Effects of Enantiopure (S)-a-Trifluoromethyl Proline Containing MIF-1`s Analogue on Stress-induced Analgesia

The present study was undertaken to investigate the effects of novel CF3-(MIF-1) analogue on stress-induced analgesia after restraint and cold stress in rats. The analgesic effect of CF3-(MIF-1) has been evaluated by paw pressure (PP) and hot plate (HP), and compared to the native tripeptide MIF-1. Our hypothesis was that incorporation of the trifluoromethyl group (CF3) in the novel MIF-1 analogue molecule would increase its antagonizing effect in both tests and after both kinds of stress compared to the native tripeptide MIF-1. The results obtained showed that both the non-fluorinated and the fluorinated peptides significantly decreased SIA in PP and HP tests during the whole investigated period and CF3-(MIF-1) had a stronger anti-opioid effect compared to MIF-1. *Corresponding author: Adriana Bocheva, Department of Pathophysiology, Faculty of Medicine, Medical University, Sofia, Bulgaria, E-mail: adriana_bocheva@abv.bg Received April 20, 2013; Accepted May 17, 2013; Published May 20, 2013 Citation: Bocheva A, Nocheva H, Jlalia I, Lensen N, Chaume G, et al. (2013) Effects of Enantiopure (S)-a-Trifluoromethyl Proline Containing MIF-1`s Analogue on Stress-induced Analgesia. Med chem 3: 206-209. doi:10.4172/21610444.1000140 Copyright: © 2013 Bocheva A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Melanocyte-stimulating hormone release inhibition factor-1 (MIF-1), also known as PLG based on its peptide chain (Pro-Leu-Gly-NH 2 ), is an endogenous brain peptide that belongs to the Tyr-MIF-1 family of peptides. It has been isolated from bovine hypothalamus and human parietal cortex and has been shown to be involved in a wide spectrum of physiological processes, including the development of stress [1][2][3].
MIF-1 represents a class of naturally occurring opiate antagonists. It does not bind to opiate receptors and it is the first peptide shown to exert anti-opioid effects [3]. In particular, MIF-1 binds to its own non-opiate sites and it is able to block the analgesic effect of morphine in paw-pressure and tail-flick tests [3] and enkephalin in a radiant heat tail-flick assay [4]. In a double-blind human study the peptide antagonized the effects of morphine [5]. MIF-1 also significantly inhibits the expression of some forms of stress-induced analgesia (SIA) in various species [2,6].
MIF-1 has the simplest structure among all Tyr-MIF-1 peptides and its relevance in activating and or modulating different CNS pathways has been demonstrated [7][8][9]. It is also regarded as a promising starting point for development of novel pharmaceutical agents [10].
A trifluoromethyl group containing MIF-1`s analogue has been recently synthesized by our research group. Our attention has been attracted by cyclic amino acids like prolines [11][12][13][14], pyroglutamic acids [15,16] and pseudoproline [17,18] derivatives. Literature data confirm that prolines improve stability of the molecules they are incorporated in and modulate their biological activities [19]. Pyroglutamic acid represents free amino acids metabolite and contributes to the maintenance of the mechanical integrity of the skin [20]. Pseudoprolines can be used to examine the bioactive conformation of prolyl-containing therapeutic targets [21]. It's known that biological activity depends on peptides` conformation. Our previously data showed that incorporation of CF3 group enhances the analgesic effect of MIF-1`s analogue in PP-test probably due to the increased lipophilicity. The involvement of opioid receptors and the nitric oxide system in this newly synthesized peptide's analgesia has also been demonstrated [22].
Our research group's interest is also focused on evaluation of the antinociceptive properties of compounds that may represent starting points for new analgesics development. Pain is an unpleasant experience accompanying many pathological processes. It is manifested as acute as well as chronic pain. Different acute stress models were proved to induce analgesia -named stress-induced analgesia (SIA). We adopt different acute pain models for experimental investigation of changes in SIA by substances with putative analgesic importance.
Two models of acute pain (restraint and cold exposure) were utilized to ascertain whether the novel CF3-(MIF-1) analogue could change SIA.
Given its preliminary antinociceptive activity and the opioidergic and nitricoxidergic systems involvement in its mechanisms of action, our suggestion was that CF3-(MIF-1) might affect SIA ( Figure 1).

Animals
The experiments were carried out on male Wistar rats (180-200 g), housed at 12 h light/dark cycle. Food and water were available ad libitum. All experiments were carried out between 09.00 and 12.00 a.m.. Each group included 5-8 rats.

Nociceptive tests
Paw-pressure test (Randall-Selitto test): The changes in rats` mechanical nociceptive threshold were measured using an analgesimeter (Ugo Basile). Increasing pressure was applied to the hind-paw of an animal; the pressure (g) required to elicit nociceptive response, such as squeak, and struggle, was taken as the mechanical nociceptive threshold. A cut-off value of 500 g was observed to prevent damage of the paw.

Hot plate test:
The latency of response to pain was measured from the moment of placing an animal on a metal plate (heated to 55 ± 0.5°C) to the first signs of pain (paw licking, jumping). The cut-off time observed to prevent paw damage was 30 s.

Stress procedures
Acute model of restraint stress (RS): The animals were immobilized for one hour using plastic tubes with adjustable plaster tapes on the outside. Holes were left for breathing. A control group was not submitted to restraint.

Acute model of cold stress (CS):
The animals were placed for one hour in a refrigerating chamber at 4°C.
Drugs and treatment: MIF-1 (obtained from Tocris) and the newly synthesized CF3-(MIF-1) analogue were both applied at a dose of 1 mg/kg, and injected intraperitoneally (i.p.), dissolved in a sterile saline (0.9% NaCl) solution immediately after the termination of the stress-procedure.
All experimental procedures were carried out in accordance to the institutional guidance and general recommendations on the use of animals for scientific purposes.

Statistical analysis:
The results were statistically assessed by one way analysis of variance ANOVA with an additional Student`s test. Data were represented mean ± S.E.M. Values of P<0.05 were considered statistically significant.
Applied immediately after 1hRS both peptides showed a statistically relevant decrease of R-SIA evaluated on the 15 th (p<0.001) and the 30th min (p<0.001) of the experiment. On the 45 th min both peptides led to no statistically relevant change in pain thresholds compared to RS (Figure 2).
Evaluation of HP-latencies showed that CF3-(MIF-1) had no statistically relevant analgesic effect compared to controls, while MIF-1 increased the HP-latency (p<0.05) (Figure 3 and 5).  Evaluation by HP-test showed for both the peptides a statistically relevant (p<0.001) decrease in HP-latency on the 15 th and the 30 th min ( Figure 5).
According to our expectations the newly synthesized MIF-1`s analogue affected both restraint-and cold-SIA, proved by both PP-and HP-test (Figures 2-5).

Discussion
Stress is known to influence neuroendocrine, autonomic, hormonal, and immune functioning. It has debilitating effects on different body systems [23,24]. Various stress models (immobilization, foot shock, hot and cold exposure) have been reported to induce analgesia, known as stress-induced analgesia (SIA) [25]. Two forms of SIA were commonly distinguished: an opioid-mediated and a non-opioid one. The opioid form of SIA is reversed by naloxone or naltrexone, whereas the nonopioid analgesia is insensitive to these opioid receptor antagonists [26,27].
One mechanism known to play a part in the organism response to stress, is activation of the endogenous opioid system [28]. Endogenous opioid peptides are produced in the body and take part in various functions as hormones or neuromodulators. The Tyr-MIF-1`s family of peptides, including MIF-1, are neuropeptides, neuromodulators, able to inhibit the expression of some forms of SIA [3]. They possess opioid and anti-opioid properties.
Several criteria have to be fulfilled for a peptide to be an anti-opioid one [28]: 1) The compound must bind to a specific receptor, encoded by its own specific gene, or, alternatively, it should bind with high affinity to opioid receptors; 2) The compound should counteract the effects of opioids, morphine or L-NAME; 3) The compound could play a role in opiate tolerance and dependence by counteracting the effects of chronic opioids; 4) The compound could counteract stress-induced analgesia.
Our previously data showed that the newly synthesized MIF-1`s analogue with CF3 group had a significant Nal-reversible analgesic effect and significantly decreased the strong analgesic effect of L-NAME in both tests applied. These results suggest the involvement of the opioidergic and the NO-ergic systems in the analgesic effects of CF3-(MIF-1).
The results proved CF3-(MIF-1)`s anti-opioid effects. CF3-(MIF-1) significantly decreased SIA in two stress models evaluated by both PP and HP nociceptive tests -thus another criteria for anti-opioid peptides was fulfilled.
In conclusion we assume that the incorporation of CF3 group in MIF-1`s molecule results in increased suppressive effect of the newly synthesized analogue in both tests applied and after both kinds of stress compared to the native tripeptide MIF-1. CF3-MIF-1 has a stronger anti-opioid effect compared to MIF-1.