CN112402428B

CN112402428B - Application of remazolin in preparation of medicine for treating postoperative hyperalgesia induced by opioid

Info

Publication number: CN112402428B
Application number: CN202011344050.3A
Authority: CN
Inventors: 李依泽; 张麟临; 于泳浩; 王国林; 李晶; 丁冉; 董贝贝; 陈怡�
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2022-02-25
Anticipated expiration: 2040-11-26
Also published as: CN112402428A

Abstract

The invention relates to application of remazolam in treating postoperative hyperalgesia induced by opioid. The invention proves that remimazolam can inhibit remifentanil-induced postoperative hyperalgesia by promoting GABAa receptor expression. The research result of the invention provides a novel treatment way for hyperalgesia caused by opioid, and the invention has good application prospect clinically.

Description

Application of remazolin in preparation of medicine for treating postoperative hyperalgesia induced by opioid

Technical Field

The invention belongs to the field of biological medicine, and relates to application of remazolam in treatment of postoperative hyperalgesia induced by opioid.

Background

Opioids are the most important analgesic drugs for clinical treatment of acute and chronic pain and cancer pain, and the clinical dosage is very large, but they can activate the in vivo nociceptive mechanism while relieving pain, which is manifested by enhanced responsiveness of the body to nociceptive stimuli and increased demand of analgesic drugs, i.e. opioid-induced hyperalgesia (OIH). Remifentanil is a super-short-acting mu-opioid receptor agonist, and is widely applied to analgesia in clinical operation due to the advantages of quick response, quick clearance, no accumulation, metabolism independent of liver and kidney functions and the like. However, remifentanil-induced hyperalgesia (RIH) occurs at a much higher rate than other opioid analgesics, up to 85%. Another study found that patients with surgery times longer than 2 hours had an incidence of RIH of 32.7% and cumulative infusion rates exceeding 30 μ g/kg even as high as 41.8%. RIH is mainly characterized in that remifentanil is infused at the speed of 0.05-0.3 mu g/kg/min for 60-90 min, the degree and range of postoperative incision pain are increased, and the requirement of opioid analgesics is increased. RIH not only reduces the analgesic effect of the medicine, but also promotes pain perception, produces abnormal pain, even causes postoperative chronic pain, and the patient has more and more large demand for opioid dosage, which not only increases hospitalization time, medical expenses and occupies medical resources, but also increases physical and psychological trauma of the patient, aggravates the pain of the patient and seriously affects the life quality of the patient. Currently, there is no clinically effective treatment, mainly because the mechanism of its occurrence is not yet clear, and it is therefore imperative to deeply elucidate the pathogenesis of remifentanil-induced hyperalgesia and to find effective therapeutic strategies.

Disclosure of Invention

The invention aims to provide a new pharmaceutical application of remimazolam, and particularly provides an application of remimazolam in preparing a medicine for preventing and treating remifentanil-induced postoperative hyperalgesia.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides the use of an agent which inhibits excitability of spinal cord dorsal horn neurons in the manufacture of a medicament for the prevention or treatment of opioid-induced post-operative hyperalgesia. Preferably, the opioid is remifentanil.

Further, the agent includes an agent that inhibits translocation of GluN2B basement membrane of NMDAR, an agent that inhibits phosphorylation of Ser845-GluA1 subunit of AMPAR, or an agent that promotes expression of GABAa receptor.

Still further, the agent that promotes GABAa receptor expression is remazolam.

Further, the opioid-induced postoperative hyperalgesia includes incision hyperalgesia after opioid infusion and hyperalgesia induced after simple opioid infusion.

Further, post opioid infusion incision hyperalgesia includes post-operative mechanical hyperalgesia and thermal hyperalgesia.

Further, hyperalgesia induced after a mere opioid infusion is thermal hyperalgesia.

Further, the medicament comprises an agent for inhibiting excitability of spinal dorsal horn neurons as an active ingredient and a pharmaceutically acceptable ingredient.

Furthermore, the ratio of the agent for inhibiting excitability of spinal cord dorsal horn neurons in the medicament of the invention as an active ingredient is 0.1-99% w/w, and the ratio of the pharmaceutically acceptable ingredient is 0.1-99% w/w.

Furthermore, the dosage form of the medicine is any pharmaceutically acceptable dosage form. Including but not limited to tablets (including dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, effervescent tablets, etc.), hard capsules (including enteric-coated capsules), soft capsules, granules, pills, micro-pills, dropping pills, dry suspensions, oral solutions, dry syrups, powders, oral suspensions, oral quick-release or slow-release or controlled-release dosage forms, injections (including sterile powder injections for injection and freeze-dried powder injections), aqueous solution injections, ointments, gels, emulsions, patches, suppositories, gels, and the like.

The medicament of the invention can be applied by oral route, and can also be administrated by intravenous, intramuscular, intradermal or subcutaneous injection route.

The agents of the invention may be used alone or in combination with other agents for the treatment of opioid-induced post-operative hyperalgesia.

Pharmaceutically acceptable ingredients of the present invention include, but are not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, buffers, pH adjusting agents, preservatives, antioxidants, bacteriostats, stabilizers, suspending agents, solubilizers, surfactants (e.g., wetting agents), colorants, and isotonic (i.e., which renders the formulation isotonic with the blood or other relevant bodily fluids of the subject patient). Suitable carriers, diluents, excipients and the like can be found in standard pharmaceutical books. See, e.g., the handbook of Pharmaceutical Additives (handbook), second edition (editors m.ash and i.ash), 2001(SynapseInformation Resources, inc., endiott, New York, USA); remington's pharmaceutical Science, 18 th edition, Mack Publishing Company, Easton, Pa., 1990; and the Handbook of pharmaceutical Excipients (Handbook of pharmaceutical Excipients), second edition, 1994.

The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, and the like, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue undesirable toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

A "patient" of the invention can be an animal, a mammal, a placental mammal, a rodent (e.g., guinea pig, hamster, rat, mouse), a murine (e.g., mouse), a lagomorph (e.g., rabbit), a canine (e.g., dog), a feline (e.g., cat), an equine (e.g., horse), a porcine (e.g., pig), an ovine (e.g., sheep), a bovine (e.g., cow), a primate, an ape (e.g., monkey or ape), a simian (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or a human.

The invention provides a method of inhibiting excitability of spinal dorsal horn neurons in vitro comprising administering an agent that inhibits membrane translocation of the GluN2B subunit of NMDAR, an agent that inhibits phosphorylation of the Ser845-GluA1 subunit of AMPAR, or an agent that promotes GABAa receptor expression.

Further, the agent that promotes GABAa receptor expression is remazolam.

The invention provides a medicament for preventing or treating opioid-induced post-operative hyperalgesia, comprising an agent that inhibits excitability of spinal cord dorsal horn neurons.

Preferably, the agent comprises an agent that inhibits translocation of the GluN2B basement membrane of NMDAR, an agent that inhibits phosphorylation of the Ser845-GluA1 subunit of AMPAR, or an agent that promotes GABAa receptor expression.

More preferably, the agent that promotes GABAa receptor expression is remazolam.

The invention is as defined above for the above-mentioned drugs.

The remazolam can be remazolam and a medicinal derivative of remazolam, and the medicinal derivative comprises pharmaceutically acceptable salt or ester and the like.

"prevention" as used herein refers to the prevention or reduction of the development of post-operative hyperalgesia after use in the presence of possibly post-operative hyperalgesia-inducing factors.

"treating" in the context of the present invention means to reduce the degree of postoperative hyperalgesia, or to normalize postoperative hyperalgesia, or to slow the progression of postoperative hyperalgesia in the liver.

The invention has the advantages and beneficial effects that:

the invention discloses remazolam for treating remifentanil-induced postoperative hyperalgesia for the first time, and a novel treatment way of hyperalgesia caused by opioid is found.

Drawings

Figure 1 shows a graph of the results of remimazolam inhibition of notched hyperalgesia following remifentanil infusion, where a: mechanical pain sensation; b: thermal pain sensation, wherein n is 6; p < 0.001; p < 0.05; $ P < 0.01; $ P < 0.001;

figure 2 shows a graph of the results of remimazolam inhibition of hyperalgesia induced following simple remifentanil infusion, where a: mechanical pain sensation; b: thermal pain sensation, wherein n is 6; p < 0.001; $ P < 0.01; $ P < 0.001;

figure 3 is a graph showing the results of remazolam's effect on the expression level of GABAa receptor, GluN2B membrane protein, Ser845GluA1 phosphorylation, where a: immunoblot; b: a statistical histogram in which n is 6; p < 0.001; $ P < 0.01; $ P < 0.001;

figure 4 shows a graph of the results of remazolam effect on GABAa receptor expression levels, where a: immunohistochemistry; b: a statistical histogram in which n is 6; p < 0.001; $ P < 0.001.

Detailed Description

The invention will be more readily understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to limit the scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 Effect of Remazolam on opioid-induced notched hyperalgesia

1. Grouping experiments: 42 male SD rats with age of 1 month and weight of 100-120 g are purchased from the experimental animal center of military medical science institute of liberty military of China. Dividing into 7 groups by adopting a random number table method (n is 6):

a normal saline group (NS group) which is infused with normal saline with the volume equal to that of remifentanil for 60min through tail vein;

remifentanil + incisional pain group (RI group), remifentanil (batch number: A010919, Yichang Fuyao Co., Ltd.) was infused via tail vein at 1. mu.g.kg^-1·min^-1Setting up incision pain model for 60 min;

remazolin 1 (batch number: Jiangsu Henrie pharmaceuticals GmbH) + remifentanil + incisional pain group (R1RI group) was infused via tail vein with remazolin 1mg kg^-1·h^-1Remifentanil 1 ug kg^-1·min^-1Setting up incision pain model for 60 min;

2 mg-kg of remimazolam is infused into the remimazolam 2+ remifentanil + incisional pain group (R2RI group or RRI group) through tail vein^-1·h^-1Remifentanil 1 ug kg^-1·min^-1For 60min, incision pain is establishedA model;

the remimazolam 4+ remifentanil + incisional pain group (R4RI group) is infused with 4 mg.kg of remimazolam through the tail vein^-1·h^-1Remifentanil 1 ug kg^-1·min^-1Setting up incision pain model for 60 min;

remifentanil (R group), 1 ug kg remifentanil through tail vein infusion^-1·min^-1The total time is 60 min;

remazolam 2+ remifentanil (RR group) via tail vein infusion of 2mg kg of Remazolam^-1·h^-1Remifentanil 1 ug kg^-1·min^-1The total time is 60 min.

2. Making an incision pain model: the rats were anesthetized by 2% sevoflurane inhalation, the left hind paw was disinfected, a longitudinal incision of about 1cm in length was made from the proximal 0.5cm of the sole to the toe, the skin was incised, the plantar muscles were lifted with ophthalmologic forceps and separated longitudinally to the periosteum, keeping the muscles intact and attached. After hemostasis was applied, the skin was sutured with 4-0 silk. The incised skin cannot overlap, invert, split. The postoperative wound is disinfected by iodophor, and a small amount of erythromycin ointment is smeared to prevent infection.

3. Behavioral experiments: the heat stimulated withdrawal latency (PWL) and mechanical stimulated withdrawal threshold (PWT) were determined 24h before remifentanil infusion (T0), 2, 6, 24, and 48h after infusion stopped (T1-4), with laboratory temperatures 18-22 ℃ and rest. The method comprises the steps of measuring PWL by adopting an YLS-6B intelligent hot plate instrument (Huaibei Zhenghua biological instrument equipment Co., Ltd.), recording the time from the contact of the right rear foot with the hot plate to the occurrence of any reaction of retraction, tiptoe standing, struggle, hoarse and foot licking as PWL, continuously measuring for 3 times at intervals of 5min, and taking the average value as PWL (sec). To prevent scalding of the rat paw, the PWL was limited to 20 s. Rats were placed in 20cm by 20cm metal cages and after 30min, the bones of the right hind paw were stimulated between the 2 and 3 phalanges with BSEVF3 von Frey cellosilk (Harvard Apparatus, USA), pressure was applied vertically, the appearance of a rapid paw withdrawal response, licking of the right paw or pressure at the time of whooshing was recorded, measurements were made 3 times in succession at 5min intervals and the mean value was PWT (g). To prevent damage to the rat paw, the maximum pressure was set at 50g, and rats above this pressure were excluded from the experiment.

4. Western blot: after the last 1 behavioral determination, the rats were sacrificed, and the spinal cord dorsal horn L4-5 segment was taken and Western blot was used to determine the expression of the spinal cord dorsal horn GABAa receptor, Ser845-GluA1, GluN2B and GAPDH proteins. Adding precooled tissue protein lysate into dorsal horn tissue of spinal cord, and grinding into tissue homogenate. Centrifuging the homogenate at 4 deg.C for 5min at 12000rpm with a centrifugation radius of 10cm to obtain supernatant as total protein of spinal cord tissue. Membrane proteins were extracted using a membrane protein extraction kit (Thermo, USA) according to the instructions. The extracted protein was denatured at 95 ℃ for 5 min. Transferring the protein onto a PVDF membrane, adding 5% skimmed milk powder, sealing at room temperature for 2h, washing the membrane, adding a primary antibody, an anti-rabbit GABAa receptor antibody, an anti-rabbit Ser845-GluA1 antibody, an anti-rabbit GluN2B antibody and an anti-rabbit GAPDH antibody (all 1:1000, Abcam company, USA) respectively, incubating at 4 ℃ overnight, washing TBST for 3 times, each time for 5min, adding a goat anti-rabbit secondary antibody or a goat anti-mouse secondary antibody (dilution 1: 2000, Beijing Zhonghua Kingjin Qiaoji organism, Ltd.) to incubate at room temperature for 2h, washing the TBST for 3 times, each time for 5min, adding a luminescent reagent in a dark room, and scanning and imaging. And analyzing the band gray value by using Gene Tools image analysis software, and reflecting the target protein expression level by using the ratio of the target protein band gray value to the GAPDH band gray value.

5. Immunohistochemistry: rats were anesthetized with sevoflurane, the L4-5 spinal cord segment was removed, fixed in freshly prepared 4% paraformaldehyde solution for 6h, paraffin sections (4 μm thick) were prepared conventionally, and the distribution of GABAa receptors in the dorsal horn of the spinal cord was examined by immunohistochemical staining. The method comprises the following specific steps:

dewaxing: baking the paraffin sections in an oven (60 ℃, 2h), and removing surface paraffin; and (3) dehydrating: placing the dewaxed slices in xylene (I, II) and gradient ethanol (100%, 95%, 90%, 80%, 70%, 50%, 30%) solution cylinders for dehydration, respectively soaking for 5min, and washing for 3 times with 1 XPBS; antigen retrieval: soaking the dehydrated slices in citric acid buffer solution (0.01M), heating at high temperature for boiling for 3min (95 deg.C), naturally cooling to room temperature, heating again for 2min, and naturally cooling; dropping a proper amount of peroxidase blocking agent on the section, incubating for 15min (room temperature) to block non-specific staining caused by endogenous catalase, and then washing for 3 times by 1 XPBS; dripping 5% Bovine Serum Albumin (BSA) on the slices, incubating for 20min (room temperature), removing the BSA, and naturally drying; the sections were added dropwise to 100. mu.l of primary antibody to GABAa receptor (1:500 dilution) and incubated overnight at 4 ℃; discarding the GABAa receptor primary antibody on the section, washing with 1XPBS for 3 times, dripping 100 μ l (1:1000 dilution) of horseradish peroxidase-labeled secondary antibody, incubating for 60min (room temperature), and washing with 1XPBS for 3 times; dropwise adding fresh DAB working solution (A, B mother solution 500 μ l each) onto the slices, immediately observing the slices under a microscope of 100 times, and washing the slices with tap water for 3 times when the slices are found to be yellow brown (about 5 min); dropping appropriate amount of hematoxylin staining solution on the slice for 5min to stain cell nucleus; dropping appropriate amount of 1% hydrochloric acid alcohol on the slices for color separation, and cleaning the hydrochloric acid alcohol with tap water after staying for 5 seconds; placing the slices in gradient ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%) solution jar and xylene (I, II) in sequence for dehydration, transparency (each for 5min), washing with 1XPBS for 2 times, dripping appropriate amount of neutral gum, sealing, and air drying; and (4) microscopic observation: DAB appeared brown-yellow, positive cells. 5 sections of each specimen are randomly drawn, each section is observed under a light microscope (X200), 5 high-power fields which are not overlapped with the dorsal horn of the spinal cord of a rat are selected, positive nerve cells are marked, and the protein expression condition of the GABAa receptor is evaluated by integral optical density. All stained images were analyzed using Image J Image analysis software.

6. Statistical analysis: SPSS 18.0 statistical software is adopted for analysis, and normally distributed measurement data are averaged to be +/-standard deviation

Showing that the measurement data comparison of random block design adopts one-factor analysis of variance, the measurement data comparison of repeated measurement design adopts the analysis of variance of repeated measurement design, P<A difference of 0.05 is statistically significant.

7. Results

(1) Remifentanil infusion exacerbates post-operative mechanical and thermal hyperalgesia

With raw materialsInfusion of saline (NS) group compared to remifentanil + incisional pain (RI) group at 1. mu.g.kg^-1·min^-160 minutes resulted in a significant decrease in the withdrawal threshold (PWT) and withdrawal latency (PWL) from 2h to 48h (all P)<0.05). The infusion of saline for 60 minutes, followed by the Incision pain model group (NS + Incision) resulted in a significant decrease in PWT and PWL from 2h to 48h (all P<0.01), the degree of reduction was close to the level of the Remi group at each time point (all P)>0.05). These results show that the dose is 1. mu.g.kg^-1·min^-1Rate of infusion of remifentanil increased the opioid-induced incision burning pain and mechanical hyperalgesia. Hypersensitivity reactions to thermal and mechanical pain caused by remifentanil infusion and Incision pain models (Remi + inclusion) can last from 2 hours to 48 hours (fig. 1). Additionally, remifentanil (R) infusion alone also increased thermal and mechanical hyperalgesia (fig. 2). These results indicate that the incision after remifentanil infusion developed thermal and mechanical hyperalgesia 2h post-operatively and peaked at 48h (all P)<0.01) (fig. 1).

(2) Remifentanil infusion and incision reduced spinal cord dorsal horn GABAa receptor expression, increased expression of Ser845-GluA1 and membrane GluN2B

Pain-related protein changes in the dorsal spinal cord angle following remifentanil infusion are critical to the development of postoperative OIH. At 48h after remifentanil and incision pain model, the rats were sacrificed to remove the dorsal horn of the spinal cord and a significant decrease in GABAa receptor protein expression was found in Western blot results (P <0.05, fig. 3). The RI group was reduced by 0.51 times (P ═ 0.005), respectively, compared to the NS group. However, the dorsal spinal angle Ser845-GluA1 and the supramembranous GluN2B in the RI group were increased by 1.95-fold and 2.03-fold, respectively, compared to the NS group (<0.01, fig. 3). The above results indicate that hyperalgesia following remifentanil infusion is associated with decreased GABAa receptor expression in the dorsal horn of the spinal cord and increased Ser845-GluA1 and membrane GluN 2B.

(3) Remazolam infusion can reduce postoperative hyperalgesia caused by remifentanil

Rats in the NS group showed no change in mechanical or thermal pain threshold (P) at each time point>0.05, fig. 2A, B). The Von Frey test shows that compared to NS ratsRI can cause PWT to drop rapidly (<2 hours) and continuously decreased (>48 hours) (P<0.05, fig. 2A), indicating persistent mechanical allodynia. In addition, the hot pain behavior test showed that the RI group also resulted in a decrease in PWL (P) for at least 48 hours after surgery<0.01, fig. 2B), indicating that remifentanil infusion can cause post-operative thermal hyperalgesia. Interestingly, we observed that remazolin (divided into three concentrations of 1mg kg) was injected via the tail vein^-1·h^-1，2mg·kg^-1·h^-1， 4mg·kg^-1·h^-1) And remifentanil 1. mu.g.kg^-1·min^-1After 60min, and the incision pain model is established, remifentanil and postoperative mechanical and thermal hyperalgesia caused by incision are 2 mg-kg of remimazolam^-1·h^-1(group R2 RI), 4 mg. kg^-1·h^-1(group R4 RI) was significantly reduced, as evidenced by recovery of PWT and PWL (P)<0.01, FIG. 1), but 1mg kg of remazolam^-1·h^-1(group R2 RI) was ineffective, suggesting that the lowest effective dose of remazolam for treating remifentanil-induced post-operative hyperalgesia in rats was 2 mg-kg^-1·h^-1Remimazolam, however, had a significant reduction in remifentanil-induced hyperalgesia after 2 hours, lasting over 48 hours. Behavioral experiments further confirmed the role of remimazolam in hyperalgesia induced by simple remifentanil (group R) infusion, as evidenced by 2 mg-kg remimazolam infusion into the tail vein^-1·h^-1(group RR) reduced PWT and PWL reduction following remifentanil infusion.

(4) Remazolam reduces central sensitization caused by RIH by increasing GABAa receptor expression and reducing Ser845-GluA1 and GluN2B expression

Our previous studies found that remifentanil-induced hyperalgesia not only exhibited a reduced pain threshold in behavioral experiments, but also affected the expression of excitatory glutamate receptors that mediate pain transmission, e.g., increased expression of phosphorylation of Ser845-GluA1 subunit of AMPAR, increased GluN2B sub-basal membrane translocation of NMDAR. The role of GABAa receptor in remifentanil hyperalgesia has not been explored, and to investigate whether remimazolam also affected GABAa receptor expression, we infused 2mg kg of remimazolam via the tail vein^-1·h^-1A remifentanil incision pain model was then made and GABAa receptor membrane protein expression was measured using L4/5 spinal cord dorsal horn.

The RI group was found to have a 0.51 fold decrease in GABAa receptor membrane protein (P) compared to NS<0.01, FIG. 3), and 2 mg.kg of remazolam^-1·h^-1(RRI) can increase the expression level of GABAa receptor membrane protein to the level of NS (NS vs. RRI, 1: 0.89, P)>0.05, fig. 3), the results of GABA a receptor immunohistochemistry were very similar to those of Western blot (fig. 4), suggesting that 2 mg-kg of remazolam^-1·h^-1Can inhibit the down-regulation of GABAa receptor membrane protein caused by remifentanil incision pain model.

On the other hand, 2 mg/kg of remazolen^-1·h^-1The RI-induced phosphorylation of Ser845-GluA1 subunit of AMPAR was reduced (NS vs. rri, P ═ 0.239; fig. 3). Remazolam also reduced RI-induced GluN2B basement membrane translocation of NMDAR to a level close to the NS group (NS with RRI, P0.328; fig. 3). These results indicate that remazolam not only mediates GABAa receptor expression, but is also involved in phosphorylation of the Ser845-GluA1 subunit of spinal cord dorsal horn AMPAR and membrane translocation of the GluN2B subunit of NMDAR. Suggesting that the increased excitability of RIH-associated dorsal horn neurons can be reversed by increasing GABAa receptor expression.

In summary, our findings indicate that decreased GABAa receptor expression on spinal cord dorsal horn neuronal membranes may promote mechanical and thermal sensitization following remifentanil infusion and incision. Pharmacological activation of the GABAa receptor apparently prevented remifentanil-induced postoperative hyperalgesia. Our studies suggest that the GABAa receptor agonist, remimazolam, is a novel treatment for opioid-induced hyperalgesia.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. Use of remimazolam for the preparation of a medicament for the prevention or treatment of remifentanil-induced post-operative hyperalgesia, characterized in that the remifentanil-induced post-operative hyperalgesia is post-operative mechanical or thermal hyperalgesia.