Cardiorespiratory/sedative effects of a peptide identified in crotalic venom compared to acepromazine and xylazine in horses

ABSTRACT The purpose of this study was to investigate whether intravenous crotalphine produces significant sedation, as well as physiological changes, in healthy standing horses. Six mares, aged 8 years and weighing 415kg underwent three different treatments in a crossover design: TA (acepromazine: 50μg.kg-1), TC (crotalphine: 0.01μg.kg-1) and TX (xylazine: 1000μg.kg-1), intravenously. At various time points over 60 minutes, physiologic variables were recorded: heart rate, respiratory rate, and rectal temperature. The head height from the ground (HHG) was evaluated in centimeters. Data were analyzed using ANOVA followed by Dunnett’s test or Friedman followed by Dunn’s test, under 5% significance. Heart rate decreased significantly at M5 and M10 compared with Mb in TX (28±7, 26±6 and 40±8 beats/minute-1, respectively; p=0.0004). Respiratory rate and rectal temperature did not differ among groups or time points. The HHG significantly decreased in all groups compared with Mb at various time points (p<0.0001). In conclusion, crotalphine did not produce reliable and durable sedation in healthy standing mares and did not influence cardiorespiratory variables in a clinically relevant manner.


INTRODUCTION
In the routine of veterinary practice in large animals, some drugs have a significant role, such as tranquilizers and sedatives, since the use of these substances as components of an anesthetic protocol provides advantages in the approach and management of the patient.
Among the drugs used, acepromazine is a sedative that promotes muscle relaxation, free of analgesic effects (Rankin, 2015), with vasodilating and hypotensive properties (Steffey et al., 1985), however, it allows a compensatory response with an increase in cardiac rate (Muir et al., 1979;Coulter et al., 1981).
Another widely used drug is xylazine, which has sedative, analgesic, and muscle relaxant effects.As it belongs to the class of α-2 adrenergic receptor agonists and these receptors are distributed throughout the body, vasoconstriction (transitory) followed by bradycardia and hypotension are observed as side effects (Biaggioni and Robertson, 2018).
There are reports by Vital Brazil, between 1930 and 1940, suggesting the presence of an analgesic factor in rattlesnake venom, since those caused by crotalic accidents did not report severe pain, but paresthesia (Souza e Silva et al., 1996;Picolo et al., 2003).The analgesic compound present in crotalic venom was isolated, synthesized, and then named "crotalphine".Crotalphine is a 14-amino acid peptide (EFSPENCQGESQPC) containing a disulfide bridge and a pyroglutamic acid, synthesized from the sequence of the analgesic compound purified from the snake venom Crotalus durissus terrificus (Konno et al., 2008).Crotalphine can induce potent and long-lasting analgesic effects in acute and chronic pain models (including cancer pain and neuropathic pain), an effect estimated in rodents (Gutierrez et al., 2008;Konno et al., 2008).The action mechanism of this effect involves the specific peripheral activation of CB2 receptors, which, once activated, release endogenous peptides, particularly dynorphin A, which is the endogenous agonist of kappa receptors, which causes antinociception by action of the receptors (Machado, 2014).Some opioids drugs can cause excitement in horses due to their agonistic action at the mu (μ) receptor.For this, the association of sedatives with these opioid agents is used in veterinary anesthesiology.However, kappa (κ) receptor agonies induce, in addition to analgesia, a considerable degree of sedation and, particularly in horses, are used as a mildly acting sedative (Taylor and Clarke, 2009).Notwithstanding the known analgesic effects of crotalphine, to date there are no prospective studies addressing the sedative effects of this peptide in domestic animals.
Therefore, the purpose of this study was to investigate whether intravenous crotalphine produces significant sedation, as well as physiological changes, in healthy standing horses.It is not an objective of this study to assess the analgesic effects of crotalphine in horses.

MATERIALS AND METHODS
This study was carried out at the "Roque Quagliato" Veterinary Hospital of the University Centre of Ourinhos -UNIFIO, after approval by the local Ethics Committee under protocol number 002/2016.Crotalphine dose extrapolation.The dosage of Crotalphine for horses (0.01µg.kg. - ) was established through allometric extrapolation, (Pachaly, 2006) from the dose performed in rats of approximately 200g, (0.048 to 6μg.kg -1 ) being the equine the target animal and rat the known animal (Brigatte et al., 2013).We use the lowest dosage due to unknown effects.From the metabolic weight, basal and specific metabolic rate of known and target animals, it was possible to identify the dosage of crotalphine.The final dosage of this allometry was carried out based on the information on the doses used and which did not cause significant side effects in studies already carried out.
Crotalphine solution.From the dosage established by the allometric method, the initial solution in lyophilized form at a temperature of -20°C was diluted in a laminar flow hood.For this dilution, the handler was dressed in sterile gloves, cap, and mask, in order to avoid contamination during the procedure.The vial with the solution containing 1mg (lyophilized) was diluted in 1mL of sterile water.After, the final dilution remained 1:10 maintaining the final concentration of 100µg.ml - .These 10mL were divided into 10 aliquots in Eppendorf's each being 1 ml.All aliquots were kept isolated in a freezer at -20°C and the temperature checked every 12 hours every day.
Animals.Six female horses of various breeds, aged between 3 and 15 years (average 8 years), average weight of 415kg, belonging to the Experimental Farm of University Centre of Ourinhos -UNIFIO were used in this study.Only animals that were considered healthy according to a physical evaluation that included heart rate (HR), respiratory rate (RR), rectal temperature and oral mucosa color, and complete cell count were included.Exclusion criteria were any hematological alterations, clinical alterations, patients undergoing treatment in progress or pregnant females.The animals were randomly subjected to three treatments, by crossover and blind, respecting an interval between them of seven days, as described below: • TA (acepromazine treatment) -50μg.kgHead heigh from the ground was assessed using a measuring tape fixed to the restraint trunk, from the animal's lower lip.Ataxia and sedation score were performed using a table with scores (Table 1).All differences were considered significant when p<0.05.

RESULTS AND DISCUSSION
In TX, the HR values in M 5 and M 10 were respectively decreased to 28±7 and 26±6 beats/minute -1 in relation to M b (40±8 beats/minute -1 ).This can be explained by the fact that xylazine is an alpha-2 adrenergic receptor agonist drug, acting to inhibit the release of noradrenaline in the synaptic cleft, with promotes negative chronotropism due to the increase in peripheral vascular resistance as described by Valverde (2010).Muir and Mason (1993), when evaluating the effect of acepromazine on HR, observed a significant increase as in this study, corroborating the findings of Muir et al. (1979);Coulter et al. (1981) who an increase in HR that was compensatory to the decrease in systemic vascular resistance and blood pressure.
To measure the head heigh from the ground (HHG), the authors used a measuring tape attached to a physical restraint trunk for large animals, similar to the method used by Queiroz Neto et al. (2001);Christovão et al. (2006);Carregaro et al. (2007).Thus, it can be observed that HHG, in TA, decreased to 100±23cm, 102±13cm and 92±2-cm in M 10 , M 30 and M 60 respectively when compared to baseline (127±8cm).In the treatment of crotalphine, HHG decreased to 109±7 cm in M 10 compared to baseline (127±7cm), and in TX, compared to baseline (126±3cm), it decreased to 53±42cm, 52±37cm and 58±26cm in M 5 , M 10 and M 15 , respectively (Table 2).Short (1998) describes that the tranquilizing and sedative effects of acepromazine were more pronounced between 15 and 20 minutes after the administration of doses between 0.02 and 0.06mg.kg - (IV).In the present study, a maximum measurable effect was observed between 10 and 60 minutes on the mean distance from the head to the ground, after application of acepromazine at a dose of 0.05mg.kg - .It is already known that alpha-2 agonists cause more reliable sedation than phenothiazines, in addition to being analgesics, which does not occur with acepromazine (Muir and Hubbell, 1991), a fact that can be observed in the present study, corroborating the reports by Clarke et al. (1991) and England et al. (1992).
No excitation was observed in TA and TC.Castro (1981), reports that 0.3mg.kg - (IV) of acepromazine caused mild or severe excitation in 11 of 20 animals tested, which did not occur in the present study, although the authors used a lower dose.(0.05mg.kg -1 ).Christovão et al. (2006) and Roscoe (2007) report that calm horses can mask the effect of the medications administered.In the present study, half of the mares used were calm, which could fit into these animals mentioned by the authors, which disguise the effect of the drug in comparison with M b .
In addition to the cardiorespiratory parameters and the others already other signs were observed, such as: exchange of support, urination, defecation, yawning, flatulence, and sweating.The animals' mean exchange of support during the evaluation period was 17.3 in TX, 25.5 in TA and 18.8 in TC.
Sweating in the TX was negative in four animals, whereas in the TA and TC sweating was negative in all of the animals.There was no relationship between the presence of sweating and the treatments studied (p=0.1054).Elfenbein et al. (2009) andSteffey et al. (2000) mentioned that, as occurred in this study, sweating in animals that received xylazine was present.
The occurrence of lip and eyelid ptosis together with drowsiness may indicate mild sedation in horses (Castro and Eisenach 1989;Rossi et al., 2003).Lip ptosis was observed in five animals in the TX.In the TA, it was obtained in three animals.On TC only one animal had this event and the relationship between treatments and lip ptosis was not significant (p=0.0695).Muir and Hubbell (1991); Wilson et al. (2002) reported the occurrence of lip ptosis with the use of acepromazine and xylazine in horses, as happened in the present study in animals using TA and TX.
In TX, five animals urinated, while in the other treatments this was not observed (p=0.0010).The occurrence of urination after the application of alpha-2 agonist drugs has also been described by other authors in other species, as observed by Ribeiro et al. (2012) in cattle that received intravenously xylazine or detomidine and by Thurmon et al. (1984);Trim and Hanson (1986) on mares and ponies, respectively.Such an event can be explained by the effect of xylazine on one or more water retention mechanisms (inhibition of antidiuretic hormone synthesis or excretion and/or its actions on distal renal tubules (Thurmon et al., 1978).
Only at TA, three animals had flatulence during the evaluation period after administration of acepromazine (p=0.0273).At TC, all the animals defecated after the application of crotalphine (p=0.0036)during the evaluation period and TX and TA only one animal defecated.Guirro (2008) reports that, after the administration of crotalphine, 83% of the animals in their study defecated, corroborating the findings in the present study, however, Roger et al. (1994) mention that kappa receptor agonist opioids reduce defecation.
Limitations of the study were the low number of animals that participated in the project and its management, in which there should have been an adaptation period, since none of the animals was used to staying for an hour in the containment trunk after going through a period of fasting, causing half the horses to remain restless during the evaluation period.

CONCLUSION
Crotalphine did not produce reliable and durable sedation in healthy standing mares and did not influence cardiorespiratory variables in a clinically relevant manner.Defecation suggests increased gastrointestinal motility, but more studies need to be carried out to investigate this effect.

Table 1 .
Descriptive numerical scale representation for measuring the degrees of ataxia (Guirro et al,

Table 2 .
Heart rate (HR), respiratory rate (RR), rectal temperature (RT) and head height from the ground (HHG) of six mares at baseline (M b ) and following intravenous administration of acepromazine (TA), crotalphine (TC) or xylazine (TX), over 60 minutes (M 1 to M 60 )