Daily peritoneal administration of sodium pyrophosphate in a dialysis solution prevents the development of vascular calcification in a mouse model of uraemia.

BACKGROUND: The high rate of cardiovascular mortality in patients with end-stage renal disease (ESRD) is a significant barrier to improved life expectancy. Unique in this population is the marked development and aggressive worsening of vascular calcification (VC). Pyrophosphate (PPi), an endogenous molecule, appears to naturally inhibit soft tissue calcification, but may be depressed in chronic kidney disease (CKD) and ESRD. Although once thought to be a promising therapeutic, PPi's very short half-life in circulation curtailed earlier studies. We tested the possibility that a slow, continuous entry of PPi into the circulation and prevention of VC might be achieved by daily peritoneal dialysis (PD). METHODS: Pharmacokinetic studies were first carried out in rats with renal impairment resulting from a 5/6 nephrectomy. Efficacy studies were then performed in the apolipoprotein E gene knockout mouse model overlaid with CKD. PPi was delivered by means of a permanent peritoneal catheter in a solution simulating PD, but without the timed removal of spent dialysate. von Kossa's staining followed by semiquantitative morphological image processing, with separation of inside (intimal) and outside (presumed medial) lesions, was used to determine aortic root calcification. RESULTS: In comparison to an intravenous bolus, delivery of PPi in a PD solution resulted in a slower, extended delivery over >4 h. Next, the efficacy studies showed that a 6-day/week PD-simulated administration of PPi resulted in a dose-dependent inhibition of aortic calcification in both intimal and medial lesions. A dose-response effect on total aortic calcification was also documented, with a full inhibition seen at the highest dose. A limited peritoneal catheter-related inflammation was observed, as expected, and included the placebo-treated control groups. This inflammatory response could have masked a lower level PPi-specific adverse effect, but none was observed. CONCLUSIONS: Our findings suggest potential for PPi, administered during PD, to prevent the development of VC and to potentially extend the life of ESRD patients.

Itraconazole IV nanosuspension enhances efficacy through altered pharmacokinetics in the rat.

The goal of this research was to evaluate an intravenous itraconazole nanosuspension dosage form, relative to a solution formulation, in the rat. Itraconazole was formulated as a nanosuspension by a tandem process of microcrystallization followed by homogenization. Acute toxicity, pharmacokinetics, and distribution were studied in the rat, and compared with a solution formulation of itraconazole. Efficacy was studied in an immunocompromised rat model, challenged with a lethal dose of either itraconazole-sensitive or itraconazole-resistant C. albicans. Itraconazole nanosuspension was tolerated at significantly higher doses compared with a solution formulation. Pharmacokinetics of the nanosuspension were altered relative to the solution formulation. C(max) was reduced and t(1/2) was much prolonged. This occurred due to distribution of the nanosuspension to organs of the monocyte phagocytic system (MPS), followed by sustained release from this IV depot. The higher dosing of the drug, enabled in the case of the nanosuspension, led to higher kidney drug levels and reduced colony counts. Survival was also shown to be superior relative to the solution formulation. Thus, formulation of itraconazole as a nanosuspension enhances efficacy of this antifungal agent relative to a solution formulation, because of altered pharmacokinetics, leading to increased tolerability, permitting higher dosing and resultant tissue drug levels.

Interconversion pharmacokinetics of eplerenone, a selective aldosterone blocker, and its lactone-ring open form.

The interconversion pharmacokinetics of eplerenone and its lactone-ring open form, SC-70303, were examined in dogs using a stable isotope method. [13CD3]EP and SC-70303 were coadministered orally (10 mg/kg) and intravenously (5 mg/kg) as aqueous solutions under fasted conditions. After I.V. administration of [13CD3]EP, the mean AUC of [13CD3]EP was 16.0 h. microg/mL, while the C(max), T(max), and AUC for [13CD3]SC-70303 acid were 0.744 microg/mL, 0.5 h, and 3.49 h. microg/mL, respectively. After I.V. administration of SC-70303, the AUC for SC-70303 acid was 6.36 h. microg/mL, while the C(max), T(max), and AUC for EP were 2.26 microg/mL, 0.5 h, and 9.48 h. microg/mL, respectively. After oral administration of [13CD3]EP, the C(max), T(max), and AUC for [13CD3]EP were 6.01 microg/mL, 0.5 h, and 27.7 h. microg/mL, respectively, and the corresponding values for [13CD3]SC-70303 acid were 0.972 microg/mL, 0.75 h, and 5.52 h. microg/mL, respectively. After oral administration of SC-70303, the C(max), T(max), and AUC for EP were 1.38 microg/mL, 0.83 h, and 9.29 h. microg/mL, respectively, and the corresponding values for SC-70303 acid were 0.330 microg/mL, 0.67 h, and 2.19 h. microg/mL, respectively. The systemic availability was 90% for [13CD3]EP and 17.5% for SC-70303 acid. EP and SC-70303 acid were rapidly interconvertible in the dog. The percentage of dose converted to [13CD3]SC-70303 acid following I.V. administration of [13CD3]EP was 55.0%, while the percentage of dose converted to EP following I.V. administration of SC-70303 was 60.2%.

Atypical dose-route-dependent food effects of eplerenone in the dog: presence of food effects following intravenous dosing and lack of food effects of following oral dosing.

This study was conducted to investigate why a food effect was observed following an intravenous dose of eplerenone (EP) in the dog, but not following oral dosing. Three female dogs were implanted with a chronic portal vein access port and received radiolabeled EP doses orally (15 mg/kg in solution) and intravenously (7.5 mg/kg via cephalic and portal veins) under fasted and fed conditions. Mean AUC values for EP after infusion through the cephalic vein were 23.0 +/- 2.7 and 18.2 +/- 1.1 h.microg/mL under fasted and fed conditions, respectively. Corresponding values after infusion through the portal vein were 20.7 +/- 3.2 and 12.9 +/- 1.3 h.microg/mL, respectively. After oral administration, EP was absorbed 82.0 +/- 6.9 and 98.0 +/- 8.3% under fasted and fed conditions; corresponding mean AUC values were 32.0 +/- 2.0 and 30.8 +/- 3.6 h.microg/mL, respectively. The AUC value for SC-70303 acid (the open lactone form of EP) was lower under fed conditions after cephalic vein infusion, but was greater under fed conditions after portal vein infusion or oral solution administration. The hepatic first-pass effect of EP was 12.6 +/- 6.3% under fasted conditions and 27.1 +/- 6.0% under fed conditions. Pharmacokinetic analysis of EP concentrations after portal vein infusion and oral administration showed that under fed conditions the rate constants for bile excretion and for liver metabolism and urinary excretion were increased while the rate constant for elimination and/or metabolism in the gastrointestinal tract was reduced. In conclusion, the apparent lack of food effect after oral administration was observed because enhanced clearance was compensated by increased absorption.

Nanosuspension formulation of itraconazole eliminates the negative inotropic effect of SPORANOX in dogs.

Previously, it was observed that a nanosuspension formulation of itraconazole was more efficacious and yet less acutely toxic in rats as compared with the conventional solution formulation, SPORANOX (itraconazole) Injection. The present study compares the two formulations with respect to specifically myocardial contractility in conscious dogs. Motivation for doing so is highlighted by the black-box warning in the package insert for SPORANOX (itraconazole) Injection, which warns of negative inotropic effects. Conscious dogs, instrumented with a high-fidelity pressure transducer in the left ventricle, were placed in a sling for dosing and cardiac monitoring. Test and control articles were administered intravenously via a peripheral vein, and left ventricular parameters were measured continuously through 60 min from the start of dosing. As expected, SPORANOX (itraconazole) Injection caused a significant reduction in myocardial contractility as determined by the contractility index. In contrast, the itraconazole nanosuspension administered at twice the dose and at twice the rate of infusion did not result in significant changes in myocardial contractility. A novel formulation technology applied to itraconazole completely prevented the negative inotropic effect observed in conscious dogs as compared with SPORANOX (itraconazole) Injection.

Differential enantioselectivity and product-dependent activation and inhibition in metabolism of verapamil by human CYP3As.

In vitro studies of enantioselective metabolism of R-(+)- and S-(-)verapamil (VER) were conducted using human cDNA-expressed CYP3A isoforms, CYP3A4, CYP3A5, and CYP3A7. N-dealkylated products nor-VER [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] and D617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile] were the major metabolites for all CYP3A isoforms regardless of enantiomer. Enantioselectivity of CYP3A4 and CYP3A7 was most similar among the three isoforms. This coincides with the degree of homology of amino acids at the active sites and in the total amino acid sequences of the enzymes. Biphasic substrate inhibition was observed for the formation of nor-VER and D617, whereas simple biphasic kinetics were observed for the formation of O-demethylated products for both enantiomers with CYP3A4. The biphasic substrate inhibition was observed only for nor-VER, and simple biphasic kinetics were observed for D617 and O-demethylated products for both enantiomers with CYP3A5. However, with CYP3A7, D617 and O-demethylated products showed typical Michaelis-Menten kinetics, and only nor-VER displayed substrate (monophasic) inhibition. When metabolic rates of VER were determined in the presence of three different effectors, midazolam, testosterone, and nifedipine, activation, inhibition, or activation and inhibition of VER metabolism was observed depending on the enantiomers, metabolites, effectors, and cytochrome P450 isoforms. Addition of anti-CYP3A4 antibody inhibited formation of all metabolites for both CYP3A4 and CYP3A5. The atypical phenomena (biphasic substrate inhibition, activation, and inhibition depending on product formation) of VER kinetics could be adequately explained by introducing the concept of steric interaction into a two binding-site model.

Involvement of human UGT2B7 and 2B15 in rofecoxib metabolism.

O-Glucuronidation of 5-hydroxyrofecoxib is the major biotransformation pathway of rofecoxib in human, rat, and dog. The glucuronide conjugate is also involved in the reversible metabolism of rofecoxib in rat and human. Atypical bimodal phenomena were observed in their plasma concentration-time curves with a large variability among different human subjects. It is unclear which family members of human UDP-glucuronosyltransferases (UGT) are involved in the formation of the glucuronide. O-Glucuronidation of 5-hydroxyrofecoxib by human liver microsomes and eight cDNA-expressed human UGT isoforms were investigated. Human liver microsomes formed 5-hydroxyrofecoxib glucuronide with apparent V(max) value of 1736 pmol/min/mg of protein and K(m) value of 44.2 microM. Eight individual cDNA-expressed human UGT isozymes (1A1, 1A3, 1A4, 1A6, 1A8, 1A9, 2B7, and 2B15) were evaluated for glucuronidation of 5-hydroxyrofecoxib. Among them UGT2B15 exhibited the highest metabolism rate with apparent V(max) value of 286 pmol/min/mg of protein and K(m) value of 16.1 microM, whereas UGT2B7 showed apparent V(max) value of 47.1 pmol/min/mg of protein and K(m) value of 41.6 microM. These results indicated that human UGT2B15 has the highest level of activity for catalyzing the glucuronidation of 5-hydroxyrofecoxib. Because polymorphisms have been identified in human UGT2B7, 2B15 genes and O-glucuronidation of 5-hydroxyrofecoxib plays a major role in biotransformation of rofecoxib, it is possible that human UGT2B7 and 2B15 polymorphisms for O-glucuronidation of 5-hydroxyrofecoxib are responsible for the high variability in bimodal patterns in human plasma concentration-time curves.

Pharmacokinetics and metabolism of [14C]eplerenone after oral administration to humans.

A pharmacokinetics and metabolism study was conducted in eight healthy human volunteers. After oral administration of [14C]eplerenone (EP) at a dose of 100 mg per person as an aqueous solution, blood, saliva, breath, urine, and fecal samples were collected at various time points. All matrices were analyzed for total radioactivity and/or for EP and its open-lactone-ring form (EPA). EP was well absorbed, and a mean EP Cmax of 1.72 mug/ml was achieved 1.2 h postdose. After the Cmax, plasma concentrations of EP declined with a half-life of 3.0 h. Plasma concentrations of EPA were much lower than EP concentrations, and the area under the plasma-concentration time curve (AUC) for EPA was only 4% of the EP AUC. Plasma protein binding was moderate (33-60%) but concentration-dependent over the therapeutic concentration range. EP and its metabolites did not preferentially partition into the red blood cells and blood concentrations of total radioactivity were lower than plasma concentrations. Approximately 66.6% and 32.0% of the radioactive dose were excreted in urine and feces, respectively. The majority of urinary and fecal radioactivity was due to metabolites, indicating extensive metabolism of EP. The major metabolic pathways were 6beta- and/or 21-hydroxylation and 3-keto reduction. There was no evidence for any alteration of the 9,11-epoxide ring or the methyl ester. As a percentage of dose, the primary metabolic products excreted in urine and feces included 6beta-hydroxy-EP (6beta-OHEP) (32.0%), 6beta,21-OHEP (20.5%), 21-OHEP (7.89%), and 2alpha,3beta,21-OHEP (5.96%). The amounts of the other metabolites excreted were less than 5% each.

Involvement of CYP3A in the metabolism of eplerenone in humans and dogs: differential metabolism by CYP3A4 and CYP3A5.

In vitro studies were conducted to identify the major metabolites of eplerenone (EP) and the cytochrome p450 (p450) isozymes involved in its primary oxidative metabolism in humans and dogs. The major in vitro metabolites were identified as 6 beta-hydroxy EP and 21-hydroxy EP in both humans and dogs. EP was metabolized by cDNA-expressed human CYP3A4 and dog CYP3A12 but only minimally by human CYP3A5. In human microsomes, inhibition of total metabolism by the CYP3A-selective inhibitors ketoconazole, troleandomycin, and 6',7'-dihydroxybergamottin, each at 10 micro M concentration, was 83 to 95%, whereas inhibition with inhibitors selective for other p450 isozymes was minimal. In dog liver microsomes, the percentages of inhibition were 53 to 76% with the CYP3A-selective inhibitors. A monoclonal anti-CYP3A4 antibody inhibited EP metabolism by 84%, whereas other monoclonal antibodies had minimal effects. The formation of 6 beta-hydroxy and 21-hydroxy metabolites in human liver microsomes was best correlated with CYP3A-selective dextromethorphan N-demethylation and testosterone 6 beta-hydroxylation activities. EP moderately inhibited only CYP3A (testosterone 6 beta-hydroxylase) activity in human liver microsomes by 23, 34 and 45% at concentrations of 30, 100, and 300 micro M, respectively. With human microsomes, the V(max) and K(m) for 6 beta-hydroxylation and 21-hydroxylation were 0.973 nmol/min/mg and 217 micro M, and 0.143 nmol/min/mg and 211 micro M, respectively. The human hepatic clearance calculated from total in vitro EP metabolism was 2.30 ml/min/kg, which agrees with in vivo data. In conclusion, 6 beta- and 21-hydroxylation of EP is primarily catalyzed by CYP3A4 in humans and CYP3A12 in dogs. Also, it is unlikely that EP would substantially inhibit the metabolism of other drugs that are metabolized by CYP3A4 or other p450 isoforms.