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Research Article Open Access
Pharmacokinetics of Diminazene Aceturate in Buffalo Calves
Hemant Kumar Pandey1, Kaushal Kumar Singh2, Birendra Kumar Roy3* and Suruchi Kumari4
1Dept. of Pharmacology and Toxicology, Faculty of Veterinary Science & A.H, Birsa Agricultural University, Ranchi - 834006, India
2Dept. of Pathology, Faculty of Veterinary Science & A.H, Birsa Agricultural University, Ranchi - 834006, India
3Dept. of Pharmacology and Toxicology, Faculty of Veterinary Science & A.H, Birsa Agricultural University, Ranchi - 834006, India
4Research Scholor, Dept. of Pharmacology and Toxicology, Faculty of Veterinary Science & A.H, Birsa Agricultural University, Ranchi - 834006, India
*Corresponding author: Birendra Kumar Roy,
University Professor& Chairman Dept. of Pharmacology and Toxicology,
Faculty of Veterinary Science & A.H,
Birsa Agricultural University, Ranchi - 834006, India,
Tel: 91-651- 2450759;
Fax: 91-651-2450759
 
Received February 03, 2010; Accepted March 12, 2010; Published March 12, 2010
 
Citation: Pandey HK, Singh KK, Roy BK, Kumari S (2010) Pharmacokinetics of Diminazene Aceturate in Buffalo Calves. J Bioanal Biomed 2: 013- 016. doi: 10.4172/1948-593X.1000015
 
Copyright: © 2010 Pandey HK, 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.
 
Abstract
 
The pharmacokinetic study of diminazene aceturate (DMZ) was carried out in two separate groups of 4 each clinically healthy female Murrah buffalo calves after single dose i.v. (8 mg/kg) and i.m. (16 mg/kg) administration. The mean free peak serum concentration of DMZ (CSmax) after i.v. (26.28±0.67μg/ml) and i.m. (8.41±2.43 μg/ml) administrations were obtained at tmax of 5 and 30 min respectively. The DMZ serum concentrations time data were best fitted to the two compartment open model. The calculated serum half life (t½ β) values of DMZ were 15.099±2.504 and 14.225±2.682 h after i.v. and i.m. administration respectively. The mean values of total body clearance rate of DMZ (ClB) after i.m. (3.785±1.119 ml/kg/min) was significantly higher (P<0.05) as compared with the i.v. (0.537±0.063 ml/kg/min). DMZ was highly bound (77.14 to 94.40%) to buffalo calf plasma protein and its penetration into erythrocytes increased with increasing concentrations in blood (7.60 to 33.00μg/ml). Based on pharmacokinetic profiles, the satisfactory dosage regimens of diminazene aceturate in buffalo calves were derived (2 mg/kg, i.v. and 12mg/kg, i.m). In case of emergent disease conditions to ensure high DMZ serum concentrations, i.v. route may be preferred over i.m. route.
 
Keywords
 
Diminazene aceturate; Buffalo calves; Pharmacokinetics
 
Abbreviations
 
A&B: Zero time drug concentration in serum during distribution / absorption and elimination phases respectiely; C°s: Theoretical drug concentration in serum at zeero time; t½: Half life of drug in distribution, absorption and eliminationphases respectively; AUC: Total area under curve; K12, K21 & K2: Micro rate concentration for drug transfer from central to peripheral, peripheral to central and elimination frm the central compartment respectively; FC: Fraction of drug available for elimination from the central compartment; T/P: Tissue to Plasma concentration ration of the drug; Vdc, VdB and Vdarea: The apparent volume of dristribution, based on elimination and based on total area under curve respectively of the drug; ClB: Total body clearance rate of the drug
 
Introduction
 
Diminazene aceturate (DMZ), an aromatic diamidine is the drug of choice for babesiosis in livestock. Babesiosis poses a major health problem for cattle, buffalo, sheep and goats in all the tropical and subtropical regions of the world. Its usefulness and tolerance in animals have been documented (Sardar et al., 1994).

The pharmacokinetic studies on DMZ have been carried out in sheep (Aliu and Odegaard, 1985), goats (Aliu et al.,1984), bovine calves (Sardar et al., 1995), cow (Kellner et al.,1985), rats (Raether et al.,1972) and rabbits (Gilbert, 1983). The information on the disposition kinetics of DMZ in buffalo although important to be known is not available. This was possibly due to the fact that buffalo being an unique species of Indian subcontinent did not attract much attention of workers abroad. It may be mentioned here that in Chotanagpur plateau as well as in other hilly tracts of India, the buffalos are the principal milch and draft animals. In view of the above, the present work was designed to explore and identify the disposition kinetics of DMZ in buffalo calves in order to facilitate its proper clinical usages.
 
Materials and Methods
 
Animals
 
The pharmacokinetic study of diminazene aceturate after i.v. and i.m. administrations was conducted on 2 groups of 4 each clinically healthy 1.5 - 2 years female Murrah buffalo calves (150 -160 kg) of Ranchi Veterinary College dairy farm. The experimental animals were kept at room temperature in the faculty dairy farm were fed on standard rations, partial grazing (Panday and Roy,1998) and has free access to water.

Drugs used
 
Diminazene aceturate (Dimaze®, Merind Limited, Mumbai, India). DMZ granules 5G was dissolved in 30 ml water for injection and was injected as a single i.v. (8 mg/kg) and i.m. dose (16 mg/kg) in the right jugular vein and gluteal muscles respectively to the experimental buffalo calves of the corresponding animal's group.
 
Collection of experimental samples
 
Blood samples (5 ml) were collected from the left jugular vein by veinipuncture in clean test tubes from each experimental buffalo calf at 5, 10, 15, 30 min and 1, 2, 3, 6, 12, 24 and 48 h after single dose i.v. (8 mg/kg) and at 5, 10, 15, 20, 30min and 1, 2, 3, 4, 6, 8, 12, 24 and 48 h after i.m. (16 mg/kg) administration. Serum was separated after keeping the tubes in slanted position for 4- 5 h at room temperature. The predrug administration blood samples in sufficient quantity from each experimental buffalo calf were collected and serum separated. These serum samples served as analytical controls and were also used for preparation of DMZ standards.

Predrug administration (0 h) blood sample 3 to 5 ml from each experimental buffalo calf were also collected in heparinized test tubes for separation of plasma for DMZ plasma protein binding study. Plasma from these blood samples were separated by centrifugation at 2,500 G for 10 min.
 
Analytical methods
 
The analysis of DMZ in serum samples was carried out by the colorimetric method reported by Raether et al., 1972. The limits of detection was 0.05 μg/ml diminazene aceturate.
 
Pharmacokinetic calculations
 
The pharmacokinetic parameters of DMZ in buffalo calves were calculated by the Personal Computor following the method described by (Gibaldi et al., 1969; Notari, 1980; Baggot, 1977).
 
In-vitro Plasma Protein Binding
 
The in-vitro plasma protein binding of DMZ in buffalo calves was determined by the equilibrium dialysis technique (Davis, 1943).
 
In-vitro Erythrocyte up-take
 
The in-vitro erythrocyte up-take of DMZ in buffalo calves blood was studied by the method of Michel, (1949) modified by Oser, (1965).
 
Statistical Analysis
 
Data are presented as mean ±S.E. Significance of the observed differences in the pharmacokinetic parameters between i.v. and i.m. groups was assessed by students unpaired t-test (Snedecor and Cochran, 1964). A value of P<0.05 was considered statistically significant.
 
Results
 
The mean comparative total and free serum concentrations of DMZ and percent metabolite in buffalo calves after single dose i.v. and i.m. administrations are presented in Table 1. The mean comparative pharmacokinetic values of DMZ after single dose i.v. and i.m. administrations are presented in Table 2. The invitro plasma protein binding and up-take of diminazene aceturate by buffalo calf erythrocytes have been shown in Table 3 and Table 4 respectively.
 
Table1: Comparative mean (n=4) serum concentration (μg/ml) of diminazene in buffalo calves after i.v. (8mg/kg) and i.m. (16mg/kg) administration.
 
Table 2: Comparative mean values (n=4) of kinetic parameters of diminazene in buffalo calves after single i.v. (8mg/kg) and i.m. (16mg/kg) administration.A & B, Zero time drug concentration in serum during distribution / absorption and elimination phases respectiely; Cos ,Theoretical drug concentration in serum at zeero time; t1/2 b, Half life of drug in distribution, absorption and elimination phases respectively; AUC, Total area under curve; K12, K21 & K2,Micro rate concentration for drug transfer from central to peripheral, peripheral to central and elimination frm the central compartment respectively; FC, Fraction of drug available for elimination from the central compartment ; T/P, tissue to plasma concentration ration of the drug; Vdc, VdB and Vdarea,The apparent volume of dristribution, based on elimination and based on total area under curve respectively of the drug; ClB, Total body clearance rate of the drug.
 
Discussion
 
DMZ serum concentrations
 
The mean free serum concentrations of diminazene aceturate obtained after single i.v. dose in buffalo calves showed higher value of Csmax as compared to the i.m. administration (Table 1). The higher value of Csmax after i.v. dose was expected because of complete bio-availability of the compound into the systemic circulation (Baggot, 1977). The capillary endothelial tissue forms a barrier against drug entry into systemic circulation after i.m. administration. However, in this experiment, the aquous solution of DMZ (7.5%) was injected i.m. into the gluteal muscle. The rate of absorption of DMZ administered i.m. in buffalo calves in this study would have mainly depended on the vascularity of the injection site. Other factors governing the rate of absorption include the degree of ionization and lipid solubility of the compound, molecular size of the lipid insoluble substances and the area over which the injection is given (Schou, 1961; Sund and Schou, 1964). Absorption of compounds from aquous solutions injected i.m. is relatively rapid and the peak concentration in serum is usually reached within 30 min. to 1 h. The results obtained in this study also evidenced the rapid absorption of diminazene with a peak serum concentration at 30 minute (Table 1).The calculated value obtained for absorption rate constant (Ka) for free DMZ (4.377±0.526 h-1 ) also strengthened the above statement that DMZ was rapidly absorbed in buffalo calves. Sardar et al. (1994) reported a Cpmax of 30.25±1.37μg/ml after single dose i.v. (16mg/kg) administration of DMZ in bovine calves. The Csmax (8.41±2.43 μg/ml) obtained in this experiment after single i.m. dose (16 mg/kg) in buffalo calves was higher than that reported by Mamman and Peregrine, (1994) in goats after single i.m. dose (3.5 mg/kg). The results obtained after single i.m. dose showed that diminazenz was rapidly absorbed and produced required therapeutic concentration at 5 minute (1.33±0.07μg/ml).
 
Acetylation
 
The rate of acetylation as observed from the % metabolite at various DMZ serum concentration time data in this study (Table 1) did not increase or decrease in the mathematical order. However, lower DMZ total serum concentrations obtained at later points of time both after i.v. and i.m. administrations in this study could have been due to increased rate of acetylation. Thus the rate constants of acetylation of DMZ in buffalo did not obey consistently the "0" or "1" order kinetics model in this study. Such observation with respect to acetylation of sulphonamides in dairy cattle have been also reported (Stowe and Sisodia, 1963)..
 
Pharmacokinetics
 
The DMZ serum concentration time data obtained in buffalo calves in this study were best fitted to a 2-compartment open pharmacokinetic model. The Cos value (41.60±3.50 μg/ml) of free DMZ obtained after single i.v. dose was approximately 8 times higher as compared with (5.28±0.51 μg/ml) i.m. admin-istration. However, the Csmax (28.15±0.47 μg/ml) obtained after i.v. was approximately 3 times higher as compared to the Csmax (8.41±2.43 μg/ml) after i.m. injection in buffalo calves. It may be mentioned that the Cos is a hypothetical parameter being the sum of A and B, can differ with a little variation in selection of points pertaining to elimination phase. The higher value obtained for Ka (4.377±0.526 /h) along with a corresponding lower value of absorption half life (t½ Ka) shows that DMZ was rapidly absorbed after i.m. injection in buffalo calves and could maintain therapeutic concentration upto 48 h. The data of t½ Ka shows that half life of the amount of DMZ injected by i.m. route was absorbed in 10 minute.

The higher values obtained for α (8.951±1.465 h-1) and Ka (4.377±0.526 h-1) and the subsequent lower value for t½ α (0.083±0.011 h) and t½ Ka (0.165±0.020 h) denoted a rapid distribution and absorption of DMZ in the body fluids of buffalo calves. Mallick et al. (1998) have reported the values for α and t½ α of DMZ in goats as 2.39±0.12 h-1 and 0.29±0.25 h respectively. The rapid distribution and absorption of DMZ was further strengthened by the higher sum of K12 and K21 than the K2 as depicted in Table 2.

The apparent volume of distribution, based on elimination and total area under curve of DMZ for i.v. and i.m.routes in buffalo calves derived were 0.196±0.016, 0.673±0.043 and 0.664±0.042 and 3.113±0.299, 5.455±0.301 and 5.499±0.287 L Kg-1 respectively. The consistant higher values obtained for the apparent volume of distribution showed that this compound perfused well in to the body spaces of buffalo species. The volume of distribution (Vd) of DMZ in goats has been reported to be 1.464±0.053 L Kg-1 (Mallick et al., 1998). The derived ClB of DMZ after i.v. and i .m. routes were 0.537±0.063 and 3.785±1.119 ml/kg/min respectively. Sardar et al. (1994) have reported the ClB value of this compound after i.m. dose in bovine calves as 0.564±0.090 ml/kg/min.

The approximately equal values of t½ β obtained after i.v. (15.099±2.50 h) and i.m. (14.225±2.682 h) envisaged first-order kinetics of DMZ in buffalo species. Sardar et al. (1994) reported t½ β value equal to 31.604±2.296 h in bovine calves after single i.m. dose (16mg/kg) which was twice the value obtainedin this experiment for buffalo calves. Mallick et al. (1998) reported t½ β value of DMZ equal to 13.49±1.26 h in goats after single i.v. dose (8mg/kg) which is closer to the value of t½ β obtained in this experiment.The overall kinetic variables of total DMZ for i.v. and i.m. routes derived in buffalo calves in this study were comparatively higher than their respective values for free DMZ. Based on the pharmacokinetic variables obtained in this study, the satisfactory dosage regimen of diminazene aceturate in buffalo was derived (2mg/kg i.v. and i.m.).
 
Plasma Protein Binding
 
Diminazene was observed to be highly bound to plasma proteins of buffalo calves (77 to 94.4%) in the concentration range of 6.25 to 100 μg/ml (Table 3).
 
Table3: In-vitro plasma protein binding value of diminazene in buffalo calves.
 
Erythrocytes' uptake
 
The uptake of DMZ by buffalo calves erythrocytes has been found to range between 7 to 33 μg/ml in 8.5 × 106/ml at the blood concentration range of 6.25 to 100 μg/ml. DMZ showing a sustained rise with concentration with a maximum at 100 μg/ ml. The passage of DMZ into erythrocytes and its accumulation there in provides an added benefit from the clinical point ofview against intra erythrocytic protozoan parasites susceptible to this compound (Table 4).
 
Table 4: The up-take of diminazene by buffalo calf erythrocytes (Total count of RBC in buffalo calf blood was 8.15x106/ml).
 
Acknowledgements
 
Authors acknowledge Dr.N.N.singh, Vicechancellor for providing the fund for this study. Dean, R.V.C. and Officer-incharge, R.V.C. dairy farm are also acknowledged for sparing experimental animals. Merind Limited, Mumbai is also acknowledged for generous supply of drug.
 
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