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Persistent hyperglycemia in type 1 diabetes triggers numerous signaling pathways, which may prove deleterious to the endothelium. As hyperglycemia damages the endothelial layer via multiple signaling pathways, including enhanced oxidative stress, downregulation of angiotensin-converting enzyme 2 signaling, and exacerbation of endoplasmic reticulum (ER) stress, it becomes difficult to prevent injury using monotherapy. Thus, the present study was conceived to evaluate the combined effect of ER stress inhibition along with angiotensin-converting enzyme 2 activation, two major contributors to hyperglycemia-induced endothelial dysfunction, in preventing endothelial dysfunction associated with type 1 diabetes. Streptozotocin-induced diabetic animals were treated with either diminazene aceturate (5 mg·kg -1 per day, p.o.) or tauroursodeoxycholic acid, sodium salt (200 mg·kg -1 per day i.p.), or both for 4 weeks. Endothelial dysfunction was evaluated using vasoreactivity assay, where acetylcholine-induced relaxation was assessed in phenylephrine pre-contracted rings. Combination therapy significantly improved vascular relaxation when compared with diabetic control as well as monotherapy. Restoration of nitrite levels along with prevention of collagen led to improved vasodilatation. Moreover, there was an overall reduction in aortic oxidative stress. We conclude that by simultaneously inhibiting ER stress and activating angiotensin-converting enzyme 2 deleterious effects of hyperglycemia on endothelium were significantly alleviated. This could serve as a novel strategy for the prevention of endothelial dysfunction.

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., (© 2020 Wiley Periodicals, Inc.)

Although renin-angiotensin system (RAS) imbalance is manifested in cardiomyopathies with different etiologies, the impact of RAS effectors on Chagas cardiomyopathy and skeletal myositis is poorly understood. Given that diminazene aceturate (DMZ) shares trypanocidal, angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) stimulatory effects, we investigated the impact of DMZ on cardiomyocytes infection in vitro, renin-angiotensin system, Chagas cardiomyopathy and skeletal myositis in vivo. Cardiomyocytes and T. cruzi were used to evaluate DMZ toxicity in vitro. The impact of 20-days DMZ treatment (1 mg/kg) was also investigated in uninfected and T. cruzi-infected mice as follows: control uninfected and untreated, uninfected treated with DMZ, infected untreated and infected treated with DMZ. DMZ had low toxicity on cardiomyocytes, induced dose-dependent antiparasitic activity on T. cruzi trypomastigotes, and reduced parasite load but not infection rates in cardiomyocytes. DMZ increased ACE2 activity and angiotensin-(1-7) plasma levels but exerted no interference on angiotensin-converting enzyme (ACE) activity, ACE, ACE2 and angiotensin II levels in uninfected and infected mice. DMZ treatment also reduced IFN-γ and IL-2 circulating levels but was ineffective in attenuating parasitemia, MCP-1, IL-10, anti-T. cruzi IgG, nitrite/nitrate and malondialdehyde production, myocarditis and skeletal myositis compared to infected untreated animals. As the antiparasitic effect of DMZ in vitro did not manifest in vivo, this drug exhibited limited relevance to the treatment of Chagas disease. Although DMZ is effective in upregulating angiotensin-(1-7) levels, this molecule does not act as a potent modulator of T. cruzi infection, which can establish heart and skeletal muscle parasitism, lipid oxidation and inflammatory damage, even in the presence of high concentrations of this RAS effector., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We have previously demonstrated that the phosphorylation of p38 MAPK, through spinal AT 1 receptor activation, is involved in formalin-induced nociception and follows accompanied by the increase in spinal angiotensin (Ang) II levels. We have also found that Ang (1-7), an N-terminal fragment of Ang II generated by ACE2, prevents the Ang II-induced nociceptive behavior via spinal MAS1 and the inhibition of p38 MAPK phosphorylation. Here, we examined whether the ACE2 activator diminazene aceturate (DIZE) can prevent the formalin-induced nociception in mice. The i.t. administration of DIZE attenuated the second, but not the first phase of formalin-induced nociceptive response. An increase in the activity of spinal ACE2 was measured following DIZE administration. The inhibitory effect of DIZE on nociception was abolished by the i.t. co-administration of the MAS1 antagonist A779. The i.t. administration of Ang (1-7) showed a similar effect on the second phase of the response which was also attenuated by A779. Furthermore, DIZE and Ang (1-7) each inhibited the formalin-induced phosphorylation of p38 MAPK on the dorsal lumbar spinal cord. This inhibition was again prevented by A779. ACE2 was expressed in neurons and microglia but absent from astrocytes in the superficial dorsal horn. Our data show that the i.t.-administered DIZE attenuates the second phase of the formalin-induced nociception which is accompanied by the inhibition of p38 MAPK phosphorylation. They also suggest the involvement of MAS1 activation on spinal neurons and microglia in response to the increase in Ang (1-7) following ACE2 activation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Neprilysin inhibitors (NEPi) are assisting the renin-angiotensin system (RAS) inhibitors in halting diabetic cardiomyopathy (DCM). Away from conventional tactic, a recent report revealed the renoprotective potential of NEPi and angiotensin-converting enzyme (ACE2) activator combination therapy against diabetic nephropathy. However, this combination so far not evaluated against DCM, thus the present investigation aiming the same., Methods: Streptozotocin-induced (55 mg/kg, ip) type 1 diabetic (T1D) male Wistar rats were treated with either monotherapy of thiorphan (0.1 mg/kg/day, po) or diminazene aceturate (5 mg/kg/day, po), or their combination therapy, for four weeks. After hemodynamic measurements, all the rats' heart and plasma were collected for biochemistry, ELISA, histopathology, and immunoblotting., Results: Metabolic perturbations and failing cardiac functions associated with diabetes were markedly attenuated by combination therapy. Besides, unfavourable alterations in RAS and natriuretic peptides system (NPS) were corrected by combination therapy. Interestingly, combination therapy significantly increased plasma and heart cGMP levels compared to T1D and monotherapy receiving rats. Moreover, rats receiving combination therapy exhibited significant inhibition of activated NF-κB, TGF-β and apoptotic signalling, and a notable reduction in cardiac fibrosis when compared to T1D rats. Expressions of posttranslational histone modifications markers; H3K4Me2 and its methyltransferases (SET7/9 and RBBP5) were significantly enhanced in T1D hearts, which were significantly reduced by combination therapy., Conclusions: The NEPi and ACE2 activator combination therapy effectively prevented DCM by normalising RAS and NPS activities, increasing cGMP, inhibiting inflammatory, pro-fibrotic and apoptotic signalling, and reversing H3K4Me2 and its methyl transferases expressions., (Copyright © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Background: Globally, working equines have a continued and growing socioeconomic role in supporting the livelihoods of between 300-600 million people in low income countries which is rarely recognised at a national or international level. Infectious diseases have significant impact on welfare and productivity in this population and equine trypanosomiasis is a priority disease due to its severity and prevalence. Strategies are required to improve the prevention, diagnosis, management and treatment of trypanosomiasis in equines and more data are required on the efficacy and safety of current trypanocidal drugs., Methods: A prospective randomised, open-label non-inferiority trial was performed in The Gambia on horses and donkeys that fulfilled 2/5 clinical inclusion criteria (anaemia, poor body condition, pyrexia, history of abortion, oedema). Following randomised trypanocidal treatment (diminazene diaceturate, melarsomine dihydrochloride or isometamidium chloride), animals were observed for immediate adverse drug reactions and follow-up assessment was performed at 1 and 2 weeks. Blood samples underwent PCR analysis with specific Trypanosoma sp. primers. Treatment efficacy was assessed by measuring changes in clinical parameters, clinicopathological results and PCR-status post-treatment after evaluating for bias. Using PCR status as the outcome variable, non-inferiority of isometamidium treatment was determined if the upper bound limit of a 2-sided 95% CI was less than 10%., Results: There was a significant beneficial effect upon the Trypanosoma sp. PCR positive population following trypanocidal treatment for all groups. The findings of clinical evaluation and PCR status supported a superior treatment effect for isometamidium. Melarsomine dihydrochloride efficacy was inferior to isometamidium. There were immediate, self-limiting side effects to isometamidium in donkeys (26%). Diminazene had the longest duration of action as judged by PCR status., Conclusions: The data support the continued use of isometamidium following careful dose titration in donkeys and diminazene for trypanosomiasis in equines using the doses and routes of administration reported., Competing Interests: The authors have declared that no competing interests exist.

Background: Developing new antibabesial drugs with a low toxic effect to the animal and with no resistance from Babesia parasites is in urgent demand. In this concern, the antimalarial, anticancer and antioxidant effect of thymoquinone (TQ), a phytochemical compound found in the plant Nigella sativa, has been reported. Therefore, in the present study, the antibabesial effect of this compound was evaluated on the growth of piroplasm parasites., Results: Significant inhibition (P < 0.05) of the in vitro growth of piroplasm parasites were observed after treatment by TQ with IC 50 values of 35.41 ± 3.60, 7.35 ± 0.17, 0.28 ± 0.016, 74.05 ± 4.55 and 67.33 ± 0.94 μM for Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi and Babesia caballi, respectively. The in vitro inhibitory effect of TQ was significantly enhanced (P < 0.05) when used in combination with either diminazene aceturate on bovine Babesia and equine Babesia and Theileria cultures. In B. microti-infected mice, oral and intraperitoneal administrations of TQ showed significant (P < 0.05) inhibition of parasite growth at a dose of 70 mg/kg and 50 mg/kg, respectively, compared to the control group., Conclusions: The obtained results indicate that thymoquinone might be a promising medicinal compound for use in the treatment of animal piroplasmosis.

Background: Trypanosoma evansi is mechanically transmitted by biting flies and affects camels, equines, and other domestic and wild animals in which it causes a disease called surra. At least two types of Trypanosoma evansi circulate in Ethiopia: type A, which is present in Africa, Latin America and Asia, and type B, which is prevalent in Eastern Africa. Currently, no information is available about the drug sensitivity of any Ethiopian T. evansi type., Methodology/principal Findings: This study was conducted with the objective of determining the in vivo drug sensitivity of two T. evansi type A and two type B stocks that were isolated from camels from the Tigray and Afar regions of Northern Ethiopia. We investigated the efficacy of four trypanocidal drugs to cure T. evansi infected mice: melarsamine hydrochloride (Cymelarsan), diminazene diaceturate (Veriben and Sequzene), isometamidium chloride (Veridium) and homidium chloride (Bovidium). Per experimental group, 6 mice were inoculated intraperitoneally with trypanosomes, treated at first peak parasitemia by daily drug injections for 4 consecutive days and followed-up for 60 days. Cymelarsan at 2 mg/kg and Veriben at 20 mg/kg cured all mice infected with any T. evansi stock, while Sequzene at 20 mg/kg caused relapses in all T. evansi stocks. In contrast, Veridium and Bovidium at 1 mg/kg failed to cure any T. evansi infection in mice., Conclusions/significance: We conclude that mice infected with Ethiopian T. evansi can be cured with Cymelarsan and Veriben regardless of T. evansi type. In contrast, Veridium and Bovidium are not efficacious to cure any T. evansi type. Although innate resistance to phenanthridines was previously described for T. evansi type A, this report is the first study to show that this phenomenom also occurs in T. evansi type B infections., Competing Interests: The authors have declared that no competing interests exist.

Background: Trypanocidal drugs have been used to control African animal trypanosomosis for several decades. In Ethiopia, these drugs are available from both authorized (legal) and unauthorized (illegal) sources but documentation on utilization practices and quality of circulating products is scanty. This study looked at the practices of trypanocidal drug utilization by farmers and the integrity of active ingredient in trypanocides sold in Gurage zone, south western Ethiopia. The surveys were based on a structured questionnaire and drug quality determination of commonly used brands originating from European and Asian companies and sold at both authorized and unauthorized markets. One hundred farmers were interviewed and 50 drug samples were collected in 2013 (Diminazene aceturate = 33 and Isometamidium chloride = 17; 25 from authorized and 25 from unauthorized sources). Samples were tested at the OIE-certified Veterinary Drug Control Laboratory (LACOMEV) in Dakar, Senegal, by using galenic standards and high performance liquid chromatography., Results: Trypanosomosis was found to be a major threat according to all interviewed livestock keepers in the study area. Diminazene aceturate and isometamidium chloride were preferred by 79% and 21% of the respondents respectively, and 85% of them indicated that an animal receives more than six treatments per year. About 60% of these treatments were reported to be administered by untrained farmers. Trypanocidal drug sources included both unauthorized outlets (56%) and authorized government and private sources (44%). A wide availability and usage of substandard quality drugs was revealed. Twenty eight percent of trypanocidal drugs tested failed to comply with quality requirements. There was no significant difference in the frequency of non-compliance between diminazene-based and isometamidium chloride products (P = 0.87) irrespective of the marketing channel (official and unofficial). However, higher rates of non-compliant trypanocides were detected for drugs originating from Asia than from Europe (P = 0.029)., Conclusion: The findings revealed the presence of risk factors for the development of drug resistance, i.e. wide distribution of poor quality drugs as well as substandard administration practices. Therefore, it is strongly recommended to enforce regulatory measures for quality control of veterinary drugs, to expand and strengthen veterinary services and to undertake trypanocidal drug efficacy studies of wider coverage.

Background and Purpose: One of the protective actions of angiotensin converting enzyme-2 (ACE2) is the inactivation of angiotensin II. Expression and activity of ACE2 was reduced in glomeruli of diabetic patients and in animal models of diabetes. Recently the potential role of recombinant ACE2 administration in preventing diabetic nephropathy (DN) has been shown. Here we have tested the effects of the ACE2 activator, diminazene aceturate (DIZE), in a model of DN., Experimental Approach: Male Wistar rats were rendered diabetic using a single dose of streptozotocin (55 mg·kg -1 , i.p.). After 4 weeks, diabetic animals were divided into experimental groups and treated with DIZE, at a low dose (5 mg·kg -1 ·day -1 ), a high dose (15 mg·kg -1 ·day -1 ) and the high dose with of the AT 2 receptor antagonist PD123319 (10 mg·kg -1 ·day -1 ). At the end of the treatment , kidneys from all the groups were collected and processed separately for glomerular isolation, protein isolation, mRNA extraction and for immunohistochemical studies., Key Results: Treatment with DIZE restored ACE2 expression in glomeruli and increased expression of AT 2 receptors in whole kidney and isolated glomeruli of diabetic animals. DIZE administration reduced angiotensin II levels and increased angiotensin-(1-7) levels in diabetic kidney. However, PD123319 treatment reversed all these actions of DIZE., Conclusions and Implications: DIZE treatment reduced diabetes-induced renal damage as shown by reduction of fibrosis and apoptosis. These protective actions of DIZE were blocked by the AT 2 receptor antagonist. Taken together, these results suggest that DIZE protected against DN through the ACE2/angiotensin-(1-7)/ AT 2 receptor axis., (© 2017 The British Pharmacological Society.)

African trypanosomosis is a parasitic disease affecting both humans (sleeping sickness) and animals (nagana). In murine trypanosomosis, the B-cell compartment is rapidly destroyed after infection. In addition, B-cell lymphopoiesis in the bone marrow is abrogated, B-cell subsets in the spleen are irreversibly depleted, and B-cell memory is destroyed. Here, we investigated the effect of cure of infection on the B-cell compartment. Suramin and diminazene aceturate were used in this study as these drugs exhibit different modes of uptake and different mechanisms of trypanocidal action. Curative drug treatment of trypanosomosis infection led to the re-initiation of B-cell lymphopoiesis in the bone marrow, and to the repopulation of splenic B-cell subsets, independent of the drug used. Neither of these drugs by itself induced measurable effects on B-cell lymphopoiesis in the bone marrow or B-cell homoeostasis in the spleen in healthy, naïve animals., (© 2015 John Wiley & Sons Ltd.)

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.

This study aimed to verify the effect of the treatment with A. satureioides essential oil (free and nanoencapsulated forms) and diminazene aceturate on hematological and biochemical variables in rats infected by Trypanosoma evansi. The 56 rats were divided into seven groups with eight rats each. Groups A, C and D were composed by uninfected animals, and groups B, E, F and G were formed by infected rats with T. evansi. Rats from groups A and B were used as negative and positive control, respectively. Rats from the groups C and E were treated with A. satureioides essential oil, and groups D and F were treated with A. satureioides nanoencapsulated essential oil. Groups C, D, E and F received one dose of oil (1.5 mL kg(-1)) during five consecutive days orally. Group G was treated with diminazene aceturate (D.A.) in therapeutic dose (3.5 mg kg(-1)) in an only dose. The blood samples were collected on day 5 PI for analyses of hematological (erythrocytes and leukocytes count, hemoglobin concentration, hematocrit, mean corpuscular and mean corpuscular hemoglobin concentration) and biochemical (glucose, triglycerides, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, urea and creatinine) variables. A. satureioides administered was able to maintain low parasitemia, mainly the nanoencapsulated form, on 5 days post infection. On the infected animals with T. evansi treated with A. satureioides essential oil (free and nanocapsules) the number of total leucocytes, lymphocytes and monocytes present was similar to uninfected rats, and different from infected and not-treated animals (leukocytosis). Treatment with A. satureioides in free form elevated levels of ALT and AST, demonstrating liver damage; however, treatment with nanoencapsulated form did not cause elevation of these enzymes. Finally, treatments inhibited the increase in creatinine levels caused by infection for T. evansi. In summary, the nanoencapsulated form showed better activity on the trypanosome; it did not cause liver toxicity and prevented renal damage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the effect of treatment with liposomal (L-DMZ) and conventional (C-DMZ) diminazene aceturate formulations on hepatic and renal functions of rats, experimentally infected with Trypanosoma evansi. For this purpose, 72 Wistar rats (Rattus norvegicus) were divided into six groups (A, B, C, D, E, and F). Each group was subdivided into two other subgroups in order to assess the biochemical and histological results on days 7 and 40 post-treatment (PT). Treatments were carried out based on two different therapeutic protocols: L-DMZ and C-DMZ at 3.5mg/kg(-1), single dose (groups C and D), and five successive doses within intervals of 24h (groups E and F). Groups A and B corresponded to uninfected and infected (without treatment) animals, respectively. Sample collections were held on days 7 and 40 PT for the assessment of hepatic [alkaline phosphatase (AP), alanine transferase (ALT), albumin, gamma glutamil transferase (GGT) and renal functions (creatinine and urea). Additionally, the histology of fragments of liver, kidney, and spleen was performed. Animals in group B showed a significant increase in AP, GGT, ALT, and urea when compared with group A. On day 7 post-inoculation (PI), the biochemical analysis showed a reduction (P<0.05) of AP and GGT, while the levels of urea were increased in groups C, D, E, F. On day 40 PT, ALT was increased in these same groups when compared with group A. In histopathology, changes in liver samples were observed on day 7 PT in groups D and F, especially regarding the area and density of the hepatocytes. Renal analysis exhibited changes in glomerular space, glomerular, and corpuscular areas in group E. Therefore, these results allowed us to conclude that the treatment with L-DMZ and C-DMZ led to variable biochemical changes, which defined the functions of the liver and kidneys of treated animals, since the main histopathology alterations were observed in animals treated with liposomes, at their higher dosages. Thus, treatments with L-DMZ and C-DMZ in five consecutive doses were effective although being followed by liver toxicity., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Objective: To investigate the effect of diminazene aceturate (DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei., Methods: Thirty adult male albino rats, randomly assigned into 6 groups (A-F) of 5 rats each were used. They were either infected with 1×10(6) trypanosomes intraperitoneally (groups A-E) or uninfected (group F). The different groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B-3.5 mg/kg DA and 7.5 mg/kg levamisole; group C-3.5 mg/kg DA and 100 mg/kg vitamin C; and group D-3.5 mg/kg DA and 7.5 mg/kg levamisole and 100 mg/kg vitamin C. Group E was left untreated. Parameters assessed include: rectal temperature, body weight changes, packed cell volume (PCV), Haemoglobin concentration (Hb), total leucocyte count (TLC) differential leucocyte count (DLC), parasitaemia, clinical signs and survivability., Results: Average pre-patent period of 5 days was recorded. Parasites in the blood were cleared in all treated groups (A-D) within 48 hours post treatment (PT). Untreated rats in group E died between 25 and 32 days post infection (PI). Relapse was not recorded in all the treated groups (A-D). The initial reduction in PCV, Hb, TLC and increases in rectal temperature following infection were reversed by the treatments. The rats that received drug combinations (groups B, C and D) showed faster and higher recovery rates than the uninfected control and group A., Conclusions: Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei., (Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Purpose: Uveitis is a common cause of vision loss. The renin angiotensin system (RAS), which plays a vital role in cardiovascular system, is a potent mediator of inflammation and has been implicated in the pathogenesis of uveitis. A newly identified axis of RAS, ACE2/Ang-(1-7)/Mas, has emerged as a novel target because it counteracts the deleterious effect of angiotensin II. The purpose of this study was to investigate the effect of endogenous ACE2 activation in preventing endotoxin-induced uveitis (EIU) in mice., Methods: ACE2 activator diminazene aceturate (DIZE) was administered both systemically and locally. For systemic administration, female BALB/c mice received intraperitoneal injection of DIZE (60 mg/kg body weight [BW]) for 2 days prior to lipopolysaccharide (LPS) intravitreal injection (125 ng) to induce uveitis. For local study, DIZE was given at 0.5, 0.1, and 0 mg/mL as eyedrops six times per day for 2 days before LPS injection. The anterior segment of the mice was examined at 12, 24, 48, and 72 hours after LPS injection, and clinical scores were determined at the same time. Morphology and infiltrating inflammatory cells were evaluated after 24 hours. The mRNA levels of inflammatory cytokines were analyzed by real-time RT-PCR. ACE2 activity was determined using a self-quenching fluorescent substrate., Results: At 24 hours, the clinical score of mice treated with DIZE systemically was significantly lower (mean, ∼1.75) than the saline vehicle group (mean, ∼4) (P < 0.001). Histological examination showed 63.4% reduction of infiltrating inflammatory cells in the anterior segment and 57.4% reduction in the posterior segment of DIZE-treated eyes. The number of CD45(+) inflammatory cells in the vitreous of the DIZE-treated group was decreased (43.3%) compared to the vehicle group (P < 0.01). The mRNA levels of inflammatory cytokines were significantly reduced in the DIZE-treated group (P < 0.01, P < 0.001). The number of infiltrating inflammatory cells was also significantly reduced in eyes that received topical administration of DIZE: 73.8% reduction in the 0.5 mg/mL group and 51.7% reduction in the 0.1mg/mL group compared to the control group. DIZE treatment resulted in significantly increased ACE2 activity in the retina (P < 0.001)., Conclusions: Endogenous ACE2 activation by DIZE has a preventive effect on LPS-induced ocular inflammation in the EIU mouse model. These results support the notions that RAS plays a role in modulating ocular immune response and that enhancing ACE2 provides a novel therapeutic strategy for uveitis., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

This study aimed to develop and test the in vitro and in vivo effectiveness of diminazene aceturate encapsulated into liposomes (L-DMZ) on Trypanosoma evansi. To validate the in vitro tests with L-DMZ, the efficacy of a commercial formulation of diminazene aceturate (C-DMZ) was also assessed. The tests were carried out in culture medium for T. evansi, at concentrations of 0.25, 0.5, 1, 2 and 3 μg mL(-1) of L-DMZ and C-DMZ. A dose-dependent effect was observed for both formulations (L-DMZ and C-DMZ), with the highest dose-dependent mortality of trypomastigotes being observed at 1 and 3 h after the onset of tests with L-DMZ. The results of in vivo tests showed the same effects in the animals treated with L-DMZ and C-DMZ in single doses of 3.5 mg kg(-1) and for 5 consecutive days (3.5 mg kg(-1) day(-1)). It was possible to conclude that T. evansi showed greater in vitro susceptibility to L-DMZ when compared with C-DMZ. In vivo tests suggest that treatment with the L-DMZ and C-DMZ showed similar efficacy in vivo. The potential of the formulation developed in this study was clearly demonstrated, as it increased the efficacy of the treatment against trypanosomosis, but more studies are needed to increase the effectiveness in vivo.

Background: There is an urgent need for the development of new, cheap, safe and highly effective drugs against African trypanosomiasis that affects both man and livestock in sub-Saharan Africa including Ethiopia. In the present study the exudate of Aloe gilbertii, an endemic Aloe species of Ethiopia, aloin, aloe-emodin and rhein were tested for their in vitro and in vivo antitrypanosomal activities against Trypanosoma congolense field isolate. Aloin was prepared from the leaf exudate of A. gilbertii by acid catalyzed hydrolysis. Aloe-emodin was obtained by oxidative hydrolysis of aloin, while rhein was subsequently derived from aloe-emodin by oxidation. In vitro trypanocidal activity tests were conducted on parasites obtained from infected mice, while mice infected with T. congolense were used to evaluate in vivo antitrypanosomal activity of the test substances., Results: Results of the study showed that all the test substances arrested parasites motility at effective concentration of 4.0 mg/ml within an incubation period ranging from 15 to 40 min. Moreover, the same concentration of the test substances caused loss of infectivity of the parasites to mice during 30 days observation period. Among the tested substances, rhein showed superior activity with minimum inhibitory concentration (MIC) of 0.4 mg/ml. No adverse reactions were observed when the test substances were administered at a dose of 2000 mg/kg. Rhein at doses of 200 and 400 mg/kg, and the exudate, aloin and aloe-emodin at a dose of 400 mg/kg reduced the level of parasitaemia significantly (P < 0.05) and improved anaemia., Conclusion: The results obtained in this investigation indicate that aloin and its derivatives particularly rhein have the potential to be used as a scaffold for the development of safe and cost effective antitrypanosomal drugs that can be useful in the continuing fight against African trypanosomiasis.

Cytauxzoon felis is a hemoprotozoan parasite of cats. While many infected cats die of acute illness, some enter a chronic carrier state. To date, no treatment has been documented to clear the chronic carrier state, leaving recovered cats to act as a potential indirect source of infection via a tick vector. Diminazene diaceturate is an anti-protozoal therapy that has been suggested for use in the treatment of acute cytauxzoonosis, but which failed to clear the carrier state at the dose used in acute illness. We hypothesized that a dose-intensified regimen of diminazene could reduce or eliminate parasitemia from five domestic cats naturally infected with C felis. Cats were administered 4 mg/kg of diminazene diaceturate intramuscularly for 5 consecutive days. Clearance of the organism was assessed via semi-quantitative polymerase chain reaction and light microscopy 1, 3, 6 and 10 weeks after starting treatment. Additionally, cats were monitored for adverse drug reactions by daily observation and examination. Complete blood count, biochemical profile and urinalysis were performed at 1, 3 and 10 weeks. Adverse events were common and included profuse salivation and nausea at the time of injection, monoparesis in the injected leg, proteinuria and potential hepatotoxicity. Severity of parasitemia was not reduced. Diminazene diaceturate cannot be recommended for elimination of the carrier state of C felis infection.

The experiment was performed with the aim of investigating the effect of a flavonoid mixture, Daflon® 500 mg (DF) on the erythrocyte fragility and lipoperoxidative changes, induced by Trypanosoma brucei brucei infection in Wistar rats. Fifty adult male rats randomly divided into five groups of 10 animals each were used. Rats in the control group were administered (1 mL/kg) distilled water only, while the other groups were infected with T. brucei brucei and treated with Daflon® 500 mg and/or Diminazene aceturate. At the end of 5 weeks, EDTA-blood samples and serum samples were collected from the rats, and were used to determine erythrocyte osmotic fragility (EOF) and serum malondialdehyde (MDA) concentration respectively. The results showed that EOF and MDA concentration significantly (P<0.05) increased in the infected untreated group when compared to the treatment groups. Treatment with Daflon® 500 mg and Diminazene aceturate significantly (P<0.05) reduced trypanosome-induced increases in EOF and lipoperoxidative changes, suggesting possible antioxidant properties of Daflon® 500 mg and its therapeutic value in trypanosomosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We have previously demonstrated that diminazene aceturate (DIZE), a putative angiotensin 1-7 converting enzyme activator, protects rats from monocrotaline (MCT)-induced pulmonary hypertension (PH). The present study was conducted to determine if the beneficial effects of DIZE are associated with improvements in autonomic nervous system (ANS) modulation. PH was induced in male rats by a single subcutaneous injection of MCT (50 mg/kg). A subset of MCT rats were treated with DIZE (15 mg/kg/day) for a period of 21 days, after which the ANS modulation was evaluated by spectral and symbolic analysis of heart rate variability (HRV). MCT administration resulted in a significant (P<0.001) increase in the right ventricular systolic pressure (62 ± 14 mmHg) when compared with other experimental groups (Control: 26 ± 6; MCT + DIZE: 31 ± 7 mmHg), while DIZE treatment was able to decrease this pressure. Furthermore MCT-treated rats had significantly reduced total power of HRV than the controls. On the other hand, although not significant, a trend towards increased HRV was observed in the MCT + DIZE group (Control: 108 ± 47; MCT: 12 ± 8.86 and MCT + DIZE: 40 ± 14), suggesting an improvement of the cardiac autonomic modulation. This observation was further confirmed by the low-frequency/high-frequency index of spectral analysis (Control: 0.74 ± 0.62; MCT: 1.45 ± 0.78 and MCT + DIZE: 0.34 ± 0.49) which showed that DIZE treatment was able to recover the ANS imbalance observed in the MCT-induced pulmonary hypertensive rats. Collectively, our results demonstrate that MCT-induced PH is associated with a significant increase in sympathetic modulation and a decrease in HRV, which are markedly improved by DIZE treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Diminazene aceturate and Antipyrine combination therapy is widely used in veterinary medicine. A simple reverse HPLC method for the analysis of samples of a ready injectable formulation containing a mixture of active ingredients and inactive excipients has been developed. The HPLC analysis was carried out using a reversed phase (RP)-C18 (250 mm×4.0 mm, 5 μm) column. The isocratic mobile phase consisted of a mixture of acetonitrile, methanol, phosphate buffer and hexane sulfonate; the flow rate was 0.6 mL/min and ultraviolet detection was at 291 nm. This method was validated in accordance with FDA and ICH guidelines and showed good linearity, accuracy, precision, selectivity and the system suitability results were within the acceptance criteria. A stability-indicating study was also carried out and indicated that this method could be used for purity and degradation evaluation of these formulations., (© Georg Thieme Verlag KG Stuttgart · New York.)

Clinical, epidemiological, and pathological aspects of trypanosomiasis caused by Trypanosoma vivax in calves were reported for the first time in northeast Brazil. Clinical and epidemiological data, packed cell volumes (PCV), and parasitemia were assessed in 150 calves in May 2009 (rainy season-survey 1) and in 153 calves in November 2009 (dry season-survey 2) in three farms (A, B, and C). Prevalence of T. vivax in calves examined in the survey 1 was 63.3%, 65.0%, and 80.0% in farms A, B, and C, respectively. Morbidity varied from 63.3% to 80%, mortality from 15% to 30% and lethality from 23% to 37.5%. In survey 1, for all farms, high parasitemia (from 30.3 to 26.2 × 10(6) parasites/mL), fever (from 39.8 to 40.3°C), low PCV (from 15.7% to 18.1%), and body score (from 2.5 to 3.5) were detected. Calves showed depression, weight loss, pale mucous membranes, enlarged lymph nodes, edema of the dewlap, cough, coryza, and diarrhea. The animals from farms A and B were treated with diminazene aceturate. Six months after, in survey 2, non-treated calves from farm C showed values for prevalence (81.82), morbidity (81.82), mortality (12.73), and lethality (15.55) similar to those in survey 1 (P > 0.05). Also in survey 2, four calves aging merely 1-3 days old presented high parasitemia levels (from 32 × 10(6) to 74 × 10(6) parasites/mL), suggesting transplacental transmission. In conclusion, trypanosomiasis by T. vivax constitutes high prevalent disease for calves raised in Brazilian semiarid and may have transplacental transmission.

There is a real need to develop new therapeutic strategies for African trypanosomiasis infections. In our study, we developed a new drug delivery system of diminazene (DMZ), a trypanocidal drug registered for veterinary use. This drug candidate presents a limited efficacy, a poor affinity for brain tissue and instability. The development of colloidal formulations based on a porous cationic nanoparticle with an oily core ((70)DGNP(+)), has potentially two advantages: stabilization of the drug and potential targeting of the parasite. We analyzed two processes of drug loading: in process (DMZ was added during the preparation of (70)DGNP(+) at 80 °C) and post-loading (DMZ was mixed with a (70)DGNP(+) solution at room temperature). Poor stability of the drug was observed using the in process technique. When using the post-loading technique over 80% drug entrapment efficiency was obtained at a ratio of DMZ:phospholipids (wt:wt) < 5%. Moreover, DMZ loaded into (70)DGNP(+) was found to be protected against oxidation and was stable for at least six months at 4 °C. Finally, in vitro tests on T.b. brucei showed an increased efficacy of DMZ loaded in (70)DGNP(+).

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are predominantly expressed in the nervous system. ASICs are involved in a number of neurological diseases such as pain, ischemic stroke and multiple sclerosis but limited tools are available to target these channels and provide probes for their physiological functions. Here we report that the anti-protozoal diarylamidines, 4',6-diamidino-2-phenylindole (DAPI), diminazene, hydroxystilbamidine (HSB) and pentamidine potently inhibit ASIC currents in primary cultured hippocampal neurons with apparent affinities of 2.8 microM, 0.3 microM, 1.5 microM and 38 microM, respectively. These four compounds (100 microM) failed to block ENaC channels expressed in oocytes. Sub-maximal concentrations of diminazene also strongly accelerated desensitization of ASIC currents in hippocampal neurons. Diminazene blocked ASIC1a, -1b -2a, and -3 currents expressed in CHO cells with a rank order of potency 1b > 3 > 2a >or= 1a. Patchdock computational analysis suggested a binding site of diarylamidines on ASICs. This study indicates diarylamidines constitute a novel class of non-amiloride ASIC blockers and suggests that diarylamidines may be developed as therapeutic agents in treatment of ASIC-involved diseases., ((c) 2010. Published by Elsevier Ltd. All rights reserved.)

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.

Two groups of Trypanosoma congolense isolates collected from cattle in 1996 (n=39) and 2003 (n=38) in the Eastern Province of Zambia were analyzed by BclI-PCR-RFLP to assess the evolution of diminazene aceturate (DA) resistance over a period of seven years. The results show a significant increase of DA resistance in this relatively short period of time. In 1996, among the 39 isolates, 61.5% were found sensitive, 12.8% resistant and 25.7% had a mixed BclI-PCR-RFLP profile. In 2004, among the 38 isolates, 10.5% were found sensitive, 63.2% were resistant and 26.3% showed a mixed BclI-PCR-RFLP profile. In vivo tests in mice showed that isolates with a sensitive or mixed RFLP profile were sensitive to DA whereas isolates with a resistant RFLP profile were resistant. Since there are no indications that the drug pressure has increased between 1996 and 2003, it is suggested that genetic exchange of resistance genes might explain the increased frequency of resistance to DA.

A series of platinium(II) complexes of formula [Pt2L4(berenil)2]Cl4.4HCl.2H2O where L is piperidine (1), 4-picoline (2), 3-picoline (3) or isopropylamine (4) was prepared and their cytotoxicity have been tested against the growth of human breast cancer cells. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than cisplatin. Data from the ethidium displacement assay indicated that these compounds show moderate specificity for AT base pairs of DNA. Compounds 1-4 were also potent topoisomerase II inhibitors, with 50% inhibitory concentrations (IC50) ranging from 5 to 50 microM.

The chemotherapeutic efficacy of diminazene aceturate (Berenil)--a standard veterinary trypanocide and pentamidine isethionate (PMI)--a human trypanocide was compared in dogs experimentally infected with Trypanosoma brucei brucei. Also, the activities of the drugs on some serum liver enzymes were evaluated before and after treatment to ascertain the relative safety of the drugs. Fifteen local dogs (mongrels) were used for the study. Three of the dogs were uninfected controls, and twelve were infected with a stock of T. brucei brucei. Three of the infected dogs were untreated controls, three were given diminazene aceturate (DA) at 7 mg/kg body weight intramuscularly (i/m), another three received pentamidine isethionate (PMI) at 4 mg/kg i/m on days 14, 17, 19, 27, 29, and 31 post infection (PI) and the remaining three dogs were also given same dose of PMI on days 14, 16, 18, 20, 22, 24 and 26 PI. Both trypanocides effectively cleared the parasites from the blood of the infected treated dogs. However, the infection subsequently relapsed at day 42 PI in one of the dogs in the DA treated group which later died at day 70 PI. Relapse infection was not recorded with the PMI treated groups although two dogs died in the PMI treated group II (treatment at days 14, 17, 19, 27, 29, and 31 PI) without showing relapsed parasitaemia. The packed cell volume (PCV), red blood cell (RBC) count, and haemoglobin (Hb) level which decreased significantly following infection, were reversed by the trypanocidal treatment. The reversal in the red cell values was faster in the PMI treated groups than in the DA treated group. The serum alkaline phosphate (SAP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels increased following infection and drug administration. The increase in the enzyme levels was greater in the DA treated groups than PMI treated groups. It was thus concluded that PMI given at 4 mg/kg i/m at days 14, 16, 18, 20, 22, 24, and 26 PI constituted a safe and efficient trypanocide and exhibited a superior trypanocidal action than DA in T. brucei brucei infected dogs.

Homeopathic Crotalus horridus 200C was evaluated in 13 clinical cases of babesiosis in dogs, compared with another 20 clinical cases treated with diminazine. Babesiosis is an important tropical tick-borne haemoprotozoan disease in dogs clinically manifested by anorexia, dehydration, temperature, dullness/depression, diarrhoea/constipation, pale mucosa, hepatomegaly, vomiting/nausea, splenomegaly, distended abdomen/ascites, yellow coloured urine, emaciation/weight loss, and occular discharge. The diagnosis of babesiosis was based on cytological evidence of Babesia gibsoni in freshly prepared blood smears. The dogs were treated with oral C. horridus 200C, 4 pills four times daily for 14 days (n=13) or diminazine aceturate 5 mg/kg single intramuscularly dose (n=20). All the dogs were administered 5% Dextrose normal saline at 60 ml/kg intravenously for 4 days. Initial clinical scores were similar in both groups and showed similar progressive improvement with the two treatments over 14 days. Parasitaemia also improved in both groups, but haematological values showed no change. No untoward reactions were observed. It appears that C. horridus is as effective in causing clinical recovery in moderate cases of canine babesiosis caused by Babesia gibsoni as the standard drug diminazine. Large scale randomized trials are indicated for more conclusive results.

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.

A comparison of two wavelet approaches, Daubechies and reverse Biorthogonal, is described for the quantitative resolution of a binary mixture of diminazene aceturate (DIMA) and phenazone (PHE) in veterinary granules for injection without any chemical separation. These two approaches were specified as db4 (a = 180) and rbior3.7 (a = 125) respectively, after testing the signal analysis parameters for the overlapping absorption spectra and ratio spectra. In the first step db4 (a = 180) was applied to the original absorbance data vector of DIMA and PHE. In the second step rbio3.7 (a = 125) was applied to the ratio spectra data vectors of DIMA using the divisor PHE. The same approach was also subjected to the ratio spectra of PHE using the divisor DIMA. The db4 (a = 180) and rbior3.7 (a = 125) calibration graphs were constructed using the transformation values obtained in the wavelet domain. In the method validation, the wavelet calibration functions were tested using synthetic mixtures and the standard addition technique. The simultaneous quantitative analysis of DIMA and PHE in the commercial veterinary preparation was achieved by the elaborated methods. The assay results were compared with each other and good agreement was observed.

A novel functionalised copolymer with three polymeric components, poly(ethylene glycol)-block-poly(aspartic acid-stat-phenylalanine), PEG-P(asp-phe), was synthesised and investigated for its potential to form micelles via ionic interactions with a model water-soluble drug, diminazene aceturate. Drug-free solutions of structurally related PEG-P(asp-phe) 5:6:4 and PEG-P(asp-phe) 5:4:6 copolymers indicated polymeric aggregation into micellar-type constructs. The size of PEG-P(asp-phe) 5:6:4 micelles was found to be pH and drug content-dependent. The drug-loaded systems existed as discreet units and were fairly uniform in size and shape. More drug could be included in the PEG-P(asp-phe) 5:6:4 micelles as compared to if only interaction with carboxyl groups from aspartic acid units was responsible for micelle formation, indicating the augmentative role of phenylalanine moieties in drug-incorporation. The slower in vitro drug release from PEG-P(asp-phe) 5:6:4 micelles as compared to PEG-Pasp (AB) micelles indicated the role of the phenylalanine moiety in controlling drug release. This study, therefore, confirmed the potential of a novel tri-component copolymer structure, PEG-P(asp-phe), for the formation of polyionic micelles for drug delivery.

The objective of this study was to apply transition models to distinguish between factors associated with both incident and persistent trypanosome infections. Data collected from 1561 cattle were analyzed from a long-term study involving 8 herds in which both trypanosome infections (a total of 56,931 cattle sampling-months) and tsetse (Glossina spp.) challenge were monitored monthly from March 1986 to March 1998. Both pour-on and insecticide-target tsetse control programs and mass treatment with diminazene aceturate before tsetse control were associated with significant decreases in both incidence and persistence of trypanosome infection relative to noncontrol periods, as were seasonal and sex effects. The magnitudes of the effects were, however, often different for new and persistent infections. For persistence of infection, there were 2 trends. In general, the duration of infection increased during the study, despite the regular treatment with diminazene aceturate. The transition model had 2 major benefits. The first was to identify an increasing duration of infections with time, taking into account other factors associated with increasing infection risk. The second was to highlight different patterns in the effects of certain factors on new and persistent trypanosome infections.

Trypan, a diamidine based drug, was tested as an antileishmanial agent. Duplicate cultures of both Leishmania major and Leishmania donovani promastigotes in M199 medium and Trypan at various concentrations were tested. The cultures were incubated at 25 degrees C and parasites counted at 48 h interval, and the data generated was used to establish growth inhibition curves. Drug-free cultures were included to serve as control. In the in vivo study, a total of 40 BALB/c mice were divided into five groups of 8 mice each. They were infected with 2 x 10(6) promastogotes on the left footpad. Two groups were treated with 70 microg/ml of Trypan, a total of 500 microl used immediately after infection, one group by topical application and the other administered intraperitoneally. The treatments were repeated for the two other groups 10 weeks post infection, one by topical application and the other administered intraperitoneally. One group was not treated and thus served as control. Footpad sizes were measured using Vernier calliper every 2 weeks for 21 weeks. In the in vitro studies, Trypan inhibited growth of either L. major or L. donovani promastigotes in all the concentrations tested with more dramatic inhibition in high concentrations. Based on the in vivo studies, it was evident that Trypan had effect on L. major infected lesions when applied topically immediately after infection. However, there was no effect when treatment commenced after the lesions were established. The data is discussed.

The trypanocide berenil was assessed for chemical stability over the pH range 1-8 at 37 degrees C and 0.2 M ionic strength. It was found to be sufficiently unstable under acid conditions that its therapeutic efficacy is most likely severely compromised when administered orally. At pH 3, the half-life was 35 min, decreasing to 1.5 min at pH 1.75. Reaction rate constants were corrected for the effects of buffer catalysis and were found to range from 2.00 min(-1) at pH 1 to 6.1 x 10(-6) min(-1) at pH 8. The pH-rate profile displayed a region (pH 1-4) where specific acid catalysis was dominant, followed by a transitional region (pH 5-7), and finally a region (pH >7) where uncatalysed degradation was most important. It is recommended that berenil be enteric coated for formulations to be used in treating Third World parasitic diseases.

Effects of diminazene aceturate and ceftriaxone disodium were evaluated on sperm quality of rams. Daily intramuscular injections of diminazene (6 mg/kg) or ceftriaxone (28.5 mg/kg) were given to each of seven Akkaraman rams assigned per drug for two days. Semen samples were collected from the rams at post-treatment 1, 4, 24, 48, 72, 144, 288 and 336 h and examined for sperm characteristics and hyaluronidase activity. Results showed that use of ceftriaxone and diminazene caused significant (P<0.01) decreases in sperm concentration, volume and motility compared to control group within 288 h post-treatment. In addition, hyaluronidase activity increased significantly (P<0.01) in semen of rams treated with ceftriaxone while remained unchanged in those received diminazene. In conclusion, diminazene aceturate and ceftriaxone disodium did not have any deleterious effect on hyaluronidase enzyme. However, both drugs caused impairment of sperm in rams during the 288 h.

It has been demonstrated that the attenuated organisms used in the unfrozen South African Babesia bovis and B. bigemina (redwater) vaccines are susceptible for longer periods to the residual effect of the anti-babesial drugs diminazene and imidocarb dipropionate than the virulent field strains. Reports of vaccine failures in some animals vaccinated with the frozen South African redwater vaccines after prophylactic treatment with imidocarb dipropionate have led us to reinvestigate the validity of the recommended prescribed waiting periods. Results indicated that waiting periods before administration of the frozen B. bovis and B. bigemina vaccines in animals that have been treated with diminazene at 3.5 mg/kg live weight, compare favorably with results initially obtained for the unfrozen vaccines at 4 and 8 weeks, respectively. However, the inhibitory effect of imidocarb dipropionate at 3.0 mg/kg live weight on the infectivity of both frozen B. bovis and B. bigemina vaccines is longer than previously anticipated and necessitated changing the minimum waiting periods before administration of these vaccines from 8 to 12 weeks and 16 to 24 weeks, respectively.

The objective of the present study was to incorporate the hydrophilic drug diminazenediaceturate at a high loading into lipid nanoparticles by creating nanoparticles from lipid-drug conjugates (LDC). IR and DSC data showed that the antitrypanosomal drug diminazene is able to react with fatty acids to form water-insoluble salts like diminazenedistearate and -dioleate. The salts could be transformed into nanoparticles using high-pressure homogenization technique, established for solid lipid nanoparticles (SLN). By using polysorbate 80 as surfactant, physically stable LDC nanoparticle dispersions of both salts could be obtained. The mean PCS diameters and polydispersity indices were 364 nm and 0.233 for diminazenedistearate and 442 nm and 0.268 for diminazenedioleate, respectively. Due to the composition of the LDC bulk materials, nanoparticles with a high drug load of 33% (w/w) were obtained even for this highly water-soluble drug diminazenediaceturate. The new carrier system of LDC nanoparticles overcomes one limitation of SLN, i.e. the limited loading capacity for hydrophilic drugs. Transforming water-soluble hydrophilic drugs into LDC and formation of nanoparticles allows prolonged drug release and targeting to specific sites by i.v. injection. These results provide a first basis of using LDC-polysorbate 80 nanoparticles for brain delivery of diminazene to treat second stage human African trypanosomiasis (HAT).

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.

In Zimbabwe, studies were made of the effect of host behaviour on the feeding success of Glossina pallidipes Austen and G. morsitans morsitans Westwood (Diptera: Glossinidae) attracted to cattle of different age and sex. The mean feeding rates for male and female G. pallidipes attracted to oxen were 60% and 58%, respectively, compared to 33% and 53% for male and female G. m. morsitans. The feeding rate of G. pallidipes varied between oxen and was inversely correlated with a host's rate of defensive leg movements, which, in turn, was positively correlated with the density of Stomoxys spp. (Diptera: Muscidae) caught in the vicinity of the host. Tsetse were significantly less successful in feeding from young cattle. For G. pallidipes, the feeding rate on calves (<6 months) was 11%, whereas for male and female G. m. morsitans the rates were 12% and 20%, respectively. Significantly lower feeding rates were apparent for cattle aged up to 2 years, when the feeding rate for G. pallidipes (31%) was still significantly less than that on mature oxen (68%). Feeding rates for G. pallidipes on adult female cattle were lower than those on oxen (45% vs. 61%). The lower feeding rates in young animals were attributed to higher rates of defensive movements. The results suggest that higher rates of defensive activities by young cattle reduce the risk of them contracting trypanosomiasis.

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.

Bobcats (Lynx rufus) are the reservoir hosts for Cytauxzoon felis, the causative agent of cytauxzoonosis. Cytauxzoonosis is a highly fatal tickborne blood protozoal disease of domestic and exotic cats. Treatment of clinically affected cats has generally been unrewarding. In our report, 6 of 7 cats had signs of illness and laboratory findings indicative of cytauxzoonosis and were successfully treated with 2 i.m. injections of diminazene aceturate or imidocarb dipropionate (2 mg/kg [0.9 mg/lb] of body weight, each). One cat died after the first injection of diminazene. Additional treatment with isotonic fluids i.v. and heparin s.c. were used as supportive care for dehydration and disseminated intravascular coagulation that were detected by laboratory testing between diminazene or imidocarb treatments. Atropine was effective in recovery and preventing adverse reactions associated with imidocarb treatment of 1 cat.

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.

From June to August in 1988, a total of 64 pure-bred Landrace, Yorkshire (large white) and Inner Mongolian Black Swine from a farm in Huhehot were affected with a disease, 13 died. The pathogen was confirmed primarily to be Babesia perroncitoi. The drugs used for treatment were Berenil and Acaprin. The intramuscular dose for Berenil was 3 mg per kilogram weight. The subcutaneous dose for Acaprin was 0.8 mg per kilogram weight. All of which had satisfactory results. In the same season in the second year and the third year, the disease was also reported, but all sick animals were cured.

Ten trypanosome isolates were collected at random from cattle at Ghibe, Ethiopia, in February 1993 and all shown to be savannah-type Trypanosoma congolense. When inoculated into naïve Boran (Bos indicus) calves, all 10 isolates were resistant to diminazene aceturate (Berenil), isometamidium chloride (Samorin) and homidium chloride (Novidium) at doses of 7.0 mg/kg body weight (b.w.), 0.5 mg/kg b.w. and 1.0 mg/kg b.w., respectively. In order to determine whether this multiple-drug resistance was expressed by individual trypanosomes, clones were derived from two of the isolates and characterised in mice for their sensitivity to the three compounds; by comparison to drug-sensitive populations, the two clones expressed high levels of resistance to all 3 trypanocides. In experiments to characterise the uptake kinetics of [14C]-Samorin, the maximal rates of uptake (Vmax) for 4 Ghibe isolates ranged from 9.2 to 15.0 ng/10(8) trypanosomes/min. In contrast, Vmax for the isometamidium-sensitive clone T. congolense IL 1180 was 86.7 +/- 8.6 ng/10(8) trypanosomes/min. Lastly, molecular karyotypes were determined for eight isolates: seven different chromosome profiles were observed. These data indicate that in February 1993 there was a high prevalence of drug-resistant trypanosome populations with different chromosome profiles in cattle at Ghibe. Since a similar situation existed at the same site in July 1989, this suggests that the drug-resistance phenotype of trypanosomes at Ghibe had not altered over a 4 year period.

As part of a study on livestock productivity under trypanosomosis risk in the region of Boundiali, northern Côte d'Ivoire, 21 herds of cattle (N'Dama, Baoulé and Zebu crosses) and 20 flocks of Djallonké and Djallonké x Sahel sheep were monitored monthly for body weight, packed red cell volume and trypanosomal parasitaemia over various periods between January 1984 and December 1992. A tsetse control campaign using biconical traps impregnated with alpha-cypermethrin started in December 1987. Tsetse control reduced the relative tsetse density by over 95% between 1988 and 1992, and this was associated with reductions in the prevalence of Trypanosoma congolense over the same period of over 90% both in sheep and cattle. Average reductions in the prevalence of T. vivax were lower, on average 68% in adults and 85% in young animals. Attempts were made in the design of the study to allow comparisons between controlled and uncontrolled areas; however, there were too many confounding and uncontrollable factors to allow such comparisons to be made. It was necessary, therefore, to compare data collected from all herds and flocks before and after the intervention, with the consequential difficulties in accounting for uncontrollable year-to-year variations in factors affecting trypanosome prevalence in livestock.

The trypanocidal value of liposomal diminazene was examined. Three hundred mice were divided randomly into six groups of 50 mice each. Groups 1, 2 and 3 were pretreated 2 h before Trypanosoma brucei evansi challenge with 0.2 ml saline solution, 0.2 ml diminazene solution (5 mg ml-1) and 0.2 ml liposomal solution (5 mg diminazene per ml), respectively. Mice in Group 1 all died within 6 days, 33 in Group 2 and four in Group 3 died within 15 days after challenge, respectively. Groups 4, 5, and 6 were treated 4 days after T. b. evansi challenge with 0.2 ml saline solution 0.2 ml diminazene solution (5 mg ml-1) and 0.2 ml liposomal solution (5 mg diminazene per ml), respectively. Mice in Group 4 all died within 6 days, 19 in Group 5 and two in group 6 died within 15 days after challenge, respectively. The remaining mice survived for more than 30 days, were symptom-free and behaved normally. No adverse effects associated with treatment were noted.

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.