BLT2 AGONISTS FOR THE TREATMENT OF PAIN

Abstract

The present invention pertains to novel analgesics useful for treating pain. BLT2 agonists were found to desensitize TRPV1 mediated signaling in sensory neurons. Thus the invention provides BLT2 agonists as novel pain therapeutics. Additional aspects of the invention pertain to combinations of BLT2 agonists with BLT1 antagonists for treating pain in subjects. Pharmaceutical compositions and kits comprising the new analgesics of the invention are furthermore provided.

Claims

1. A Leukotriene B4 receptor 2 (BLT2) agonist for use in the inhibition of a neurological sensation in a subject, preferably the prevention or treatment of pain, wherein the neurological sensation is mediated by the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1).

2. The BLT2 agonist for use according to claim 1, which is CAY10583.

3. The BLT2 agonist for use according to claim 1, wherein said BLT2 agonist is used in combination with at least one additional compound inhibiting TRPV1-sensitization.

4. The BLT2 agonist for use according to claim 1, wherein said inhibition of a neurological sensation in a subject comprises the administration of the BLT2 agonist to the subject suspected to or experiencing the neurological sensation.

5. The BLT2 agonist according to claim 1, wherein said prevention or treatment of pain further comprises the administration of at least one additional compound inhibiting TRPV1-sensitization to the subject suspected to suffer from pain or suffering from pain.

6. The BLT2 agonist for use according to claim 3, wherein the at least one additional compound inhibiting TRPV1-sensitization is a Leukotriene B4 receptor 1 (BLT1) antagonist or inhibitor.

7. A combination for use in the prevention or treatment of pain, the combination comprising a BLT2 agonist and a BLT1 antagonist or inhibitor.

8. The BLT2 agonist for use according to claim 1, or the combination for use in the prevention or treatment of pain, the combination comprising a BLT2 agonist and a BLT1 antagonist or inhibitor, wherein said TRPV1 mediated sensation is a taste sensation, itching of the skin, a burning sensation, or a pain sensation.

9. The BLT2 agonist for use, or the combination for use according to claim 8, wherein the pain is inflammatory pain, inflammatory hyperalgesia, hyperalgesia, neuropathic pain, migraine, cancer pain, visceral pain, osteoarthritis pain, chronic pain and postsurgical pain.

10. A pharmaceutical composition for use in the prevention or treatment of pain, comprising a BLT2 agonist according to claim 1, or a combination for use in the prevention or treatment of pain, the combination comprising a BLT2 agonist and a BLT1 antagonist or inhibitor, wherein said TRPV1 mediated sensation is a taste sensation, itching of the skin, a burning sensation, or a pain sensation.

11. The pharmaceutical composition for use according to claim 10, further comprising a pharmaceutically acceptable carrier and/or excipient.

12. An in-vitro method for desensitization of TRPV1 in a cell, the method comprising the step of activation of BLT2.

13. The method according to claim 12, wherein the activation of BLT2 in the cell is affected by contacting the cell with an effective amount of a BLT2 agonist.

14. The method according to claim 12, wherein the cell is a neuronal cell, preferably a peripheral neuronal cell.

Description

[0073] The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

[0074] FIG. 1: LTB4 dose dependently sensitizes TRPV1 in DRG neurons. (A) DRG neurons were double stimulated with capsaicin (200 nM, 15 s each) and incubated with either vehicle or LTB4 (100 nM) for two minutes prior to the second capsaicin stimulation. (B) Dose-dependent difference in ratio between the first and the second capsaicin response using the same protocol as described in (A). Data represent the mean±SEM of the following number of neurons (n=20-86); ANOVA Kruskal-Wallis test; Dunn's multiple comparisons test *p<0.05, ****p<0.0001 student's t-test.

[0075] FIG. 2: BLT1 and 2 are co-expressed in peripheral sensory neurons. Representative images of a MELC analysis from cultured DRG neurons 1 day after preparation. Images for BLT1, 2, the lectin IB4 and CGRP are shown in false colors. The white bar represents xx μm. Percentage of IB4- (n=179) or CGRP-positive neurons (n=111) expressing BLT1 or 2.

[0076] FIG. 3: BLT1 mediates TRPV1-sensitization and BLT2 mediates TRPV1-desensitization by LTB4. (A) DRG neurons were double stimulated with capsaicin (200 nM, 15 s each) and incubated with either vehicle or the indicated compounds for two minutes prior to the second capsaicin stimulation. TRPV1 sensitization by LTB4 (100 nM or 1 μM) can be reduced by preincubation with a BLT1 antagonist (U73502, 1 μM) or by a BLT2 agonist (CAY10583, 400 nM) while a BLT2 antagonist (LY2552833, 10 μM) increases TRPV1 sensitization. Data represent mean±SEM 20-105 cells. ANOVA Kruskal-Wallis test; Dunn's multiple comparisons test *p<0.05, **p<0.01, ****p<0.0001 student's t-test; n.s. not significant.

[0077] FIG. 4: BLT2 activation inhibits TRPV1-desensitization by EP4 and Bradykinin. (A) DRG neurons were double stimulated with capsaicin (200 nM, 15 s each) and incubated with either the EP4 agonist ONOae329 (500 nM) or with ONOae329 and CAY10583 (400 nM) together for two minutes prior to the second capsaicin stimulation. (B) Difference in ratio between the first and the second capsaicin response using the same protocol as described in panel A. Data represent the mean±SEM of the following number of neurons: 52 (vehicle), 109 (ONOae329), 106 (ONOae329+CAY10583); One-Way ANOVA, Dunn's multiple comparisons test; **p<0.0096. (C+D) same as panel A+B except that 500 nM bradykinin was used for TRPV1 sensitization. Data represent the mean±SEM of the following number of neurons: 33 (vehicle), 83 (bradykinin), 93 (bradykinin+CAY10583); One-Way ANOVA, Holm-Sidak's multiple comparisons test; *p=0.0126

[0078] FIG. 5: BLT2 activation decreases thermal pain thresholds in mice. CAY10583 (10 μl, 5 μM) or vehicle was administered intraplantarly. Thermal thresholds were determined at the indicated time points. Data are presented as mean±S.E.M. of 6 animals. Two way ANOVA/Bonferroni *P<0.05, **P<0.005, ****P<0.0005.

EXAMPLES

[0079] Materials and Methods

[0080] Animals:

[0081] All animal experiments were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the local Ethics Committees for Animal Research (Darmstadt). For all behavioral experiments the inventors used only 6-12 weeks old male C57BL/6N mice purchased from commercial breeding companies (Charles River, Sulzfeld, Germany, Janvier, Le Geneset-Saint-Isle, FR). To compare mechanical thresholds the inventors used age and sex matched littermates as control.

[0082] Behavioral Tests:

[0083] For the determination of mechanical allodynia or thermal hypersensitivity, mice were kept in test cages on an elevated grid for at least 2 hours to allow accommodation. Baseline measurements were performed using a Hargreaves Apparatus (Ugo Basile, Comerio, VA, Italy) detecting withdrawal latency of the hind paws after mechanical stimulation for determination of thermal thresholds, mice were kept in test cages on a warmed glass plate (32° C.) for at least 2 hours on the first day to allow accommodation. Then, the mid-plantar region of the paws was stimulated with a radiant heat device, consisting of a high intensity projector lamp, until withdrawal occurred. The non-injected and injected paws were measured alternately in intervals of 5-10 min. For all behavioral tests the investigator was blinded for treatment or genotype of the mice. 10 μl of LTB4 (5 μM), CAY10583 (5 μM) or vehicle were injected intraplantarly.

[0084] Primary Dorsal Root Ganglia (DRG) Cultures:

[0085] Murine DRGs were dissected from spinal segments and directly transferred to ice cold HBSS with CaCl.sub.2 and MgCl.sub.2 (Invitrogen, Carsbad, Calif., USA). Next, isolated DRGs were incubated with collagenase/dispase (500 U/ml Collagenase; 2.5 U/ml Dispase) in neurobasal medium containing L-glutamine [2 mM] penicillin (100 U/ml), streptomycin (100 μg/ml), B-27 and gentamicin (50 μg/ml) (all from Invitrogen, Carlsbad, Calif., USA) at 37° C. for 75 min. After removal of the collagenase/dispase-solution, cells were washed twice with neurobasal medium containing 10% FCS and incubated for 10 min with 0.05% trypsin (Invitrogen, Carlsbad, Calif., USA). The washing steps were repeated and the cells were mechanically dissociated with a 1 ml Gilson pipette. Finally, the neurons were plated on poly-l-lysine (Sigma, Deisenhofen, Germany) coated glass cover slips and incubated with neurobasal medium containing L-glutamine [2 mM] penicillin (100 U/ml), streptomycin (100 μg/ml), B-27 and gentamicin (50 μg/ml) over night until assessment by calcium imaging.

[0086] Calcium Imaging Experiments:

[0087] Calcium-Imaging experiments were performed as published previously. Briefly, Leica Calcium-imaging setup was used, consisting of a Leica DMI 4000 b inverted microscope equipped with a DFC360 FX (CCD-) camera, Fura-2 filters and an N-Plan 10×/0.25 Ph1 objective (all from Leica Microsystems, Wetzlar, Germany). Images were taken every 2 seconds and processed with the LAS AF-software. For each experiment the inventors chose an area with large cell numbers and monitored 40-110 cells simultaneously. Calcium-Imaging experiments were performed using DRG-neurons 24-48 hours after preparation. Cells were loaded with 5 μM fura-2-AM-ester and 0.02% Pluronic F-127 (both Biotium, Hayward, Calif. and incubated for 30 to 60 min. at 37° C. Then, the cells were washed with external solution (containing in mM: NaCl [145], CaCl.sub.2 [1.25], MgCl.sub.2 [1], KCl [5], D-glucose [10], HEPES [10]; adjusted to pH 7.3). Baseline measurements were performed in external solution at a flow rate of 1-2 ml/min. Stimulation of the neurons were done using Capsaicin (200 nM). Where indicated a 2 minute pretreatment with LTB.sub.4 (100-1000 nM), BLT2 Agonist CAY10583 (400 nM), BLT2 Antagonist LY2552833 (10 μM), or BLT1 Antagonist U75302 (1 μM) (all Cayman Chem. Ann Arbor, Mich.) was performed.

[0088] Multi Epitope Ligand Cartography (MELC):

[0089] The MELC technology has been described previously (Pierre et al., 2008; Pierre, 2010). 10 μl LTB4, CAY10583 or vehicle were injected in the hindpaw. Animals were killed at the indicated time points. Paw edemas were embedded in tissue freezing medium (Leica microsystems Nussloch, Germany), cryosections of 10 μm thickness were sliced using the Leica CM 3050S cryostat (−20° C.; Leica, Wetzlar, Germany) and applied on silane-coated coverslips. The tissue was fixed in 4% paraformaldehyde in PBS, permeabilized with 0.1% triton in PBS and blocked with 3% BSA in PBS for 1 h at room temperature. The sample was placed on the stage of an inverted wide-field fluorescence microscope (Leica DM IRE2; ×63 oil lens NA 1.32). A picture before the application of antibodies was taken. By a robotic process, first the slices were incubated for 15 min with predetermined fluorescence-labeled antibodies (supplementary data 1) and rinsed with PBS. Afterwards, the phase contrast and fluorescence signals were imaged by a cooled charge-coupled device camera (Apogee KX4; Apogee Instruments, Roseville, Calif., 2× binning results in images of 1024×1024 pixels; final pixel size was 286×286 nm.sup.2). To delete the specific signal of the antibody before addition of the next, a bleaching step was performed. A postbleaching image was recorded and subtracted from the following fluorescence tag image during the data analysis. Using the corresponding phase contrast images, fluorescence images produced by each antibody were aligned pixel-wise. Images were corrected for illumination faults using flat-field correction.

[0090] Data Analysis and Statistics:

[0091] All data are presented as mean±S.E.M. To determine statistically significant differences in all behavioral experiments analysis of variance (ANOVA) for repeated measures was used followed by post hoc Bonferroni correction using GraphPad Prism. For in vitro experiments comparing only two groups, student's t-test was carried out. P<0.05 was considered as statistically significant.

Example 1

Increasing LTB4 Concentrations Reducing LTB4-Mediated TRPV1 Sensitization

[0092] LTB4 has been shown to activate at high concentrations TRPV1. Here, the inventors investigated whether or not LTB4 is able to sensitize TRPV1 activation at lower concentrations. Therefore the inventors stimulated primary cultures of murine DRG neurons twice with the selective TRPV1-agonist capsaicin and incubated the cells prior the second stimulation with increasing LTB4 concentrations. The inventors found that preincubation with 100 nM LTB4 doubled the capsaicin-induced intracellular calcium increases (FIG. 1A,B). Surprisingly, the enhanced response to LTB4 was reduced when using higher LTB4 concentrations (200-1000 nM; FIG. 1B).

[0093] The narrow concentration range for LTB4-induced TRPV1 sensitization led us to hypothesize that both LTB4 receptors, the high affinity receptor BLT1 and the low affinity receptor BLT2, mediate opposing effects on TRPV1 sensitization. Thus, the sensitization seen at low LTB4 concentrations (100 nM) might be mediated by the high affinity LTB4 receptor BLT1. With increasing LTB4 concentrations the low affinity LTB4 receptor BLT2 might become activated and decrease the TPV1 sensitization.

Example 2

Expression of BLT2 in Sensory Neurons

[0094] In the next step, the expression of BLT1 and BLT2 in DRG neurons was determined. Serial immunohistochemistry using the MELC system showed that BLT1 and 2 are colocalized in murine DRG neurons (FIG. 2). The BLT1 and 2 expressing neurons were sensory neurons, since they also expressed calcitonin gene related protein (CGRP), a marker for peptigergic sensory neurons, or isolectin B4 (IB4), a marker for non-peptigergic sensory neurons (FIG. 2). In total around 40% of all IB4 and CGRP-expressing neurons were also expressing BLT1 and 2.

Example 3

BLT2 Agonist CAY10583 Abolishes LTB4-Mediated TRPV1 Sensitization

[0095] To investigate whether or not BLT1 mediates TRPV1 sensitization, the inventors tested effect of the BLT1 antagonist U75302 on LTB4-induced TRPV1 sensitization. Indeed, U75302 was able to abolish TRPV1-sensitization induced by 100 nM LTB4 (FIG. 3). Next, the inventors studied the effect of the BLT2 antagonist LY2552833 on TRPV1 sensitization. In accordance with the notion that BLT2 decreases TRPV1 sensitization, LY2552833 had no significant effect on TRPV1 sensitization by 100 nM LTB4, but increased TRPV1 sensitization by 1000 nM LTB4 (FIG. 3). Finally, the BLT2 agonist CAY10583 by itself had no effect on capsaicin-induced TRPV1 activation, but abolished TRPV1 sensitization by 100 nM LTB4 (FIG. 3).

Example 4

BLT2 Agonist CAY10583 Abolishes ONOae329- and PKC-Mediated TRPV1 Sensitization

[0096] Next, the effect of the BLT2 agonist CAY10583 on TRPV1 sensitization by receptors activating signaling pathways known to sensitize TRPV1 was studied. Therefore, TRPV1 was sensitized using the PGE2 receptor 4 (EP4) ligand ONOae329. Here, sensitization is achieved through increased cAMP synthesis, subsequent protein kinase A (PKA) activation and TRPV1 phosphorylation (Bhave et al., 2002). Pretreatment of murine DRG neurons with ONOae329 induced a strong sensitization of TRPV1 responses to capsaicin which was abolished in presence of the BLT2 agonist CAY10583 (FIG. 4A,B). Likewise, bradykinin-induced phosphorylation and sensitization of TRPV1 through PKC was significantly decreased in presence of CAY10583 (FIG. 4C,D).

Example 5

BLT2 Agonist CAY10583 Reduces Pain Sensation in Rats

[0097] To study potential analgesic effects of BLT2 activation, the BLT2 agonist CAY10583 or vehicle were injected in hind paws of mice and the thermal pain thresholds were determined. In accordance with the inventor's data showing decreased TRPV1 sensitization in vitro, CAY10583 was able to significantly increase thermal pain thresholds as compared to mice receiving only vehicle (FIG. 5).

REFERENCES

[0098] Andoh T, Kuraishi Y (2005) Expression of BLT1 leukotriene B4 receptor on the dorsal root ganglion neurons in mice. Brain research Molecular brain research 137:263-266.

[0099] Bhave G, Zhu W, Wang H, Brasier D J, Oxford G S, Gereau R W t (2002) cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation. Neuron 35:721-731.

[0100] Hwang S W, Cho H, Kwak J, Lee S Y, Kang C J, Jung J, Cho S, Min K H, Suh Y G, Kim D, Oh U (2000) Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances. Proc Natl Acad Sci USA 97:6155-6160.

[0101] Iizuka Y, Yokomizo T, Terawaki K, Komine M, Tamaki K, Shimizu T (2005) Characterization of a mouse second leukotriene B4 receptor, mBLT2: BLT2-dependent ERK activation and cell migration of primary mouse keratinocytes. J Biol Chem 280:24816-24823.

[0102] Okubo M, Yamanaka H, Kobayashi K, Fukuoka T, Dai Y, Noguchi K (2010) Expression of leukotriene receptors in the rat dorsal root ganglion and the effects on pain behaviors. Mol Pain 6:57.

[0103] Pierre S, Maeurer C, Coste O, Becker W, Schmidtko A, Holland S, Wittpoth C, Geisslinger G, Scholich K (2008) Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing. Mol Cell Proteomics 7:2475-2485.

[0104] Pierre S, and Scholich, K. (2010) Toponomics: Studying protein-protein interactions and protein networks in intact tissue. Molecular BioSystems 6:641-647.

[0105] Scholich K, Geisslinger G (2006) Is mPGES-1 a promising target for pain therapy? Trends Pharmacol Sci 27:399-401.

[0106] Sisignano M, Park C K, Angioni C, Zhang D D, von Hehn C, Cobos E J, Ghasemlou N, Xu Z Z, Kumaran V, Lu R, Grant A, Fischer M J, Schmidtko A, Reeh P, Ji R R, Woolf C J, Geisslinger G, Scholich K, Brenneis C (2012) 5,6-EET Is Released upon Neuronal Activity and Induces Mechanical Pain Hypersensitivity via TRPA1 on Central Afferent Terminals. J Neurosci 32:6364-6372.

[0107] Yokomizo T, Izumi T, Chang K, Takuwa Y, Shimizu T (1997) A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387:620-624.

[0108] Yokomizo T, Kato K, Terawaki K, Izumi T, Shimizu T (2000) A second leukotriene B(4) receptor, BLT2. A new therapeutic target in inflammation and immunological disorders. J Exp Med 192:421-432.