RU2429874C1 - Analgesic agent for visceral pain management - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is offered is application of amide N-(6-phenylhexanoyl)glycin-L-triptophane (GB-115, dipeptide, previously known as a psychotropic substance) as an agent for visceral pain management. Evident analgesic activity of the compound is shown in a visceral pain model (writhing test). An analgesic effect of GB-115 is exceeded in efficiency by morphine, nevertheless is comparable in antinociception intensity to the nonsteroidal anti-inflammatory drug diclofenac. Naltrexone iodide, a nonselective peripheral opioid receptor antagonist completely prevents GB-115 from displaying analgesic properties that testifies to GB-115 interaction with peripheral opioid receptors. The k-opioid receptor blockade by norbinaltorphimine, a selective k-receptor antagonist, arrests pharmacological effects of GB-115.

EFFECT: drug is characterised by a unique spectrum of receptor reactions and the absence of side effects, practically nontoxic, does not cause sedation and respiratory depression peculiar to opioid analgesics, does not exhibit ulcerogenic effects and action of a hematosis process inherent in NSAIDs.

6 dwg, 3 tbl

Description

The invention relates to medicine, in particular to pharmacology, and relates to a new use of the known substance - the amide N- (6-phenylhexanoyl) glycine-L-tryptophan (GB-115) (RF Patent No. 2227144, Bull. No. 11, 2004) as analgesic for the relief of visceral pain.

Visceral pain occurs when the nociceptors of the internal organs are irritated and differs from somatic pain in the diffuse distribution pattern, difficulty in localization, and associated autonomous reflex reactions. It is generally accepted that pain in diseases of internal organs can be caused by impaired blood flow in organs, spasm or convulsive contractions of smooth muscles, stretching of the walls of hollow organs, and inflammatory changes in organs and tissues. The density of nociceptors is especially high in the peritoneum, with inflammation of which acute and intense pain occurs. The pathophysiology and mechanism of the occurrence of visceral pain is still not completely clear, and the existing therapeutic approaches for treating this type of pain are not optimal (Olesenet AE et al. World J Gastroenterol. 2009. January 14; 15 (2): 177-181 ) The use of traditional opioid analgesics, agonists (µ receptors, is not always justified and is complicated by the presence of side effects of central origin (euphoria, sedation, nausea).

Currently, antagonists of 5-HT ₃ receptors (Gershon MD, et al. Gastroenterology 2007; 132: 397-414), probiotics (Quigley EM, Neurogastroenterol Motil 2007; 19: 19) are potential agents for treating abdominal pain in clinical trials. 166-172), agonists of α2 and β3 adrenergic receptors (Camilleri M., et al. Clin Gastroenterol Hepatol 2003; 1: 111-121; Celiek S. et al. Gastroenterology 2007; 133: 175-183), CRF ₁ antagonists receptors (Sagami Y. et al. Gut 2004; 53: 958-964), antagonists of NMDA receptors (Strigo IA, Pain 2005; 113: 255-264) and antidepressants (Peghini PL et al. Gut 1998; 42: 807- 813). One of the most promising pharmacological targets for treating visceral pain is peripheral opioid κ receptors (Bradesi S. et al. Curr Opin Pharmacol. 2008 December; 8 (6): 697-703).

For the first time, the existence of k-opioid receptors (COR), functionally different from µ- and δ-opioid receptors, was shown in the 70s of the XX century (Martin et al., J. Pharmacol. Exp. Ter. 1976; 197: 517- 532). Using radioligand research methods, the heterogeneity of KOR and the existence of k ₁ and k ₂ receptors were subsequently proved, each of which is subdivided into (k _1a -, k _2a -) and (k _1b -, k _2b -) subtypes (Rothman et al. , Peptides. 1990; 11: 311-331). However, to date, only KOP ₁ has been able to clone, the pharmacology of which is identical to the previously described k ₁ opioid receptor (Simonin et al., Proc. Natl. Acad. Sci, USA 1995; 92: 7006-7010). Most currently available k-agonists are optimized for k ₁ -binding site or cloned KOP ₁ .

The first KOR agonists exhibited pronounced analgesic properties on models of visceral pain that persisted under a variety of experimental conditions regardless of the type of animal (mouse or rat), target organs (duodenum, colon, gall bladder, peritoneum), the nature of the damaging agent (sprain or chemical irritation), anesthetized or conscious animals, such as pain (primary or inflammatory), as well as the chemical nature of k-agonists (Diop et al., Eur J Pharmacol. 1994 Dec 12; 271 (1): 65-71; Burton & Gebhart, J Pharmac ol Exp Ther. 1998 May; 285 (2): 707-15; Sengupta et al., Pain. 1999 Feb; 79 (2-3): 175-85; Joshi SK. et al. The Journal of Neuroscience, August 1 2000, 20 (15): 5874-5829; Sandner-Kiesling et al., Pain. 2002 Mar; 96 (l-2): 13-22; Kamp EH et al. Am J Physiol Gastrointest Liver Physiol. 2003 Mar; 284 (3): G434-44), without causing euphoria, respiratory depression, or gastrointestinal function (Rivie're PJM and Junien, JL In: Drug Development, Molecular Targets for GI Diseases, eds. Gaginella, TS & Guglietta A. Totowa, NJ: Humana Press. 2000, 203-238). Experimental visceral inflammation reduces the pain threshold by increasing the response to pain and enhancing the analgesic potential of k-agonists (Langlois et al., Eur J Pharmacol. 1997 Apr 18; 324 (2-3): 211-7; Burton & Gebhart, J Pharmacol Exp Ther. 1998 May; 285 (2): 707-15; Sengupta et al., Pain. 1999 Feb; 79 (2-3): 175-85). The visceral pain models used suggest that k ₁ receptors mediating the response of KOR agonists are located on the periphery, since their effects are blocked by peripheral opioid antagonists that do not penetrate the BBB (Sandner-Kiesling et al., Pain. 2002 Mar; 96 (1 -2): 13-22).

Currently, there is relatively little information on the effectiveness of k-agonists in clinical trials in the treatment of visceral pain.

Fedotozine significantly exceeded the placebo effect in reducing abdominal pain in non-ulcer dyspepsia (Fraitag et al., Dig Dis Sci. 1994 May; 39 (5): 1072-7) and irritable bowel syndrome (CPK) (Dapoigny M. et al. Digest Dis Sci 1995; 40: 2244-2249), and also increased the pain threshold for intestinal distension in patients with IBS (Delvaux et al. Gastroenterology. 1999, 116 (1): 38-45). A series of works by Gebhart's (1998) showed that the analgesic effect of fedotozine is mediated by inhibition of Na channels on primary afferent neurons, along with central effects on MOP and KOR (Su X. et al. J Neurophysiol 1998; 80: 3112-3119). After receiving negative results in the second phase of clinical trials, further development of the drug stopped.

The data on the antinociceptive activity of asimadoline are very contradictory: in some works, it turned out to be an effective tool for relieving abdominal pain in IBS (Delvaux et al., Aliment Pharmacol Ther. 2004 Jul 15; 20 (2): 237-46), but in others ( Szarka LA et al., Clin Gastroenterol Hepatol 2007; 5: 1268-1275). In a large-scale (596 patients) study, asimadoline for 4 months significantly reduced pain and discomfort in IBS in patients with moderate pain (Mangel A.W. et al. Aliment Pharmacol Ther 2008; 28: 239-249). Other authors have noted the ability of asimadoline to cause hyperalgesia in the treatment of non-visceral postoperative pain (Machelska H. et al. J Pharmacol Exp Ther 1999; 290: 354-61).

Pilot clinical trials have revealed the ability of acetamide derivatives to penetrate the BBB, cause dysphoria, sedation and hyperalgesia (Rivie're, PJM and Junien, JL In: Drug Development, Molecular Targets for GI Diseases, eds. Gaginella, TS & Guglietta A., Totowa, NJ: Humana Press. 2000, 203-238), which stopped the use of first generation KOR agonists and initiated the search for selective peripheral k-agonists among peptide compounds. The new strategy was based on the idea of creating larger peptide molecules that do not penetrate the BBB and do not cause central side effects (Table 1). Not all attempts, however, have been successful. So, E-2078, a synthetic L-amino acid analog of dinorphin _1-8 , showed central effects and caused analgesia, including non-opioid nature (Butelman ER et al. Psychopharmacology (Beri) 1999; 143: 190-6). Experimental studies of FE200041, a D-amino acid tetrapeptide, have demonstrated the presence of antinociceptive properties that are realized through interaction with peripheral KOR (Vanderah TW et al. J Pharmacol Exp Ther 2004; 310: 326-33). A subsequent evaluation of its modified analogue, FE200665 (CR665), revealed peripheral antinociceptive activity in a wide range of doses in experimental models of visceral pain. Currently, CR665 has been referred to clinical trials (Vanderah TW et al. Eur J Pharmacol 2008; 583: 62-72).

Thus, the peptide agonists of KOR show high analgesic activity in various models of visceral pain. These effects are mediated through peripheral KOR and, possibly, as an additional mechanism, through sodium channels located at the peripheral ends of primary sensitive afferents. The analgesic potential of k-agonists in visceral pain is enhanced in the presence of inflammation (Riviere PJM, Junien JL In Dmg Development, Molecular Targets for GI Diseases 2000. Totowa, NJ: Humana Press; 203-238.eds. Gaginella, TS & Guglietta A. )

In general, the pharmacological profile of k-agonists suggests that peripheral selective k-agonists should be effective in treating visceral pain of various etiologies, including abdominal surgery associated with postoperative pain and bowel obstruction, pancreatic pain, dysmenorrhea, and functional disorders such as irritable bowel syndrome and dyspepsia.

The experiments were carried out on outbred male mice weighing 24-29 g. The animals were kept for 6 days before the start of the experiment in a vivarium of 20 animals per cage with the provision of water and standard ad libitum briquetted food under a normal 12-hour light regime. The experiments were carried out from 09:00 to 15:00 h.

For the study we used: compound GB-115 (0.05-20.0 mg / kg, p / o, for 40 min), a retropeptide analog of the endogenous tetrapeptide cholecystokinin (synthesized at the V.V.Zakusov Research Institute of Pharmacology RAMS), an opioid agonist of morphine hydrochloride (3.0 and 5.0 mg / kg, ip, for 20 min) (Sigma Chemical Co.), diclofenac sodium (20.0 mg / kg, p / o, 40 min) and indomethacin (25.0 mg / kg, p / o , over 40 min) as a reference drug (Sigma Chemical Co.), non-selective opioid antagonist of naloxone hydrochloride (1.0 mg / kg, s / c, over 20 MHH) (Sigma Chemical Co.), peripheral opioid receptor antagonist naltrexone iodide 10.0 mg / kg, s / c, over 30 min) (synthesized at the V.V.Zakusov Research Institute of Pharmacology RAMS), selective antagonist of k-opioid receptors norbinaltorfimin (5.0 mg / kg, b / w, over 20 min ), an agonist of k-opioid receptors and a mu-opioid receptor antagonist pentazocine (2.0 mg / kg, s / c, over 30 min) (Sigma Chemical Co.). All substances were dissolved in distilled water, with the exception of GB-115, which was dissolved in distilled water with the addition of Tween-80.

The cramp test was performed in accordance with the previously described methodology (Collier N.O. et al. Pharmacol.Chemother., 1968, 32, 295-310). Animals were injected with morphine (5.0 mg / kg), GB-115, pentazocine, as well as naloxone, naltrexone iodide and norbinaltorfimine 15 minutes before morphine and GB-115, and distilled water as a control (p / o, 40 minutes ) before intraperitoneal (i / b) injection of 0.75% acetic acid solution. The number of lordoses (“cramps”) was counted in each animal for 15 minutes immediately after the introduction of acetic acid.

The hot plate test was used to determine the latency of the reaction. All animals were selected 1 hour before testing on the basis of basic reactivity in this model with the removal of mice left on a copper plate heated to 56 ± 0.5 ° C for more than 15 seconds. A latency of 30 seconds was regarded as 100% analgesia. The time of occurrence of the avoidance reaction (jump and / or licking of the hind paw) was recorded in mice after administration of morphine, GB-115 (10.0 mg / kg, p / o, 40 min), naloxone (15 min before morphine and GB-115) , naltrexone iodide (15 minutes before morphine and GB-115) and distilled water as a control.

The tail-flick test. Using the analgesia test “analgesia test, tail flick type 812” from Hugo Sachs Electronic (Germany), the tail pullback time from the moment of application of local heat was recorded. The studied substances were administered in the same doses and terms as in the hot plate test. All animals were selected in 24 hours according to the results of background testing in this setup, with the exception of those individuals who remained without reaction for more than 4 seconds. A latency of 30 seconds was regarded as 100% analgesia.

Data are presented in% of the maximum possible effect (MVE) = (latent reaction period after administration of the compound minus the background latent reaction period) / (maximum exposure time minus the background latent reaction period) × 100% (Le Bars D., M. Gozariu M. and Cadden SW Pharmacol. Rev., 2001; 53 (4), 597-652).

All preparations were administered in a volume of 0.1 ml / 10 g of animal weight.

Statistical data processing was performed using one-way ANOVA analysis of variance, followed by the use of the non-parametric Mann-Whitney U Test criterion for independent groups.

Radioligand analysis. The ability of GB-115 to displace high affinity neuroreceptor ligands from binding sites has been studied in accordance with the protocols of Serer (France) (http: //www/cerep.com).

The interaction of GB-115 with recombinant human µ-opioid receptors (MPAs) was studied on the HEK-293 cell line (Wang L. et al., J Neurochem. 1994 Nov; 63 (5): 1726-30) using [ ³ H] DAMGO at a concentration of 0.08 nM as the labeled ligand and reference substance - DAMGO (IC ₅₀ = 1.3 · 10 ^-9 M, Ki = 5.5 · 10 ^-10 M, nН = 1.1). Nonspecific binding was determined in the presence of 10 μM naloxone. The incubation time with the cell line was 120 min at a temperature of 22 ° C.

The interaction of GB-115 with recombinant human δ ₂ opioid receptors (DOPs) was studied in the CHO cell line (Simoni M. et al., J Endocrinol. 1994 May; 141 (2): 3 59-67) using labeled ligand [ ³ H] DADLE at a concentration of 0.5 nM. The comparison substance is DPDPE (IC ₅₀ = 1.6 · 10 ^-9 M, Ki = 9.8 · 10 ^-10 M, nН = 1.3). Nonspecific binding was determined in the presence of 10 μM naltrexone. The incubation time with the cell line was 120 min at a temperature of 22 ° C.

The interaction of GB-115 with recombinant rat κ-opioid receptors (CBS) was studied on the CHO cell line (Meng et al., Proc Nati Acad Sci USA. 1993 Nov 1; 90 (21): 9954-8). [ ³ H] 69593 at a concentration of 1.0 nM was used as a labeled ligand. The comparison substance is U 50488 (IC ₅₀ = 1.0 · 10 ^-9 M, Ki = 6.7 · 10 ^-10 M, nН = 1.2). Nonspecific binding was determined in the presence of 10 μM naloxone. The incubation time with the cell line was 60 min at a temperature of 22 ° C.

IC ₅₀ values and the Hill coefficient (nН) were determined using a nonlinear regression analysis of competitive displacement curves with approximation of the curve by the Hill equation. The Ki value was calculated according to the Cheng-Prussoff equation (Y. Cheng, WHPrusoff, Biochemical pharmacology, 1973; 22 (23), 3099-3108).

The invention is illustrated by the following examples:

Example 1. Antinociceptive properties of GB-115 with prolonged stimulation.

The cramp test allows you to evaluate both the peripheral and central mechanisms of antinociception and is recommended as a simple method for screening compounds that affect nociceptive processes induced by inflammation (Vogel HG, Vogel WH Dmg discovery and evaluation. Pharmacological assays. Berlin, Heidelberg: Springer -Veriag; 1997. p. 360-420). Acetic acid 0.75% upon intraperitoneal administration irritates the serous membranes and provokes stereotyped behavior in mice and rats, which is characterized by abdominal contractions, movements of the whole body (especially the hind limbs) and a decrease in motor activity. Such behavior is considered reflex and is considered evidence of visceral pain (Ness T.J., Gebhart G.F. Pain. 1990; 41: 167-234). This method is sensitive, predictable, and allows you to capture the antinociceptive effects of even weak analgesics (Hunskaar S. J Neurosci Methods. 1985 Jun; 14 (1): 69-76).

In the cramp test, it was found that the GB-115 compound in the dose range of 0.1–20.0 mg / kg has pronounced analgesic activity in mice during chemical stimulation due to intraperitoneal administration of acetic acid (Fig. 1). The development of the effect of GB-115 is noted with increasing dose, reaching at a dose of 10.0 mg / kg the maximum inhibition of reactions by 76.3% compared with the control (p <0.0001). A subsequent increase in the dose of dipeptide to 20.0 mg / kg leads to a weakening of analgesia to 43.9% (p <0.05).

The effect of the compound GB-115 at a dose of 10.0 mg / kg is inferior in strength to morphine (5.0 mg / kg), which reduces nociceptive manifestations by 95.5% (p <0.001), and is comparable with the analgesic activity of the non-steroidal anti-inflammatory drug diclofenac (20.0 mg / kg) (figure 2).

The analgesic effect of morphine is completely stopped by naloxone (Fig. 3A) and does not depend on the blockade of peripheral opioid receptors by naltrexone iodide (Fig. 3B). On the other hand, preliminary blockade of opioid receptors by naloxone partially (by 29.8%) removes the antinociceptive effect of compound GB-115 compared to the control (Fig. 3A). Naltrexone iodide prevents the manifestation of the analgesic activity of GB-115, restoring the threshold of pain sensitivity to control values (pigv).

It was found that the blockade of KOR by the selective antagonist of KOR by norbinalthorfimine (5.0 mg / kg, i.p.) prevented the manifestation of the antinociceptive activity of GB-115 (10.0 mg / kg) in the cramp test in mice. Pentazocine (2.0 mg / kg, s.c.), a KOR agonist selected as the reference drug, also statistically significantly reduced the number of abdominal contractions in animals (p <0.01). This effect was eliminated against the background of preliminary blockade of KOR with norbinaltorfimine.

Example 2. Antinociceptive properties of GB-115 under conditions of short-term thermal stimulation.

In case of short-term thermal irritation, as a rule, skin integuments are stimulated, and visceral and musculoskeletal tissues are not involved, which makes it possible to differentiate the analgesic properties of the studied compound not only by the modality of the stimulus, but also by the nature of the pain caused.

When animals are placed on a metal plate heated to a constant temperature, a two-component behavioral reaction occurs, which is expressed in “paw licking” and “jumping”. Such avoidance is regarded as a supraspinal response. It is known that opioids predominantly influence “licking,” and weaker analgesics (for example, paracetamol and acetylsalicylic acid) increase the latent period of the “jump” reaction. To increase the specificity and sensitivity of this test, we measured the latent period of the first avoidance reaction, regardless of its nature (Carter, Pain. 1991 Nov; 47 (2): 211-20).

In response to thermal stimulation in the “hot plate” test, the GB-115 compound statistically significantly weakened the nociceptive reaction only at the most studied dose (20.0 mg / kg), (Fig. 5), significantly inferior to morphine in these conditions by the force of action (Table. one).

To identify possible antinociceptive properties of the GB-115 compound at the spinal level, the tail tail test was used, based on the application of a heat beam to the limited surface of the mouse tail, which provokes a sharp pull back. An increase in tail holding time is interpreted as an analgesic effect. It is believed that this test is effective primarily for the detection of opioid analgesics (Le Bars D., M. Gozariu M. and Cadden S.W. Pharmacol. Rev., 2001; 53 (4), 597-652).

It was found that the connection of GB-115 with increasing dose gradually statistically significantly increases the latent period of the tail-flick test in the tail flick test (up to 281.2% at a dose of 10.0 mg / kg), also inferior to morphine in action (Fig. 5, Table 1). When the dose is increased to 20.0 mg / kg, the GB-115 compound does not exhibit an antinociceptive effect in this test (Fig. 6).

Table 1 The effect of compound GB-115 on nociceptive reactions under conditions of short-term thermal stimulation compared with diclofenac and morphine Substances Doses (mg / kg) Route of administration Latent reaction period,% of MVE. Hot plate test Tail pull test The control - per os 3.9 ± 5.6 1.6 ± 0.5 Diclofenac 20.0 per os 5.3 ± 3.9 4.3 ± 1.4 Morphine 3.0 i.p. 51.3 ± 9.7 *** 7.1 ± 1.7 *** GB-115 10.0 per os 20.6 ± 9.2 4.5 ± 0.5 *** Note: data are presented as M ± SEM; * - p <0.05, *** - p <0.001 statistically significant differences compared with the control group, ### - p <0.001 compared with the morphine group, xp <0.05 compared with the GB-115 group, Marm- Whitney U test; the number of animals in the group is from 6 to 14.

Preliminary non-selective blockage of opioid receptors by naloxone prevents the manifestation of morphine-induced analgesia and partially suppresses the analgesic effect of GB-115 in the tail-tail test compared to the control group (Table 2). Naltrexone iodide, an antagonist of peripheral opioid receptors, does not affect the antinociceptive properties of morphine, while the effectiveness of GB-115 is statistically significantly reduced compared to the independent action of the dipeptide (p <0.05) and does not differ from the control group.

table 2 The effect of blockade of central and peripheral opioid receptors on the manifestation of nociceptive effects of GB-115 Substances Doses (mg / kg) Route of administration Latent reaction period,% of MVE. Hot plate test Tail pull test Water distill. (Control) - by 3.9 + 5.6 1.6 + 0.5 Water +
Morphine - by 5.0 in / b 64.0 + 12.5 *** 46.4 + 15.0 *** Naloxone +
morphine 1.0 PC 5.0 in / b -6.7 + 9.0 ### -0.2 + 9.0 ### Naltrexone iodide + Morphine 10.0 PC 47.7 + 16.7 ** 33.8 + 12.9 *** 5.0 in / b Water +
GB-115 - by 10.0 by 20.6 + 9.2 4.5 + 0.5 *** Naloxone +
GB-115 1.0
10.0 PC
by 10.9 + 4.4 3.1 + 0.3 *, x Naltrexone iodide + GB-115 10.0 PC 14.4 + 13.6 2.6 + 0.8 x 10.0 by Note: data are presented as M + SEM; * - p <0.05, *** - p <0.001 statistically significant differences compared with the control group, ### - p <0.001 compared with the water + morphine group, x - p <0.05 compared with the water + GB- group 115, Mann-Whitney U test; the number of animals in the group is from 6 to 14.

Example 3. Neuroreceptor mechanisms of action of GB-115

As a result of radioligand analysis, it was found that GB-115 in the range of micromolar concentrations interacts with k-opioid receptors (KOR): IC ₅₀ = 1.6 · 10 ^-5 M, Ki = 1.0 · 10 ^-5 M, nН = 0.5

When studying the interaction of GB-115 with µ- (MOP) and δ ₂ (DOP) - opioid receptors, the following receptor interaction parameters were established: IC ₅₀ = 1.3 · 10 ^-4 M, Ki = 5.5 · 10 ^-5 M, nH = 0.4 (MOP) and IC ₅₀ = 1.7 · 10 ^-4 M, Ki = 1.0 · 10 ^-4 M, nH = 0.8 (DOP), respectively. The values of the parameters IC ₅₀ and Ki for GB-115 are in the region of high concentrations, which indicates the absence of specific interaction of MOP and DOP receptors.

The next stage of the study was devoted to cellular functional analysis, during which the agonistic properties of GB-115 with respect to KOR in the concentration of EC ₅₀ = 1.6 · 10 ^-4 were revealed, for the standard KOR agonist U 50488, the value of EC ₅₀ = 1.0 · 10 ^-9

Thus, the results obtained in the study of the interaction of GB-115 with opioid receptors in vitro are fully consistent with the established pharmacological effects of GB-115, reveal the primary mechanisms of its action as an analgesic, and determine further prospects for pharmacological study and use in pharmacotherapy.

Example 4. Comparative characteristics of the connection of GB-115 with other analgesics for the presence of side effects.

1. Acute toxicity

When comparing the toxic properties of GB-115 with comparison drugs (diclofenac and pentazocine), it has significant advantages: LD ₅₀ for GB-115 is not determined in doses of more than 3500 mg / kg in rats and more than 6000 mg / kg in mice.

A drug Animal species Route of administration LD ₅₀ , mg / kg References Diclofenac rats by 62.5 Arzneimittel-Forschung. Drug Research. Vol.30, Pg. 1398, 1980. the mouse by 170.0 Pharmazie Vol. 37, Pg. 148, 1982. in / b 345.0 Arzneimittel-Forschung. Drug Research. Vol. 43, Pg. 44, 1993. Pentazocine rats by 1110.0 Kiso to Rinsho. Clinical Report. Vol. 4, Pg. 2145, 1970. the mouse by 305.0 Acta Medicia Okayama. Vol. 35, Pg. 179, 1981. in / b 85.0 Chemical and Pharmaceutical Bulletin. Vol.24, Pg. 2912, 1976. GB-115 rats by <3500 Determination of LD _{50 was} not possible due to the absence of animal death. the mouse by <6000 Determination of LD _{50 was} not possible due to the absence of animal death.

About 30% of patients taking non-steroidal anti-inflammatory drugs (NSAIDs) are hospitalized due to various complications (Wilson Let al. Curr Ther Res 2004; 62: 835-50). According to the results of clinical studies, the formation of numerous ulcers (ulcerogenesis), bleeding and inflammation in the digestive tract are the main side effects associated with the use of NSAIDs in medical practice, including diclofenac sodium, indomethacin and aspirin (Nakashima S. et al. Aliment Pharmacol Ther 2005 ; 21 Suppi 2: 60-66; Ramirez-Alcantara V et al. Am J Physiol Gastrointest Liver Physiol. 2009 Aug 27).

When studying the chronic toxicity of GB-115 with its daily oral administration for six months at doses of 0.1 and 10.0 mg / kg, it was found that this compound does not have a damaging effect on the blood system and does not cause pathologically significant changes in the biochemical parameters of blood serum in the studied animals and physicochemical properties of urine in rats; does not cause morphological and histological changes in the organs and tissues of rats and rabbits; does not have a local irritant effect.

2. Impact on the function of the respiratory system

According to the literature, unlike μ-opioid receptor agonists, selective κ-agonists cause analgesia, accompanied by slight respiratory depression (Bowdle T.A. Drug Saf. 1998 Sep; 19 (3): 173-89). In vitro experiments have shown that an κ receptor agonist, in particular U50,488, inhibits both the frequency and amplitude of respiration (Takita K.et al. Brain Res. 1997 Dec 5; 778 (1): 233-41).

A study of the effect of GB-115 on the respiratory rate was carried out on male white rats weighing 250-300 g, anesthetized with urethane (1300 mg / kg, ip). The respiratory rate was recorded using a special sensor mounted on the chest of animals and connected through a Biopack device (USA) to a Pentium IV computer. GB-115 was administered at a dose of 50.0 mg / kg, ip. According to the obtained curve, the respiratory rate was calculated. The experimental data were processed statistically, the significance of the differences was determined using the non-parametric Friedman test and the Dannett's test multiple comparisons method.

The experiments showed that the GB-115 compound in the studied dose did not cause pathological forms of respiration: apnea, Cheyne-Stokes, Biot, Kusmaul, agonal breathing. At the same time, the substance caused a statistically significant decrease in the frequency of respiratory movements 30-60 minutes after administration, however, this indicator did not go beyond the reference values of the norm for anesthetized rats (60-140 respiratory movements per minute) (Table 3).

Thus, the GB-115 compound has an undoubted advantage over existing κ receptor agonists without affecting the respiration function in animals.

Table 3 The effect of compounds GB-115 (50 mg / kg, ip) on respiratory rate in anesthetized rats Source. level The time after the introduction of GB-115, min 5 10 twenty thirty 60 114.5
100.0 ÷ 117.0 107.5
101.0 ÷ 114.0 105.0
92.0 ÷ 111.0 101.0
87.0 ÷ 114.0 97.0 *
84.0 ÷ 103.0 89.5 *
80.0 ÷ 101.0 Note: data are presented as median, 25% and 75% percentiles, the number of animals n = 6; * p≤0.05 - in relation to the initial level.

3. Impact on motor activity

There are many reports in the scientific literature about the effect of κ-agonists on motor activity. So, pentazocine caused a slight sedation in patients with bipolar disorders (Cohen B.M. and Murphy B. Int J Neuropsychopharmacol. 2008 Mar; 11 (2): 243-7). A quick but short violation of the coordination of movement was observed in the experiment under the influence of a synthetic κ-agonist U69.593 (Fantegrossi WE et al. Behav Pharmacol. 2005 Dec; 16 (8): 627-33). Asimadoline, a selective κ-opioid receptor agonist, caused dysphoria and sedation (M. Camilleri Neurogastroenterol Motil. 2008 Sep; 20 (9): 971-979).

To assess impaired coordination of movement, gait and balance, the test “rotating rod” was used. Mice were placed on a horizontal rod with a diameter of 2.5 cm and a rotation speed of 10 rpm, and the retention time on the rod was fixed. The inability of animals to maintain balance on the rod for 120 seconds under the influence of the studied compounds was considered as a manifestation of impaired coordination of movements. GB-115 at doses of 0.1, 1.0, 10.0 and 20.0 mg / kg and distilled water as a control was administered orally 40 minutes before testing. (Todd W. Vanderah et al. Journal of Pharmacology and Experimental Therapeutics; 2004; 310: 326-333).

It was found that during the observation period (120 sec) all animals retained the ability to stay on a rotating rod. Thus, in the range of analgesic doses, GB-115 does not cause a sedative effect and impaired coordination of movement in animals.

Thus, the compound GB-115 is a new peptide analgesic for oral administration, mainly aimed at stopping visceral pain and realizing its action through peripheral k-opioid receptors.

Brief Description of the Drawings

Figure 1 shows the effect of GB-115 on the number of abdominal contractions in mice induced by acetic acid 0.75%. Data are presented as M ± SEM; along the abscissa axis: doses of GB-115, mg / kg, along the ordinate axis: the number of cramps; statistically significant differences compared with the control group are shown at * -p <0.05, *** - p <0.001 according to the non-parametric Mann-Whitney U test; the number of animals in the group is from 9 to 25.

Figure 2 shows the effect of GB-115 (10.0 mg / kg, p / o), morphine (5.0 mg / kg, ip) and diclofenac (20.0 mg / kg, p / o) on the behavior of animals in the cramp test ". Data are presented as M ± SEM; along the abscissa axis: 1st column - control, 2nd column - morphine, 3rd column - diclofenac, 4th column - GB-115; ordinate: number of writhing; *** - p <0.001 statistically significant differences compared with the control; Mann-Whitney U test; the number of animals in the group is from 9 to 21.

Figure 3 shows that in the cramp test, the non-selective blocking of opioid receptors by naloxone completely suppresses the effects of morphine (5.0 mg / kg, ip) and partially GB-115 (10.0 mg / kg, ip) (fig. A ); blockade of peripheral opioid receptors with naltrexone iodide prevents the manifestation of the antinociceptive activity of GB-115 without affecting the effect of morphine in mice (Fig. B). Data are presented as M ± SEM. On figa along the abscissa axis: 1st column - control, 2nd column - morphine, 3rd column - morphine + naloxone, 4th column - GB-115, 5th column - GB-115 + naloxone ; ordinate: the number of cramps. On figv on the abscissa: 1st column - control, 2nd column - morphine, 3rd column - morphine + naltrexone iodide, 4th column - GB-115, 5th column - GB-115 + naltrexone iodide; ordinate: number of writhing; * -p <0.05, *** - p <0.001 statistically significant differences compared with the control, xp <0.05 compared with the GB-115 group, Mann-Whitney U test; the number of animals in the group is from 10 to 15.

Figure 4 shows the effect of κ-opioid receptor blockade by norbinalthorfimine on the manifestation of the antinociceptive activity of GB-115 (10.0 mg / kg, ip) in the cramp test in mice. Data are presented as M ± SEM; along the abscissa axis: 1st column - control, 2nd column - GB-115, 3rd column - norbinaltorfimin + GB-115, 4th column - pentazocine, 5th column - norbinaltorfimin + pentazocine; ordinate: number of writhing; ** - p <0.01 significant differences compared with the control group, ## - p <0.01 compared with the GB-115 group, xp <0.05 compared with the pentazocine group, Mann-Whitney U test; the number of animals in the group is from 10 to 15.

Figure 5 shows the effect of GB-115 (10.0 mg / kg, ip) on the latent period of the avoidance reaction in the hot plate test in mice. Data are presented as M ± SEM; abscissa: doses of GB-115, mg / kg; along the ordinate axis: maximum possible effect (MVE),%; * p <0.05 statistically significant differences compared with controls, Mann-Whitney U test; the number of animals in the group is from 9 to 15.

Figure 6 shows the effect of GB-115 (10.0 mg / kg, ip) on the latent period of the avoidance reaction in the tail tail test in mice. Data are presented as M ± SEM; abscissa: doses of GB-115, mg / kg; along the ordinate axis: maximum possible effect (MVE),%; ** - p <0.01, *** - p <0.001 statistically significant differences compared with the control, Mann-Whitney U test; the number of animals in the group is from 9 to 15.

Claims (1)

The use of N- (6-phenylhexanoyl) glycine-L-tryptophan amide as an analgesic for the relief of visceral pain.

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