Inactivation of human immunodeficiency virus by gamma radiation and its effect on plasma and coagulation factors.

The inactivation of HIV by gamma-radiation was studied in frozen and liquid plasma; a reduction of the virus titer of 5 to 6 logs was achieved at doses of 5 to 10 Mrad at -80 degrees C and 2.5 Mrad at 15 degrees C. The effect of irradiation on the biologic activity of a number of coagulation factors in plasma and in lyophilized concentrates of factor VIII (FVIII) and prothrombin complex was examined. A recovery of 85 percent of the biologic activity of therapeutic components present in frozen plasma and in lyophilized coagulation factor concentrates was reached at radiation doses as low as 1.5 and 0.5 Mrad, respectively. As derived from the first-order radiation inactivation curves, the radiosensitive target size of HIV was estimated to be 1 to 3 MDa; the target size of FVIII was estimated to be 130 to 160 kDa. Gamma radiation must be disregarded as a method for the sterilization of plasma and plasma-derived products, because of the low reduction of virus infectivity at radiation doses that still give acceptable recovery of biologic activity of plasma components.

Determination of drug protein-binding by high-performance liquid chromatography using a chemically bonded bovine albumin stationary phase.

A liquid chromatographic method for the determination of the degree of protein-binding of drugs has been established, using a stationary phase to which bovine serum albumin has been bonded chemically. In a structurally heterogeneous group of compounds, results of the method correlate well with protein-binding data obtained by equilibrium dialysis (r = 0.89, n = 23, p less than 0.001). Within a series of analogous piperazines a good correlation is found (r = 0.981, n = 11, p less than 0.001). The chromatographic method allows automation of the measurement of protein-binding of large series of compounds with protein-binding ranging between 10 and 99%. The method is not expensive and is less time consuming than equilibrium dialysis. Only 1 mg of technical-grade material is required to determine the protein-binding, and radioactive labelling of the material is not necessary.

Kinetics of rate controlled rectally administered ranitidine to male volunteers.

Ranitidine hydrochloride solution was given rectally to six healthy volunteers by means of an osmotic pump (Osmet) at a zero-order rate for 8 h. Quite constant steady-state drug concentrations were achieved in the range of 64-123 ng/ml plasma (mean Css: 100.6 ng/ml). The mean absorption time was 0.45 h (range 0-I.02 h), indicating that absorption was not always instantaneous. It is concluded that the Osmet system together with the rectal route offer the possibility for achieving steady-state concentrations of ranitidine, enabling the determination of pharmacodynamic effects. In addition, the rectal route can be considered as an alternative to oral and intravenous administration.

Correlation between the metabolism of hexobarbital and aminopyrine in vivo in rats.

Two model substrates for oxidative hepatic enzyme activity, namely hexobarbital and aminopyrine, were simultaneously orally administered to rats, and blood concentrations of the substrates measured by g.l.c. The apparent intrinsic clearances of hexobarbital (Cl*int.HB) and of aminopyrine (Cl*int,AM) were correlated in untreated rats, and in rats pretreated with phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls or carbon tetrachloride. Cl*int,HB and Cl*int,AM were both increased by phenobarbital and polychlorinated biphenyl pretreatment. Pretreatment with 3-methylcholanthrene had hardly any effect, and carbon tetrachloride caused a strong diminution of Cl*int.HB and Cl*int.AM. When the dose of aminopyrine was decreased, both Cl*int,HB and Cl*int,AM increased. This indicated that the primary metabolite of aminopyrine, monomethylaminopyrine, inhibits cytochrome P-450. The correlation coefficient for all clearance data was 0.92 (N = 36). It was concluded that both hexobarbital and aminopyrine are metabolized in vivo by the same or closely related cytochrome P-450 isozymes, and both may be used as model substrates in vivo for metabolic conversions primarily mediated by the major phenobarbital-inducible cytochrome P-450 subspecies.

Conical precolumn as loading buffer for the main column.

A conical high-performance liquid chromatographic precolumn was developed to cope with the problems that arise during the processing of large volumes of biological samples. The shape of the column was designed so as to offer a large loading capacity at the front of the precolumn. The stainless-steel construction, which is pressure resistant up to 40 MPa, can be fully integrated into high-performance systems. In the present work, the precolumn arrangement was used in the assay of pamoic acid in human plasma and in the isolation of radioactive metabolites from pools of animal urine and of supernatants from liver homogenates. Apart from extremely polar compounds, which were not retained on the precolumn, recovery of metabolites was practically complete. Almost the same resolution was obtained with the equivalent of 900 ml of urine, purified and enriched on the precolumn, as with a 2-ml sample of the original urine. Likewise, the chromatographic metabolite pattern of 650 ml of supernatant from homogenized liver was similar to that of a deproteinized sample of 2 ml. It is suggested that the precolumn is usable for all chromatographic problems involving enrichment of small amounts of substances in large amounts of complex matrices.

Fluvoxamine: metabolic fate in animals.

The metabolic fate in animals of the antidepressant compound fluvoxamine was investigated. The 14C-labeled drug was administered orally to dogs, rats, hamsters, and mice, and excretion in urine and feces was measured. Chromatographic patterns of the urines were developed by high performance liquid chromatography. These patterns were used as guides in the isolation of the metabolites, its initial step consisting of concentration of the radioactivity in the urine pools in a conical precolumn, followed by separation in the same HPLC system as used for the metabolite patterns. Altogether, 32 radioactive substances were isolated from the urine pools of the four animal species. They were all identified by the combined use of proton nuclear magnetic resonance and mass spectrometry, and by information obtained from chromatographic behavior and color reactions. Several of the 32 compounds were identical, leaving a total of 11 different metabolites in the four species. In all the animal species, the main focus of fluvoxamine degradation was its aliphatic methoxyl group. In three species, this resulted in the corresponding carboxylic acid as the main metabolite, but in the mouse the corresponding alcohol, in glucuronidated form, was at least as important. In mouse and hamster, the methyl ester was a minor metabolite. Products of acetylation or oxidative removal of the primary amino group accounted for only minor proportions of the metabolite patterns. While fluvoxamine itself has the (E)-configuration, several metabolites occurred both in the (E)- and the (Z)-form. The parent compound was isolated only from the urine of dogs, it accounted for less than 10% of the urinary radioactivity.

Dydrogesterone: metabolism in animals.

The metabolic pattern of dydrogesterone was investigated in the rat, dog, mouse, rabbit and rhesus monkey. The drug was administered orally in 3H-labelled form. Following enzymatic hydrolysis of conjugates the radioactive metabolites were extracted from the urine, and in rat and dog also from bile. The separation method used for the development of the metabolite patterns was reversed-phase high performance liquid chromatography. Dydrogesterone and 4 derivatives, known or suspected to be metabolites, were used as marker substances. In all the species a substantial portion of the urinary or biliary radioactivity was too polar to be extracted, or it was not resolved in the chromatographic system used. The radioactivity which did develop into a pattern coincided with two or more of the marker substances. Only in the monkey, the pattern contained a peak of some substance which did not coincide with any marker. The urinary patterns of rat, dog, and mouse differed substantially, from each other as well as from those of rabbit and monkey. The patterns for the latter two animals showed certain similarities, both to each other and to the human urinary pattern as reconstructed from previous studies. It is concluded that with regard to the metabolic fate of dydrogesterone, the rabbit resembles man more than does any other species.

Dydrogesterone: metabolism in man.

An investigation of the urinary metabolites of the oral progestational agent dydrogesterone in healthy women of childbearing age is reported. The drug was administered in 3H-labelled form and the urine of the first 8 h, containing on average 38% of the radioactivity administered, was used as the source of the metabolites. It was fortified with urine collected during the first 8 h of a similar study with nonlabelled dydrogesterone. After enzymatic hydrolysis of conjugated metabolites, 43 different chemical species were isolated by means of extraction, followed by column and thin layer chromatography. Three of these metabolites, constituting about 70% of the urinary radioactivity, were positively identified as 20 alpha-hydroxy-9 beta, 10 alpha-pregna-4, 6-diene-3-one (52%), 21-hydroxy-9 beta, 10 alpha-pregna-4, 6-diene-3, 20-dione (18%) and 16 alpha-hydroxy-9 beta, 10 alpha-4, 6-diene-3, 20-dione (1%). Of the remainder, 20 (13%) were tentatively characterized as various products of oxidative attack, all probably having the 4, 6-diene-3-one configuration intact. It is concluded that the 4, 6-diene-3-one configuration is metabolically stable in combination with the 9 beta, 10 alpha configuration. This finding may explain why dydrogesterone, in contrast no progesterone, is orally effective, and lacks estrogenic properties.