Julian J. Jaffe

The effects of phenobarbital, chloral betaine, and glutethimide administration on warfarin plasma levels and hypoprothrombinemic responses in man

Ten male volunteers received phenobarbital, glutethimide, chloral betaine, and a placebo at bedtime for 3 week periods. Plasma warfarin levels and prothrombin times were determined after a single oral dose of warfarin prior to and immediately following placebo or hypnotic drug treatment. Glutethimide and phenobarbital lowered the plasma warfarin concentration and reduced the half‐life of warfarin by nearly 50 per cent. Chloral betaine had less effect but differed significantly from control values. Phenobarbital and glutethimide interfered with the hypoprothrombinemic effect of warfarin; chloral betaine and placebo did not. The findings suggest increased metabolic inactivation of warfarin by induction of liver microsomal enzymes.

Glutathione S-transferase in adult Dirofilaria immitis and Brugia pahangi.

Glutathione S-transferase (EC 2.5.1.18) was detected in the cytosolic and microsomal fractions of adult Dirofilaria immitis females at respective levels of 30 nmol and 3 nmol min-1 (mg protein)-1 activity with the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The transferase activity in the cytosolic fraction of adult Brugia pahangi females was 10 nmol min-1 mg-1 with CDNB; determination of its activity in the microsomal fraction of this filariid was not attempted. These filarial glutathione S-transferases were further characterized after their purification by glutathione-affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytosolic transferase from D. immitis, molecular weight 47000, yielded a single subunit of around 28 kDa. The cytosolic and microsomal transferases from D. immitis differed in their activity with CDNB, 1,2-dichloro-4-nitrobenzene, 4-benzylchloride and ethacrynic acid. The cytosolic transferase from B. pahangi was distinguished by its high activity with ethacrynic acid. Both glutathione S-transferases from D. immitis also functioned as a glutathione peroxidase, strongly preferring cumene hydroperoxide as a substrate over hydrogen peroxide. Both were equiactive inhibitors of malonaldehyde formation in the NADPH-microsomal lipid peroxidation system. Thus, in addition to the ability of filarial glutathione S-transferases to detoxify electrophilic xenobiotics, at least those from D. immitis also exhibited selenium-independent glutathione peroxidase activity. Their glutathione S-transferase function suggests a potential role for these enzymes in the leukotriene synthetic pathway, if filariae can form such eicosanoids from arachidonate. Functioning as a glutathione peroxidase, they could serve to protect filarial membrane lipids from peroxidation.

Two types of pyruvate kinase in schistosomes and filariae

Abstract 1. 1. Pyruvate kinase (PK) from the trematode Schistosoma mansoni closely resembles PK from rabbit muscle in its response to various purine and pyrimidine nucleoside diphosphates and divalent cations, in its insensitivity to activation by FDP and relative insensitivity to inhibition by ATP and in its somewhat higher affinity for PEP. 2. 2. PKs from the filarial nematodes Litomosoides carinii and Dirofilaria immitis closely resemble PK from rabbit liver in the pattern of their substrate and cofactor specificities, by being strongly activated by FDP and inhibited by ATP, and by showing a relatively lower affinity for PEP. 3. 3. The properties of PK from S. mansoni miracidia were indistinguishable from those of PK from the adult forms, and this was also the case for the PKs from D. immitis microfilariae and adults. 4. 4. The molecular weights of PKs from S. mansoni , D. immitis and rabbit muscle were all around 270,000±10 per cent.

Dihydrofolate reductases within the genus Trypanosoma

Dihydrofolate reductase activity was detected in extracts of rat-adapted bloodstream forms of Trypanosoma (Trypanozoon) brucei, T. (T.)rhodesiense, T. (T.)equiperdum, T.(Duttonella)vivax, T.(Nannomonas) congolense (section Salivaria); T.(Herpetosoma)lewisi, and T.(Schizotrypanum)cruzi (section Stercoraria). This enzyme was also detected in extracts of culture forms of T.(T.)rhodesiense, T.(H.)lewisi, and T.(S.)cruzi. The trypanosomal dihydrofolate reductases shared the general properties of the analogous enzyme from diverse genera in their requirement for dihydrofolic acid as substrate and reduced nicotinamide adenine dinucleotide phosphate as cofactor. They also showed the characteristic extreme sensitivity to the inhibitory action of 4-amino analogs of folic acid such as methotrexate. On the other hand, the trypanosomal reductases exhibited a pattern of susceptibility to inhibition by certain 2,4-diaminopyrimidines and related heterocyclic compounds which was different from the patterns of susceptibility to these agents that have been described for reductases from bacterial and mammalian sources. Superimposed upon this distinctive pattern, moreover, was a further differential response, whereby the drug sensitivities of the reductases of salivarian trypanosomes were almost identical and, as a group, clearly distinguishable from those of the stercorarian species. Those properties of dihydrofolate reductases from culture and bloodstream forms of T.(T.)rhodesiense, T.(H.) lewisi, or T.(S.)cruzi that were compared were closely similar.

Comparative properties of trypanosomal and mammalian thymidine kinases.

Abstract 1. 1. Thymidine kinase was isolated from a rat-adapted bloodstream form of Trypanosoma ( Trypanozoon ) Brucei rhodesiense , from an epimastigotic form of T. ( T. ) Brucei rhodesiense , and from regenerating rat liver 2. 2. Characterization revealed that the trypanosomal kinases had identical properties. 3. 3. Trypanosomal and liver kinases were similar regarding the ability of IUdR, F 3 -TdR, FUdR, UdR and UR to act as competitive substrates of thymidine. Both enzymes showed equal sensitivity to a non-competitive inhibition by suramin, and no sensitivity to a modulating effect by cytidine nucleotides. 4. 4. Trypanosomal and liver kinases, in order of decreasing efficacy, used magnesium, manganese and cobalt ions as cofactors. Both were inactive with calcium or barium ions. Liver kinase also used zinc ion effectively. 5. 5. Liver thymidine kinase had higher K m values for thymidine and ATP than did trypanosomal kinase. 6. 6. Trypanosomal thymidine kinase was versatile in its requirement for phosphate donor; liver kinase was very rigid. 7. 7. Trypanosomal thymidine kinase was tenfold less sensitive to dTTP feedback inhibition than liver kinase.

Trypanocidal properties of 5′-O-sulfamoyladenosine, a close structural analog of nucleocidin☆

Abstract A rodent-adapted strain of Trypanosoma (Trypanozoon) rhodesiense , challenged in vivo by a series of purine and pyrimidine sulfamoyl nucleosides, was only sensitive to those containing the adenosine nucleus. The most active compound tested, 5′- O -sulfamoyladenosine, which differs structurally from the trypanocidal antibiotic nucleocidin only by the lack of a fluorine atom, was 100% curative with minimal toxicity to mice with early infections at a single orally administered dose of 2 mg/kg or at a dose of 0.1 mg/kg injected intraperitoneally daily for 3 consecutive days. The rate of cures fell if treatment was delayed for 24 hours or longer. 5′- O -Sulfamoyladenosine is less toxic to mice than nucleocidin, and its therapeutic index could be raised by manipulating the dose regimen. In vitro , 5′- O -sulfamoyladenosine inhibited by 50% the uptake of adenine-8- 14 C by bloodstream form T.(T.) rhodesiense at a concentration of 1 × 10 −8 M , and the incorporation of 14 C-phenylalanine into protein at 5 × 10 −8 M . In this concentration range, glucose utilization by the trypanosomes was inhibited only slightly, but progressively greater inhibition of glucose utilization was observed at higher concentrations.

Nucleoside analogs as antiparasitic agents.

In considering the potential usefulness of nucleoside analogs as antiparasitic agents, it is appropriate to recall the words of Ehrlich:’ “What we want is a Chemotherapia specifics, that is, we are looking for chemical agents which, on the one hand, are taken up by certain parasites and are able to kill them and, on the other hand, in the quantities necessary for this lethal action, are tolerated by the host without too great damage.” There are two general means by which antiparasitic agents can be selectively toxic for the pathogenic organisms. They can exploit physiological or biochemical differences between the parasites and their hosts, and/or they can be administered in a manner that could exploit peculiarities of habitat and behavior exhibited by the parasites. The following are cited as illustrative examples: Piperazine (diethylenediamine) produces a flaccid paralysis of the musculature of the parasitic nematode Ascaris lumbricoides, either by neuromuscular blockade’ or by hyperpolari~ation,~ but this drug exerts no such action on mammalian muscle. An outstanding property of plasmodial dihydrofolate reductases is their much greater sensitivity to inhibition by pyrimethamine, trimethoprim, and related diaminopyrimidines than the analogous mammalian enzyme?*’ A close correlation was found between the concentrations of these drugs required to inhibit plasmodia1 dihydrofolate reductases by 50% in a cell-free system and the minimum concentration required to inhibit the growth of these parasites in uitro and in uivo. Niclosamide (N-(2’-chloro-4-nitrophenyl)J-chlorosalicylamide) is very effective in the treatment of various tapeworm infestations of man following its oral administration in relatively large doses6 Under these circumstances, these intestinal parasites are exposed to lethal concentrations of the drug, yet niclosamide is well tolerated and very safe. Its low host toxicity is attributed to the fact that very little is absorbed from the gastrointestinal tract and that it has no direct irritant effect.’ The selective toxicity of tubercidin (7-deazaadenosine) for the blood flukes Schistosoma mansoni and s. japonicum in vivo can be increased when it is administered, after its prior absorption into a portion of the hosts’ total erythrocytes in vitro, by transfusing the drug-laden erythrocytes back into each infected donor.8-1’ Schistosomes feed on erythrocytes,” which renders these cells useful vehicles for delivery of antischistosoma1 agents such as tubercidin, which can be sequestered within them. Because tubercidin is a purine nucleoside analog with antiparasitic activity, it will be discussed later in more detail. Evidence is now at hand to indicate that there are peculiarities in the biochemistry and physiology of diverse kinds of parasites that theoretically are exploitable for chemotherapy by nucleoside analogs.

Sensitivity of trypanosoma equiperdum to the action of tumor-inhibitory antibiotics in vitro ☆

Abstract Trypanosoma equiperdum , a member of the brucei-evansi group of African trypanosomes, is sensitive to the following tumor-inhibitory antibiotics: actinomycin D, mitomycin C, porfiromycin, phleomycin, and pactamycin. Only pactamycin inhibited trypanosomal carbohydrate metabolism in vitro , an action that might have contributed to the ability of this antibiotic to depress protein synthesis and various pathways of nucleic acid metabolism. The inhibitory effects of the other antibiotics were restricted to trypanosomal nucleic acid metabolism. Actinomycin D, 1.6 × 10 −8 M, inhibited the incorporation of 3 H-adenine and 3 H-uracil into RNA by approximately 50%, but had no effect at this concentration upon the incorporation of derivatives of these isotopes into DNA. Higher concentrations had no effect upon the incorporation of 3 H-adenine and 3 H-thymidine into DNA, but did inhibit the incorporation of derivatives of 3 H-uracil into DNA. The distribution of radioactivity in the nucleotide components of the acid-soluble fractions suggested that the predominant action of actinomycin D was to inhibit RNA polymerase. Mitomycin C and its methyl congener, porfiromycin, 2.2 × 10 −4 M and 5.7 × 10 −4 M, respectively, inhibited the incorporation of derivatives of 3 H-adenine and 3 H-uracil into RNA and DNA by approximately 50%. At 7.5 × 10 −6 M and 1.1 × 10 −5 M, mitomycin C and porfiromycin inhibited the incorporation of 3 H-thymidine into DNA by approximately 50%, and at 7.5 × 10 −5 M and 7.2 × 10 −5 M, the evolution of 14 CO 2 derived from orotic acid- 14 COOH. The ability of the mitosane antibiotics, at relatively low concentrations, to inhibit the incorporation of thymidine into DNA suggested that interference with DNA replication was a prominent feature of this group. The data also indicated that at higher concentrations, mitomycin C and porfiromycin inhibited RNA polymerase—whether by direct action or as a consequence of their effects upon DNA synthesis remains unsettled. Phleomycin, 1.3 × 10 −4 M, inhibited the incorporation of 3 H-adenine and 3 H-uracil into RNA by approximately 50%. At this concentration, phleomycin inhibited the incorporation of derivatives of 3 H-uracil into DNA to a greater extent than the derivatives of 3 H-adenine. At 3.2 × 10 −6 M, phleomycin inhibited the incorporation of 3 H-thymidine into DNA by approximately 50%. The action of phleomycin seemed to be qualitatively similar to that of the mitosane antibiotics, except for its inability to inhibit orotidylic acid decarboxylase.

Comparative activity and properties of lactate dehydrogenase, xanthine dehydrogenase, and dihydrofolate reductase in normal and Brugia pahangi infected Aèdes aegypti.

The amount of xanthine dehydrogenase (XDH), dihydrofolate reductase (DHFR), and lactate dehydrogenase (LDH) in crude extracts of 4- to 5-day-old adult Aedes aegypti was determined, and the properties of these enzymes were partially characterized. It was then found that the amount and other selected characteristics of XDH and LDH in extracts of female Ae. aegypti processed 5 to 7 days and 12 to 14 days after they had fed upon either normal or Brugia pahangi-infected jirds were indistinguishable from those of these two enzymes in extracts of female mosquitoes that did not have a blood meal. Under the same circumstances, the selected characteristics of DHFR were also unaffected. However, there was a suggestion that the amount of DHFR was slightly increased in extracts of female Ae. aegypti processed 5 to 7 days after they had fed upon B. pahangi-infected jirds; by 12 to 14 days after the blood meal, there was a consistent 30% to 60% increase in the amount of DHFR inextracts of infected mosquitoes. DHFR activity could not be detected in a similarly prepared extract of 4,000 to 5,000 infective (L-3) B. pagangi larvae, the approximate number present in the infected mosquito extracts. It would appear, therefore, that the increased amount of turnover of DHFR in the mosquito host occurs in response to advanced infection with B. pahangi.

Isolation, partial purification and some properties of two acid proteases from adult Dirofilaria immitis

Two acid proteases were isolated from the soluble extracts of adult Dirofilaria immitis, the filarial heartworm of canines. Activity of these proteases was detected using 3H-labeled bovine alpha-casein as substrate, and they were designated Fp-I and Fp-II in order of their elution from a CM-cellulose column. The molecular weight of partially purified Fp-I was approximately 170000, and it was active between pH 4.6-5.8. The activity of Fp-I doubled in the presence of various sulfhydryl reagents at 5 mM, and it was inhibited 50-60% by the sulfhydryl inhibitors p-hydroxymercuribenzoate and iodoacetate at 1 mM, the heavy metal chelating agent o-phenanthroline at 1 mM and the peptide aldehyde protease inhibitors pepstatin (10 microM), leupeptin, antipain and chymostatin (50 microM). The molecular weight of the more extensively purified Fp-II is approximately 48000. This protease was active between pH 2.6-3.4 and was highly sensitive to inhibition by pepstatin (80% inhibition at 10 nM). Fp-II was not significantly affected by sulfhydryl reagents, sulfhydryl inhibitors, metal chelating agents or peptide aldehyde protease inhibitors other than pepstatin. These properties of dirofilarial Fp-II resemble those of mammalian cathepsin D.