Development and Validation of a UHPLC UV Method for the In-Process Control of Bosentan Monohydrate Synthesis.

Bosentan monohydrate (4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]benzene-1-sulfonamide monohydrate) is a dual endothelin receptor antagonist (ERA) applied in the treatment of pulmonary arterial hypertension. To achieve effective process control of the bosentan monohydrate synthesis, it was necessary to develop a selective and not highly time-consuming method for ultra-high performance liquid chromatography (UHPLC). The method is characterized by adequate sensitivity, reproducibility and selectivity for the determination of bosentan monohydrate and related compounds from all synthetic stages. The UHPLC separation was carried out by reversed phase chromatography on the Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm) with a mobile phase composed of solvent A (0.1 %, v/v, acetic acid in water) and solvent B (methanol), in the gradient mode at the flow rate of 0.4 mL min-1. Limits of detection and quantification for the compounds were ≤0.1 µg mL-1 and 0.3 µg mL-1, respectively. The linearity for all related compounds was investigated as in the range for the active pharmaceutical ingredient (API) and as in the range for the in-process control. The developed method was validated according to the current guidelines, proving the suitability of the method for its intended purpose.

THE COMPARISON OF THE STABILITY INDICATING TWO HPLC METHODS AND THEIR APPLICATION FOR THE DETERMINATION OF BOSENTAN IN COATED TABLETS.

Due to the raising requirements of drug quality, there is an increasing need for fast liquid separations of pharmaceutical substances with high efficiency and good resolution. The ultra-high pressure liquid chromatography (UHPLC) has been considered to meet this challenge. However, it was found that this fast method has also serious disadvantages. The range of applications of the UHPLC in the analysis of pharmaceutical substances and dosage forms is currently extensively discussed. In this study we investigated the consequences of the shortening of the analysis time of the liquid chromatographic method. Bosentan, a non-peptide antagonist of human endothelin receptors, was chosen as an example in this study, due to its therapeutic importance and lack of the reported analytical methods of the drug product. Two high-performance, reversed phase liquid chromatography methods with UV detection at 220 nm were developed for this purpose. Both methods were validated and the resulting performance characteristics were compared. The first separation (method A) was achieved on Kinetex column, (2.6 µ C18 1OOA, 150 x 4.6 mm), the second-fast (method B) employed Kinetex column, (1.7 µ XB-C18 100A 50 x 3.0 mm). Both methods were performed with a buffered mobile phase containing 0.1% of triethylamine in water brought to the pH 2.5 with phosphoric acid and methanol as the solvent A and acetonitrile as the solvent B. Gradient program was used and flow rate of 0.8 mL/min and 0.4 mL/min, for the methods A and B, respectively. The methods were validated according to the ICH guidelines for specificity, precision on the specified and LOQ limits, intermediate precision, accuracy, linearity (correlation co-efficient =0.999) and robustness. The robustness was confirned using four factors: the mobile phase pH, the flow rate of the mobile phase, column temperature and the second column of the same kind. The limits of detection and quantification were established as 0.0132 and 0.1505 µg/mL for methods A and B, respectively. Both validated methods complied with the acceptance criteria. The method B was 3.5 times faster than the method A, but the method A showed much better sensitivity. The resolution between compound B and bosentan was 3.39 and 1.75 for methods A and B, respectively. The lower sensitivity limits the use of Method B, especially in the analyses at low levels of active substances (e.g., bioanalysis, validation of the cleaning procedures) and makes method A more suitable for this purpose.

Identification and physicochemical characteristics of temozolomide process-related impurities.

In this article the crystal structures of the starting material TZ-5 and the key intermediate TZ-6 of temozolomide (TZ-7), an anticancer therapeutic agent, are presented, together with their spectroscopic and thermal characteristics. Both compounds crystallize in the triclinic P-1 space group. X-ray crystallography studies proved that the compound TZ-6 exists as a monohydrate. A complete structural assignment was obtained for the signals in the 1H-, 13C- and 15N-nuclear magnetic resonance spectra and the structures were confirmed by Fourier-Transform infrared and Raman spectroscopy. The article describes the importance of the high purity of TZ-6 during the small-scale plant production of TZ-7 in a desired polymorphic form III with the purity higher than 99.50%, according to an HPLC method.

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors.

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity.

Biological evaluation of omega-(dialkylamino)alkyl derivatives of 6H-indolo[2,3-b]quinoline--novel cytotoxic DNA topoisomerase II inhibitors.

A series of novel 6H-indolo[2,3-b]quinoline derivatives, substituted at C-2, C-9 or N-6 position with dialkyl(alkylamino)alkyl chains differing in the number of methylene groups, was prepared. These compounds were evaluated in vitro for their antimicrobial and cytotoxic activity against several cell lines of different origin and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. Liphophilic and calf thymus DNA-binding properties of these compounds were also investigated. All the compounds tested inhibited the growth of Gram-positive bacteria and fungi at MIC values ranging between 0.25 and 1 mM. They also showed cytotoxic activity against KB (human cervix carcinoma) cells (ID50 varied from 2.1 to 9.0 microM) and were able to overcome multidrug resistance in colorectal adenocarcinoma LoVo/DX, uterine sarcoma MES-SA/DX5 and promyelocytic leukemia HL-60/MX2 cells (the values of the resistance index RI fell between 0.54 and 2.4). The compounds induced G2M-phase cell cycle arrest in Jurkat T-cell leukemia cells, revealed DNA-binding properties and inhibited topoisomerase II activity.

Synthesis of 6-substituted 6H-indolo[2,3-b]quinolines as novel cytotoxic agents and topoisomerase II inhibitors.

A systematic investigation into the impact of the substituents introduced into the indolo[2,3-b]quinoline system is described. The findings clearly demonstrate that the compounds bearing a methyl group or a longer aliphatic chain at the N-6 position are inactive against prokaryotic and eukaryotic cells. The introduction of alkyl-amino-alkyl substituent at the N-6 position of indolo[2,3-b]quinoline accounts for the appearance of the antimicrobial and.cytotoxic properties. The cytotoxicity against oral epidermoid carcinoma KB (ID50) is in the range from 2.0 to 9.0 microM, and the antimicrobial activity (MIC) falls between 0.03 and 0.50 mM. The structural relation within 6H-indolo[2,3-b]quinolines, concerning their antimicrobial and cytotoxic activity, corresponds well with their ability to bind DNA and to inhibit topoisomerase II activity.

Synthesis, physicochemical and biological evaluation of technetium-99m labeled lapatinib as a novel potential tumor imaging agent of Her-2 positive breast cancer.

Tumors that are Her-2-positive tend to grow and spread more quickly than other types of breast cancer. Overexpression of Her-2 can be a predictive biomarker for stratification of patients for therapy with Herceptin (containing humanized IgG1 monoclonal antibody trastuzumab) or Tykerb (containing lapatinib di-p-toluenesulfonate) drug. Usually, Her-2 status is determined by immunohistochemical (IHC) as well as fluorescent or chromogenic in situ hybridisation (FISH or CISH) analysis of biopsy material. The objective of the present work was to standardize the conjugation of anti-cancer drug lapatinib (which recognizes selectively the Her-2 extracellular domain) with technetium-99m complex, of type '4+1', to obtain (99m)Tc(NS3)(CN-lapatinib) conjugate for use as in vivo tracer of the Her-2 expression in breast cancer. The conjugate (99m)Tc(NS3)(CN-lapatinib) was formed with high yield, high radiochemical purity and specific activity within the range 25-30 GBq/µmol. The biological in vitro and in vivo studies of the conjugate showed its high affinity to Her-2 receptor (Kd = 3.5 ± 0.4 nM, Ki = 2.9 ± 0.5 nM, Bmax = 2.4 ± 0.3 nM, approximate number of 2.4 × 10(6) binding sites per cell, IC50 = 41.2 ± 0.4 nM) and also pointed out to the clearance through the hepatic and renal route in comparable degree. Basing on these results one can conclude that (99m)Tc(NS3)(CN-lapatinib) conjugate could be a promising radiopharmaceutical for in vivo diagnosis of the Her-2 status in breast with impact on treatment planning.

Stability of sample solution as a crucial point during HPLC determination of chemical purity of temozolomide drug substance.

An HPLC method for determination of related substances in temozolomide drug substance was developed. Particular attention was paid to the stability studies due to the fact that temozolomide is unstable in a solution and quickly decomposes to its main degradation product 5-amino-4-imidazolecarboxamide (AIC). A mixture of diluted acetic acid and acetonitrile (4:1, v/v) as a diluent guaranteed lowering the decomposition of temozolomide in the solution. As it is not practically possible to fully eliminate the decomposition of temozolomide during an analysis, the mathematical correction of the results was proposed which allows to analyse almost five times more samples per week, comparing to the procedure without the application of the correction. The accuracy of the correction procedure was proved by investigating the recovery of AIC spiked to temozolomide solutions at different levels. Recoveries equalled 90-108% for AIC concentrations contained in the range of 0.30-1.80 µg ml(-1). The developed method was validated according to the current guidelines, proving the suitability of the method for its intended purpose.