Drug Exposure Time Research Articles

Ecotoxicological properties of ketoprofen and the S(+)-enantiomer (dexketoprofen): Bioassays in freshwater model species and biomarkers in fish PLHC-1 cell line.

The increased use of nonsteroidal anti-inflammatory drugs (NSAIDs) has resulted in their ubiquitous presence in the environment. The toxicological properties of these 2 widely prescribed NSAIDs, namely racemic ketoprofen and its enantiomer S(+)-ketoprofen (dexketoprofen), were evaluated, firstly, by acute and chronic toxicity tests using 3 representative model organisms (Vibrio fischeri, Pseudokirchneriella subcapitata, and Ceriodaphnia dubia) and, secondly, by evaluating the responses of biotransformation systems and multidrug resistance-associated proteins (MRP1/MRP2) using the Poeciliopsis lucida hepatocellular carcinoma 1 (PLHC-1) fish hepatic cell line. Toxicity data from both acute and chronic dexketoprofen exposure indicated higher sensitivity through inhibition of bioluminescence and algal growth and through increased mortality/immobilization compared to racemic ketoprofen exposure. The growth inhibition test showed that racemic ketoprofen and dexketoprofen exhibited different effect concentration values (240.2 and 65.6 μg/L, respectively). Furthermore, racemic ketoprofen and dexketoprofen did not exert cytotoxic effects in PLHC-1 cells and produced compound-, time-, and concentration-specific differential effects on cytochrome P450 1A (CYP1A) and glutathione S-transferase levels. For CYP1A, the effects of racemic ketoprofen and dexketoprofen differed at the transcriptional and catalytic levels. Exposure to racemic ketoprofen and dexketoprofen modulated MRP1 and MRP2 mRNA levels, and these effects were also dependent on compound, exposure time, and concentration of the individual drug. The present study revealed for the first time the interactions between these NSAIDs and key detoxification systems and different sensitivity to the racemic mixture compared to its enantiomer. Environ Toxicol Chem 2018;37:201-212. © 2017 SETAC.

A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles

Delivery of biomacromolecular drugs into the inner ear is challenging, mainly because of their inherent instability as well as physiological and anatomical barriers. Therefore, protein-friendly, hydrogel-based delivery systems following local administration are being developed for inner ear therapy. Herein, biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing interferon α-2 b (IFN α-2 b) were loaded in chitosan/glycerophosphate (CS/GP)-based thermosensitive hydrogel for IFN delivery by intratympanic injection. The injectable hydrogel possessed a physiological pH and formed semi-solid gel at 37 °C, with good swelling and deswelling properties. The CS/GP hydrogel could slowly degrade as visualized by scanning electron microscopy (SEM). The presence of NPs in CS/GP gel largely influenced in vitro drug release. In the guinea pig cochlea, a 1.5- to 3-fold increase in the drug exposure time of NPs-CS/GP was found than those of the solution, NPs and IFN-loaded hydrogel. Most importantly, a prolonged residence time was attained without obvious histological changes in the inner ear. This biodegradable, injectable, and thermosensitive NPs-CS/GP system may allow longer delivery of protein drugs to the inner ear, thus may be a potential novel vehicle for inner ear therapy.

Abstract 1085: Time dependent analysis for the combination of standardized Annona cherimola and Solanum nigrum extracts with 5-FU against head and neck cancer cells

Abstract Phytochemicals of natural origin constitutes very initiative field of anticancer drug discovery. Annona sp. and Solanum nigrum are folk herbs with reported use for the treatment of several neoplasias. Natural herb products and folk medicine might not be potent anticancer remedies and are recommended for combination therapy. In the current work, we mathematically evaluated the influence of combining standardized extracts of A. cherimola and S. nigrum to the cytotoxic profile of 5-FU against head and neck cancer cells (HEP-2). Cytotoxicity assessment and combination analysis were calculated after MTT assays followed by fitting to Emax model and calculating the combination index (CI-value). After 24 h exposure, both A. cherimola and S. nigrum extracts were more potent than 5-FU with IC50 of 29.4±4.4, 7.3±1.3 and 3.4±4.4 µg/ml, respectively. Both A. cherimola and S. nigrum extracts significantly decreased the IC50 of 5-FU to 5.2±0.55 and 7.6±0.85 µg/ml, respectively; however the CI-values were indicative of additive and antagonistic interactions, respectively (0.91 and 2.49, respectively). After 48 h of exposure, the IC50 of 5-FU, A. cherimola and S. nigrum extracts were 3.1±0.45, 0.87±0.22 and 7.2±0.65 µg/ml, respectively. Similarly, combination of A. cherimola and S. nigrum extracts with 5-FU showed additive and antagonistic interaction with CI-values of 1.05 and 3.37, respectively. After 72 h of exposure, the IC50 of 5-FU, A. cherimola and S. nigrum extracts were 1.9±0.1, 2.1±0.24 and 2.1±0.1 µg/ml, respectively. Similarly, combination of A. cherimola and S. nigrum extracts with 5-FU showed additive and antagonistic interaction with CI-values of 1.2 and 2.21, respectively. Then, CnT model was used to assess the relative influence of drug concentration and exposure time on the cytotoxic profile of treatments under investigation. For S. nigrum and its combination with 5-FU, n-values were 0.9 and 0.63 which is indicative of relatively high influence of drug concentration. On the other hand, n-value for 5-FU treatment was 0.37; this indicates higher influence of drug exposure time compared to other treatment. The n-values of A. cherimola and its combination with 5-FU were 0.51 and 0.54, respectively; this indicate comparable influence of both drug concentration and exposure time. As a confirmatory measure, expression of the apoptotic gene, BAX, was increased after 5-FU combination with A. cherimola and decreased after combination with S. nigrum. In conclusion, some natural herb remedies such as A. cherimola might influence the cytotoxic profile of 5-FU and particularly after short exposure time; while on the other hand, some herb remedies such as S. nigrum significantly antagonizes its cytotoxic activity against head and neck cancer cells. Citation Format: Serag Eldin I. Elbehairi, Aly F. Mohamed, Nahla G. Mohamed, Mohammad I. Basuony, Mahmoud M. Salem, Metwally M. Montaser, Mohammad Y. Alfaifi, Ayman M. Noreddin, Ahmed M. Al-Abd. Time dependent analysis for the combination of standardized Annona cherimola and Solanum nigrum extracts with 5-FU against head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1085. doi:10.1158/1538-7445.AM2017-1085

Structural recognition of tubulysin B derivatives by multidrug resistance efflux transporters in human cancer cells.

Multidrug resistance (MDR) is a major hindrance to curative chemotherapy of various human malignancies. Hence, novel chemotherapeutics must be evaluated for their recognition by MDR efflux transporters. Herein we explored the cytotoxic activity of synthetic tubulysin B (Tub-B, EC1009) derivatives (Tub-B-hydrazide/EC0347 and Tub-B bis-ether/EC1820), and their recognition by the MDR efflux transporters P-glycoprotein 1 (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). Originally isolated from Myxobacteria, tubulysins exhibited potent cytotoxic activity via microtubule depolymerization, and evaded recognition by these MDR efflux pumps. We show that subtle modifications in the natural Tub-B structure enhance its cytotoxicity and drug efflux efficiency. Whereas increasing the lipophilicity of Tub-B drugs enhanced their diffusion into the cell and consequently decreased the IC50 values (≥ 0.27 nM), increasing drug polarity enhanced their recognition by P-gp (>200-fold resistance in P-gp-overexpressing cells). Furthermore, restricting drug exposure time to the clinically relevant 4 h pulse, markedly enhanced efflux by P-gp, resulting in a 1000-fold increased resistance, which was further enhanced upon increased P-gp levels (i.e. an additional 3-fold increase in P-gp levels resulted in >6,000-fold resistance). The unique ability of EC1009 to evade recognition by MDR efflux pumps warrants drug development of tubulysin B derivatives as potent antitumor agents which overcome MDR in cancer.

On the chemotherapeutic agents localization in tissue by means of snake venoms

The efficiency of anti-tumour drug strongly depends on its dose. Higher drug doses and exposure times usually result in better treatment. It is why the implementation of high-dose treatment is always attractive. However, most of the drug delivery techniques meet essential limitations. In isolated regional perfusion a tumour can be exposed to high-dose therapeutic influence but the target organ may be isolated from the rest of circulatory system only for a relatively short period of time. During systemic injection of anti-tumour agents dose limitations are dictated by side toxicity danger. Viperidae venoms are known to cause local stagnation of blood flow and blood-tissue exchange processes in the place of snakebite. In present paper we suggest to use Viperidae snake venoms in addition to anti-tumour drugs for regional anti-cancer therapy. We suppose that Viperidae venoms will assist in drug localization. We state that their usage will help in high-dosage therapy implementation.

Optimization of liposomal topotecan for use in treating neuroblastoma.

The purpose of this work was to develop an optimized liposomal formulation of topotecan for use in the treatment of patients with neuroblastoma. Drug exposure time studies were used to determine that topotecan (Hycamtin) exhibited great cytotoxic activity against SK‐N‐SH, IMR‐32 and LAN‐1 neuroblastoma human cell lines. Sphingomyelin (SM)/cholesterol (Chol) and 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC)/Chol liposomes were prepared using extrusion methods and then loaded with topotecan by pH gradient and copper‐drug complexation. In vitro studies showed that SM/Chol liposomes retained topotecan significantly better than DSPC/Chol liposomes. Decreasing the drug‐to‐lipid ratio engendered significant increases in drug retention. Dose‐range finding studies on NRG mice indicated that an optimized SM/Chol liposomal formulation of topotecan prepared with a final drug‐to‐lipid ratio of 0.025 (mol: mol) was better tolerated than the previously described DSPC/Chol topotecan formulation. Pharmacokinetic studies showed that the optimized SM/Chol liposomal topotecan exhibited a 10‐fold increase in plasma half‐life and a 1000‐fold increase in AUC 0–24 h when compared with Hycamtin administered at equivalent doses (5 mg/kg). In contrast to the great extension in exposure time, SM/Chol liposomal topotecan increased the life span of mice with established LAN‐1 neuroblastoma tumors only modestly in a subcutaneous and systemic model. The extension in exposure time may still not be sufficient and the formulation may require further optimization. In the future, liposomal topotecan will be assessed in combination with high‐dose radiotherapy such as 131I‐metaiodobenzylguanidine, and immunotherapy treatment modalities currently used in neuroblastoma therapy.

New insights into the intracellular distribution pattern of cationic amphiphilic drugs

Cationic amphiphilic drugs (CADs) comprise a wide variety of different substance classes such as antidepressants, antipsychotics, and antiarrhythmics. It is well recognized that CADs accumulate in certain intracellular compartments leading to specific morphological changes of cells. So far, no adequate technique exists allowing for ultrastructural analysis of CAD in intact cells. Azidobupramine, a recently described multifunctional antidepressant analogue, allows for the first time to perform high-resolution studies of CADs on distribution pattern and morphological changes in intact cells. We showed here that the intracellular distribution pattern of azidobupramine strongly depends on drug concentration and exposure time. The mitochondrial compartment (mDsRed) and the late endo-lysosomal compartment (CD63-GFP) were the preferred localization sites at low to intermediate concentrations (i.e. 1 μM, 5 μM). In contrast, the autophagosomal compartment (LC3-GFP) can only be reached at high concentrations (10 μM) and long exposure times (72 hrs). At the morphological level, LC3-clustering became only prominent at high concentrations (10 μM), while changes in CD63 pattern already occurred at intermediate concentrations (5 μM). To our knowledge, this is the first study that establishes a link between intracellular CAD distribution pattern and morphological changes. Therewith, our results allow for gaining deeper understanding of intracellular effects of CADs.

Activity of rifampicin and linezolid combination in Mycobacterium tuberculosis

BackgroundLinezolid (LZD) is not commonly used for treating tuberculosis (TB), but in some patients with drug-resistant TB it is being used. However, the in vitro LZD activity, in combination with rifampicin (RIF) against Mycobacterium tuberculosis has not been fully elucidated. AimsThe aim of this study was to evaluate the in vitro activity of RIF/LZD combination against M. tuberculosis clinical isolates. Materials and methodsThe activity of the RIF/LZD combination was firstly determined in M. tuberculosis H37Rv, 14 susceptible, 9 isoniazid nonresistant and 14 multi-drug resistant (MDR) M. tuberculosis clinical isolates by modified checkerboard assay, Resazurin Drugs Combination Microtiter Assay (REDCA). After, the Time Kill Curve Assay, at 0.5 × MIC of drugs, in combination and alone, was performed in M. tuberculosis H37Rv and 8 (20.5%) of those clinical isolates, which the RIF/LZD combination showed to have synergistic effect by the checkerboard assay. Results and conclusionBy Time Kill Curve Assay, we could observe in M. tuberculosis H37Rv and susceptible isolates, that LZD alone, at sub inhibitory concentration, has poor effect on the bacillus death. In some cases, the bacillus growth stayed constant while in others showed regrowth at the eighth day of drug exposure. RIF alone exhibits potent concentration-dependent bactericidal activity, and was strongly dependent by the drug exposure time. The RIF/LZD combination accomplished a bacteriostatic effect in the reference strain and susceptible isolates. For the RIF resistant isolates, the RIF/LZD combination did not enhance the effect in killing bacillus. In this sense, additional, in vitro and in vivo studies are needed to evaluate the effect of RIF/LZD combination in order to better understand the adjunctive action of LZD in the treatment of TB and prevent the emergence of mutants with resistance to the available anti-TB drugs.

Drug exposure in early pregnancy might be related to the effects of increased maternal progesterone in implantation period

Aim: This short communication aims to evaluate the relation in between drug exposure time and early pregnancy regarding gestational weeks.Methods: The study covers the referrals made to the Department of Pharmacology for a teratogenic consultation in a 3-year period. From the recordings of pregnant women, the last menstrual period and the starting date of medication were used to determine the time of prescription with regard to gestational weeks.Results: In all of the three years, potentially teratogenic medication was prescribed more frequently in the 3rd, 4th and 5th gestational weeks (in between 15–35 days of pregnancy). Approximately 75% of the pregnant women in the study were prescribed with drugs, most frequently with analgesics, antibiotics, gastrointestinal drugs and antidepressants, in these gestational weeks.Conclusions: The timing of prescriptions in early pregnancy frequently coincides with the increased levels of maternal progesterone in implantation period. Progesterone may lead to negative mood symptoms of an increased pain perception, anxiety, irritability and aggression in some of the pregnant women and therefore causes an increased stress condition which in turn may result in pain, infection and inflammation in the individual. Taking the frequently used medications into consideration, the reason for prescriptions in this period might be related to the symptoms originating from the effects of progesterone. Future studies are needed to better demonstrate this association of drug exposure and effects of maternal progesterone in early pregnancy.

Abstract A14: Predicting the response of triple negative breast cancer to doxorubicin

Abstract Introduction: The goal of this study is to establish a data-driven model of cytotoxic therapy that predicts in vitro cell population dynamics in response to a single doxorubicin dose. Whereas traditional therapy response assays are evaluated at a single timepoint following constant exposure to drug, this abstract focuses on a series of fluorescence microscopy experiments that characterize the temporal dynamics of SUM-149PT cell populations, a model of triple negative breast cancer, in response to time-resolved doxorubicin treatments. These data are then used to train a model to predict population dynamics following exposure to doxorubicin. Experimental Design: SUM-149PT cells, labeled with a nuclear fluorescent marker, were seeded in microtiter plates and allowed to reach a steady proliferation rate. Doxorubicin was then introduced at concentrations ranging from 10 nM to 2.5 μM and subsequently removed via media replacement after 6, 12, or 24 hours. These cells were imaged daily via fluorescent microscopy for 25 days following application of doxorubicin. Nuclei were segmented and counted to quantify cell population. Six replicates of each of the 28 treatment conditions were collected. A logistic growth model modified by a set of time-dependent growth functions was employed for this study. Briefly, two piecewise growth functions were implemented: a constant function and a monoexponential function. The constant function assumed an immediate transition from the pre-treatment growth rate to a stable, post-treatment rate following doxorubicin exposure. The monoexponential function allowed for a smooth induction of drug effect, while ultimately allowing for recovery of the cell population. Both models were fit to each treatment condition independently using MATLAB (Natick, MA). The area under the concentration of drug versus time curve (AUC) was used to summarize each treatment condition, allowing for comparison of data from all exposure times. The ability of the model to predict population changes following treatment at new concentrations and exposure times was then evaluated. A leave-one-out approach was employed for these predictions. For each treatment condition in the test set, the Akaike information criterion (AIC) was used to select the appropriate growth model, and the appropriate parameters were learned from the training data. The average percent error of both the best fit and predicted models are reported. Results: SUM-149PT cellular response to doxorubicin was tightly linked to both drug concentration and exposure time. Higher doses (>1 μM) induced rapid cell death. Smaller doses (<1 μM) induced a concentration-dependent nonlinear response defined by an initial increase in population size that, depending on exposure time, was followed by a protracted decrease in cell number and subsequent regrowth of the population. The half-maximal effect of doxorubicin was observed at an AUC of 1136 (±151) nM hr. The constant model was selected at both AUC extremes (AUC < 250 and AUC > 15000 nM–hr). The monoexponential model was selected at intermediate AUC's. The mean percent error of the AIC-selected models across all treatment conditions was 12.4 (±11.0) while the mean percent error of the predicted models was 14.9 (±13.0). Conclusion: These time-resolved treatment protocols replicate the clinically observed pharmacokinetics of cytotoxic therapies more closely than the constant concentrations in previous dose-response assays. With this data-rich approach, the in vitro response of a triple negative breast cancer cell line to doxorubicin can be predicted. Of note, different models are selected over the range of AUC's to reflect the various drug mechanisms of action. This approach should allow for predictions of tumor response in animal models and, eventually, personalized, computationally-optimized treatment regimens that maximize tumor control with doxorubicin. Citation Format: Matthew T. McKenna, Stephanie L. Barnes, Jared A. Weis, Abigail M. Searfoss, Darren R. Tyson, Erin C. Rericha, Michael I. Miga, Vito Quaranta, Thomas E. Yankeelov. Predicting the response of triple negative breast cancer to doxorubicin. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A14.

Incidence of tuberculosis among patients with rheumatoid arthritis using TNF blockers in Brazil: data from the Brazilian Registry of Biological Therapies in Rheumatic Diseases (Registro Brasileiro de Monitoração de Terapias Biológicas – BiobadaBrasil)

ObjectivesTo assess the incidence of tuberculosis and to screen for latent tuberculosis infection among Brazilians with rheumatoid arthritis using biologics in clinical practice. Patients and methodsThis cohort study used data from the Brazilian Registry of Biological Therapies in Rheumatic Diseases (Registro Brasileiro de Monitoração de Terapias Biológicas – BiobadaBrasil), from 01/2009 to 05/2013, encompassing 1552 treatments, including 415 with only synthetic disease-modifying anti-rheumatic drugs, 942 synthetic DMARDs combined with anti-tumor necrosis factor (etanercept, infliximab, adalimumab) and 195 synthetic DMARDs combined with other biologics (abatacept, rituximab and tocilizumab). The occurrence of tuberculosis and the drug exposure time were assessed, and screening for tuberculosis was performed. Statistical analysis: Unpaired t-test and Fisher's two-tailed test; p<0.05. ResultsThe exposure times were 981 patient-years in the controls, 1744 patient-years in the anti-TNF group (adalimumab=676, infliximab=547 and etanercept=521 patient-years) and 336 patient-years in the other biologics group. The incidence rates of tuberculosis were 1.01/1000 patient-years in the controls and 2.87 patient-years among anti-TNF users (adalimumab=4.43/1000 patient-years; etanercept=1.92/1000 patient-years and infliximab=1.82/1000 patient-years). No cases of tuberculosis occurred in the other biologics group. The mean drug exposure time until the occurrence of tuberculosis was 27(11) months for the anti-TNF group. ConclusionsThe incidence of tuberculosis was higher among users of synthetic DMARDs and anti-TNF than among users of synthetic DMARDs and synthetic DMARDs and non-anti-TNF biologics and also occurred later, suggesting infection during treatment and no screening failure.

Incidência de tuberculose em pacientes com artrite reumatoide em uso de bloqueadores do TNF no Brasil: dados do Registro Brasileiro de Monitoração de Terapias Biológicas BiobadaBrasil

ObjectivesTo assess the incidence of tuberculosis and to screen for latent tuberculosis infection among Brazilians with rheumatoid arthritis using biologics in clinical practice. Patients and methodsThis cohort study used data from the Brazilian Registry of Biological Therapies in Rheumatic Diseases (Registro Brasileiro de Monitoração de Terapias Biológicas–BiobadaBrasil), from 01/2009 to 05/2013, encompassing 1,552 treatments, including 415 with only synthetic disease‐modifying anti‐rheumatic drugs, 942 synthetic DMARDs combined with anti‐tumor necrosis factor (etanercept, infliximab, adalimumab) and 195 synthetic DMARDs combined with other biologics (abatacept, rituximab and tocilizumab). The occurrence of tuberculosis and the drug exposure time were assessed, and screening for tuberculosis was performed. Statistical analysis: Unpaired t‐test and Fisher's two‐tailed test; p<0.05. ResultsThe exposure times were 981 patient‐years in the controls, 1744 patient‐years in the anti‐TNF group (adalimumab=676, infliximab=547 and etanercept=521 patient‐years) and 336 patient‐years in the other biologics group. The incidence rates of tuberculosis were 1.01/1,000 patient‐years in the controls and 2.87 patient‐years among anti‐TNF users (adalimumab=4.43/1,000 patient‐years; etanercept=1.92/1,000 patient‐years and infliximab=1.82/1,000 patient‐years). No cases of tuberculosis occurred in the other biologics group. The mean drug exposure time until the occurrence of TB was 27(11) months for the anti‐TNF group. ConclusionsThe incidence of tuberculosis was higher among users of synthetic DMARDs and anti‐TNF than among users of synthetic DMARDs and synthetic DMARDs and non‐anti‐TNF biologics and also occurred later, suggesting infection during treatment and no screening failure

Alkaloids of fascaplysin are effective conventional chemotherapeutic drugs, inhibiting the proliferation of C6 glioma cells and causing their death in vitro.

Glioblastoma multiforme is an invasive malignant glial brain tumor with a poor prognosis for patients. The primary reasons that lead to the development of treatment resistance are associated with tumor cells infiltrating the brain parenchyma and the specific properties of tumor stem cells. A crucial research area in medical science is the search for effective agents that are able to act on these targets. Fascaplysin alkaloids possess potent antitumor activity. Modern methods for the targeted delivery of drugs reveal extensive possibilities in terms of the clinical use of these compounds. The aim of the present study was to establish effective concentrations of fascaplysin that inhibit the growth and kill the cells of glial tumors, as well as to perform a comparative analysis of fascaplysin's effectiveness in relation to other chemotherapy drugs. C6 glioma cells were utilized as an optimal model of glioblastoma. It was established that fascaplysin at 0.5 µM has a strong cytotoxic effect, which is subsequently replaced by tumor cell death via apoptosis as the length of drug exposure time is increased. Fascaplysin kills glioma cells at a dose higher than 0.5 µM. The efficiency of fascaplysin was observed to significantly exceed that of temozolomide. Therefore, a significant feature of fascaplysin is its ability to inhibit the growth of and kill multipotent tumor cells.

Twice Daily Dosing with the Highly Specific BTK Inhibitor, Bgb-3111, Achieves Complete and Continuous BTK Occupancy in Lymph Nodes, and Is Associated with Durable Responses in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL)

Twice Daily Dosing with the Highly Specific BTK Inhibitor, Bgb-3111, Achieves Complete and Continuous BTK Occupancy in Lymph Nodes, and Is Associated with Durable Responses in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL)

Identification of Target Pathways Induced By the Multiple Myeloma Tumor Microenvironment Using Activity-Based Protein Profiling and Ex Vivo Protein Kinase Inhibitor Screening

Multiple myeloma (MM) is a heterogeneous plasma cell neoplasm that remains all but incurable despite recent advances in treatment. Indeed, nearly all patients eventually experience disease progression or relapse due to a reservoir of residual myeloma cells that appear to persist through pro-survival signaling from interactions with the tumor microenvironment (TME), leading to eventual clonal expansion. Thus, identifying targets that are induced in MM by the TME may reveal new and important targets amenable to therapeutic intervention. To develop a non-biased method to screen bone marrow specimens from myeloma patients for activated targets throughout the course of disease, we used a combination of activity-based protein profiling (ABPP) and a high-throughput protein kinase inhibitor (PKI) screen using a platform that recapitulates the TME. Target validation was then performed using ex vivo functional screens of pathways using MM patient specimens.The MM cell lines MM1.S, H929, and OPM2 were grown in mono-culture or co-culture with HS5 bone marrow stroma cells for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe (ActivX, Thermo). Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer). Using this method, 176, 136, and 85 kinases out of a total of 1511, 1409, and 1281 proteins were preferentially enriched by 2-fold change from MM1.S, H929, and OPM2 myeloma cells grown in co-culture conditions with HS5 bone marrow stroma, respectively. Of these, 42 kinases were common to all three and 87 were common to two of three MM cell lines. Kinases were chosen for target validation after pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database to identify signaling networks.To identify functionally relevant signaling networks identified via ABPP experiments, the same MM cell lines were simultaneously screened with 30 protein kinase inhibitors (PKIs) in a novel high throughput viability assay. This label-free method measures the viability of MM cells grown in a collagen matrix with bone marrow stroma cells in 384-well plates to simulate the TME by capturing brightfield images every 30 minutes for 96h using a motorized microscope equipped with an incubation chamber. Digital image analysis software measures live cell numbers by detecting membrane motion and generates viability curves as a function of drug concentration and exposure time (Khin et al. Cancer Research 2014). This functional screen confirming known MM survival networks, validated 12 kinases/PKIs in the context of the TME and highlighted novel targetable pathways.To provide an additional level of screening, the same PKIs were tested in CD138-MACS-selected cells from 15 MM patient specimens in a high-throughput viability assay. Eight PKIs targeting IGFR, PLK1, Abl, mTOR, FAK/Pyk2, ALK, Akt, and Casein Kinase-1δ (CK1δ)/CK1ε also showed significant activity in the 15 primary MM specimens. Our three-tiered pharmaco-proteomic screen identified eight kinases critical to MM survival in the context of the TME. Notably, a highly specific in-house inhibitor of Casein Kinase 1δ/CK1ε, SR-3029, which targets the Wnt/β-catnenin pathway, was identified as the most effective compound assessed as a single agent in our ex vivo viability assay in all patients with an average 36h LD50 of 290nM. This compound is under further investigation in MM (Submitted Abstract: Burger, et al, ASH 2016). Additional studies are underway to functionally interrogate the pathways identified in this screen, including ErbB1/EGFR, EphA1 and AMPK. Future work will optimize this method for evaluation of primary bone marrow specimens with ABPP followed by functional validation to better predict potential clinical response at different disease stages. We anticipate that this iterative "at the moment of care" approach is critical because drug resistant tumor phenotypes fluctuate with therapy, and this strategy can track and define clinically relevant changes in tumor cells in situ after the selection pressures applied by exposure to therapy. DisclosuresShain:Novartis: Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Signal Genetics: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Myelodysplastic Syndromes and Acute Myelogenous Leukemia Resulting from Therapy for Autoimmune Disease, a Case-Control Cohort Study of 40,011 Patients

▪BackgroundMyeloid neoplasms (MN) occurring secondary to treatment of non-malignant disease, especially autoimmune diseases (AID), and the overlap between them, is an increasing clinically important observation. Confounding variables for myeloid transformation include the nature of autoimmune disease as an incessant, chronically activated inflammatory cascade that has been associated with an increased risk of myeloid malignancy, and disease modifying therapies, often with cytotoxic and immunosuppressive drugs. Thus, the addition of therapeutic agents for treatment of AID theoretically amplifies the opportunity for MN to develop in genetically susceptible individuals. To our knowledge, there is no large study evaluating a comprehensive primary AID population that developed MDS or AML with detailed assessment of therapeutic interventions for AID. The presented study queries the association of cytotoxic, anti-inflammatory, and immune-modulating agents to treat AID as risk factors for developing myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML).MethodsIn a 10-year period from 2004-2014, a total of 40,011 patients with an ICD-9 coded diagnosis of AID were seen at Mayo Clinic Arizona and Florida campuses; 311 patients from this population had a concomitant coded diagnosis of MDS and/or AML. In an IRB approved, retrospective chart review, we confirmed 86 cases meeting strict inclusion criteria for AID and confirmed MDS or AML. We performed a case-control match from the same primary autoimmune population for age, gender, and autoimmune specific diagnosis at a 2:1 ratio querying for therapeutic interventions.ResultsMyeloid neoplasm specifics in our case cohort of 86 patients were 55 MDS, 21 de novo AML, and 10 AML with history of MDS. Average age was 72 years with a slight male predominance (57%). Rheumatoid arthritis (26.4%), psoriasis (20.7%), systemic lupus (13.8%), was the most common autoimmune profiles. Median onset of autoimmune disease to diagnosis of myeloid neoplasm was 6 years (range 1-54 years). Up to 26% controls documented no systemic agent for treatment of autoimmune disease and similarly 12.8% of the case cohort (p=0.142). Case and control populations treated with systemic therapies had similar exposures by category for immune-modulating agents (33.7% and 37.8% respectively) and cytotoxic therapies (47.7 and 44.8% respectively). A total of 57/86 cases (66.3%) received either a cytotoxic or immune-modulating agent. In comparison, 105/172 (61.1%) controls received either agent; p=0.495. Azathioprine was the only statistically significant agent exposure observed more frequently in the study cohort than controls with an OR of 7.05 (p= < 0.001). There was no increased incidence of MDS or AML in TNF-antagonist treated patients. No significant correlation for duration of agent exposure by drug category was observed.DiscussionIn a large primary AID population, azathioprine exposure was associated with a 7 fold risk of MDS or AML (p=< 0.001). Notable, but not statistically significant case cohort trends among cytotoxic agents was exposure to cyclophosphamide (OR 3.58) followed by mitoxantrone (OR 2.73). Methotrexate, mercaptopurine, and mycophenolate had favorable odd ratios, but these were not statistically significant. No association was found for TNF-inhibitory agents. Finally, there was no association of drug exposure time to the incidence in development of MDS or AML. DisclosuresDueck:Bayer: Honoraria. Mesa:Gilead: Research Funding; Novartis: Consultancy; Celgene: Research Funding; Incyte Corporation: Research Funding; Galena: Consultancy; Ariad: Consultancy; CTI: Research Funding; Promedior: Research Funding. Al-Kali:Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding. Foran:Millennium Pharmaceuticals, Inc.: Research Funding; novartis: Honoraria; karyopharm: Honoraria; medscape: Honoraria; pfizer: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding.

Anti-Invasive and Anti-Proliferative Synergism between Docetaxel and a Polynuclear Pd-Spermine Agent.

The present work is aimed at evaluating the antitumour properties of a Pd(II) dinuclear complex with the biogenic polyamine spermine, by investigating: i) the anti-angiogenic and anti-migration properties of a Pd(II) dinuclear complex with spermine (Pd2Spm); ii) the anti-proliferative activity of Pd2Spm against a triple negative human breast carcinoma (MDA-MB-231); and finally iii) the putative interaction mediated by combination of Pd2Spm with Docetaxel. Anti-invasive (anti-angiogenic and anti-migratory) as well as anti-proliferative capacities were assessed, for different combination schemes and drug exposure times, using the CAM assay and VEGFR2 activity measurement, the MatrigelTM method and the SRB proliferation test. The results thus obtained evidence the ability of Pd2Spm to restrict angiogenesis and cell migration: Pd2Spm induced a marked inhibition of migration (43.8±12.2%), and a higher inhibition of angiogenesis (81.8±4.4% for total length values, at 4 μM) as compared to DTX at the clinical dosage 4x10-2 μM (26.4±14.4%; n = 4 to 11). Combination of Pd2Spm/DTX was more effective as anti-invasive and anti-proliferative than DTX or Pd2Spm in sole administration, which is compatible with the occurrence of synergism: for the anti-angiogenic effect, IC50(Pd2Spm/DTX) = 0.5/0.5x10-2 μM vs IC50(DTX) = 1.7x10-2 μM and IC50(Pd2Spm) = 1.6 μM. In conclusion, the reported effects of Pd2Spm on angiogenesis, migration and proliferation showed that this compound is a promising therapeutic agent against this type of breast cancer. Moreover, combined administration of Pd2Spm and DTX was found to trigger a substantial synergetic effect regarding angiogenesis inhibition as well as anti-migratory and anti-proliferative activities reinforcing the putative use of Pd(II) complexes in chemotherapeutic regimens. This is a significant outcome, aiming at the application of these combined strategies towards metastatic breast cancer (or other type of resistant cancers), justifying further studies that include pre-clinical trials.

AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function.

Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury.

Abstract 776: Multiscale treatment response model for triple-negative breast cancer linking drug pharmacokinetics to tumor cell population dynamics

Abstract Introduction The goal of this study is to establish a predictive model of cytotoxic therapy that incorporates in vitro drug pharmacokinetics and cell-scale therapy response data, on a cell-line specific basis. We report on a series of time-resolved fluorescence microscopy experiments to characterize the uptake of doxorubicin and its effect on the population dynamics of MDA-MB-231 cells, a model of triple negative breast cancer. Experimental Design We leveraged the intrinsic fluorescence of doxorubicin to measure its uptake by MDA-MB-231 cells. Cells, labeled with a fluorescent nuclear marker, were seeded in microtiter plates and incubated with doxorubicin concentrations ranging from 10 nM to 10 μM for 6, 12, or 24 hours. These plates were imaged daily via bright field and fluorescent microscopy after addition of doxorubicin. Nuclei were segmented and automatically counted to quantify cell population size. Counts were normalized to population size at time of treatment and converted to population doublings. On a separate channel, extracellular, cytoplasmic, and nuclear doxorubicin fluorescence were quantified. A compartment model describing the movement of doxorubicin from the extracellular space into cells was fit to these data. We then constructed a cell treatment response model and fit it, coupled with the compartment model, to the population data using MATLAB. Results MDA-MB-231 cellular response to doxorubicin was tightly linked to both drug concentration and exposure time. Higher doses (&amp;gt; 1 μM) invariably induced rapid cell death. Smaller doses (&amp;lt; 1 μM) induced a concentration-dependent nonlinear response defined by an initial increase in population size that, depending on exposure time, was followed by a protracted decrease in cell number. For example, when treated with 156 nM for 6, 12, and 24 hours, we observed, respectively, an average of 2.6, 2.1, and 0.67 population doublings over the first 150 hours after treatment (p &amp;lt; 0.05 among groups). These populations then either held stable or receded out to 400 hours, when we observed net population doublings of 2.6, 1.7, and 0.037, respectively (p &amp;lt; 0.05). Untreated cells followed a logistic growth pattern, with an average total of 4.4 population doublings. Conclusion These time-resolved treatment protocols replicate clinically observed pharmacokinetics of cytotoxic therapies more closely than the constant concentrations in previous dose-response assays. By explicitly considering both drug and population dynamics, our mathematical model enables exploration, in silico, of treatment protocols intractable experimentally. Predictions from model simulations can then be tested experimentally, hopefully allowing for computationally-optimized and experimentally validated treatment regimens that maximize cytotoxic effects of doxorubicin. Citation Format: Matthew T. McKenna, Stephanie L. Barnes, Abigail Searfoss, Darren R. Tyson, Erin Rericha, Vito Quaranta, Thomas E. Yankeelov. Multiscale treatment response model for triple-negative breast cancer linking drug pharmacokinetics to tumor cell population dynamics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 776.

Abstract 2169: High content screening in MCF7 and MCF10A cells show differential responses depending on oxygen levels and mechanistic readout for viability

Abstract Oxygen levels in typical cell culture conditions do not accurately reflect the oxygen levels cells are exposed to within the body. Furthermore, oxygen levels can vary within the tumor microenvironment. These variances can affect how cells respond to a variety of drugs and small molecules often used during cancer treatment. Previous studies have shown altered drug sensitivities in MCF7 cells depending on the oxygen levels the cells are grown in. To further understand how oxygen levels affect drug sensitivity, the response of tumorigenic MCF7 cells were compared to non-malignant MCF10A cells, cultured under low and high oxygen. The goal of this study was to examine the differences in the sensitivity of MCF7 and MCF10A cells to drugs from the Tocriscreen Total compound library when cultured under low and high oxygen levels and for different drug exposure time periods. Cells were grown using standard cell culture conditions (19% oxygen) or low oxygen (5%). Using high-content imaging and high-throughput analysis methods, viability was assessed using two different mechanistic readouts; cellular metabolic activity and membrane permeability. Post-screening analysis was performed to confirm positive hits by performing dose-responses and determining IC50 concentrations using the same reagents as in the screen, and reagents to assess oxidative stress and cellular proliferation. Results showed that viability differed depending on mechanistic readout and platform method used to determine hits as well as duration of exposure to compound. In addition, the response of MCF10A cells was not always identical to that of the MCF7 cells. Post-screening analysis of “hits” indicated different potencies of compounds tested depending on oxygen level and cell type. These data suggest that some drugs may affect MCF7 and MCF10A cells differently depending on environmental oxygen levels and mechanistic readout used to determine cellular health. Citation Format: Michelle Yan, Michael O’Grady, Anderson April, Scott Clarke, Quentin Low, Carolyn DeMarco, Veronica Calderon, Kathy Kihn, Leticia Montoya, Carmen Finnessy, Marcy Wickett. High content screening in MCF7 and MCF10A cells show differential responses depending on oxygen levels and mechanistic readout for viability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2169.