A case study on the in silico absorption simulations of levothyroxine sodium immediate-release tablets.

The aim of this case study was to develop a drug-specific absorption model for levothyroxine (LT4) using mechanistic gastrointestinal simulation technology (GIST) implemented in the GastroPlus™ software package. The required input parameters were determined experimentally, in silico predicted and/or taken from the literature. The simulated plasma profile was similar and in a good agreement with the data observed in the in vivo bioequivalence study, indicating that the GIST model gave an accurate prediction of LT4 oral absorption. Additionally, plasma concentration-time profiles were simulated based on a set of experimental and virtual in vitro dissolution data in order to estimate the influence of different in vitro drug dissolution kinetics on the simulated plasma profiles and to identify biorelevant dissolution specification for LT4 immediate-release (IR) tablets. A set of experimental and virtual in vitro data was also used for correlation purposes. In vitro-in vivo correlation model based on the convolution approach was applied in order to assess the relationship between the in vitro and in vivo data. The obtained results suggest that dissolution specification of more than 85% LT4 dissolved in 60 min might be considered as biorelevant dissolution specification criteria for LT4 IR tablets.

An investigation into the influence of experimental conditions on in vitro drug release from immediate-release tablets of levothyroxine sodium and its relation to oral bioavailability.

The aim of this study was to investigate the influence of experimental conditions on levothyroxine sodium release from two immediate-release tablet formulations which narrowly passed the standard requirements for bioequivalence studies. The in vivo study was conducted as randomised, single-dose, two-way cross-over pharmacokinetic study in 24 healthy subjects. The in vitro study was performed using various dissolution media, and obtained dissolution profiles were compared using the similarity factor value. Drug solubility in different media was also determined. The in vivo results showed narrowly passing bioequivalence. Considering that levothyroxine sodium is classified as Class III drug according to the Biopharmaceutics Classification System, drug bioavailability will be less sensitive to the variation in its dissolution characteristics and it can be assumed that the differences observed in vitro in some of investigated media probably do not have significant influence on the absorption process, as long as rapid and complete dissolution exists. The study results indicate that the current regulatory criteria for the value of similarity factor in comparative dissolution testing, as well as request for very rapid dissolution (more than 85% of drug dissolved in 15 min), are very restricted for immediate-release dosage forms containing highly soluble drug substance and need further investigation. The obtained results also add to the existing debate on the appropriateness of the current bioequivalence standards for levothyroxine sodium products.

Adenosine rescues glioma cells from cytokine-induced death by interfering with the signaling network involved in nitric oxide production.

We investigated the influence of adenosine on inducible nitric oxide (NO) synthase (iNOS)-dependent NO synthesis and viability of cytokine-treated C6 rat glioma cells. Adenosine significantly inhibited interferon-gamma (IFN-gamma)+interleukin-1beta (IL-1beta)-induced synthesis of iNOS mRNA/protein and subsequent production of NO in C6 cells. The uptake of adenosine into glioma cells was not required for the suppression of iNOS induction, as confirmed by the inability of the adenosine transport blocker nitrobenzylthyoinosine to block the observed effect. Adenosine also blocked the IFN-gamma+IL-1beta-triggered expression of mRNA for the proinflammatory cytokine TNF-alpha, while it significantly enhanced the accumulation of cyclooxygenase-2 (COX-2) mRNA in glioma cells. However, blockade of TNF-alpha action and COX-2 activity with anti-TNF-alpha antibodies and indomethacin, respectively, revealed that modulation of TNF-alpha and COX-2 was not involved in adenosine-mediated iNOS suppression. Adenosine significantly inhibited cytokine-induced activation of mitogen-activated protein kinase (MAPK) family members p38 MAPK, p42/44 MAPK and c-Jun N-terminal kinase (JNK) in C6 cells. The levels of transcription factors IRF-1 and c-Fos, as well as the phosphorylation of c-Jun were also reduced in adenosine-treated C6 cells, while the activation of NF-kappaB was enhanced via increased phosphorylation of its inhibitory unit IkappaB. Importantly, adenosine-mediated suppression of NO release rescued glioma cells from NO-dependent cytokine cytotoxicity. These data suggest a possible role for adenosine-mediated inhibition of glial NO synthesis in regulation of the inflammatory CNS damage and brain cancer progression.

Synthesis and biological activity of some new 5'-O-acyl tiazofurin derivatives.

Three new 5'-O-acyl tiazofurin derivatives 2-4 were synthesized and evaluated for their antiproliferative activity against different tumour cell lines as well as for their ability to induce apoptosis in C6 cells in vitro. Apart of the antitumour assays, the cell membrane permeation of 2-4 and their intracellular metabolism in C6 cells in vitro was also studied in order to evaluate their potential as possible tiazofurin bioisosteres or prodrugs.

The efflux of purine nucleobases and nucleosides from the rat brain.

The efflux of purine nucleobases and their nucleosides from the rat brain was investigated using the brain efflux index (BEI) method. Calculated BEI values showed that purine nucleobases had very rapid initial efflux after the intracerebral injection, which was followed by the slower efflux due to the intracellular trapping of labelled molecules and confirmed by the capillary depletion technique. The efflux of ribonucleosides was much slower than the efflux of nucleobases and the structure of the sugar moiety seemed to be important, since a significant difference in the efflux velocity between ribo- and deoxyribonucleosides was observed. The results of self- and cross-inhibition studies suggested that the efflux of test molecules was saturable and that purines shared the same transport system on the abluminal side of the blood-brain barrier.

The linkage of glucose to tiazofurin decreases in vitro uptake into rat glioma C6 cells.

The aim of this study was to analyse the uptake of the synthetic nucleoside tiazofurin and glucoso-linker-tiazofurin conjugate (GLTC) into rat C6 glioma cells in vitro. Results indicated that C6 cells accumulated [3H] tiazofurin slowly with time and that accumulation was reduced by the presence of unlabelled GLTC in the medium which implies that GLTC competes with tiazofurin for transport sites. Uptake of [14C] 2 deoxy-glucose into these cells was very rapid and was not affected by the presence of unlabelled GLTC. To prove the true rate of uptake, the HPLC analysis of cellular extract was performed. After the 360 min of incubation in medium that contained 0.15 mM of tiazofurin, the sum of the concentration of tiazofurin and it's metabolite thiazole-adenine dinucleotide (TAD) in the cells was a total of approximately 4.8% of the amount added to each flask. After the same period of incubation in medium which contained 0.15 mM of GLTC, the sum of concentrations of conjugate, free tiazofurin and TAD represented less than 1/3 of the total concentration measured after the incubation with free tiazofurin and was further reduced in the presence of dipyridamole. Therefore, it can be concluded that GLTC shows some affinity for the nucleoside transporter, but the actual rate of uptake is low.

HPLC determination of tiazofurin in the rat brain.

A sensitive, selective and reproducible HPLC method for determination of tiazofurin in rat brain was developed and validated. The method allowed determination and quantification of nanomolar concentrations of tiazofurin in brain and its regions (hippocampus, cortex and striatum) of treated animals. Separation of tiazofurin from other peaks from brain tissue was achieved by isocratic elution on reverse phase chromatographic column. The mobile phase consisted of 0.05 M sodium acetate pH 4.6. Run time was 15 min.

Simultaneous LC determination of tiazofurin, its acetyl and benzoyl esters and their active metabolite thiazole-4-carboxamide adenine dinucleotide in biological samples.

A rapid and sensitive HPLC-RP method for simultaneous determination of tiazofurin, its 5'-O acetyl and benzoyl esters and their active metabolite thiazole-4-carboxamide adenine dinucleotide was developed and validated. The method allowed determination and quantification of nanomolar quantities of these substances in cell extracts of treated cells, and was also used in kinetic studies of cellular uptake of tiazofurin and its esters from the cultivation medium. Separation of the analyzed substances from unidentified peaks from both biological materials was achieved by gradient elution, thus reducing the possibility of interference. The mobile phase consisted of a 0.1 M sodium-hydrogen phosphate, pH 5.1 and methanol. Run time was 22 min, with 5 min equilibration time.

Purine nucleoside analog--sulfinosine modulates diverse mechanisms of cancer progression in multi-drug resistant cancer cell lines.

Achieving an effective treatment of cancer is difficult, particularly when resistance to conventional chemotherapy is developed. P-glycoprotein (P-gp) activity governs multi-drug resistance (MDR) development in different cancer cell types. Identification of anti-cancer agents with the potential to kill cancer cells and at the same time inhibit MDR is important to intensify the search for novel therapeutic approaches. We examined the effects of sulfinosine (SF), a quite unexplored purine nucleoside analog, in MDR (P-gp over-expressing) non-small cell lung carcinoma (NSCLC) and glioblastoma cell lines (NCI-H460/R and U87-TxR, respectively). SF showed the same efficacy against MDR cancer cell lines and their sensitive counterparts. However, it was non-toxic for normal human keratinocytes (HaCaT). SF induced caspase-dependent apoptotic cell death and autophagy in MDR cancer cells. After SF application, reactive oxygen species (ROS) were generated and glutathione (GSH) concentration was decreased. The expression of key enzyme for GSH synthesis, gamma Glutamyl-cysteine-synthetase (γGCS) was decreased as well as the expression of gst-π mRNA. Consequently, SF significantly decreased the expression of hif-1α, mdr1 and vegf mRNAs even in hypoxic conditions. SF caused the inhibition of P-gp (coded by mdr1) expression and activity. The accumulation of standard chemotherapeutic agent--doxorubicin (DOX) was induced by SF in concentration- and time-dependent manner. The best effect of SF was obtained after 72 h when it attained the effect of known P-gp inhibitors (Dex-verapamil and tariquidar). Accordingly, SF sensitized the resistant cancer cells to DOX in subsequent treatment. Furthermore, SF decreased the experssion of vascular endothelial growth factor (VEGF) on mRNA and protein level and modulated its secretion. In conclusion, the effects on P-gp (implicated in pharmacokinetics and MDR), GSH (implicated in detoxification) and VEGF (implicated in tumor-angiogenesis and progression) qualify SF as multi-potent anti-cancer agent, which use must be considered, in particular for resistant malignancies.

Justification of metformin hydrochloride biowaiver criteria based on bioequivalence study.

The Biopharmaceutics Classification System (BCS) represents the framework for predicting the intestinal drug absorption based on its solubility and intestinal permeability. Recent research has lead to the use of in vitro tests to waive additional in vivo bioequivalence studies for some pharmaceutical products (i.e., biowaiver). The current regulations permit waivers for BCS Class I (highly soluble/highly permeable) drug substances, which represent up to 25% of the drugs. Efforts in both the science and regulatory bodies are being made to extend biowaivers to certain Class II and III products, which would represent more than 50% of all drugs coming to the market. The aim of this study was to investigate the influence of experimental conditions on metformin hydrochloride (CAS 1115-70-4) release from two immediate-release tablet formulations with proven bioequivalence and justify the biowaiver request for dissolution profile similarity in three pH media. The results obtained indicate that differences in drug dissolution observed in vitro were not reflected in vivo. Such data support the existing idea that BCS Class III drugs are eligible biowaiver candidates, even if a very rapid dissolution criterion is not fulfilled.

Characterization of 9L glioma model of the Wistar rat.

The aim of our study was to develop and characterize solid brain tumors in Wistar rats, which could be used in investigations concerning the molecular mechanisms that lay beneath the genesis of the gliomas as well as in the testing of curative potentials of various therapeutics. The tumors were induced by intracerebral inoculation of 9L glioma cells and characterized by morphometrical, histological and immunohistochemical analysis after 7, 14 and 21 postimplantation days. Immunohistochemical characterization included detection of the nuclear antigene Ki-67 as the proliferative cell marker, GFAP as a tracer of reactive gliosis surrounding the tumor mass, and CD4/CD8 and ED1 antigens, as markers of the immunological response. Our results showed that after 7 days all experimental animals developed solid, well-circumcised tumors, which were clearly separated from the surrounding brain tissue. Tumors showed progressive growth from the 7th to the 21st day despite the observed immunological response starting after 14 days. Histologically tumors were hypercellular with neovascularization and necrosis. These results indicate that reproducible morphometric evaluation can be performed on 9L tumors growing in immunocompetent Wistar rats, enabling its use as an animal tumor model for the evaluation of various therapeutic approaches.