RU2823374C1 - Dimeric dipeptide mimetic bdnf as agent for treating and preventing opioid addiction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to pharmacology, and concerns using the known bis-monosuccinyl-methionyl-serine heptamethylenediamide as an agent for treating and preventing opioid addiction. Bis-monosuccinyl-methionyl-serine heptamethylenediamide (GSB-214) in doses of 0.1–5.0 mg/kg with a single intraperitoneal administration dose-dependently prevents morphine withdrawal-induced abrupt drop in body weight and reduces severity of behavioral signs of morphine withdrawal syndrome in outbred male rats, in particular, reducing the total WS index of morphine withdrawal by 41.5% and reducing diarrhea by 96.7% in the most effective dose of 1.0 mg/kg, as well as eliminating ptosis in all studied doses.

EFFECT: compound GSB-214 in dose of 1 mg/kg with subchronic intraperitoneal introduction for 6 days prevents formation of tolerance to analgesic action of morphine with thermal stimulation.

1 cl, 3 dwg, 1 tbl, 3 ex

Description

The invention relates to medicine, in particular to pharmacology, and concerns the use of the known heptamethylenediamide bis-monosuccinyl-methionyl-serine (compound GSB-214) as a means for the treatment and prevention of opioid addiction.

Opioids are widely used to treat severe acute pain due to trauma, surgery or major burns, and to treat chronic combat syndromes. One of the main problems associated with long-term use of opioids is the high risk of developing drug dependence [Benyamin R, et al. Pain Physician. 2008; 11(2 Suppl): S105-20; Vowles KE et al. Pain. 2015; 156(4): 569-76; Volkow ND, McLellan AT. N Engl J Med. 2016; 374(13): 1253-63; Henningfield JE, et al. Prev Med. 2019; 128: 105852]. The rapid development of tolerance to the analgesic effects of opioids, requiring increased dosage to maintain adequate analgesia, contributes to this risk. Pharmacological approaches to overcoming tolerance to the analgesic effects of narcotic analgesics, especially in those with established opioid dependence, include continued use of conventional medications, short-term use, sometimes significantly higher than average doses, of additional opioids and the prescription of non-opioid drugs aimed at improving pain relief and reducing tolerance and/ or hyperalgesia induced by opioid drugs [Huxtable CA, et al. Anaesth Intensive Care. 2011; 39(5): 804-23; Yu EH et al. Anaesthesia. 2016; 71(11): 1347-62; Volkow ND, 2016]. Identifying innovative strategies to reduce the development of opioid tolerance may help reduce the risks of opioid abuse associated with chronic opioid use, providing important benefits in the treatment of chronic pain.

Long-term use of narcotic analgesics leads to the formation of an opioid withdrawal syndrome, which includes somatic, autonomic, neurological and psychological manifestations, which is a classic sign of opioid use disorder. Prevention of withdrawal symptoms is one of the most common reasons for continued use of opioid drugs [Hutcheson D, et al. Nat Neurosci. 2001; 4: 943], therefore pharmacotherapy is usually aimed at minimizing the severity of withdrawal symptoms.

Current medications used to treat withdrawal symptoms include opioid agonists, such as methadone and buprenorphine [Amato L et al. Cochrane Database Syst Rev. 2013; 2013(2): CD003409; Reed LJ et al. J Clin Psychopharmacol. 2007; 27(2): 188-92], which directly suppress aversive signs of withdrawal, as well as non-opioid drugs, for example, clonidine [Gold M, et al. The Lancet. 1978; 312: 599-602], which reduce the severity of individual symptoms.

In several countries, the synthetic μ-opioid receptor agonist methadone has been used for the treatment of pain, as well as for the treatment, maintenance of opioid withdrawal, and prevention of withdrawal symptoms secondary to the intended therapeutic effects of opioids for more than 60 years [Layson-Wolf C et al. J Pain Palliat Care Pharmacother. 2002; 16(1): 29-59]. The pharmacokinetic properties of methadone, which cause large individual differences among patients, create difficulties in developing recommendations for its use. The lack of a weaning strategy from a standardized dose of methadone often results in increased exposure and/or withdrawal symptoms [Walters RA, et al. J Pharm Pract. 2021; 34(3): 417-22; Glasper A et al. Pharmacology. 2008; 81(2): 92-6), which may be accompanied by psychosis [Levinson I, et al. J Clin Psychiatry. 1995; 56(2): 73-6]. The partial μ-opioid receptor agonist and κ-opioid receptor antagonist buprenorphine is considered an effective treatment for opioid dependence, but it also has significant variability in response to pharmacotherapy among patients. For example, studies of CYP3A4 gene variants have shown that the ultra-rapid metabolizer phenotype requires higher doses of buprenorphine, therefore, additional genotyping is necessary to tailor buprenorphine therapy to individual patients [Meaden CW et al. Pharmacogenomics J. 2021; 21(2): 128-39]. Features of the pharmacological profile of buprenorphine indicate an increased risk of accelerated opioid withdrawal when patients begin treatment with buprenorphine, namely, when switching from long-acting opioids [Oakley B et al. Drug Alcohol Rev. 2021; 40(4): 567-71]. The long-acting opioid receptor antagonist naltrexone (XR-NTX) is another treatment approach aimed at preventing relapse, and XR-NTX is noninferior to buprenorphine in the maintenance treatment of opioid use disorder [Tanum L et al. JAMA psychiatry. 2017; 74: 1197–205], although its use requires successful completion of a supervised withdrawal protocol.

It is known that drug treatment patients vary significantly in the manifestations of withdrawal syndrome, which makes it possible to identify clinically significant phenotypes of response to opioid withdrawal [Chopra MP et al. Pharmacology Biochemistry and Behavior. 2008; 90: 787-96; Sun J et al. Addict Behav. 2012; 37: 1138-44; Dunn KE, et al. Addict Biol. 2020; 25(1): e12680], which often cause an inadequate response to pharmacotherapy. Existing methods of cognitive-behavioral therapy for opioid addiction do not fully solve the problem of treating addictive disorders, so the development of new drugs that eliminate the clinical manifestations of withdrawal syndrome and are not opioid receptor agonists continues to be important.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and interacts with tropomyosin tyrosine kinase receptor B (TrkB receptor), which prevents damage to various populations of neurons [Hyman C et al. Nature. 1991; 350 (6315): 230-2; Mamounas LA et al. J Neurosci. 1995; 15(12): 7929-39]. BDNF may also play a role in the molecular and cellular adaptations underlying opioid abuse [Heberlein A et al. J Psychopharmacol. 2011; 25 (11): 1480-84). In animals, morphine increases the expression of BDNF in certain areas of the brain [Hatami H et al. Neuropeptides. 2007; 41(5): 321-8; Mashayekhi FJ et al. Neurochem Res. 2012; 37(7): 1517-23]. Physical symptoms of opioid withdrawal may be accompanied by neurodegeneration, which has been experimentally proven in vivo, where morphine withdrawal negatively affected synaptic plasticity and neuronal survival [Bandaru VV et al. J Neuroimmune Pharmacol. 2011; 6 (4): 640-9; Campbell LA et al. Neuropharmacology. 2015; 92: 98-107]. According to the researchers, this phenomenon may be associated with the neurotoxic effects of proBDNF [Teng HK et al. J Neurosci. 2005; 25(22): 5455-63] and once again points to the need to relieve acute opioid withdrawal states in persons with established dependence. Since experimental data indicate that BDNF mediates behavioral adaptations underlying opioid addiction [Bolanos CA, Nestler EJ. Neuromolecular Med. 2004; 5(1): 69-83], it can be considered as a promising pharmacological target for therapeutic intervention in opioid addiction. Unlike full-length BDNF, small-molecule BDNF analogues readily cross the blood-brain barrier after systemic administration, which is essential for their potential clinical use. This is confirmed by the results of a study of the anti-addictive properties of a low molecular weight mimetic of the 4th loop of BDNF (compound GSB-106), which activates MAPK-ERK and PI3K/AKT post-receptor cascades after interaction with TrkB receptors, which, with “prophylactic” subchronic administration, prevented the formation of reinforcing effects of opioids, and also weakened the development of somatic signs of morphine withdrawal, preventing the increase in the content of the transcription factor ΔFosB in the striatum and nucleus accumbens in rats [Kolik LG et al. Bull Expert. Biol. and Med. 2020; 170(7): 40-4].

Based on the original hypothesis about the interaction of neurotrophins with tyrosine kinase Trk receptors using the most exposed areas of their loop structures associated with the implementation of certain biological effects, a dimeric dipeptide mimetic of the 1st loop of BDNF heptamethylenediamide bis-monosuccinyl-methionyl-serine (HSB compound) was designed and synthesized -214) (Fig. 1), which in in vitro experiments interacts with TrkB receptors with subsequent activation of the predominantly post-receptor PI3K/AKT signaling pathway [Gudasheva TA et al. Bioorg Khim. 2012; 38(3): 280-90]. In in vitro and in vivo experiments, the GSB-214 compound demonstrated neuroprotective [Gudasheva TA, 2012] and antidiabetic properties [Yagubova SS et al. Bulletin Expert Biol. and Med. 2020; 169(6): 712-5] in the absence of anxiolytic [Kolik LG et al. Expert and Clin Pharmacol. 2020; 83(11): 3-7), antidepressant-like (Mezhlumyan AG et al. Pharmacokinetics and Pharmacodynamics. 2020; 1: 11-7) and antinociceptive effects (unpublished data) per se upon acute administration.

However, data on the nature of the effect of heptamethylenediamide bis-monosuccinyl-methionyl-serine (GSB-214) on the manifestation of signs of morphine withdrawal and the formation of tolerance to the analgesic effect of morphine against the background of formed opioid dependence are currently lacking.

The essence of the invention is the use of heptamethylenediamide bis-monosuccinyl-methionyl-serine as a means for the treatment and prevention of opioid addiction.

The purpose of the invention is to develop new drugs with anti-addictive potential that are not opioid receptor agonists for pharmacotherapy and prevention of opioid addiction. This goal was achieved using bis-monosuccinyl-methionyl-serine heptamethylenediamide.

The technical result of the invention is to expand the arsenal of tools for use in drug treatment practice for the purpose of treating and preventing the formation of opioid addiction.

The implementation of the present invention is demonstrated by the following non-limiting examples.

This study was carried out on 90 outbred white male rats (FGBNU Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency, Stolbovaya branch). The animals were kept 6 individuals per cage in the vivarium conditions of the V.V. Research Institute of Pharmacology. Zakusova" (temperature 21-23°C, relative humidity 40-60%) in natural light and free access to water and briquetted feed for 10 days before testing in accordance with SP 2.2.1.3218-14 "Sanitary and epidemiological requirements to the design, equipment and maintenance of experimental biological clinics (vivariums)” dated August 29, 2014 No. 51. The experiments were approved by the Commission on Biomedical Ethics of the V.V. Research Institute of Pharmacology. Zakusova."

Morphine hydrochloride (Minmedbioprom association "Chimkentbiopharm", substance) was dissolved in water for injection and administered intraperitoneally (i.p.) at the rate of 0.1 ml/100 g of rat weight. Animals in the control group received water for injection. Compound GSB-214 (bis-monosuccinyl-methionyl-serine heptamethylenediamide) in the form of a substance, synthesized at the Federal State Budgetary Institution “Research Institute of Pharmacology named after V.V. Zakusov” in doses of 0.1, 1.0 and 5.0 mg/kg were dissolved in water for injection and administered intravenously at the rate of 0.1 ml/100 g of rat weight.

Formation of dependence on opiates and assessment of somatic manifestations of morphine withdrawal syndrome.

Rats developed dependence on morphine and their behavior was assessed for the presence of specific signs of withdrawal syndrome (WS) in accordance with the standard scheme described previously [Kolik L.G. et al. Bulletin Expert Biol. and Med. 2020; 170(7): 40-4]. In order to reduce the level of stress and adapt to the experimental conditions, the animals were subjected to a “handling” procedure for 3-4 days before the start of the research. The rats were placed in an experimental room with natural light 1 day before the start of the experiment. Animals were weighed every other day, starting from the 1st day of morphine administration. The average values of changes in body weight of rats in groups were calculated according to the days of the experiment. To obtain animals dependent on morphine, the drug was administered to animals in increasing doses of 10-20 mg/kg, 2 times a day every 8 hours for 5 days: 1st day total - 10 and 10 mg/kg, 2nd day - 10 and 20 mg/kg, 3rd day - 20 and 20 mg/kg, 4th day - 20 and 20 mg/kg, 5th day - 20 mg/kg. Bis-monosuccinyl-methionyl-serine heptamethylenediamide, i.p., was administered once 30 minutes before morphine on the 5th day of the experiment. Testing of animals for the presence of specific signs of SO morphine was carried out on the 5th day of the experiment for 5 minutes in an “open field” (illuminated circular arena with a diameter of 80 cm) 15 minutes after the administration of the opiate receptor antagonist naloxone (Du Pont De Nemours Int. S.A. , Swiss") at a dose of 1.0 mg/kg to provoke CO. Control animals were given water for injection every day for 5 days, once in the morning, and on the 5th day of the experiment, before testing, they were given naloxone at a dose of 1.0 mg/kg. For all groups, the behavioral reactions of animals (locomotor activity (LA), stance, grooming, defecation) and specific signs of morphine CO (16 indicators) were recorded. Discrete signs of withdrawal (diarrhea - in points, shaking and grinding of teeth - according to the number of acts) were assessed quantitatively and alternatively, the rest - in an alternative form according to the “yes” - “no” principle. The total index (SI) of the severity of SI for each animal and the average values for the experimental and control groups were calculated based on alternative signs with the maximum possible SI value equal to 16 points. The average value of the severity of OM in the morphine group (active control) was taken as 100%.

Formation of tolerance to the analgesic effect of morphine and assessment of pain thresholds during thermal stimulation of nociceptors.

The nociceptive response in rats was assessed using the “tail flick” test. First of all, the background pain threshold was determined. The animals were placed on the working surface of the Ugo Basile analgesimeter (Italy), fixed with one hand, while the lower third of the animals' tail was located above a heated infrared emitter with a diameter of 6 mm. The emitter power is set in the device to a value corresponding to the latent period of the reaction of 3-5 seconds, which corresponds to the emitter values of 35-37 conventional units. units The maximum reaction time is limited to 20 seconds. The initial average values of pain thresholds were determined based on 4 measurements with an interval of 10 minutes, the average value of the pain threshold for each rat (sec) was taken as 100%.

To develop tolerance to the analgesic effect, morphine at a dose of 2.0 mg/kg was administered intravenously 2 times a day with an interval of at least 8 hours for 6 days in accordance with a previously described method [Konstantinopolsky MA et al. Pharmacokinetics and pharmacodynamics. 2016. 2: 40-5]. Compound GSB-214 at a dose of 1.0 mg/kg was administered intravenously once a day 45 minutes before the morphine injection. The analgesic effect was recorded on the 1st, 3rd and 6th days of the experiment once 45 minutes after administration of the GSB-214 compound. The difference between the values of pain thresholds on the 1st and 6th days of the experiment, expressed as a percentage (%) for each group of animals, means a change in tolerance over time. Comparison of the corresponding indicators for the “morphine” group (active control) and each of the experimental groups showed the presence or absence of tolerance for this effect in comparison with the “morphine” group.

Statistical processing of the obtained results was carried out using one-way analysis of variance (ANOVA), Duncan's test for independent groups, Kruskall-Wallis test and Wilcoxon test for dependent groups. The normality of data distribution was checked using the Shapiro-Wilks test. Critical significance level α=0.05.

Example 1. Effect of single-administration hexamethylenediamide bis-(N-monosuccinyl-L-seryl-L-lysine) on morphine withdrawal-induced changes in body weight in rats

The experiments were performed on outbred male rats weighing 240-250 g (n=40). 5 experimental groups were formed: group 1 “water for injection + water for injection + naloxone” (control); group 2 “morphine + water for injection + naloxone” (active control); group 3 “morphine + GSB-214 at a dose of 0.1 mg/kg + naloxone”; group 4 “morphine + GSB-214 at a dose of 1 mg/kg + naloxone”; group 5 “morphine + GSB-214 at a dose of 5 mg/kg + naloxone.”

Changes in body weight of experimental animals were recorded throughout the experiment. In accordance with figure 2, already by the 3rd day there was a sharp decrease in body weight by 7%, 13%, 9% and 15%, respectively, for animals of groups 2, 3, 4 and 5 receiving morphine (p <0.001, p <0.001, p<0.01 and p<0.001) compared to day 1. After a single injection of the GSB-214 compound on the 6th day of the experiment, 24 hours after the last injection of morphine, a significant decrease in body weight loss was recorded in the groups receiving GSB-214 at doses of 0.1 and 1 mg/kg, compared with the morphine group "(p<0.01, p<0.01), whereas in the group receiving GSB-214 at a dose of 5 mg/kg, the decrease in body weight did not differ from the morphine group and amounted to 13%. Thus, GSB-214 in doses of 0.1 and 1 mg/kg with a single administration against the background of established opioid dependence prevented the loss of body weight in animals.

Example 2. Effect of hexamethylenediamide bis-(N-monosuccinyl-L-seryl-L-lysine) with a single administration on the manifestation of somatic signs of morphine “withdrawal syndrome” in rats

The experiments were performed on outbred male rats weighing 240-250 g (n=40). 5 experimental groups were formed: group 1 “water for injection + water for injection + naloxone” (control); group 2 “morphine + water for injection + naloxone” (active control); group 3 “morphine + GSB-214 at a dose of 0.1 mg/kg + naloxone”; group 4 “morphine + GSB-214 at a dose of 1 mg/kg + naloxone”; group 5 “morphine + GSB-214 at a dose of 5 mg/kg + naloxone.”

Testing of animals for the presence of specific signs of CO morphine was carried out on the 5th day of the experiment for 5 minutes in an “open field”. Control animals received water injections daily for 5 days according to the above scheme, as animals from the experimental groups, and on the 5th day 1st day of experiment before testing - naloxone at a dose of 1 mg/kg. Changes in the severity of individual signs of SO morphine under the influence of the dipeptide GSB-214 are presented in the table.

According to the data obtained, GSB-214 exhibited dose-dependent activity in relieving somatic signs of morphine withdrawal with a maximum effective dose of 1 mg/kg, as evidenced by a decrease in the total index of morphine withdrawal SD by 41.5% compared to the active control group (morphine). With a single administration of GSB-214 at a dose of 1 mg/kg in animals with a developed dependence on morphine, the most pronounced effect was observed, namely, a decrease in diarrhea by 96.7% (p<0.001), a decrease in body posture disorders by 70% (p< 0.001), ptosis by 60% (p<0.01), convulsive reactions by 75% (p<0.01), absence of teeth grinding (p<0.05). There was virtually no effect of GSB-214 on indicators such as rhinorrhea, dyspnea, the number of writhings, attempts to escape and vocalization. With an increase in dose to 5 mg/kg, the effectiveness of the GSB-214 compound noticeably decreased, which is consistent with the known data on the dome-shaped dose-effect relationship characteristic of peptide compounds (table).

Example 3. Effect of hexamethylenediamide bis-(N-monosuccinyl-L-seryl-L-lysine) with subchronic administration on the formation of tolerance to the analgesic effect of morphine in rats

The compound GSB-214, when administered independently, did not have an analgesic effect, as evidenced by the absence of statistically significant changes in pain response thresholds on the 1st, 3rd and 6th days of the experiment in the GSB-214 group compared to the control group (Fig. .3 A, B). The analgesic effect of morphine in the “tail flick” test with thermal stimulation of nociceptors was most pronounced on day 1 (p < 0.0001, 182%) compared to the control group, gradually disappearing by day 6 of observations compared to the control group (p>0.05) and with the 1st day of observation (p<0.0001) (Fig. 3 A, B)

With the preliminary administration of the compound GSB-214 (group “GSB-214 + morphine”) 45 minutes after the injection of morphine on the 1st day of the experiment, a statistically significant increase in the latent period of the tail-flick reaction was noted (p<0.0001, 179%) compared with the control group, comparable to the effect of morphine per se (Fig. 3 A, B). On the 3rd day in the GSB-214 + morphine group, the analgesic effect was more pronounced than in the morphine group, 148 and 127% (p<0.01 and p<0.05), respectively, compared to the control group. On the 6th day in the GSB-214 + morphine group, the analgesic effect remained at the level of 143% and was statistically significantly different from the morphine group (p<0.05, 106%) and the control group (p<0.05 , 108%) (Fig. 3 A, B).

Description of drawings

Fig. 1. Structure of the BDNF homodimer showing loops 1-4 and design diagram of a dimeric mimetic of the 1st loop of BDNF.

Fig. 2. The effect of the compound GSB-214 upon single administration on

change in body weight in rats with established opioid dependence.

The x-axis is the days of the experiment.

The y-axis is the body mass of animals in grams.

Experimental groups:

1 - “control” - gray dotted line,

2 - “morphine” - solid black line,

3 - “M + GSB-214 at a dose of 0.1 mg/kg” - solid gray line,

4 - “M + GSB-214 at a dose of 1 mg/kg” - solid light gray line,

5 - “M + GSB-214 at a dose of 5 mg/kg” - solid dark gray line.

^^ - p<0.01, ^^^ - p<0.001 - statistically significant differences compared to the control group; ** - p<0.01 - statistically significant differences with the indicators of the “morphine” group (active control), according to ANOVA and Duncan’s test; ## - p<0.01 when comparing on day 1 with subsequent days of the experiment according to the Kruskall-Wallis test. The number of animals in each group is 10. Data are presented as average values (mean).

Fig. 3. Effect of the compound GSB-214 upon subchronic administration on the formation of tolerance to the analgesic effect of morphine

3A

The x-axis is the days of the experiment, the y-axis is time, sec.

Experimental groups:

1 - “control” is indicated by a dark gray column;

2 - “GSB-214” is indicated by a light gray column;

3 - “morphine” is indicated by a gray column;

4 - “GSB-214 at a dose of 1 mg/kg + morphine” is indicated by a black column.

* - p<0.05, ** - p<0.01, *** - p<0.0001 statistically significant differences compared to the control group; # - p<0.05 statistically significant differences compared to the “morphine” group; xxx - p<0.0001 statistically significant differences when compared between days 1 and 6 within each group, according to one-way analysis of variance ANOVA, Duncan test and Wilcoxon test for dependent groups. There are 8 animals in each group. Data are presented as mean values and standard error of the mean (SEM).

3B

The x-axis is the days of the experiment, the y-axis is % of the initial level.

Experimental groups:

1 - “control” is indicated by a dark gray column;

2 - “GSB-214” is indicated by a light gray column;

3 - “morphine” is indicated by a gray column;

4 - “GSB-214 at a dose of 1 mg/kg + morphine” is indicated by a black column.

* - p<0.05, ** - p<0.01, *** - p<0.0001 statistically significant differences compared to the control group; # - p<0.05 statistically significant differences compared to the “morphine” group; xxx - p<0.0001 statistically significant differences when compared between days 1 and 6 within each group, according to one-way analysis of variance ANOVA, Duncan test and Wilcoxon test for dependent groups. There are 8 animals in each group. Data are presented as average values (mean).

Claims (1)

The use of heptamethylenediamide bis-monosuccinyl-methionyl-serine as a means for the treatment and prevention of opioid addiction.