CN110917190B

CN110917190B - Preparation for inhibiting adverse reaction of opioid analgesic and application thereof

Info

Publication number: CN110917190B
Application number: CN201911212238.XA
Authority: CN
Inventors: 周培岚; 苏瑞斌; 张艺馨; 陈铭
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2022-12-23
Anticipated expiration: 2039-11-29
Also published as: CN110917190A

Abstract

The invention relates to a preparation for inhibiting adverse reactions of opioid analgesic drugs and application thereof, wherein the preparation contains 17-N-allylamino-17-demethoxygeldanamycin or/and one or more of homologues thereof, and the opioid analgesic drugs comprise: morphine hydrochloride tablet, morphine sulfate sustained release tablet, codeine phosphate tablet, morphine hydrochloride injection, pethidine hydrochloride injection and fentanyl injection or a combination of more than one of them. By adopting the technical scheme of the invention, the 17-AAG can obviously inhibit the symptoms of prompting withdrawal reaction along with chronic administration of morphine, can obviously inhibit physical tolerance and mental dependence of morphine, and simultaneously has double effects of resisting tumors and inhibiting tolerance and dependence of opioid analgesic drugs by reasonably using the 17-AAG and homologs thereof and the opioid analgesic drugs, thereby reducing the use of other drugs.

Description

Preparation for inhibiting adverse reaction of opioid analgesic and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a preparation for inhibiting opioid analgesic adverse reactions and application thereof.

Background

Cancer patients are afflicted by pain, opioid analgesic drugs are generally adopted for analgesic treatment clinically to relieve pain feeling of the cancer patients, but the opioid drugs are easy to generate tolerance and dependence.

The loss of tolerance, i.e. analgesic efficacy, is one of the common complications of opioid therapy, leading to an ever increasing dosage requirement and a reduced effectiveness over time.

Dependence is the development of an altered physiological state revealed by opioid withdrawal syndrome involving autonomic nerves and physical hyperactivity.

Opioid tolerance and dependence are well-known physiological phenomena, and a large number of researches find that opioid adverse reactions comprise tolerance and dependence with rather complicated mechanisms and involve various neurotransmitters such as excitatory amino acid, 5-hydroxytryptamine, dopamine, norepinephrine and acetylcholine. At present, except for the adoption of an opioid receptor antagonist, no medicament for obviously inhibiting opioid dependence and tolerance is clinically used, but the side effect of the use of the opioid receptor antagonist is obvious, namely, the analgesic and sedative effects of the opioid are completely inhibited while the opioid dependence tolerance is inhibited. The international pharmaceutical industry has many researches on the generation and inhibition technology of opioid adverse reactions, for example, the "analgesic tolerance inhibitor" with patent number 200980119667.0, applied by japan synergetics and zymophyte Kabushiki Kaisha, aims to develop a preparation capable of inhibiting opioid adverse reactions.

The inhibition of the tolerance and dependence of opioid analgesics is particularly important, because opioid analgesics are important drugs for relieving cancer pain of cancer patients, especially patients with advanced cancer, and the dosage of opioid analgesics is larger and the effect is worse with the prolonging of the medication time, and the patient is caused to have dependence, which is not different from frostbite for patients with advanced cancer. Therefore, it is important to develop a preparation capable of suppressing the tolerance and dependence of opioid analgesics, so that it can be used in the treatment of patients requiring long-term analgesia.

Disclosure of Invention

The invention aims to provide a preparation for inhibiting opioid analgesic adverse reactions, which solves the problems in the background art.

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

a preparation for inhibiting adverse reaction of opioid analgesic drugs, which contains 17-N-allylamino-17-demethoxygeldanamycin or/and one or more of its homologues.

Further, the molecular formulas of the 17-N-allylamino-17-demethoxygeldanamycin and its homologue are as shown in (I) to (VI):

wherein: (I) In the formula, when R is OCH ₃ When (I) is geldanamycin;

when R is NHCH ₂ CH＝CH ₂ When (I) is 17-N-allylamino-17-demethoxygeldanamycin, abbreviated as 17-AAG;

when R is NHCH ₂ CH ₂ N(CH ₃ ) ₂ When (I) is 17-demethoxy-17- [ [2- (dimethylamino) ethyl ] ethyl]Amino group]Geldanamycin, abbreviated 17DMAG.

Preferably, the formulation contains 17-AAG.

Further, the opioid analgesic is a drug which acts on a mu opioid receptor to exert an analgesic effect.

Further, the opioid analgesic includes: morphine hydrochloride tablet, morphine sulfate sustained release tablet, codeine phosphate tablet, morphine hydrochloride injection, pethidine hydrochloride injection and fentanyl injection or a combination of more than one of them.

Specifically, the effects of the preparation comprise reducing analgesic tolerance and reducing opioid dependence of cancer patients when using opioid.

Preferably, the effective amount of 17-AAG or/and homologues thereof is 0.1-1000nmol in the medicament to be administered intracerebroventricularly.

Preferably, the effective amount of 17-AAG or/and homologues thereof is 0.1-1000nmol in the amount of the drug to be administered via the abdomen.

The 17-AAG or/and the homologues thereof are applied to the medicines for inhibiting the adverse reaction of opioid analgesic medicines used by cancer patients.

The invention has the beneficial effects that:

1. the 17-AAG is simultaneously administrated with morphine, the analgesic effect of morphine can be inhibited when the morphine is administrated acutely, but the tolerance of morphine can be obviously inhibited when the morphine is administrated chronically: as can be seen from example 2, the inhibition (tolerance) slope of analgesic effect of experimental mice on morphine was significantly reduced by the simultaneous administration of 17-AAG with morphine for 7 days, and the withdrawal was promoted by naloxone (10 mg/kg) 7 days after the administration, and the symptoms of the withdrawal-promoting reaction were significantly inhibited by the chronic administration of 17-AAG with morphine.

2. The 17-AAG is simultaneously administrated along with morphine, and can obviously inhibit physical tolerance and mental dependence of morphine: example 3 can see that morphine-combined 17-AAG drug group mice can not form conditional site preference in conditional training, while morphine-combined vehicle group and morphine group can form obvious conditional site preference, which shows that 17-AAG can obviously inhibit not only opioid physical tolerance and dependence, but also opioid mental dependence.

3. The 17-AAG and the homologues thereof have the characteristics of inhibiting the growth of tumor cells and the adverse reaction of opioid analgesic drugs, and can reduce the treatment cost of cancer patients and the adverse reaction caused by the mixed use of a plurality of drugs: mu-opioid receptors (MOR) play an important role in opioid-induced analgesia, tolerance, dependence, and the like. Heat shock protein 90 (HSP 90) is a promising target in cancer treatment, tumor cells have obvious dependence on the Hsp90, the content of the Hsp90 in the tumor cells is 2 to 3 times of that of normal cells, and the Hsp90 has an enhancement effect on MOR phosphorylation. The research at present considers that MOR phosphorylation receptor endocytosis can be one of the biological mechanisms of opioid drug dependence tolerance, and the invention discovers that 17-AAG can obviously inhibit the enhancement effect of Hsp90 beta on MOR phosphorylation (example 4) and can obviously inhibit the enhancement effect of Hsp90 beta on extracellular signal-regulated kinase (ERK) phosphorylation (example 5). Because cancer patients generally need to take opioid for a long time to relieve pain, adverse reactions can be generated, particularly dependence and tolerance are caused, and the analgesic effect of opioid is influenced, the cancer patients need to take drugs which can inhibit the adverse reactions of opioid analgesic drugs at the same time. Under the technical scheme of the invention, the compound preparation reasonably uses 17-AAG and homologues thereof and opioid analgesic drugs, and has double effects of resisting tumors and inhibiting tolerance and dependence of the opioid analgesic drugs, thereby reducing the use of other drugs.

Drawings

FIG. 117-Effect of AAG and geldanamycin on analgesia by morphine upon acute administration

FIG. 217-Effect of AAG on the analgesic Rate of Morphorphine upon Chronic dosing

FIG. 317 Effect of AAG on analgesic tolerance to Morphorphine upon chronic dosing

FIG. 417-Effect of chronic administration of AAG on morphine withdrawal response

FIGURE 517-Experimental time-Schedule of conditional site preference for morphine by chronic administration of AAG

FIG. 617-grading of morphine conditional site preference for chronic administration of AAG

FIG. 717-distance of movement of medication accompanying case preferred by chronic administration of AAG for morphine conditioned place

FIG. 8 electrophoretic patterns of Hsp90 β capable of significantly promoting MOR phosphorylation

FIG. 917-electrophoretogram of significant inhibition of the enhancement of MOR phosphorylation by Hsp90 β by AAG

FIG. 10 electrophoretogram of geldanamycin also capable of significantly inhibiting the enhancement of MOR phosphorylation by Hsp90 β

FIG. 11 is an electrophoretogram of Hsp90 beta capable of obviously promoting ERK phosphorylation

FIG. 1217-electrophoretogram of the effect of significantly inhibiting the enhancement of ERK phosphorylation by Hsp90 β by AAG

FIG. 13 electrophoretogram of geldanamycin also capable of significantly inhibiting the enhancement of ERK phosphorylation by Hsp90 β

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1: simultaneous administration of 17-AAG with morphine, test and results of acute administration

A total of 40C 57/B6 mice were randomly divided into 4 groups, and the basal pain zone of the animals was determined on a hot plate (55 ℃) before administration, followed by intracerebroventricular administration of 5 microliters of 17-AAG (10 nmol), vehicle (solvetent), 17-AAG (10 nmol) and geldanamycin (10 nmol), respectively, 15 minutes later followed by subcutaneous administration of physiological saline (10 ml/kg), morphine (Mor, 10 mg/kg), and the pain zone of the animals was determined on a hot plate 30min after administration. Maximum percent analgesia (% MPE) = (post-administration pain domain-pre-administration pain domain)/(60-pre-administration pain domain) × 100%. It can be seen from Table 1 and FIG. 1 that 17-AAG has no analgesic effect on mice, morphine has a significant analgesic effect on mice, and 17-AAG (10 nmol) and geldanamycin (10 nmol) can significantly inhibit the analgesic effect of morphine.

TABLE 1 Effect of AAG and geldanamycin on analgesia by morphine upon acute administration

Note: n =10, mean ± sem,. P <0.001, compared to the 17-AAG (10 nmol) + saline group; # P <0.05, compared to vehicle + morphine (10 mg/kg).

In addition, in other strains of mice (Kunming, ICR), it was observed that 17-AAG and its homolog Geldanamycin (Geldaramycin) both when administered intracerebroventricularly and intraperitoneally significantly inhibited the analgesic effect of acute morphine administration.

Example 2 experiment and results of Simultaneous administration of AAG with morphine and Chronic administration

A total of 30C 57/B6 mice were kept in the experimental environment for 2-3 days, randomized into 3 groups, dosed twice daily, starting at 8 AM. In 6 pm, animals in group 00 were administered morphine (100 mg/kg) continuously and tested in the pain area for 7 days, naloxone (10 mg/kg) was administered by intraperitoneal injection 2 hours after the last administration, and immediately after administration, the mice were placed in a withdrawal observation box, and withdrawal symptoms (withdrawl signs) such as jumping, writhing, head-shaking, tremor, hair-combing, etc. within 15min were observed and scored. As can be seen from Table 2 and FIG. 2, the analgesic percentage of morphine (100 mg/kg) after the first day of administration is as high as 94%, while 17AAG (10 nmol) can significantly inhibit the analgesic effect of morphine, reducing the analgesic rate to 78.6%, but the analgesic effect of morphine after the administration of the vehicle in the ventricle is not significantly affected, and the analgesic rate is 98%. However, with the prolonged administration time, the analgesic effect of morphine is reduced, namely morphine tolerance is achieved, the analgesic rate is 33.69% by day 7, 17AAG can obviously inhibit the tolerance effect of morphine, the analgesic rate is 51.16% by day 7, and as can be seen from figure 3, the analgesic reduction slope of the 17AAG + morphine group is obviously lower than that of the vehicle + morphine group and the single morphine group. As can be seen from table 3 and fig. 4, 17AAG was able to significantly suppress morphine physical dependence withdrawal symptoms after naloxone was used to promote withdrawal.

TABLE 2 Effect of AAG on analgesic tolerance to morphine upon chronic administration

Note: * P <0.001, P <0.01, # P <0.05 vs. first day of the same group, # P <0.05, 17-AAG pretreated group compared to vehicle pretreated or morphine alone, two-way anova.

TABLE 3 scoring of morphine withdrawal response upon chronic administration of AAG

Note: # P <0.05, 17-AAG pretreated vs vehicle pretreated or morphine alone, one-way ANOVA.

In conclusion, on a morphine chronic administration physical dependence promotion withdrawal model, compared with a morphine combined solvent group, the analgesic effect of a morphine combined 17-AAG drug group is obviously enhanced from the 2 nd day to the 7 th day after administration, and the 17-AAG can obviously inhibit the physical dependence and tolerance of morphine. The definite molecular biology and neural loop mechanisms of 17-AAG for inhibiting morphine tolerance and dependence are still under further study, but it is possible that the transmitters are subjected to stress inhibition in different brain regions, normal transmitter balance is maintained, after opioid drugs act on central or peripheral MOR, GABAergic interneurons are inhibited, V-aminobutyric acid release is reduced, and after downstream transmitter release is de-inhibited, some adverse reactions are caused.

Example 3 Experimental and results of Effect of concurrent administration of AAG with morphine on mental dependence

The experimental time design for conditional site-preference for morphine for chronic administration of 17-AAG is shown in FIG. 5, with 50C 57/B6 mice, animals raised in the experimental environment for 2-3 days, pre-acclimation period (d 1-d 3): and (3) adapting for 1 time every day for 15min every time after 3 days, recording the residence time of two sides within 15min after two times, and screening out unqualified animals by taking the average value of the two times of d2 and d3 as a previous measurement result.

Animal elimination criteria: the difference of the residence time of the total animals in the box bodies on the two sides of the black box and the white box exceeds 100s, natural bias is considered to exist, and individuals with the residence time close to that in the box bodies on the two sides are removed; the difference of the residence time of the overall animals in the boxes on the two sides does not exceed 100s, and the individuals with the residence time exceeding 560s in the boxes on any one side are removed. Individuals with poor basic status are removed, and saline is injected subcutaneously after each preadaptation is taken out, so that the animals are familiar with the operation.

Conditional training period (d 4-d 8): based on the previous measurements, 40 animals were randomly divided into 4 groups of 10 animals each. Selecting bias or non-bias program, if bias, carrying medicine on non-bias side; if it is not biased, a counter-balanced design is used, i.e., half of the animals are accompanied by drugs in the black box and the other half are accompanied by drugs in the white box. Each training was started once every day at 8 am and at 14 pm. The partition is inserted to confine the animal in a black or white box. Each animal receives task training twice a day for 30 min/time; alternatively administering sterilized water for injection or corresponding medicine for training, and balancing the training sequence. The specific process is as follows: animals of each group were trained on the non-concomitant side after administration of sterile water for injection in the morning and on the concomitant side after administration of the corresponding drug in the afternoon. And by analogy, training is continuously carried out for 5 days. 8 in the morning of each day, the training time is 00-12, the training time is 30-19 in the afternoon. The test group of morphine 10mg/kg with physiological saline was administered subcutaneously, and the test group of 17AAG (10 nmol/5. Mu.l) and vehicle (5. Mu.l) was administered intracerebroventricularly.

Test period (d 9): the test was performed the next day after the training period, and the animals were not dosed and the residence time on the concomitant side and on the non-concomitant side was recorded within 15 min. The mouse residence time in the white box-the black box residence time is the conditional place preference score.

TABLE 4 Effect of AAG on conditional location preference for morphine

Note: # P <0.05, 17-AAG pretreated group compared to vehicle pretreated morphine group, one-way anova.

As can be seen from table 4 and fig. 6 and 7, apart from the distance of the concomitant medication box movement and the conditional position preference score, on the mouse conditional position preference model (CPP), the morphine-combined 17-AAG drug group mouse failed to develop effective conditional position preference in conditional training, whereas the morphine-combined vehicle group developed significant conditional position preference. The 17-AAG can obviously inhibit not only the physical dependence of the opioid, but also the mental dependence of the opioid. The 17-AAG and the analogues thereof are undergoing an anti-tumor phase II clinical test, and the opioid is a commonly used cancer analgesic, so when the two medicines are taken together, the 17-AAG and the analogues thereof can obviously reduce the dependence, tolerance and addiction of the opioid. Of course, 17-AAG and its analogs can also exert similar effects when non-cancerous analgesia is required, requiring long-term administration of opioids.

Example 4: experiments and results of 17-AAG inhibition of HSP90 protein to enhance phosphorylation of mu-opioid receptor (MOR)

In the experiment, MOR-CHO is adopted to transiently transfect pcDNA3.1myc/hisB-Hsp90 beta or pcDNA3.1myc/hisB (vector) by a liposome method, then 17-AAG (10 mu M) or a solvent (solvent) is given for 20h, and finally MOR receptor agonist DAMGO (10 mu M) is given to act on cells for 5, 15 and 30min respectively. Cells were lysed with RIPA cell lysate on ice for 30Min. After centrifugation at 14000rpm (rpm) for 15min, cell supernatants were collected, 5 × Loading buffer was added, boiling water denaturation was performed for 5min, proteins were electrophoresed for 90min with 10% SDS-PAGE gel 100V, 160mA was transferred to PVDF membrane, blocked with 5% skim milk powder, MOR phosphorylation was detected using anti-MOR phosphorylation antibody (1: 1000), MOR was detected using anti-MOR phosphorylation antibody (1: 1000), and Hsp90 β was detected using anti-Myc antibody (1: 2000).

From figure 8, it can be seen that Hsp90 β is able to significantly promote MOR phosphorylation, as evidenced by a significantly deepened MOR phosphorylated protein band. After the administration of 17-AAG (10. Mu.M) for 20 hours, the increase of MOR phosphorylation by Hsp90 beta was significantly inhibited and the protein band was significantly reduced, as compared with the vehicle (solvent) (FIG. 9). Similarly, geldanamycin (10 μ M) was also able to significantly inhibit the enhancement of MOR phosphorylation by Hsp90 β after 20 hours of action (figure 10). The 17-AAG and the analogues thereof can inhibit phosphorylation of M0R, and probably is a molecular mechanism that the 17-AAG and the analogues thereof can inhibit adverse reactions such as analgesic tolerance, dependence and the like of opioid drugs.

Example 5: experiment and result of using 17-AAG to inhibit HSP90 protein to enhance ERK phosphorylation effect

In the experiment, 24h after instantly transfecting pcDNA3.1myc/hisB-Hsp90 beta or pcDNA3.1myc/hisB (vector) by adopting an MOR-CH0 liposome method, 17-AAG (10 mu M) or a solvent (solvent) is given for acting for 20h, and finally MOR receptor agonist DAMGO (10 mu M) is given for acting on cells for 5, 15 and 30Min respectively, and the cells are cracked by 30Min on ice by using RIPA cell lysate. After centrifugation at 14000rpm for 15min, cell supernatants were collected, 5 Xloading buffer was added, denaturation in boiling water for 5min, proteins were electrophoresed with 10% SDS-PAGE gel 100V for 90min, 160mA was transferred to PVDF membrane, blocked with 5% skim milk powder, ERK phosphorylation was detected with anti-ERK phosphorylation antibody (1: 1000), ERK was detected with anti-ERK antibody (1: 1000), and Hsp 90. Beta. Was detected with anti-Myc antibody (1: 2000).

From fig. 11, hsp90 β can significantly promote ERK phosphorylation, which is indicated by a significantly deeper band of ERK-phosphorylated protein. After the administration of 17-AAG (10 μ M) for 20 hours, the increase of ERK phosphorylation by Hsp90 β can be inhibited significantly compared with the vehicle (solvent), which is shown by the fact that the band of ERK phosphorylation protein is reduced significantly (FIG. 12). Similarly, geldanamycin (10 μ M) was also able to significantly inhibit the enhancement of ERK phosphorylation by Hsp90 β after 20 hours of exposure (fig. 13). After the action of an agonist, a phosphorylation receptor of MOR is endocytosed to recruit beta-arresting and activate a downstream MAPK pathway, so that the phosphorylation of ERK is up-regulated, which is one of biological mechanisms of opioid dependence tolerance, and the phosphorylation of ERK can be inhibited by 17-AAG and analogues thereof, and probably is a molecular mechanism that the analgesic tolerance and the psychotropic behavior of opioid can be inhibited by 17-AAG and analogues thereof.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

Use of 17-N-allylamino-17-demethoxygeldanamycin in the manufacture of a medicament for inhibiting the psychotropic dependence of an opioid analgesic for use by a patient suffering from cancer.