Level Of Protein Binding Research Articles

Utility of Physiologically Based Pharmacokinetic Modeling to Investigate the Impact of Physiological Changes of Pregnancy and Cancer on Oncology Drug Pharmacokinetics.

The treatment of cancer during pregnancy remains challenging with knowledge gaps in drug dosage, safety, and efficacy due to the under-representation of this population in clinical trials. Our aim was to investigate physiological changes reported in both pregnancy and cancer populations into a PBPK modeling framework that allows for a more accurate estimation of PK changes in pregnant patients with cancer. Paclitaxel and docetaxel were selected to validate a population model using clinical data from pregnant patients with cancer. The validated population model was subsequently used to predict the PK of acalabrutinib in pregnant patients with cancer. The Simcyp pregnancy population model reasonably predicted the PK of docetaxel in pregnant patients with cancer, while a modified model that included a 2.5-fold increase in CYP2C8 abundance, consistent with the increased expression during pregnancy, was needed to reasonably predict the PK of paclitaxel in pregnant patients with cancer. Changes in protein binding levels of patients with cancer had a minimal impact on the predicted clearance of paclitaxel and docetaxel. PBPK modeling predicted approximately 60% lower AUC and Cmax for acalabrutinib in pregnant versus non-pregnant patients with cancer. Our results suggest that PBPK modeling is a promising approach to investigate the effects of pregnancy and cancer on the PK of oncology drugs and potentially inform dosing for pregnant patients with cancer. Further evaluation and refinement of the population model are needed for pregnant patients with cancer with additional compounds and clinical PK data.

Plasma protein binding and tissue retention kinetics influence the rate and extent of nicotine delivery to the brain

Nicotine from inhaled combustible cigarette smoke is delivered rapidly to the brain, and sufficient unbound nicotine concentrations exert pharmacological effects. In addition to nicotine, combustible cigarette smoke also contains a significant number of toxicants that trigger perturbations, leading to an altered steady state due to differential expression of proteins. In this study, a physiologically based pharmacokinetic (PBPK) model for inhaled nicotine was used to simulate the influence of lysosomal change-driven tissue retention and plasma protein binding levels on nicotine pharmacokinetics (PK). A 3 × increase in tissue lysosomal volumes lowered the nicotine brain maximum concentration (Cmax) by 20.8%. Similarly, a 50% increase in plasma protein binding also lowered the unbound plasma arterial nicotine Cmax by 39.4%. Such fundamental changes in nicotine disposition due to physiological changes in combustible cigarette smokers will lead to altered nicotine consumption and exposure-responses of other weakly basic drugs. Literature reports indicate that nicotine consumed from non-combustible products do not alter drug exposures, indicating fewer or less severe toxicant-driven perturbations with the use of these products. Although several other parameters influence nicotine PK, this PBPK modeling study shows that increased tissue retention and plasma protein binding reduce nicotine delivery to the brain and could lead to differential consumption of combustible cigarettes. These differences in physiological states among combustible cigarette smokers need to be evaluated and should be considered during therapeutic interventions.

Validation of Methods for in Vitro-in Vivo Extrapolation Using Hepatic Clearance Measurements in Isolated Perfused Fish Livers.

In vitro biotransformation assays using hepatocytes or liver subcellular fractions, combined with in vitro-in vivo extrapolation (IVIVE) models, have been proposed as an alternative to live fish bioconcentration studies. The uncertainty associated with IVIVE approaches to date has been attributed to assay protocols, model assumptions, or variability of in vivo data. An isolated perfused trout liver model that measures hepatic clearance has been proposed for validating IVIVE predictions in the absence of other confounding factors. Here, we investigated the hepatic clearances of five chemicals (pyrene, phenanthrene, 4-n-nonlyphenol, deltamethrin, and methoxychlor) in this model and compared measured rates to values predicted from published in vitro intrinsic clearances for validation of IVIVE models. Additionally, we varied protein concentrations in perfusates to test binding assumptions of these models. We found that measured and predicted hepatic clearances were in very good agreement (root mean squared error 16.8 mL h-1 g-1) across three levels of protein binding and across a more diverse chemical space than previously studied within this system. Our results show that current IVIVE methods can reliably predict in vivo clearance rates and indicate that discrepancies from measured bioconcentration factors might be driven by other processes, such as extrahepatic biotransformation, etc., and help streamline optimization efforts to the processes that truly matter.

ESR1 inhibits ionizing radiation-induced ferroptosis in breast cancer cells via the NEDD4L/CD71 pathway

Ferroptosis is the name given to the type of non-apoptotic cell death that is caused by iron accumulation and subsequent lipid peroxidation. However, how ionizing radiation (IR)-induced ferroptosis is regulated in estrogen receptor-positive (ER+) breast cancer cells remains unclear. To attempt to resolve this issue, bioinformatics analysis was performed to evaluate the prognostic value of estrogen receptor 1 (ESR1) in breast cancer tissues. A total of four breast cancer cell lines and an MCF10A non-malignant counterpart were used. Western blotting was used to analyze the levels of protein expression, whereas immunoprecipitation (IP) and ubiquitination experiments were used to test protein binding and ubiquitination levels, respectively. Flow cytometry was subsequently used to analyze cell death and lipid peroxidation levels. The results showed that a high expression level of ESR1 was significantly correlated with poor overall survival in breast cancer. ESR1 knockdown significantly enhanced IR-induced ferroptosis and increased the CD71 protein level. The IP results showed that ESR1 enhanced the binding of the E3 ubiquitin ligase NEDD4L to CD71, promoting the ubiquitination and degradation of CD71, suggesting that CD71 expression was regulated by both ESR1 and NEDD4L. Taken together, the findings in the present study have demonstrated a regulatory relationship between ESR1 and NEDD4L/CD71 in IR-induced ferroptosis. In addition, the ESR1/NEDD4L/CD71 axis may be a potential target for the radiotherapy of breast cancer.

Mosloflavone-Resveratrol Hybrid TMS-HDMF-5z Exhibits Potent In Vitro and In Vivo Anti-Inflammatory Effects Through NF-κB, AP-1, and JAK/STAT Inactivation.

TMS-HDMF-5z is a hybrid of the natural products mosloflavone and resveratrol. It was discovered to show potent inhibitory effects against lipopolysaccharide (LPS)-induced production of inflammatory mediators in RAW 264.7 macrophages. However, its mechanism of action is unknown. Hence this study aimed to demonstrate and explore in vitro and in vivo anti-inflammatory effects of TMS-HDMF-5z and its mechanism of action employing RAW 264.7 macrophages and carrageenan-induced hind paw edema. This work revealed that TMS-HDMF-5z suppressed the LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein, mRNA, and promoter binding levels and tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and interferon-β (IFN-β) at the mRNA expression in RAW 264.7 macrophages. The results showed that TMS-HDMF-5z reduced the transcription and DNA binding activities of nuclear factor-κB (NF-κB) through inhibiting nuclear translocation of p65 and phosphorylation of κB inhibitor α (IκBα), IκB kinase (IKK), and TGF-β activated kinase 1 (TAK1). Additionally, TMS-HDMF-5z attenuated the LPS-induced transcriptional and DNA binding activities of activator protein-1 (AP-1) by suppressing nuclear translocation of phosphorylated c-Fos, c-Jun, and activating transcription factor 2 (ATF2). TMS-HDMF-5z also reduced the LPS-induced phosphorylation of Janus kinase 1/2 (JAK1/2), signal transducers and activators of transcription 1/3 (STAT1/3), p38 mitogen-activated protein kinase (MAPK), and MAPK-activated protein kinase 2 (MK2). In rats, TMS-HDMF-5z alleviated carrageenan-induced hind paw edema through the suppressing iNOS and COX-2 via NF-κB, AP-1, and STAT1/3 inactivation. Collectively, the TMS-HDMF-5z-mediated inhibition of NF-κB, AP-1, and STAT1/3 offer an opportunity for the development of a potential treatment for inflammatory diseases.

Retention of hemostatic and immunological properties of frozen plasma and COVID-19 convalescent apheresis fresh-frozen plasma produced and freeze-dried in Canada.

Randomized clinical trial data show that early plasma transfusion may save lives among trauma patients. Supplying plasma in remote environments is logistically challenging. Freeze-dried plasma (FDP) offers a possible solution. A Terumo BCT plasma freeze-drying system was evaluated. We compared pooled frozen plasma (FP) units with derived Terumo BCT FDP (TFDP) units and pooled COVID-19 convalescent apheresis fresh-frozen plasma (CC-AFFP) with derived CC-TFDP units. Parameters measured were: coagulation factors (F) II; V; VII; VIII; IX; XI; XIII; fibrinogen; Proteins C (PC) and S (PS); antithrombin (AT); α2 -antiplasmin (α2 AP); ADAMTS13; von Willebrand Factor (vWF); thrombin-antithrombin (TAT); D-dimer; activated complement factors 3 (C3a) and 5 (C5a); pH; osmolality; prothrombin time (PT); and activated partial thromboplastin time (aPTT). Antibodies to SARS-CoV-2 in CC-AFFP and CC-TFDP units were compared by plaque reduction assays and viral protein immunoassays. Most parameters were unchanged in TFDP versus FP or differed ≤15%. Mean aPTT, PT, C3a, and pH were elevated 5.9%, 6.9%, 64%, and 0.28 units, respectively, versus FP. CC-TFDP showed no loss of SARS-CoV-2 neutralization titer versus CC-AFFP and no mean signal loss in most pools by viral protein immunoassays. Changes in protein activities or clotting times arising from freeze-drying were <15%. Although C3a levels in TFDP were elevated, they were less than literature values for transfusable plasma. SARS-CoV-2-neutralizing antibody titers and viral protein binding levels were largely unaffected by freeze-drying. In vitro characteristics of TFDP or CC-TFDP were comparable to their originating plasma, making future clinical studies appropriate.

CEFOVECIN PROTEIN BINDING AS A PREDICTOR FOR EXTENDED DURATION OF ACTION: A REVIEW OF CURRENT LITERATURE AND IN VITRO ANALYSIS IN MULTIPLE ZOOLOGICAL SPECIES.

Cefovecin is a third-generation cephalosporin antibiotic with an efficacy of 2 wk following a single injection in domestic dogs and cats. A high degree of plasma protein binding to cefovecin has been proposed as one of the mechanisms responsible for the long elimination half-life, but protein binding has not been evaluated extensively in nondomestic species. In this study, a review of the current literature was conducted, and pharmacokinetic data were compiled for species in which cefovecin has been evaluated thus far. Additionally, in vitro cefovecin protein binding was evaluated in plasma from 22 nondomestic species representing a broad range of taxa. Animals of the order Carnivora demonstrated protein-binding levels of >98%, which is supportive of the long elimination half-life seen in related species. Protein binding was highly variable in Artiodactyl and Perissodactyl species, with dolphins (Tursiops truncatus) and barasingha (Rucervus duvaucelii) displaying high protein binding (99.12% to >99%), but not gazelles (Eudorcas thomsonii) or equids (91.76-92.70%). Cefovecin was not highly bound in any reptiles or birds, corresponding to short half-lives reported for these taxa. These results suggest that a high percentage of plasma protein binding in vitro may predict in which species cefovecin may exhibit a long half-life and duration of action in vivo. These findings may aid in selecting species for cefovecin pharmacokinetic research and for empirical treatment of infections caused by susceptible bacteria.

Interpreting 2D Materials Bio‐Nano Interactions: Influence of Aggregation Status, Protein Corona, Cell Culture Media, and Cell Types (Adv. Mater. Interfaces 17/2021)

Interpreting 2D Materials Bio-Nano Interactions In article number 2100251, Khuloud T. Al-Jamal and co-workers demonstrate that the interpretation of bio-nano interactions for 2D nanomaterials requires extra care. Minor surface chemistry changes on 2D nanomaterials result in various serum protein binding levels, colloidal stabilities, and aggregation status in biological media. Thus, using label-free approaches such as Synchrotron radiation-based Fourier-transform infrared spectromicroscopy (SR-FTIR) provides an alternative gateway to study the bio-nano interfaces in the naïve form.

Preliminary Data on a Phase 1/2A First in Human Study of the Menin-KMT2A (MLL) Inhibitor KO-539 in Patients with Relapsed or Refractory Acute Myeloid Leukemia

Preliminary Data on a Phase 1/2A First in Human Study of the Menin-KMT2A (MLL) Inhibitor KO-539 in Patients with Relapsed or Refractory Acute Myeloid Leukemia

Using chemiluminescence imaging of cells (CLIC) for relative protein quantification

Cell physiology and cellular responses to external stimuli are partly controlled through protein binding, localization, and expression level. Thus, quantification of these processes is pivotal in understanding cellular biology and disease pathophysiology. However, it can be methodologically challenging. Immunofluorescence is a powerful technique, yet quantification by this method can be hampered by auto-fluorescence. Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding and internalization. Moreover, through specific membrane permeabilization we were able to quantify both cytosolic and nuclear proteins, and their translocation. We validated the CLIC quantification method by performing parallel experiments with other quantification methods like ELISA, qPCR, and immunofluorescence microscopy. The workflow of the immunoassay was found to be advantageous in certain instances when compared to these quantification methods. Since the reagents used for CLIC are common to other immunoassays with no need for specialized equipment, and due to the good linearity, dynamic range and signal stability inherent to chemiluminescence, we suggest that this assay is suitable for both small scale and high throughput relative protein quantification studies in whole cells.

HPLC-MS/MS coupled with equilibrium dialysis method for quantification of free drug concentration of pazopanib in plasma

BackgroundThe selective occurrence of hepatotoxicity observed with use of pazopanib may be attributed to its high level of plasma protein binding and low hepatic extraction ratio. The primary objective was to investigate changes in free drug concentration amongst patients with varying albumin concentrations. MethodsA HPLC-MS/MS method using C18 column (4.6 × 150 mm, 5 μm) with ESI source in positive mode had been developed and validated for the quantitative determination of free pazaopanib concentration in human plasma. Prior to sample preparation, patient samples were subjected to 6-hour equilibrium dialysis with molecular weight cut-off set at 8000 Da. ResultsThe calibration curves were linear over the range of 5–1000 ng/mL, with a lower limit of quantification of 5 ng/mL. The intra-day and inter-day precisions and accuracies were all within ± 15 %, at 3 different quality controls. Higher median fraction unbound of pazopanib were observed in patients (n = 17) with lower than normal albumin concentrations. ConclusionWith the developed assay, monitoring of plasma free concentrations may be evaluated as an indicator of pazopanib exposure in patients.

The Effects of Special Patient Population Plasma on Pharmacokinetic Quantifications Using LC-MS/MS.

Clinical development of lesinurad, a selective uric acid reabsorption inhibitor, required analysis of lesinurad in plasma from special patient populations. EMA and FDA bioanalytical method validation guidance have recommended studying matrix effects on quantitation if samples from special patient populations are to be analyzed. In addition to lesinurad (plasma protein binding 98.2%), the matrix effects from special population plasma on the quantitation of verapamil (PPB 89.6%), allopurinol and oxypurinol (PPB negligible) were also investigated. The plasma from special population patients had no matrix effects on the three quantification methods with stable isotope labeled internal standard, protein precipitation extraction, and LC-MS/MS detection. The validated lesinurad plasma quantification method was successfully applied for the pharmacokinetic evaluations to support the clinical studies in renal impaired patients. Special population plasma did not affect quantitation of drugs with a wide range of plasma protein binding levels in human plasma. With the confirmation that there is no impact on quantification from the matrix, the bioanalytical method can be used to support the pharmacokinetic evaluations for clinical studies in special populations.

Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI.

Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.

Influence of binding affinity and blood plasma level on cerebral pharmacokinetics and PET imaging characteristics of two novel xanthine PET radioligands for the A1 adenosine receptor

IntroductionThe suitability of novel positron emission tomography (PET) radioligands for quantitative in vivo imaging is affected by various physicochemical and pharmacological parameters. In this study, the combined effect of binding affinity, lipophilicity, protein binding and blood plasma level on cerebral pharmacokinetics and PET imaging characteristics of three xanthine-derived A1 adenosine receptor (A1AR) radioligands was investigated in rats. MethodsA comparative evaluation of two novel cyclobutyl-substituted xanthine derivatives, 8-cyclobutyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CBX) and 3-(3-[18F]fluoropropyl)-8-(1-methylcyclobutyl)-1-propylxanthine ([18F]MCBX), with the reference A1AR radioligand 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX) was conducted. This evaluation included in vitro competition binding assays, in vitro autoradiography and in vivo PET imaging. Differences in cerebral pharmacokinetics and minimal scan duration required for quantification of cerebral distribution volume (VT) were assessed. ResultsMeasured Ki values of non-labeled CBX, MCBX and CPFPX were 10.0 ± 0.52 nM, 3.3 ± 0.30 nM and 1.4 ± 0.15 nM, respectively (n = 3–4). In vitro autoradiographic binding patterns in rat brain were comparable between the radioligands, as well as the fraction of non-specific binding (1.0–1.9%). In vivo cerebral pharmacokinetics of the novel cyclobutyl-substituted xanthines differed considerably from that of [18F]CPFPX. Brain uptake and VT of [18F]CBX were substantially lower despite the higher concentration of radiotracer in plasma. [18F]MCBX showed comparable uptake and VT, but faster cerebral kinetics than [18F]CPFPX. However, the faster kinetics of [18F]MCBX did not enable the quantification of cerebral VT in a shorter scan time. ConclusionsThe combined effect of individual physicochemical and pharmacological properties of a radiotracer on its PET imaging characteristics cannot be readily predicted. In vivo performance of the xanthine A1AR radioligands was mainly influenced by binding affinity; plasma concentrations and cerebral kinetics were of secondary importance.

Quantification of fenoprofen in human plasma using UHPLC-tandem mass spectrometry for pharmacokinetic study in healthy subjects.

A rapid, simple and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed to quantify fenoprofen, a nonsteroidal anti-inflammatory drug in human plasma for a pharmacokinetic study in healthy subjects. Owing to high levels of protein binding, protein precipitation followed by solid-phase extraction was employed for the extraction of fenoprofen and fenoprofen-d3 (used as internal standard) from 200 μL human plasma. Separation was performed on a BEH C18 (50 × 2.1 mm, 1.7 μm) column using methanol-0.2% acetic acid in water (75:25, v/v) under isocratic elution. Electrospray ionization was operated in the negative mode for sample ionization. Ion transitions used for quantification in the selected reaction monitoring mode were m/z241/197 and m/z244/200 for fenoprofen and fenoprofen-d3, respectively. Under the optimized conditions, fenoprofen showed excellent linearity in the concentration range 0.02-20 μg/mL (r2 ≥ 0.9996), adequate sensitivity, favorable accuracy (96.4-103.7%) and precision (percentage coefficient of variation ≤4.3) with negligible matrix effect. The validated method was successfully applied to a pharmacokinetic study of fenoprofen in healthy subjects. The significant features of the method include higher sensitivity, small plasma volume for processing and a short analysis time.

Synthesis and Evaluation of Bicyclic Hydroxypyridones as Inhibitors of Catechol O-Methyltransferase.

A series of bicyclic pyridones were identified as potent inhibitors of catechol O-methyltransferase (COMT). Substituted benzyl groups attached to the basic nitrogen of the core scaffold gave the most potent inhibitors within this series. Rat pharmacokinetic studies showed medium to high levels of clearance for this series, but with high free fraction due to remarkably low levels of protein and tissue binding. In rat biomarker studies, levels of unbound drug exposure are seen in the brain, which exceed their respective IC50s, leading to changes in the levels of dopamine metabolites in a manner consistent with COMT inhibition.

Microdialysis Determination of Cefquinome Pharmacokinetics in Murine Thigh From Healthy, Neutropenic, and Actinobacillus pleuropneumoniae-Infected Mice.

This study was aimed at applying microdialysis to explore cefquinome pharmacokinetics in thigh and plasma of healthy, neutropenic, and Actinobacillus pleuropneumoniae-infected mice. The relative recoveries (RRs) were tested in vitro by dialysis and retrodialysis and in vivo by retrodialysis. ICR mice were randomly divided into four groups: H-40 (healthy mice receiving cefquinome at 40 mg/kg), H-160, N-40 (neutropenic mice), and I-40 mg/kg (thigh infected-mice with A. pleuropneumoniae). After cefquinome administration, plasma was collected by retro-orbital puncture and thigh dialysate was collected by using a microdialysis probe with Ringer’s solution at a perfusion rate of 1.5 μL/min. Plasma and thigh dialysate samples were assessed by HPLC–MS/MS and analyzed by a non-compartment model. The mean in vivo recoveries in the thigh were 39.35, 38.59, and 37.29% for healthy, neutropenic, and infected mice, respectively. The mean plasma protein-binding level was 16.40% and was independent of drug concentrations. For all groups, the mean values of the free AUCinf in plasma were higher than those in murine thigh, while the elimination T1/2β for plasma were lower than those for murine thigh. Cefquinome penetration (AUCthigh/AUCplasma) from the plasma to thigh was 0.76, 0.88, 0.47, and 0.98 for H-40, N-40, I-40, and H-160 mg/kg, respectively. These results indicated that infection significantly affected cefquinome pharmacokinetics in murine thigh. In conclusion, we successfully applied a microdialysis method to evaluate the pharmacokinetics of cefquinome in murine thigh of healthy, neutropenic, and A. pleuropneumonia-infected mice and the pharmacokinetics of cefquinome was obviously affected by infection in thigh.

Structural optimization and additional targets identification of antisense oligonucleotide G3139 encapsulated in a neutral cytidinyl-lipid combined with a cationic lipid in vitro and in vivo

Antisense oligonucleotides (ASOs) usually contain a fully phosphorothioate (PS) backbone, which possibly interact with many genes and proteins under intracellular conditions. G3139 is an ASO that targets Bcl-2 mRNA and induces cell apoptosis. Here, we report a kind of cytidinyl-lipid combined with a cationic lipid (DNCA/CLD, molar ration, 28:3, named mix), which may interact with oligonucleotides via H-bond formation, pi-stacking and electrostatic interaction, accompanied by low zeta potentials. The IC50 value of G3139 delivered by mix-lipid reduced from above 20 μM to 0.158 μM for MCF-7/ADR, and exhibited stronger antiproliferation upon other cancer cell lines. In addition, PS modification in the 3′-half of G3139 (especially at positions 13-16) enhanced serum stability, target specificity and anticancer activity. Also, a locked nucleic acid (LNA) gapmer G3139 (LNA-G3139) showed superior antiproliferation (78.5%) and Bcl-2 mRNA suppression effects (85.5%) at 200 nM, mainly due to its high complementary RNA affinity. More apoptosis-associated targets were identified, and a lower level of non-specific protein binding (HSA) revealed that both antisense and aptamer mechanisms might simultaneously exist. A combination of a new delivery system and chemical modifications, such as in LNA-G3139, may have potential clinical application prospects in the future.

Effect of Plasma Protein Binding on the Anti-Hepatitis B Virus Activity and Pharmacokinetic Properties of NVR 3-778.

High plasma protein binding (PPB) levels not only affect drug-target engagement but can also impact exposure of hepatocytes to antivirals and thereby affect antiviral activity. In this study, we assessed the effect of PPB on the antiviral activity of NVR 3-778, a sulfamoylbenzamide capsid assembly modulator (CAM). To this end, primary human hepatocyte (PHH) medium was spiked with plasma proteins. First, the effect of plasma proteins on the hepatitis B virus (HBV) infection assay was evaluated. The addition of plasma proteins neither decreased cell viability nor affected HBV DNA secretion or intracellular HBV RNA accumulation. In contrast, the secretion and intracellular amount of HBV proteins were induced with increasing amounts of plasma proteins. Next, the antiviral activity of NVR 3-778 was demonstrated by multiple assays while PPB and the time-dependent disappearance of the parent drug were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma proteins strongly decreased the free fraction of NVR 3-778, resulting in a physiologically relevant in vitro hepatocyte exposure. NVR 3-778 displayed a high PPB level, while the antiviral activity was reduced approximately only 4-fold. The disconnect between the high PPB level and the only moderate shift of the antiviral activity was explained by the rapid hepatic clearance of NVR 3-778 in the absence of plasma proteins. This study highlights the use of PHHs as a model to accurately determine the antiviral activity by capturing PPB, clearance, and liver distribution. It is advantageous to consider both pharmacokinetics and pharmacodynamics for selection of HBV antiviral drug candidates and for successful extrapolation of in vitro data to clinical studies.

Binding of tylosin, tilmicosin and oxytetracycline to proteins from honeybees, larvae and beehive products

American Foulbrood (AFB) caused by the spore-forming bacterium Paenibacillus larvae is the most serious disease of bacterial origin affecting larvae and pupae of honeybees. Antibiotics are used in many countries for the control of AFB in high incidence areas, but their misuse may lead to antibiotic resistance of bacterial strains and honey contamination. The objective of the present work was to determine, through a biological method, the protein binding of tylosin, tilmicosin and oxytetracycline to worker jelly; honey; pollen; adult bees and larvae in order to propose their kinetic routes. The sensitivity limit of the technique used was 0.05 μg/ml for tylosin and tilmicosin and 0.01 μg/ml for oxytetracycline, respectively. The method had intra and inter-assay correlation coefficients over 0.90, respectively and a coefficient variation of intra-and inter-assay for all antibiotics and processed samples under 5%. Tylosin and oxytetracycline presented lower percentages of protein binding in tissues and hive products (average 15%) in relation to those observed for tilmicosin (29%). In conclusion, tylosin is useful for AFB control in honey bee colonies due to its chemical characteristics, antimicrobial activity and levels of protein binding in bees, larvae, and beehive products.