Quantitative Analysis of Abamectin, Albendazole, Levamisole HCl and Closantel in Q-DRENCH Oral Suspension Using a Stability-Indicating HPLC-DAD Method.

Combination therapy of many anthelmintic drugs has been used to achieve fast animal curing. Q-DRENCH is an oral suspension, containing four different active drugs against GIT worms in sheep, commonly used in Australia and New Zeeland. The anti-parasitic drugs are Albendazole (ALB), Levamisole HCl (LEV), Abamectin (ABA), and Closantel (CLO). The main purpose of this study is to present a new simultaneous stability-indicting HPLC-DAD method for the analysis of the four drugs. The recommended liquid system was 1 mL of Triethylamine/L water, adjusting the pH to 3.5 by glacial acetic acid: acetonitrile solvent (20:80, v/v). Isocratic elusion achieved the desired results of separation at a 2 mL/min flow rate using Zorbax C-18 as a stationary phase. Detection was performed at 210 nm. The linearity ranges were 15.15 to 93.75 µg/mL for ALB, 25 to 150 µg/mL for LEV, 30 to 150 µg/mL for ABA, and 11.7 to 140.63 µg/mL for CLO. Moreover, the final greenness score was 0.62 using the AGREE tool, which reflects the eco-friendly nature. Moreover, the four drugs were determined successfully in the presence of their stressful degradation products. This work presents the first chromatographic method for simultaneous analysis for Q-DRENCH oral suspension drugs in the presence of their stressful degradation products.

Salicylanilide Analog Minimizes Relapse of Clostridioides difficile Infection in Mice.

Clostridioides difficile infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and pseudomembranous colitis. Although antibiotics for CDI exist, they are either expensive or cause recurrence of the infection due to their altering the colonic microbiota, which is necessary to suppress the infection. Here, we leverage a class of known membrane-targeting compounds that we previously showed to have broad inhibitory activity across multiple Clostridioides difficile strains while preserving the microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized, and the most potent analog was selected through an in vitro inhibitory assay to evaluate its pharmacokinetic parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and diminished disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way for novel therapy that can potentially target the cell membrane of C. difficile to minimize relapse in the recovering patient.

Understanding the main route of drug entry in adult Fasciola hepatica: Further insights into closantel pharmacological activity.

Closantel (CLS) is highly effective against adult liver flukes after its oral or subcutaneous (sc) administration in ruminants. Trans-tegumental diffusion and oral ingestion are the two potential routes available for the entry of drugs into Fasciola hepatica. The work reported here contributes to improve the understanding of CLS pharmacology. The main goals of were: I) to determine the pattern of in vivo CLS accumulation into adult F. hepatica and relevant tissues in CLS-treated sheep; II) to investigate the influence of the physicochemical composition of the incubation medium on the CLS diffusion process into adult F. hepatica; III) to assess the ovicidal activity of CLS against F. hepatica eggs; and IV) to investigate the in vivo effect of CLS treatment on glutathione S-transferases activity in adult liver flukes exposed to CLS. Fourteen healthy sheep were each orally infected with 75 F. hepatica metacercariae. Sixteen (16) weeks after infection, animals were treated with CLS by oral (n = 6, 10 mg/kg) or sub-cutaneous (sc) (n = 6, 5 mg/kg) route. At 12, 24 and 36 h post-treatment, animals were sacrificed (n = 2) and samples of blood, bile and adult F. hepatica were collected. In addition, flukes recovered from non-treated sheep (n = 2) were ex vivo incubated (60 min) in the presence of CLS in either RPMI or bile as incubation medium. CLS concentration was measured by HPLC. The ovicidal activity of CLS was investigated using eggs obtained from the bile of untreated sheep. Finally, glutathione S-transferase activity in F. hepatica recovered from untreated and CLS-treated sheep was assessed. In the in vivo studies, the highest CLS concentrations were measured in plasma and adult liver flukes. A positive correlation was observed between CLS concentration in plasma and in F. hepatica. Results obtained in the current work indicate that the in vivo accumulation of CLS into adult liver flukes occurs mainly by the oral route. After ex vivo incubation, the uptake of CLS by the parasite was markedly diminished in the presence of bile compared with that observed in the presence of RPMI as incubation medium. CLS lacks ovicidal activity at therapeutically relevant concentrations. Lastly, CLS significantly increased glutathione S-transferase activity in flukes recovered at 12 h (oral treatment) and 24 h (sc treatment), compared to the control liver flukes.

Host pharmacokinetics and drug accumulation of anthelmintics within target helminth parasites of ruminants.

Anthelmintic drugs require effective concentrations to be attained at the site of parasite location for a certain period to assure their efficacy. The processes of absorption, distribution, metabolism and excretion (pharmacokinetic phase) directly influence drug concentrations attained at the site of action and the resultant pharmacological effect. The aim of the current review article was to provide an overview of the relationship between the pharmacokinetic features of different anthelmintic drugs, their availability in host tissues, accumulation within target helminths and resulting therapeutic efficacy. It focuses on the anthelmintics used in cattle and sheep for which published information on the overall topic is available; benzimidazoles, macrocyclic lactones and monepantel. Physicochemical properties, such as water solubility and dissolution rate, determine the ability of anthelmintic compounds to accumulate in the target parasites and consequently final clinical efficacy. The transcuticular absorption process is the main route of penetration for different drugs in nematodes and cestodes. However, oral ingestion is a main route of drug entry into adult liver flukes. Among other factors, the route of administration may substantially affect the pharmacokinetic behaviour of anthelmintic molecules and modify their efficacy. Oral administration improves drug efficacy against nematodes located in the gastroinestinal tract especially if parasites have a reduced susceptibility. Partitioning of the drug between gastrointestinal contents, mucosal tissue and the target parasite is important to enhance the drug exposure of the nematodes located in the lumen of the abomasum and/or small intestine. On the other hand, large inter-animal variability in drug exposure and subsequent high variability in efficacy is observed after topical administration of anthelmintic compounds. As it has been extensively demonstrated under experimental and field conditions, understanding pharmacokinetic behaviour and identification of different factors affecting drug activity is important for achieving optimal parasite control and avoiding selection for drug resistance. The search for novel alternatives to deliver enhanced drug concentrations within target helminth parasites may contribute to avoiding misuse, and prolong the lifespan of existing and novel anthelmintic compounds in the veterinary pharmaceutical market.

In vitro biological evaluation of new antimycobacterial salicylanilide-tuftsin conjugates.

Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen that can survive in host cells, mainly in macrophages. An increase of multidrug-resistant tuberculosis qualifies this infectious disease as a major public health problem worldwide. The cellular uptake of the antimycobacterial agents by infected host cells is limited. Our approach is to enhance the cellular uptake of the antituberculars by target cell-directed delivery using drug-peptide conjugates to achieve an increased intracellular efficacy. In this study, salicylanilide derivatives (2-hydroxy-N-phenylbenzamides) with remarkable antimycobacterial activity were conjugated to macrophage receptor specific tuftsin based peptide carriers through oxime bond directly or by insertion of a GFLG tetrapeptide spacer. We have found that the in vitro antimycobacterial activity of the salicylanilides against M. tuberculosis H37Rv is preserved in the conjugates. While the free drug was ineffective on infected macrophage model, the conjugates were active against the intracellular bacteria. The fluorescently labelled peptide carriers that were modified with different fatty acid side chains showed outstanding cellular uptake rate to the macrophage model cells. The conjugation of the salicylanilides to tuftsin based carriers reduced or abolished the in vitro cytostatic activity of the free drugs with the exception of the palmitoylated conjugates. The conjugates degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked salicylanilide-amino acid fragment as the smallest active metabolite.

Scavenging of highly reactive gamma-ketoaldehydes attenuates cognitive dysfunction associated with epileptogenesis.

Cognitive dysfunction is a major comorbidity of the epilepsies; however, treatments targeting seizure-associated cognitive dysfunction, particularly deficits in learning and memory are not available. Isoketals and neuroketals, collectively known as gamma-ketoaldehydes are formed via the non-enzymatic, free radical catalyzed oxidation of arachidonic acid and docosahexaenoic acid, respectively. They are attractive candidates for oxidative protein damage and resultant cognitive dysfunction due to their formation within the plasma membrane and their high proclivity to form cytotoxic adducts on protein lysine residues. We tested the hypothesis that gamma-ketoaldehydes mechanistically contribute to seizure-associated memory impairment using a specific gamma-ketoaldehyde scavenger, salicylamine in the kainic acid and pilocarpine rat models of temporal lobe epilepsy. We show that gamma-ketoaldehydes are increased following epileptogenic injury in hippocampus and perirhinal cortex, two brain regions imperative for learning and memory. Treatment with an orally bioavailable, brain permeable scavenger, salicylamine attenuated 1) spatial memory deficits 2) reference memory deficits and 3) neuronal loss and astrogliosis in two mechanistically distinct models of epilepsy without affecting the epileptogenic injury or the development of chronic epilepsy. We have previously demonstrated that reactive oxygen species and the lipid peroxidation biomarkers, F2-isoprostanes are produced following status epilepticus. However, which reactive species specifically mediate oxidative damage to cellular macromolecules remains at large. We provide novel data suggesting that memory impairment occurs via gamma-ketoaldehyde production in two models of epilepsy and that treatment with a gamma-ketoaldehyde scavenger can protect vulnerable neurons. This work suggests a novel target and therapy to treat seizure-induced memory deficits in epilepsy.

Repositioning organohalogen drugs: a case study for identification of potent B-Raf V600E inhibitors via docking and bioassay.

Drug repositioning has been attracting increasingly attention for its advantages of reducing costs and risks. Statistics showed that around one quarter of the marketed drugs are organohalogens. However, no study has been reported, to the best of our knowledge, to aim at efficiently repositioning organohalogen drugs, which may be attributed to the lack of accurate halogen bonding scoring function. Here, we present a study to show that two organohalogen drugs were successfully repositioned as potent B-Raf V600E inhibitors via molecular docking with halogen bonding scoring function, namely D(3)DOCKxb developed in our lab, and bioassay. After virtual screening by D(3)DOCKxb against the database CMC (Comprehensive Medicinal Chemistry), 3 organohalogen drugs that were predicted to form strong halogen bonding with B-Raf V600E were purchased and tested with ELISA-based assay. In the end, 2 of them, rafoxanide and closantel, were identified as potent inhibitors with IC50 values of 0.07 µM and 1.90 µM, respectively, which are comparable to that of vemurafenib (IC50: 0.17 µM), a marketed drug targeting B-Raf V600E. Single point mutagenesis experiments confirmed the conformations predicted by D(3)DOCKxb. And comparison experiment revealed that halogen bonding scoring function is essential for repositioning those drugs with heavy halogen atoms in their molecular structures.

Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta.

Closantel (CLS) is currently used in programs for the strategic control of gastrointestinal nematodes. CLS is extralabel used in different dairy goat production systems. From available data in dairy cows, it can be concluded that residues of CLS persist in milk. The current work evaluated the concentration profiles of CLS in plasma and milk from lactating orally treated dairy goats to assess the residues pattern in dairy products such as cheese and ricotta. Six (6) female Saanen dairy goats were treated orally with CLS administered at 10 mg/kg. Blood and milk samples were collected between 0 and 36 days post-treatment. The whole milk production was collected at 1, 4, 7, and 10 days post-treatment to produce soft cheese and ricotta. CLS concentrations in plasma, milk, cheese, whey, and ricotta were determined by HPLC. The concentrations of CLS measured in plasma were higher than those measured in milk at all sampling times. However, the calculated withdrawal time for CLS in milk was between 39 and 43 days postadministration to dairy goats. CLS residual concentrations in cheese (between 0.93 and 1.8 µg/g) were higher than those measured in the milk used for its production. CLS concentrations in ricotta were sixfold higher than those in the milk and 20-fold higher than those in the whey used for its production. The persistent and high residual concentrations of CLS in the milk and in the cheese and ricotta should be seriously considered before issuing any recommendation on the extralabel use of CLS in dairy goat farms.

Pharmacokinetics of a novel closantel/ivermectin injection in cattle.

Two studies are described on the pharmacokinetics of a combination anthelmintic consisting of ivermectin and closantel for use in cattle. In the first, the pharmacokinetics of both active drugs in the combination were compared with the formulation with either ivermectin or closantel removed. No differences in the pharmacokinetics were observed, indicating that neither the absorption nor distribution of ivermectin or closantel in the combination were influenced by the presence of the other. In the second study the pharmacokinetics of ivermectin and closantel in the combination product were compared with control formulations of each. No difference was found between the closantel formulations. With ivermectin it was noted that absorption and excretion were more rapid and Cmax higher in the combination, although the AUC of both formulations were not significantly different.

[Concentration in plasma and excretion in milk of lactating cows after oral administration of tribromsalan, oxyclozanide and bromofenofos].

The fasciolicides tribromsalan (TBS), oxyclozanide (OCZ) and bromofenofos (BFF) were orally administered to three lactating cows. The concentrations of TBS, OCZ and the BFF metabolite dephosphate bromofenofos (DBFF) in plasma, and the excretion of these compounds in milk were determined by high-performance liquid chromatography. In plasma, the concentrations of TBS, OCZ and DBFF reached maximum at about 1.0 day and the compounds remained detectable until 5.7, 7.4 and 15.1 days after administration, respectively. The detection limits of these compounds in plasma were 10, 2 and 2 ppb, respectively. In milk, the concentrations of TBS, OCZ and DBFF reached maximum at about 24 hours and the compounds remained detectable until 30-47, 30-47 and 78-119 hours after administration, respectively. The detection limits of these compounds in milk were 5.1 and 1 ppb, respectively. The residence times of TBS and BFF were very close to the withdrawal times of the fasciolicides.

Stability and partitioning of closantel and rafoxanide in ruminal fluid of sheep.

The stability and the partitioning of closantel and rafoxanide in ruminal fluid (RF) was examined in vitro. Stability was evaluated in two studies in a ruminal fluid-artificial saliva (RF-AS) mixture containing either drug. Drug concentrations were measured in samples collected sequentially from four batches of RF-AS fortified with either closantel or rafoxanide in one study and in four separately incubated aliquots of a RF-AS mixture of each drug in the second study at the start and at various intervals during a 24 h incubation period. The viability of the in vitro RF-AS incubation model was validated by the presence of digoxin degradation (T1/2 of 39,1 +/- 13 h) and by the absence of significant time related differences (P> 0,5) in volume of gas produced, pH and methylene blue reduction time of the RF-AS drug mixture. Partitioning of closantel and rafoxanide was determined by measuring the relative drug concentration of the fluid and particulate phases in RF fortified with either drug at different concentrations. Closantel and rafoxanide were shown to be stable in a RF-AS mixture and were not subjected to any significant biodegradation. An initial marked reduction in drug concentration measured in the RF-AS mixture during the first 2 h of incubation was attributed to the attachment of both drugs onto particulate matter. This was subsequently confirmed in the partitioning study. More than 80% of closantel and rafoxanide was shown to be associated with the particulate phase of RF

Quantitative determination of Closantel residues in milk by high-performance liquid chromatography with fluorescence detection.

A HPLC method with fluorescence detection for quantitative determination of Closantel residues in milk has been developed and validated. The proposed cleaning procedure with acetonitrile and acetone extraction, and solid-phase clean-up with Florisil enables concentrations of Closantel below 50 micrograms/l to be determined. The method was shown to be sufficient, precise, accurate, selective and rugged. The method was applied in the regular monitoring of Closantel residues in milk and of the pharmacokinetic behavior of Closantel in sheep.

The pharmacology of halogenated salicylanilides and their anthelmintic use in animals.

The halogenated salicylanilides are a large group of compounds developed mainly for their antiparasitic activity in animals. Several halogenated salicylanilides with potent antiparasitic activity have been synthesised of which only closantel, niclosamide, oxyclozanide, rafoxanide and resorantel are commercially available. Closantel and rafoxanide, which represent the most important drugs in the group, are used extensively for the control of Haemonchus spp. and Fasciola spp. infestations in sheep and cattle and Oestrus ovis in sheep in many parts of the world. Niclosamide is used extensively for its anticestodal activity in a wide range of animals. Antiparasitic activity of the halogenated salicylanilides has also been demonstrated against a large number of other internal parasites, in particular haematophagous helminths, and external parasites including ticks and mites, in a variety of animal species. Several cases of toxicity and mortality have been reported for closantel and rafoxanide in sheep and goats. Their unique pharmacokinetic behaviour appears to play an important role in the efficacy and safety of these compounds. The chemical and physical characteristics, mode of action, pharmacokinetics, antiparasitic activity and toxicity of the halogenated salicylanilides in animals are reviewed.

Intravascular plasma disposition and salivary secretion of closantel and rafoxanide in sheep.

The plasma and salivary disposition of closantel and rafoxanide were examined following intravenous administration in adult sheep. Two studies were conducted with rafoxanide at 7.5 mg/kg and 1 with closantel using 2 doses (5 and 15 mg/kg). The pharmacokinetic profile of both drugs in plasma were best described by a 2-compartmental model with 1st-order rate constants. Plasma disposition of closantel and rafoxanide were characterised by a rapid distribution (t1/2(alpha)) of <30 min), long elimination half-life (t1/2(beta)) of 17.0 +/- 4.0 days for closantel and 7.2 +/- 0.6 days for rafoxanide), small apparent volume of distribution (V(SS) of <0.15 l/kg) and a slow rate of total body clearance (Cl of <0.01 ml/min/kg). The area under the drug plasma concentration curve (AUC) of closantel at 5 mg/kg was nearly twice as large as that of rafoxanide at 7.5 mg/kg resulting from the slower t1/2(beta) observed with closantel compared to rafoxanide. Large individual differences were observed in the rate measurements of distribution (k12, k21 and t1/2(alpha)), whereas the parameters of elimination (k10, t1/2(beta) and Cl), were more consistent between animals. A dose proportional increase in AUC was observed for closantel administered at 5 and 15 mg/kg. A low, constant salivary concentration of closantel (mean of 0.04 +/- 0.05 microg/mL) and rafoxanide (mean of 0.07 +/- 0.04 microg/mL) was observed during the 24-h examination period after dosing.

The effect of feed intake level on the pharmacokinetic disposition of closantel in sheep.

Closantel (CLS), containing a trace of [14C]CLS, was administered intraruminally to sheep whose feed intake was maintained at either 800 or 400 g day-1. The kinetic disposition of [14C]metabolites was determined in rumen and abomasal fluid and particulate digesta and of CLS per se in plasma. The slower digesta flow rate in the sheep on low, compared with high, feed intake resulted in the proportion of the dose passing through the abomasum being reduced from 60 to 45%. Increased absorption of CLS from the rumen of sheep on low feed intake resulted in both higher maximum CLS concentration and greater area under CLS plasma concentration versus time curve, although the elimination half-life was independent of feed intake. Not only are the higher plasma CLS concentrations likely to increase efficacy against Haemonchus contortus, the threshold concentrations that are considered to inhibit the establishment of ingested H. contortus larvae were extended by 10-14 days. The extended CLS presence after reduced feeding, when integrated with parasite treatment programmes, provides an opportunity to reduce the impact of H. contortus infection.

Haemonchus contortus: the uptake and metabolism of closantel.

Closantel is an anthelmintic which associates with plasma albumin and is useful for the control of sheep parasites, such as Haemonchus contortus, that ingest blood. However, the utility of closantel for parasite control has been threatened by the emergence of resistance. The mechanisms of resistance are unknown. A closantel-resistant and a closantel-susceptible isolate of H. contortus were compared with respect to the distribution and metabolism of closantel. Neither strain appeared to metabolise closantel in vitro or in vivo. Following treatment of infected sheep with radioactively labelled closantel, isotope levels in closantel-resistant adult H. contortus were significantly lower than in susceptible worms. This reduced accumulation of drug could contribute to closantel resistance by mechanisms such as reduced feeding, failure to dissociate the drug-albumin complex in the gut or increased efflux of closantel from resistant worms.

Comparative pharmacokinetic disposition of closantel in sheep and goats.

The pharmacokinetic disposition of closantel was examined following intraruminal (i.r.) or intramuscular (i.m.) administration to adult Merino sheep and to adult and 3-month-old, suckling Angora goats. In adult goats the maximum concentration (Cmax) and area under the plasma concentration with time curve (AUC) following 3.75, 7.5 and 15.0 mg closantel/kg given i.r. increased with dose however the time of Cmax (Tmax = 2.6d) in plasma was unaffected by dose rate. The elimination phase (K10) of closantel was monoexponential with a half-life (t1/2) of 4.7d again unaffected by dose rate. Apart from a more rapid absorption phase and earlier Tmax following 3.75 mg closantel/kg i.m., pharmacokinetic behaviour was similar to that following i.r. administration at 3.75 or 7.5 mg/kg. Although absorption rate was more rapid in kids after i.r. administration at 7.5 mg/kg, pharmacokinetic disposition of closantel was otherwise similar to that in adult goats. No closantel was detected in milk of treated doses or in the plasma of their kids. I.R. closantel at 7.5 mg/kg was more slowly absorbed in goats than in sheep but Cmax was similar in both species. However, K10 t1/2 was significantly shorter in goats (4d) than in sheep (14d). Faster elimination resulted in an almost three-fold lowering of AUC in goats and could dramatically reduce the sustained action of closantel in this species compared with sheep.

Steady-state and transient membrane potentials in human red cells determined by protonophore-mediated pH changes.

Protonophores have been used frequently to determine changes in membrane potential in suspensions of red cells, since such changes are reflected by changes in extracellular pH, due to proton and consequently protonophore reequilibration. In a previous paper (Bennekou, P. 1988, J. Membrane Biol. 102:225-234) a kinetic model for the translocation of a protonophore, CCCP, across the human red cell membrane was established. This model accounts for the protonophore reequilibration following abrupt changes in membrane potential. In this paper the limitations of the method with regard to the estimation of transient membrane potentials are examined, using the transport model to simulate changes in extracellular pH in response to noninstantaneous changes in membrane potential. The temperature and time resolution calculated from the model are reported. Furthermore, it is shown that the transport model established for CCCP is valid for another protonophore, TCS, thus indicating the general validity of the transport scheme for the entire class of protonophores.

Flukicidal action of closantel against immature and mature Fasciola hepatica in experimentally infected rats and sheep.

The relative importance of peak level- and residual level-related flukicidal activity of closantel against immature and mature Fasciola hepatica was evaluated in a comparative efficacy trial using two animal species with a different plasma elimination pattern, that is, the rat and the sheep with an elimination half-life of less than one week and of two to three weeks, respectively. The rats were dosed orally with closantel at 20 mg kg-1 at two, four, six, eight and 10 weeks; the sheep at 10 mg kg-1 at eight, 10 and 12 weeks after artificial infection. Necropsy was performed either one week after treatment or 12 weeks after infection. Efficacy rates and the length of the recovered flukes were evaluated. It was demonstrated that the flukicidal effect of closantel is directly related to its peak plasma levels and less to its residual plasma concentrations. In the rat, a high efficacy (P less than 0.001) could be demonstrated against immature stages of four weeks or older. The two-week immature stages were less markedly affected. No significant differences in efficacy and size of the flukes were noted between the animals autopsied one week after treatment and those autopsied 12 weeks after infection. In the sheep, the efficacy against six-week and eight-week-old immature stages varied between 70.3 and 76.8 per cent and between 92.8 and 96.5 per cent, respectively. As in the rats, no marked differences in efficacy were noted between the animals autopsied one week after treatment and those autopsied 12 weeks after infection.(ABSTRACT TRUNCATED AT 250 WORDS)