Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells.

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B-/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B-/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

Stability of calcium levofolinate reconstituted in syringes and diluted in NaCl 0.9% and glucose 5% polyolefin/polyamide infusion bags.

PURPOSE: Calcium levofolinate (CaLev) for intravenous administration is commercially available as a powder that must be reconstituted for injection or reconstituted and then diluted before administration. The lack of stability data on CaLev solutions renders necessary extemporaneous manual preparation, preventing the use of automated/semi-automated systems, with a consequent loss in terms of quality and safety. METHODS: The present work assessed the chemical-physical and microbiological stability of CaLev prepared in sodium chloride 0.9%, glucose 5% and water for injections and stored in polyolefin/polyamide bags and polypropylene syringes at 2-8°C protected from light. For this purpose, we developed and validated a new rapid High Performance Liquid Chromatography with Ultra Violet Diode-Array Detection (HPLC-UV-DAD) method. RESULTS: The samples tested were stable for 14 days, retaining >95% of their initial concentration and showing no change in colour, turbidity or pH. Microbiological tests performed on the samples were negative. CONCLUSIONS: Our results confirmed the analytical stability of CaLev in NaCl 0.9%, glucose 5% and water for injection at concentrations used in clinical practice at our institute. This enables our centralized laboratory to organize the preparation of this drug in advance and the use of robots rather than manual preparation reduces the workload and the risk of preparation errors.

Validation of RP-HPLC method to assess the compatibility of metoclopramide and midazolam intravenous mixture used in patients with cancer.

BACKGROUND: The delivery of intravenous medication by continuous infusion is necessary and widespread for treatment of patients with advanced cancer. Few scientific papers have focused on assessment of the chemical compatibility of these therapeutic mixtures. An analytical assessment of the physical and chemical compatibility of these combinations is needed. OBJECTIVES: To determine the chemical and physical compatibility of binary mixtures of metoclopramide (MET) and midazolam (MID). METHODS: Mixtures of drugs were prepared under aseptic conditions in 0.9% sodium chloride at concentrations used in our clinical practice for continuous infusion. The samples were stored in polyethylene bags at room temperature in the presence of light for 15 days. Chemical compatibility was evaluated by high-performance liquid chromatography (HPLC). Physical compatibility was tested by visual inspection (for evidence of precipitation and colour change) and by pH determination. RESULTS: No changes in colour, precipitation of components, measurable losses of volume or notable changes in pH were seen. The combinations tested were compatible for 15 days (retained >95% of their initial concentration). CONCLUSIONS: This study confirms the analytical compatibility of MET and MID, when mixed in 0.9% sodium chloride at concentrations employed in our clinical practice.