Safety evaluation of diminazene aceturate on dogs

EFFECT OF DIMINAZENE ACETURATE ON CANINE LEFT VENTRICULAR FUNCTIONS

A PRE-DATA SEMINAR
(VCJ 697)

PRESENTED BY
OGUNBIYI TOLUWALOPE .O
MATRIC NUMBER: 20133223

DEPARTMENT OF VETERINARY PHYSIOLOGY AND PHARMACOLOGY
COLLEGE OF VETERINARY MEDICINE
FEDERAL UNIVERSITY OF AGRICULTURE, ABEOKUTA
SUPERVISOR: DR. AJIBOLA E.S.

                                                                                                                                   MAY, 2018.

INTRODUCTION
The prevalence of Canine haemoparasitic diseases in Abeokuta and Nigeria at large is quite high (Opara et al., 2017). The treatment of these diseases is challenging and has relied heavily on the use of a wide range of different classes of anti-protozoan drugs (Irwin, 2009). Diminazene aceturate (DMZ) has been used widely by Nigerian veterinarian and has proven to be effective at the recommended dose of 3.5mg/kg (Anene et al., 2001). There are however concern about its safety. The drug is known to have a narrow safety margin and has been associated with CNS toxicity in dogs (Losos and Crockett, 1969; Donghyun et al., 2014). This toxicity is known to occur at either the normal therapeutic dose or at supra-threapeutic dose. Although direct toxicity to the heart has not been well characterized in dogs, there are reports that DMZ has a blood pressure lowering effect when administered to dogs at 3.5mg/kg (Wien 1943). The report that pentamidine, a synthetic analogue of DMZ could cause CNS and cardiac toxicity in humans Whartom et al., 1987) has further heightened the prospect of DMZ complicating the existing haemodynamic perturbations in canine patients suffering from canine babesiosis and other haemoparasitic diseases (Jacobson et al., 2000; Ajibola et al., 2016) )
Although there had been some attempts in the past to understand the mechanism of the purported hypotension caused by DMZ in dogs, there had not been a direct attempt at evaluating the cardiac functional changes that may be associated with DMZ therapy in dogs.
In this current study, Echocardiography, a non-invasive technique that evaluates both cardiac structure and function (Allen, 1982; Bonagura, 1983) will be used in evaluating the haemodynamic changes associated with DMZ therapy in dogs. This technique has been used in both research and clinical practice. M-Mode echocardiography has been used in preclinical drug testing and indices obtained from this technique have been found to be reliable in cardiac function evaluation (Hanton, 2014). The aim of this study shall be to determine and compare the effects of either 3.5mg/kg and 7.0mg/kg of DMZ on cardiac function indices of dogs. The study will also correlate the changes in cardiac function indices and serum levels of DMZ in dogs administered with either of the dose.
Justification
The reported toxicity of diminazene aceturate to the central nervous system of dogs could cause arrhythmia in dogs by affecting the autonomic innervation to the heart. This is school of thought is further strengthened beacuse pentamidine, an anti-protozoan drug used in human and a synthetic analogue of diminazene aceturate has been reported to have pro-arrhythmic effects. The possibility of the arrhythmia causing haemodynamic disturbances is high in diminazene aceturate treated dogs. This is further worsened in dogs suffering from haemoprotozoan diseases with cardiac complications. The mechanism of the blood pressure lowering effect reported in DMZ treated dogs is not known. Since cardiac toxicity is not well characterized in dogs and there had not been a previous study evaluating the direct effect of diminazene aceturate of the heart of dogs, this study intends to use echocardiography in evaluating left ventricular functions such as cardiac-output, ejection fraction, fractional-shortening and stroke volume in dogs administered with either 3.5mg/kg or 7.0mg/kg of diminazene aceturate

GENERAL OBJECTIVES
To evaluate the effect of diminazene aceturate on left ventricular function of dogs

Specific objectives.

1. To evaluate serial changes in the cardiac output, stroke volume, ejection fraction and
2. fractional shortening in dogs treated with either 3.5mg/kg or 7.0mg/kg
3. To correlate the changes in serum concentration of diminazene aceturate with the cardiac function indices when dogs are treated with either of the tested dosesTo compare the effects of the two doses of diminazene aceturate on the cardiac function indices studied
   MATERIALS AND METHODS
   Materials
   • Ten adult male dogs
   • 7% diminazene aceturate ()
   • Needles and syringes
   • Ultrasound scanner (LEIDAL MEDICAL, England) with convex probe of frequency 5MHz.
   • Enzyme Linked Immunosorbent Assay test kit
   Experimental design
   The dogs will be assigned into two treatment groups comprising of five dogs each in a randomized cross-over design. Each of the dogs will be administered with either 3.5mg/kg or 7.0mg/kg of 7% Diminazene Aceturate (Hoest, England) intramuscularly. A period of 4weeks will be allowed between each treatment to allow for a complete wash-out of the drug.

Echocardiography
Echocardiogram will be obtained in each of the dogs using both the long and short axis para-sternal approach. Individual recordings will be made by placing the dog on the right lateral recumbency at 30minutes and just before the administration of the drug. The echocardiogram are then recorded serially at 5, 15, 30, 45, 60, and 120 minutes after the drug administration.
Echocardiographic examination will be conducted with ultrasound scanner (LEIDAL MEDICAL, England) with convex probe of frequency 5MHz. The echocardiogram will be taken in standing position without anesthesia. An acoustic gel will be applied at the interface between the transducer and echo window. A 2-D guided M- mode measurement of the left ventricle at the level of papillary muscle will obtained from the short axis right parasternal view. The following indices will be obtained using the inbuilt caliper of the echocardiograph; Left ventricular internal diameter in diastole (LVIDd), Left ventricular internal diameter in systole(LVIDs), Septum wall thickness in diastole (SWTd), Septum wall thickness in systole(SWTs),Posterior wall thickness in diastole (PWTd), Posterior wall thickness in systole(PWTs), left ventricular end diastolic and systolic measurements will be taken at the largest and at the smallest dimensions between the interventricular septum and the left ventricular free wall respectively. Five individual measurement of each variable will be obtained and averaged. All measurements will be made according to standard convention. These indices will be derived according to the method of Bonagura (1983); Fractional shortening (FS) = LVIDd - LVIDs/LVIDd; Ejection Fraction (EF) = LVIDd3- LVIDs3/LVIDd3.
End diastolic volume (EDV) and End systolic volume (ESV) will be derived from the formula of Teicholz (1976) as follows: EDV =7(LVIDd) 3/2.4 +LVIDd; ESV =7(LVIDs) 3/ 2.4 +LVIDs. Stroke volume (SV) in cm3 = EDV-ESV.

Serum Diminazene aceturate assay
The test samples would be analysed for diminazene aceturate by a competitive ELISA. Microtitre plates would be coated by placing 100 ml of the antibody optimally diluted in the coating buffer to every well. The plates would then be left at 4 0C overnight after which they would be frozen at -200C until required. When required, the plates would be thawed, washed six times with PBST(phosphate buffered saline containing Tween 20) and blotted dry. Samples would be diluted tenfold in a solution of enzyme conjugate, diluted optimally in phosphate buffered saline containing Tween 20(PBST). The diluted samples would then be loaded onto wells of antibody-coated ELISA plates,followed by overnight incubation at 4 8C. The plates would be washed as above. Colour development would be achieved by adding 100 ml of a 1:1 mixture of chromogen (TMB; Kirkengaard and Perry, Maryland USA):substrate (H2O2) per well and incubating at 370C for 10 min on a shaker incubator (Dynatech, Guernsey, Britain). The HRPO would catalyse the oxidation of TMB to a blue coloured complex. The reaction would be stopped by adding 100 ml of 1 M orthophosphoric acid per well resulting in a yellow colour. The optical densities (ODs) of the yellow solution would be measured at 450nm using a multichannel microplate reader (Bio-Tek instruments Wincoski, USA) inter-faced to a personal computer (Compaq Presario 7234, Ulsan, Korea). The drug concentrations of unknown samples would be calculated using the Eiaquik program designed by Eisler et al.(1993), from a calibration curve fitted using a fourparameter logistic regression of calibration standards.

REFERENCES
Ajibola, E.S, Oyewale JO, Oke, B.O (2016): Arterial blood pressure changes in acute Trypanosoma brucei infection of dogs. Bulletin of Animal Health and Production in Africa, 64(4): 73-79
Allen D.G (1982): Echocardiography as a research and clinical tool in veterinary medicine. Canadian Veterinary Journal, 23, 313-316
Anene BM., Onah, D.N., Nawa, Y (2001): Drug resistance in pathogenic African trypanosomes: What hopes for the future? Veterinary Parasitology, 96:83-100
Bonagura, J.D (1983): M- mode Echocardiography, Basic principles. Veterinary Clinics of North America: Small Animal Practice, 12:299-319
Donghyun H., WongKyoung, Y., Changbiag, H (2014): Cerebellar encephalopathy from diminazene aceturate toxicity in a dog. Korean Journal of Veterinary Research, 54(3)193-196

Eisler, M.C., Gault, E.A., Smith, H.V., Peregrine, A.S.,Holmes, P.H., 1993. Evaluation and improvement of enzyme-linked immunosorbent assay for the detection of isometamidium in bovine serum. Ther. Drug Monit. 15,236_/242

Hanton G (2014): Echocardiography, a non-invasive method for investigating preclinical drug toxicity and safety. Journal of Clinical Toxicology, 4(3); 200
Irwing P.J (2009): Canine Babesiosis; from molecular taxonomy to control. Parasitic Vectors,(suppl 1)S4
Jacobson, LS, Lobetti, RG, and Vaughan-Scott, T (2000): Blood pressure changes in Dogs with Babesiosis. Journal of South African Veterinary Association, 71(1):14-20
Losos, G.C., Crockett, E (1969): Toxicity of Berenil in the dog. Veterinary Records, 85; 196

Murilla, G.A., Eisler, M.C., Peregrine, A.S., Ndug’u, J.M.,Holmes, P.H., 1999. Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the trypanocidal drug homidium in serum of treated cattle. J. Vet. Pharmacol. Ther. 22, 301_/307
Opara, M.N., Adewunmi, T.S., Mohammed, B.R., Obeta, S.S., Simon, MK, Jegede, OC., Agbede, RIS(2017) Investigations on the haemoprotozoan parasites of Nigerian local breeds of dogs in Gwagwalada Federal capital territory (FCT) Nigeria. Research Journal of Parasitology, 12:1-7

       Teichholz L.E, Kreulen T.K, Herman M.V and Gorlin R (1976): Problems in echocardiographic volume determinations: echocardiographic angiographic correlations in the presence of absence of synergy.  American Journal of Cardiology, 37: 7-11.

       Whartom J.M., Demopulos, PA., Goldschlager, N (1987): Torsade de pointes during administration of pentamidine Isethionate. American Journal of Medicine, 83; 571.