Methods of treating chronic and neuropathic pain mediated by N-type neuronal calcium channels using D-enantiomeric peptides

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. A method of treating pain mediated by 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 both of peptides “RD2” (SEQ ID NO: 1) and “D3” (SEQ ID NO: 2) and/or a polymer comprising one or both of RD2 (SEQ ID NO: 1) and D3 (SEQ ID NO: 2), thereby reducing the pain mediated by NCCs in the subject when compared to no treatment with the composition.

2. The method of claim 1, wherein chronic pain is treated.

3. The method of claim 1, wherein neuropathic pain is treated.

4. The method of claim 1, wherein NCCs the N-type neuronal calcium channels (NCCs) are blocked.

5. The method of claim 1, wherein the peptides are composed essentially of D-enantiomers.

6. The method of claim 1, wherein the composition comprises RD2 (SEQ ID NO: 1) and/or a polymer comprising RD2 (SEQ ID NO: 1).

7. The method of claim 1, wherein the composition comprises D3 (SEQ ID NO: 2) and/or a polymer comprising D3 (SEQ ID NO: 2).

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

9. The method of claim 1, wherein the composition is administered by one or more of intravenous, subcutaneous, intraperitoneal, intranasal or oral administration.

10. The method of claim 1, wherein the composition is administered orally.

11. The method of claim 4, wherein the composition has an IC.sub.50 value of 1 nanomolar to 1 millimolar for N-type NCCs.

12. A method of reducing the release of neurotransmitters associated with pain and mediated by N-type neuronal calcium channels (NCCs) in a subject in need thereof, wherein the method comprises contacting the NCCs in the subject with a composition consisting of or comprising one or both of peptides RD2 (SEQ ID NO: 1) and D3 (SEQ ID NO: 2) and/or a polymer comprising one or both of RD2 (SEQ ID NO: 1) and D3 (SEQ ID NO: 2) for use as an analgesic in the subject in need thereof, thereby reducing the release of neurotransmitters associated with pain and mediated by NCCs in the subject when compared to no treatment with the composition.

13. The method of claim 12, wherein a calcium influx through the NCC in the subject is reduced compared to a control subject with no treatment with the composition.

14. The method of claim 13, wherein the function of L-type NCCs in the subject is not modified when compared to a control subject with no treatment with the composition.

15. A method of inhibiting an N-type NCC in a subject in need thereof, wherein the method comprises contacting the N-type NCC in the subject in need thereof with a composition consisting of or comprising one or both of peptides RD2 (SEQ ID NO: 1) and D3 (SEQ ID NO: 2) and/or a polymer comprising one or both of RD2 (SEQ ID NO: 1) and D3 (SEQ ID NO: 2), thereby inhibiting the N-type NCC in the subject when compared to no treatment with the composition.

16. The method of claim 15, wherein the function of L-type NCCs in the subject is not modified when compared to a control subject with no treatment with the composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the accompanying drawings,

(2) FIG. 1A is a representative graph of current intensity mediated by a CaV2.2 channel as set forth in Example 1 below;

(3) FIG. 1B is a graph representing a course over time of inhibition, taken in a representative cell, as set forth in Example 1 below;

(4) FIG. 1C represents the average ratio of current intensity to voltage of cells that express CaV2.2 in the presence and absence of RD2 as set forth in Example 1 below;

(5) FIG. 1D shows the fraction of activated channels vs. the voltage plot for the same cells as in FIG. 1C as set forth in Example 1 below;

(6) FIG. 2A is a representative image of current intensity mediated by a CaV1.2 channel as set forth in Example 1 below;

(7) FIG. 2B represents the change in current intensity on perfusion with several substances as set forth in Example 1 below;

(8) FIG. 2C represents the average ratio of current intensity to voltage of cells that express CaV1.2 in the presence and absence of RD2 as set forth in Example 1 below;

(9) FIG. 2D shows the fraction of activated channels vs. the voltage plot for the same cells as in FIG. 2C as set forth in Example 1 below;

(10) FIG. 3 represents the capacity of RD2 to block ion currents mediated via the CaV2.2 and CaV1.2 channels as set forth in Example 1 below;

(11) FIG. 4A is a representative image of current intensity mediated by a CaV2.2 channel as set forth in Example 2 below;

(12) FIG. 4B shows the inhibition induced by D3 after perfusion with Ringer's solution as set forth in Example 2 below;

(13) FIG. 4C shows a current to voltage plot for D3 with respect to CaV2.2 mediated ion currents as set forth in Example 2 below;

(14) FIG. 4D shows the fraction of activated channels vs. the voltage plot for the same cells as in FIG. 4C as set forth in Example 2 below;

(15) FIG. 5 represents the capacity of D3 to block ion currents mediated via the CaV2.2 channel a set forth in Example 2 below;

(16) FIG. 6 represents the capacity of cD3 and cRD2 to block ion currents mediated via the CaV1.2 and CaV2.2 channels a set forth in Example 3 below;

(17) FIG. 7A is a representative image of current intensity mediated by a CaV2.2 channel before and after exposure to cD3r as set forth in Example 3 below;

(18) FIG. 7B shows the average current intensity to voltage ratio of CaV2.2 expressing cells with and without presence of cD3r as set forth in Example 3 below;

(19) FIG. 7C shows representative ion current discharges mediated by the CaV2.1 channel before and after exposure to cD3r as set forth in Example 3 below;

(20) FIG. 7D shows the average current density to voltage ratio with and without presence of cD3r and the activation curve of CaV2.1 expressing cells as set forth in Example 3 below;

(21) FIG. 8A is a representative image of current discharges mediated by a CaV2.2 channel before and after exposure to cRD2r as set forth in Example 3 below; expressing cells with and without presence of cRD2r and fraction of activated channels versus voltage as set forth in Example 3 below;

(22) FIG. 8C shows representative ion current discharges mediated by the CaV2.1 channel before and after exposure to cRD2r and the course over time of the blockade in a representative cell during successive voltage pulses as set forth in Example 3 below; and

(23) FIG. 8D shows the average current density to voltage ratio with and without presence of cRD2r and the activation curve of CaV2.1 expressing cells as set forth in Example 3 below.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(24) The further examples can be carried out with any of the above-described peptides.

EXAMPLES

(25) 1. RD2 (SEQ ID NO: 1) (C-terminal amidated)

(26) A. Protein Constructs:

(27) 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).

(28) B: Cell Transfection:

(29) 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.

(30) C. Electrophysiology:

(31) 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 MΩ 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 values±SEM. Ion currents were corrected using the P/4 protocol (leak subtraction).

(32) In order to investigate the pharmacological effect of RD2 (SEQ ID NO: 1), 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 (SEQ ID NO: 1) 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 μM) and omega-conotoxin (1 nM).

(33) D. Results:

(34) Effect of RD2 (SEQ ID NO: 1) on CaV2.2-mediated ion currents:

(35) 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 (SEQ ID NO: 1) (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 (SEQ ID NO: 1); 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 (SEQ ID NO: 1) (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 (SEQ ID NO:

(36) 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).

(37) Effect of RD2 (SEQ ID NO: 1) on CaV1.2-mediated ion currents:

(38) 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 (SEQ ID NO: 1) (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 (SEQ ID NO: 1). FIG. 2B: No change in current intensity on perfusion with 150 nM of RD2 (SEQ ID NO: 1)). In contrast, perfusion with 10 μM 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 (SEQ ID NO: 1) (FIGS. 2C and 2D).

(39) E: Conclusion

(40) RD2 (SEQ ID NO: 1) 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 (SEQ ID NO: 1) blocks the CaV2.2 channel, while the CaV1.2 channel is insensitive to RD2 (SEQ ID NO: 1) in the tested concentration (FIG. 3).

(41) 2. D3 (SEQ ID NO: 2) (C-terminal amidated)

(42) A. Protein Constructs:

(43) 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.

(44) B: Cell Transfection:

(45) 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.

(46) C. Electrophysiology:

(47) 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 values±SEM. Ion currents were corrected using the P/4 protocol (leak subtraction).

(48) In order to investigate the pharmacological effect of D3 (SEQ ID NO: 2), 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 (SEQ ID NO: 2) 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).

(49) D. Results:

(50) Effect of D3 (SEQ ID NO: 2) on CaV2.2-mediated ion currents:

(51) 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 (SEQ ID NO: 2) (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 (SEQ ID NO: 2); 4B: The inhibition induced by D3 (SEQ ID NO: 2) 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 (SEQ ID NO: 2) 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).

(52) E: Conclusion

(53) D3 (SEQ ID NO: 2) blocks the CaV2.2 channel (FIG. 5).

(54) 3. cD3 (cyclized D3 (SEQ ID NO: 2)) and cRD2 (cyclized RD2 (SEQ ID NO: 1))

(55) The experiments were carried out analogously to examples 1 and 2.

(56) 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. **p≤0.01

(57) Effect of cD3r on the CaV2.2 and the CaV1.2 Channel (FIG. 7).

(58) 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).

(59) Effect of cRD2r on the CaV2.2 and the CaV1.2 Channel (FIG. 8).

(60) 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).