The LC-MS/MS-Based Measurement of Isopimaric Acid in Rat Plasma and Application of Pharmacokinetics.

Isopimaric acid (IPA) exhibits a diverse array of pharmacological activities, having been shown to function as an antihypertensive, antitumor, antibacterial, and hypocholesterolemic agent. However, few studies of the pharmacokinetics of IPA have been performed to date, and such analyses are essential to explore the in vivo mechanisms governing the biological activity of this compound. As such, we herein designed a selective LC-MS approach capable of quantifying serum IPA levels in model rats using an Agilent HC-C18 column (250 mm × 4.6 mm, 5 µm) via isocratic elution with a mobile phase composed of methanol 0.5% formic acid (91 : 9, v/v) at a 1 mL/min flow rate. Ion monitoring at m/z 301.2 [M-H]- was used to quantify IPA levels in plasma samples from these rats, while internal standard (IS) levels were assessed at m/z 455.3 [M-H]-. After validation, this approach was employed to conduct a pharmacokinetic analysis of rats administered IPA via the oral (p.o. 50, 100, or 200 mg/kg) and intravenous (i.v. 5 mg/kg) routes. Analyses of noncompartmental pharmacokinetic parameters revealed that IPA underwent secondary absorption following oral administration to these animals, with the two tested oral doses (50 and 100 mg/kg) being associated with respective absolute bioavailability values of 11.9% and 17.5%. In summary, this study may provide a foundation for future efforts to explore the mechanistic basis for the pharmacological activity of IPA, offering insights to guide its subsequent clinical utilization.

A zwitterionic near-infrared fluorophore for real-time ureter identification during laparoscopic abdominopelvic surgery.

Iatrogenic injury of the ureters is a feared complication of abdominal surgery. Zwitterionic near-infrared fluorophores are molecules with geometrically-balanced, electrically-neutral surface charge, which leads to renal-exclusive clearance and ultralow non-specific background binding. Such molecules could solve the ureter mapping problem by providing real-time anatomic and functional imaging, even through intact peritoneum. Here we present the first-in-human experience of this chemical class, as well as the efficacy study in patients undergoing laparoscopic abdominopelvic surgery. The zwitterionic near-infrared fluorophore ZW800-1 is safe, has pharmacokinetic properties consistent with an ideal blood pool agent, and rapid elimination into urine after a single low-dose intravenous injection. Visualization of structure and function of the ureters starts within minutes after ZW800-1 injection and lasts several hours. Zwitterionic near-infrared fluorophores add value during laparoscopic abdominopelvic surgeries and could potentially decrease iatrogenic urethral injury. Moreover, ZW800-1 is engineered for one-step covalent conjugatability, creating possibilities for developing novel targeted ligands.

Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine (8-hydroxyquinoline) as an ionophore.

The ionophore 8-hydroxyquinoline (oxine) has been used to radiolabel cells and liposomal medicines with 111In and, more recently, 89Zr, for medical nuclear imaging applications. Oxine has also shown promising ionophore activity for the positron-emitting radionuclide 52Mn that should allow imaging of labelled cells and nanomedicines for long periods of time (>14 days). However, to date, the radiometal complex formed and its full labelling capabilities have not been fully characterised. Here, we provide supporting evidence of the formation of [52Mn]Mn(oxinate)2 as the metastable complex responsible for its ionophore activity. The cell labelling properties of [52Mn]Mn(oxinate)2 were investigated with various cell lines. The liposomal nanomedicine, DOXIL® (Caelyx) was also labelled with [52Mn]Mn(oxinate)2 and imaged in vivo using PET imaging. [52Mn]Mn(oxinate)2 was able to label various cell lines with moderate efficiency (15-53%), however low cellular retention of 52Mn (21-25% after 24 h) was observed which was shown not to be due to cell death. PET imaging of [52Mn]Mn-DOXIL at 1 h and 24 h post-injection showed the expected pharmacokinetics and biodistribution of this stealth liposome, but at 72 h post-injection showed a profile matching that of free 52Mn, consistent with drug release. We conclude that oxine is an effective ionophore for 52Mn, but high cellular efflux of the isotope limits its use for prolonged cell tracking. [52Mn]Mn(oxinate)2 is effective for labelling and tracking DOXIL in vivo. The release of free radionuclide after liposome extravasation could provide a non-invasive method to monitor drug release in vivo.

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.

A phase I study of the metal ionophore clioquinol in patients with advanced hematologic malignancies.

UNLABELLED: Clioquinol is a small-molecule metal ionophore that inhibits the proteasome through a metal-dependent mechanism. Here, we report a phase I study of clioquinol in patients with refractory hematologic malignancies. Neuropathy and abdominal pain were dose-limiting toxicities. Minimal pharmacodynamic effects were observed, and there were no clinical responses. BACKGROUND: Clioquinol is a small-molecule metal ionophore that inhibits the enzymatic activity of the proteasome and displays preclinical efficacy in hematologic malignancies in vitro and in vivo. Therefore, we conducted a phase I clinical trial of clioquinol in patients with refractory hematologic malignancies to assess its safety and determine its biological activity in this patient population. METHODS: Patients with refractory hematologic malignancies were treated with increasing doses of oral clioquinol twice daily for 15 doses. Plasma and intracellular levels of clioquinol were measured. Enzymatic activity of the proteasome was measured before and after drug administration. RESULTS: Sixteen cycles of clioquinol were administered to 11 patients with 5 patients reenrolled at the next dose level as per the permitted intrapatient dose escalation. Dose-limiting neurotoxicity and abdominal pain were observed at a dose of 1600 mg twice daily. Intracellular drug levels were low. Minimal inhibition of the proteasome was observed. No clinical responses were observed. CONCLUSION: In patients with refractory hematologic malignancies, the maximal tolerated dose of clioquinol was determined. Minimal inhibition of the proteasome was observed at tolerable doses, likely due to low intracellular levels of the drug.

Pyrithione and 8-hydroxyquinolines transport lead across erythrocyte membranes.

Acute and chronic lead poisoning remains a significant health problem. Although chelating agents can bind to plasma lead, they cannot cross cell membranes where the total body lead burden resides, and are thus inefficient at reducing the total body lead burden. Recently, calcium and sodium ionophores have been shown to transport lead across cell membranes providing a novel method for reducing total body lead stores. We recently found that clioquinol, an 8-hydroxyquinoline derivative, can act as a zinc ionophore. We postulated that zinc ionophores might also be able to transport lead across biological membranes. To study this, we loaded lead in vitro into human erythrocytes and then studied the ability of zinc ionophores to transport lead into the extracellular space, where it was trapped with a lead chelator. Using inductively coupled plasma mass spectrometry (ICP-MS), we found that several 8-hydroxyquinoline derivatives, as well as the zinc and sodium salts of pyrithione (N-hydroxypyridine-2-thione), reduced erythrocyte lead content. The water-soluble compound, sodium pyrithione, was able to reduce lead in citrated whole blood, without partitioning into the erythrocytes. These results indicate that two classes of zinc ionophores can transport lead across a biological membrane, and they confirm that these ionophores are not cation-specific. Lead ionophores may prove useful in mobilizing lead into the extracellular space, thereby improving the efficacy of chelation therapy, in vivo or ex vivo.

Microprocessor-controlled iontophoretic drug delivery of 5-fluorouracil: pharmacodynamic and pharmacokinetic study.

PURPOSE: The purpose of this study was to fabricate monolithic 5-fluorouracil (5-FU) transdermal patch with microprocessor- controlled iontophoretic delivery, to evaluate the pharmacodynamic effects on Dalton's lymphoma ascites (DLA) induced in Balb/c mice, and to study pharmacokinetics in rabbits. MATERIALS AND METHODS: The transdermal patches were prepared by solvent casting method; a reprogrammable microprocessor was developed and connected to the patches. DLA cells were injected to the hind limb of Balb/c mice (10 animals/group). In the first group of mice 5-FU was administered i.v. (12 mg/kg). In the second group of mice, transdermal patches (20 mg/patch/animal) were installed and kept for 10 consecutive days, while the third (control) group was kept without any treatment. The tumor diameter was measured every 5th day for 30 days, and the animal survival time and death pattern were studied. The electric current density protocol of 0.5 mA/cm(2) for 30 min was used in the pharmacokinetic study in rabbits. RESULTS: There was a significant reduction in tumor volume in the animals treated with monolithic matrix 5-FU transdermal patch compared to untreated controls and i.v. therapy. Tumor volume of the control animals was 5.8 cm(3) on the 30th day, while in 5-FU with transdermal patch delivery animals it was only 0.23 cm(3) (p <0.05). DLA cells tumor-bearing mice treated with 5-FU with transdermal patch had significantly increased lifespan (ILS). Control animals survived only 21+/-1 days after the tumor inoculation, while i.v. 5-FU and 5-FU patches animals survived 24+/-2.7 days and 39.5+/-1.87 days with ILS of 25.58% and 88.09%, respectively (p <0.01). There was significant sustained release of 5-FU through microprocessor-controlled patches and half-life was significantly higher (p <0.05) compared to the i.v. route. CONCLUSION: Cytotoxic concentration of 5-FU can be achieved through the transdermal drug delivery and effective therapeutic drug concentration can be maintained up to 24 h, with less toxicity. A new generation of transdermal drug delivery systems based on microprocessor-controlled iontophoresis is in the late stages of development and promises to enhance the treatment of local and systemic medical conditions. The incorporation of microprocessor into these systems has been an important advancement to ensure safe and efficient administration of a wide variety of drugs.

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.

Oxidative monensin metabolism and cytochrome P450 3A content and functions in liver microsomes from horses, pigs, broiler chicks, cattle and rats.

The oxidative metabolism of monensin, an ionophore antibiotic extensively used in veterinary practice as a coccidiostat and a growth promoter, was studied in hepatic microsomal preparations from horses, pigs, broiler chicks, cattle and rats. As assayed by the measurement of the amount of the released formaldehyde, the rate of monensin O-demethylation was nearly of the same order of magnitude in all species, but total monensin metabolism, which was estimated by measuring the rate of substrate disappearance by a high-performance liquid chromatography (HPLC) method, was highest in cattle, intermediate in rats, chicks and pigs, and lowest in horses. When expressed as turnover number (nmol of metabolized monensin/min nmol cytochrome P450-1), the catalytic efficiency (chick >> cattle >> pig approximately rat > horse) was found to correlate inversely with the well known interspecies differences in the susceptibility to the toxic effects of the ionophore, which is characterized by an oral LD50 of 2-3 mg/kg bodyweight (bw) in horses, 50-80 mg/kg bw in cattle and 200 mg/kg bw in chicks. Chick and cattle microsomes also displayed both the highest catalytic efficiency toward two P450 3A dependent substrates (erythromycin and triacetyloleandomycin) and the highest immunodetectable levels of proteins cross-reacting with anti rat P450 3A1/2. Further studies are required to define the role played by this isoenzyme in the oxidative biotransformation of the drug in food producing species.

Efeitos da monensina em bezerros holandeses submetidos a estresse pelo calor / Monesin effects in dutch dairy cattle submitted to heat stress

Este trabalho teve como objetivo estudar a ação da monensina em animais em estresse térmico sobre o desempenho, digestibilidade do alimento, parâmetros ruminais, concentrações de Ca, P, Mg, Na, Zn e Cu no soro sangüíneo e retenção e absorção destes minerais. Foram utilizados 24 bezerros machos da raça Holandesa (peso médiio 82 kg), estrutua de tramentos de fatorial 2 x 2, dois níveis de suplemntaç+o: 0 mg de monensina e 85 mg de mononsina/animal/dia e duas temperaturas: temperatura ambiente (24,3 ºC) e de estresse térmico (33,2 ºC)...

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes.

Cytoplasmic concentration of Mg(2+) ([Mg(2+)](i)) was measured with a fluorescent indicator furaptra in ventricular myocytes enzymatically dissociated from rat hearts (25 degrees C). To study Mg(2+) transport across the cell membrane, cells were treated with ionomycin in Ca(2+)-free (0.1 mM EGTA) and high-Mg(2+) (10 mM) conditions to facilitate passive Mg(2+) influx. Rate of rise of [Mg(2+)](i) due to the net Mg(2+) influx was significantly smaller in the presence of 130 mM extracellular Na(+) than in its absence. We also tested the extracellular Na(+) dependence of the net Mg(2+) efflux from cells loaded with Mg(2+). After [Mg(2+)](i) was raised by ionomycin and high Mg(2+) to the level 0.5-0.6 mM above the basal value ( approximately 0.7 mM), washout of ionomycin and lowering extracellular [Mg(2+)] to 1.2 mM caused rapid decline of [Mg(2+)](i) in the presence of 140 mM Na(+). This net efflux of Mg(2+) was completely inhibited by withdrawal of extracellular Na(+) and was largely attenuated by imipramine, a known inhibitor of Na(+)/Mg(2+) exchange, with 50% inhibition at 79 microM. The relation between the rate of net Mg(2+) efflux and extracellular Na(+) concentration ([Na(+)](o)) had a Hill coefficient of 2 and [Na(+)](o) at half-maximal rate of 82 mM. These results demonstrate the presence of Na(+) gradient-dependent Mg(2+) transport, which is consistent with Na(+)/Mg(2+) exchange, in cardiac myocytes.

Spectroscopic studies of PhTX facilitated cation movement across membranes.

Philanthotoxins, noncompetitive inhibitors of the nicotinic acetylcholine receptor and various glutamate receptors, were found to be capable of mediating cation transport across lipid bilayers. With respect to the relatively weak binding constants of these amphiphilic polyamines to neuronal receptor proteins, this finding implies that their interaction with cell membranes might have to be considered in addition to that with protein receptors to fully understand the molecular mechanism of these neurotoxins.

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.

Comparación del efecto protector de una vacuna y un ionóforo contra coccidias en pollos / Comparison between vaccine protector effect and a ionofore against coccidiosis in chickens

Se realizó un estudio con pollos de engorda alojados en piso, con el objeto de comparar la eficacia de una vacuna con Eimeria tenella, E. necatrix, E. acervulina y E. máxima contra el ionóforo narazina en el alimento en la prevención de coccidiosis. Se emplearon 320 pollos, divididos en cuatro lotes, cada uno con 4 repeticiones de 20 aves. El lote a recibió alimento sin coccidiostato. A las tres semanas de iniciado el experimento, los tres fueron confrontados con un inóculo formado con una mezcla de E. tenella, E. necatrix, E. acervulina y E. máxima. El lote D fue testigo negativo. Las variables registradas fueron peso, consumo de alimentos, índice de ooquistes en heces y cama, lesiones, mortalidad y pigmentación de los tarsos. Los resultados mostraron diferencias significativas entre las 0-4 semanas en la ganancia de peso y conversión alimenticia, la mejor ganancia de peso correspondió al grupo D, seguida del A, B y C, respectivamente. La conversión alimenticia fue más eficiente para el grupo D. Los datos de 4 a 7 y de 0 a 7 semanas no mostraron diferencias estadísticas en ninguno de los parámetros analizados. El índice anticoccidial fue el mejor para el grupo A seguido del B y C, respectivamente, lo cual indica que el inmunógeno proporcionó una adecuada protección contra la coccidiosis aviar

CP-72,588, a semisynthetic analog of the polyether ionophore UK-58,582 with increased anticoccidial potency.

We have employed semisynthesis to enhance the anticoccidial potency of a polyether ionophore. CP-72,588 is the alpha-methyl analog of the fermentation-derived polyether ionophore UK-58,852. The parent ionophore required a dose of 15 ppm to achieve anticoccidial efficacy in chickens equivalent to that of salinomycin at 60 ppm. CP-72,588 demonstrated substantially improved potency, with efficacy at 5 to 7.5 ppm. The intrinsic antimicrobial potencies of the two ionophores are similar; however, CP-72,588 was found in chicken tissues at higher levels than those of the parent ionophore when each was administered at the same dose (8 ppm). The enhanced potency of CP-72,588 may be partially due to enhanced uptake into tissues.

How to evaluate the distribution of an "invisible" amphiphile between biological membranes and water.

To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzyme's activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range Förster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.