Biological In Vitro Evaluation of PIL Graft Conjugates: Cytotoxicity Characteristics.

In vitro cytotoxicity of polymer-carriers, which in the side chains contain the cholinum ionic liquid units with chloride (Cl) or pharmaceutical anions dedicated for antituberculosis therapy, i.e., p-aminosalicylate (PAS) and clavulanate (CLV), was investigated. The carriers and drug conjugates were examined, in the concentration range of 3.125-100 µg/mL, against human bronchial epithelial cells (BEAS-2B) and adenocarcinomic human alveolar basal epithelial cells (A549) as an experimental model cancer cell line possibly coexisting in tuberculosis. The cytotoxicity was evaluated by MTT test and confluency index, as well as by the cytometric analyses, including Annexin-V FITC apoptosis assay. The polymer systems showed supporting activity towards the normal cells and no tumor progress, especially at the highest concentration (100 µg/mL). The analysis of cell death did not show meaningful changes in the case of the BEAS-2B, whereas in the A549 cell line, the cytostatic activity was observed, especially for the drug-free carriers, causing death in up to 80% of cells. This can be regulated by the polymer structure, including the content of cationic units, side-chain length and density, as well as the type and content of pharmaceutical anions. The results of MTT tests, confluency, as well as cytometric analyses, distinguished the polymer systems with Cl/PAS/CLV containing 26% of grafting degree and 43% of ionic units or 46% of grafting degree and 18% of ionic units as the optimal systems.

Aldophosphamide-thiazolidine (NSC-613060) an oxazaphosphorine cytostatic that crosses the blood brain barrier.

The pharmacologically active metabolite of cyclophosphamide is aldophosphamide. With cysteine, aldophosphamide forms stable aldophosphamide-thiazolidine which under physiological pH and temperature conditions hydrolyzes to aldophosphamide and cysteine. Aldophosphamide-thiazolidine was synthesized and tested for its ability as a cytostatic. The LD50 after a single intraperitoneal injection in mice was determined to be 2162 mg/kg, but after intravenous bolus administration of 500 mg/kg or in chronic toxicity tests with daily intraperitoneal injections, neurological side effects were observed. Antitumor activity was determined in therapy experiments in CD2F1 mice bearing subcutaneously transplanted P388 mouse leukemia cells. Administration of 100 mg/kg (less than 5% LD50) days 1-5 after tumor transplantation yielded an ILS of 100%. Organ distribution studies showed that aldophosphamide-thiazolidine is evenly distributed in all tissues examined, including brain tissue. The possibilities to increase the antitumor activity of aldophosphamide-thiazolidine by modulating the alkylating function are discussed.

Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG ß-subunit.

Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for the biopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-α2b), which belongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, shows a poor stability in solution and an extremely short plasma half-life which determines a strict therapeutic regimen comprising high and repeated doses. In this work, we have used a strategy based on the fusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) ß-subunit, bearing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-α2b. Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were efficiently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferative bioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through this approach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a 19-fold decreased plasma apparent clearance compared to the wild-type cytokine. Moreover, CTP-IFN-CTP demonstrated a significant increase in in vitro thermal resistance and a higher stability against plasma protease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by the CTP moiety. These results constitute the first report that postulates CTP as a tag for improving both the in vitro and in vivo stability of rhIFN-α2b which, in turn, would positively influence its in vivo bioactivity.

Microparticles prepared from biodegradable polyhydroxyalkanoates as matrix for encapsulation of cytostatic drug.

Microparticles made from degradable polyhydroxyalkanoates of different chemical compositions a homopolymer of 3-hydroxybutyric acid, copolymers of 3-hydroxybutyric and 4-hydroxybutyric acids (P3HB/4HB), 3-hydroxybutyric and 3-hydroxyvaleric acids (P3HB/3HV), 3-hydroxybutyric and 3-hydroxyhexanoic acids (P3HB/3HHx) were prepared using the solvent evaporation technique, from double emulsions. The study addresses the influence of the chemical compositions on the size and ξ-potential of microparticles. P3HB microparticles loaded with doxorubicin have been prepared and investigated. Their average diameter and ξ-potential have been found to be dependent upon the level of loading (1, 5, and 10 % of the polymer mass). Investigation of the in vitro drug release behavior showed that the total drug released from the microparticle into the medium increased with mass concentration of the drug. In this study mouse fibroblast NIH 3T3 cells were cultivated on PHA microparticles, and results of using fluorescent DAPI DNA stain, and MTT assay showed that microparticles prepared from PHAs of different chemical compositions did not exhibit cytotoxicity to cells cultured on them and proved to be highly biocompatible. Cell attachment and proliferation on PHA microparticles were similar to those on polystyrene. The cytostatic drug encapsulated in P3HB/3HV microparticles has been proven to be effective against HeLa tumor cells.

Carbon nanotubes enhance the internalization of drugs by cancer cells and decrease their chemoresistance to cytostatics.

Etoposide is a semisynthetic, chemotherapeutic drug widely recommended to treat an extensive range of human cancers. Our studies indicate that, while etoposide is capable of killing human cancer cells, exposure to single-walled carbon nanotubes (SWCNTs) and etoposide results in enhanced cell death that appears to be synergistic and not merely additive. In this study, we used high pressure liquid chromatography and mass spectrometry to quantify the internal effective dose of etoposide when the human pancreatic cancer cell (PANC-1) was exposed to the combination of these agents. Our results unequivocally indicate that SWCNTs improve etoposide uptake and increase its capacity to kill cancer cells. We suggest that a combination of SWCNTs and etoposide may prove to be a more efficient chemotherapeutic protocol, especially because of the potential to lower toxic drug doses to levels that may be useful in decreasing adverse side effects, as well as in lowering the probability of inducing chemoresistance in exposed cancer cells.

[Update in the management of extravasations of cytocytostatic agent]. / Actualización del manejo de extravasaciones de agentes citostáticos.

OBJECTIVE: To present current developments in the specific management of extravasations of antineoplastic agents after the extravasation. METHOD: We conducted a search in PubMed, Medline and IDIS-Iowa to identify papers written in English or Spanish that described new specific measures for the management of extravasations. We also reviewed the references given in these papers and recent tertiary sources related to oncology or cytostatic agents. The search covered the period between 1997 and 2010. RESULTS: There are only specific measures for the treatment of extravasations of 22 cytostatic agents. These measures are presented for each cytostatic agent, according their drug group. CONCLUSIONS: Although currently there is no general consensus on the specific management of antineoplastic agents after extravasation, this review outlines the information collected and published so far, so that it may be of use to any national health centre where cytostatic drugs are prescribed, handled or administered.

Cellular pharmacokinetic/pharmacodynamic relationship of platinum cytostatics in head and neck squamous cell carcinoma evaluated by liquid chromatography coupled to tandem mass spectrometry.

Cisplatin (diaminodichloroplatinum) is the favored platinum (Pt) drug for the treatment of head and neck squamous cell carcinoma (HNSCC). However, Pt drug alternatives such as carboplatin (diaminoplatinum-cyclobutan-1,1-dicarboxylate) or oxaliplatin [oxalato[(1R,2R)-cyclohexanediamino]platinum] have not been comprehensively investigated in HNSCC. Moreover, little data reveal the decisive efficacy determinant and whether Pt drug efficacy is truly concentration-dependent. Using five human HNSCC cell lines, we determined the concentrations of cisplatin, carboplatin, and oxaliplatin leading to 50% inhibition of cell proliferation (IC(50)). Concurrently we quantified cellular drug uptake by liquid chromatography coupled to tandem mass spectrometry and evaluated mRNA expression of drug transporters involved in Pt drug uptake by quantitative real-time polymerase chain reaction. Mean IC(50) among the five cell lines was 6.2 ± 1.9 µM for cisplatin and 11.6 ± 4.2 µM for oxaliplatin, whereas carboplatin showed significantly lower proliferation inhibition (IC(50) 107.5 ± 21.2 µM). In agreement with this finding carboplatin poorly accumulated in HNSCC cells, compared with cisplatin and oxaliplatin. HNSCC cell lines expressed Pt drug transporters. Taken together, the results demonstrate: 1) carboplatin was less effective and was poorly taken up; 2) a high individuality among cell lines was found concerning the accumulation of cisplatin and oxaliplatin despite similar in vitro efficacy; and 3) distinct expression of SLC22A2 and ABCC2 accompanies strong uptake and cytotoxicity of Pt drugs. In conclusion, we demonstrate that in vitro efficacy of cisplatin and oxaliplatin in HNSCC is concentration-independent because they exhibited different uptake characteristics but similar efficacies, suggesting oxaliplatin as a promising alternative against HNSCC that needs further evaluation in clinical trials.

A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin ß chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

[Growth modulating and cytotoxic effects of the peptide from hemolymph of blow fly Calliphora vicina (Diptera, Calliphoridae) in vitro].

Biological activity of alloferon (AF), peptide, consisting of 13 a. a. isolated from hemolymph of experimentally infected blow fly Calliphora vicina was studied. AF in concentrations form 1 x 10(-5) to 250 microg/ml was added into the culture medium of the target cell lines K562, J-96, P388DI, Hep22a and 3T3B-SV40. First two days the peptide in concentrations 1 x 10(-5) and 1 x 10(-3) microg/ml in most cases stimulated the cell proliferative activity and suppressed the cell growth when applied in concentrations 10 and 100 microg/ml. Trend in growth modulating effect was dependent on duration of AF treatment. The peptide did not expressed cytotoxic effect with the exception of destruction of P388D1 cells that was registered after 96 h incubation in the medium initially contained 100 microg/ml AF. Simultaneously, cytotoxic and growth modulating effects of doxorubicin and cytosinarabinoside, as well as hybrid molecules, AF--cytosinarabinoside (cytal) and AF--doxorubicin (doxal) have been studied. Doxorubicin and cytosinarabinoside expressed greater growth inhibition effect compared to the hybrid molecules and AF itself. The results obtained with mass cell cultures were supported by experiments where P388D1 cells clonogenic capacity was tested. Besides, it has been demonstrated that AF rapidly penetrates into cytoplasm of J-96 cells and concentrates mainly into and around the cell nuclei.

The antiproliferative cytostatic effects of a self-activating viridin prodrug.

Although viridins like wortmannin (Wm) have long been examined as anticancer agents, their ability to self-activate has only recently been recognized. Here, we describe the cytostatic effects of a self-activating viridin (SAV), which is an inactive, polymeric prodrug. SAV self-activates to generate a bioactive, fluorescent viridin NBD-Wm with a half-time of 9.2 hours. With cultured A549 cells, 10 micromol/L SAV caused growth arrest without inducing apoptosis or cell death, a cytostatic action markedly different from other chemotherapeutic agents (vinblastine, camptothecin, and paclitaxel). In vivo, a SAV dosing of 1 mg/kg once in 48 hours (i.p.) resulted in growth arrest of an A549 tumor xenograft, with growth resuming when dosing ceased. With a peak serum concentration of SAV of 2.36 micromol/L (at 2 hours post i.p. injection), the concentration of bioactive NBD-Wm was 41 nmol/L based on the partial inhibition of neutrophil respiratory burst. Therefore, SAV was present as an inactive prodrug in serum (peak = 2.36 micromol/L), which generated low concentrations of active viridin (41 nmol/L). SAV is a prodrug, the slow release and cytostatic activities of which suggest that it might be useful as a component of metronomic-based chemotherapeutic strategies.