CN112516284B

CN112516284B - Application of short peptide in preparation of product with morphine tolerance elimination effect

Info

Publication number: CN112516284B
Application number: CN202011458379.2A
Authority: CN
Inventors: 唐琼瑶; 张赭; 陈晨; 唐明希; 滕月; 周楠; 柯少溪; 董平
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-12-21
Anticipated expiration: 2040-12-11
Also published as: CN112516284A

Abstract

The research of the invention finds that the short peptide P10581 can basically and completely turn over the inflammatory pain threshold induced by carrageenan, and the analgesic effect lasts for more than 12h, which indicates that the short peptide P10581 does not produce tolerance to the analgesic effect of mechanical pain. And the low dose of the synthetic short peptide P10581 can also eliminate the tolerance of analgesic effect of morphine.

Description

Application of short peptide in preparation of product with morphine tolerance elimination effect

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of short peptide in preparation of a product with a morphine tolerance eliminating effect.

Background

Morphine (Morphine), an opioid receptor agonist, is present in an amount of 4% to 21% of the opioid, with an average of about 10%. The IUPAC name is 7,8-Didehydro-4, 5-epoxy-17-methyl-morphine-3, 6-diol. The molecular formula is C17H19NO3, and the molecular weight is 285.3. In 1806 German chemist Zeerdina first isolated it from opium and named morphine using the name Greek dream Morpheus. Morphine hydrochloride, a derivative thereof, is a common anesthetic in clinic, has strong analgesic effect and good analgesic effect selectivity, is used for treating severe pain caused by trauma, operation, burn and the like, is also used for treating angina caused by myocardial infarction, and can be used as an analgesic, antitussive and antidiarrheal agent.

Morphine is an important choice in clinical pain management, but repeated use can produce physiological tolerance and dependence. The clinical manifestations are mainly that the analgesic effect is reduced, and the analgesic effect is gradually weakened or even disappears after the opioid is continuously given, and the same analgesic effect can be obtained only by increasing the dosage of the opioid.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made in view of the above-mentioned technical drawbacks.

Therefore, the invention provides an application of a short peptide in preparing a product with the function of eliminating morphine tolerance, wherein the amino acid sequence of the short peptide is as follows: WKCNPNDDKCCRPKLKC are provided.

As a preferred scheme of the application of the short peptide in the preparation of products with the function of eliminating morphine tolerance, the short peptide comprises the following components in percentage by weight: the short peptide is low-dose short peptide, and the action concentration of the short peptide is more than 0.05 mu g/Kg.

As a preferred scheme of the application of the short peptide in the preparation of products with the function of eliminating morphine tolerance, the short peptide comprises the following components in percentage by weight: the action concentration of the low-dose short peptide is 0.05-0.1 mu g/Kg.

As a preferred scheme of the application of the short peptide in the preparation of products with the function of eliminating morphine tolerance, the short peptide comprises the following components in percentage by weight: when the action concentration of the short peptide is more than 2 mug/Kg, the short peptide can play a role of analgesia without generating tolerance.

As a preferred scheme of the application of the short peptide in the preparation of products with the function of eliminating morphine tolerance, the short peptide comprises the following components in percentage by weight: the product can be administered by intraperitoneal injection, intravenous injection, or intrathecal injection.

The invention has the beneficial effects that: the research of the invention finds that the short peptide P10581 can basically completely reverse the inflammatory pain threshold induced by carrageenan, and the analgesic effect can last for more than 12h, which indicates that the short peptide P10581 does not produce tolerance to the analgesic effect of mechanical pain. And the low dose of the synthetic short peptide P10581 can also eliminate the tolerance of analgesic effect of morphine.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a graph showing the comparison of tolerance of synthetic short peptide P10581 and morphine to rat hyperalgesia inhibitory effect.

FIG. 2 is a graph showing the effect of low dose of short peptide P10581 on rat mechanical hyperalgesia.

Figure 3 is a graph of the tolerance of low dose of short peptide P10581 to the analgesic effect of morphine.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Experimental animals, instruments and reagents:

healthy adult male SD rats (over 8 weeks) weighing 180-220 g were selected and purchased from the laboratory animal center of Xuzhou university of medicine.

Carrageenan (abbreviated as Carr) is a product of Sigma corporation of America, and 1% Carrageenan (0.1g Carrageenan dissolved in 10ml of 0.9% physiological saline solution and dispensed into 10 EP tubes) was prepared, stored at-20 deg.C, and taken out and placed on ice half an hour before use.

38500-PAM pressure application measurement System (Ugo basic Biological Research Apparatus, Commerio-valve, Italy) is composed of two parts, one part is a handheld unit, and the other part is an integrated electronic unit. The handheld unit is a force sensor designed according to the Randall-Selitto pain tester, and the integrated electronic unit can automatically record the maximum mechanical foot-shortening reflex threshold value applied to the sole of the left hind foot of the rat.

Preparation of rat inflammatory pain model:

6 μ l 1% carrageenan inflammatory pain model: according to the literature (Alessandri-Haber N, et al Neurosci. Vol 29(19), pp 6217-6288, 2009); the method described prepares a carrageenan model of inflammatory pain. The method is used for detecting the analgesic effect of the medicine in a plantar injection administration mode. The rats were injected with 1% carrageenan in an amount of 6 μ l from between the 2, 3 toes of the left hind paw, at a concentration that induces hyperalgesia, i.e.: swelling and a decrease in pain threshold occurred in the soles of the rats within 1 hour after injection. The rats with a mechanical paw withdrawal reflex threshold below 100gf were successfully modeled as carrageenan-inflammatory pain. The rats in the inflammatory model with an inflammatory threshold above 100gf were given knockouts.

And (3) detecting mechanical hyperalgesia:

experiment one: the tolerance of synthetic short peptide P10581 and morphine to the rat hyperalgesia-inhibiting effect was determined. 180g-220g healthy adult male SD rats were selected and placed in the same cage (18 total) after the tail numbering. After 2h of room adaptation, the rats were allowed to rest and their mechanical foot-retracting thresholds were measured at a constant force rate (30gf/s) with the tapered tip of an 38500-PAM pressure application measurement system (Ugo basic Biological Research Apparatus, commercial-valve, Italy) aligned to the central part of the plantar region of the left hind paw of the rat, and the maximum measurement time was set to 15s (when the measurement value was greater than 450gf, the rat was injured). The mechanical paw withdrawal threshold was recorded as the left hind paw withdrawal of the rat and measurement was stopped if the paw withdrawal reflex did not appear after 15s in the test rat. The measurement was repeated 3-5 times per rat, each time at 5-10min intervals, and the average value was taken. After the measurement, 6 μ l of 1% carrageenan was injected intradermally into the sole of the foot to establish a rat inflammatory pain model. The mechanical hind paw withdrawal threshold of the left hind paw of the rat was examined 1 hour after carrageenan injection, and rats with abnormal threshold size were rejected (molding failed). The rats with higher inflammatory threshold values and lower inflammatory threshold values are mixed and averagely divided into 3 groups, 6 rats are arranged in each group, so that the inflammatory threshold values of two groups of rats are similar in distribution, the group A is injected with normal saline, the group B is injected with 5mg/Kg morphine, the group C is injected with 2 mu g/Kg P10581 in the left and rear plantar skin, the mechanical paw withdrawal threshold value of each group of rats when the left and rear paw is withdrawn is recorded after 1 hour, each rat is tested 3-5 times repeatedly (the time interval of each test is 5-10min), and the average value of the paw withdrawal threshold values is taken as the actual paw withdrawal threshold value (the unit is: g) of the rat. Thereafter, the drug was injected every 2h at the above-mentioned dose and administration time, and the mechanical paw withdrawal threshold of the rat was recorded for a total of 6 times.

Experiment two: the low dose of short peptide P10581 was determined to have no inhibitory effect on rat mechanical hyperalgesia. The procedure of the method for measuring the inhibition effect of the low-dose short peptide P10581 on rat mechanical hyperalgesia is similar to that of the experiment I:

(1) in the second experiment, a total of 12 healthy adult SD rats 180g-220g are selected, after molding is carried out by 1% carrageenan, rats with failed molding are removed, the rest rats are divided into 2 groups, 6 rats are respectively injected in the group A, and 0.1 microgram/Kg of synthetic short peptide P10581 is injected in the left and the rear sole of the foot of the group B.

(2) The drug was injected every 2h at the first dose and the mechanical paw withdrawal threshold was recorded for a total of 7 repeated doses.

Experiment three: the effect of low dose synthetic short peptide P10581 on tolerance to the analgesic effect of morphine was determined. The method steps for determining the effect of low dose of synthetic short peptide P10581 on the tolerance of the analgesic effect of morphine were similar to those of experiment one, but with several differences:

(1) experiment three, a total of 12 180g-220g healthy adult SD rats are selected, after 1% carrageenan is molded, the rats are randomly divided into 2 groups, 6 rats are selected in each group, A, B groups of rats are injected with 0.1 mu g/Kg P10581 and 0.05 mu g/Kg P10581 in the left and rear plantar skins respectively, after 30min, two groups of rats are injected with 5mg/Kg morphine in the left and rear plantar skins, after 1h, the mechanical foot-shrinking threshold value of each group of rats during left and rear foot-shrinking is recorded, and each rat is tested for 3-5 times (the time interval of each test is 5-10 min).

(2) The drug was injected every 2h according to the first dose and time of administration and the mechanical paw withdrawal threshold was recorded for a total of 7 times in rats.

The experimental design follows the principle of random and double-blind experimental design.

The short peptide sequence of the invention: WKCNPNDDKCCRPKLKC (named P10581) with 17 amino acid sequences (as in FIG. 1), derived from spider toxin GsMTx4 located in two loop domains of loop2 and loop 3. FIG. 1 is a graph showing the comparison of tolerance of synthetic short peptide P10581 and morphine to rat hyperalgesia inhibitory effect. Wherein, Panel A shows the sequence of synthetic peptide P10581; B. comparison of the analgesic effects of synthetic peptide P10581 and morphine on rat hyperalgesia, the polypeptide and morphine were administered by intraplantar injection every 2 hours. C.p10581 and morphine for standardized tolerance comparisons to rat hyperalgesia and analgesia; the experimental result shows that the short peptide P10581(2 mu g/kg) can basically and completely reverse the inflammatory pain threshold induced by carrageenan, and the analgesic effect lasts for more than 12h, which indicates that the short peptide P10581 does not produce tolerance to the analgesic effect of rat mechanical pain. However, the analgesic effect of morphine (5mg/kg) gradually decreased with the increase of the number of injections, i.e., morphine was gradually resistant to the analgesic effect.

FIG. 2 shows that low dose of short peptide P10581 has no effect on rat mechanical hyperalgesia. The short peptide P10581(0.1 mug/Kg) is administrated by intraplantar injection every 2 hours, and the result shows that the low dose P10581(0.1 mug/Kg) has no analgesic effect on rat hyperalgesia and has no obvious analgesic effect all the time along with the increase of the administration times.

Figure 3 shows that low dose of synthetic short peptide P10581 abrogated the tolerance of analgesic effects of morphine. Rats were induced in an inflammatory pain model by injecting 6 μ l of 1% carrageenan (Carr) into the left hind sole of the foot beforehand, then injecting synthetic short peptide P10581 at a low dose (0.1 μ g/Kg or 0.05 μ g/Kg) into the swollen part of the left sole of the foot of the rat, injecting morphine (5mg/Kg) into the left hind sole of two groups of rats after 30min, and recording the mechanical foot-shortening threshold value of each group of rats when the left hind foot is contracted after 1 h. Thereafter, the drug was injected every 2h at the above-mentioned dose and administration time, and the mechanical paw withdrawal threshold of the rat was recorded for a total of 7 times. The results show that the tolerance of morphine analgesic effect is almost completely eliminated by synthesizing the short peptide P10581 at low dose (0.1 mu g/Kg or 0.05 mu g/Kg), and the effect of the short peptide is continuously existed in the experimental process. FIG. 3 comparison of the standardized tolerance of low dose synthetic short peptide P10581 and morphine to the analgesic effect of rat hyperalgesia, it can be seen that: under the condition of no co-administration of the polypeptide P0581, the pain effect of morphine is continuously reduced, which indicates that the analgesic effect of morphine generates tolerance; however, the analgesic effect of morphine is kept unchanged under the condition of co-administration of low dose of polypeptide P10581 (0.1. mu.g/Kg or 0.05. mu.g/Kg), which shows that the polypeptide P10581 can almost completely eliminate the analgesic tolerance of morphine.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. The application of the short peptide as a tolerance inhibitor of morphine and the synergistic effect of morphine on preparing a compound analgesic product is characterized in that: the amino acid sequence of the short peptide is as follows: WKCNPNDDKCCRPKLKC, respectively; the compound analgesic product is used in modes of intraperitoneal injection, intravenous injection and intrathecal injection, and the short peptide is low-dose short peptide with the action concentration of 0.05-0.1 mug/Kg.