William A. Creasey

L-Asparaginase: Clinical, Biochemical, Pharmacological, and Immunological Studies

Abstract Forty patients with acute leukemia were treated with Intravenous L-asparaginase in doses ranging from 10 to 1,000 international units/kg body weight per day for 2 to 20 days. Most had long...

Biochemical and pharmacological studies with 1-β-d-arabinofuranosylcytosine in man

Abstract Cytosine arabinoside, labeled with tritium, was administered to five patients with far-advanced neoplasms. The analog was rapidly deaminated to uracil arabinoside; the latter accounted for between 86 and 96 per cent of the radioactivity excreted in the urine. Incorporation of tritium-labeled cytidine into DNA by suspensions of human leukemic leukocytes was inhibited in the presence of cytosine arabinoside. Although administration of cytosine arabinoside at therapeutic levels depressed the ability of leukemic leukocytes to incorporate cytidine into DNA in vitro , there was no correlation between the degree and duration of this effect and the clinical response to the drug. Tritium-labeled cytosine arabinoside entered human leukemic leukocytes very rapidly when incubated with the cells in vitro ; there was a small but significant incorporation of the analog into both DNA and RNA.

Biochemical effects of the vinca alkaloids II. A comparison of the effects of colchicine, vinblastine and vincristine on the synthesis of ribonucleic acids in Ehrlich ascites carcinoma cells

Abstract The synthesis of RNA was studied in Ehrlich ascites carcinoma growing in the peritoneal cavities of mice treated with either colchicine, vinblastine or vincristine. In all cases, treatment with drug inhibited the incorporation of [ 3 H]uridine into phenol-extractable RNA by about 50%, the duration of the inhibition being in the range of 15–24 h. Separation of the RNA into soluble, ribosomal and rapidly-labelling fractions, indicated that the primary effect of these drugs is on soluble RNA, the doses in mg per kg needed for 50% inhibition being 0.3 for vincristine, I.O for colchicine and 0.8 for vinblastine. At higher dose levels, smaller changes were produced in the synthesis of ribosomal and rapidly-labelling interphase RNA. Pretreatment with massive doses of glutamic acid (0.9–2.7 g per kg) prevented, to varying degress, the inhibition in RNA synthesis produced by the alkaloids. Uptake of [I- 14 C]valine into tumor cell proteins also was depressed by treatment. The significance of these findings for the problem of control of cell division is discussed.

Colchicine, vinblastine and griseofulvin: Pharmacological studies with human leukocytes☆

Abstract Some aspects of the interactions of the vinca alkaloids, colchicine and griseofulvin with human leukocytes in suspension have been explored. Tritiated colchicine, vinblastine and griseofulvin enter the cells where significant binding by a supernatant protein takes place. Glutamate competitively inhibits the entry of vinblastine into the leukocytes when present in large excess. Inhibition of the incorporation of valine-14C into leukocyte protein occurred in the presence of vinblastine and vincristine at concentrations down to 6 × 10−6 M; colchicine and griseofulvin did not affect this incorporation until levels of 3 × 10−4 M were attained. Concentrations of these agents as high as 1 × 10−4 M did not inhibit the incorporation of uridine-3H NA or of acetate-14C into lipid. The data were considered to support the concept that the antimitotic and anti-inflammatory effects of vinca alkaloids, colchicine and griseofulvin result from binding to precursor units of the microtubules.

Biochemical effects of the vinca alkaloids: III. The synthesis of ribonucleic acid and the incorporation of amino acids in Ehrlich ascites cells in vitro

Abstract The incorporation of [ 3 H]uridine into RNA by Ehrlich ascites cells in vitro , was inhibited by colchicine, vinblastine and vincristine, the percentage reductions for a drug level of 0.2 μmole/ml being 20, 58 and 26, respectively. Addition of glutamic acid at 1–8 μmoles/ml reduced or prevented this depression; lesser antagonistic effects were observed with aspartic or α-ketoglutaric acids, while arginine and glutamine were inactive at comparable levels. The incorporation of [ 14 C]glutamic acid, but not of [ 14 C]glycine, into the acid-soluble fraction was markedly reduced (40 % at 0.2 μmole/ml) by vinblastine and vincristine, but only slightly depressed by colchicine. Cells harvested from mice treated 2–13 h before sacrifice with vinblastine (2 mg/kg) exhibited a reduced rate of uptake of [ 14 C]glutamic acid into the acid-soluble fraction. Some reduction in the amount of [ 14 C]glutamic acid entering cellular proteins was also observed after treatment with vinblastine both in vivo and in vitro . Development of resistance to vinblastine was accompanied by elevation in the baseline rate of uptake of [ 3 H]uridine into RNA, and a profound reduction in the rate of entry of [ 14 C]glutamic acid into the acid-soluble fraction; both these parameters were less sensitive to vinblastine treatment in vitro .

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly☆

Abstract Griseofulvin—shown previously to disrupt the mitotic apparatus in vivo —inhibited the in vitro microtubule assembly reaction completely at 8 × 10−4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.

BIOCHEMICAL EFFECTS OF THE VINCA ALKALOIDS--I. EFFECTS OF VINBLASTINE ON NUCLEIC ACID SYNTHESIS IN MOUSE TUMOR CELLS.

Abstract The uptake of 3 H-uridine into the soluble RNA of Ehrlich ascites cells derived from mice treated with vinblastine is depressed about 60 per cent as compared with that of controls. Since the specific activity of both uridine and cytidine nucleotides is reduced it appears that this alkaloid inhibits the synthesis of the whole chain of soluble RNA. Synthesis of ribosomal RNA is not affected. The incorporation of 3 H-thymidine into DNA is reduced by high concentrations of vinblastine in vitro but not by therapeutic doses of this agent in vivo .

The administration of 5-fluorouracil by mouth

When 5‐fluorouracil was administered by mouth, concentrations of the drug in blood and urine were comparable to and more sustained than those achieved after parenteral treatment in four of seven subjects. This finding serves as evidence that the same precautions against drug side effects are indicated with oral as with parenteral treatment. Patients with metastases to the bowel or congestive heart failure had poorer absorption of 5‐fluorouracil. The findings are evidence that intravenous and oral administration of the drug can result in different levels of 5‐fluorouracil in blood. It may be worthwhile evaluating the relationship among blood levels, different patterns of absorption, and therapeutic response as a means of determining the optimal mode of therapy. One patient who failed to respond to intravenous 5‐fluorouracil responded to oral 5‐fluorouracil. Objective regression of hepatic and other metastases were observed in four patients treated with 5‐fluorouracil by mouth.

Biochemical effects of d-tetrandrine and thalicarpine

Abstract The benzylisoquinoline alkaloids d -tetrandrine and thalicarpine inhibit the biosynthesis of DNA, RNA and proteins, when incubated with S180 cells in vitro . Oxidation of glucose[ 14 C] to 14 CO 2 was not affected by either alkaloid at levels up to 100 μg/ml in vitro . Incorporation of labeled acetate into lipids was inhibited only by thalicarpine at 100 μg/ml. Inhibition of the incorporation of thymidine into DNA was also observed in vivo after treatment with these drugs at 30–120 mg/kg; under these conditions, the synthesis of RNA and protein was not inhibited. In an attempt to elucidate the mechanism for inhibition of nucleic acid synthesis, the interaction of DNA, RNA and polynucleotides with the alkaloids was studied by gel filtration and dialysis. The two drugs associated with both DNA and RNA, but exhibited different affinities for the five polynucleotides examined. Both alkaloids were bound by polyguanylic and polyadenylic acids, but whereas d -tetrandrine associated only poorly with polythymidylic acid and not at all with polyuridylic acid, it was polycytidylic acid that showed no affinity for thalicarpine.

INVESTIGATIONS WITH 5-AZAOROTIC ACID, AN INHIBITOR OF THE BIOSYNTHESIS OF PYRIMIDINES DE NOVO.

Preclinical and clinical metabolic investigations of 5‐azaorotic acid, an inhibitor of the biosynthesis de novo of pyrimidine nucleotides, have suggested its possihle use as an antitumor agent in the therapy of liver and the kidney tumors. Tissue slice experiments indicated extensive utilization of orotic acid by the liver and kidney, and inhihition of this utilization by 5‐azaorotic acid. Tissue distribution studies in rats showed localization of the drug in these tissues. Metabolism to a compound resembling N‐formylbiuret tuas observed in human studies, and rapid urinary cxcmtion of unchanged 5‐azaorotic acid occurred. Effective blockade of the utilization of exogenous orotic acid by 5‐azaorotic acid was sustained in patients for 6 hours after intravenous administration of the compound. Renal toxicity, possibly secondary to crystalline deposits in the tubules, may restrict the clinical use of the compound.