Population pharmacokinetics of phenobarbital by mixed effect modelling using routine clinical pharmacokinetic data in Japanese neonates and infants: an update.

WHAT IS KNOWN AND OBJECTIVE: Optimal use of phenobarbital in the neonatal population requires information regarding the drug's pharmacokinetics and the influence of various factors, such as different routes of administration, on the drug's disposition. However, because of sampling restrictions, it is often difficult to perform traditional pharmacokinetic studies in neonates and infants. This study was conducted to establish the role of patient characteristics in estimating doses of phenobarbital for neonates and infants using routine therapeutic drug monitoring data. METHODS: The population pharmacokinetics of phenobarbital was evaluated using 109 serum concentration measurements obtained from routine phenobarbital monitoring of 70 neonates and infants. The data were analysed using the non-linear mixed effects model. A one-compartment pharmacokinetic model with first-order elimination was used. Covariates screened were current total bodyweight (TBW), gestational age, postnatal age (PNA), post-conceptional age, gender and neonates-infants clearance factor (serum concentration of phenobarbital; Conc). RESULTS AND DISCUSSION: The final pharmacokinetic parameters were CL/F (mL/h) = (5.95.TBW (kg) +1.41.PNA (weeks)) Conc (serum phenobarbital concentration >50 µg/mL)(-0.221),Vd/F(L) =1.01.TBW (kg), and F = 0.483 for oral administration and F = 1 was assumed for suppository. Conc(-0.221) is 1 for phenobarbital concentration <50 µg/mL. The important variables for predicting phenobarbital clearance in this study were TBW, PNA and Conc. Phenobarbital clearance increases proportionately with increasing TBW, and an older newborn was expected to have a higher rate of clearance than a younger newborn of equal bodyweight. Moreover, the clearance of phenobarbital decreased nonlinearly with increasing serum concentration of phenobarbital >50 µg/mL (Conc(-0.221) ). WHAT IS NEW AND CONCLUSION: We developed a new model for neonate and infant dosing of phenobarbital with good predictive performance. Clinical application of our model should permit more accurate selection of initial and maintenance doses to achieve target phenobarbital concentrations in Japanese neonates and infants, thereby enabling the clinician to achieve the desired therapeutic effect. A similar approach can be used to validate our model for use in other neonate and infant populations.

Antioxidant status and lipid peroxidation in diabetic rats under hyperbaric oxygen exposure.

Hyperglycemia is known to cause oxidative stress that leads mainly to enhanced production of mitochondrial reactive oxygen species (ROS). It has been demonstrated that hyperbaric oxygen (HBO) treatment also increases the formation of ROS. There are, however, no comprehensive evaluations of such oxidative effects in diabetes which requires HBO treatment. The purpose of this study is to investigate the influence of a clinically-recommended HBO treatment on glucose homeostasis and oxidative stress in rats with streptozotocin (STZ)-induced diabetes. Under the clinically-used HBO exposure protocol, the levels of blood glucose, thiobarbituric acid reactive substances (TBARS) as a lipid peroxidation marker, and the activity of superoxide dismutase (SOD) as an antioxidant enzyme marker were investigated in the erythrocytes, liver, pancreas, skeletal muscle, and brain of rats with STZ induced diabetes. The levels of blood glucose and TBARS increased significantly (p<0.05), and the activity of SOD decreased significantly (p<0.05) in the erythrocytes and all organs of rats with diabetes subjected to HBO exposure. These results suggested that HBO exposure might boost glucose autoxidation and increase ROS production in STZ-induced diabetes as side-effects of administering HBO treatment for the first time.

Leptin levels recover normally in healthy older adults after acute diet-induced weight loss.

OBJECTIVES: Involuntary weight loss affects 20% of community dwelling older adults. The underlying mechanism for this disorder is unknown. Objective is to determine if failure of older persons to regain weight is associated with elevated pro-inflammatory cytokine and leptin levels. DESIGN: Prospective diet intervention study. SETTING: University of Washington Medical Center from 2001-2005. PARTICIPANTS: Twenty-one younger (18-35 years old) and nineteen older (>or= 70 years old) men and women. INTERVENTION: Each subject was placed for two weeks on a weight-maintaining diet, followed in sequence by 2 weeks of 30% caloric restriction, then 4 weeks of ad libitum food intake. MEASUREMENTS: Plasma leptin levels, fasting serum pro-inflammatory cytokine levels, and peripheral blood mononuclear cell cytokine levels were measured. RESULTS: Leptin levels in the two cohorts decreased after caloric restriction and increased after ad-libitum food consumption resumed. Plasma TNF alpha levels were higher in older subjects compared to younger adults. However, there was no association between changes in TNF alpha levels and changes in AUC leptin. CONCLUSION: Leptin levels in healthy older individuals responded appropriately in a compensatory manner to changes in body weight. These data do not support a cytokine dependent elevation in leptin levels as being responsible for the failure of older adults to regain weight.

Population pharmacokinetics of digoxin in Japanese patients: a 2-compartment pharmacokinetic model.

OBJECTIVE: To clarify the observed variability of digoxin disposition by performing a population pharmacokinetic analysis in a Japanese population. DESIGN: Retrospective analysis of clinical pharmacokinetic data. PATIENTS AND PARTICIPANTS: Data were obtained from 106 patients with heart failure and atrial fibrillation (43 males and 63 females). METHODS: Digoxin concentrations in serum were measured by fluorescence polarisation immunoassay. Population pharmacokinetic analysis was performed using a 2-compartment open pharmacokinetic model with the computer program NONMEM. RESULTS: 246 serum concentrations were obtained. Final pharmacokinetic parameters were: CL (L/h) = (0.036 x TBW + 0.112 x CL(CR)) x 0.77SPI x 0.784CCB, V1 = 1.83 L/kg, V2 = 22.6 L/kg and Q = 0.629 L/h/kg, where CL is total body clearance, V1 and V2 are the apparent volumes of distribution in the central and peripheral compartments, Q is intercompartmental clearance, TBW is total bodyweight (in kg), CL(CR) is creatinine clearance (in ml/min), SPI = 1 for concomitant administration of spironolactone (and zero otherwise) and CCB = 1 for concomitant administration of calcium antagonists (and zero otherwise). Concomitant administration of digoxin and spironolactone resulted in a 23% decrease in digoxin clearance. Concomitant administration of digoxin and calcium antagonists (diltiazem, nicardipine, nifedipine or verapamil) resulted in a 21.6% decrease in digoxin clearance. CONCLUSIONS: The estimated population parameter values may assist clinicians in the individualisation of digoxin dosage regimens.

Expression of oncogene products and growth factors in early gallbladder cancer, advanced gallbladder cancer, and chronic cholecystitis.

The expression of oncogene products and growth factors (epidermal growth factor, transforming growth factor-beta, erbB-2, ras p 21, and c-myc) in gallbladder cancer and chronic cholecystitis was measured by immunohistochemical staining on paraffin-embedded serial sections. Expression of these products was graded according to staining intensity in an area of positively stained cells. This study reports the detection of oncogene products and growth factors in cholecystitis as well as in early and late gallbladder cancer. The multiexpression of oncogene products and growth factors was greater for both gallbladder cancer groups as compared with the cholecystitis group. The percentage of epidermal growth factor positivity diminished with increased proportion of interstitial tissue and, conversely, the percentage of transforming growth factor positivity increased with increased proportion of interstitial tissue. The proportion of ras positivity was significantly greater in both early and advanced cholecystic cancer as compared with cholecystitis, but also was considerable even for cholecystitis. These results suggest that various oncogenes may have significant roles in gallbladder cancer and that collagen synthesis is reduced by epidermal growth factor and enhanced by transforming growth factor-beta.

Acute epiglottitis. An 18-year experience in Rhode Island.

OBJECTIVE: To assess the incidence, clinical characteristics, management, and outcome of epiglottitis in a defined population over an 18-year period. DESIGN: Case series. SETTING: The state of Rhode Island, 1975 through 1992. PATIENTS OR OTHER PARTICIPANTS: Cases who met predetermined criteria for acute epiglottitis identified from hospital discharges and the State Medical Examiner's log of prehospitalization deaths. MAIN OUTCOME MEASURES: Incidence by year and age, clinical presentation, results of diagnostic evaluations, management, and outcome. RESULTS: Four hundred seven cases were identified, 134 in children and 273 in adults. Incidence in children dropped from 38 cases in the first 3 years of the study to 1 case in the last 3 years (p < 0.001). Adult cases increased from 17 in the first 3 years to 69 in the last 3 years (p < 0.001). Seventy-nine percent of adults and 32% of children were treated without an artificial airway. Factors associated with airway obstruction included symptomatic respiratory difficulty, stridor, drooling, shorter duration of symptoms, enlarged epiglottis on radiograph, and Haemophilus influenzae bacteremia (p < 0.001 for each). Twelve patients died (3 children and 9 adults), with all cases of fatal respiratory obstruction occurring within 12 h of presentation. CONCLUSIONS: There have been significant changes in the clinical epidemiology of epiglottitis, which now occurs almost exclusively in adults, often with less severe symptoms and a lower incidence of H influenzae infection. While careful observation is indicated for all patients, the data suggest that those with certain clinical characteristics can be treated safely without an immediate artificial airway.

Purification and characterization of endogenous protein activator of human platelet proteasome.

An endogenous activator of 20S proteasome was purified from human platelets and its effect on three peptidase activities of proteasome was studied. This activator had a molecular weight of 170 kDa, and was composed of 32 kDa polypeptides as determined by SDS-PAGE. It was highly labile upon heat treatment (56 degrees C, 20 s) and proteinase (pronase CB) digestion. Suc-LLVY-MCA degrading activity of the platelet proteasome showed positive cooperativity between two or more catalytic sites because the coefficient was 1.54 when analyzed by use of the Hill plot. The endogenous activator increased Vmax and caused a loss of cooperativity. The plot of reaction velocity as a function of activator concentration yielded a saturation curve, implying the binding of the activator to proteasome. Boc-LTR-MCA degrading activity followed Michaelis-Menten kinetics. The activator enhanced the activity by increasing Vmax and decreasing Km. In contrast, CBz-LLE-2NA degrading activity could not be analyzed according to any kinetic scheme reported so far. The activator stimulated this activity at lower substrate concentrations (below 200 microM), while it inhibited the activity at higher substrate concentrations (400-800 microM). It is concluded from these findings that the endogenous protein activator may regulate the intracellular proteasome activity by functioning as a positive allosteric effector.

Crystal structures of modified myoglobins. I. Heme orientation and structural changes around heme in myoglobins reconstituted with isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme.

The crystal structures of sperm whale metmyoglobins reconstituted with four modified hemes, isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.217, 0.218, 0.213, and 0.222, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The structural changes of the modified myoglobin from the native myoglobin were examined on difference Fourier maps; the orientation of 4-ethyldeuteroheme in the heme pocket is such that the heme is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other three hemes are the same as that of the protoheme in the native myoglobin. The changes of the structures around the heme become greater in the order of isopemptoheme, 2-ethyldeuteroheme less than pemptoheme less than 4-ethyldeuteroheme. The magnitudes of the changes seem to be related to the oxygen affinities of these four reconstituted myoglobins.

Abnormalities of acetylcholinesterase in Alzheimer's disease with special reference to effect of acetylcholinesterase inhibitor.

In brains from Alzheimer's disease patients, a high activity of acetylcholinesterase (AChE) was detected in the senile plaque-rich fraction and its isozyme pattern was mainly type A, containing a collagen-like tail. AChE inhibitors, including physostigmine, E-2020, amiridin, tetrahydroaminoacridine (THA) and Nicergoline had a poor effect on AChE present in the senile plaque-rich fraction isolated from Alzheimer brain than that either in the soluble fraction of Alzheimer brain or in the control brain. However, AChE purified from rat skeletal muscle (type A) was significantly more susceptible to AChE inhibitors than that purified from rat brain (G4 form) or from human erythrocytes (G2 form). E-2020 inhibited all 3 types of isozymes more effectively than physostigmine, amiridine, Nicergoline or THA. The inhibitory effect of AChE inhibitors on AChE solubilized from senile plaque was also small as compared with AChE in normal human brain, rat brain, human erythrocytes or rat skeletal muscle. These results suggest that the characteristics of AChE present in senile plaques are abnormal or different from that in normal brain or skeletal muscle. As AChE in the Alzheimer brain seems to contain a higher degree of glycosylation, the hydrophobic property of anomalous AChE may serve a seed of amyloid fibril in senile plaques.

Potential etiologic role of PAF in two major septic complications; disseminated intravascular coagulation and multiple organ failure.

A possible role of platelet-activating factor (PAF) in the occurrence of the two septic complications, i.e., disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) was investigated, employing a rabbit model and a novel PAF antagonist E5880. By an instillation of fecal suspension into the common bile duct of the rabbit, manifestations of DIC and MOF were observed with high reproducibility by 9 hours after the septic insult. E5880 was intravenously administered to 12 rabbits for 1 hour after the septic insult at dose of 1 mg/kg (n = 6) or 3mg/kg (n = 6). All the rabbits were subjected to observation of vital signs and serial determination of laboratory tests for 9 hours and then lung, liver and kidney were removed for histological examination. Blood endotoxin level increased significantly by 9 hours after the septic insult. Although administration of E5880 did not affect the endotoxemia, the antagonist attenuated in a dose related manner laboratory manifestation of DIC such as thrombocytopenia and prolonged prothrombin time as well as that of MOF such as increase in serum bilirubin and creatinine level. The beneficial effect of E5880 on MOF was also confirmed by the histological evaluation. These observations indicated that PAF is deeply involved in the occurrence of DIC and MOF due to sepsis and E5880 may be one of the modalities to treat or prevent these two major septic complications.

Expression of transcription factors and stem cell factor precedes hepatocyte differentiation in rat pancreas.

Multiple foci of morphologically and functionally differentiated hepatocytes are induced in the pancreas of adult rats subjected to a copper depletion-repletion regimen. Differentiation of hepatocytes in pancreas is preceded by irreversible depletion of over 90% of pancreatic acinar cells. Progressive acinar cell loss during 4-6 weeks of copper deficiency results in the proliferation of oval cells, some of which may serve as the hepatocyte precursor or stem cells. Albumin mRNA is detected in oval cells at 5 and 6 weeks by in situ hybridization at which time no morphologically identifiable hepatocytes are evident in the pancreas. Immunocytochemical analysis demonstrated the presence of stem cell factor (SCF) in proliferating oval cells during 6 weeks of copper depletion, and Northern blot analysis revealed the expression of liver-enriched transcription factors in the rat pancreas during this 4-6-week period of copper deficiency. CCAAT/enhancer binding protein alpha (C/EBP alpha) mRNA was detected first at 4 weeks of copper deficiency. By 5 and 6 weeks of copper deficiency, the expression of mRNAs of C/EBP alpha, beta, and delta, and hepatocyte nuclear factor-3 factor (HNF-3 beta) was markedly enhanced. This enhanced expression of liver-enriched transcription factors and the SCF during oval cell proliferation in the pancreas preceding the expression of albumin mRNA and subsequent differentiation of hepatocyte phenotype further supports the identity of these oval cells as hepatocyte precursors or stem cells.

Induction of radical scavenging ability and protection against radiation-induced damage to microsomal membranes following low-dose irradiation.

PURPOSE: To investigate changes in rat liver cytosolic radical scavenging ability and in microsomal membrane function following low doses of radiation. MATERIALS AND METHODS: Wistar rats were irradiated with 1-50 cGy of X-rays and liver cytosolic radical scavenging ability was determined using DPPH, a stable free radical. Liver microsomal drug metabolizing activity was determined using hexobarbital as a substrate. Cytosolic antioxidants and microsomal enzymes were spectrophotometrically determined. RESULTS: Irradiation of rats at around 5-10 cGy induced liver cytosolic radical scavenging ability and a considerable increase in this ability at 5 cGy was observed for 3 days after irradiation. Glutathione reductase was suggested as a candidate of cytosolic antioxidants. Radiation-induced damage to liver microsomal drug metabolizing enzyme activity was suppressed by preirradiation with 5 cGy, mainly by protecting cytochrome P-450. CONCLUSIONS: Low doses of radiation increased cytosolic radical scavenging ability and resulted in protection of microsomal membrane function which is easily damaged by radiation-induced free radicals.

Susceptibility of Mongolian gerbils (Meriones unguiculatus) to leptospires and the protective effect of vaccination.

Mongolian gerbils, Meriones unguiculatus, were shown to be highly susceptible to Leptospira interrogans serovars such as icterohaemorrhagiae, copenhageni, canicola, autumnalis, javanica, pyrogenes and hebdomadis as compared to guinea pigs and hamsters. Mortality with signs of haemorrhage and jaundice was recorded in all experimental rodents after intraperitoneal inoculation with all strains of the serovars indicated. However, Mongolian gerbils were comparatively susceptible to strains which were of low virulence to guinea pigs and hamsters. Use of leptospiral vaccination proved effective in protecting the animals against inoculum challenges. The Mongolian gerbil is a species that may be selectively and preferentially useful for assays on the protective effects of leptospiral vaccination.

Effect of Bacillus subtilis spore administration on activation of macrophages and natural killer cells in mice.

The effect of Bacillus subtilis (strain A102) spores on the activation of murine macrophages and natural killer cells (NK) was examined. The macrophage activity and NK activity were enhanced by oral administration of A102 spores, and slightly enhanced by oral administration of culture supernatant. There was no difference in the results of macrophage activity and NK activity using other live or dead spores. The NK activity and macrophage activity were increased with increments of concentration up to 0.1 g per mouse, and both activities were decreased at concentration of more than 0.15 g per mouse. The NK activity was increased 1 and 2 days after oral administration of A102 spores, and the activity level 2 days after administration was about 3-fold higher than the level prior to treatment. Macrophage activity was also increased from 1 to 3 days after oral administration of A102 spores, and the activity level 3 days after administration was about 3-fold higher than the level prior to treatment. The induction of interferons at 1 day after oral administration in mouse serum was 5-fold higher than that in controls. These findings indicate that oral administration of A102 gave rise to the induction of interferons, and it is likely that macrophages and NK cells were activated by interferons.

Protective effects of serum thymic factor to Leptospira interrogans serovar Copenhageni infection in Mongolian gerbils.

The susceptibility to Leptospira interrogans serovar copenhageni in Mongolian gerbils treated with 10 micrograms of serum thymic factor (FTS) 1 day before infection was examined. Susceptibility of gerbils treated 5 times with 10 micrograms of FTS was also investigated. Mortality of FTS-treated gerbils was significantly lower than that of controls when small challenge doses were used. To analyse the FTS-induced resistance to leptospiral infection, natural killer (NK) cell activity and macrophage activity were studied. Macrophage activity was unaltered but NK cell activity was enhanced in FTS-treated gerbils, with or without leptospiral infection. Since no side-effects of FTS were observed, this compound should be considered for the treatment of leptospirosis.

Impairment of macrophage function in Mongolian gerbils.

Chemiluminescence studies on superoxide generation by phagosomes using opsonized zymosan showed the highest fluorescence in murine splenic macrophages among four different kinds of splenic or peritoneal macrophages from mice or gerbils. Murine splenic macrophages phagocytized two to three times more latex particles than gerbil splenic macrophages, but peritoneal macrophages did not show a significant difference in phagocytic activity between mice and gerbils. Phagocytosis by macrophages was determined by a technique based on measurement of the release of hydrogen peroxide and myeloperoxidase from phagosomes using microspheres conjugated with 3-(p-hydroxyphenyl) propionic acid (HPPA-MS). HPPA is a substrate of lysosomal myeloperoxidase. The fluorescence of HPPA-HPPA-MS produced by phagocytized HPPA-MS was measured with an immunoreaction analysis system (IMRAS), and the enzyme activities of the four different kinds of peritoneal or splenic macrophages from mice and gerbils were compared. All four kinds of macrophages produced HPPA-HPPA-MS in their phagosomes during phagocytosis and murine splenic macrophages showed the highest level of enzyme activity.

Determination of radionuclides produced by neutrons in heavily exposed workers of the JCO criticality accident in Tokai-mura for estimating an individual's neutron fluence.

In the Tokai-mura criticality accident, three workers were heavily exposed. Biological materials, such as blood, urine, vomit and hair, were collected from the workers and analyzed for radioactivities, produced by the neutron irradiation. Activation products. such as 24Na, -K and 82Br, were found in these materials by gamma-ray spectrometry. The radionuclide of the highest activity observed in biological materials was 24Na, e.g. the concentrations of this nuclide in the blood samples from the three patients at the accident time were 169, 92 and 23 Bq/ml, respectively. The concentrations of stable sodium in the same samples were determined by ICP-AES to obtain specific activities of 24Na (concentration ratio between the produced 24Na and stable 23Na), which are essential for estimating the neutron fluences and radiation doses. The specific activities of 24Na obtained for the three patients through the blood analysis were 8.2 x 10(4),4.3 x 10(4) and 1.2 x 10(4) Bq24Na/g23Na. Based on these values, individual's neutron fluences were estimated to be 5.7 x 10(11), 3.0 x 10(-1) and 0.85 x 10(11) cm(-2), respectively.

Bioassay for neutron-dose estimations of three patients in the JCO criticality accident in Tokai-mura by measuring beta-ray emitters.

The measurement of beta-emitters in biological samples (hair, urine and bone) from three patients in the JCO criticality accident was performed to assess the neutron dose to individuals. The result of the measurements of 32P in hair and urine collected immediately after the accident showed that sufficient 32P activities had been induced in the hair by fast neutrons and in the urine by thermal neutrons to know the severity of the exposure to the individuals and to the position. From the measurement of 32P and 45Ca in bone from various anatomical parts of two patients who died 82 and 210 days after the accident, it was suggested that the distribution of the induced beta-emitters activities could prove the position and posture of the patients at the moment of exposure.

Borrelidin inhibits a cyclin-dependent kinase (CDK), Cdc28/Cln2, of Saccharomyces cerevisiae.

We identified borrrelidin, a member of macrolide antibiotic, as an inhibitor of a cyclin-dependent kinase of the budding yeast, Cdc28/Cln2. A 50% inhibition concentration (IC50) of borrelidin for Cdc28/Cln2 was 24 microM. In addition, borrelidin arrests both haploid and diploid cells in G1 phase at the point indistinguishable from that of alpha-mating pheromone, at concentrations not affecting the gross protein synthesis. Although the inhibition of CDK activity may not be a solo cause of the G1 arrest, our results indicate that borrelidin is a potential lead compound for developing novel CDK inhibitors of higher eukaryotes.