Antiparasitic Agent Ivermectin Research Articles

Overcoming flumatinib resistance in chronic myeloid leukaemia: Insights into cellular mechanisms and ivermectin's therapeutic potential.

Chronic myeloid leukaemia (CML) is a haematological malignancy characterized by the constitutive tyrosine kinase activity of the BCR-ABL1 fusion protein. Flumatinib, a second-generation tyrosine kinase inhibitor, has exhibited superior clinical efficacy compared to its precursor, imatinib. However, with increased clinical use, resistance to flumatinib has emerged as a significant challenge. To investigate the mechanisms of flumatinib resistance in CML, we induced the human CML cell line K562 using a flumatinib concentration gradient method invitro, successfully establishing a flumatinib-resistant K562/FLM cell line. This cell line exhibited cross-resistance to imatinib and doxorubicin, but remained sensitive to the antiparasitic agent ivermectin, which possesses antitumoural effects. Through cellular experimentation, we explored the resistance mechanisms, which indicated that K562/FLM cells evade flumatinib cytotoxicity by enhancing autophagy, increasing the expression of membrane transport proteins, particularly P-glycoprotein, ABCC1 and ABCC4, as well as enhancing phosphorylation of p-EGFR, p-ERK and p-STAT3 proteins. Moreover, it was found that ivermectin effectively suppressed the expression of autophagy and transport proteins in K562/FLM cells, reduced the activity of the aforementioned phosphoproteins, and promoted apoptotic cell death. Collectively, the increased autophagy, higher expression of drug-efflux proteins and hyperactivation of the EGFR/ERK/STAT3 signalling pathway were identified as pivotal elements promoting resistance to flumatinib. The significant effects of ivermectin might offer a novel therapeutic strategy to overcome flumatinib resistance and optimize the treatment outcomes of CML.

Improvement of in vitro dissolution profile of poorly aqueous soluble anti-parasitic agent ivermectin using novel hydrophilic polymeric carriers

Ivermectin (IVM), a BCS Class II drug with weak water solubility, has minimal oral absorption and dissolution. This study aims to enhance the dissolution profile of IVM by performing solid dispersion methods using four hydrophilic polymers: Kollicoat IR, Kollidon 90F, poloxamer 407, and hydroxypropyl methylcellulose (HPMC). The solid dispersion formulations (SDF) were made through physical mixing, solvent evaporation, and melt solvent/fusion. Using three ratios of IVM and hydrophilic carriers (1:1, 1:2, and 1:3), the cumulative release rate of IVM from formulations formed by physical mixing, solvent evaporation, and fusing was much larger than pure IVM (10%). IVM release rates from formulations including Poloxamer 407, Kollicoat IR, and HPMC polymers were 76% (fusion technique), 69% (physical mixing), and 47% (solvent evaporation method). The research demonstrated that fusion optimized drug solubility better than physical mixing and solvent evaporation. The research found that SD of weakly water-soluble IVM with Kollicoat IR/Poloxamer 407 improves its in vitro dissolving profile much better. Bangladesh J. Sci. Ind. Res. 58(4), 209-220, 2023

In-Hospital Receipt of Ivermectin Is Associated With No Benefit in Mortality, Disease Progression, or Length of Stay for Patients With Mild to Moderate COVID-19

Background Use of the antiparasitic agent ivermectin as a potential treatment for early coronavirus disease 2019 (COVID-19) is not recommended, yet inpatient administration continues to be seen. Methods This was a retrospective analysis of more than 100,000 patients admitted to a large health system of community hospitals for COVID-19 to assess the association of ivermectin administration, either early in disease course or at higher severity, with mortality and disease progression (intensive care unit admission or use of mechanical ventilation). Propensity match analysis was used to account for covariates. Results Of the 122,002 patients hospitalized with COVID-19 included in this analysis, 1.1% (1380) received ivermectin while at mild to moderate severity (on room air or nasal cannula), and 0.2% (n = 293) received the drug while severe (noninvasive or mechanical ventilation). Among those who received it with mild to moderate severity, there was no effect on relative risk (RR) of mortality (RR, 1.01; 95% confidence interval [CI], 0.802–1.27), intensive care unit admission (RR, 0.984; 95% CI, 0.865–1.12), or use of mechanical ventilation (RR, 0.976; 95% CI, 0.777–1.23), but an increase in average length of stay (mean days, 8.0 vs 7.5; P < 0.001). Of those who received their first dose of ivermectin with more severe COVID-19, there was an increase in the RR of mortality (RR, 1.63; 95% CI, 1.34–1.99; P < 0.001). Conclusion Among hospitalized patients admitted with mild to moderate COVID-19, in-hospital administration of ivermectin provides no benefit in mortality or disease progression when administered early in the disease course, and may contribute to excess mortality when administered to patients at more severe disease states.

Repurposing Potential of the Antiparasitic Agent Ivermectin for the Treatment and/or Prophylaxis of COVID-19.

Due to the rapid, vast, and emerging global spread of the Coronavirus Disease 2019 (COVID-19) pandemic, many drugs were quickly repurposed in a desperate attempt to unveil a miracle drug. Ivermectin (IVM), an antiparasitic macrocyclic lactone, was tested and confirmed for its in vitro antiviral activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in early 2020. Along with its potential antiviral activity, the affordability and availability of IVM resulted in a wide public interest. Across the world, trials have put IVM to test for both the treatment and prophylaxis of COVID-19, as well as its potential role in combination therapy. Additionally, the targeted delivery of IVM was studied in animals and COVID-19 patients. Through this conducted literature review, the potential value and effectiveness of the repurposed antiparasitic agent in the ongoing global emergency were summarized. The reviewed trials suggested a value of IVM as a treatment in mild COVID-19 cases, though the benefit was not extensive. On the other hand, IVM efficacy as a prophylactic agent was more evident and widely reported. In the most recent trials, novel nasal formulations of IVM were explored with the hope of an improved optimized effect.

Review of the Emerging Evidence Demonstrating the Efficacy of Ivermectin in the Prophylaxis and Treatment of COVID-19.

Background:After COVID-19 emerged on U.S shores, providers began reviewing the emerging basic science, translational, and clinical data to identify potentially effective treatment options. In addition, a multitude of both novel and repurposed therapeutic agents were used empirically and studied within clinical trials.Areas of Uncertainty:The majority of trialed agents have failed to provide reproducible, definitive proof of efficacy in reducing the mortality of COVID-19 with the exception of corticosteroids in moderate to severe disease. Recently, evidence has emerged that the oral antiparasitic agent ivermectin exhibits numerous antiviral and anti-inflammatory mechanisms with trial results reporting significant outcome benefits. Given some have not passed peer review, several expert groups including Unitaid/World Health Organization have undertaken a systematic global effort to contact all active trial investigators to rapidly gather the data needed to grade and perform meta-analyses.Data Sources:Data were sourced from published peer-reviewed studies, manuscripts posted to preprint servers, expert meta-analyses, and numerous epidemiological analyses of regions with ivermectin distribution campaigns.Therapeutic Advances:A large majority of randomized and observational controlled trials of ivermectin are reporting repeated, large magnitude improvements in clinical outcomes. Numerous prophylaxis trials demonstrate that regular ivermectin use leads to large reductions in transmission. Multiple, large “natural experiments” occurred in regions that initiated “ivermectin distribution” campaigns followed by tight, reproducible, temporally associated decreases in case counts and case fatality rates compared with nearby regions without such campaigns.Conclusions:Meta-analyses based on 18 randomized controlled treatment trials of ivermectin in COVID-19 have found large, statistically significant reductions in mortality, time to clinical recovery, and time to viral clearance. Furthermore, results from numerous controlled prophylaxis trials report significantly reduced risks of contracting COVID-19 with the regular use of ivermectin. Finally, the many examples of ivermectin distribution campaigns leading to rapid population-wide decreases in morbidity and mortality indicate that an oral agent effective in all phases of COVID-19 has been identified.

The History of Methylprednisolone, Ascorbic Acid, Thiamine, and Heparin Protocol and I-MASK+ Ivermectin Protocol for COVID-19.

An alliance of established experts on critical care, Front Line COVID-19 Critical Care Alliance (FLCCC), has published two protocols for treatment of COVID-19. The first one, methylprednisolone, ascorbic acid, thiamine, and heparin (MATH+), is intended for hospital and intensive care unit treatment of pulmonary phases of the disease. It is based on affordable, commonly available components: anti-inflammatory corticosteroids (methylprednisolone, "M"), high-dose vitamin C infusion (ascorbic acid, "A"), vitamin B1 (thiamine, "T"), anticoagulant heparin ("H"), antiparasitic agent ivermectin, and supplemental components ("+") including melatonin, vitamin D, elemental zinc, and magnesium. The MATH+ protocol has received scarce attention due to the World Health Organization (WHO) advising against the use of corticosteroids in the beginning of the pandemic. In addition, randomized controlled clinical trials were required as a condition for adoption of the protocol. As the hospital mortality rate of MATH+ treated patients was approximately a quarter of the rate of patients receiving a standard of care, the authors of the protocol considered performing such trials unethical. Other parties have later performed clinical trials with corticosteroids and anticoagulants, which has led to a more widespread adoption of these components.In October 2020, ivermectin was upgraded from an optional component to an essential component of the protocol. According to the authors, ivermectin is considered the first agent effective for both prophylaxis (prevention) of COVID-19 and for treatment of all phases of COVID-19 including outpatient treatment of the early symptomatic phase. Therefore, at the end of October 2020, a separate ivermectin-based I-MASK+ protocol for prophylaxis and early outpatient treatment of COVID-19 was published.

Ivermectin, a therapeutic against RNA viruses and an emerging candidate for COVID-19 patients: A Short Review

A highly contagious Coronavirus disease 2019 (COVID-19) continues to spread across the globe as scientists still search for an efficacious treatment to cure those infected and to curb this pandemic crisis. Despite several clinical trials being underway to test the possible therapies to combat COVID-19, the global response to the outbreak remains profoundly limited to monitoring and containment. In recent clinical trials, the broad-spectrum antiparasitic agent Ivermectin was found to be efficacious against Severe Acute Respiratory Coronavirus 2 (SARS CoV-2); the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), in vitro. The researchers reported Vero/hSLAM cells infected with SARS CoV-2, treated with Ivermectin, showing a 93% to 99% reduction in viral replication within 24-48 hours of administration. Since Ivermectin plays a pivotal role in several biological mechanisms, it may serve as a potential candidate in the treatment of a wide range of viruses including COVID-19 along with other types of positive-sense singlestranded RNA viruses. Previous clinical trials of ivermectin have also exhibited antiviral potential against several RNA viruses, such as Flaviviruses (Zika virus, Chikungunya virus(CHIK-V), Dengue virus(DENV), Yellow Fever Virus(YFV), West Nile Virus(WNV) and others), Human immunodeficiency virus – 1(HIV-1) and Influenza Virus. However, recent pharmacokinetic data pertinent to ivermectin attained from excessive dosing clinical studies demonstrates that the inhibitory concentrations of SARS-CoV-2 are not likely to be attainable in humans. To safeguard patients from these deleterious effects due to excessive ivermectin dosing in COVID-19 patients, it is imperative to conduct a further investigation through clinical trials to appraise the possible antiviral benefits of ivermectin for SARS CoV-2 infection in humans

MATH+ and I-MASK+ Protocols for Prevention and Treatment of All Phases of COVID-19 – A Short Review

An alliance of established experts on critical care, Front Line Covid-19 Critical Care Alliance (FLCCC), has published two protocols for treatment of COVID-19. The first one, MATH+, is intended for hospital and intensive care unit treament of pulmonary phases of the disease. It is based on affordable, commonly available components: anti-inflammatory corticosteroids (methylprednisolone, M), high-dose vitamin C infusion (ascorbic acid, A), vitamin B1 (thiamine, T), anticoagulant heparin (H), antiparasitic agent ivermectin, and supplemental components (+) including melatonin, vitamin D, elemental zinc and magnesium. The MATH+ protocol has received scarce attention due to the World Health Organization advising against the use of corticosteroids in the beginning of the pandemic. In addition, randomized controlled clinical trials were required as a condition for adoption of the protocol. As the hospital mortality rate of MATH+ treated patients was less than a quarter of the rate of patients receiving a standard of care, the authors of the protocol considered performing such trials unethical. Later, other parties have performed clinical trials with e.g. corticosteroids and anticoagulants which has led to their more widespread adoption. Other essential components of the protocol remain unadopted. In October 2020, ivermectin was upgraded from an optional component to an essential component of the protocol. According to the authors, ivermectin is considered the first agent effective for both prophylaxis (prevention) of COVID-19 and for treatment of all phases of COVID-19 including outpatient treatment of the early symptomatic phase. Therefore, at the end of October 2020, a separate ivermectin-based I-MASK+ protocol for prophylaxis and early outpatient treatment of COVID-19 was published.

SILAC quantitative proteomics analysis of ivermectin-related proteomic profiling and molecular network alterations in human ovarian cancer cells.

The antiparasitic agent ivermectin offers more promises to treat a diverse range of diseases. However, a comprehensive proteomic analysis of ivermectin-treated ovarian cancer (OC) cells has not been performed. This study sought to identify ivermectin-related proteomic profiling and molecular network alterations in human OC cells. Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics was used to study the human OC TOV-21G cells. After TOV-21G cells underwent 10 passages in SILAC-labeled growth media, TOV-21G cells were treated with 10 ml of 20 μmol/L ivermectin in cell growing medium for 24 h. The SILAC-labeled proteins were digested with trypsin; tryptic peptides were identified with mass spectrometry (MS). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to mine signaling pathway alterations with ivermectin-related proteins in TOV-21G cells. Gene ontology (GO) analysis was used to explore biological functions of ivermectin-related proteins, including biological processes (BPs), cellular components (CCs), and molecular functions (MFs). The protein-protein interaction network was analyzed with molecular complex detection (MCODE) to identify hub modules. In total, 4,447 proteins were identified in ivermectin-treated TOV-21G cells. KEGG analysis revealed 89 statistically significant signaling pathways. Interestingly, the clustering analysis of these pathways showed that ivermectin was involved in various cancer pathogenesis processes, including modulation of replication, RNA metabolism, and translational machinery. GO analysis revealed 69 statistically significant CCs, 87 MFs, and 62 BPs. Furthermore, MCODE analysis identified five hub modules, including 147 hub molecules. Those hub modules involved ribosomal proteins, RNA-binding proteins, cell-cycle progression-related proteins, proteasome subunits, and minichromosome maintenance proteins. These findings demonstrate that SILAC quantitative proteomics is an effective method to analyze ivermectin-treated cells, provide the first ivermectin-related proteomic profiling and molecular network alterations in human OC cells, and provide deeper insights into molecular mechanisms and functions of ivermectin to inhibit OC cells.

Ivermectin as a potential COVID-19 treatment from the pharmacokinetic point of view: antiviral levels are not likely attainable with known dosing regimens

The broad-spectrum antiparasitic agent ivermectin has been very recently found to inhibit SARS-CoV-2 in vitro and proposed as a candidate for drug repurposing in COVID-19. In the present report the in vitro antiviral activity end-points are analyzed from the pharmacokinetic perspective. The available pharmacokinetic data from clinically relevant and excessive dosing studies indicate that the SARS-CoV-2 inhibitory concentrations are not likely to be attainable in humans.

Abstract 3962: Antitumor effects of the antiparasitic agent ivermectin via inhibition of yes-associated protein 1 expression in gastric cancer

Abstract [Background] Yes-associated protein 1 (YAP1) acts as an oncogene through dephosphorylation and nuclear translocation, and nuclear accumulation of YAP1 is associated with poor prognosis in gastric cancer (GC). We previously identified ivermectin, an antiparasitic drug, as a YAP1 inhibitor. Here, we aimed to clarify whether ivermectin had antitumor effects on GC through inhibition of YAP1. [Result] First, we evaluated the antiproliferative effects of ivermectin on human GC cells using in vitro proliferation assays and a xenograft mouse model. YAP1-knockdown assays were performed to assess whether the sensitivity to ivermectin depended on YAP1 expression. Next, we explored the mechanism through which ivermectin regulated YAP1 expression or localization by immunoblotting and reverse transcription-quantitative polymerase chain reaction for YAP1 and the downstream gene CTGF. Finally, the clinical significance of YAP1 expression was examined using three independent GC datasets. We found that MKN1 GC cells were most sensitive to ivermectin, whereas MKN7 cells were most resistant. In MKN1 xenografts, ivermectin suppressed tumor growth, and the sensitivity of MKN1 cells to ivermectin was decreased by YAP1 knockdown. Ivermectin inhibited YAP1 nuclear expression and CTGF expression in MKN1 cells but not MKN7 cells. Moreover, ivermectin decreased YAP1 mRNA expression, thereby inhibiting nuclear accumulation of YAP1 in MKN1 cells. In survival analysis, low YAP1 mRNA expression was associated with a better prognosis in three independent GC datasets. [Conclusion] Ivermectin suppressed the growth of GC in vitro and in vivo by inhibiting YAP1 expression. Furthermore, GC with low YAP1 expression had favorable clinicopathological features and good prognosis. These findings provided insights into the antiproliferative effects of ivermectin as a YAP1 inhibitor and established a theoretical basis for preclinical evaluations of ivermectin for management of GC. Citation Format: Shotaro Kuramitsu, Sho Nanbara, Qingjiang Hu, Yousuke Kuroda, Takaai Masuda, Shuhei Ito, Hidetoshi Eguchi, Yoshihiko Mehara, Koshi Mimori. Antitumor effects of the antiparasitic agent ivermectin via inhibition of yes-associated protein 1 expression in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3962.

Antitumor effects of the antiparasitic agent ivermectin via inhibition of Yes-associated protein 1 expression in gastric cancer.

Yes-associated protein 1 (YAP1) acts as an oncogene through dephosphorylation and nuclear translocation, and nuclear accumulation of YAP1 is associated with poor prognosis in gastric cancer (GC). We previously identified ivermectin, an antiparasitic drug, as a YAP1 inhibitor. Here, we aimed to clarify whether ivermectin had antitumor effects on GC through inhibition of YAP1. First, we evaluated the antiproliferative effects of ivermectin on human GC cells using in vitro proliferation assays and a xenograft mouse model. YAP1-knockdown assays were performed to assess whether the sensitivity to ivermectin depended on YAP1 expression. Next, we explored the mechanism through which ivermectin regulated YAP1 expression or localization by immunoblotting and reverse transcription-quantitative polymerase chain reaction for YAP1 and the downstream gene CTGF. Finally, the clinical significance of YAP1 expression was examined using three independent GC datasets. We found that MKN1 GC cells were most sensitive to ivermectin, whereas MKN7 cells were most resistant. In MKN1 xenografts, ivermectin suppressed tumor growth, and the sensitivity of MKN1 cells to ivermectin was decreased by YAP1 knockdown. Ivermectin inhibited YAP1 nuclear expression and CTGF expression in MKN1 cells but not MKN7 cells. Moreover, ivermectin decreased YAP1 mRNA expression, thereby inhibiting nuclear accumulation of YAP1 in MKN1 cells. In survival analysis, low YAP1 mRNA expression was associated with a better prognosis in three independent GC datasets. In conclusion, we identified ivermectin as a potential antitumor agent and found a promising novel therapeutic strategy for inhibition of GC progression by blocking YAP1 expression.

Antibiotic ivermectin preferentially targets renal cancer through inducing mitochondrial dysfunction and oxidative damage

Renal cell carcinoma (RCC) is the most aggressive type of genitourinary cancer and highly resistant to current available therapies. In this work, we investigated the effects and mechanism of anti-parasitic agent ivermectin in RCC. We show that ivermectin significantly inhibits proliferation and induces apoptosis in multiple RCC cell lines that represent different histological subtypes and various mutation status. Importantly, ivermectin is significantly less or ineffective in normal kidney cells compared with RCC cells, demonstrating the preferential toxicity of ivermectin to RCC. Ivermectin also significantly inhibits RCC tumor growth in vivo. Mechanistically, ivermectin induces mitochondrial dysfunction via decreasing mitochondrial membrane potential, mitochondrial respiration and ATP production. As a consequence of mitochondrial dysfunction, oxidative stress and damage is detected in ivermectin treated RCC cells and xenograft mouse model. The rescue of ivermectin's effect by acetyl-l-Carnitine (ALCAR, a mitochondrial fuel) or antioxidant N-acetyl-l-cysteine (NAC) confirms mitochondria as the target of ivermectin in RCC cells. Compared to normal kidney cells, RCC cells have higher mitochondrial mass and respiration, and ATP production, which might explain the preferential toxicity of ivermectin to RCC. Our work suggest that ivermectin is a promising candidate for RCC treatment and targeting mitochondrial metabolism is an alternative therapeutic strategy for RCC.

Automated thin-film microextraction coupled to a flow-through cell: somewhere in between passive and active sampling.

A prototype for the automated thin-film microextraction of pharmaceuticals from aqueous solutions has been developed and is presented here for the first time. With a software-controlled setup, extraction methods for ivermectin and iohexol have been developed. The widely used antiparasitic agent ivermectin is non-polar and has a high tendency to sorb to surfaces. In contrast to this, the nonionic but polar iodinated X-ray contrast agent iohexol is freely water soluble. With these two substances, a wide range of polarity is covered. Sorption kinetics and thermodynamics of ivermectin and iohexol were studied. With the presented passive sampling approach, it was possible to extract up to 96.2% ivermectin with a C18-phase within 1h and up to 74.6% of iohexol with a PS-DVB phase within 36h out of water. Using abamectin as internal standard, it was possible to quantitatively follow dissipation of ivermectin in a simulated surface water experiment. Predominantly, the newly developed prototype can be used for automated and time-resolved extraction of xenobiotics from waterbodies under field conditions, for the extraction of substances under laboratory conditions as an alternative to the elaborate solid-phase extraction, and for the automated control of chemical reaction kinetics.

Analysis and dissipation of the antiparasitic agent ivermectin in cattle dung under different field conditions.

Cattle treated with the veterinary parasiticide ivermectin fecally excrete residues. The authors report the exposition and dissipation characteristics of these residues in dung of ivermectin-treated cattle and in soil beneath this dung on pastures in Canada, France, Switzerland, and The Netherlands. Residues were quantified for dung collected from cattle after 3 d, 7 d, 14 d, and 28 d posttreatment and subsequently exposed in the field for up to 13 mo. The authors optimized a high-performance liquid chromatography-fluorescence detection method to detect ivermectin residues in dung and soil matrices. They showed that a solid phase extraction and purification step generally can be eliminated to reduce the time and cost of these analyses. They also found that the addition of water to relatively dry samples improves the extraction efficiency of residues. They then analyzed the field samples to document differences in ivermectin dissipation in cattle dung among sites, with 50% dissipation times of up to 32 d and 90% dissipation times >396 d. They further showed that the dissipation characteristics of residues are comparable between dung of ivermectin-treated cattle and dung to which ivermectin has been added directly. Lastly, they report the first use of a desorption electrospray ionization-high-resolution-mass spectrometric method to detect residues of metabolites in a dung matrix. Environ Toxicol Chem 2016;35:1924-1933. © 2016 SETAC.

Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death.

Overexpression of P2X7 receptors correlates with tumor growth and metastasis. Yet, release of ATP is associated with immunogenic cancer cell death as well as inflammatory responses caused by necrotic cell death at sites of trauma or ischemia-reperfusion injury. Using an FDA-approved anti-parasitic agent Ivermectin as a prototype agent to allosterically modulate P2X4 receptors, we can switch the balance between the dual pro-survival and cytotoxic functions of purinergic signaling in breast cancer cells. This is mediated through augmented opening of the P2X4/P2X7-gated Pannexin-1 channels that drives a mixed apoptotic and necrotic mode of cell death associated with activation of caspase-1 and is consistent with pyroptosis. We show that cancer cell death is dependent on ATP release and death signals downstream of P2X7 receptors that can be reversed by inhibition of NADPH oxidases-generated ROS, Ca2+/Calmodulin-dependent protein kinase II (CaMKII) or mitochondrial permeability transition pore (MPTP). Ivermectin induces autophagy and release of ATP and HMGB1, key mediators of inflammation. Potentiated P2X4/P2X7 signaling can be further linked to the ATP rich tumor microenvironment providing a mechanistic explanation for the tumor selectivity of purinergic receptors modulation and its potential to be used as a platform for integrated cancer immunotherapy.

The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells

AbstractTo identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

Activation of Chloride Channels with the Anti-Parasitic Agent Ivermectin Induces Membrane Hyperpolarization and Cell Death in Leukemia Cells.

Abstract 410FDA-approved drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. To identify such compounds, we compiled a library of known drugs with high maximal tolerated doses and well-known toxicity profiles. We screened this library in a dose-response manner in 4 leukemia cell lines to identify cytotoxic compounds as measured by the MTS assay. From these screens, we identified the anti-parasitic agent ivermectin (IVM) that induced cell death at low micromolar concentrations in all four leukemia lines tested. IVM is a derivative of avermecin B1 and licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis). To evaluate the effects of IVM as a potential anti-cancer agent, leukemia and myeloma (n = 9) cell lines were treated with increasing concentrations of IVM. 72 hours after incubation, cell viability was determined by the MTS assay. IVM decreased the viability of 3/5 leukemia cell lines with an LD50 < 5uM and all other tested malignant cell lines with an LD50 < 10uM. Cell death was confirmed by Trypan blue staining and Annexin V staining. In clonogenic growth assays, 6uM IVM reduced clonogenic growth by ≥ 40% in 3/6 primary AML samples, but <15% in 3/3 samples of normal hematopoietic mononuclear cells. Given the effects of IVM in cell lines, we evaluated the drug in mouse models of leukemia. Here, K562, OCI-AML2, and MDAY-D2 leukemia cells were implanted subcutaneously into the flanks of sublethally irradiated NOD/SCID mice. One week after implantation, when the tumors were palpable, mice were treated with IVM at 3mg/kg/day by oral gavage or buffer control. Compared to control, IVM decreased tumor volume and weight in all 3 xenograft models by up to 72% without observable toxicity. Of note, a dose of 3mg/kg in mice translates to a dose of 0.24 mg/kg in humans based on scaling of body weight and surface area. This dose is readily achievable in human as patients routinely receive 0.2mg/kg for the treatment of parasitic disease and overdoses of up to 6g were not toxic. As an anti-parasitic, IVM binds and activates chloride ion channels, so we tested the effects of IVM on chloride flux in leukemia cells. OCI AML2 cells were treated with increasing concentrations of IVM and changes in intracellular chloride were measured using the fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium and flow cytometry. IVM increased intracellular chloride ion concentrations within 30 minutes of treatment. Chloride influx was accompanied by plasma membrane hyperpolarization within 1 hr of treatment, but no change in mitochondrial membrane potential was noted up to 24 hours after treatment. Plasma membrane hyperpolarization was dependent on chloride influx, as treatment with IVM in chloride-free media did not induce membrane hyperpolarization. Alternations in intracellular chloride and membrane hyperpolarization can lead to increased reactive oxygen species (ROS) generation. Therefore, we measured changes in ROS after treatment with IVM. IVM (6uM) increased ROS generation in OCI-AML2 cells up to 2 ± 0.2 fold within 4 hours of treatment. Increased ROS generation appeared functionally important for IVM-induced cell death as pre-treatment with the antioxidant N-acetyl-L-cysteine (NAC) inhibited IVM-induced cell death. Further supporting a mechanism of cell death related to increased ROS, IVM treatment increased expression of STAT1, IFIT3, OAS1, and TRIM22, members of the STAT1 signaling pathway that are known to be upregulated upon ROS generation. Cytarabine and daunorubicin are used in the treatment of AML and increase ROS production through mechanisms related to DNA damage. Therefore, we evaluated the combination of IVM and cytarabine and daunorubicin. By isobologram analysis, IVM synergized with cyatarabine (CI=0.51, 0.58, 0.65 at ED25, ED50, ED75, respectively) and daunorubicin (CI=0.48, 0.51, 0.54 at ED25, ED50, ED75, respectively). Thus, in summary, IVM activates chloride channels in leukemia cells leading to membrane hyperpolarization and increased ROS generation. In addition, it demonstrated preclinical activity in this disease at pharmacologically achievable concentrations. Therefore, IVM could be rapidly repurposed for the treatment of leukemia and highlights a potential new therapeutic strategy for this disease. Disclosures:Off Label Use: Ivermectin is an antiparasitic agent, licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis).

Ivermectin potentiates ATP-induced ion currents in cortical neurones: Evidence for functional expression of P2X4 receptors?

We examined the effects of an antiparasitic agent ivermectin on ATP- and alpha,beta-methylene-ATP (alpha,beta-me-ATP)-activated currents in neurones acutely isolated from slices of somato-sensory cortex of 17-22 days old CBL57 mice. Membrane currents were monitored using whole-cell patch clamp combined with a "concentration-clamp" fast agonist application system. Ivermectin potentiated membrane currents induced by both ATP and alpha,beta-me-ATP applied alone or in the presence of broad P2X antagonist PPADS. Ivermectin also significantly increased amplitude and frequency of spontaneous P2X-mediated EPSCs recorded from cortical slices incubated with mixture of glutamate (CNQX, D-APV, SYM2081), GABA (picrotoxin), nicotinic cholinoreceptor (hexamethonium) antagonists and sodium channel blocker tetrodotoxin. These results indicate functional expression of P2X4 receptors in cortical neurones and their involvement in purinergic synaptic transmission in cortex.

Determination of ivermectin in bovine liver by optical immunobiosensor

A rapid and sensitive biosensor immunoassay was developed for residues of the antiparasitic agent ivermectin in bovine liver. A detection limit of 19.1 ng g −1 was achieved. The sensor employed was a Biacore optical instrument based on surface plasmon resonance. 5- O-succinoylivermectin-apo-transferrin conjugate was used to produce monoclonal antibody while a second derivative, ivermectin-oxime, was immobilised onto the surface of a sensor chip. A range of assay parameters (flow rate, injection time, temperature) and extraction techniques were investigated. In the final assay procedure, ivermectin was extracted with acetonitrile followed by C 8 SPE clean-up. Matrix effect was minimised by increasing the flow rate to 25 μl min −1 and reducing the sample injection time to 2 min. The average value for liver samples spiked at 100 ng g −1 (the MRL for the drug) and 50 ng g −1 concentrations were 93.7 and 43.2 ng g −1, respectively.