CN115475162B

CN115475162B - Application of 4-isobutyl-2-pyrrolidone in preparing analgesic drug and analgesic drug

Info

Publication number: CN115475162B
Application number: CN202211269786.8A
Authority: CN
Inventors: 蒋卫; 高天乐; 沈斌彬; 蒋建东; 戴冕; 韩燕星; 李秀君; 王璐璐
Original assignee: ZHEJIANG ZHENYUAN PHARMACEUTICAL CO Ltd
Current assignee: ZHEJIANG ZHENYUAN PHARMACEUTICAL CO Ltd
Priority date: 2022-10-18
Filing date: 2022-10-18
Publication date: 2023-06-02
Anticipated expiration: 2042-10-18
Also published as: CN115475162A

Abstract

The invention belongs to the technical field of analgesic drugs, and in particular relates to application of 4-isobutyl-2-pyrrolidone in preparing analgesic drugs and an analgesic drug. The 4-isobutyl-2-pyrrolidone has analgesic activity and can be used for preparing analgesic drugs. The 4-isobutyl-2-pyrrolidone and the gamma-aminobutyric acid medicines are combined, the 4-isobutyl-2-pyrrolidone can reduce the transmembrane resistance of MDCK-MDR1 cells, increase the permeability among cells, promote the transmembrane transport of gamma-aminobutyric acid medicines, increase the medicine concentration of the gamma-aminobutyric acid medicines reaching target tissues, and enhance the analgesic effect of the gamma-aminobutyric acid medicines.

Description

Application of 4-isobutyl-2-pyrrolidone in preparing analgesic drug and analgesic drug

Technical Field

The invention belongs to the technical field of analgesic drugs, and in particular relates to an application of 4-isobutyl-2-pyrrolidone in preparing an analgesic drug and an analgesic drug.

Background

Pain is an unpleasant sensory and emotional experience that occurs when the body is damaged, and is a complex clinical manifestation of pathological and physiological changes, and can be local or systemic manifestations of diseases, and people will always refer to diseases with the above "pain" as the main symptom as "pain syndrome". Currently, GABA drugs such as opioid analgesics, non-steroidal anti-inflammatory drugs, gabapentin and pregabalin, and tricyclic antidepressants are classical clinical analgesics and have a long history of use in pain therapy. However, the clinical application of the traditional Chinese medicine composition also has a plurality of defects and defects, and adverse reactions are daunting. For example, some strong opioid analgesics clinically used for severe pain caused by postoperative, wound, cancer and the like are easy to generate dependence in clinical application, and can generate a certain degree of tolerance, and once the mental dependence appears, the strong opioid analgesics bring about greater problems for clinical medication; non-gamma-aminobutyric acid (GABA) medicines such as gabapentin and pregabalin have limited therapeutic effects on chronic pain; tricyclic antidepressants generally have only mild effects and slow onset of action.

Therefore, how to make up the shortages of the analgesic effect of the existing compounds, and how to find, develop and develop safe and powerful clinical intervention drugs is urgent. There is no description of the analgesic effect of 4-isobutyl-2-pyrrolidone (4-isobutyl-2-pyrrolidone).

Disclosure of Invention

The invention aims to make up the defects of the prior art, provide compounds related to analgesic effect and strengthen the analgesic effect of gamma-aminobutyric acid medicaments.

The invention provides an application of 4-isobutyl-2-pyrrolidone in preparing analgesic drugs.

The invention also provides application of the 4-isobutyl-2-pyrrolidone in preparing products for enhancing analgesic effect of gamma-aminobutyric acid medicines.

The invention also provides application of the 4-isobutyl-2-pyrrolidone combined gamma-aminobutyric acid medicines in preparing products for treating pain.

Preferably, the molar concentration ratio of the 4-isobutyl-2-pyrrolidone to the gamma-aminobutyric acid medicine in the product is (1-4): (1-4).

Preferably, the gamma-aminobutyric acid medicine comprises pregabalin and/or gabapentin.

Preferably, the drug includes one of an external drug, an internal drug, and an injection drug.

Preferably, the pain comprises peri-operative pain and/or chronic pain.

The invention also provides an analgesic, the main components of which are 4-isobutyl-2-pyrrolidone and gamma-aminobutyric acid medicines.

Preferably, the molar concentration ratio of the 4-isobutyl-2-pyrrolidone and the gamma-aminobutyric acid medicine in the analgesic is (1-4): (1-4).

The invention provides an application of 4-isobutyl-2-pyrrolidone in preparing analgesic drugs. The chemical formula of the 4-isobutyl-2-pyrrolidone is C ₈ H ₁₅ NO, which belongs to pyrrolidone compounds, has analgesic activity and can be used for treating analgesic drugs.

Further, 4-isobutyl-2-pyrrolidone and gamma-aminobutyric acid drugs, such as pregabalin or gabapentin, are used in combination, the gamma-aminobutyric acid drugs exert analgesic effect by penetrating the blood brain barrier into the central system, and 4-isobutyl-2-pyrrolidone can reduce the transmembrane resistance of MDCK-MDR1 cells, increase the permeability among cells, promote the transmembrane transport of gamma-aminobutyric acid drugs, and increase the drug concentration of gamma-aminobutyric acid drugs reaching target tissues. The peripheral neuropathic pain model shows that 4-isobutyl-2-pyrrolidone can enhance the inhibition effect of pregabalin on mechanical pain and cold pain of a mouse with the peripheral neuropathic pain model; the incision pain model shows that 4-isobutyl-2-pyrrolidone can increase the inhibition effect of pregabalin on mechanical pain and thermal pain of incision pain mice. 4-isobutyl-2-pyrrolidone can enhance the analgesic effect of pregabalin.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIGS. 1-1 and 1-2 show the effects of mechanical pain (foot-shortening threshold) in mice model of peripheral neuropathic pain in different treatment groups, wherein P <0.05, <0.01 (vs. baseline BL of the individual), #P <0.01 (vs. 0.12mmol/kg of 4-isobutyl-2-pyrrolidone) and # - [ P ] <0.01 (vs. 0.12mmol/kg of pregabalin);

FIG. 2 is a graph showing the area under the curve (AUC) comparison of the effects of treatment on mechanical pain in mice model of peripheral neuropathic pain in different treatment groups, wherein P <0.01 (compared to the blank control group) and #P <0.01 (compared to the 4-isobutyl-2-pyrrolidone 0.12mmol/kg group);

FIGS. 3-1 and 3-2 are effects (response time) of different treatment groups on cryopain sensitivity in mice model of peripheral neuropathic pain;

FIG. 4 shows the area under the curve (AUC) comparison of the effects of treatment on cryopain in mice model of peripheral neuropathic pain in different treatment groups, wherein P <0.01 (comparison to the blank control group) and #P <0.05 (comparison to the 4-isobutyl-2-pyrrolidone 0.12mmol/kg group);

FIGS. 5-1 and 5-2 are effects of different treatment groups on mechanical pain (footwell threshold) in incision pain model mice, wherein P <0.05, # P <0.01 (vs. self baseline BL), # P <0.05, # P <0.01 (vs. 4-isobutyl-2-pyrrolidone 0.12mmol/kg group), and & & P <0.01 (vs. pregabalin 0.12mmol/kg group);

FIG. 6 is a graph showing the area under the curve (AUC) comparison of mechanical pain in mice treated with different treatment groups versus incision pain model, wherein P <0.001 (compared to the blank) and # # # P <0.001 (compared to the 4-isobutyl-2-pyrrolidone 0.12mmol/kg group);

FIGS. 7-1 and 7-2 are graphs of the effect (response time) of different treatment groups on cold pain in incision pain model mice;

FIG. 8 is a graph showing the area under the curve (AUC) comparison of the effect of treatment on pain in mice with incision pain models for different treatment groups;

fig. 9-1 and 9-2 effect of different treatment groups on incision pain model mice thermal pain (response time), where P <0.05, <0.01 (compared to self baseline BL);

FIG. 10 is a graph showing the area under the curve (AUC) comparison of the effect of treatment on pain in incision pain model mice for different treatment groups;

FIG. 11 shows the results of cell viability assays;

FIG. 12 is a graph showing the results of transmembrane resistance measurements, wherein #P <0.05, #P <0.01 (as compared to the Blank group);

FIG. 13 is a graph showing the results of transmembrane transport assays, wherein #P <0.05, #P <0.01 (as compared to the pregabalin 100 μg/ml group).

Detailed Description

The chemical formula of the 4-isobutyl-2-pyrrolidone is also known as 4-isobutyl-2-pyrrolidone ₈ H ₁₅ NO, which is an OP compound, has a CAS number of 61312-87-6. The structural formula of the 4-isobutyl-2-pyrrolidone is as follows:

the 4-isobutyl-2-pyrrolidone has analgesic activity, and the experiment of a peripheral neuropathic pain model shows that the 4-isobutyl-2-pyrrolidone can inhibit mechanical pain sensitivity of a peripheral neuropathic pain model mouse in a dose-dependent manner, is ineffective to cold pain sensitivity, and can be used for preparing analgesic drugs. The invention has no strict requirement on the source of the 4-isobutyl-2-pyrrolidone, and can be purchased conventionally.

The invention also provides application of the 4-isobutyl-2-pyrrolidone in preparing products for enhancing analgesic effect of gamma-aminobutyric acid medicines. The gamma-aminobutyric acid medicine preferably comprises pregabalin and/or gabapentin, further comprises pregabalin or gabapentin, and more preferably comprises pregabalin. The product of the present invention preferably comprises one of an external, an internal and an injection. The 4-isobutyl-2-pyrrolidone can reduce the transmembrane resistance of MDCK-MDR1 cells, increase the permeability among cells, promote the transmembrane transport of gamma-aminobutyric acid drugs, and increase the drug concentration of the gamma-aminobutyric acid drugs reaching target tissues; the experimental result of mice shows that 4-isobutyl-2-pyrrolidone can remarkably increase the inhibitory effect of pregabalin on mechanical pain sensitivity of mice with peripheral neuropathic pain models, and enhance the inhibitory effect of pregabalin on mechanical pain sensitivity and thermal pain sensitivity of mice with incision pain. Therefore, 4-isobutyl-2-pyrrolidone can enhance the analgesic effect of gamma-aminobutyric acid medicines.

In view of the above, the invention also provides application of the 4-isobutyl-2-pyrrolidone combined with gamma-aminobutyric acid in preparing a product for treating pain. The molar concentration ratio of 4-isobutyl-2-pyrrolidone to pregabalin in the product of the present invention is preferably (1 to 4): (1 to 4), more preferably 1:1. the product of the present invention preferably comprises one of an external, an internal and an injection; the pain preferably comprises peri-operative pain or chronic pain, further preferably one or more of neuropathic pain, central pain and cancer pain, more preferably one or more of cold pain, thermal pain and mechanical pain. In the present invention, the mass of 4-isobutyl-2-pyrrolidone per 1g of the product is preferably 25 to 300mg, more preferably 30 to 250mg, still more preferably 50 to 200mg, most preferably 100 to 150mg; the mass of the gamma-aminobutyric acid based drug per 1g of the product is preferably 25 to 300mg, more preferably 30 to 250mg, still more preferably 50 to 200mg, most preferably 100 to 150mg. In the specific implementation process, 1g of the medicine/person, namely 25-300 mg of 4-isobutyl-2-pyrrolidone/person and 25-300 mg of gamma-aminobutyric acid medicine/person are taken, so that pain can be effectively treated.

The gamma-aminobutyric acid medicine preferably comprises pregabalin and/or gabapentin, further comprises pregabalin or gabapentin, and more preferably comprises pregabalin. The chemical name of pregabalin, namely (S) -3-aminomethyl-5-methylhexanoic acid, is C ₈ H ₁₇ NO ₂ Is an antiepileptic drug, CAS accession no: 148553-50-8. The invention has no strict requirement on the source of the pregabalin, and can be purchased conventionally. The invention combines the 4-isobutyl-2-pyrrolidone with the gamma-aminobutyric acid medicines to enhance the analgesic effect of the gamma-aminobutyric acid medicines.

The invention also provides an analgesic drug which mainly comprises 4-isobutyl-2-pyrrolidone and gamma-aminobutyric acid drugs. The gamma-aminobutyric acid medicine preferably comprises pregabalin and/or gabapentin, further comprises pregabalin or gabapentin, and more preferably comprises pregabalin. The molar concentration ratio of 4-isobutyl-2-pyrrolidone and gamma-aminobutyric acid in the analgesic drug of the present invention, and the sources of 4-isobutyl-2-pyrrolidone and pregabalin are the same as above, and are not described herein.

For further explanation of the present invention, the application of 4-isobutyl-2-pyrrolidone provided in the present invention to analgesia, an analgesic drug, is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Animal experiment

1. Main experiment materials

4-isobutyl-2-pyrrolidone (from Ding Yuan (Tianjin) Biotechnology Co., ltd.), pregabalin (from Beijing carboline technology Co., ltd.), C57BL/6 female mice (from Beijing Vitrenlhua laboratory animal technology Co., ltd.) of 5-6 weeks week old and 20-30 g weight.

2. Animal experiment

2.1 establishing a model mouse for peripheral neuropathic pain

(1) After anesthetizing the mice with chloral hydrate (5%), the skin of the left hind limb of the mice was cut with scissors and the biceps femoris was cut along the femur, exposing the sciatic nerve and its three terminal branches: sural nerve, common sural nerve and tibial nerve. The total fibular and tibial nerves were axonally severed using butterfly scissors and the lower residual nerves were removed 2mm, taking care not to contact or pull the distal fibular nerve. The torn muscle and skin were replaced and sutured using suture No. 5. In operation, mice were placed on a heating pad to maintain body temperature between 35-38 ℃. After the operation, the hind paws on the same side of the mice can generate mechanical pain sensitivity and cold pain sensitivity reaction within 10 days, and the drug effect detection can be carried out after the pain sensitivity reaction.

The method for measuring the mechanical pain sensitivity comprises the following steps: the mice were placed in plastic cages with metal mesh at the bottom, left hind paw soles of the mice were stimulated with increasing force using standard-compliant von frey filaments after resting for 1 hour until hind paw of the mice was retracted. Applying 5 (10) times of pressure at a frequency of 1 time/s at the time of testing, and reaching a response threshold when the hind paw of the mouse is retracted at least 3 (6) times;

the method for measuring cold pain sensitivity is to test mice for cold pain sensitivity by dripping acetone on the plantar surface of the hind paw. The mice were observed for immediate response to the application of acetone, the response time was recorded and the extent of the response was scored. The reaction time is the time corresponding to the reactions of lifting feet, rapid shaking, licking soles and the like after the stimulation of the acetone.

(2) The mice from step (1) were randomly and equally divided into a blank group, a medium dose 4-isobutyl-2-pyrrolidone-treated group, a high dose 4-isobutyl-2-pyrrolidone-treated group, a pregabalin-treated group and a 4-isobutyl-2-pyrrolidone-combined pregabalin-treated group, each group having 6 to 7 animals, and administered in the manner of table 1.

TABLE 1 modes of administration

(3) After the administration according to step (2), mechanical pain and cold pain were measured for 0min (baseline, BL) for the administration of the blank control group, the medium dose 4-isobutyl-2-pyrrolidone-treated group and the high dose 4-isobutyl-2-pyrrolidone-treated group, respectively, 30min,60min,120min,240min,6h,8h,24h, specifically, the mechanical pain threshold of mice at each time point was measured, and the results were shown in fig. 1-1, fig. 1-2, fig. 2 and table 2; the cold stimulus response time of mice at each time point was measured, and the results are shown in FIG. 3-1, FIG. 3-2, FIG. 4 and Table 3.

TABLE 2 treatment effects of combination of 4-isobutyl-2-pyrrolidone and pregabalin on mechanical pain in peripheral neuropathic pain mice

Note that: data are presented with mean±sem; * P <0.05, # P <0.01 (vs. self baseline BL), # P <0.01 (vs. OP 0.12), $ P <0.01 (vs. P0.12), $ P <0.01 (vs. blank).

Table 3.4-isobutyl-2-pyrrolidone and pregabalin combination treatment effect data on cold pain in peripheral neuropathic pain mice

Note that: data are presented with mean±sem; p <0.01 (compared to the placebo group) and #p <0.05 (compared to the OP0.12 group).

From FIGS. 1-1-, 1-2, 2 and Table 2, it can be seen that 4-isobutyl-2-pyrrolidone produced an inhibitory effect on mechanical pain in mice model of peripheral neuropathic pain at a dose of 0.24mmol/kg (significant anti-mechanical pain effects compared to baseline at the 4 hour time point) (grey shaded portion represents normal mechanical pain threshold range for intact (normal) animals). And in the observation time range after administration, 0.12mmol/kg of 4-isobutyl-2-pyrrolidone has no obvious inhibition effect on mechanical pain of the peripheral nerve pathological pain model mice. 4-isobutyl-2-pyrrolidone can significantly increase the inhibitory effect of pregabalin on mechanical pain in peripheral neuropathic pain mice (0.12 mmol/kg of 4-isobutyl-2-pyrrolidone was combined with 0.12mmol/kg of pregabalin at each time point within 0.5-4 hours, and the analgesic effect was significantly better than that of the 0.12mmol/kg pregabalin administration group).

From figures 3-1, 3-2, 4 and table 3, it can be seen that at a single time point, 4-isobutyl-2-pyrrolidone at a dose of 0.12mmol/kg or 0.24mmol/kg had no significant inhibitory effect on the cold pain of the peripheral neuropathic pain model mice (no significant difference from Baseline (BL) or blank control at each time point; grey shaded portions represent the range of normal thresholds for intact animals). However, by comparing the area under the curve (AUC) with values from 0 to 8 hours integrated together, 4-isobutyl-2-pyrrolidone significantly increased the inhibitory effect of pregabalin on cold pain in peripheral neuropathic pain mice (0.12 mmol/kg of 4-isobutyl-2-pyrrolidone in combination with 0.12mmol/kg of pregabalin, exhibited significant differences compared to the blank or 0.12mmol/kg of 4-isobutyl-2-pyrrolidone group; the analgesic effect of the combination group was superior to that of the 0.12mmol/kg pregabalin administration group).

1-1, 1-2, 3-1, 3-2, 4 and tables 2-3, it can be seen that 4-isobutyl-2-pyrrolidone can dose-dependently inhibit mechanical nociception in a peripheral neuropathic pain model mouse, but has no significant inhibitory effect on nociception in a peripheral neuropathic pain model mouse. In addition, the 4-isobutyl-2-pyrrolidone can obviously increase the inhibition effect of pregabalin on mechanical pain sensitivity and cold pain sensitivity of peripheral neuropathic pain mice.

2.2 establishing a model of incision pain mice

(1) After the mice were anesthetized with chloral hydrate (5%), a 5mm longitudinal incision was made along the central axis at the left hind paw sole of the mice, 3mm near the heel, using a scalpel, the incision penetrating the skin, fascia and muscle. The bent forceps are taken to penetrate the muscles and tendons exposed from the lower part, and are pulled open by force (the integrity of the muscles and tendons is not destroyed). The torn muscle and skin were replaced and the wound was closed using suture No. 5. In operation, mice were placed on a heating pad to maintain body temperature between 35-38 ℃. The periwound mechanical pain threshold is detected and compared with the preoperative mechanical pain threshold. The mechanical pain sensitivity and cold and hot pain sensitivity reaction of the incision side hind paws reach the peak value after the operation for about 24 hours, and the drug effect detection is carried out in the period of time.

Wherein the mechanical pain sensitivity and cold pain sensitivity measuring method is the same as the above, and the thermal pain sensitivity measuring method comprises the following steps: thermal pain was measured by placing a radiant heat source (Ugo basic, italy) on the plantar surface of the hind paw. The machine will automatically record the hind paw withdrawal latency. The stimulus intensity was adjusted so that the baseline latency period of the normal animals was set at 2-6 seconds, the deadline of the rats was set at 20 seconds, and the deadline of the mice was set at 15 seconds.

(2) Grouping and dosing according to step 2.1 (3) following dosing according to step (2), mechanical pain and cold pain were measured in the blank, medium dose 4-isobutyl-2-pyrrolidone treatment, pregabalin treatment and 4-isobutyl-2-pyrrolidone combined pregabalin treatment for 0min (baseline, BL), 30min,60min,120min,240min,6h, and specifically, mechanical pain thresholds were measured for mice at each time point, and the results are shown in fig. 5-1, fig. 5-2, fig. 6 and table 4; the cold stimulus response time of mice at each time point was measured and scored according to scoring criteria (score 0 = no response; 1 = startle response, no significant hindpaw retraction; 2 = stimulated hindpaw retraction; 3 = stimulated hindpaw continuous retraction with tremor and licking of hindlimb) as shown in figures 7-1, 7-2, 8 and table 5; the mice heat stimulus response time was measured at each time point and the results are shown in FIG. 9-1, FIG. 9-2, FIG. 10 and Table 6.

TABLE 4 treatment effects of mechanical pain sensitivity in cut mice with 4.4-isobutyl-2-pyrrolidone and pregabalin combination

Note that: data are presented with mean±sem; * P <0.01 (compared to self baseline BL), #p <0.05, #p <0.01, #p <0.001 (compared to OP 0.12), P <0.05, & P <0.01 (compare to P0.12) and P <0.001 (compare to blank).

TABLE 5 treatment effects of 4-isobutyl-2-pyrrolidone and pregabalin combination on Cold pain in cut mice

Note that: data are presented with mean±sem.

Table 6 4 therapeutic Effect of combination of isobutyl-2-pyrrolidone and pregabalin on thermal pain in incision-pain mice

Note that: data are presented with mean±sem, P <0.05, P <0.01 (as compared to self baseline BL).

From FIGS. 5-1, 5-2, 6 and Table 4, it can be seen that 4-isobutyl-2-pyrrolidone was associated with a dose of 0.24mmol/kg, at the 3 hour time point, with significant anti-mechanical sensitization compared to baseline. And, 4-isobutyl-2-pyrrolidone in combination with pregabalin (1-4 hours compared to baseline) produced an inhibitory effect on mechanical pain in cut pain mice (grey shaded portions represent the range of normal mechanical pain thresholds in intact animals). And in the observation time range, the drug effect of the 4-isobutyl-2-pyrrolidone combined pregabalin group with the concentration of 0.12mmol/kg is obviously better than that of the pregabalin group with the concentration of 0.12mmol/kg (the analgesic effect of pregabalin is obviously lower than that of the combined drug in the statistical sense in 1, 3 and 4 hours).

As can be seen from fig. 7-1, 7-2, 8 and table 5, 0.12mmol/kg of pregabalin did not exhibit significant anti-cold pain effects after administration. 0.12mmol/kg of 4-isobutyl-2-pyrrolidone in combination with pregabalin had no statistically significant significance, although it had a tendency to reduce cold pain.

From fig. 9-1, 9-2, 10 and table 6, it can be seen that 0.12mmol/kg of pregabalin does not exhibit a significant effect of thermal pain sensitivity during the observation time, whereas 0.12mmol/kg of 4-isobutyl-2-pyrrolidone in combination with pregabalin significantly increases the response time of incision pain mice to thermal stimulation within 2-6 hours, thereby exhibiting a significant analgesic effect.

It can be seen in conjunction with figures 5-1, 5-2, 6, 7-1, 7-2, 8, 9-1, 9-2, 10 and the contents of tables 4-6 that 4-isobutyl-2-pyrrolidone can increase the inhibitory effect of pregabalin on mechanical and thermal pain in incision pain mice, but does not significantly increase the inhibitory effect of pregabalin on cold pain in incision pain mice.

Example 2

Cell experiment

1. Main experiment materials

4-isobutyl-2-pyrrolidone (available from Ding Yuan (Tianjin) Biotechnology Co., ltd.), pregabalin (available from Beijing carboline technology Co., ltd.), MDCK-MDR1 cells, DMEM medium (available from Beijing Fine biology Co., ltd.), fetal bovine serum, penicillin, streptomycin, glutamine and HBSS solution.

2. Experimental procedure

2.1MDCK-MDR1 cell culture and plating

(1) Cell culture

Adding fetal bovine serum, penicillin, streptomycin and glutamine into a DMEM culture medium as a basic culture medium to enable the content of the fetal bovine serum, the penicillin, the streptomycin and the glutamine to reach 10% v/v, 100U/mL and 1wt.% in sequence, so as to obtain a complete culture medium;

MDCK-MDR1 cells are processed according to the ratio of 2 to 3 multiplied by 10 ⁵ cells/cm ² Is cultured in complete DMEM medium at 37deg.C under a relative humidity of 95% and a gas composition of 5% CO ₂ Culturing in a constant temperature incubator with +95% air, and passaging every 2-3 days. The cells were stored frozen and resuscitated by conventional methods.

(2) Culture in culture plate

Taking one bottle of MDCK-MDR1 cells growing in a T75 cell bottle, discarding the culture medium when the growth is observed under a mirror to be not less than 80%, rinsing with PBS, adding 1mL of PBS containing 0.02% EDTA and 0.25% pancreatin for soaking, and standing at 37 ℃ for 5min. The cells should be detached from the flask as viewed under a microscope. Neutralizing with 5mL of complete culture medium, blowing to separate cells from the wall of the culture flask, and displaying the floating state of single cells;

a12-well 0.4 μm polycarbonate membrane Transwell plate (bottom area 1.12 cm) ² ) Each well was multiplied by 2X 10 according to the bottom area of the polyester film Transwell plate ⁵ cells/cm ² MDCK-MDR1 cells were added in an amount of 0.5mL per well, and 1.5mL of complete DMEM medium was added outside the scaffolds, calculated from individual cells. Culturing in a constant temperature incubator with 37 deg.C, 5% CO2/95% air and relative humidity of 95% for 7 days, and changing liquid every day during culturing.

2.2 Experimental procedure

(1) Cytotoxicity assays

Cell viability was measured by CCK-8, in particular: MDCK-MDR1 cells in logarithmic growth phase were cultured at 1.0X10 ⁵ /cm ² Is plated in 96-well plates, equally divided into a blank control group, a low dose 4-isobutyl-2-pyrrolidone-treated group and a high dose 4-isobutyl-2-pyrrolidone-treated group, and each of which is dosed in the manner of table 7, and 6 replicates are obtained.

Table 7 mode of administration

After the end of the dosing, the fresh medium was changed and 100. Mu.L of 10% CCK-8 was added to each well and incubated for 60min at 37 ℃. OD values were measured at 450nm by Infinite M200 microplate reader, and the ratio (%) of the number of surviving cells to the blank was calculated, and the results are shown in FIG. 11.

From FIG. 11, it can be seen that there was no significant difference in cell viability between the 1% w/v OP or 2% w/v OP dosing group and the placebo group, indicating that 1% w/v OP or 2% w/v OP dosing did not cause significant apoptosis, thereby disrupting the integrity of the membrane composed of MDCK-MDR1 cells used in the transmembrane transport experiments below, thereby affecting transmembrane resistance TEER.

(2) Step 2.1MDCK-MDR1 cells were equally divided into a blank control group, a low dose 4-isobutyl-2-pyrrolidone-treated group and a high dose 4-isobutyl-2-pyrrolidone-treated group, and were measured after 7 days of incubation using a Millipore MERS00002 Millicell-ERS cell transmembrane resistance meter, and were measured once every 20 minutes based on the time of the first measurement for 5 hours, and immediately dosed as 9 when the 4 th measurement was ended (i.e., at 60 minutes), with 4 replicates for each treated group.

Table 9 mode of administration

The measurement results of the cell transmembrane resistor are shown in FIG. 12. As can be seen from fig. 12, 4-isobutyl-2-pyrrolidone administration group (1% w/v and 2% w/v) significantly reduced transmembrane resistance TEER and increased intercellular permeability within 80-300 minutes after administration compared with the blank group.

(3) The transmembrane transport experiments were carried out with reference to "Effect of Sinomenine on the In Vitro Intestinal Epithelial Transport of Selected Compounds" (Lu Z, chen W, viljoen A, et al Effect of sinomenine on the in vitro intestinal epithelial transport of selected compounds [ J ]. Phytotherapy Research: an International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives,2010,24 (2): 211-218.) and ensure that the transmembrane resistance of each small well was >1000Ω, in particular dosing was carried out in the manner of Table 10, in 7 replicates per treatment group.

Table 10 mode of administration

The transmembrane transport efficiency was measured by rapidly sampling 50. Mu.L (after sampling at each time point, re-replenishing 50. Mu.L of fresh HBSS solution and then rapidly inserting back into the original well plate) from the AP side (small hole) and BL side (large hole) at 0, 30, 60, 90 and 120min after the administration according to Table 10, referring to "micro dialysate sampling of free sinomenine, ligustrazine, gabapentin, paracetamol, pregabalin and amitriptyline in rat blood and brain tissue fluid and HPLC-MS/MS quantitative method study (pharmaceutical journal, 9 th 2198-2206 of 2020, 9 th page total)", and the results are shown in Table 11 and FIG. 13.

TABLE 11 transmembrane transport efficiency of 4-isobutyl-2-pyrrolidone and pregabalin combination

Note that: data were shown with mean±sem, #p <0.05, #p <0.01 (compared to pregabalin treated group).

From table 11 and fig. 13, it can be seen that the transmembrane transport rate of pregabalin is significantly increased 60-120 minutes after administration in the presence of 1% w/v 4-isobutyl-2-pyrrolidone compared to the pregabalin control group, and that 4-isobutyl-2-pyrrolidone, when combined with pregabalin, promotes the transmembrane transport of pregabalin and increases the drug concentration of pregabalin to the target tissue.

According to the embodiment, the 4-isobutyl-2-pyrrolidone has analgesic activity, can reduce the transmembrane resistance of MDCK-MDR1 cells, increase the permeability among cells, promote the transmembrane transport of pregabalin, increase the drug concentration of pregabalin reaching target tissues, and enhance the analgesic effect of pregabalin.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

Application of 4-isobutyl-2-pyrrolidone in preparing analgesic medicine is provided.
Application of 2, 4-isobutyl-2-pyrrolidone in preparing medicines for enhancing analgesic effect of gamma-aminobutyric acid medicines;

the gamma-aminobutyric acid medicine is pregabalin.
Application of 3, 4-isobutyl-2-pyrrolidone combined gamma-aminobutyric acid medicines in preparing medicines for treating pain;

the gamma-aminobutyric acid medicine is pregabalin and/or gabapentin.
4. The use according to claim 3, wherein the molar concentration ratio of 4-isobutyl-2-pyrrolidone to gamma-aminobutyric acid in the medicament is (1-4): (1-4).
5. The use according to any one of claims 2 to 4, wherein the medicament comprises one of an external, an internal and an injection.
6. The use according to any one of claims 2 to 4, wherein the pain comprises peri-operative pain and/or chronic pain.
7. An analgesic drug, characterized in that the active ingredients of the analgesic drug are 4-isobutyl-2-pyrrolidone and gamma-aminobutyric acid drugs;

the molar concentration ratio of the 4-isobutyl-2-pyrrolidone and the gamma-aminobutyric acid medicine in the analgesic is (1-4): (1-4);

the gamma-aminobutyric acid medicine is pregabalin.