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.

Soil Behaviour of the Veterinary Drugs Lincomycin, Monensin, and Roxarsone and Their Toxicity on Environmental Organisms.

Lincomycin, monensin, and roxarsone are commonly used veterinary drugs. This study investigated their behaviours in different soils and their toxic effects on environmental organisms. Sorption and mobility analyses were performed to detect the migration capacity of drugs in soils. Toxic effects were evaluated by inhibition or acute toxicity tests on six organism species: algae, plants, daphnia, fish, earthworms and quails. The log Kd values (Freundlich model) of drugs were: lincomycin in laterite soil was 1.82; monensin in laterite soil was 2.76; and roxarsone in black soil was 1.29. The Rf value of lincomycin, roxarsone, monensin were 0.4995, 0.4493 and 0.8348 in laterite soil, and 0.5258, 0.5835 and 0.8033 in black soil, respectively. The EC50 for Scenedesmus obliquus, Arabidopsis thaliana, Daphnia magna and LC50/LD50 for Eisenia fetida, Danio rerio, and Coturnix coturnix were: 13.15 mg/L,32.18 mg/kg dry soil,292.6 mg/L,452.7 mg/L,5.74 g/kg dry soil and 103.9 mg/kg (roxarsone); 1.085 mg/L, 25 mg/kg dry soil, 21.1 mg/L, 4.76 mg/L, 0.346 g/kg dry soil and 672.8 mg/kg (monensin); 0.813 mg/L, 35.40 mg/kg dry soil, >400 mg/L, >2800 mg/L, >15 g/kg dry soil, >2000 mg/kg (lincomycin). These results showed that the environmental effects of veterinary drug residues should not be neglected, due to their mobility in environmental media and potential toxic effects on environmental organisms.

A physiologically based pharmacokinetic model for chickens exposed to feed supplemented with monensin during their lifetime.

We developed a flow-limited physiologically based pharmacokinetic model for residues of monensin in chickens and evaluated its predictive ability by comparing it with an external data set describing concentration decays after the end of treatment. One advantage of this model is that the values for most parameters (34 of 38) were taken directly from the literature or from field data (for growth and feed intake). Our model included growth (changes in body weight) to describe exposure throughout the life of the chicken. We carried out a local sensitivity analysis to evaluate the relative importance of model parameters on model outputs and revealed the predominant influence of 19 parameters (including three estimated ones): seven pharmacokinetic parameters, five physiological parameters and seven animal performance parameters. Our model estimated the relative bioavailability of monensin as feed additive at 3.9%, which is even lower than the absolute bioavailability in solution (29.91%). Our model can be used for extrapolations of farming conditions, such as monensin supplementation or building lighting programme (which may have a significant impact for short half-life molecules such as monensin). This validated PBPK model may also be useful for interspecies extrapolations or withdrawal period calculations for modified dosage regimens.

Serum, milk, and tissue monensin concentrations in cattle with adequate and potentially toxic dietary levels of monensin: pharmacokinetics and diagnostic interpretation.

Used in both beef cattle and dairy cows, monensin can provide many health benefits but can, when unintended overexposures occur, result in adverse effects. Information on serum and tissue concentrations following overexposure and/or overt toxicosis which may aid in diagnostics and clinical outcome is lacking. The aim of this study was to determine concentrations of monensin in biological specimens following oral exposure for 10 days to an approved dose (1 mg/kg) and a higher dose (5 mg/kg) of monensin given daily on a body weight basis to 10 dairy cows. No deaths were reported; cows receiving 5 mg/kg showed early signs of toxicosis including depression, decreased feed intake, and diarrhea after 4 days of exposure. Histopathological findings were minimal in most cows. Pharmacokinetic modeling of the detected serum concentrations for the 1 and 5 mg/kg dose groups determined the Cmax , Tmax, and t1/2λ to be 0.87 and 1.68 ng/mL, 2.0 and 1.0 h, and 1.76 and 2.32 days, respectively. Mixed regression models showed that the dose level and days since last dose were significantly associated with monensin concentrations in all four tissues, and with cardiac troponin levels. The high dose resulted in a significant elevation of monensin in tissues at approximately 4.7 times compared to the monensin concentrations in the tissues of animals from the low-dose group. The cTnI concentrations in the high-dose group were 2.1 times that of cTnI in the low-dose group. Thus, the ability to diagnose monensin overexposure and/or toxicosis will improve from knowledge of biological monensin concentrations from this study.

Stearylamine Liposomal Delivery of Monensin in Combination with Free Artemisinin Eliminates Blood Stages of Plasmodium falciparum in Culture and P. berghei Infection in Murine Malaria.

The global emergence of drug resistance in malaria is impeding the therapeutic efficacy of existing antimalarial drugs. Therefore, there is a critical need to develop an efficient drug delivery system to circumvent drug resistance. The anticoccidial drug monensin, a carboxylic ionophore, has been shown to have antimalarial properties. Here, we developed a liposome-based drug delivery of monensin and evaluated its antimalarial activity in lipid formulations of soya phosphatidylcholine (SPC) cholesterol (Chol) containing either stearylamine (SA) or phosphatidic acid (PA) and different densities of distearoyl phosphatidylethanolamine-methoxy-polyethylene glycol 2000 (DSPE-mPEG-2000). These formulations were found to be more effective than a comparable dose of free monensin in Plasmodium falciparum (3D7) cultures and established mice models of Plasmodium berghei strains NK65 and ANKA. Parasite killing was determined by a radiolabeled [(3)H]hypoxanthine incorporation assay (in vitro) and microscopic counting of Giemsa-stained infected erythrocytes (in vivo). The enhancement of antimalarial activity was dependent on the liposomal lipid composition and preferential uptake by infected red blood cells (RBCs). The antiplasmodial activity of monensin in SA liposome (50% inhibitory concentration [IC50], 0.74 nM) and SPC:Chol-liposome with 5 mol% DSPE-mPEG 2000 (IC50, 0.39 nM) was superior to that of free monensin (IC50, 3.17 nM), without causing hemolysis of erythrocytes. Liposomes exhibited a spherical shape, with sizes ranging from 90 to 120 nm, as measured by dynamic light scattering and high-resolution electron microscopy. Monensin in long-circulating liposomes of stearylamine with 5 mol% DSPE-mPEG 2000 in combination with free artemisinin resulted in enhanced killing of parasites, prevented parasite recrudescence, and improved survival. This is the first report to demonstrate that monensin in PEGylated stearylamine (SA) liposome has therapeutic potential against malaria infections.

Biodegradation of veterinary ionophore antibiotics in broiler litter and soil microcosms.

Ionophore antibiotics (IPAs) are polyether compounds used in broiler feed to promote growth and control coccidiosis. Most of the ingested IPAs are excreted into broiler litter (BL), a mixture of excreta and bedding material. BL is considered a major source of IPAs released into the environment as BL is commonly used to fertilize agricultural fields. This study investigated IPA biodegradation in BL and soil microcosms, as a process affecting the fate of IPAs in the environment. The study focused on the most widely used IPAs, monensin (MON), salinomycin (SAL), and narasin (NAR). MON was stable in BL microcosms at 24-72% water content (water/wet litter, w/w) and 35-60 °C, whereas SAL and NAR degraded under certain conditions. Factor analysis was conducted to delineate the interaction of water and temperature on SAL and NAR degradation in the BL. A major transformation product of SAL and NAR was identified. Abiotic reaction(s) were primarily responsible for the degradation of MON and SAL in nonfertilized soil microcosms, whereas biodegradation contributed significantly in BL-fertilized soil microcosms. SAL biotransformation in soil microcosms yielded the same product as in the BL microcosms. A new primary biotransformation product of MON was identified in soil microcosms. A field study showed that MON and SAL were stable during BL stacking, whereas MON degraded after BL was applied to grassland. The biotransformation product of MON was also detected in the top soil layer where BL was applied.

Effect of three polyether ionophores on pharmacokinetics of florfenicol in male broilers.

Drug-drug interactions (DDIs) may adversely affect the prevention and cure of diseases. The effects of three polyether ionophore antibiotics, salinomycin (SAL), monensin (MON), and maduramycin (MAD) on the pharmacokinetics of florfenicol (FFC) were investigated in broilers. The chickens were fed rations with or without SAL (60 mg/kg feeds), MON (120 mg/kg feeds), or MAD (5 mg/kg feeds) for 14 consecutive days. FFC was given to the chickens either intravenously (i.v.) or orally (p.o.) at a single dose of 30 mg/kg body weight. Blood samples were taken from each chicken at 0-24 h postadministration of FFC. The plasma concentration of FFC was detected by high-performance liquid chromatography. The plasma concentration of FFC decreased with i.v. or p.o. co-administration of SAL, MON, or MAD in broilers, implying occurrence of DDIs during the co-administration of FFC with these ionophores. Our findings suggest that more attention should be given to the use of FFC to treat bacterial infections in chickens supplemented with polyether ionophore antibiotics.

pH-sensitive capsules as intracellular optical reporters for monitoring lysosomal pH changes upon stimulation.

The concept of a long-term sensor for ion changes in the lysosome is presented. The sensor is made by layer-by-layer assembly of oppositely charged polyelectrolytes around ion-sensitive fluorophores, in this case for protons. The sensor is spontaneously incorporated by cells and resides over days in the lysosome. Intracellular changes of the concentration of protons upon cellular stimulation with pH-active agents are monitored by read-out of the sensor fluorescence at real time. With help of this sensor concept it is demonstrated that the different agents used (Monensin, Chloroquine, Bafilomycin A1, Amiloride) possessed different kinetics and mechanisms of action in affecting the intracellular pH values.

Comparison of the oral bioavailability and tissue disposition of monensin and salinomycin in chickens and turkeys.

The current study describes the pharmacokinetic parameters of two carboxylic polyether ionophores: monensin in turkeys and salinomycin in chickens. These data can be used to understand and predict the occurrence of undesirable residues of coccidiostats in edible tissues of these animal species. Special attention is paid to the distribution of residues between the different edible tissues during and at the end of the treatment period. For the bioavailability studies, monensin was administered to turkeys intravenously, in the left wing vein, at a dose of 0.4 mg /kg and orally at a dose of 20 mg /kg. Salinomycin was administered to chickens intravenously, in the left wing vein, at a dose of 0.25 mg /kg and orally at a dose of 2.5 mg /kg. Residue studies were carried out with supplemented feed at the rate of 100 mg /kg of feed for monensin in turkeys and 70 mg /kg for salinomycin in chickens, respectively. Coccidiostats had a low bioavailability in poultry (around 30% for monensin in chickens, around 1% for monensin in turkeys and around 15% for salinomycin in chickens). Monensin in chickens had a longer terminal half-life (between 3.07 and 5.55 h) than both monensin in turkeys (between 1.36 and 1.55 h) and salinomycin in chickens (between 1.33 and 1.79 h). The tissue /plasma partition coefficients showed a higher affinity of both monensin and salinomycin for fat, followed by liver and muscle tissue. The depletion data showed a fairly rapid elimination of coccidiostats in all the tissues after cessation of treatment. According to the results of depletion studies, a withdrawal period of 1 day seems sufficient to avoid undesirable exposure of consumers.

Bioavailability, distribution and depletion of monensin in chickens.

The pharmacokinetics of monensin including apparent volume of distribution, total body clearance, systemic bioavailability, partition coefficients and tissue residues were determined in chickens. The drug was given by intravenous injection in the left wing vein at the dose of 0.46 mg/kg and by intracrop administration at the dose of 4 mg/kg according to a destructive sampling. The pharmacokinetic variables were compared after noncompartmental, naïve averaged, naïve pooled and nonlinear mixed-effects modelling analyses. Partition coefficients and tissue residues were determined after a treatment with feed additives (125 mg/kg of feed) of 33 days. The clearance, volume of distribution and bioavailabilty were approximately 2.2 L/h/kg, approximately 9 L/kg and approximately 30% respectively except with nonlinear mixed effects models that presented values of 1.77 L/h/kg, 14.05 L/kg and 11.36% respectively. Tissue/plasma partition coefficients were estimated to 0.83, 3.39 and 0.51 for liver, fat and thigh muscle respectively. Monensin residues after treatment were not detected 6 h after withdrawal except for fat where monensin was still quantifiable 12 h after. Pharmacokinetic variables seem to be inaccurate when assessed with non linear mixed-effects modelling associated to destructive sampling in chickens. Values varied slightly with noncompartmental, naïve averaged and naïve pooled analyses. The absorption, elimination and partition parameters will be incorporated into a physiologically based pharmacokinetic model and the depletion study will be used to test the ability of this model to describe monensin residues in edible tissues under different dosage regimens.

Liquid chromatography-electrospray tandem mass spectrometric method for quantification of monensin in plasma and edible tissues of chicken used in pharmacokinetic studies: applying a total error approach.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for use in pharmacokinetic studies in order to determine the concentrations of monensin in plasma and edible tissues of chicken. Two sample preparations were performed, one for determining monensin concentrations in plasma using acetonitrile for protein precipitation and another one for determining monensin concentrations in muscle, liver, and fat using methanol-water followed by a clean up on a solid-phase extraction cartridge. Sample extracts were injected into the LC-MS/MS system, and a gradient elution was performed on a C18 column. Narasin was used as internal standard. The LC-MS/MS method was validated using an approach based on accuracy profiles, and applicability of the method was demonstrated for the determination of monensin in chicken plasma, muscle, liver, and fat in a pharmacokinetic study.

Monensin in lipid emulsion for the potentiation of ricin A chain immunotoxins.

The utilization of carboxylic ionophores such as monensin for immunotoxin potentiation may be hampered by the poor solubility and short in vivo half-life of these highly lipophilic compounds. Therefore, the use of monensin formulated in a lipid/water emulsion was investigated for the in vitro and in vivo potentiation of immunotoxins. Monensin in emulsion or in buffer was equally effective for the in vitro potentiation of the cytotoxicity of both anti-human transferrin receptor and anti-carcinoembryonic antigen immunotoxins against target cells. In mice, buffer and lipid emulsion were compared as vehicles for the i.p. administration of monensin. The half-life of monensin in the peritoneal cavity of BALB/c x DBA/2 F1 (CD2F1) mice was increased 20-fold by inclusion in lipid emulsion (13 min versus 0.75 min). Treatment i.p. with anti-human transferrin receptor immunotoxin or anti-carcinoembryonic antigen immunotoxin and monensin emulsion prolonged the survival of mice with macroscopic i.p. tumor xenografts of H-Meso-1 mesothelioma and LS174T colorectal carcinoma (200-250% increased length of median survival). The in vivo antitumor effect of the cell-specific immunotoxin plus monensin emulsion was superior to immunotoxin alone or to immunotoxin plus monensin in buffer (P less than 0.03; Mann-Whitney U test). This indicates that delivery of monensin in preformed lipid emulsion may produce a reservoir effect of the ionophore in the peritoneal cavity of tumor-bearing mice. Nonspecific control immunotoxin plus monensin emulsion produced no increase in survival. Long-term tumor-free survival (greater than 150 days versus a median survival of 25 days for controls) of mice bearing microscopic LS174T xenografts was obtained by treatment with anti-human transferrin receptor immunotoxin plus monensin emulsion. Administration of either monensin in buffer or monensin in emulsion without immunotoxin had no significant effect on survival. Monensin in this pharmacologically available form significantly improved the in vivo efficacy of both anti-human transferrin receptor immunotoxin and anti-carcinoembryonic antigen immunotoxin when used as regional therapy.

Carrier protein-monensin conjugates: enhancement of immunotoxin cytotoxicity and potential in tumor treatment.

We have investigated the potentiation of transferrin [Tfn]-toxin [Tfn-ricin toxin A chain (RTA) and Tfn-So6 saporin toxin] and monoclonal antibody-RTA conjugates by monensin (Mo) and by a human serum albumin (HSA)-monensin conjugate in vitro. The in vivo survival and in vitro and in vivo toxicity of HSA-Mo were also studied; monensin was chemically linked to HSA carrier protein via a disulfide bridge. HSA-Mo was 2-13-fold less toxic than Mo for cells in vitro. HSA-Mo was active in the same concentration range as Mo in potentiating mAb-RTA and Tfn-toxin conjugates reactive with Tfn receptors expressed by different cell lines in monolayer cell cultures. Multicell tumor spheroid cultures were used to investigate the target cell killing effect of cytotoxic conjugates and HSA-Mo in three-dimensional structures mimicking the properties of nonvascularized micrometastases. Spheroids 300-400 microns were as sensitive to Tfn-RTA and HSA-Mo in combination as monolayer cells. After 24 h incubation at 37 degrees C in human serum about 2% HSA-Mo molecules remained available for immunotoxin potentiation and about 10% after 24 h incubation in human cerebrospinal fluid. BALB/c mice tolerated injections of 2 mg/kg HSA-Mo i.v. and of 16 mg/kg i.p. The HSA-Mo half-life in the serum of BALB/c mice was 0.5 h. Following i.v. injection about 0.5% of the initial HSA-Mo persisted in the circulation at 24 h.

In vivo potentiation of ricin toxicity by monensin delivered through liposomes.

Monensin, a carboxylic ionophore, which is known to raise intravesicular pH, was intercalated in liposomes and its effect on the toxicity of ricin in mice was studied. The toxicity of ricin in vivo was found to be significantly enhanced by the administration of monensin intercalated in liposomes (liposomal monensin). The observed enhancement of the toxicity of ricin by monensin was highly dose-dependent and was maximal when ricin was injected within 60 min of monensin injection. The survival time was found to be reduced in the range of 8-20 h, depending on the dose of ricin used, by liposomal monensin. Stability of liposomes containing monensin as inferred from the release of entrapped calcein or FITC-dextran under both in vivo and in vitro conditions was comparable to that observed for liposomes without monensin. Liposomal monensin remains in circulation for 2 h and was cleared from the blood stream after 4 h. In contrast, 15 min was required for the clearance of monensin when administered in free form. Studies on the distribution of liposomal monensin and 125I-ricin in various tissues have revealed that monensin is mainly localized in the liver and spleen which are also the major sites for ricin accumulation. Our observation on the substantial enhancement of ricin toxicity in vivo by liposomal monensin strongly supports the potential usefulness of the latter as a potentiating agent in the enhancement of the toxicity of immunotoxin or hormonotoxin for selective elimination of cancer cells.

Milk residues and performance of lactating dairy cows administered high doses of monensin.

Milk residues and performance were evaluated in lactating cows that were fed up to 10 times the recommended dose of monensin. Following an acclimatization period of 14 d, during which cows were fed a standard lactating cow total mixed ration containing 24 ppm monensin, 18 lactating Holstein dairy cows were grouped according to the level of feed intake and then randomly assigned within each group to 1 of 3 challenge rations delivering 72, 144, and 240 ppm monensin. Outcome measurements included individual cow daily feed intakes, daily milk production, body weights, and monensin residues in composite milk samples from each cow. There were no detectable monensin residues (< 0.005 microg/mL) in any of the milk samples collected. Lactating cows receiving a dose of 72 ppm monensin exhibited up to a 20% reduction in dry matter intake, and a 5% to 15% drop in milk production from the pre-challenge period. Cows receiving doses of 144 and 240 ppm monensin exhibited rapid decreases in feed intake of up to 50% by the 2nd d and milk production losses of up to 20% and 30%, respectively, within 4 d. Lactating cows receiving up to 4865 mg monensin per day had no detectable monensin residues (< 0.005 microg/mL) in any of the milk samples collected. Results of this study confirm that food products derived from lactating dairy cattle receiving monensin at recommended levels are safe for human consumption.

Stealth monensin liposomes as a potentiator of adriamycin in cancer treatment.

Small unilamellar stealth monensin liposomes (SMLs) were prepared from multilamellar liposomes (MLVs). The MLVs were prepared by using dipalmitoyl phosphatidylcholine (DPPC), cholesterol, distearoyl glycerophosphoethanolamine coupled to poly(ethylene glycol) (DSPE-PEG) and stearylamine in the molar ratio of 10:5:1.4:1.4 (32.8 mM total lipid). The encapsulation efficiencies of monensin in MLVs and small unilamellar vesicles (SUVs) was 6x10++(-6) and 10(-7) M, respectively. The stability of SMLs was studied at 4 degrees C. The amount of leakage of monensin from SMLs was less than 20% after four weeks of storage. The in vitro release of monensin from SMLs in human serum was determined, and t1/2 was found to be 10 h. Pharmacokinetic studies on SMLs were carried out in BALB/c mice. More than 20% of SMLs remained in blood circulation after 24 h. SMLs increased the uptake of adriamycin (AM) in HL-60-resistant cells by more than two fold, compared to monensin in solution. SMLs potentiated the effect of AM against both sensitive and resistant HL-60 cells (six- and tenfold potentiation, respectively) and human LOVO tumor cells (four- and 200-fold potentiation, respectively). However, the highest potentiation was observed against resistant human breast tumor MCF7 cells, and was found to be 2400 times in comparison to AM alone. Transmission electron microscopic (TEM) studies carried out with HL-60-resistant tumor cells incubated with SMLs showed that SMLs caused dilation of the golgi of tumor cells within 10 min. The dilation of golgi was reversible after reincubation of the cells in fresh medium. SMLs showed considerable potential as a potentiator in combination with AM in overcoming drug resistance.

Long-circulating monensin nanoparticles for the potentiation of immunotoxin and anticancer drugs.

The carboxylic ionophore monensin was formulated into long-circulating nanoparticles with the help of polyethylene glycol/poly (DL-lactide-co-glycolide) diblock copolymers, in an attempt to enhance the cytotoxicity of a ricin-based immunotoxin, anti-My9, and anticancer drugs like adriamycin and tamoxifen. This study looked into various aspects involving the preparation (using a homogenizer and an EmulsiFlex homogenizer-extrusion device) and lyophilization of long-circulating monensin nanoparticles (LMNP) of particle size < 200 nm in diameter. The particle size of LMNP was reduced from 194 nm to 160 nm by passing the nanoparticles through an EmulsiFlex, before freeze-drying. There was a 4.8-83.7% increase in the particle size of LMNP after freeze-drying, which was dependent upon the manufacturing conditions such as use of the EmulsiFlex for size reduction before freeze-drying, the freezing method (rapid/slow) and the concentration of lyoprotectant (mannitol or trehalose) employed for freeze-drying. LMNP freeze-dried with 2.4% of trehalose showed minimal size change (< 9%) after freeze-drying. Further, the freezing method was found to have negligible effect on the particle size of LMNP freeze-dried with trehalose in comparison with mannitol. The entrapment efficiency of monensin in LMNP was found to be 14.2 +/- 0.3%. The LMNP were found to be spherical in shape and smooth in surface texture as observed by atomic force microscopy. In-vitro release of monensin from LMNP in phosphate buffered saline (PBS) pH 7.4 or PBS supplemented with 10% human serum indicated that there was an initial rapid release of about 40% in the first 8 h followed by a fairly slow release (about 20%) in the next 88 h. In-vivo studies conducted with Sprague-Dawley rats showed that 20% of monensin remained in circulation 4-8 h after the intravenous administration of LMNP. An in-vitro dye-based cytotoxicity assay (MTS/PMS method) showed that there was 500 times and 5 times potentiation of the cytotoxicity of anti-My9 immunotoxin by LMNP (5 x 10(-8) M of monensin) in HL-60 sensitive and resistant human tumour cell lines, respectively. Further, LMNP (5 x 10(-8) M of monensin) potentiated the cytotoxicity of adriamycin in MCF 7 and SW 620 cell lines by 100 fold and 10 fold, respectively, and that of tamoxifen by 44 fold in MCF 7 cell line as assessed by crystal violet dye uptake assay. Our results suggest that it is possible to prepare LMNP possessing appropriate particle size (< 200 nm), monensin content and in-vitro and in-vivo release characteristics with the help of a homogenizer and an EmulsiFlex homogenizer-extrusion device. LMNP can be freeze-dried with minimal increase in particle size by using a suitable concentration of a lyoprotectant like trehalose. Furthermore, LMNP could potentiate the cytotoxicity of immunotoxin, adriamycin and tamoxifen by 5-500 fold in-vitro, which will be further investigated in-vivo in a suitable animal model.

Inhibition of resistance plasmid transfer in Escherichia coli by ionophores, chlortetracycline, bacitracin, and ionophore/antimicrobial combinations.

Medicinal feed additives bacitracin, chlortetracycline (CTC), laidlomycin, lasalocid, and salinomycin inhibited the transfer of multiresistance-conferring plasmid pBR325 (Tet(r) Amp(r) Cp(r), 6.0 kb) into selected gram-negative strains with the use of an in vitro model. High concentrations of ampicillin-sensitive competence-pretreated Escherichia coli HB 101 cells were exposed to 10% (v/v) of 1:10 dimethyl sulfoxide/agent : water containing test mixtures for 0.5 hr prior to plasmid addition and transforming conditions. Transformation was inhibited for all antimicrobials and showed a positive association wich higher concentration. Additional testing of ionophore compounds separately and in combination with bacitracin, chlortetracycline, lincomycin, roxarsone, tylosin, and virginiamycin at representative feed concentrations demonstrated 80.6% to >99.9% inhibition (P < 0.001) of resistance transfer. Bacitracin alone inhibited transformation within the range of 50-500 ppm. No increase in resistance transfer was observed when poultry-derived and reference gram-negative isolates having low or no transformation efficiency were additionally tested. The results suggest that these compounds, at relevant concentrations used in animal feed, may interfere with cell envelope-associated DNA uptake channels or other transformation competence mechanisms. Through these mechanisms, ionophores and cell membrane-interactive feed agents such as CTC and bacitracin may act to inhibit resistance transfer mechanisms within poultry and livestock.

Effect of ionophorous anticoccidials on invasion and development of Eimeria: comparison of sensitive and resistant isolates and correlation with drug uptake.

Prophylactic levels of three ionophorous antibiotics, monensin, salinomycin, and lasalocid, were administered to groups of chickens and turkeys. All three ionophores markedly inhibited invasion of cecal tissues by sporozoites of ionophore-sensitive (IS) Eimeria tenella. Monensin and salinomycin also reduced invasion in turkeys by sporozoites of E. adenoeides, but lasalocid only minimally inhibited invasion. Invasion of ceca of monensin-medicated chickens was significantly greater by sporozoites of ionophore-resistant (IR) E. tenella than of the IS isolate. Concomitant experiments showed significant differences in [14C]monensin accumulation among IS and IR isolates of E. tenella. The decreased uptake of monensin by the IR isolates appeared to be accompanied by a decrease in responsiveness to the activity of monensin as well as to two other ionophores, salinomycin and narasin in cell culture. The amount of monensin, salinomycin or narasin required to inhibit development of E. tenella by 50% was 20 to 40 times higher for the IR isolates than for the IS ones. Collectively, the data suggest that differences in ionophore accumulation by IS and IR isolates of E. tenella might reflect differences in membrane chemistry and that these differences are responsible for the expressions of resistance that were observed in these studies. This expression of resistance appears to be common to all ionophores tested.

Studies on the toxic interaction between monensin and tiamulin in rats: toxicity and pathology.

The characteristics of the toxic interaction between monensin and tiamulin were investigated in rats. A three-day comparative oral repeated-dose toxicity study was performed in Phase I, when the effects of monensin and tiamulin were studied separately (monensin 10, 30, and 50 mg/kg or tiamulin 40, 120, and 200 mg/kg body weight, respectively). In Phase II, the two compounds were administered simultaneously to study the toxic interaction (monensin 10 mg/kg and tiamulin 40 mg/kg b.w., respectively). Monensin proved to be toxic to rats at doses of 30 and 50 mg/kg. Tiamulin was well tolerated up to the dose of 200 mg/kg. After combined administration, signs of toxicity were seen (including lethality in females). Monensin caused a dose-dependent cardiotoxic effect and vacuolar degeneration of the skeletal muscles in the animals given 50 mg/kg. Both compounds exerted a toxic effect on the liver in high doses. After simultaneous administration of the two compounds, there was a mild effect on the liver (females only), hydropic degeneration of the myocardium and vacuolar degeneration of the skeletal muscles. The alteration seen in the skeletal muscles was more marked than that seen after the administration of 50 mg/kg monensin alone.