Protoxin-ii variants and methods of use

Abstract

The present invention relates to Protoxin-II variants, polynucleotides encoding them, and methods of making and using the foregoing.

1 . An isolated Protoxin-II variant, wherein the Protoxin-II variant inhibits human Nav1.7 activity with an IC 50  value of about 1×10 −7  M or less, wherein the IC 50  value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7, wherein the Protoxin-II variant has a W7Q and/or a W30L substitution, wherein residue numbering is according to SEQ ID NO: 1. 
     
     
         2 . The isolated Protoxin-II variant of  claim 1 , comprising the sequence X 1 X 2 X 3 CX 4 X 5 WX 6 QX 7 CX 8 X 9 X 10 X 11 X 12 CCX 13 X 14 FX 15 CX 16 LWCX 17 KKLL (SEQ ID NO: 432), wherein
 X 1  is G, P, A or deleted;   X 2  is P, A or deleted;   X 3  is S, Q, A, R or Y;   X 4  is Q, R, K, A or S;   X 5  is K, S, Q or R;   X 6  is M or F;   X 7  is T, S, R, K or Q;   X 8  is D or T;   X 9  is S, A or R;   X 10  is E, R, N, K, T or Q;   X 11  is R or K;   X 12  is K, Q, S or A;   X 13  is E, Q or D;   X 14  is G or Q;   X 15  is V or S;   X 16  is R or T; and   X 17  is K or R;   optionally having an N-terminal extension or a C-terminal extension,   wherein the polypeptide inhibits human Nav1.7 activity with an IC 50  value of about 1×10 −7  M or less, wherein the IC 50  value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.   
     
     
         3 . The Protoxin-II variant of  claim 2 , wherein the N-terminal extension comprises the amino acid sequence of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 or 385 and/or the C-terminal extension comprises the amino acid sequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 or 397. 
     
     
         4 . (canceled) 
     
     
         5 . The Protoxin-II variant of  claim 3 , wherein the N-terminal and/or the C-terminal extension is conjugated to the Protoxin-II variant via a linker. 
     
     
         6 . The Protoxin-II variant of  claim 5 , wherein the linker comprises the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402. 
     
     
         7 . The isolated Protoxin-II variant of  claim 1 , comprising the amino acid sequence of SEQ ID NOs: 30, 40, 44, 52, 56, 56, 59, 65, 78, 109, 110, 111, 114, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 162, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 178, 179, 180, 182, 183, 184, 185, 186, 189, 190, 193, 195, 197, 199, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 224, 226, 227, 231, 232, 243, 244, 245, 247, 249, 252, 255, 258, 261, 263, 264, 265, 266, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 332, 334, 335, 336, 337, 339, 340, 341, 342, 346, 351, 358, 359, 364, 366, 367, 368, 369, 370, 371, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430 or 431. 
     
     
         8 . The isolated Protoxin-II variant of  claim 1 , that inhibits human Nav1.7 activity with an IC 50  value of about 3×10 −8  M or less. 
     
     
         9 . The isolated Protoxin-II variant of  claim 8  that inhibits human Nav1.7 activity with an IC 50  value of between about 3×10 −8 M to about 1×10 −9  M. 
     
     
         10 . The isolated Protoxin-II variant of  claim 8 , comprising the amino acid sequence GPQCX 1 X 2 WX 3 QX 4 CX 5 X 6 X 7 X 8 X 9 CCX 10 X 11 FX 12 CX 13 LWCX 14 KKLL (SEQ ID NO: 433), wherein
 X 1  is Q, R, K, A or S;   X 2  is K, S, Q or R;   X 3  is M or F;   X 4  is T, S, R, K or Q;   X 5  is D or T;   X 6  is S, A or R;   X 7  is E, R, N, K, T or Q;   X 8  is R or K;   X 9  is K, Q, S or A;   X 10  is E, Q or D;   X 11  is G or Q;   X 12  is V or S;   X 13  is R or T; and   X 14  is K or R.   
     
     
         11 . The isolated Protoxin-II variant of  claim 1 , comprising the amino acid sequence of SEQ ID NOs: 56, 78, 111, 114, 117, 118, 119, 122, 123, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 158, 159, 165, 172, 173, 175, 177, 178, 183, 184, 185, 186, 189, 190, 193, 197, 199, 207, 210, 211, 216, 217, 224, 266, 273, 282, 335, 408, 409, 410, 422, 424, 425, 426, 427 and 428. 
     
     
         12 . An isolated Protoxin-II variant comprising the amino acid sequence that is 90%, identical to the amino acid sequence of SEQ ID NO: 422 (GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLL-COOH); wherein
 the Protoxin-II variant has Q at position 7 and L at position 30, when residue numbering is according to SEQ ID NO: 1; and   the polypeptide inhibits human Nav1.7 activity with an IC 50  value of about 30×10 −9  M or less, wherein the IC 50  value is measured using a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −6  M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.   
     
     
         13 . The isolated Protoxin-II variant of  claim 1 , having a free C-terminal carboxylic acid, amide, methylamide or butylamide group. 
     
     
         14 . An isolated fusion protein comprising the Protoxin-II variant of  claim 1  conjugated to a half-life extending moiety. 
     
     
         15 . The fusion protein of  claim 14 , wherein the half-life extending moiety is human serum albumin (HSA), albumin binding domain (ABD), Fc or polyethylene glycol (PEG). 
     
     
         16 . An isolated polynucleotide encoding the Protoxin-II variant of  claim 12 . 
     
     
         17 . A vector comprising the isolated polynucleotide of  claim 16 . 
     
     
         18 . A host cell comprising the vector of  claim 17 . 
     
     
         19 . A method of producing the isolated Protoxin-II variant, comprising culturing the host cell of  claim 18  and recovering the Protoxin-II variant produced by the host cell. 
     
     
         20 . A pharmaceutical composition comprising the isolated Protoxin-II variant or fusion protein of  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         21 . A method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject in need thereof an effective amount of the Protoxin-II variant or the fusion protein of  claim 1  to treat the pain. 
     
     
         22 . The method of  claim 21 , wherein pain is chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, post-operative pain, thermal pain, phantom limb pain, or pain associated with inflammatory conditions, primary erythemalgia (PE), paraoxysmal extreme pain disorder (PEPD), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis, fibromyalgia, painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN), trigeminal neuralgia (TN), spinal cord injuries or multiple sclerosis. 
     
     
         23 . The method of  claim 22 , wherein the Protoxin-II variant is administered peripherally. 
     
     
         24 . The method of  claim 23 , wherein the Protoxin-II variant is administered locally to a joint, spinal cord, surgical wound, sites of injury or trauma, peripheral nerve fibers, urogenital organs, or inflamed tissues. 
     
     
         25 . The method of  claim 24 , wherein the subject is a human. 
     
     
         26 .- 29 . (canceled)