RU2731217C2 - Analogue of alpha-conotoxin rgia for treating pain - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: present invention relates to a novel peptide compound capable of blocking pain signal transmission. Invention object is an analogue of conotoxin RgIA, in which disulphide bonds are modified to thioester bonds. Novel analogue shows high biological and physiological activity.

EFFECT: invention can be used in preparing drug preparations for treating pain of various aetiology.

2 cl, 8 dwg, 1 tbl, 3 ex

Description

The technical field to which the invention relates.

The present invention relates to biochemistry, in particular to a new peptide compound capable of blocking the passage of a pain signal. More specifically, this invention relates to the use of this peptide for the treatment of pain of various etiologies.

State of the art.

Chronic pain usually means pain, the duration of the sensation of which obviously exceeds the time it takes to heal from the cause that caused it. According to etiology, chronic pain is divided by many specialists into nociceptive and neuropathic. Nociceptive chronic pain is caused by the activation of pain receptors in organs and tissues, for example, as a result of an inflammatory response. Chronic neuropathic pain is caused by disturbances in the functioning of the nervous system and can be of both peripheral origin (somatic nerves, the nervous system of the digestive tract) and central (disorders in the spinal cord or brain).

Chronic pain is an extremely urgent problem in modern medicine. According to various estimates, from 10.1% to 55.2% of people in the world suffer from various manifestations of chronic pain (Harstall C, Ospina M. How Prevalent Is Chronic Pain? June 2003 volume XI issue2 Pain Clinical Updates, International Association for the Study of Pain. Pages = 1-4). It should be noted that, according to various sources, up to 67% of visits to surgical personnel are somehow associated with the feeling of nonspecific pain in the body, which can be attributed to chronic pain. In the UK, for example, the negative economic impact associated with such chronic pain is estimated at around £ 100 million per year (Chronic Abdominal and Visceral Pain: Theory and Practice. Pankaj Jay Pasricha, William D. Willis, G.F. Gebhart). Despite the great importance of the problem, there is no single approach to the treatment of chronic pain: the range of methods extends from psychological or cognitive therapy to the use of opioid analgesics. Moreover, each of the approaches has certain disadvantages, which confirms the presence of an unmet demand for a new generation of analgesic for the treatment of chronic pain.

Opioid analgesics are very effective in relieving symptoms of chronic pain. However, their use is associated with the development of addiction and a decrease in effectiveness with prolonged use. At the same time, the phenomenon of chronic pain lies precisely in the duration of pain. Thus, even highly effective opioids are not the optimal treatment for chronic pain (Chronic Abdominal and Visceral Pain: Theory and Practice. Pankaj Jay Pasricha, William D. Willis, G.F. Gebhart).

However, relatively recently, the peptide calcium channel antagonist "Ziconotide", which is an omega-conotoxin, has begun to be used to treat chronic pain. The main advantage of this conotoxin over opioid analgesics is the absence of many side effects, such as the development of dependence, a gradual decrease in the analgesic effect, and insomnia. At the same time, "Ziconotide" has a number of side effects, including paradoxical reactions - increased pain under the action of the drug (Sanford, Mark. "Intrathecal ziconotide: a review of its use in patients with chronic pain refractory to other systemic or intrathecal analgesics." CNS drugs 27.11 (2013): 989-1002.).

To date, several alpha-conotoxins capable of suppressing neuropathic pain have been isolated and investigated from the venom of sea snails. So the recently discovered representative of the 4/3 subgroup of α-conotoxins - RgIA, in contrast to the related ImI and ImII, turned out to be a highly active and selective blocker of α9α10 nAChR [Ellison, M. et al (2006) Biochemistry, 45, 1511-1517.]. Of particular interest in this peptide and Vc1.1 α-conotoxin is due to their ability to suppress neuropathic pain in in vivo models [Vincler, M. et al. Proc. Natl. Acad. Sci. USA 103,17880-17884]. However, the involvement of α9α10 nAChR in the anti-pain effect is currently being actively debated [Nevin, S.T., et al Mol. Pharmacol., 72, 1406-1410], in particular because the natural prototype of α-conotoxin Vc1.1 (Vela carrying two post-translational modifications [Jakubowski, JA, et al J. Mass Spectrom., 39, 548- 557]), is also a potent antagonist of α9α10 nAChR, but has no anti-pain activity.

The drug is a synthetic version of a natural compound, the RgIA peptide, first discovered in the cDNA library of the venomous glands of the sea mollusk Conus regius. The original peptide RgIA is a member of the alpha-conotoxin family - short 10-15-membered peptides that inhibit nicotinic acetylcholine receptors (nAhR). A natural source of alpha-conotoxins is the venom of the Conus shellfish. RgIA has two fundamentally different types of molecular targets.

The first type of molecular target for RgIA is potential sensitive calcium channels. This type of ion channel plays a key role in the release of neurotransmitters responsible for pain. Blocking voltage-gated calcium channels reduces the release of pain neurotransmitters from presynaptic endings, which contributes to the achievement of an analgesic effect (Fig. 1). Let's look at the process in stages. At the first stage (1), a signal encoding pain sensations arrives at the end of the effector process (axon) of a sensitive neuron. Under normal conditions, this leads to the activation of voltage-dependent calcium channels (3) and the entry of calcium ions into the cytoplasm of the neuron. Calcium ions bind to the protein synaptotagmin, which triggers the fusion of the membranes of synaptic vesicles with the presynaptic membrane (4) and the release of neurotransmitters into the synaptic cleft (5). To reduce the efficiency of pain signal transmission, it is sufficient to reduce the activity of voltage-sensitive calcium channels. One of the ways of this decrease in activity is the activation of metabotropic receptors for gamma-aminobutyric acid (GABAB). RgIA activates GABAB, which in turn triggers a signaling cascade leading to a decrease in calcium channel activity (2). As a result of this effect, the number of "painful" neurotransmitters in the synaptic cleft decreases and, as a result, the intensity of pain sensations decreases.

The second type of target is nAhR alpha9 / alpha10 subtypes, for which the peptide has the highest affinity (100-200 nM). A feature of the receptors of these subtypes is that they are involved in the conduct of pain signals of various etiologies, mainly neuropathic and inflammatory. In particular, model animals knocked out for the alpha9 nAhR gene are less susceptible to mechanically induced hyperalgesia, one of the components of neuropathic pain, which certainly confirms the relevance of the use of ligands of this nAhR as pain modulators (doi: 10.1186 / 1744-8069-10-64) ...

A feature of this mechanism of analgesia is that nAhR alpha9 / alpha10 subtype are themselves ligand-gated calcium channels (Fig. 2). Therefore, their activation leads to the entry of calcium ions into the cytoplasm of the neuron and stimulation of the release of "pain" neurotransmitters into the synaptic cleft. RgIA directly inhibits nAxR and prevents calcium ion entry, which reduces neurotransmitter release and pain transmission.

Both types of targets - GABAB and alpha9 / alpha10 nAhR - are expressed in neurons of the dorsal root ganglion (DRG). The neurons of these ganglia are involved in the transmission of sensory information from peripheral organs to the spinal cord, in particular, signals transmitted through unmyelinated class C fibers that transmit pain signals (Fig. 3). Pain signals along the "C" fibers enter the spinal cord through the dorsal roots. At the same time, in the ganglia of these roots, a signal is transmitted from a sensitive neuron to an intercalary neuron. The molecular targets of interest to us are expressed in the synaptic contact of these neurons: metabotropic receptors of gamma-aminobutyric acid (GABAB), coupled with voltage-sensitive calcium channels, as well as ionotropic acetylcholine receptors of the α9 / α10 type.

Thus, when RgIA interacts with receptors on the surface of neurons in the dorsal root ganglia, pain signals are inhibited as a result of suppressing the release of neurotransmitters from presynaptic terminals of sensory neurons. This leads to the fact that the efficiency of information transmission through such a synapse becomes much lower. Consequently, signals of lower intensity enter the brain, which is interpreted as a decrease in pain.

It should be noted that RgIA and its analogs compare favorably with various low-molecular-weight analgesics on the market. Being peptide compounds, they poorly penetrate the blood-brain barrier, which excludes the development of "central" side effects. On the other hand, the main target of their action is receptors on the surface of neurons of the dorsal root ganglia to a lesser extent isolated from the systemic blood flow, which makes it possible to use traditional methods of drug administration instead of complex systems with the introduction of an analgesic into the cerebrospinal fluid.

At the same time, one of the obstacles for the introduction of conotoxins, like other disulfide-containing peptides, into medical practice as drugs is the instability of disulfide bonds (doi: 10.1016 / j.ijpharm.2015.04.041, doi: 10.1089 / ars.2009.3068) ... As a result of thiol-disulfide exchange or reduction reactions, the original molecules lose their structure and, as a consequence, their affinity for their targets in the body, the physiological effect decreases or disappears completely. Therefore, researchers are making significant efforts to create RgIA and Vc1.1 based on conotoxins as therapeutically interesting, more stable molecules (doi: 10.1021 / jm501126u, doi: 10.1002 / anie.201409678, doi: 10.1002 / bip.22699, doi: 10.1038 / srep13264). All known attempts refer to either the complete replacement of the disulfide bond, or to its modification. The main disadvantage of the obtained compounds is a decrease in their activity. Thus, the task of developing stable and active analogs of conotoxins for their use as analgesics of a new generation, devoid of the drawbacks of existing drugs, is very urgent. The closest analogue of the subject of the invention is the natural alpha-conotoxin RgIA.

Disclosure of the invention.

The present invention solves the problem of obtaining a stable molecule that can be used in drugs for the treatment of pain. The technical result is achieved by changing the natural structure of the alpha-conotoxin RgIA molecule by modifying the disulfide bond, which consists in its partial desulfurization.The object of the invention is a peptide consisting of the same amino acid residues as natural RgIA, but containing two intramolecular thioether (sulfide) bonds between cysteine residues 2 and 8, 3 and 12. Amino acid sequence of peptide Rg2S (SEQ ID: 1): H-Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Arg-Tyr-Arg-Cys-Arg- OH. Such a change in structure makes it possible to solve the problem of the stability of the molecule, since the sulfide bond is not reduced under normal conditions by thiol agents (for example, dithiothreitol). This in turn results in a rigid structure that is not cleaved by enzymes. For example, under the influence of trypsin, only the C-terminal arginine is cleaved from the peptide SEQ ID: 1, the rest of the molecule remains stable. At the same time, the change in structure did not affect the biological activity of the molecule. Thus, in the hot plate test it was shown (Table 1) that, firstly, the peptide SEQ ID: 1 exhibits greater activity compared to natural conotoxins VC1.1 and Rg1A, and, secondly, the activity of the claimed peptide is dose-dependent.

Experiments on natural targets of conotoxin Rg1A showed that the activity of the Rg2S peptide was preserved or slightly decreased. Thus, in experiments on electrophysiological testing of the excitability of neurons in the dorsal root ganglia, it was found that the application of modified alpha-conotoxins RgIA causes a decrease in the excitability of neurons (an increase in the rheobase index of Fig. 4)

Talice 1. Time of withdrawing and licking the mouse paw in the hot plate test. Means ± unbiased standard deviation.

Full-cell recording of the membrane potential of neurons in the dorsal root ganglia was performed in the current fixation mode. Electrode micropipettes were made using an automatic Narishige puller (pipette resistance not less than 5 MΩ) and filled with an intracellular solution (140 mM CsCl, 6 mM CaCl2, 2 mM MgCl2, 2 mM MgATP, 0.4 mM NaGTP, 10 mM HEPES / CsOH, 20 mM BAPTA / KOH; pH 7.3). Measurements are carried out at room temperature (23-25 ° C) using a patch-clamp amplifier HEKA. In this experiment, the steps of the current injected into the cell were sequentially fed in steps of increasing amplitude with a step of 50 picoA and a duration of 500 ms. As a result of the experiment, the rheobase of the neuron was determined before and after exposure to the modified conotoxin RgIA at a concentration of 1 μM. An increase in the rheobase indicates inhibition of the neuron, and, consequently, the difficulty of conducting a pain signal through this neuron. As previously established in the literature (doi: 10.1136 / gutjnl-2015-310971), these effects are most likely mediated by the activation of metabotropic receptors for gamma-aminobutyric acid.

To study the activity of the peptide Rg2S on alpha9 / alpha10 nAxR, Xenopus oocytes expressing this receptor were used. The measurements were carried out using a TURBO TEC-03X differential amplifier (DruMMond, Germany) by the method of two-electrode potential clamping (TEVC, two-electrode voltage сlamp) at -60 mV. The volume of the chamber containing the oocyte was 50 μL. The ionic currents of the oocyte were recorded by applying a 25 μM solution of acetylcholine iodide (SigMa-Aldrich, United States) prepared on the basis of Barth's buffer containing calcium ions. The volume of the introduced ligand is 100 μl, washing with buffer is 1 ml. There was a pause of 5 minutes between ligand applications. To study the effect of conotoxins on α9α10 nAChR, oocytes were preincubated with solutions of RgIa (1 μM) and its analog (1 μM, 10 μM, 100 μM) for 5 minutes before applying the agonist.

A solution of the Rg2S-2 analog at a concentration of 1-100 μM blocks the ion current of α9α10 expressing oocytes caused by a 25 μM acetylcholine solution (Fig. 5). At the same time, for RgIA, the blocking effect was demonstrated at a toxin concentration of 1 μM or less, which indicates a lower analogue affinity for this nAhR subtype.

Experiments on the study of the activity of the Rg2S peptide (SEQ ID: 1) showed that it is able to affect the pathways of transmission of pain impulses and suppress pain by interacting with various targets.

The invention is illustrated in FIG. 1-3:

Fig 1. Mechanism of action of conotoxin RgIA via metabotropic receptors of gam-aminobutyric acid.

Fig 2. The mechanism of action of conotoxin RgIA through ionotropic acetylcholine receptors (nAhR).

Fig 3. Simplified diagram of the transmission of pain signals with the participation of the ganglia of the dorsal roots of the spinal cord.

Fig 4. Increase in rheobase of neurons of the dorsal root ganglia under the influence of a modified analogue of alpha-conotoxin RgIA. As a result of the action of the tested peptides, the excitability of neurons decreases, the generation of the action potential is blocked. The arrow marks the presence of an action potential in the control (peak) and the absence of Rg2S after application.

Fig 5. Electrophysiological testing of the effect of the conotoxin analog RgIA on the activity of α9α10 nAhR.

Figure 6. Reaction mixture during oxidation of the linear precursor of the Rg2S peptide.

Fig 7. Analytical chromatogram of the Rg2S peptide.

Fig 8. Mass spectrum of the Rg2S peptide.

Examples of implementation of the invention.

Example 1. Chemical synthesis of the peptide SEQ ID NO: 1: H-Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Arg-Tyr-Arg-Cys-Arg-OH

Attachment of the first amino acid. Preparation of Fmoc-Arg (Pbf) -P (Fmoc = 9-fluorenylmethyloxycarbonyl).

200 mg of 2-chlorotrityl chloride polymer with a hydroxyl group content of 1.0 mmol / g is washed with anhydrous methylene chloride. 713 mg (1.1 mmol) of Fmoc-Arg (Pbf) -OH and 374 μl (2.2 mmol) of diisopropylethylamine are dissolved in 5 ml of methylene chloride, the solution is stirred for 5 min and then poured onto the polymer. The reaction is carried out for 1 hour at room temperature and stirring. At the end of the reaction, the polymer is filtered off and washed three times successively with methylene chloride, ethyl alcohol, dimethylformamide.

Description of one synthetic cycle of polypeptide chain extension.

Obtaining Fmoc-Cys (Trt) -Arg (Pbf) -P:

a) The peptidyl polymer obtained in the previous step is treated for 20 min with a 20% piperidine solution in dimethylformamide. The polymer is washed sequentially with 5 ml of the following solvents: dimethylformamide - 3 times for 2 minutes, with a mixture of dioxane-water (2: 1) - 2 times for 5 minutes, with dimethylformamide - 5 times for 2 minutes.

b) In 5 ml of dimethylformamide, 644 mg (1.1 mmol) of Fmoc-Cys (Trt) -OH, 150 mg (1.1 mmol) of 1-hydroxybenzotriazole, and 172 μl (1.1 mmol) of N, N'- diisopropylcarbodiimide, the solution is stirred for 10 min at 0 ° C and then poured onto the suspended polymer. The reaction is carried out for 4 hours with periodic stirring. After the completion of the reaction, the polymer is filtered off, washed with dimethylformamide, and treated with 5 ml of a mixture of Ac ₂ O-pyridine-dimethylformamide (20:20:60) for 1 hour, after which the polymer is successively washed with dimethylformamide, isopropanol, dimethylformamide.

The synthesis of the polypeptide chain is carried out manually in a glass flow reactor (2 × 20 cm) according to the following protocol for each synthetic cycle (based on 8-10 ml of solvent per 400 mg of the starting polymer), when carrying out the condensation reaction (operation 6), the volume of the reaction mixture is used 5 -7 ml:

1. DMF (dimethylformamide) (5 × 2 min.);

2.20% piperidine in DMF (20 min.);

3. DMF (3x2 min.);

4. dioxane-water, 2: 1, (2 × 5 min.);

5. DMF (5 x 2 min.);

6. Condensation reaction: 5 molar equivalents of activated Fmoc-amino acid (4 hours);

7. DMF (3 x 2 min.);

8.acylation: Ac ₂ O-pyridine-dimethylformamide 20:20:60 (1 hour);

9. DMF (3 x 2 min.);

10.isopropanol (3 x 2 min.);

For activation of Fmoc-derivatives of amino acids using a DIPCDI / HOBT mixture (N, N'-diisopropylcarbodiimide / 1-hydroxybenzotriazole) to a solution of 1.1 mmol (5 equivalents) of Fmoc-protected amino acid and 150 mg (1.1 mmol, 5 equivalents) HOBt in 4 ml DMF, 170 μl (1.1 mmol, 5 equivalents) of DIPCDI was added, the solution was stirred for 10 min.

The completeness of the condensation reaction is monitored using ninhydrin or, in the case of N-terminal proline, isatin tests after operation 6 of the synthetic protocol.

The following amino acid derivatives are used for synthesis: Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Pro-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Arg ( Pbf) -OH, Fmoc-Tyr (tBu) -OH.

Cleavage of the peptide from the polymer.

For the reaction of cleavage of the peptide from the polymer and the simultaneous unblocking of the protecting groups of the side chains of amino acids, 800 mg of the peptidyl polymer are taken. 15 ml of a mixture of TFA (trifluoroacetic acid) _- H2O in a volume ratio of 97.5: 2.5 are added to the peptidyl polymer, the suspension is stirred for 2 h, then the resulting peptide solution is filtered from the polymer, washed with 5 ml of TFA, and the excess TFA is evaporated at reduced pressure. The peptide is precipitated with 100 ml of ethyl ether, filtered off and washed with ether (5 × 20 ml). The precipitate is dissolved in 5 ml of 10% acetic acid for 20 minutes, filtered off and washed with 5 ml of 10% acetic acid. The resulting peptide solution is lyophilized and desalted on a column (2.5 × 60 cm) with Sephadex G-10 in 0.1 M acetic acid. The peptide is purified using reverse phase HPLC in an acetonitrile gradient (from 10% to 35% in 75 min.) In 0.1% TFA at an eluent flow rate of 3 ml / min; the absorption of the eluate is recorded at a wavelength of 226 nm. Fractions corresponding to the main peak in the chromatogram are collected and lyophilized. The molecular weight of the peptide, established by mass spectrometry, was 1574.6 Da, the theoretical calculated molecular weight was 1574.8 Da.

Obtaining sulfide bonds between cysteines in the linear conotoxin RgIA.

In 80 ml of a mixture of acetonitrile / water (1/1) with stirring, dissolve 40 mg of the linear peptide RgIA, then add 300 μl of diisopropylethylamine (pH≥12). The reaction is carried out in an open flask with stirring for two days. Then add acetic acid to pH = 5, the solution is frozen and lyophilized. From the resulting mixture (Fig. 6), the main peak is isolated (Fig. 7). The molecular weight of the peptide, established by mass spectrometry, was 1506.8 Da (Fig. 8), the theoretical calculated molecular weight was 1506.8 Da.

Example 2. Activity of SEQ ID NO: 1 peptide in hot plate test.

An experimental male white mouse weighing 20-25 g was placed on a thermostatted plate (53.5 ± 0.3 deg C). The time of the first withdrawal and licking of the hind leg was recorded, after which the mouse was transferred to a separate container. Three measurements were taken before drug injection. The interval between measurements was 7.5 minutes. The test substance at the indicated dose (see Table 1) in an aqueous solution with 0.9% sodium chloride was injected into the same individual intraperitoneally in a volume of no more than 0.3 ml. 7.5 minutes after injection, four measurements of the time of the first withdrawal and licking of the hind paw were made at intervals of 7.5 minutes. The mean time for withdrawing and licking the hind paw of the mouse before and after injection was calculated.

Example 3. Two-electrode fixation of the membrane potential of Xenopus laevis oocytes

Plasmids containing genes of α9 and α10 subunits of rat nAChR (vector pGEMNE) were linearized with restriction endonuclease NheI (NEB, USA). The resulting linearized plasmids were isolated using the QIAquick PCR Purification Kit (Quiagen, Netherlands). Then, using the T7 MMessage mMaxine transcription kit (Ambion Inc., Austin, TX, USA), the mRNA of α9 and α10 nAChR subunits is synthesized in vitro. To purify the resulting mRNA, we used the RNeasy MinElute Cleanup kit (Quiagen, Netherlands).

For subsequent transfection, stage IV and V Xenopus laevis oocytes are selected, obtained by direct dissection of the female abdominal cavity with the isolation of the ovaries on one side. The frog is placed in a cold solution of para-aminobenzoate 0.5 g / l (Potaba, Germany) for 15-30 minutes and then transferred to ice. With cooled scissors, an incision is made about 10 mm long, from where a fragment of the ovary is removed. The isolated ovaries were treated with type A collagenase (4 mg / ml, Worthington, USA) in Barth's calcium-free buffer (88.0 mM NaCl, 1.1 mM KCl, 2.4 mM NaHCO3, 0.8 mM MgSO4, 15.0 mM HEPES / NaOH, pH 7.6) in within 4 hours. About 7 ng of mRNA of α9 and α10 nAChR subunits are injected in the region of the animal pole of oocytes using a NanoJect-2 vacuum nanoinjector (Drummond, Germany) in a volume of 23 nL per oocyte. The injected oocytes are then incubated for 72-120 hours at a constant temperature of + 17 ° C in Barth's buffer containing calcium ions (88.0 mM NaCl, 1.1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca (NO3) 2, 0.4 mM CaCl2 , 0.8 mM MgSO4, 15.0 mM HEPES / NaOH, pH 7.6) with the addition of 100 μg / ml apmicillin and 40 μg / ml gentamicin.

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Sequence listing

<110> Limited Liability Company "Syneuro"

<120> An analogue of alpha-conotoxin RgIA to treat pain.

<130> Rg2S

<150>

<151> 2016-11-24

<160> 1

<210> 1

<211> 13

<212> PRT

<213> Art

<220>

<223> Sulfide bond between Cys2 / Cys8 and Cys3 / Cys12

<400> 1

Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Arg-Tyr-Arg-Cys-Arg

1 5 10

<---

Claims (3)

1. Compound of general formula (I):

... 2. Use of a compound according to claim 1 for the treatment of pain.

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