Richard L. Cysyk

Kinetics of cis‐dichlorodiammineplatinum

The cancer chemotherapeutic cis‐dichlorodiammineplatinum (cis‐DDP) was administered to 8 patients (1‐hr intravenous infusion) at a dose of 70 mg/m2. Plasma and urine concentrations of platinum were determined by flame less atomic absorption spectrometry. Measured plasma platinum concentrations revealed a biphasic clearance of platinum with half‐life values of 23 min and 67 hr. Platinum values obtained 3 wk after the infusion indicated that a third excretory phase might be present. Urinary measurements showed 17 ± 2.7% of the administered dose excreted in the first 4 hr and 23 ± 3.9% excreted in the first 24 hr. Renal excretion appears to be predominantly by glomerular filtration. Non‐protein‐bound plasma platinum values were calculated and the non‐protein‐bound platinum was found to be rapidly and biphasically cleared from the plasma with half‐life values of 8 to 10 min and 40 to 45 min.

Determination of serum and plasma uridine levels in mice, rats, and humans by high-pressure liquid chromatography

Abstract Serum and plasma uridine levels in mice, rats, and humans were conveniently measured by reverse-phase high-pressure liquid chromatography. Human serum levels were in the range of 1.9 to 8.4 nmol/ml, rat serum levels ranged from 3.7 to 9.4 nmol/ml, and mouse serum levels measured 8.0 to 11.8 nmol/ml. Levels of uridine were the same in human plasma and serum; however, plasma from mice and rats was somewhat lower than serum in uridine content ranging from 1.7 to 4.1 nmol/ml in rats and 1.5 to 4.7 nmol/ml in mice. There was some variation in the individual human serum and rat plasma uridine levels throughout the day, but the values were within the normal range, and the variations had no set pattern. Withholding food for 16–24 h had no observable effect on serum uridine levels in mice and humans or on rat plasma levels. These results suggest that uridine levels are regulated and are not a direct reflection of dietary intake of uridine.

Isolation and characterization by electrospray-ionization mass spectrometry and high-performance anion-exchange chromatography of oligosaccharides derived from hyaluronic acid by hyaluronate lyase digestion: Observation of some heretofore unobserved oligosaccharides that contain an odd number of units

Hyaluronic acid was degraded with hyaluronate lyase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus), and the resulting oligosaccharides up to dp 16 were characterized by electrospray-ionization mass spectrometry (ESIMS) and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). In accordance with the known regiospecificity of the enzyme, the products included even-numbered oligosaccharides of structure beta-D-4en-thrHexpA-(1-->3)-[beta-D-GlcpNAc-(1-->4)-beta-D- GlcpA]n-(1-->3)-D-GlcpNAc. Minor amounts of novel and unexpected odd-numbered oligomers, having the structure beta-D-4en-thrHexpA-(1-->3)-[beta-D-GlcpNAc-(1-->4)-D-Glc pA]n, were also isolated and characterized. This study, in addition to others beginning to appear in the literature, demonstrates the usefulness of ESIMS and HPAEC-PAD in the analysis and characterization of anionic glycosaminoglycan-type oligosaccharides.

Effect of 5-benzylacyclouridine, a potent inhibitor of uridine phosphorylase, on the metabolism of circulating uridine by the isolated rat liver.

5-Benzylacyclouridine (BAU) is a specific inhibitor of uridine phosphorylase, the first enzyme in the catabolism of uridine. It was found that 20 and 100 microM BAU dramatically reduced the rapid clearance of trace amounts of either [14C]uridine or hyperphysiologic concentrations of non-labeled uridine by the isolated rat liver perfused with an artificial oxygen carrier. In the absence of exogenously added uridine, non-treated livers maintained circulating concentrations of 1-2 microM uridine. In the presence of 20 microM BAU, these concentrations were increased 2- to 3-fold higher than physiologic levels (1.4 +/- 0.6 microM) and remained elevated for the duration of the experiment (120-160 min). In the presence of 100 microM BAU, uridine concentrations rose continuously at rates of between 80 and 150 nmoles per hr per g of liver, and the clearance of a single radioactive spike of uridine was reduced extensively. The half-life of a uridine spike was extended 2-fold in the presence of 20 microM BAU and 5- to 6-fold in the presence of 100 microM BAU. Exogenously added uridine (15 and 40 microM) was cleared rapidly by nontreated livers, with a half-life of approximately 10 min. However, BAU at a concentration of 20 microM increased the half-life of 15 or 40 microM uridine added to the perfusate by approximately 10-fold. A 100 microM concentration of BAU inhibited the removal of 40 microM circulating uridine, but with 15 microM uridine there was a continuous increase in the circulating concentration similar to that seen in the absence of added uridine. We conclude that extensive inhibition of uridine phosphorylase occurs at 100 microM BAU and partial inhibition at 20 microM BAU. These data indicate independent catabolic and excretory functions of the rat liver with respect to uridine.

Uptake of 2-β-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) and analogues by the facilitated transport mechanism of erythrocytes

Tiazofurin (TR), a new antitumor agent, enters human erythrocytes by utilizing their facilitated nucleoside transport system. TR competes with endogenous nucleosides for this transport mechanism, thereby reducing nucleoside uptake into the cells. Pre-incubation of erythrocytes for 10 min at 22 degrees C with 100 microM and 500 microM TR reduced the transport of 14C-uridine into the cells by 27% and 74%, respectively. Simultaneous exposure of cells to TR and [14C]uridine did not alter the inhibitory effect of TR. Furthermore, the transport inhibitory effect of TR was lost when cells were washed twice with Hanks basal salt solution following a 10-min pre-incubation with TR. The Km and Vmax (+/- S.E.) for radiolabeled TR transport into erythrocytes are 170 +/- 26 microM and 55 +/- 13 nmol/h per 10(6) cells, respectively, which is similar to the kinetic constants measured for uridine transport into erythrocytes (Km = 168 +/- 37 microM and Vmax = 61 +/- 16 nmol/h per 10(6) cells). The Ki (+/- S.E.) of TR for uridine transport is 178 +/- 11 microM and for thymidine transport is 102 +/- 59 microM. Three analogues of TR (its selenium isostere (SR), and Ara (Ara-TR) and Xylo (Xylo-TR) derivatives) were compared with TR for their ability to compete with and inhibit uridine transport, as these analogues were not available in a radiolabeled form for direct measurement of their transport into the cell. SR had similar kinetic characteristics of inhibition of uridine transport to TR (Ki = 145 +/- 15 microM) but Ara-TR had a Ki = 1.04 +/- 0.13 mM while Xylo-TR inhibited uridine transport with a Ki = 1.57 +/- 0.67 mM. Thus, TR is transported into erythrocytes with the same velocity and affinity for the carrier as uridine and competitively inhibits nucleoside transport into the cell. Of 3 other C-nucleoside derivatives examined, SR is of similar potency to TR but Ara-TR and Xylo-TR are much less effective at competing with uridine for the nucleoside transporter.

Attempts to increase intratumoral blood flow in the rat solid walker 256 tumor by the use of the perfluorocarbon emulsion fluosol-DA

We have examined the effect of the perfluorocarbon emulsion, Fluosol-DA 20% (FDA), on blood flow in rats bearing an advanced solid Walker 256 tumor implanted s.c. Blood-FDA exchange in unanesthetized rats maintained under 100% oxygen was accomplished by simultaneous arterial withdrawal and i.v. infusion until the hematocrit was less than 4%. Control rats were maintained under 100% oxygen but did not undergo any exchange. Regional blood flow studies in tumors of control and FDA-exchanged rats were performed using [14C]iodoantipyrine and quantitative autoradiography. FDA-blood exchange did not increase flow to the whole tumor. Similarly, the pattern of regional flow within the tumor, which was determined in histologically distinct areas--including dense and normocellular, necrotic and peripheral zones invading into muscle and connective tissue--was not substantially altered. Flow to cerebral tissue was increased two-fold, although flow to normal tissues including temporalis muscle, skin and diaphragm was not altered. These results show that FDA-blood exchange does not enhance vascular flow in solid Walker 256 tumor implanted s.c. in the rat.

Influence of ammonium ions on hepatic de novo pyrimidine biosynthesis

Carbamyl phosphate (CP) is synthesized in the liver by two separate enzymes, CPS I and CPS II. CPS I, an intramitochondrial enzyme involved in ureogenesis, has a relative activity of 500- to 1000-fold greater than CPS II, a cytoplasmic enzyme which initiates the sequence of reactions for pyrimidine biosynthesis. The contributions of NH4Cl (substrate for CPS I) ang glutamine (substrate for CPS II) as precursors for pyrimidine biosynthesis in isolated hepatocytes were compared by measuring their effect on uracil nucleotide pool size, the incorporation of NaH14CO3 into these pools, and the accumulation of orotic acid. Physiological concentrations of NH4Cl caused a marked stimulation of incorporation of radioactivity into uracil nucleotides (6-fold increase at 0.5 mM NH4Cl), and radioactive orotate appeared in both the cells and the medium. In contrast, glutamine (at concentrations up to 10 mM) had no effect on the incorporation of radioactivity into uracil nucleotides, and no orotic acid was detected. Uracil nucleotide pools were expanded up to 50% by low levels of NH4Cl, but there was no expansion of this pool in the presence of added glutamine. NH4Cl-driven pyrimidine de novo biosynthesis was insensitive to feedback inhibition by an expanded uracil nucleotide pool, to galactosamine treatment, and to acivicin treatment, indicating that NH+4 stimulated pyrimidine biosynthesis as a result of CP synthesis by mitochondrial CPS I. The consequence of intramitochondrially produced CP being available for pyrimidine biosynthesis is that the controlling step of this pathway (CPS II) is bypassed. The appearance of orotic acid following NH4Cl stimulation indicated that the rate-controlling step of hepatic de novo pyrimidine synthesis under these conditions was orotate phosphoribosyl transferase. These data indicate that, at physiological concentrations of NH+4, the majority of uracil nucleotides synthesized in isolated rat hepatocytes was derived from intramitochondrially generated CP. The effect of NH4Cl on the output of uridine by the isolated perfused rat liver was examined. In the presence of a single addition of 20 mM NH4Cl, the excretion of uridine was increased from 100-200 to 375 nmol h-1 g-1 liver and orotic acid was released into the circulating perfusate reaching a maximum of 2 microM (in 220 ml of perfusate) after 2 h. With 40 mM NH4Cl, uridine export was increased to 450 nmol h-1 g-1 and a maximum of 5 microM orotic acid was released into the perfusate after 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction and development of mouse liver glutathione S-transferase activity

Mouse liver glutathione S-transferase activity at birth was 1/10 that of adults, and increased steadily with each successive week of age until adult values were reached at 8 weeks. Activity was inducible with phenobarbital; however, the percentage increase in activity was dependent upon substrate. 2 distinct peaks of transferase activity were obtained on CM-cellulose chromatography. The ratios of transferase activity observed for each peak demonstrated that glutathione S-transferase activity in mouse liver is associated with at least 2 distinct proteins with differing substrate specificities.

A fluorescence assay for 4′-(9-acridinylamino)methanesulfon-m-anisidide, a new antitumor agent

Abstract A sensitive fluorescent method is described for the detection of 4′-(9-acridinylamino)-methanesulfon- m -anisidide (AMSA) in serum. The assay is based on the alkaline hydrolysis of AMSA into 9(10 H )-acridone. While AMSA has negligible fluorescence, 9(10 H )-acridone fluoresces brilliantly (excitation 266 nm, emission 470 nm). The assay is shown to be linear from 0.01 to 1.0 μ m . In addition, the assay is shown to be useful, in conjunction with an ethyl acetate extraction, in distinguishing serum levels of parent AMSA from metabolized or protein-bound AMSA.

Non-steroidal anti-inflammatory drugs and tumor progression: inhibition of fibroblast hyaluronic acid production by indomethacin and mefenamic acid

The antitumor effects of non-steroidal anti-inflammatory drugs (NSAIDs) have been documented in a variety of both clinical and experimental settings, although the mechanisms responsible remain unclear. In the present study, we show that the NSAIDs indomethacin and mefenamic acid inhibit the calf serum-stimulated production of hyaluronic acid (HA) in murine Swiss 3T3 fibroblasts, at concentrations where DNA synthesis is unaffected. HA is an extracellular matrix glycosaminoglycan associated with cell migration and tumor invasion. Our data suggest that one mechanism whereby NSAIDs inhibit tumor progression may be to inhibit the synthesis of HA by host fibroblasts, and that the eicosenoid pathway may represent an important control point in the growth-factor-mediated production of HA in fibroblasts. Thus the use of an agent which inhibits HA synthesis may be a novel approach to alter the invasive and metastatic properties of tumor cells in a non-cytotoxic fashion.