Exosome-mediated delivery platform of biomacromolecules into the brain: Cetuximab in combination with doxorubicin for glioblastoma therapy.

Monoclonal antibodies (mAbs) have become the predominant treatment modality for various diseases due to their high affinity and specificity. Although antibodies also have great potential for neurological diseases, they couldn't fully meet the therapeutic requirements due to their high molecular weight and limitations in crossing the blood-brain barrier (BBB). Herein, an innovative strategy based on exosomes (Exos) platform was developed to enhance the delivery of cetuximab (CTX) into the brain, and in combination with doxorubicin (DOX) for the synergistic targeted therapy of glioblastoma (GBM). The in vitro/vivo experiments have shown that exosomes could effectively promote BBB penetration and increase the content of CTX in glioma cells and brain lesions. Cytotoxicity and wound healing experiments have shown that CTX-Exo-DOX could significantly inhibit the proliferation of tumor cells. Finally, in vivo results showed that CTX-Exo-DOX significantly prolonged the survival time of tumor-bearing rats to 28 days, which was 1.47 times that of the DOX group. In summary, exosomes could deliver more antibodies into the brain, and CTX-Exo-DOX is a promising co-delivery system for the treatment of GBM. The results of this study will also provide a prospective strategy for antibody drugs in the treatment of neurological diseases.

Evaluation of herb-drug interactions between compound Danshen dripping pills and clopidogrel based on the pharmacokinetics and pharmacodynamics in rats.

Compound Danshen dripping pills (CDDP), a well-known traditional Chinese medicine, is widely used to prevent and treat cardiovascular diseases. CDDP is usually prescribed in combination with clopidogrel (CLP), but the herb-drug interactions are rarely reported. This study evaluated the effects of CDDP on the pharmacokinetics and pharmacodynamics of coadministered CLP, and ensured the safety and efficacy of their usage. The trial design included a single-dose administration and multidose test for 7 consecutive days. Wistar rats received CLP alone or CLP combined with CDDP. After the final dose, plasma samples were collected at various time points, and the active metabolite H4 of CLP was analyzed by ultrafast liquid chromatography coupled with triple quadrupole tandem mass spectrometry. The main pharmacokinetic parameters of Cmax (maximum [or peak] serum concentration), Tmax (peak plasma time), t1/2 (half-time), AUC0-∞ (area under the concentration-time curve from dosing (time 0) to infinite time), and AUC0-t (area under the concentration-time curve from dosing [time 0] to time t) were calculated using the non-compartment model. In addition, prothrombin time, activated partial thromboplastin time, bleeding time, and adenosine diphosphate-induced platelet aggregation were evaluated for anticoagulation and antiplatelet aggregation activity. In this study, we found that CDDP had no significant effect on the metabolism of CLP in rats. In pharmacodynamic studies, the combination group showed significant synergistic antiplatelet activity compared with the CLP or CDDP groups alone. Based on pharmacokinetic and pharmacodynamic results, CDDP and CLP have synergistic effects on antiplatelet aggregation and anticoagulation.

In Silico and In Vivo Pharmacokinetic Evaluation of 84-B10, a Novel Drug Candidate against Acute Kidney Injury and Chronic Kidney Disease.

Acute kidney injury (AKI) and chronic kidney disease (CKD) have become public health problems due to high morbidity and mortality. Currently, drugs recommended for patients with AKI or CKD are extremely limited, and candidates based on a new mechanism need to be explored. 84-B10 is a novel 3-phenylglutaric acid derivative that can activate the mitochondrial protease, Lon protease 1 (LONP1), and may protect against cisplatin-induced AKI and unilateral ureteral obstruction- or 5/6 nephrectomy [5/6Nx]-induced CKD model. Preclinical studies have shown that 84-B10 has a good therapeutic effect, low toxicity, and is a good prospect for further development. In the present study, the UHPLC-MS/MS method was first validated then applied to the pharmacokinetic study and tissue distribution of 84-B10 in rats. Physicochemical properties of 84-B10 were then acquired in silico. Based on these physicochemical and integral physiological parameters, a physiological based pharmacokinetic (PBPK) model was developed using the PK-Sim platform. The fitting accuracy was estimated with the obtained experimental data. Subsequently, the validated model was employed to predict the pharmacokinetic profiles in healthy and chronic kidney injury patients to evaluate potential clinical outcomes. Cmax in CKD patients was about 3250 ng/mL after a single dose of 84-B10 (0.41 mg/kg), and Cmax,ss was 1360 ng/mL after multiple doses. This study may serve in clinical dosage setting in the future.

Cobalt/calcium bimetallic oxides based on bio-waste eggshells for the efficient degradation of norfloxacin by peroxymonosulfate activation.

Cobalt-calcium bimetallic oxide (ECCO) was successfully prepared using eggshells and cobalt nitrate, which could be used to activate peroxymonosulfate (PMS) to remove norfloxacin (NOR). The compositional structure and surface properties of the catalysts were explored by various characterization analysis. The degradation efficiency of ECCO was 7.86 and 440 times higher than that of cobalt oxide and calcium oxide, respectively. The prepared ECCO (0.1 g/L) had a high degradation efficiency of over 90% against NOR (10 mg/L, 100 mL) by activating the PMS (0.15 g/L) at a wide pH range of 3.0-9.0 in 35 min. With a NOR removal efficiency of 91.4% after five cycles, the catalyst showed good reusability. The high degradation efficiency of NOR was resulted from enhanced electron transfer capability, the low point of zero charge and diversified reactive oxygen species (ROS). The ROS were identified as SO4•-, •OH and 1O2, which were produced by activating PMS on the active sites of Co and oxygen vacancies. It is the first report of the use of eggshells to synthesize cobalt-calcium bimetallic oxides ECCO for the activation of PMS to eliminate NOR, which is important for the development of green and efficient catalysts.

Capture and purification of an untagged nanobody by mixed weak cation chromatography and cation exchange chromatography.

Nanobodies (Nbs) are single-domain antibodies, which have potential application value in tumor-targeted therapy, immunotherapy, diagnostic probe, and molecular imaging. Typically, Nbs are captured by affinity chromatography via the addition of specific fusion tags at their N or C terminus. Nerve growth factor (NGF), which regulates the growth and development of peripheral and central neurons, maintains neuronal survival and plays a key role in both arthritis and acute and chronic pain. In this study, a method for capture and purification of an untagged Nb (anti-NGF Nb) by mixed weak cation chromatography and cation exchange chromatography was established. First, pH 4.0-5.0 was demonstrated to be the optimal loading condition for Capto MMC to capture anti-NGF by the design of experiments (DOE). Furthermore, high purity and yield products can be obtained at laboratory scale and commercial production scale by adjusting the protein pH. Additionally, direct capture of anti-NGF Nb using Capto MMC without adjusting anti-NGF Nb harvest pH was investigated. The anti-NGF Nb captured by Capto MMC was 67.2% yield, 94.5% monomer purity, and host cell protein (HCP) was reduced from 74,931 ppm to 484 ppm. The anti-NGF Nb that were further purified using Capto S ImpAct achieved 84.5% yield and 99.2% purity and 77 ppm of HCP. The scaling-up process was consistent with the results of the optimized process, further demonstrating the feasibility of this method. This outcome provides a highly promising and competitive alternative to affinity chromatography-based processing strategies for Nbs.

Anti-Colorectal Cancer Effects of a Novel Camptothecin Derivative PCC0208037 In Vitro and In Vivo.

Colorectal cancer is one of the most common malignancies, and the topoisomerase inhibitor irinotecan (CPT-11)-based chemotherapeutic regimen is currently the first-line treatment with impressive therapeutic efficacy. However, irinotecan has several clinically significant side effects, including diarrhea, which limit its clinical utility and efficacy in many patients. In an effort to discover better and improved pharmacotherapy against colorectal cancer, we synthesized a novel topoisomerase inhibitor, PCC0208037, examined its anti-tumor efficacy and related molecular mechanisms, and characterized its toxicity and pharmacokinetic profiles. PCC0208037 suppressed colorectal cancer cell (CRC) proliferation and increased cell cycle arrest, which may be related to its effects on up-regulating DNA damage response (DDR)-related molecules and apoptosis-related proteins. PCC0208037 demonstrated robust anti-tumor activity in vivo in a colorectal cancer cell xenograft model, which was comparable to or slightly better than CPT-11. In a preliminary toxicology study, PCC0208037 demonstrated much weaker tissue damage to colorectal tissue than CPT-11, and its impacts on food intake and body weight loss were more transient and recovered faster than CPT-11 in mice. This could be partially explained by the pharmacokinetic findings, which showed that PCC0208037 and its active metabolite, SN-38, were more accumulated in tumor tissue than in the intestine, as compared to CPT-11. Taken together, these results described a novel Topo I inhibitor with a comparative advantage over the standard treatment of colorectal cancer CPT-11 and could be a promising candidate compound for the treatment of colorectal cancer that warrants further investigation.

Clozapine affects the pharmacokinetics of risperidone and inhibits its metabolism and P-glycoprotein-mediated transport in vivo and in vitro: A safety attention to antipsychotic polypharmacy with clozapine and risperidone.

Antipsychotic polypharmacy (APP), as one maintenance treatment strategy in patients with schizophrenia, has gained popularity in real-world clinical settings. Risperidone (RIS) and clozapine (CLZ) are the most commonly prescribed second-generation antipsychotics, and they are often used in combination as APP. In this study, the pharmacokinetics of RIS and CLZ in rats were examined after co-administration to explore the reliability and rationality of co-medication with RIS and CLZ. In addition, the effects of CLZ on RIS metabolism and transport in vitro were investigated. The results illustrated that in the 7-day continuous administration test in rats, when co-administered with CLZ, the area under curve and peak concentrations of RIS were increased by 2.2- and 3.1-fold at the first dose, respectively, increased by 3.4- and 6.2-fold at the last dose, respectively. The metabolite-to-parent ratio of RIS was approximately 22% and 33% lower than those of RIS alone group at the first and last doses, respectively. Moreover, CLZ significantly increased RIS concentrations in the brain (3.0-4.8 folds) and cerebrospinal fluid (2.1-3.5 folds) in rats, which was slightly lower than the impact of verapamil on RIS after co-medication. Experiments in vitro indicated that CLZ competitively inhibited the conversion of RIS to 9-hydroxy-RIS with the inhibition constants of 1.36 and 3.0 µM in rat and human liver microsomes, respectively. Furthermore, the efflux ratio of RIS in Caco-2 monolayers was significantly reduced by CLZ at 1 µM. Hence, CLZ may affect the exposure of RIS by inhibiting its metabolism and P-glycoprotein-mediated transport. These findings highlighted that APP with RIS and CLZ might increase the plasma concentrations of RIS and 9-hydroxy-RIS beyond the safety ranges and cause toxic side effects.

Detection of a New Tert-Leucinate Synthetic Cannabinoid 5F-MDMB-PICA and Its Metabolites in Human Hair: Application to Authentic Cases.

Methyl 2 -[ [ 1- (5- fluoropentyl) indole - 3- carbonyl] amino] -3, 3- dimethyl - butanoate (5F-MDMB-PICA) is a new synthetic cannabinoid characterized by valinate or tert-leucinate moieties. In recent years, 5F-MDMB-PICA has been abused in the form of "spice-like" herbal incenses or electronic cigarette oil. A UHPLC-MS/MS method was developed to detect 5F-MDMB-PICA and its metabolites in human hair. Approximately 20 mg of hair was weighed and pulverized with methanol below 4°C. After ultrasonication, centrifugation and filtration, 200 µL of supernatant was placed into an autosampler vial and analyzed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm particle size) using an acetonitrile-20 mmol/L ammonium acetate (0.1% formic acid, 5% acetonitrile) gradient with a run time of 8 min. The limit of detection (LOD) ranged from 0.5 to 5 pg/mg, and the lower limit of quantitation (LLOQ) ranged from 1 to 5 pg/mg. The method was shown to be linear over a concentration range of 1-200 pg/mg. The linear correlation (R 2) of the calibration curves for all analytes was >0.999. The accuracy varied from 95.4 to 107.4%, while the intra- and inter-day precision RSD values were 0.7-10.6% and 1.7-12.2%, respectively. Recoveries were within the range of 61.1-93.3%, and matrix effects were in the range of 19.1-102.6%. The validated method was successfully applied to the identification and quantification of 5F-MDMB-PICA and its metabolites in hair from authentic forensic cases.

Liquid chromatography tandem mass spectrometry method for determination of fulvestrant in rat plasma and its application to pharmacokinetic studies of a novel fulvestrant microcrystal.

Fulvestrant ('Faslodex'), an estrogen receptor antagonist, is available for the treatment of advanced breast cancer. The oil-based vehicle of Faslodex can lead to various adverse effects. A novel fulvestrant microcrystal (aqueous suspension) was developed in this study to eliminate these adverse effects. A sensitive and robust liquid chromatography tandem mass spectrometry method was developed and validated for the determination of fulvestrant in rat plasma using supported-liquid extraction. The separation of fulvestrant was achieved on an Agilent SB-C18 column (2.1 × 50 mm, 3.5 µm) with isocratic elution using fulvestrant-d3 as internal standard. Mass spectrometric detection was conducted in negative multiple reaction monitoring mode. Ion transitions were at m/z 605.5 → 427.5 for fulvestrant and m/z 608.5 → 430.5 for fulvestrant-d3. The excellent linearity was demonstrated over the range 0.05-100.0 ng/ml (r2 = 0.99). The lower limit of quantitation was 0.05 ng/ml, which was superior to that reported in literature The method validation was evaluated by selectivity, accuracy, precision, recovery and matrix effect in agreement with the US Food and Drug Administration guidance. The method was successfully applied to a pharmacokinetic study of a novel fulvestrant microcrystal in rats after intramuscular administration. It revealed that the rate of absorption increases and the extent of absorption is constant with a decrease in microcrystal diameter.

Mechanism exploration on the aluminum supplementation coupling the electrokinetics-activating geopolymerization that reinforces the solidification of the municipal solid waste incineration fly ashes.

Traditional cementation technique is insufficient in making municipal solid waste incineration (MSWI) fly ashes meet the permissible leaching threshold. Aluminum supplementation and electrokinetic (EK) activation were combinedly incorporated into the traditional solidification pathway to enhance the geopolymerization and the immobilization of Pb and Cd in MSWI fly ashes in this study. The aluminum addition remarkably affected the geopolymer formation. The minimum toxicity leaching as well as the maximum compressive strength were achieved at the combination of the voltage gradient of 1.0 V/cm, the proposing time of 48 or 72 h, the mass ratio of alkali activator to fly ash of 11.5%, and the modulus of 2.1. Chloride reduction in the mortar obtained during the EK process increased the negative charge relativity of oligomers. The leaching concentrations of Pb and Cd from the geopolymer were successfully predicted by a linear model based on the compressive strengths at 28 d. Higher reaction degree was found in the EK-activated mortar in the geopolymerization kinetics. Aluminum supplementation had induced the production of some amorphous aluminosilicate minerals including Al6Si2O13, CaAl2Si2O8·4H2O, Ca2Al3(Si3O12)OH, Ca2Al(OH)7·3H2O, and Ca4Al2O6Cl2·10H2O during the EK process. Larger particles observed in the EK-treated specimen directly verified the EK-activated pozzolanic reactions.

A novel LC-MS/MS approach to the pharmacokinetic study of free and bound aflibercept simultaneously.

To comprehensively evaluate the pharmacokinetic (PK) characteristics of aflibercept, we established a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to determine the concentration of vascular endothelial growth factor (VEGF)-A-bound aflibercept and free aflibercept. A specific sample preparation method of nano-surface and molecular-orientation limited (nSMOL) proteolysis was performed to extract both free and bound aflibercept from plasma. The tryptic peptides unique to aflibercept and VEGF-A were selected to quantify the amounts of total aflibercept and aflibercept-VEGF complex, respectively. The method was validated by evaluating its selectivity, linearity, precision, accuracy, extraction recovery, matrix effect, and stability. It was then successfully used to quantify total and bound aflibercept concentrations in cynomolgus monkey plasma, while indirectly obtaining the concentration of free aflibercept by subtraction. The PK results of this LC-MS/MS method are comparable to the traditional enzyme-linked immunosorbent assay (ELISA) results. It is thus a reliable and complementary method for the PK evaluation of aflibercept. Graphical abstract.

Pharmacokinetics, bioavailability, excretion, and metabolic analysis of Schisanlactone E, a bioactive ingredient from Kadsura heteroclita (Roxb) Craib, in rats by UHPLC-MS/MS and UHPLC-Q-Orbitrap HRMS.

Schisanlactone E (SE) is a bioactive ingredient extracted from the stem of Kadsura heteroclita (Roxb) Craib. SE has various pharmacological activity such as anti-tumor and anti-leukemia effects. However, its absorption, distribution, metabolism, and excretion have rarely been examined. In this study, new quali-quantitative analytical methods were developed for metabolic and pharmacokinetic studies of SE in rats. A UHPLC-MS/MS method was developed to determine SE in rat plasma, urine, and feces. Samples were precipitated with methanol and analyzed in multiple reaction monitoring mode. The established method was validated and applied to the pharmacokinetics, bioavailability, and excretion analysis of SE after oral (6 mg/kg) or intravenous (2 mg/kg) administration. The absolute oral bioavailability of SE was approximately 79.3%. After oral administration, SE was mainly excreted via feces with a rate of 41.7% for 48 h. SE could not be detected in urine. Furthermore, a UHPLC-Q-Orbitrap HRMS method was developed for the metabolite screening of SE in rat plasma, urine, and feces. Metabolites were extracted by solid phase extraction and analyzed with full MS/dd-MS2 scan mode. As a result, 15 metabolites including 11 phase I and 4 phase II metabolites were identified by a three-step analytical strategy. The carboxyl group, the five membered ring, and the six membered α,ß-unsaturated lactone ring of SE could be predicted as the main metabolic sites. This study provides comprehensive insights into the pharmacokinetic and metabolic profiles of SE, and would be valuable for future development and utilization of SE and Kadsura heteroclita.

Identification and Quantification of Bioactive Compounds in Diaphragma juglandis Fructus by UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS.

Diaphragma juglandis fructus is the dry wooden diaphragm inside walnuts and a byproduct in food processing of walnut kernels. The purpose of our research is to enrich the information on compounds in Diaphragma juglandis fructus to further discover and exploit its potential nutritional value. In this study, new quali-quantitative analytical approaches were developed to identify and determine bioactive compounds in Diaphragma juglandis fructus. Two-hundred compounds, including hydrolyzable tannins, flavonoids, phenolic acids, and quinones, were identified by UHPLC-Q-Orbitrap HRMS, more than 150 of which were first discovered in Diaphragma juglandis fructus. Among them, 21 major dietary polyphenols with health-promoting effects were successfully quantified using UHPLC-MS/MS, with total contents of 2.88-6.18 mg/g. This successful characterization and quantification of bioactive compounds in Diaphragma juglandis fructus gives a better understanding of its potential nutritional value and supports efficiently developing and reusing it instead of discarding it as agrofood waste.

A simple MS method to characterize the higher order structures of antibody therapeutics.

A scheme with enhanced throughput using the hydroxyl radical foot-printing technique to map the higher order structures (HOSs) of antibody therapeutics was developed. This method takes advantage of the simplicity of multiple reaction monitoring (MRM) by monitoring the remaining unmodified peptides, thus overcoming the complexity of the data analysis of the conventional free radical foot-printing technique, wherein various oxidized forms of the peptides are measured. In this study, a preliminary method validation was carried out to show that the technique has good specificity, sensitivity, and repeatability. Finally, we were able to use the method to differentiate the HOSs of the disulfide isoforms of the IgG2 antibody, provide site-specific structural differences between IgG1 and its deglycosylated counterpart, and determine similarities between a biosimilar candidate and the originator product.

Synthesis, Characterization and In Vitro Evaluation of Dual pH/Redox Sensitive Marine Laminarin-Based Nanomedicine Carrier Biomaterial for Cancer Therapy.

In order to improve the anti-cancer therapy efficiency of hydrophobic drugs such as curcumin (Cur), a novel dual pH/redox sensitive marine laminarin-based nanomedicine carrier biomaterial with photo-dynamic therapy (PDT) was synthesized in this study. The new synthetic chemical structure, named as Hematin-Laminarin-Dithiodipropionic Acid-MGK (HLDM), was characterized by 1H-NMR and IR. The Cur-loaded micelles were then prepared via dialysis method. The HLDM could self-assemble into micelles in water with hydrodynamic diameter of 135±15 nm. The particle size, zeta potential and morphology of micelles were detected by transmission electron microscope (TEM). Interestingly, the in vitro release experiment showed that the release amount of Cur-loaded HLDM micelles could reach 80% in the pH and redox sensitive environment. Furthermore, cell study showed that the Cur-loaded HLDM micelles had stronger cellular uptake and cytotoxicity to MCF-7 cells than that of HLDM. The multifunctional marine laminarin based nanomedicine carrier biomaterial can be used for new drug delivery systems with dual pH/redox sensitivity for cancer therapy.

Multivesicular liposomes for sustained release of bevacizumab in treating laser-induced choroidal neovascularization.

Bevacizumab is an anti-vascular endothelial growth factor drug that can be used to treat choroidal neovascularization (CNV). Bevacizumab-loaded multivesicular liposomes (Bev-MVLs) have been designed and developed to increase the intravitreal retention time of bevacizumab and reduce the number of injection times. In this study, Bev-MVLs with high encapsulation efficiency were prepared by double emulsification technique, and antibody activity was determined. The results revealed that 10% of human serum albumin (HSA) could preserve the activity of bevacizumab. In vitro release of Bev-MVLs appeared to be in a more sustained manner, the underlying mechanisms of Bev-MVLs indicated that bevacizumab was released from MVLs through diffusion and erosion. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that bevacizumab could retain its structural integrity after being released from MVLs in vitro. In vivo imaging was used to evaluate the retention time of antibody in rat eyes, while pharmacokinetic analysis was performed on rabbit eyes. These results indicated that Bev-MVLs exhibited sustained release effects as compared to bevacizumab solution (Bev-S). Bev-MVLs could effectively inhibit the thickness of CNV lesion as compared to Bev-S at 28 days after treatment. Furthermore, these data suggest that Bev-MVLs are biologically feasible to increase the retention time of bevacizumab in vitreous humor. This novel Bev-MVLs may therefore serve as a promising sustained release drug delivery system for the treatment of CNV.

In Vivo Evaluation of Reduction-Responsive Alendronate-Hyaluronan-Curcumin Polymer-Drug Conjugates for Targeted Therapy of Bone Metastatic Breast Cancer.

Many cancers, such as human breast cancer and lung cancer, easily metastasize to bones, leading to the formation of secondary tumors in advanced stages. On the basis of the CD44-targeted effect of oHA and the bone-targeted effect of ALN, we prepared a reduction-responsive, CD44 receptor-targeting and bone-targeting nanomicelle, called CUR-loaded ALN-oHA-S-S-CUR micelles. In this study, we aimed to evaluate the antitumor activity and bone-targeting ability of CUR-loaded ALN-oHA-S-S-CUR micelles. The in vivo experiment results showed that a larger number of micelles was gathered in the bone metastatic tumor tissue and reduced the bone destruction. The CUR-loaded ALN-oHA-S-S-CUR micelles markedly inhibited the tumor growth. So the CUR-loaded ALN-oHA-S-S-CUR micelles constitute a promising drug delivery system for bone tumor therapy.

Enhanced In Vitro and In Vivo Anticancer Properties by Using a Nanocarrier for Co-Delivery of Antitumor Polypeptide and Curcumin.

In this paper, a novel pH and redox dual-sensitive nanocarrier loaded with curcumin (Cur) and anticancer polypeptide (AP) was developed for dual targeting mitochondrial and CD44 receptor. The amphiphilic block copolymer was prepared by triphenylphosphonium (TPP)/oligomeric hyaluronic acid (oHA)/disulfide-menthone 1,2-glycerol ketal (SM), hereinafter referred to as TPP-oHSM. The TPP targeted the mitochondria, pH/redox dual-sensitive SM served as a hydrophobic part, and the CD44 receptor targeting oHA worked as a hydrophilic part. The chemical structure of the TPP-oHSM was identified using 1H NMR and FTIR technologies. Cur and AP were loaded into the TPP-oHSM micelles by self-assembly and denoted as C/A@TM. The C/A@TM prepared in this study exhibited an approximately spherical structure, with a mean diameter of 191.3 ± 3.1 nm and a negative zeta potential of -26.10 ± 0.45 mV. The in vitro release study and cellular uptake test revealed that the C/A@TM targeted the mitochondria and CD44 receptor, as well as it showed sensitivity towards pH and redox. In addition, the C/A@TM demonstrated satisfactory cytotoxic effects against MDA-MB-231 cells and MCF-7 cells. Finally, the in vivo application of the C/A@TM showed excellent therapeutic effects. The C/A@TM developed in this study exhibited promising multifunctional properties as a co-delivery carrier of polypeptide and chemical drug for an effective clinical therapy for cancer.

Constituents of Mediterranean Spices Counteracting Vascular Smooth Muscle Cell Proliferation: Identification and Characterization of Rosmarinic Acid Methyl Ester as a Novel Inhibitor.

SCOPE: Aberrant vascular smooth muscle cell (VSMC) proliferation is involved in atherosclerotic plaque formation and restenosis. Mediterranean spices have been reported to confer cardioprotection, but their direct influence on VSMCs has largely not been investigated. This study aims at examining rosmarinic acid (RA) and 11 related constituents for inhibition of VSMC proliferation in vitro, and at characterizing the most promising compound for their mode of action and influence on neointima formation in vivo. METHODS AND RESULTS: RA, rosmarinic acid methyl ester (RAME), and caffeic acid methyl ester inhibit VSMC proliferation in a resazurin conversion assay with IC50 s of 5.79, 3.12, and 6.78 µm, respectively. RAME significantly reduced neointima formation in vivo in a mouse femoral artery cuff model. Accordingly, RAME leads to an accumulation of VSMCs in the G0 /G1 cell-cycle phase, as indicated by blunted retinoblastoma protein phosphorylation upon mitogen stimulation and inhibition of cyclin-dependent kinase 2 in vitro. CONCLUSION: RAME represses PDGF-induced VSMC proliferation in vitro and reduces neointima formation in vivo. These results recommend RAME as an interesting compound with VSMC-inhibiting potential. Future metabolism and pharmacokinetics studies might help to further evaluate the potential relevance of RAME and other spice-derived polyphenolics for vasoprotection.

Novel strategy using tryptic peptide immunoaffinity-based LC-MS/MS to quantify denosumab in monkey serum.

AIM: Denosumab is a recombinant fully human IgG2 that has a high affinity and specificity for human RANKL. Commercially available RANKL labeled with an Fc fragment cannot be used to establish an indirect ELISA. To characterize denosumab pharmacokinetic a robust and accuracy method should be developed urgently. RESULTS: In this study, an immunoaffinity enrichment method coupled with LC-MS/MS was established. The LC-MS/MS method acquired a linear range from 0.1 to 30 µg/ml. The intra- and inter-run precision (CV%) was within 11.5 and 10.5%, respectively. More importantly, the LC-MS/MS pharmacokinetic data were consistent with ELISA. CONCLUSION: This approach accelerated the quantification, reduced the costs and provided an alternative in case of lacking the special antigen to denosumab or a RANKL-biotinylated reagent.