D-ENANTIOMERIC PEPTIDES FOR THE THERAPY OF CHRONIC AND NEUROPATHIC PAIN

Abstract

The present invention relates to a composition consisting of or containing peptides selected from the group consisting of or containing RD2, D3, homologs having at least 50% identity and derivatives of RD2 or D3 and also polymers containing or consisting of RD2/D3 homologs having at least 50% identity and derivatives of RD2 and under D3 for use as an analgesic, for use in pain therapy, for use in the treatment of chronic and/or neuropathic pain and/or for inhibiting N-type neuronal calcium channels (NCCs).

Claims

1.-15. (canceled)

16. A method of treating pain and/or of inhibiting N-type neuronal calcium channels (NCCs), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition consisting of or comprising one or more peptides selected from RD2, D3, homologs having at least 50% identity and derivatives of RD2 or D3, and also polymers comprising or consisting of RD2, D3, homologs having at least 50% identity and derivatives of RD2 and D3.

17. The method of claim 16, wherein chronic pain is treated.

18. The method of claim 16, wherein neuropathic pain is treated.

19. The method of claim 16, wherein NCCs are blocked.

20. The method of claim 16, wherein the peptides are composed essentially of D-enantiomers.

21. The method of claim 16, wherein the one or more peptides comprise one or more peptides selected from RD2, homologs having at least 50% identity and derivatives of RD2, and also polymers comprising or consisting of RD2, homologs having at least 50% identity and derivatives of RD2.

22. The method of claim 16, wherein the one or more peptides comprise one or more peptides selected from D3, homologs having at least 50% identity and derivatives of D3, and also polymers comprising or consisting of D3, homologs having at least 50% identity and derivatives of D3.

23. The method of claim 16, wherein the peptides are administered in a dose of from 1 g to 1 g per kilo of body weight.

24. The method of claim 16, wherein the composition is administered by one or more of intravenous, subcutaneous, intraperitoneal, intranasal or oral administration.

25. The method of claim 16, wherein the composition is administered orally.

26. The method of claim 19, wherein the composition has an IC.sub.50 value of 1 nanomolar to 1 millimolar for N-type NCCs.

27. The method of claim 16, wherein a derivative of RD2 or D3 is administered.

28. The method of claim 27, wherein the derivative is a cyclized peptide.

29. The method of claim 27, wherein the derivative is an amidated peptide.

30. The method of claim 16, wherein the peptide is linked to one or more of amino acids, linkers, spacers, functional groups and/or further substances.

31. A method of reducing the release of neurotransmitters compared to a control in a subject in need thereof, wherein the method comprises contacting a composition consisting of or comprising peptides selected from RD2, D3, homologs having at least 50% identity and derivatives of RD2 or D3, and also polymers comprising RD2, D3, homologs having at least 50% identity and derivatives of RD2 and/or D3 for use as an analgesic in the subject in need thereof with an N-type NCC.

32. The method of claim 31, wherein a calcium influx through an NCC is reduced compared to a control.

33. The method of claim 32, wherein a function of L-type NCCs is not modified with respect to a control.

34. A method of inhibiting an N-type NCC compared to a control, wherein the method comprises contacting the N-type NCC in a subject in need thereof with a composition consisting of or comprising peptides selected from RD2, D3, homologs having at least 50% identity and derivatives of RD2 or D3, and also polymers containing RD2, D3, homologs having at least 50% identity and derivatives of RD2 and/or D3.

35. The method of claim 34, wherein a function of L-type NCCs is not modified with respect to a control.

Description

EXAMPLES

[0081] 1. RD2 (C-Terminal Amidated)

[0082] A. Protein Constructs:

[0083] The coding regions of the two different alpha1 pore-forming units (CaValpha1) of the voltage-dependent calcium channel were fused in frame (C-terminal fusion) with the fluorescent proteins as follows: the unit of rabbit CaV1.2 (UniProtKB: P15381) was fused with YFP (CaV1.2-YFP), while the human unit CaV2.2 (UniProtKB: Q00975-1) was fused with GFP (CaV2.2-GFP). The beta-subunit CaVbeta2e (UniProtKB: Q8VGC3-2) was linked to mRFP (CaVbeta2e-mRFP), and CaVbeta4 (UniProtKB: 000305.2) was linked to mCherry (CaVbeta4-mCherry).

[0084] B: Cell Transfection:

[0085] As the normal function and surface expression of the CaValpha1 subunit requires association with the CaVbeta subunit, tsA201 cells were transiently co-transfected with either CaV1.2-YFP and CaVbeta2e-mRFP or CaV2.2-GFP and CaVbeta4-mCherry. The transfection was carried out using Lipofectamine 2000 (Invitrogen), and the successfully transfected cells were identified by means of fluorescent signals. Electrophysiological discharges were carried out 24-48 hours after transfection.

[0086] C. Electrophysiology:

[0087] Ion currents were measured using the whole cell patch clamp technique with an EPC-10 amplifier with implemented PatchMaster software (HEKA Elektronik). Barium was used as a carrier. Borosilicate glass pipettes with resistance values of 0.9-2 M were pulled on a Sutter P-1000 puller (Harvard Apparatus), and their tips were subjected to surface heat-polishing using a Narishige MF-830 microforge. External measuring solution used: 140 mM TEA-MeSO.sub.3, 10 mM BaCl.sub.2, and 10 mM HEPES (pH 7.3); internal measuring solution used: 135 mM Cs-MeSO.sub.3, 10 mM EGTA, 5 mM CsCl.sub.2, 1 mM MgCl.sub.2, 4 mM MgATP, 0.4 mM Na2GTP and 10 mM HEPES (pH 7.3). Data analysis was carried out using a combination of the software FitMaster (HEKA), Origin (OriginLab) and Excel (Microsoft). All data are shown as mean valuesSEM. Ion currents were corrected using the P/4 protocol (leak subtraction).

[0088] In order to investigate the pharmacological effect of RD2, cells were detached and transferred to a perfusion flow that either contained or did not contain the test substance. The observations were conducted under constant perfusion in order to ensure a constant concentration of the test substance. RD2 was dissolved in DMSO with a final concentration of 1 mM and dissolved in the external measuring solution to 150 nM shortly before use. Control experiments were conducted with the known CaV1.2 and CaV2.2 calcium channel blockers nimodipine (10 M) and omega-conotoxin (1 nM).

[0089] D. Results:

[0090] Effect of RD2 on CaV2.2-Mediated Ion Currents:

[0091] CaV2.2 is localized in the presynaptic nerve endings and mediates neurotransmission in central synapses. CaV2.2-mediated ion currents were taken up by tsA201 cells that express CaV2.2/CaVbeta4. Perfusion of the cells with an external measuring solution comprising RD2 (150 nM), but not with the external measuring solution alone, resulted in a significant reduction in the ion current (FIG. 1A, B; where A: Representative graph of current intensity mediated by a CaV 2.2 channel, produced during a pulse of 40 MS, based on a holding potential of 90 mV to +20 mV before and after contact with composition comprising 150 mM of RD2; B: Course over time of inhibition, taken in a representative cell, during successive pulses of 90 mV to +20 mV, every five seconds). The current-to-voltage plot (I-V) showed a reduction in the ion current at all tested voltages of up to 55% (FIG. 1C: Average ratio of current intensity to voltage of cells that express CaV 2.2 in the presence and absence of RD2 (n=7)). As a blockade of the ion current was not accompanied by a change in voltage-dependent activation of the channel, a pore-blocking mechanism of RD2 is assumed (FIG. 1D: Fraction of activated channels vs. the voltage plot (activation curve) for the same cells as in FIG. C.; as a control; there is therefore no change in the function or mechanism of the channel, as the curves are on top of each other and not displaced). Ion currents are interrupted after administration of 1 nM of the CaV2.2 blocker omega-conotoxin, which confirms that they were mediated via the CaV2.2 channel. (FIG. 1A insert).

[0092] Effect of RD2 on CaV1.2-Mediated Ion Currents:

[0093] CaV1.2 is the L-type calcium channel primarily expressed in the heart and responsible for coupling of the electrical activation of the cardiomyocytes with the myofilament contraction. The ion currents were taken up by CaV1.2 and CaVbeta2e co-expressing cells and remained virtually unchanged after exposure to 150 nM of RD2 (FIG. 2A: Representative image of current intensity mediated by a CaV 1.2 channel, produced during a pulse of 40 ms, based on a holding potential of 90 mV to +10 mV before and after contact with a composition comprising 150 mM of RD2. FIG. 2B: No change in current intensity on perfusion with 150 nM of RD2). In contrast, perfusion with 10 M of nimodipine, a known blocker of the CaV1.2 channel, led to almost complete blockade of the ion current. The voltage dependency of activation of the CaV1.2 channel was therefore not affected by the presence of RD2 (FIGS. 2C and 2D).

[0094] E: Conclusion

[0095] RD2 was tested at a concentration of 150 nM for its capacity to block ion currents mediated via the CaV2.2 and CaV1.2 channel. The tests were conducted in CaV2.2 and CaV1.2 channel-expressing tsA201 cells using the whole cell patch clamp technique. RD2 blocks the CaV2.2 channel, while the CaV1.2 channel is insensitive to RD2 in the tested concentration (FIG. 3).

[0096] 2. D3 (C-Terminal Amidated)

[0097] A. Protein Constructs:

[0098] The coding region of the human alpha1 pore-forming units (CaValpha1) of the voltage-dependent calcium channel CaV2.2 (UniProtKB: Q00975-1) was fused with GFP to form CaV2.2-GFP. The beta-subunit CaVbeta4 (UniProtKB: 000305.2) was fused with mCherry to form CaVbeta4-mCherry.

[0099] B: Cell Transfection:

[0100] As the normal function and surface expression of the CaValpha1 subunit requires association with the CaVbeta subunit, tsA201 cells were transiently co-transfected with CaV2.2-GFP and CaVbeta4-mCherry. The transfection was carried out using Lipofectamine 2000 (Invitrogen), and the successfully transfected cells were identified by means of fluorescent signals. Electrophysiological discharges were carried out 24-48 hours after transfection.

[0101] C. Electrophysiology:

[0102] Ion currents were measured using the whole cell patch clamp technique with an EPC-10 amplifier with implemented PatchMaster software (HEKA Elektronik). Barium was used as a carrier. Borosilicate glass pipettes with resistance values of 0.9-2 MC were pulled on a Sutter P-1000 puller (Harvard Apparatus), and their tips were subjected to surface heat-polishing using a Narishige MF-830 microforge. External measuring solution used: 140 mM TEA-MeSO.sub.3, 10 mM BaCl.sub.2, and 10 mM HEPES (pH 7.3); internal measuring solution used: 135 mM Cs-MeSO.sub.3, 10 mM EGTA, 5 mM CsCl.sub.2, 1 mM MgCl.sub.2, 4 mM MgATP, 0.4 mM Na2GTP and 10 mM HEPES (pH 7.3). Data analysis was carried out using a combination of the software FitMaster (HEKA), Origin (OriginLab) and Excel (Microsoft). All data are shown as mean valuesSEM. Ion currents were corrected using the P/4 protocol (leak subtraction).

[0103] In order to investigate the pharmacological effect of D3, cells were detached and transferred to a perfusion flow that either contained or did not contain the test substance. The observations were conducted under constant perfusion in order to ensure a constant concentration of the test substance. D3 was dissolved in DMSO with a final concentration of 1 mM and dissolved in the external measuring solution to 150 nM shortly before use. Control experiments were conducted with the known CaV2.2 calcium channel blocker omega-conotoxin (1 nM).

[0104] D. Results:

[0105] Effect of D3 on CaV2.2-Mediated Ion Currents:

[0106] CaV2.2-mediated ion currents were taken up by tsA201 cells that express CaV2.2/CaVbeta4. Perfusion of the cells with an external measuring solution comprising D3 (150 nM), but not with the external measuring solution alone, resulted in a significant reduction in the ion current (FIG. 4A, B; where A: Representative image of current intensity mediated by a CaV 2.2 channel produced during a pulse of 40 ms, based on a holding potential of 90 mV to +20 mV before and after contact with a composition comprising 150 mM of D3; 4B: The inhibition induced by D3 is reversible after reperfusion with an external Ringer's solution. The current intensity was recorded during successive pulses of 90 mV +20 mV, every 5 sec.). The current-to-voltage plot (I-V) showed a reduction in the ion current at all tested voltages of up to 54% (FIG. 4C). As a blockade of the ion current was not accompanied by a change in voltage-dependent activation of the channel, a pore-blocking mechanism of D3 is assumed (FIG. 4D). Ion currents are interrupted after administration of 1 nM of the CaV2.2 blocker omega-conotoxin, which confirms that they were mediated via the CaV2.2 channel (FIG. 4A, insert).

[0107] E: Conclusion

[0108] D3 Blocks the CaV2.2 Channel (FIG. 5).

[0109] 3. cD3 (Cyclized D3) and cRD2 (Cyclized RD2)

[0110] The experiments were carried out analogously to examples 1 and 2.

[0111] Summary of the effects of cD3r and cRD2r on the Cav2.2 and Cav1.2 channel (FIG. 6). The two bar charts summarize the average ion currents taken from the current intensity-voltage diagrams at +20 mV for the Cav2.2 and +10 mV for the Cav1.2. **p0.01

[0112] Effect of cD3r on the CaV2.2 and the CaV1.2 Channel (FIG. 7).

[0113] Representative ion current discharges mediated by the CaV2.2 channel were triggered during a 40 ms pulse of 90 mV to +20 mV before and after exposure to 150 nM of cD3r (7A, left diagram). The right diagram 7A shows the course over time of the blockade in a representative cell during successive pulses of 90 mV to +20 m every five seconds. 7B: Average current intensity to voltage ratio of CaV2.2 expressing cells with and without the presence of cD3r (n=8) and fraction of activated channels versus voltage (activation curve). 7C: Representative ion current discharges mediated by the CaV2.1 channel were triggered during a 40 ms pulse of 90 mV to +20 mV before and after exposure to 150 nM of cD3r (7C, left diagram). The right diagram 7C shows the course over time of the blockade in a representative cell during successive pulses of 90 mV to +20 m every five seconds. 7D: Average current intensity to voltage ratio with and without the presence of cD3r (n=8) and activation curve of CaV2.1 expressing cells (n=7).

[0114] Effect of cRD2r on the CaV2.2 and the CaV1.2 Channel (FIG. 8).

[0115] 8A: Representative ion current discharges mediated by the CaV2.2 channel were triggered during a 40 ms pulse of 90 mV to +20 mV before and after exposure to 150 nM cRD2r (8A, left diagram). The right diagram 8A shows the course over time of the blockade in a representative cell during successive pulses of 90 mV to +20 m every five seconds. 8B: Average current intensity to voltage ratio of CaV2.2 expressing cells with and without the presence of cRD2r (n=7) and fraction of activated channels versus voltage (activation curve). 8C: Representative ion current discharges mediated by the CaV2.1 channel were triggered during a 40 ms pulse of 90 mV to +20 mV before and after exposure to 150 nM of cRD2r (left diagram). The right diagram 8C shows the course over time of the blockade in a representative cell during successive pulses of 90 mV to +20 m every five seconds. 8D: Average current intensity to voltage ratio with and without the presence of cRD2r (n=8) and activation curve of CaV2.1 expressing cells (n=6).