William Trager

Acquired Immunity to Ticks.

It is well known that the American dog tick, Dermacentor variabilis Say, can be reared through its entire life cycle on guinea pigs. In the course of permitting successive batches of larvae of this tick to engorge on the same guinea pig, it was repeatedly observed that, while the first batch always gave a large number of engorged larvae, the following batches gave few or none. This suggested that guinea pigs, after one infestation with larvae of D. variabilis, acquire an effective immunity against these ectoparasites. The experiments reported in this paper show that such an immunity is indeed developed, and that it may be solid enough to prevent larvae from engorging and to reduce the amount of blood taken by nymphs and adults.

Plasmodium falciparum in culture: use of outdated erythrocytes and description of the candle jar method.

Plasmodium falciparum has been cultured continuously in a simplified technique using plastic petri dishes in a candle jar. This method has the advantage of being readily adaptable to many experiments, especially those requiring numerous replicates. Experiments using this method have allowed examination of some factors responsible for variations in parasite multiplication rates. Generally, fetal calf serum is not comparable to human serum, modifications to the RPMI 1640 medium are usually deleterious, and erythrocytes aged in citrated plasma are better for malarial cultures than freshly obtained cells; thus, erythrocytes outdated by blood-bank standards could provide a readily available supply of cells for large-scale production of malarial antigens. Continuous culture of Plasmodium falciparum in vitro was first obtained by a method providing for a slow flow of medium over a thin settled layer of human erythrocytes (Trager and Jensen, 1976; Trager, 1976). This method lends itself to partial automation and provides for continuous production of parasite material. It is not convenient, however, for the study of the many different factors that might affect growth of the parasites. For this purpose we developed a simplified method using plastic petri dishes in a candle jar. The essentials of this petri dish method have already been briefly described (Trager and Jensen, 1976). Here we present full details of the method and its application in demonstrating the feasibility of using outdated erythrocytes and for testing certain other modifications of the culture conditions. MATERIALS AND METHODS

Warfarin STEREOCHEMICAL ASPECTS OF ITS METABOLISM AND THE INTERACTION WITH PHENYLBUTAZONE

An examination of the metabolic fate of the R and the S isomers of warfarin revealed that the two isomers were metabolized by different routes. R warfarin was oxidized to 6-hydroxywarfarin and was reduced to the (R,S) warfarin alcohol. In contrast, S warfarin was oxidized to 7-hydroxywarfarin and was reduced to the (S,S) warfarin alcohol. S warfarin was also oxidized to 6-hydroxywarfarin. These observations suggested that interactions between warfarin and other drugs might be manifest stereo-specifically, i.e., have a different effect on the isomers of warfarin, so a series of experiments were conducted with each isomer of warfarin, before and after phenylbutazone. The plasma clearance of S warfarin was slowed from 3.1 to 1.1% per h in one subject and from 2.3 to 1.6% per h in another. In contrast, the clearance of R warfarin was increased from 1.5 to 3.0% per h and from 0.9 to 1.6% per h in two subjects after phenylbutazone. The rate of clearance of racemic warfarin was unaffected by phenylbutazone; the depression of the rate of clearance of the S isomer masked the stimulation of the clearance of the R isomer. Since S warfarin is five times more potent an anticoagulant than R warfarin, it is concluded that inhibition of the metabolism of S warfarin provides one mechanism for the augmented anticoagulation which follows phenylbutazone.

The mechanism of the interaction between amiodarone and warfarin in humans

Amiodarone decreased the total body clearance of both (R)‐ and (S)‐warfarin in normal subjects but did not change volumes of distribution. Warfarin excretion products were quantified and clearance and formation clearance values calculated. Amiodarone and metabolites inhibited the reduction of (R)‐warfarin to (R,S)‐warfarin alcohol‐1 and the oxidation of both (R)‐ and (S)‐warfarin to phenolic metabolites. Inhibition of warfarin hydroxylation by amiodarone in human liver microsomes was compared with the in vivo results. In agreement, the in vitro data indicates that amiodarone is a general inhibitor of the cytochrome P450 catalyzed oxidation of both enantiomers of warfarin, but the metabolism of (S)‐warfarin is more strongly inhibited than that of (R)‐warfarin. These data suggest that the enhanced anticoagulant effect observed when amiodarone and warfarin are coadministered is attributable to inhibition of P4502C9, the isozyme of P‐450 primarily responsible for the conversion of (S)‐warfarin to its major metabolite, (S)‐7‐hydroxywarfarin.

Human malaria parasites in continuous culture. 1976.

Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38°C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. They have remained infective to Aotus.

Interaction of amiodarone with racemic warfarin and its separated enantiomorphs in humans

To evaluate a stereoselective interaction for amiodarone and racemic warfarin, we performed a prospective study with its separated enantiomorphs. Single oral doses of racemic warfarin, 1.5 mg/kg, were administered to six normal subjects, with and without oral amiodarone, 400 mg daily, for the hypoprothrombinemic duration. Both the hypoprothrombinemia (P < 0.001) and plasma warfarin concentrations (P < 0.01) were significantly increased. The experiments were repeated separately with the R‐ and S‐warfarin enantiomorphs. S‐warfarin with amiodarone significantly increased both the hypoprothrombinemia (P < 0.001) and plasma warfarin concentrations (P < 0.01). R‐warfarin with amiodarone significantly increased both the hypoprothrombinemia (P < 0.001) and plasma warfarin concentrations (P < 0.001). Thus amiodarone augmented the anticoagulant effect nonstereoselectively by reduced metabolic clearance of both warfarin enantiomorphs. Amiodarone and racemic warfarin can be a dangerous combination, particularly when either drug is added to a stabilized regimen of the other drug, unless the prothrombin times are monitored carefully.

Plasmodium falciparum: Microaerophilic requirements in human red blood cells

Abstract The respiratory requirements of Plasmodium falciparum were studied in vitro in continuous cultivation. The cultures were held in petri dishes containing the parasites incubated in different gas mixtures for periods of 72 to 144 hr with daily media changes. Atmospheres were combinations of 0.5 to 21% O2 mixed with 1 to 5% CO2 diluted with N2. Gas concentrations and the pH of media were measured with an O 2 CO 2 analyzer. Best growth was realized in all cases at 3% O2 and 1 to 2% CO2. The culture appeared to be selfperpetuating in O2 concentrations as low as 0.5% providing the CO2 was not over 2%. Oxygen concentrations of 21% proved deleterious to growth. The parasite however, failed to grow in the highly reducing atmosphere of anaerobic “Brewer Jars,” suggesting that P. falciparum is an obligate microaerophile.

Warfarin metabolism in man: identification of metabolites in urine

After administration of the coumarin anticoagulant racemic warfarin to normal humans, seven fluorescent compounds were chromatographically separated from extracts of their urine. Four of these were identified using mass spectrometry, thin-layer chromatography, and ultraviolet absorption spectroscopy. One metabolic pathway, reduction of the acetonyl side chain of warfarin, resulted in the formation of a second asymmetric carbon atom, and two diastereoisomer alcohols were identified. These warfarin alcohols are structurally similar to pharmacologically active coumarin derivatives. They have not been reported in animal studies. In addition, 6- and 7-hydroxywarfarin were identified. These are the first studies to document the metabolic fate of warfarin in the normal human.

Continuous culture of Plasmodium falciparum: its impact on malaria research.

The methods developed by us in 1976 for the continuous culture of the erythrocytic stages of Plasmodium falciparum make this organism available to a large variety of scientists. As a result, much has been learned about P. falciparum during the past 20 years. Here we attempt to emphasize recent developments in the diverse aspects for which the culture method has been particularly useful: chemotherapy; drug resistance; vaccine development; pathogenesis; export of proteins into the host cell; cell biology, the mitochondrion and the plastid; innate resistance involving mutant human erythrocytes; gametocytogenesis; genetics, transfection; molecular biology; biochemistry; extracellular cultivation.

Influence of stiripentol on cytochrome P450-mediated metabolic pathways in humans: In vitro and in vivo comparison and calculation of in vivo inhibition constants

The spectrum of cytochrome P450 inhibition of stiripentol, a new anticonvulsant, was characterized in vitro and in vivo.