Intrinsic radiosensitivity of human pancreatic tumour cells and the radiosensitising potency of the nitric oxide donor sodium nitroprusside.

A panel of eight human pancreatic tumour cell lines displayed high intrinsic radioresistance, with mean inactivation doses between 2.4 and 6.5 Gy, similar to those reported for melanoma and glioblastoma. The radiosensitising potency of sodium nitroprusside, a bioreductive nitric oxide donor, was assessed in a model of metabolism-induced hypoxia in a cell micropellet. Sodium nitroprusside at 0.1 mM revealed a radiosensitising effect with an overall enhancement ratio of 1.9 compared with 2.5 for oxygen. Radiosensitising activity correlated with the enhancement of single-strand DNA breakage caused by radiation. In suspensions with cell densities of between 3% and 30% (v/v), the half-life of sodium nitroprusside decreased from 31 to 3.2 min, suggesting a value of around 1 min for micropellets. Despite this variation, the radiosensitising activity was similar in micropellets and in diluted cell suspensions. S-nitroso-L-glutathione was found to possess radiosensitising activity, consistent with a possible role of natural thiols in the storing of radiobiologically active nitric oxide adducts derived from sodium nitroprusside. As measured by a nitric oxide-specific microsensor, activation of sodium nitroprusside occurred by bioreduction, whereas S-nitroso-L-glutathione showed substantial spontaneous decomposition. Both agents appear to exert radiosensitising action through nitric oxide as its scavenging by carboxy phenyltetramethylimidazolineoxyl N-oxide (carboxy-PTI0) and oxyhaemoglobin resulted in attenuated radiosensitisation. Sodium nitroprusside was at least 10-fold more potent than etanidazole, a 2-nitroimidazole used as a reference. Our data suggest that sodium nitroprusside, a drug currently used for the treatment of hypertension, is a potential tumour radioresponse modifier

Radiosensitization of hypoxic tumour cells by S-nitroso-N-acetylpenicillamine implicates a bioreductive mechanism of nitric oxide generation

The radiosensitizing activity of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, was assessed in a model of non-metabolic hypoxia achieved in an atmosphere of 95% nitrogen–5% carbon dioxide. A 10 min preincubation of hypoxic EMT-6 cells (10 × 106 ml−1) with 0.1 and 1 mM SNAP before radiation resulted in an enhancement ratio of 1.6 and 1.7 respectively. The level of spontaneous NO release, measured by a NO specific microsensor, correlated directly with the concentration of SNAP and was enhanced 50 times in the presence of cells. Dilution of the cell suspension from 10 to 0.1 × 106 ml−1 resulted in a 16-fold decline in NO release, but only a twofold decrease in radiosensitization was observed. Preincubation of hypoxic cells with SNAP for 3 min up to 30 min caused an increasing radiosensitizing effect. Extended preincubation of 100 min led to the loss of radiosensitization although the half-life of SNAP is known to be 4–5 h. Taken together, these observations suggest that SNAP generates NO predominantly by a bioreductive mechanism and that its biological half-life is unlikely to exceed 30 min. The lack of correlation between free NO radical and radiosensitizing activity may reflect a role of intracellular NO adducts which could contribute to radiosensitization as well. © 1999 Cancer Research Campaig

NF-κB inhibition impairs the radioresponse of hypoxic EMT-6 tumour cells through downregulation of inducible nitric oxide synthase

Hypoxic EMT-6 tumour cells displayed a high level of inducible nitric oxide synthase (iNOS) and an increased radiosensitivity after a 16 h exposure to lipopolysaccharide, a known activator of nuclear factor-κB (NF-κB). Both iNOS activation and radioresponse were impaired by the NF-κB inhibitors phenylarsine oxide and lactacystin. Contrasting to other studies, our data show that inhibition of NF-κB may impair the radioresponse of tumour cells through downregulation of iNOS. © 2003 Cancer Research UK.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Application of 3D printing photopolymerization technology in fabrication of pharmaceutical dosage forms for pediatric population

Tродимензионалнa (3Д) штампа заснована je на производњи виртуелних објеката механизмом наношења материјала „слој по слој“, на основу дигиталног дизајна 3Д објекта.Threedimensional (3D) printing is based on producing virtually designed 3D objects in a layer-by-layer manner

DNA breakage, cytotoxicity, drug accumulation and retention in two human ovarian tumor cell lines AZ224 and AZ364 treated with adriamycine, modulated by verapamil.

We investigated the cytotoxicity of adriamycin in two human ovarian tumor cell lines, AZ224 and AZ364, by the MTT-test and we analysed the formation of DNA single-strand (SSB) and double-strand breaks (DSB) by means of the alkaline and neutral elution technique. The AZ364 cell line was 15 times more resistant to ADR (ID 50 = 10.0 μg/ml) than the AZ224 cell line (ID 50 = 0.66 μg/ml) after 1 hr of drug exposure. Immediately after exposure, we observed a biphasic dose response for SSB in the AZ224 cells over a concentration range of 0.1 to 32.0 μg/ml, while practically no DSB were found. Upon drug removal and incubation in drug-free medium, full repair of SSB was observed for an ADR concentration of 1 μg/ml. On the contrary, the DSB became significantly increased for all tested concentrations and persisted as observed after 3 hr and 7 hr in drug-free medium. The resistant cell line AZ 364 showed consistently less DNA breakage than the AZ 224 cell line. This inherent difference in sensitivity to ADR could, however, not be explained on the basis of the cellular pharmacokinetics of the drug. Verapamil induced a 3 to 4 fold potentiation of the ADR cytotoxicity in both cell lines after continuous exposure and was associated with an increase in DNA-breakage. The results of our study confirm that there is a lack of correlation between cytotoxicity of ADR and DNA strand breakage immediately after 1 hr of drug exposure. Instead, we emphasize the importance of the formation, extent and persistence of protein-associated DSB upon drug removal to the cytotoxic action of ADR in vitro.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Chronic hypoxia modulates tumour cell radioresponse through cytokine-inducible nitric oxide synthase

Chronic hypoxia up-regulated the mRNA and protein expression of inducible nitric oxide synthase (iNOS) in EMT-6 tumour cells exposed to interferon (IFN)-gamma and interleukin (IL)-I beta, Low concentrations of cytokines (1 unit ml-1) in 1% but not in 21% oxygen induced a remarkable increase in NO production and a 1.8-fold hypoxic cell radiosensitization. Therefore, chronic hypoxia may potentially be exploited to increase tumour cell radioresponse through the cytokine-inducible iNOS pathway. © 2001 Cancer Research Campaign.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Fun-shaped oral dosage forms for the pediatric population fabricated by digital light processing (DLP) 3D printing technique

Introduction Three-dimensional (3D) printing as an innovative technology in the field of drug manufacturing has attracted a lot of attention from the scientific and professional public in recent years. Classified into seven main categories, all 3D printing techniques are based on the same layer-by-layer printing mechanism, where the structure of an object is created from a digital 3D file using computer-aided design (CAD) software or imaging techniques (Trenfield et al., 2018). 3D printing techniques have the potential to provide drug dosage forms of precise geometry and variety of shapes, with tendency to revolutionize the way drugs are designed and manufactured (Trenfield et al., 2018). 3D printing also pretends to play an important role in the concept of personalized medicine, allowing dose adjustment according to individual patient needs based on their own characteristics, requirements and conditions of the disease, in order to achieve the most suitable therapeutic outcomes. The approach of "one size fits all" could be changed by using 3D printing techniques in the manufacturing of small batches of patient-tailored medicines (Zema et al., 2017). In this study, digital light processing (DLP), also known as photopolymerization technique which utilizes light irradiation to create solid objects from photoreactive liquid resin, was used to fabricate fun-shaped oral dosage forms with an aim to achieve flexible dose adjustment of atomoxetine hydrochloride (AH), according to the specific needs of pediatric patients. Materials and methods Materials Poly(ethylene glycol)diacrylate (PEGDA, average MW 250) was obtained from Sigma-Aldrich, Japan. Poly(ethylene glycol) (PEG 400, average MW 400) was purchased from Fagron B.V., The Netherlands. Mannitol Parteck® M 200 was obtained from Merck, Germany. AH was kindly donated by Hemofarm AD, Vrsac, Serbia. Diphenyl(2,4,6-trimethylbenzoyl)phosphineoxide (DPPO) was purchased from Sigma-Aldrich, Germany. Preparation of photoreactive suspensions and 3D printing process Content of AH was 5% (w/w, formulation F1) or 10% (w/w, formulation F2). PEGDA and PEG 400 were used in a constant ratio of 3:1. Both formulations contained 0.50% of mannitol and 0.10% of DPPO. The water content was 5% (w/w, F1) or 10% (w/w, F2), depending on the amount of the active substance. Fun-shaped 3D models (Mickey Mouse, Ring, Pentagon and Cylinder) were designed in Autodesk fusion software version 2.0.8809 (Autodesk Inc, USA), exported as a stereolithography file (.stl) into the 3D printer software (Chitubox, version 1.7.0) and printed with Wanhao Duplicator 8 printer (Wanhao, China). 3D models of Mickey Mouse and Ring were printed from formulation F1, while 3D models of Pentagon and Cylinder were printed from formulation F2. Mass, dimensions and drug content determination 3D-printed dosage forms (n = 10) were weighed on an analytical balance (Kern & Sohn, Germany) and measured (length/diameter and thickness) using a digital caliper (Vogel Germany GmbH & Co. KG, Kevelaer, Germany). The drug content was determined UV spectrophotometrically (Evolution 300, Thermo Fisher Scientific, USA) at the wavelength of 270 nm. For standard preparation, 10 mg of AH was dissolved in 10 mL of absolute ethanol, shaken in an ultrasonic bath for 60 min at room temperature, cooled and then filtered through 0.45 μm filters (Millipore, USA). For test preparation one dosage form of each formulation was crushed and all samples underwent the same procedure as described for standard preparation. In vitro drug release testing The dissolution test was performed with a USP-I Erweka DT 600 (Erweka, Germany) apparatus, in 500 mL of distilled water at 37 ± 0.5 °C, until a plateau was reached. The basket speed was fixed at 100 rpm, aliquots (5 mL) were withdrawn at time intervals of 15, 30, 45, 60, 120, 180, 240, 300, 360 and 420 min, respectively, filtered through 0.45 μm filters and the amount of AH released was determined at 270 nm. Measurements were performed in triplicate, for each formulation and each dosage form. Differential Scanning Calorimetry (DSC) and Polarized Light Microscopy DSC was performed on a DSC 1 instrument (Mettler Toledo, Germany). Samples were subjected to heating at 10 °C/min in the range from 0 to 200 °C under constant nitrogen gas flow of 50 mL/min. The obtained data were analyzed in the STARe software (version 12.10, Mettler, Toledo). An Olympus BX53-P polarized microscope (Olympus, Japan) was used for visual examination of the internal structure, as well as for crystal detection. Photos were acquired using cellSens Entry Version 1.14 software (Olympus, Japan). Results and discussion Fun-shaped 3D models were successfully printed and printing time mainly depended on the geometry of the defined 3D model (on average, 10 minutes for 6 dosage forms), confirming the suitability of DLP technique for obtaining drugs of various shapes and sizes in a short period of time (Stanojević et al., 2021). All of the fabricated dosage forms had a smooth surface and a uniform shape. The dimensions and mass of the printed dosage forms varied to some extent, which was expected due to the phenomenon of light scattering caused by suspended drug particles (Stanojević et al., 2021). The drug content depended on the amount of AH in the initial formulation and the geometry of the 3D model - 3.19 mg (Cylinder, F2), 4.42 mg (Ring, F1), 8.31 mg (Mickey Mouse, F1) and 26.51 mg (Pentagon, F2), respectively, which indicates the potential of the DLP technique to provide dosage forms with the possibility of "dose tailoring" and individualization of therapy. The results of the dissolution test showed a prolonged release of AH from printed dosage forms. The Ring model exhibited the highest dissolution rate, which was consistent with its high surface area-to-volume ratio, while the Pentagon model exhibited the slowest drug release. DSC analysis showed broad endotherms between 60 and 80 °C, and the absence of sharp melting peak of AH. The drug crystals might have been dissolved during the heating process and therefore, samples were further analyzed by polarized light microscopy. Cross-sections indicated the presence of AH crystals, before and after the dissolution test, due to incomplete drug release from polymeric matrix. The layered structure was also observed confirming the fact that dosage forms were printed in a layer-by-layer manner. Conclusion Fun-shaped oral dosage forms with AH were successfully printed with DLP 3D printer. DLP 3D printing technique offers simple and fast way to fabricate innovative drug dosage forms, enabling flexible dose adjustments by varying the amount of incorporated active substance and the geometric shape of the created 3D models, as well.Short paper accepted by the Scientific Committee for the presentation at the 7th Congress of Pharmacy in Macedonia with international participation, Ohrid, North Macedonia, October 5-9 2022