Pharmacokinetics of butafosfan after intravenous and intramuscular administration in piglets.

The pharmacokinetics and bioavailability of butafosfan in piglets were investigated following intravenous and intramuscular administration at a single dose of 10 mg/kg body weight. Plasma concentration-time data and relevant parameters were best described by noncompartmental analysis after intravenous and intramuscular injection. The data were analyzed through WinNolin 6.3 software. After intravenous administration, the mean pharmacokinetic parameters were determined as T1/2λz of 3.30 h, Cl of 0.16 L kg/h, AUC of 64.49 ± 15.07 µg h/mL, Vss of 0.81 ± 0.44/kg, and MRT of 1.51 ± 0.27 h. Following intramuscular administration, the Cmax (28.11 µg/mL) was achieved at Tmax (0.31 h) with an absolute availability of 74.69%. Other major parameters including AUC and MRT were 48.29 ± 21.67 µg h/mL and 1.74 ± 0.29 h, respectively.

Effect of food on the bioavailability of lesogaberan given as an oral solution or as modified-release capsules in healthy male volunteers.

The novel Type B gamma-aminobutyric acid (GABAB)-receptor agonist lesogaberan (AZD3355) has been evaluated as an add-on to proton pump inhibitor treatment for gastroesophageal reflux disease, but the effect of food on the bioavailability of this compound has not been assessed. In this openlabel crossover study, healthy males received single 100 mg doses of lesogaberan (oral solution (A) or oral modified release (MR) capsules with a dissolution rate of 50% (B) or 100% (C) over 4 h) with and without food. Blood plasma concentrations of lesogaberan were assessed over 48 h. A log-transformed geometric mean Cmax and AUC ratio within the 90% confidence interval (CI) range (0.80 - 1.25) was defined as excluding a clinically relevant food effect. Overall, 57 subjects completed the study. Only the oral lesogaberan solution had a fed/fasting Cmax ratio outside the 90% CI range (Cmax ratio: 0.76). AUC ratios were within the 90% CI limits for all three lesogaberan formulations. The only substantial change in tmax associated with food intake was observed for the oral solution (1.0 h without food, 1.8 h with food). In conclusion, a clinically relevant food effect could be excluded for the lesogaberan MR formulations, but not for the oral lesogaberan solution.

The effect of intravenous magnesium hypophosphite in calcium borogluconate solution on the serum concentration of inorganic phosphorus in healthy cows.

The goal of this study was to determine the effect of intravenous (IV) administration of phosphite on the serum concentration of inorganic phosphorus in cows. Twelve clinically healthy cows were divided into four groups of three. All cows received 600 mL of a 40% calcium borogluconate solution; three cows each received this as a rapid (20 min) IV infusion with and without 6% magnesium hypophosphite, and three other cows each received this as a slow IV infusion (8 h) with and without 6% magnesium hypophosphite. Samples of blood were collected for the determination of serum concentrations of calcium, inorganic phosphorus and magnesium before and 10, 20, 40, 60 and 90 min and 2, 3, 4, 5, 6, 7, 8, 24, 48 and 72 h after the start of treatment. The concentration of calcium increased after treatment in all cows but the increase was most rapid in cows that received the rapid infusion. In cows that received the rapid IV infusion containing magnesium hypophosphite, the mean concentration of inorganic phosphorus decreased significantly 3-4 h after treatment compared with initial serum levels. The serum concentration of inorganic phosphorus did not change significantly in cows that received the rapid IV solution without magnesium hypophosphite or the slow IV infusion with or without magnesium hypophosphite. The serum concentration of magnesium increased after treatment in all cows receiving magnesium hypophosphite but remained unchanged in the others. The rapid infusion of calcium borogluconate without magnesium hypophosphite made all three cows anorexic and hypercalcaemic and the slow infusion made 1/3 anorexic. It is concluded that the IV administration of a calcium solution containing magnesium hypophosphite does not increase the serum concentration of inorganic phosphorus.

Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: kinetics, crystal structure and pharmacokinetics.

FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284-15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with K(i) values of 41 and 12 nM respectively. A high-resolution (1.35 A; 1 A=0.1 nm) crystal structure of the FAH.CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 mumol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation.

The temperature dependence of human erythrocyte transport of phosphate, phosphite and hypophosphite.

The temperature dependence of the erythrocyte anion transport protein (Band 3 or AE1) mediated influx of three nonspherical substrates, the divalent anions phosphate and phosphite, and the monovalent hypophoshite, were determined. Phase transitions were found in the temperature dependence of the influxes of all three anions. The 95% confidence limits for the transition temperatures were: 34.6-38.1 degrees C, 7.4-9.1 degrees C and 6.7-9.7 degrees C for phosphate, phosphite and hypophosphite, respectively, while the critical influx rates at the transitions were 29-50, 64-102 and 26-58 ions/s per carrier, respectively. That the critical rates rather than the transition temperatures are of similar magnitude indicates that the transitions are related to transport mechanisms rather than to thermal protein conformational changes. These critical rates are two orders of magnitude lower than those reported for the self-exchange of Cl- and Br- (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). The critical rate of monovalent hypophosphite is similar to that of divalent phosphate and phosphite, but not to that of Cl- indicating that this effect is mediated by the structure of the substrate rather than by its charge. The disparity in the rates rc at which phase transitions occur in AE1-mediated transport of spherical and nonspherical anions indicates a difference in the interaction between the two classes of anions and the protein.

Microviscosity of human erythrocytes studied with hypophosphite and 31P-NMR.

A 31P-NMR method, which complements earlier 13C-NMR procedures for probing the intra-erythrocyte microenvironment, is described. Hypophosphite is an almost unique probe of the erythrocyte microenvironment, since it is rapidly transported into the cell via the band 3 protein, and intra- and extracellular populations give rise to distinct resonances in the 31P-NMR spectrum. Relaxation mechanisms of the 31P nucleus in the hypophosphite ion were shown to be spin-rotation and dipole-dipole. Analysis of longitudinal relaxation rates in human erythrocytes, haemolysates and concentrated glycerol solutions allowed the determination of microviscosity using the Debye equation. Bulk viscosities of lysates and glycerol solutions were measured using Ostwald capillary viscometry. Translational diffusion coefficients were then calculated from the viscosity estimates using the Stokes-Einstein equation. The results with a range of solvent systems showed that 'viscosity' is a relative phenomenon and that bulk (i.e., macro-) viscosity is therefore not necessarily related to the NMR-determined viscosity. The intracellular NMR-determined viscosities from red cells, ranging in volume from 65.5 to 100.1 fl, varied from 2.10 to 2.67 mPa s. This is consistent with the translational diffusion coefficients of the hypophosphite ion altering by only 20%, whereas the values determined from bulk viscosity measurements conducted on lysates of these cells are consistent with a 230% change.

Hypophosphite ion as a 31P nuclear magnetic resonance probe of membrane potential in erythrocyte suspensions.

Hypophosphorus acid has a single pKa of 1.1 and at physiological pH values it is therefore present almost entirely as the univalent hypophosphite ion. When added to a red cell suspension the ion crosses the cell membrane rapidly, via the anion exchange protein, and the intra- and extracellular populations of the ion give rise to separate 31P NMR resonances. From a single 31P NMR spectrum it was possible to determine the relative amounts of hypophosphite in the intra- and extracellular compartments and thereby estimate the corresponding concentrations. The ratio of intracellular to extracellular hypophosphite concentration was independent of the total hypophosphite concentration for cells suspended in NaCl solutions and was independent of hematocrit. The hypophosphite distribution ratio increased as extracellular NaCl was replaced iso-osmotically with citrate or sucrose, through it remained very similar to the corresponding hydrogen ion distribution ratio. Incorporation of the hypophosphite distribution ratio into the Nernst equation yielded an estimate of the membrane potential. For cells suspended in NaCl solutions the estimated potential was consistently around -10 mV.

The contribution of magnetic susceptibility effects to transmembrane chemical shift differences in the 31P NMR spectra of oxygenated erythrocyte suspensions.

Triethyl phosphate, dimethyl methylphosphonate, and the hypophosphite ion all contain the phosphoryl functional group. When added to an oxygenated erythrocyte suspension, the former compound gives rise to a single 31P NMR resonance, whereas the latter compounds give rise to separate intra- and extracellular 31P NMR resonances. On the basis of experiments with intact oxygenated cell suspensions (in which the hematocrit was varied) and with oxygenated cell lysates (in which the lysate concentration was varied), it was concluded that the chemical shifts of the intra- and extracellular populations of triethyl phosphate differ as a consequence of the diamagnetic susceptibility of intracellular oxyhemoglobin but that this difference is averaged by the rapid exchange of the compound across the cell membrane. The difference in the magnetic susceptibility of the intra- and extracellular compartments contributes to the observed separation of the intra- and extracellular resonances of dimethyl methylphosphonate and hypophosphite. The magnitude of this contribution is, however, substantially less than that calculated using a simple two-compartment model and varies with the hematocrit of the suspension. Furthermore, it is insufficient to fully account for the transmembrane chemical shift differences observed for dimethyl methylphosphonate and hypophosphite. An additional effect is operating to move the intracellular resonances of these compounds to a lower chemical shift. The effect is mediated by an intracellular component, and the magnitude of the resultant chemical shift variations depends upon the chemical structure of the phosphoryl compound involved.