Kazuko Sagawa

Pharmacokinetics and bioavailability of the isoflavone biochanin A in rats

Biochanin A(BCA) is a dietary isoflavone present in legumes, most notably red clover, and in many herbal dietary supplements. BCA has been reported to have chemopreventive properties and is metabolized to the isoflavone genistein (GEN), BCA conjugates, and GEN conjugates. The metabolites may contribute to the chemopreventive effects of BCA. The absorption, metabolism, and disposition of BCA have not been determined in rats. Our objective was to evaluate the pharmacokinetics and metabolism of BCA in rats. Male Sprague-Dawley rats were administered BCA by intravenous injection (1 and 5 mg/kg), by intraperitoneal injection (5 and 50 mg/kg), and orally (5 and 50 mg/kg). Plasma and bile samples were enzymatically hydrolyzed in vitro to determine conjugate concentrations for BCA and GEN. Equilibrium dialysis was used to determine protein binding. The BCA and GEN concentrations in plasma, urine, and bile were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pharmacokinetic parameters of BCA were analyzed by noncompartmental analysis. Significant levels of BCA conjugates and GEN conjugates were detected in plasma and bile. Both BCA and GEN were found to have a high clearance and a large apparent volume of distribution; the bioavailability of both was poor (<4%). Reentry peaks were evident after oral administration of both BCA and GEN, suggesting enterohepatic cycling. The free fraction of BCA in rat plasma was 1.5%. A2-compartment model that included both linear and nonlinear clearance terms and enterohepatic recirculation best described the plasma data. This represents the first evaluation of the dose-dependent pharmacokinetics and metabolism of BCA in rats.

Fed and fasted gastric pH and gastric residence time in conscious beagle dogs.

The gastric pH values are controversial in the literature. Some suggest the dog gastric pH is higher than human and dog gastric pH after fed with particular diet is uncertain. Gastric pH in 16 male beagle dogs was measured using Bravo pH telemetry system. For the fed study, the dogs received 10 or 200 g of dog dry food (5L18) 15 min before dosing the Bravo pH capsule, followed by a 50 mL of water to aid in swallowing. It was surprising to find a small, but statistically significantly lower pH in the fed compared to the fasted stomach. The average gastric pH in fasted dogs was 2.05 and 1.08 and 1.26 for 10 and 200 g fed dogs. The average gastric emptying time of the capsule was 1.4, 9.4 and 20 h for fasted, 10 g fed and 200 g fed dogs, respectively. The inter-individual variability was higher in fasted dogs than in fed dogs. The results showed the gastric pH in each colony of dogs can be different from reported values in the literature. It emphasizes that the importance of measuring the pH in each colony when dogs are used to evaluate pharmacokinetics of pH sensitive drugs or formulations.

Effect of experimentally induced hypothyroidism on sulfate renal transport in rats

Decreased serum sulfate concentrations are observed in hypothyroid patients. However, the mechanism involved in thyroid hormone-induced alterations of renal sulfate homeostasis is unknown. The objectives of this investigation were to determine the effect of 6-propyl-2-thiouracil (PTU)-induced hypothyroidism in rats on 1) the in vivo serum concentrations, renal clearance, and renal reabsorption of sulfate, 2) the in vitro renal transport in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, and 3) the cellular mechanism of the hypothyroid-induced alteration in sulfate renal transport. Serum sulfate concentrations, renal fractional reabsorption of sulfate, and creatinine clearance were decreased significantly in the hypothyroid group. The V max values for sodium-sulfate cotransport in BBM were significantly decreased in the kidney cortex from the hypothyroid animals (0.90 ± 0.31 vs. 0.49 ± 0.08 nmol ⋅ mg-1 ⋅ 10 s-1, n = 5-6, P < 0.05) without changes in K m. There were no significant differences in V max and K m for sulfate/anion exchange transport in BLM. Sodium-dependent sulfate transporter (NaSi-1) mRNA and protein levels were significantly lower in the kidney cortex from hypothyroid rats. Hypothyroidism did not alter the membrane motional order (fluidity) in BBM and BLM, which indicates that the changes in the membrane fluidity do not represent the mechanism for the altered renal transport. These results demonstrate that PTU-induced hypothyroidism decreases sodium-sulfate cotransport by downregulation of the NaSi-1 gene.

Interactions between the flavonoid biochanin A and P-glycoprotein substrates in rats: In vitro and in vivo

The purpose of this study was to investigate the in vitro and in vivo interactions between flavonoids and P-glycoprotein (P-gp) substrates. The inhibitory effects of flavonoids on P-gp were determined by accumulation studies in P-gp-overexpressing MCF-7/ADR cells using daunomycin (DNM) as a model substrate. Morin, phloretin, biochanin A, chalcone, and silymarin significantly increased DNM accumulation by greater than 2.5-fold, suggesting they are P-gp inhibitors. To explore potential in vivo interactions of flavonoids with P-gp, the effect of biochanin A on the pharmacokinetics of the P-gp substrates doxorubicin, cyclosporine A, and paclitaxel was investigated. In contrast to the in vitro results, intraperitoneal or oral administration of biochanin A did not significantly change the pharmacokinetics of doxorubicin and cyclosporine A. Moderate interaction was observed between biochanin A and paclitaxel, resulting in lower AUC values after both i.v. and oral administration of paclitaxel. The disconnect between the in vitro and in vivo data suggests that P-gp interactions mediated by biochanin A may be limited due to its poor bioavailability and rapid clearance. It is also possible that other transporters or metabolizing enzymes are more important in the in vivo disposition of doxorubicin, cyclosporine A, and paclitaxel than P-gp.

In vitro and in vivo characterization of amorphous, nanocrystalline, and crystalline ziprasidone formulations

Ziprasidone, commercially available as Geodon capsules, is an atypical antipsychotic used in the treatment of schizophrenia and bipolar disorder. It is a BCS Class II drug that shows up to a 2-fold increase in absorption in the presence of food. Because compliance is a major issue in this patient population, we developed and characterized solubilized formulations of ziprasidone in an effort to improve absorption in the fasted state, thereby resulting in a reduced food effect. Three formulations utilizing solubilization technologies were studied: (1) an amorphous inclusion complex of ziprasidone mesylate and a cyclodextrin, (2) a nanosuspension of crystalline ziprasidone free base, and (3) jet-milled ziprasidone HCl coated crystals made by spray drying (CCSD) the drug with hypromellose acetate succinate. The formulations were characterized by in vitro methods appropriate to each particular solubilization technology. These studies confirmed that ziprasidone mesylate - cyclodextrin was an amorphous inclusion complex with enhanced dissolution rates. The ziprasidone free base crystalline nanosuspension showed a mean particle size of 274 nm and a monomodal particle size distribution. In a membrane permeation test, the CCSD showed a 1.5-fold higher initial flux compared to crystalline ziprasidone HCl. The three formulations were administered to fasted beagle dogs and their pharmacokinetics compared to Geodon capsules administered in the fed state. The amorphous complex and the nanosuspension showed increased absorption in the fasted state, indicating that solubilized formulations of ziprasidone have the potential to reduce the food effect in humans.

Hormonal Regulation of Sodium/Sulfate Co-Transport in Renal Epithelial Cells

Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17beta-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.

Effects of acute caffeine ingestion and menopause on sulfate homeostasis in women

Inorganic sulfate is a physiological anion which is utilized in the metabolism of both endogenous compounds and xenobiotics. Its homeostasis is maintained predominantly by facilitated reabsorptive processes in the kidneys. The objectives of the present investigation were to evaluate the effects of menopausal status and caffeine ingestion on the serum concentrations and clearance of inorganic sulfate. Thirty-nine women who were classified as premenopausal, postmenopausal with or without estrogen treatment, and postmenopausal with osteoporosis participated in the study. The women were studied on two separate occasions following the ingestion of a decaffeinated beverage to which 6 mg caffeine/kg lean body mass or no caffeine was added. All women were habitual caffeine users (mean ingestion of 588 mg caffeine per day) but abstained from all caffeine sources for 2 weeks prior to the control study day. Postmenopausal women with estrogen supplementation exhibited significantly lower sulfate serum concentrations (0.24 +/- 0.02 mM vs. 0.32 +/- 0.04 mM in premenopausal women, mean +/- SD, p < 0.05) and a decreased renal reabsorption of sulfate for the control (no caffeine) period. There was no difference in serum sulfate or sulfate reabsorption in estrogen supplemented postmenopausal women, compared with women not taking estrogen. Postmenopausal women with osteoporosis had significantly lower creatinine and sulfate clearances than postmenopausal women with estrogen supplementation which may be related to their older age, or factors related to the disease process. The 6 mg/kg dose of caffeine caused a diuresis, but no change in GFR, as indicated by urine volume and creatinine clearance values, respectively. Caffeine administration resulted in an increase in the sulfate excretion rate; there was no change in sulfate serum concentrations. The results of this investigation indicate that menopause results in decreased sulfate serum concentrations that may be the consequence of a decreased renal reabsorption of sulfate. Secondly, this investigation demonstrated that caffeine ingestion increases the urinary excretion of sulfate, an effect that may be related to the diuretic effect of caffeine or due to a caffeine-induced alteration in the renal reabsorption of sulfate.

Detection and quantitation of a sodium-dependent sulfate cotransporter (NaSi-1) by sandwich-type enzyme-linked immunosorbent assay.

Abstract The sodium-dependent sulfate transporter (NaSi-1) DNA has been recently identified from rat kidney cortex. The objective of this study was to develop a quantitative assay for the NaSi-1 transporter protein. The NaSi-1 antigen was prepared by fusion protein techniques following analysis of the primary sequence for antigenicity. Polyclonal and monoclonal antibodies against the NaSi-1 antigen were raised in rabbits and mice, respectively. The specificity of the raised antibodies was examined by Western analysis using brush-border membrane (BBM) and basolateral membrane (BLM) purified from rat kidney cortex. Both NaSi-1 polyclonal and monoclonal antibodies detected a 69-kDa protein in the BBM. Using the purified monoclonal antibody as the capture antibody and the polyclonal antibody as the detecting antibody, a simple and sensitive sandwich-type enzyme-linked immunosorbent assay was developed to quantitate NaSi-1 transporter protein levels in tissue. The specificity of the assay was examined using BBM, BLM and NaSi-1-transfected Madin-Darby canine kidney cells. The assay was capable of detecting NaSi-1 at levels as low as 6.58 fmol. The concentration of NaSi-1 transporter protein in crude membrane isolated from rat kidney cortex was 0.094±0.014 fmol/µg protein (mean±SD of three preparations).

Molecular mechanisms of renal sulfate regulation.

Inorganic sulfate is an important physiological anion that is a required cofactor for sulfate conjugation reactions of both endogenous and exogenous compounds. It is necessary for the detoxification of xenobiotics and endogenous compounds (catecho-lamines, steroids, bile acids), for the synthesis of structural components of membranes and tissues (sulfated glycosaminoglycans), and for the biologic activity of endogenous compounds (heparin and cholecystokinin). Inorganic sulfate homeostasis is largely maintained by reabsorption in the renal proximal tubule. Sodium-dependent sulfate cotrans-port in the brush border membrane is of primary importance in the regulation of plasma inorganic sulfate concentrations. Altered renal reabsorption of sulfate has been observed under different physiological (age, pregnancy, low dietary intake), pathological (hypo-thyroidism, trace metal excess), and pharmacological conditions (treatment with non-steroidal antiinflammatory agents). The recent identification of the sulfate transporter genes has allowed the investigation of the molecular mechanisms of altered sulfate transport. Although the regulation of sulfate homeostasis is not fully understood, recent investigations have explored the cellular mechanisms of some of these alterations. In this review, the physiological importance of inorganic sulfate, the availability of this anion, and the regulation of sulfate homeostasis are discussed.

Pharmacokinetics and Disposition of the Thiouracil Derivative PF-06282999, an Orally Bioavailable, Irreversible Inactivator of Myeloperoxidase Enzyme, Across Animals and Humans.

The thiouracil derivative PF-06282999 [2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide] is an irreversible inactivator of myeloperoxidase and is currently in clinical trials for the potential treatment of cardiovascular diseases. Concerns over idiosyncratic toxicity arising from bioactivation of the thiouracil motif to reactive species in the liver have been largely mitigated through the physicochemical (molecular weight, lipophilicity, and topological polar surface area) characteristics of PF-06282999, which generally favor elimination via nonmetabolic routes. To test this hypothesis, pharmacokinetics and disposition studies were initiated with PF-06282999 using animals and in vitro assays, with the ultimate goal of predicting human pharmacokinetics and elimination mechanisms. Consistent with its physicochemical properties, PF-06282999 was resistant to metabolic turnover from liver microsomes and hepatocytes from animals and humans and was devoid of cytochrome P450 inhibition. In vitro transport studies suggested moderate intestinal permeability and minimal transporter-mediated hepatobiliary disposition. PF-06282999 demonstrated moderate plasma protein binding across all of the species. Pharmacokinetics in preclinical species characterized by low to moderate plasma clearances, good oral bioavailability at 3- to 5-mg/kg doses, and renal clearance as the projected major clearance mechanism in humans. Human pharmacokinetic predictions using single-species scaling of dog and/or monkey pharmacokinetics were consistent with the parameters observed in the first-in-human study, conducted in healthy volunteers at a dose range of 20–200 mg PF-06282999. In summary, disposition characteristics of PF-06282999 were relatively similar across preclinical species and humans, with renal excretion of the unchanged parent emerging as the principal clearance mechanism in humans, which was anticipated based on its physicochemical properties and supported by preclinical studies.