Long-term responders and survivors on pazopanib for advanced soft tissue sarcomas: subanalysis of two European Organisation for Research and Treatment of Cancer (EORTC) clinical trials 62043 and 62072.

BACKGROUND: Pazopanib recently received approval for the treatment of certain soft tissue sarcoma (STS) subtypes. We conducted a retrospective analysis on pooled data from two EORTC trials on pazopanib in STS in order to characterize long-term responders and survivors. PATIENTS AND METHODS: Selected patients were treated with pazopanib in phase II (n = 118) and phase III study (PALETTE) (n = 226). Combined median progression-free survival (PFS) was 4.4 months; the median overall survival (OS) was 11.7 months. Thirty-six percent of patients had a PFS ≥ 6 months and were defined as long-term responders; 34% of patients survived ≥18 months, defined as long-term survivors. Patient characteristics were studied for their association with long-term outcomes. RESULTS: The median follow-up was 2.3 years. Patient characteristics were compared among four subgroups based on short-/long-term PFS and OS, respectively. Seventy-six patients (22.1%) were both long-term responders and long-term survivors. The analysis confirmed the importance of known prognostic factors in metastatic STS patients treated with systemic treatment, such as performance status and tumor grading, and additionally hemoglobin at baseline as new prognostic factor. We identified 12 patients (3.5%) remaining on pazopanib for more than 2 years: nine aged younger than 50 years, nine females, four with smooth muscle tumors and nine with low or intermediate grade tumors at initial diagnosis. The median time on pazopanib in these patients was 2.4 years with the longest duration of 3.7 years. CONCLUSIONS: Thirty-six percent and 34% of all STS patients who received pazopanib in these studies had a long PFS and/or OS, respectively. For more than 2 years, 3.5% of patients remained progression free under pazopanib. Good performance status, low/intermediate grade of the primary tumor and a normal hemoglobin level at baseline were advantageous for long-term outcome. NCT00297258 (phase II) and NCT00753688 (phase III, PALETTE).

Solid-substrate fermentation ofKloeckera apiculata andCandida utilis on apple pomace to produce an improved stock-feed.

Apple pomace was used in solid-substrate fermentation with the yeastsKloeckera apiculata orCandida utilis Y15. A total crude protein content of 7.5% (w/w) was achieved after 72 h for each yeast. The concentration of essential amino acids in the modified apple pomace was more than twice that in the control, enhancing its nutritive value as a stock-feed supplement. The fermentation of pomace as described can be used to reprocess this waste material into a useful value-added product for the agricultural sector.

Use of isotopically chiral [4'-13C]famciclovir and 13C NMR to identify the chiral monoacetylated intermediates in the conversion of famciclovir to penciclovir by human intestinal wall extract.

Famciclovir is the oral form of the potent antiherpesvirus agent, penciclovir. Hydrolysis of one of the acetyl ester groups of famciclovir creates a chiral centre leading to the possible formation of (R)- and (S)-enantiomers. During its conversion to penciclovir, famciclovir forms two chiral metabolites, namely monoacetyl-6-deoxy-penciclovir and monoacetyl-penciclovir. The absolute configuration and stereospecificity of the monoacetyl metabolites of famciclovir, produced in human intestinal wall extract, were determined using isotopically chiral famciclovir and 13C NMR spectroscopy of the isolated metabolites. 13C NMR showed that the esterase(s), in human intestinal wall extract, hydrolysed the acetyl group preferentially from the pro-(S)-acetoxymethyl group of famciclovir. The specificity of esterase action in forming monoacetyl-6-deoxy-penciclovir and monoacetyl-penciclovir was about 77 and 72%, respectively.

Use of isotopically chiral [4'-13C]penciclovir and 13C NMR to determine the specificity and absolute configuration of penciclovir phosphate esters formed in HSV-1 and HSV-2 infected cells and by HSV-1-encoded thymidine kinase.

Penciclovir is a potent antiherpesvirus agent which is highly selective due to its phosphorylation only in virus infected cells. Phosphorylation of one of the hydroxymethyl groups of penciclovir (PCV) creates a chiral centre leading to the possible formation of (R)- and (S)-enantiomers. The absolute configuration and stereospecificity of the PCV-phosphates produced in cells infected with herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), as well as by HSV-1-encoded thymidine kinase, were determined using isotopically chiral [4'-13C]PCV precursors and 13C NMR spectroscopy of the isolated metabolites. The absolute configuration of penciclovir-triphosphate (PCV-TP) produced in HSV-1 infected cells was shown to be S with an enantiomeric purity of greater than 95%. However, in contrast fo HSV-1-infected cells in which none of the (R) enantiomer was detected, about 10% of (R)-PCV-TP was produced in HSV-2-infected cells. Phosphorylation of PCV by HSV-1-encoded thymidine kinase was found to give 75% (S)- and 25% (R)-PCV-monophosphate. The proportion of the (S)-isomer appears to be amplified in the subsequent phosphorylations leading to the triphosphate.

Mode of antiviral action of penciclovir in MRC-5 cells infected with herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus.

The metabolism and mode of action of penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were studied and compared with those of acyclovir. In uninfected MRC-5 cells, low concentrations of the triphosphates of penciclovir and acyclovir were occasionally just detectable, the limit of detection being about 1 pmol/10(6) cells. In contrast, in cells infected with either herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV), penciclovir was phosphorylated quickly to give high concentrations of the triphosphate ester. Following the removal of penciclovir from the culture medium, penciclovir-triphosphate remained trapped within the cells for a long time (half-lives, 20 and 7 h in HSV-2- and VZV-infected cells, respectively). In HSV-2-infected cells, acyclovir was phosphorylated to a lesser extent and the half-life of the triphosphate ester was only 1 h. We were unable to detect any phosphates of acyclovir in VZV-infected cells. (S)-Penciclovir-triphosphate inhibited HSV-1 and HSV-2 DNA polymerase competitively with dGTP, the Ki values being 8.5 and 5.8 microM, respectively, whereas for acyclovir-triphosphate, the Ki value was 0.07 microM for the two enzymes. Both compounds had relatively low levels of activity against the cellular DNA polymerase alpha, with Ki values of 175 and 3.8 microM, respectively. (S)-Penciclovir-triphosphate did inhibit DNA synthesis by HSV-2 DNA polymerase with a defined template-primer, although it was not an obligate chain terminator like acyclovir-triphosphate. These results provide a biochemical rationale for the highly selective and effective inhibition of HSV-2 and VZV DNA synthesis by penciclovir and for the greater activity of penciclovir than that of acyclovir when HSV-2-infected cells were treated for a short time.

In vitro expression of Lac-PTS and tagatose 1,6-bisphosphate aldolase genes from Lactococcus lactis subsp. cremoris plasmid pDI-21.

A 4.4-kb EcoR1-EcoR1 DNA fragment from the Lactococcus lactis subsp. cremoris plasmid pDI-21 encoded the tagatose 1,6-bisphosphate (TBP) aldolase gene and the Lac-PTS genes. In vitro transcription-translation using Escherichia coli S30 extract showed the synthesis of 41,000-, 23,000- and 12,000-dalton proteins which correspond to the TBP-aldolase, Lac-PTS enzyme II, and factor III proteins respectively.

Selection of an oral prodrug (BRL 42810; famciclovir) for the antiherpesvirus agent BRL 39123 [9-(4-hydroxy-3-hydroxymethylbut-l-yl)guanine; penciclovir].

The limited oral absorption in rodents of the antiherpesvirus agent 9-(4-hydroxy-3-hydroxymethylbut-l-yl)guanine (BRL 39123 [penciclovir; British approved name]) prompted a search for oral prodrugs. The 6-deoxy derivative of penciclovir (BRL 42359) and the corresponding diacetyl and dipropionyl 6-deoxy derivatives (BRL 42810 [famciclovir; British approved name] and BRL 43599) were tested as oral prodrugs. The in vivo absorption (dose, 0.2 mmol/kg) and the conversion to the active compound, penciclovir, were determined in rats. Compared with the sodium salt of penciclovir given intravenously, the bioavailabilities of penciclovir from orally administered penciclovir, BRL 42359, famciclovir, and BRL 43599 were 1.5, 9, 41, and 27%, respectively. These prodrugs and 6-deoxyacyclovir were tested for stability in rat duodenal contents and for metabolism in rat intestinal wall homogenate, liver homogenate, and blood and in the corresponding human fluids and tissues. Famciclovir was much more stable than BRL 43599 in human duodenal contents (half-lives, greater than 2 h and 7 min, respectively) yet was efficiently converted to penciclovir by the tissue homogenates. The major metabolic pathway was by deacetylation followed by oxidation at the 6 position. The rate of oxidation was comparable to that of 6-deoxyacyclovir, which is known to be converted efficiently to acyclovir in humans. Famciclovir was selected for further evaluation and progression to studies in humans. These subsequent studies confirmed that, after oral dosing with famciclovir, more than half the dose was absorbed and rapidly converted to penciclovir.

Prodrugs of the selective antiherpesvirus agent 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (BRL 39123) with improved gastrointestinal absorption properties.

Potential oral prodrugs of the antiherpesvirus acyclonucleoside 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (1, BRL 39123) have been synthesized and evaluated for bioavailability of 1 in the blood of mice. Reduction of 9-[4-acetoxy-3-(acetoxymethyl)but-1-yl]-2-amino-6-chloropurine (13) using ammonium formate and 10% palladium on carbon afforded the 2-aminopurine 14, which was hydrolyzed to the monoacetate 15 and to 2-amino-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purine (5). The 2-aminopurine 5 was subsequently converted to additional monoester (17, 21-23) and diester (16, 24) derivatives and to its di-O-isopropylidene derivative 18. Both 5 and its esters (14-17, 21, 22) and also 18 were well absorbed after oral administration and converted efficiently to 1, the diacetyl (14) and dipropionyl (16) esters providing concentrations of 1 in the blood that were more than 15-fold higher than those observed after dosing either 1 or its esters (25-27). Some 6-alkoxy-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purines (8-10), the preparation of which has been reported previously, also showed improved absorption properties, but their conversion to 1 was less efficient than for the 2-aminopurine derivatives. On the basis of these results and subsequent experiments involving determinations of rates of conversion to 1 in the presence of rat and human tissue preparations, 9-[4-acetoxy-3-(acetoxymethyl)but-1-yl]-2-aminopurine (14, BRL 42810) was identified as the preferred prodrug of 1. Oral bioavailability studies in healthy human subjects confirmed 14 as an effective prodrug, and this compound is now being evaluated in clinical trials.

Mode of action of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) against herpes simplex virus in MRC-5 cells.

The metabolism and mode of action of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) were studied in herpes simplex virus type 1 (HSV-1)-infected and uninfected MRC-5 cells and compared with those of acyclovir. In uninfected cells incubated with 10 microM acyclonucleoside for 4 h, no phosphorylation of either BRL 39123 or acyclovir was detected. In contrast, in HSV-1-infected cells, both BRL 39123 and acyclovir were phosphorylated up to the triphosphate esters. Phosphorylation of BRL 39123 occurred much more rapidly and proceeded to a greater extent than did that of acyclovir. Furthermore, following the removal of acyclonucleoside from the culture medium, the intracellular triphosphate ester of BRL 39123 was much more stable than was that of acyclovir, the half-lives being about 10 and 0.7 h, respectively. BRL 39123 treatment effectively inhibited the formation of HSV-1 DNA in infected MRC-5 cells, 50% inhibitory concentrations of BRL 39123 and acyclovir being 0.04 microgram/ml (0.16 microM) and 0.15 microgram/ml (0.67 microM), respectively. In addition, BRL 39123 was shown to be more effective than acyclovir at inhibiting viral DNA synthesis following short treatment times, presumably reflecting the greater stability of BRL 39123 triphosphate. Neither BRL 39123 nor acyclovir inhibited cellular DNA synthesis in uninfected cells at concentrations of up to 100 micrograms/ml.