Cheryl L. Golas

Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS180

The Ah (aromatic hydrocarbon) receptor mediates induction of aryl hydrocarbon hydroxylase (AHH; an enzyme activity associated with cytochrome P450IA1) by polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene (MC) and benzo[a]pyrene (BP) and the halogenated toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Until recently the AhR seemed to be present only at very low levels in human cells and tissue. With a modified assay (the presence of sodium molybdate and a reduction in the amount of charcoal used to adsorb excess ligand) we found that cytosol from LS180 cells contains a high concentration of AhR (400-500 fmol/mg cytosolic protein) when detected by [3H]TCDD or [3H]MC. Cytosolic receptor also was detected with [3H]BP but at a level that was 35% of that detected with [3H]TCDD or [3H]MC. These levels are similar to those found in mouse Hepa-1 hepatoma cells in which AhR has been extensively characterized. The apparent binding affinity (Kd) of the cytosolic receptor for [3H]TCDD and for [3H]MC was about 5 nM. As with Hepa-1, the human LS180 cytosolic AhR sedimented at about 9 S on sucrose gradients when detected with [3H]TCDD, [3H]BP or [3H]MC. The nuclear-associated ligand.receptor complex recovered from cells incubated in culture with [3H]TCDD sedimented at about 6.2 S. The 9.8 S cytosolic form corresponds to a multimeric protein of a relative molecular mass (Mr) of about 285,000 whereas the 6.2 S nuclear receptor corresponds to a multimeric protein of Mr 175,000. The smallest specific ligand-binding subunit (detected by sodium dodecyl sulfate-polyacrylamide electrophoresis under denaturing conditions of receptor photoaffinity labeled with [3H]TCDD) was about Mr 110,000. AHH activity was induced in cells exposed in culture to TCDD or benz[a]anthracene (BA). The EC50 was 4 x 10(-10) M for TCDD and 1.5 x 10(-5) M for BA. For both inducers the EC50 in LS180 cells was shifted about one log unit to the right as compared to the EC50 for AHH induction in mouse Hepa-1 cells. The lower sensitivity of the LS180 cells to induction of AHH activity by TCDD or BA is consistent with the lower affinity of TCDD and MC for binding to human AhR. The ligand-binding properties, physicochemical properties, and mode of action of the AhR in this human cell line are therefore very similar to those of the extensively characterized AhR in rodent cells and tissues.

Pharmacokinetics and adverse effects of amphotericin B in infants and children

The pharmacokinetics and safety of amphotericin B infusion were studied in 13 infants and children (age range 3 weeks to 18 years; median age 11 years) treated with the drug for proved (n = 11) or suspected (n = 2) fungal infections. The dose during the first day was 0.5 mg/kg, followed by a daily dose of 1 mg/kg for the rest of the treatment period in most patients. The drug was infused over 4 to 6 hours. During the first day, serum concentrations were above the target therapeutic level of 0.3 microgram/ml in all patients at 2 and 6 hours from the start of the infusion, in 12 of 13 patients at 12 hours, but in only 6 of 13 patients at 24 hours. On the third day, all concentrations were greater than 0.3 microgram/ml throughout the 24-hour period, and in 12 of 13 patients were greater than 0.5 microgram/ml. The same kinetic profile prevailed on days 7 to 10 of therapy, with a tendency for increasing concentrations. Elimination half-life was 9.93 +/- 1.5 hours (mean +/- SEM), clearance rate 26 +/- 5 ml/kg.hr, and distribution volume 378 +/- 25 ml/kg. The half-life inversely correlated with patients age. Pharmacokinetic values calculated during the first day were not different from those calculated on day 3. Significant decreases in hemoglobin, platelets, and serum potassium concentration were recorded along with significant increases in serum creatinine, urea, and aspartate transaminase values. Because of the large pharmacokinetic variability and the high rate of serious adverse effects, individualized dosing of amphotericin B based on therapeutic drug monitoring should be considered.

Measurement of amphotericin b in serum or plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the analysis of amphotericin B in 25 microliters of serum or plasma is described. The procedure involves the addition of the internal standard, p-nitrobenzyloxyamine, to the sample followed by a precipitation of protein with acetonitrile. The supernatant is directly injected into a chromatograph attached to a reversed-phase mu Bondapak (Waters) column containing C18 packing. The mobile phase is a 60 : 40 mixture of a sodium acetate buffer (10 mM, pH 7.0)--acetonitrile, and we employ a flow-rate of 1.5 ml/min and a detection wavelength of 405 nm. Total analysis time per sample is 10 min. Coefficients of variation were found to be less than 4% for concentrations less than 2 mg/l. Analytical recoveries were between 75 and 80%. No drug or drug metabolite interference was found. The method will be used to study pharmacokinetic and pharmacodynamic data in a pediatric population.

Competitive binding of 7-substituted-2,3-dichlorodibenzo-p-dioxins with human placental Ah receptor—A QSAR analysis

The competitive binding affinities of thirteen 7-substituted-2,3-dichlorodibenzo-p-dioxins to the human placental cytosolic aryl hydrocarbon (Ah) receptor were determined using [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as the radioligand. Multiple parameter linear regression analysis of the competitive binding C50 values for these compounds gave the following equation: pEC50 (M) = 6.246 + 1.632 pi - 1.764 sigma 0m + 1.282 HB where pi, sigma m and HB are the physiochemical parameters for substituent lipophilicity, meta-directing electronegativity, and hydrogen bonding capacity respectively. The 7-t-butyl- and 7-phenyl-2,3-dichlorodibenzo-p-dioxins were treated as outliers for the derivation of this equation, and these results suggest that only substituents with van der Waals volumes less than 40 cm3/mol were accommodated in the receptor binding site. The equations previously derived from the binding of the 7-substituted-2,3-dichlorodibenzo-p-dioxins to the rat, mouse, guinea pig, and hamster hepatic cytosolic receptor were different than the correlation obtained using human placental receptor and provide further evidence for the interspecies differences in the molecular and binding properties of the Ah receptor protein.

Chloramphenicol pharmacokinetics in the newborn

We studied pharmacokinetics of chloramphenicol in 9 neonates having a mean gestational age of 31.2 +/- 1.9 weeks (mean +/- SEM). The studied dose was the final dose of treatment in 8 of these and the first dose in 2 of these. 1 neonate was studied twice. Concentrations of chloramphenicol and its 3-monosuccinate and 1-monosuccinate esters were measured in serum by high performance liquid chromatography. Apparent total body clearance of chloramphenicol correlated with postnatal age (r = 0.81, p less than 0.01). Mean apparent clearance was 1.1 ml X min-1 X kg-1. Serum concentrations of succinate esters were below assay sensitivity after 6 h postdose. Factors leading to excessive chloramphenicol concentrations (greater than 25.0 mg/l) were evaluated in another 44 newborns. Instability of the patients clinical condition was an important cause of excessive serum concentrations during ongoing therapy.

Initiation of chloramphenicol therapy in the newborn infant

To evaluate the ability of a loading dose of chloramphenicol succinate to rapidly, achieve adequate serum concentrations of chloramphenicol, we compared two intravenously administered dosages of chloramphenicol succinate given to initiate treatment. Thirteen premature neonates received an initial dose of 12.5 mg/kg; 26 received a loading dose of 20 mg/kg. Capillary blood samples were obtained at two, four, and 12 hours after the first dose. After the dose of 12.5 mg/kg, 45% of the neonates did not achieve serum concentrations greater than 10 mg/L. After the loading dose of 20 mg/kg, all neonates achieved concentrations greater than 10 mg/L. The peak chloramphenicol concentrations after the 12.5 mg/kg dose was 8.8 +/- 11.2 mg/L (+/- SEM) and after the 20 mg/kg loading dose, 15.9 +/- 0.7 mg/L. The disposition of chloramphenicol was age dependent. Chloramphenicol concentration peaked at four hours in neonates less than or equal to 2 days postnatal age and at two hours in neonates 3 to 55 days postnatal age. Chloramphenicol succinate concentrations were greater in younger than in older neonates at both two and four hours after the dose. We conclude that a loading dose is appropriate when using chloramphenicol succinate in neonates.

7-Substituted-2,3-dichlorodibenzo-p-dioxins as competitive ligands for the Ah receptor: Quantitative structure-activity relationships (QSARs) and a comparison of human receptor with Ah receptor from rodents

Abstract The competitive binding affinities of thirteen 7-substituted-2,3,-dichlorodibenzo-p-dioxins to the human Ah placental cytosolic Ah receptor were determined versus [ 3 H ]-2,3,7,8- tetrachlorodibenzo -p- dioxin (TCDD) as the radioligand. Multiple parameter linear regression analysis of the competitive binding EC50 values for these compounds gave the following equation: pEC 50 (M) =6.246+1.632 π − 1.764σ m° + 1.282 HB were π is the substituent lipophilicity, σm° the meta-directing electronegativity and HB the hydrogen binding capacity. The equation obtained using human placental receptor was different than correlations previously derived for the binding of the same series of compounds to the rat, mouse, guinea pig or hamster cytosolic Ah receptor, providing further evidence for interspecies differences in the properties of the Ah receptor protein.

Toward optimization of therapy in the neonate

Clinical Pharmacology and Therapeutics (1983) 33, 551–555; doi:10.1038/clpt.1983.75

The high performance liquid chromatographic measurement of chloramphenicol and its succinate esters in serum

We describe a reliable HPLC procedure for the simultaneous measurement of chloramphenicol and its succinate esters in 30 μL of serum or plasma. The procedure has been applied to assess both the optimum dosing regimen and clinical pharmacokinetics in premature infants.