CN114796446B - Use of orphan peptides for treating ketamine addiction - Google Patents
Use of orphan peptides for treating ketamine addiction Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
- A61P25/36—Opioid-abuse
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
According to the invention, an effective dose of orphan peptide is delivered to the central nervous system in the brain through the circulation of cerebrospinal fluid by adopting a non-human primate ketamine addiction model in a intrathecal administration mode to enhance the drug self-management of rhesus, so that the orphan peptide has a remarkable inhibition effect on the forcible drug-foraging behavior of ketamine, the problem of the reduction of the expression quantity of the orphan peptide in the brain caused by ketamine addiction can be rapidly and effectively solved, the orphan peptide enters the brain through intrathecal administration, the negative emotions such as dysphoria, irritability and the like caused by forced withdrawal of ketamine can be relieved, the drug craving is reduced, and meanwhile, the function of hypothalamus-pituitary-adrenal (HPA) axis is restored, and the orphan peptide can be used for treating ketamine addiction.
Description
Technical Field
The invention belongs to the field of biomedicine, and in particular relates to a method for treating ketamine addiction by using orphan peptide.
Background
At present, novel synthetic drugs are increasingly flooded, the harm is extremely large, and no effective drug is clinically used for drug rehabilitation treatment and rehabilitation. Ketamine (Ketamine, KET) is used as novel drug K powder to enter public sight, and can rapidly cross blood brain barrier along with blood circulation to stimulate brain to generate strong euphoria, so that the ketamine has a magic effect. Ketamine abuse causes serious physical injury to the smoker and psychological dependence. The low cost, strong irritability and addiction make ketamine the "first mouth milk" for the lawless persons to lure the victim to take the poison. Forced withdrawal from ketamine produces a strong craving for relapse in drug addicts, resulting in a dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis.
Methadone is the most effective drug that has been developed for use. While methadone successfully reduces the craving and re-absorption rate of drugs by patients, effectively relieves withdrawal symptoms, it has drug tolerance and drug dependence. The orphan peptide is proved to be an analgesic drug with analgesic effect and no human abuse risk in various tests after intrathecal administration of non-human primate. Some researchers prove that the orphan peptide aggravates withdrawal symptoms, which is not beneficial to the drug self-management of alcohol addicted mice; another part of the study showed that orphan peptides were able to reduce alcohol intake in alcohol addicted mice.
Compared to mice, the phylogenetic, anatomic, physiological, neurochemical and behavioral of non-human primate (NHP) are more similar to humans, and rhesus drug addiction models can share complex emotional functions, genetic, neurological and phenotypic basis with humans. Therefore, the use of the rhesus monkey addiction model for researching the use of the orphanin for treating ketamine addiction by intrathecally injecting the orphanin is reliable. At present, no study has been conducted to verify that orphan peptides can be used to treat ketamine addiction using intrathecal administration.
Disclosure of Invention
The invention adopts a non-human primate ketamine addiction model for the first time to deliver an effective dose of orphan peptide to the central nervous system in the brain through cerebrospinal fluid circulation in a intrathecal administration mode to enhance the drug self-management of rhesus monkeys. The invention proves that the orphan peptide has remarkable inhibition effect on the forcible foraging behavior of ketamine, can quickly and effectively solve the problem of reduced expression quantity of the orphan peptide in brain caused by ketamine addiction, can relieve the negative emotions such as violence, irritability and the like caused by forcible withdrawal of ketamine by intrathecal administration, reduces the medication craving, and simultaneously restores the function of hypothalamus-pituitary-adrenal (HPA) axis, and can be used for treating ketamine addiction, thereby completing the invention.
According to one aspect, the invention relates to the use of orphan peptides in the manufacture of a medicament for the treatment of ketamine addiction in primates.
According to another aspect of the invention, the invention relates to a method of treating ketamine addiction in a primate, characterized by administering to the primate in need thereof a therapeutically effective amount of a orphan peptide.
According to another aspect, the invention relates to the use of orphan peptides in the manufacture of a medicament for the combined treatment of ketamine addiction in primates with other drugs.
According to another aspect of the invention, the invention relates to a method of treating ketamine addiction in a primate, characterized by administering to the primate in need thereof a therapeutically effective amount of a orphan peptide and other drugs.
It will be appreciated by those skilled in the art that other drugs used in combination with orphan peptides for the treatment of ketamine addiction in primates are drugs that can be used to treat ketamine addiction, such as fluoxetine, venlafaxine, imipramine, and the like.
According to another aspect, the invention relates to a pharmaceutical composition for the treatment of primate ketamine addiction, characterized by comprising orphan peptide and a pharmaceutically acceptable carrier.
According to the present invention, the above pharmaceutical composition may further contain other drugs, for example, drugs which can be used for treating ketamine addiction, such as fluoxetine, venlafaxine, imipramine, etc.
According to the invention, preferably, the orphan peptide and pharmaceutical composition are administered intrathecally.
Those skilled in the art will appreciate that a variety of pharmaceutically acceptable carriers known in the art, particularly those useful for intrathecal administration, may be employed in the present invention.
According to the invention, the orphan peptide is present in the pharmaceutical composition in 0.1-99%, preferably 1-90%, more preferably 5-50%. The administration amount of orphan peptide is, for example, 0.1 to 50mg/kg, preferably 1 to 10mg/kg, more preferably 0.5 to 5mg/kg.
According to the invention, the primate is preferably a human, rhesus.
The invention provides a rhesus monkey ketamine addiction model effective dose of orphanin by intrathecal injection, the ketamine content in the rhesus monkey body is higher before the orphanin is administrated, and the reward system is activated, so that the concentration of the orphanin in the brain is low and the cortisol content in the blood plasma is too high. Intrathecal injection of orphan peptide increases the concentration of orphan peptide in cerebrospinal fluid, and orphan peptide plays a sedative role in brain, and at this time, the behavior test result shows that the addiction degree of rhesus monkeys is reduced, the cortisol content in plasma is reduced, and even the normal range can be restored.
The invention can obtain a pharmacokinetics curve by measuring the concentration of the orphan peptide in the rhesus monkey before and after administration, and can also verify the drug effect macroscopically by matching the detection of the concentration of the drug in the rhesus monkey with the behavior test. The detection result of the concentration of the plasma cortisol can verify the efficacy of the medicine on a microscopic scale.
Several key features and advantages of the present invention: first, intrathecal injection achieves remarkable efficacy of a therapeutically effective amount of orphan peptide, can inhibit rhesus addictive behaviors in a macroscopic view, and can restore physiological functions of an HPA axis in a microscopic view; second, the withdrawal effect of the orphan peptide to a therapeutically effective amount is not dose dependent and drug tolerating. To demonstrate that intrathecal administration of orphan peptides has withdrawal efficacy, the present invention was validated using three examples.
Drawings
FIG. 1 is a bar graph of rhesus monkey addictive behavior scores for the model and experimental groups before and after a single low dose administration
FIG. 2 is a bar graph of cortisol concentration in rhesus plasma in model and experimental groups before and after a single low dose administration
FIG. 3 is a bar graph of the variation of the concentration of orphan peptides in the cerebrospinal fluid and plasma of rhesus monkeys of the experimental group 1 before and after a single low dose administration
FIG. 4 is a bar graph of the variation of the concentration of orphan peptides in the cerebrospinal fluid and plasma of rhesus monkeys of the experimental group 2 before and after a single low dose administration
FIG. 5 is a bar graph of the variation of the concentration of orphan peptides in the cerebrospinal fluid and plasma of rhesus monkeys in experimental group 3 before and after single low dose administration
FIG. 6 is a bar graph of rhesus monkey addictive behavior scores for the model and experimental groups before and after a single high dose administration
FIG. 7 is a bar graph of cortisol concentration in rhesus plasma in model and experimental groups before and after single high dose administration
FIG. 8 is a histogram of variation in concentration of orphan peptides in cerebrospinal fluid and plasma of rhesus monkeys of Experimental group 1 before and after single high dose administration
FIG. 9 is a histogram of variation in concentration of orphan peptides in cerebrospinal fluid and plasma of rhesus monkeys of Experimental group 2 before and after single high dose administration
FIG. 10 is a bar graph of rhesus monkey addictive behavior scores for the model and experimental groups before and after continuous low dose dosing
FIG. 11 is a bar graph of cortisol concentration in rhesus monkey plasma in model and experimental groups before and after continuous low dose dosing
FIG. 12 is a bar graph of the variation of the concentration of orphan peptides in cerebrospinal fluid and plasma of rhesus monkeys of the experimental group 1 before and after administration of a continuous low dose
FIG. 13 is a bar graph of variation in concentration of orphan peptides in rhesus cerebrospinal fluid and plasma of test group No. 2 before and after continuous low dose administration
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications of the invention will become apparent to those skilled in the art upon reading the description herein, and such equivalents are intended to fall within the scope of the invention as defined by the appended claims.
EXAMPLE 1 Single Low dose administration
A rhesus model of ketamine addiction was established by the following method:
Rhesus monkeys were individually fed 3 times daily with sufficient monkey feed and 2 times daily with fruit, free drinking water, and intramuscular injection of ketamine hydrochloride injection to create a rhesus model of ketamine addiction, comprising the steps of:
(a) Acute molding phase dosing phase: the ketamine hydrochloride injection is injected into the muscle of the rhesus monkey at constant doses of 8mg/kg body weight twice a day for 14 consecutive days;
(b) Transition period of acute molding stage: injecting ketamine hydrochloride injection into the muscle of the rhesus at the dosage of the step (a) in the morning and injecting an equal volume of physiological saline into the muscle of the rhesus in the afternoon on the following 1 day;
(c) Forced withdrawal period in acute molding stage: the rhesus is intramuscular injected with an equal volume of physiological saline twice daily for 3 days;
(d) Consolidating model phase dosing period: injecting ketamine hydrochloride injection into rhesus muscle for 5 days twice a day according to the dosage of the step (a);
(e) Consolidating model phase transition period: injecting ketamine hydrochloride injection into the muscle of the rhesus at the dosage of the step (a) in the morning and injecting an equal volume of physiological saline into the muscle of the rhesus in the afternoon on the following 1 day;
(f) Consolidating the forced withdrawal period of the model stage: the rhesus is intramuscular injected with an equal volume of physiological saline twice daily, 5 days later.
Behavioral testing was performed on rhesus monkeys in the untreated control group and the addicted model group after modeling: the operator holds the syringe with needle sheath for 5 seconds, touches but does not pull the pull rod, and if the rhesus mates, presses with the syringe with needle sheath to perform a sham injection.
Comparing the behavior difference of the rhesus monkeys in the untreated control group with that of the rhesus monkeys in the model group to judge whether the rhesus monkeys in the model group have drug withdrawal, irritability, attack and avoid dangerous addictive behaviors; and recording the occurrence times of the addictive behaviors, correcting the percentage of the occurrence times of the addictive behaviors to the total test times by using the duration time of the rope taking and the dangerous behavior avoiding, and obtaining a behavior test score, wherein the score is increased by 1 minute when the duration time of the rope taking is increased by 1 second, and the score is decreased by 1 minute when the duration time of the dangerous behavior avoiding is increased by 2 seconds. The minimum score of the behavioral test score is 0, the score of the behavioral test score is 100, the behavioral test score is 0 to 25 (excluding) and is non-addictive, the behavioral test score is 25 to 50 (excluding) and is slightly addictive, the behavioral test score is 50 to 75 (excluding) and is moderately addictive, and the behavioral test score is 75 to 100 (excluding) and is severely addictive.
The drug cord of rhesus monkeys in the addictive model group exhibited any one or more of the following behaviors as compared to rhesus monkeys in the untreated control group:
after an operator holds the injector to touch the pull rod, the buttocks are actively displayed to be static;
An operator holds the injector to touch the pull rod and then approaches the injector immediately, and stands sideways to close to the front end of the cage;
the injector is seen and immediately approaches after the operator touches the pull rod, leaning sideways down to the front of the cage.
The challenge of rhesus monkeys in the addictive model group was shown to be any one or more of the following compared to rhesus monkeys in the untreated control group:
After receiving the false injection, the user can jump and jump the foot up and down;
after receiving the false injection, the teeth are threaded, the eyebrows are wrinkled, and the rocking cage makes sound;
the forelimbs are extended out of the cage to grasp the injector after receiving the sham injection.
According to the invention, the challenge of rhesus monkeys in the addictive model group, compared to rhesus monkeys in the untreated control group, was shown to be the following behaviour:
the forelimbs were extended out of the cage after receiving the sham injection to attack the operator.
According to the invention, the risk of avoiding the craving of the rhesus monkeys in the addictive model group is represented by any one or more of the following behaviors, compared to the rhesus monkeys in the untreated control group:
the twisting head is intended to avoid the decrease of the behavior of directly facing the syringe to the side;
the low head directly avoids the behavior reduction of the direct-view injector;
behavior of hiding the rear of the cage away from the syringe is reduced;
reduced syringe body recline and retrograde behavior is seen.
The results after the behavioral tests show that the rhesus monkeys in the addicted model group show higher frequency of rope medicine behaviors compared with the non-treated control group, and the behavior is specifically one or more of the following behaviors: after an operator holds the injector to touch the pull rod, the buttocks are actively displayed to be static; an operator holds the injector to touch the pull rod and then approaches the injector immediately, and stands sideways to close to the front end of the cage; the injector is seen and immediately approaches after the operator touches the pull rod, leaning sideways down to the front of the cage. Rhesus monkeys in the addictive model group were more challenged than rhesus monkeys in the untreated control group, and exhibited any one or more of the following behaviors: after receiving the false injection, the user can jump and jump the foot up and down; after receiving the false injection, the teeth are threaded, the eyebrows are wrinkled, and the rocking cage makes sound; the forelimbs are extended out of the cage to grasp the injector after receiving the sham injection. Rhesus monkeys in the addicted model group were more likely to develop aggression compared to rhesus monkeys in the untreated control group, as evidenced by stretching the forelimbs out of the cage after receiving the sham injection to attack the operator. The reduced risk of avoidance in rhesus monkeys in the addicted model group compared to rhesus monkeys in the untreated control group is exhibited by any one or more of the following behaviors: the twisting head is directed sideways to avoid the reduction of the behavior of the injector directly facing; the low head directly avoids the reduced behavior of the direct-view injector; behavior of hiding the rear of the cage away from the syringe is reduced; reduced syringe body recline and retrograde behavior is seen. .
The rhesus models of 4 ketamine addiction were randomized into 2 groups after the end of modeling, wherein: experiment group 3 rhesus monkey numbers 1,2, 3; model group 1 rhesus monkeys. All rhesus monkeys were fed individually, with 3 times daily sufficient monkey feed and 2 times fruit, with free drinking.
The orphin was administered at a dose of 0.5mg/kg to the experimental group, and was dissolved in 500. Mu.l of physiological saline for injection and then injected intrathecally, and the model group was administered with an equal volume of physiological saline for injection.
All rhesus monkeys were tested for behavior, cortisol (CRF) concentration and orphan peptide concentration before and after administration of orphan peptide or physiological saline. The behavior test can directly observe the addiction degree of rhesus monkeys. This plasma cortisol concentration assay can assess whether orphan peptides have the potential to restore HPA function by measuring cortisol concentration in plasma. The detection of the concentration of the orphan peptide comprises detection of the concentration of the orphan peptide drug in cerebrospinal fluid and the concentration of the orphan peptide drug in plasma, so as to prove that the orphan peptide acts along with the cerebrospinal fluid entering the ventricle through intrathecal administration and determine the residence time of the drug in vivo.
The sampling time points for the rhesus performance test of experimental group 1,2 and 3 rhesus performance test of model group are the day 1, day 4, day 7 and day 14 before administration (day 0). The results of the behavioral tests were divided into addiction levels with the lowest score of 0 and the full score of 100, with the following criteria: 0 to 25 (no) are non-addictive; 25 to 50 (no) mild addiction; 50 to 75 (no) are moderate addiction; 75 to 100 points (inclusive) are severe addiction. The time points for detecting cortisol concentration in plasma of model group, experimental group 1,2 and 3 are before administration (day 0), 1 day after administration, 3 days, 4 days and 7 days. The sampling time points for detecting the concentration of the orphan peptide in the cerebrospinal fluid and the blood plasma of the rhesus monkeys of the experimental groups 1 and 2 are before administration (day 0), 1 day, 4 days and 7 days after administration, and the sampling time points for detecting the concentration of the orphan peptide in the cerebrospinal fluid and the blood plasma of the rhesus monkeys of the experimental group 3 are before administration (day 0), 1 day, 3 days, 5 days and 7 days after administration. The specific time for sample collection was 8:00 am to 9:00 am. During behavior test, a video camera is used for recording and collecting data. Samples for orphan peptide concentration detection and cortisol concentration detection were derived from cerebral spinal fluid and plasma collected from centrifugal venous blood of rhesus monkeys. Orphan peptide concentration in rhesus cerebrospinal fluid and plasma was detected using a orphan enzyme-linked immunosorbent assay (NOC ELISA). Cortisol concentrations in rhesus monkey plasma were detected using a cortisol enzyme-linked immunosorbent assay (CRF ELISA) to determine the effect of orphan peptides on improving HPA axis imbalance.
The addiction score (mean±sem) statistics of the behavioral tests are as follows: model group rhesus monkeys had a score of 100 (severe addiction) before injection of equal volume of normal saline to day 7 after injection, and a score of 75.13 (severe addiction) on day 14; the scores of the rhesus monkey addiction in the experimental group 3 were 94.42 +/-5.58 (heavy addiction) before the administration, 39.42+/-12.05 (light addiction) on the 1 st day after the administration, 30.89+/-16.39 (light addiction) on the 4 th day, 47.22+/-29.00 (light addiction) on the 7 th day and 31.48+/-19.85 (light addiction) on the 14 th day. This indicates that the orphan peptide can reduce drug use thirst and excessive stress behaviors such as violence, aggression and the like in withdrawal period after administration. See fig. 1.
The results of the measurement of cortisol concentration (mean±sem) in plasma are as follows: model group rhesus monkey plasma cortisol levels were consistently above the normal concentration range (50.75-228.36 ng/ml), model group rhesus monkeys injected with equal volume of normal saline before 303.98ng/ml, day 1, 392.67ng/ml, day 3, 316.88ng/ml, day 4, 268.51ng/ml, day 7, 339.46ng/ml; the rhesus monkeys of the experimental group were pre-327.19 + -23.97 ng/ml, 203.34 + -6.98 ng/ml at day 1, 200.87 + -14.99 ng/ml at day 3, 214.52 + -7.11 ng/ml at day 4, 225.37 + -2.24 ng/ml at day 7 after administration. Plasma cortisol concentrations in both pre-dosing and model groups were above the normal range, with the plasma cortisol concentrations in the experimental groups decreasing and returning to normal levels from day 1 to day 4 after dosing and the plasma cortisol concentrations in day 7 after dosing rising to near the upper limit of the normal concentration regime (228.36 ng/ml). This indicates that the single low dose administration of orphan peptide was able to restore HPA axis function to normal within 7 days. See fig. 2.
The results of rhesus monkey orphan peptide concentration detection in experimental group 1 are as follows: the drug concentration of cerebrospinal fluid and plasma before administration is 0.0ng/ml, the drug concentration of cerebrospinal fluid on the 1 st day after administration is 371.6ng/ml, the drug concentration of plasma is 22.2ng/ml, the drug concentration of cerebrospinal fluid on the 4 th day after administration is 284.0ng/ml, the drug concentration of plasma is 2.5ng/ml, the drug concentration of cerebrospinal fluid on the 7 th day is 16.0ng/ml, and the drug concentration of plasma is 3.2ng/ml; the drug concentration in the cerebrospinal fluid of rhesus monkey No.1 was higher than that in the plasma on day 1 after administration. This indicates that after intrathecal injection of orphan peptide in rhesus monkeys of experimental group 1, the drug enters the cerebrospinal fluid and circulates into the brain with the cerebrospinal fluid to exert effect, alleviating withdrawal symptoms. Furthermore, drug concentration in rhesus cerebrospinal fluid was extrapolated in combination with rhesus cortisol concentration in the 7 th day experimental group and rhesus cerebrospinal fluid in the 7 th day 1 experimental group: a therapeutically effective amount may not be achieved when the drug concentration in the cerebrospinal fluid is below 16.0 ng/ml. The results of rhesus monkey orphan peptide concentration detection in experimental group No. 2 are as follows: the drug concentration of cerebrospinal fluid and plasma before administration is 0.0ng/ml, the drug concentration of cerebrospinal fluid on the 1 st day after administration is 124.0ng/ml, the drug concentration of plasma is 21.0ng/ml, the drug concentration of cerebrospinal fluid on the 4 th day after administration is 0.0ng/ml, the drug concentration of plasma is 9.3ng/ml, the drug concentration of cerebrospinal fluid on the 7 th day is 0.0ng/ml, and the drug concentration of plasma is 3.1ng/ml; the drug concentration in the cerebrospinal fluid of rhesus monkey No. 2 was higher than that in plasma on day 1 after administration. This indicates that after intrathecal injection of orphan peptide in rhesus monkeys of experimental group No. 2, the drug enters the cerebrospinal fluid and circulates into the brain with the cerebrospinal fluid to exert effect, alleviating withdrawal symptoms. The rhesus monkey No. 2 has lighter weight, lower dosage and faster metabolism. The results of rhesus monkey orphan peptide concentration detection in experimental group 3 are as follows: the drug concentration of cerebrospinal fluid and plasma before administration is 0.0ng/ml, the drug concentration of cerebrospinal fluid on the 1 st day after administration is 87.6ng/ml, the drug concentration of plasma is 10.9ng/ml, the drug concentration of cerebrospinal fluid on the 3 rd day after administration is 42.4ng/ml, the drug concentration of plasma is 0.0ng/ml, the drug concentration of cerebrospinal fluid on the 5 th day is 0.0ng/ml, the drug concentration of plasma is 1.4ng/ml, the drug concentration of cerebrospinal fluid on the 7 th day is 0.0ng/ml, and the drug concentration of plasma is 1.4ng/ml; the concentration of the drug in the cerebrospinal fluid of rhesus monkey No. 3 was higher than that in plasma on day 1 after administration. This indicates that after intrathecal injection of orphin in rhesus monkey No. 3, the drug enters the cerebrospinal fluid and circulates into the brain with the cerebrospinal fluid to exert effects, alleviating withdrawal symptoms. See fig. 3-5.
In conclusion, single intrathecal administration of low-dose orphanin can effectively inhibit craving, aggression and agitation behavior of rhesus monkey in withdrawal period, and the orphanin can inhibit the re-absorption desire caused by withdrawal of drug from monarch and change agitation caused by withdrawal of drug from monarch; single low-dose intrathecal administration of the orphan peptide can reduce the concentration of cortisol in rhesus monkeys to a normal level, and the orphan peptide is suggested to be helpful for recovering the normal function of the HPA axis of the human body; the in vivo concentration test of the orphan peptide shows that the administration dosage of 0.5mg/kg has an abstinence effect, the rhesus monkey can improve abnormal behaviors within 14 days, the dosage can maintain normal physiological functions within 7 days, and the orphan peptide is suggested to help human beings to fix own volition and improve own physiological functions within 7 days.
EXAMPLE 2 Single high dose administration
The rhesus models of 3 ketamine addiction were randomized into 2 groups after the end of modeling, wherein: experimental group 2 rhesus numbers 1, 2; model group 1 rhesus monkeys. All rhesus monkeys were fed individually, with 3 times daily sufficient monkey feed and 2 times fruit, with free drinking.
The orphan peptide was administered to the experimental group at a dose of 5mg/kg, dissolved in 500. Mu.l of physiological saline for injection, and injected intrathecally, and the model group was administered with an equal volume of physiological saline for injection.
All rhesus monkeys were tested for behavior, cortisol (CRF) concentration and orphan peptide concentration before and after administration of orphan peptide or physiological saline. The behavior test can directly observe the addiction degree of rhesus monkeys. This plasma cortisol concentration assay can assess whether orphan peptides have the potential to restore HPA function by measuring cortisol concentration in plasma. The detection of the concentration of the orphan peptide comprises detection of the concentration of the orphan peptide drug in cerebrospinal fluid and the concentration of the orphan peptide drug in plasma, so as to prove that the orphan peptide acts along with the cerebrospinal fluid entering the ventricle through intrathecal administration and determine the residence time of the drug in vivo.
The sampling time points for the rhesus performance test of the experimental group 1 and the rhesus performance test of the model group 2 are the day 1, the day 4, the day 7 and the day 14 before the administration (day 0). The results of the behavioral tests were divided into addiction levels with the lowest score of 0 and the full score of 100, with the following criteria: 0 to 25 (no) are non-addictive; 25 to 50 (no) mild addiction; 50 to 75 (no) are moderate addiction; 75 to 100 points (inclusive) are severe addiction. The time points for detecting cortisol concentration in plasma of model group, experimental group 1 and 2 are before administration (day 0), after administration, 1 day, 2 days, 3 days, 4 days and 7 days. The sampling time points for detecting the concentration of the orphan peptide in the cerebrospinal fluid and the plasma of the rhesus monkeys (1 and 2) are before administration (0 day), 1 day after administration, 4 days and 7 days. The specific time for sample collection was 8:00 am to 9:00 am. During behavior test, a video camera is used for recording and collecting data. Samples for orphan peptide concentration detection and cortisol concentration detection were derived from cerebral spinal fluid and plasma collected from centrifugal venous blood of rhesus monkeys. Orphan peptide concentration in rhesus cerebrospinal fluid and plasma was detected using a orphan enzyme-linked immunosorbent assay (NOC ELISA). Cortisol concentrations in rhesus monkey plasma were detected using a cortisol enzyme-linked immunosorbent assay (CRF ELISA) to determine the effect of orphan peptides on improving HPA axis imbalance.
The addiction score (mean±sem) statistics of the behavioral tests are as follows: model group rhesus monkeys had 100 (severe addiction) craving scores from day 14 before injection of equal volume of normal saline to day 14 post injection; the scores of the 2 rhesus monkeys in the experimental group are 58.50 +/-16.67 (moderate addiction), 15.33+/-13.67 (non-addiction) on the 1 st day after administration, 6.05+/-6.05 (non-addiction) on the 4 th day, 7.89+/-7.89 (non-addiction) on the 7 th day, and 12.98+/-12.98 (non-addiction) on the 14 th day. This indicates that the orphan peptide can reduce drug use thirst and excessive stress behaviors such as violence, aggression and the like in withdrawal period after administration. See fig. 6.
The results of the measurement of cortisol concentration (mean±sem) in plasma are as follows: model group rhesus monkey plasma cortisol levels were consistently above the normal concentration range (50.75-228.36 ng/m 1), 232.30ng/ml before injection of equal volume of normal saline, day 1, 245.04ng/ml, day 2, 244.13ng/ml, day 3, 370.68ng/ml, day 4, 305.13ng/ml, day 7, 338.81ng/ml; the rhesus monkeys in the experimental group were pre-366.38 + -13.49 ng/ml, 152.85 + -55.06 ng/ml after administration on day 1, 234.33 + -15.18 ng/ml on day 2, 164.13 + -1.12 ng/ml on day 3, 211.91 + -10.68 ng/ml on day 4, 261.88 + -4.50 ng/ml on day 7. Plasma cortisol concentrations in the pre-dosing experimental group and the model group were higher than the normal range, and on days 1 to 4 after dosing, the plasma cortisol concentrations in the experimental group were reduced and restored to normal levels, and on day 7 after dosing, the plasma cortisol concentrations were increased, and on day 7, the HPA axis function was presumed to be abnormal. This indicates that the single high dose administration of orphan peptide was able to restore HPA axis function to normal within 7 days. See fig. 7.
The results of rhesus monkey orphan peptide concentration detection in experimental group 1 are as follows: the drug concentration of cerebrospinal fluid and plasma before administration is 0.0ng/ml, the drug concentration of cerebrospinal fluid on the 1 st day after administration is 363.3ng/ml, the drug concentration of plasma is 17.7ng/ml, the drug concentration of cerebrospinal fluid on the 4 th day after administration is 40.0ng/ml, the drug concentration of plasma is 0.0ng/ml, the drug concentration of cerebrospinal fluid on the 7 th day is 0.0ng/ml, and the drug concentration of plasma is 0.0ng/ml; the drug concentration in the cerebrospinal fluid of rhesus monkey No. 1 was higher than that in the plasma on day 1 after administration. This indicates that after intrathecal injection of orphan peptide in rhesus monkeys of experimental group 1, the drug enters the cerebrospinal fluid and circulates into the brain with the cerebrospinal fluid to exert effect, alleviating withdrawal symptoms. The results of rhesus monkey orphan peptide concentration detection in experimental group No. 2 are as follows: the drug concentration of cerebrospinal fluid and plasma before administration is 0.0ng/ml, the drug concentration of cerebrospinal fluid on the 1 st day after administration is 692.6ng/ml, the drug concentration of plasma is 14.0ng/ml, the drug concentration of cerebrospinal fluid on the 4 th day after administration is 62.7ng/ml, the drug concentration of plasma is 12.8ng/ml, the drug concentration of cerebrospinal fluid on the 7 th day is 61.3ng/ml, and the drug concentration of plasma is 8.0ng/ml; the drug concentration in the cerebrospinal fluid of rhesus monkey No. 2 was higher than that in plasma on day 1 after administration. This indicates that after intrathecal injection of orphan peptide in rhesus monkeys of experimental group No. 2, the drug enters the cerebrospinal fluid and circulates into the brain with the cerebrospinal fluid to exert effect, alleviating withdrawal symptoms. See fig. 8 and 9.
In conclusion, single high-dose intrathecal administration of the orphan peptide can effectively inhibit the craving, aggression and agitation behaviors of the rhesus monkey in the withdrawal period, and the orphan peptide can be suggested to inhibit the re-absorption desire caused by withdrawal of the monarch drug and change the agitation caused by withdrawal of the monarch drug; single high-dose intrathecal administration of the orphan peptide can reduce the concentration of cortisol in rhesus monkeys to a normal level, and the orphan peptide is suggested to be helpful for recovering the normal function of the HPA axis of the human body; the in vivo concentration test of the orphan peptide shows that the administration dose of 5mg/kg has the effect of stopping addiction, the rhesus monkey can improve abnormal behaviors within 14 days, the dose is the same as the effect of single administration of low dose, and normal physiological functions can be maintained within 7 days, so that the single administration of the high dose of the orphan peptide can help people to fix own mind and improve own physiological functions within 7 days, but the stopping addiction of the orphan peptide does not have dose dependency.
EXAMPLE 3 continuous Low dose administration
The rhesus model of 3 ketamine addiction in example 2 was again constructed as a stable ketamine addiction model after a recovery period of 3 months and the grouping set up in example 2 was repeated after the end of the modeling. 3 ketamine addicted rhesus monkeys were divided into 2 groups, wherein: experimental group 2 rhesus monkeys maintained numbers 1, 2 in example 2; model group rhesus 1. All rhesus monkeys were fed individually, with 3 times daily sufficient monkey feed and 2 times fruit, with free drinking.
The orphin was administered at a dose of 0.5mg/kg to the experimental group, and was dissolved in 500. Mu.l of physiological saline for injection and then injected intrathecally, and the model group was administered with an equal volume of physiological saline for injection. The administration time interval is 7 days, and the administration is 4 times, and the administration is respectively carried out on the 0 th day, the 7 th day, the 14 th day and the 21 st day.
All rhesus monkeys were tested for behavior, cortisol (CRF) concentration and orphan peptide concentration before and after administration of orphan peptide or physiological saline. The behavior test can directly observe the addiction degree of rhesus monkeys. The plasma cortisol concentration detection can be used for evaluating whether the orphan peptide has the potential of restoring HPA function by detecting the cortisol concentration. The detection of the concentration of the orphan peptide comprises detection of the concentration of the orphan peptide drug in cerebrospinal fluid and the concentration of the orphan peptide drug in plasma, so as to prove that the orphan peptide acts along with the cerebrospinal fluid entering the ventricle through intrathecal administration and determine the residence time of the drug in vivo.
The rhesus monkey behavior test sampling time points of the experimental groups 1 and 2 are the day before administration (day 0), the day 1, the day 7, the day 8, the day 14, the day 15, the day 21, the day 22 and the day 28 after administration. The results of the behavioral tests were divided into addiction levels with the lowest score of 0 and the full score of 100, with the following criteria: 0 to 25 (no) are non-addictive; 25 to 50 (no) mild addiction; 50 to 75 (no) are moderate addiction; 75 to 100 points (inclusive) are severe addiction. The time points for measurement of cortisol concentration in plasma of experimental groups 1 and 2 were pre-dose (day 0), 1 day after dose, 7 days, 8 days, 14 days, 15 days, 21 days, 22 days, and 28 days. Since rhesus monkeys in the model group unexpectedly died during the post-operation wakefulness of day 21, the model group behavior test and the time point for detecting the concentration of cortisol in plasma were the pre-administration (day 0), the 1 st, 7 th, 8 th, 14 th, 15 th, and 21 th days after administration. The sampling time points for detection of the solitary peptide concentration in the cerebrospinal fluid and the plasma of rhesus monkeys (1 and 2) are before administration (day 0), 1 day after administration, 7 days, 8 days, 14 days, 15 days, 21 days, 22 days and 28 days. The specific time for sample collection was 8:00 am to 9:00 am. During behavior test, a video camera is used for recording and collecting data. Samples for orphan peptide concentration detection and cortisol concentration detection were derived from cerebral spinal fluid and plasma collected from centrifugal venous blood of rhesus monkeys. Orphan peptide concentration in rhesus cerebrospinal fluid and plasma was detected using a orphan enzyme-linked immunosorbent assay (NOC ELISA). Cortisol concentrations in rhesus monkey plasma were detected with a cortisol enzyme-linked immunosorbent assay (cortisol ELISA) to determine the effect of orphan peptides on improving HPA axis imbalance.
The addiction score (mean±sem) statistics of the behavioral tests are as follows: model group rhesus monkeys had 100 (severe addiction) craving scores from day 22 before injection of equal volume of normal saline to day 22 post injection; the scores of the 2 rhesus monkeys for addiction were 90.60±9.40 (severe addiction), 4.40±4.40 (non-addiction) on day 1, 31.25±20.65 (mild addiction) on day 7 (first, and before second), 0.0±0.0 (non-addiction) on day 8 (1, after second), 14 (7, and before third), 17.63 ± 17.63 (non-addiction), 11.02±11.02 (non-addiction) on day 15 (1, after third), 11.02±11.02 (non-addiction), 21 (7, after third, and before fourth), 32.25±6.25 (mild addiction), 12.50±12.50 (non-addiction) on day 22 (1, and 11.05±11.05 (non-addiction) on day 28 (7, after fourth). This indicates that the orphan peptide can reduce drug use thirst and excessive stress behaviors such as violence, aggression and the like in withdrawal period after drug administration; combining the addiction score of example 2 with the dosing dose, the same individual was treated with the high dose drug and then again with the low dose drug, which also reduced the addictive behavior, indicating that orphan peptides did not have drug tolerance problems in improving addictive behavior. See fig. 10.
The results of the measurement of cortisol concentration (mean±sem) in plasma are as follows: model group rhesus monkey plasma cortisol levels were consistently above the normal concentration range (50.75-228.36 ng/ml), model group rhesus monkeys injected with equal volume of normal saline prior to 287.29ng/ml, day 1, 320.51ng/ml, day 7, 325.09ng/ml, day 8, 283.85ng/ml, day 14, 345.71ng/ml, day 15, 401.84ng/ml, day 21, 298.74ng/ml, day 22, 350.29ng/ml; the experimental group rhesus monkeys were given 384.09 + -9.73 ng/ml prior to administration, 264.16 + -70.10 ng/ml at day 1, 206.81 + -28.22 ng/ml at day 7, 191.79 + -14.11 ng/ml at day 8, 195.88 + -20.03 ng/ml at day 14, 189.05 + -5.01 ng/ml at day 15, 227.68 + -52.74 ng/ml at day 21, 192.70 + -5.92 ng/ml at day 22, 162.65 + -12.29 ng/ml at day 28. The plasma cortisol concentrations in the experimental and model groups were above the normal range prior to the first dose, and after the dose the plasma cortisol concentrations in the experimental group were reduced and restored to normal levels, with the plasma cortisol concentrations remaining at normal levels on days 7 to twenty-eight. This indicates that the continuous low dose administration of orphan peptide helps to restore the function of the HPA axis thoroughly; meanwhile, by combining the plasma cortisol concentration detection result of the embodiment 2 with the administration dosage, the same individual can also recover the cortisol concentration to the normal concentration range after receiving the high-dose drug treatment and receiving the low-dose drug treatment again, which proves that the orphan peptide has no drug tolerance problem in recovering the normal function of the HPA axis. See fig. 11.
The results of rhesus monkey orphan peptide concentration detection in experimental group 1 are as follows: the drug concentration of the cerebrospinal fluid and the blood plasma before administration is 0.0ng/ml, the drug concentration of the cerebrospinal fluid on 1 day is 218.1ng/ml, the drug concentration of the blood plasma is 4.8ng/ml, the drug concentration of the cerebrospinal fluid on 7 days is 58.2ng/ml, the drug concentration of the blood plasma is 3.0ng/ml, the drug concentration of the cerebrospinal fluid on 8 days is 165.3ng/ml, the drug concentration of the blood plasma is 4.1ng/ml, the drug concentration of the cerebrospinal fluid on 14 days is 61.0ng/ml, the drug concentration of the blood plasma is 4.1ng/ml, the drug concentration of the cerebrospinal fluid on 15 days is 64.8ng/ml, the drug concentration of the blood plasma is 5.0ng/ml, the drug concentration of the cerebrospinal fluid on 21 days is 0.0ng/ml, the drug concentration of the blood plasma is 4.2ng/ml, the drug concentration of the cerebrospinal fluid on 22 days is 82.7ng/ml, the drug concentration of the blood plasma is 4.1ng/ml, the drug concentration of the cerebrospinal fluid on 28 days is 5.4ng/ml, and the drug concentration of the blood plasma is 4.6ng/ml. See fig. 12.
Rhesus 1 had higher drug concentrations than plasma in cerebrospinal fluid on days 1,8, 15, and 22. This indicates that after intrathecal injection of orphan peptide in rhesus monkeys of experimental group 1, the drug enters the cerebrospinal fluid and circulates into the brain with the cerebrospinal fluid to exert effect, alleviating withdrawal symptoms.
The results of rhesus monkey orphan peptide concentration detection in experimental group No. 2 are as follows: the drug concentration in cerebrospinal fluid before administration was 1.0ng/ml, the drug concentration in plasma was 0.0ng/ml, the drug concentration in cerebrospinal fluid on day 1 was 118.5ng/ml, the drug concentration in plasma was 5.3ng/ml, the drug concentration in cerebrospinal fluid on day 7 was 0.0ng/ml, the drug concentration in plasma was 5.0ng/ml, the drug concentration in cerebrospinal fluid on day 8 was 323.8ng/ml, the drug concentration in plasma was 5.2ng/ml, the drug concentration in cerebrospinal fluid on day 14 was 68.2ng/ml, the drug concentration in plasma was 2.5ng/ml, the drug concentration in cerebrospinal fluid on day 15 was 260.2ng/ml, the drug concentration in plasma was 4.9ng/ml, the drug concentration in cerebrospinal fluid on day 21 was 0.0ng/ml, the drug concentration in plasma was 4.2ng/ml, the drug concentration in cerebrospinal fluid on day 22 was 128.9ng/ml, the drug concentration in plasma was 6.0ng/ml, the drug concentration in cerebrospinal fluid on day 28 was 0.0ng/ml, the drug concentration in cerebrospinal fluid on day 10.0 ng. See fig. 13.
Rhesus No. 2 had higher drug concentrations than plasma in cerebrospinal fluid on day 1, day 8, day 15, and day 22. This indicates that after intrathecal injection of orphin in rhesus monkeys of experimental group No. 2, the drug enters the cerebrospinal fluid and circulates into the brain with the cerebrospinal fluid to exert effects, thereby alleviating withdrawal symptoms.
In conclusion, the continuous low-dose intrathecal administration of the orphan peptide can effectively inhibit the craving, aggression and violence of the rhesus monkey in the withdrawal period in behavior for four weeks, and the orphan peptide is suggested to possibly inhibit the withdrawal desire caused by withdrawal of the monarch drug and change the violence caused by withdrawal of the monarch drug; continuous four weeks of intrathecal administration of low-dose orphan peptide can reduce the concentration of cortisol in rhesus monkeys to a normal level, and the continuous administration of orphan peptide is also indicated to be helpful for completely recovering the normal function of human HPA axis; the in vivo concentration test of the orphan peptide shows that the low-dose administration treatment is carried out again after the high-dose administration, so that the abnormal behavior caused by addiction can be improved, the concentration of the cortisol is reduced to the normal level, and the effect of abstinence is achieved, and the effect that the orphan peptide reaches the effective concentration can exert the effect of abstinence, and the abstinence symptom is improved, and the drug tolerance and the dose dependency are avoided.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The use of orphan peptides in the manufacture of a medicament for the treatment of primate ketamine addiction.
2. The use of claim 1, wherein the orphan peptide is administered intrathecally.
3. The use of claim 1, wherein the primate is a human or rhesus.
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