CN115400141B - Application of STING agonist in preparation of medicine for treating chronic pruritus - Google Patents

Abstract

The invention discloses an application of STING agonist in preparing a medicine for treating chronic pruritus. The invention finds a common path between analgesia and itch relief of the STING agonist ADU-S100 and develops a new indication of the STING agonist DU-S100.

Description

Application of STING agonist in preparation of medicine for treating chronic pruritus

Technical Field

The invention belongs to the field of biological medicine, and relates to application of STING agonists in preparation of medicines for treating chronic pruritus.

Background

Opioids are the most important analgesic drugs for clinical treatment of acute and chronic pain. Morphine is a typical opioid, which is still clinically recognized as the central flow column for analgesic therapy for the relief of severe pain and pain. It triggers analgesia by stimulating opioid receptors, a G protein-coupled receptor that is highly expressed in the central nervous system. However, opioids may cause side effects such as nausea, vomiting and more importantly, itching, especially after epidural or intrathecal administration. While itch caused by intrathecal morphine injection has longer duration and difficult treatment. Intrathecal administration of the opioid reached peak concentrations in cerebrospinal fluid almost immediately, as compared to epidural administration. It is counted that the occurrence rate of pruritus in patients treated by systemic opioid is 2-10%, while the occurrence rate of pruritus in patients treated by intrathecal opioid is increased to 30% -60%. It has been observed that pregnant women are more prone to itch after spinal cord opioid treatment, with an incidence of 60% to 100%, and appear to exhibit dose dependence, possibly due to interaction of estrogen with opioid receptors. Severe itching can sometimes be more unpleasant for the patient than the pain itself, and severe one can even affect the patient's postoperative recovery. The mechanism of opioid-related itching is not completely understood. Many different drugs with different potential mechanisms of action have been used to prevent or treat established itch. Their relative efficacy, optimal dose and possibility of adverse reactions are not yet clear. For example, naloxone, while widely used to control opioid-related itching, may reverse the analgesic effect of opioids; while antihistamines that are effective in alleviating acute itching are unlikely to play any role in preventing itching caused by opioids within the spinal canal. The fact that opioid-related itching can be reversed from opioid receptor antagonists (naloxone, nalbuphine) suggests that there may be an opioid receptor-mediated central mechanism. In summary, although itch following opioid use is a common side effect, few studies to date have elucidated the mechanisms, mediators, neural pathways, and pathophysiology of this symptom. Therefore, it is urgent to deeply elucidate the pathogenesis of opioid-induced acute pruritus and to find effective therapeutic strategies.

Disclosure of Invention

One of the purposes of the present invention is to provide the use of STING agonists in the preparation of a medicament for the treatment of chronic itch.

It is a second object of the present invention to provide pharmaceutical compositions comprising STING agonists.

In order to achieve the above purpose, the present invention adopts the following specific scheme:

according to one aspect of the invention, there is provided the use of a STING agonist in the manufacture of a medicament for the treatment of chronic itch.

Further, the chronic itch is opioid-induced itch.

Further, the opioid comprises morphine, fentanyl, pethidine.

In a specific embodiment of the invention, the opioid is morphine.

STING agonists, which may also be referred to as promoters, refer to any substance that increases STING protein activity, increases STING gene or protein stability, upregulates STING protein expression, increases STING protein useful duration, or promotes STING gene transcription and translation, which may be useful in the present invention as a substance for upregulating STING, and thus may be useful in preventing or treating chronic itching. Examples of the promoter include a nucleic acid promoter and a protein promoter. Such promoters include, but are not limited to, vectors that increase expression of STING, STING proteins, or active peptides thereof.

Many suitable vectors are known to those skilled in the art of molecular biology, the choice of which depends on the desired function. Non-limiting examples of vectors include plasmids, cosmids, viruses, bacteriophages and other vectors conventionally used, for example, in genetic engineering. Methods well known to those skilled in the art can be used to construct a variety of plasmids and vectors.

The vector may be a virus. Viral vectors are used to introduce non-endogenous nucleic acid sequences encoding target-specific polypeptides. The viral vector may be a retroviral vector or a lentiviral vector. Viral vectors may also include nucleic acid sequences encoding transduction markers.

Viral vectors suitable for use with the compositions of the present invention include those identified for use in human gene therapy applications. Suitable viral vectors include RNA virus-based vectors, such as retroviral-derived vectors, such as moloney Murine Leukemia Virus (MLV) -derived vectors, and more complex retroviral-derived vectors, such as lentiviral-derived vectors. HIV-1 derived vectors belong to this class.

Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and sendai viruses), positive strand RNA viruses (e.g., picornaviruses and A viruses) and double stranded DNA viruses, including adenoviruses, herpesviruses (e.g., type 1 and type 2 herpes simplex viruses and Epstein-Barr viruses and cytomegalovirus) and poxviruses (e.g., vaccinia, chicken pox and canary pox). Other viruses include, but are not limited to, norwalk viruses, togaviruses, flaviviruses, reoviruses, papillomaviruses, hepatitis viruses and hepatitis viruses, examples of retroviruses include avian leukemias, mammalian type C, type B viruses, type D viruses, HTLV group, lentiviruses and foamy viruses.

The vector may be an expression vector. Expression vectors according to the invention are capable of directing replication and expression of the nucleic acid molecules of the invention in a host.

Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45 a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV-dhfr, pZD 35, okayama-Berg cDNA expression vectors DV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pcA 3.1, pSBRO 1, pSBRO 26, pS-36, pSHi-36, and pEIR-36 (Biotechnology), and Biotechnology-67. Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen). Another vector suitable for expression of proteins in Xenopus (Xenopus) embryos, zebra fish embryos, and a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2+.

In particular embodiments of the invention, STING agonists include agents that promote the expression of STING genes, agents that enhance the activity of STING proteins.

Further, the reagent for promoting the expression of STING gene includes a reagent for promoting the expression of STING gene mRNA and a reagent for promoting the expression of STING protein.

Further, the STING agonist is ADU-S100.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising an opioid and a STING agonist.

Further, the opioid is morphine and the STING agonist is ADU-S100.

Further, morphine is present in a unit dose of 0.5 μg and ADU-S100 is present in a unit dose of at least 1001nmol of ADU-S.

Further, the unit dose of morphine was 0.5. Mu.g, and the unit dose of ADU-S100 was as small as ADU-S100100nmol.

The pharmaceutical compositions of the present invention comprise a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carrier includes, but is not limited to, diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, disintegrants.

Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and its salts, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose and the like; surfactants such as polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride of stearic acid, cetyl alcohol, etc.; wetting agents such as glycerin, starch, and the like; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauryl saccharate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powdery), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrants such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH controlling agents, surfactants, and the like.

Wherein the stabilizer comprises human serum protein, L-amino acid, sugar and cellulose derivative. The L-amino acid may also include any one of glycine, cysteine and glutamic acid. Sugars include monosaccharides such as glucose, mannose, galactose, fructose, and the like; sugar alcohols such as mannitol, cellosolve, xylitol, and the like; disaccharides such as sucrose, maltose, lactose, and the like; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfide, hyaluronic acid, and the like, and derivatives thereof. Cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose and sodium hydroxymethyl cellulose. Surfactants include ionic or nonionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, fatty acid glycerides. The additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and the corresponding salts (their alkali or alkaline rare earth metal salts, such as sodium, potassium, calcium and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars and glycerol. The chelating agent comprises sodium ethylenediamine tetraacetate and citric acid.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or by an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical compositions of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.

The dosage of the pharmaceutical composition of the present invention may be adjusted during the course of treatment according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

The present invention also provides a method of treating chronic itch, said method comprising the steps of: administering to a subject in need thereof a STING agonist as described herein before.

Further, opioid was injected following administration of the STING agonist described previously.

Preferably, the opioid is morphine.

Further, morphine was dosed at 0.5 μg, and ADU-S100 was dosed at least 1nmol of ADU-S100.

Further, morphine was dosed at 0.5. Mu.g and ADU-S100 was dosed at ADU-S100100nmol.

The invention has the advantages and beneficial effects that:

the invention discovers a new pharmacological action of STING agonist ADU-S100, namely prevention and treatment of opioid morphine-induced pruritus;

the present invention has found a common pathway between the STING agonist ADU-S100 in analgesia and itch relief.

Drawings

FIG. 1 shows a graph of the effect of ADU-S100 on analgesic effects resulting from intrathecal injection of morphine, wherein A: tail flick latency period; b: the percentage of maximum analgesic effect; NS group: a sheath physiological saline injection group; m groups: intrathecal injection of morphine 0.5 μg; A1M group: morphine 0.5 μg+ADU-S100 nmol; A2M group: morphine 0.5 μg+ADU-S100 nmol; A3M group: morphine 0.5 μg+ADU-S100 100nmol; n=8; in comparison to NS group, #p <0.01, two-way ANOVA;

FIG. 2 shows a graph of the effect of ADU-S100 on itch induced by intrathecal injection of morphine, wherein A: the number of harassments; b: total harassment times for 30 min; NS group: a sheath physiological saline injection group; m groups: intrathecal injection of morphine 0.5 μg; A1M group: morphine 0.5 μg+ADU-S100 nmol; A2M group: morphine 0.5 μg+ADU-S100 nmol; A3M group: morphine 0.5 μg+ADU-S100 100nmol; n=8; compared to NS group, #p<0.01; compared with group M, P<0.01; in contrast to the A1M group, ^& P<0.01; one-way or two-way ANOVA;

FIG. 3 shows a graph of the effect of ADU-S100 on STING expression, wherein A: immunoblot image; b: immunoblotting results statistics; NS group: a sheath physiological saline injection group; m groups: intrathecal injection of morphine 0.5 μg; A3M group: morphine 0.5 μg+ADU-S100 100nmol; n=4; compared to NS group, #p <0.01; p <0.01 compared to group M; one-way ANOVA;

fig. 4 shows a graph of immunohistochemical experimental results, wherein a: immunohistochemical map; b: an immunohistochemical result statistical graph; NS group: a sheath physiological saline injection group; m groups: intrathecal injection of morphine 0.5 μg; A3M group: morphine 0.5 μg+morphine 0.5 μg+ADU-S100 100nmol; n=4; compared to NS group, #p <0.01; p <0.01 compared to group a; one-way ANOVA.

Detailed Description

The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. The experimental procedure, without specific conditions noted in the examples, is generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring HarborLaboratory Press, 1989) or as recommended by the manufacturer.

Example ADU-S100 is effective in inhibiting itch induced by intrathecal morphine injection

1. Experimental procedure

1. Experimental materials

Male C57BL/6 were purchased from laboratory animal center of the national institute of medical science, the military, and the national institutes of the free army, 30, 6-8 weeks old, and 10-20g in weight. The random number table method was used to divide into 5 groups (n=8):

2. experimental grouping

Saline group (NS group): intrathecal injection of physiological saline;

morphine group (M group): intrathecal injection of 0.5 μg morphine;

ADU-s100+morphine group (A1M group): immediately after intrathecal injection of ADU-S100 nmol, 0.5 μg of morphine was intrathecally injected;

ADU-s100+morphine group (A2M group): immediately after intrathecal injection of ADU-S100 nmol, 0.5 μg of morphine was intrathecally injected;

ADU-s100+morphine group (A3M group): immediately after intrathecal injection of ADU-S100100nmol, 0.5 μg of morphine was intrathecally injected.

1. Mouse intraspinal morphine pruritus model

The mice were placed in a glass sealed jar containing a small amount of isoflurane, immediately removed after anesthesia and placed in the prone position. A piece of disinfection gauze is arranged on the side cover of the mouse head, the mouse body is held by the left palm, and the two sides of ilium are fixed by the forefinger and the thumb. The middle finger of the right hand is positioned on the back of the mouse, the iliac crest level of the mouse corresponds to the L6 spinous process, and the gap between L5 and 6 can be reached upwards, namely the needle inserting position. The right hand holds a 10 mu L microinjector, the needle is inserted at an angle of about 80-90 degrees with the spine, and after the needle touches the bone, the needle is inclined to an angle of about 30 degrees with the spine to continue to slowly advance, so that the sudden lateral swing of the rat tail is taken as a success sign. Stopping needle insertion after the lateral swing of the rat tail occurs, slowly injecting the medicine (volume is 5 mu l), and slowly withdrawing the needle while rotating after the injection is completed, wherein the injection time is not shorter than 2 s. After the injection was completed, the mice were placed in a transparent observation cage (15×15×20 cm) in a closed environment, and the behavioral manifestations of the mice within 30min after intrathecal injection were recorded using a camera (the mice were placed in the observation cage for at least 1h before imaging, and allowed to adapt to the environment). The scratching behavior is defined as one or more successive scratching of the back, neck, behind the ear by the hind paw of the mouse, licking, biting or landing the hind paw, and is noted as scratching once. According to the video playback, statistics of the number of scratching by unknowns were performed, and the number of scratching by the hind paw of the mouse was recorded every 5min. The results were statistically analyzed to compare whether there was a statistical difference between the groups.

2. Analgesic behavioural assessment

The experiment adopts a hot water (52.0+/-0.5 ℃) tail-flick method to evaluate the analgesic effect after the intraspinal administration. The tail 1/2 of the mice was immersed in hot water and the time from the tail entry to the tail rapid flick off the water surface was recorded with a stopwatch, which is the hot water tail flick latency period (tail flicking latency, TFL) of the mice. The tail of the mice stays in hot water for 10 seconds (cut-off time) at maximum time so as not to cause damage to the tail. The post-injection incubation period (post-drug incubation) was measured 3 times in succession, 15s apart, and an average was taken. The mice were trained 2, 3 times before formally starting the measurement, and then the basal tail flick latency of the mice before injecting the drug was determined, as well as the tail flick latency of the mice at time points of 30min, 60min, 90min, 120min after intrathecal injection of the different drugs. The percentage of maximum analgesic effect (percent of maximal possible potential effect,% MPE),% mpe= (post-dose latency-basal tail flick latency)/(Cut-off tail flick latency-basal tail flick latency) ×100% was calculated. The% MPE was used as an evaluation index of analgesic effect. The results were statistically analyzed and differences in analgesic effect between groups were compared.

3、Western blot

After the behavioural assay is finished, the mice are sacrificed, spinal cord dorsal horn L4-5 segments are taken, and the expression of spinal cord dorsal horn STING and GAPDH proteins is measured by a Western blot method. Adding precooled tissue protein lysate into dorsal horn tissue of spinal cord, and grinding into tissue homogenate. Centrifuging the homogenate at 4 ℃ for 5min at 12000rpm with a radius of 10cm, and obtaining the supernatant as the total protein of the spinal cord tissue. The extracted protein was denatured at 95℃for 5min. Separating by 10% SDS-PAGEd electrophoresis, transferring protein to PVDF membrane with loading amount of 15 μl, adding 5% skimmed milk powder, sealing at room temperature for 2 hr, washing, adding primary antibody, anti-rabbit STING antibody and anti-rabbit GAPDH antibody (1:1000, abcam company, USA), incubating at 4deg.C overnight, washing with TBST for 3 times, 5min each time, adding goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (dilution 1:2000, ab-gold bridge biological Co., beijing) for 2 hr at room temperature, washing with TBST for 3 times, adding luminescent agent in darkroom for exposure, scanning, and imaging. The band gray value is analyzed by using Gene Tools image analysis software, and the expression level of the target protein is reflected by the ratio of the target protein band gray value to the STING band gray value.

4. Immunohistochemistry

The mice are anesthetized by isoflurane, L4-6 spinal cord segments are taken out, immersed in a freshly prepared 4% paraformaldehyde solution for fixing for 6 hours, paraffin sections (4 mu m in slice thickness) are prepared conventionally, and the distribution expression condition of STING in the dorsal horn of the spinal cord is detected by adopting an immunohistochemical staining method. The method comprises the following specific steps: dewaxing: baking paraffin sections in an oven (60 ℃ for 2 hours) to remove surface paraffin; dehydrating: sequentially placing the dewaxed slices into xylene (I, II) and gradient ethanol (100%, 95%, 90%, 80%, 70%, 50%, 30%) solution cylinders for dehydration, respectively soaking for 5min, and finally washing for 3 times by using 1 XPBS; antigen retrieval: immersing the dehydrated slices in citric acid buffer solution (0.01M), boiling at high temperature for 3min (95 ℃), naturally cooling the slices to room temperature, then heating for 2min again, and naturally cooling the slices; dripping a proper amount of peroxidase blocking agent on the slice, incubating for 15min (room temperature) to block nonspecific staining caused by endogenous catalase, and then flushing for 3 times by 1 XPBS; dropwise adding 5% Bovine Serum Albumin (BSA) on the slice, incubating for 20min (room temperature), discarding the BSA, and naturally airing; 100 μl of STING primary antibody (1:500 dilution) was added dropwise to the sections and incubated overnight at 4deg.C; discarding STING primary antibody on the slice, washing 3 times by using 1XPBS, dripping 100 mu L of horseradish peroxidase-labeled secondary antibody (diluted 1:1000), incubating for 60min (room temperature), and washing 3 times by using 1 XPBS; the freshly prepared DAB working solution (A, B mother liquor 500. Mu.L each) was added dropwise to the sections, the sections were immediately placed under a 100-fold microscope for observation, and when the sections were found to be yellow brown (about 5 min), the sections were washed 3 times with tap water; dropping a proper amount of hematoxylin dye solution onto the slice for about 5min to dye the cell nucleus; dropping a proper amount of 1% hydrochloric acid alcohol on the slice for color separation, and washing the hydrochloric acid alcohol with tap water after 5 seconds of residence; sequentially placing slices into gradient ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%) solution cylinder and xylene (I, II) for dehydration and transparency (5 min each), washing with 1XPBS for 2 times, dripping appropriate amount of neutral gum, sealing, and air drying; and (3) observing by a microscope: DAB color shows brown yellow, namely positive cells. Each specimen was randomly extracted with 5 sections, each section was observed under a light microscope (X200), 5 high-power fields were selected in which the dorsal horn of the spinal cord of the rat was not overlapped, positive nerve cells were labeled, and protein expression of STING was evaluated by integrating optical density. All stained images were analyzed using Image J Image analysis software.

6. Statistical analysis: the SPSS 23.0 statistical software is adopted for analysis, the normal distribution measurement data is expressed by mean ± standard deviation (±s), the measurement data of the random block design is compared by single factor analysis of variance, the measurement data of the repeated measurement design is compared by repeated measurement design analysis of variance, and P <0.05 is the difference and has statistical significance.

2. Experimental results

1. Intra-spinal morphine injection can relieve pain, but induce itching

The analgesic effect of intrathecal injection of morphine in mice was first evaluated for its behavioral changes (fig. 1A and B). The basal tail flick latency period of each group of mice is 2-4 s. The% MPE values for each group of mice were calculated based on the hot water tail flick latency. The% MPE values of mice prior to intrathecal injection were all 0, the basal level. NS group mice had no significant change in% MPE at each time point, both before and within 2 hours after intrathecal administration. Mice in the group of morphine injection are obviously higher than those in the group of NS in hot water tail flick latency period of 30min and 60min after the intrathecal injection and% MPE, and P is less than 0.01, so that 0.5 mug of morphine injection in the sheath is indicated to have obvious analgesic effect.

It was then investigated whether morphine could cause the itching behaviour of mice (fig. 2A and B). According to video playback, no significant scratching was observed in the NS group of mice, and the number of scratching times for 30min was about 3 to 7, which served as a baseline level of scratching behavior of the mice. Mice with intrathecal morphine injection had significantly increased number of scratching compared to NS, P <0.01. And it was found by analysis that the scratching response after intrathecal morphine injection was time dependent in mice. The itching symptoms of the mice begin to appear 0-5 min after the administration, the itching is most obvious 10-15 min, the scratching times are slightly reduced within 15-20 min, and the scratching times are rapidly reduced after the intrathecal administration for 20 min.

2. ADU-S100 infusion can reduce pruritus caused by intraspinal morphine injection in a dose-dependent manner, and does not affect analgesic effect of morphine

To investigate whether intrathecal injection of ADU-S100 infusion could reduce itching due to intrathecal injection of morphine, mice were randomly divided into NS groups, 5 total groups of 0.5 μg intrathecal injection of morphine, 0.5 μg of morphine+adu-S100 nmol, 0.5 μg of morphine+adu-S100 10nmol, and 0.5 μg of morphine+adu-S100 100nmol (n=6).

In terms of pruritus behavioural (fig. 2A and B), NS intrathecal injection did not cause scratching in mice. Intrathecal injection of 0.5 μg of morphine resulted in itching in mice as previously described. By combining morphine with ADU-S100 with different dosages, the ADU-S100 can effectively relieve skin itch caused by injecting morphine into a vertebral canal through statistics and analysis of scratching times.

The total number of scratching in the 0.5 μg+ADU-S100 nmol group was still higher than that in the NS group at each time period, and the P was less than 0.05. However, the number of scratching was not significantly different from that of morphine 0.5 μg group (P > 0.05) in the corresponding period after administration.

Increasing ADU-S100 to 10nmol, in combination with morphine, after evaluation and statistical analysis of the itching behaviour of mice in the same way, mice in the group 0.5 μg+adu-S100 nmol had significantly reduced scratching times in 0 to 5min, 5 to 10min and 10 to 15min after dosing compared to the 0.5 μg group of morphine in the corresponding time period, P <0.01, but showed no significant difference (P > 0.05) in scratching times in 15 to 20min after dosing compared to the 0.5 μg group of morphine in the corresponding time period. Furthermore, mice in the morphine 0.5 μg+ADU-S100 nmol group had statistically significant scratching times, P <0.01, over time periods of 0-5 min, 5-10 min and 10-15 min post-dose compared to the NS group. Thus, the combined use of 0.5 μg of morphine and 100nmol of ADU-S can significantly reduce the scratching times of mice with morphine in the spinal canal, but the scratching reaction is still obviously increased compared with that of the control group, and the itching is not completely relieved.

After evaluation and statistical analysis of the pruritus behaviour of mice with the same method, by increasing ADU-S100 to 100nmol in combination with morphine, it was found that mice in the group with 0.5 μg+adu-S100 dose of morphine did not show significant scratching behaviour during each period after administration, and that there was no significant difference compared to the NS group during the same period. The scratching times of mice in the 0.5 mug+ADU-S100 100nmol group of morphine are obviously lower than those of mice in the 0.5 mug group of morphine in the time period within 0-5 min and 5-10 min, the statistical significance is achieved, P is less than 0.01, and the times of the mice in the 0.5 mug+ADU-S100 100nmol group of morphine in the 5-10 min, 10-15 min and 15-20 min are obviously reduced compared with those in the 0.5 mug+ADU-S100 nmol group of morphine, P is less than 0.01. Counting the total scratching times for 30min can obtain that the 0.5 mug+ADU-S100 nmol group of morphine and the 0.5 mug+ADU-S100 100nmol group of morphine are obviously reduced compared with the 0.5 mug group of morphine, and P is less than 0.01, wherein the reduction of the 0.5 mug+ADU-S100 100nmol group of morphine is more obvious. Thus, 0.5 μg of morphine combined with ADU-S100100nmol resulted in complete alleviation of cutaneous pruritus in intraspinal morphine mice, indicating that ADU-S100 treatment of morphine pruritus is dose dependent, whereas ADU-S100100nmol is the optimal therapeutic dose for cutaneous pruritus in morphine mice.

Furthermore, compared to intrathecal injection of morphine 0.5 μg (FIGS. 1A and B), 0.5 μg+ADU-S1001nmol of morphine, 0.5 μg+ADU-S100 10nmol of morphine and 0.5 μg+ADU-S100 100nmol of small sheath injection followed by 30min and 60min hot water tail flick latency and% MPE were not statistically different (P > 0.05), suggesting that intrathecal injection of ADU-S100 does not affect the analgesic effect of morphine.

3. ADU-S100 may reduce itching caused by intrathecal morphine injection by increasing STING expression

To study STING expression, mice were sacrificed after the end of the behavioural assay, spinal cord dorsal horn L4-5 segments were taken, and Western blot was used to determine spinal cord dorsal horn STING and GAPDH protein expression.

Studies have found (FIGS. 3A and B) that intrathecal injection of morphine in the 0.5 μg group significantly reduced (P < 0.01) expression of STING protein compared to NS, whereas the STING agonist ADU-S100100nmol significantly increased (P > 0.05) expression levels of STING, and that the results of STING immunohistochemistry (FIGS. 4A and B) were very similar to those of Western blot, suggesting that ADU-S100100nmol could inhibit down-regulation of STING by the morphine pruritus model.

Taken together, it was found that reduced STING expression on dorsal horn neurons of the spinal cord may be involved in morphine-induced pruritus. Pharmacological activation of STING agonist ADU-S100 significantly prevented morphine-induced itching. Studies suggest that STING agonist ADU-S100 may be a novel approach in the treatment of opioid-induced itch.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims (2)

1. Use of adu-S100 in the manufacture of a medicament for treating chronic itch, which is opioid-induced itch.
2. 2. The use according to claim 1, wherein the opioid is morphine.