Spontaneous locomotor activity was recorded for 3 min after administration of the investigated substances in a computerized device which counted photobeam interruptions (OPTO-VARIMEX (Columbus Instruments, Columbus, OH, USA) and ATM3 automatic system using Auto-Track software version 4.2).

In the test, a hot plate at a temperature of 55 °C (Columbus Instruments, Columbus, OH, USA) by the reaction of jerking and/or licking the hind legs, sensitivity to thermal effects was studied.

A suspended emulsion, of complete Freund’s adjuvant (CFA) and saline solution (1:1), was injected into the plantar surface of the left hind leg of mice (20 µl/paw). Control mice received 20 µl of saline solution (i.p.). 24 h after CFA injection, the time of paw withdrawal in response to thermal stimulation (53 °C) was recorded.

Experimental groups were administered 0.6% of acetic acid in saline solution (10 ml/kg−1 intraperitoneally (i.p.)). After injection, the mice were placed in a transparent cylinder, and the number of writhes was counted for 15 minutes.

For registration of body temperature a rectal probe MLT1404 and PowerLab software (Adinstruments Inc., Colorado Springs, CO, USA) were used. When recording the temperature, the mobility of the animals in boxes MLA5018 (ADInstruments Inc., Colorado Springs, Colorado, USA) was limited. Body temperature was recorded for 100 minutes after administration, adaptation – for 20 minutes.

The experiments were partially carried out using the equipment provided by the Center of Shred Facilities of IBCh, supported by The Russian Ministry of Education and Science, Grant RFMEFI62117X0018).

There was an in vivo study conducted to evaluate the pharmacological effects of polypeptide modulators APHC1, APHC2, APHC3, and A13. At the beginning of the study, the possibility of the studied polypeptides influencing the central nervous system was excluded. The evaluation was carried out in the motor activity test, since compounds capable of reducing motor activity can distort the results of behavioral tests used to test pharmacological effects, especially in pain tests. One one dosage of polypeptides, 0.1 mg/kg, was used, which was close to the effective dose of the polypeptides studied in further experiments in vivo. There was no change in motor activity for any of the polypeptides at a dose of 0.1 mg/kg. The tests revealed comparable results in terms of distance traveled and points. Thus, the results of the efficacy of the studied polypeptides in pain tests will not be the result of impaired motor activity or sedative effect.

The hot plate test is the easiest for inhibitors of TRPV1 because TRPV1 plays an important role in the perception of exogenous temperature stimuli. The dose-effect of the studied polypeptides on changes in behavioral reactions associated with analgesic action was estimated (Fig. 1).

Figure 1.

Time (sec) of the first reaction in the hot plate test after administration of polypeptides APHC1, 2, 3 and A13 in the hot plate test (n = 9 for each group). Note: * - The results are presented as the mean ± s.e.; * -p<0.05 versus saline group (ANOVA followed by Tukey’s test).

The studied APCH 1-3 substances significantly increased the time spent on a thermostated surface (Fig. 1), at a dose of 0.1 mg/kg. A further increase in the dose to 1 mg/kg showed no development of analgesic effect, except of APCH 3 which increased the analgesic effect, but with an increase in the dose to 10 mg/kg decreased to the control group. A13 at a dose of 0.01 mg/kg showed analgesic effect; however, a further increase in the dose had no effect on the development of analgesic effect, and was comparable with the control group.

The results showed that the investigated polypeptides exhibit a dome-shaped dose-response relationship. This effect is typical for substances whose action involves peptidergic mechanisms (McNamara et al. 2005, Wu et al. 2010). Thus, a further increase in the dose will cause no increase in the biological effect of the studied substances.

Previously, it was shown that the knockout animals exhibit analgesic activity in the hot plate test by the TRPV1 gene (Caterina et al. 2000). The results of the study of analgesic activity of polypeptides APHC1-3 and A13 in the hot plate test well coincide with the literature data and indicate the development of analgesic activity through receptors – TRPV1. The control in the study of analgesic activity was the homologue of polypeptides APHC1-3, Aprotinin, the most powerful inhibitor of serine proteases. Aprotinin showed comparable results of analgesic activity with the control group (not shown) at doses of 0.1 and 1 mg/kg. Thus, the analgesic activity of the investigated polypeptides is not the result of the ability to partially inhibit serine proteases, but the modulation of TRPV1 channels.

CFA-induced thermal hyperalgesia depends on the activation of TRPV1; this was shown in the TRPV1-knockout mice and using TRPV1antagonists (Davis et al. 2000). CFA-induced thermal hyperalgesia is a complex process in which various inflammatory mechanisms reduce the temperature threshold through TRPV1 and affect thermal sensitivity (Jara-Oseguera et al. 2008). In the mice, which had been administered CFA, thermal hyperalgesia was observed, which was manifested by a decreased time of the paw withdrawal in response to thermal effects. To study analgesic activity, the studied polypeptides were tested in the CFA test (Fig. 2).

Figure 2.

Time (sec) of the first reaction to CFA-induced hypersensitivity after administration of polypeptides APHC1, 2, 3 and A13 in the hot plate test (n = 9 for each group). Note: * - The results are presented as the mean ± s.e.; * - p < 0.05 versus saline group (ANOVA followed by Tukey’s test).

All the investigated polypeptides showed a significant increase in the presence on the thermostated surface from the control group. At a dose of 0.01 mg/kg, there is a comparable activity of the studied polypeptides APHC1-3, ~21%, and A13 to ~35% relative to the control, and they are significantly different from the “parent” polypeptides APHC1 and 3. A further increase in the dose to 0.1 mg/kg showed that APHC2 and 3 did not increase their activity and remained at the same level ~22% relative to the control. APHC1 and A13 did not differ significantly from each other and increased their activity to ~56% and ~65% relative to the control. At a dose of 1 mg/kg, all the studied polypeptides lost their pharmacological activity, polypeptides APHC2 and 3 did not differ significantly from the control, APHC1 and A13 reduced their activity by ~30% relative to the dose of 0.1 mg/kg. Hybrid polypeptide A13, consisting of APHC1 and APHC3, showed the similar activity of APHC3.

Activation of TRPV1 can be triggered by low pH values. Intraperitoneal administration of acetic acid activates TRPV1 receptors, causing specific behavior (writhes) of the experimental animals, which is characterized as visceral pain (Ikeda et al. 2001, Le Bars et al. 2001, Tang et al. 2007). According to the results of the study in hot plate and CFA-induced hypersensitivity tests, further study of pharmacological activity was carried out at a dose of 0.1 mg/kg. The experimental animals were administered the investigated polypeptides 15 minutes before the injection of acetic acid. Polypeptides АРНС1 and АРНС2 showed a significant decrease in writhes, by ~26 and ~27% relative to the control group. АРНС3 and A13 reduced the number of writhes significantly more (by ~50 and ~67% relative to the control group) and significantly differed from АРНС1 and АРНС2. Conventional molecules that inhibit (Tang et al. 2007) and activate (Lehto et al. 2008) pH-induced TRPV1 currents exhibit similar effects in in vivo experiments. They significantly reduce the number of writhes after the introduction of acetic acid. These data were confirmed by the results of the current study, APHC3 and A13 significantly reduced the number of writhes.

TRPV1 is involved in thermoregulation, and almost all its known agonists and antagonists change core body temperature (Garami et al. 2010, Romanovsky et al. 2009). Hyperthermia is a critical side effect of TRPV1 antagonists, so the effect of polypeptides on body temperature was evaluated. As a control group, the antagonist of TRPV1, AMG9810 (30 mg/kg) and Aprotinin (0.1 mg/kg), was used, which is an inhibitor of serine proteases and acts as a homologue of the studied substances. Introduction of saline solution, which was used as solvent of AMG9810 (10% DMSO in normal saline), did not change the body temperature of the experimental animals. The introduction of AMG9810 (as the main antagonist of TRPV1) contributed to an increase in the body temperature of mice by 1.6 °C, as previously reported (Gavva et al. 2007). Serine proteinase inhibitor, polypeptide Aprotinin, caused an increase in body temperature (0.4–0.5 °C), which was not significantly different from the control group. APHC1 and 3 caused a decrease in body temperature of the experimental animals. APHC1 resulted in a more rapid effect of lowering the temperature by -0.8 °C within 30 minutes after injection. The reduced temperature remained throughout the period of observation of the animals in the experiment. APHC3 caused a slow decrease in body temperature, reaching -0.6 °C from the 60^th minute after administration. The results obtained were not significantly different from the control group. APHC2 did not cause changes in body temperature. A13 at a dose of 0.1 mg/kg increased body temperature by 2.3±0.2 °C from the 15^th minute after administration. A hyperthermal response to A13 administration was observed during 90 min of temperature registration (not shown) and was similar to the effect from low-molecular antagonist AMG9810. Antagonists potentiating pH-induced activation of TRPV1 either reduce or do not change body temperature (Lehto et al. 2008, Romanovsky et al. 2009).

Antagonists interacting with the intracellular TRPV1 channel exhibit a hyperthermal effect if they are able to inhibit pH-induced TRPV1 currents (Honore et al. 2009, Lehto et al. 2008). The hyperthermal reaction that occurs when an antagonist is administered is that the antagonist’s ability to inhibit the permanently activated abdominal channels of TRPV1 provokes a cold defence response (Gavva et al. 2007, Steiner et al. 2007). The factors maintaining the channels in the activated state have not yet been identified. PH modulation was proposed as one of the most likely factors (Garami et al. 2010).

Thus, the investigated polypeptide A13 obviously inhibits TRPV1 and affects body temperature as a classic antagonist of this receptor. Consequently, the antagonistic properties of A13 became different from the properties of both initial analgesic peptides.