Influence of microsomal concentration on apparent intrinsic clearance: implications for scaling in vitro data.

The influence of microsomal concentration on unbound fraction (fu(mic)), half-life (t(1/2)), apparent intrinsic clearance (CL(int,app)) and apparent Michaelis-Menten constant (K(m,app)) was examined for two compounds, one representative of high nonspecific binding to microsomes (compound A) and one representative of low (compound B). Kinetic parameters were estimated for the two probe compounds at two human microsomal protein concentrations (0.46 and 2.3 mg/ml) and cytochrome P450 concentrations (0.20 and 1.0 microM), representing a 5-fold difference in microsomal concentration. For compound A, fu(mic) and CL(int,app) were inversely proportional to microsomal concentration. Conversely, the K(m,app) of compound A was proportional to microsomal concentration and the half-life was unchanged. For compound B, half-life was inversely proportional to microsomal concentration. In this case, fu(mic), CL(int,app), and K(m,app) were not proportionally influenced. The experimental observations were entirely consistent with that predicted by a mathematical relationship between microsomal concentration, fu(mic), t(1/2), CL(int,app), and K(m,app). These results demonstrate that when nonspecific binding is extensive, CL(int,app) is dependent on the arbitrary choice of microsomal concentration included in the incubation.

Disposition and metabolism of the novel macrolide antibiotic CP-163505 in cattle.

The plasma pharmacokinetics, lung tissue to plasma concentration ratios, and depletion profiles in edible tissue (liver, muscle, kidney, fat and injection site) for a single subcutaneous dose of a novel macrolide antibiotic, CP-163505 (20-[3-dimethylaminopropyl(L-alanyl)amino]-20-deoxo-repromicin), were investigated in crossbred beef cattle. Mean peak plasma concentration of 2.5 +/- 0.4 micrograms/mL, occurring at 0.5 h, was found for CP-163505 following a 5 mg/kg dose (n = 5). The pharmacokinetic profile consisted of a distribution phase, followed by an extended terminal elimination phase (t1/2 of 19 h). The disposition of CP-163505 was characterized by distribution from the plasma into the tissue resulting in lung to plasma ratios of 103 and 87 at 72 h following a single 5 or 10 mg/kg dose, respectively. The depletion of CP-163505 from edible tissues was determined following administration of tritiated CP-163505 at a dose of 10 mg/kg. On day 42, the liver contained the highest mean concentration of total tritium residues, 5.9 +/- 3.4 micrograms/g. CP-163505 was determined to be a significant component of the total residues in liver with 72% on day 3 and 50% on day 42. Three metabolites of CP-163505 were identified by liquid chromatography with mass spectrometry (LC/MS/MS) in liver samples: loss of alanine, formation of an hydroxyl derivative, and sulfate addition to the lactone ring.

Simultaneous determination of danofloxacin and N-desmethyldanofloxacin in cattle and chicken edible tissues by liquid chromatography with fluorescence detection.

A rugged, simple, and selective method for the determination of danofloxacin and its primary metabolite, N-desmethyldanofloxacin, in cattle (liver, muscle, kidney, and fat) and chicken (liver and muscle) tissues was developed. The method is selective for danofloxacin and N-desmethyldanofloxacin over other veterinary important fluoroquinolones, such as enrofloxacin, ciprofloxacin, norfloxacin, and ofloxacin. Selectivity is achieved through a combination of extraction, chromatography, and fluorescence detection. The analytes were extracted from homogenized tissues using a methanol-perchloric-phosphoric acid solution. After centrifugation, direct injection of extraction supernate was possible. The limit of quantitation was 20 pg on column. Separation was achieved on an Inertsil C8 (5 microns, 100 A) column with dimensions of 250 x 4.6 mm I.D. The mobile phase consisted of 0.05 M phosphate buffer (pH 3.5)-acetonitrile (88:12). A fluorescence detector was utilized with an excitation wavelength of 280 nm and an emission wavelength of 440 nm. The assay was accurate and reproducible within the range of 10 to 500 ng/g for both danofloxacin and N-desmethyldanofloxacin. Intra-assay accuracy was between 98 and 101%, and precision was less than 4%. Inter-assay accuracy was between 99 and 102%, while precision was less than 2%. Recoveries for both analytes over the dynamic range were greater than 90% for all the tissues.

Electrophoretically mediated micro-assay of alkaline phosphatase using electrochemical and spectrophotometric detection in capillary electrophoresis.

Electrophoretically mediated micro-assays of alkaline phosphatase (ALP) are demonstrated in capillary electrophoretic systems using both electrochemical (ED) and spectrophotometric (UV) detection. In the ED mode, p-aminophenylphosphate was used as the substrate and p-aminophenol (pAP) was monitored at a carbon fiber electrode held at +180 mV vs. Ag/AgCl. Spectrophotometric detection was achieved using the substrate p-nitrophenylphosphate, and monitoring the product p-nitrophenol (pNP) at 405 nm. The detection limit for pAP by ED was determined to be 100-fold lower than for pNP using UV detection. In the determination of ALP, both methods were found to be linear. The detection limit for ALP using zero potential assays with UV detection was determined to be 1.8.10(-6) mg/ml compared to 1.8.10(-7) mg/ml using ED.

Pharmacokinetic studies using microdialysis probes in subcutaneous tissue: effects of the co-administration of ethanol and acetaminophen.

Loop geometry microdialysis probes with membrane lengths of 40-60 mm were used to monitor the effects of acute and chronic doses of ethanol on acetaminophen pharmacokinetics in awake, freely-moving rats. Microdialysis probes used in this configuration provide very high concentration recoveries and good precision at flow rates below 2 microliters min-1. The ability of microdialysis to monitor pharmacokinetics in subcutaneous tissue and blood vessels is compared. Dialysates acquired simultaneously from both blood vessels and subcutaneous tissue showed corresponding disposition for acetaminophen. Acute intraperitoneal doses of ethanol (1 ml kg-1) are shown to increase the relative bioavailability, measured as AUC, by 40%, elimination half-life by 24%, and changes in CL and Vd were also observed. Larger doses of ethanol, up to 2 ml kg-1, had a similar incremental effect on the pharmacokinetic parameters in some animals, but apparent decreased abdominal blood flow in others caused diminished absorption and drastically altered pharmacokinetic parameters. Chronic doses of ethanol (5% in drinking water for 14 days) caused an increase in bioavailability and other pharmacokinetic parameters, but changes were not as significant as following acute doses. Acute doses of ethanol (1 ml kg-1) were also observed to change the pharmacokinetics of acetaminophen at hepatotoxic levels of the drug. However, acute intraperitoneal doses of acetaminophen (10 mg kg-1) were observed not to have an effect on ethanol pharmacokinetics.

Determination of endogenous ions in intercellular fluid using capillary ultrafiltration and microdialysis probes.

Capillary ultrafiltration probes are novel sampling tools for continuously monitoring small molecules in the extracellular fluid of awake animals. Capillary ultrafiltration uses a vacuum applied to hydrophilic membrane fibres and extracts intercellular fluid and quantitatively recover many small hydrophilic molecules. The effects of continuously removing a small amount of fluid from the interstitial space are not known. The concentration of sodium, potassium, calcium and inorganic phosphorus were determined in the collected ultrafiltrates from subcutaneous tissue. These values were compared to literature values and to concentrations determined for the same animals using microdialysis. The concentrations of sodium, potassium, calcium and inorganic phosphorous were found to be 140 +/- 4, 3.7 +/- 0.1, 1.1 +/- 0.1 and 1.7 +/- 0.1 mM, respectively, in the subcutaneous ultrafiltrates obtained from rats. These corresponded very well with literature values and microdialysates, obtained, using pure water as the perfusate, in subcutaneous tissue. The concentration of sodium and potassium were determined to be 142 +/- 2 mM and 3.6 +/- 0.2 mM, respectively, for the dialysates. Hyperinsulinemic-induced decrease in intercellular potassium levels under a euglycemic clamp were monitored using capillary ultrafiltration probes in rats to further validate this technique for monitoring small molecule dynamics in the intercellular space. The intercellular level of potassium in rats decreased from 3.6 +/- 0.5 to 2.6 +/- 0.3 mM after an acute dose of pork insulin.

Pharmacokinetic studies using micellar electrokinetic capillary chromatography with in vivo capillary ultrafiltration probes.

Micellar electrokinetic capillary chromatography (MECC) is shown to be a quantitative method for the determination of theophylline in capillary ultrafiltrates of biological systems. MECC exhibits reproducibility in migration times of 1.3% relative standard deviation (R.S.D.) (n = 31) and peak heights of 3.0% R.S.D. (n = 28). MECC and reversed-phase liquid chromatography (LC) are shown to be complementary techniques for the determination of theophylline in ultrafiltrate samples. In vivo sampling of awake, freely moving rats is achieved using capillary ultrafiltration probes implanted in subcutaneous tissue. The ability of MECC coupled with in vivo capillary ultrafiltration to determine theophylline pharmacokinetics is demonstrated. The half-life of elimination for a 15 mg/kg intraperitoneal dose of theophylline was determined to be 3.1 +/- 0.4 h for MECC and 3.2 +/- 0.4 h for LC (n = 4, mean +/- standard error of the mean). Concurrent results for derived pharmacokinetic parameters (area under the curve, volume of distribution, concentration at time zero and clearance) were obtained for MECC and LC.

Pharmacokinetic monitoring in subcutaneous tissue using in vivo capillary ultrafiltration probes.

Capillary ultrafiltration probes were utilized for in vivo sampling of therapeutic drugs in awake rats. Capillary ultrafiltration probes implanted into subcutaneous tissue were able to follow the disposition of acetaminophen and theophylline. Ultrafiltration probes provided samples at a rate of 2-3 microL/min. Ultrafiltrates were analyzed by liquid chromatography with either UV or electrochemical detection. Simultaneous ultrafiltration and microdialysis probes and multiple ultrafiltration probes were used in individual animals to validate the technique. The pharmacokinetics of two well-established drugs, acetaminophen and theophylline, were monitored in awake, freely moving rats to demonstrate the viability of the technique. The half-life for acetaminophen was determined to be 20.9 +/- 1.0 min (n = 6) for a 2 mg/kg dosing. The half-life of elimination for theophylline was determined to be 3.0 +/- 0.1 hr (n = 4) for a 4 mg/kg dose. The capillary ultrafiltration probes exhibited a constant flow rate of 2.4 +/- 0.1 microL/min and removed 50 nL/min/mm of fluid from the extracellular space. Capillary ultrafiltration sampling is shown to be an excellent tool for in vivo monitoring of drug disposition and a suitable method for determining pharmacokinetic parameters in awake animals.

Capillary ultrafiltration: in vivo sampling probes for small molecules.

Capillary ultrafiltration is a novel sampling method convenient for low molecular weight substances in living biological systems. By application of a negative pressure across a hydrophilic membrane capillary, small molecules are actively "pulled" across the membrane and collected. By elimination of large molecules and cellular matter, the ultrafiltrate collected is well suited for further analysis by liquid chromatography, capillary electrophoresis, or mass spectrometry. Ultrafiltration probes (UF probes) provide a simple means to obtain a small-volume sample from subcutaneous tissue, blood, saliva, or any other biological fluid in vivo. The dependence of recovery on flow rate, temperature, membrane dimensions, and vacuum magnitude are considered. The relative merits of capillary ultrafiltration probes and microdialysis probes are considered. UF probe applications presented include in vivo monitoring of drug disposition in human saliva and in the subcutaneous space of awake, freely moving rats.

In vivo sampling using loop microdialysis probes coupled to a liquid chromatograph.

Microdialysis probes with longer membranes (20-100 mm) provide increased relative recovery over traditional shorter probes (1-4 mm) developed for neuroscience applications. The characterization and optimization of "straight through" or "loop type" probes for use in subcutaneous tissue are considered. Membrane area, probe size, inlet and outlet tubing dimensions, and flow-rate are examined for their effects on relative recovery, the total collection rate, and bulk flow through the membrane wall. Polyacrylonitrile and regenerated cellulose membrane fibers with different geometries were compared. Sampling probes used fibers 3-10 cm long. Inlet and outlet tubing was varied from 25 to 110 microns I.D. with lengths of 10 to 50 cm. Probe configurations optimized for relative recovery, flow-rate, and utility for in vivo use are presented. Utilizing microdialysis probes with large membrane surface areas results in relative concentration recovery of greater than 50% at flow-rates of greater than 5 microliters/min. Therapeutic drug monitoring in subcutaneous tissue of awake animals is explored.