Giovanni Codacci-Pisanelli

Thymidylate synthase and drug resistance

Thymidylate synthase is an important target for both fluorinated pyrimidines and for new folate analogues. Resistance to 5-fluorouracil (5FU) can be related to insufficient inhibition of thymidylate synthase. The 5FU-nucleotide FdUMP induces inhibition of thymidylate synthase which is enhanced and retained for longer in the presence of increased folate pools, for which leucovorin is a precursor. In a murine model system, 5FU treatment caused a 4-fold induction of thymidylate synthase levels which may have contributed to resistance. Addition of leucovorin to this treatment prevented this induction and increased the antitumour effect 2-3-fold. In the clinical setting, 5FU administration to patients resulted in approximately 50% inhibition of TS after 48 h. The combination with leucovorin resulted in a more pronounced inhibition after 48 h (approximately 70%). A significant relationship was observed with outcome of treatment; when thymidylate synthase levels were high and inhibition was low, no response was observed. A separate study showed that low thymidylate synthase levels appeared to be an independent prognostic factor for adjuvant therapy.

Epigenetic treatment of solid tumours: a review of clinical trials.

Epigenetic treatment has been approved by regulatory agencies for haematological malignancies. The success observed in cutaneous lymphomas represents a proof of principle that similar results may be obtained in solid tumours. Several agents that interfere with DNA methylation-demethylation and histones acetylation/deacetylation have been studied, and some (such as azacytidine, decitabine, valproic acid and vorinostat) are already in clinical use.The aim of this review is to provide a brief overview of the molecular events underlying the antitumour effects of epigenetic treatments and to summarise data available on clinical trials that tested the use of epigenetic agents against solid tumours. We not only list results but also try to indicate how the proper evaluation of this treatment might result in a better selection of effective agents and in a more rapid development.We divided compounds in demethylating agents and HDAC inhibitors. For each class, we report the antitumour activity and the toxic side effects. When available, we describe plasma pharmacokinetics and pharmacodynamic evaluation in tumours and in surrogate tissues (generally white blood cells).Epigenetic treatment is a reality in haematological malignancies and deserves adequate attention in solid tumours. A careful consideration of available clinical data however is required for faster drug development and possibly to re-evaluate some molecules that were perhaps discarded too early.

Antitumour activity, toxicity and inhibition of thymidylate synthase of prolonged administration of 5-fluorouracil in mice

Continuous infusions of 5-fluorouracil (5-FU) are increasingly used in the treatment of cancer. Their optimal use, however, has still to be determined since the availability of suitable animal models is limited. We studied continuous infusions in mice using subcutaneously implanted pellets that release 5-FU over a period of 3 weeks. At the maximum tolerated dose (MTD) (based on the systemic toxicity in healthy animals) we assessed the antitumour activity, haematological toxicity, inhibition of thymidylate synthase (TS) in tumours and the concentration of 5-FU in plasma during the 3-week period. We also studied the addition of leucovorin in different schedules. The dose-limiting toxicity was weight loss, and at the MTD of 10 mg of 5-FU released in 21 days per mouse myelosuppression was tolerable (nadir for leucocytes and thrombocytes was approximately 40% of pretreatment levels). In several independent experiments using the 5-FU-resistant Colon 26 tumour, a good antitumour activity was observed during the first part of the infusion, but thereafter the growth of the tumours resumed; the overall effect of continuous infusions was thus comparable to that of bolus injections. Coadministration of leucovorin did not enhance the therapeutic results; depending on the schedule used, it proved ineffective or only increased toxicity. Similar results were obtained with head and neck squamous cell carcinomas and with the 5-FU-sensitive tumour Colon 38. In Colon 26 tumours the TS activity (FdUMP-binding assay) initially decreased to 20-30% of controls and returned to normal after 11 days. In the catalytic TS assay a slight inhibition was observed for the continuous infusion, followed after 11 days by a marked (4-fold) increase in activity. 5-FU plasma levels varied from 0.1 to 1 microM following a circadian rhythm (with a peak at 6 h after light onset), and were maintained during the entire period. Subcutaneously implanted pellets represent a suitable model to study prolonged administration of 5-FU in mice and to evaluate the effect of modulating agents in laboratory animals before transferring data obtained in vitro to the clinic.

Chemotherapy, targeted agents, antiemetics and growth-factors in human milk: How should we counsel cancer patients about breastfeeding?

An increasing number of women are diagnosed with cancer during pregnancy and lactation. Women are usually advised to interrupt breastfeeding during systemic anticancer treatment for fear of serious adverse effects to the nursed infant. However, the issue is poorly addressed in the literature and very few studies have evaluated the safety of breastfeeding during or after cytotoxic drugs or target agents administration. In this review we will analyze the available evidence that addresses the issue of anticancer drugs, targeted agents, antiemetics and growth-factors excretion in human milk. This could serve as a unique resource that may aid physicians in the management of breastfeeding cancer patients interested in maintaining lactation during treatment.

High-Dose 5-Fluorouracil with Uridine-Diphosphoglucose Rescue Increases Thymidylate Synthase Inhibition but Not 5-Fluorouracil Incorporation into RNA in Murine Tumors

5-Fluorouracil (5FU) shows a steep dose response curve in several experimental systems, but the clinical use of high doses is hampered by the toxic side effects of this drug. Uridine diphosphoglucose (UDPG) rescue allows an increase in the maximum tolerated dose of 5FU in mice from 100 (FU100) to 150 mg/kg (5FU150+UDPG) and the higher dose is more effective than the standard treatment against several tumors. In the present paper we report on the effect of high-dose 5FU on thymidylate synthase (TS) levels and on 5FU incorporation into RNA. In the resistant murine tumor (Colon 26A) high-dose 5FU inhibited TS catalytic activity 8 h after treatment (4-fold; p = 0.00041) and the inhibition persisted until day 3 (p < 10–4). Standard-dose 5FU did not significantly inhibit TS activity. In a relatively sensitive tumor (Colon 26-10), there was no difference in the initial extent of TS inhibition by the two 5FU doses, but TS was still inhibited (2-fold) on day 3 after (5FU150+UDPG) while it was within the normal range after 5FU100. In both tumor types TS activity showed an impressive rebound (3-fold) on days 3–7, and this occurred after both 5FU doses. In Colon 26A, however, a new 5FU injection on day 7 was still able to inhibit TS but not as effectively as the first dose. 5FU incorporation into RNA reached similar peak values (8 pmol/µg RNA) after the two 5FU doses, but the clearance was faster in mice receiving UDPG rescue. We conclude that UDPG does not interfere with the extent of TS inhibition by 5FU, but UDPG allows the use of a higher dose of 5FU resulting in enhanced TS inhibition. UDPG, however, increases 5FU clearance from RNA. In this experimental system the inhibition of TS seems essential in order to obtain a good antitumor activity, while 5FU incorporation into RNA does not seem to play a role in the antitumor activity of 5FU. Since preliminary results indicate that UDPG is well tolerated by patients, the use of higher 5FU doses may improve the response rate of human tumors.

Chemotherapy and Target Therapy in the Management of Adult High- Grade Gliomas

Adult high grade gliomas (HGG) are the most frequent and fatal primary central nervous system (CNS) tumors. Despite recent advances in the knowledge of the pathology and the molecular features of this neoplasm, its prognosis remains poor. In the last years temozolomide (TMZ) has dramatically changed the life expectancy of these patients: the association of this drug with radiotherapy (RT), followed by TMZ alone, is the current standard of care. However, malignant gliomas often remain resistant to chemotherapy (CHT). Therefore, preclinical and clinical research efforts have been directed on identifying and understanding the different mechanisms of chemo-resistance operating in this subset of tumors,in order to develop effective strategies to overcome resistance. Moreover, the evidence of alterations in signal transduction pathways underlying tumor progression, has increased the number of trials investigating molecular target agents, such as anti-epidermal growth factor receptor (EGFR) and anti- vascular endothelial growth factor (VEGF) signaling. The purpose of this review is to point out the current standard of treatment and to explore new available target therapies in HGG.

Pharmacokinetics of bolus 5-fluorouracil: relationship between dose, plasma concentrations, area-under-the-curve and toxicity

Abstract The pharmacokinetics of 5-fluorouracil (5FU) have been related to toxicity and antitumor activity, in particular for continuous infusion schedules, but to a lesser extent for frequently used bolus injections. The use of intensive sampling schedules limits the application of pharmacokinetics to optimize individual dosing or to define the ideal combination with other drugs. We therefore reanalyzed a pharmacokinetic study in order to develop a limited sampling schedule. Patients received escalating doses of 5FU at 500, 600 and 720 mg/m2 as a bolus until toxicity developed. Blood samples were analyzed until 24 h after administration. The area under the concentration time curve from 0-90 min (AUC0-90) was strongly correlated with dose and also with toxicity (p = 0.0009). The 5FU concentrations at 30 and 60 min were correlated to the AUC30-240 and to that of the AUC0-90 (r2= 0.970). The use of limited sampling (30, 60, 90 min) in a patient given 353 mg/m2 5FU with severe toxicity at initial dosing at 500 mg/m2 revealed that the AUC0-90 at 353 mg/m2 was higher than the normal AUC0-90 for 500 mg/m2. This patient appeared to have an 8-fold lower activity of the 5FU degradation enzyme dihydropyrimidine dehydrogenase. Limited sampling will allow us to define potential aberrant kinetics of pharmacokinetic interaction of 5FU with other drugs being developed for treatment of colorectal cancer.

5-Flugrouracil (FU) and mitomycin c (MMC) in the management of colorectal carcinoma. part ii.in vitro activity of the two drugs in short term tumor cultures

Eighty-seven colorectal adenocarcinomas from untreated patients were investigated by short term tumor cultures to testin vitro sensitivity to 5- fluorouracil and mitomycin C. This study reports the preliminary results of a multistep program aimed at the prospective clinical application of the assay. At present this in vitro experience was performed in parallel with a clinical trial carried out with the same drugs. The in vitro activity of the two anticancer agents is in agreement with the response rate reported in monochemotherapy; our data would suggest an increase of responses using the combination of fluorouracil and mitomycin in comparison to single drug therapy. A low cosensitivity rate and a high number of cases sensitive to one drug but resistant to the other, account for the use of this test as screening of active drugs in the individual patient.

“The only thing I know is that I know nothing”: 5-fluorouracil in human milk

Women are usually advised to interrupt breastfeeding during chemotherapy for concerns of serious side-effects to the infant. However, the passage of cytotoxic drugs in human milk has been poorly studied. A 36-year-old woman was diagnosed with rectal cancer while she was still breastfeeding her 9-month-old son. The tumor was staged as cT3N1M0 and neoadjuvant 5-flourouracil (5-FU)-based chemoradiotherapy (CRT) was planned. She was advised to stop nursing, however, she expressed her desire to resume breastfeeding after treatment. After a multidisciplinary discussion, the patient was advised to pump her breasts twice daily during CRT in order to maintain milk production. To decide when breastfeeding could be safely resumed, we evaluated the pharmacokinetics of 5-FU in milk. The patient received standard CRT with 5-FU 200 mg/m/day as i.v. continuous infusion concurrently with pelvic radiotherapy. Samples of peripheral blood were taken before the start of 5-FU and after 1, 2 and 5 weeks. Milk samples were obtained before, during and up to 10 days following the completion of therapy. 5-FU concentrations were measured using an highperformance liquid chromatography (HPLC) method [1] modified and optimized for plasma and milk samples. Four plasma samples were collected and analyzed. The concentration of 5-FU in plasma ranged between 11.14 and 114.95 lM (Figure 1A and B). Thirty-three milk samples were collected and tested. 5-FU levels were undetectable in milk at any time during and after CRT treatment. To validate the HPLC methodology used, we carried out parallel analyses on human milk spiked with different concentrations of 5-FU, which were stored, extracted and measured in the same conditions. 5-FU was detected in the validation samples with a limit of detection at 0.5 lM (Figure1C and D). The patient completed the planned treatment and eventually underwent radical surgery without complications. Ten days after surgery, when pharmacokinetics results were available, the patient tried to resume breastfeeding, but her infant refused to latch. Breastfeeding is an essential physiologic process that provides nutrition and protects the child against infection, immunologic disorders and some types of cancer during adulthood [2, 3]. Nevertheless and despite the lack of evidence, breastfeeding is usually not recommended during maternal chemotherapy because of the potential toxic effects to the infant. In this case, we report for the first time that 5-FU was undetectable at any time during and following CRT, although maternal plasma concentrations reflected normal pharmacokinetics. The amount of a drug or its metabolites excreted into milk is dependent upon several factors: lipid solubility, molecular size, ionization, protein binding and halflife in maternal plasma [4]. 5-FU has a short half-life and a high protein bound in serum [5]; these factors probably accounted for the undetectable levels of 5-FU in milk. Our results are reassuring, but genetic background and plasma levels variability might influence 5-FU distribution into milk. Collecting breast milk for drug assays during and after chemotherapy, as impractical as this approach may seem, could provide valuable information to counsel patients receiving chemotherapy who are willing to resume breastfeeding after the end of treatment.

Activity of Ruboxyl, a Nitroxyl Derivative of Daunorubicin, on Experimental Models of Colorectal Cancer Metastases

We evaluated the activity of ruboxyl (Rbx), a nitroxyl analogue of daunorubicin (Dauno), in experimental models of hepatic metastases from colorectal carcinoma (CRC) and compared it with its parent compound and with 5-fluorouracil (5FU). In mice treated by intraperitoneal injections Rbx and 5FU proved more effective than Dauno: the Index of Inhibition of Metastases in comparison with controls was 43% for Dauno, 70% for 5FU and 84% for Rbx. In BDIX rats implanted with the syngeneic cell line DHD K12/TRb, both Rbx and 5FU, administered as a continuous intravenous infusion for 7 days, reduced the development of liver metastases from a median of 23.8 ± 2.16 for controls to 3.2 ± 1.3 for 5FU and 1.0 ± 1.4 for Rbx (p < 0.0001 versus controls for both treatments): the comparison of Rbx and 5FU showed a trend in favour of this new anthracycline. Median survival was prolonged from 40.6 ± 3.4 days in controls to 56.0 ± 5.8 days with Rbx and 58.0 ± 4.69 days with 5FU. Considering that in a phase I study Rbx showed only minor and manageable toxic side effect, its activity in the clinical treatment of CRC metastases may deserve further attention.