Comparison of solubility profiles for pioneer and generic monensin premixes in biorelevant simulated intestinal fluid based on shake flask extractions.

In the United States, a generic Type A medicated article product can gain the FDA approval by demonstrating bioequivalence (BE) to the pioneer product by successfully conducting a blood level, pharmacodynamic, or clinical BE study. A biowaiver can be granted based on several criteria, assuming the dissolution of the test and reference products represents the only factor influencing the relative bioavailability of both products. Monensin is practically insoluble in H2O per the USP definition. Previously published data from a comparison study of monensin dissolution profiles from the pioneer product and four generic products using biorelevant media showed that generic monensin products demonstrated different dissolution profiles to the pioneer product in these USP biorelevant rumen media. This follow-up study compared the solubility profiles in simulated intestinal fluid (cFaSSIF, pH 7.5) for the pioneer product and four generic products. The generic monensin products demonstrated different in vitro dissolution profiles to the pioneer product in biorelevant media. The differences demonstrated in solubility and dissolution profiles are of concern regarding the potential efficacy of generic monensin in cattle. There are also additional concerns for the potential development of Eimeria resistance in cattle receiving a sub-therapeutic dose of monensin from a less soluble generic product.

Novel Cerium(IV) Coordination Compounds of Monensin and Salinomycin.

The largely uncharted complexation chemistry of the veterinary polyether ionophores, monensic and salinomycinic acids (HL) with metal ions of type M4+ and the known antiproliferative potential of antibiotics has provoked our interest in exploring the coordination processes between MonH/SalH and ions of Ce4+. (1) Methods: Novel monensinate and salinomycinate cerium(IV)-based complexes were synthesized and structurally characterized by elemental analysis, a plethora of physicochemical methods, density functional theory, molecular dynamics, and biological assays. (2) Results: The formation of coordination species of a general composition [CeL2(OH)2] and [CeL(NO3)2(OH)], depending on reaction conditions, was proven both experimentally and theoretically. The metal(IV) complexes [CeL(NO3)2(OH)] possess promising cytotoxic activity against the human tumor uterine cervix (HeLa) cell line, being highly selective (non-tumor embryo Lep-3 vs. HeLa) compared to cisplatin, oxaliplatin, and epirubicin.

Polyether Cyclization Cascade Alterations in Response to Monensin Polyketide Synthase Mutations.

The targeted manipulation of polyketide synthases has in recent years led to numerous new-to-nature polyketides. For type I polyketide synthases the response of post-polyketide synthases (PKS) processing enzymes onto the most frequently polyketide backbone manipulations is so far insufficiently studied. In particular, complex processes such as the polyether cyclisation in the biosynthesis of ionophores such as monensin pose interesting objects of research. We present here a study of the substrate promiscuity of the polyether cyclisation cascade enzymes in monensin biosynthesis in the conversion of redox derivatives of the nascent polyketide chain. LC-HRMS/MS2 -based studies revealed a remarkable flexibility of the post-PKS enzymes. They acted on derivatized polyketide backbones based on the three possible polyketide redox states within two different modules and gave rise to an altered polyether structure. One of these monensin derivatives was isolated and characterized by 2D-NMR spectroscopy, crystallography, and bioactivity studies.

Synthesis and evaluation of antibacterial and trypanocidal activity of derivatives of monensin A.

The synthesis and biological evaluation of eleven derivatives of the natural polyether ionophore monensin A (MON), modified at the C-26 position, is presented. Eight urethane and three ester derivatives were tested for their antimicrobial activity against different strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. In addition, their antiparasitic activity was also evaluated with bloodstream forms of Trypanosoma brucei. The majority of the modified ionophores were active against a variety of Gram-positive bacterial strains, including methicillin-resistant S. epidermidis, and showed better antibacterial activity than the unmodified MON. The phenyl urethane derivative of MON exhibited the most promising antibacterial activity of all tested compounds, with minimal inhibitory concentration values of 0.25-0.50 µg/ml. In contrast, none of the MON derivatives displayed higher antitrypanosomal activity than the unmodified ionophore.

Housefly larvae (Musca domestica) significantly accelerates degradation of monensin by altering the structure and abundance of the associated bacterial community.

Vermicomposting of livestock manure using housefly larvae is a promising biotechnology for waste reduction and control of antibiotic pollution. Monensin (MON), an ionophore polyether antibiotic (IPA), is widely used in broiler feed to control coccidiosis. However, MON residues in litter have become a major source of pollution in the environment. In this work, we studied the efficiency of housefly larvae (Musca domestica) on monensin attenuation during a 12-day laboratory scale vermicomposting experiment. We observed a 94.99% reduction in MON concentration after four days in treatment groups, while it took twelve days to remove more than 94.71% of MON in the control group. We found that the bacterial community composition of the substrate was reshaped by housefly larvae. From the treatment groups, three MON-degrading bacterial strains were isolated and identified as Acinetobacter sp., Stenotrophomonas sp. and Alcaligenes sp. based on 16 S rRNA gene sequence analysis. These three strains were among dominant the bacteria in treated substrates, showing between 52.80% and 89.25% degradation of MON in mineral salt medium within 28 days. Furthermore, two MON-degrading bacteria (Stenotrophomonas sp. and Alcaligenes sp.) were more abundant in treatment groups and larvae gut groups compared with those in control groups. The abundance enhancement of MON-degrading bacteria was related to the change in ambient temperature and pH in the substrates, which were affected by housefly larvae activities. Our results confirm that housefly larvae can significantly accelerate degradation of MON in chicken manure by increasing the abundance of MON-degrading bacteria.

Prediction of Transformation Products of Monensin by Electrochemistry Compared to Microsomal Assay and Hydrolysis.

The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation.

Electrogenic and nonelectrogenic ion fluxes across lipid and mitochondrial membranes mediated by monensin and monensin ethyl ester.

Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczynski et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108-2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin.

Treatment of influenza virus with beta-propiolactone alters viral membrane fusion.

Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. Although BPL has been described to chemically modify nucleic acids, its effect on viral proteins, potentially affecting viral infectivity, remains poorly studied. Here, a H3N2 strain of influenza virus was submitted to treatment with various BPL concentrations (2-1000µM). Cell infectivity was progressively reduced and entirely abolished at 1mM BPL. Virus fusion with endosome being a critical step in virus infection, we analyzed its ability to fuse with lipid membrane after BPL treatment. By monitoring calcein leakage from liposomes fusing with the virus, we measured a decrease of membrane fusion in a BPL dose-dependent manner that correlates with the loss of infectivity. These data were complemented with cryo transmission electron microscopy (cryoTEM) and cryo electron tomography (cryoET) studies of native and modified viruses. In addition, a decrease of leakage irrespective of BPL concentration was measured suggesting that the insertion of HA2 fusion peptide into the target membrane was inhibited even at low BPL concentrations. Interestingly, mass spectrometry revealed that HA2 and M1 matrix proteins had been modified. Furthermore, fusion activity was partially restored by the protonophore monensin as confirmed by cryoTEM and cryoET. Moreover, exposure to amantadine, an inhibitor of M2 channel, did not alter membrane fusion activity of 1mM BPL treated virus. Taken together these results show that BPL treatment inhibits membrane fusion, likely by altering function of proteins involved in the fusion process, shedding new light on the effect of BPL on influenza virus.

Anti-proliferative activity of Monensin and its tertiary amide derivatives.

New tertiary amide derivatives of polyether ionophore Monensin A (MON) were synthesised and their anti-proliferative activity against cancer cell lines was studied. Very high activity (IC50=0.09 µM) and selectivity (SI=232) of MON against human biphenotypic myelomonocytic leukemia cell line (MV4-11) was demonstrated. The MON derivatives obtained exhibit interesting anti-proliferative activity, high selectivity index and also are able to break the drug-resistance of cancer cell line.

Photodegradation of veterinary ionophore antibiotics under UV and solar irradiation.

The veterinary ionophore antibiotics (IPAs) are extensively used as coccidiostats and growth promoters and are released to the environment via land application of animal waste. Due to their propensity to be transported with runoff, IPAs likely end up in surface waters where they are subject to photodegradation. This study is among the first to investigate the photodegradation of three commonly used IPAs, monensin (MON), salinomycin (SAL) and narasin (NAR), under UV and solar irradiation. Results showed that MON was persistent in a deionized (DI) water matrix when exposed to UV and sunlight, whereas SAL and NAR could undergo direct photolysis with a high quantum yield. Water components including nitrate and dissolved organic matter had a great impact on the photodegradation of IPAs. A pseudosteady state kinetic model was successfully applied to predict IPAs' photodegradation rates in real water matrices. Applying LC/MS/MS, multiple photolytic transformation products of IPAs were observed and their structures were proposed. The direct photolysis of SAL and NAR occurred via cleavage on the ketone moiety and self-sensitized photolysis. With the presence of nitrate, MON was primarily degraded by hydroxyl radicals, whereas SAL showed reactivity toward both hydroxyl and nitrogen-dioxide radicals. Additionally, toxicity tests showed that photodegradation of SAL eliminated its antibiotic properties against Bacillus subtilis.

Inhibition and biotransformation potential of veterinary ionophore antibiotics under different redox conditions.

Veterinary ionophore antibiotics (IPAs) are polyether compounds used extensively in the livestock industry to promote animal growth and prevent coccidia infection. However, the environmental fate and impact of IPAs are not fully understood. In this study, the inhibition and biotransformation potential of the most commonly used IPAs, monensin (MON) and salinomycin (SAL), were investigated under well-defined aerobic, nitrate-reducing, fermentative/sulfate-reducing, and fermentative/methanogenic conditions. Batch assays were conducted with mixed cultures developed from poultry litter (PL), PL-fertilized soil, and municipal anaerobic sludge. Significant transformation of MON and SAL was observed in aerobic, low-buffer capacity culture series as a result of abiotic acid-catalyzed IPAs hydrolysis induced by nitrification. Biotransformation of IPAs was the main transformation process in aerobic, high-buffer capacity culture series. MON persisted under fermentative/sulfate-reducing conditions, whereas SAL was transformed by fermentative bacteria. Both MON and SAL were stable under nitrate-reducing and methanogenic conditions. At IPAs concentrations up to 1 mg/L, MON inhibited only methanogenesis, whereas SAL did not impact any of the biological processes investigated in this study. Multiple, new primary IPA biotransformation products were observed on LC/MS, and their molecular structures were tentatively identified by analyzing LC/MS/MS fragmentation patterns. Overall, MON and SAL exhibited different inhibition and biotransformation patterns at each redox condition tested, which could greatly influence their fate and impact upon their release into the environment as a result of agricultural activities.

Monensin A acid complexes as a model of electrogenic transport of sodium cation.

New Monensin A acid complexes with water molecule, sodium chloride and sodium perchlorate were obtained and studied by X-ray and (1)H, (13)C NMR and FT-IR methods as well as ab initio calculations. The crystal structure of the complexes indicates the complexation of the water molecule and Na(+) cation in the pseudo-cycle conformation of the Monensin acid molecule stabilised by intramolecular hydrogen bonds. Important for stabilisation of this structure is also the intermolecular hydrogen bonds with water molecule or the coordination bonds with Na(+) cation. It is demonstrated that the counterions forming intermolecular hydrogen bonds with OH groups influence the strength of the intramolecular hydrogen bonds, but they have no influence on the formation of pseudo-cyclic structure. Spectroscopic studies of the complexes in dichloromethane solution have shown that the pseudo-cyclic structure of the compounds is conserved. As follows from the ab initio calculations, the interactions between the Na(+) cation and the electronegative oxygen atoms of Monensin acid totally change the molecular electrostatic potential around the supramolecular Monensin acid-Na(+) cationic complex relative to that of the neutral Monensin acid molecule.

Acid-catalyzed transformation of ionophore veterinary antibiotics: reaction mechanism and product implications.

Ionophore antibiotics (IPAs) are polyether antimicrobials widely used in the livestock industry and may enter the environment via land application of animal waste and agricultural runoff. Information is scarce regarding potential transformation of IPAs under environmental conditions. This study is among the first to identify the propensity of IPAs to undergo acid-catalyzed transformation in mildly acidic aquatic systems and characterize the reactions in depth. The study focused on the most widely used monensin (MON) and salinomycin (SAL), and also included narasin (NAR) in the investigation. All three IPAs are susceptible to acid-catalyzed transformation. MON reacts much more slowly than SAL and NAR and exhibits a different kinetic behavior that is further evaluated by a reversible reaction kinetic model. Extensive product characterization identifies that the spiro-ketal group of IPAs is the reactive site for the acid-catalyzed hydrolytic transformation, yielding predominantly isomeric and other products. Toxicity evaluation of the transformation products shows that the products retain some antimicrobial properties. The occurrence of IPAs and isomeric transformation products is also observed in poultry litter and agricultural runoff samples. Considering the common presence of mildly acidic environments (pH 4-7) in soils and waters, the acid-catalyzed transformation identified in this study likely plays an important role in the environmental fate of IPAs.

Disruption of lysosome function promotes tumor growth and metastasis in Drosophila.

Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras(V12) cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras(V12) cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras(V12) cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.

Expanding the antibacterial selectivity of polyether ionophore antibiotics through diversity-focused semisynthesis.

Polyether ionophores are complex natural products capable of transporting cations across biological membranes. Many polyether ionophores possess potent antimicrobial activity and a few selected compounds have the ability to target aggressive cancer cells. Nevertheless, ionophore function is believed to be associated with idiosyncratic cellular toxicity and, consequently, human clinical development has not been pursued. Here, we demonstrate that structurally novel polyether ionophores can be efficiently constructed by recycling components of highly abundant polyethers to afford analogues with enhanced antibacterial selectivity compared to a panel of natural polyether ionophores. We used classic degradation reactions of the natural polyethers lasalocid and monensin and combined the resulting fragments with building blocks provided by total synthesis, including halogen-functionalized tetronic acids as cation-binding groups. Our results suggest that structural optimization of polyether ionophores is possible and that this area represents a potential opportunity for future methodological innovation.

Greenhouse gas, animal performance, and bacterial population structure responses to dietary monensin fed to dairy cows.

The present study investigated the effects of a feed additive and rumen microbial modifier, monensin sodium (monensin), on selected variables in lactating dairy cows. Monensin fed cows (MON, 600 mg d(-1)) were compared with untreated control cows (CON, 0 mg d(-1)) with respect to the effects of monensin on the production of three greenhouse gases (GHG), methane (CH(4)), nitrous oxide (N(2)O), and carbon dioxide (CO(2)), along with animal performance (dry matter intake; DMI), milk production, milk components, plasma urea nitrogen (PUN), milk urea nitrogen (MUN), and the microbial population structure of fresh feces. Measurements of GHG were collected at Days 14 and 60 in an environmental chamber simulating commercial dairy freestall housing conditions. Milk production and DMI measurements were collected twice daily over the 60-d experimental period; milk components, PUN, and MUN were measured on Days 14 and 60. The microbial population structure of feces from 6 MON and 6 CON cows was examined on three different occasions (Days 14, 30, and 60). Monensin did not affect emissions of methane (CH(4)), nitrous oxide (N(2)O), and carbon dioxide (CO(2)). Over a 24-h period, emissions of CH(4), N(2)O, and CO(2) decreased in both MON and CON groups. Animal performance and the microbial population structure of the animal fresh waste were also unaffected for MON vs. CON cows.

Forty years of monensin for the control of coccidiosis in poultry.

In July 1971, the polyether ionophorous antibiotic monensin was introduced in the United States for the control of coccidiosis in poultry. At that time, prospects for new anticoccidial agents were not good. Amprolium had enjoyed several years of use, but many other compounds had been abandoned as resistance to them developed. After the introduction of monensin, most commercial broilers were medicated with the drug and it is still widely used for this purpose today. Apart from in poultry, monensin is also used to control coccidiosis in game birds, sheep, and cattle. Indeed, more animals have been medicated with ionophores, such as monensin, for control of disease than any other medicinal agents in the history of veterinary medicine. In this review, we discuss the discovery, mode of action, and efficacy of monensin, together with matters of importance to the poultry industry such as commercial use, drug resistance, toxicity, pharmacology and residues, host immunity to coccidiosis, and effects in other avian species.

Sorption and abiotic transformation of monensin by iron and manganese oxides.

Monensin, an ionophore antibiotic, is commonly administered as a feed additive to cattle and poultry. A large percentage of the administered dose is excreted in animal waste, which is often applied to agricultural fields as fertilizer. The objective of this work is to gain insight into the fate of monensin in soil by investigating the interactions between monensin and common soil minerals, including sorption and transformation to unmonitored partial oxidation products. Batch sorption experiments across varying conditions (i.e., pH, ionic strength) and desorption experiments (i.e., methanol, PO43-, methyl tert-butyl ether) were used to determine the extent to which a selection of common redox-active soil minerals [birnessite (δ-MnO2), goethite (α-FeOOH), hematite (α-Fe2O3)] can bind and transform monensin. Monensin was bound by hematite (pH < 7.5, up to 7.5 mmol kg-1), goethite (pH < 7.5, up to 3.4 mmol kg-1), and birnessite (pH < 7, up to 0.1 mmol kg-1). Combined sorption and transformation were the greatest for hematite and the lowest for birnessite. Sorption to hematite was more reversible than to goethite. Each desorption from goethite recovered <10% of sorbed monensin, whereas desorption from hematite recovered up to 69% of sorbed monensin, dependent on the solution. The potential for iron and manganese (hydr)oxides to abiotically transform monensin through reductive dissolution to partial oxidation products was evaluated by mass spectral analysis following sorption experiments. Additionally, the dominant sorption mechanism was inferred through ATR-FTIR spectroscopy, via examination of the carboxylate peak separation differences, on goethite and hematite to be bridging bidentate.

Calcium salts of long-chain fatty acids from linseed oil decrease methane production by altering the rumen microbiome in vitro.

Calcium salts of long-chain fatty acids (CSFA) from linseed oil have the potential to reduce methane (CH4) production from ruminants; however, there is little information on the effect of supplementary CSFA on rumen microbiome as well as CH4 production. The aim of the present study was to evaluate the effects of supplementary CSFA on ruminal fermentation, digestibility, CH4 production, and rumen microbiome in vitro. We compared five treatments: three CSFA concentrations-0% (CON), 2.25% (FAL) and 4.50% (FAH) on a dry matter (DM) basis-15 mM of fumarate (FUM), and 20 mg/kg DM of monensin (MON). The results showed that the proportions of propionate in FAL, FAH, FUM, and MON were increased, compared with CON (P < 0.05). Although DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) digestibility decreased in FAL and FAH compared to those in CON (P < 0.05), DM digestibility-adjusted CH4 production in FAL and FAH was reduced by 38.2% and 63.0%, respectively, compared with that in CON (P < 0.05). The genera Ruminobacter, Succinivibrio, Succiniclasticum, Streptococcus, Selenomonas.1, and Megasphaera, which are related to propionate production, were increased (P < 0.05), while Methanobrevibacter and protozoa counts, which are associated with CH4 production, were decreased in FAH, compared with CON (P < 0.05). The results suggested that the inclusion of CSFA significantly changed the rumen microbiome, leading to the acceleration of propionate production and the reduction of CH4 production. In conclusion, although further in vivo study is needed to evaluate the reduction effect on rumen CH4 production, CSFA may be a promising candidate for reduction of CH4 emission from ruminants.

Synthesis and Anticancer Activity of Dimeric Polyether Ionophores.

Polyether ionophores represent a group of natural lipid-soluble biomolecules with a broad spectrum of bioactivity, ranging from antibacterial to anticancer activity. Three seem to be particularly interesting in this context, namely lasalocid acid, monensin, and salinomycin, as they are able to selectively target cancer cells of various origin including cancer stem cells. Due to their potent biological activity and abundant availability, some research groups around the world have successfully followed semi-synthetic approaches to generate original derivatives of ionophores. However, a definitely less explored avenue is the synthesis and functional evaluation of their multivalent structures. Thus, in this paper, we describe the synthetic access to a series of original homo- and heterodimers of polyether ionophores, in which (i) two salinomycin molecules are joined through triazole linkers, or (ii) salinomycin is combined with lasalocid acid, monensin, or betulinic acid partners to form 'mixed' dimeric structures. Of note, all 11 products were tested in vitro for their antiproliferative activity against a panel of six cancer cell lines including the doxorubicin resistant colon adenocarcinoma LoVo/DX cell line; five dimers (14-15, 17-18 and 22) were identified to be more potent than the reference agents (i.e., both parent compound(s) and commonly used cytostatic drugs) in selective targeting of various types of cancer. Dimers 16 and 21 were also found to effectively overcome the resistance of the LoVo/DX cancer cell line.