A Novel Strategy for Liposomal Drug Separation in Plasma by TiO2 Microspheres and Application in Pharmacokinetics.

Purpose: Liposomes are nano-scale materials with a biofilm-like structure. They have excellent biocompatibility and are increasingly useful in drug delivery systems. However, the in vivo fate of liposomal drugs is still unclear because existing bioanalytical methods for quantitation of total and liposomal-encapsulated drugs have limits. A novel strategy for liposomal-encapsulated drug separation from plasma was developed via the specific coordinate binding interaction of TiO2 microspheres with the phosphate groups of liposomes. Methods: Liposomal-encapsulated docetaxel was separated from plasma by TiO2 microspheres and analyzed by the UPLC-MS/MS method. The amount of TiO2, pH of the dilutions, plasma dilution factors and incubation time were optimized to improve extraction recovery. The characterization of the adsorption of liposome-encapsulated drugs by TiO2 microspheres was observed by electron microscopy. For understanding the mechanism, pseudo-first and the pseudo-second order equations were proposed for the adsorption process. The study fully validated the method for quantitation of liposomal-encapsulated in plasma and the method was applied to the pharmacokinetic study of docetaxel liposomes. Results: The encapsulated docetaxel had a concentration range of 15-4000 ng/mL from the plasma sample using a TiO2 extraction method. Successful method validation proved the method was sensitive, selective and stable, and was suitable for quantitation of docetaxel liposomes in plasma samples. Extraction recovery of this method was higher than that of SPE method. As shown in electron microscopy, the liposomes adsorbed on TiO2 microspheres were intact and there was no drug leakage. The study proposed pseudo-first and the pseudo-second order equations to facilitate the adsorption of liposomal drugs with TiO2 microspheres. The proposed strategy supports the pharmacokinetic study of docetaxel liposomes in rats. Conclusion: TiO2 extraction method was stable, reproducible, and reliable for quantitation of encapsulated docetaxel. Because of versatility of lipids, it is expected to a universal bioanalysis method for the pharmacokinetic study of liposomes.

Bioequivalence and safety evaluation of two preparations of metformin hydrochloride sustained-release tablets (Boke® and Glucophage®-XR) in healthy Chinese volunteers: a randomized phase I clinical trial.

BACKGROUND: As per the National Medical Products Administration (NMPA) requirements, the quality and efficacy of generic drugs must be consistent with those of the innovator drug. We aimed to evaluate the bioequivalence and safety of generic metformin hydrochloride sustained-release (MH-SR) tablets (Boke®) developed by Beijing Wanhui Double-crane Pharmaceutical Co. Ltd., China and the innovator product metformin hydrochloride extended-release tablets (Glucophage®-XR) manufactured by Bristol-Myers Squibb Company, New York, NY, in healthy Chinese volunteers. MATERIALS AND METHODS: We performed a bioequivalence and safety assessment of MH-SR (500 mg/tablet) and Glucophage®-XR (500 mg/tablet) tablets in a randomized, open-label, two-period, two-sequence crossover, single-dose oral study in 48 healthy Chinese adult participants under fasting conditions (Chinese Clinical Trial Registration No. CTR20171306). The washout period was seven days. Bioequivalence (80.00-125.00%) was assessed using adjusted geometric mean ratios (GMRs) and two-sided 90% confidence intervals (CIs) of the area under the curve (AUC) and maximum concentration (Cmax) for each component. RESULTS: The 90% CIs of the test/reference preparation for key pharmacokinetic parameters were 97.36-108.30% for AUC0→t, 97.26-108.09% for AUC0→∞ and 96.76-111.37% for Cmax. No severe adverse events (AEs) were observed. However, 38 adverse drug reactions (ADRs) occurred, including metabolic or nutritional conditions (n = 8), infections (n = 2), gastrointestinal conditions (n = 10) and abnormal inspection (n = 18). No significant difference was observed between MH-SR (23 ADRs, 10 participants) and Glucophage®-XR (15 ADRs, 12 participants) (p = .500). Bioequivalence was concluded since the 90% CIs of the main pharmacokinetic parameters were within the equivalence interval (80.00-125.00%). CONCLUSIONS: MH-SR (500 mg/tablet) and Glucophage®-XR (500 mg/tablet) were found to be bioequivalent and safe under fasting conditions in healthy Chinese participants. Thus, the market demand for MH-SR tablets (500 mg/tablet) can be met using the generic alternative.KEY MESSAGESGeneric MH-SR tablets (500 mg, Beijing Wanhui Double-crane Pharmaceutical Co. Ltd., Beijing, China) and innovator MH-SR tablets (Glucophage®-XR, 500 mg, Bristol-Myers Squibb Company, New York, NY, USA) were bioequivalent and safe in healthy Chinese volunteers under single-dose administration and fasting conditions.The main goal of this study is to support an increase in the supply of MH-SR tablets in China by proving the efficacy and safety of a generic alternative.Although no sugar was administered in the BE trial of the MH-SR tablets under fasting conditions, no hypoglycaemic event occurred. The method used in this study is expected to serve as a reference for BE studies of different MH-SR formulations.

A New Strategy for High-Efficient Tandem Enrichment and Simultaneous Profiling of N-Glycopeptides and Phosphopeptides in Lung Cancer Tissue.

N-glycosylation and phosphorylation, two common posttranslational modifications, play important roles in various biological processes and are extensively studied for biomarker and drug target screening. Because of their low abundance, enrichment of N-glycopeptides and phosphopeptides prior to LC-MS/MS analysis is essential. However, simultaneous characterization of these two types of posttranslational modifications in complex biological samples is still challenging, especially for tiny amount of samples obtained in tissue biopsy. Here, we introduced a new strategy for the highly efficient tandem enrichment of N-glycopeptides and phosphopeptides using HILIC and TiO2 microparticles. The N-glycopeptides and phosphosites obtained by tandem enrichment were 21%-377% and 22%-263% higher than those obtained by enriching the two PTM peptides separately, respectively, using 160-20 µg tryptic digested peptides as the starting material. Under the optimized conditions, 2798 N-glycopeptides from 434 N-glycoproteins and 5130 phosphosites from 1986 phosphoproteins were confidently identified from three technical replicates of HeLa cells by mass spectrometry analysis. Application of this tandem enrichment strategy in a lung cancer study led to simultaneous characterization of the two PTM peptides and discovery of hundreds of differentially expressed N-glycosylated and phosphorylated proteins between cancer and normal tissues, demonstrating the high sensitivity of this strategy for investigation of dysregulated PTMs using very limited clinical samples.

Pharmacokinetics and Bioequivalence of Single-Oral-Dose Linagliptin: A Randomized, 2-Period Crossover Trial in Chinese Healthy Subjects Under Fasting and Fed Conditions.

The bioequivalence of the reference and test linagliptin formulations was assessed in healthy Chinese subjects under fasting and fed conditions. The study was designed as a single-dose, randomized, open-label, 2-period crossover study with a 35-day washout period between 2 administrations. Forty-eight healthy subjects received 5 mg of test and reference linagliptin formulation orally under fasting condition. The geometric mean of the maximum observed linagliptin concentration (Cmax ) for the test formulation was 4.9 ng/mL (reference, 5.0 ng/mL), the area under the plasma concentration-time curve from 0 to 72 hours (AUC0-72 ) was 154.7 ng · h/mL (reference, 157.4 ng · h/mL). Thirty-six subjects received 5 mg of test and reference linagliptin formulation orally under fed conditions. The geometric mean of Cmax for the test linagliptin formulation was 2.8 ng/mL (reference, 2.8 ng/mL), AUC0-72 was 133.5 ng · h/mL (reference, 136.6 ng · h/mL). The 90%CIs for the test/reference ratio for Cmax and AUC0-72 met the bioequivalence criteria (80%-125%). The test and reference formulations of linagliptin were well tolerated and bioequivalent under fasting and fed conditions.

Phase I, randomized, double-blind, single-dose study to assess the pharmacokinetics, safety, and immunogenicity of the proposed biosimilar SCT630 and adalimumab in healthy Chinese subjects.

We aimed to compare the pharmacokinetics, safety, and immunogenicity of the adalimumab biosimilar SCT630 with those of its reference (adalimumab, Humira®). This study involved a randomized, double-blind, parallel-controlled design; healthy subjects (N = 146) were randomly distributed into two groups to receive a single-dose subcutaneous injection of 40 mg SCT630 or 40 mg adalimumab, with a 71-day follow up. The bioequivalence of the primary pharmacokinetic parameters (AUC0-t) and maximum observed serum concentration (Cmax) between SCT630 and adalimumab were the primary endpoints; safety and immunogenicity of SCT630 compared with those of adalimumab were the secondary endpoints. The geometric mean Cmax ratio of SCT630 to adalimumab and its 90% confidence interval (CI) were 116.02% and 108.66%-123.88%, AUC0-t ratio and 90% CI were 109.47% and 99.80%-120.08%, and AUC0-∞ ratio and 90% CI were 109.24% and 99.80%-120.78%. These PK parameters fulfilled the equivalence criterion of 80.00%-125.00%. Treatment-emergent adverse events (TEAEs) occurred in 62 (84.9%) and 61 (83.6%) subjects; mild and moderate drug-related TEAEs were observed in 60 (82.2%) and 59 (80.8%) subjects in the adalimumab and SCT630 groups, respectively. On day 71, 69 (95.8%) subjects in the adalimumab group and 66 (93%) in the SCT630 group reported positive anti-drug antibodies. Among them, 15 (21.7%) and 11 (16.7%) subjects showed positive neutralizing antibodies, with no significant difference. SCT630 was well tolerated and demonstrated PK and safety profiles similar to adalimumab. The profiles support the initiation of further confirmatory study to demonstrate the clinical similarity of SCT630 to adalimumab.

A multicenter randomized trials to compare the bioequivalence and safety of a generic doxorubicin hydrochloride liposome injection with Caelyx ® in advanced breast cancer.

Purpose: To compare the pharmacokinetic (PK) bioequivalence (BE) and safety of a generic pegylated liposomal doxorubicin (PLD) formulation with the reference product Caelyx®. Methods: A multicenter, single-dose, open-label, randomized, two-way crossover study was conducted in patients with breast cancer. For each period, the patients were administered with the test or the reference PLD intravenously at a dose of 50 mg/m2. Cmax, AUC0-t and AUC0-∞ for free, and encapsulated doxorubicin (doxorubicin) and partial AUC (AUC0-48h, AUC48h-t) for encapsulated doxorubicin were evaluated in 17 blood samples taken predose, and increasing time intervals over the following 14 days in each period. A washout period of 28-35 days was observed before crossing over. Results: 48 patients were enrolled and randomised, of which 44 were included and analysed in bioequivalence set (BES). The 90% confidence intervals (CIs) of the geometric mean ratio (GMR) of Cmax, AUC0-t and AUC0-∞ for free doxorubicin and encapsulated doxorubicin all fall within the bioequivalent range of 80% to 125%. The 90% CIs of GMR of partial AUC (AUC0-48h, AUC48h-t) for encapsulated doxorubicin also fall within the bioequivalent range. 48 patients were all included in the safety set (SS). The incidence of treatment-emergent adverse events (TEAEs) related to T and R was 95.8% (46/48) and 97.8% (45/46) respectively. The highest incidence of TEAEs was various laboratory abnormalities. 2 patients withdrew due to T-drug-related AEs. Only one patient experienced serious adverse events and no death occurred in this study. There were no significant differences between the safety profiles of the generic formulation and Caelyx®. Conclusions: Bioequivalence between the test and the reference products was established for free and encapsulated doxorubicin. Clinical trial registration: http://www.chinadrugtrials.org.cn, identifier [CTR20210375].

Synthesis of zwitterionic polymer modified graphene oxide for hydrophilic enrichment of N-glycopeptides from urine of healthy subjects and patients with lung adenocarcinoma.

As one of the most common and important post-translational modifications, protein N-glycosylation plays essential roles in many biological processes and have long been considered closely correlated with the occurrence and progression of multiple diseases. Systematic characterization of these disease-related protein N-glycosylation is one of the most convenient ways for new diagnostic biomarker and therapeutic drug target discovering. However, the biological samples are extremely complex and the abundance of N-glycoproteins are especially low, which make highly efficient N-glycoprotein/glycopeptide enrichment before mass spectrometry analysis a prerequisite. In this work, a new type of hydrophilic material (GO-pDMAPS) was prepared by in situ growth of linear zwitterionic polymer chains on the surface of GO and it was successfully applied for N-glycopeptide enrichment from human urine. Due to the excellent hydrophilicity and the facilitate interactions between this GO-pDMAPS and the targets, a total of 1426 N-glycosylated sites corresponding to 766 N-glycoproteins as well as 790 N-glycosylation sites corresponding to 470 N-glycoproteins were enriched and identified from urine of healthy subjects and patients with lung adenocarcinoma, respectively. Among which, 27 N-glycoproteins were expressed exclusively and 4 N-glycoproteins were upregulated at least 3 times comparing with the healthy group, demonstrating the tremendous potential of this new hydrophilic material for large scale and in depth N-glycoproteome research.

Pharmacokinetics and Disposition of Heparin-Binding Growth Factor Midkine Antisense Oligonucleotide Nanoliposomes in Experimental Animal Species and Prediction of Human Pharmacokinetics Using a Physiologically Based Pharmacokinetic Model.

Encapsulating the antisense oligonucleotide drug MK-ASODN with nanoliposomes greatly improved its potency and targeting to the heparin-binding growth factor midkine. The disposition and pharmacokinetic (PK) parameters of MK-ASODN nanoliposomes were studied in monkeys and rats, and the human PK parameters were predicted based on preclinical data using a physiologically based pharmacokinetic (PBPK) model. Following intravenous injection, the drug plasma concentration rapidly declined in a multiexponential manner, and the drug was rapidly transferred to tissues from the circulation. The terminal t1/2 in plasma was clearly longer than that of the unmodified antisense nucleic acid drug. According to the AUC,MK-ASODN nanoliposomes were mainly distributed in the kidney, spleen, and liver. . MK-ASODN nanoliposomes were highly plasma protein bound, limiting their urinary excretion. Very little MK-ASODN nanoliposomes were detected in urine or feces. The plasma disposition of MK-ASODN nanoliposomes appeared nonlinear over the studied dose range of 11.5-46 mg kg-1. The monkey PBPK model of MK-ASODN nanoliposomes was well established and successfully extrapolated to predict MK-ASODN nanoliposome PK in humans. These disposition and PK data support further development in phase I clinical studies.

Safety, Tolerability, Pharmacokinetics, and Immunogenicity of a Monoclonal Antibody (SCTA01) Targeting SARS-CoV-2 in Healthy Adults: a Randomized, Double-Blind, Placebo-Controlled, Phase I Study.

SCTA01 is a novel monoclonal antibody with promising prophylactic and therapeutic potential for COVID-19. This study aimed to evaluate the safety, tolerability, pharmacokinetics (PK) and immunogenicity of SCTA01 in healthy adults. This was a randomized, double-blind, placebo-controlled, dose escalation phase I clinical trial. Healthy adults were randomly assigned to cohort 1 (n = 5; 3:2), cohort 2 (n = 8; 6:2), cohort 3, or cohort 4 (both n = 10; 8:2) to receive SCTA01 (5, 15, 30, and 50 mg/kg, respectively) versus placebo. All participants were followed up for clinical, laboratory, PK, and immunogenicity assessments for 84 days. The primary outcomes were the dose-limiting toxicity (DLT) and maximal tolerable dose (MTD), and the secondary outcomes included PK parameters, immunogenicity, and adverse events (AE). Of the 33 participants, 18 experienced treatment-related AEs; the frequency was 52.0% (13/25) in participants receiving SCTA01 and 62.5% (5/8) in those receiving placebo. All AEs were mild. There was no serious AE or death. No DLT was reported, and the MTD of SCTA01 was not reached. SCTA01 with a dose range of 5 to 50 mg/kg had nearly linear dose-proportional increases in Cmax and AUC parameters. An antidrug antibody response was detected in four (16.0%) participants receiving SCTA01, with low titers, between the baseline and day 28, but all became negative later. In conclusion, SCTA01 up to 50 mg/kg was safe and well-tolerated in healthy participants. Its PK parameters were nearly linear dose-proportional. (This study has been registered at ClinicalTrials.gov under identifier NCT04483375.).

A first-in-human, randomized, double-blind, single- and multiple-dose, phase I study of recombinant human thymosin ß4 in healthy Chinese volunteers.

The study evaluated the safety, tolerability, pharmacokinetics (PK) and anti-drug antibody (ADA) of the recombinant human thymosin ß4 (NL005) for single and multiple intravenous injections in healthy subjects. Seven cohorts, with 54 healthy subjects, were given a single intravenous dose of NL005 or placebo and were observed for 28 days. The cohorts received ascending doses of either 0.05, 0.25, 0.5, 2.0, 5.0, 12.5 or 25.0 µg/kg in the single-dose trial. A total of 30 healthy subjects were randomly enrolled in the multiple-dose trial, and 3 cohorts (0.5, 2.0 and 5.0 µg/kg) were administered once human thymosin ß4 daily for 10 days and observed for 28 days. The adverse events were mild to moderate in intensity. There were no dose-limiting toxicities or serious adverse events. The plasma concentration, maximum peak concentration (Cmax ) and AUC of each dose group increased with the increase in the dose. The tendency of terminal clearance in each dose group was consistent, and there was no obvious accumulation after continuous administration. Thus, the drug can be concluded to be well tolerated and safe in healthy people and suitable for use in a clinical study for the treatment of acute myocardial infarction.

Development and validation of an ultrasensitive LC-MS/MS method for the quantification of cetagliptin in human plasma and its application in a microdose clinical trial.

This study established and validated an LC-MS/MS method for the ultrasensitive determination of cetagliptin in human plasma. Sample pretreatment was achieved by liquid-liquid extraction with ethyl acetate, and chromatographic separation was performed on an XB-C18 analytical column (50 × 2.1 mm, 5 µm) with gradient elution (0.1% formic acid in acetonitrile and 0.1% formic acid) at a flow rate of 1.0 mL/min. For mass spectrometric detection, multiple reaction monitoring was used, and the ion transitions monitored were m/z 421.2-86.0 for cetagliptin and m/z 424.2-88.0 for cetagliptin-d3. Method validation was performed according to the U.S. Food and Drug Administration Bioanalytical Method Validation Guidance, for which the calibration curve was linear in the range of 50.0-2000 pg/mL. All of the other results, such as selectivity, lower limit of quantitation, precision, accuracy, matrix effect, recovery, and stability, met the acceptance criteria. The validated method was successfully applied in a microdose clinical trial to systematically investigate the pharmacokinetic profile of cetagliptin in healthy subjects. Both rapid absorption and prolonged duration demonstrate the potential value of cetagliptin for diabetes treatment.

A facile "one-material" strategy for tandem enrichment of small extracellular vesicles phosphoproteome.

Small extracellular vesicles (SEVs), are cell-derived, membrane-enclosed nanometer-sized vesicles that play vital roles in many biological processes. Recent years, more and more evidences proved that small EVs have close relationship with many diseases such as cancers and Alzheimer's disease. The use of phosphoproteins in SEVs as potential biomarkers is a promising new choice for early diagnosis and prognosis of cancer. However, current techniques for SEVs isolation still facing many challenges, such as highly instrument dependent, time consuming and insufficient purity. Furthermore, complex enrichment procedures and low microgram amounts of proteins available from clinical sources largely limit the throughput and the coveage depth of SEVs phosphoproteome mapping. Here, we synthesized Ti4+-modified magnetic graphene-oxide composites (GFST) and developed a "one-material" strategy for facile and efficient phosphoproteome enrichment and identification in SEVs from human serum. By taking advantage of chelation and electrostatic interactions between metal ions and phosphate groups, GFST shows excellent performance in both SEVs isolation and phosphopeptide enrichment. Close to 85% recovery is achieved within a few minutes by simple incubation with GFST and magnetic separation. Proteome profiling of the isolated serum SEVs without phosphopeptide enrichment results in 515 proteins, which is approximately one-fold more than those otained by ultracentrifugation or coprecipitation kits. Further application of GFST in one-material-based enrichment led to identification of 859 phosphosites in 530 phosphoproteins. Kinase-substrate correlation analysis reveals enriched substrates of CAMK in serum SEVs phosphoproteome. Therefore, we expect that the low instrument dependency and the limited sample requirement of this new strategy may facilitate clinical investigations in SEV-based transportation of abnormal kinases and substrates for drug target discovery and cancer monitoring.

Pharmacokinetics and Bioequivalence of Rasagiline Tablets in Chinese Healthy Subjects Under Fasting and Fed Conditions: An Open, Randomized, Single-Dose, Double-Cycle, Two-Sequence, Crossover Trial.

Objective: This study evaluated the pharmacokinetics, safety, and bioequivalence (BE) of two formulations of rasagiline tablets in healthy Chinese subjects under fasting and fed conditions. Methods: An open, randomized, single-dose, double-cycle, two-sequence, self-crossover pharmacokinetic study in healthy Chinese subjects under fasting and high-fat postprandial conditions was performed. A total of 108 healthy subjects (36 in the fasting group and 72 in the postprandial group) were recruited. In each cycle of the study under both conditions, subjects received a single oral dose of 1 mg of a test or reference preparation of rasagiline tablets (1 mg each). A washout period of 3 days was observed. Blood samples were obtained up to 10 h post-intake. Primary endpoints were the BE of major pharmacokinetic parameters (AUC0-t and AUC0-∞) and the maximum observed serum concentration (Cmax). Secondary endpoints were safety parameters. Results: The 90% confidence interval (CI) of the geometric mean ratio (GMR) of the test drug vs. the reference drug for rasagiline was 94.16-105.35% for the AUC0-t under fasting conditions and 99.88-107.07% under postprandial conditions. The 90% CIs for the AUC0-∞ were 93.55-105.01% and 99.59-107.05% under fasting and postprandial conditions, respectively. The 90% CIs for the Cmax were 88.26-108.46% and 89.54-118.23% under fasting and postprandial conditions, respectively. The 90% CIs for the test/reference AUC ratio and Cmax ratio were within the acceptable range (0.80-1.25) for BE. In this BE study, there were no serious adverse events (AEs). Conclusion: BE between the test and the reference products was established in both fasting and postprandial conditions. The two formulations of rasagiline showed good tolerability and a similar safety profile. Clinical Trial Registration: chinaDrugtrials.org.cn, identifier CTR20181466.

Bioequivalence Study Comparing Fixed-Dose Combination of Clopidogrel and Aspirin with Coadministration of Individual Formulations in Chinese Subjects Under Fed Conditions: A Phase I, Open-Label, Randomized, Crossover Study.

INTRODUCTION: Simultaneous administration of acetylsalicylic acid (ASA) and clopidogrel has demonstrated efficacy in the treatment of acute coronary syndrome. Clopidogrel + ASA in a fixed-dose combination (FDC) provides a pharmaceutical option to enhance adherence to the coadministration of dual antiplatelet therapy (DAPT). Herein, we evaluate the bioequivalence of enteric ASA and clopidogrel in an FDC compared with simultaneous administration of the individual formulations. METHODS: This study is a randomized, single-center, open-label, three-sequence, three-period, two-treatment, crossover study conducted in healthy Chinese male and female subjects under fed conditions. Subjects were randomized to receive, in each period, a single dose of (1) a combination tablet containing 75-mg clopidogrel and 100-mg enteric ASA (test formulation) or (2) coadministration of one 75-mg clopidogrel tablet and one 100-mg enteric-coated ASA tablet (reference formulations) under fed conditions. Plasma samples were analyzed for ASA, salicylic acid, clopidogrel, and the clopidogrel metabolite SR26334. For ASA, the reference-scaled average bioequivalence (RSABE) analysis was conducted for Cmax of ASA because within-subject standard deviation (SDW) was ≥ 0.294 for log-transformed Cmax. RESULTS: The point estimate (test/reference geometric mean ratio) was between 0.80 and 1.25, and the upper one-sided 95% confidence interval (CI) for the scaled average bioequivalence metric was ≤ 0 (-0.08). AUC of ASA as SDW was < 0.294 for log-transformed AUClast and AUC. Estimates of 90% CIs for log-transformed AUClast and AUC ratios were within the bioequivalence range of 0.80 to 1.25 (0.98-1.08 and 1.00-1.10, respectively). For clopidogrel, the 90% CIs for the ratios comparing log-transformed Cmax, AUClast, and AUC ratios of clopidogrel following administration of test versus reference formulation were calculated using the ABE method and were well within the acceptable range of 0.80 to 1.25 (1.02-1.12, 0.92-0.99, and 0.92-0.98, respectively). CONCLUSION: FDC of ASA and clopidogrel was bioequivalent to the simultaneous administration of the individual formulations in healthy Chinese subjects under fed conditions. TRIAL REGISTRATION: CTR20190376.

Development of a Gold Nanoparticle-Functionalized Surface Plasmon Resonance Assay for the Sensitive Detection of Monoclonal Antibodies and Its Application in Pharmacokinetics.

As a prominent human therapeutic, therapeutic monoclonal antibodies (mAbs) have attracted increasing attention in the past decade due to their high-targeting specificity, low toxicity, and prolonged efficacy. Systematic pharmacokinetic analysis of mAbs not only largely facilitates the understanding of their biologic functions but also promotes the development of therapeutic drug discovery, early clinical trial implementation, and therapeutic monitoring. However, the extremely complex nature of biomatrices and the especially low dosages of mAbs make their detection in biomatrices and further pharmacokinetic analysis highly challenging. Therefore, a method capable of reliably, quickly, and sensitively quantifying mAbs in biomatrices is urgently needed. In this work, we developed and evaluated an gold nanoparticle-functionalized surface plasmon resonance assay for cetuximab (C225) detection and pharmacokinetic analysis in rhesus monkeys. Combining its advantages of label-free pretreatment and amplified signal response, the lower limit of quantitation of C225 in monkey serum was reduced to 0.0125 µg/ml, and the linear range had an order of magnitude comparable to that of an ELISA-based method. Furthermore, the pharmacokinetics of C225 in rhesus monkeys was studied after intravenous infusions of single doses at 7.5, 24, and 75 mg/kg. The concentration of C225 in monkey serum was detectable after dosing for 720 hours. We believe that this new strategy will be applicable as a general protocol for mAb quantification, pharmacokinetic characteristic determination, and toxicokinetic analysis during drug development.

Role of the Ebola membrane in the protection conferred by the three-mAb cocktail MIL77.

MIL77, which has a higher manufacturing capacity than ZMapp, comprises MIL77-1, MIL77-2, and MIL77-3. The mechanisms by which these antibodies inhibit glycoprotein are unclear. Infection by viruses with lipid-bilayer envelopes occurs via the fusion of the viral membrane with the membrane of the target cell. Therefore, the interaction between the antibodies and the EBOV membrane is crucial. We examined the interactions between MIL77 and the viral membrane using SPR. MIL77-1 selectively binds to viral membranes, while MIL77-2 and MIL77-3 do not. MIL77-1's ability to screen the more rigid domains of the membranes results in a locally increased concentration of the drug at the fusion site. Although MIL77-2 recognizes an epitope of GP, it is not necessary in the MIL77 cocktail. These results highlight the importance of EBOV membrane interactions in improving the efficiency of a neutralizing antibody. Furthermore, the viral membrane may be an important target of antibodies against EBOV.

Development a hydrazide-functionalized thermosensitive polymer based homogeneous system for highly efficient N-glycoprotein/glycopeptide enrichment from human plasma exosome.

As one of the most common post-translational modifications, protein N-glycosylation precipitates in many important biological processes and has closely correlations with the occurrence and progression of multiple diseases. Plasma exosomes secreted by cells contain various bioactive N-glycoproteins which may serve as potential biomarkers for early disease diagnosis and treatment. However, the protein N-glycosylation profile in human plasma exosome is largely unknown, due to the technical challenges in glycoprotein identification. Signals of the rare N-glycoproteins/N-glycopeptides are severely suppressed by the abundant coexisting non-glycosylated counterparts in mass spectrometry analysis. Therefore, specific enrichment of N-glycoprotein/glycopeptide is a prerequisite for large scale N-glycosylation profiling. In this work, we developed a hydrazide functionalized thermosensitive polymer for efficient enrichment and in-depth identification of protein N-glycosylation in human plasma exosome by mass spectrometry. The polymer chains completely dissolve in the enrichment system to form a homogeneous solution. Therefore, efficient covalent coupling between the N-glycoprotein/glycopeptide and the polymer chain is achieved, due to the reduced interfacial mass transfer resistance and the densely packed accessible functional groups on the polymer chains. Furthermore, the thermosensitive polymer can be easily precipitated and recovered by simply rising the system temperature to above 34 °C. As a result, 329 N-glycosylation sites corresponding to 180 N-glycoproteins were enriched and identified from plasma exosomes of glioma patients and healthy subjects using the thermosensitive polymer. By quantitative comparison, we found 26 N-glycoproteins significantly changed between the glioma patients and the healthy subjects, demonstrating the potential of this new strategy for N-glycoproteome research of plasma exosome and biomarker discovery.

Preparation of polymer brushes grafted graphene oxide by atom transfer radical polymerization as a new support for trypsin immobilization and efficient proteome digestion.

Highly efficient protein digestion is one of the key issues in the "bottom-up" strategy-based proteomic studies. Compared with the time-consuming solution-based free protease digestion, immobilized protease digestion offers a promising alternative with obviously improved sample processing throughput. In this study, we proposed a new immobilized protease digestion strategy using two kinds of polymer-grafted graphene oxide (GO) conjugated trypsin. The polymer brush grafted GO was prepared using in situ polymer growth on initiator-functionalized GO using surface-initiated atom transfer radical polymerization (SI-ATRP) and characterized by AFM, TEM, TGA, and XPS. The polymer brush grafted GO supports three-dimensional trypsin immobilization, which not only increases the loading amount but also improves accessibility towards protein substrates. Both of the two types of immobilized trypsin provide 700 times shorter digestion time, while maintaining comparable protein/peptide identification scale compared with that of free trypsin digestion. More interestingly, combined application of the two types of immobilized trypsin with different surface-grafted polymers leads to at least 18.3/31.3% enhancement in protein/peptide identification compared with that obtained by digestion using a single type, indicating the potential of this digestion strategy for deeper proteome coverage using limited mass spectrometer machine hour. We expect these advantages may find valuable application in high throughput clinical proteomic studies, which often involve processing of a large number of samples. Graphical abstract Preparation of polymer brushes grafted and trypsin immobilized graphene oxide and its application in proteome digestion and mass spectrometry identification.

Synthesis of hydrazide-functionalized hydrophilic polymer hybrid graphene oxide for highly efficient N-glycopeptide enrichment and identification by mass spectrometry.

Protein N-glycosylation is one of the most important post-translational modifications, participating in many key biological and pathological processes. Large-scale and precise identification of N-glycosylated proteins and peptides is especially beneficial for understanding their biological functions and for discovery of new clinical biomarkers and therapeutic drug targets. However, protein N-glycosylation is microheterogeneous and low abundant in living organisms, therefore specific enrichment of N-glycosylated proteins/peptides before mass spectrometry analysis is a prerequisite. In this work, we developed a new type of polymer hybrid graphene oxide (GO) by in situ growth of hydrazide-functionalized hydrophilic polymer chains on the GO surface (GO-PAAH) for selective N-glycopeptide enrichment and identification by mass spectrometry. The densely attached and low steric hindrance hydrazide groups as well as the highly hydrophilic nature of GO-PAAH facilitate N-glycopeptide enrichment by the combination of hydrazide capturing and HILIC interaction. Taking advantage of the unique features of GO-PAAH, all of the three N-glycopeptides of bovine fetuin were successfully enriched and identified with significantly enhanced signal intensities from a digest mixture of bovine fetuin and bovine serum albumin at a mass ratio of 1:100, demonstrating the excellent enrichment selectivity of GO-PAAH. Furthermore, a total of 507 N-glycosylation sites and 480 N-glycopeptides in 232 N-glycoproteins were enriched and identified from 10µL of human serum by three replicates using this novel enrichment material, which is nearly two times higher than the commercial hydrazide resin based method (280 N-glycosylation sites, 261 N-glycopeptides and 144 N-glycoproteins in three experiments). Among the identified, 95 N-glycosylation sites were not reported in the Uniprot database, and 106 N-glycoproteins were disease related in the Nextprot database, indicating the potential of this new enrichment material in global mapping of protein N-glycosylation.

Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O-GlcNAc Proteins by Mass Spectrometry.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of proteins in eukaryotic cells. Despite their low abundance, O-GlcNAc-modified proteins play many important roles in regulating gene expression, signal transduction, and cell cycle. Aberrant O-GlcNAc proteins are correlated with many major human diseases, such as Alzheimer's disease, diabetes, and cancer. Because of the extremely low stoichiometry of O-GlcNAc proteins, enrichment is required before mass spectrometry analysis for large-scale identification and in-depth understanding of their cellular function. In this work, we designed and synthesized a novel thermosensitive immobilized triarylphosphine reagent as a convenient tool for efficient enrichment of azide-labeled O-GlcNAc proteins from complex biological samples. Immobilization of triarylphosphine on highly water-soluble thermosensitive polymer largely increases its solubility and reactivity in aqueous solution. As a result, facilitated coupling is achieved between triarylphosphine and azide-labeled O-GlcNAc proteins via Staudinger ligation, due to the increased triarylphosphine concentration, reduced interfacial mass transfer resistance, and steric hindrance in homogeneous reaction. Furthermore, solubility of the polymer from complete dissolution to full precipitation can be easily controlled by simply adjusting the environmental temperature. Therefore, facile sample recovery can be achieved by increasing the temperature to precipitate the polymer-O-GlcNAc protein conjugates from solution. This novel immobilized triarylphosphine reagent enables efficient enrichment and sensitive detection of more than 1700 potential O-GlcNAc proteins from HeLa cell using mass spectrometry, demonstrating its potential as a general strategy for low-abundance target enrichment.