Effect of the Combination of Synthetic Anthelmintics with Carvacryl Acetate in Emulsions with and without a Sodium Alginate Matrix on Haemonchus contortus.

The present study aimed to evaluate the effect of nanoemulsions using combined synthetic anthelmintics, thiabendazole (TBZ), levamisole (LEV), and ivermectin (IVM), with carvacryl acetate (CA) against Haemonchus contortus, and also tested the presence and absence of alginate (ALG). The anthelmintic effect of the CA/TBZ nanoemulsion was evaluated in the egg hatch test (EHT). The effects of CA/IVM and CA/LEV nanoemulsions were evaluated in the larval development test (LDT). The emulsions CA/TBZ/ALG and CA/TBZ showed a multimodal profile, with most particles on the nanometric scale. The encapsulation efficiency in CA/TBZ/ALG was 80.25%, and that in CA/LEV/ALG was 89.73%. In the EHT, CA/TBZ and CA/TBZ/ALG showed mean combination indices (CIs) of 0.55 and 0.36, respectively, demonstrating synergism in both. In LDT, CA/IVM had an average CI of 0.75, and CA/LEV and CA/LEV/ALG showed CI values of 0.4 and 0.93, respectively. It was concluded that CA/TBZ showed a synergistic interaction, and CA/TBZ/ALG showed an enhanced effect. In addition, the matrix brought stability to the product, encouraging its improvement to obtain higher efficacy.

Antineutrophil cytoplasmic antibody in children with nephrotic syndrome treated with levamisole: a cross-sectional cohort study.

BACKGROUND: Levamisole is a commonly used steroid-sparing agent (SSA), but the reported incidence of antineutrophil cytoplasmic antibody (ANCA) positivity has been concerning. METHODS: Observational cross-sectional study wherein children aged 2 to 18 years with frequently relapsing/steroid dependent nephrotic syndrome (FRNS/SDNS) on levamisole for ≥ 12 months were tested for ANCA. RESULTS: A total of 210 children (33% female), median age of 7.3 (IQR: 5.6-9.6) years, and a median duration of levamisole exposure of 21 (IQR: 15-30) months were tested. ANCA was positive in 18% (n = 37): 89% (n = 33) perinuclear ANCA (pANCA), 3% (n = 1) cytoplasmic ANCA (cANCA), and 8% (n = 3) both. Of ANCA-positive children, none had reduced eGFR or abnormal urinalysis. The majority of these children were asymptomatic (81%, n = 30). Rash was more common among ANCA-positive children [6/37 (16%) vs. 3/173 (2%), p = 0.0001]. On multivariate analysis, higher age (OR = 1.02, [95th CI: 1.01 to 1.03], p = 0.007) and longer duration of levamisole exposure (OR = 1.05, [95th CI: 1.02 to 1.08], p = 0.0007) were associated with ANCA positivity. Levamisole was stopped in ANCA-positive children with the resolution of any clinical manifestations if present. Repeat ANCA testing was performed in 54% (20/37), and all were ANCA negative by 18 months. CONCLUSIONS: Children with FRNS/SDNS on longer duration of levamisole were associated with increasing prevalence of ANCA positivity, but most of these children were clinically asymptomatic. Prospective studies are required to determine the chronology of ANCA positivity and its clinical implication.

Granulomatosis with polyangiitis or its mimic? A case report.

Differentiation between granulomatosis with polyangiitis (GPA) limited to the upper airways and cocaine-induced midline destructive lesion (CIMDL) may be particularly difficult because of their common histopathologic features and antineutrophil cytoplasmic antibody (ANCA) profiles. We herein present a case involving a young woman with an initial diagnosis of GPA based on upper and lower airway manifestations and constitutional symptoms, histopathologic evidence of granulomas, a positive cytoplasmic ANCA indirect immunofluorescent test result, and proteinase 3 positivity by enzyme-linked immunosorbent assay (ELISA). CIMDL was confirmed based on the appearance of a hard palate perforation, positivity for methylecgonine on urine toxicology, a positive perinuclear ANCA indirect immunofluorescent test result, and subsequent human neutrophil elastase (HNE) ANCA positivity by ELISA. Finally, based on the coexistence of CIMDL, constitutional symptoms, and lower airway manifestations, the diagnosis was modified to cocaine-induced GPA mimic. Urine toxicology for cocaine and HNE ELISA are indicated in young patients with GPA who develop limited airway disease to check for the presence of CIMDL and cocaine-/levamisole-induced ANCA-associated vasculitis. Continued abstinence from cocaine is the first-choice therapy for both CIMDL and cocaine-induced GPA mimic.

Cocaine-Induced Toxic Leukoencephalopathy: A Case Report.

We present a case here where a 57-year-old South Asian male with disturbed mental status developed multifocal leukoencephalopathy, which we believe was caused by cocaine usage. Cocaine was detected in the urine toxicological sample. Non-acute CT head, with a follow-up brain MRI demonstrating hyperintensity of the T2 FLAIR signal corresponding to diffusion restriction throughout the whole supratentorial white matter, involving semiovale and subcortical U fibres in the occipital lobes as well as posterior frontal and parietal centrum. It was less likely that the patient had posterior reversible encephalopathy syndrome (PRES), which can potentially manifest similarly in a clinical and imaging context because there was no abrupt rise of blood pressure at presentation or during the patient's stay. Extensive examinations were conducted to exclude additional factors that may contribute to the patient's appearance, including autoimmune, vasculitis, and infectious diseases. Levamisole, a significant chemical that is frequently used to increase the volume of cocaine samples and has been linked to neuronal damage, should be examined in individuals who use cocaine and exhibit these kinds of clinical symptoms. The patient was prescribed 250 mg of methylprednisolone twice daily for five days after it was determined that cocaine-induced neuronal toxicity was the cause of his symptoms. Although no improvement was seen right away, over the course of the next few days, he did exhibit a gradual, albeit slight, improvement in his mental status while residing in the nursing home. It is crucial to comprehend the possible connection between cocaine usage, a commonly abused drug, and people exhibiting similar clinical symptoms. To have a better understanding of the pathophysiology and possible treatment approach, more research is necessary as there is now no recommended therapy regimen.

An open label non-inferiority randomized controlled trial evaluated alternate day prednisolone given daily during infections vs. levamisole in frequently relapsing nephrotic syndrome.

Initial therapies for children with frequently relapsing nephrotic syndrome include alternate-day prednisolone that is given daily during infections, or levamisole. In this open label, non-inferiority trial, 160 patients, 2 to 18-years-old with frequent relapses, were randomly assigned to receive either prednisolone (0.5-0.7 mg/kg/alternate-day, given daily during infections), or levamisole (2-2.5 mg/kg/alternate-days) for one-year. Patients with relapses on alternate day prednisolone at over 1 mg/kg, prior use of potent steroid-sparing therapies, eGFR under 60 ml/min/1.73 m2 and significant steroid toxicity were excluded. Primary outcome was the proportion of patients with frequent relapses, defined as three-relapses in one-year, or two-relapses within six-months if associated with significant steroid toxicity or loss to follow up. Eighty patients each were randomized to receive prednisolone and levamisole. Baseline features showed preponderance of young patients presenting within two-years of disease onset. On intention-to-treat analysis, frequent relapses were more common in patients administered prednisolone (40% versus 22.5%; risk difference 17.5%; 95% confidence interval 3.4-31.6%). Prednisolone was not non-inferior to levamisole in preventing frequent relapses. However, the two groups showed similar proportions of patients in sustained remission, comparable frequency of relapses, and low frequency of adverse events. The decline in steroid requirement from baseline was higher in the levamisole group. Per-protocol analysis showed similar results. These results have implications for choice of therapy for frequently relapsing nephrotic syndrome. Although therapy with alternate-day prednisolone was not non-inferior to levamisole in preventing frequent relapses, both therapies were effective in other outcome measures. Thus, levamisole was relatively steroid-sparing and may be preferred in patients at risk of steroid toxicity.

Concurrent use of two dual-combination drenches containing monepantel/abamectin and oxfendazole/levamisole in sheep: effect on marker residues 21 and 28 days after administration.

AIMS: To determine the concentration, in comparison with the maximum residue limit (MRL), of anthelmintic marker residues in the target tissues (liver and fat) of sheep treated concurrently with two oral drenches, one containing monepantel and abamectin and the other oxfendazole and levamisole. METHODS: On day 0 of the study, 12 sheep (six male and six female; 8-9-months old) were dosed according to individual body weight determined the day prior. Zolvix Plus (dual-active oral drench containing 25 g/L monepantel and 2 g/L abamectin) was administered to all animals prior to administration of Scanda (dual-active oral drench containing 80 g/L levamisole hydrochloride and 45.3 g/L oxfendazole). Six sheep (three male and three female) were slaughtered 21 and 28 days after treatment and renal fat and liver samples were collected.Using validated methods, analyses for monepantel sulfone, abamectin, levamisole and oxfendazole (expressed as total fenbendazole sulfone following conversion of the combined concentrations of oxfendazole, fenbendazole and fenbendazole sulfone) were performed on liver samples while renal fat specimens were analysed for monepantel sulfone and abamectin residues only. Detected concentrations were compared to the established MRL in sheep for each analyte determined by the Ministry for Primary Industries. RESULTS: All residues detected in samples of liver and fat collected 21 and 28 days after treatment were below the MRL for each analyte. All liver samples collected on day 21 had detectable monepantel sulfone (mean 232 (min 110, max 388) µg/kg) and oxfendazole (mean 98.7 (min 51.3, max 165) µg/kg) residues below the MRL (5,000 and 500 µg/kg, respectively). Monepantel sulfone (mean 644 (min 242, max 1,119) µg/kg; MRL 7,000 µg/kg) residues were detected in 6/6 renal fat samples. Levamisole residues were detected in 3/6 livers (mean 40.0 (min 14.3, max 78.3) µg/kg; MRL 100 µg/kg), and abamectin residues in 1/6 livers (0.795 µg/kg; MRL 25 µg/kg) and 2/6 fat samples, (mean 0.987 (min 0.514, max 1.46) µg/kg; MRL 50 µg/kg) 21 days after treatment. CONCLUSION AND CLINICAL RELEVANCE: These results suggest that concurrent administration of Zolvix Plus and Scanda to sheep is unlikely to result in an extended residue profile for any of the active ingredients, with all analytes measured being under the approved New Zealand MRL 21 days after treatment. This work was not completed in line with guidance for establishing official residue profiles, nor is it sufficient to propose a new withholding period.

Functional validation of novel levamisole resistance marker S168T in Haemonchus contortus.

Recently, a S168T variant in the acetylcholine receptor subunit ACR-8 was associated with levamisole resistance in the parasitic helminth Haemonchus contortus. Here, we used the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiology to measure the functional impact of this S168T variant on the H. contortus levamisole-sensitive acetylcholine receptor, L-AChR-1.1. Expression of the ACR-8 S168T variant significantly reduced the current amplitude elicited by levamisole compared to acetylcholine, with levamisole changing from a full to partial agonist on the recombinant L-AChR. Functional validation of the S168T mutation on modulating levamisole activity at the receptor level highlights its critical importance as both a mechanism and a marker of levamisole resistance.

The proof is in the poo-ding: Benefits of the longitudinal molecular surveillance of drug resistance demonstrated in a New South Wales cattle herd.

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019-2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes.

Toxicity and Toxicokinetics of a Four-Week Repeated Gavage of Levamisole in Male Beagle Dogs: A Good Laboratory Practice Study.

Levamisole (LVM) is considered an immunomodulatory agent that has the potential to treat various cancer and inflammation diseases. However, there is still much debate surrounding the toxicokinetic and toxicological information of LVM. Therefore, it is crucial to assess its toxicity to provide useful data for future human LVM risk assessments. In this study, a barrier environment was established under the guidance of good laboratory practice (GLP) at the Fujian Center for New Drug Safety Evaluation. Male beagle dogs were orally administered with 5, 15, and 30 mg/kg of LVM daily for four weeks. Toxicity assessment was based on various factors such as mortality, clinical signs, food and water consumption, body weight, body temperature, electrocardiogram, ophthalmological examination, hematology, serum biochemistry, organ/body coefficients, histopathological study, and toxicokinetic analysis. The results of this study showed that LVM did not exhibit any significant toxicological effects on beagle dogs at the exposure levels tested. A no observed adverse effect level (NOAEL) of LVM was set at 30 mg/kg/day for male beagle dogs, which is equivalent to a 12-fold clinical dose in humans. Moreover, the repeated exposure to LVM for four weeks did not lead to any bioaccumulation. These findings provide valuable insights for future human LVM risk assessments.

Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

Efficacy of levamisole, ivermectin and moxidectin against Capillaria spp. in European hedgehogs (Erinaceus europaeus).

This randomised study aimed to assess and compare the efficacy of treatment protocols containing levamisole, ivermectin, or moxidectin against Capillaria spp. in naturally infected European hedgehogs (Erinaceus europaeus) presented to a British wildlife rehabilitation centre. Faecal analysis, consisting of wet mount and flotation, was performed for 229 hedgehogs weighing ≥200g. Animals testing positive for Capillaria spp. (81%), excluding pregnant females, were randomly allocated a treatment protocol. Initially, hedgehogs (n = 50) received one of six 'pilot' protocols, whereas the remaining animals (n = 97) received one of three 'main' protocols. Faecal analysis was repeated on day 8 and day 12 after treatment initiation. Efficacy of each treatment was assessed based on Capillaria reduction rate (CRR), weight gain, presence of respiratory clinical signs, and outcome. Pilot protocols containing only moxidectin had a significantly lower CRR (≥28.1%) compared to those with levamisole or ivermectin (≥86.6%), whereas the main protocols containing levamisole had a significantly higher CRR (≥93.0%) compared to those containing only ivermectin (≥69.3%). Clinical parameters did not differ significantly between treatments, but animals with respiratory clinical signs at the end of the trial were significantly more likely to have lower CRR and test positive for Crenosoma striatum. C. striatum often appeared refractory to treatment, and managing these infections requires additional anthelmintic therapy. Based on the formulations and dosages trialled, moxidectin is not recommended for treating capillariosis in European hedgehogs, whereas levamisole given orally for two consecutive days at 25-35 mg/kg is suggested as the treatment of choice.

The role of ANCA in the management of cocaine-induced midline destructive lesions or ENT pseudo-granulomatosis with polyangiitis: a London multicentre case series.

OBJECTIVE: In this multicentric study involving three London hospitals, we compared ANCA-positive and ANCA-negative cocaine-induced midline destructive lesions (CIMDL) patients to assess how presence of antineutrophil cytoplasmic antibodies (ANCA) may correlate with disease severity. Our secondary aims are to better classify etiology centered around ANCA positivity and, consequently, better disease management. METHODS: A retrospective review was performed to identify patients with CIMDL seen between January 2019 and December 2022. Population data including age, sex, presentation, endoscopic findings, duration of cocaine use and active use of cocaine, type of treatment, laboratory (including ANCA serology), radiological, and histological findings were collected. RESULTS: Forty CIMDL patients (25 male, median age of 42 years) were identified. The majority of them (72.5%) presented with either a septal perforation, a saddle nose deformity (22.5%), and/or a palatal fistula (20.0%). ANCA was positive in 71.1% of cases (66.7% p-ANCA). No statistically significant differences in the general characteristics, type of treatment, laboratory results, radiological or histological findings were observed when comparing ANCA-positive and ANCA-negative CIMDL patients or when comparing p-ANCA and c-ANCA patients. Similarly, no statistically significant difference was obtained when comparing the pattern of distribution of lesions between the two groups. CONCLUSIONS: A large percentage of CIMDL patients showed positive ANCA test (71.1%) and in the majority of the cases a p-ANCA pattern specifically targeting PR3 (p-ANCA, PR3 + MPO-). However, ANCA positivity or presence of a specific ANCA pattern was not associated with more severe presentation or more aggressive disease. Given its similarities to granulomatosis with polyangiitis (GPA), we recommend the use of the term "cocaine-induced ENT pseudo-GPA" instead of CIMDL. LEVEL OF EVIDENCE: IV Laryngoscope, 2023.

Multidrug resistance in Haemonchus contortus in sheep - can it be overcome?

Introduction: Gastrointestinal nematodes pose a threat to animal health and affect farmers by negatively impacting farm management. Material and Methods: The study was conducted on a sheep farm with suspected reductions in the efficacies of anthelmintics. Efficacy was determined using in vivo faecal egg count reduction, in vitro egg hatch (EHT) and larval development (LDT) tests. In the first phase, 60 sheep were equally split into six groups. Group 1 received the recommended dose of albendazole (ALB), group 2 received the same after fasting for 24 h, group 3 received the dose divided into two halves at 6 h intervals, group 4 received a double dose of ALB, and group 5 received the recommended dose of ivermectin (IVM). Group 6 served as a control. The second phase of the experiment had two groups: one treated with levamisole (LEV) and a control group. Faecal samples were collected from all sheep. Results: No reduction of egg output was observed in the groups treated with single, double, or divided doses of ALB, but one of 13.7-16.9% was noted in the fasting group. Efficacy in the IVM group ranged from 31.50 to 39.97%. The mean concentrations sufficient to prevent 50% of the eggs from hatching in the in vitro EHT and the mean concentrations in which the development of larvae to the L3 stage was inhibited by 50% in the LDT exceeded established thresholds for benzimidazoles and IVM. Haemonchus contortus was the only species identified after treatment. The LDT did not indicate the presence of resistance to LEV. All animals treated with LEV were negative for eggs 10 d after treatment. Conclusion: Resistance to ALB and IVM in Haemonchus contortus was confirmed. Alternative approaches to improve the efficacies of benzimidazole did not sufficiently increase the efficacy, but LEV was an efficient anthelmintic treatment.

The pipeline for drugs for control and elimination of neglected tropical diseases: 2. Oral anti-infective drugs and drug combinations for off-label use.

In its 'Road map for neglected tropical diseases 2021-2030', the World Health Organization outlined its targets for control and elimination of neglected tropical diseases (NTDs) and research needed to achieve them. For many NTDs, this includes research for new treatment options for case management and/or preventive chemotherapy. Our review of small-molecule anti-infective drugs recently approved by a stringent regulatory authority (SRA) or in at least Phase 2 clinical development for regulatory approval showed that this pipeline cannot deliver all new treatments needed. WHO guidelines and country policies show that drugs may be recommended for control and elimination for NTDs for which they are not SRA approved (i.e. for 'off-label' use) if efficacy and safety data for the relevant NTD are considered sufficient by WHO and country authorities. Here, we are providing an overview of clinical research in the past 10 years evaluating the anti-infective efficacy of oral small-molecule drugs for NTD(s) for which they are neither SRA approved, nor included in current WHO strategies nor, considering the research sponsors, likely to be registered with a SRA for that NTD, if found to be effective and safe. No such research has been done for yaws, guinea worm, Trypanosoma brucei gambiense human African trypanosomiasis (HAT), rabies, trachoma, visceral leishmaniasis, mycetoma, T. b. rhodesiense HAT, echinococcosis, taeniasis/cysticercosis or scabies. Oral drugs evaluated include sparfloxacin and acedapsone for leprosy; rifampicin, rifapentin and moxifloxacin for onchocerciasis; imatinib and levamisole for loiasis; itraconazole, fluconazole, ketoconazole, posaconazole, ravuconazole and disulfiram for Chagas disease, doxycycline and rifampicin for lymphatic filariasis; arterolane, piperaquine, artesunate, artemether, lumefantrine and mefloquine for schistosomiasis; ivermectin, tribendimidine, pyrantel, oxantel and nitazoxanide for soil-transmitted helminths including strongyloidiasis; chloroquine, ivermectin, balapiravir, ribavirin, celgosivir, UV-4B, ivermectin and doxycycline for dengue; streptomycin, amoxicillin, clavulanate for Buruli ulcer; fluconazole and isavuconazonium for mycoses; clarithromycin and dapsone for cutaneous leishmaniasis; and tribendimidine, albendazole, mebendazole and nitazoxanide for foodborne trematodiasis. Additional paths to identification of new treatment options are needed. One promising path is exploitation of the worldwide experience with 'off-label' treatment of diseases with insufficient treatment options as pursued by the 'CURE ID' initiative.

In vivo anthelmintic activity of Eleusine indica extracts against gastrointestinal nematodes of goats.

Objective: The study aimed to determine the potential anthelmintic activity of the ethyl acetate extract of Eleusine indica that will result in an effective reduction in fecal egg per gram (EPG) counts in naturally infected goats compared to the commercial anthelmintic levamisole. Materials and Methods: The experimental animals were 21 goats naturally infected with gastrointestinal nematodes. The goats were divided into groups that were given a single dose of E. indica extract. Five concentrations of E. indica were tested for anthelmintic activity: 100, 200, 300, 400, and 500 mg extract/kg body weight. Fecal sample collection was done before treatment, during the first treatment, and every week thereafter for 28 days post-treatment (dpt). A modified McMaster technique was used to determine the EPG of feces, and the mean efficacies of the extracts were compared with those of the commercial anthelmintic levamisole. Results: As early as 7 dpt, there was an observed reduction in the epg counts after the administration of E. indica extracts across all concentrations. Administering 500 mg of extract/kg body weight resulted in a maximum efficacy of 56.21%. However, the efficacy achieved was lower than that of levamisole (96.83%). Conclusion: The results show that the E. indica extract can reduce the fecal EPG counts of naturally infected goats, thus creating a potential natural anthelmintic that can be developed further.

Reproductive and margin of safety of a fixed-dose combination injectable endectocide (0.2 mg/kg doramectin; 6.0 mg/kg levamisole hydrochloride) in cattle.

We present a fixed-dose combination injectable (FDCI) solution for cattle formulated for a single subcutaneous administration at a dose rate of 1 ml/25 kg of body weight to deliver a dose of 0.2 mg/kg of doramectin and 6.0 mg/kg of levamisole hydrochloride (5.1 mg/kg base equivalent). This drug product is marketed in the United States under the tradename Valcor® and in Australia and New Zealand under the tradename Dectomax V®. Both levamisole and doramectin have histories of safe and effective use in ruminants, with safety margins of 3X and 25X, respectively. Three studies were conducted to demonstrate the safety of the new FDCI: margin of safety (Study 1), and reproductive safety in sexually nulliparous beef heifers (Studies 2 and 3). In Study 1, 3-month-old sexually intact male and female calves were given either saline (control) or 1X, 2X, or 3X FDCI on Days 0, 14, and 28. General health, clinical, and neurological observations were made throughout the study, and clinical and pathology evaluations were made at study end. Studies 2 and 3 demonstrated the reproductive safety of the FDCI on sexually nulliparous beef heifers using estrus synchronization and timed artificial insemination. Treatments of either saline (control) or 3X FDCI were administered to coincide with either folliculogenesis, implantation, organogenesis, early gestation, or late gestation. Reproductive safety was demonstrated by evaluating rates of conception, calving, abortion, and stillbirth, dystocia scores, and calf health. In all studies, the FDCI at 1X, 2X, or 3X dosages was well tolerated. In the margin of safety study, 3X calves showed increased incidence of salivation for up to 8 h post-dosing compared to other groups. Injection sites were palpable post-dosing in all three FDCI groups but resolved by Day 28 in all but one animal each in 2X and 3X. In the reproductive safety studies, the FDCI had no effect on conception, pregnancy, fetal development, or postnatal viability. Injection site swelling was increased in frequency and duration compared to controls. The studies demonstrate the safety of the new FDCI in cattle from 3 months of age and in reproducing heifers during all reproductive stages from folliculogenesis through gestation and up to a month post-partum.

Levamisole Modulation of Podocytes' Actin Cytoskeleton in Nephrotic Syndrome.

Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.

Simultaneous analysis of the of levamisole with triclabendazole in pharmaceuticals through developing TLC and HPLC-PDA chromatographic techniques and their greenness assessment using GAPI and AGREE methods.

Two simple and rapid chromatographic methods were developed and validated for the analysis of levamisole and triclabendazole simultaneously in pure and pharmaceutical products. The first method is thin-layer chromatography (TLC) with densitometry, and the second method is high-performance liquid chromatography with PDA detection (HPLC-PDA). A Hypersil BDS C18 column with dimensions of 4.6 × 150 mm and a particle size of 5 µm was used in the HPLC-PDA method. An isocratic condition was used to carry out the separation, and the mobile phase was made up of acetonitrile and a 0.03 M potassium dihydrogen phosphate buffer in double-distilled water. The ratio of the mobile phase preparation was 70:30 (v/v), and the flow rate was 1 mL/min. A wavelength of 215 nm was employed for analyte detection. Precoated silica gel 60 F254 aluminium plates were used for the TLC method's separation. Mobile phase was made of ethyl acetate, hexane, methanol, and ammonia (69:15:15:1) for the separation. The detection wavelength selected was 215 nm. According to the International Council for Harmonization (ICH) guidelines, the proposed methods were validated and it was found that the two chromatographic methods are accurate, precise, and linear for both compounds in the range of 3.75-37.5 and 6-60 mg/L for the HPLC method for levamisole and triclabendazole, respectively and in the range of 2-14 µg/spot for the TLC method. The developed methods greenness profile was assessed using AGREE and ComplexGAPI tools.

Dose confirmation of a novel fixed dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) against naturally acquired gastrointestinal nematodes in US cattle.

Macrocyclic lactone (ML) resistance in cattle gastrointestinal nematodes (GINs) is an increasing problem. Concurrent combination anthelmintic therapy incorporating an existing ML with a second drug class has been proposed to control cattle GINs while slowing the development of ML resistance. Two dose confirmation studies were conducted to investigate the efficacy of a new fixed-dose combination injectable (FDCI) anthelmintic against common cattle GINs known to negatively impact production. The FDCI is formulated with 5 mg/ml doramectin and 150 mg/ml levamisole hydrochloride (HCl). Cattle enrolled in the two studies were sourced from either the Southern (Study 1, n = 30) or Midwest (Study 2, n = 36) United States. Animals with GIN infections confirmed by fecal egg count (FEC) were randomly allocated to one of three treatment groups. On Day 0, cattle with positive FECs on Day -5( ± 2) were weighed and administered a single subcutaneous injection of either saline (0.9% sodium chloride) at 0.04 ml/kg, 10 mg/ml doramectin at 0.02 ml/kg (to provide 0.2 mg/kg doramectin) or the FDCI at 0.04 ml/kg (to provide 0.2 mg/kg doramectin and 6.0 mg/kg levamisole HCl). On Day 14, fecal samples were collected, animals were euthanized, and worms were collected from the intestinal tract of each animal. Treatment efficacy was calculated using worm burdens and the fecal egg count reduction test (FECRT). Pre-treatment (Day -5, Study 1; Day -3, Study 2) mean FECs were 999.4-1136.2 eggs per gram (EPG) in Study 1 and 137.1-226.6 EPG in Study 2. The FDCI was active against cattle GIN populations in both studies, with FECRT ≥ 99.98% in both studies. Compared to saline-treated cattle, FDCI-treated cattle had significantly fewer adult and immature worms of all identified species on Day 14. In Study 1, Day 14 efficacy of the FDCI was 96.9% for Cooperia spp. (C. oncophora (99.7%) and C. punctata (95.9%)), 99.1% for Nematodirus helvetianus, and 99.8% for Ostertagia spp. In Study 2, the FDCI provided 100% efficacy against all adult GIN species identified, including all GINs identified in Study 1 and Trichostrongylus axei. The FDCI also provided 95.5% efficacy against immature Ostertagia spp. and 100% efficacy against immature Cooperia spp. (Study 2). Doramectin was effective against all adult cattle GINs (except N. helvetianus) in Study 2 but was only effective against adult Ostertagia spp. in Study 1. Additionally, doramectin was only effective against immature Cooperia spp. (and not immature Ostertagia spp.) in Study 2. A single administration of the doramectin + levamisole HCl FDCI provides a new and effective approach to the treatment and control of common cattle GINs, including those exhibiting decreased susceptibility to doramectin alone.

Exploring the Efficacy and Safety of Levamisole Hydrochloride against Microcotyle sebastis in Korean Rockfish (Sebastes schlegelii): An In Vitro and In Vivo Approach.

Parasitic infections pose significant challenges in aquaculture, and the increasing resistance to conventional anthelmintics necessitates the exploration of alternative treatments. Levamisole hydrochloride (HCl) has demonstrated efficacy against monogenean infections in various fish species; however, research focused on Microcotyle sebastis infections in Korean rockfish (Sebastes schlegelii) remains limited. Therefore, this study aimed to evaluate the efficacy of levamisole HCl against M. sebastis infections in Korean rockfish with the goal of optimizing anthelmintic usage in aquaculture. In this study, we first assessed the susceptibility of M. sebastis to levamisole HCl in vitro. Subsequently, in vivo evaluations were conducted to assess the drug's efficacy, safety, and to identify optimal administration methods. In vitro experiments revealed concentration-dependent sensitivity of M. sebastis to levamisole HCl, with a minimum effective concentration (MEC) of 100 mg/L. In vivo experiments employed oral administration, intraperitoneal injection, and immersion treatments based on the MEC. Oral administration proved to be a safe method, yielding efficacy rates of 27.3% and 41.6% for 100 mg/kg and 200 mg/kg doses, respectively, in contrast to the immersion and injection methods, which induced symptoms of abnormal swimming, vomiting, and death. Biochemical analyses conducted to assess the safety of levamisole HCl revealed a transient, statistically significant elevation in the levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) on day three post-administration at 20 °C. Following this, no substantial differences were observed. However, at 13 °C, the enzyme levels remained relatively consistent, emphasizing the role of water temperature conditions in influencing the action of levamisole HCl. Our research findings substantiate the efficacy of levamisole HCl against M. sebastis in Korean rockfish, underscoring its potential for safe oral administration. These results provide valuable insights for developing parasite control strategies involving levamisole HCl in Korean rockfish populations while minimizing adverse impacts on fish health and the environment. However, this study bears limitations due to its controlled setting and narrow focus. Future research should expand on these findings by testing levamisole HCl in diverse environments, exploring different administration protocols, and examining wider temperature ranges.