Effect of aphidicolin on DNA synthesis in HSV-1 infected and uninfected Vero cells

The effect of aphidicolin on DNA synthesis in herpes simplex virus type 1 (HSV-1) infected and uninfected Vero cells was determined by isodensity banding of [³²P]-labelled DNA. A 50% inhibition of HSV-1 DNA synthesis was observed at 0.07 μM aphidicolin while 2.1 and 1.3 μM were required to inhibit the cellular DNA synthesis to 50% in infected and uninfected Vero cells, respectively. When the viral DNA synthesis was totally inhibited by 10 μM aphidicolin, the cellular DNA synthesis was inhibited to about 90% in both infected and uninfected cells. Aphidicolin inhibited the cellular DNA synthesis in HSV-1 infected and uninfected Vero cells remaining in the presence of 250 μM foscarnet to the same extent as the DNA synthesis in the absence of foscarnet.     

Antiviral activity of 5-thiocyanatopyrimidine nucleosides.

The antiviral activity of the 5-thiocyanatopyrimidine nucleosides 5-NCSrU¹, 5-NCSdU, 5-NCSaraU and tri-O′-acetyl-5-NCSrU has been evaluated in primary rabbit kidney (PRK) cell cultures challenged with either DNA (vaccinia, herpes simplex) or RNA (vesicular stomatitis) viruses. 5-NCSdU inhibited vaccinia virus multiplication at 10 μg/ml, and vaccinia and herpes simplex virus induced cytopathogenicity at 4 μg/ml. Tri-O′-acetyl-5-NCSrU inhibited vesicular stomatitis virus-induced cytopathogenicity at 1–10 μg/ml. None of the compounds had profound effects on host cell RNA or DNA synthesis, even at 200 μg/ml, as monitored by [³H]uridine and [³H]thymidine incorporation respectively, except 5-NCSdU, which brought about a 10–30-fold increase of [³H]thymidine incorporation at 200 μg/ml. The inhibitory effect of 5-NCSdU on vaccinia virus replication and its stimulatory effect on [³H]thymidine incorporation were almost completely reversed by thymidine at concentrations 100 times lower than that of the thiocyanato derivative. When treated with dithiothreitol, the 5-thiocyanatopyrimidine nucleosides also lost a significant part of their biological activity, presumably due to reduction to the corresponding 5-mercapto analogs.     

Antiherpes activity of [E]-5-(1-propenyl)-2′-deoxyuridine and 5-(1-propenyl)-1-β-d-arabinofuranosyluracil

5-(1-Propenyl)-1-β-d-arabinofuranosyluracil has been synthesized, and this compound and [E]-5-(1-propenyl)-2′-deoxyuridine have been tested for inhibition of herpes virus multiplication. Only [E]-5-(1-propenyl)-2′-deoxyuridine was found to be an active inhibitor reducing by 50% the plaque formation of herpes simplex virus type 1 (HSV-1) at about 1 μM. A comparison to the bromovinyl derivatives showed the following order of decreasing activity; $\text{[}\text{E}\text{]-5-(2-}\text{bromovinyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(2-}\text{bromovinyl}\text{)-1-β-}\text{d}\text{-}\text{arabinofuranosyluracil}\text{≥ [}\text{E}\text{]-5-(1-}\text{propenyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(1-}\text{propenyl}\text{)-1-β-}\text{arabinofuranosyluracil}$. HSV-1 mutants lacking thymidine kinase or resistant against acycloguanosine were resistant against [E]-5-(1-propenyl)-2′-deoxyuridine. All compounds seemed to be phosphorylated by HSV-1 thymidine kinase in a cell-free assay. The compounds were phosphorylated to a lower extent by cellular or HSV-2 thymidine kinase, and the HSV-2 strains tested were inhibited by less than 50% at 100 μM in plaque assays. A selective inhibition of HSV-1 DNA synthesis by [E]-5-(1-propenyl)-2′-deoxyuridine was observed in infected cells indicating an effect on viral DNA polymerase. [E]-5-(1-Propenyl)-2′-deoxyuridine had a low cellular toxicity and a therapeutic effect when applied topically to HSV-1-infected guinea pig skin.     

The effects of chlorambucil on the utilization of exogenous thymidine by tumour cells.

Treatment of an alkylating agent-sensitive strain of the Yoshida ascites sarcoma with chlorambucil resulted in an inhibition of the incorporation of [³H]thymidine into DNA, which could be overcome by incubating cells in high extracellular concentrations of thymidine. Increase in cellular DNA content and the dilution of specific radioactivity in pre-labelled DNA indicated that DNA synthesis was continuing at times when [³H]thymidine incorporation was inhibited. Uptake and phosphorylation of thymidine were not impaired by the treatment and the reduced incorporation of [³H]thymidine into DNA is attributable to a block in the utilization of TTP derived from exogenous nucleoside.     

Antiherpetic activity and mechanism of action of 9-(4-hydroxybutyl)guanine

9-(4-Hydroxybutyl)guanine was synthesized and tested for antiherpes activity. In cell cultures, different strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were inhibited by 50% at 2–14 μM of 9-(4-hydroxybutyl)guanine, while a HSV-1 mutant lacking thymidine kinase (HSV-1 TK^-) was resistant. Linear competitive inhibition of purified HSV-1-induced thymidine kinase (TK) with thymidine as a variable substrate was observed for 9-(4-hydroxybutyl)guanine with an apparent K_i value of 2.06 μM while the corresponding K_i value for the cellular TK was > 250 μM. By using high performance liquid chromatography, the formation of 9-(4-hydroxybutyl)guanine monophosphate by HSV-1 TK was measured and the rate of product formation was found to be about 10% of that found by using thymidine as a substrate. A selective inhibition of HSV-1 DNA synthesis by 9-(4-hydroxybutyl)guanine was observed in infected Vero cells. 9-(4-Hydroxybutyl)guanine had a low cellular toxicity. A weak therapeutic effect on herpes keratitis in rabbits was observed whereas cutaneous HSV-1 infection in guinea pigs and systemic HSV-2 infection in mice were not affected by this compound.     

Reversal of the cytotoxicity of 3'-amino-3'-deoxythymidine by pyrimidine deoxyribonucleosides.

Mammalian cell replication is strongly inhibited by 3′-amino-3′deoxythymidine (3′-aminothymidine). This cytotoxieity can be specifically prevented or reversed by pyrimidine 2′-deoxyribonucleosides. The addition of 50 μM 2′-deoxycytidine to L1210 cells treated with 10 μM 3′ the population doubling time from about 38 hr to 17 hr. The control cells doubled every 13 hr. Another cytotoxic effect produced by 3′-aminothymidine is a dose- and time-dependent increase in cell volume. 2′-Deoxycytidine can effectively prevent and reverse this increase. 3′-Aminothymidme appears to be a potent selective inhibitor of DNA synthesis in L1210 cells. The incorporation of [³H]thymidine into DNA was inhibited by 50 per cent at 1 μM 3′-aminothymidine, a concentration which reduced L1210 replication by about 65 per cent. The rate of incorporation of [³H] adenine into DNA, another measure of DNA synthesis, was reduced similarly by 3′-aminothymidine. and 2′-deoxycytidine eliminated this inhibition as well. An effect on RNA or protein synthesis was not detected. The incorporation of [³H] uridine or [³H] adenine into RNA, or of tritiated amino acids into protein, was not reduced by 25 μM 3′-aminothymidine. These results suggest that selective disruption of DNA metabolism may account for the cytotoxicity of 3′-aminothymidine.     

Antiviral activity of 2,3-dihydro-1H-imidazo[1,2-b]pyrazole in herpes simplex virus type 1-infected mammalian cells.

2,3-Dihydro-1H-imidazo[1,2-b]pyrazole (IMPY), a known inhibitor of DNA synthesis, has been shown to be a useful drug for the synchronization of mammalian cells in culture. Recent studies in our laboratory indicate that IMPY may possess significant antiviral activity against herpes simplex virus (HSV) type 1. IMPY. at a concentration of 80 μg/ml or 0.73 mM, reduced syncytia formation approximately 80 per cent. A 50 per cent inhibitory dose was calculated for each drug in order to compare potency in syncytia reduction of IMPY with that of arabinosyladenine (ara-A) and arabinosyl-hypoxanthine (ara-H). Our results indicated that the antiviral potencies of the three drugs were ranked in the order ara-A > ara-H > IMPY, the 50 per cent inhibitory doses being 22, 195 and 309 μM respectively. Utilizing the microplate procedure of Sidwell and Huffman [Appl. Microbiol.22, 797 (1971)], inhibition of viral cytopathic effect was rated against drug cytotoxicity and a virus rating (VR) established. A virus rating of 0.68 was calculated for IMPY. In comparison, VR values of 0.84 and 0.66 were obtained for ara-A and ara-H respectively. In contrast to the syncytia reduction studies, IMPY appeared to possess antiviral activity equivalent to that of ara-H according to the criteria of the virus rating assay. A technique was developed for evaluating the degree of selectivity (°S) of a drug with respect to its differential effect on viral and cellular DNA synthesis. IMPY was found to possess a negative selectivity at all concentrations studied, reflecting the fact that it inhibited cellular DNA synthesis more than viral DNA synthesis. In contrast, ara-A and ara-H both expressed positive degrees of selectivity in that they inhibited viral DNA synthesis more extensively than cellular DNA synthesis.     

Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.     

Cytotoxic Effects of Tris (2,3-dibromopropyl) phosphate and 2,4-diaminoanisole

Abstract The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and the hair-dye component 2,4-diaminoanisole (2,4-DAA) were studied for possible cytotoxic effects in rat hepatoma cells grown in culture and in suspensions of isolated rat hepatocytes. Cell growth of Reuber cells was inhibited by 50% at 50 μg/ml Tris-BP and 20 μg/ml 2,4-DAA, respectively. At 200 μg/ml Tris-BP protein synthesis in Reuber cells was reduced by 40%, whereas 50% inhibition of protein synthesis in isolated hepatocytes was seen at 100 μg/ml. IC50 of 2,4-DAA with respect to protein synthesis was found at 400 μg/ml in Reuber cells and at 3600 μg/ml in MH₁C₁ cells, whereas in the isolated hepatocytes IC50 was 650 μg/ml. DNA synthesis was inhibited by 50% at 225 μg/ml Tris-BP in Reuber cells. At 500 μg/ml 2,4-DAA DNA synthesis in Reuber and MH₁C₁ cells was inhibited by more than 80%.     

Effects of complex of 3,6-di-(dimethylamino)-dibenzopyriodonium with praseodymium dicitrate on the syntheses of DNA, RNA, protein, nucleoprotein and ATP of leukemia L 7712 cells in mice

The incorporations of [3H] thymidine, [3H]uridine and [3H]leucine into DNA, RNA and protein synthesis in leukemia 7712 cells were inhibited by the complex of 3,6-di-(dimethylamino)-dibenzopyriodonium with praseodymium (Pr, rare earth element) dicitrite 34 micrograms/ml for 3-24 h. The degree of inhibition increased in proportion to the incubation time. After being treated with [C17H20N2I]3[Pr(C6H5O7)2] 34 micrograms/ml for 3, 6, 12 and 24 h, the incorporation of [32P]Na2HPO4 into the nucleoprotein of leukemia 7712 cells was inhibited by 49, 57, 65 and 85%, while those into ATP were inhibited by 43, 59, 65 and 83%, respectively. The ID50 of [C17H20N2I]3[Pr(C6H5O7)2] on DNA synthesis in leukemia 7712 cells at 24 h was 22 micrograms/ml. After the complex was removed from the medium entirely, the rate of DNA synthesis decreased with time over 3-12 h. This result indicated that the inhibition mechanism was likely due to damage to the DNA template.     

Inhibition of herpes simplex virus DNA replication by ara-tubercidin

Preliminary studies of the biochemical basis for the antiviral activity of the pyrrolo[2,3-d]pyrimidine nucleoside ara-tubercidin were conducted. Herpes simplex virus DNA synthesis was 3-fold more sensitive to inhibition by ara-tubercidin than was cellular DNA synthesis. Partially purified herpes DNA polymerases were more sensitive to inhibition by ara-tubercidin 5'-triphosphate than were cellular polymerases alpha and beta. Inhibition of viral DNA polymerase was competitive with dATP and noncompetitive with dTTP. The results suggest that the viral DNA polymerase plays a significant role in the antiviral activity of ara-tubercidin.     

Effects of cryptolepine on collagen-induced aggregation and on the mobilization and metabolism of arachidonic acid by rabbit platelets

1. We examined the effect of cryptolepine on collagen-induced aggregation and on the mobilization, and metabolism of arachidonic acid in rabbit platelets.2. Preincubation of platelets with cryptolepine (50–100 μM) did not affect the primary wave of aggregation but resulted in a dose-dependent, surmountable inhibition of the secondary wave of aggregation induced by collagen (5 μg/ml). The inhibition by cryptolepine was greater when cryptolepine was incubated with the platelets after the peak of the primary wave of aggregation.3. Cryptolepine (50–100 μM) dose-dependently inhibited thrombin (1.5 U/ml) and A23187 (2.5 μM)-induced release of ¹⁴C[AA] from platelet membrane phospholipid pools. The percentage inhibition of A23187-induced ¹⁴C[AA] release was 31.3 ± 4.3% (50 μM) and 79.3 ± 5.4% (100 μM), while thrombin-induced release was inhibited by 39.2 ± 2.4% (50 μM) and 68.2 ± 3.6% (100 μM).4. At near maximal concentration (100 μM) which significantly inhibited secondary aggregatory response and ¹⁴C[AA] release, cryptolepine had no effect on the platelet metabolism of ¹⁴C[AA] to thromboxane B₂, HHT and 12 HETE.5. The present findings suggest that cryptolepine inhibited collagen-induced secondary aggregation through a selective antiphospholipase-like activity. There was not effect on platelet cyclooxygenase and lipoxygenase activities of platelets.     

Biochemical effects of d-tetrandrine and thalicarpine.

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[¹⁴C] to ¹⁴CO₂ 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.     

Biosynthesis of deoxyribonucleic acid, ribonucleic acid and protein in vivo by neonatal mice after a toxic dose of cyclophosphamide

Swiss-Webster mice received 80 $\text{mg}\text{kg}$ of cyclophosphamide 1 day after birth and were allowed to develop or were given 100 $\text{μCi}\text{kg}$ of [¹⁴C]labeled thymidine, uridine or leucine at various times afterwards. Growth retardation and increased mortality were noted during the postnatal period. DNA synthesis, as measured by [¹⁴C]thymidine incorporation, was inhibited in the liver, brain and carcass for at least 3 days after cyclophosphamide treatment. DNA synthesis was reduced 1 day after treatment to 8.4, 26 and 25 per cent of control in the liver, brain and carcass respectively. RNA synthesis, as measured by [¹⁴C]uridine incorporation, was reduced only in the liver and brain. Liver RNA synthesis was reduced to 36 and 64 per cent of control at 1 and 5 days after cyclophosphamide treatment respectively. Brain RNA synthesis was reduced to 78 and 64 per cent of control 1 and 4 days after treatment. [¹⁴C]leucine incorporation, taken as a measure of protein synthesis, was not affected in a manner which would indicate that drug treatment altered this parameter of differentiation. The data suggested that cyclophosphamide neonatal toxicity may be related to a prolonged inhibition of DNA and RNA synthesis during the first 5 days of life. This observation was correlated with the slow rate of cyclophosphamide activation and excretion in neonatal mice.     

Incorporation into nucleic acids of the antiherpes guanosine analog buciclovir, and effects on DNA and protein synthesis

Using cells expressing herpes simplex virus (HSV) thymidine kinase, we investigated the metabolism of the acyclic antiherpes guanosine analog buciclovir, in relation to the effects of the drug on viral DNA and protein synthesis. In these cells the predominant metabolite of buciclovir was its triphosphate, as in the HSV-1 infected Vero cells investigated in parallel. Further metabolism of buciclovir led to incorporation into RNA and DNA. Buciclovir inhibited DNA synthesis, not RNA synthesis, and prevented an increase in the size of newly synthesized DNA. To study the relative effects of BCV on cellular and viral DNA synthesis, human TK-cells transformed to a TK+ phenotype with HSV-2 DNA, were infected with HSV-1. In these HSV-1 infected cells buciclovir-triphosphate caused a preferential inhibition of viral DNA synthesis. Despite incorporation of buciclovir into RNA, and the presence of buciclovir-triphosphate from the time of infection onwards, no effect was observed on the synthesis of the beta proteins ICP-6 and ICP-8. Presumably as a consequence of inhibition of viral DNA synthesis, the synthesis of a beta gamma protein (gD) and a gamma protein (gC) were inhibited, and synthesis of the beta proteins (ICP-6 and ICP-8) was not shut-off. Glycosylation of gC that was still synthesized, was not inhibited. Thus, the biological effects of buciclovir can be explained by its inhibition of DNA synthesis.     

The effect of external deoxyribonucleosides on deoxyribonucleoside triphosphate concentrations in human lymphocytes.

The effects of deoxyribonucleosides on the intracellular levels of deoxyribonucleoside triphosphates (dNTP) and on the rate of labelled thymidine incorporated into DNA of human phytohaemag-glutinin-stimulated lymphocytes have been studied. Thymidine (10^?2–10^?6M) expanded the dTTP and reduced dATP and dCTP levels. Deoxycytidine (10^?3M) expanded the dCTP level and caused inhibition of [³H] thymidine incorporation into DNA but had no detectable effect on the other dNTP concentrations. Deoxyadenosine (10^?3 M) expanded the dATP level, and reduced the other dNTP levels and deoxyguanosine (10^?4M) expanded the dGTP level and reduced the dCTP level; both inhibited [³H] thymidine incorporation into DNA. The sensitivity of these cells to the addition of deoxynucleosides to their culture medium indicates that the plasma and tissue levels of nucleosides may profoundly influence DNA synthesis by human cells in vivo.     

On the complex nature of the antiviral activity of coumermycin A1: its interference with the replication of herpes simplex virus type 1

The mechanism of inhibition of the replication of herpes simplex virus type 1 (HSV-1) by coumermycin A1 (CA1), an inhibitor of bacterial DNA gyrase, has been investigated. Concentrations of antibiotic slightly higher than those needed for 50% inhibition of viral growth were able to inhibit viral DNA synthesis in infected cells. This effect was accompanied by a depressed synthesis of viral polypeptides. Protein synthesis was also inhibited in uninfected cells, especially after long exposure to the drug, but not in a cell-free system. In vitro assays of highly purified HSV-1 DNA polymerase in the presence of the drug, provided evidence that the enzyme was a target of CA1. The viral polymerase was in fact inhibited by the antibiotic to an extent comparable to that of viral DNA synthesis in intact cells. In contrast, DNA polymerase alpha, the enzyme involved in chromosomal DNA replication, was relatively insensitive to CA1. The drug was also shown to bind to protein and to viral and cellular DNA.     

Biological activities and modes of action of 9-alpha-D-arabinofuranosyladenine and 9-alpha-D-arabinofuranosyl-8-azaadenine

From earlier studies it is known that 9-α-d-arabinofuranosyladenine (α-araA) and 9-α-d-arabinofuranosyl-8-azaadenine (α-ara-8-azaA) bave in vitro antiviral activity, are cytotoxic, and are metabolized in mammalian cells to the triphosphates. This study was designed to compare the in vivo antiviral activities of these compounds and their loci of action with those of 9-β-d-arabinofura-nosyladenine (β-araA). the latter compound selectively inhibits DNA synthesis in intact cells, and its triphosphate is a known inhibitor of DNA polymerases and ribonucleotide reductase. Whereas β-araA was significantly effective in the treatment of systemic herpes simplex virus type 1 (HSV-1) infections in mice, α-araA and α-ara-8-azaA were therapeutically ineffective. α-AraATP at a concentration of ~1 mM did not inhibit (1) DNA polymerases present in crude extracts of cultured H.Ep.-2 cells; (2) DNA polymerases present in extracts of KB cells; (3) partially purified DNA polymerase-α from mouse embryo cells; or (4) DNA polymerases induced by HSV-1 and HSV-2. DNA polymerase-β from mouse embryo cells was inhibited to a small extent by 10^?4 M α-araATP. In contrast, all of these enzymes were inhibited by β-araATP at a concentration of 10^?5M (as shown in these or in earlier studies). the reductions of CDP and UDP by ribonucleotide reductase from L1210 cells were not inhibited by αaraATP (~10^?3M), whereas β-araATP produced 70–80 per cent inhibition at this concentration. In cultured H.Ep.-2 cells, α-ara-8-azaA inhibited the incorporation of thymidine, uridine, and formate into macromolecules, but it was without effect on the incorporation of adenine and hypoxanthine, and produced marginal inhibition of the incorporation of leucine. α-Ara-8-azaA produced a dose-dependent inhibition of the accumulation of [¹⁴C] formyl-glycinamide ribonucleotide in H.Ep.-2 cells treated with azaserine and [¹⁴C] formate. These results indicate that the α-nucleosides inhibit nucleic acid synthesis by mechanisms different from those of β-araA.     

Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β-d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10^?6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose (id₅₀) of XA for [³H]uridine and [³H]thymidine incorporation into RNA and DNA respectively. The id₅₀ for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.     

A fully automated synthesis of [18F]‐FEAU and [18F]‐FMAU using a novel dual reactor radiosynthesis module

2′‐Deoxy‐2′‐[¹⁸F]fluoro‐5‐substituted‐1‐β‐D ‐arabinofuranosyluracils, including 2′‐deoxy‐2′‐[¹⁸F]fluoro‐5‐methyl‐1‐β‐D ‐arabinofuranosyluracil [¹⁸F]FMAU and [¹⁸F]FEAU are established radiolabeled probes to monitor cellular proliferation and herpes simplex virus type 1 thymidine kinase (HSV1‐tk) reporter gene expression with positron emission tomography. For clinical applications, a fully automated CGMP‐compliant radiosynthesis is necessary for production of these probes. However, due to multiple steps in the synthesis, no such automated synthetic protocols have been developed. We report here a fully automated synthesis of [¹⁸F]‐FEAU and [¹⁸F]‐FMAU on a prototype dual reactor module TRACERlab FX FN. The synthesis was performed by using a computer‐programmed standard operating procedure, and the product was purified on a semipreparative high‐performance liquid chromatography (HPLC) integrated with the synthesis module using 12% EtOH in 50 mM Na₂HPO₄. Finally, the percentage of alcohol was adjusted to 7% by adding Na₂HPO₄ and filtered through a Millipore filter to make dose for human. The radiochemical yield on the fluorination was 40±10% (n=10), and the overall yields were 4±1% (d. c.), from the end of the bombardment; [¹⁸F]FEAU (n=7) and [¹⁸F]FMAU (n=3). The radiochemical purity was >99%, specific activity was 1200–1300 mCi/µmol. The synthesis time was 2.5 h. This automated synthesis should be suitable for production of [¹⁸F]FIAU, [¹⁸F]FFAU, [¹⁸F]FCAU, [¹⁸F]FBAU and other 5‐substitued thymidine analogues. Copyright © 2009 John Wiley & Sons, Ltd.