New antiglaucomatous agent for the treatment of open angle glaucoma: Polymeric inserts for drug release and in vitro and in vivo study.

A benzamidine derivative from diminazene was tested for a novel activity: treatment of primary open-angle glaucoma. This drug was incorporated into mucoadhesive polymeric inserts prepared using chitosan (Chs) and chondroitin sulfate (CS). Of current interest is the mucoadhesion, which increases the contact time with the ocular surface, resulting in improved bioavailability; also, the inserts are made to act as a prolonged release system. In the present work the inserts were prepared by the solvent casting method using different polymeric proportions (30:70, 50:50, 75:25% w/w Chs:CS and 100% Chs). Thermal analysis and infrared spectroscopy both demonstrated physical dispersion of the active drug. The most promising was the 50:50% Chs:CS which demonstrated that it was not fragile and has an in vitro release profile of up to 180 minutes. In addition, it presented greater adhesion strength in relation to the other formulations. These physicochemical results corroborate the in vivo tests performed. In this sense, we also demonstrated that the treatment with the 50:50% insert can control the intraocular pressure (IOP) for at least 3 weeks and prevents damage to the retinal ganglion cells (RGCs) compared to the placebo insert. Thus, this indicates thus that the new drug is quite viable and promising in glaucoma treatment.

Ocular Inserts for Sustained Release of the Angiotensin-Converting Enzyme 2 Activator, Diminazene Aceturate, to Treat Glaucoma in Rats.

The aim of this study was to develop and evaluate the effects of chitosan inserts for sustained release of the angiotensin-converting enzyme 2 (ACE2) activator, diminazene aceturate (DIZE), in experimental glaucoma. Monolayer DIZE loaded inserts (D+I) were prepared and characterized through swelling, attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and in vitro drug release. Functionally, the effects of D+I were tested in glaucomatous rats. Glaucoma was induced by weekly injections of hyaluronic acid (HA) into the anterior chamber and intraocular pressure (IOP) measurements were performed. Retinal ganglion cells (RGC) and optic nerve head cupping were evaluated in histological sections. Biodistribution of the drug was accessed by scintigraphic images and ex vivo radiation counting. We found that DIZE increased the swelling index of the inserts. Also, it was molecularly dispersed and interspersed in the polymeric matrix as a freebase. DIZE did not lose its chemical integrity and activity when loaded in the inserts. The functional evaluation demonstrated that D+I decreased the IOP and maintained the IOP lowered for up to one month (last week: 11.0 ± 0.7 mmHg). This effect of D+I prevented the loss of RGC and degeneration of the optic nerve. No toxic effects in the eyes related to application of the inserts were observed. Moreover, biodistribution studies showed that D+I prolonged the retention of DIZE in the corneal site. We concluded that D+I provided sustained DIZE delivery in vivo, thereby evidencing the potential application of polymeric-based DIZE inserts for glaucoma management.

Biodynamic parameters of micellar diminazene in sheep erythrocytes and blood plasma.

In this work, we used a preparation of diminazene, which belongs to the group of aromatic diamidines. This compound acts on the causative agents of blood protozoan diseases produced by both flagellated protozoa (Trypanosoma) and members of the class Piroplasmida (Babesia, Theileria, and Cytauxzoon) in various domestic and wild animals, and it is widely used in veterinary medicine. We examined the behavior of water-disperse diminazene (immobilized in Tween 80 micelles) at the cellular and organismal levels. We assessed the interaction of an aqueous and a water-disperse preparation with cells of the reticuloendothelial system. We compared the kinetic parameters of aqueous and water-disperse diminazene in sheep erythrocytes and plasma. The therapeutic properties of these two preparations were also compared. We found that the surface-active substances improved intracellular penetration of the active substance through interaction with the cell membrane. In sheep blood erythrocytes, micellar diminazene accumulated more than its aqueous analog. This form was also more effective therapeutically than the aqueous analog. Our findings demonstrate that use of micellar diminazene allows the injection dose to be reduced by 30%.

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.

The effectivity analysis of accumulation of liposomal, micellar, and water-soluble forms of diminazene in cells and in organs.

The study was aimed at evaluating the effectiveness of liposomal, micellar, and water-soluble drug forms of diminazene for its localization in cells and selective accumulation at the sites of aggregation of pathogenic organisms. Pharmacological and dynamic properties of a new injection micellar diminazene preparation were experimentally determined. These properties were compared with the same parameters obtained for the water-soluble and liposomal diminazene aceturate drug forms. The drug forms studied may be arranged in the following order of decreasing effectiveness of accumulation of diminazene in red and white blood cells and in murine organs: liposome form, micellar form, and water-soluble form.

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.

Pharmacokinetics, urinary excretion and dosage regimen of diminazene in crossbred calves.

The pharmacokinetics, urinary excretion and dosage regimen of diminazene were investigated in crossbred male calves following a single intramuscular dose (3.5 mg x kg-1). Following intramuscular administration, the pharmacokinetics of diminazene was described with a one-compartment open model. The absorption rate constant and absorption half-life were 9.86 +/- 3.06 h-1 and 0.121 +/- 0.40 h, respectively. The value of elimination half-life was 107.5 +/- 8.50 h. The apparent volume of distribution was 0.74 +/- 0.07 L x kg-1. Systemic availability following intramuscular administration was 91.7%. Approximately 65% of the administered dose of diminazene was eliminated in the urine within 24 h of its intramuscular administration. Diminazene was bound to plasma proteins to the extent of approximately 32%. The satisfactory intramuscular dosage regimen of diminazene for calves would be 2.24 mg x kg-1 followed by 1.5 mg x kg-1 at 7 days.

Comparative pharmacokinetics of diminazene in lactating goats and sheep.

The pharmacokinetic aspects of diminazene aceturate were studied in lactating goats and sheep after single intravenous and intramuscular administrations of 3.5 mg/kg b.wt. Plasma and milk concentrations were determined by use of reversed phase high-performance liquid chromatography (HPLC) after ion-pair extraction. Following intravenous injection, the disposition of diminazene in goats and sheep conformed to a two-compartment model with rapid distribution and slower elimination phases. Values of (t1/2 beta) were obtained indicating a slower final disappearance of the drug from plasma of sheep (21.17 h) than in goats (16.39 h). Diminazene concentrations were maintained for more than 4 days in the plasma of goats and sheep. In both species of animals, diminazene was rapidly absorbed following intramuscular administration of 3.5 mg/kg b.wt. The peak plasma concentrations (Cmax) were 7.00 and 8.11 micrograms/ml and were attained at (Tmax) 0.92 and 1.12 hours in goats and sheep, respectively. The elimination half-life (t1/2el) of diminazene after intramuscular administration was shorter in goats (16.54 h) than in sheep (18.80 h). Systemic bioavailabilities (F%) of diminazene after intramuscular administration were 94.94% and 82.64% in goats and sheep, respectively. Diminazene could be detected in milk of goats and sheep within 10 min post-injection. Milk concentrations of the drug were lower in goats than in sheep and were detected for 5 and 6 days following both routes of administration, respectively.

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.

The effects of hyperosmolar agents lithium chloride and sucrose on the brain concentration of diminazene aceturate in rats.

The concentrations of diminazene aceturate in the brain of Trypanosoma brucei brucei infected and uninfected rats treated with diminazene aceturate (3.1 mg/kg, im) and either LiCl (2.5, 5.0 and 10 micrograms/kg) or sucrose (0.25, 0.5 and 1.0 g/kg) were determined. When diminazene aceturate was administered at a standard dose of 3.1 mg/kg (im), the addition of LiCl (10 micrograms/kg, im) increased significantly (P < 0.05) the concentration of the drug in the brains of both trypanosome infected and normal infected rats. The addition of sucrose (1.0 g/kg, im) instead of LiCl failed to give any significant increase in diminazene aceturate levels in the brain. The diminazene aceturate levels were significantly (P < 0.05) higher in the organs (brain, kidney, liver and spleen) of trypanosome infected compared to uninfected rats. The concentration of diminazene aceturate in the organs increased significantly (P < 0.05) with increasing concentrations of LiCl.

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.

Apparent rarity of diminazene-resistant trypanosomes in goats infected with a diminazene-resistant population of Trypanosoma congolense.

Experiments were carried out in goats to determine the frequency with which diminazene-resistant trypanosomes occur in parasite populations before and after the intramuscular treatment of the goats with diminazene aceturate. Trypanosoma congolense IL 3274, a diminazene-resistant clone, was used to initiate infections in three groups of five goats. The goats in the first group were treated with diminazene aceturate at a dose of 7.0 mg kg-1 bodyweight within 10 seconds of infection; one of the goats was cured. All of the second group, which received no treatment, became parasitaemic. The third group of goats received the same dose of drug as the first group but three days after all of them were first detected parasitaemic; trypanosomes reappeared in all the five goats. When this third group was treated, the frequency of trypanosomes resistant to the drug dosage was estimated to be less than 1 in 10(3). The parasites which reappeared after the treatment of these animals were used to infect two additional groups of five goats intravenously. The goats in one group were treated with the same dose of drug as before, within 10 seconds of infection and were all cured. In contrast, the five goats in the second, untreated, group became parasitaemic. Finally, when the goats in which the infections had relapsed were retreated with diminazene aceturate at the same dose rate, the level of parasitaemia temporarily decreased by at least 10(3) trypanosomes ml-1. These findings suggest that diminazene-resistant T congolense occur at low levels in trypanosome populations despite attempts to select for a population resistant to the dose of drug used.

A bioequivalence and pharmacokinetic evaluation of two commercial diminazene aceturate formulations administered intramuscularly to cattle.

The bioequivalence of the diminazene formulation Veriben (Centaur) was determined in cattle (n = 10) by means of a single-dose, randomized cross-over experiment. The results of nine statistical procedures commonly used for bioequivalence evaluation are discussed. Veriben was found to be equivalent to Berenil (Hoechst) with respect to the area under the plasma concentration versus time curve, but not in terms of the maximum plasma drug concentration and the time to maximum plasma drug concentration. Pharmacokinetic parameters were calculated in which bioequivalence data (n = 10) together with data from an additional four cattle were used. A two-compartment model best described the pharmacokinetic behaviour of diminazene in cattle. Peak concentrations of diminazene (3.24 +/- 0.16 micrograms/ml) were reached 49.8 (+/- 7.6) min after intramuscular injection of 3.5 mg/kg drug, with absorption proceeding rapidly (t1/2 alpha = 1.93 +/- 0.95 h). Diminazene was slowly eliminated (t1/2 beta = 222 h), resulting in a mean residence time of 13.27 d. The safe interval necessary between successive treatments of diminazene or before live babesia vaccines should be administered, and a recommended pre-slaughter withdrawal period are also discussed.

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).

Pharmacokinetics of diminazene in female Boran (Bos indicus) cattle.

The disposition kinetics and bioavailability of diminazene in five healthy heifers were determined after single intravenous (i.v.) and intramuscular (i.m.) administration of the drug in sequence with a wash-out period between administrations of 6 weeks. Intact diminazene in plasma, whole blood and urine samples was analysed using high-performance liquid chromatography. Nonlinear regression analysis of the i.v. and i.m. data indicated that, for either route, the plasma disappearance curves of diminazene were best described by triexponential equations. The i.v. bolus was followed by rapid and biphasic distribution with half-life values of 0.04 h and 0.58 h, Vd(ss) was 1.91 +/- 0.42 l/kg, elimination half-life was 31.7 h while Cl averaged 1.74 +/- 0.40 ml/min/kg. Within 30 min of the i.v. dose, the erythrocyte/plasma partition ratio of diminazene was 0.30 +/- 0.15. Diminazene was rapidly absorbed following i.m. administration; t1/2ka was 0.60 h. Cmax, 4.68 +/- 1.12 micrograms/ml, was attained in 10-15 min and systemic availability was 102.42 +/- 7.25%. The half-life of the terminal disappearance phase was 145.48 h. About 8.26% of the i.m. dose was excreted intact in the urine within the first 24 h of treatment. In vitro, diminazene was bound to bovine plasma albumin to the extent of 38.01-91.10%.