Pharmaceutical compositions for treating pain

Abstract

The present invention discloses a novel use of a pharmaceutical composition in the preparation of a medicament in the treatment of opioid-induced tolerance and addiction, in particular to the use of AC1 inhibitor NB001 and AC1&8 mixed inhibitors NB010 and NB011 in the preparation of a medicament in the treatment of opioid-induced tolerance and addiction.

Claims

1. A method of reducing morphine tolerance in a human patient, comprising the steps of: i) repetitively administering morphine to the human patient; and ii) administering to the human patient an analgesically effective dose of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol (NB001) or a pharmaceutically acceptable salt or solvate thereof, wherein the NB001 or pharmaceutically acceptable salt or solvate thereof suppresses AC1 activity and increases analgesia of the morphine, and wherein the human patient has osteoarthritis, rheumatoid arthritis, or a toothache.

2. The method of claim 1, wherein the NB001 or the pharmaceutically acceptable salt or solvate thereof is administered in combination with 6-amino-9-(2-p-methoxy-ethyl)-9H-purine-8-thiol (NB010).

3. The method of claim 2, wherein the NB001 or the pharmaceutically acceptable salt or solvate thereof administered in combination with NB010 is further administered in combination with 6-morpholin-4-yl-7H-purine (NB011).

4. The method of claim 1, wherein the NB001 or pharmaceutically acceptable salt or solvate thereof is administered orally as tablets containing 0.01, 10.0, 50.0, 100, 150, 200, 250, or 500 milligrams of NB001 or a pharmaceutically acceptable salt or solvate thereof.

5. A method of reducing morphine tolerance in a human patient, comprising the steps of: i) repetitively administering morphine to the human patient; and ii) administering to the human patient a pharmaceutical composition comprising an analgesically effective dose of 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol (NB001) or a pharmaceutically acceptable salt or solvate thereof, wherein the NB001 or pharmaceutically acceptable salt or solvate thereof suppresses AC1 activity and increases analgesia of the morphine, and wherein the human patient has osteoarthritis, rheumatoid arthritis, a migraine, a headache, a toothache, or a burn.

6. The method of claim 5, wherein the NB001 or pharmaceutically acceptable salt or solvate thereof is administered in combination with 6-amino-9-(2-p-methoxy-ethyl)-9H-purine-8-thiol (NB010).

7. The method of claim 6, wherein the NB001 or pharmaceutically acceptable salt or solvate thereof administered in combination with NB010 is further administered in combination with 6-morpholin-4-yl-7H-purine (NB011).

8. The method of claim 5, wherein the NB001 or pharmaceutically acceptable salt or solvate thereof is administered orally as tablets containing 0.01, 10.0, 50.0, 100, 150, 200, 250, or 500 milligrams of NB001 or a pharmaceutically acceptable salt or solvate thereof.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Inhibition of pre-LTP and post-LTP in ACC using AC1 & 8 inhibitor NB010.

(2) FIG. 2 The effect of inhibition of AC1/8 by different reagents on pre-LTP in ACC.

(3) FIG. 3 Morphine tolerance induced by repeated injections of morphine (10 mg/kg) was reduced by using the AC1 inhibitor NB001 (5 mg/kg).

(4) FIG. 4 NB010 and NB011 produced significant effects in reversing morphine tolerance.

(5) FIG. 5 The result of the conditioned place preference experiment.

(6) FIG. 6 The effect of different agents on anxiety caused by nerve injury.

(7) FIG. 7 The result of Itching response test.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(8) NB001, 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol, has molecular formula C12H20N6O, and molecular weight 264.33. Available from Sigma 686301-48-4.

(9) NB010, 6-amino-9-(2-p-methoxy-ethyl)-9H-purine-8-thiol has the following chemical structure:

(10) ##STR00013##

(11) A product available from Asinex LTD under BAS03384.

(12) NB01 1,4-(9H-purin-6-yl)morpholine (also known as 6-morpholin-4-yl-7H-purine) has the following chemical structure:

(13) ##STR00014##

(14) A product available from Maybridge company under JFD02793.

(15) Pharmaceutically acceptable salts of the compounds having the indicated chemical formulae fall, within the scope of the present invention, into the definitions of Compound A and Compound B.

Example 1. Screening for AC1 & 8 Mixed Inhibitors

(16) A set of biochemical screening tests were performed to search for potential AC1 and AC8 co-inhibitors (or referred to as AC1 & 8 inhibitors). AC1 or AC8 expressing cell lines were subject to cAMP assays, gene activation (pCREB) assays, and integrative physiological experiments to screen for potential compounds A and B acting on AC1 and AC8. It was found that both NB010 and NB011 produced significant inhibition of AC1 as well as AC8 activity at 100 μM (see Table 1). Regarding test methods, see Wang, H. S., et al., Identification of an Adenylyl Cyclase Inhibitor for Treating Neuropathic and Inflammatory Pain. Science Translational Medicine, 2011. 3(65).

(17) TABLE-US-00001 TABLE 1 Effects of NB010 and NB011 on the activity of adenylyl cyclase subtype 8 and 1 Percent Inhibition AC8 AC1 NB010 (100 μM) 88 ± 2% inhibition 96 ± 4% NB011 (100 μM) 86 ± 2 91 ± 3%

Example 2. Evaluation of NB010 and NB011 on Anxiety-Related Cortical LTP

(18) Previous studies have indicated that AC1 and AC8 activity is required for synaptic LTP induced by repetitive stimulation (Zhuo, 2016). To evaluate the effects of NB010 and NB011 on AC1 and AC8 activity in neurons, two forms of LTP in ACC cortical slices of adult mice were recorded.

(19) As above-mentioned, 6 burst stimulation induced potentiation of synaptic responses for at least 40 min (FIG. 1A, n=8 slices). Induction of ACC LTP can be blocked by pretreatment with NB010 (25 μM) (FIG. 1A, n=5 slices). Similar results were found with NB011 (FIG. 2, n=6 slices). In contrast, a non-selective AC inhibitor SQ22356 in the same dose did not cause a significant decrease in pre-LTP (FIG. 2, n=5). In contrast, pre-LTP was not blocked by using GABApention (25 μM) (n=5).

(20) A recent report for ACC (Chen et al., 2014) also found that GABApention does not affect ACC LTP. Interestingly, forskolin (10 μM) resulted in a slight increase in pre-LTP (FIG. 2), indicating that AC activation is critical for pre-LTP. These findings indicate that both NB010 and NB011 can inhibit AC1 and AC8 activity in adult mouse neurons. This proves that NB010 and NB011 may be used to treat anxiety caused by injury.

(21) Recent studies have indicated that anxiety caused by injury may be related to pre-LTP (Koga et al., 2015; Zhuo, 2016), therefore the present inventors decided to test the effect of NB010 and NB011 on pre-LTP. And both compounds were found to inhibit pre-LTP (FIG. 1). For detailed test process, see Koga, K., et al., Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain. Neuron, 2015. 85(2): p. 377-389.

(22) FIG. 1 Inhibition of pre-LTP and post-LTP in ACC using AC1 & 8 inhibitor NB010, where (A) Post-LTP was induced in the control slices (filled circles; n=8), whereas post-LTP was blocked by the application of NB010 (25 μM) solution (n=5); (B) Pre-LTP was induced in the control slices (filled circles; n=8); whereas pre-LTP was blocked by the application of NB010 (25 μM) solution (n=5). FIG. 2 showed the effect of inhibition of AC1/8 by different reagents on pre-LTP in ACC. where, the data showed that the induction of pre-LTP was enhanced in the last 5 minutes during 45 minutes. * indicates a significant difference compared to the control group (where NB010 and NB011 resulted in a decrease while forskolin resulted in an increase).

Example 3. Behavioral Effects of AC1 and AC1&8 Inhibitors on Morphine Tolerance

(23) First, the present inventors intended to examine whether AC1 knockout mice will affect the morphine tolerance caused by repetitive injections of morphine. According to previous reports by the inventors, morphine was injected (10 mg/kg, s.c.) daily for 7 days to measure morphine-induced tolerance. It was found that in AC1 knockout mice, morphine produced significant great analgesic effects as compared with saline-treated wild-type mice (n=5-8 mice: day 7: control mice, MPE 18±6%, AC1 KO, 40±8%; P<0.05). These results indicate that inhibiting AC1 activity may be beneficial for reducing morphine tolerance. Next a selective AC1 inhibitor NB001 (5 mg/kg, i.p.) was used to treat wild-type mice with morphine. Both nociceptive hotplate and tail-flick response latencies were recorded. It was found that NB001 co-application produced a significant effect on response latencies after chronic morphine treatment (p<0.005) (FIG. 3).

(24) Previous studies from AC1 and AC8 double knockout mice found that morphine tolerance was significantly reduced (Li et al., 2006). The inventors expected that AC1 &8 inhibitors may also produce beneficial effects in reducing morphine tolerance. Similarly, pretreatment with NB010 or NB011 (5 mg/kg, i.p.) both produced significant greater analgesic effects at 7 days after morphine injections (n=5-8 mice, p<0.05 as compared with control treatment in each case). For detailed test methods, see Li, S., Lee, M L, Bruchas, M R, Chan, G C, Storm, D R, and Chavkin, C. (2006). Calmodulin-stimulated adenylyl cyclase gene deletion effects morphine responses. Mol Pharmacol 70, 1742-1749.

(25) FIG. 3 showed that morphine tolerance induced by repeated injections of morphine (10 mg/kg) was reduced by using the AC1 inhibitor NB001 (5 mg/kg).

(26) Accordingly, the inventors expected that both the AC1 inhibitor NB001 and the AC1&8 inhibitors NB010 and NB011 may be useful for enhancing morphine analgesic effect, thereby enable the repetitive use of morphine.

(27) As shown in FIG. 4, NB010 and NB011 produced significant effects in reversing morphine tolerance at 7 days after morphine injection (10 mg/kg) (n=5-7 mice). In contrast, 100 mg/kg of GABApentin did not produce a significant effect (n=6 mice). In addition, the effect of the non-selective AC inhibitor SQ22356 was also tested and no significant effect was found at the same dose, indicating that selective inhibition of AC1/AC8 is critical.

(28) To further test the role of AC1/AC8 in morphine tolerance, AC activator forskolin was applied and it was found that morphine produced tolerance faster in mice (n=3 mice) 3 days after repetitive administration of morphine.

(29) For the AC1 inhibitor NB001 (5 mg/kg) and the AC1/AC8 inhibitor NB010 or NB011 (5 mg/kg), morphine tolerance induced by repetitive injections of morphine (10 mg/kg) 7 days after repetitive morphine injections reduced. GABApentin (100 mg/kg) or SQ22356 (5 mg/kg) did not produce any significant effect.

(30) To test possible dose-related effects, the NB compound were applied at a lower dose of 1 mg/kg; no significant effect was found with NB001/NB010/NB011, indicating that the effect produced was dose-related.

Example 4. Conditioned Place Preference (CPP) to Morphine

(31) Conditioned Place Preference (CPP) was used to verify the reduced response of CREB mutant mice to the beneficial effects of morphine (Walters and Blendy, 2001). Morphine CPP was subsequently induced in saline-treated and inhibitor treated mice. It was found that mice treated with NB001 (5 mg/kg, ip) spent significantly less time exploring the morphine-paired side of the chamber than saline-treated mice, indicating that AC1 played a role in the enhanced properties of morphine (p<0.05). There is no significant difference in their initial preference for either side of the chamber. For the detailed test methods, see Walters C L, Blendy J A (2001) Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse. J Neurosci 21:9438-9444.

(32) For the conditioned place preference experiment of morphine, the inventors found that NB00I caused a significant reduce in morphine-induced preference. Both NB010 and NB011 (5 mg/kg) are similar to NB001 (FIG. 5). In contrast, a non-selective AC inhibitor SQ22356 in the same dose did not produce any inhibition (FIG. 5). It indicated that AC1/AC8 inhibition is selective. The effect of GABApention at 100 mg/kg was also tested. As a result shown in FIG. 5, morphine (10 mg/kg) was repeatedly used to induce place preference behavior after 9 days, and the effects of applying the AC1 inhibitor NB001, AC1/8 inhibitor NB010 or NB011 (5 mg/kg) were significant. GABApentin (100 mg/kg) or SQ22356 (5 mg/kg) did not produce any significant effect.

(33) In addition, the test results also showed (not shown in the figure) that when the dose of NB001/NB010/NB011 was reduced to 1 mg/kg, the expected effect was not produced, so the effect of NB001/NB010/NB011 was dose-dependent (n=3-5 mice). Forskolin (5 mg/kg) did not produce any inhibitory effect, and the results also showed a slight increase in selection time (n=3 mice).

Example 5. Anxiety Behavior Test

(34) As previously mentioned, the inventors found that nerve injury caused anxiety behavior in the EPM test (Koga et al., 2015; see FIG. 6). The inventors therefore examined whether NB010 or NB011 can reduce anxiety and other behaviors since both inhibitors prevent anxiety-related pre-LTP. As expected, NB010 or NB011 (5 mg/kg) produced significant reduction in anxiety responses caused by nerve injury (P<0.05 in each case, n=6 mice for each agent) (see FIG. 6).

(35) At lower doses (1 mg/kg), NB010 (see FIG. 6) did not significantly decrease. Similar result was obtained with NB011 of 1 mg/kg. Neither GABApentin (100 mg/kg, i.p.) nor SQ22356 (5 mg/kg) significantly reduced behavioral anxiety (FIG. 3). The use of forskolin (5 mg/kg) did not further increase anxiety, which may be due to the production of saturation effect (FIG. 6).

(36) For detailed experimental methods, see Koga, K., et al., Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain. Neuron, 2015. 85(2): p. 377-389.

(37) FIG. 6 shows the effect of different agents on anxiety caused by nerve injury. The use of AC1 & 8 inhibitor NB010/NB011 (5 mg/kg, i.p.) reduced anxiety caused by nerve injury (7 days after injury). At low doses of 1 mg/kg, neither NB010 nor NB011 affected anxiety. The use of GABApentin (100 mg/kg) or SQ22356 (5 mg/kg) had no significant effect. EPM test was used in this example to assess the anxiety state.

(38) Itching Response

(39) It is well known that itching is often caused by a variety of diseases, including the chronic use of opioids. To evaluate if the inhibition of AC1 and AC8 may reduce itching response, the inventors evaluated the effect of NB010 and NB011 on behavioral itching response to injection of Compound A. A specific test method refers to U.S. Patent Publication No. US20110098295 A1, and as a result, it was found that the total scratching responses induced by intradermal injection of compound 48/80 (100 μg/50 μl) was found to be significantly reduced in mice receiving the injection of NB010 or NB011 pretreatment (n=6 mice) as compared with wild-type mice (n=7) (FIG. 7). The application of GABApention (100 mg/kg) did not cause a significant reduction in the itching response (FIG. 7). SQ22356 also did not show significantly reduction at the dose tested (5 mg/kg, ip). In contrast, the application of forskolin (5 mg/kg) significantly enhanced the behavioral itching response (FIG. 7). FIG. 7 shows the effect of different agents that affect the cAMP pathway on behavioral itching responses in mice. The application of NB010 or NB011 significantly reduced the itching response induced by histamine releasing compound 40/80 in adult mice. In contrast, neither GABApentin nor SQ22356 produce any significant effect. Forskolin is an AC activator that significantly enhances the itching response. *P<0.05 compared to control.

(40) Experimental Animals

(41) AC1 and AC8 KO mice, see Wei F, Qui C S, Kim S J, et al. Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclase. Neuron. 2002; 36(4): 713-726, were bred for several generations (F8-F12) on C57B1/6 background. Control wild-type (WT) mice were adult male (8-12 weeks old) C57B11/6 mice from Charles River. At the end of the experiment, animals were treated with excess inhalation of anesthetic (halothane). Animals were housed on a 12 h: 12 h light: dark cycle with food and water available ad libitum.

(42) Biochemical Screening Tests for Novel Inhibitors for AC8 and AC1

(43) For the AC8 expression vector pcDNA3-AC1 transfection, HEK293 cells were plated onto a 60 mm diameter petri dish at a density of 1×10.sup.6 per plate, grown overnight, and transfected with pcDNA3-AC1 (0.8 μg DNA per plate) by Lipofectamine 2000 (Invitrogen). Stable transfected clones were selected and maintained in media containing 0.8 mg/ml G418 (Invitrogen, CA). For transient expression of other AC isoforms in HEK293 cells, HEK293 cells were seeded in 96-well tissue culture dishes and transfected with AC5 plasmid respectively, and subject to experiment 48 hours after transfection.

(44) HEK293 cells expressing ACs were harvested and lysed in 0.1 M HCl after different treatments. Direct cAMP measurements were performed using a direct cAMP enzyme immunoassay kit (Assay Designs, MI) and optical density values were measured at 405 nm by a microplate reader. Phosphodiesterase was inhibited by the addition of 1 mM 3-isobutyl-1-methylxanthine (Sigma, MO) to cultures.

(45) CRE Luciferase Reporter Gene Assay

(46) HEK293 cells were subcultured into 96-well plates in the absence of antibiotics and grown overnight, and transfected with pGL3-CRE firefly luciferase and pGL3-CMV-adrenergic luciferase constructs (0.25 μg DNA per well) by Lipofectamine 2000 reagent. The transfected cells were incubated overnight and the medium was changed to DMEM containing 10% fetal calf serum. After 48 hours, cells were treated with 10 μM forskolin, 10 μM A23187 and 2 mM CaCl2), or a combination of 10 μM forskolin, 10 μM A23187 and 2 mM CaCl2, in the absence or presence of each chemical tested at the concentration of 100 μM. At the end of 6 hours, the cultured cells were harvested and assayed for luciferase activity by Dual Luciferase Reporter Assay System (Promega). Relative light units were measured using a SIRIUS luminometer.

(47) Whole Cell Patch Clamp Recording

(48) Coronal brain sections (300 μm) at ACC level were prepared using standard methods (Wu et al., 2005). The sections were transferred to an immersion recovery chamber containing oxygen (95% 02 and 5% CO2) artificial cerebrospinal fluid (ACSF) (containing mM: 124 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 25 NaHCO3, 1 NaH2PO4, 10 glucose) at room temperature for at least 1 hour. Experiments were performed in a recording chamber on a stage of a BX51W1 microscope equipped with infrared DIC optics for visualization. Excitatory postsynaptic currents (EPSCs) were recorded from layer II/III neurons with an Axon 200B amplifier (Axon Instruments, CA), and the stimulations were delivered by a bipolar tungsten stimulating electrode placed in layer V of the ACC. Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated EPSCs were induced by repetitive stimulations at 0.05 Hz and neurons were voltage clamped at −70 mV in the presence of AP5 (50 μM). The recording pipettes (3-5 MΩ) were filled with a solution containing (in mM): 145 K-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na3-GTP and 10 phosphocreatine disodium and was adjusted to pH 7.2 with KOH. The internal solution (in mM: 140 cesium methanesulfonate, 5 NaCl, 0.5 EGTA, 10 HEPES, 2 MgATP, 0.1 Na3GTP, 0.1 spermine, 2 QX-314 bromide and 10 phosphocreatine disodium (adjusted to pH 7.2 with CsOH) was used in the rectification of AMPA receptor-mediated transmission experiment. For miniature EPSC (mEPSC) recordings, 0.5 μM TTX was added in the perfusion solution. Picrotoxin (100 μM) was always present to block GABAA receptor-mediated inhibitory synaptic currents in all experiments. Access resistance was 15-30 MΩ and monitored throughout the experiment. Data were discarded if access resistance changed by more than 15% during the experiment. Data were filtered at 1 kHz, and digitized at 10 kHz.

(49) Conditioned Place Preference Experiment

(50) A chamber with two distinct contextual environments (different walls, floor and smell) was used (MED-associates, St. Albans, Vt.). On the first day of test, animals were allowed to freely explore both sides of the chamber for 30 min and data were used to divide animals into groups of approximately equal bias. Over the next eight days, each animal was given either 10 mg/kg morphine or an equivalent volume of saline on alternate days at different sides of the chamber. Animals were confined to a specific side of the chamber for 30 minutes. After conditioning, all animals were injected with saline and allowed to freely explore both sides of the chamber for 30 minutes. Place preference was defined as an increase in the time spent in the morphine-paired side after conditioning as compared to before.

(51) Neuropathic Pain Model

(52) A model of neuropathic pain was induced by connecting a common peroneal nerve (CPN) as previously described (Li et al., 2010). Briefly, mice were anesthetized by intraperitoneal injection of a mixture saline of ketamine (0.16 mg/kg; Bimeda-MTC) and xylazine (0.01 mg/kg; Bayer). CPN was visible between the anterior and posterior groups of muscles running almost transversely. The chronic CPN was slowly connected to the chromic gut suture 5-0 (Ethicon) until the contraction of the dorsiflexors of the foot was visible as twitching of digits. Mechanical allodynia test was performed on 3rd, 7th and 14th postoperative day. The experiment was blinded and different individuals were responsible for the surgery and the measurements of the mechanical sensitivity of mice.

(53) Mechanical Allodynia Test

(54) Mice were placed in a circular container and adapted for 30 minutes before test. Mechanical allodynia was evaluated based on the responsiveness of hind paws to the application of von Frey filaments (Stoelting) to the bending point. Positive responses included licking, biting, and sudden withdrawal of the hind paws. Experiments were conducted to characterize the threshold stimulus. Mechanical pressure (force, 0.008 g) from 1.65 filaments was found to be harmless in naïve mice. This filament was then used to test mechanical allodynia.