Patent Grant
10975129
The instant application contains a Sequence Listing which has been submitted in the parent application in ASCII format via EFS-WEB and is hereby incorporated herein by reference in its entirety for use as the ACII copy in this application. Said ASCII copy, created on Oct. 23, 2014, is named U.S. Ser. No. 14/505,592.txt—and is 331.27 kilobytes in size and contains no new matter.
The present invention relates to Protoxin-II variants, synthetic polynucleotides encoding them, and methods of making and using the foregoing.
Voltage-gated sodium channels (VGSC) are present in all excitable cells including cardiac and skeletal muscle cells and central and peripheral neurons. In neuronal cells, sodium channels are responsible for amplifying sub-threshold depolarizations and generating the rapid upstroke of the action potential. As such, sodium channels are essential to the initiation and propagation of electrical signals in the nervous system. Aberrant sodium channel function is thought to underlie a variety of medical disorders (Hibner and Jentsch, Hum Mol Genet 11:2435-45, 2002), including epilepsy (Yogeeswari et al., Curr Drug Targets 5:589-602, 2004), arrhythmia (Tfelt-Hansen et al., J Cardiovasc Electrophysiol 21:107-15, 2010), myotonia (Cannon and Bean, J Clin Invest 120:80-3, 2010), and pain (Cregg et al., J Physiol 588:1897-904, 2010). Sodium channels are typically a complex of various subunits, the principle one being the pore-forming alpha-subunit, which is alone sufficient for function.
Nine known members of the family of voltage-gated sodium channel alpha subunits exist in humans, Nav1.1-Nav1.9. The Nav1.x subfamily can be pharmacologically subdivided into two groups, the tetrodotoxin (TTX)-sensitive and TTX-resistant. Nav1.7, (a.k.a. PN1 or hNE) is encoded by the SCN9A gene, is TTX-sensitive and is primarily expressed in peripheral sympathetic and sensory neurons. Nav1.7 accumulates at nerve fiber endings and amplifies small sub-threshold depolarizations and acts as a threshold channel that regulates excitability.
Nav1.7 function is implicated in various pain states, including acute, inflammatory and/or neuropathic pain. In man, gain of function mutations of Nav1.7 have been linked to primary erythermalgia (PE), a disease characterized by burning pain and inflammation of the extremities (Yang et al., J Med Genet 41:171-4, 2004), and paroxysmal extreme pain disorder (PEPD)(Fertleman et al., Neuron 52:767-74, 2006). Consistent with this observation, non-selective sodium channel blockers lidocaine, mexiletine and carbamazepine can provide symptomatic relief in these painful disorders (Legroux-Crespel et al., Ann Dermatol Venereol 130:429-33, 2003; Fertleman et al., Neuron 52:767-74, 2006).
Loss-of-function mutations of Nav1.7 in humans cause congenital indifference to pain (CIP), a rare autosomal recessive disorder characterized by a complete indifference or insensitivity to painful stimuli (Cox et al., Nature 444:894-8, 2006; Goldberg et al, Clin Genet 71:311-9, 2007; Ahmad et al., Hum Mol Genet 16:2114-21, 2007).
Single nucleotide polymorphisms in the coding region of SCN9A have been associated with increased nociceptor excitability and pain sensitivity. For example, a polymorphism rs6746030 resulting in R1150W substitution in human Nav1.7 has been associated with osteoarthritis pain, lumbar discectomy pain, phantom pain, and pancreatitis pain (Reimann et al., Proc Natl Acad Sci USA 107:5148-53, 2010). DRG neurons expressing the R1150W mutant Nav1.7 display increased firing frequency in response to depolarization (Estacion et al., Ann Neurol 66:862-6, 2009). A disabling form of fibromyalgia has been associated with SCN9A sodium channel polymorphism rs6754031, indicating that some patients with severe fibromyalgia may have a dorsal root ganglia sodium channelopathy (Vargas-Alarcon et al., BMC Musculoskelet Disord 13:23, 2012).
In mice, deletion of the SCN9A gene in nociceptive neurons leads to reduction in mechanical and thermal pain thresholds and reduction or abolition of inflammatory pain responses (Nassar et al., Proc Natl Acad Sci USA 101:12706-11, 2004). Ablating SCN9A in all sensory neurons abolished mechanical pain, inflammatory pain and reflex withdrawal responses to heat. Deleting SCN9A in both sensory and sympathetic neurons abolished mechanical, thermal and neuropathic pain, and recapitulated the pain-free phenotype seen in humans with Nav1.7 loss-of-function mutations (Minett et al., Nat Commun 3:791, 2012). Nav1.7 inhibitors or blockers may therefore be useful in the treatment of a wide range of pain associated with various disorders.
Spider venoms are known to contain a large number of sodium channel blocking peptides, including Huwentoxin-IV (HwTx-IV) (Peng et al., J Biol Chem 277:47564-71, 2002), Protoxin-I, Protoxin-II (Middleton et al., Biochemistry 41:14734-47, 2002) and Phrixotoxin-III (Bosmans et al., Mol Pharmacol 69:419-29, 2006). There is a need for identification of additional Nav1.7 blockers for treatment of a wide range of pain indications. In particular, there is a need for new Nav1.7 blockers with selectivity for Nav1.7 over other voltage gated sodium channel isoforms.
One embodiment of the invention is an isolated Protoxin-II variant comprising the sequence
wherein
Another embodiment of the invention is a polynucleotide encoding the Protoxin-II variant of the invention.
Another embodiment of the invention is a vector comprising the isolated polynucleotide of the invention.
Another embodiment of the invention is a host cell comprising the vector of the invention.
Another embodiment of the invention is a method for producing the Protoxin-II variant of the invention, comprising culturing the host cell of the invention and recovering the Protoxin-II variant from the cell.
Another embodiment of the invention is a pharmaceutical composition comprising the isolated Protoxin-II variant of the invention and a pharmaceutically acceptable excipient.
Another embodiment of the invention is a method of treating Nav1.7-mediated pain in a subject, comprising administering to a subject an effective amount of the Protoxin-II variant of the invention to treat the Nav1.7-mediated pain.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.
As used herein and in the claims, the singular forms “a,” “and,” and “the” include plural reference unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which an invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, exemplary compositions and methods are described herein.
The term “polypeptide” means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as “peptides”. Polypeptides may also be referred as “proteins”.
The term “polynucleotide” means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNAs and RNAs are typical examples of polynucleotides.
The term “complementary sequence” means a second isolated polynucleotide sequence that is antiparallel to a first isolated polynucleotide sequence and that comprises nucleotides complementary to the nucleotides in the first polynucleotide sequence.
The term “vector” means a non-natural polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain a cDNA encoding a protein of interest and additional elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.
The term “expression vector” means a vector that can be utilized in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
The term “variant” as used herein refers to a polypeptide or a polynucleotide that differs from wild type Protoxin-II polypeptide of SEQ ID NO: 1 or the polynucleotide encoding the wild type Protoxin-II having the sequence of SEQ ID NO: 107 by one or more modifications for example, substitutions, insertions or deletions of nucleotides or amino acids.
Throughout the specification, residues that are substituted in the Protoxin-II variants are numbered corresponding to their position in the wild-type Protoxin-II of SEQ ID NO: 1. For example, “Y1A” in the specification refers to the substitution of tyrosine at residue position that corresponds to the position 1 in the wild type Protoxin-II of SEQ ID NO:1 with alanine.
“Complementary DNA” or “cDNA” refers to a well-known synthetic polynucleotide that shares the arrangement of sequence elements found in native mature mRNA species with contiguous exons, with the intervening introns present in genomic DNA are removed. The codons encoding the initiator methionine may or may not be present in cDNA. cDNA may be synthesized for example by reverse transcription or synthetic gene assembly.
“Synthetic” or “non-natural” as used herein refers to a polynucleotide or a polypeptide molecule not present in nature.
“Nav1.7” (also referred to as hNE or PN1) or “hNav1.7” as used herein refers to the well-known human sodium channel protein type 9 subunit alpha having a sequence shown in GenBank accession number NP_002968.1 and in SEQ ID NO: 79.
The term “wild type Protoxin-II” or “wild type ProTx-II” as used herein refers to the tarantula Thrixopelma pruriens (Peruvian green velvet tarantula) toxin peptide having the amino acid sequence YCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH (SEQ ID NO: 1) as described in Middleton et al., Biochemistry 41(50):14734-47, 2002.
The term “recombinant Protoxin-II” or recombinant ProTx-II” as used herein refers to the recombinant Protoxin-II obtained from expression and subsequent cleavage of a Protoxin-II fusion protein having the sequence of GPYCQKWMWTCDSERKCCEGMVCRLWCKKKLW-OH as shown in SEQ ID NO: 2. Recombinant Protoxin-II incorporates a two amino acid N-terminal extension (residues G and P) when compared to the wild type Protoxin-II.
“Blocks human Nav1.7 activity” or “inhibits human Nav1.7 activity” as used herein refers to the ability of the Protoxin-II variant of the invention to reduce membrane depolarization induced by veratridine (3-veratroylveracevine) with an IC50 value of about 1×10−7 M or less in a FLIPR® Tetra membrane depolarization assay using fluorescence resonance energy transfer (FRET), where veratridine-induced depolarization is measured as a reduction in FRET signal using DISBAC2(3) ([bis-(1,3-diethylthiobarbituric acid) trimethine oxonol]) as an acceptor and PTS18 (trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate) as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm in a cell line stably expressing human Nav1.7.
“FLIPR® Tetra membrane depolarization assay” as used herein is the assay described in Example 3.
The term “substantially identical” as used herein means that the two Protoxin-II variant amino acid sequences being compared are identical or have “insubstantial differences” Insubstantial differences are substitutions of 1, 2, 3, 4, 5, 6, or 7 amino acids in the Protoxin-II variant amino acid sequence that do not adversely affect peptide properties. Amino acid sequences substantially identical to the Protoxin-II variants disclosed herein are within the scope of the application. In some embodiments, the sequence identity can be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Percent identity can be determined for example by pairwise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carslbad, Calif.). The protein sequences of the present invention may be used as a query sequence to perform a search against public or patent databases, for example, to identify related sequences. Exemplary programs used to perform such searches are the XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov), or the GenomeQuest™ (GenomeQuest, Westborough, Mass.) suite using the default settings.
Conventional one and three-letter amino acid codes are used herein as shown in Table 1.
The present invention provides isolated Protoxin-II (ProTx-II) variant polypeptides that inhibit human Nav1.7 activity, polynucleotides encoding them, vectors, host cells, and methods of using the polynucleotides and polypeptides of the invention. The polypeptides of the invention inhibit depolarization resulting from Nav1.7 activation, and therefore may be useful in the treatment of various conditions associated with pain and conditions associated with sensory or sympathetic neuron dysfunction.
The variants of the invention are potent inhibitors of Nav1.7. The current invention is based, at least in part, on the finding that certain residue substitutions in Protoxin-II enhance selectivity, synthetic yield and/or homogeneity without adversely affecting the potency of the generated Protoxin-II variants, specifically W7 and M19, and additionally residues Y1 and S11, and further additionally residues E12, R22 and (residue numbering according to SEQ ID NO: 1).
One embodiment of the invention is an isolated Protoxin-II variant, wherein the Protoxin-II variant inhibits human Nav1.7 activity with an IC50 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 IC50 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.
Another embodiment of the invention is an isolated Protoxin-II variant comprising the sequence
wherein
In some embodiments, the N-terminal extension comprises the amino acid sequences of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 or 385.
In some embodiments, 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.
In some embodiments, the N-terminal and/or the C-terminal extension is conjugated to the Protoxin-II variant via a linker.
In some embodiments, the linker comprises the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.
In some embodiments, the N-terminal extension consists of the amino acid sequences of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384 or 385.
In some embodiments, the C-terminal extension consists of the amino acid sequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396 or 397.
In some embodiments, the linker consists of the amino acid sequence of SEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.
The Protoxin-II variants of the invention are potent Nav1.7 inhibitors. Recombinant Protoxin-II (SEQ ID NO: 2) has an IC50 value of about 4×10−9 M for human Nav1.7 in a veratridine-induced depolarization inhibition assay measuring decline in FRET (fluorescence resonance energy transfer) in cells stably expressing Nav1.7 using FLIPR® Tetra instrument (Molecular Devices) using experimental details described in Example 3. A Protoxin-II variant is “a potent” Nav1.7 inhibitor when the IC50 value in the assay described above and in Experiment 3 is about 30×10−9 M or less i.e. within 10 fold of recombinant Protoxin-II. For clarity, an IC50 of 30×10−9 M is identical to IC50 of 3.0×10−8 M.
The Protoxin-II variant polypeptides of the invention may be produced by chemical synthesis, such as solid phase peptide synthesis, on an automated peptide synthesizer. Alternatively, the polypeptides of the invention may be obtained from polynucleotides encoding the polypeptides by the use of cell-free expression systems such as reticulocyte lysate based expression systems, or by recombinant expression systems. Those skilled in the art will recognize other techniques for obtaining the polypeptides of the invention. In an exemplary method, the Protoxin-II variants of the invention are generated by expressing them as human serum albumin (HSA) fusion proteins utilizing a glycine-rich linker such as (GGGGS)4 (SEQ ID NO:80) or (GGGGS)6 (SEQ ID NO: 81) coupled to a protease cleavable linker such as a recognition sequence for HRV3C protease (Recombinant type 14 3C protease from human rhinovirus) LEVLFQGP (HRV3C linker) (SEQ ID NO: 82), and cleaving the expressed fusion proteins with the HRV3C protease to release the recombinant Protoxin-II variant peptides. Hexahistidine (SEQ ID NO: 108) or other tags may be used to facilitate purification using well known methods.
Protoxin-II variants of the invention may be purified using methods described herein. In an exemplary method, Protoxin-II variants of the invention expressed as HSA fusion proteins and cleaved with HRV3C protease may be purified using sold phase extraction (SPE) as described herein.
Generation of the Protoxin-II variants optionally having N-terminal and/or C-terminal extensions, and Protoxin-II variant fusion proteins is typically achieved at the nucleic acid level. The polynucleotides may be synthesized using chemical gene synthesis according to methods described in U.S. Pat. Nos. 6,521,427 and 6,670,127, utilizing degenerate oligonucleotides to generate the desired variants, or by standard PCR cloning and mutagenesis. Libraries of variants may be generated by standard cloning techniques to clone the polynucleotides encoding the Protoxin-II variants into the vector for expression.
The Protoxin-II variants may incorporate additional N- and/or C-terminal amino acids when compared to the wild type Protoxin-II of SEQ ID NO: 1, for example resulting from cloning and/or expression schemes. For example, cleavage from HSA after expression of the variant as HSA-linker-HRV3C cleavable peptide-Protoxin-II variant fusion protein may result in the incorporation of additional two residues to the N-terminus of each Protoxin-II variant, such as G and P.
The Protoxin-II variants of the invention are tested for their ability to inhibit human Nav1.7 using methods described herein. An exemplary assay is a veratridine-induced depolarization inhibition assay measuring decline in FRET (fluorescence resonance energy transfer) in cells stably expressing Nav1.7. Another exemplary assay employs electrophysiological recordings to measure changes in Nav1.7-mediated currents using well known patch clamp techniques and as described herein.
Another embodiment of the invention is an isolated Protoxin-II variant comprising the amino acid sequence of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 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, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 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, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 35, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368 367, 370 or 371.
The Protoxin-II variants of the invention may inhibit human Nav1.7 with an IC50 value of about 1×10−7 M or less, about 1×10−8 M about 1×10−9 or less, about 1×10−10 M or less, about 1×10−11 M or less, or about 1×10−12 M or less. Exemplary variants demonstrating the range of IC50 values are variants having amino acid sequences shown in SEQ ID NOs: 30, 40, 44, 52, 56, 56, 59, 65, 78, 109, 110, 111, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 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, or 368.
Table 2 and Table 3 show the sequences of select Protoxin-II variants.
In some embodiments, the isolated Protoxin-II variant inhibits human Nav1.7 activity with an IC50 value of about 3×10−8 M or less.
In some embodiments, the isolated Protoxin-II variant inhibits human Nav1.7 activity with an IC50 value of between about 3×10−8 M to about 1×10−9 M.
Another embodiment of the invention is an isolated Protoxin-II variant comprising the amino acid sequence
wherein
Exemplary Protoxin-II variants that inhibit human Nav1.7 activity with an IC50 value of about 30×10−9 M or less are variants comprising the amino acid sequences 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 or 335.
In some embodiments, the isolated Protoxin-II variant selectively inhibits human Nav1.7. The Protoxin-II variants of the invention may be more selective towards Nav1.7 when compared to the recombinant Protoxin-II (SEQ ID NO: 2). In the QPatch electrophysiology assay, recombinant Protoxin-II has an IC50 of about 2.2×10−9 M for Nav1.7 and an IC50 of about 62×10−9 M for Nav1.6, and therefore the ratio of IC50 for Nav1.6 to IC50 for Nav1.7 about 28 fold. “Selectivity” or “selective” or “more selective” or “selectively blocks” or “selectively inhibits” when used herein refers to a Protoxin-II variant that has a ratio of IC50 for Nav1.6 to IC50 for Nav1.7 (IC50 (Nav1.6)/IC50 (Nav1.7)) equal or over about 30. IC50 for Nav1.6 may be assayed in a QPatch electrophysiology assay using cell lines stably expressing Nav1.6 using similar methods to those described for Nav1.7.
Residue positions in Protoxin-II that can be mutagenized to improve selectivity include residues 7 and 19, and optionally residues 1 and 11, and further optionally 12, 20, 22 and 26 (residue numbering according to SEQ ID NO: 1). Exemplary substitutions to improve selectivity are Y1Q, W7Q, S11R, S11A, E12T, M19F, V20S, R22T, and K26R. Exemplary Protoxin-II variants with improved selectivity are variants of SEQ ID NOs: 56, 59, 65, 78, 111, 114, 117, 118, 119, 121, 122, 123, 129, 130, 133, 150, 190, 217, 281, 324, 325 or 326.
Another embodiment of the invention is an isolated Protoxin-II variant comprising the sequence
wherein
In some embodiments, the isolated Protoxin-II variant comprises the sequence
wherein
Another embodiment is an isolated Protoxin-II variant comprising the amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 78
wherein
In some embodiments, the isolated Protoxin-II variant has a free C-terminal carboxylic acid, amide, methylamide or butylamide group, which are generated via routine synthetic methods.
Another embodiment of the invention is an isolated fusion protein comprising the Protoxin-II variant of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 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, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 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, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 35, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368 367, 370 or 371. Such second polypeptides may be well known leader or secretory signal sequences, or synthetic sequences resulting for example from cloning steps, or tags such as hexahistidine tag (SEQ ID NO: 108). Such second polypeptide may be a half-life extending moiety. In one embodiment, the isolated fusion protein comprises the Protoxin-II variant of the invention conjugated to a half-life extending moiety.
Exemplary half-life extending moieties that can be used include well known human serum albumin, transthyretin (TTR), a thyroxine-binding globulin (TGB), albumin-binding domains, or an Fc or fragments thereof. Biologically suitable polymers or copolymers may also be used, for example ethylene glycol or polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20000, dextran, polylysine, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, octane, or carbohydrates (dextran, cellulose, oligo- or polysaccharides). These moieties may be direct fusions with the Protoxin-II variant polypeptides and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced Protoxin-II variants of the invention.
In another embodiment, the half-life extending moiety of the fusion protein of the invention is human serum albumin, albumin binding domain (ABD), or polyethylene glycol (PEG).
In another embodiment, the half-life extending moiety of is conjugated to the Protoxin-II variant via a linker. Suitable linkers are well known and include linkers having the sequence shown in SEQ ID NOs: 80 or 81.
Exemplary fusion proteins incorporating Protoxin-II variants of the invention are those having the polypeptide sequence of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 or 103.
Protoxin-II variants of the invention incorporating additional moieties may be compared for functionality by several well-known assays. For example, pharmacokinetic properties of Protoxin-II variants coupled to PEG may be evaluated in well known in vivo models.
Additional Protoxin-II variants and Protoxin-II variant fusion proteins are within the scope of the invention. Additional substitutions to the Protoxin-II variants of the invention can be made as long as the resulting variant or the fusion protein retains similar characteristics when compared to the parent peptide. Exemplary modifications are for example conservative substitutions that will result in Protoxin-II variants with similar characteristics to those of the parent molecules. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Alternatively, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981). Non-conservative substitutions can be made to the Protoxin-II variants that involve substitutions of amino acid residues between different classes of amino acids to improve properties of the Protoxin-II variants and Protoxin-II variant fusion proteins. Whether a change in the amino acid sequence of a polypeptide or fragment thereof results in a functional homolog can be readily determined by assessing the ability of the modified polypeptide or fragment to produce a response in a fashion similar to the unmodified polypeptide or fragment using the assays described herein. Peptides, polypeptides or proteins in which more than one replacement takes place can readily be tested in the same manner.
Another embodiment of the invention is an isolated synthetic polynucleotide comprising a polynucleotide encoding the Protoxin-II variant of the invention.
Certain exemplary synthetic polynucleotides are disclosed herein, however, other synthetic polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the Protoxin-II variants and Protoxin-II variant fusion proteins of the invention are also within the scope of the invention. Exemplary synthetic polynucleotides are for example polynucleotide sequences shown in SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96, 98, 100, 102 and 104, which encode the Protoxin-II variant fusion proteins of the invention. Those skilled in the art can readily identify the polynucleotide segments in the fusion proteins that encode the Protoxin-II variant itself. The synthetic polynucleotide sequences encoding the Protoxin-II variants or fusion proteins of the invention can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended host cell. The synthetic polynucleotide may be a cDNA.
The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer. Alternatively, the polynucleotides of the invention may be produced by other techniques such as PCR based duplication, vector based duplication, or restriction enzyme based DNA manipulation techniques. Techniques for producing or obtaining polynucleotides of known sequences are well known.
The polynucleotides of the invention may also comprise at least one non-coding sequence, such as transcribed but not translated sequences, termination signals, ribosome binding sites, mRNA stabilizing sequences, introns and polyadenylation signals. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids. These additional polynucleotide sequences may, for example, encode a marker or well-known tag sequences such as a hexa-histidine (SEQ ID NO: 108) or a HA tag which facilitate the purification of fused polypeptides.
Another embodiment of the invention is a vector comprising the polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotide of the invention into a given organism or genetic background by any means. For example, polynucleotides encoding the Protoxin-II variants or the Protoxin-II variant fusion proteins of the invention are inserted into an expression vector and may be operably linked to control sequences in the expression vector to ensure efficient expression, such as signal sequences, promoters (e.g. naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are chosen to be compatible with the host cell chosen to express the Protoxin-II variant or the Protoxin-II variant fusion protein of the invention. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the proteins encoded by the incorporated polynucleotides.
Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences.
Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. For expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 PGK1, ENO or PYK1 promoter and the like, or a regulatable promoter such as a GAL1 or GAL10 promoter. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNHl8a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).
An exemplary vector for expression of the Protoxin-II variants or Protoxin-II variant fusion proteins is a vector having ampicillin-resistance selection marker, CMV promoter, CMV intron, signal peptide, neomycin resistance, f1 origin of replication, SV40 polyadenylation signal, and cDNA encoding the Protoxin-II variant or the Protoxin-II variant fusion protein of the invention.
Another embodiment of the invention is a host cell comprising the vector of the invention. The term “host cell” refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells or archeal cells.
Escherichia coli, bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species, are examples of prokaryotic host cells. Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics, Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.
Introduction of a polynucleotide, such as a vector, into a host cell can be effected by methods well known to those skilled in the art. Exemplary methods are calcium phosphate transfection, DEAE-Dextran mediated transfection, microinjection, cationic lipid-mediated transfection and electroporation.
Another embodiment of the invention is a method for producing the Protoxin-II variant of the invention comprising the steps of providing a host cell of the invention; and culturing the host cell under conditions sufficient for the expression of at least one Protoxin-II variant of the invention.
Host cells can be cultured under any conditions suitable for maintaining or propagating a given type of host cell and sufficient for expressing a polypeptide. Culture conditions, media, and related methods sufficient for the expression of polypeptides are well known in the art. For example, many mammalian cell types can be aerobically cultured at 37° C. using appropriately buffered DMEM media while bacterial, yeast and other cell types may be cultured at 37° C. under appropriate atmospheric conditions in LB media.
In the methods of the invention, the expression of the Protoxin-II variant can be confirmed using a variety of well-known methods. For example, expression of a polypeptide can be confirmed using detection reagents, such as using SDS-PAGE or HPLC.
Another aspect of the invention is a method of modulating the activity of Nav1.7 in a biological tissue, the method comprising contacting the biological tissue expressing Nav1.7 with a Nav1.7-modulating amount of the Protoxin-II variant of the invention.
Methods of Treatment
Protoxin-II variants of the invention may be utilized in any therapy where it is desired to treat, reduce or alleviate symptoms of pain or other disorders of sensory or sympathetic neuron dysfunction.
Pain treated with the Protoxin-II variants of the invention may be any type of pain, such as chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, pain associated with inflammatory conditions, post-operative pain, thermal pain or pain associated with disease and degeneration.
Pain treated with the Protoxin-II variants of the invention may be Nav1.7-mediated pain.
Nav1.7-mediated pain as used herein refers to pain resulting at least partially from increased Nav1.7 channel activity.
The methods of the invention may be used to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.
The pain and/or Nav1.7-mediated pain may result from one or more causes, such as peripheral neuropathy, central neuropathy, nerve compression or entrapment syndromes such as carpal tunnel syndrome, tarsus tunnel syndrome, ulnar nerve entrapment, compression radiculopathy, lumbar spinal stenosis, sciatic nerve compression, spinal root compression, intercostal neuralgia, compression radiculopathy and radicular lower back pain, spinal root lesions, neuritis, autoimmune diseases, general inflammation, chronic inflammatory conditions, arthritis, rheumatic diseases, lupus, osteoarthritis, general gastrointestinal disorders, colitis, gastric ulceration, duodenal ulcers, inflammatory bowel disorders, irritable bowel syndrome, pain associated with diarrhea, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, skin complaints with inflammatory components, sunburn, carditis, dermatitis, myositis, neuritis, collagen vascular diseases, inflammatory pain and associated hyperalgesia and allodynia, neuropathic pain and associated hyperalgesia and allodynia, multiple sclerosis, demyelinating diseases, diabetes, diabetic neuropathy pain, causalgia, pain resulting from amputation or abscess, phantom limb pain, fracture pain, bone injury, direct trauma, HIV infection, acquired immune deficiency syndrome (“AIDS”), small pox infection, herpes infection, exposure to toxins or other foreign particles or molecules, invasive cancer, cancer, chemotherapy, radiotherapy, hormonal therapy, burns, congenital defect, dental pain, gout pain, fibromyalgias, encephalitis, chronic alcoholism, hypothyroidism, uremia and vitamin deficiencies, trigeminal neuralgia, stroke, thalamic pain syndrome, general headache, migraine, cluster headache, tension headache, mixed-vascular and non-vascular syndromes, sympathetically maintained pain, deafferentation syndromes, asthma, epithelial tissue damage or dysfunction, disturbances of visceral motility at respiratory, genitourinary, gastrointestinal or vascular regions, wounds, burns, allergic skin reactions, pruritis, vasomotor or allergic rhinitis, or bronchial disorders, dysmenorrhoea, pain during labor and delivery, dyspepsia, gastroesophageal reflux, pancreatitis, and visceralgia.
Other disorders of sensory or sympathetic neuron dysfunction that may be alleviated by the Protoxin-II variants of the invention include itch, cough and asthma. In mice, global deletion of the SCN9A gene leads to complete insensitivity to histamine-induced itch (Gingras et al., American Pain Society Meeting Abstract 2013 and U.S. Pat. Publ. No. 2012/0185956). This finding suggests that peptide Nav1.7 blockers may have utility in the treatment of itch, which may arise from various sources, such as dermatological or inflammatory disorders; or inflammatory disorders such as renal or hepatobiliary disorders, immunological disorders, medication reactions and unknown/idiopathic conditions, including dermatitis, psoriasis, eczema, insect sting or bite. Nav1.7 is also expressed in sensory nerves innervating the airways (Muroi et al., J Physiol. 2011 Dec. 1; 589(Pt 23):5663-76; Muroi et al., Am J Physiol Regul Integr Comp Physiol. 2013 Apr. 10), suggesting that peptide Nav1.7 blockers may be beneficial in the treatment of cough e.g., acute or chronic cough, or cough caused by irritation from gastroesophageal reflux disease, and inflammatory diseases of the airways such as asthma and allergy-related immune responses, bronchospasm, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, and hiccups (hiccoughs, singultus). Silencing Nav1.7 in vivo in nodose ganglia of guinea pigs using shRNA nearly abolished the cough reflex induced by mechanical probing (Muroi et al., Am J Physiol Regul Integr Comp Physiol. 2013 Apr. 10).
One aspect of the invention is a method of alleviating or treating itch, cough or asthma in a subject by administering a therapeutically effective amount of the Protoxin-II variant of the invention to a subject in need thereof for a time sufficient to alleviate the itch, cough or asthma.
Another aspect of the invention is a method of alleviating or treating Nav1.7-mediated itch, Nav1.7-mediated cough or Nav1.7-mediated asthma in a subject by administering a therapeutically effective amount of the Protoxin-II variant of the invention to a subject in need thereof for a time sufficient to alleviate the itch, cough or asthma.
Nav1.7-mediated itch as used herein refers to itch resulting at least partially from increased Nav1.7 channel activity.
Nav1.7-mediated cough as used herein refers to cough resulting at least partially from increased Nav1.7 channel activity.
Nav1.7-mediated asthma as used herein refers to asthma resulting at least partially from increased Nav1.7 channel activity.
Protoxin-II variants of the invention may be tested for their effect in reducing or alleviating pain and/or Nav1.7-mediated pain using animal models described herein, and models such as the rat spinal nerve ligation (SNL) model of neuropathic pain, carageenan induced allodynia model, the Freund's complete adjuvant (CFA)-induced allodynia model, the thermal injury model, the formalin model and the Bennett Model, and other models as described in U.S. Pat. Appl. No. 2011/0124711 and U.S. Pat. No. 7,998,980. Carageenan induced allodynia and CFA-induced allodynia are models of inflammatory pain. The Bennett model provides an animal model for chronic pain including post-operative pain, complex regional pain syndrome, and reflex sympathetic dystrophy.
Any of the foregoing animal models may be used to evaluate the efficacy of Protoxin-II variants of the invention inhibitor in treating pain and/or NAv1.7-mediated pain. The efficacy of the Protoxin-II variants of the invention may be compared to a no treatment or placebo control. Additionally or alternatively, efficacy may be evaluated in comparison to one or more known pain-relieving medicaments.
The present invention provides methods of treating Nav1.7-mediated pain using the Protoxin-II variants of the invention. It has been discovered in the pending application by the inventors (U.S. Patent Application No. 61/781,276) that administration of Nav1.7 blocking peptides are efficacious in treating and/or alleviating pain in various animal models of pain, contrary to what was disclosed and suggested in the literature. While peptide inhibitors of Nav1.7 have been shown to be potent and/or selective towards Nav1.7 in in vitro cell culture models using overexpressed Nav1.7 or on isolated neurons in which the blood-nerve barrier is subverted through desheathing or hypertonic saline injection, they have so far proven non-efficacious in in vivo animal models of pain, where the lack of efficacy has been reported to result from the inability of the peptides to pass the blood-nerve barrier. Several publications describe lack of efficacy of Nav1.7 blocking peptides in animal models of pain or in isolated nerves. For example Hackel et al., Proc Natl Acad Sci 109:E2018-27, 2012, describes the inability of ProTx-II to inhibit action potential firing in isolated nerves unless the perineural barrier, which provides a diffusion barrier in this model, is compromised. ProTx-II was found non-efficacious in rodent models of acute and inflammatory pain; a likely explanation stated the inability of ProTx-II to cross the blood-nerve barrier (Schmalhofer et al., Mol Pharmacol 74:1476-1484, 2008). It has been proposed that Nav1.7 peptide toxin blockers have poor oral bioavailability and they are difficult to deliver to nerve endings, implying that their use as therapeutic agents remain limited (Dib-Hajj et al., Nature Rev Neuroscience 14, 49-62, 2013).
Nav1.7 is expressed in the peripheral nervous system e.g., in nociceptive dorsal root ganglions (DRG), most notably in nociceptive small-diameter DRG neurons, in particular in peripheral terminals in the skin, with little representation in the brain. Nav1.7 distribution (e.g. sensory ending) and physiology predispose it to a major role in transmitting painful stimuli.
One embodiment of the invention is a method of treating Nav1.7-mediated pain by administering a therapeutically effective amount of the Protoxin-II variant of the invention to a subject in need thereof for a time sufficient to treat the Nav1.7-mediated pain.
The Protoxin-II variants of the invention Nav1.7 may be utilized in any therapy where it is desired to treat Nav1.7-mediated pain or other disorders of sensory or sympathetic neuron dysfunction. “Treat” or “treatment” of pain is meant to include partially or completely to prevent, stop, inhibit, reduce, or delay the perception of pain.
In some embodiments, the Nav1.7-mediated 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, or pain associated with disease and degeneration.
Neuropathic pain includes for example painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN) or trigeminal neuralgia (TN). Other causes of neuropathic pain include spinal cord injuries, multiple sclerosis, phantom limb pain, post-stroke pain and HIV-associated pain. Conditions such as chronic back pain, osteoarthritis and cancer may also result in the generation of neuropathic-related pain and thus are potentially suitable for treatment with the Protoxin-II variants of the invention.
In another embodiment, the Nav1.7-mediated pain is associated with primary erythemalgia (PE), paraoxysmal extreme pain disorder (PEPD), osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis or fibromyalgia.
In the methods of the invention, the Protoxin-II variants of the invention may be conjugated to a second polypeptide to form a fusion protein. Such fusion proteins are for example the well-known Fc fusions or fusions to human serum albumin to extend half-life of the peptide inhibitors. The conjugation may be a direct conjugation via a linker, such as a glycine-serine rich linker. Such linkers are well known in the art. The Protoxin-II variants of the invention incorporating additional moieties may be compared for their Nav1.7 blocking ability and efficacy in treatment or reducing pain using well known methods and those described herein.
Other disorders of sensory or sympathetic neuron dysfunction that can be treated with the Protoxin-II variants of the invention, including asthma, cough, heart-burn, itch, dermatitis, bladder instability, and Reynaud's disease.
Pharmaceutical Compositions
The Protoxin-II variants of the invention may be formulated in a pharmaceutically acceptable vehicle or carrier. One embodiment of the invention is a pharmaceutical composition comprising the isolated Protoxin-II variant of the invention and a pharmaceutically acceptable excipient.
A suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. These solutions are sterile and generally free of particulate matter, and may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable excipients as required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. Suitable vehicles and their formulation and packaging are described, for example, in Remington: The Science and Practice of Pharmacy (21st ed., Troy, D. ed., Lippincott Williams & Wilkins, Baltimore, Md. (2005) Chapters 40 and 41).
In the methods of the invention, the Protoxin-II variants of the invention may be administered by peripheral administration. “Peripheral administration” or “administered peripherally” means introducing an agent into a subject outside of the central nervous system. Peripheral administration encompasses any route of administration other than direct administration to the spine or brain.
Peripheral administration can be local or systemic. Local administration may be used to concentrate the therapeutic to the site of action, such as local administration to joints, spinal cord, surgical wounds, sites of injury/trauma, peripheral nerve fibers, various organs (GI, urogenital, etc) or inflamed tissues. Systemic administration results in delivery of a pharmaceutical composition to essentially the entire peripheral nervous system of the subject and may also result in delivery to the central nervous system depending on the properties of the composition.
Routes of peripheral administration encompass, without limitation, topical administration, intravenous or other injection, and implanted mini-pumps or other extended release devices or formulations.
Pharmaceutical compositions of the invention include formulations involving the Protoxin-II variants of the invention in sustained- or controlled-delivery formulations. These formulations may be achieved through use of for example injectable microspheres, bio-erodible particles, microemulsions, nanoparticles, nanocapsules, macroemulsions, polymeric compounds (such as polyesters, polyamino acids, hydrogels, poly(lactic acid), polyglycolic acid or ethylene vinylacetate copolymers), beads or liposomes, hyaluronic acid or implantable drug delivery devices.
The Protoxin-II variants of the invention may be prepared for use for parenteral (subcutaneous, intramuscular or intravenous), intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intra-arterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices, or any other administration, particularly in the form of liquid solutions or suspensions; for buccal or sublingual administration such as in the form of tablets or capsules; or intranasally such as in form of powders, nasal drops or aerosols or certain agents; transdermally in a form of a gel, ointment, lotion, cream or dusting powder, suspension or patch delivery system with chemical enhancers to either modify the skin structure or to increase the drug concentration in the transdermal patch, or with agents that enable the application of formulations containing proteins and peptides onto the skin (Int. Pat. Publ. No. WO98/53847), or applications of electric fields to create transient transport pathways such as electroporation, or to increase the mobility of charged drugs through the skin such as iontophoresis, or application of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402). The composition also may be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
In certain embodiments, where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
The concentration of the Protoxin-II variants of the invention or other peptide inhibitors of Nav1.7 in such pharmaceutical formulation can vary widely, for example from about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or between 2% to 5%, up to as much as 15%, 20%, 30%, 40%, 50%, 60% or 70% by weight and will be selected primarily based on fluid volumes, viscosities and other factors, according to the particular mode of administration selected. The Protoxin-II variants of the invention can be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective with conventional protein preparations. Lyophilization and reconstitution techniques are well known in the art.
An exemplary pharmaceutical compositions of the present invention may comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further include sorbitol, sucrose, Tween-20 and/or a suitable substitute thereof.
The appropriate therapeutically effective dose may be determined readily by those skilled in the art. An effective dose refers to an amount or dosage sufficient to produce a desired result, i.e. to partially or completely prevent, stop, inhibit, reduce, or delay the perception of pain associated with any painful medical condition. The effective amount may vary depending on the specific vehicle and the Protoxin-II variants of the invention selected, and is also dependent on a variety of factors and conditions related to the subject to be treated and the severity of the pain. For example, factors such as age, weight and health of the subject to be administered with the pharmaceutical compositions of the invention as well as dose response curves and toxicity data obtained in preclinical animal work could be among those considered. A determined dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician or other person skilled in the relevant art (e.g. nurse, veterinarian, or veterinary technician) during the treatment period. The determination of an effective amount or a therapeutically effective amount for a given agent is well within the ability of those skilled in the art.
Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 ml sterile buffered water, and between about 1 ng to about 100 mg, about 50 ng to about 30 mg or about 5 mg to about 25 mg of a Protoxin-II variant of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 mg to about 30 mg or about 5 mg to about 25 mg of the Protoxin-II variants of the invention. Actual methods for preparing parenterally administrable compositions are well known and are described in more detail in, for example, “Remington's Pharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.
The present invention will now be described with reference to the following specific, non-limiting examples.
Protoxin-II single position limited amino acid scanning library substitution was designed to assess to what degree selectivity, peptide yield, and homogeneity can be improved.
Protoxin-II variants were designed as HRV3C protease cleavable HSA fusion proteins in the following format from N- to C-terminus: 6×His-HSA-linker-HRV3C cleavable peptide-Protoxin-II variant (“6×His” disclosed as SEQ ID NO: 108); linker being (GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 80, HSA having the sequence of SEQ ID NO: 106, HRV3C cleavable peptide having the sequence of SEQ ID NO: 82). Each Protoxin-II variant, after cleavage from HSA had a residual N-terminal GP from the cleavage site.
The variants were characterized in membrane depolarization assays using FLIPR® Tetra as described in Example 3 FLIPR® Tetra membrane depolarization assay, and in whole cell patch clamp experiments using the QPatch assay as described in Example 3.
Combinatorial libraries were designed to test for additive effects of select single position hits in an attempt to generate Nav1.7 antagonists with further improved potency and selectivity profile compared to the native peptide.
Construction of the Expression Vectors
The designed Protoxin-II variant genes were generated using synthetic gene assembly technology as described in U.S. Pat. No. 6,521,427. The amino acid sequences of the designed peptide variants were back-translated to DNA sequences using human high-frequency codons. The DNA sequence of each variant gene, together with a portion of vector DNA including the DNA cloning sites, was synthesized as multiple oligonucleotides, some of which contained degenerate codons, and assembled into full-length DNA fragments. The assembled DNA fragments were amplified by PCR and PCR products were subsequently cloned as a pool. Pooled PCR products were digested with the appropriate restriction enzymes and cloned into the designed expression vector in such a manner as to fuse each toxin variant gene to the signal peptide and the fusion partner (6×His-HSA-linker-HRV3C cleavable peptide (“6×His” disclosed as SEQ ID NO: 108) contained in the vector. Standard molecular biology techniques were used to identify a positive clone for each designed variant. The plasmid DNA from these positive clones was purified and sequence confirmed before expressing the Protoxin-II peptide variant fusion proteins using standard methods.
Protein Expression
HEK 293-F cells were maintained in 293 Freestyle™ media (Invitrogen Cat #12338) and split when the cell concentration was between 1.5 and 2.0×106 cells per ml. The cells were grown in suspension, shaking at 125 RPM in a humidified incubator set at 37° C. and 8% CO2. HEK 293F cells were transiently transfected using a DNA/lipid complex after they were diluted to 1.0×106 cells per ml. To generate the complex, 1.25 μg DNA per ml of transfection was diluted in 1.0 ml of OptiPro media (Invitrogen Cat #12309) and 1.25 ml of Freestyle™ Max transfection reagent (Invitrogen Cat #16447) was diluted in 1.0 ml of OptiPro media. The DNA and Max transfection reagent were mixed together and incubated for 10 minutes at room temperature before adding to the cells. Transfected cells were placed in a humidified incubator set at 37° C. and 8% CO2 for 4 days shaking at 125 RPM. The supernatant was separated from the cells by centrifugation at 5,000×g for 10 minutes and filtered through a 0.2 μm filter (Corning; Cat #431153), then concentrated 10 and 50 fold using an Amicon Ultra Concentrator 10K (Cat #UFC901096), and centrifuging for approximately 10 minutes at 3,750×g.
Protoxin-II variants were expressed as HSA fusion proteins as indicated in Example 1 and the Protoxin-II variant peptides were cleaved with HRV3C protease prior to purification. Two methodologies were tested for efficient purification of the Protoxin-II variants.
Protein Purification
Purification of Protoxin-II Variants by RP-HPLC
The secreted proteins were purified from the expression supernatants via IMAC using 1 ml HisTrap HP columns (GE Healthcare Cat #17-5247-01). The chromatography method was run using an AKTA Xpress and protein was eluted from the column using a step gradient of Imidazole. Peak fractions were pooled and digested overnight with HRV 3C protease (1 μg protease/150 μg fusion).
Cleaved peptide-fusion pools were further purified using a Dionex HPLC system with a reverse phase Phenomenex Luna 5 μm C18(2) column (Cat #00B-4252-PO-AX). Samples were eluted from the column with a 0-68% Acetonitrile (0.05% TFA) linear gradient. Elution fractions were pooled, lyophilized overnight and reconstituted in HEPES buffered saline, pH 7.4 (10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl2, 1 mM MgCl2).
Table 4 shows yields of Protoxin-II variants purified by RP-HPLC. The average mg yield/L was 0.01615.
Purification of Protoxin-II Variants by Solid Phase Extraction (SPE)
The secreted proteins were purified from the expression supernatants via IMAC using 1 ml HisTrap HP columns (GE Healthcare Cat #17-5247-01). The chromatography method was run using an AKTA Xpress and protein was eluted from the column using a step gradient of Imidazole. Peak fractions were pooled and digested overnight with HRV3C protease (1 μg protease/150 μg fusion). The cleaved sample was loaded into a 50 kDa molecular weight cut off centrifugal filter unit (Millipore UFC805096) and cleaved peptide collected in the filtrate fraction.
Peptide pools were loaded onto a 96-well solid phase extraction block (Agilent Bond Elut Plexa A3969030) for further purification, desalting, and concentration. Blocks were used in conjunction with a vacuum manifold (Whatman). Peptide samples were loaded and washed in 0.05% TFA in water and eluted with a step gradient of acetonitrile with 0.05% TFA in water. Elution fractions were then lyophilized overnight and reconstituted in HEPES buffered saline, pH 7.4 (10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl2, 1 mM MgCl2).
Peptides were reconstituted in supplemented HEPES buffered saline, pH 7.4 (10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl2, 1 mM MgCl2) and absorbance was measured at 280 nm. Concentration values were then calculated using each sample's extinction coefficient. 2 μg of each peptide were loaded onto an Invitrogen NuPAGE® Novex® Bis-Tris Gel 15 well gel and run in MES buffer non-reduced.
Samples were analyzed on Agilent 1100 HPLC using 4-80% acetonitrile in 0.05% TFA linear gradient with a Phenomenex Luna C18(2) analytical column (Cat #00A-4041-B0). Concentrations of all peptides were normalized and 10 μl of each were injected for a total of 1.3 μg per sample. Absorbance at 220 nm was monitored and chromatograms analyzed were using Chromeleon software.
Table 5 shows yields (mg) of Protoxin-II variants purified by SPE. The average mg yield/L was 0.05353.
The benefits of the SPE purification process are ease and throughput of purification since samples are processed in parallel in a 96-well block rather than serially on RP-HPLC, and improvement in yield. There was, on average, more than 3-fold higher yield (mg/L) for variants purified by SPE versus RP-HPLC.
Select Protoxin-II variants were characterized in membrane depolarization and whole cell patch clamp assays to assess their potency and selectivity towards Nav1.7.
FLIPR® Tetra Membrane Depolarization Assay
The ability of the generated peptides to inhibit membrane depolarization induced by Nav1.7 agonist veratridine (3-Veratroylveracevine; Biomol, Catalog #NA125) was measured with a FRET (fluorescence resonance energy transfer) assay on FLIPR® Tetra using DISBAC2(3) (Invitrogen, K1018) as an electron acceptor and PTS18 (Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate) (Sigma) as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm.
HEK293 cells stably expressing human Nav1.7 were cultured in DMEM/F-12 media (1:1), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 400 μg/mL geneticin and 100 μM NEAAs (all reagents from Invitrogen). 50 μL of harvested cells were plated at 25,000 cells/well into poly-lysine coated 384-well black clear bottom plates. The plates were incubated at room temperature (RT) for 15 min followed by an overnight incubation at 37° C. All incubations were done in the dark unless otherwise stated. The next day, the wells were washed 4 times with assay buffer (137 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM Glucose, 10 mM HEPES, pH 7.4), and resuspended in 25 μL of assay buffer. 2× stock (6 μM) of the PTS18 dye was prepared by suspending the dye in 10% pluronic F127 in DMSO at 1:1 (v/v ratio). 25 μL of the 2×PTS18 stock was added into the wells and the cells were stained for 30 min at RT, after which the dye was washed off with the assay buffer. Peptides were suspended at 3× their final concentration in the assay buffer containing 10 μM DISBAC2(3) and 400 μM VABSC-1 to suppress background fluorescence (Sigma, cat #201987). 25 μL/well of the suspended peptides were added into each well, and incubated for 60 minutes at RT. Depolarization was induced by 25 μM final concentration of veratridine (by adding 25 μL/well of 75 μM (3×) stock solution), and the reduction in the mean intensity of FRET dye fluorescence was measured 30-100 seconds after adding the agonist. A 1.3× dilution of each measured peptide occurred after adding veratridine by convention, the concentration at the beginning of the FLIPR® Tetra assay is reported.
Concentration-response curves of synthetic Protoxin-II (Peptide International) were constructed in each experimental series and were used as controls. Fluorescence counts for each well were converted to % response by normalizing the signal to the difference between negative control (response to agonist veratridine alone) and positive control (response to veratridine in the presence of 10 μM tetracaine) values. For measurements, “spatial uniformity correction” (all fluorescence traces are normalized to the average initial starting intensity) and “subtract bias value” (subtract the initial starting intensity from each trace) were turned on in FLIPR® Tetra. Each data point represented the response in an individual well. All individual data points were used in a non-linear least-squares fitting procedure to find the best fit to a Hill function using Origin (Microcal). IC50 values were extracted from the resultant fitted curve. The mean responses of the positive (P) and negative (N) controls were used to calculate the % response in a well as follows: % response=100*(N−R)/(N−P).
Assay plates were accepted if the potency of control antagonists for that day were within ±0.5 log units of their historical mean.
QPatch Assay
HEK293 cells stably expressing human Nav1.5 (SEQ ID NO: 105), Nav1.7 (SEQ ID NO: 79) or Nav1.6 (SEQ ID NO: 407) were cultured in DMEM/F-12 media (1:1), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 400 μg/mL Geneticin and 100 μM NEAAs (all reagents from Invitrogen). Cells were maintained at 37° C. and in 5% CO2 and assayed upon reaching 50-90% confluency. CHO cells stably expressing human Nav1.6 in a tetracycline-inducible manner (SEQ ID NO: 407) were cultured in HAMs F12, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 10 μg/mL Blasticidin and 400 μg/mL Zeocin. Cells were maintained at 37° C. and in 5% CO2, and assayed upon reaching 50-90% confluency. Nav1.6 expression was induced with 1 μg/ml of tetracycline, 24-48 h prior to an experiment.
Before testing in QPatch HT (Sophion), cells were first dissociated using 0.05% trypsin (5 min at 37° C.), resuspended in CHO-S-SFM media (Life Technologies) and gently triturated to break up cell clumps. Cell density was adjusted to 1-2×106/mL with the same media and cells were the transferred to a cell “hotel” in QPatch HT and used in experiments for several hours. For giga-ohm seal formation and whole-cell patch clamp recording, the extracellular solution contained 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 10 mM HEPES, pH=7.4 and osmolarity=315 mOsm. The intracellular solution contained 135 mM CsF, 10 mM CsCl, 5 mM EGTA, 5 mM NaCl and 10 mM HEPES, pH=7.3 and osmolarity=290 mOsm. The voltage protocol used in the assay was as follows. From a holding potential of −75 mV (Nav1.7), −60 mV (Nav1.6), or −105 mV (Nav1.5) cells were first hyperpolarized to −120 mV for 2 sec and then depolarized to 0 mV for 5 ms before returning to the holding potential. This protocol was repeated once every 60 sec during liquid applications (see below). Cells were otherwise held at the holding potential when the above voltage protocol was not executed. Upon establishment of the whole-cell recording configuration, a total of five applications of the extracellular solution (all containing 0.1% bovine serum albumin (BSA) with or without test compound, except for the last application, which contained 1 μM TTX or 10 mM lidocaine as a positive control) were made on to cells being recorded. The first liquid application contained only the control buffer (5 μl). The voltage protocol was executed 10 times (for a total duration of 10 min) five sec after the application. The next three liquid applications (5 μl each) contained a test compound (same compound at the same concentration for all three applications) or control buffer (for control cells only). Five seconds after each of these applications, the voltage protocol was again executed 10 times (also once per min). The last application contained positive (composed of three 10 μl sub-applications, each separated by 2 sec), five seconds after which the same voltage protocol was executed twice to obtain the baseline current. Currents were sampled at 25 kHz and filtered at 5 kHz with an 8-pole Bessel filter. The series resistance compensation level was set at 80%. For each cell, the peak current amplitude at 0 mV for each current trace in the first four liquid applications was first subtracted from that of the last trace in the presence of positive control and then normalized to that of the last trace in the first (control buffer) application as % inhibition. To control for current rundown, this (% inhibition) value for each cell in the presence of a test compound was further normalized to the average % inhibition value for control (typically 5-6) cells in the same experiment. The mean of the last two such values in the last compound application (i.e., the corrected % inhibition value for each concentration of a test compound) were taken as the % inhibition value for each cell at the particular compound concentration tested. The % inhibition values for all cells tested at each compound concentration were averaged and used in concentration response calculations. All experiments were performed at room temperature (22° C.). Data are expressed as mean±se. Wild type Protoxin-II was included in each experiment as a positive control. Data were accepted only if the potency of Protoxin-II was within ±0.5 log units of its historical mean.
IC50 values for Nav1.7 for select Protoxin-II variants obtained using the FLIPR® Tetra are shown in Table 6.
Select Protoxin-II variants were tested for selectivity against human Nav1.5 using QPatch. IC5, values for both Nav1.7 and Nav1.5 for select peptides obtained using QPatch are shown in Table 7.
Table 7.
Combinatorial libraries were designed to test for additive effects of select single position hits in an attempt to generate Nav1.7 antagonists with further improved potency and selectivity profile compared to the native peptide using several approaches.
A limited amino acid scan was conducted at all non-cysteine Protoxin-II positions using A, D, Q, R, K and S for diversification. In these experiments, Protoxin-II was expressed and tested as monovalent Fc fusion protein as described in Example 1. From this scan, substitutions Y1Q, W7Q, S11A, were identified that improved potency and/or selectivity of the resulting variants.
A full amino acid scan (excluding cys and trp) at positions M6 and M19 was also conducted. M19F substitution was identified from this scan that improved potency and/or selectivity of the resulting variants.
Protoxin-II/Huwentoxin-IV single position chimeras were designed bidirectionally. The purpose of this library was to obtain Protoxin-II variants that retained potency and selectivity profile of the wild type Protoxin-II and would achieve beneficial refolding properties associated with Huwentoxin-IV. Substitutions R22T and E12N were identified from this scan.
Peptide NV1G1153 was further engineered by diversifying position Y1 by a limited amino acid scan using R, K, T, A, D, E, Q and S, and by charge cluster engineering, where all sets of charged residues in the three-dimensional structure of the peptide (D10/E12, K4/E17, D10/E12/R13) were mutated.
N- and C-terminal extensions were introduced to select peptides, including NV1G1153 with the purpose of improving peptide distribution to the site of action and of improving half-life of the peptides without significantly increasing the molecular weight of the resulting peptide. The N- and C-terminal extensions that were used are shown in Table 8 and 9, respectively, and are described in Oi et. al., Neuroscience Letters 434, 266-272, 2008; Whitney et. al., Nature Biotechnology 2011 29:4, 352-356; Sockolosky et. al., (2012) 109:40, 16095-16100. Cell penetrating peptides HIV Tat and polyarginine were also used. Various linkers were used to couple the Protoxin-II variant to the N- and/or C-terminal extensions. The linkers used are shown in Table 10.
Protoxin-II variants from each campaign were tested for their potency and selectivity for Nav1.7 using methods described in Example 3. The amino acid sequences of the variants that inhibited Nav1.7 with an IC50 value of 200 nM or less are shown in Table 3. Table 11 shows the amino acid substitutions in select variant when compared to the wild type Protoxin-II, and the IC50 values for Nav1.7 inhibition in the FLIPR Tetra assay.
The wild-type Protoxin-II inhibits Nav1.7 with an IC50 value of about 4 nM in FLIPR assay as described in Example 3. Variants retaining significant Nav1.7 potency were characterized further.
Select Protoxin-II variants were tested for their inhibition of Nav1.7 and for their selectivity against human Nav1.6 using QPatch. IC50 values for both Nav1.7 and Nav1.6 for select peptides obtained using QPatch are shown in
The amino acid sequences of the peptides shown in
The protoxin-II variants were expressed and purified as described in Example 1, or synthesized by standard solid phase synthesis methods. The yields of the recombinant or synthetic peptides were compared to the yields of the wild-type protoxin. Table 12 shows that the yields of the select protoxin-II variants were significantly higher than that of protoxin-II, indicating improved folding properties of the variants. The scale of the solid-phase synthesis was 0.5 mmol.
Materials and Methods
Animals Male C57Bl/6 mice (24-26 g), ordered from Charles River and housed individually, were used for this study.
Behavioral Tests
Von Frey Test: Mechanical (tactile) threshold was assessed by Von Frey Hairs following the Up-Down method (Dixon, 1980, Chaplan et al., 1994). 7 graded stimuli (von Frey filaments: 0.03, 0.07, 0.16, 0.4, 0.6, 1, 2 g; Stoelting, Wood Dale, Ill.) were used. Von Frey hairs were presented perpendicularly against the center plantar area (between toris) on a hindpaw. Sufficient force was applied to bend the filament slightly and held for 3 seconds. Per the Chaplan paper, a positive response can be either 1) a sharp withdrawal or 2) immediate flinching upon removal of the filament. See Chaplan et al for more details. Mice were acclimated to the wire mesh in the testing chamber for 30-60 minutes prior to testing.
Hargreaves Test: A modified Hargreaves box was used to measure thermal paw withdrawal latency (PWL) (Hargreaves et al., 1988, Pain, 32:77-88; Dirig et al., 1997, J Neurosci. Methods, 76:183-191). This box consists of a chamber with a raised glass floor maintained at a constant temperature (27° C.). The thermal nociceptive stimulus originates from a projection bulb light beam below the glass surface. The light beam is aimed at the area between toris (center plantar). The “start” button will turn on the light and start the timer. Movements (such as a sudden withdrawal) of the stimulated paw will trigger the switch to turn off the light and stop the timer. The latency in seconds is displayed. If no movement occurs, the bulb will be turned off after 20 seconds (cutoff) to prevent tissue injury. The animals were allowed to habituate on the glass surface for 30-60 minutes before PWL measurement. Constant amperage was used throughout the study, which resulted in Pre-test paw withdrawal latencies between 8-12 seconds when averaged over 3 to 6 read-outs taken at least 5 minutes apart.
MPE % Calculation: Percent maximum possible effect (MPE %)=(T1−T0)/(Tc−T0)×100%. T0: threshold on day0 (post-CFA, pre-pump); T1: threshold on day1 post pump implantation; Tc: cut-off of the test (20 s for the Hargreaves test and 2 g for the Von Frey test)
Hotplate Test: Animals were placed on a 10″×10″ metal plate surrounded by 4 Plexiglas walls (15 inches high). The plate was maintained at a temperature of either 50 or 55° C. The response latency (time when the animal first flinches or licks its hind paw, jumps, or vocalizes) was measured and the animal removed from the plate. Animals showing no response were removed from the plate after 40 s (50° C.) or 20 s (55° C.) to prevent any possible tissue damage. This trial was repeated 2-5 times every 15-60 minutes in a day.
Inflammatory Pain Models
CFA Model: Animals were anesthetized with isoflurane (4% induction and 2% maintenance) and 20 μL of 100% Complete Freund's Adjuvant (CFA; Sigma-Aldrich; Saint Louis, Mo.) was injected into the center plantar area on one hind paw using a 27 gauge needle attached to a 50 μL Hamilton syringe.
Carrageenan model: Animals were anesthetized with isoflurane (4% induction and 2% maintenance) and 25 μL of 2% A-carrageenan (Sigma-Aldrich; Saint Louis, Mo.) dissolved in normal saline was injected into the center plantar area on hind paws using an insulin syringe (BD; Franklin Lakes, N.J.).
Implantation of Mini Pumps
Alzet micro-osmotic mini pumps (Durect Corporation Model 1003D and 2001D) were filled and primed per manufacturer's guide. Mice were anesthetized with isoflurane (5% induction; 2% maintenance). Their backs were shaved, wiped down with isopropyl alcohol and povidone iodine, and a small incision was made between the scapulae. Using a pair of forceps or hemostat, a small pocket was formed by spreading the subcutaneous connective tissues apart. The pump was inserted into the pocket with the flow moderator pointing away from the incision. The skin incision was then closed using 7 mm staples and the animals were allowed to recover in their home cages.
Data Analysis
Data are represented as mean±s.e.m. Prism (Graphpad Software Inc., La Jolla, Calif.) was used for graphing and statistical analysis. For comparison of threshold values over time, a two-way ANOVA followed by Bonferroni's multiple comparison test was used with a significance level of p<0.05. Hotplate and MPE % data were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test.
Results
Efficacy of variants NV1D3034-OH (NV1D3034-COOH), NV1D3368-OH (NV1D3368-COOH) and NV1D2775-OH (NV1D2775-COOH) was studied in the CFA model, a commonly used model of inflammatory pain. The injection of CFA in the hindpaw induced paw edema (not shown) and hypersensitivity to thermal stimuli (thermal hyperalgesia), as indicated by the lowered thermal latency in the injected paw on day0 (
NV1D3368-OH fully reversed CFA-induced thermal hyperalgesia at 684 and 1824 μg/day (
CFA-induced thermal hyperalgesia was readily reversed by two additional peptides, NV1D3368-amide (NV1D3368-NH2) and NV1D3034-N-methylamide (NV1D3034-NHMe). Thermal MPE % from the experiments is summarized in Table 13.
NV1D2775-OH also exhibited strong, dose-dependent efficacy in the hotplate test (
The efficacy of NV1D2775-OH was evaluated in another model of inflammatory pain, the carrageenan model. Animals were implanted with NV1D2775-OH or PBS pumps. Thermal withdrawal latencies were measured pre- and on day1 post-pump. A-carrageenan was injected into the hindpaws and thermal latencies were measured again on 2, 3 and 4 hr following carrageenan. NV1D2775-OH at 1824 μg/day produced significant analgesia (
The instant application is a continuation of U.S. application Ser. No. 14/505,592, filed on Oct. 3, 2014, which claims priority of the benefits of the filing of U.S. Provisional Application Ser. No. 61/886,100, filed Oct. 3, 2013. The complete disclosures of the aforementioned related U.S. patent applications are hereby incorporated herein by reference for all purposes.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4309989 | Fahim | Jan 1982 | A |
| 4767402 | Kost et al. | Aug 1988 | A |
| 6103495 | Mehta et al. | Aug 2000 | A |
| 6521427 | Evans | Feb 2003 | B1 |
| 6670127 | Evans | Dec 2003 | B2 |
| 6881725 | Yerxa et al. | Apr 2005 | B2 |
| 7998980 | Moran et al. | Aug 2011 | B2 |
| 9102751 | Chen et al. | Aug 2015 | B2 |
| 9102757 | Chen et al. | Aug 2015 | B2 |
| 9624280 | Flinspach et al. | Apr 2017 | B2 |
| 20030148273 | Dong et al. | Aug 2003 | A1 |
| 20070212685 | MacDonald et al. | Sep 2007 | A1 |
| 20110065647 | Meir et al. | Mar 2011 | A1 |
| 20110124711 | Sah et al. | May 2011 | A1 |
| 20120185956 | Gingras | Jul 2012 | A1 |
| 20130296247 | Park et al. | Nov 2013 | A1 |
| 20150087596 | Eckert et al. | Mar 2015 | A1 |
| 20150099705 | Flinspach et al. | Apr 2015 | A1 |
| 20160257726 | Flinspach et al. | Sep 2016 | A1 |
| 20160287666 | Flinspach et al. | Oct 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| 2013262630 | Dec 2014 | AU |
| 2014329454 | Jan 2019 | AU |
| 112016007062 | Dec 2017 | BR |
| 2873860 | Nov 2013 | CA |
| 101979411 | Feb 2011 | CN |
| 102859003 | Jan 2013 | CN |
| 103635593 | Mar 2014 | CN |
| 104768568 | Jul 2015 | CN |
| 2013825986 | Jul 2015 | CN |
| 105793277 | Jul 2016 | CN |
| 108220406 | Jun 2018 | CN |
| 2852397 | Mar 2016 | EP |
| 3052519 | Aug 2016 | EP |
| 2015518836 | Jul 2015 | JP |
| 2016519872 | Jul 2016 | JP |
| 2016-533710 | Nov 2016 | JP |
| 2018-512123 | May 2018 | JP |
| 9853847 | Dec 1998 | WO |
| 2006116156 | Nov 2006 | WO |
| 2007109324 | Sep 2007 | WO |
| 2008088422 | Feb 2009 | WO |
| 2010028089 | Mar 2010 | WO |
| 2010104115 | Sep 2010 | WO |
| 2012004664 | Jan 2012 | WO |
| 2012004664 | Jan 2012 | WO |
| 2013173706 | Nov 2013 | WO |
| 2014016673 | Jan 2014 | WO |
| 2014165277 | Oct 2014 | WO |
| 2015051216 | Apr 2015 | WO |
| 2016140859 | Sep 2016 | WO |
| 2016161100 | Oct 2016 | WO |
| Entry |
|---|
| Communication pursuant to Article 94(3) EPC issued in European Application No. 14 790 422.1-1120 dated Sep. 10, 2018; 6 pages. |
| Examination report No. 2 issued in Australian Application No. 2014329454 dated Sep. 17, 2018; 2 pages. |
| English translation of Reexamination Decision No. 162864 issued by the Patent Reexamination Board of National Intellectual Property Administration, P.R. China in Chinese Application No. 201380025986.1 dated Oct. 23, 2018; 25 pages. |
| English translation of Decision of Rejection issued in Japanese Application No. 2015-512885 dated Oct. 30, 2018; 16 pages. |
| Examination Report issued by the Government of Pakistan Intellectual Property Organization the Patent Office in Patent Application No. 189/2016 dated Nov. 9, 2018; 2 pages. |
| Whitney et al., “Fluorescent peptides highlight peripheral nerves during surgery in mice”, nature biotechnology, vol. 29, No. 4, (2011), pp. 352-358. |
| Yang et al., “Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia”, J Med Genet, (2004), 41, pp. 171-174. |
| Yogeeswari et al., “Ion Channels as Important Targets for Antiepileptic Drug Desgin”, Current Drug Targets, (2004), 5, pp. 589-602. |
| Dib-Hajj et al., “The NaV1.7 sodium channel: from molecule to man” Nature Reviews Neuroscience, vol. 14, (2013), pp. 49-62. |
| Park et al., “Studies Examining the Relationship between the Chemical Structure of Protoxin II and Its Activity on Voltage Gated Sodium Channels”, Journal of Medicinal Chemistry, 57, (2014), pp. 6623-6631. |
| International Search Report of International Application No. PCT/US2014/058972 dated Mar. 26, 2015. |
| Smith et al, “Molecular Interactions of the Gating Modifier Toxin, ProTx-II, with Nav1.5: implied existence of a novel toxin binding site coupled to activation”, The American Society for Biochemistry and Molecular Biology, Inc. (2007) vol. 282, No. 17, pp. 12687-12697. |
| Yang et al., “Discovery of a selective NaV1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models”, Proceedings of the National Academy of Sciences (20013), vol. 110, No. 43, pp. 17534-17539. |
| International Application No. PCT/US2016/019549 filed Feb. 25, 2016. |
| International Application No. PCT/US2016/025247 filed Mar. 31, 2016. |
| U.S. Appl. No. 15/060,158, filed Mar. 3, 2016. |
| U.S. Appl. No. 15/090,328, filed Apr. 4, 2016. |
| Revel et al., “Potency optimization of Huwentoxin-IV onhNav1.7: A neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spiderSelenocosmia huwena”, Peptides, Elsevier, Amsterdam, NL, (2013), vol. 44, pp. 40-46, XP028551011. |
| Berjanskii et al., “Predictor: A Web Server for Predicting Protein Torsion Angle Restraints”, Nucleic Acids Research, vol. 34, pp. W63-W69 (2006). |
| Cheung et al., “Dangle: A Bayesian Inferential Method for Predicting Protein Backbone Dihedral Angels and Secondary Structure”, Journal of Magnetic Resonance, vol. 202, pp. 223-233 (2010). |
| Clare, et al., “Voltage-Gated Sodium Channels as Therapeutic Targets”, Therapeutic Focus, vol. 15, No. 11, pp. 506-520 (2000). |
| Delaglio et al., “NMR Pipe: A Multidimensional Spectral Processing System Based on UNIX Pipes”, Journal of Biomolecular NMR, vol. 6, pp. 277-291 (1995). |
| Genbank:ABY77745.1, “HWTX-IVb precursor [Haplopelma schmidti]”, Protein—NCBI (2007). |
| Guntert, et al., “Torsion Angle Dynamics for NMR structure Calculation with the New Program Dyana”, JMB, vol. 273, pp. 283-298 (1997). |
| Hubner, et al., “Ion Channel Diseases”, Human Molecular Genetics, vol. 11, No. 20, pp. 2435-2445 (2002). |
| Humphrey, et al., “VMD: Visual Molecular Dynamics”, Journal of Molecular Graphics, vol. 14, pp. 33-38 (1996). |
| Jeener, et al., Investigation of Exchange Processes by Two-Dimensional NMR Sectroscopy, J. Chem. Phys., vol. 71, No. 11, pp. 4546-4553 (1979). |
| Examination report No. 1 issued in Australian Patent Application No. 2014329454 dated Jan. 4, 2018. |
| Office Action issued in U.S. Appl. No. 14/399,088 dated Jun. 15, 2018. |
| Office Action issued in U.S. Appl. No. 15/090,328 dated Jun. 6, 2018. |
| Office Action issued in U.S. Appl. No. 15/583,793 dated May 18, 2018. |
| Examination Report issued by the Government of Pakistan Intellectual Property Organization the Patent Office in Patent Application No. 129/2016 dated Apr. 25, 2018. |
| Notice of Reexamination issued in Chinese Patent Application No. 201380025986.1 dated May 8, 2018. |
| Invitation pursuant to Rule 137(4) EPC and Article 94(3) EPC issued in European Application No. 14790422.1-1120 dated May 17, 2018. |
| Communication pursuant to Article 94(3) EPC issued in European Application No. 13791454.5-1120 dated May 17, 2018. |
| International Preliminary Report on Patentability; PCT/US2016/025247; dated Apr. 4, 2017. |
| International Preliminary Report on Patentability; PCT/US2016/019549; dated Sep. 5, 2017. |
| Notice of Allowance issued in U.S. Appl. No. 14/505,592, dated Aug. 9, 2016. |
| Notice of Allowance issued in U.S. Appl. No. 13/833,555, dated Feb. 27, 2015. |
| Gingras et al., Global Nav1.7 KO mice recapitulate phenotype of congenital indifference to pain reported in humans; American Pain Society Meeting, The Journal of Pain, Abstract 2013. |
| Remington's Pharmaceutical Sciences, 15th Edition. Edited under the direction of Arthur Osol and John E. Hoover. Mack Publishing Co., Easton, PA 18042, 1975. |
| Minassian et al., “Functional Studies of the Interaction Between Huwentoxin-IV and the Voltage-Gated Sodium Channel Nav1.7”, Janssen—Pharmaceutical Companies of Johnson and Johnson, Janssen Research and Development, San Diego, CA, Poster. |
| Office Action issued in U.S. Appl. No. 15/583,793 dated Dec. 14, 2017. |
| Office Action issued in U.S. Appl. No. 13/833,555 dated Sep. 23, 2011. |
| Office Action issued in U.S. Appl. No. 13/833,555 dated Jan. 8, 2014. |
| Office Action issued in U.S. Appl. No. 13/833,555 dated Jul. 17, 2014. |
| Office Action issued in U.S. Appl. No. 13/833,555 dated Nov. 3, 2014. |
| Notice of Allowance issued in U.S. Appl. No. 14/505,592, dated Dec. 23, 2016. |
| Chen et al., “Synthesis, Folding and Bioactivity Analysis of K27A-HWTX-IV: A mutant of the TTX-sensitive Sodium Channel Inhibitor, Huwentoxin-IV”, Jour Nat Scie Hunan Naun Univ, Sep. 2003, vol. 26, No. 3, pp. 2-7, English Abstract. |
| Dib-Hajj et al., “Sodium Channels in Normal and Pathological Pain”, Annual Review Neuroscience, (2010), vol. 33, pp. 325-347. |
| U.S. Appl. No. 13/833,555, filed Mar. 15, 2013. |
| European Communication pursuant to Article 94(3) EPC issued in corresponding EP Application No. 14 790 422.1-1410 dated May 4, 2017. |
| European Communication pursuant to Article 94(3) EPC, issued in European Patent Application No. 14 790 422.1-410 dated Nov. 16, 2017. |
| Patent Examination Report No. 1 issued in corresponding Australian Patent Application No. 2013262630 dated Nov. 2, 2016. |
| English translation of First Office Action issued in corresponding Chinese Patent Application No. 201380025986.1 dated Jan. 27, 2016. |
| English translation of Second Office Action issued in corresponding Chinese Patent Application No. 201380025986.1 dated Sep. 26, 2016. |
| European Communication pursuant to Article 94(3) EPC; EP Application No. 13 791 454.5-1402; dated Jul. 11, 2017. |
| English translation of Notice of Reasons for Rejection issued in corresponding Japanese Patent Application No. 2015-512885 dated Mar. 14, 2017. |
| English translation of Notice of Reasons for Rejection issued in corresponding Japanese Patent Application No. 2015-512885 dated Dec. 12, 2017. |
| Deng et al., Life Science Research,vol. 17, No. 2 (Apr. 2013) pp. 111-115. (English Abstract Only). |
| Cavanagh et al., Protein NMR Spectroscopy: Principles and Practice, 2nd Edition, 2006 Academic Press. (Description Only). |
| Bax et al., MLEV-17 Based Two-Dimensional Homonuclear Magnetization Transfer Spectroscopy. Journal of Magnetic Resonance, vol. 65, (1985) pp. 355-360. |
| Minett et al., “Pain without Nociceptors? Nav1.7-Independent Pain Mechanisms”, Cell Reports, (2014), vol. 6, No. 2301-302. |
| Wang et al., “Expression and characterization of Huwentoxin-XI (HWTX-XI) and its mutants”, Chinese Journal of Biotechnology, (2011), vol. 27, No. 2, pp. 262-268, English Abstract. |
| Xiao et al., “The Tarantula Toxins ProTx-II and Huwentoxin-IV Differentially Interact with Human Nav1.7 Voltage Sensors to Inhibit Channel Activation and Inactivation”, Molecular Pharmacology, (2010), vol. 78, No. 6, pp. 1124-1134. |
| Office Action issued in U.S. Appl. No. 14/505,592 dated Mar. 16, 2016. |
| Office Action issued in U.S. Appl. No. 14/505,592 dated Nov. 3, 2015. |
| Office Action issued in U.S. Appl. No. 14/399,088 dated Mar. 13, 2017. |
| Office Action issued in U.S. Appl. No. 15/090,328 dated Jun. 23, 2017. |
| Office Action issued in U.S. Appl. No. 15/090,328 dated Oct. 13, 2017. |
| Australian Office Action issued in Application No. 2016226443 dated Oct. 25, 2019, 3 pages. |
| International Search Report and Written Opinion of related International Application No. PCT/US2013/041572 dated Dec. 13, 2013. |
| International Preliminary Report on Patentability of related International Application No. PCT/US2013/041572 dated Nov. 1, 2014. |
| International Search Report and Written Opinion of International Application No. PCT/US2016/025247 dated Jul. 12, 2016. |
| International Search Report and Written Opinion of related International Application No. PCT/US2014/058972 dated Mar. 26, 2015. |
| International Preliminary Report on Patentability of related International Application No. PCT/US2014/058972 dated Apr. 5, 2016. |
| International Search Report and Written Opinion of International Application No. PCT/US2016/019549 dated Aug. 30, 2016. |
| Office Action dated Dec. 24, 2015 from U.S. Appl. No. 14/399,088. |
| Office Action dated Oct. 7, 2016 from U.S. Appl. No. 14/399,088. |
| U.S. Appl. No. 61/648,871, filed May 18, 2012. |
| U.S. Appl. No. 61/702,538, filed Sep. 18, 2012. |
| U.S. Appl. No. 61/781,276, filed Mar. 14, 2013. |
| Extended European Search Report issued in European Application No. 16774187.5 dated Nov. 15, 2018. |
| Communication pursuant to Rules 70(2) and 70a(2) EPC issued in European Application No. 16774187.5 dated Dec. 4, 2018. |
| Notice of Allowance issued in U.S. Appl. No. 15/090,328 dated Dec. 26, 2018. |
| Office Action issued in U.S. Appl. No. 15/583,793 dated Jan. 4, 2019. |
| Notice of Allowance issued in U.S. Appl. No. 14/399,088 dated Nov. 21, 2018. |
| English translation of Examination Report issued by the Patent Office of the Cooperation Council for the Arab States of the Gulf in Application No. GC 2016-30933 dated Jan. 10, 2019. |
| Frank Bosmans et al., “Four Novel Tarantula Toxins as Selective Modulators of Voltage-Gated Sodium Channel Subtypes”, Molecular Pharmacology, vol. 62, No. 2, (2006), pp. 419-429. |
| Stephen C. Cannon et al., “Sodium channels gone wild: resurgent current from neuronal and muscle channelopathies”, The Journal of Clinical Investigation, vol. 120, No. 1, (2010), pp. 80-84. |
| James J. Cox et al., “An SCN9A channelopathy causes congenital inability to experience pain”, Nature PublishingGroup, vol. 444, (2006), pp. 894-898. |
| Roman Gregg et al., “Pain channelopathies”, The Journal of Physiology, vol. 588, No. 11, (2010), pp. 1897-1904. |
| David M. Dirig et al., “Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli”, Journal of Neuroscience Methods, No. 76, (1997), pp. 183-191. |
| Mark Estacion et al., “A Sodium Channel Gene SCN9A Polymorphism That Increases Nociceptor Excitability”, Annals of Neurology, vol. 66, No. 6, (2009), pp. 862-866. |
| Sultan Ahmad et al., “A stop codon mutation in SCN9A causes lack of pain sensation”, Human Molecular Genetics, vol. 16, No. 17, (2007), pp. 2114-2121. |
| Caroline R. Fertleman et al., “SCN9A Mutations in Paroxysmal Clinical Study Extreme Pain Disorder: Allelic Variants Underlie Distinct Channel Defects and Phenotypes”, Neuron 52, (2006), pp. 767-774. |
| Goldberg YP et al., “Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations”, Clinical Genetics, (2007), pp. 311-319. |
| Dagmar Hackel et al.,“Transient opening of the perineurial barrier for analgesic drug delivery”, PNAS, (2012) pp. 2018-2027. |
| K. Hargreaves et al., “A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia”, Elsevier, (1998), pp. 77-88. |
| Christian A. Hubner, “Ion channel diseases”, Human Molecular Genetics, vol. 11, No. 2, (2002), pp. 2435-2445. |
| Julie K. Klint et al., “Spider-venom peptides that target voltage-gated sodium channels: Pharmacological tools and potential therapeutic leads”, Toxicon, 60, (2012), pp. 478-491. |
| E. Legroux-Crespel et al., “Traitement de l'erythermalgie familiale par I;association lidocaine-mexiletine”, Articles Scientifiques, (2003), vol. 130, pp. 429-433. |
| Richard E. Middleton et al., “Two Tarantula Peptides Inhibit Activation of Multiple Sodium Channels”, Biochemistry, (2002), pp. 14734-14747. |
| Michael S. Minett et al., “Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons”, Nature communications, (2012), pp. 1-9. |
| Yukiko Muroi et al.,“Selective silencing of NaV1.7 decreases excitability and conduction in vagal sensory neurons”, The Journal of Physiology, 589/3, (2011), pp. 5663-5676. |
| Niassar et al., “Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain”, PNAS, vol. 101, No. 34, (2004), pp. 12706-12711. |
| Oi et al., “Isolation of specific peptides that home to dorsal root ganglion neurons in mice”, Neuroscience Letters, 434, (2008), pp. 266-272. |
| Peng et al., “Function and Solution Structure of Huwentoxin-IV, a Potent Neuronal Tetrodotoxin (TTX)-sensitive Sodium Channel Antagonist from Chinese Bird Spider Selenocosmia huwena”, The Journal of Biological Chemistry, vol. 277, No. 49, Issue of Dec. 2002, pp. 47564-47671. |
| Reimann et al., “Pain perception is altered by a nucleotide polymorphism in SCN9A”, PNAS, vol. 107, No. 11, (2010), pp. 5148-5153. |
| Schmalhofer et al., “ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors”, Molecular Pharmacology, vol. 74, No. 6, (2008), pp. 1476-1484. |
| Sockolosky et al., “Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions”, PNAS, vol. 109, No. 40, (2012), pp. 16095-16100. |
| Tfelt-Hansen et al., “Inherited Cardiac Diseases Caused by Mutations in the Nav1.5 Sodium Channel”, Journal of Cardiovascular Electrophysiology, vol. 21, No. 1, (2010), pp. 107-115. |
| Hagman, “Sterilization”, Pharmaceutical Manufacturing, Lippincott Williams & Wilkins, Baltimore, MD (2005) Chapters 40, pp. 776-801. |
| Vargas-Alarcon et al., “A SCN9A gene-encoded dorsal root ganglia sodium channel polymorphism associated with severe fibromyalgia”, BMC Musculoskeletal Disorders, (2012),13:23, pp. 5 pages. |
| Chinese Search Report of Chinese Application No. 201380025986.1 dated Jan. 19, 2016. |
| European Search Report of European Application No. 13791454.5, dated Jan. 27, 2016. |
| Minassian et al., “Functional Studies of the Interaction Between Huwentoxin-IV and the Voltage-Gated Sodium Chanel NAv 1.7”, Johnson & Johnson Pharmaceutical Research & Development, San Diego, CA (2011). |
| Minassian, N., et al., “Analysis of the Structural and Molecular Basis of Voltage-Sensitive Sodium Channel Inhibition by the spider Toxin, Huwentoxin-IV (μ-TRTX-Hh2a)”, The Journal of Biological Chemistry, online Jun. 12, 2013. |
| Jiang et al., “Molecular diversification based on analysis of expressed sequence tags from the venom glands of the Chinese bird spider Ornithoctonus huwena” Toxicon, 2008, vol. 51, pp. 1479-1489. |
| Liu et al., “Isolation and Characterization of Hainantoxin-IV, a Novel Antagonist of Tetrodotoxin-Sensitive Sodium Channels From the Chinese Bird Spider Selenocosmia hainana”, Cellular and Molecular Life Sciences, vol. 60 (5), 2003, pp. 972-978. |
| Mackerell et al., “Extending the Treatment of Backbone Energetics in Protein Force Fiels: Limitations of Gas-Phase Quantum Mechanics in Reproducing Protein Conformational Distributions in Molecular Dynamics Simulations” Improved Force Fields for Proteins, 2004, pp. 1401-1415. |
| Liu, “Screening of peptide-based modulators of voltage-gated sodium channels using QPatch”, Janssen Research and Development, 2012, pp. 1-22. |
| Xiao et al., “Common Molecular Determinants of Tarantula Huwentoxin-IV Inhibition of NA Channel Voltage Sensors on Domains II and IV”, Journal of Biological Chemistry, 2011, pp. 27301-27334. |
| Xiao et al., “Tarantula Huwentoxin-IV Inhibits Neuronal Sodium Channels by Binding to Receptor Site 4 and Trapping the Domain II Voltage Sensor in the Closed Configuration”, Journal of Biological Chemistry, 2008, pp. 27300-27313. |
| Spassov et al., “Introducing and Implicit Membrane in Generalized Born/Solvent Accessibility Continuum Solvent Models”, J. Phys. Chem, 2002, vol. 106, pp. 8726-8738. |
| Shih et al., “Functional Studies of the interaction between Huwentoxin-IV and the Voltage-gated sodium channel Na 1.7”, Janssen, pp. 1. (2012). |
| Phillips et al., “Scalable Molecular Dynamics with NAMD”, Journal of Computational Chemistry, 2005, vol. 26, pp. 1781-1802. |
| Xuan “A Study of the Interactive Mechanism between Huwentoxin-IV and Sodium Channel”, Chinese Master's Theses Full Test Database, Medicine and Health Sciences, No. S1, pp. E079-E241, Dec. 15, 2011. |
| Bodenhausen, G., et al., Natural Abundance Nitrogen-15 NMR by Enhanced Heteronuclear Spectroscopy:, vol. 69, No. 1, Jan. 1980 pp. 185-189. |
| Zhang, M., et al., “Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels”, Journal of Biological Chemistry, vol. 282, No. 42, pp. 30699-30706 (2007). |
| Kumer et al., “A Two-Dimensional Nuclear Overhauser Enhancement (2D NOE) Experiment for the Elucidation of Complete Proton-Proton Cross-Relaxation Networks in Biological Macromolecules”, Biochemical and Biophysical Research Communications, vol. 95, No. 1, Jul. 16, 1980, pp. 1-6. |
| Muroi et al., “Selective Inhibition of Vagal Afferent Nerve Pathways Regulating Cough using Na 1.7 shrnA Silencing in Guinea Pig Nodose Ganglia”, Am. J. Physiol regul Integr Comp Physiol, vol. 304, pp. R1017-R1023 (2013). |
| M. Flinspach et al., “Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor”, Scientific Reports, Jan. 3, 2017, pp. 1-16, vol. 7. |
| Justin K. Murray et al., “Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the Nav1.7 Sodium Channel”, Journal of Medicinal Chemistry, 2015, pp. 2299-2314, vol. 58, American Chemical Society, ACS Publications. |
| Extended European Search Report issued in European Patent Application No. 16759288.0 dated Jul. 20, 2018. |
| Communication pursuant to Rules 70(2) and 70a(2) EPC issued in European Patent Application No. 16759288.0 dated Aug. 7, 2018. |
| English Translation of Notice of Reasons for Rejection issued in Japanese Patent Application No. 2016-519872, dated Aug. 28, 2018. |
| Office Action issued in U.S. Appl. No. 15/090,328 dated Sep. 11, 2018. |
| Translation of Notification of Defects for related Israeli Patent Application No. 254599, dated Dec. 17, 2019, 3 pages. |
| Translation of Notification of Defects for related Israeli Patent Application No. 254273, dated Dec. 18, 2019, 3 pages. |
| Office Action for related U.S. Appl. No. 16/673,750, dated Mar. 19, 2020, 36 pages. |
| Office Action for related Chinese Patent Application No. 201480066344.0, dated Jan. 22, 2020, 11 pages. |
| Office Action for related European Patent Application No. 16744187.5-1111, dated Mar. 2, 2020, 4 pages. |
| Notice of Reasons for Rejection for related Japanese Patent Application No. 2017-551164, dated Feb. 19, 2020, 14 pages. |
| Annex to EPO Form 2004, Communication pursuant to Rule 71(3) EPC; Communication under Rule 71(3) EPC for related European Patent Application No. 14 790 422.1-1118, dated Apr. 24, 2020, 906 pages. |
| Office Action for related Canadian Patent Application No. 2,882,123, dated Mar. 13, 2020, 3 pages. |
| Notice of Reasons for Rejection for related Japanese Patent Application No. 2019-036383, dated Dec. 24, 2019, 4 pages. |
| Schmalhofer, et al., “ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels,Blocks Action Potential Propagation in Nociceptors” Molecular Pharmacology, 74:1476-1484, 2008. |
| Murray et al., “Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel” J. Med. Chem. 2015, 58, pp. 2299-2314. |
| Minassian et al., “Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (_-TRTXHh2a)” The Journal of Biological Chemistry vol. 288, No. 31, dated Aug. 2, 2013, pp. 22707-22720. |
| Office Action for the Taiwanese Patent Application No. 105110361, dated May 14, 2020, 16 pages. |
| Office Action for the Taiwanese Patent Application No. 105106183, dated May 14, 2020, 16 pages. |
| Japanese Office Action for related Japanese Patent Application No. 2015-512885 dated Apr. 7, 2020, 6 Pages. |
| Canadian Office Action for related Canadian Patent Application No. 2873860 dated Apr. 27, 2020, 3 Pages. |
| European Office Action for related European Patent Application No. 16759288.0-1118 dated May 12, 2020, 6 Pages. |
| Office Action for the related Argentina Patent Application No. 20160100893, dated Jul. 23, 2020, 6 pages. |
| Office Action for the related European Patent Application No. 13791454.5-1118, dated Jul. 28, 2020, 3 pages. |
| Office Action for the related Argentina Patent Application No. 20160100558 dated Jul. 7, 2020, 15 pages. |
| Flinspach et al., “Insensitivity to pain induced by a potent selective closed-state Nav1. 7 inhibitor. Scientific reports, 2017, vol. 7, No. 1, pp. 1-16.” |
| Office Action for the related Chinese Patent Application No. 201680026028.X, dated Jul. 17, 2020, 27 pages. |
| Office Action for the related Japanese Patent Application No. 2017-551164, dated Sep. 29, 2020, 7 pages. |
| Preliminary Office Action for the related Brazilian Patent Application No. BR112017018834-1, dated Oct. 8, 2020, 5 pages. |
| Preliminary Office Action for the related Brazilian Patent Application No. BR112017020896-2, dated Sep. 14, 2020, 5 pages. |
| Office Action for the related European Patent Application No. 16774187.5-1111, dated Sep. 14, 2020, 4 pages. |
| Office Action for the related Japanese Patent Application No. 2019-036383, dated Nov. 4, 2020, 16 pages. |
| Chen et al., Synthesis, folding and bioactivity analysis of K27A-HWTX-IV: a mutant of the TTX-sensitive sodium channel inhibitor, Hu wentoxin-IV., Hunan Shifan Daxue Ziran Kexue Xuebao, 26(3) (2003), pp. 67-72, 6 pages. |
| Office Action for the related Pakistan Patent Application No. 18912016, dated Nov. 5, 2020, 2 pages. |
| Office Action for the related Canadian Patent Application No. 2926052, dated Oct. 6, 2020, 5 pages. |
| Office Action for the related U.S. Appl. No. 15/583,793, dated Sep. 10, 2020, 81 pages. |
| Office Action for the related U.S. Appl. No. 16/673,750, dated Sep. 15, 2020, 38 pages. |
| Office Action for the related Chinese Patent Application No. 2020110901278310, dated Nov. 12, 2020, 12 pages. |
| Office Action for the related U.S. Appl. No. 16/673,750, dated Jan. 8, 2021, 37 pages. |
| Office Action for the related Japanese Patent Application No. 2017-546100, dated Jan. 12, 2021, 14 pages. |
| Office Action for the related Japanese Patent Application No. 2020-013560, dated Jan. 26, 2021, 7 pages. |
| Office Action for the related Taiwanese Patent Application No. 105106183, dated Jan. 12, 2021, 14 pages. |
| Office Action for the related Taiwanese Patent Application No. 105110361, dated Jan. 12, 2021, 15 pagges. |
| Number | Date | Country | |
|---|---|---|---|
| 20200048316 A1 | Feb 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61886100 | Oct 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14505592 | Oct 2014 | US |
| Child | 15489714 | US |
