The intravenous pharmacokinetics of diminazene in healthy dogs.

Diminazene remains one of South Africa's most commonly used antiprotozoal agents for the management of babesiosis in dogs. Although the drug has been on the market for over 40 years, its intravenous pharmacokinetics are poorly known. To better understand the pharmacokinetics of the drug Berenil, it was reconstituted in sterile water and administered intravenously to 6 adult German shepherd dogs. All 6 dogs demonstrated the previously described secondary peak in the plasma concentration versus time profile. The plasma pharmacokinetics for diminazene are described by both non-compartmental and compartmental models. From non-compartmental analysis, the area under curve to the last sample point (AUClast), clearance (CL) and volume of distribution (Vz) were 4.65 +/- 1.95 ng/ml/h, 0.77 +/- 0.18 l/kg/h and 2.28 +/- 0.60 l/kg, respectively. For compartmental modelling, the plasma concentrations were fitted to both a 2-compartmental open model and a recirculatory enterohepatic model. From the recirculation model, the rate of release and re-entry into the central compartment varied markedly with the rate of release from the gall bladder (Ttom) being estimated at 27 +/- 20.90 h. Once released, drug re-entry into the central compartment was variable at 9.70 +/- 5.48 h. With normal biliary excretion time being about 2 h, this indicates that the redistribution cannot be occurring physiologically from the bile. Although it was not possible to identify the site from which sequestration and delayed release is occurring, it is believed that it is most likely from the liver. The study therefore showed that the secondary peak described for the pharmacokinetics of intramuscular administered diminazene in the dog is not related to biphasic absorption.

Preparation of polyclonal antibodies to diminazene and its detection in animal blood plasma.

This work was undertaken to examine the possibility of preparing polyclonal antibodies to diminazene by using colloidal gold nanoparticles as an antigen carrier and adjuvant. The antibodies prepared by us were used for immunodot determination of diminazene in calf-blood plasma. The immunochemical results agreed with those obtained by high-performance liquid chromatographic estimation of diminazene content in animal biological fluids.

Confirmation of diminazene diaceturate in bovine plasma using electrospray liquid chromatography-mass spectrometry.

Diminazene diaceturate is used as a trypanocide for cattle in tropical regions. This paper describes a LC-MS(n) method to confirm the presence of diminazene in bovine plasma. Bound diminazene in plasma samples was freed with dilute phosphoric acid, then concentrated on a bonded C(18) SPE cartridge. The LC-MS(n) method utilized electrospray ionization coupled with an ion trap mass spectrometer. Ions observed in MS(2) and MS(3) product ion spectra, as well as those from the MS(1) spectrum, were monitored. The method was validated with plasma samples fortified with diminazene diaceturate (4-100ng/mL). Diminazene was confirmed in samples fortified with diminazene diaceturate at levels of 6.4ng/mL or higher.

The pharmacokinetics of diminazene aceturate after intramuscular administration in healthy dogs.

The pharmacokinetics of diminazene aceturate following intramuscular (i.m.) administration at 4.2 mg/kg was evaluated in 8 healthy German Shepherd dogs. Blood samples were collected at 19 intervals over a period of 21 days. Diminazene plasma concentrations were measured using a validated HPLC method with UV detection and a sensitivity of 25 ng/ml. The in vitro and in vivo binding of diminazene to blood elements was additionally determined. Diminazene pharmacokinetics showed a large inter-individual variation after i.m. administration. It had a short absorption half-life (K01-HL of 0.11 +/- 0.18 h), resulting in a C(max) of 1849 +/- 268.7 ng/ml at T(max) of 0.37 h and a mean overall elimination half-life (T1/2beta) of 5.31 +/- 3.89 h. A terminal half-life of 27.5 +/- 25.0 h was measured. At 1 h after i.m. injection, 75% of the diminazene in whole blood was in the plasma fraction. The results of this study indicate that diminazene is rapidly distributed and sequestered into the liver, followed by a slower terminal phase during which diminazene is both redistributed to the peripheral tissues and/or renally excreted. It is recommended that diminazene administered i.m. at 4.2 mg/kg should not be repeated within a 21-day period.

A competitive enzyme-linked immunosorbent assay for diminazene.

Diminazene aceturate has remained a very important therapeutic drug for trypanosomosis in cattle, sheep and goats since its introduction into the market in 1955. Despite its continued use, the methods available for its detection in body fluids are lengthy and inefficient for routine monitoring of drug levels in treated animals. A competitive enzyme linked immunosorbent assay (ELISA) has now been developed and optimized for the detection of diminazene in bovine serum. In the assay, diminazene in the test samples and that in a newly developed diminazene-horseradish peroxidase conjugate compete for antibodies to diminazene raised in rabbits and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H(2)O(2)) is used as chromogen-substrate system. The assay has a detection limit of 0.8 ng/ml of serum with a high specificity for diminazene. Cross-reactivity with either homidium bromide and quinapyramine sulphate/chloride of 0.0004% is negligible while that with isometamidium chloride is 0.71%. The assay was able to detect diminazene levels in normal Boran steers for at least two weeks after intramuscular injection with the drug at a dose of 3.5 mg/kg bw. The assay will be useful in monitoring diminazene use, and development of resistance in trypanosomosis endemic areas.

Determination by capillary zone electrophoresis of berenil, phenamidine, diampron and dibromopropamidine in serum and urine.

A quick, simple and reliable analysis method has been developed in order to determine berenil, phenamidine, diampron and dibromopropamidine by capillary zone electrophoresis in samples of serum and urine. In order to define the operation parameters in CZE, we have carried out a study on how the apparent electrophoretic mobility (mu(app)) varies when pH, buffer concentration, voltage and temperature are modified. Ohm's law plot has been studied, too. With the data obtained from this study we have determined the optimum work conditions, which are: citrate buffer 25 mM, pH=3.70, 14 kV, 30 degrees C, wavelength of the UV detector: 200 nm, capillary tube: 570 mm x 75 microm. Under these conditions, all the products appear in times between: 7.6 min phenamidine and 8.8 min dibromopropamidine, limits of detection being: berenil: 0.50, phenamidine: 0.25, diampron: 0.40 and dibromopropamidine: 0.80 microg ml(-1). We have carried out a recovery study with three kinds of extraction cartridges: Sep-pak C-18 plus, Sep-pak C-8 plus and Oasis HBL for each one of the products in blood and urine.

Pharmacokinetics of diminazene in plasma and lymph of goats.

OBJECTIVE: To characterize the pharmacokinetics of diminazene in plasma and pseudo-afferent lymph of East Africa X Galla goats. DESIGN: The efferent prescapular lymphatic duct of 3 goats was cannulated 8 weeks after surgical removal of the lymph node. Thereafter, 3.5 mg of diminazene base/ kg of body weight was administered to these goats and to 3 noncannulated goats. PROCEDURE: Using high-performance liquid chromatography, concentration of diminazene was determined in plasma and lymph collected up to 96 hours after treatment. RESULTS: Maximal concentrations of diminazene in plasma of noncannulated goats (median [range], 4.30 [4.28 to 5.01] micrograms/ml), plasma of cannulated goats (3.94 [2.94 to 4.06] micrograms/ml), and lymph (1.06¿0.73 to 1.86] micrograms/ml) were significantly different (P < 0.05); values in lymph were considerably lower than those in plasma from noncannulated and cannulated animals. Time to reach maximal concentration did not differ significantly between lymph and plasma of noncannulated and cannulated goats. Over the first 24 hours after drug administration, concentration of diminazene in plasma of noncannulated goats was generally higher than that in lymph, but thereafter was similar. Apparent volume of distribution of diminazene in the plasma of noncannulated (2.57 [1.93 to 2.60] L/kg) and cannulated (2.30 [1.04 to 2.40] L/kg goats did not differ significantly. Penetration ratio of diminazene into lymph, compared with plasma, of cannulated goats was 1.69:1. CONCLUSIONS: Disposition of diminazene in goats is characterized by higher concentration in plasma than in lymph. However, the drug persists longer in lymph than in plasma. CLINICAL RELEVANCE: The longer persistence of diminazene in lymph than in plasma may account for the enhanced therapeutic efficacy of diminazene in the early stage, compared with later stages, of a tsetse fly-transmitted trypanosome infection.

Disposition of diminazene aceturate (Berenil) in trypanosome-infected pregnant and lactating cows.

Three cows were repeatedly infected with different strains of Trypanosoma congolense and treated intramuscularly each time with a different dose of diminazene aceturate (Berenil). Biphasic decline was observed of the maximal plasma drug levels, which were attained at 15 min after the first treatment and at 30 min after the second and third treatments. The rate constants for the distribution and terminal phases depended on the period of exposure to parasitaemia of the animal at the time of treatment. Maximal diminazene aceturate residue levels were found in milk 8 h post treatment and declined biexponentially to 4.56 ng ml-1 and 8.76 ng ml-1 at 21 days post treatment after 3.5 mg kg-1 and 7.0 mg kg-1 doses, respectively. In the three cows, higher drug residues were found in the kidney (7.04, 3.92 and 7.99 micrograms g-1) than in liver (3.26, 2.87 and 1.24 micrograms g-1) and heart (1.79, 1.25 and 1.03 micrograms g-1). The results of this study indicate that the level of parasitaemia (degree of anaemia) in the animal at the time of treatment affects the distribution, disposition and elimination of diminazene aceturate in the animal. Furthermore, the residue level in milk after treatment depends on the treatment dose and could easily be bioavailable to the consumer.

Paired-ion extraction and high-performance liquid chromatographic determination of diminazene in cattle plasma: a modified method.

The high-performance liquid chromatographic method published by Aliu & Odegaard (1983) was found to give poor peak separation when used to determine plasma diminazene concentrations in cattle. Before bioequivalence studies could be carried out, the method had to be modified. Solid-phase extraction with acetonitrile/0.025 M Na-octane sulphonate and 2% acetic acid as eluent, followed by sample concentration, gave recoveries of > 90% for diminazene and the internal standard. A mobile phase of acetonitrile/0,005 M Na-octane sulphonate, 0.1% triethylamine, pH 3.2 with acetic acid on a Nova Pak C18 column was used for the analysis. Wavelength switching was used to determine the internal standard (imidocarb) and diminazene at their respective wavelengths of maximum absorbance, resulting in a fivefold increase in the limit of detection for diminazene. The modified method attained a detection limit of 2 ng.m.-1 (peak 4x baseline noise), limit of quantitation of 10 ng.m.-1 (coefficient of variation < 15%) and an accuracy of > 96% over the range from 10-5000 ng.m.-1.

Pharmacokinetics of diminazene in plasma and cerebrospinal fluid of goats.

The pharmacokinetics of diminazene in the cerebrospinal fluid (CSF) and plasma of five uninfected goats treated with single intramuscular doses of 3.5 mg diminazene base kg-1 bodyweight was investigated. The concentrations of the drug were determined by high performance liquid chromatography, and were three to four times lower in CSF than in plasma. The kinetics of the drug in CSF and plasma differed significantly with respect to Cmax, tmax, AUC0-48h, AUMC0-48h, Cl and Vd(ss).

Time-dose-response of Trypanosoma brucei brucei to diminazene aceturate (Berenil) and in vitro simulation of drug-concentration-time profiles in cattle plasma.

Bloodstream form Trypanosoma brucei brucei of axenically growing populations were incubated in the presence of 10.0, 1.0 or 0.1 micrograms/ml diminazene aceturate (Berenil) at 37 degrees C for various periods and, subsequently, either inoculated into mice or further propagated in vitro in drug-free medium. Growth was monitored for 10 days. The ability of trypanosomes of drug-sensitive CP 2137 (clone 1) to grow in vitro was irreversibly damaged after short incubation (< 1 min) with 10.0 micrograms/ml or after 15 min with 1.0 micrograms/ml diminazene aceturate. In contrast, drug-resistant CP 2469 (clone 1) trypanosomes tolerated incubation with 10 micrograms/ml of drug for up to 6 h and 1.0 micrograms/ml of drug for up to 24 h. Differences in drug susceptibility were also detected regarding infectivity to mice and changes in trypanosome cell volume. The results demonstrated that less than 1 min exposure to diminazene aceturate at concentrations as seen in bovine plasma at the initial peak after diminazene aceturate treatment is enough to irreversibly damage drug-sensitive trypanosomes. However, these concentrations were not sufficient to completely eliminate drug-resistant trypanosomes after exposure for 1-6 h; trypanosomes continued to grow for 48 h before the majority of them died and only a few organisms survived to revive the cultures. When drug-sensitive trypanosomes were exposed in vitro for 24 h to diminazene aceturate at the level of concentrations found in cattle after treatment with 3.5 mg/kg, most of the trypanosomes died and none of the surviving parasites could be propagated in vitro in the absence of drug for more than 2 days. However, a small population of drug-resistant trypanosomes was not irreversibly damaged and a few surviving trypanosomes were able to establish growing cultures. The addition of feeder layer cells did not change the outcome of these experiments.

Comparative pharmacokinetics of diminazene in noninfected Boran (Bos indicus) cattle and Boran cattle infected with Trypanosoma congolense.

The pharmacokinetics of diminazene in five female Boran (Bos indicus) cattle before and then during acute and chronic phases of experimental infections with Trypanosoma congolense were investigated. A 7.0% (wt/vol) solution of diminazene aceturate (Berenil) was used in all three phases of the study and administered as a single intramuscular dose of 3.5 mg of diminazene base per kg of body weight. There were no significant differences between the values of pharmacokinetic parameters for the noninfected cattle and the values for cattle with a chronic T. congolense infection. However, the maximum concentration of the drug in plasma during the acute phase of infection (8.25 +/- 1.72 micrograms/ml) was significantly (P < 0.01) greater than that during chronic infection (5.04 +/- 0.26 micrograms/ml) and that in the noninfected state (4.76 +/- 0.76 micrograms/ml). Similarly, the time to maximum concentration of the drug in plasma when diminazene was administered during the acute phase of infection (18.00 +/- 6.71 min) was significantly (P < 0.02) shorter than that for noninfected cattle (36.00 +/- 8.22 min) and that during chronic infection (33.75 +/- 7.50 min). The volume of distribution at steady state during acute infection (1.01 +/- 0.31 liter/kg) was significantly (P < 0.01) smaller than that in the noninfected state (1.37 +/- 0.17 liter/kg) and that in chronic infection (1.51 +/- 0.24 liter/kg). Eight hours after the drug had been administered, the concentration-time data profiles for each of the three study phases were very similar. Mean concentrations of diminazene in plasma 48 h after administration of the drug were 0.43 +/- 0.07 microgram/ml in noninfected cattle, 0.43 +/- 0.11 microgram/ml during the acute phase of trypanosome infection, and 0.44 +/- 0.09 microgram/ml during the chronic phase of the infection. Results of the present study indicate that the area under the concentration-time curve for diminazene in trypanosome-infected cattle did not differ significantly for noninfected cattle. It, therefore, appears that the total amount of diminazene attained and maintained in the plasma of cattle is not significantly altered during infection with T. congolense.

Effects of the combination of DL-alpha-difluoromethylornithine and diminazene aceturate in Trypanosoma congolense infection of dogs.

The therapeutic activity of a combination of difluoromethylornithine (DFMO) with diminazene aceturate was investigated in mongrel dogs experimentally infected with Trypanosoma congolense. The criteria used in the assessment of the trypanocidal effect of the therapy include the examination of the blood for parasites, as well as clinical and haematological changes at intervals following treatment. Diminazene aceturate and DFMO alone and in combination produced intermittent aparasitaemia in the dogs. Although relapse infection occurred with all three treatment regimes, the drug combination gave the best result. The packed red cell volume, haemoglobin concentrations and red blood cell values decreased significantly following parasite inoculation but increased after treatment. The total leucocyte counts decreased in all the infected dogs but improved with treatment, and the differential leucocyte counts indicated neutropenia in all the infected animals prior to treatment.

Studies in cattle on the disposition of the anti-trypanosomal drug diminazene diaceturate (Berenil).

After intramuscular injection of ca. 3.5 mg kg-1 diminazene diaceturate-(bis-phenyl-U-14C) to two healthy male calves weighing 185 and 180 kg, levels of radioactivity were determined in blood, plasma, urine, faeces, and in edible tissues. The maximal blood level of 4.6 micrograms and 4.7 micrograms equivalents of diminazene diaceturate ml-1 (calculated from total radioactivity) occurred 15 min (calf C1) and 45 min (calf C2) after administration. The decrease in concentration followed a biphasic process with half lives of 2 and 188 hours. Seven days after treatment 47.1% of the dose had been excreted in the urine and 7.1% in the faeces. The respective values were 72.2% and 10.3% after 20 days. The half lives were similar to those in blood. The main product in urine was unchanged diminazene. Distribution studies showed concentrations which were low in general in edible tissues, i.e. in skeletal musculature and fat (below 1 microgram equivalent g-1), but higher in organs with excretory functions. There were 75.5 micrograms equivalents g-1, corresponding to 22% of the dose, in the liver 7 days after injection. This had decreased to 24.4 micrograms equivalents g-1, corresponding to 15% of the dose, at day 20. Only unchanged deminazene was detected in the liver extracts by thin-layer chromatography. Relay-bioavailability studies showed that the total liver residues were only partially available (mean = 23% of the dose) when fed to rats.

Gas chromatography chemical ionization mass spectrometric analysis of diminazene in plasma.

A quantitative and sensitive method was developed for the determination of diminazene in plasma. The assay involves the reduction of diminazene to 4-aminobenzamidine and 4-hydrazinobenzamidine. The latter is further reduced to give an additional mole of 4-aminobenzamidine which is extracted, acetylated and condensed with hexafluoroacetylacetone to form a volatile derivative that is subsequently analyzed by gas chromatography chemical ionization mass spectrometry. 4-Aminobenzylamidine was synthesized and used as an internal standard. The method is reproducible and its sensitivity limit using 1 ml of plasma is 0.1 microgram diminazene ml-1. This sensitivity limit is sufficient to detect plasma levels in cattle following therapeutic doses of the drug.