Modifications and uses of conotoxin peptides

Abstract

The present disclosure describes analog conotoxin peptides of the alpha-conotoxin peptide RgIA. These analog conotoxin peptides block the alpha 9 alpha 10 subtype of the nicotinic acetylcholine receptor (nAChR) and can be used for treating pain and inflammation including inflammatory pain, cancer related pain, and neuropathic pain. The RgIA analogs described in the present invention include a variety of sequence modifications and chemical modifications that are introduced to improve the drug-like characteristics of RgIA analogs and thereby increase their therapeutic value.

Description

[0001] Cross reference of related applications

[0002] This application claims the priority of U.S. Provisional Patent Application No. 62 / 123,123 filed on November 7, 2014, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention provides modified sequences of conotoxin peptides, pharmaceutical compositions of conotoxin peptides, and methods of using them to treat pain and other disorders. Background technique

[0004] Predatory marine snails in the genus Conus have venom rich in neuropharmacologically active peptides (conotoxin peptides or cone snail protein "CSP"). There are approximately 500 species of Cono, and among those examined so far, the conservative feature is the presence of α-conotoxin peptide in the venom. The natural α-conotoxin peptide is a highly disulfide cross-linked peptide with C1-C3 and C2-C4 disulfide bonds.

[0005] Due to the high sequence variability of its non-cysteine ​​residues, α-conotoxin is extre...

Examples

example 1

[0196] Example 1. Amidation of the C-terminus increases the stability of RgIA analogs.

[0197] For the two RgIA analogs, an amide group was used to replace the hydroxyl group in the C-terminal carboxyl group of the peptide to increase stability. CSP-2 (SEQ ID NO: 3) is significantly more stable, such as when the C-terminal is amidated (ie, NH 2 When added to the C-terminus), a higher percentage of remaining peptides in the biomatrix compared to the original carboxyl group to prove ( Figure 2A ). A similar discovery was obtained using CSP-4 (SEQ ID NO: 5) and shown in Figure 2B in.

[0198] Illustrative peptide sequences where the selected C-terminus was amidated to increase stability are set out in Table 8.

[0199] Table 8. C-terminal amidated peptide sequence

[0200] sequence

SEQ ID NO.

GCCSDPRCRYRCR-amide

21

GCCSDPRCRX12RCR-amide

22

GCCTDPRCX11X12QCR-amide

23

GCCTDPRCX11X12QCRRR-amide

24

GX28X28TDPRX28X11X12QX28Y-amide

25

GX13CTDPRX13X11X12QCR-amide

26

GCCTDPRCRX1...

example 2

[0205] Example 2. Lipidation of conotoxin peptides.

[0206] The lipidated succinimidyl valerate was coupled with CSP-4-NH2 (SEQ ID NO: 25). Make 5-10mg of conotoxin peptide and lipidated succinimidyl valerate with a molecular weight ratio of 1.5:1 in the dark at room temperature in 0.25mL anhydrous dimethylformamide in 0.0026mL N, The reaction was stirred for 16 hours in the presence of N-diisopropylethylamine. The reaction completion degree and the concentration of lipidated conotoxin peptide were measured by reversed-phase chromatography using Proscher C18 column. The lipidated conotoxin peptide was purified in dimethylformamide by reversed-phase chromatography via Hypersep C18 column using gravity feed. Load the sample onto the calibrated column at 95% H 2 O / 5% methanol / 0.1% formic acid. Load the column in the same buffer. Place the sample in four bed volumes of 95% methanol / 5% H 2 Elute in the fraction of O / 0.1% formic acid. The fractions containing lipidated conotoxin ...

example 3

[0208] Example 3. Evaluation of lipidated CSP-4 in the capsaicin model.

[0209] The capsaicin model of neuropathic pain was used to evaluate the therapeutic potential of RgIA analogs in the treatment of neuropathic pain. In this model, 30 μg of capsaicin was injected intraplantarly into the hind paws of rats to produce capsaicin-induced pain in the rats. Thermal hyperalgesia (a measure of sensitivity to pain as measured by the Hargreaves test; Hargreaves et al., 1988) was performed 15min, 30min, and 45min after the capsaicin injection. Paw withdrawal latency (baseline) was measured before capsaicin injection. C12-CSP-4-NH2, C16-CSP-4-NH2, or a vehicle without peptides were injected subcutaneously 2 hours to 3 hours before the capsaicin injection. Such as Figure 7A with 7B It can be seen that the injection of lipidated C12-CSP-4-NH2 and C16-CSP-4-NH2 resulted in a decrease in capsaicin-induced thermal hyperalgesia.

[0001] Cross reference of related applications

[0002] This application claims the priority of U.S. Provisional Patent Application No. 62 / 123,123 filed on November 7, 2014, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention provides modified sequences of conotoxin peptides, pharmaceutical compositions of conotoxin peptides, and methods of using them to treat pain and other disorders. Background technique

[0004] Predatory marine snails in the genus Conus have venom rich in neuropharmacologically active peptides (conotoxin peptides or cone snail protein "CSP"). There are approximately 500 species of Cono, and among those examined so far, the conservative feature is the presence of α-conotoxin peptide in the venom. The natural α-conotoxin peptide is a highly disulfide cross-linked peptide with C1-C3 and C2-C4 disulfide bonds.

[0005] Due to the high sequence variability of its non-cysteine ​​residues, α-conotoxin is extre...

Examples

example 1

[0196] Example 1. Amidation of the C-terminus increases the stability of RgIA analogs.

[0197] For the two RgIA analogs, an amide group was used to replace the hydroxyl group in the C-terminal carboxyl group of the peptide to increase stability. CSP-2 (SEQ ID NO: 3) is significantly more stable, such as when the C-terminal is amidated (ie, NH 2 When added to the C-terminus), a higher percentage of remaining peptides in the biomatrix compared to the original carboxyl group to prove ( Figure 2A ). A similar discovery was obtained using CSP-4 (SEQ ID NO: 5) and shown in Figure 2B in.

[0198] Illustrative peptide sequences where the selected C-terminus was amidated to increase stability are set out in Table 8.

[0199] Table 8. C-terminal amidated peptide sequence

[0200] sequence

SEQ ID NO.

GCCSDPRCRYRCR-amide

21

GCCSDPRCRX12RCR-amide

22

GCCTDPRCX11X12QCR-amide

23

GCCTDPRCX11X12QCRRR-amide

24

GX28X28TDPRX28X11X12QX28Y-amide

25

GX13CTDPRX13X11X12QCR-amide

26

GCCTDPRCRX1...

example 2

[0205] Example 2. Lipidation of conotoxin peptides.

[0206] The lipidated succinimidyl valerate was coupled with CSP-4-NH2 (SEQ ID NO: 25). Make 5-10mg of conotoxin peptide and lipidated succinimidyl valerate with a molecular weight ratio of 1.5:1 in the dark at room temperature in 0.25mL anhydrous dimethylformamide in 0.0026mL N, The reaction was stirred for 16 hours in the presence of N-diisopropylethylamine. The reaction completion degree and the concentration of lipidated conotoxin peptide were measured by reversed-phase chromatography using Proscher C18 column. The lipidated conotoxin peptide was purified in dimethylformamide by reversed-phase chromatography via Hypersep C18 column using gravity feed. Load the sample onto the calibrated column at 95% H 2 O / 5% methanol / 0.1% formic acid. Load the column in the same buffer. Place the sample in four bed volumes of 95% methanol / 5% H 2 Elute in the fraction of O / 0.1% formic acid. The fractions containing lipidated conotoxin ...

example 3

[0208] Example 3. Evaluation of lipidated CSP-4 in the capsaicin model.

[0209] The capsaicin model of neuropathic pain was used to evaluate the therapeutic potential of RgIA analogs in the treatment of neuropathic pain. In this model, 30 μg of capsaicin was injected intraplantarly into the hind paws of rats to produce capsaicin-induced pain in the rats. Thermal hyperalgesia (a measure of sensitivity to pain as measured by the Hargreaves test; Hargreaves et al., 1988) was performed 15min, 30min, and 45min after the capsaicin injection. Paw withdrawal latency (baseline) was measured before capsaicin injection. C12-CSP-4-NH2, C16-CSP-4-NH2, or a vehicle without peptides were injected subcutaneously 2 hours to 3 hours before the capsaicin injection. Such as Figure 7A with 7B It can be seen that the injection of lipidated C12-CSP-4-NH2 and C16-CSP-4-NH2 resulted in a decrease in capsaicin-induced thermal hyperalgesia.

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