Effects of diminazene aceturate on flip-flop plasma pharmacokinetics of piroxicam in dogs

2.1 Drugs

Diminazene aceturate and piroxicam produced by Hambet, Shandong, China were used for the studies at single dose of 3.5 mg/kg body weight.

2.2 Determination of therapeutic dose of piroxicam

Human equivalent dose formula was used to determine therapeutic dose of piroxicam (3.5 mg/kg) in dogs (Saganuwan, 2012).

$\mathrm{H}\mathrm{u}\mathrm{m}\mathrm{a}\mathrm{n}\mathrm{E}\mathrm{q}\mathrm{u}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{D}\mathrm{o}\mathrm{s}\mathrm{e}(\mathrm{H}\mathrm{E}\mathrm{D})=\frac{\mathit{Animal}Dose\times AnimalKm}{\mathit{HumanKm}}$ $$\mathrm{W}\mathrm{h}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{M}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{s}\mathrm{m}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{t}({\mathrm{k}}_{\mathrm{m}})=\frac{\mathit{Body}SurfaceArea}{\mathit{Body}weight}$$

But: Human BSA = H^0.528 × W^0.528 × K.

Dog BSA = H^0.528 × W^0.528 × K (multiply height by 2).

K_m = metabolic constant; BSA = Body surface area; K (constant)=0.14; H =height; W = weight.

2.3 Experimental animals

The study was conducted in the Department of Veterinary Phamacology and Toxicology laboratory, Collage of Veterinary Medicine, Federal University of Agriculture Makurdi, Nigeria. Ten apparently healthy Nigerian indigenous dogs of both sexes, aged 8 ± 2 months, and weighing 10 ± 0.5 kg were purchased from dog market in Makurdi, and used for the experiment. The animals divided into two groups of 5 each, were kept in a clean kernel, and fed normal rice, beans, semovita, fish and meat thrice daily. Clean water was provided ad libitun. The dogs acclamatized for 14 days were handled according to the international guiding principles on biomedical research involving the use of animals (CIONs and ICLAS, 2012), as approved by the Ethical Committee, Collage of Veterinary Medicine, Federal University of Agriculture Makurdi, Nigeria.

2.4 Experimental design

Modified randomized cross-over controlled design was adopted for this study. Each of the animals was randomly picked for administration of the drugs. The group of dogs administered piroxicam was used as control (Akogwu et al., 2017a).

2.5 Drug administration and sampling

Ten dogs (5males; 5 females) were injected piroxicam (3.5 mg/kg) in the thigh muscles. After one month, the same group of dogs was injected in the separate thigh muscles, diminazene aceturate (3.5 mg/kg), followed by piroxicam at 3.5 mg/kg body weight. Modified arithmetic method was used for blood sampling (Akogwu et al., 2017b). Blood samples were collected ten minutes before drug administration, from the cephalic vein using a 23G needle, and 5 ml syringe into ethylene diamine tetra acetate (EDTA) bottles at 0.0, 0.08, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, 96 h, respectively, for analysis of piroxicam. The samples collected were immediately centrifuged at 5000 revolution per minute (rpm) for 5 min. Plasma was obtained using a micropipette, placed in cryogenic vials, and stored at −20 °C until analyzed.

2.6 Preparation of standard

Piroxicam (1,000 mg/ml) stock solution was diluted with acetonitrile to obtain serial dilutions containing 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg of piroxicam in 1 ml of solution. One millitre of each was again diluted with 5 ml of acetonitrile. One millitre of plasma was added to each of the solutions. The tubes were centrifuged at 2500 rpm for 15 min, and 1 ml of supernatant was removed and 0.1 ml of 1.47 M aqueous perchloric acid (HCLO₄) added. A spectrophotometer at 330 nm was used to measure the absorbance against a blank prepared in same manner with piroxicam and piroxicam/diminazene aceturate. The absorbance was plotted against concentration of piroxicam and diminazene aceturate (Akogwu et al., 2017a,b).

2.7 Analysis of piroxicam

The revised method of Nagabhushanam and Sudha (2010) was adopted. Acetonitrile 2.5 ml was added to 1 ml of serum in centrifuge tubes, mixed gently and properly. The tubes were centrifuged at 2500 rpm for 15 min, and 1 ml of the supernatant was added to 0.1 of 1.47 M aqueous perchloric acid solution. The mixture was properly shaken and measured at 330 nm using a spectrophotometer, to obtain the absorbance against a blank prepared in the same manner with plasma. Minimum limit of detection was 2 µg/ml (Akogwu et al., 2017a,b).

2.8 Calculation of pharmacokinetic parameters

The pharmacokinetic parameters for individual animals were calculated manually using established non-compartmental pharmacokinetic equations (Baggot, 2001) as modified by Saganuwan (2020). The fractions of doses absorbed were determined according to the method of Saganuwan (2012).

1. C_maxwas determined from graph

2. T_maxwas determined from graph

3. Absorption rate constant (α) was calculated as follows;

1. Absorption half-life (T_1/2) = $\frac{0.693}{a}hr$

2. Mean absorption time (MAT)= $\frac{T1/2a}{0.693}hr$

3. Elimination rate constant (β) was calculated as: β= $\frac{\mathit{InCmax}-InCmin}{\mathit{Tmin}-Tmax}\left(\frac{1}{h}\right)$

4. The values of microconstant were used to determine the following parameters; Elimination half- life (T_1/2β) = $\frac{0.0693}{\beta }\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$\mathit{hr}$}\right.$

5. Area under curve: AUC_0-n = $\sum$ {C_p1 + C_p2 $\times$ (t₂-t₁) +{C_p2 + C_p3 $\times$ (t₃-t₂)} +……..(mg/L/hr)

6. Area under the curve Zero to infinity (AUC_0-∞) = AUC_0-96 + $\frac{\mathit{Cplast}}{\beta }$ (mg/L/h)

7. Body clearance (Clb = Vd × β (L/kg/hr)

8. Volume of distribution area (Vd) = $\frac{\mathit{Clb}(L/kg)}{\beta }$

9. Mean residence time (MRT) = $\frac{\mathit{Vd}}{\mathit{Clb}}(hr)$

10. Area under moment curve = MRT $\times$ AUC (mg/L)

11. Fraction of absorbed drug (Fad) = $\frac{\mathit{Amount}ofdrugabsorbed}{\mathit{Vd}}$  =  $(\alpha \times \beta \times AUC0-9$ 6 h)

Fractions of piroxicam absorbed from zero to 96 h and zero to infinity were calculated and translated to percent drug absorbed.

• Xv. Bioavalability of absorbed fraction (Faf) =. $\frac{\mathit{AUC}piroxicam/diminazene(iv)}{\mathit{AUC}piroxicam(iv)}$

2.9 Statistical analysis

Plasma kinetic data were presented both in graphical and tabular form. Plasma concentrations and pharmacokinetic parameters for piroxicam and piroxicam/diminazene aceturate were presented as mean $\pm$ standard error of mean (SEM). Kinetic parameters were analyzed using students t test paired at 5% level of significance. The concentrations of piroxicam and piroxicam/diminazene aceturate were analyzed using two-way analysis of variance (ANOVA), and least significant difference was detected at 5% level (Zar, 2008).

3.1 Plasma concentration–time profile of piroxicam alone and piroxicam co-administered with diminazene aceturate in Nigerian indigenous dogs following intramuscular administration

The concentration of piroxicam and piroxicam/diminazene aceturate over a range of time is presented in Table 1. The concentrations were not different, significantly (p > 0.05) at 0.08 and 0.25 h. However, the concentration was significantly higher (p < 0.05) at 0.5 h (8.270 ± 0.98), 2 h (8.870 ± 1.47), 3 h (10.260 ± 0.36 µg/ml) in piroxicam/ diminazene aceturate group as compared to 4.70 ± 0.95, 5.500 ± 1.49 and 6.680 ± 0.87 µg/ml in the piroxicam group respectively. Nevertheless at 12 h, the concentration of piroxicam /diminazene aceturate dropped significantly (p < 0.05) to 5.780 ± 1.2 µg/ml as compared to piroxicam group (7.730 ± 1.65 µg/ml), respectively (Table1). Plasma concentration–time profile of piroxicam alone (Fig. 1), piroxicam/diminazene aceturate (Fig. 2) and combination of piroxicam and piroxicam/diminazene aceturate (Fig. 3) obey two compartmental model of kinetics that is flip-flop in nature.

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Fig. 1

Mean plasma concentration–time curve of 3.5 mg/kg intramuscular piroxicam alone in dogs (n = 10).

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Fig. 2

Mean plasma concentration–time curve of 3.5 mg/kg intramuscular piroxicam coadministered with intramuscular diminazene aceturate (3.5 mg/kg) in dogs (n = 10).

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Fig. 3

Mean plasma concentration–time curve of 3.5 mg/kg intramuscular piroxicam, and 3.5 mg/kg of piroxicam/diminazene aceturate (3.5 mg/kg) in dogs respectively (n = 20).

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3.2 Pharmacokinetic prarameters of intramuscular piroxicam and piroxicam/diminazene aceturate in Nigerian indigenous dogs

The calculated pharmacokinetic parameters of piroxicam and piroxicam/diminazene aceturate are presented in Table 2. Plasma concentration maximum (C_max = 12.659 ± 0.85 µg/ml), time peak (T_max = 13.675 ± 9.21 h), absorption half-life (T_1/2α = 0.016 ± 0.04 h), elimination rate constant (β = 0.137 ± 0.10 h⁻¹), area under curve from zero to infinity (AUC_0-∞=778.885 ± 66.99 mg/L/h) and area under moment curve (AUMC = 98200.140 ± 5.33 mg/h²/L) were significantly lower (p < 0.05) in piroxicam/diminazene aceturate treated group as compared to C_max (18.560 ± 2.97 µ/mg), T_max(45000 ± 11.49 h), T_1/2α(0.250 ± 0.00 h), β(0.935 ± 0.75 h⁻¹) AUC_0-∞(814.472 ± 86.43 mg/L/h) and AUMC(36274640 ± 9010 mg/h²/L) in the piroxicam treated group respectively. However, absorption rate constant (α = 7.405 ± 1.75 h⁻¹), mean absorption time (MAT = 0.658 ± 0.45 h), area under curve AUC_0-96h (734.832 ± 63.20 mg/L/h), volume of distribution (Vd = 0.311 ± 0.16 L/kg) and mean residence time (MRT = 106.561 ± 54.84 h) were significantly higher (p < 0.005) in piroxicam/diminazene aceturate group as compared with α(5.823 ± 1.46 h⁻¹), MAT (0.361 ± 0.11 h) Vd (0.206 ± 0.08 L/kg) and MRT (49.793 ± 13.43 h) in the group treated with piroxicam alone respectively (Table 2).