Isometamidium chloride alters redox status, down-regulates p53 and PARP1 genes while modulating at proteomic level in Drosophila melanogaster.

As trypanocide, several side effects have been reported in the use of Isometamidium chloride. This study was therefore, designed to evaluate its ability to induce oxidative stress and DNA damage using D. melanogaster as a model organism. The LC50 of the drug was determined by exposing the flies (1-3 days old of both genders) to six different concentrations (1 mg, 10 mg, 20 mg, 40 mg, 50 mg and 100 mg per 10 g of diet) of the drug for a period of seven days. The effect of the drug on survival (28 days), climbing behavior, redox status, oxidative DNA lesion, expression of p53 and PARP1 (Poly-ADP-Ribose Polymerase-1) genes after five days exposure of flies to 4.49 mg, 8.97 mg, 17.94 mg and 35.88 mg per 10 g diet was evaluated. The interaction of the drug in silico with p53 and PARP1 proteins was also evaluated. The result showed the LC50 of isometamidium chloride to be 35.88 mg per 10 g diet for seven days. Twenty-eight (28) days of exposure to isometamidium chloride showed a decreased percentage survival in a time and concentration-dependent manner. Isometamidium chloride significantly (p < 0.05) reduced climbing ability, total thiol level, Glutathione-S-transferase, and Catalase activity. The level of H2O2 was significantly (p < 0.05) increased. The result also showed significant (p < 0.05) reduction in the relative mRNA levels of p53 and PARP1 genes. The in silico molecular docking of isometamidium with p53 and PARP1 proteins showed high binding energy of -9.4 Kcal/mol and -9.2 Kcal/mol respectively. The results suggest that isometamidium chloride could be cytotoxic and a potential inhibitor of p53 and PARP1 proteins.

Factors associated with persistence of African animal trypanosomiasis in Lango subregion, northern Uganda.

African animal trypanosomiasis (AAT) continues to inflict heavy losses on livestock production especially cattle in terms of decreased production and productivity in Uganda. AAT is a disease complex caused by tsetse fly-transmitted Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma congolense, and Trypanosoma vivax. The disease is most important in cattle but also known to cause serious losses in pigs, camels, goats, and sheep. Several control measures including live bait technology, mass treatment of cattle with trypanocidal drugs, and deployment of tsetse traps have been used in the past 10 years, but the problem still persists in some areas. This necessitated an exploration of the factors associated with continued trypanosome infections in cattle, which are also known reservoirs for the zoonotic trypanosomiasis. A structured questionnaire was administered to 286 animal owners from 20 villages purposively selected from Lira, Kole, and Alebtong districts of Lango subregion to obtain information on the factors associated with persistence of infection. Over 50% of the respondents reported trypanosomiasis as a major challenge to their livestock. Land ownership (P = 0.029), type of livestock kept (P = 0.000), disease control strategy employed (P = 0.000), source of drugs (P = 0.046), and drug preparation (P = 0.017) were associated with persistent AAT infection. We recommend continued farmer sensitization on the threat of AAT and the available prevention and control options. The use of isometamidium chloride for prophylaxis against trypanosomiasis is highly recommended. There is also a need to foster qualified private veterinary drug supply in the region.

Use of physiological biomarkers in diagnosis along with field trials of different trypanisidal drugs in camels of Cholistan desert.

The point prevalence of trypanosomiasis with different physiological biomarkers along with evaluation of the most responsive trypanosidal drug against trypanosomiasis under field conditions was studied. For this purpose a total of 300 free range camels were selected at different grazing and watering point in Cholistan desert. The study population of camels included 150 clinically suspected camels for trypanosomiasis and 150 healthy camels with normal temperature, pulse and respiration. For therapeutic trials 36 positively diagnosed animals were randomly divided into three experimental groups for therapeutic trials. Group A was treated with Imidocarb dipropionate (ID) @ 1.2 mg kg-1 body weight; Group B was treated with Diaminazine aceturate (DA) @ 3.5 mg kg-1 body weight and Group C was treated with Isometamidium chloride hypochloride (IC) @ 0.75 mg kg-1 body weight of camels. Data on risk factors of age,sex, ectoparasites, housing was also collected. Results revealed an overall 15% point prevalence of trypanosomiasis. There was significant (P < 0.05) decline in the values of physiological biomarkers of total erythrocyte counts, hemoglobin concentration, packed cell volume, serum total proteins and albumin while erythrocyte sedimentation rate was increased in infected camels as compared to healthy ones. Different hepatic enzymes including aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and alkaline phosphatase were also significantly increased in the infected animals. Therapeutic trials indicated that Isometamidium chloride hypochloride (IC) was more effective than Imidocarb dipropionate (ID) and Diaminazine aceturate (DA). It is concluded that haemato-biochemical parameters were important physiological biomarkers and IC was the most responsive therapeutic agent against trypanosomiasis in camels in field conditions. The risk factors analysis showed older camels (>5 years) showed highest infection while infection was found to be lowest in less than 1 year age group.

Nanoformulation of a Trypanocidal Drug Isometamidium Chloride Ameliorates the Apurinic-Apyrimidinic DNA Sites/Genotoxic Effects in Horse Blood Cells.

Isometamidium chloride (ISM) is a trypanocide for the prophylactic and therapeutic use against vector-borne animal trypanosomosis (mainly Surra caused by Trypanosoma evansi) and African animal trypanosomosis caused by T. congolense/T. vivax/T. brucei). ISM was found to be an efficient trypanocide for therapeutic/prophylactic use against trypanosomosis; however, it produces some local and systemic detrimental effects in animals. We synthesized isometamidium chloride-loaded alginate gum acacia nanoformulation (ISM SANPS) to lessen the detrimental side effects of isometamidium chloride (ISM) while treating trypanosomal diseases. We intended to determine the cytocompatibility/toxicity, and DNA deterioration/chromosomal structural or number changes (genotoxicity) of ISM SANPs using mammalian cells in a concentration-dependent manner. Apurinic/apyrimidinic (AP) sites are one of the major types of DNA lesions formed during base excision and repair of oxidized, deaminated, or alkylated bases. The intensity of the cellular AP site is an excellent marker of the deterioration of DNA quality. We thought it pertinent to quantify the AP sites in ISM SANPs-treated cells. Our investigations established a dose-dependent cyto-compatibility or toxicity and DNA impairment (genotoxicity) in ISM SANPs-treated horse peripheral blood mononuclear cells. ISM SANPs were biocompatible at various concentrations tested on the mammalian cells.

In vivo analysis of trypanocidal drug resistance in sahelian goats infected by Trypanosoma vivax strains collected in northern Togo.

Trypanosoma (T.) vivax is one of the animal trypanosomes species causing calf mortality and economic losses in Togo. Despite its importance as the most widely distributed trypanosome species, T. vivax has received little attention because it is difficult to cultivate most field isolates in rodents. No molecular diagnostic tools for the identification of drug-resistant in T. vivax are currently available. Herein, four field isolates of T. vivax from Togo were cryopreserved and assessed for susceptibility to diminazene aceturate (DA) and isometamidium chloride (ISM) in goats. For field isolate preparation, 1 ml of blood from an infected goat was diluted in 111 µl of phosphate-buffered-saline and stored in liquid nitrogen. The in vivo experiment drug test was performed using twenty Sahelian goats with six-month of age and weighing 14.5 ± 1.6 kg. These experimental goats were purchased from a tsetse free-area Dori, a Sahelian region of Burkina Faso. The cryopreserved T. vivax isolates with unknowns, DA, and ISM sensitivity was inoculated to five goats and one goat was used as control. Each animal was inoculated by intravenously route 1 × 105 trypanosomes from the donor goat. Relapses were earlier in the first phase of treatment (14.85 ± 1.08 days) compared with the second phase (20 ± 3.39 days). The overall mean PCV of the control group decreased from 32% to 17% at day-60 (P-value < 0.001). Three isolates were phenotypically resistant to 0.5 mg per kg body weight (BW) ISM and one for 3.5 mg per kg BW of DA. There were no relapses with the 7 mg per kg BW dose DA. This study shows the resistance of T. vivax to two main trypanocidal drugs in different villages of Mango. The results suggest the extension of surveillance strategies to remote villages in Togo and will guide the veterinarian or herder in choosing a mass treatment strategy. Further studies will be needed to better understand the molecular basis of the observed resistance.

Efficacy of diminazene diaceturate and isometamidium chloride hydrochloride for the treatment of Trypanosoma evansi in mice model.

Diminazene diaceturate (DIM) and isometamidium chloride hydrochloride (ISMM) have been widely used for the treatment of animal trypanosomosis. We evaluated the efficacy of standard doses of DIM and ISMM followed by their double doses for the treatment of Trypanosoma evansi in experimentally infected mice. A T. evansi strain obtained from a naturally infected camel in Afar was used. 25 swiss white mice randomly divided in to five groups were inoculated with 0.2 mL of blood containing 103 trypanosomes. At the peak of parasitemia (≈ 2 weeks post infection), groups A and B were treated with the standard dose (3.5 mg/kg body weight [BWT]) of DIM; groups C and D were treated with the standard dose (0.5 mg/kg BWT) of ISMM; and group E served as infected control. In the DIM standard dose groups, relapses and peak parasitemia were observed 20- and 25-days post treatment respectively. Similarly, relapses and peak parasitemia were observed 21- and 27-days post treatment in the ISMM standard dose groups. All mice in the control group died within two weeks post infection. Following relapses, mice were treated with the double doses of DIM (7 mg/kg BWT) or ISMM (1 mg/kg BWT). Parasitemia was not detected for 3 months following the double dose treatments. Following dexamethasone administration for 7 days, all but one mouse in the DIM group remained negative for another month. In general, although the T. evansi strain was resistant to the standard doses of DIM and ISMM their double doses completely cleared the infection.

Development of cationic Isometamidium chloride loaded long-acting lipid nanoformulation: optimization, cellular uptake, pharmacokinetics, biodistribution, and immunohistochemical evaluation.

The aim of the present work involved the development and evaluation of long-acting Isometamidium chloride (ISMM)-Docusate sodium (DS) complex loaded lipid nanoparticles (LA ISMM-DS LNP). The development involved screening various anionic complexing agents, including DS, dextran sulphate, and sodium alginate. Anionic DS was selected to synthesize hydrophobic ionic complex (ISMM-DS HIC), which was loaded into lipid nanoparticles (LA ISMM-DS LNP) by in situ complexation followed by the solvent evaporation method. 35-5-folds increase in the drug loading of hydrophilic cationic ISMM within nanoparticles was observed due to ISMM-DS HIC. The LA ISMM-DS LNP were non-hemolytic (0-2.52%), cytocompatible (80.6-47.5% cell viability), and enhanced THP-1 cellular uptake (2.3-folds higher) compared with free ISMM. The LA ISMM-DS LNP engender protracted in vivo plasma drug concentration for seven days with enhanced AUC0-ꝏ, MRT0-ꝏ, and t1/2, along with reduced Cl compared with free ISMM. Interestingly, the amount of ISMM was 2.9-, 4.2- and 2.0-folds higher in target reticuloendothelial (RES) organs like liver (Kupffer cells), spleen (spleenotropic macrophages and 15% T-lymphocytes), and lymph nodes (75% T-lymphocytes), respectively in LA ISMM-DS LNP group compared with free ISMM. Furthermore, LA ISMM-DS LNP caused higher peripheral blood mononuclear cells (PBMC) infiltration with diminished toxicity and inflammation. Therefore, the in vitro and in vivo studies predicted enhanced safety and efficacy of LA ISMM-DS LNP compared with free ISMM. To conclude, successfully developed LA ISMM-DS LNP would elicit a tremendous clinical potential for treatment and prevention against trypanosomiasis.

Molecular identification of diminazene aceturate-resistant strains of Trypanosoma congolense in naturally infected domestic animals of Yoko in the centre region of Cameroon.

African animal trypanosomiases (AAT) remain the major constraint for livestock production, agriculture and food security in Africa. Although several control measures have been developed to fight AAT, the use of trypanocides remains the main strategy in most affected poor and rural communities. However, several studies have highlighted drug-resistant-trypanosome infections in many African countries, though this phenomenon is still not well described. This study aims to detect trypanosome species and the molecular profiles of drug-resistant-trypanosomes in naturally infected domestic animals of Yoko in the centre region of southern Cameroon. Therefore, in October 2017, 348 animals were blood sampled. The level of packed cell volume (PCV) was evaluated in each animal and trypanosome infections were investigated with the capillary tube centrifugation technique (CTC). Thereafter, DNA was extracted from blood samples and different trypanosome species were identified by PCR. The resistant/sensitive molecular profiles of trypanosomes for diminazene aceturate (DA) and isometamidium chloride (ISM) were investigated by PCR-RFLP. About 18.4% (64/348) of animals analyzed by PCR were found with trypanosome infections including Trypanosoma vivax, Trypanosoma brucei s.l. and Trypanosoma congolense forest and savannah. Trypanosoma congolense savannah was the predominant species with an infection rate of 15.2%. Between villages, significant (p˂0.0001) differences were found in the overall trypanosome infection rates. No molecular profile for ISM resistant-trypanosomes was identified. Conversely, about 88.9% (40/45) of T. congolense positive samples have shown molecular profiles of DA-resistant strains while the remaining 11.1% (5/45) showed mixed molecular profiles of resistant/sensitive strains. Results showed that the molecular profiles of DA-resistant strains of T. congolense in domestic animals of Yoko were widespread. This data needs to be confirmed by testing in vivo the drug susceptibilities of the trypanosome strains herein detected. In conclusion, appropriate future control measures are required. In addition to the intensification of vector control, ISM is advised for the treatment of animals infected by trypanosomes.

Comparison of therapeutic efficacy of different drugs against Trypanosoma vivax on experimentally infected cattle.

In this study, we evaluated the therapeutic efficacy of diminazene diaceturate at a dose of 7 mg/kg (DA), imidocarb dipropionate at 4.8 mg/kg (IMD), isometamidium chloride at 0.5 and 1.0 mg/kg (ISM 0.5 and ISM 1.0) and combinations applied through different methods to treat Trypanosoma vivax in experimentally infected calves. Thirty male Girolando calves were kept indoors and infected intravenously with T. vivax trypomastigotes (approximately 1 × 106). On D-1, the calves were randomized based on the quantity of infecting parasites per animal, yielding six groups of five animals each: G1: positive control group without treatment; G2 animals treated with DA on Day 0 intramuscularly (IM); G3 animals treated with IMD on Day 0 and D + 14 subcutaneously; G4 animals treated with ISM 0.5 on Day 0 IM; G5 animals treated with ISM 1.0 on Day 0 IM; G6 animals received DA on Day 0 and ISM 1.0 on D + 14, both IM. Throughout 180 days, blood samples were collected for the evaluation of T. vivax using the Woo, Brener and PCR methods. The results indicated that the treatment protocols with DA and/or ISM 0.5 and ISM 1.0 had high efficacy (100 %) against T. vivax. Interestingly, cattle that received ISM remained free of parasites until D + 180. In contrast, animals treated with IMD had relapsed T. vivax detected on the 10th and 14th days post-treatment (DPT). Cattle that received ISM 1.0 did not exhibit relapsed T. vivax in the blood, even after reinfection performed on the 50th DPT. However, treatment with DA on Day 0 failed to prevent a new infection of T. vivax on the 50th DPT. The animals that received ISM 1.0 had a transient decrease in packed cell volume similar to that found in the control group. The reappearance of T. vivax in herds in Brazil treated with DA likely occurred due to the short half-life of the drug and not necessarily due to T. vivax resistance to DA.

Genomic analysis of Isometamidium Chloride resistance in Trypanosoma congolense.

Isometamidium Chloride (ISM) is one of the principal drugs used to counteract Trypanosoma congolense infection in livestock, both as a prophylactic as well as a curative treatment. However, numerous cases of ISM resistance have been reported in different African regions, representing a significant constraint in the battle against Animal African Trypanosomiasis. In order to identify genetic signatures associated with ISM resistance in T. congolense, the sensitive strain MSOROM7 was selected for induction of ISM resistance in a murine host. Administered ISM concentrations in immune-suppressed mice were gradually increased from 0.001 mg/kg to 1 mg/kg, the maximal dose used in livestock. As a result, three independent MSOROM7 lines acquired full resistance to this concentration after five months of induction, and retained this full resistant phenotype following a six months period without drug pressure. In contrast, parasites did not acquire ISM resistance in immune-competent animals, even after more than two years under ISM pressure, suggesting that the development of full ISM resistance is strongly enhanced when the host immune response is compromised. Genomic analyses comparing the ISM resistant lines with the parental sensitive line identified shifts in read depth at heterozygous loci in genes coding for different transporters and transmembrane products, and several of these shifts were also found within natural ISM resistant isolates. These findings suggested that the transport and accumulation of ISM inside the resistant parasites may be modified, which was confirmed by flow cytometry and ex vivo ISM uptake assays that showed a decrease in the accumulation of ISM in the resistant parasites.

[Simultaneous determination of diminazene aceturate and isometamidium chloride residues in cattle tissues by high performance liquid chromatography with solid-phase extraction].

A method for the simultaneous determination of trypanocidal diminazene aceturate (DIM) and isometamidium chloride (ISM) that containing benzamidine groups in cattle tissues was developed by high performance liquid chromatography (HPLC) with solid-phase extraction (SPE). The tissue samples were extracted with different proportions of water-acetonitrile, then were cleaned up by Oasis WCX cartridges. DIM and ISM were separated by HPLC with a Spherisorb CN column (250 mm×4.6 mm, 5 µm). Acetonitrile-0.05 mol/L ammonium formate solution (pH 2.4) was used as mobile phases with gradient elution. The detection wavelength of UV was set at 380 nm. The limits of detection (LODs) and the limits of quantification (LOQs) of DIM and ISM in cattle tissues were 0.01 mg/kg and 0.025 mg/kg, respectively. The correlation coefficients (r) of DIM and ISM in cattle tissues were not less than 0.9993. The average recoveries of DIM and ISM at three spiked levels were 82.2%-97.6% with the intra-day relative standard derivations (RSDs) of 0.3%-5.2% (n=5) and inter-day RSDs of 1.3%-5.2% (n=15). The method was successfully applied to the analysis of DIM and ISM in cattle tissues. The method is rapid, sensitive and repeatable for the determination of diminazene aceturate and isometamidium chloride in cattle tissues.