Protoxin-II variants and methods of use

Patent metadata

Field	Value
Publication number	US-11083776-B2
Application number	US-201916673750-A
Country	US
Kind code	B2
Filing date	Nov 4, 2019
Priority date	Apr 2, 2015
Publication date	Aug 10, 2021
Grant date	Aug 10, 2021

Abstract

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

First claim

We claim:

1. A method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject in need thereof an effective amount of an isolated Protoxin-II variant or a fusion protein thereof to treat the pain, wherein the Protoxin-II variant has at least one amino acid substitution selected from the group consisting of W7Q and W30L; wherein residue numbering is according to SEQ ID NO: 1.

2. A method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject in need thereof an effective amount of an isolated Protoxin-II variant or a fusion protein thereof, wherein the Protoxin-II variant inhibits human Nav1.7 activity with an IC 50 value of about 1×10 −7 M or less, about 1×10 −8 M or less, about 1×10 −9 M or less, about 1×10 −10 M or less, about 1×10 −11 M or less, or about 1×10 −12 M or less, wherein the IC 50 value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −8 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.

3. The method of claim 1 , wherein the isolated Protoxin-II variant comprises 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 X 15 X 16 CX 17 LWCX 18 KKLX 19 (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, S, or Y;
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, T or asparagyl-4-aminobutane;
X 9 is S, A R, I, or V;
X 10 is E, R, N, K, T, Q, Y or glutamyl-4-aminobutane;
X 11 is R or K;
X 12 is K, Q, S, A or F;
X 13 is E, Q, D, L, N or glutamyl-4-aminobutane;
X 14 is G, Q or P;
X 15 is M or F;
X 16 is V or S;
X 17 is R, T or N-omega methyl-L-arginine;
X 18 is K or R; and
X 19 is W or L,

optionally having an N-terminal extension or a C-terminal extension.

4. The method of claim 1 , wherein the isolated Protoxin-II variant has a substitution at one or more residue positions Y 1 , W 7 , S 11 , E 12 , K 14 , E 17 , G 18 , R 22 and L 29 , when residue numbering is according to SEQ ID NO: 1.

5. The method of claim 1 , wherein the isolated Protoxin-II variant is a derivative of the sequence YCQKWMQTCDSERKCCEGMVCRLWCKKKLW-OH (SEQ ID NO: 424); wherein residue Y 1 , S 11 , E 12 , K 14 , E 17 , G 18 , R 22 and/or L 29 is substituted with
a) any other amino acid selected from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;
b) a non-natural amino acid; and
c) W30 is substituted by L.

6. The method of claim 1 , wherein the isolated Protoxin-II variant is a derivative of the sequence YCQKWMQTCDSERKCCEGMVCRLWCKKKLL-OH (SEQ ID NO: 425); wherein residue Y 1 , S 11 , E 12 , K 14 , E 17 , G 18 , M 19 and/or L 29 is substituted with
a) any other amino acid selected from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; or
b) a non-natural amino acid.

7. The method of claim 1 , wherein the isolated Protoxin-II variant comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 422 (GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLL-COOH); wherein the residue numbering is according to SEQ ID NO: 1.

8. The method of claim 1 , wherein the isolated Protoxin-II variant comprises the sequence X 1 X 2 X 3 CQKWMQTCDX 4 X 5 RX 6 CCX 7 X 8 X 9 VCRLWCKKKX 10 X 11 (SEQ ID NO: 737); 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 S, A, R, I or V;
X 5 is E, R, N, K, T, Q, Y or glutamyl-4-aminobutane;
X 6 is K, Q, S, A or F;
X 7 is E, Q, D, L, N or glutamyl-4-aminobutane;
X 8 is G, Q or P;
X 9 is M or F;
X 10 is L, V; and
X 11 is W or L.

9. The method of claim 1 , wherein the isolated Protoxin-II variant further comprises an N-terminal extension and 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.

10. The method of claim 1 , wherein the isolated Protoxin-II variant further comprises a C-terminal extension and wherein 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.

11. The method of claim 1 , wherein the isolated Protoxin-II variant further comprises an N-terminal extension and/or a C-terminal extension and wherein the N-terminal and/or the C-terminal extension is conjugated to the Protoxin-II variant via a linker.

12. The method of claim 11 , wherein the linker comprises the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.

13. The method of claim 1 , wherein the isolated Protoxin-II variant inhibits human Nav1.7 activity with an IC 50 value of about 3×10 −8 M or less, when the IC 50 value is measured using a veratridine-induced depolarization inhibition assay using fluorescence resonance energy transfer (FRET) in the presence of 25×10 −8 M 3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.

14. The method of claim 13 , wherein the isolated Protoxin-II variant inhibits human Nav1.7 activity with an IC 50 value of between about 3×10 −8 M to 1×10 −9 M.

15. The method of claim 1 , wherein the isolated Protoxin-II variant inhibits Nav1.7 activity by at least 25%, when the Nav1.7 activity is measured using a patch clamp assay using an extracellular solution containing 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 ), 5 mM glucose, and 10 mM HEPES, pH=7.4, and osmolarity=315 mOsm, an intracellular solution containing 135 mM CsF, 10 mM CsCl, 5 mM EGTA, 5 mM NaCl and 10 mM HEPES, pH=7.3 and osmolarity=290 mOsm and a holding potential of −75 mV.

16. The method of claim 1 , wherein the isolated Protoxin-II variant comprises 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 X 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.

17. The method of claim 1 , wherein the isolated Protoxin-II variant comprises the amino acid sequence of SEQ ID NOs: 30, 40, 44, 52, 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, 3

Assignees

• Janssen Biotech Inc

Inventors

• Flinspach Mack
• Wickenden Alan
• Fellows Ross
• Xu Qinghao
• Piekarz Andrew

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• A61P21/00

  Drugs for disorders of the muscular or neuromuscular system · CPC title

• A61P1/18

  for pancreatic disorders, e.g. pancreatic enzymes · CPC title

• A61P25/04

  Centrally acting analgesics, e.g. opioids · CPC title