Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Evaluation of edonerpic maleate as a CRMP2 inhibitor for pain relief.

We have previously reported that the microtubule-associated collapsin response mediator protein 2 (CRMP2) is necessary for the expression of chronic pain. CRMP2 achieves this control of nociceptive signaling by virtue of its ability to regulate voltage-gated calcium and sodium channels. To date, however, no drugs exist that target CRMP2. Recently, the small molecule edonerpic maleate (1 -{3-[2-(1-benzothiophen-5-yl)ethoxy]propyl}azetidin-3-ol maleate), a candidate therapeutic for Alzheimer's disease was reported to be a novel CRMP2 binding compound with the potential to decrease its phosphorylation level in cortical tissues in vivo. Here we sought to determine the mechanism of action of edonerpic maleate and test its possible effect in a rodent model of chronic pain. We observed: (i) no binding between human CRMP2 and edonerpic maleate; (ii) edonerpic maleate had no effect on CRMP2 expression and phosphorylation in dorsal root ganglion (DRG) neurons; (iii) edonerpic maleate-decreased calcium but increased sodium current density in DRG neurons; and (iv) edonerpic maleate was ineffective in reversing post-surgical allodynia in male and female mice. Thus, while CRMP2 inhibiting compounds remain a viable strategy for developing new mechanism-based pain inhibitors, edonerpic maleate is an unlikely candidate.

Synthesis and evaluation of styrene-maleic acid copolymer conjugated amphotericin B.

Amphotericin B (AmB), which plays a central role in the treatment of systemic fungal infections, is difficult to formulate because it's sparingly soluble in water and organic solvents. We previously prepared AmB-loaded micelles using styrene-maleic acid copolymer (SMA). Although solubilization was achieved by this formulation, stability in the blood circulation was as insufficient as that of Fungizone®, which is a conventional formulation of AmB. Meanwhile, it is well known that polymer-drug conjugates are more stable in circulation than drug-loaded micelles. Therefore, in this study, we developed covalently conjugated SMA-AmB (SMA-AmB conjugate). The SMA-AmB conjugate was found to be soluble and present as micelles in aqueous solution. Furthermore, it was revealed that this micelle behaves as a larger molecule by forming a complex with albumin. The circulation in the blood increased significantly compared to that of Fungizone®, which was suggested to be due to this complex-forming ability. Although in vitro and in vivo antifungal activity of the SMA-AmB conjugate against Saccharomyces cerevisiae was reduced by 1/3 compared to that of Fungizone®, hemolysis decreased to 1/40 or less, and the LD50 decreased to 1/10. In conclusion, it is expected that the SMA-AmB conjugate can be a polymer-therapeutic agent with high antifungal selectivity.

Tissue Distribution and Systemic Toxicity Evaluation of Raloxifene Targeted Polymeric Micelles of Poly (Styrene-Maleic Acid)-Poly (Amide- Ether-Ester-Imide)-Poly (Ethylene Glycol) Loaded With Docetaxel in Breast Cancer Bearing Mice.

BACKGROUND: Due to the low water solubility of Docetaxel (DTX), it is formulated with ethanol and Tween 80 with lots of side effects. For this reason, special attention has been paid to formulate it in new drug nano-carriers. OBJECTIVE: The goal of this study was to evaluate the safety, antitumor activity and tissue distribution of the novel synthesized Raloxifene (RA) targeted polymeric micelles. METHODS: DTX-loaded RA-targeted polymeric micelles composed of poly(styrene-maleic acid)- poly(amide-ether-ester-imide)-poly(ethylene glycol) (SMA-PAEE-PEG) were prepared and their antitumor activity was studied in MC4-L2 tumor-bearing mice compared with non-targeted micelles and free DTX. Safety of the micelles was studied by Hematoxylin and Eosin (H&E) staining of tumors and major organs of the mice. The drug accumulation in the tumor and major organs was measured by HPLC method. RESULTS: The results showed better tumor growth inhibition and increased survival of mice treated with DTX-loaded in targeted micelles compared to the non-targeted micelles and free DTX. Histopathological studies, H&E staining of tumors and immunohistochemical examination showed the potential of DTX-loaded RA-targeted micelles to inhibit tumor cells proliferation. The higher accumulation of the DTX in the tumor tissue after injection of the micelles compared to the free DTX may indicate the higher uptake of the targeted micelles by the G-Protein-Coupled Estrogen Receptors (GPER). CONCLUSION: The results indicate that RA-conjugated polymeric micelles may be a strong and effective drug delivery system for DTX therapy and uptake of the drug into tumor cells, and overcome the disadvantages and side effects of conventional DTX.

A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure.

Oxidative stress leads to the activation of the Nuclear factor-erythroid-2-related factor 2 (Nrf2) pathway. While most studies have focused on the activation of the Nrf2 pathway after single chemical treatment, little is known about the dynamic regulation of the Nrf2 pathway in the context of repeated exposure scenarios. Here we employed single cell live imaging to quantitatively monitor the dynamics of the Nrf2 pathway during repeated exposure, making advantage of two HepG2 fluorescent protein reporter cell lines, expressing GFP tagged Nrf2 or sulfiredoxin 1 (Srxn1), a direct downstream target of Nrf2. High throughput live confocal imaging was used to measure the temporal dynamics of these two components of the Nrf2 pathway after repeated exposure to an extensive concentration range of diethyl maleate (DEM) and tert-butylhydroquinone (tBHQ). Single treatment with DEM or tBHQ induced Nrf2 and Srxn1 over time in a concentration-dependent manner. The Nrf2 response to a second treatment was lower than the response to the first exposure with the same concentration, indicating that the response is adaptive. Moreover, a limited fraction of individual cells committed themselves into the Nrf2 response during the second treatment. Despite the suppression of the Nrf2 pathway, the second treatment resulted in a three-fold higher Srxn1-GFP response compared to the first treatment, with all cells participating in the response. While after the first treatment Srxn1-GFP response was linearly related to Nrf2-GFP nuclear translocation, such a linear relationship was less clear for the second exposure. siRNA-mediated knockdown demonstrated that the second response is dependent on the activity of Nrf2. Several other, clinically relevant, compounds (i.e., sulphorophane, nitrofurantoin and CDDO-Me) also enhanced the induction of Srxn1-GFP upon two consecutive repeated exposure. Together the data indicate that adaptation towards pro-oxidants lowers the Nrf2 activation capacity, but simultaneously primes cells for the enhancement of an antioxidant response which depends on factors other than just Nrf2. These data provide further insight in the overall dynamics of stress pathway activation after repeated exposure and underscore the complexity of responses that may govern repeated dose toxicity.

Cyclodextrin-grafted poly(anhydride) nanoparticles for oral glibenclamide administration. In vivo evaluation using C. elegans.

The aim of this work was to prepare and evaluate cyclodextrins-modified poly(anhydride) nanoparticles to enhance the oral administration of glibenclamide. A conjugate polymer was synthesized by incorporating hydroxypropyl-ß-cyclodextrin to the backbone of poly(methylvinyl ether-co-maleic anhydride) via Steglich reaction. The degree of substitution of anhydride rings by cyclodextrins molecules was calculated to be 4.9% using H-NMR spectroscopy. A central composite design of experiments was used to optimize the preparative process. Under the optimal conditions, nanoparticles displayed a size of about 170 nm, a surface charge of -47 mV and a drug loading of 69 µg GB/mg. X-ray diffraction studies confirmed the loss of the crystalline structure of GB due to its dispersion into the nanoparticles, either included into cyclodextrin cavities or entrapped in the polymer chains. Glibenclamide was mainly release by Fickian-diffusion in simulated intestinal fluid. GB-loaded nanoparticles produced a hypolipidemic effect over C. elegans N2 wild-type and daf-2 mutant. The action mechanism included daf-2 and daf-28 genes, both implicated in the insulin signaling pathway of C. elegans. In summary, the covalent linkage of cyclodextrin to the poly(anhydride) backbone could be an interesting strategy to prepare nanoparticles for the oral administration of glibenclamide.

Dissolving polymeric microneedle arrays for enhanced site-specific acyclovir delivery.

Acyclovir is widely indicated for the treatment of herpes labialis (cold sores), typically caused by the herpes simplex virus type 1 (HSV-1). However, topical acyclovir has poor efficacy, due to its low skin permeability. The purpose of this study was, therefore, to evaluate the ability of dissolving polymeric microneedle (MN) arrays to improve the local delivery of acyclovir. Acyclovir-loaded dissolving MN arrays (0.49 cm2) were formulated from aqueous blends of Gantrez® S-97 with 361 needles per array (589 ±â€¯9.29 µm height). MN penetrated excised neonatal porcine skin, showing sufficient mechanical strength to resist compression and maintained their appearance after application of a 0.089 N per needle force for 30 s. Dissolution of the needles was observed within 15 min after application to skin and the needles had completely dissolved at 2 h in vitro. In vitro skin permeation studies revealed that the percentage of total acyclovir loading which permeated the skin over a 24 h period using MNs was approximately 45 times higher than that of a commercial cream formulation (Lipsore®). The accumulation of acyclovir at the basal epidermis, the target site of the herpes simplex virus, using MNs was a total of 21.5 µg/cm3in vitro, which is approximately 5 times greater than the 99% inhibition of viral cytopathic effect (ID99) required for HSV infections. This level was also 16 times higher than that obtained using the cream formulation. An in vivo study showed that the use of acyclovir-loaded dissolving MN arrays successfully provided intradermal delivery of acyclovir over a 48 h period and the drug levels in the skin delivered using MN arrays (45.09 ±â€¯13.28 µg/cm3) were superior to those generated by the cream formulation (4.55 ±â€¯1.37 µg/cm3). Accordingly, acyclovir-loaded dissolving MN arrays could be a promising approach for effective local delivery of acyclovir.

Salt formation improved the properties of a candidate drug during early formulation development.

The purpose of this study was to investigate if AZD5329, a dual neurokinin NK1/2 receptor antagonist, is a suitable candidate for further development as an oral immediate release (IR) solid dosage form as a final product. The neutral form of AZD5329 has only been isolated as amorphous material. In order to search for a solid material with improved physical and chemical stability and more suitable solid-state properties, a salt screen was performed. Crystalline material of a maleic acid salt and a fumaric acid salt of AZD5329 were obtained. X-ray powder diffractiometry, thermogravimetric analysis, differential scanning calorimetry and dynamic vapor sorption were used to investigate the physicochemical characteristics of the two salts. The fumarate salt of AZD5329 is anhydrous, the crystallization is reproducible and the hygroscopicity is acceptable. Early polymorphism assessment work using slurry technique did not reveal any better crystal modification or crystallinity for the fumarate salt. For the maleate salt, the form isolated originally was found to be a solvate, but an anhydrous form was found in later experiments; by suspension in water or acetone, by drying of the solvate to 100-120 °C or by subjecting the solvate form to conditions of 40 °C/75%RH for 3 months. The dissolution behavior and the chemical stability (in aqueous solutions, formulations and solid-state) of both salts were also studied and found to be satisfactory. The compound displays sensitivity to low pH, and the salt of the maleic acid, which is the stronger acid, shows more degradation during stability studies, in line with this observation. The presented data indicate that the substance fulfils basic requirements for further development of an IR dosage form, based on the characterization on crystalline salts of AZD5329.

Poly(methyl vinyl ether-co-maleic acid) for enhancement of solubility, oral bioavailability and anti-osteoporotic effects of raloxifene hydrochloride.

Raloxifene HCl (RH) has poor water solubility and due to its extensive first pass metabolism; its bioavailability is only 2%. The purpose of the present study was to enhance the aqueous solubility, oral bioavailability and anti-osteoporotic effects of RH by electro-sprayed nanoparticles (NPs) in ovariectomized rats. NPs containing RH and different ratio of poly(methyl vinyl ether-co-maleic acid) (PMVEMA) were electrosprayed. The voltage, distance of needle to the collector, flow rate of the solution and polymeric percentage were optimized according to the size of NPs and drug solubility. The optimized formulation was characterized by SEM, XRD, DSC, and FTIR. The pharmacokinetic parameters were studies by oral administration of a single dose of 15mg/kg in Wistar rats. The anti-osteoporotic effects were studied in female ovariectomized rats. Animals were treated with 6mg/kg/day for 2months then serum calcium, phosphorous and alkaline phosphatase levels were measured. RH loaded electrosprayed NPs showed 10-fold enhanced solubility compared to the free drug. Moreover, the XRD and SEM tests displayed an amorphous state of drug in the NPs. FTIR and DSC tests revealed no interaction between the polymer and the drug. Serum calcium, phosphorous and alkaline phosphatase levels were significantly decreased in ovariectomized rats receiving oral RH NPs (P<0.05). No significant difference was detected between RH NPs and estradiol groups (P>0.05). Oral bioavailability of NPs showed 7.5-fold increase compared to the pure drug. The electrosprayed PMVEMA nanoparticles can enhance solubility, bioavailability and antiosteoporotic effects of RH.

Bioequivalence study of two formulations of flupirtine maleate capsules in healthy male Chinese volunteers under fasting and fed conditions.

AIM: This study developed a high-performance liquid chromatography-tandem mass spectrometry method to simultaneously determine the concentrations of flupirtine and its major active metabolite D-13223 in human plasma in order to assess the bioequivalence (BE) of two flupirtine maleate capsules among healthy male Chinese volunteers under fasting and fed conditions. MATERIALS AND METHODS: There were two single-center, randomized, single-dose, open-label, laboratory-blinded, two-period, cross-over studies which included 24 healthy male Chinese volunteers under fasting and fed conditions, respectively. Plasma samples were collected prior to and up to 48 h after dosing. The concentrations of flupirtine and its major active metabolite D-13223 in plasma samples were determined by a validated method, that is, high-performance liquid chromatography coupled with a tandem mass spectrometry detector. Pharmacokinetic metrics of area from time zero to the last measurable concentration (AUC0-t), area under the plasma concentration-time curve from administration to infinite time (AUC0-∞), and Cmax were used for BE assessment. RESULTS: Forty-eight healthy volunteers who met the criteria were enrolled and completed the study. According to the observation of vital signs and laboratory measurement, no volunteers had any adverse reactions. Under fasting condition, the geometric mean ratios (90% CI) of the test/reference drug for flupirtine were 103.0% (98.1%-108.2%) for AUC0-t, 102.9% (98.2%-107.9%) for AUC0-∞, and 97.0% (85.9%-109.5%) for Cmax. Under fed condition, the geometric mean ratios (90% CI) of the test/reference drug for flupirtine were 101.7% (98.4%-105.1%) for AUC0-t, 101.6% (98.5%-104.8%) for AUC0-∞, and 103.5% (94.7%-113.0%) for Cmax. The difference between test and reference formulations, Tmax, was not statistically significant. The 90% CIs of the test/reference AUC ratio and Cmax ratio of D-13223 were also within the acceptance range for BE both under fasting and fed conditions. CONCLUSION: The two formulations of flupirtine maleate capsule were bioequivalent (the test and the reference products) under fasting and fed conditions, and thus both can be used interchangeably in the clinical setting.

Discovery of DS79182026: A potent orally active hepcidin production inhibitor.

Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hepcidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the synthesis and structure-activity relationships (SARs) of a series of benzisoxazole compounds as orally active hepcidin production inhibitors. The optimization study of multi kinase inhibitor 1 led to a potent and bioavailable hepcidin production inhibitor 38 (DS79182026), which showed serum hepcidin lowering effects in a mouse IL-6 induced acute inflammatory model.

Poly (methyl vinyl ether-co-maleic acid) - Pectin based hydrogel-forming systems: Gel, film, and microneedles.

Cross-linking of natural and synthetic polymers is widely explored to achieve the desired material properties (mechanical strength, drug loading capacity, swelling and erosion rates). However, the potential of polymers produced by crosslinking poly (methyl vinyl ether-co-maleic acid) (PMVE/MA) and pectin (PE) in pharmaceutics is mainly unexplored so far. We have investigated the effect of various esterification conditions and pectin content on the physicochemical properties. Materials have been characterized by fourier transform infrared, differential scanning calorimetry and scanning electron microscopy. In addition, swelling and bioadhesive features of PMVE/MA-PE hydrogel systems were investigated. A band shift for the carbonyl group from 1706 to 1776cm-1, and glass transition (Tg) increased from 55.4±0.9°C to 119.5±0.3°C confirmed the formation of esterification reaction within the cross-linked films. Cross-linked PMVE/MA:PE films with a ratio of 5 demonstrated a superior mass increase when compared to 2.5, 3.125, 3.75, 6.25, and 7.5 ratios of the same hydrogel film. Formulations containing PMVE/MA and pectin with a ratio of 3.75 showed superior bioadhesive features. For the first time, we engineered three-dimensional printing based swell-able microneedle arrays made out of cross-linked PMVE/MA-PE. Microneedle arrays height and aspect ratio were ranged from 702.5±11.9µm to 726±23.3µm and 3.12±0.20 to 3.29±0.21, respectively. Cross-linked PMVE/MA-PE Microneedle arrays (10-2, 24h) indicated the least height loss, 22.33±4.15%, during axial compression test; whilst, transverse failure of cross-linked PMVE/MA-PE Microneedle arrays was varied from 0.15±0.05 to 0.25±0.04N/needle. In conclusion, we obtained a novel cross-linked polymer system with promising features of drug delivery and bio-analytical applications.

Non-invasive imaging of the levels and effects of glutathione on the redox status of mouse brain using electron paramagnetic resonance imaging.

Glutathione (GSH) is the most abundant non-protein thiol that buffers reactive oxygen species in the brain. GSH does not reduce nitroxides directly, but in the presence of ascorbates, addition of GSH increases ascorbate-induced reduction of nitroxides. In this study, we used electron paramagnetic resonance (EPR) imaging and the nitroxide imaging probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), to non-invasively obtain spatially resolved redox data from mouse brains depleted of GSH with diethyl maleate compared to control. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of the redox status in vivo and mapped as a "redox map". The obtained redox maps from control and GSH-depleted mouse brains showed a clear change in the brain redox status, which was due to the decreased levels of GSH in brains as measured by a biochemical assay. We observed a linear relationship between the reduction rate constant of MCP and the level of GSH for both control and GSH-depleted mouse brains. Using this relationship, the GSH level in the brain can be estimated from the redox map obtained with EPR imaging.

The Effects of Long-Term Chaetomellic Acid A Administration on Renal Function and Oxidative Stress in a Rat Model of Renal Mass Reduction.

Purpose. This study aimed to evaluate the effect of chronic treatment with chaetomellic acid A (CAA) on oxidative stress and renal function in a model of renal mass reduction. Methods. Male Wistar rats were subjected to 5/6 nephrectomy (RMR) or sham-operated (SO). One week after surgery, rats have been divided into four experimental groups: RMR: RMR rats without treatment (n = 14); RMR + CAA: RMR rats treated with CAA (n = 13); SO: SO rats without treatment (n = 13); and SO + CAA: SO rats treated with CAA (n = 13). CAA was intraperitoneally administered in a dose of 0.23 µg/Kg three times a week for six months. Results. RMR was accompanied by a significant reduction in catalase and glutathione reductase (GR) activity (p < 0.05) and a decrease in reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. CAA administration significantly increased catalase and GR activity (p < 0.05) and increased GSH/GSSG ratio, but no significant difference between the treated and nontreated groups was found in this ratio. No significant differences were found between the RMR groups in any of the parameters of renal function. However, CAA administration slightly improves some parameters of renal function. Conclusions. These data suggest that CAA could attenuate 5/6 RMR-induced oxidative stress.

Miconazole Nitrate-loaded Microparticles For Buccal Use: Immediate Drug Release and Antifungal Effect.

BACKGROUND: Miconazole nitrate has been widely employed in treatment of oral mycoses, however your immediate bio-availability and location in the affected area is critical. OBJECTIVE: The aim of this study was to prepare and evaluate Eudragit® L100 and Gantrez MS-955 microparticles containing miconazole nitrate for oral delivery. METHODS: Microparticles were prepared by spray-drying method to achieve high encapsulation efficiency and increase the drug solubility. The microparticles were formed containing 10% and 20% of drug on polymer Eudragit® L100 (E10 and E20), Gantrez MS-955 (G10 and G20) or their combination (EG10 and EG20). The influence of formulation factors (polymer:drug ratio, type of polymer) on yield percent, encapsulation efficiency, particle size, Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction, differential scanning calorimetry, in vitro drug release and antifungal activity were investigated. RESULTS: Acceptable yield, micrometer-sized and drug-loading efficiencies higher than 89% were obtained. No change in FTIR assignments was recorded after the microencapsulation procedure. X-ray and differential scanning calorimetry studies revealed amorphous/non-crystalline formulations. Miconazole nitrate-microparticles provided a remarkable increase of dissolution rate of the drug. Miconazole nitrate and G10, G20 and EG20 microparticles fitted to biexponential kinetic model, and E10, E20 and EG10 microparticles, monoexponential kinetic model. The antifungal activity test demonstrated that miconazole nitrate-microparticles possessed the same anti-Candida albicans activity as the pure drug. CONCLUSION: These results indicate that miconazole nitrate-microparticles are feasible carriers for increased release of miconazole at oral environment.

Polycationic Nanofibers for Nucleic Acid Scavenging.

Dying cells release nucleic acids (NA) and NA-containing complexes that activate inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation frequently encountered in autoimmune and inflammatory diseases. In this study, grafting of cationic polymers onto a nanofibrous mesh enabled local scavenging of negatively charged pro-inflammatory molecules in the extracellular space. Nucleic acid scavenging nanofibers (NASFs) formed from poly(styrene-alt-maleic anhydride) conjugated with 1.8 kDa bPEI resulted in nanofibers of diameters 486 ± 9 nm. NASFs inhibited the NF-κB response stimulated by the negatively charged agonists, CpG and poly(I:C), in Ramos-blue cells but not Pam3CSK4, a nonanionic agonist. Moreover, NASFs significantly impeded NF-κB activation in cells stimulated with damage-associated molecular pattern molecules (DAMPs) released from doxorubicin killed cancer cells. In vivo application of NASFs to open wounds demonstrated nucleic acid scavenging in wounds of diabetic mice infected with Pseudomonas aeruginosa, suggesting the in vivo efficacy of NASFs. This simple technique of generating NASF results in effective localized anti-inflammation in vitro and local nucleic acid scavenging in vivo.

A combination of sorafenib and nilotinib reduces the growth of castrate-resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. Several tyrosine kinases have been implicated in the development and growth of CRPC, as such targeting these kinases may offer an alternative therapeutic strategy. We established the combination of two tyrosine kinase inhibitors (TKIs), sorafenib and nilotinib, as the most cytotoxic. In addtion, to improve their bioavailability and reduce their metabolism, we encapsulated sorafenib and nilotinib into styrene-co-maleic acid micelles. The micelles' charge, size, and release rate were characterized. We assessed the effect of the combination on the cytotoxicity, cell cycle, apoptosis, protein expression, tumor spheroid integrity, migration, and invasion. The micelles exhibited a mean diameter of 100 nm, a neutral charge, and appeared highly stable. The micellar TKIs promoted greater cytotoxicity, decreased cell proliferation, and increased apoptosis relative to the free TKIs. In addition, the combination reduced the expression and activity of several tyrosine kinases and reduced tumor spheroid integrity and metastatic potential of CRPC cell lines more efficiently than the single treatments. The combination increased the therapeutic potential and demonstrated the relevance of a targeted combination therapy for the treatment of CRPC. In addition, the efficacy of the encapsulated drugs provides the basis for an in vivo preclinical testing.

Pharmacokinetics and bioavailability of oral single-dose maleic acid in biofluids of Sprague-Dawley rats.

Maleic acid (MA) was purposefully adulterated in an array of starch-based foods in Taiwan, inciting a food safety incident. Due to limited data on the pharmacokinetics and bioavailability of ingested MA, we studied pharmacokinetic (PK) parameters in serum and urine of Sprague Dawley rats. Three groups of male and female rats were given three doses of MA by oral gavage; biofluid samples were collected accordingly. Data demonstrated that a non-compartment model best described MA's linear kinetic behavior upon ingestion. The mean residence life of maleic acid in serum was 17.58 h and 9.84 h for low-dosed male and female rats, whereas 8.24 h and 4.17 h for high-dosed male and female rats, respectively. Our results revealed oral bioavailability ranged from 30.8 to 41.0% for males and 32.2-39.1% for females. The data confirmed that ingested MA is absorbed and metabolized rapidly, along with low bioavailability. Future pathological studies may determine whether prolonged and low-level exposures of MA produce nephrotoxicity. These data provide additional contribution to current understanding of the kinetics of MA in a rat model and enable the development of a physiologically based model, which is essential to form the basis of evidenced-based food safety guidelines.

Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice.

Selol is a semi-synthetic compound containing selenite that is effective against cancerous cells and safer for clinical applications in comparison with other inorganic forms of selenite. Recently, we have developed a formulation of poly(methyl vinyl ether-co-maleic anhydride)-shelled selol nanocapsules (SPN), which reduced the proliferative activity of lung adenocarcinoma cells and presented little deleterious effects on normal cells in in vitro studies. In this study, we report on the antitumor activity and systemic effects induced by this formulation in chemically induced lung adenocarcinoma-bearing mice. The in vivo antitumor activity of the SPN was verified by macroscopic quantification, immunohistochemistry and morphological analyses. Toxicity analyses were performed by evaluations of the kidney, liver, and spleen; analyses of hemogram and plasma levels of alanine aminotransferase, aspartate transaminase, urea, and creatinine; and DNA fragmentation and cell cycle activity of the bone marrow cells. Furthermore, we investigated the potential of the SPN formulation to cause hemolysis, activate the complement system, provoke an inflammatory response and change the conformation of the plasma proteins. Our results showed that the SPN reduced the area of the surface tumor nodules but not the total number of tumor nodules. The biochemical and hematological findings were suggestive of the low systemic toxicity of the SPN formulation. The surface properties of the selol nanocapsules point to characteristics that are consistent with the treatment of the tumors in vivo: low hemolytic activity, weak inflammatory reaction with no activation of the complement system, and mild or absent conformational changes of the plasma proteins. In conclusion, this report suggests that the SPN formulation investigated herein exhibits anti-tumoral effects against lung adenocarcinoma in vivo and is associated with low systemic toxicity and high biocompatibility.