Mean global DNA methylation serves as independent prognostic marker in IDH-wildtype glioblastoma.

BACKGROUND: The IDH-wildtype glioblastoma (GBM) patients have a devastating prognosis. Here, we analyzed the potential prognostic value of global DNA methylation of the tumors. METHODS: DNA methylation of 492 primary samples and 31 relapsed samples, each treated with combination therapy, and of 148 primary samples treated with radiation alone were compared with patient survival. We determined the mean methylation values and estimated the immune cell infiltration from the methylation data. Moreover, the mean global DNA methylation of 23 GBM cell lines was profiled and correlated to their cellular radiosensitivity as measured by colony formation assay. RESULTS: High mean DNA methylation levels correlated with improved survival, which was independent from known risk factors (MGMT promoter methylation, age, extent of resection; P = 0.009) and methylation subgroups. Notably, this correlation was also independent of immune cell infiltration, as higher number of immune cells indeed was associated with significantly better OS but lower mean methylation. Radiosensitive GBM cell lines had a significantly higher mean methylation than resistant lines (P = 0.007), and improved OS of patients treated with radiotherapy alone was also associated with higher DNA methylation (P = 0.002). Furthermore, specimens of relapsed GBM revealed a significantly lower mean DNA methylation compared to the matching primary tumor samples (P = 0.041). CONCLUSIONS: Our results indicate that mean global DNA methylation is independently associated with outcome in glioblastoma. The data also suggest that a higher DNA methylation is associated with better radiotherapy response and less aggressive phenotype, both of which presumably contribute to the observed correlation with OS.

Patient-individual phenotypes of glioblastoma stem cells are conserved in culture and associate with radioresistance, brain infiltration and patient prognosis.

Identification of prognostic or predictive molecular markers in glioblastoma resection specimens may lead to strategies for therapy stratification and personalized treatment planning. Here, we analyzed in primary glioblastoma stem cell (pGSC) cultures the mRNA abundances of seven stem cell (MSI1, Notch1, nestin, Sox2, Oct4, FABP7 and ALDH1A3), and three radioresistance or invasion markers (CXCR4, IKCa and BKCa ). From these abundances, an mRNA signature was deduced which describes the mesenchymal-to-proneural expression profile of an individual GSC culture. To assess its functional significance, we associated the GSC mRNA signature with the clonogenic survival after irradiation with 4 Gy and the fibrin matrix invasion of the GSC cells. In addition, we compared the molecular pGSC mRNA signature with the tumor recurrence pattern and the overall survival of the glioblastoma patients from whom the pGSC cultures were derived. As a result, the molecular pGSC mRNA signature correlated positively with the pGSC radioresistance and matrix invasion capability in vitro. Moreover, patients with a mesenchymal (>median) mRNA signature in their pGSC cultures exhibited predominantly a multifocal tumor recurrence and a significantly (univariate log rank test) shorter overall survival than patients with proneural (≤median mRNA signature) pGSCs. The tumors of the latter recurred predominately unifocally. We conclude that our pGSC cultures induce/select those cell subpopulations of the heterogeneous brain tumor that determine disease progression and therapy outcome. In addition, we further postulate a clinically relevant prognostic/predictive value for the 10 mRNAs-based mesenchymal-to-proneural signature of the GSC subpopulations in glioblastoma.

Repurposing Disulfiram for Targeting of Glioblastoma Stem Cells: An In Vitro Study.

Mesenchymal glioblastoma stem cells (GSCs), a subpopulation in glioblastoma that are responsible for therapy resistance and tumor spreading in the brain, reportedly upregulate aldehyde dehydrogenase isoform-1A3 (ALDH1A3) which can be inhibited by disulfiram (DSF), an FDA-approved drug formerly prescribed in alcohol use disorder. Reportedly, DSF in combination with Cu2+ ions exerts multiple tumoricidal, chemo- and radio-therapy-sensitizing effects in several tumor entities. The present study aimed to quantify these DSF effects in glioblastoma stem cells in vitro, regarding dependence on ALDH1A3 expression. To this end, two patient-derived GSC cultures with differing ALDH1A3 expression were pretreated (in the presence of CuSO4, 100 nM) with DSF (0 or 100 nM) and the DNA-alkylating agent temozolomide (0 or 30 µM) and then cells were irradiated with a single dose of 0-8 Gy. As read-outs, cell cycle distribution and clonogenic survival were determined by flow cytometry and limited dilution assay, respectively. As a result, DSF modulated cell cycle distribution in both GSC cultures and dramatically decreased clonogenic survival independently of ALDH1A3 expression. This effect was additive to the impairment of clonogenic survival by radiation, but not associated with radiosensitization. Of note, cotreatment with temozolomide blunted the DSF inhibition of clonogenic survival. In conclusion, DSF targets GSCs independent of ALDH1A3 expression, suggesting a therapeutic efficacy also in glioblastomas with low mesenchymal GSC populations. As temozolomide somehow antagonized the DSF effects, strategies for future combination of DSF with the adjuvant standard therapy (fractionated radiotherapy and concomitant temozolomide chemotherapy followed by temozolomide maintenance therapy) are not supported by the present study.

Potassium Channels in Cancer.

Neoplastic transformation is reportedly associated with alterations of the potassium transport across plasma and intracellular membranes. These alterations have been identified as crucial elements of the tumourigenic reprogramming of cells. Potassium channels may contribute to cancer initiation, malignant progression and therapy resistance of tumour cells. The book chapter focusses on (oncogenic) potassium channels frequently upregulated in different tumour entities, upstream and downstream signalling of these channels, their contribution to the maintenance of cancer stemness and the formation of an immunosuppressive tumour microenvironment. In addition, their role in adaptation to tumour hypoxia, metabolic reprogramming, as well as tumour spreading and metastasis is discussed. Finally, we discuss how (oncogenic) potassium channels may confer treatment resistance of tumours against radiation and chemotherapy and thus might be harnessed for new therapy strategies, for instance, by repurposing approved drugs known to target potassium channels.

The Tumor Suppressor MTUS1/ATIP1 Modulates Tumor Promotion in Glioma: Association with Epigenetics and DNA Repair.

Glioblastoma (GBM) is a highly aggressive brain tumor. Resistance mechanisms in GBM present an array of challenges to understand its biology and to develop novel therapeutic strategies. We investigated the role of a TSG, MTUS1/ATIP1 in glioma. Glioma specimen, cells and low passage GBM sphere cultures (GSC) were analyzed for MTUS1/ATIP1 expression at the RNA and protein level. Methylation analyses were done by bisulfite sequencing (BSS). The consequence of chemotherapy and irradiation on ATIP1 expression and the influence of different cellular ATIP1 levels on survival was examined in vitro and in vivo. MTUS1/ATIP1 was downregulated in high-grade glioma (HGG), GSC and GBM cells and hypermethylation at the ATIP1 promoter region seems to be at least partially responsible for this downregulation. ATIP1 overexpression significantly reduced glioma progression by mitigating cell motility, proliferation and facilitate cell death. In glioma-bearing mice, elevated MTUS1/ATIP1 expression prolonged their survival. Chemotherapy, as well as irradiation, recovered ATIP1 expression both in vitro and in vivo. Surprisingly, ATIP1 overexpression increased irradiation-induced DNA-damage repair, resulting in radio-resistance. Our findings indicate that MTUS1/ATIP1 serves as TSG-regulating gliomagenesis, progression and therapy resistance. In HGG, higher MTUS1/ATIP1 expression might interfere with tumor irradiation therapy.

γH2AX foci assay in glioblastoma: Surgical specimen versus corresponding stem cell culture.

AIM: To assess radiation response using γH2AX assay in surgical specimens from glioblastoma (GB) patients and their corresponding primary gliosphere culture. To test the hypothesis that gliospheres (stem cell enriched) are more resistant than specimens (bulky cell dominated) but that the interpatient heterogeneity is similar. MATERIAL AND METHODS: Ten pairs of specimens and corresponding gliospheres derived from patients with IDH-wildtype GB were studied. Specimens and gliospheres were irradiated with graded doses and after 24 h the number of residual γH2AX foci was counted. RESULTS: Gliospheres showed a higher Nestin expression than specimens and exhibited two different phenotypes: free floating (n = 7) and attached (n = 3). Slope analysis revealed an interpatient heterogeneity with values between 0.15 and 1.30 residual γH2AX foci/Gy. Free-floating spheres were more resistant than their parental specimens (median slope 0.13 foci/Gy versus 0.53) as well as than the attached spheres (2.14). The slopes of free floating spheres did not correlate with their corresponding specimens while a trend for a positive correlation was found for the attached spheres and the respective specimens. Association with MGMT did not reach statistical significance. CONCLUSION: Consistent with the clinical phenotype and our previous experiments, GB specimens show low radiation sensitivity. Stem-cell enriched free-floating gliospheres were more resistant than specimens supporting the concept of radioresistance in stem cell-like cells. The lack of correlation between specimens and their respective gliosphere cultures needs validation and may have a profound impact on future translational studies using γH2AX as a potential biomarker for personalized radiation therapy.

Against Repurposing Methadone for Glioblastoma Therapy.

Methadone, which is used as maintenance medication for outpatient treatment of opioid dependence or as an analgesic drug, has been suggested by preclinical in vitro and mouse studies to induce cell death and sensitivity to chemo- or radiotherapy in leukemia, glioblastoma, and carcinoma cells. These data together with episodical public reports on long-term surviving cancer patients who use methadone led to a hype of methadone as an anti-cancer drug in social and public media. However, clinical evidence for a tumoricidal effect of methadone is missing and prospective clinical trials, except in colorectal cancer, are not envisaged because of the limited preclinical data available. The present article reviews the pharmacokinetics, potential molecular targets, as well as the evidence for a tumoricidal effect of methadone in view of the therapeutically achievable doses in the brain. Moreover, it provides original in vitro data showing that methadone at clinically relevant concentrations fails to impair clonogenicity or radioresistance of glioblastoma cells.

Physiologically based pharmacokinetic model outputs depend on dissolution data and their input: Case examples glibenclamide and dipyridamole.

A plethora of dissolution tests exists for oral dosage forms, with variations in selection of the dissolution medium, the hydrodynamics and the dissolution equipment. This work aimed at determining the influence of media composition, the type of dissolution test and the method for entering the data into a PBPK model on the ability to simulate the in vivo plasma profile of an immediate release formulation. Using two rDCS IIa substances, glibenclamide and dipyridamole, housed in immediate-release formulations as model dosage forms, dissolution tests were performed in USP apparatus II with the biorelevant media FaSSGF, FaSSIF V1, V2 and V3 using both single-stage and two-stage test designs. The results were then integrated into the PBPK software SimcypⓇ either as the observed release profile (dissolution rate model, DRM) or using a semi-mechanistic model (diffusion layer model, DLM) and compared with in vivo plasma profiles. The selection of the FaSSIF version did not appear to have any relevant influence on the dissolution of the weakly basic dipyridamole, while the weakly acidic glibenclamide was sensitive to the difference in pH between FaSSIF V1, V2 and FaSSIF V3. Since both compounds have pKa values close to the pH of biorelevant media representing conditions in the small intestine, these results may be specific to compounds with similar ionization behavior. Single-stage and two-stage testing led to equivalent simulations for glibenclamide. Only results from the single-stage test in FaSSGF led to a close simulation of the pharmacokinetic profile of dipyridamole when data were inputted using the DRM, while simulations from two-stage testing were most similar to the observed pharmacokinetic profile when DLM with selection of a dynamic pH profile in the small intestine was selected as the data input method. These results emphasize the importance of data input to the simulation results.

hERG K+ Channels Promote Survival of Irradiated Leukemia Cells.

Many tumor cells express highly elevated activities of voltage-gated K+ channels in the plasma membrane which are indispensable for tumor growth. To test for K+ channel function during DNA damage response, we subjected human chronic myeloid leukemia (CML) cells to sub-lethal doses of ionizing radiation (0-8 Gy, 6 MV photons) and determined K+ channel activity, K+ channel-dependent Ca2+ signaling, cell cycle progression, DNA repair, and clonogenic survival by whole-cell patch clamp recording, fura-2 Ca2+ imaging, Western blotting, flow cytometry, immunofluorescence microscopy, and pre-plating colony formation assay, respectively. As a result, the human erythroid CML cell line K562 and primary human CML cells functionally expressed hERG1. Irradiation stimulated in both cell types an increase in the activity of hERG1 K+ channels which became apparent 1-2 h post-irradiation. This increase in K+ channel activity was paralleled by an accumulation in S phase of cell cycle followed by a G2/M cell cycle arrest as analyzed between 8 and 72 h post-irradiation. Attenuating the K+ channel function by applying the hERG1 channel inhibitor E4031 modulated Ca2+ signaling, impaired inhibition of the mitosis promoting subunit cdc2, overrode cell cycle arrest, and decreased clonogenic survival of the irradiated cells but did not affect repair of DNA double strand breaks suggesting a critical role of the hERG1 K+ channels for the Ca2+ signaling and the cell cycle control during DNA damage response.

Dissolution behavior of various drugs in different FaSSIF versions.

Biorelevant media have proven to be useful in predicting the performance of poorly soluble drugs in the gastrointestinal tract. Several versions of fasted state simulated intestinal fluids have been published and compared with respect to their physical chemical properties and solubilization of drugs. However, to date there have been no reports in the literature comparing dissolution of poorly soluble drugs in these media. In this study eleven BCS Class II compounds (five nonionized compounds, three weak bases and three weak acids) were investigated with respect to their thermodynamic solubility and dissolution behavior in three biorelevant media simulating conditions in the small intestine (FaSSIF V1, FaSSIF V2 and FaSSIF V3). It was shown that the maximum percentage release of drugs from their commercial formulations can differ from the results for the thermodynamic solubility of the pure drug; these differences can be largely attributed to API presentation, composition of the formulation and manufacturing effects. The results were additionally compared with data for solubility in HIF taken from the literature in order to determine which version of FaSSIF most closely corresponds to the physiological conditions. The different versions of FaSSIF are able to achieve solubility results similar to those in HIF, with closest results generally achieved in FaSSIF V1. The magnitude of solubility/dissolution differences among the three FaSSIF versions is dependent on the drug's characteristics. In the case of weakly basic compounds, the differences among the FaSSIF versions are minor. For weakly acidic compounds the behavior in the different versions is primarily pH dependent and influenced by the lipid composition of the FaSSIF only to a minor extent. The differences in solubility and dissolution of the nonionized compounds among the three versions of FaSSIF becomes apparent above a log P value of 2.5, with larger differences among the versions at high log P values.

Biorelevant dissolution testing of a weak base: Interlaboratory reproducibility and investigation of parameters controlling in vitro precipitation.

Following a previous study which aimed to determine the interlaboratory reproducibility of biorelevant dissolution testing in the USP 2 apparatus for commercial formulations of two weak acids (ibuprofen and zafirlukast), this study attempts to determine the interlaboratory reproducibility using a similar protocol for a commercially available formulation of a weak base, indinavir. Fourteen partners including twelve industrial and two academic partners participated in this study. To ensure uniformity, all partners were provided with a standardized protocol to perform (i) a single medium dissolution test in fasted state simulated gastric and intestinal fluids (FaSSGF and FaSSIF, respectively) and (ii) a two-stage dissolution experiment simulating gastrointestinal transfer. Optionally, partners could run a single-stage dissolution test in fed state simulated intestinal fluid (FeSSIF). For each dissolution test, one Crixivan® capsule (containing 400 mg indinavir as its sulfate salt) was added as dose of interest. For the single medium dissolution test in FaSSIF, all partners observed rapid release of indinavir resulting in supersaturated concentrations, followed by precipitation to equilibrium solubility. The degree and period of supersaturation varied among the participating laboratories. Average dissolution profiles in FeSSIF appeared to be highly reproducible with dissolved concentrations remaining lower than the thermodynamic solubility of indinavir in FeSSIF. For the two-stage dissolution test, most partners observed supersaturated concentrations in the intestinal compartment; two partners observed no supersaturation due to immediate precipitation. Given the fact that a high interlaboratory but low intralaboratory variability was observed when supersaturation/precipitation occurred, an undefined factor was hypothesized as a potential cause of the variability in precipitation. Hence, the impact of several experimental factors on the supersaturation and precipitation behavior of indinavir was investigated in a next step. The investigation indicated that variability is likely attributable to a combination of factors, especially, the time elapsed between sampling and dilution of the sample with the mobile phase. Therefore, when designing a test in which supersaturation and precipitation is anticipated, stringent control of the test methodology, especially regarding sampling and dilution, is needed.

Alternating Electric Fields (TTFields) Activate Cav1.2 Channels in Human Glioblastoma Cells.

Tumor treating fields (TTFields) represent a novel FDA-approved treatment modality for patients with newly diagnosed or recurrent glioblastoma multiforme. This therapy applies intermediate frequency alternating electric fields with low intensity to the tumor volume by the use of non-invasive transducer electrode arrays. Mechanistically, TTFields have been proposed to impair formation of the mitotic spindle apparatus and cytokinesis. In order to identify further potential molecular targets, here the effects of TTFields on Ca2+-signaling, ion channel activity in the plasma membrane, cell cycle, cell death, and clonogenic survival were tested in two human glioblastoma cell lines in vitro by fura-2 Ca2+ imaging, patch-clamp cell-attached recordings, flow cytometry and pre-plated colony formation assay. In addition, the expression of voltage-gated Ca2+ (Cav) channels was determined by real-time RT-PCR and their significance for the cellular TTFields response defined by knock-down and pharmacological blockade. As a result, TTFields stimulated in a cell line-dependent manner a Cav1.2-mediated Ca2+ entry, G1 or S phase cell cycle arrest, breakdown of the inner mitochondrial membrane potential and DNA degradation, and/or decline of clonogenic survival suggesting a tumoricidal action of TTFields. Moreover, inhibition of Cav1.2 by benidipine aggravated in one glioblastoma line the TTFields effects suggesting that Cav1.2-triggered signaling contributes to cellular TTFields stress response. In conclusion, the present study identified Cav1.2 channels as TTFields target in the plasma membrane and provides the rationale to combine TTFields therapy with Ca2+ antagonists that are already in clinical use.

Potential Role of CXCR4 Targeting in the Context of Radiotherapy and Immunotherapy of Cancer.

Cancer immunotherapy has been established as standard of care in different tumor entities. After the first reports on synergistic effects with radiotherapy and the induction of abscopal effects-tumor shrinkage outside the irradiated volume attributed to immunological effects of radiotherapy-several treatment combinations have been evaluated. Different immunotherapy strategies (e.g., immune checkpoint inhibition, vaccination, cytokine based therapies) have been combined with local tumor irradiation in preclinical models. Clinical trials are ongoing in different cancer entities with a broad range of immunotherapeutics and radiation schedules. SDF-1 (CXCL12)/CXCR4 signaling has been described to play a major role in tumor biology, especially in hypoxia adaptation, metastasis and migration. Local tumor irradiation is a known inducer of SDF-1 expression and release. CXCR4 also plays a major role in immunological processes. CXCR4 antagonists have been approved for the use of hematopoietic stem cell mobilization from the bone marrow. In addition, several groups reported an influence of the SDF-1/CXCR4 axis on intratumoral immune cell subsets and anti-tumor immune response. The aim of this review is to merge the knowledge on the role of SDF-1/CXCR4 in tumor biology, radiotherapy and immunotherapy of cancer and in combinatorial approaches.

KCa3.1 Channels and Glioblastoma: In Vitro Studies.

BACKGROUND: Several tumor entities including brain tumors aberrantly overexpress intermediate conductance Ca2+ activated KCa3.1 K+ channels. These channels contribute significantly to the transformed phenotype of the tumor cells. METHOD: PubMed was searched in order to summarize our current knowledge on the molecular signaling upstream and downstream and the effector functions of KCa3.1 channel activity in tumor cells in general and in glioblastoma cells in particular. In addition, KCa3.1 expression and function for repair of DNA double strand breaks was determined experimentally in primary glioblastoma cultures in dependence on the abundance of proneural and mesenchymal stem cell markers. RESULTS: By modulating membrane potential, cell volume, Ca2+ signals and the respiratory chain, KCa3.1 channels in both, plasma and inner mitochondrial membrane, have been demonstrated to regulate many cellular processes such as migration and tissue invasion, metastasis, cell cycle progression, oxygen consumption and metabolism, DNA damage response and cell death of cancer cells. Moreover, KCa3.1 channels have been shown to crucially contribute to resistance against radiotherapy. Futhermore, the original in vitro data on KCa3.1 channel expression in subtypes of glioblastoma stem(-like) cells propose KCa3.1 as marker for the mesenchymal subgroup of cancer stem cells and suggest that KCa3.1 contributes to the therapy resistance of mesenchymal glioblastoma stem cells. CONCLUSION: The data suggest KCa3.1 channel targeting in combination with radiotherapy as promising new tool to eradicate therapy-resistant mesenchymal glioblastoma stem cells.

Cellular Effects of the Antiepileptic Drug Valproic Acid in Glioblastoma.

BACKGROUND/AIMS: Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug is used to treat epileptic seizure of glioblastoma patients. Besides its antiepileptic activity, VPA has been attributed further functions that improve the clinical outcome of glioblastoma patients. Those comprise the inhibition of some histone deacetylase (HDAC) isoforms which reportedly may result in radiosensitization. Retrospective analysis of patient data, however, could not unequivocally confirm a prolonged survival of glioblastoma patients receiving VPA. The present study aimed to identify potential VPA targets at the cellular level. METHODS: To this end, the effect of VPA on metabolism, Ca2+-, biochemical and electro-signaling, cell-cycling, clonogenic survival and transfilter migration was analyzed in three human glioblastoma lines (T98G, U-87MG, U251) by MTT assay, Ca2+ imaging, immunoblotting, patch-clamp recording, flow cytometry, delayed plating colony formation and modified Boyden chamber assays, respectively. In addition, the effect of VPA on clonogenic survival of primary glioblastoma spheroid cultures treated with temozolomide and fractionated radiation was assessed by limited dilution assay. RESULTS: In 2 of 3 glioblastoma lines, clinical relevant concentrations of VPA slightly slowed down cell cycle progression and decreased clonogenic survival. Furthermore, VPA induced Ca2+ signaling which was accompanied by pronounced K+ channel activity and transfilter cell migration. VPA did not affect metabolic NAD(P)H formation or radioresistance of the glioblastoma lines. Finally, VPA did not impair clonogenic survival or radioresistance of temozolomide-treated primary spheroid cultures. CONCLUSIONS: Combined, our in vitro data do not propose a general use of VPA as a radiosensitizer in anti-glioblastoma therapy.

TRPM8 is required for survival and radioresistance of glioblastoma cells.

TRPM8 is a Ca2+-permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca2+ signaling and resulting K+ channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy.

Validation of Dissolution Testing with Biorelevant Media: An OrBiTo Study.

Dissolution testing with biorelevant media has become widespread in the pharmaceutical industry as a means of better understanding how drugs and formulations behave in the gastrointestinal tract. Until now, however, there have been few attempts to gauge the reproducibility of results obtained with these methods. The aim of this study was to determine the interlaboratory reproducibility of biorelevant dissolution testing, using the paddle apparatus (USP 2). Thirteen industrial and three academic laboratories participated in this study. All laboratories were provided with standard protocols for running the tests: dissolution in FaSSGF to simulate release in the stomach, dissolution in a single intestinal medium, FaSSIF, to simulate release in the small intestine, and a "transfer" (two-stage) protocol to simulate the concentration profile when conditions are changed from the gastric to the intestinal environment. The test products chosen were commercially available ibuprofen tablets and zafirlukast tablets. The biorelevant dissolution tests showed a high degree of reproducibility among the participating laboratories, even though several different batches of the commercially available medium preparation powder were used. Likewise, results were almost identicalbetween the commercial biorelevant media and those produced in-house. Comparing results to previous ring studies, including those performed with USP calibrator tablets or commercially available pharmaceutical products in a single medium, the results for the biorelevant studies were highly reproducible on an interlaboratory basis. Interlaboratory reproducibility with the two-stage test was also acceptable, although the variability was somewhat greater than with the single medium tests. Biorelevant dissolution testing is highly reproducible among laboratories and can be relied upon for cross-laboratory comparisons.

Justification of disintegration testing beyond current FDA criteria using in vitro and in silico models.

Drug product performance testing is an important part of quality-by-design approaches, but this process often lacks the underlying mechanistic understanding of the complex interactions between the disintegration and dissolution processes involved. Whereas a recent draft guideline by the US Food and Drug Administration (FDA) has allowed the replacement of dissolution testing with disintegration testing, the mentioned criteria are not globally accepted. This study provides scientific justification for using disintegration testing rather than dissolution testing as a quality control method for certain immediate release (IR) formulations. A mechanistic approach, which is beyond the current FDA criteria, is presented. Dissolution testing via United States Pharmacopeial Convention Apparatus II at various paddle speeds was performed for immediate and extended release formulations of metronidazole. Dissolution profile fitting via DDSolver and dissolution profile predictions via DDDPlus™ were performed. The results showed that Fickian diffusion and drug particle properties (DPP) were responsible for the dissolution of the IR tablets, and that formulation factors (eg, coning) impacted dissolution only at lower rotation speeds. Dissolution was completely formulation controlled if extended release tablets were tested and DPP were not important. To demonstrate that disintegration is the most important dosage form attribute when dissolution is DPP controlled, disintegration, intrinsic dissolution and dissolution testing were performed in conventional and disintegration impacting media (DIM). Tablet disintegration was affected by DIM and model fitting to the Korsmeyer-Peppas equation showed a growing effect of the formulation in DIM. DDDPlus was able to predict tablet dissolution and the intrinsic dissolution profiles in conventional media and DIM. The study showed that disintegration has to occur before DPP-dependent dissolution can happen. The study suggests that disintegration can be used as performance test of rapidly disintegrating tablets beyond the FDA criteria. The scientific criteria and justification is that dissolution has to be DPP dependent, originated from active pharmaceutical ingredient characteristics and formulations factors have to be negligible.

Ion Channels in Brain Metastasis.

Breast cancer, lung cancer and melanoma exhibit a high metastatic tropism to the brain. Development of brain metastases severely worsens the prognosis of cancer patients and constrains curative treatment options. Metastasizing to the brain by cancer cells can be dissected in consecutive processes including epithelial-mesenchymal transition, evasion from the primary tumor, intravasation and circulation in the blood, extravasation across the blood-brain barrier, formation of metastatic niches, and colonization in the brain. Ion channels have been demonstrated to be aberrantly expressed in tumor cells where they regulate neoplastic transformation, malignant progression or therapy resistance. Moreover, many ion channel modulators are FDA-approved drugs and in clinical use proposing ion channels as druggable targets for future anti-cancer therapy. The present review article aims to summarize the current knowledge on the function of ion channels in the different processes of brain metastasis. The data suggest that certain channel types involving voltage-gated sodium channels, ATP-release channels, ionotropic neurotransmitter receptors and gap junction-generating connexins interfere with distinct processes of brain metastazation.