CN120346203A - Use of GluK receptor agonists in the treatment of chronic pain - Google Patents

Abstract

The invention belongs to the fields of neurobiology and pain treatment, and particularly relates to application of GluK receptor agonists in preparation of medicines for treating chronic pain. The GluK receptor agonist is ATPA or a pharmaceutically acceptable salt and solvate thereof. The present inventors have found that the rhodopsin receptor mediated synaptic plasticity in the Anterior Cingulate Cortex (ACC) is significantly impaired in chronic pain states, manifested by a loss of long-term inhibitory capacity, whereas selective activation of rhodopsin receptor subtype GluK1 may reverse the abnormality. Up-regulation of GluK1 expression in ACC following chronic pain, activation of GluK1 with the GluK1 receptor agonist ATPA has significant therapeutic effects on neuropathic pain and inflammatory pain. The results indicate that targeted activation of GluK a may produce analgesic effects by restoring homeostasis of synaptic plasticity within ACC, gluK a receptor agonists may be useful in the treatment of chronic pain.

Description

Use of GluK receptor agonists in the treatment of chronic pain

Technical Field

The invention belongs to the fields of neurobiology and pain treatment, and particularly relates to application of GluK receptor agonists in preparation of medicines for treating chronic pain.

Background

Chronic pain is a disease that severely jeopardizes human health, and about 20% of the population worldwide suffers from chronic pain for a long period of time. At present, the treatment of chronic pain is always a clinical problem, and the treatment medicine mainly takes opium medicines such as morphine and the like as main medicines. However, long-term opioid administration can lead to tolerance, addiction, and severe withdrawal reactions. The existing treatment methods have limited effects on chronic pain and lack analgesic drugs which are effective for a long time and have small side effects. The reason for this is mainly that we lack sufficient knowledge of the cause and regulation mechanism of chronic pain. In the pain sensation transmission loop, the anterior cingulate Area (ACC) of the cerebral cortex is considered to have a very important role in both pain perception and mood regulation. Numerous clinical human brain imaging and animal electrophysiology studies have shown that both peripheral neuralgia and emotional pain stimuli can activate the ACC region. Overactivation of ACC is closely associated with a number of chronic pain conditions, which are often accompanied by fear, anxiety and cognitive dysfunction.

The mechanism of chronic pain generation involves peripheral and central sensitization, synaptic plasticity abnormalities, neuroinflammation, and the like. Central sensitization, such as long-term potentiation of synaptic transmission (LTP), is a key cellular mechanism that undergoes long-term changes in physiological and pathological conditions in the mammalian central nervous system. In contrast, studies confirm that impaired long-term inhibition (LTD) function of glutamatergic synapses within ACC is an important marker of chronic pain sensitization. Activating or amplifying LTD production within the ACC helps in pain relief and produces analgesia. There are no analgesic drugs currently directed to targeting LTD signaling pathways.

In recent years, studies have shown that the neurotransmitter glutamate (Glutamate) plays a central role in pain signaling and regulation. Wherein the dysfunction of the rhodopsin (KA) type glutamate receptor GluK subtype (formerly GluR 5) is closely related to the maintenance of chronic pain. GluK1 receptors are widely distributed in the central nervous system, and are highly expressed particularly in the Anterior Cingulate Cortex (ACC), which is not only the integral center of pain affective components, but also a key regulatory node of pain-mood co-morbid. GluK1 receptors are involved in the dynamic balance of synaptic plasticity in ACC by modulating presynaptic glutamate release and postsynaptic neuronal excitability. For example, in a model of nerve injury, down-regulation or inhibition of function of GluK receptor expression may result in overactivation of the ACC glutamatergic loop, thereby exacerbating pain affective components and anxiety-like behavior. However, the prior art intervention strategies for GluK receptor have focused on antagonist development, which can relieve pain but often accompanies side effects such as cognitive dysfunction or emotional suppression, and limit the clinical application. Since GluK receptor is expressed on both excitatory and inhibitory neurons, the development of targeted analgesic drugs against GluK receptor agonists is of great prospect and significance.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of GluK receptor agonist in preparing medicines for treating chronic pain.

The present inventors have found that in chronic pain conditions, the synaptic plasticity mediated by the rhodopsin receptor subtype GluK1 in ACC is significantly impaired, manifested by a loss of capacity of the LTD induced by KA, whereas selective activation of the GluK1 receptor reverses this abnormality. Up-regulation of GluK1 expression in ACC following chronic pain, activation of GluK1 with the GluK1 receptor agonist ATPA has significant therapeutic effects on neuropathic pain and inflammatory pain. The results indicate that targeted activation of KA receptor subtype GluK1 may produce analgesia by restoring homeostasis of synaptic plasticity in ACC, gluK1 receptor agonists can be used to treat chronic pain.

Further, the GluK receptor agonist is ATPA or pharmaceutically acceptable salt and solvate thereof, wherein the ATPA is (RS) -2-amino-3- (3-hydroxy-5-tert-butyl isoxazol-4-yl) propionic acid, and the structural formula is as follows:。

Further, the medicament takes GluK receptor agonist as the only effective component.

Still further, the medicament comprises an active ingredient for enteral or parenteral administration, and a suitable pharmaceutically organic or inorganic inert carrier material.

Still further, the pharmaceutically suitable organic or inorganic inert carrier material is selected from one or more of water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.

Further, the dosage form of the medicine is a solid dosage form or a liquid dosage form.

Still further, the solid dosage form is a tablet, coated tablet, suppository or capsule.

Still further, the liquid dosage form is a solution, suspension or emulsion.

Further, the medicament comprises suitable pharmaceutical excipients.

Still further, the adjuvant is selected from one or more of a preservative, a stabilizer, a wetting agent, an emulsifier, an osmotic pressure regulator and a buffer.

Further, the chronic pain is neuropathic pain or inflammatory pain.

The invention has the following beneficial effects:

1. Targeting regulatory mechanisms the present invention for the first time reveals a specific analgesic effect of ACC region GluK receptor activation (via ATPA) in chronic neuropathic pain by enhancing long-term inhibition (LTD) of synaptic transmission in the ACC brain region.

2. Dose-dependent analgesia the invention significantly improves the mechanical foot contraction threshold (PWT) and the thermal pain response latency of mice with sciatic nerve branch ligation (SNI) neuropathic pain model and Complete Freund's Adjuvant (CFA) inflammatory pain model by microinjection of ATPA (10, 50 and 150 ng/kg), the effect is dose-dependent, and takes effect 5 minutes after single administration for about 20 minutes.

3. The behavior specificity separation is that the analgesic effect of ATPA is not accompanied with the improvement of anxiety-like behavior unlike the traditional analgesic drugs (such as opioids), which shows that the ATPA regulates pain perception through independent channels and provides direct evidence for an analgesic-emotional decoupling treatment strategy.

4. The clinical transformation potential is that ATPA has no obvious influence on normal animals, does not induce behavior change in a sham operation group, and indicates that the effect of ATPA has pathological state selectivity, can avoid systemic side effects, and is suitable for the accurate intervention of chronic pain patients.

Drawings

FIG. 1 shows the results of analysis of the correlation of defects in the function of the GluK receptor-mediated LTD in ACC with chronic pain sensitization. Wherein A is an experimental schematic diagram of the MED64 multichannel system for recording the field potential (fEPSP) after ACC excitatory synapse. The model B is an SNI operation schematic diagram, namely, the common fibular nerve (CPN) and the Tibial Nerve (TN) are ligated and cut off, and the Sural Nerve (SN) is reserved, C is the comparison of the mechanical foot contraction threshold (PWT) of the SNI group mice and the Sham group mice (0.066+/-0.02482 g vs. The Sham operation group is 0.88+/-0.1744 g, p < 0.01), D is the LTD of the Sham group mice ACC brain slice after KA stimulation for different time, D is the LTD of the SNI group mice ACC brain slice after KA stimulation for different time, and E is the comparison of the LTD of the Sham group mice and the SNI group mice ACC brain slice after KA stimulation for 180 min.

Fig. 2 is the expression of GluK1 in the SNI chronic pain model. Wherein a is a representative western blot band pattern and B is a quantitative statistical pattern of GluK and GluK2/3 protein expression levels in ACC of SNI mice (< 0.05 p; n=3/group based on double-sided unpaired student t test).

Figure 3 is the analgesic effect of ACC microinjection GluK of the receptor agonist ATPA in the SNI model. Wherein A is a statistical graph of mechanical foot shrinkage threshold (PWT) of a Sham group mouse and a SNI group mouse after ATPA injection of 50 ng/kg of ACC, B is a statistical graph of hotplate latency of a Sham group mouse and a SNI group mouse after ATPA injection of 50 ng/kg of ACC, C is a graph of change of mechanical foot shrinkage threshold (PWT) of a Sham group mouse and a SNI group mouse after ATPA injection of different concentrations of ACC with time, and D is a graph of change of hotplate latency of a Sham group mouse and a SNI group mouse after ATPA injection of different concentrations of ACC with time.

Figure 4 is the analgesic effect of ACC microinjection GluK of the receptor agonist ATPA in the CFA model. Wherein, A is a schematic diagram of a CFA induced inflammatory pain mouse model. B is the analgesic effect of ATPA (10, 50, 150 ng/kg) with different concentrations on mechanical hyperalgesia after injection into CFA mouse ACC. C is the change in analgesic effect over time after a single injection of ATPA.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1

1. Methods and materials

1.1 Experimental materials

(1) Animals

The experimental animals were adult male C57BL/6 mice (6-8 weeks old) purchased from the university of Western An traffic medical laboratory animal center. Raising in the animal house of the front scientific and technical institute of western traffic university, keeping the room temperature at 24+/-1 ℃, keeping the air humidity at 55% -60%, and taking food with free drinking water with the illumination period of 12h (8:30 am to 8:30 pm). The animal experiment program in the embodiment is licensed by the animal and management committee of the western traffic university, and animal experiment and animal ethics specifications are strictly complied with in the experiment process.

(2) Medicine

KAINIC ACID (KA, CAS number: 487-79-6) and (RS) -2-amino-3- (3-hydroxy-5-t-butylisoxazol-4-yl) propionic acid (ATPA, CAS number: 140158-50-5) were purchased from Tocris Cookson (Bristol, UK) and dissolved in physiological saline (0.9% sodium chloride injection). Complete Freund's adjuvant CFA (CAS number 9007-81-2) was purchased from Sigma-Aldrich (USA) and dissolved in physiological saline for use.

2. Experimental method

(1) Preparation of ACC ex vivo brain tablet

Preparation method reference of coronary brain tablet containing ACC ：Koga, K., G. Descalzi, T. Chen, H. G. Ko, J. S. Lu, S. Li, J. Son, T. Kim, C. Kwak, R. L. Huganir, M. G. Zhao, B. K. Kaang, G. L. Collingridge and M. Zhuo (2015). "Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain (vol 85, pg 377, 2015)." Neuron86(4): 1109-1109.

First, an artificial cerebrospinal fluid (ACSF) and a high sugar slicing solution were prepared, each of which was oxygenated (mixed gas 95% O ₂ and 5% CO ₂) by 50 mL, 30min, before slicing. When slicing, mice were anesthetized with isoflurane and sacrificed in cervical dislocation, brain tissue was acutely isolated and immersed in a high sugar slice solution with sufficient oxygen, cooled 1 min, and then the whole brain was removed and trimmed. The front end was fixed up with glue to a tray of a vibrating microtome (Leica, VT 1200S). The isolated brain slices containing ACC were cut coronally, and the thickness of the brain slices was 300. Mu.m. Brain pieces were then transferred to ACSF and recording was started after incubation at room temperature for at least 1 h.

(2) Multichannel field potential recording

1) Preparation of microelectrode array probes

This example uses the MED64 multichannel recording system (P515A, panasonic Alpha-MED SCIENCES). The MED64 probe had 64 planar microelectrode arrays arranged in an 8X 8 pattern with an electrode spacing of 150 μm. For better attachment of brain slices to the MED64 probe. The MED64 probe required hydrophilic treatment prior to use, was soaked in 0.1% polyethylenimine (P-3143, sigma-Aldrich) borate buffer (pH 8.4), and protected from light overnight at room temperature. The probe surface was then rinsed with sterile distilled water at least three times.

2) Multichannel field potential recording

After 1 hour incubation, the brain pieces containing ACC were placed in the MED64 probe (fig. 1A), and after positioning, the grid and anchors were carefully placed on the brain pieces to ensure stability of the brain pieces during experimental recording. The whole experiment process uses a perfusion pump to continuously perfuse fresh ACSF (95% O ₂ and 5% CO ₂) with mixed gas into brain slices at a speed of 2-3 mL/min. Brain pieces were incubated on the probes for at least 1 hour before recording was started. A channel was selected in the deep layer (V-VI layer) as the stimulation site. By Mobius software, bi-directional constant current pulses (10-20 μa,0.2 ms) were continuously stimulated to the stimulation sites and induced around the excitatory postsynaptic field potential (fEPSP). The stimulus was recorded at intervals 1 min, and samples were taken on average every 5 minutes during analysis. The "slope" parameter represents the average slope of each fEPSP recorded by the active channel. After 0.5 hours of baseline stabilization recording, KA was added to the perfusate and rinsed after 20 min hours. The antagonist or inhibitor was administered 10 minutes prior to KA administration and rinsed with KA. The fEPSP response of 2.5 h was then recorded.

(3) Chronic neuropathic pain model (SNI)

Chronic neuropathic pain is induced by ligature of the sciatic nerve. Mice were anesthetized with isoflurane. Shaving and iodic disinfection are carried out in the left hind limb operation area, longitudinal incision is made in the middle femur, muscle layers are separated in a blunt manner, and sciatic nerve and three branches thereof are exposed. The common fibular nerve (CPN) and Tibial Nerve (TN) were cut and tightly ligated with 5-0 silk. Sural Nerve (SN) was left intact (fig. 1B), and then the muscles and skin were sutured layer by layer. The Sham group (Sham) only exposed the sciatic nerve, but did not damage any nerve. After surgery, all animals were placed on the thermal pad for at least 30 minutes.

(4) Chronic complete Freund's adjuvant induced inflammatory pain model (CFA)

Complete Freund's adjuvant, CFA; sigma-Aldrich, USA) was mixed with 0.9% sterile saline at a 1:1 volume ratio to prepare a homogeneous emulsion by vortex shaker. A microinjector was used to accurately withdraw 10 μl of CFA emulsion, needle the central plantar region of the left hind paw of the mouse, slowly inject into subcutaneous tissue, stop the needle 20 s after injection to ensure emulsion deposition, then slowly rotate the withdrawal needle, avoiding fluid extravasation.

(5) ACC cannula implantation and microinjection of drugs

Cannula implantation and microinjection references ：Zhao, M. G., H. Toyoda, Y. S. Lee, L. J. Wu, S. W. Ko, X. H. Zhang, Y. Jia, F. Shum, H. Xu, B. M. Li, B. K. Kaang and M. Zhuo (2005). "Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory." Neuron47(6): 859-872.

After anesthetizing the mice with isoflurane, the mice were fixed on a stereotactic apparatus to ensure head stability, shaved head hair and sterilized with iodophor. Cutting the top skin of the cranium, leveling the cranium, exposing the Bregma point, determining the ACC coordinates (AP+0.9 mm, ML + -0.3 mm and DV-1.5 mm) of the mouse according to the brain map by taking the Bregma point as an origin, drilling holes at the positioning points, slowly inserting a sleeve to a target depth, fixing the sleeve by dental cement, waiting for the dental cement to solidify (about 15 minutes), removing the clamp, inserting a catheter cap, screwing the clamp, and placing the clamp on a heat preservation pad until the mouse is completely awake.

Microinjection drug and behavioral testing were performed 7 days after SNI surgery. A solution of ATPA (dissolved in physiological saline, tocris Cookson, bristol, UK) was prepared at a concentration of 10, 50 ng/kg. Microinjection was performed using a syringe pump (Razel Scientific Instruments, stamford, CT, USA) and Hamilton syringe (Hamilton, reno, NY, USA), with the injection tube and syringe connected by a polyethylene tube. The drug was injected at a slow rate (0.5 μl/min), 0.5 μl was injected on each side, and the needle was left for 1 minute after injection, ensuring drug diffusion. Behavior testing was performed 5 minutes after microinjection was completed.

(5) Behavior testing

1) Mechanical foot-reduction threshold test

The left hindlimb foot contraction threshold (PWT) of mice was measured by the Up-Down method using Von Frey fiber filaments. A series of filaments had different bending forces including 0.008 g, 0.02 g, 0.04 g, 0.16 g, 0.4g, 0.6 g, 1 g, 1.4 g, and 2.0 g. The mice were placed in a plexiglas cage with a metal mesh at the bottom, and after they were calm (no exploratory or combing action) the test was started, starting with 0.4g of fiber filaments, vertically tapping the central area of the hind limb plantar (avoiding the foot pad edge) of the mice, to a slight bend and hold for 3-6 seconds. If there is no response, the next coarser filament is used until a positive response occurs, including licking, biting or suddenly withdrawing the hind limb. If a positive reaction occurs at 0.4g, the next finer filament is used until no reaction occurs. The test was repeated 5 times to give results, with a1 minute interval between each positive result.

2) Hotplate testing

The mice were placed on a hot plate tester at 55.+ -. 1 ℃. The latency of the first positive response in the hind paw of the mouse is recorded in reference ：Zhou, Z., W. Shi, K. Fan, M. Xue, S. Zhou, Q. Y. Chen, J. S. Lu, X. H. Li and M. Zhuo (2021). "Inhibition of calcium-stimulated adenylyl cyclase subtype 1 (AC1) for the treatment of neuropathic and inflammatory pain in adult female mice." Mol Pain17: 17448069211021698.. Positive responses include rapid paw lifting, paw licking and jumping. To avoid burn in mice, the test time was up to 30 seconds.

2. Experimental results

(1) Post-SNI ACC GluK receptor mediated LTD impairment

To explore the change in GluK receptor-mediated LTD in chronic pain, this example recorded the excitatory postsynaptic field potential (fEPSP) of mouse ACC brain slices in sham-operated and SNI model groups by MED64 multichannel system. The results show that sham mice ACC brain slices induced stable long-term inhibition LTD (baseline slope reduced to 58.19±8.23%, n=5 brain slices/5 mice, p < 0.001) under KA (5 μm) stimulation, whereas the LTD induction of SNI mice was significantly impaired (baseline slope maintained at 93.49±15.08%, n=5 brain slices/5 mice, p > 0.05) (fig. 1E-1F). This result suggests that chronic nerve damage results in GluK receptor-mediated synaptic plasticity dysfunction within ACC, suggesting that GluK receptor-mediated LTD dysfunction may be a key mechanism for pain sensitization.

(2) Up-regulation of GluK receptor expression following chronic pain model

To further verify whether GluK's 1 receptor function was altered following chronic pain, we examined the expression of GluK protein following SNI by Western Boltting experiments. As shown in fig. 2, both GluK protein and GluK2/3 protein expression were up-regulated (n=3) following the SNI chronic pain model, suggesting that GluK receptor function was enhanced following chronic pain.

(3) Dose-dependent generation of analgesic effects by microinjection of ATPA with ACC in SNI model

In SNI model mice, bilateral microinjection GluK a receptor-specific agonist ATPA significantly improved mechanical allodynia (fig. 3A) and thermal hyperalgesia (fig. 3B), with a significant increase in mechanical Paw Withdrawal Threshold (PWT) of 50 ng/kg ATPA to 0.291± 0.0538 g (p <0.001, tukey test) compared to the saline control group (PWT: 0.051±0.0067 g), whereas 10 ng/kg and 150 ng/kg reached 0.065±0.005 g (p < 0.05) and 0.32±0.0506 g (p < 0.01), respectively, 5 minutes after administration (fig. 3C), indicating a dose-dependent analgesic effect of ATPA. In the hot plate test, the reaction latency of 50 ng/kg ATPA group was prolonged from 6.249 + -0.4442 s to 8.955 + -0.5114 s (p < 0.01) in the physiological saline group. Further analysis showed that a single injection of 50 ng/kg ATPA was effective for 5 minutes, with analgesic effect lasting about 20 minutes (fig. 3D), suggesting a rapid and reversible onset of action.

(4) Dose-dependent generation of anti-inflammatory pain effects by microinjection of ACC ATPA in CFA model

In the CFA-induced inflammatory pain model, the mechanical foot-shrinking threshold (PWT) of CFA-induced inflammatory pain mice was significantly increased after ACC injection of different concentrations of ATPA (10, 50, and 150 ng/kg). PWTs of 10, 50, and 150 ng/kg ATPA groups were elevated to 0.28±0.06 g (p < 0.01), 0.9±0.06 g (p < 0.001), and 0.95±0.05 g (p < 0.001), respectively, compared to Saline control group (Saline group, 0.07±0.01 g), indicating concentration-dependent analgesic effect of ATPA (fig. 4B). Furthermore, the analgesic effect exhibited a time-dependent change after a single ACC injection of ATPA. The onset of drug effect was 5 minutes after injection, and the analgesic effect was continued for about 20 minutes, followed by gradual regression (fig. 4C). In conclusion, ATPA is effective in alleviating CFA-induced hyperalgesia, and the analgesic effect is concentration and time dependent.

It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

Claims (10)

1. The application of GluK1 receptor agonist in preparing medicine for treating chronic pain is characterized in that GluK receptor agonist is ATPA or pharmaceutically acceptable salt and solvate thereof, wherein the ATPA is (RS) -2-amino-3- (3-hydroxy-5-tert-butyl isoxazol-4-yl) propionic acid, and the structural formula is:。
2. 2. The use of GluK a receptor agonist according to claim 1 for the manufacture of a medicament for the treatment of chronic pain, wherein the medicament comprises GluK a receptor agonist as the sole active ingredient.
3. 3. Use of GluK receptor agonist according to claim 2 in the manufacture of a medicament for the treatment of chronic pain, wherein the medicament comprises an active ingredient for enteral or parenteral administration and a suitable pharmaceutically inert organic or inorganic carrier material.
4. 4. Use of GluK receptor agonist according to claim 3 in the manufacture of a medicament for the treatment of chronic pain wherein the suitable pharmaceutically organic or inorganic inert carrier material is selected from one or more of water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.
5. 5. The use of GluK a receptor agonist according to claim 3 in the manufacture of a medicament for the treatment of chronic pain, wherein the medicament is in the form of a solid or liquid dosage form.
6. 6. The use of GluK a receptor agonist in the manufacture of a medicament for the treatment of chronic pain according to claim 5 wherein the solid dosage form is a tablet, coated tablet, suppository or capsule.
7. 7. The use of GluK a receptor agonist in the manufacture of a medicament for the treatment of chronic pain according to claim 5 wherein the liquid dosage form is a solution, suspension or emulsion.
8. 8. Use of a GluK receptor agonist according to claim 3 in the manufacture of a medicament for the treatment of chronic pain, wherein the medicament comprises suitable pharmaceutical excipients.
9. 9. The use of GluK a receptor agonist according to claim 8, wherein the adjuvant is selected from one or more of a preservative, a stabilizer, a wetting agent, an emulsifier, an osmotic pressure regulator and a buffer.
10. 10. The use of GluK a receptor agonist according to claim 1 in the manufacture of a medicament for the treatment of chronic pain, wherein the chronic pain is neuropathic pain or inflammatory pain.