Milton T. Bush

Studies on the metabolic fate of [14C]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the mouse.

Studies on the Metabolic Fate of [14C]2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in the Mouse. KOSHAKJI, R. P., HARBISON, R. D., and BUSH, M. T. (1984). Toxicol. Appl. Pharmacol. 73, 69-77. After a single po dose (135 micrograms/kg; 62 microCi/kg) of 14C-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male ICR/Ha Swiss mice, 67 to 76% of the administered dose was eliminated via the feces and 1 to 2% in the urine during the first 24 hr following treatment. It seems likely that most of this material was simply not absorbed. Much of the remaining chemical was then excreted slowly in the urine (2%) and feces (7%) during the next 10 days, partly as the unchanged compound and partly as metabolites. One of the metabolites (Fraction II) appears to be a single polar, acidic metabolite characterized in urine (0.4 +/- 0.1%) and feces (2.2 +/- 0.2%), and is also likely excreted as a glucuronide conjugate. The rest of the radioactivity was in the form of unchanged TCDD in the animal body (17 +/- 2%). Steady rates of decline in the concentrations of the 14C as well as of the unchanged TCDD were reached in the feces and urine after the fifth day following the administration of the chemical. Based on this steady rate, the half-life of the radioactivity in the body was approximately 20 days. Urine, feces, and whole body were analyzed by solvent extraction, 14C counting, thin-layer chromatography, and countercurrent distribution.

CHARACTERIZATION OF DRUG-PROTEIN INTERACTIONS BY CLASSIC METHODS*

In this brief review, we will consider dialysis, ultrafiltration, and gel filtration. These “classical” methods for estimating the degree of binding of drug molecules by proteins may be considered to involve variations of one fundamental principle: the physical separation (of some) of the relatively small free drug molecules from the large particles of protein and drug-protein complex. Such a separation is accomplished by the use of a barrier or membrane that has pores large enough to permit passage of the small molecules but not large enough to allow passage of the large protein molecules or those of the drugprotein complex. A typical experimental situation is illustrated in FIGURE 1. The problem of the determination of the concentration of free drug molecules has commonly been approached by one or more of three procedures. By far the most common and most classical has been that of equilibrium dialysis. The other procedures, which have been devised or come into more or less frequent use mostly within the last decade, are ultrafiltration and gel filtration. These last two methods have been proposed as having considerable advantages over conventional equilibrium dialysis, especially economy of time and material. It is something of an irony, however, that over this same period of time, actually beginning as early as 1955, techniques based on dialysis have been brought to a much higher degree of sophistication than that of the conventional technique, although biologists, for the most part, seem to have remained unaware of these developments. The situation now seems to be that dialysis, in addition to yielding the data by which the validity of many other methods is o can be

Metabolism of bufotenine-2'-14C in human volunteers.

The major ultimate metabolites of bufotenine-2′-14C in two volunteers have been identified and quantitated in the urine after intravenous administration of doses of 0.2 mg and 1 mg (10 μc/mg). Both subjects excreted over 90% of the 14C within 12 hours. Most of this was in the form of 5-hydroxyindoleacetic acid (68% and 74%). Only small amounts of bufotenine itself were excreted (1% and 6%). The remaining radioactive materials in the urine were not identified.

Brain barbital levels and anesthesia as influenced by physostigmine and epinephrine

Abstract The potentiating effect of physostigmine on barbital anesthesia has been attributed to a modification of the permeability of the blood-brain barrier as a result of cholinesterase inhibition. The rate of penetration of barbital into mouse brain and its possible alteration by pharmacological means were investigated. Individual brain barbital levels were determined by an improved analytical technique and correlated with alterations in the lag in onset of barbital anesthesia. Barbital with physostigmine produced signs of depression much sooner and much more intense than barbital alone. However, these signs could not be attributed to increased brain barbital concentrations because in animals receiving physostigmine brain barbital levels were not increased over the controls. The early ataxia, loss of righting reflex, produced by physostigmine could be prevented by pretreatment with atropine methyl nitrate without an alteration in brain barbital levels. Animals receiving barbital with epinephrine displayed immediate signs of depression and became anesthetized much sooner than controls. These animals had brain barbital concentrations above the control from 2 to 20 min. The epinephrine effect, however, cannot be attributed to increased brain barbital levels alone, because at 10 min the controls showed no signs of depression with the same brain barbital levels as the ‘enesthetized’ epinephrine-treated animals. There were no apparent differences in behavior of barbital plus norepinephrinetreated mice from that of controls although a small increase in brain barbital levels was noted. For the experimental conditions chosen it is concluded that: (1) Physostigmine does not alter the rate of penetration of barbital into the brain. The early loss in righting reflex produced by the combination of barbital and physostigmine is probably mediated peripherally. (2) Epinephrine contributes to a greatly increased rate of onset and intensity of ‘anesthesia’ by some mechanism(s) in addition to increasing the rate of entry of barbital into the brain.

Ultra-short-acting barbiturates as oral hypnotic agents in man

Administration to man of about 10 mg. per kilogram of thiopental sodium, hexobarbital sodium or methohexital sodium can produce rapid, intense sedation of only an hour or twos duration, with substantial recovery, including return of mental clarity within 3 to 4 hours. Quantitative determinations of barbiturate concentrations in the blood are closely correlated with clinical effects. From these and other results, we infer that to achieve the rapid absorption necessary for these results, the stomach must be empty at the time of administration and the drug must be in solution or in a form which is qUickly soluble in the aqueous medium in the stomach. Correlations between chemical structure and physical properties, on the one hand, and lipid solubility and rapidity of absorption on the other are discussed.

Extraction and identification of barbiturates

Abstract The principles and practice of the application of fractional extraction to the isolation and identification of drugs and their metabolic products have been discussed with special reference to the barbiturates. The measurement of partition coefficients in dilute solutions (about 5 μg./ml.) with the aid of ultraviolet spectrophotometry is described, and implemented as an aid in identification by the presentation of original data for a dozen common barbiturates and several other common drugs. It is suggested that refinement and extension of the data and technique should make this a more and more useful tool in both toxicological and research investigations.

Quantitative determination of primidone in biological material.

Abstract A quantitative chemical method is described for oxidizing primidone (with chromic acid) to phenobarbital. A mixture of the two drugs in urine or blood is analyzed by extracting both with ether, removing the phenobarbital from this by extraction with 0.1M ethanolamine, then evaporating the ether and oxidizing the primidone, which is left. The separation, and the determination of phenobarbital in both fractions is practically quantitative for samples of 50 μg to 500 μg. If present, p-hydroxy-phenobarbital can be quantitatively eliminated by chromic acid treatment.

Diazomethane: Improved analytical and distillation techniques for small quantities and some synthetic applications

Abstract An improved design of apparatus for the small-scale (about 5 μmol to about 50 mmol) preparation of diazomethane is described. The diazomethane is generated from commonly used precursors and distilled by aeration in a glass apparatus connected by Teflon tubing and without a condenser. A new simple and reasonably accurate procedure for assay of diazomethane is described. This depends on reaction with excess [ 14 C]benzoic acid in toluene followed by quantitative removal of the excess acid by partitioning with pH 10 aqueous buffer and assaying the methyl [ 14 C]benzoate in the toluene by liquid scintillation counting. Examples are given of the use of accurately known amounts of diazomethane and [ 14 C] diazomethane for the preparation of methylated derivatives of [2- 14 C]barbital, 4′-hydroxy-[2- 14 C]phenobarbital, and mephobarbital. Small amount(s) of dimethyl-barbital ( O -methyl) were separated and partly characterized by gas chromatography/mass spectroscopy and NMR.

Enhancement of Cesium-137 Excretion by Rats Treated with Acetazolamide

Acetazolamide (10 mg/kg, intraperitoneally) increases the urinary excretion of cesium-137 in the rat. Meralluride (6.8 mg of Hg per kilogram, subcutaneously) blocks the effect of acetazolamide on the cesium-137 excretion without any influence on the urine volume and pH of the urine. This indicates that acetazolamide increases the urinary excretion of cesium-137 by increasing its secretion through the renal tubule in the same manner as it increases the excretion of potassium and rubidium-86.

Synthesis of millimole amounts of14C- and2D-labelled primidone, phenylethylmalonamide and phenobarbital for high-sensitivity detection in biological materials

SummarySyntheses of millimole quantities of14C- and2D-labelled primidone, phenylethylmalonamide and phenobarbital are described. The labels are incorporated in positions that permit quantitative recovery in the metabolites. Methods are given for isolating the compounds by countercurrent distribution and thin-layer chromatography. Mass spectra of2D-labelled and non-labelled primidone are presented. Detection, identification and quantitation of the compounds and their metabolites in biological material by radiotracer and GC-MS procedures are discussed.ZusammenfassungSynthesen von Millimolen14C- und2D-markierten Primidons, Phenyläthylmalonamids und Phenobarbitals wurden beschrieben. Die Markierung erfolgte an Stellen, die eine quantitative Auffindung in den betreffenden Metaboliten ermöglichen.Methoden zur Isolierung der genannten Verbindung durch Gegenstromverteilung und Dünnschichtchromatographie wurden angegeben Massenspektren von2D-markiertem und nicht markiertem Primidon wurden angegeben. Nachweis, Identifizierung und Bestimmung der Verbindungen und ihrer Stoffwechselprodukte in biologischem Material mit Radiotracern bzw. durch Gaschromatographie und Massenspektrometrie wurden erörtert.