Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes.

Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents. Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective. A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages. This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol. To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy. The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively. ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels. Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system. The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages.

Iontophoretic transport of oligonucleotides across human epidermal membrane: a study of the Nernst-Planck model.

The objective of this study was to investigate the transport behavior of a series of oligonucleotides with human epidermal membrane (HEM) and to examine the applicability of the modified NERNST-PLANCK model to transdermal iontophoresis of these macromolecules. Iontophoretic transport experiments were first carried out in a synthetic model membrane system (Nuclepore membranes) with a four-electrode potentiostat to examine the baseline modified NERNST-PLANCK model. The modified NERNST-PLANCK model derived from the Einstein relation and the Stokes-Einstein equation taken from previous work did not hold for the oligonucleotides. Results obtained in the Nuclepore studies were, however, consistent with predictions of the modified NERNST-PLANCK model using the experimentally determined electromobilities and diffusion coefficients. The electromobilities of the oligonucleotides (determined by capillary electrophoresis) were found to be more than a factor of two smaller than expected from the Einstein relation between electromobilities and diffusion coefficients (the latter determined in diffusion cell experiments). A correlation between these electromobilities and the theoretical electromobilities estimated by considering the effects of counterion binding and the effects of mobility reduction according to colloid theory was also observed. These results suggest that the modified NERNST-PLANCK model predictions are satisfactory only when the electromobilities and the effective molecular size of the oligonucleotides are known and are used directly to predict the iontophoretically enhanced transport. Results with the HEM experiments generally agreed with model predictions based on the experimental electromobilities. The oligonucleotide HEM flux data also suggest the existence of pores with effective pore radii greater than the effective radii estimated in previous studies with small molecular weight model permeants.

Intercellular adhesion molecule-1 suppression in skin by topical delivery of anti-sense oligonucleotides.

We topically applied 20 nucleotide phosphorothioate intercellular adhesion molecule-1 anti-sense oligodeoxynucleotide in a cream formulation. It effectively inhibited tumor necrosis factor-alpha-induced expression of intercellular adhesion molecule-1 in human skin transplanted on severe compromised immunodeficient mice. The effects were concentration dependent, sequence specific, and resulted from reduction of intercellular adhesion molecule-1 mRNA levels in the skin. Intravenous administration of the drug did not show pharmacologic effects, probably due to insufficient drug concentrations in skin. Topical delivery, however, produced a rapid and a significantly higher accumulation of oligodeoxynucleotide in the epidermis and dermis. The results strongly suggest that topically applied anti-sense oligonucleotides can be delivered to target sites in the skin and may be of considerable value in the treatment of psoriasis and other inflammatory skin disorders.

Improvement of the encapsulation efficiency of oligonucleotide-containing biodegradable microspheres.

The objective of this study was to encapsulate an oligonucleotide drug within poly(lactide) microparticles with high encapsulation efficiencies at high theoretical drug loadings by the solvent evaporation method. With the conventional W/O/W method, the encapsulation efficiency decreased with increasing internal water content, increasing stirring time prior to filtration of the microparticles and increasing drug loading. The encapsulation was improved by replacing methylene chloride with ethyl acetate, by using micronized drug powder instead of an internal aqueous phase or by adding electrolytes or nonelectrolytes to the external phase. With ethyl acetate, a pre-emulsification step into a smaller volume of external aqueous phase was necessary in order to avoid premature polymer precipitation and to obtain microparticles. The addition of salts (NaCl or MgCl(2)) or sorbitol to the external aqueous phase significantly improved the encapsulation efficiency, even at high theoretical drug loadings. The microparticles had a denser structure with a smooth, pore-free surface.

Microencapsulation of antimicrobial ceftiofur drugs.

Polymeric microparticles containing two ceftiofur salts as antimicrobial agents for intramammary application in dry cows were prepared by modified o/w-solvent evaporation methods (dispersion or cosolvent method) or by a w/o/w-multiple emulsion solvent evaporation method. The microspheres were characterized with respect to drug loading, drug release, and morphological properties. The three methods resulted in high encapsulation efficiencies. The choice of organic solvent/solvent mixture strongly affected the structure of the microparticles; both matrix and reservoir-type structures with different porosities were obtained. Scaling up to larger batch sizes resulted in microspheres with a faster drug release. The addition of water-miscible cosolvents to the water-immiscible polymer solution allowed the preparation of microparticles from a drug solution rather than a drug dispersion. Microparticles prepared by the cosolvent method could be separated after shorter time intervals from the aqueous phase; the microspheres had a denser matrix with finely dispersed drug crystals and a slower drug release when compared with microspheres prepared by the dispersion method, which had a more porous structure with larger embedded drug crystals. The cosolvent and dispersion methods present a simple alternative to the w/o/w-solvent evaporation method for the encapsulation of water-soluble drugs with an external water phase.

Effects of an infusion of dopamine on the cardiopulmonary effects of Escherichia coli endotoxin in anaesthetised horses.

Horses with colic may be endotoxaemic and subsequently develop hypotension during anaesthesia for surgical operation. The aim of this study was to evaluate the efficacy of dopamine as a means to improve cardiovascular function in anaesthetised endotoxaemic horses. Nine horses (five in group 1 and four in group 2) were anaesthetised with thiopentone and guaifenesin and anaesthesia was maintained with halothane. After approximately one hour, facial artery pressure, heart rate, pulmonary artery pressure, cardiac output, temperature, pHa, PaCO2, PaO2, base excess, packed cell volume, plasma protein concentration and white cell count were measured (time 0). Escherichia coli endotoxin was infused intravenously over 15 minutes in both groups. Group 2 horses were given an intravenous infusion of dopamine (5 micrograms kg-1 min-1) starting five minutes after the start of the endotoxin infusion and continuing for 60 minutes. Measurements were made at 15 minute intervals for 120 minutes. In group 1, one horse died during the endotoxin infusion and in two other horses mean facial artery pressures decreased to 50 mm Hg. Total pulmonary vascular resistance and packed cell volume were significantly increased. Cardiac output, cardiac index and change in mean arterial pressure were significantly greater in group 2 horses than in group 1 horses. Conversely, diastolic pulmonary artery pressure, total vascular resistance and total pulmonary resistance were significantly less in group 2 than in group 1. PaO2, base excess and white blood cell count were significantly decreased in both groups. It was concluded that dopamine improved cardiovascular function in the presence of endotoxaemia and attenuated the rate of haemoconcentration, but had no effect on the development of decreased PaO2 or metabolic acidosis.

Efeitos de baixas doses de Flunixin Meglumine na endotoxemia experimental em cavalos / Low dose Flunixin Meglumine: effects on experimental endotoxaemia in horses

A aplicaçäo de Flunixin Meglumine antes do desafio endotoxínico causou significante supressäo na geraçäo plasmática de tramboxane e 6-Keto-prostaglandina F1 alfa em eqüinos. Na dose de 0,25 mg/kg de peso vivo, o produto provocou, em cavalos pré-medicados, significativa supressäo nos índices elevados de lactado sangüíneo. A reduçäo dos sintomas clínicos de endotoxemia por açäo do Flunixin Meglumine foi dependente, facilitando a avaliaçäo clínica do animal

HPLC determination and pharmacokinetics of thiabendazole and its major metabolite 5-OH thiabendazole in equine plasma.

Separate high performance liquid chromatographic methods were developed for thiabendazole (TBZ) and 5-hydroxy thiabendazole (5-OH-TBZ) determination in horse plasma using 1-methyl-2-phenyl benzimidazole (MPBZ) as an internal standard. In both methods TBZ and 5-OH-TBZ were extracted from plasma using organic solvents, injected on to a C-18 column, and eluents monitored by a fluorescence detector. However, mobile phase composition, extraction solvent as well as detector wavelength differed in the two methods. The linear range for TBZ was 0.02 to 0.77 microgram ml-1 while that for 5-OH-TBZ was 0.96 to 8.0 micrograms ml-1. A commercially available TBZ oral suspension was administered to four thoroughbred horses in the following manner: days 1 and 2, 44 mg kg-1; days 4 and 5, 440 mg kg-1. Blood samples were collected during the 24 hours after administration and then analysed for TBZ and 5-OH-TBZ. Half-lives (t1/2), maximum plasma concentrations (Cmax), area under plasma concentration time curves (AUC O-alpha), and relative apparent bioavailability (F), were determined using pharmacokinetic equations. The pharmacokinetic parameters varied in the following manner: 1.16 to 13.63 hours (t1/2), 12 to 131 micrograms ml-1 X hours (AUC O-alpha), 3.33 to 8.90 micrograms ml-1 (Cmax), 1.38 to 0.12 (F) after 44 mg kg-1 and 440 mg kg-1 doses, respectively. The ratios of concentrations of TBZ to 5-OH-TBZ after oral administration of TBZ, were significantly lower for 44 mg kg-1 than 440 mg kg-1 doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Low dose flunixin meglumine: effects on eicosanoid production and clinical signs induced by experimental endotoxaemia in horses.

The efficacy of low doses of flunixin meglumine in reducing eicosanoid generation and clinical signs in response to experimentally induced endotoxaemia was investigated. Thromboxane B2 and 6-keto-prostaglandin F1 alpha were measured in serum and plasma by radioimmunoassay. Plasma flunixin concentrations were determined by high performance liquid chromatography and pharmacokinetic parameters derived non-compartmentally. In horses administered flunixin meglumine before endotoxin challenge, a significant suppression in plasma thromboxane B2 and 6-keto-prostaglandin F1 alpha generation was observed. Elevations in blood lactate were significantly suppressed in horses pretreated with 0.25 mg/kg bodyweight flunixin meglumine. Reduction of the clinical signs of endotoxaemia by flunixin meglumine was dose dependent. Low doses of flunixin inhibited eicosanoid production without masking all of the physical manifestations of endotoxaemia necessary for accurate clinical evaluation of the horse's status.

Hypertension in bulls and steers anesthetized with guaifenesin-thiobarbiturate-halothane combination.

Eight bulls and steers (research animals) and 18 bulls (surgical patients) were anesthetized with guaifenesin and thiopental or thiamylal and for 90 minutes with halothane. Arterial blood pressure and heart rate were recorded in all animals. Cardiac output, plasma glucose and lactate concentrations, PCV, plasma proteins and plasma thromboxane B2 values were determined before (control) and every 15 minutes during anesthesia in the research animals. Plasma catecholamine concentrations were measured in 3 of the research animals and 3 of the surgical patients. Arterial pressure, heart rate, and plasma thromboxane B2 and catecholamine concentrations were also measured immediately after the trachea was intubated. All animals, except one, were hypertensive during anesthesia. Heart rate during anesthesia was significantly increased, compared with control measurements, and cardiac output was decreased. Plasma glucose and lactate values significantly increased when the animals were restrained on their sides. Plasma glucose concentrations remained increased during anesthesia, but lactate decreased. Packed cell volume and plasma proteins were unchanged by the induction of anesthesia. Plasma norepinephrine concentration was unchanged during anesthesia, and epinephrine concentration was decreased. Endotracheal intubation caused a transient significant increase in arterial pressure, heart rate, and thromboxane B2 and a nonsignificant increase in norepinephrine.

Effects of flunixin meglumine, phenylbutazone and a selective thromboxane synthetase inhibitor (UK-38,485) on thromboxane and prostacyclin production in healthy horses.

The efficacy of three agents which alter the metabolism of arachidonic acid was investigated in normal, conscious horses. A dose response evaluation was made of flunixin meglumine and phenylbutazone, two cyclo-oxygenase inhibitors, and of a selective thromboxane synthetase inhibitor, UK-38,485. Radioimmunoassay of thromboxane B2 (TxB2) and 6-keto prostaglandin F1 alpha (PGF1 alpha) was used to assess the concentrations of thromboxane A2 (TxA2) and prostacyclin (PGI2) respectively, in serum. Flunixin was the most potent inhibitor of serum TxB2 and 6-keto PGF1 alpha production. UK-38,485 also decreased serum TxB2 generation while significantly increasing serum 6-keto PGF1 alpha levels, thus confirming its selectivity as a thromboxane synthetase inhibitor.

Modulation of arachidonic acid metabolism in endotoxic horses: comparison of flunixin meglumine, phenylbutazone, and a selective thromboxane synthetase inhibitor.

Two cyclooxygenase inhibitors (flunixin meglumine and phenylbutazone) and a selective thromboxane synthetase inhibitor were assessed in the management of experimental equine endotoxemia. Drugs or saline solution were administered to 16 horses 15 minutes before administration of a sublethal dose of endotoxin (Escherichia coli 055:B5). Plasma concentrations of thromboxane B2 (TxB2), prostacyclin (6-keto PGF1 alpha), plasma lactate, and hematologic values and clinical appearance were monitored for 3 hours after endotoxin administration. Pretreatment with flunixin meglumine (1 mg/kg of body weight) prevented most of the endotoxin-induced changes and correlated with a significant decrease in plasma TxB2 and 6-keto PGF1 alpha concentrations, compared with concentrations in nontreated horses (ie, pretreated with saline solution). Pretreatment with phenylbutazone (2 mg/kg) attenuated the effects of endotoxin and was associated with a brief, early, significant increase in plasma TxB2 concentrations, but not in plasma 6-keto PGF1 alpha concentrations. Pretreatment with the thromboxane synthetase inhibitor did not appear to clinically benefit the horses involved; however, arachidonic acid metabolism was redirected to prostacyclin production.

Flunixin meglumine given in small doses: pharmacokinetics and prostaglandin inhibition in healthy horses.

The pharmacokinetics and inhibition of prostaglandin synthesis in conscious horses given various dosages of flunixin meglumine were studied. Plasma concentrations of flunixin were measured by high-performance liquid chromatography, and serum thromboxane B2 and 6-keto prostaglandin F1 alpha were quantitated by radioimmunoassay. Within the dosage range studied, linear pharmacokinetics were achieved. After IV administration of flunixin (1.1 mg/kg, 0.25 mg/kg, 0.1 mg/kg), significant suppression of serum thromboxane generation persisted for 12, 4, and 3 hours, respectively. Repeated administrations of flunixin (0.25 mg/kg) once every 8 hours maintained significant suppression of thromboxane generation for the duration of treatment. After treatment with flunixin was stopped, serum thromboxane generation exceeded base line (pretreatment values). Among the groups, significant alteration of 6-keto prostaglandin F1 alpha production was not observed.

Pharmacokinetics of flunixin meglumine in the cow.

Plasma levels of flunixin were measured in heifers after a single intravenous injection (1.1 mg kg-1), using high performance liquid chromatography. Plasma concentration versus time curves were best described by a two compartment model. The distribution phase (alpha) half-life was 0.294 hours, the elimination phase (beta) half-life was 8.12 hours and the volume of distribution was 1050 ml kg-1.

Cardiopulmonary effects of prostacyclin infusion in anesthetized horses.

Prostacyclin was infused IV into 6 horses anesthetized with halothane. Three dosage rates (10, 30, and 100 ng/kg of body weight/min) were evaluated in each horse. Facial and pulmonary artery pressures, heart rate, cardiac output, blood temperature, and arterial and mixed venous pH, PCO2, and PO2 were measured. Arterial blood was collected for determination of glucose, lactate, and PCV. Mixed venous blood was sampled for assay of 6-keto-prostaglandin F1 alpha and catecholamines. Infusion of prostacyclin at 10 ng/kg/min had no effect on the variables measured, whereas the 30 ng/kg/min dosage decreased diastolic and mean arterial pressure at 15 and 30 minutes and PaO2 at 15 minutes (P less than 0.05). Prostacyclin infusion at 100 ng/kg/min significantly decreased arterial pressure, total vascular resistance, and total pulmonary resistance. Heart rate increased slightly, and cardiac output increased by 44%. Arterial PO2 decreased from 311 mm of Hg to 137 and 135 mm of Hg at 15 and 30 minutes, respectively. Blood glucose was increased. Prostacyclin infusions of 30 and 100 ng/kg/min increased blood concentrations of 6-keto-prostaglandin F1 alpha by factors of 5 and 40, respectively. Significant changes in catecholamine concentrations did not occur.

Pharmacokinetics of flunixin meglumine in dogs.

The pharmacokinetics of flunixin meglumine, a potent nonsteroidal anti-inflammatory agent, were studied in 6 intact, awake dogs. Plasma samples were obtained up to 12 hours after IV administration of flunixin meglumine. Flunixin concentration was determined, using high performance liquid chromatography. Plasma data best fit a 2-compartment model. Distribution half-life was 0.55 hour; elimination half-life was 3.7 hours; volume of distribution (area) was 0.35 L/kg; volume of distribution at steady state was 0.18 L/kg; volume of the central compartment was 0.079 L/kg; and total body clearance was 0.064 L/hr/kg. Flunixin concentrations obtained over a 6-hour period in 3 dogs with septic peritonitis did not differ significantly from those obtained from healthy dogs.

Pheochromocytoma in the horse and measurement of norepinephrine levels in horses.

Ten cases of pheochromocytoma in horses were obtained from the literature and a computer search of medical records. The clinical, laboratory and pathological features of pheochromocytoma in horses were reviewed. Pheochromocytoma is a catecholamine secreting tumor which tends to occur in older horses without breed or sex predisposition. It is usually unilateral adrenal medullary in location and benign. Malignancy was present in one horse. The most common clinical signs were sweating, tachycardia, tachypnea, muscle tremor and anxiety; however the tumor may be asymptomatic. Clinical signs were nonspecific and could be confused with other diseases, especially abdominal pain. Hyperglycemia is a consistent finding. Venous norepinephrine levels were measured in normal horses. Norepinephrine measurements may prove to be a diagnostic aid in horses with pheochromocytoma.

Tobacco smoke xenobiotic compound appearance in mothers' milk after involuntary smoke exposures. I. Nicotine and cotinine.

In the development of the extraction procedure for the analysis of nicotine and cotinine from a single breast milk sample, nicotine and cotinine were detected in 3 of 10 nonsmoking mothers' milk samples. Interviews of these women revealed the presence of nonsmoking husbands and households but of tobacco smoke exposures during the working day. Clinically these levels may be considered inconsequential in regard to the threat to the mother and nursing infant but may be important in studies designed to monitor tobacco smoke xenobiotic compound appearance in breast milk.