Use of d-enantiomeric peptides for the therapy of neuropathic pain

Abstract

The invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, as well as to homologs, fragments or parts thereof.

Description

[0001] Use of D-enantiomeric peptides for the treatment of neuropathic pain

[0002] Description

[0003] The invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, homologs, fragments and parts thereof, as well as such a peptide for use in the treatment of neuropathic pain.

[0004] One of the aims of the present invention was to develop a specific inhibitor of the voltage-gated N-type calcium channel (Ca v 2.2), for the treatment of neuropathic pain. The Ca v2.2, which is mainly located at presynaptic nerve endings of neuronal cells in the brain and spinal cord, mediates the release of neurotransmitters involved in pain processing. The binding of a Ca v 2.2 Inhibitors inhibit the influx of calcium into the nerve cells, which reduces the release of neurotransmitters and ultimately prevents the transmission of the pain signal.

[0005] Pain disorders, like diabetes and hypertension, are among the most common diseases and have enormous impacts on the quality of life of those affected, as well as social and economic consequences. The prevalence of chronic neuropathic pain is 7 to 10% in the general population (van Hecke et al., 2014). This corresponds to approximately 8 million patients in Germany alone.

[0006] Chronic neuropathic pain develops following damage to somatosensory nerve structures in the peripheral or central nervous system due to injuries (spinal cord injuries, stroke) or various diseases (e.g., multiple sclerosis, diabetic polyneuropathy, postherpetic neuralgia). Due to its severity, it severely limits the quality of life of those affected, making normal daily life almost impossible. Clinically, these syndromes are characterized by sensory deficits and persistent burning pain, including attacks and evoked pain, so effective pain therapy must be initiated as early and as intensively as possible. However, it must also consider the side effects that may occur, which, with long-term use, as required for neuropathic pain, can significantly outweigh the benefits of the treatment.In general, neuropathic pain exhibits chronic, severe, and often opioid-resistant characteristics, making its clinical treatment a major challenge. The high addictive potential of existing medications is particularly problematic. If one wishes to avoid opiates and other highly addictive medications, there are currently no suitable active substances available for neuropathic pain. Approximately 20-40% of patients respond inadequately or not at all to currently available drug therapies (so-called "non-responders").

[0007] In addition to antidepressants (tricyclic / tetracyclic antidepressants, selective serotonin / norepinephrine reuptake inhibitors), long-acting opioids, anticonvulsants that act on neuronal sodium channels (e.g., carbamazepine), and topical therapeutics (lidocaine patches, high-dose capsaicin patches), pharmacological therapies that act on neuronal calcium channels, such as gabapentin, pregabalin, and ziconotide (Prialt®), are used. Gabapentin and pregabalin are specific P / Q-type calcium channel inhibitors, and ziconotide is the only approved specific N-type calcium channel inhibitor. Ziconotide is used in patients suffering from extremely severe pain for whom all other treatment options have failed.

[0008] The approval of Ziconotide / Prialt is based on the results of three Phase III clinical trials involving more than 1,200 patients. In all studies, Ziconotide / Prialt significantly reduced pain compared to placebo, including severe, treatment-resistant chronic pain resulting from cancer or AIDS. Among other things, the additional intrathecal use of Ziconotide / Prialt reduced the required amount of opioid. Compared to morphine, Ziconotide / Prialt causes fewer adverse effects.

[0009] However, the intrathecal (i.t.) use of ziconotide / Prialt carries the risk of potentially serious infections such as meningitis, which can be life-threatening. Meningitis due to the penetration of organisms along the catheter or accidental contamination of the infusion system is a known complication of intrathecal drug administration, particularly with external systems. Furthermore, ziconotide / Prialt causes severe central nervous system side effects in 88% of patients, including dizziness, nausea, nystagmus, confusion, unsteady gait, memory impairment, blurred vision, headache, asthenia, vomiting, and somnolence.

[0010] Despite the very complex and risky method of application and the strong side effects, Ziconotide / Prialt is used for pain therapy because it is the only approved specific N-type calcium channel inhibitor that shows success even in the treatment of very severe pain, e.g. caused by tumors or AIDS, where even morphine no longer provides relief.

[0011] The only approved specific Ca v 2.2 Inhibitor (Ziconotide / Prialt) causes severe central nervous system side effects such as dizziness, nausea, nystagmus, confusion, unsteady gait, memory impairment, blurred vision, headache, asthenia, vomiting, and somnolence in 88% of patients. This can only be explained if one assumes that Ziconotide / Prialt is used in addition to Ca v2.2 further "off targets" are encountered. For the purposes of this application, "off target" refers to the accumulation or release of a drug at one or more unintended sites of action, which may reduce the drug's efficacy and / or cause systemic side effects.

[0012] Furthermore, the only approved route of administration for Ziconotide / Prialt is intrathecal injection, as it does not cross the blood-brain barrier (BBB). However, intrathecal administration can lead to significant complications and side effects, such as spinal nerve or spinal cord injuries, local or systemic infections, and cerebrospinal fluid (CSF) leakage, which are not expected with systemic administration via oral (po), subcutaneous (sc), or intravenous (iv). Furthermore, Ziconotide / Prialt binds with extremely high affinity to the Ca v2.2 (Ki = 17.4 pM). Therefore, an overdose cannot be easily reversed and is virtually irreversible over hours and possibly days. This is further exacerbated by the fact that ziconotide / prialt is not efficiently eliminated from the central nervous system, as it does not cross the BBB, as previously described. Ziconotide / prialt has severe effects on heart rate, blood pressure, and the baroreceptor heart rate reflex following systemic administration.

[0013] The object of the present invention was therefore to develop new peptides which specifically inhibit the voltage-gated N-type calcium channel (Ca v 2.2) can inhibit.

[0014] This object is achieved by a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, as well as homologs, fragments and parts thereof.

[0015] Compared to L-enantiomeric peptides such as ziconotide, D-peptides are highly stable in vivo against protease degradation, thus potentially offering the possibility of oral administration. Furthermore, they offer the advantage of being generally non-immunogenic.

[0016] Surprisingly, safety pharmacology studies in preparation for a Phase I clinical trial for the D-enantiomeric peptide RD2, which was developed for the treatment of Alzheimer's dementia, revealed off-target effects. Among other things, RD2 was able to bind radiolabeled conotoxin from Ca v2.2 in cell homogenate with an IC50 of approximately 150 nM. A more detailed electrophysiological study confirmed the inhibitory effect of RD2 on Ca v 2.2. This inhibition is specific for Ca v 2.2, the L-type calcium channel (Cavl.2) is not inhibited. This is important because this would lead to cardiovascular side effects.

[0017] Since RD2 is the validated target Ca v 2.2 clearly does not target the off-target receptors that cause the side effects of ziconotide / Prialt, but was a serendipitous discovery. RD2 was further optimized through rational design with respect to its IC50 concentration and binding kinetics. Its advantageous properties, such as Ca v 2.2, stability and thus oral administration, are retained in the optimized RD2 derivatives.

[0018] In contrast to the only approved drug without addictive potential, the trade name Prialt, the peptides obtained according to the invention have the advantage that they can be taken orally and do not have to be administered intrathecally into the spinal canal, which can lead to additional severe side effects such as post-puncture headaches or infections after surgical implantation of the pump. Therefore, a systemically applicable (preferably orally) specific Ca v 2.2 Inhibitor of enormous value.

[0019] A further advantage over Prialt is that the peptides according to the invention inhibit Ca v 2.2 channel only reversibly, thus preventing overdose. Furthermore, the L-type calcium channel Cavl.2 is not affected, which is very important, as this would lead to, among other things, serious heart problems.

[0020] Further preferred embodiments are defined in the dependent claims.

[0021] In the following, the term "comprise" shall also include "consisting of".

[0022] The peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 were found using rational design. In this way, 10 D-enantiomeric peptides were identified that specifically bind to Ca v 2.2 channel inhibit, developed.

[0023] SEQ ID 1: RD2A psvfthnrrrrr

[0024] SEQ ID 2: RD2B psvfshnrrrrr

[0025] SEQ ID 3: RD2C psvftrnrrrrr

[0026] SEQ ID 4: RD2D psvfthsrrrrr

[0027] SEQ ID 5: RD2E psvfthqrrrrr

[0028] SEQ ID 6: RD2CRD2C psvftrnrrrrrpsvftrnrrrrr

[0029] SEQ ID 7: RD2CRD2B psvftrnrrrrrpsvfshnrrrrr

[0030] SEQ ID 8: RD2BRD2C psvfshnrrrrrpsvftrnrrrrr

[0031] SEQ ID 9: RD2CRD2D psvftrnrrrrrpsvfthsrrrrr

[0032] SEQ ID 10: RD2DRD2C psvfthsrrrrrpsvftrnrrrrr

[0033] Sequence processing of SEQ ID 1 to 10 (e.g. sequence variation) makes it possible to develop further therapeutically applicable substances.

[0034] The present invention may also relate to other peptides that can be identified by the method disclosed above.

[0035] The object of the invention is also achieved by a peptide containing homologs, fragments and parts of the amino acid sequence according to SEQ ID NO: 1 to 10.

[0036] "Homologous sequences" or "homologues" in the context of the invention means that an amino acid sequence has an identity with one of the above-mentioned amino acid sequences of the monomers of at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%. 80% and 90% are preferred. Instead of the term "identity," the terms "homologous" and "homology" are used synonymously in the present description. The identity between two nucleic acid sequences or polypeptide sequences is calculated by comparison using the BESTFIT program based on the algorithm of Smith, TF and Waterman, M. S (Adv. Appl. Math. 2: 482-489 (1981)) setting the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acids: gap creation penalty: 50 and gap extension penalty: 3.Preferably, the identity between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence / polypeptide sequence over the entire sequence length, as calculated by comparison using the GAP program based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Biol. 48: 443-453) setting the following parameters for amino acids: Gap creation penalty: 8 and Gap extension penalty: 2; and the following parameters for nucleic acids: Gap creation penalty: 50 and Gap extension penalty: 3.

[0037] Two amino acid sequences are identical for the purposes of the present invention if they have the same amino acid sequence.

[0038] In a further variant, the peptides according to the invention have sequences that differ from the specified sequences by up to two or three amino acids.

[0039] Furthermore, sequences containing the above-mentioned sequences can also be used as peptides.

[0040] The peptide according to the invention is further preferably characterized in that at the free C-terminus, instead of the carboxyl group, there is an acid amide group (CONH2 group) or a COH group, COCl group, COBr group, CONH-alkyl radical or a CONH-alkyl-amine radical, or the peptide is cyclized.

[0041] This particularly advantageously solves the additional problem of providing a peptide without a negative charge at the C-terminus. This advantageously ensures that it can bind to the target molecule with higher affinity than a peptide that has a carboxyl group at the free C-terminus. Peptides with a free, unmodified carboxyl group have a negative charge at this end in the physiological state.

[0042] In one embodiment of the invention, the peptide according to the invention is modified in the physiological state, in particular at pH 6-8, in particular 6.5-7.5, in particular at pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9 or pH 8.0 such that the C-terminus does not carry a negative charge, but is instead neutral or has one or more positive charges.

[0043] In one embodiment, the peptide is characterized by the presence of an amide group at the free C-terminus instead of the carboxyl group. Thus, instead of the carboxyl group (-COOH group), an amide group (-CONH2 group) is located at the C-terminus.

[0044] The peptide is therefore particularly advantageously amidated at the free C-terminus.

[0045] The peptide is therefore particularly advantageously amidated at the free C-terminus and unmodified at the free N-terminus.

[0046] This particularly advantageously solves the further problem of providing a peptide without excess negative charge, which can bind more affinely to the target molecule and is easily obtainable.

[0047] In a further embodiment of the disclosure, the following further groups are present instead of the carboxyl group: COH, COCl, COBr, CONH-alkyl radical, CONH-alkyl-amine radical (positive net charge), etc., although this is not limited to the technical teaching of the main claim.

[0048] The peptide according to the invention is further preferably characterized in that it contains 2, 3, 4, 5, 6, 7, 8, 9 or more copies of the sequences with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5.

[0049] Variants are also conceivable, wherein the peptide contains 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0050] Particularly preferred are dimers of the sequences with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, where the two monomers are peptides with the same SEQ ID or with a different SEQ ID. The peptide according to the invention is further preferably characterized in that the peptide consists essentially of D-amino acids.

[0051] For the purposes of the present invention, the term "essentially composed of D-enantiomeric amino acids" means that the monomers to be used according to the invention are composed of at least 50%, 55%, 60%, 65%, 70%, preferably 75%, 80%, particularly preferably 85%, 90%, 95%, in particular 96%, 97%, 98%, 99%, 100% D-enantiomeric amino acids.

[0052] The peptide according to the invention is further preferably characterized in that it consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0053] The peptide according to the invention is further preferably characterized in that the peptide is linked to another substance.

[0054] For the purposes of the invention, the linkage is a chemical bond as defined in Römpp Chemie Lexikon, 9th edition, volume 1, page 650 ff, Georg Thieme Verlag Stuttgart, preferably a main valence bond, in particular a covalent bond.

[0055] In one variant, the substances are medicinal products or active ingredients, as defined in accordance with Section 2 or Section 4 (19) of the German Medicines Act (Medicinal Products Act), as of September 2012. In another variant, active ingredients are therapeutically active substances used as medicinally effective substances. Anti-inflammatories are preferred.

[0056] In another variant, the substances are compounds that enhance the effect of the peptides.

[0057] Alternatively, the peptides according to the invention have any desired combination of at least two or more features of the variants, embodiments, and / or alternatives described above. The peptide according to the invention is further preferably characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 are covalently or non-covalently linked to one another.

[0058] A covalent connection or linkage of the peptide units exists within the meaning of the invention if the peptides are linked head to head, tail to tail or head to tail linearly, with or without linkers or linker groups inserted therebetween.

[0059] The peptide according to the invention is further preferably characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 are linked to one another without a linker, i.e. directly to one another, or with a linker group.

[0060] A non-covalent linkage within the meaning of the invention exists if the peptides are linked to one another, for example, via biotin and streptavidin, in particular streptavidin tetramer.

[0061] The peptide according to the invention is further preferably characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 are linearly linked to one another.

[0062] In a variant of the present invention, the peptides may be linked to one another linearly, in particular as described above.

[0063] The peptides can be produced, for example, via chemical synthesis or peptide synthesis.

[0064] To analyze the effect of the derivatives as potential therapeutics for the treatment of neuropathic pain, electrophysiological studies were performed on the stable Cav2.2 HEK cell line, which expresses Ca v 2.2 olB-1, ß3a and o2böl subunits were overexpressed. Patch-clamp experiments showed that the derivatives specifically block the Cav2.2 channel in single-digit micromolar concentrations, while the Cav1.2 channel (L-type calcium channel) is not inhibited at all in this concentration range. The present invention therefore also relates to the peptides according to the invention for the inhibition of N-type calcium channels (Ca v 2.2).

[0065] As explained above, the selective inhibition of N-type calcium channels (Ca v 2.2) without affecting the Cavl.2 channel (L-type calcium channel), a significant advantage of the peptides of the invention. Furthermore, the peptides of the invention have been shown to be particularly stable against degradation by proteases, thus offering the possibility of oral administration.

[0066] The present invention further relates to the peptides of the invention for use in the treatment of neuropathic pain. The invention is further illustrated below in non-limiting examples.

[0067] Examples

[0068] Example 1 - Effect of RD2C on the L-type calcium channel (Ca v 1.2)

[0069] In the following, the efficacy of the complete D-enantiomeric peptide RD2C (psvftrnrrrrr-NHz) was investigated in vitro using electrophysiological recordings. These studies aimed at inhibiting currents passing through the L-type calcium channel (Ca v 1.2) are conveyed.

[0070] Materials and Methods: For electrophysiological recordings, tsA201 (Sigma-Aldrich) mammalian cells were transiently treated with YFP-fused rabbit (r) Ca v 1.2 (UniProtKB: P15381) subunit and the Cavß2e (UniProtKB: Q8VGC3-4) auxiliary subunit were cotransfected. tsA201 cells were transfected with Lipofectamine 2000 (Thermo Fisher Scientific) and analyzed 24 to 36 hours later. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Sigma-Aldrich) at 37°C and 5% CO2.

[0071] Electrophysiology: Electrical currents were recorded using the whole-cell patch clamp technique with an EPC-10 amplifier and PatchMaster software (HEKA, Elektronik). Barium was used as the charge carrier. Borosilicate pipettes with resistances of 1.8–4.5 MΩ were pulled on a Sutter P-1000 puller (Harvard Apparatus) and fire-polished with a Narishige MF-830 Microforge. The external test solution contained (in mM): 140 TEA-MeSCh, 10 BaCl, 10 HEPES, pH adjusted to 7.3 with TEA-OH, while the internal solution contained 135 Cs-MeSO3, 10 EGTA, 5 CsCl2, 14 phosphocreatine, 1 MgCl2, 4 MgATP, 0.4 Na2GTP, and 10 HEPES; the pH was adjusted to 7.3 with CsOH. Data analysis was performed using a combination of FitMaster (HEKA), Origin (OriginLab), and Excel (Microsoft) software. All data are expressed as mean ± standard error. Currents were determined using a P / 4 protocol.

[0072] To test the pharmacological effect of the individual substances, the cells were continuously perfused with the external test solution with and without the test substance. A fresh 1 mM stock solution of all substances was prepared in H2O and diluted to 6 pM in the external test solution before use. A control experiment was conducted with nifedipine (10 pM), a Ca v 1,2-calcium channel blocker. Whole-cell currents were elicited with a depolarizing test pulse at 10 mV, which elicited the maximum inward current at a holding potential of -90 mV every 10 seconds.

[0073] Ca v 1.2 channels are expressed at high density in the heart and in the dendrites of neurons. Perfusion of the cells with the current containing 6 pM RD2C, just as with the external solution alone, did not result in a significant reduction in Ca v1.2-mediated current (Figure 1, A). In contrast, 10 pM nifedipine rapidly inhibited the L-type mediated currents, confirming that they were mediated by this channel (Figure 1, B). A summary of the results can be found in Tables 1 and 2. From the summary of the results, it can be concluded that RD2C inhibits the calcium channel Ca v 1.2 does not significantly inhibit.

[0074] Table 1: [%] of the peak current reduction caused by the application of 6 pM RD2C to Cavl.2 channels and the current restored after application of the external solution. Table 2: [%] of the peak current reduction caused by the application of 10 |jM nifedipine to Cavl.2 channels and the current restored after application of the external solution.

[0075] Example 2 - Effect of RD2C and RD2 on the N-type calcium channel (Ca v 2.2)

[0076] Subsequently, the efficacy of the fully D-enantiomeric peptides RD2C (psvftrnrrrrr-NH2) and RD2 (hptIhthnrrrrr-NH2) was investigated in vitro using electrophysiological recordings. These studies aimed at inhibiting currents mediated by the N-type calcium channel Cav2.2.

[0077] Materials and Methods: For electrophysiological recordings, HEK293 cells expressing human OIB-I (Ca v 2.2: M94172.1) splice variant and the auxiliary subunits o2bö-l (M76559.1) and ß 2a (NM_000725). The cell line was cultured at 37 °C and 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin, streptomycin, and appropriate selection antibiotics (geneticin 50 pg / ml, blasticidin 10 pg / ml, and zeocin 40 pg / ml).

[0078] Electrophysiology: Electrical currents were recorded using the whole-cell patch clamp technique with an EPC-10 amplifier and PatchMaster software (HEKA, Elektronik). Barium was used as the charge carrier. Borosilicate pipettes with resistances of 1.8–4.5 MΩ were pulled on a Sutter P1000 puller (Harvard Apparatus) and fire-polished with a Narishige MF-830 Microforge. The external test solution contained (in mM): 140 TEA-MeSCh, 10 BaCl, 10 HEPES, pH adjusted to 7.3 with TEA-OH, while the internal solution contained 135 Cs-MeSO3, 10 EGTA, 5 CsCl2, 14 phosphocreatine, 1 MgCl2, 4 MgATP, 0.4 Na2GTP, and 10 HEPES; the pH was adjusted to 7.3 with CsOH. Data analysis was performed using a combination of FitMaster (HEKA), Origin (OriginLab), and Excel (Microsoft) software. All data are expressed as mean ± standard error. Currents were determined using a P / 4 protocol.

[0079] To test the pharmacological effect of the individual substances, the cells were continuously perfused with the external test solution with and without the test substance. Fresh 1 mM stock solutions of all substances were prepared in H2O and diluted to 6 pM in the external test solution before use. A control experiment was performed with co-conotoxin-GVIA (1 pM), a Ca v 2.2 calcium channel blocker. Whole-cell currents were elicited with a depolarizing test pulse at 10 mV and / or 15 mV, with the maximum inward current elicited every 10 seconds at a holding potential of -90 mV.

[0080] Ca v2.2 channels are expressed at high density in primary somatosensory afferent neurons in the dorsal horn, particularly in superficial laminae I and II, and also in the ventral horn (Gohil et al., 1994; Yusaf et al., 2001). Perfusion of the cells with the current containing 6 pM RD2C, but not with the external solution alone, resulted in a rapid and significant reduction in Ca v 2.2-mediated current of approximately 37.41% ± 2.14% (Figure 2, B). In contrast, 6 pM RD2 caused an inhibition of 15.34% ± 1.78%. Ca v 2.2 currents were measured after application of 1 pM of Ca v 2.2-blocker co-conotoxin-GVIA, confirming that it is mediated by Ca v 2.2 channels (Figure 2, B). The data showed that the reversal of RD2C and RD2 inhibition was approximately 84.57% ± 3.74% and 87.09% ± 2.20%, respectively (Figure 2, A and C). In contrast, co-conotoxin-GVIA blocked Ca v2.2- channels irreversible (Figure 2, A and C).

[0081] A summary of the results can be found in Tables 3 to 5. From these tables it can be concluded that RD2C reduces Ca v 2.2 calcium channel in a reversible manner more strongly than RD2.

[0082] Table 3: [%] of Ca absorption by the application of 6 |jM RD2C v 2.2 channels and the current restored after application of the external solution.

[0083] Table 4: [%] of Ca absorption by application of 6pM RD2 v 2.2 channels caused peak current reduction and the current restored after application of the external solution.

[0084] Table 5: [%] of Ca ions induced by the application of 1 pM co-conotoxin-GVIA v 2.2- Channels caused peak current reduction and the current restored after application of the external solution.

[0085] Example 3 - Stability Studies The following experiment aims to test the stability of RD2 and RD2C. For this purpose, the peptides to be investigated were incubated in media simulating the gastrointestinal tract, blood, and liver.

[0086] Materials and Methods: Media preparation was performed as described in Elfgen et al. (2017 and 2019)[1, 2]. Simulated gastric and intestinal fluid (SGF & SIF) were prepared according to European Pharmacopoeia 7.0. Plasma samples were obtained from human blood from a volunteer donor. Pooled human liver microsomes were purchased from Sekisui XenoTech (Kansas City, USA; H1000). Liver microsomes were diluted in an NADPH regeneration system (NRS).

[0087] Stability studies: To test stability, 150 pM RD2 or RD2C were incubated three times in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), human plasma, or human liver microsomes at 37 °C with gentle shaking for various periods of time. To prevent microbial contamination during long-term plasma incubations, 0.1% sodium azide was added to the solutions. Long-term microsomal activity was examined by incubating microsomes without peptide after 8 and 24 h. Peptides were extracted by precipitating the proteins with trichloroacetic acid (TCA) followed by centrifugation, as described in Elfgen et al. (2017 and 2019)[1, 2]. Peptides extracted immediately from the media and precipitated media without peptides served as controls.

[0088] The samples were analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). The RP-HPLC system (Agilent Technologies, Santa Clara, USA; 1200 series) consisted of a manual injector, a quaternary pump, a thermostatted column compartment, and a variable-wavelength detector. Chromatography was performed using a C18 column (Agilent Technologies, Santa Clara, USA; ZORBAX 300SB-C18 5 pm, 4.6 x 250 mm) at 25 °C and 214 nm with a flow rate of 1 mL / min. The sample injection volume was 20 μl. The chromatograms were recorded and analyzed using Agilent ChemStation software (G2175BA; B03.01). Mobile phases were acetonitrile (A) and water (B), each supplemented with 0.15% trifluoroacetic acid (TFA) (v / v). The samples were measured isocratically at 10% solvent A for 30 min.As shown in Figure 3, RD2 and RD2C remained stable for 24 hours in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). RD2 and RD2C were metabolized very similarly in human plasma and human liver microsomes.

[0089] This suggests that RD2 and RD2C are stable in all media tested.

[0090] Example 4 - Testing cell viability using MTT

[0091] Materials and Methods: Cell viability was tested using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay to investigate the potential cytotoxicity of D-peptides on SH-SY5Y (human neuroblastoma cell line) cells.

[0092] SH-SY5Y cells (DSMZ, Germany) were cultured in DMEM medium supplemented with 20% fetal calf serum (Sigma-Aldrich, USA) at 75 cm 3large tissue culture flasks (VWR, 10062-860). The cells grew in a humidified incubator with 5% CO2 at 37°C. The medium was changed every two days, and the cells were passaged every three to five days, depending on confluence.

[0093] The MTT assay was performed according to the manufacturer's protocol (Cell Proliferation Kit I; Roche, Switzerland). SHSY-5Y cells were cultured in 96-well F, surface-treated tissue culture plates (VWR, 10062-900) at a density of 1 x 10 4 Cells were seeded in a volume of 100 μl per well and incubated for 24 hours. The cells were then treated with 1pM, 6 μM, or 12 μM D-peptides in the medium for 24 hours. The arithmetic mean of all measurements (determined in quadruplicate) per assay was calculated. The results are expressed as a percentage of MTT reduction, assuming that the absorbance of the control cells is 100%.

[0094] Figure 4 shows that the tested D-peptides have no negative effects on the cell viability of SH-SY5Y cells.

[0095] Example 5 - Testing the Effect of RD2C on Mechanical Allodynia Measured with von Frey Filaments Oxaliplatin, a platinum-based chemotherapeutic agent, is widely used to treat various types of cancer. However, the clinical value of oxaliplatin is limited by acute and chronic forms of peripheral neuropathy, including mechanical hyperalgesia, which occur as side effects in both humans (Pasetto et al., 2006) and rodents (Ling et al., 2007). There is currently no treatment for this neuropathy. In rodents, painful neuropathy is induced by administration of oxaliplatin (single ip injection of 10 mg / kg). Within one week of treatment, the animals develop long-lasting mechanical allodynia (measured with von Frey filaments).

[0096] Method:

[0097] Adult male C57BL / 6J mice, aged 35–41 days and weighing 20–25 g at baseline, were used in the study. The mice were housed in groups of 3–5 animals in ventilated cages under controlled environmental conditions, including a constant temperature (21 ± 1 °C) and a 12:12 light / dark cycle (usually 7:00 a.m. to 7:00 p.m.). Standard laboratory chow and water were provided ad libitum. To induce chemotherapy-induced neuropathy (CIN), the animals received a single intraperitoneal injection of oxaliplatin (10 mg / kg in 5% dextrose). Static mechanical (tactile) allodynia is assessed by measuring the withdrawal threshold using calibrated (force; g) von Frey monofilaments (Touch-Test Sensory Evaluator; Scientific Marketing Associates) on the plantar surface of the left hindpaw.

[0098] Figure 5 shows that on day 7, treatment with RD2C reversed mechanical allodynia after both 1 and 2 hours. Furthermore, in contrast to the vehicle-treated animals, the RD2C-treated animals showed no significant differences from baseline on days 14 and 21.

[0099] Example 6 - Testing the Effect of RD2C on Acute Pain The tail-flick test has been used in rodents since the 1940s to demonstrate the effects of anesthetics (D'Amour and Smith, 1941). It is a simple, reliable, sensitive, quantifiable, and validated measure of reflex pain that correlates highly with analgesic use in humans (Dubner, 1994; Grumbach, 1966; King et al., 1993). A radiant heat source is commonly used for the tail-flick test in rats (Cecchi et al., 2008). The rat's pain sensitivity is indicated when the animal moves its tail away from the radiant heat source. Although the tail-flick response depends on a spinal reflex, it is under the control of supraspinal structures, so it can be used in studies evaluating endogenous analgesic mechanisms such as endogenous opioids and catecholamines as well as exogenously administered analgesics.

[0100] Method: Male Sprague-Dawley rats (SD, Envigo UK) were housed in groups of 5 in cages. The animals were maintained on a 12-hour light / dark cycle and had free access to food and water. The animals were placed in individual plantar test boxes on a glass table. The radiant heat source was placed beneath the glass table, and once the animals were settled, the heat source was moved to a position where the heat beam impinged on the marked area 3 cm from the rat's tail tip. The latency time until the tail was withdrawn from the heat source was recorded. A cut-off time of 15 seconds was maintained throughout the study. Baseline 2 was conducted on the day of drug application, which was spread over two study days.On the day of drug administration (RD2C and positive control buphrenorphine), the animals were weighed, the 3-cm marker was refreshed, and the latencies for the baseline measurement were determined in the same way as for baseline measurement 1. The mean latency from both baseline measurements was used to assign the animals to the treatment groups on the day of the study. The animals were dosed and placed on the test tables (2-5 animals per table) 5 minutes before the start of the test. After the test, the animals were removed from the tables and returned to their home cage. The tail flick latencies were determined 0.5, 1, 2, and 4 hours after administration.

[0101] Figure 6 shows that RD2C has no effect on the latency period and thus on acute pain.

[0102] [1] Gohil, K., Bell, J. R., Ramachandran, J., & Miljanich, G. P. (1994), Neuroanatomical distribution of receptors for a novel voltage-sensitive calcium- channel antagonist, SNX-230 (omega-conopeptide MVIIC), Brain research, 653(1- 2), S. 258-266.

[0103] [2] Yusaf, S. P., Goodman, J., Pinnock, R. D., Dixon, A. K., & Lee, K. (2001), Expression of voltage-gated calcium channel subunits in rat dorsal root ganglion neurons, Neuroscience letters, 311(2), S.137-141.

[0104] [3] Elfgen, A., et al., Metabolic resistance of the D-peptide RD2 developed for direct elimination of amyloid-beta oligomers, Sei Rep, 2019, 9(1), S. 5715.

[0105] [4] Elfgen, A., et al., Surprisingly high stability of the Abeta oligomer eliminating all-d-enantiomeric peptide D3 in media simulating the route of orally administered drugs, Eur J Pharm Sei, 2017, 107, S. 203-207.

[0106] Figurenbeschreibung

[0107] Figure 1: Effect of RD2C on Ca v 1.2-channel

[0108] Figure 1 shows the effect of the peptide RD2C on Ca v 1.2-channel.

[0109] A: Scatter plot of Ca v 1.2-mediated currents during Ringer application and in the presence of RD2C at 6 pM at a holding potential of 90 mV.

[0110] B: Scatter plot of Ca v 1.2-mediated currents, expressed as a percentage in the presence of RD2C at 6 pM and nifedipine at 10 pM. C: Scatter plot of Ca v 1.2-mediated currents restored after washout of RD2C (6 pM) and nifedipine (10 pM) and expressed as percentage.

[0111] D: Comparison of the time course of L-type current during perfusion of RD2C (6 pM) and control nifedipine (10 pM).

[0112] The solid black line represents the exposure time to the test substance. Data are presented as mean ± standard error.

[0113] Figure 2: Comparison of the inhibition of Ca v 2.2 channels through RD2C and RD2

[0114] Figure 2 shows a comparison of the inhibition of Ca v 2.2 channels through RD2C and RD2.

[0115] A: Comparison of the time course of N-type (Ca v 2.2) current, which is inhibited during perfusion by RD2C (6 pM), RD2 (6 pM), and the control co-conotoxin GVIA (1 pM). The solid black line illustrates the exposure time to the test substance.

[0116] B: Evaluation of the inhibitory properties of RD2C (n=6), RD2 (n=5) and the control co-conotoxin-GVIA (Cgtx-GVIA) (n=3) at Ca v 2.2-mediated ion currents.

[0117] C: Restoration of Ca blockade v2.2 channels by RD2C (n = 4), RD2 (n = 5), and the control co-conotoxin GVIA (n = 3). Data are presented as mean ± standard error. Statistically significant level*** P < 0.001 using a one-way ANOVA.

[0118] Figure 3: Stability of RD2 and RD2C

[0119] Figure 3 shows the stability of RD2 and RD2C in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), human plasma, or human liver microsomes. RD2 and RD2C were incubated in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), human plasma, or human liver microsomes, respectively.

[0120] The peak areas of the non-metabolized peptides after different incubation times were normalized to the peak areas of the peptides after direct extraction from the media.

[0121] Figure 4: Cell viability of SH-SY5Y cells

[0122] Figure 4 shows the effect of D-peptides on cell viability of SH-SY5Y cells.

[0123] Cell viability was determined by the MTT change after incubation of SH-SY5Y cells with different concentrations (1 pM, 6 pM or 12 pM) of RD2, RD2B, RD2C or RD2D.

[0124] Figure 5: Effect of RD2C on mechanical allodynia in the chemotherapy-induced neuropathy (CIN) pain model

[0125] Figure 5 shows the effect of the D-peptide RD2C on mechanical allodynia (g), measured with von Frey filaments. Data were analyzed using a two-way ANOVA followed by a Dunnet post hoc analysis. Asterisks (*) indicate statistical significance compared to vehicle, while hashtags (#) denote significance compared to baseline (BL). Data are expressed as mean ± SEM.

[0126] Figure 6: Effect of RD2C on acute pain measured in the tail-flick test

[0127] Figure 6 shows the effect of the D-peptide RD2C on acute pain, measured as latency in the tail-flick test. Data were analyzed using a two-way Kruskal-Wallis ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.001 were significantly different from the vehicle treatment. Data are expressed as mean ± SEM.

Claims

Claims 1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, as well as homologues, fragments and parts thereof.

2. Peptide according to claim 1, characterized in that the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and / or SEQ ID NO: 10 or homologs having an identity of at least 80% thereof.

3. Peptide according to any one of claims 1 or 2, characterized in that it contains 2, 3, 4, 5, 6, 7, 8, 9 or more copies of the sequences with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO:

5.

4. Peptide according to any one of the preceding claims, characterized in that the peptide consists essentially of D-amino acids.

5. Peptide according to any one of the preceding claims, characterized in that the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and / or SEQ ID NO:

10.

6. Peptide according to any one of the preceding claims, characterized in that the peptide is linked to another substance.

7. Peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5 are covalently or non-covalently linked to one another.

8. Peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5 without a linker, i.e. directly linked to each other, or linked to each other with a linker group.

9. Peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5 are linearly linked to one another.

10. Peptide according to any one of the preceding claims for inhibiting N-type calcium channels (Ca v 2.2).

11. A peptide according to any one of the preceding claims for use in the treatment of neuropathic pain.

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