The Rate of Synthesis of Glycosaminoglycans and Collagen by Fibroblasts Cultured from Adult Human Liver Biopsies

Adult human liver biopsies were cultured from normal, alcoholic hepatitis, chronic active hepatitis, fibrosis plus alcoholic hepatitis (active cirrhosis), inactive cirrhosis, and drug hepatitis. The synthesis of collagen was estimated in cultures from 58 livers by measuring the conversion of [¹⁴C]proline to the [¹⁴C]hydroxyproline of collagen; that of glycosaminoglycans in cultures from 57 livers by the incorporation of [³H]acetate and ³⁵SO₄ into glycosaminoglycans (GAG). The synthesis of procollagen was increased only in cultures from alcoholic hepatitis, both in the pulse medium (P < 0.05) and in the chase medium (P < 0.02). The synthesis of insoluble collagen was increased in cultures from chronic (active) hepatitis (P < 0.01), fibrosis plus alcoholic hepatitis (active cirrhosis) (P < 0.001), and inactive cirrhosis (P < 0.05). Essentially all radioactive GAG was soluble in culture media. The predominant GAG were chondroitin-4 or -6-SO₄. The synthesis of GAG was increased only in cultures from fibrosis plus alcoholic hepatitis (active cirrhosis) both in the pulse medium (P < 0.01) and chase medium (P < 0.001).     

Ether link cleavage is the major pathway of iodothyronine metabolism in the phagocytosing human leukocyte and also occurs in vivo in the rat

These studies were performed to test the hypothesis that ether link cleavage (ELC) is an important pathway for the metabolism of thyroxine (T₄) in the phagocytosing human leukocyte. When tyrosyl ring-labeled [¹²⁵I]T₄([Tyr¹²⁵I]T₄) was incubated with phagocytosing leukocytes, 50% of the degraded label was converted into [¹²⁵I]3,5-diiodotyrosine ([¹²⁵I]DIT). Of the remaining [Tyr¹²⁵I]T₄ that was degraded, two-thirds was recovered as [¹²⁵I]-nonextractable iodine ([¹²⁵I]NEI), and one-third as [¹²⁵I]iodide. The production of [¹²⁵I]DIT was not observed when phenolic ring-labeled [¹²⁵I]T₄ ([Phen¹²⁵I]T₄) was used, although [¹²⁵I]NEI and [¹²⁵I]iodide were produced. None of these iodinated compounds were formed in leukocytes that were not carrying out phagocytosis.     

Maturational Patterns of Iodothyronine Phenolic and Tyrosyl Ring Deiodinase Activities in Rat Cerebrum, Cerebellum, and Hypothalamus

To explore the control of thyroid hormone metabolism in brain during maturation, we have measured iodothyronine deiodination in homogenates of rat cerebrum, cerebellum, and hypothalamus from 1 d postnatally through adulthood. Homogenates were incubated with ¹²⁵I-l-thyroxine (T₄) + [¹³¹I]3,5,3′-l-triiodothyronine (T₃) + 100 mM dithiothreitol. Nonradioactive T₄, T₃, and 3,3′,5′-triiodothyronine (rT₃) were included, as appropriate. The net production rate of [¹²⁵I]T₃ from T₄ in 1-d cerebral homogenates was similar to the rate in adult cerebral homogenates (9.9±2.5[SEM]% vs. 8.9±1.2% T₄ to T₃ conversion in 2 h). Production of T₃ was not detectable in 1-d cerebellar and hypothalamic homogenates. The net T₃ production rate in adult cerebellar homogenates was twice as great as, and that in adult hypothalamic homogenates similar to, the rate in cerebral homogenates.     

n-Butyrate inhibition of hyaluronate synthesis in cultured human fibroblasts.

The effects of the short-chain aliphatic carboxylic acid, n-butyrate, on glycosaminoglycan (GAG) accumulation were studied in cultured human skin fibroblasts. Normal fibroblast cultures were grown to confluence, shifted to a medium without or with n-butyrate for 24 h, labeled with either [3H]acetate or [3H]glucosamine and analyzed for [3H]GAG and [3H]hyaluronate accumulation. Accumulation was stimulated at low concentrations (0.1-1 mM) by up to 27%. Higher concentrations of n-butyrate (greater than 1 mM) inhibited [3H]GAG by up to 70-90%. This effect was maximal at 10 mM and half-maximal at 3 mM. Propionate had similar effects but was less potent. Parallel studies conducted in colonic fibroblasts revealed that n-butyrate could markedly inhibit [3H]GAG accumulation in that cell type as well. These effects were rapid, occurring within 3 h of treatment, and were reversible. Chondroitin sulfate accumulation was unaffected by the compound. A pulse-chase study failed to demonstrate any effect on [3H]GAG degradation.     

Effect of a local heating device on insulin and glucose pharmacokinetic profiles in an open-label,randomized, two-period,one-way crossover study in patients with type 1 diabetes using continuous subcutaneous insulin infusion

Background: Reducing postprandial hyperglycemia and hypoglycemia is a major challenge in the treatment of patients with diabetes. Studies suggest that heating the insulin injection site may accelerate insulin absorption. InsuPatch? (InsuLine Medical Ltd., Petach-Tikva, Israel) is a device intended to accelerate subcutaneous insulin delivery using an insulin pump by locally warming the infusion site.Objective: The aim of this study was to assess the effect of the InsuPatch device on the pharmacokinetics of short-acting insulin analogues and on postprandial glycemia.Methods: This was an open-label, randomized, 2-period, 1-way crossover study using a meal tolerance test (MTT) protocol in subjects with type 1 diabetes mellitus aged 18 to 65 years with glycosylated hemoglobin (HbA_1c) of 6% to 12%, who were using continuous subcutaneous insulin infusion (CSII) therapy (insulin lispro or aspart). A bolus of insulin 0.15 U/kg was delivered immediately before a standardized liquid meal. The effect of the device on insulin absorption and postprandial glucose excursions was tested by measuring blood glucose and insulin concentration profiles with and without the operation of the device. Adverse events (AEs) were monitored during the 2 study visits. The infusion site was visually inspected at the end of each study. All AEs were recorded in the case-report form.Results: The MTT protocol was performed in 17 white patients with type 1 diabetes (sex, male/female, 7/10; mean age, 31.8 years [range, 18–53 years]; mean body mass index, 25.6 kg/m² [range, 20.0–39.4 kg/m²]; and mean HbA_1c, 7.1% [range, 5.8%–9.3%]). The mean (SD) postprandial glucose excursion AUC at 120 minutes after the meal was significantly reduced in the group that used the device compared with the group that did not (104 [65] vs 155 [56] mg/dL/h; P < 0.005). Plasma insulin was measured randomly in 9 of the 17 subjects. Use of the device was associated with significant reductions in mean (SD) T_max (45 [28] vs 78 [35] minutes) and time to half-maximum concentration (T_50%max) (20 [11] vs 28 [10] minutes; both, P < 0.05). Insulin AUC increased significantly in the group that used the device compared with the group that did not at 30 (21 [6] vs 33 [16] mU/L/h), 60 (55 [12] vs 80 [28] mU/L/h), and 90 (93 [19] vs 118 [31] mU/L/h) minutes after administration (all, P < 0.05). Cmax also increased significantly (118 [35] vs 86 [16] mU/L [38%]; P < 0.05).Conclusion: In this pilot study, use of the InsuPatch was associated with significant decreases in T_max and T_50%max and increases in insulin AUC and C_max of subcutaneous infused rapid-acting insulin analogues, resulting in a lower postprandial glucose excursion in these patients with type 1 diabetes mellitus treated with CSII.     

Substrates for the assay of α-l-iduronidase

Procedures are described for the preparation of two disaccharides, 4-O -α -L-iduronosyl-2, 5-anhydro[³H]mannitol and $\text{3-O-α-}\text{l}\text{-iduronosyl}\text{-2,5--}\text{anhydro}\text{[}{}^3\text{H]-talitol}$, from heparin and dermatan sulfate, respectively. These disaccharides lend themselves to an easy assay of α-L-iduronidase which is based on the fractionation of the liberated neutral anhydro[³H]mannitol or anhydro[³H]talitol from the unreacted substrate by adsorption of the latter to Dowex 1.Investigation of the reaction conditions showed that the α-L-iduronidase activity (enzyme from human fibroblasts and Helix pomatia) was optimal at pH 3.6 in acetate buffer containing 0.01 M NaCl with iduronosyl-2,5-anhydro[³H]mannitol as substrate. For iduronosyl-2,5-anhydro[³H]talitol the pH optimum was 4.0 with the H. pomatia enzyme.The K_m for iduronosyl-2,5-anhydro[³H]mannitol was 0.23 mM with human fibroblasts and 0.04 mM with Helix enzyme; a KM value of 0.02 mM was determined for iduronosyl-2,5-anhydro[³H]talitol with the Helix α-l-iduronidase.     

Concentration of Myo-inositol in Skeletal Muscle of the Rat Occurs without Active Transport

The cellular uptake of nonphosphorylated myo-inositol (MI) and its incorporation into phosphoinositide in the rat epitrochlearis muscle was measured. Cellular uptake of [2-³H]MI was determined by the difference between total uptake and [2-³H]MI present in the extracellular fluid determined with [1-¹⁴C]mannitol. Cellular uptake was parabolic and directly proportional to medium MI concentrations between 25 and 3,200 μM. Saturation of a MI carrier was not evident. Moreover, uptake was not inhibited by 2 mM ouabain, 0.3 mM 2,4-dinitrophenol, or 22 mM glucose. Insulin, 100 mU/ml, was without effect on either cellular uptake of [2-³H]MI or its incorporation into phosphoinositides. In muscles that were preloaded with [2-³H]MI and then incubated in media that contained a constant amount of MI but no [2-³H]MI, 44.3% of the [2-³H]MI was released after 10 min increasing to 62.5% by 120 min. Cellular MI concentrations were 0.18 μmol/g wet tissue (four times plasma levels) in rapidly isolated and frozen epitrochlearis muscle. When muscle was incubated without MI, 48% of endogenous MI was lost rapidly. Restoration of cellular MI in 50 μM MI media occurred in two phases, a rapid uptake phase lasting 10 min and a subsequent slow phase of MI uptake.     

Genistin-rich soy isoflavone extract in substrate reduction therapy for Sanfilippo syndrome: An open-label, pilot study in 10 pediatric patients

Background: Mucopolysaccharidoses (MPSs) are a group of severe metabolic disorders caused by deficiencies in enzymes involved in the degradation of glycosaminoglycans (GAGs)—long chains of sugar carbohydrates in cells that help build bone, cartilage, tendons, corneas, skin, and connective tissue. Although enzyme replacement therapy has become available for the treatment of some types of MPS, effective treatment of neurodegenerative forms of MPS has yet to be determined. Recently, genistein (4',5,7-trihydroxyisoflavone), a specific inhibitor of protein tyrosine kinase, has been found to inhibit GAG synthesis and to reduce GAG concentrations in cultures of fibroblasts of MPS patients. Therefore, a potential substrate reduction therapy has been proposed.Objective: The aim of this study was to examine urinary GAG concentration, hair morphology, and cognitive function in patients receiving genistin treatment for Sanfilippo syndrome (MPS type III).Methods:Patients aged 3 to 14 years with a biochemically confirmed diagnosis of MPS IIIA or MPS IIIB were eligible to enroll in this open-label, pilot study. Genistin-rich soy isoflavone extract 5 mg/kg/d was administered PO for 12 months. Urinary GAG concentration, hair morphology,and cognitive function (measured using a modified version of the Brief Assessment Examination [BAE] and parent observations)were measured at baseline and after 12 months of treatment.Results: Ten patients (6 girls, 4 boys; mean age, 8 years [range,3\2-14 years];mean weight, 28 kg [range, 17\2-43 kg]) were included in the study. All patients had Sanfilippo syndrome; 5 patients had MPS IIIA and 5 had MPS IIIB. After 1 year, statistically significant improvement was found in urinary GAG concentration, hair morphology, and cognitive function. Urinary GAG concentration decreased significantly in all 5 patients with MPS IIIA and in 2 patients with MPS IIIB (P = 0.028). Hair morphology improved significantly in all 5 MPS IIIA patients and in 3 MPS IIIB patients (P = 0.012). A significant increase in the BAE score (by 2-6 points) was noted in 8 patients, while the scores of 2 patients did not change after 12 months of treatment (P = 0.012). No adverse events (AEs) considered related to treatment were reported. Moreover, no AEs not related to the treatment (apart from classical symptoms of MPS III) were noted.Conclusions: This pilot study found some improvements in GAG concentration, hair morphology, and cognitive function in these pediatric patients with Sanfilippo syndrome treated with genistin-rich soy isoflavone extract for 1 year. Clinical trials are needed to evaluate the efficacy and safety of this potential treatment.     

Effects of Flutamide on [Methyl-H]-Choline Uptake in Human Prostate Cancer-3 Cells: A Pilot Study

Background: Positron emission tomography using [methyl-¹¹C]-choline is effective in imaging many types of cancer, especially prostate cancer (PC). The antiandrogen flutamide is often used as part of the initial treatment of PC. Data on the effect of flutamide on and methylcholine incorporation into PC-3 cells are lacking in the experimental and literature work.Objectives: The aims of this study were to assess whether human PC-3 cells are susceptible to flutamide and whether the drug modulates the uptake of [methyl-³H]-choline into these cells.Methods: PC-3 cells were treated for 3 days with flutamide (≤100 nmol/L), inhibiting growth by 20% to 70% with control cells included. Two viability tests (cytotoxic analyses), the thiazole blue assay and the trypan blue exclusion method, were used to determine the median inhibitory concentration for flutamide (10 nmol/L). Control and flutamide-treated cells were incubated with [methyl-³H]-choline for 10 minutes and then in nonradioactive medium for 10 minutes to simulate the rapid blood clearance of [methyl-¹¹C]-choline tracer that occurs within 5 to 20 minutes, and then extracted using organic and aqueous solvents to determine the intracellular distribution of the tracer. Protein assay and flow-cytometry analysis were used to determine protein content and DNA synthesis in both control and treated cells. The uptake of [methyl-³H]-choline was normalized to protein content and expressed as mean (SD) dpm/1Jg protein (n = 6).Results: PC-3 cell proliferation was inhibited with flutamide treatment. After treatment of PC-3 cells with flutamide 10 nmol/L for 3 days, cells accumulated DNA during the S phase. Mean (SD) [methyl-³H]-choline uptake was found to be significantly lower with flutamide 10-nmol/L-treated cells compared with control cells (65.95 [0.72] vs 114.21 [0.57] dpm/1Jg protein; P < 0.001); the difference between the 5-nmol/L-treated cells and controls was nonsignificant.Conclusions: In this pilot study, flutamide inhibited tumor cell growth and proliferation and decreased (modulated) the uptake of [methyl-³H]-choline into androgen receptor-negative PC-3 cells. These results suggest that flutamide might inhibit proliferation by an androgen-independent mechanism.     

Effects of a concomitant single oral dose of rifampicin on the pharmacokinetics of pravastatin in a two-phase,randomized, single-blind,placebo-controlled,crossover study in healthy Chinese male subjects

Background: Pravastatin is a potent cholesterol-lowering agent; ~34% of an oral dose of pravastatin is eliminated unchanged through biliary and urinary excretion. Rifampicin is an inducer of drug metabolism enzymes, and it affects the activities of transporters involved in pravastatin disposition. Drug-drug interaction between rifampicin and pravastatin is possible because of the effects of rifampicin on the activities of drug transporters.Objective: This study was designed to investigate the effects of a single oral dose of rifampicin on the pharmacokinetics of pravastatin.Methods: Healthy Chinese male volunteers were recruited for this 2-phase, single-blind, placebo-controlled, crossover study. The subjects were randomly divided into 2 groups to receive either rifam-picin or placebo concomitantly with pravastatin. All subjects received a 20-mg oral dose of pravastatin on days 1 and 9, separated by an 8-day washout period. Subjects in the rifampicin group received a single 600-mg oral dose of rifampicin on day 1 and placebo on day 9; those in the placebo group received placebo on day 1 and a single 600-mg oral dose of rifampicin on day 9. High-performance liquid chromatography-tandem mass spectrometry was used to determine plasma concentrations of pravastatin for up to 12 hours after administration.Results: Twelve volunteers participated in the study (6 per group). The mean (SD) age of the subjects was 20 (2) years (range, 18–25 years). The mean height of the subjects was 174 (4) cm (range, 168–180 cm), and the mean weight was 69.2 (3.7) kg (range, 65–77 kg). The mean pharmacokinetic parameters for pravastatin that changed significantly were as follows (rifampicin and placebo groups, respectively): C_max (315.7 [227.2] and 115.8 [77.5] ng · mL^?1 [P = 0.009]); AUC_0–12 (604.8 [73.3] and 259.0 [133.4] ng · h · mL^?1 [P < 0.001]); AUC_0–∞) (623.3 [248.8] and 275.1 [58.5] ng · h · mL^?1 [P < 0.001]); and apparent oral clearance (CL/F) (0.52 [0.18] and 1.30 [0.58] L · h^?1 · kg^?1 [P < 0.001]). No significant changes in the T_max or t_½ of pravastatin were observed. All subjects tolerated pravastatin well during both phases of the study, with or without coadministration of rifampicin. None of the subjects withdrew from the study.Conclusion: Coadministration of a single oral dose of rifampicin significantly increased the plasma concentration of pravastatin in this group of healthy Chinese male subjects.     

Synthesis of Prostacyclin from Platelet-derived Endoperoxides by Cultured Human Endothelial Cells

We have previously shown that aspirin-treated endothelial cells synthesize prostacyclin (PGI₂) from the purified prostaglandin endoperoxide PGH₂ (1978. J. Biol. Chem.253: 7138). To ascertain whether aspirin-treated endothelial cells produce PGI₂ from endoperoxides released by stimulated platelets, [³H]arachidonic acid-prelabeled platelets were reacted in aggregometer cuvettes with the calcium ionophore A 23187, thrombin, or collagen in the presence of aspirin-treated endothelial cell suspensions. This procedure permitted thin-layer radiochromatographic quantitation of [³H]PGI₂ as [³H]6-keto-PGF_1α and [³H]thromboxane A₂ (TXA₂) as [³H]TXB₂, as well as analysis of platelet aggregation responses in the same sample. In the presence of aspirin-treated endothelial cells, platelet aggregation in response to all three agents was inhibited. [³H]6-keto-PGF_1α was recovered from the supernates of the combined cell suspensions after stimulation by all three agents. The order of PGI₂ production initiated by the stimuli was ionophore > thrombin > collagen. The amounts of platelet [³H]TXB₂ recovered were markedly reduced by the addition of aspirin-treated endothelial cells. In separate experiments, 6-keto-PGF_1α and TXB₂ were quantitated by radioimmunoassay; the results paralleled those obtained with the use of radiolabeling. The quantity of 6-keto-PGF_1α measured by radioimmunoassay represented amounts of PGI₂ sufficient to inhibit platelet aggregation. These results were obtained when 200,000 platelets/μl were combined with 3,000-6,000 aspirin-treated endothelial cells/μl. At higher platelet levels the proportion of 6-keto-PGF_1α to TXB₂ decreased and platelet aggregation occurred. Control studies indicated that aspirin-treated endothelial cells could not synthesize PGI₂ from exogenous radioactive or endogenous arachidonate when stimulated with thrombin. Therefore the endothelial cell suspensions could only have used endoperoxides from stimulated platelets.     

Familial incomplete male pseudohermaphroditism associated with impaired nuclear androgen retention. Studies in cultured skin fibroblasts

The androgen resistance syndromes are generally felt to be due to quantitative or qualitative abnormalities of the androgen receptor. Some patients with testicular feminization have no demonstrable fibroblast cytosol androgen binding, whereas others have androgen binding in cultured fibrobalsts that is thermolabile or fails to be stabilized by sodium molybdate. I describe here familial incomplete testicular feminization associated with reduced nuclear androgen retention. Fibroblasts, cultured from pubic skin biopsies of two phenotypic female 46XY siblings, were assayed for whole cell and nuclear uptake of [³H]dihydrotestosterone in dispersed, intact cells. Whole cell binding of [³H]dihydrotestosterone at 22°C in the patients' fibroblasts was in the normal range. However, no high affinity, saturable binding of [³H]dihydrotestosterone was demonstrable in crude nuclear pellets prepared from the patients' fibroblasts incubated at 37°C with the hormone. Incubating the patients' cells with [³H]methyltrienolone or examining the nuclear uptake of [³H]dihydrotestosterone in these cells at 22°C did not alter these findings. Although cytosol from the patients' cells revealed a quantitatively diminished 8S peak for [³H]dihydrotestosterone after centrifugation on sodium molybdate-containing sucrose gradients, there was no peak of ³H in the 4S region from 0.3 M KCl nuclear extracts of the patients' cells after they had been incubated with [³H]dihydrotestosterone at 37°C.     

Triiodothyronine and amiodarone effects on β3-adrenoceptor density and lipolytic response to the β3-adrenergic agonist BRL 37344 in rat white adipocytes

Summary— The β-adrenergic effects of catecholamines are potentiated by thyroid hormones in adipose tissue. Amiodarone (AM) is structurally similar to thyroid hormones and was used to explore the mechanism of the triiodothyronine (T₃) effect on β-adrenergic receptors (β-ARs) in adipose tissue. AM decreases the expression of some T₃ sensitive genes in various tissues and antagonizes the effect of T₃ on its nuclear receptors. In this study, the T₃, AM and AM + T₃ effects on the β1- and β₃-AR density were assessed on rat white adipocytes by radioligand binding using [³H]CGP 12177 after characterization of these subtypes by displacement of [³H]CGP 12177 binding by isoproterenol, BRL 37344 and noradrenaline. BRL 37344 was used to study β₃-AR lipolysis. White adipocytes from hyperthyroid rats had increased responsiveness (E_max × 2) and sensitivity (+ 38%) to BRL 37344, while those given AM alone had decreased values. Moreover, AM antagonized the T₃ effect on lipolysis. The β₁-binding characteristics (receptor density [B_max]: 45 ± 4 fmol/mg of proteins; dissociation factor [K_d]: 0.96 ± 0.10 nM) were not modified by either compound. Finally, T₃ significantly increased β₃-AR density (587 ± 69 versus 363 ± 25 fmol/mg of proteins) and K_d (38 ± 2 versus 23 ± 3 nM), while AM alone had no effect and did not antagonize the T₃ effect on β₃-AR number. In conclusion, the hyperthyroid state in the rat potentiated the lipolytic response of white adipocytes to a specific β₃-agonist and increased the β₃-AR density without changing in β₁-AR number and affinity. Furthermore, the lack of antagonism between AM and T₃ on β₃-AR expression suggests that T₃ does not work directly on the β₃-AR gene. Moreover, AM induced a functional tissular hypothyroid-like effect and its antilipolytic effect probably occurred at a postreceptor level.     

Mucolipidosis I: Studies of sialidase activity and a prenatal diagnosis

The characteristics of the sialidase (N-acetyl-α-neuraminidase) of human leukocytes, fibroblasts and amniotic fluid cell cultures were determined with a radioactive assay method utilizing neuramin-[³H]actitol as the enzyme substrate. Fibroblast cultures from patients with the inherited sialidase deficiency diseases including mucolipidosis I, sialidosis I and sialidosis II, juvenile type have less than 10% of normal sialidase activity using either this substrate, 2-(3′-methoxyphenyl)-N-acetyl-α-neuraminic acid, or 2′-(4-methylumbelliferyl)-Nacetyl-α-neuraminic acid. The total sialic acid content of fibroblasts and leukocytes from mucolipidosis I and sialidosis I patients is greatly elevated; this parameter is useful in establishing a diagnosis of sialidase deficiency. The sialic acid content of sialidosis II, juvenile type, with coexistent sialidase and β-galactosidase deficiencies, is only slightly elevated above normal levels. A patient with mucolipidosis I has 16% of normal neuramin-[³H]lactitol sialidase activity in his peripheral leukocytes. His parents were clearly distinguished from the normal range using leukocyte enzyme levels and a maternal aunt was identified as a possible carrier. The presence of this enzyme in amniotic fluid cell cultures, both fibroblastic and mixed cell type, makes possible the prenatal detection of these diseases. A pregnancy from a family at risk for having a child with mucolipidosis I was monitored by amniocentesis and subsequent sialidase measurement of the amniotic fluid cell culture.     

P-Glycoprotein Function at the Blood–Brain Barrier: Effects of Age and Gender

Purpose

P-glycoprotein (Pgp) is an efflux transporter involved in transport of several compounds across the blood?Cbrain barrier (BBB). Loss of Pgp function with increasing age may be involved in the development of age-related disorders, but this may differ between males and females. Pgp function can be quantified in vivo using (R)-[¹¹C]verapamil and positron emission tomography. The purpose of this study was to assess global and regional effects of both age and gender on BBB Pgp function.

Procedures

Thirty-five healthy men and women in three different age groups were included. Sixty minutes dynamic (R)-[¹¹C]verapamil scans with metabolite-corrected arterial plasma input curves were acquired. Grey matter time?Cactivity curves were fitted to a validated constrained two-tissue compartment plasma input model, providing the volume of distribution (V _T) of (R)-[¹¹C]verapamil as outcome measure.

Results

Increased V _T of (R)-[¹¹C]verapamil with aging was found in several large brain regions in men. Young and elderly women showed comparable V _T values. Young women had higher V _T compared with young men.

Conclusions

Decreased BBB Pgp is found with aging; however, effects of age on BBB Pgp function differ between men and women.     

Pharmacokinetics,bioequivalence, tolerability,and effects on platelet counts of two formulations of anagrelide in healthy volunteers and patients with thrombocythemia associated with chronic myeloproliferation

Background: Anagrelide hydrochloride is an anti-thrombotic agent indicated for the treatment of essential thrombocythemia (ET). In various previously published clinical trials of 2 branded formulations of anagrelide in patients with ET at high risk for thrombohemorrhagic events, the rates of adverse events and discontinuation were strikingly divergent between brands. Because the formulations and manufacturers differed, the differences in tolerability, as well as platelet counts, might have been related to differences in pharmacokinetic properties between the 2 formulations.Objectives: The present series of investigations (1) determined the pharmacokinetic profile of anagrelide and its metabolites; (2) compared the pharmacokinetic profiles of the test and reference formulations of anagrelide; (3) investigated the in vitro release of anagrelide as a marker of intragastric anagrelide release of the test and reference formulations; and (4) compared the platelet-reducing effects of the test and reference formulations in patients with thrombocythemia in 2 longitudinal studies over 4 weeks.Methods: A series of 4 in vivo studies and 1 in vitro study were conducted. In a pilot, prospective, singledose study in healthy volunteers, the pharmacokinetic properties (C_max, T_max, and AUC_0-∞) of a test formulation of anagrelide were assessed using high-performance liquid chromatography analysis of plasma samples. Based on the results from that study, a single-dose, randomized, double-blind, 2-period crossover study in healthy volunteers was conducted to determine bioequivalence of 2 formulations of anagrelide 2 mg/d (taken as 4 capsules). In vitro dissolution properties of the test or reference formulation containing 0.5 mg anagrelide as the active ingredient were studied in an assay mimicking gastrointestinal release. To test for effects on platelet counts of switching from the reference formulation (previous treatment on stable dose for 3 months) to the test formulation, two 4-week longitudinal trials were conducted: one in patients with ET (in Germany), and one in patients with thrombocythemia associated with chronic myeloproliferative disorders (CMPDs) (in Austria).Results: The pilot pharmacokinetic study of the test formulation in 16 volunteers (10 women, 6 men; mean [SD] age, 20.5 [1.5] years; weight, 69.0 [10.0 kg) suggested that anagrelide was metabolized to 3-hydroxyanagrelide (AUC_0-∞ 50% compared with anagrelide) and the inactive metabolite 2-amino-5,6-dichloro-,4-dihydroquinazolone. The subsequent bioequivalence study in 24 volunteers (14 women, 10 men; mean [SD] age, 23 [4] years; white, 100%; weight, 67.5 [10.2] kg) found that the test formulation was associated with a significantly lower C_max (point estimation [PE], 66%; 90% CI, 58%-76%; P < 0.001) and AUC_0-∞ (PE, 77%; 90% CI, 68%-86%; P = 0.001). T_max values for anagrelide and 3-hydroxyanagrelide were 1 hour longer with the test formulation compared with the reference formulation. The total number of adverse events with the reference formulation was 46; the test formulation, 29 (P = 0.05). In vitro, anagrelide from the reference formulation was immediately released (89.1% at 5 minutes), whereas there was a delayed release (93.6% at 30 minutes) from the test formulation (P < 0.05). In the last 2 studies, 2 cohorts of white patients (cohort 1, 15 patients with ET; 10 women, 5 men; mean [SD] age, 49.0 [10.7] years [range, 31-66 years]; weight, 73.2 [12.6] kg; cohort 2, 19 patients with thrombocythemia associated with CMPD; 12 women, 7 men; age, 62.6 [12.4] years [range, 38-80 years]; weight, 66.1 [13.3] kg) who had received treatment for ≥3 months with the reference formulation were switched to the same dose of the test formulation and maintained on this dose for 4 weeks. Platelet counts did not change significantly from baseline over 4 weeks and stayed within a predefined margin of 150 × 10³ cells/μL.Conclusions: The pharmacokinetic properties, adverse event rates, and in vitro dissolution profile differed between the test and reference anagrelide formulations in these healthy volunteers. In patients with ET or thrombocythemia associated with CMPD, platelet counts did not differ significantly from baseline at 4 weeks when subjects were switched from the reference to the test anagrelide formulation.     

Influx of Thyroid Hormones into Rat Liver In Vivo: DIFFERENTIAL AVAILABILITY OF THYROXINE AND TRIIODOTHYRONINE BOUND BY PLASMA PROTEINS

The transport of [¹²⁵I]thyroxine (T₄) and [¹²⁵I]triiodothyronine (T₃) into liver was investigated with a tissue sampling-portal vein injection technique in the anesthetized rat. The method allows the investigation of the effects of plasma proteins in human serum on the unidirectional influx of T₄ or T₃ into liver cells. The percent extraction of unidirectional clearance of T₃ and T₄ was 77±2% and 43±2%, respectively, after portal injection of a bolus of Ringer's solution. Cell membrane transport of T₄ or T₃ was nonsaturable because 50-μM concentrations of unlabeled hormone had no effect on transport. The addition of bovine albumin in concentrations of 1, 5, or 10 g/100 ml bound >98% of T₄ or T₃ in vitro, but had no significant effect on T₃ or T₄ transport in vivo. Conversely, 10% rabbit antisera specific for T₃ or T₄, completely abolished the intracellular distribution of thyroid hormone into liver. In the presence of rat serum, which contains albumin and thyroid hormone binding pre-albumin (TBPA), 18 and 81% of total plasma T₄ and T₃, respectively, were available for transport in vivo. The fraction of hormone available for transport in the presence of normal human serum, which contains albumin, TBPA, and thyroid hormone binding globulin (TBG) was 11% for T₄ and 72% for T₃. The fraction of hormone transported into liver after injection of serum obtained from pregnant or birth control pilltreated volunteers was 4% for T₄ (but this was not significantly different from zero) and 54% for T₃.     

Evaluation of Pancreatic VMAT2 Binding with Active and Inactive Enantiomers of [<Superscript>18</Superscript>F]FP-DTBZ in Healthy Subjects and Patients with Type 1 Diabetes

Purpose

Previous studies demonstrated the utility of [¹⁸F]fluoropropyl-(+)-dihydrotetrabenazine ([¹⁸F]FP-(+)-DTBZ) as a positron emission tomography (PET) radiotracer for the vesicular monoamine transporter type 2 (VMAT2) to quantify beta cell mass in healthy control (HC) and type 1 diabetes mellitus (T1DM) groups. Quantification of specific binding requires measurement of non-displaceable uptake. Our goal was to identify a reference tissue (renal cortex or spleen) to quantify pancreatic non-specific binding of [¹⁸F]FP-(+)-DTBZ with the inactive enantiomer, [¹⁸F]FP-(?)-DTBZ. This was the first human study of [¹⁸F]FP-(?)-DTBZ.

Procedures

Six HCs and four T1DM patients were scanned on separate days after injection of [¹⁸F]FP-(+)-DTBZ or [¹⁸F]FP-(?)-DTBZ. Distribution volumes (V_T) and standardized uptake values (SUVs) were compared between groups. Three methods for calculation of non-displaceable uptake (V_ND) or reference SUV were applied: (1) use of [¹⁸F]FP-(+)-DTBZ reference V_T as V_ND, assuming V_ND is uniform across organs; (2) use of [¹⁸F]FP-(?)-DTBZ pancreatic V_T as V_ND, assuming that V_ND is uniform between enantiomers in the pancreas; and (3) use of a scaled [¹⁸F]FP-(+)-DTBZ reference V_T as V_ND, assuming that a ratio of non-displaceable uptake between organs is uniform between enantiomers. Group differences in V_T (or SUV), binding potential (BP_ND), or SUV ratio (SUVR) were estimated using these three methods.

Results

[¹⁸F]FP-(?)-DTBZ V_T values were different among organs, and V_T(+) and V_T(?) were also different in the renal cortex and spleen. Method 3 with the spleen to estimate V_ND (or reference SUV) gave the highest non-displaceable uptake and the largest HC vs. T1DM group differences. Significant group differences were also observed in V_T (or SUV) with method 1 using spleen. SUV was affected by differences in the input function between groups and between enantiomers.

Conclusions

Non-displaceable uptake was different among organs and between enantiomers. Use of scaled spleen V_T values for V_ND is a suitable method for quantification of VMAT2 in the pancreas.

     

The Role of Phosphate in the Action of Vitamin D on the Intestine

The response of chick intestine to vitamin D and its metabolites was studied in an organ culture preparation of chick ileum explants. Both 25-hydroxycholecalciferol (25-OHD₃) at a concentration of 20 ng/ml or greater and 1,25-dihydroxycholecalciferol [1,25-(OH)₂D₃] at a concentration of 50 pg/ml or greater stimulated the rate of accumulation of [³²P]phosphate and ⁴⁵Ca by the explants and the incorporation of [³H]thymidine into DNA. The accumulation of [³²P]phosphate by the explants was against a concentration gradient and inhibited by ouabain and dinitrophenol. Two saturable mechanisms appeared to mediate the cellular accumulation of phosphate with K_a of 0.0047 and 0.125 mM, respectively. The V_max of the lower affinity transport mechanism was accelerated by 1,25-(OH)₂D₃. Actinomycin D (5.0 μg/ml) did not block the intestinal response to 1,25-(OH)₂D₃ stimulation of both [³²p]phosphate and ⁴⁵Ca accumulation. Significant stimulation of [³²P]phosphate accumulation was observed 30 min after the addition of 1,25-(OH)₂D₃, preceding the sterol-induced increase in the rate of ⁴⁵Ca uptake by 30 min and the sterol-induced increase in [³H]thymidine incorporation into DNA by 150 min. Increasing extracellular phosphate concentration to 3.0 mM increased [³H]thymidine incorporation into DNA and the rate of ⁴⁵Ca uptake by the explants. Reducing extracellular phosphate concentration to 0.05 mM attenuated the response of the explants to 1,25-(OH)₂D₃. From these observations it is postulated that the primary action of vitamin D sterols in the intestine is to enhance the ability of the mucosal cell to accumulate phosphate. The data suggest that restoration of intracellular phosphate levels may then permit expression of the cells' response to vitamin D sterols.     

Single- and multiple-dose pharmacokinetics of genistein capsules in healthy chinese subjects: A phase I, randomized, open-label study

Background: Genistein capsules are currently being developed to treat osteoporosis in China. Genistein is extracted from the fruit of Sophora japonica Leguminosae.Objective: The objective of this study was to assess the pharmacokinetics of genistein capsules after single and multiple oral doses in healthy Chinese subjects.Methods: This was a Phase I, randomized, open-label, single- and multiple- dose study in healthy Chinese adults (aged 19-40 years). In the single-dose study, subjects were randomly assigned in a 1:1:1 ratio to receive genistein 50, 100, or 300 mg (in 50-mg capsules). To assess the effect of food on the pharmacokinetics, subjects in the 50-mg group were equally randomized again into fasting and postprandial (genistein was administered after a high-fat breakfast) groups according to a 2-way cross-over design. A separate equal-sized group of subjects were administered genistein 50 mg on day 1 (single dose), received no treatment on days 2 and 3, and were administered genistein 50 mg QD for 6 days (days 4-9) to obtain a multiple-dose pharmacokinetic profile. Because genistein is converted so rapidly and completely to glucuronidated genistein after administration, plasma concentrations of glucuronidated genistein were determined using a validated high-performance liquid chromatography/ tandem mass spectrometry method. Drug tolerability was assessed by monitoring adverse events (AEs) and laboratory parameters.Results: The study enrolled 40 healthy subjects (24 men, 16 women; 10 each in the 50-, 100-, and 300-mg single-dose groups and 10 in the multiple-dose group). Three subjects voluntarily withdrew (2 in the 100-mg group and 1 in the 300-mg group) before study drug administration. Thirty-seven subjects (24 men, 13 women) completed the study and were included in the analysis. The mean (SD) values of the single-dose genistein 50-, 100-, and 300-mg groups were as follows: T_max, 6.0 (2.4), 7.4 (2.4), and 5.6 (1.2) hours, respectively; t_l/2, 13.0 (4.0), 12.6 (5.8), and 9.4 (1.1) hours; AUC_0−t, 3344 (1635), 8389 (5164), and 9361 (2428) ng/mL · h⁻¹; and C_max , 218.7 (68.6), 435.7 (202.1), and 553.4 (152.8) ng/mL. The plasma glucuronidated genistein concentrations were directly proportional to the administered dose over the range of 50 to 100 mg and increased nonproportionately with the 300-mg dose. No statistically significant differences in pharmacokinetic parameters were found in the fasting group compared with the postprandial group. In the multiple-dose group, the mean (SD) steady-state pharmacokinetic parameters on day 9 were similar to those following a single dose of genistein on day 1 (T_max, 6.0 [1.0] vs 5.9 [1.5] hours, respectively; t_l/2, 9.5 [1.5] vs 9.1 [1.5] hours; AUC_0−t, 2830 [1541] vs 2078 [1308] ng/mL · h⁻¹; C_max, 203.1 [130.9] vs 168.4 [105.7] ng/mL). All AEs were assessed as mild or moderate and resolved without treatment, with the exception of elevated alanine aminotransferase and aspartate aminotransferase activities in one subject that resolved with treatment.Conclusions: The pharmacokinetics of glucuronidated genistein appeared to fit the linear-dose range of genistein 50 to 100 mg, but not the 300-mg dose in these healthy Chinese volunteers. Food consumption did not significantly affect the pharmacokinetic properties. No significant differences were observed in the pharmacokinetic parameters after multiple doses of genistein compared with a single dose, suggesting that the drug did not accumulate after multiple doses.