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The feasibility of quantitatively tracking platinum, derived from platinum-based compounds, during subcellular fractionation was studied. Cisplatin-exposed murine Ehrlich Lettré Ascites cells were fractionated into cytosolic and crude nuclear fractions. The latter was subsequently purified. Every residue and fraction produced during the fractionation procedure were collected and the platinum content determined by inductively coupled plasma mass spectrometry. Western blotting verified that the nuclear and cytosolic fractions were pure. It was found that 18% of platinum taken up by the cells was located in the nuclear fraction while 66% was located in the cytosolic fraction. Accumulated uncertainty originating from invariable sample characteristics and giving fraction purity priority had a negative effect on platinum recovery. Thus, overall 81% (n = 3, RSD = 3.4%) of the platinum taken up by the cells was recovered in the residues and final fractions. In conclusion, a reliable intracellular localization and quantitation of platinum following administration of Cisplatin can be determined by application of the method.

Acquired resistance to chemotherapeutic drugs in cancer cells can reflect an ability to limit cellular drug availability, to repair drug induced DNA damage, and to limit initiation/progression of cell death (apoptosis). The leucine-rich-repeat-containing 8A (LRRC8A) protein is an essential component of volume sensitive channels for organic osmolytes (VSOAC) and volume regulated anion channels (VRAC), which are activated during the apoptotic process. Here we illustrate that cisplatin resistance in human ovarian cancer cells (A2780) correlates with a reduced expression of LRRC8A and copper transporter receptor 1 (CTR1), as well as a concomitant increased expression of copper-transporting P-type ATPases (ATP7A/ATP7B). We also find that cisplatin (Pt) accumulation correlates with LRRC8A protein expression and channel activity, i.e., the cellular Pt content is high when VSOAC is activated by depolarization of the plasma membrane or hypoosmotic cell swelling, and reduced when channel activity/LRRC8A expression is reduced by genetically silencing/pharmacological inhibition, or the cells have acquired a resistant phenotype with low LRRC8A protein expression. It is suggested that reduced LRRC8A expression in cisplatin-resistant A2780 cells ensures cell survival through limitation in cisplatin accumulation and a concomitant reduction in osmolytes loss via VSOAC/VRAC and hence instigation of the apoptotic process.

Analysis of peptides and proteins and their interactions with endogenous elements and metal-based drugs in biological systems demands highly efficient chromatographic systems with the possibility of performing gradient elution to achieve efficient separations. As the detector of choice in metal analysis, the ICP-MS, does not tolerate a high load of organic solvents, these should be removed from the chromatographic eluent prior to the entrance of the ICP-MS detector. The purpose of this study was to evaluate a membrane desolvation (MD) system (Aridus II) for its capability of removing organic solvents from the eluent prior to the ICP-MS introduction and at the same time study the influence on sensitivity and examine if the desolvator system jeopardized the inherent species independent sensitivity of ICP-MS. Selenium and platinum were used as model elements. The MD system was optimized regarding to sweep gas and nitrogen gas flow rates. Sensitivity was highly dependent on the combination of sweep gas flow and N2 addition, and the desolvator system should be optimized for each element. After optimization, 100% methanol and acetonitrile were tolerated by the ICP-MS with an eluent flow rate of 0.2 mL min−1. This opens the possibility of performing LC-ICP-MS analysis by gradient elution with 0–100% organic solvents. Sensitivities were generally increased by employment of the MD system, but the species independent sensitivity of ICP-MS was lost for selenium compounds (trimethylselenonium ion (TMSe), selenomethionine (SeMet), Se-methylselenocysteine (Se-MeSeCys), Se-methylseleno-N-acetyl-galactosamine (SeGalac), selenite and selenate). Sensitivities of the different Se compounds were highly dependent on the desolvator temperature. Different Pt compounds (inorganic Pt-salt, cisplatin and oxaliplatin) showed no species dependent behavior. Linearity was obtained for flow injection analysis of SeMet, TMSe and a selenopeptide in 50% methanol and of inorganic Pt, cisplatin and ox

May 1, 2014 the United States Pharmacopeia (USP) will implement two new chapters stating limit concentrations of elemental impurities in pharmaceuticals applying inductively coupled plasma methods. In the present work an inductively coupled plasma optical emission spectrometry (ICP-OES) method for quantitation of As, Cd, Cu, Cr, Fe, Hg, Ir, Mn, Mo, Ni, Os, Pb, Pd, Pt, Rh, Ru, V and Zn in tablets according to the new USP chapters was developed. Sample preparation was performed by microwave-assisted acid digestion using a mixture of 65% HNO3 and 37% HCl (3:1, v/v). Limits of detection and quantitation were at least a factor of ten below the USP limit concentrations showing that the ICP-OES technique is well suited for quantitation of elemental impurities. Excluding Os, spike recoveries in the range of 85.3-103.8% were obtained with relative standard deviations (%RSD) ranging from 1.3 to 3.2%. Due to memory effects the spike recovery and %RSD of Os were 161.5% and 13.7%, respectively, thus the method will need further development with respect to elimination of the memory effect of Os. The method was proven to be specific but with potential spectral interference for Ir, Os, Pb, Pt and Rh necessitating visual examination of the spectra. Hg memory effect was handled by using lower spike levels combined with rinsing with 0.1M HCl. The tablets had a content of Fe and Pt of 182.8±18.1 and 2.8±0.2μg/g, respectively and did therefore not exceed the limit concentration defined by USP. It is suggested that the developed method is applicable to pharmaceutical products with a composition and maximal amount of daily intake (g drug product/day) similar to the tablets used in this work.

Spray drying of pharmaceutical compounds with sticky properties is a challenging task and may require substantial time and resources. By including small-scale studies of single droplet drying kinetics a relatively high number of experiments with less material is allowed. This means one can construct a more robust design space according to Quality by Design (QbD) formulation development principles. In the current study we present a case study on the development of spray dried microparticles comprising nicotine bitartrate and hypromellose or alginate polymer, for incorporation into medicated chewing gum. By illustration of initial studies on single droplet drying kinetics, subsequent characterization of microparticles, and final characterization of compressed chewing gum this paper summarizes the entire development process.