An 1H NMR study of the cytarabine degradation in clinical conditions to avoid drug waste, decrease therapy costs and improve patient compliance in acute leukemia.

Cytarabine, the 4-amino-1-(ß-D-arabinofuranosyl)-2(1H)-pyrimidinone, (ARA-C) is an antimetabolite cytidine analogue used worldwide as key drug in the management of leukaemia. As specified in the manufacturers' instructions, once the components-sterile water and cytarabine powder-are unpackaged and mixed, the solution begins to degrade after 6 hours at room temperature and 12 hours at 4°C. To evaluate how to avoid wasting the drug in short-term, low-dose treatment regimens, the reconstituted samples, stored at 25°C and 4°C, were analyzed every day of the test week by reversed-phase HPLC and high-field NMR spectroscopy. All the samples remained unchanged for the entire week, which corresponds to the time required to administer the entire commercial drug package during low-dose therapeutic regimens. The drug solution was stored in a glass container at 4°C in an ordinary freezer and drawn with sterile plastic syringes; during this period, no bacterial or fungal contamination was observed. Our findings show that an cytarabine solution prepared and stored in the original vials retains its efficacy and safety and can, therefore, be divided into small doses to be administered over more days, thus avoiding unnecessary expensive and harmful waste of the drug preparation. Moreover, patients who require daily administration of the drug could undergo the infusion at home without need to go to hospital. The stability of the aliquots would help decrease hospitalization costs.

[Comparative costs study between ready-to-administer bag of gemcitabine and production in reconstitution unit]. / Étude comparative de coûts entre les poches de gemcitabine prêtes à l'emploi et une fabrication en unité de reconstitution.

INTRODUCTION: Dose-banding is needed to rationalise cytotoxic drugs preparations. A new step was recently crossed: gemcitabine is the first molecule to be sold in ready-to-administer bag. What's the pharmaco-economic impact of gemcitabine ready-to-administer bags versus manufacturing in preparation unit? METHODS: A retrospective analysis of gemcitabine dosage prepared over three months permitted to explain our consumption of this drug, and by modelling, to characterize the good prescription interval for each dosage. Compared costs study assessed cost of medicine, preparation kit and preparation time. RESULTS: Over the study period, for 5%, 7.5% and 10% of deviation, respectively 67%, 75% and 76% of preparations were covered by standard doses. We chose 7.5% interval. The manufacturing cost of gemcitabine infusions is around 30,000€/year for vials with solution for infusion, 32,300€ for vials with powder, versus 67,300€ for ready-to-administer bag. Approximately 7.75minutes of pharmacy technician time would be saved by preparation. DISCUSSION: Ready-to-administer bags of gemcitabine allow relevant coverage of manufacturing doses. The annual extra cost would reach 37,200€ for our establishment. But, it can be justified by expected benefits: safer medication circuit, saving time of pharmacy technician….

Preparation and in vitro assessment of wet-spun gemcitabine-loaded polymeric fibers: Towards localized drug delivery for the treatment of pancreatic cancer.

BACKGROUND/OBJECTIVES: There has been minimal improvement in the prognosis of pancreatic cancer cases in the past 3 decades highlighting the crucial need for more effective therapeutic approaches. A drug delivery system capable of locally delivering high concentrations of chemotherapeutics directly at the site of the tumor is clearly required. The aim of this study was to fabricate and characterize the biophysical properties of gemcitabine-eluting wet-spun polymeric fibers for localized drug delivery applications. METHODS/RESULTS: Fibers spun from alginate or chitosan solutions with or without the anticancer drug gemcitabine had a uniform surface area, were internally homogeneous and ranged from 50-120 µm in diameter. Drug encapsulation ranged from 13-52%, depending on the type and concentration of polymer used. Gemcitabine displayed first-order release kinetics where 64-82% of the loaded drug was rapidly released within the first 10 h followed by a sustained release over the next 134 h. A time dependent inhibition of ex vivo tumor spheroid growth and cell viability was observed after incubation with gemcitabine-loaded fibers but not control fibers. CONCLUSION: With further development these studies could lead to the manufacture of a safe and effective delivery system designed to combat non-resectable pancreatic cancer for which currently there is minimal chance of cure.

Liposomal formulation of a methotrexate lipophilic prodrug: assessment in tumor cells and mouse T-cell leukemic lymphoma.

In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG. Carcinoma cells accumulated 5 times more MTX-DG liposomes than the empty liposomes. Studies on inhibitors of liposome uptake and processing by cells demonstrated that the formulation used multiple mechanisms to deliver the prodrug inside the cell. According to the data from the present study, undamaged liposomes fuse with the cell membrane only 1.5-2 hours after binding to the cell surface, and then, the components of liposomal bilayer enter the cell separately. The study on the time course of plasma concentration in mice showed that the area under the curve of MTX generated upon intravenous injection of MTX-DG liposomes exceeded that of intact MTX 2.5-fold. These data suggested the advantage of using liposomal formulation to treat systemic manifestation of hematological malignancies. Indeed, the administration of MTX-DG liposomes to recipient mice bearing T-cell leukemic lymphoma using a dose-sparing regimen resulted in lower toxicity and retarded lymphoma growth rate as compared with MTX.

A combined experimental and computational study of novel nanocage-based metal-organic frameworks for drug delivery.

Three new metal organic frameworks (MOFs) with chemical formulae [(CH3)2NH2] [Sm3(L1)2(HCOO)2(DMF)2(H2O)]·2DMF·18H2O (1), [Cu2(L2)(H2O)2]·2.22DMA (2) and [Zn2(L1)(DMA)]·1.75DMA were synthesized and structurally characterized. 1 and 2 show a classical NbO-like topology and have two types of interconnected cages. 3 exhibits an uncommon zzz topology and has two types of interconnected cages. These MOFs can adsorb large amounts of the drug 5-fluorouracil (5-FU) and release it in a progressive way. 5-FU was incorporated into desolvated 1, 2 and 3 with loadings of 0.40, 0.42, and 0.45 g g(-1), respectively. The drug release rates were 72%, 96% and 79% of the drug after 96 hours in 1, 120 hours in 2 and 96 hours in 3, respectively. Grand Canonical Monte Carlo (GCMC) simulations were performed to investigate the molecular interactions during 5-FU adsorption to the three novel materials. The GCMC simulations reproduced the experimental trend with respect to the drug loading capacity of each material. They also provided a structural description of drug packing within the frameworks, helping to explain the load capacity and controlled release characteristics of the materials. 5-FU binding preferences to 1, 2 and 3 reflect the diversity in pore types, chemistry and sizes. The calculated drug load is more related to the molecular properties of accessible volume Vacc than to the pore size.

A validated LC-MS/MS method for rapid determination of methotrexate in human saliva and its application to an excretion evaluation study.

A sensitive and simple method for the methotrexate quantification was developed using aminopterin as internal standard. Methotrexate is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma. The compound was quantified by liquid-chromatography coupled to electrospray ionization (positive ion-mode) low-energy collision dissociation-tandem mass spectrometry. Quantitative detection was by multiple reaction monitoring of the transitions of the [M+H]+ ion of MTX to its common product ion at m/z 308.4 and of aminopterin at m/z 441.2→m/z 294.0. The method demonstrated linearity over at least three orders of magnitude and had a detection limit of 1ng/ml for methotrexate. A run time of less than 8.0min for each sample made it possible to analyze a large number of human saliva samples per day. Application of this procedure was demonstrated to a saliva excretion study of methotrexate on the samples obtained after an intravenously administration of 1mg/kg/dose of methotrexate to six patients with acute lymphoblastic leukemia.

Novel microbially triggered colon specific delivery system of 5-Fluorouracil: statistical optimization, in vitro, in vivo, cytotoxic and stability assessment.

The present study aimed to statistically optimize a colon specific formulation of 5-Fluorouracil for the treatment of colon cancer. A 3(2) full factorial design was used for optimization. The independent variables employed were amount of pectin and amount of starch paste, each at three levels. The evaluated responses were hardness, percent cumulative drug release (% CDR) at 5th h and t(90%) (time required for 90% of drug release). Drug release studies were carried out using change over media [pH 1.2, 7.4 and 6.5 in presence of 4% (w/v) rat caecal contents]. The optimized formulation was subjected to in vivo roentgenographic studies in New Zealand white rabbits to analyze the in vivo behaviour of the developed tablets. This formulation was also evaluated for cytotoxic potential using HT-29 human colon cancer cell lines. Pharmacokinetic studies in New Zealand white rabbits were conducted to determine the extent of systemic exposure provided by the developed formulation in comparison to an immediate release tablet. The optimized formulation consisting of pectin (66.67%, w/w) and starch paste (15%, w/w) released negligible amount of drug at pH 1.2 and pH 7.4 whereas significant (p < 0.05) drug release was observed at pH 6.5 in presence of 4% (w/v) rat caecal contents. Roentgenographic studies corroborated the in vitro observations, thus providing the "proof of concept". Pharmacokinetic studies revealed significant reduction in systemic exposure and cytotoxicity studies demonstrated enhanced cellular uptake of drug by the developed formulation. Shelf life of the formulation was found to be 2.83 years. The results of the study established pectin-based coated matrix tablet to be a promising system for the colon specific delivery of 5-FU so as to treat colon carcinoma.

Development and assessment of novel all-in-one parenteral formulations with integrated anticoagulant properties for the concomitant delivery of 5-fluorouracil and calcium folinate.

5-Fluorouracil in combination with its biomodulator folinic acid maintains a pivotal position in current anticancer treatment regimens. However, limitations in clinical management persist with the administration of these drugs. These limitations are associated with the use of a high pH to maintain 5-fluorouracil in solution, resulting in high rates of phlebitis and catheter blockages. Herein, we describe and compare initial studies on novel all-in-one formulations of 5-fluorouracil and folinic acid incorporating either sulfated or hydroxypropyl beta-cyclodextrins at physiological pH that potentially address these issues. All formulations markedly improved the stability of supersaturated solutions of 5-fluorouracil in the presence of folinic acid. In-vitro evaluation of the PC-3, HCT-116, MDA-MB-231, PC-14, and COLO-201 human carcinoma cell lines showed that all formulations exhibited equivalent or better cytotoxicity compared with cells exposed to 5-fluorouracil and folinic acid. Thus, these cyclodextrins do not compromise the cytotoxicity of 5-fluorouracil. Preliminary in-vivo dose tolerance profiles of the formulations were also equivalent to 5-fluorouracil and folinic acid administered separately. Furthermore, given the association between thrombosis and cancer, the potentially beneficial anticoagulant activity of the sulfated cyclodextrin-based formulations was also confirmed in vitro. Extended activated partial thromboplastin times and prothrombin times were observed for the sulfated cyclodextrins in human plasma both as individual compounds and as components of the formulations. In conclusion, these novel all-in-one formulations maintain the in-vitro potency while overcoming the accepted incompatibility of 5-fluorouracil and folinic acid, and represent improved injectable forms of 5-fluorouracil that may reduce phlebitis, catheter blockages, and thromboembolic events.

Development of a respirable, sustained release microcarrier for 5-fluorouracil I: In vitro assessment of liposomes, microspheres, and lipid coated nanoparticles.

The release rate of 5-fluorouracil (5-FU) from liposomes, microspheres, and lipid-coated nanoparticles (LNPs) was determined by microdialysis to investigate their use as a respirable delivery system for adjuvant (postsurgery) therapy of lung cancer. 5-FU was incorporated into liposomes using thin film hydration and into microspheres and LNPs by spray drying. Primary particle size distributions were measured by dynamic light scattering. Liposomes released 5-FU in 4-10 h (k(1) = 0.44-2.31/h, first-order release model). Extruded vesicles with diameters less than one micron released 5-FU more quickly than nonextruded vesicles. With poly-(lactide) (PLA) and Poly-(lactide-co-glycolide) (PLGA) microspheres, slower release rates were observed (k(1) = 0.067-0.202/h). Increasing the lactide:glycolide ratio (50:50-100:0) resulted in a progressive decrease in the release rate of 5-FU. poly-(lactide-co-caprolactone) (PLCL) microspheres released 5-FU more rapidly compared to PLGA systems (k(1) = 0.254-0.259/h). LNPs formulated with polymeric core excipients had lower release rates compared to monomeric excipients (k(1) = 0.043-0.105/h vs. k(1) = 0.192-0.345/h). Changing the lipid chain length of the shell lipid components had a relatively minor effect (k(1) = 0.043-0.129/h). Overall, these systems yielded a wide range of delivery durations that may be suitable for use as an inhalation delivery system for adjuvant therapy of lung cancer.

Epigenetic drugs: a longstanding story.

In this chapter, the development of decitabine from its synthesis in 1964 to the submission of a registration file in 2004 is reviewed. The proper application of the unique properties of decitabine took quite some time to elucidate. In addition, the practical handling in the clinic was not easy as the prolonged myelosuppression of decitabine made it difficult to determine the preferred dose and schedule. Laboratory studies on DNA methylation and cell differentiation showed possible applications in solid and hematologic malignancies. However, despite many attempts, results in solid tumors have been disappointing thus far. After thorough investigation, decitabine achieved therapeutic application in myelodysplastic syndrome (MDS), in particular in patients with a poor prognosis. Further indications may include acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), hematopoietic stem cell transplantation, sickle cell anemia, and thalassemia. Whereas most drugs are already at the end of their life cycle after 40 years, decitabine is only at the beginning. Its application will broaden with the increase in knowledge of epigenetic mechanisms and their relationship to drug therapy.