is optionally substituted with 0 or 1 Q-A;   q is independently at each occurrence 1, 2, or 3;   g and p each independently have a weighted average length of between about 2 and about 50;   m is 1 or 0;   W is C 2 -C 4 alkyl-1,2-diyl in which a hydrogen, methyl, or ethyl side chain may be present on either backbone carbon atom;   Q is a bond, O, N(H) or N(C 1 -C 4 alkyl);   A is hydrogen, C 1 -C 8 alkyl, C(O)C 1 -C 8 alkyl, C(O)N(H)C 1 -C 8 alkyl, C(O)OC 1 -C 8 alkyl, R 10 ;   and   R 11  is a carrier.   
     
     
         11 . The method of  claim 1 , wherein R 11  comprises a hydrogel comprising one or more cross-linked polymers. 
     
     
         12 . The method of  claim 1 , wherein R 11  comprises a polymer, cross-linked polymer, or hydrogel comprising one or more of hyaluronic acid, polyethylene glycol, polypropylene glycol, polyethylene oxide, polypropylene oxide, polyglutamate, polylysine, polysialic acid, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl pyrrolidone, polyoxazoline, polyiminocarbonate, polyamino acid, hydrophilic polyester, polyamide, polyurethane, polyurea, dextran, agarose, xylan, mannan, carrageenan, alginate, gelatin, collagen, albumin, cellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl starch, chitosan, nucleic acids, derivatives thereof, co-polymers thereof, or combinations thereof. 
     
     
         13 . The method of  claim 12 , wherein R 11  comprises a hydrogel comprising cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one side chain selected from —NH(W1) (O(W1)) d —V, wherein W1 is C 2 -C 4 alkyl-1,2-diyl in which a hydrogen, methyl, or ethyl side chain may be present on either backbone carbon atom;
 d is a number average of 0 to 500; and 
 V is a suitable functional group comprising azidyl, alkynyl, substituted or unsubstituted C 7 -C 12  cycloalkynyl, substituted or unsubstituted C 7 -C 12  heterocycloalkynyl, C 7 -C 12  cycloalkenyl, norbornyl, vinyl carboxyl, vinyl sulfonyl, C 2 -C 8  alkenyl, amino, thiol, C 1 -C 8  carboxyl, C 1 -C 8  carbonyl, —O—NH2, carbohydrazide, maleimide, alpha-halo carbonyl, furan, substituted or unsubstituted tetrazinyl, lysine, glutamine, cyclodextrin, or adamantanyl. 
 
     
     
         14 . A process for making a cross-linked carrier formulation, the process comprising:
 (a) functionalizing a carrier molecule, R 11 ;   (b) preparing a reactive cross-linker; and   (c) reacting the functionalized carrier molecule with the reactive cross-linker to form a cross-linked carrier by incubation for about 0.5 hours to about 48 hours at a temperature of about 4° C. to about 60° C.   
     
     
         15 . A cross-linked hydrogel obtainable using the methods of  claim 14 . 
     
     
         16 . A process for making a drug delivery system according to  claim 1 , the process comprising:
 (a) preparing a carrier molecule, R 11 , wherein R 11  is a cross-linked hydrogel;   optionally, step (a) may further comprise purifying the cross-linked hydrogel carrier molecule R 11 ;   (b) separately conjugating the traceless linker, R, to an ANGPTL3 polypeptide comprising at least one primary amine, D, thereby forming the traceless linker-D adduct;   step (b) may optionally further comprise purification of the traceless linker-D adduct,   (c) conjugating the carrier molecule, R 11 , with the traceless linker-D adduct; and   (d) purifying the drug delivery system from the reagents.   
     
     
         17 . The method of  claim 1 , wherein the disease or disorder is a musculoskeletal disease or disorder. 
     
     
         18 . A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a drug delivery system comprising Formula (III): 
       
         
           
           
               
               
           
         
         wherein ANGPTL3 comprises an ANGPTL3 polypeptide having at least 95% identity to any one of SEQ ID NO: 1 or 3-45, or combinations thereof. 
       
     
     
         19 . The method of  claim 18 , wherein ANGPTL3 is a polypeptide comprising amino acid residues 242-460 in reference to SEQ ID NO:1 and a K423Q substitution. 
     
     
         20 . The method of  claim 1 , wherein the drug delivery system or pharmaceutically acceptable salt thereof has one of the following formulae: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         21 . The method of  claim 20 , wherein each R has the following formula: 
       
         
           
           
               
               
           
         
         wherein the dashed lines indicate attachment to D and the bicyclo[6.1.0]non-4-yn-9-yl moiety, and 
         R 4  is methyl, ethyl, propyl, isopropyl, 1-methyl-cyclopropyl, or methoxymethyl. 
       
     
     
         22 . The method of  claim 1 , wherein
 Y is C(O)R 4 ;   R 4  is C 1 -C 8 alkyl or C 3 -C 7 cycloalkyl, wherein cycloalkyl is optionally substituted with 0, 1, or 2 independently selected C 1 -C 4 alkyl groups and wherein alkyl is optionally substituted by C 1 -C 4 alkoxy;   Z is N-L-A;   L is C(O)CH 2 CH 2 NH;   A is R 11 ; and   R 11  is a hydrogel derived from a cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH 2 CH 2 O) 3  CH 2 CH 2 N 3 , and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[3-(((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)propanoate], or   R 11  is a hydrogel derived from a cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH 2 CH 2 O) 3  CH 2 CH 2 N 3 , and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[1-((((1′R,8′S,9′s)-bicyclo[6.1.0]non-4′-yn-9′-yl)methoxy)carbonyl)amino-cyclopropane-1-carboxylic acid ester], or   R 11  is a hydrogel derived from a cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH 2 CH 2 O) 3  CH 2 CH 2 N 3 , and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[1-((((1′R,8′S,9′s)-bicyclo[6.1.0]non-4′-yn-9′-yl)methoxy)carbonyl)piperidine-4-carboxylic acid ester], or   R 11  is a hydrogel derived from a cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH 2 CH 2 O) 3  CH 2 CH 2 N 3 , and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[7-(((((1′R,8′S,9′s)-bicyclo[6.1.0]non-4′-yn-9′-yl)methoxy)carbonyl)amino)heptanoate], or   R 11  is a hydrogel derived from a cross-linked hyaluronic acid, wherein the hyaluronic acid comprises at least one amide-linked side chain of N(H)(CH 2 CH 2 O) 3  CH 2 CH 2 N 3 , and wherein the cross-linker used to form the hydrogel comprises PEG(2000)-bis-[2-methyl-3-(((((1′R,8′S,9′s)-bicyclo[6.1.0]non-4′-yn-9′-yl)methoxy)carbonyl)amino)propanoate].