Intracellular Drug Concentration Research Articles

Abstract P1-18-21: Neratinib reduces HER2+/luminal B breast cancer stem cell activity and inhibits resistance by blocking efflux mechanisms

Abstract Background: Breast cancer stem cells (BCSCs) have been attributed to drug resistance and recurrence of disease following treatment. BCSCs employ various mechanisms to resist chemotherapy. One such mechanism is the increase in expression of the ATPase drug efflux pumps ABCB1 and ABCG2, reducing intracellular Paclitaxel and Mitoxantrone drug concentration, respectively. Neratinib a pan-HER tyrosine kinase inhibitor was reported to block both ABCB1 and ABCG2 pump activity. We aimed to determine the effect of Neratinib on CSC activity (primary and self-renewal) and determine if the mechanism included pump inhibition. Methods: The effects of 10nM Neratinib alone and in combination with either 1µM Tamoxifen, 0.1µM Paclitaxel or 0.1µM Mitoxantrone on anoikis resistance culture was evaluated in multiple breast cancer cell lines. The mammosphere forming efficiency (MFE) and generation of secondary mammospheres (self-renewal) were calculated. Blockage of the ABCG2 by Neratinib was assessed via FAC analysis. Statistical significance was determined using unpaired t-test and one-way ANOVA. Results: Neratinib reduced CSC activity assessed by MFE in BT474 cells by 50% (P < 0.01). Combining Neratinib with Tamoxifen further reduced CSC (by 75%) than either treatment alone (P < 0.05 and P < 0.01, respectively). The combination of Neratinib and Paclitaxel also reduced the MFE by a greater extent (90%, P < 0.0001) than either treatment alone (P < 0.05 and P < 0.0001, respectively). Neratinib alone had no effect on MFE in the triple negative breast cancer cell line MDA-MB-453, combining Neratinib with Paclitaxel reduced MFE by 85%, a greater reduction than paclitaxel alone (75%), P < 0.0001. Our data also indicates that Neratinib in combination with Paclitaxel reduces CSC self-renewal in MCF7, MCF7HER2 and MDA-MB-453 cell lines. Combining Neratinib with Mitoxantrone further decreases CSC activity than either treatment alone in MCF7, MCF7HER2, SKBR3 and MDA-MB-453 cells and completely inhibited CSC self-renewal in the triple negative cell line. Neratinib also reduced the side population in BT474 and SKBR3 cells in a dose response manner, demonstrating inhibition of ABCG2 activity. Conclusions: These findings support the idea that Neratinib has the ability to overcome drug resistance caused by the ABCB1 and ABCG2 pumps. Therefore, we are currently investigating the potential of Neratinib to block the activity of the ABCB1 and ABCG2 pumps using flow cytometry to analyse the side-population and drug accumulation. They also provide insight into a role for Neratinib to enhance paclitaxel chemotherapy in triple negative CSC. Citation Format: Nathan Hull, Robert Clarke, Nigel Bundred. Neratinib reduces HER2+/luminal B breast cancer stem cell activity and inhibits resistance by blocking efflux mechanisms [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-21.

Abstract P1-06-04: SimBioSys TumorScope: Spatio-temporal modeling of the breast tumor microenvironment accurately predicts chemotherapeutic response

Abstract Background A single neuron cannot think, just as a single hepatocyte cannot filter blood. The functions of the tissues that make up our bodies are emergent, depending on both their constituent cells' internal biology, as well as how they interact with the other cells around them. Tumors are no different. They interact with nearby cells through both mechanical and molecular mechanisms, adapting their own behaviors to suit local environmental conditions, and even hijacking the functions of nearby tissues for tumor-promoting purposes. As such, predicting how a given tumor will grow or respond to treatment requires more than just an understanding of the tumor's intrinsic characteristics, it requires a spatially- and temporally-resolved description of the tumor's shape, its surrounding tissues, and the milieu of diffusible molecules that drive its behavior and interactions. Methods SimBioSys TumorScope for Breast Cancer implements a multi-scale simulation methodology that couples genome-scale metabolic modeling with reaction-diffusion and material mechanics models in order to predict how individual patients' breast tumors will respond to different chemotherapeutic regimens. The simulations explicitly track the 3D morphology of the tumor and surrounding adipose, fibroglandular, and vascular tissues (initialized using pre-treatment MRI images), as well as the concentrations of key nutrients, metabolic byproducts, and drugs as they change over time. The vasculature serves as both source and sink for the modeled chemicals; nutrients and drugs are able to diffuse out of the blood vessels, while metabolic byproducts can diffuse in. The vascular concentrations of the drugs are assumed to vary in accordance with their administration schedules and known pharmacokinetics. At each location within the simulation, the local metabolic response of the tissues to the available nutrients is modeled using flux balance analysis, yielding uptake and efflux rates for each chemical species, as well as local growth rates for the tissues. At the same time, drugs may also be taken up by the cells, leading to cell death at rates that depend on the local intracellular drug concentrations, as well as their pharmacodynamic properties. As different regions of the tissues grow or shrink in response to their microenvironmental conditions, the macroscopic tissues stretch and deform. Results SimBioSys TumorScope for Breast Cancer was retrospectively applied to over 100 patients that received neoadjuvant chemotherapy (NACT) culled from publicly-available datasets (via the TCIA). Simulations were initialized with pre-treatment MRI data, and run through the entirety of each patient's specified treatment regimen. Predicted changes in tumor volume and longest dimension were then compared against measured values at several time-points after initiation of therapy, yielding Pearson correlations of over 0.93 for both. Conclusions Through accurate spatio-temporal modeling of drug and nutrient perfusion, metabolic behavior, and the physico-chemical interactions that arise between tissues, the SimBioSys TumorScope for Breast Cancer can accurately predict the response of patients treated with NACT. Citation Format: John A Cole Jr., Joseph R. Peterson, Tyler M. Earnest, Michael J. Hallock, Eduardo Braun. SimBioSys TumorScope: Spatio-temporal modeling of the breast tumor microenvironment accurately predicts chemotherapeutic response [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-06-04.

Crizotinib sensitizes the erlotinib resistant HCC827GR5 cell line by influencing lysosomal function.

In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live‐cell microscopy. We observed additive effects in EBC‐1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR‐inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live‐cell microscopy with a pH‐sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib–erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions.

SC1.2 - Advancing predictions of tissue and intracellular drug concentrations using in vitro and PBPK modeling approaches

SC1.2 - Advancing predictions of tissue and intracellular drug concentrations using in vitro and PBPK modeling approaches

DOX-loaded peptide dendritic copolymer nanoparticles for combating multidrug resistance by regulating the lysosomal pathway of apoptosis in breast cancer cells.

Multidrug resistance (MDR) is a common phenomenon in clinical oncology and is a major obstacle to cancer chemotherapy. Many nanoparticle (NP)-based drug delivery systems have been developed to overcome MDR depending on increasing intracellular drug concentrations via increased cellular uptake and rapid drug release. The objective of this work was to investigate the performance and possible mechanisms of enzyme-sensitive mPEGylated dendron-GFLG-DOX conjugate based nanoparticles for blockading the MDR phenotype of MCF-7/ADR. In vitro, mPEGylated dendron-GFLG-DOX conjugate based nanoparticles could significantly promote cellular uptake and accumulation, potent cytotoxicity and apoptosis compared to free DOX in resistant cells. mPEGylated dendron-GFLG-DOX conjugate based nanoparticles were found to translocate across the membranes of resistant cells via active endocytic pathways leading to more DOX accumulating in the nuclei of MCF-7/ADR cells. Importantly, we found that mPEGylated dendron-GFLG-DOX conjugate based nanoparticles could induce cathepsin B in the cytoplasm and enhance lysosomal-mediated cell death compared to free DOX. Furthermore, mPEGylated dendron-GFLG-DOX conjugate based nanoparticles enhanced the drug's penetration, toxicity, and growth inhibition compared to free DOX in the three-dimensional multicellular tumor spheroid model. In vivo, mPEGylated dendron-GFLG-DOX conjugate based nanoparticles significantly improved the therapeutic efficacy against MDR xenograft tumors, and showed better biocompatibility than free DOX. These results indicated that mPEGylated dendron-GFLG-DOX conjugate based nanoparticles could be used as an alternative drug delivery system for MDR tumor treatment through initiating the lysosomal apoptosis pathway.

Nephrotoxicity of Antimicrobials and Antibiotics.

Medication-induced nephrotoxicity remains one of the most common causes of acute kidney injury (AKI) among hospitalized patients. Within the extensive group of medications associated with AKI, antibiotics and other antimicrobials are well recognized triggers of structural and functional renal impairment. Clinical manifestations range from mild forms of tubular injury to significant deterioration of kidney function requiring acute renal replacement therapy. Several mechanisms are described, although the most frequent are acute interstitial nephritis, acute tubular necrosis, intratubular crystal deposition, and proximal/distal tubulopathy with electrolyte wasting abnormalities. General risk factors for antimicrobial-induced AKI include pre-existing chronic kidney disease, and concomitant use of medication with nephrotoxic potential. Prevention and early recognition of AKI represent the standard approach to mitigate AKI and avoid morbidity.

Cetuximab enhances the efficiency of irinotecan through simultaneously inhibiting the MAPK signaling and ABCG2 in colorectal cancer cells

BackgroundThe present study sought to investigate the combined effects of cetuximab and irinotecan on colorectal cancer cells as well as the mechanisms underlying their anti-cancer effects. Material and methodsHigh performance liquid chromatography, Hoechst staining assay, and western blotting analysis were used to detect intracellular drug concentrations, cell apoptosis, and protein expression in the presence of cetuximab, irinotecan, and the combination of both. ResultsCetuximab was found to increase intracellular concentrations of irinotecan as well as cytotoxicity by inhibiting the epidermal growth factor receptor and, by extension, the downstream RAS-RAF-MEK-ERK signaling pathway. Cetuximab therefore induced apoptosis and improved the effect of irinotecan in colorectal cancer cells. It was also shown that cetuximab inhibited the drug efflux activity of ABCG2. In combination with irinotecan, cetuximab can both significantly induce cell apoptosis by inhibiting the RAS-RAF-MEK-ERK signaling pathway and improve the effects of irinotecan by decreasing drug efflux through the inhibition of ABCG2. ConclusionThese features contribute to its anti-cancer potential.

Thermosensitive Liposome-Mediated Drug Delivery in Chemotherapy: Mathematical Modelling for Spatio-temporal Drug Distribution and Model-Based Optimisation.

Thermosensitive liposome-mediated drug delivery has shown promising results in terms of improved therapeutic efficacy and reduced side effects compared to conventional chemotherapeutics. In order to facilitate our understanding of the transport mechanisms and their complex interplays in the drug delivery process, computational models have been developed to simulate the multiple steps involved in liposomal drug delivery to solid tumours. In this study we employ a multicompartmental model for drug-loaded thermosensitive liposomes, with an aim to identify the key transport parameters in determining therapeutic dosing and outcomes. The computational model allows us to not only examine the temporal and spatial variations of drug concentrations in the different compartments by utilising the tumour cord concept, but also assess the therapeutic efficacy and toxicity. In addition, the influences of key factors on systemic plasma concentration and intracellular concentration of the active drug are investigated; these include different chemotherapy drugs, release rate constants and heating duration. Our results show complex relationships between these factors and the predicted therapeutic outcome, making it difficult to identify the “best” parameter set. To overcome this challenge, a model-based optimisation method is proposed in an attempt to find a set of release rate constants and heating duration that can maximise intracellular drug concentration while minimising systemic drug concentration. Optimisation results reveal that under the operating conditions and ranges examined, the best outcome would be achieved with a low drug release rate at physiological temperature, combined with a moderate to high release rate at mild hyperthermia and 1 h heating after injection.

Validation of Cell-Based Assay for Quantification of Sesamol Uptake and Its Application for Measuring Target Exposure.

The intracellular drug concentration is needed for determination of target exposure at the site of action regarding its pharmacological action and adverse effects. Sesamol is an antiproliferative molecule from Sesamum indicum with promising health benefits. We present a method for measuring the intracellular sesamol content using reverse-phase HPLC with a UV diode array in melanoma cells. Sesamol was completely resolved by isocratic elution (4.152 ± 0.008 min) with methanol/water (70%, v/v) through a 30 °C, 5-µm C-18 column and detection at 297 nm. The present assay offers high sensitivity, fast elution, and an accurate and linear nominal concentration range of 10–1000 ng/mL (R2 = 0.9972). The % accuracy of the sesamol quality control sample was −3.36% to 1.50% (bias) with a 0.84% to 5.28% relative standard deviation (RSD), representing high repeatability and high reproducibility. The % recovery was 94.80% to 99.29%, which determined that there was no loss of sesamol content during the sample preparation. The validated method was applied to monitor intracellular sesamol concentration after treatment from 5 min to 24 h. The remaining intracellular sesamol content was correlated with its antiproliferative effect (R2 = 0.9483). In conclusion, this assay demonstrated low manipulation, quick elution, and high sensitivity, precision, accuracy, and recovery, and it was successfully applied to the quantification of sesamol in target cells.

Absorption and utilisation of epimedin C and icariin from Epimedii herba, and the regulatory mechanism via the BMP2/ Runx2 signalling pathway

Bone nonunion remains a challenge during the treatment of bone defect accompanied with infection. Epimedii herba is a widely used medicine in clinic to enhance bone healing. Epimedin C and icariin are the major flavonoid glycosides from Epimedii herba. Although the effects of these compounds have been reported, their different absorption and utilisation by osteoblasts remain unclear. In the present study, lipopolysaccharide (LPS)-induced osteoblast was adopted as the model cell to evaluate the effects of epimedin C and icariin. The intracellular and extracellular drug concentrations within 24 h were assayed by pipette tip solid-phase extraction and high-performance liquid chromatography, respectively. MTT, alkaline phosphatase (ALP) and calcified nodule staining were performed to identify and evaluate the effects of epimedin C and icariin on LPS-induced osteoblasts. The regulatory roles of epimedin C and icariin in the bone morphogenetic protein-2 (BMP-2)/Runt-related transcription factor 2 (Runx2) signalling pathway were investigated. The results revealed that epimedin C and icariin were not efficiently absorbed by LPS-induced osteoblasts. Nevertheless, they still had high utilisation efficiency after entering the cells. ALP activity, mineralisation and osteoblasts proliferation were enhanced by a high concentration of epimedin C and icariin. The suppressed expression of BMP-2 and Runx2 in LPS-induced osteoblasts was up-regulated significantly after treatment with epimedin C and icariin. These findings firstly illustrated the behavior of epimedin C and icariin from Epimedii herba on LPS-induced osteoblasts and the regulatory property on the expression of key genes and proteins of the BMP-2/Runx2 signalling pathway, which might be helpful for better understanding flavonoids’ mechanism to enhance bone repair and improving their future application.

Natural Furanocoumarin as Potential Oral Absorption Enhancer

Bioavailability of orally administered drugs can be influenced by many factors. Poor drug absorption across the intestinal membrane is one of the factors that contribute to low bioavailability of drugs. It has been suggested that the metabolism/active efflux in the small intestine is involved in the poor absorption of many drugs. Intestinal CYP3A4 and P-gp work coordinately to reduce the intracellular concentration of drugs. Recently, bioenhancers have been identified and extensively studied. The aim of this study was to evaluate natural furanocoumarins found in juices of common lime and kaffir lime as the potential enhancers for oral delivery by means of modulating CYP3A4 and/or P-gp activities. The role of isolated furanocoumarins on CYP3A4 was assessed by testosterone 6β-hydroxylation reaction, while the effect on P-gp was investigated using R123 and CAM uptake studies in Caco-2, as well as LLC-PK1and LLC-GA5-Col300. In the present study, we demonstrated that isopimpinellin isolated from common lime is the best CYP3A4 inhibitor among 4 isolated furanocoumarins, implying that isopimpinellin would possibly act as a bioenhancer by inhibiting pre-systemic metabolism. 6’,7’-Dihydroxybergamottin found in kaffir lime is a dual inhibitor of CYP3A4 and P-gp, suggest that it could potentially be used as a bioenhancer by inhibiting both pre-systemic metabolism and efflux mechanism. However,in vivostudy should be further conducted to confirm these effects in the body.

Abstract 3478: APC loss mediates doxorubicin response in breast cancer via changes in intracellular drug concentration and DNA repair pathways

Abstract Chemoresistance is one of the leading causes of breast cancer related deaths. Understanding the molecular basis for chemoresistance is essential for novel therapeutic advancement to improve patient outcome. The Adenomatous Polyposis Coli (APC) tumor suppressor is either mutated or hypermethylated in up to 70% of sporadic breast cancer; however, little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated from the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we demonstrated that APC loss decreased doxorubicin (DOX) induced apoptosis. DOX, a commonly used chemotherapeutic in breast cancer, inhibits topoisomerase IIa, resulting in double stranded DNA breaks, resulting in cell cycle arrest to allow repair or apoptosis. We made the novel observation that APC loss in MMTV-PyMT;ApcMin/+ cells activated signal transducer and activator of transcription 3 (STAT3) thereby increasing the expression of the drug efflux pump, multidrug resistance protein 1 (MDR1). Therefore, we hypothesized that APC loss prevents doxorubicin-mediated cell death through: 1) reduced intracellular DOX and 2) alterations in DNA damage and repair. To investigate the intracellular accumulation of DOX, we first used calcein incorporation assay and demonstrated that APC loss increased MDR1 activity, which was restored by an MDR1 inhibitor. In addition, MDR1 inhibition sensitized the MMTV-PyMT;ApcMin/+ cells to DOX-mediated apoptosis. To investigate the effect of APC loss on DNA damage repair pathways, we initially monitored damage recognition pathways after 24-hour DOX treatment. The MMTV-PyMT;ApcMin/+ cells exhibited decreased γH2AX and ataxia-telangiectasia mutated (ATM) phosphorylation following DOX treatment compared to controls, suggesting decreased DNA damage either through decreased intracellular DOX or enhanced DNA repair. Decreased phosphorylation of Chk1 and Chk2 was also observed in DOX-treated MMTV-PyMT;ApcMin/+ cells. Preliminary data to investigate if this decreased DNA damage was due to increased DNA damage repair, via monitoring γH2AX expression throughout treatment and recovery from DOX, suggest enhanced DNA repair in MMTV-PyMT;ApcMin/+ cells. Using the ATM inhibitor, we observed an increase in DOX sensitivity in MMTV-PyMT;ApcMin/+ cells. Alternatively, ATR inhibition did not affect DOX-mediated apoptosis. We will further investigate the efficiency of repair pathways through reporter plasmids and radiation induced damage to separate the drug transport alterations. Finally, we will test whether combination therapy of doxorubicin with an MDR1 inhibitor will reduce tumor burden in vivo. Taken together, APC loss mediates DOX resistance via reducing intracellular DOX and increasing DNA damage repair demonstrating the potential use of combination therapy to overcome chemoresistance. Citation Format: Casey Stefanski, Kaitlyn Keffler, Stephanie McClintock, Lauren Milac, Jenifer Prosperi. APC loss mediates doxorubicin response in breast cancer via changes in intracellular drug concentration and DNA repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3478.

What “The Cancer Genome Atlas” database tells us about the role of ATP-binding cassette (ABC) proteins in chemoresistance to anticancer drugs

ABSTRACTIntroduction: Chemotherapy remains the only option for advanced cancer patients when other alternatives are not feasible. Nevertheless, the success rate of this type of therapy is often low due to intrinsic or acquired mechanisms of chemoresistance. Among them, drug extrusion from cancer cells through ATP-binding cassette (ABC) proteins plays an important role. ABC pumps are primary active transporters involved in the barrier and secretory functions of many healthy cells.Areas covered: In this review, we have used The Cancer Genome Atlas (TCGA) database to explore the relationship between the expression of the major ABC proteins involved in cancer chemoresistance in the most common types of cancer, and the drugs used in the treatment of these tumors that are substrates of these pumps.Expert opinion: From unicellular organisms to humans, several ABC proteins play a major role in detoxification processes. Cancer cells exploit this ability to protect themselves from cytostatic drugs. Among the ABC pumps, MDR1, MRPs and BCRP are able to export many antitumor drugs and are expressed in several types of cancer, and further up-regulated during treatment. This event results in the enhanced ability of tumor cells to reduce intracellular drug concentrations and hence the pharmacological effect of chemotherapy.

Calcium electroporation: The bioelectrochemical treatment of spontaneous equine skin tumors results in a local necrosis

Electrochemotherapy (ECT) is an anticancer bioelectrochemical therapy where electrical field pulses (electropermeabilization) increase intracellular concentration of antitumor drugs. The procedure is very effective against skin tumors. The restrictive regulations concerning anticancer drugs in veterinary medicine limit use of ECT. Electroporation with calcium (Electroporation Calcium Therapy)(ECaT) was proved to be effective in vivo on induced tumors in laboratory animals. This study evaluated the effects of ECaT in equine sarcoids (spontaneous skin tumors) on an animal cohort. Pulse parameters for ECaT were choosen for using skin contact electrodes. ECaT was applied under general anesthesia. The tumors were removed at different days after the treatment and analyzed by histology. The study assessed the volume fraction of necrosis that was >50% for 9 of 13 sarcoids. Sixteen sarcoids in 10 horses were treated with ECaT. Macroscopic changes (a crust) were observed in 14/16 tumors. The main microscopic changes were necrosis, ulceration,hemorrhages, calcifications and thrombosis. The adverse effect was an inflammatory local reaction. Surrounding tissues were not affected. This targeted effect can be explained by its control by the field distribution in the tissue and on the interstitial diffusion of the injected Ca2+.

Methyl-\u03b2-cyclodextrin, an actin depolymerizer augments the antiproliferative potential of microtubule-targeting agents

Methyl-β-cyclodextrin (MCD), an established pharmacological excipient, depolymerizes the actin cytoskeleton. In this work, we investigated the effect of MCD-mediated actin depolymerization on various cellular phenotypes including traction force, cell stiffness, focal adhesions, and intracellular drug accumulation. In addition to a reduction in the contractile cellular traction, MCD acutely inhibits the maturation of focal adhesions. Alteration of contractile forces and focal adhesions affects the trypsin-mediated detachment kinetics of cells. Moreover, MCD-mediated actin depolymerization increases the intracellular accumulation of microtubule-targeting agents (MTAs) by ~50% with respect to the untreated cells. As MCD treatment enhances the intracellular concentration of drugs, we hypothesized that the MCD-sensitized cancer cells could be effectively killed by low doses of MTAs. Our results in cervical, breast, hepatocellular, prostate cancer and multidrug-resistant breast cancer cells confirmed the above hypothesis. Further, the combined use of MCD and MTAs synergistically inhibits the proliferation of tumor cells. These results indicate the potential use of MCD in combination with MTAs for cancer chemotherapy and suggest that targeting both actin and microtubules simultaneously may be useful for cancer therapy. Importantly, the results provide significant insight into the crosstalk between actin and microtubules in regulating the traction force and dynamics of cell deadhesion.

Alginate modified-PLGA nanoparticles entrapping amikacin and moxifloxacin as a novel host-directed therapy for multidrug-resistant tuberculosis

Polymeric nanoparticles have been widely investigated as a controlled release drug delivery platform for the treatment of tuberculosis (TB). These nanoparticles are readily internalised into macrophages and leads to high intracellular drug concentration. In this study two anti-TB drugs, amikacin and moxifloxacin were encapsulated into PLGA nanoparticles. The novelty of this work includes: (1) the efficient encapsulation of two hydrophilic second-line anti-TB drugs, and (2) intra-macrophage delivery of this combination potentially for rapid treatment of multi-drug resistant TB (MDR-TB). Two water-oil-water (w/o/w) emulsion strategies were employed in this study to produce: (1) alginate coated PLGA nanoparticles, and (2) alginate entrapped PLGA nanoparticles. The average particle size and polydispersity index (PDI) of the alginate coated PLGA nanoparticles were found to be unfavourably high with values of 640 ± 32 nm and 0.63 ± 0.09, respectively. In contrast, the alginate entrapped PLGA nanoparticles were within the desired particle size range of 312–365 nm and the PDI was 0.14– 0.31, and therefore were chosen for subsequent studies. Alginate entrapped nanoparticles exhibited an entrapment of moxifloxacin in the range of 10.1–18.7 μg/mg and an entrapment of amikacin in the range of 15–17.4 μg/mg. On the contrary, non-modified PLGA nanoparticles exhibited a significantly less amikacin loading of 6.2 μg/mg polymer and 4.2 μg/mg of moxifloxacin. The release of alginate modified PLGA nanoparticles showed slower release in comparison with the non-modified PLGA nanoparticles. The alginate entrapped PLGA nanoparticles did not show significant difference from the alginate coated PLGA nanoparticles in the release pattern. To study the macrophage uptake efficiency, both formulations of the alginate entrapped nanoparticle; non-calcium formulation and calcium, and alginate coated PLGA nanoparticles (that includes no calcium) formulation was loaded with coumarin-6 as a marker, seeded to THP-1 derived macrophages infected with Mycobacterium tuberculosis (H37Ra), and examined by confocal microscopy. Both formulations of alginate entrapped particles (calcium and non-calcium) were readily internalised into the macrophages and highly concentrated in the perinuclear region, and the non-calcium alginate entrapped formulation showed a maximum internalisation. Furthermore, the anti-mycobacterial activity of the dually entrapped drug-loaded particles (moxifloxacin and amikacin) was evaluated using M. tuberculosis-infected macrophages, which revealed an enhanced inhibition of viable bacterial count compared to single drug loaded nanoparticle formulations, and untreated cells. The dually entrapped nanoparticles showed bacterial viability of 0.6% relative to the untreated group, compared to 6.49% for amikacin alone nanoparticles and 3.27% for moxifloxacin alone nanoparticles. In conclusion, the amikacin-moxifloxacin alginate entrapped PLGA nanoparticles are promising for further in vivo studies.

Therapeutic potential of ceragenins and nanosystems containing membrane active compounds in the anti-cancer therapies

Constantly increasing morbidity and mortality of cancer, complex immunopathogenesis of tumors and variable development and severity of the disease, enforce a constant search for new therapeutic factors with anti-cancer activity. Despite the constant achievements in anti- -cancer diagnostic and therapeutic methods development, the low specificity and high toxicity of cytostatics, and the multidrug resistance expansion, still remain a considerable health problem. Currently, natural, cationic antimicrobial peptides (AMPs) and their synthetic lipid analogs from the ceragenin group (CSA) are presented as potential antineoplastic compounds. Their special features, including the membrane permeabilizing-based mechanism of action, selectivity towards tumor cells, biocompatibility and the absence of a recorded anti-AMPs resistance mechanism, make cationic antineoplastic peptides an effective alternative to modern cytostatics. Moreover, a compelling number of research confirm the possibility of using magnetic nanoparticles as highly effective and biocompatible drug carriers, ensuring the achievement of a sufficiently high intracellular concentration of drug and thus, increasing its antineoplastic activity. The results obtained so far indicate the possibility of the employment of natural AMPs and their synthetic analogs from ceragenins group in an effective eradication of cancer cells. Nevertheless, further studies aiming to elucidate the safety of proposed nanosystems and focused on the employment of ceragenin-based nanosystems in diagnostic MRI imaging and as hyperthermia inducers are needed and justified.

Rifampin Induces Expression of P-glycoprotein on the THP1 Cell–Derived Macrophages, Causing Decrease Intramacrophage Concentration of Prothionamide

Rifampin (RIF) has been widely used for the treatment of bacterial infections, including tuberculosis (TB). Treatment of drug-resistant TB is a global problem because of reduced drug efficacy. The present study determined the effect of RIF on MDR1 gene (P-glycoprotein, P-gp) expression in THP1 macrophages and analyzed the intracellular concentration of the anti-TB drug prothionamide in the presence of RIF. RIF treatment significantly induced MDR1 protein and mRNA levels in phorbol 12-myristate 13-acetate–stimulated THP1 macrophages (p < 0.001 and 0.01, respectively). The pregnane X receptor inhibitors resveratrol and ketoconazole significantly suppressed RIF-induced P-gp expression in THP1 macrophages (p < 0.05). RIF-treated THP1 macrophages also exhibited strong efflux of P-gp substrate, resulting in a reduced intracellular concentration of rhodamine-123 and prothionamide (p < 0.01 and 0.05, respectively). By contrast, the P-gp inhibitor cyclosporine A significantly increased intracellular concentration of rhodamine-123 and prothionamide (p < 0.001 and 0.05, respectively). The present results suggest that the usage of RIF together with P-gp–substrate drugs to treat TB may lead to deteriorated treatment efficacy because of the lower intracellular drug concentration. Further studies would be necessary to know the influence of RIF-induced P-gp induction on the treatment outcome of patients with TB.

Chemotherapeutic and antiangiogenic drugs beyond tumor progression in colon cancer: Evaluation of the effects of switched schedules and related pharmacodynamics

The aim of the study was to evaluate the effects and the related pharmacological mechanisms of switched schedules of antiangiogenic and chemotherapeutic drugs beyond progression after a first-line treatment in a colorectal cancer preclinical model. In vivo studies were performed in nude mice subcutaneously transplanted with colon cancer cells. The treatments included drug combinations with a switch between chemotherapeutic (i.e., irinotecan and 5-fluorouracil) and/or antiangiogenic drugs (i.e., anti-VEGF antibodies and sunitinib) at the time of tumor progression. Proliferation assays were also achieved in vitro on different colon cancer cell lines exposed to SN-38 and sunitinib alone or in combination. ABCG2 gene expression was performed with real-time PCR and SN-38 intracellular concentrations were measured.The switch in the combined treatments, at the time of tumor progression, of the chemotherapeutic (from irinotecan to 5-fluoruracil), or the antiangiogenic drug (from anti-VEGF antibodies to sunitinib) or of both drugs induced a new response. Immunohistochemistry of stromal PDGF-C, PlGF, SD1-α, Tie-2, and VEGFR-2 showed statistical differences between tumors at the time of relapse and after the switched therapy. Moreover, the combination of SN-38 and sunitinib caused synergism on colon cancer cells, with significant inhibition of the ABCG2 gene expression and an increase of SN-38 intracellular concentrations.Our observations may be of clinical relevance, suggesting the switch of single chemotherapeutic or antiangiogenic drugs beyond progression of the disease to obtain a new tumor response due to a modulation of angiogenic factors and a direct effect on tumor cells with a possible variation of intracellular drug concentrations.

In Situ-Induced Multivalent Anticancer Drug Clusters in Cancer Cells for Enhancing Drug Efficacy

Increasing intracellular drug concentration is an effective way for cancer chemotherapeutics to enhance efficacy and combat drug resistance. In this work, a series of anticancer drug conjugates were prepared by linking thiol-modified oligo( p-phenylene vinylene) with paclitaxel, vincristine, teniposide, tamoxifen, doxorubicin, or podophyllotoxin (OPV-S-Drugs) through a Michael addition reaction. These OPV-S-Drugs could undergo intracellular assembly and aggregation upon oxidation to yield multivalent anticancer drug clusters, which inhibited their diffusion from cancer cells. The intracellular aggregation of OPV-S-Drugs originates from π–π stacking and hydrophobic interactions between OPV backbones, followed by cross-linking via disulfide bond formation in the presence of reactive oxygen species (ROS). The drug clusters occur only in the cytoplasm of cancer cells expressing high ROS levels, but not in healthy mammalian cells, thus reducing the cytotoxicity to normal cells. Specifically, the super-toxicity of podophyllotoxin to normal cells was obviously suppressed while the drug efficacy was maintained through our new strategy. The diverse action mechanisms of OPV-S-Drugs toward cancer cells is proposed.