A high yield robotic synthesis of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) and 9-[3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([18F] FHPG) for gene expression imaging.

The aim of this study was to develop an automated synthesis of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) and 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) using a Scanditronix Anatech RB III robotic system. [(18)F]HF was produced via (18)O(p, n)(18)F using a Scanditronix MC17F cyclotron. On average, a typical run produced [(18)F]FHBG and [(18)F]FHPG with an uncorrected radiochemical yield of 19% and 16%, respectively, at end of synthesis (EOS) from irradiation of 95% enriched [(18)O]water. The total synthesis time was 80 min. The retention time of [(18)F]FHBG and [(18)F]FHPG (the radio-peak) was 3.9 and 4.0 min, respectively, which was consistent with the [(19)F]FHBG and [(19)F]FHPG ultraviolet peak. The radiochemical purity was greater than 97%. A robotic, automated method for [(18)F]FHBG and [(18)F]FHPG radiosynthesis is therefore feasible. The radiation burden for the operator can be reduced as much as possible. Sufficient radioactivities of [(18)F]FHBG and [(18)F]FHPG could be obtained for non-invasive monitoring the expression of transfected gene in vivo with positron emission tomography (PET).

Biosynthetic ganciclovir triphosphate: its isolation and characterization from ganciclovir-treated herpes simplex thymidine kinase-transduced murine cells.

A method is described for the preparation of ganciclovir triphosphate (GCV-TP) using murine colon cancer cells (MC38) transduced with the herpes simplex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral-tk contain required viral and host enzymes needed for complete cellular synthesis of this potent antiviral metabolite. Dose response studies showed optimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-tk cells to 300 microM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10(6) cells. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme included methanolic extraction and anion-exchange chromatography to recover the target triphosphate. Mass spectral analysis and selective enzyme degradation provided structural confirmation of the purified product. Biological activity of the purified GCV-TP was demonstrated by competitive inhibition experiments using human DNA polymerase alpha and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement with previous reports. The GCV-TP obtained was further used to enzymatically prepare GCV mono- and diphosphate in high yield. This method provides an easily scalable means of preparing milligram amounts of the triphosphates of pharmacologically active acyclic nucleosides like ganciclovir.

Phosphorylation of ganciclovir phosphonate by cellular GMP kinase determines the stereoselectivity of anti-human cytomegalovirus activity.

A racemic mixture of ganciclovir phosphonate was resolved by stereoselective phosphorylation using GMP kinase. The R-enantiomer of ganciclovir phosphonate was active against human cytomegalovirus but the S-enantiomer was less active. We show that enantiomeric selectivity of antiviral for ganciclovir phosphonate was conferred by stereoselective phosphorylations by mammalian enzymes, not by stereoselective inhibition of DNA polymerase from human cytomegalovirus.

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.