Metabolism of 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide and related five-membered heterocycles to 5'-triphosphates in human blood and L5178Y cells.

The facile metabolism of 5-amino-1-β-d-ribofuranosylimidazole-4-carboxamide (AICA ribonucleoside) and several of its structural analogs [5-fluoro-1-β-d-ribofuranosylimidazole-4-carboxamide (FICA ribonucleoside), pyrazofurin and ribavirin] to their corresponding 5′-triphosphates in human blood cells has been demonstrated in vitro. Evidence is presented that both the β- and α-anomeric forms of pyrazofurin nucleotides were present in the extracts of pyrazofurin-treated blood. Determination of the extent of incorporation of radioactivity from [U-¹⁴C]d-glucose into analog ribonucleoside 5′-triphosphates formed in human blood cells indicated that AICA ribonucleoside and ribavirin were metabolized to their 5′-monophosphates mainly (> 90 per cent) via a nucleoside kinase; however, FICA ribonucleoside appeared to be metabolized to its 5′-monophosphate both via a nucleoside kinase (ca. 67 per cent) and via phosphorolytic cleavage followed by a phosphoribosyltransferase-mediated reaction (ca. 33 per cent). The aglycones of AICA ribonucleoside and FICA ribonucleoside were also metabolized extensively to their corresponding ribonucleoside 5′-triphosphates. 5′-Aminoimidazole-4-thiocarboxamide and 3-aminopyrazole-4-thiocarboxamide were metabolized only slightly to their ribonucleoside 5′-triphosphates. [³H]ribavirin was metabolized extensively to its 5′-triphosphate in L5178Y cells but was not detectably incorporated into RNA. Ribavirin caused a substantial decrease in the pool size of GTP in L5178Y cells and a concomitant increase in the pool size of both CTP and UTP.     

Differential selectivity of 5-fluorouracil and 5'-deoxy-5-fluorouridine in cultured human B lymphocytes and mouse L1210 leukemia

The role of differential metabolic activation of a 5-fluorouracil (FU) prodrug, 5'-deoxy-5-fluorouridine (dFUR), in achieving selective cytotoxicity was investigated in cultured human (dFUR), in achieving selective cytotoxocity was investigated in cultured human B lymphocytes and murine leukemia L1210 cells. B cells were cross-sensitive to FU and dFUR. On the other hand, leukemia L1210 cells were sensitive to FU but resistant to dFUR. The difference in the biological activities of FU and dFUR in B and L1210 cells correlated with (a) the metabolism of dFUR to FU by intact B (60% conversion) and L1210 (no conversion) cells, and (b) the phosphorylase activity of B (660 nmoles converted in 2 hr per mg protein) and L1210 (undetectable) cells. The intracellular metabolism of FU and dFUR was studied using a reversed-phase ion-pair high pressure liquid chromatographic assay. FU and dFUR shared similar metabolic pathways in B cells; their anabolites included FU ribose and deoxyribose nucleosides and nucleotides. In L1210 cells, FU was anabolized to 5-fluorouridine triphosphate and 5-fluorodeoxyuridine monophosphate, whereas dFUR was present mainly as the unchanged drug. Further metabolism studies using dFUR with tritium label in either the FU moiety or the altered sugar moiety established that the metabolic pathway of dFUR to cytotoxic FU anabolites in the B cells was via phosphorolysis to FU. These data indicate that, on a cellular level, an FU prodrug such as dFUR, which is activated by cytosolic enzyme, has a different selectivity from that of FU, and that the basis of differential selectivity is the initial phosphorolysis to FU.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

5'-Methylthioadenosine phosphorylase-L. Substrate activity of 5'-deoxyadenosine with the enzyme from Sarcoma 180 cells

5′-Deoxy-5′-methylthioadenosine phosphorylase (MTA phosphorylase), an enzyme involved in the salvage of adenine moieties from 5′-deoxy-5′-methylthioadenosine (MTA) produced primarily during polyamine biosynthesis, is present in Sarcoma 180 cells (0.0026 ± 0.0002 μM units/mg cytosol protein). 5′-Deoxyadenosine (5′-dAdo), an adenosine analog previously thought not to be metabolizable, has been shown [D. Hunting and J.F. Henderson, Biochem. Pharmac. 27, 2163 (1978)] to have a number of biochemical effects on Ehrlich ascites cells. We have now found that 5′-dAdo is a substrate for the MTA phosphorylase from Sarcoma 180 cells, yielding free adenine and 5-deoxyribose-1-phosphate. The reaction was reversible and totally dependent upon phosphate. Evidence that MTA phosphorylase is responsible for 5′-dAdo phosphorylase activity includes the following: (1) Sarcoma 180 MTA phosphorylase preparations did not show additive rates of adenine production in the presence of saturating concentrations of both 5′-dAdo and MTA; (2) double-reciprocal plots of the rates of adenine formation from 5′-dAdo by Sarcoma 180 enzyme preparations in the presence of MTA displayed a pattern characteristic of alternative, competing substrates; (3) the rate of depletion of 5′-dAdo by Sarcoma 180 preparations was inhibited by the presence of MTA; (4) the K_i value of a competitive inhibitor of Sarcoma 180 MTA phosphorylase, 5′-deoxy-5′-chloroformycin, was the same when either MTA or 5′-dAdo was employed as substrate; and (5) the apparent K_m values of phosphate for both MTA and 5′-dAdo phosphorylase activities were identical (3.5mM). The K_m of Sarcoma 180 MTA phosphorylase for MTA is 4 μM; the K_m for 5′-dAdo is 23 μM (V_max relative to MTA = 180 per cent). Incubation of Sarcoma 180 cells with either 5′-dAdo or MTA caused profound elevations of adenine nucleotides, as well as an inhibition of 5-phosphoribosyl-l-pyrophosphate (PRPP) accumulation. The reaction of 5′-dAdo with MTA phosphorylase to yield free adenine, which is then salvaged to adenine nucleotides, can account for many of the previously reported biochemical effects of 5′-dAdo, such as inhibitions of PRPP accumulation, purine de novo synthesis, and glycolysis that have previously been attributed to the unmetabolized nucleoside. The other product of this reaction, 5-deoxyribose-l-phosphate, may also contribute to these effects.     

Adenosine-3′,5′-diphosphate and coenzyme a—Effects on platelet function

The effects in vitro of adenosine-3′5′-diphosphate and coenzyme A on human platelet aggregation and [¹⁴C]hydroxytryptamine release were studied. Whereas coenzyme A, at concentrations between 0.06 and 0.24 mM, inhibited the aggregation of platelets induced by ADP, adenosine-3 ′, 5 ′-disphosphate, which is part of the coenzyme A molecule, blocked both ADP- and thrombin-induced platelet aggregation. The ADP-induced platelet aggregation was inhibited at a lower adenosine-3′, 5′-diphosphate (10–20μM) concentration than was thrombin-induced aggregation (60–200 μM). Adenosine-3′,5′-diphosphate also inhibited [¹⁴C]adenosine uptake by platelets in a concentration-dependent manner (20–200 μM), but only to a maximum of 40 per cent of total [¹⁴C]adenosine radioactivity incorporated into the platelets. The inhibitory effect of adenosine-3′,5′-diphosphate and coenzyme A on the release reaction was further documented by the decrease in aggregation-induced release of [¹⁴C]5-hydroxytryptamine from prelabeled platelets into the medium. The extent of inhibition caused by coenzyme A and adenosine-3′,5′-diphosphate was found to depend upon the concentration of inhibitor and incubation time. If these agents are indeed inhibitors of platelet aggregation, then they may serve as valuable tools to study platelet function.     

Pharmacokinetics and bioavailability of oral 5'-deoxy-5-fluorouridine in cancer patients

AIMS: Oral administration of 5-fluorouracil (FUra), an important cytotoxic agent, is limited by a wide variation in bioavailability. 5'-deoxy-5-fluorouridine (dFUrd), a masked form of FUra, has shown promise clinically when given intravenously or orally as a solution or tablet. This study investigates the efficacy of an oral capsule formulation of dFUrd in generating continuous systemic levels of this compound in cancer patients. METHODS: Six patients with advanced intestinal or ovarian malignancies were given three cycles of dFUrd, days 1-5, at intervals of 4 weeks. The doses of dFUrd were 600 mg m-2 three times daily, 800 mg m-2 three times daily, and 1000 mg m-2 three times daily, on cycles one, two and three, respectively (total dose 36 g m-2 ). The initial dose in each cycle was given as a slow intravenous injection over 10 min, and the remainder orally. Plasma and urine levels of dFUrd and two of its metabolites, FUra and 5,6-dihydro-5-fluorouracil (FUraH2 ), were monitored in six patients at each dose level. RESULTS: All six patients completed the study, receiving three different doses over a 3 month period, following which one had achieved a partial response, one had stable disease, and four had developed progressive disease. Side-effects were negligible, and only two instances of transient diarrhoea WHO grade 1 were seen. Total body clearance (CLtot) of intravenous dFUrd decreased with increasing dose; 2.7, 2.0 and 1.3 l min-1 m-2, following doses of 600, 800 and 1000 mg m-2, respectively. The mean elimination half-life of intravenous dFUrd increased with the dose from 15 to 22 min. Oral dFUrd was rapidly absorbed with a lag time of less than 20 min. The mean elimination half-life (t1/2, z ) of oral dFUrd was 32-45 min in the dose range 600-1000 mg m-2. The AUC of FUra and FUraH2 increased overproportionally with increasing intravenous doses of dFUrd. The mean systemic bioavailability of oral dFUrd was 34-47%. CONCLUSIONS: dFUrd, which selectively releases the antimetabolite FUra in tumour cells, can be given orally at doses of 600-1000 mg m-2 three times daily for 5 days. The systemic levels achieved are equivalent to those seen following continuous infusions of dFUrd or FUra. Toxicity is tolerable, and further clinical investigation of oral dFUrd is warranted.     

Human platelet 5HT receptors: Characterisation and functional association

Normal human blood platelets in plasma were incubated at 2°C with tritiated 5-hydroxytryptamine ([³H]5HT), and the specific receptor binding was displaced by the addition of unlabelled 5HT. The kinetic parameters of this binding were established and a two-site model for the platelet 5HT receptor demonstrated. Site A has a K_D of 0.5–1 nM and capacity of 6–10 fmol/10^* platelets, and site B a K _D of 15–36 nM and capacity of 100–150 fmol/10⁸ platelets. Non-specific or non-receptor binding of [³H]5HT to platelets at 2°C was resolved into a passive linear component and an active saturable component sensitive to metabolic inhibition. Binding to the lower affinity 5HT receptor site was inhibited by drugs of the tricylic antidepressant type with IC₅₀ values similar to those against the active uptake component of non-specific binding as described. Isomers of the neuroleptic drug flupenthixol showed a differential and competitive antagonism of high affinity [³H]5HT binding. The 5HT antagonist methysergide, and pizotifen and mianserin also were competitive inhibitors at this site. The rank order of potency of these drugs correlated with thier action as inhibitors of 5HT induced platelet aggreation. It is conclude that bindin of [³H]5HT to intact human platelets satisfies all the critical for specific binding and that the two sites demonstrated, of high and lower affinity, are concerned with the functions of 5HT induced aggregation and 5HT uptake respectively.     

Phosphorolysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted-2'-deoxyuridines by purified human thymidine phosphorylase and intact blood platelets

Various 5-substituted-2'-deoxyuriclines (dUrd), inclucling 5-ethyl-,5-propyl-, 5-trifluoromethyl-, 5-hydroxymethyl-, 5-formyl-, 5-vinyl-, (E)-5-(2-chlorovinyl)-, (E)-5-(2-bromovinyl)-, 5-fluoro-. 5-chloro-. 5-bromo-. 5-iodo-, 5-cyano-, 5-thiocyano-, 5-nitro- and 5-amino-dUrd. were shown to be effective substrates for the thymidine (dThd) phosphorylase isolated from human blood platelets. Some of dUrd analogs, i.e. the highly potent and selective antiherpes agent (E)-5-(2-bromovinyl)-dUrd. were degraded more rapidly than the natural substrates, dUrd and dThd. All dUrd analogs were also readily catabolised by intact human blood platelets. The potent inhibitors of thymicline phosphorylase, 6-amino-thymine and 6-amino-5-bromo-uracil, strongly inhibited the phosphorolysis of (E)-5-(2-bromovinyl)-dUrd by both purified enzyme and intact platelets.     

In vitro and in vivo studies of a promising antileukemic thymidine analogue, 5-hydroxymethyl-2' deoxyuridine

The toxicity and metabolism of a thymidine analogue, 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) were studied with human leukemia cells (HL-60) and with human platelets. 3 X 10(-5) M 5HmdUrd caused a 50% inhibition in the proliferation of HL-60 cells. The compound was hydrolyzed to 5-hydroxymethyluracil (5HmUra) by the enzyme thymidine phosphorylase (EC 2.4.2.4) present in leukemia cells; this catabolic product was non-toxic. The catabolism of 5HmdUrd by human platelet thymidine phosphorylase could be inhibited by 6-aminothymine. The toxicity of 5HmdUrd was effectively reversed by deoxycytidine and 5HmdUrd increased the incorporation of deoxycytidine into dCTP and DNA several fold. The two latter phenomena are explicable in terms of a feedback action to ribonucleotide reductase, resulting in deoxycytidylate starvation, which is a known effect of excess thymidine. We report here also our preliminary observations that 5HmdUrd is active against mouse leukemia in vivo.     

The human blood platelet: A cellular model to study the degradation of thymidine and its inhibition

Intact platelets catabolize extracellular thymidine into thymine. Studies of the concentration dependent degradation of thymidine by intact platelets indicate a Michaelis mechanism with an apparent K_m of about 0.12mM and a V_max of 2.5 nmoles/min for 3 × 10⁸ platelets. This degradation process is inhibited by various nucleosides, pyrimidine bases and C-5 or C-6 substituted uracils. Cytidine, deoxycytidine, adenosine and deoxyadenosine seem to inhibit thymidine degradation by reducing the intracellular transport of thymidine. Uridine inhibits both the thymidine transport and the activity of the phosphorolytic enzyme, thymidine phosphorylase (EC 2.4.2.4). Some substituted uracils are specific inhibitors of thymidine phosphorylase activity. 6-Amino-5-bromouracil, the most active of them, either with acellular extracts or purified thymidine phosphorylase, is also the best inhibitor of thymidine degradation in intact human platelets. Platelets constitute a new model to study the efficiency of specific inhibitors on thymidine catabolism in an ‘human intact cell’ which contains only one pyrimidine nucleoside phosphorylase, the thymidine phosphorylase.     

Enhancement of 5-fluorouracil incorporation into human lymphoblast ribonucleic acid

Various drug combinations have been explored as a means of enhancing the incorporation of 5-fluorouracil (5-FU) into RNA from the human T-lymphoblast CEM line. The relative incorporation of [³H]FU into newly synthesized alkali-sensitive RNA was determined by concurrent ³²P-incubation and calculation of the ratio of [³H]FU/³²P.$\text{N-(}\text{Phosphonacetyl}\text{)-}\text{L}\text{-}\text{aspartate}$ (PALA) was employed at concentrations of 0.1 mg/ml for 16 hr prior to the addition of [³H]FU; it enhanced the formation of (5-FU) RNA by 3- to 5-fold. The effects of methotrexate (MTX) and 6-methylmercaptopurine riboside (MMPR) on the formation of (5-FU)RNA and phosphoribosyl-l-pyrophosphate (PRPP) were also monitored. Increases of 10- to 15-fold in PRPP occurred following a 6-hr exposure to either MTX or MMPR at concentrations of 1 μM and resulted in up to a 5-fold enhancement in the [³H]FU/³²P ratio. Exposure of cells to combinations of PALA with MTX or MMPR resulted in up to 20-fold increases in (5-FU)RNA formation. These drug combinations were more than additive with 5-FU in their inhibitory effects as determined by assays monitoring cell growth. These studies should be applicable in expanding ongoing clinical trials of combination chemotherapy with PALA and 5-FU.     

177Lu‐5‐Fluorouracil a potential theranostic radiopharmaceutical: radiosynthesis,quality control,biodistribution, and scintigraphy

The aim of this study is to develop ¹⁷⁷Lu‐5‐Flourouracil as a potential cancer therapeutic radiopharmaceutical. 5‐Flourouracil (5‐FU) is widely accepted as an anticancer drug of broad spectrum fame. The labeling of 5‐FU was carried out at different set of experimental conditions using high specific activity of ¹⁷⁷LuCl₃. The optimum conditions for maximum radiochemical yield was set: 5‐FU (5 mg), ¹⁷⁷LuCl₃ (185 MBq), diethylenetriaminepentaacetic acid (10 µg), reaction volume (2 mL), pH (5.5), temperature (80°C), and reaction time (20 min). The radiochemical labeling was assessed with Whatman No. 2 paper, instant thin layer chromatographic, and radio‐HPLC, which revealed >94% labeling results with sufficient stability up to 6 h. Serum stability study also showed ¹⁷⁷Lu‐5‐FU promising stability. Biodistribution study in normal rats and rabbits showed liver, stomach, kidney, and heart as area of increased tracer accumulation just after injection, which decreased to 1.4%, 0.4%, 0.2%, and 0.39% ID/g, respectively, after 72 h. Glomerular filtration rate and cytotoxicity study results of ¹⁷⁷Lu‐5‐FU showed it had no adverse effect on renal function and nontoxic to blood cells. The promising characteristics of ¹⁷⁷Lu‐5‐FU, that is, clever elimination from kidney and nontoxic nature toward blood cells make it the radiopharmaceutical for further testing in patients for therapeutic purposes.     

Pyrimidine acyclonucleosides,inhibitors of uridine phosphorylase

A new class of nucleoside analogs, the pyridimine acyclonucleosides, are competitive inhibitors of uridine phosphorylase but have no effect on thymicline phosphorylase, uridine kinase or thymidine kinase. The most potent of the series is acyclothymidine [5-methyl-1-(2′-hydroxyethoxymethyl)uracil] with a K_i value of 3 μM. K_i values of less than 30 μM were estimated for other analogs substituted at the 5-position of the pyrimicline ring. Extracts of xenografts of six human tumors were assayed for tissue levels of uricline phosphorylase and thymicline phosphorylase and for inhibition of 5-fluoro-2′-deoxyuridine (FUdR) phosphorolytic activity by acyclouridine [1-(2′-hydroxyethoxymethyl) uracil]. FUdR cleavage was inhibited most in those tissues in which the ratio of thymidine phosphorylase to uricline phosphorylase was low. Potential usage of these uricline phosphorylase inhibitors with the chemotherapeutic agent FUdR is discussed.     

Metabolic fate of 1-hexylcarbamoyl-5-fluorouracil after oral administration in mice

1. The metabolic fate of a new antitumour agent, 1-hexylcarbamoyl-5-fluoro[6-14C]uracil (¹⁴C-HCFU) was compared with that of 5-fluoro[6-14C]uracil (¹⁴C-FU) after oral administration to mice.

2. 1-(5-Hydroxyhexylcarbamoyl)-5-fluorouracil (5-hydroxy-HCFU) and 1-(5-oxohexylcarbamoyl)-5-fluorouracil (5-keto-HCFU) were found as major intermediate metabolites of ¹⁴C-HCFU and were produced by ω-1 oxidation.

3. FU was detected in plasma 180min after oral administration of ¹⁴C-HCFU, whereas unchanged FU disappeared within 60min after ¹⁴C-FU.

4. ¹⁴C-HCFU and resulting FU were retained in tissues for a long period after oral administration, while administered ¹⁴C-FU was rapidly degraded.     

Possible Involvement of Oxidative Stress in 5‐Fluorouracil‐Mediated Myelosuppression in Mice

Abstract: Certain chemotherapeutic agents subject cells to oxidative stress, thereby promoting adverse effects. However, the molecular machinery governing 5‐fluorouracil (5‐FU)‐mediated myelotoxicity is obscure. The purpose of this study was to clarify whether 5‐FU‐induced myelotoxicity is a cause of oxidative stress. Treatment of mice with 5‐FU (75 mg/kg, i.p.) caused a significant induction of haem oxygenase‐1 and a decrease in glutathione contents in bone marrow cells, both of which are the indicators of oxidative stress. The 5‐FU‐mediated decrease in the myeloid colony formation was intensified in Nrf2^?/? mice, in which antioxidant proteins were down‐regulated. N‐Acetylcysteine reversed the 5‐FU‐induced decreases in the glutathione content, number of bone marrow cells per femur and myeloid colony formation. Results from the present study reveal that 5‐FU induces oxidative stress in bone marrow, which is involved, at least in part, in myelotoxicity in mice. Therefore, Nrf2‐dependent genes as well as glutathione levels in bone marrow could be therapeutic targets for decreasing such side‐effects in 5‐FU‐based chemotherapy.     

Uptake of 14C-5-hydroxytryptamine by human and rat platelets and its pharmacological inhibition

Summary In order to approach the uptake of ¹⁴C-5HT by platelets as a first-order process, experimental conditions were selected in which accumulation of the amine either by diffusion or by other passive nonsaturable processes could be excluded. These conditions included an incubation period of ¹⁴C-5HT with human or rat platelets of 4 min or 30 s, respectively and the use of substrate concentrations around the calculated apparent K _m values (0.25–2.0 M). While the apparent K _m values were rather similar for human and rat platelets, V _max was about 5 times higher in rat than in human platelets.The kinetic model adopted in this study was used to evaluate the relative potency and the type of inhibition of ¹⁴C-5HT uptake exhibited by imipramine, chlorimipramine and (+)-fenfluramine. All 3 compounds inhibited ¹⁴C-5HT uptake by platelets. Chlorimipramine was about 10 times more effective than imipramine both in rat and in human platelets. Both drugs were more potent inhibitors on human than on rat platelets. (+)-Fenfluramine was almost as active as imipramine on rat but 30 times less potent than imipramine on human platelets. Both imipramine and chlorimipramine inhibited ¹⁴C-5HT uptake by an apparent non-competitive mechanism, whereas (+)-fenfluramine appeared to act as a competitive inhibitor. No differences were found in this respect between human and rat platelets.Pharmacological or therapeutic doses of these drugs usually result in plasma concentrations similar to those found in this study to effectively inhibit platelet ¹⁴C-5HT uptake.     

Inhibition of nucleoside phosphorylase cleavage of 5-fluoro-2'-deoxyuridine by 2,4- pyrimidinedione derivatives

Several 2,4-pyrimidinedione (uracil) derivatives were evaluated as inhibitors of the pyrimidine nucleoside phosphorylases that cleave 5-fluorn-2'-deoxyuridine (FUdR) to 5-fluorouracil. Pyrimidinediones substituted at either N-1 or C-5, or both, markedly inhibited the phosphorolysis of FUdR by the uridine-deoxyuridine phosphorylases of Ehrlich ascites and Novikoff hepatoma cells. The most potent inhibitors were 5-benzyluracil derivatives substituted with alkoxy groups on the meta-position of the benzyl moiety; the most active of these was 5-{[3-(phenylmethoxy)phenyl]methyl}uracil. The same derivatives, however, did not inhibit the phosphorolysis of FUdR by the thymidine phosphorylases of murine liver, human leukocytes and HeLa (S3) cells. 6-Anilino and 6-(1-naphthylmethylamino) derivatives of uracil, which have been shown by others to inhibit the cleavage of FUdR by the thymidine phosphorylase activity of Escherichia coli, did not inhibit any of the mammalian thymidine or uridinedeoxyuridine phosphorylase activities. By contrast, pyrimidinediones substituted with smaller, non-hydrophobic groups at either C-5 or C-6, or both, inhibited the cleavage of FUdR by both the mammalian thymidine and uridine-deoxyuridine phosphorylases. The most active of these, 6-aminothymine, was also the best inhibitor of thymidine phosphorylase. Our results demonstrate differences in the active sites of the various pyrimidine nucleoside phosphorylases, and should provide a basis for the design of more potent and specific inhibitors of the nucleoside phosphorylase(s) responsible for the cleavage of FUdR in man.     

Effect of glucagon, dibutyryl adenosine 3',5'-cyclic monophosphate and phosphodiesterase inhibitors on rat liver phosphorylase activity and adenosine 3',5'-cyclic monophosphate levels

Theophylline, papaverine and 1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazole-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester HCl (SQ 20,009) were found to inhibit both high K_m (200 μM) and low K_m(3 μM) cyclic 3′,5′-AMP phosphodiesterase activity in 100,000 g supernatant fraction from rat liver. Theophylline was the least potent and SQ 20,009 the most potent inhibitor of both activities. In the liver slice preparation, papaverine (5 × 10^?4), theophylline (10^?3 M) and SQ 20,009 (10^?3 M) failed to elevate cyclic 3′,5′-AMP, while glucagon (10^?6 M) produced a significant (P < 0·05) elevation in the tissue levels of the cyclic nucleotide. All three phosphodiesterase inhibitors significantly (P < 0·05) augmented the ability of glucagon to elevate cyclic 3′,5′-AMP levels. Glucagon and papaverine stimulated phosphorylase activity, while theophylline inhibited both basal and glucagon-stimulated phosphorylase activity. SQ 20,009 was without effect. It is concluded that no cause and effect relationship can be assumed between an ability to inhibit phosphodiesterase and to alter phosphorylase activity.     

Nature of Lowered 5-Hydroxytryptamine Uptake by Blood Platelets of Patients with Down's Syndrome

Abstract: Disturbance of central serotoninergic system has been suggested in Down's syndrome (DS). In this syndrome the 5-HT concentration in blood platelets is below normal, but the mechanism behind this has been controversial. Recently. evidence has accumulated indicating a decreased active transport of 5-HT possibly due to lowered activity of Mg⁺⁺-dependent Na⁺-K⁺ -stimulated adenosine triphosphatase. In the present study the kinetic analysis of 5-HT uptake by blood platelets from DS patients revealed a decreased V_max suggesting decreased transport function but an unchanged affinity to the uptake receptors, as indicated by normal K_m values. The controls were mentally retarded patients from the same institute. The uptake continued in a linear fashion up to 20 min., suggesting an unchanged storage of 5-HT. Also the effect of zinc on the 5-HT uptake was studied; plasma zinc levels have been noted to be lowered in DS infants. In vitro zinc caused a dose-dependent inhibition ofuptake at 10^?4 an M. In vivo. after 2 weeks treatment 135 mg/day orally no significant effect was noted.     

Action of immobilized neuraminidase on the accumulation of 5-hydroxytryptamine by human platelets

The accumulation of 5-HT was studied in human platelets following graded desialylation with neuraminidase immobilized on macrobeads. Platelets were removed from the soluble sialylglyco-proteins of the plasma by gel filtration on Sepharose 2B; the uptake of 5-HT by gel filtered platelets over four hours was similar to that found using platelet rich plasma. Platelet aggregation induced by 10^?5M 5-HT and 10^?6M ADP was decreased following gel filtration and the use of Sepharose 2B insolubilized apyrase did not ameliorate this effect. Removal of up to 30 per cent of the platelet sialic acid, which results in changes in the glycoprotein pattern as demonstrated by two dimensional gel electrophoresis, appears to accelerate the rate of uptake of 5-HT. This is followed by a significantly decreased rate once 50% desialylation has been achieved. Native platelets and those from which 30 per cent of the sialic acid had been removed were refractory to resialylation with exogenous rat liver sialyltransferase though this enzyme preparation catalyses the resialylation of soluble asialoglycoprotein acceptor.