Development, validation and application of a UHPLC-MS/MS method for quantification of the adiponectin-derived active peptide ADP355 in rat plasma.

A UHPLC-MS/MS method for the quantification of ADP355, an adiponectin-derived active peptide, was developed and validated. The extraction method employed simple protein precipitation using methanol and chromatographic separation was achieved on anAccucore™ RP-MS C18 column (100 × 2.1 mm, 2.6 µm, 80 Å), using 0.1% formic acid in both water and acetonitrile with gradient elution at the flow rate of 400 µl/min within 4.0 min. Detections were performed under positive ion mode with multiple reaction monitoring ion transitions m/z 1109.2 → 309.8 and 871.4 → 310.1 for ADP355 and Jt003 respectively at unit resolution. The linearity range of the calibration curve was 2-1,000 ng/ml with a lower limit detection of 0.5 ng/ml. The selectivity, linearity, precision, accuracy, recovery, matrix effect and stability were validated, and all items met the requirement of US Food and Drug Administration guidance. This method was successfully applied to an intravenous pharmacokinetic study of ADP355 in rats and the in-vitro stability in rat serum, plasma and whole blood was also assessed.

Elevated Serum Neuropeptide FF Levels Are Associated with Cognitive Decline in Patients with Spinal Cord Injury.

BACKGROUND: Spinal cord injury (SCI) has high incidence globally and is frequently accompanied by subsequent cognitive decline. Accurate early risk-categorization of SCI patients for cognitive decline using biomarkers can enable the timely application of appropriate neuroprotective measures and the development of new agents for the management of SCI-associated cognitive decline. Neuropeptide FF is an endogenous neuropeptide with a multitude of functions and is associated with neuroinflammatory processes. This prospective study investigated the predictive value of serum neuropeptide FF levels measured after acute SCI for subsequent cognitive decline. METHODS: 88 patients presenting with acute SCI without preexisting neurological injury, brain trauma, or severe systemic illness and 60 healthy controls were recruited. Serum neuropeptide FF levels, clinical, and routine laboratory variables including low-density lipoprotein, high-density lipoprotein, fasting blood glucose, total triiodothyronine (TT3), total thyroxine (TT4), and thyroid-stimulating hormone (TSH) levels collected from all subjects were assessed. Montreal cognitive assessment (MoCA) was performed 3 months after enrollment. SCI patients were grouped according to quartile of serum neuropeptide FF level and MoCA scores were compared using ANOVA. Additionally, multivariate linear regression with clinical and laboratory variables was performed to predict MoCA scores. RESULTS: SCI patients displayed significantly higher baseline serum neuropeptide FF levels than healthy controls (38.5 ± 4.1 versus 23.4 ± 2.0 pg/ml, p < 0.001∗∗). SCI patients in higher quartiles of baseline serum neuropeptide FF displayed significantly lower MoCA scores at 3 months. Linear regression analysis indicated serum neuropeptide FF levels as a significant independent predictor of worse MoCA scores after SCI (r = 0.331, p = 0.034∗). CONCLUSION: Early serum neuropeptide FF levels significantly and independently predicted cognitive decline after acute SCI among patients without preexisting neurological disorders.

Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV-vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.

Serum C-reactive protein metabolite (CRPM) is associated with incidence of contralateral knee osteoarthritis.

The heterogeneous nature of osteoarthritis (OA) and the need to subtype patients is widely accepted in the field. The biomarker CRPM, a metabolite of C-reactive protein (CRP), is released to the circulation during inflammation. Blood CRPM levels have shown to be associated with disease activity and response to treatment in rheumatoid arthritis (RA). We investigated the level of blood CRPM in OA compared to RA using data from two phase III knee OA and two RA studies (N = 1591). Moreover, the association between CRPM levels and radiographic progression was investigated. The mean CRPM levels were significantly lower in OA (8.5 [95% CI 8.3-8.8] ng/mL, n = 781) compared to the RA patients (12.8 [9.5-16.0] ng/mL, n = 60); however, a significant subset of OA patients (31%) had CRPM levels (≥ 9 ng/mL) comparable to RA. Furthermore, OA patients (n = 152) with CRPM levels ≥ 9 ng/mL were more likely to develop contra-lateral knee OA assessed by X-ray over a two-year follow-up period with an odds ratio of 2.2 [1.0-4.7]. These data suggest that CRPM is a blood-based biochemical marker for early identification OA patients with an inflammatory phenotype.

On the road of dried blood spot sampling for antidoping tests: Detection of GHRP-2 abuse.

Dried blood spots (DBSs) sampling is gaining support by the antidoping community because of simplicity and cost-effective characteristics, especially in collection, transport, and storage. Nevertheless, DBS applicability demands specific studies for each of the analytes proposed for testing. Here, GHRP-2 has been selected as a representing member of the growth hormone-releasing peptides (GHRPs) family to provide further evidence of DBS suitability for GHRPs abuse detection in sport testing. An analytical procedure to extract GHRP-2 and its main metabolite (AA-3) from DBS and to detect them by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. The method has been validated for the detection of GHRP-2. Specificity and identification capabilities have been assessed in agreement with antidoping guidelines. The low AA-3 levels found in DBS samples prevented its effective application for the determination of this metabolite. The limit of detection (LoD) for GHRP-2 has been established at 50 pg/ml. Long-term stability (>2 years) has been confirmed. The procedure has been successfully applied to actual DBS samples from an administration study with a single intravenous dose of GHRP-2 (100 µg) being detected up to 4 h after drug injection. GHRP-2 concentrations have been higher in venous blood DBS than in capillary blood DBS. Despite the observed differences, a similar detection window has been achieved independently of the type of blood used. In summary, this study provides specific evidence supporting DBS usefulness to detect GHRP-2, and potentially other GHRPs family members, for antidoping tests.

Conjugating MMAE to a novel anti-HER2 antibody for selective targeted delivery.

OBJECTIVE: To investigate the target delivery properties of RC48-ADC, a novel antibody drug conjugate (ADC) comprising cytotoxic monomethyl auristatin E (MMAE) and an anti-human epidermal growth factor receptor 2 (HER2) antibody tethered via valine-citrulline linker, in vitro and in vivo. MATERIALS AND METHODS: Dissociation rate of MMAE from RC48-ADC was used as an estimate of its stability in serum. Cytotoxicity of the antibody and RC48-ADC towards multiple cell lines was measured. Subcellular distribution of the drug was determined by fluorescence imaging. The mechanism of lysosome targeting was verified. Endocytic pathways of RC48-ADC were assessed by the cellular fluorescence intensity of fluorescently-labelled drugs. Intracellular and extracellular distribution of MMAE was analysed after RC48-ADC or MMAE administration to characterize MMAE release. The serum and tumour concentration of MMAE was compared after tail-vein injection of RC48-ADC into tumour-bearing mice. RESULTS: RC48-ADC was highly stable in human serum. HER2-overexpressed cell line SK-BR-3 proliferation was stronger when suppressed by RC48-ADC than by the naked antibody. Both RC48-ADC and naked antibody were internalized via caveolae-mediated and clathrin-mediated endocytosis and concentrated in lysosomes. Higher HER2 expression was associated with enhanced uptake and intracellular release of conjugated MMAE; free MMAE could kill tumour cells via the bystander effect. Although serum RC48-ADC concentration was higher than that in tumours, exposure of MMAE in tumours was ~200 times higher than in serum, which rationalized the reduced toxicity of RC48-ADC. CONCLUSIONS: In vitro and in vivo experiments confirmed the targeted transport and release of RC48-ADC; it could selectively deliver MMAE to the targeted HER2-positive cell or tumour tissue, which could reduce off-target toxicity and enhance anti-tumour potency in humans.

Physiologically Based Pharmacokinetic Model-Informed Drug Development for Polatuzumab Vedotin: Label for Drug-Drug Interactions Without Dedicated Clinical Trials.

Model-informed drug development (MIDD) has become an important approach to improving clinical trial efficiency, optimizing drug dosing, and proposing drug labeling in the absence of dedicated clinical trials. For the first time, we developed a physiologically based pharmacokinetic (PBPK) model-based approach to assess CYP3A-mediated drug-drug interaction (DDI) risk for polatuzumab vedotin (Polivy), an anti-CD79b-vc-monomethyl auristatin E (MMAE) antibody-drug conjugate (ADC). The model was developed and verified using data from the existing clinical DDI study for brentuximab vedotin, a similar vc-MMAE ADC. Analogous to the brentuximab vedotin clinical study, polatuzumab vedotin at the proposed labeled dose was predicted to have a limited drug interaction potential with strong CYP3A inhibitor and inducer. Polatuzumab vedotin was also predicted to neither inhibit nor induce CYP3A. The present work demonstrated a high-impact application using a PBPK MIDD approach to predict the CYP3A-mediated DDI to enable drug labeling in the absence of any dedicated clinical DDI study. The key considerations for the PBPK report included in the Biologics License Application/Marketing Authorization Application submission, as well as the strategy and responses to address some of the critical and challenging questions from the health authorities following the submission are also discussed. Our experience and associated perspective using a PBPK approach to ultimately enable a drug interaction label claim for polatuzumab vedotin in lieu of a dedicated clinical DDI study, as well as the interactions with the regulatory agencies, further provides confidence in applying MIDD to accelerate the registration and approval of new drug therapies.

Thromboelastographic Research of Arginine-, Leucine and Lysine-Containing Peptides.

Anticoagulant effects of 12 short peptides of the glyproline series - Arg-Glu-Arg-Pro-Gly-Pro (RERPGP), Arg-Glu-Arg-Val-Gly-Pro (RERVGP), Arg-Glu-Arg-Gly-Pro (RERGP), Arg-Pro-Gly-Pro (RPGP), Pro-Leu-Pro (PLP), Pro-Leu-Pro-Ala (PLPA), Pro-Gly-Pro-Leu (PGPL), Phe-Pro-Leu-Pro-Ala (FPLPA), Pyr-Arg-Pro (PyrRP), Lys-Lys-Arg-Arg-Pro-Gly-Pro (KKRRPGP), Arg-Lys-Lys-Arg-Pro-Gly-Pro (RKKRPGP), and Lys-Arg-Lys-Pro-Gly-Pro (KRKPGP) in concentrations of 10-3 and 10-2 mg/ml in vitro was demonstrated by the thromboelastographic method. The effects of 6 peptides ((RERPGP, RPGP, PLP, PLPA, RKKRPGP, and KKRRPGP) were also observed in vivo after intranasal or oral administration. Changes in the studied thromboelastographic parameters towards hypocoagulation in comparison with the control group were noted. Arginine and leucine glyprolines produced the maximum anticoagulant effect.

Aging-associated changes in CD47 arrangement and interaction with thrombospondin-1 on red blood cells visualized by super-resolution imaging.

CD47 serves as a ligand for signaling regulatory protein α (SIRPα) and as a receptor for thrombospondin-1 (TSP-1). Although CD47, TSP-1, and SIRPα are thought to be involved in the clearance of aged red blood cells (RBCs), aging-associated changes in the expression and interaction of these molecules on RBCs have been elusive. Using direct stochastic optical reconstruction microscopy (dSTORM)-based imaging and quantitative analysis, we can report that CD47 molecules on young RBCs reside as nanoclusters with little binding to TSP-1, suggesting a minimal role for TSP-1/CD47 signaling in normal RBCs. On aged RBCs, CD47 molecules decreased in number but formed bigger and denser clusters, with increased ability to bind TSP-1. Exposure of aged RBCs to TSP-1 resulted in a further increase in the size of CD47 clusters via a lipid raft-dependent mechanism. Furthermore, CD47 cluster formation was dramatically inhibited on thbs1-/- mouse RBCs and associated with a significantly prolonged RBC lifespan. These results indicate that the strength of CD47 binding to its ligand TSP-1 is predominantly determined by the distribution pattern and not the amount of CD47 molecules on RBCs, and offer direct evidence for the role of TSP-1 in phagocytosis of aged RBCs. This study provides clear nanoscale pictures of aging-associated changes in CD47 distribution and TSP-1/CD47 interaction on the cell surface, and insights into the molecular basis for how these molecules coordinate to remove aged RBCs.

Imunofan-RDKVYR Peptide-Stimulates Skin Cell Proliferation and Promotes Tissue Repair.

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today's science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name "Imunofan" (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30-40% and 20-50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

The systemic inflammatory response syndrome in acute antipsychotic poisoning.

The purpose of this study was to investigate the mutual effect of systemic inflammatory response syndrome (SIRS) accompanied with fibrinolysis, endotoxemia, and coagulation in severe cases of antipsychotic poisoning. A total of 199 patients were examined, of which 71 were men and 128 were women. The age of the patients was from 22 to 63 years, (45.3 ± 6.1 years on average). According to the results of the course of therapy, the patients were divided into two groups. In the blood plasma, the content of C-reactive protein, fibrinogen and its proteolysis products (oligopeptides, D-dimers), interleukin-6 were determined. In the first 1 to 3 days, in group 1, the level of interleukin-6 decreased and approached the normal level (P ≤ .05). The opposite trend continued throughout the observation of patients from group 2-their levels of interleukin-6 increased day by day (P ≤ .05). The concentration of D-dimer already in 1 day after admission to intensive care in patients from group 2 exceeded the norm by 14 times (P ≤ .05). The level of D-dimer correlated with the level of oligopeptides in blood plasma upon admission, as well as for 3 and 5 days after admission to intensive care: 0.36, 0.76 at P ≤ .05, 0.94 at P ≤ .01, respectively. Similar correlations were obtained for the content of oligopeptides in urine and the level of D-dimer: 0.55, 0.85 at P ≤ .05, 0.93 at P ≤ .01. In this regard, the most pronounced correlation is that between the SIRS score, plasma D-dimer level, and the plasma level of the D-dimer derivatives, oligopeptides.

Structure-activity relationships of tubulysin analogues.

The tubulysins are an emerging antibody-drug conjugate (ADC) payload that maintain potent anti-proliferative activity against cells that exhibit the multi-drug resistant (MDR) phenotype. These drugs possess a C-11 acetate known to be hydrolytically unstable in plasma, and loss of the acetate significantly attenuates cytotoxicity. Structure-activity relationship studies were undertaken to identify stable C-11 tubulysin analogues that maintain affinity for tubulin and potent cytotoxicity. After identifying several C-11 alkoxy analogues that possess comparable biological activity to tubulysin M with significantly improved plasma stability, additional analogues of both the Ile residue and N-terminal position were synthesized. These studies revealed that minor changes within the tubulin binding site of tubulysin can profoundly alter the activity of this chemotype, particularly against MDR-positive cell types.

Degradation of thymic humoral factor γ2 in human, rat and mouse blood: An experimental and theoretical study.

The degradation of the immunomodulatory octapeptide, thymic humoral factor γ2 (THF-γ2, thymoctonan) has been studied in whole blood samples from human, rat and mouse. The peptide, Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu, was shown to be rapidly degraded by peptidases. The half-life of the intact peptide was less than 6 min at 37 °C in blood from the three species tested. The main fragments formed from THF-γ2 were found to be Glu-Asp-Gly-Pro-Lys-Phe-Leu (2-8), Asp-Gly-Pro-Lys-Phe-Leu (3-8) and Glu-Asp-Gly-Pro-Lys (2-6) in human and in rat blood and 2-8 and 2-6 in mouse blood. Analysis of the time course of degradation revealed a sequential removal of single amino acids from the N-terminus (aminopeptidase activities) in a process that was apparently unable to cleave the Gly-Pro bond (positions 4-5 in the peptide) together with an independent cleavage of the Lys-Phe bond (positions 6-7 in the peptide) to release the dipeptide Phe-Leu. This behaviour and the effects of inhibitors showed the involvement of metallo-exopeptidases in the N-terminal digestion and a phosphoramidon-sensitive metallo-endopeptidase in the cleavage of the Lys-Phe bond. The degradation patterns in human blood were modelled in terms of the competing pathways involved approximating to first-order kinetics, and an analytical solution obtained via the method of Laplace Transforms. The half-life of THF degradation in whole rat blood sample was found to be significantly lower than in human or mouse.

ß2-Homo-Amino Acid Scan of µ-Selective Opioid Tetrapeptide TAPP.

TAPP (H-Tyr-d-Ala-Phe-Phe-NH2) is a potent, µ-selective opioid ligand. In order to gain further insights into pharmacophoric features of this tetrapeptide, we have performed a ß2-Homo-amino acid (ß2hAA) scan of the TAPP sequence. To this aim, 10 novel analogues have been synthesized and evaluated for µ-opioid and δ-opioid receptor affinity as well as for stability in human plasma. The derivatives included compounds in which a (R)- or (S)-ß2-Homo-Homologue replaced the amino acids in the TAPP sequence. The derivatives with (R)- or (S)-ß2hPhe4 turned out to bind µOR with affinities equal to that of the parent. ß2hAAs in position 1 and 3 resulted in rather large affinity decreases, but the change differed depending on the stereochemistry. ß2-Homologation in the second position gave derivatives with very poor µOR binding. According to molecular modelling, the presented α/ß-peptides adopt a variety of binding poses with their common element being an ionic interaction between a protonable amine of the first residue and Asp147. A feature required for high µOR affinity seems the ability to accommodate the ring in the fourth residue in a manner similar to that found for TAPP. Contrary to what might be expected, several compounds were significantly less stable in human plasma than the parent compound.

Improved targeting of an anti-TAG-72 antibody drug conjugate for the treatment of ovarian cancer.

INTRODUCTION: Ovarian cancer has only a 17% 5-year survival rate in patients diagnosed with late stage disease. Tumor-associated glycoprotein-72 (TAG72), expressed in 88% of all stages of ovarian cancer, is an excellent candidate for antibody-targeted therapy, as it is not expressed in normal human adult tissues, except in the secretory endometrium. METHODS: Using the clinically relevant anti-TAG72 murine monoclonal antibody CC49, we evaluated antibody drug conjugates (ADCs) incorporating the highly potent, synthetic antimitotic agent monomethylauristatin E (MMAE). MMAE was conjugated to CC49 via reduced disulfides in the hinge region, using three different types of linker chemistry, vinylsulfone (VS-MMAE), bromoacetamido (Br-MMAE), and maleimido (mal-MMAE). RESULTS: The drug antibody ratios (DARs) of the three ADCs were 2.3 for VS-MMAE, 10 for Br-MMAE, and 9.5 for mal-MMAE. All three ADCs exhibited excellent tumor to blood ratios on PET imaging, but the absolute uptake of CC49-mal-MMAE (3.3%ID/g) was low compared to CC49-Br-MMAE (6.43%ID/g), at 142 hours. Blood clearance at 43 hours was 38% for intact CC49, about 24% for both CC49-VS-MMAE and CC49-Br-MMAE, and 7% for CC49-mal-MMAE. CC49-VS-MMAE was not further studied due to its low DAR, while CC49-mal-MMAE was ineffective in the OVCAR3 xenograft likely due to its rapid blood clearance. In contrast, CC49-Br-MMAE treated mice exhibited an average of a 15.6 day tumor growth delay and a 40% increase in survival vs controls with four doses of 7.5 or 15 mg/kg of CC49-Br-MMAE. CONCLUSION: We conclude that CC49-Br-MMAE with a high DAR and stable linker performs well in a difficult to treat solid tumor model.

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS.

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone-releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized "hematocrit effect." Graphical abstract.

Liquid chromatography-high resolution mass spectrometric method for the quantification of monomethyl auristatin E (MMAE) and its preclinical pharmacokinetics.

MMAE is a potent antimitotic drug used as payload of an antibody-drug conjugate which shows potent activity in preclinical and clinical studies against a range of lymphomas, leukemia and solid tumors. Liquid chromatography-high resolution mass spectrometric method was developed for the quantification of MMAE and its preclinical pharmacokinetics. The method consisted of protein precipitation using acetonitrile (ACN) for sample preparation and liquid chromatography - quadrupole - time-of-flight - tandem mass spectrometry (LC-qTOF-MS/MS) analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2 ), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 1.01-2,200 ng/mL for MMAE. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. Recovery was 42.84%. The dilution integrity was determined for 5-fold dilution and the accuracy and precision ranged within ±25%. The stability results indicated that MMAE was stable for the following conditions: short-term (4 h), long-term (4 weeks), freeze/thaw (3 cycles) and post-preparative stability (12 h). This qualified method was successfully applied to a pharmacokinetic study of MMAE in rat as a preclinical animal model. The PK results suggest that MMAE has moderate CL and low BA.Also, these results would be helpful in having a comprehensive understanding of the PK characteristics of MMAE and developing ADC in future.

Self-Assembled Monolayer Epitope Bridges for Molecular Imprinting and Cancer Biomarker Sensing.

The research in biomedicine, cell signaling, diagnostics, and biocatalysis rely on selective protein binders that specifically capture a protein in a complex medium for either preparative or analytical use. These molecules are generally of biological origin and exposed to instability, denaturation, high cost, and inherently low binding capability. Imprinted polymers, serving as the artificial protein binders, demonstrate good potential to overcome these drawbacks. In this study, a novel epitope imprinting strategy is reported by employing double-cysteine-modified peptides as the templates and adsorbing the templates on a gold surface by means of forming self-assembled monolayer bridges, followed by electropolymerization to create a polymer network. The imprinted surface was initially designed to demonstrate specific affinity toward a short peptide (i.e., the epitope) or a target protein (i.e., neuron specific enolase) in buffer. This surface was subsequently used to measure the cancer biomarker in human serum that allows detecting 12 times lower concentration than threshold level of the biomarker. The molecular receptors exhibited a Kd < 65 pM for their respective target protein and low cross-reactivity with four nonspecific molecules. As compared to current strategies for the epitope imprinting, for example, through traditional, vertically adsorbed, or histidine-modified peptides, such a molecularly tunable system based on a surface-imprinting process may provide more efficient sensing systems with desirable affinity, sensitivity, and specificity in diagnostics applications.

A phase 1 study evaluating safety and pharmacokinetics of losatuxizumab vedotin (ABBV-221), an anti-EGFR antibody-drug conjugate carrying monomethyl auristatin E, in patients with solid tumors likely to overexpress EGFR.

Losatuxizumab vedotin (formerly ABBV-221) is a second-generation antibody-drug conjugate targeting epidermal growth factor receptor (EGFR). In this multicenter phase 1 study, eligible patients with EGFR-dependent solid tumors received losatuxizumab vedotin (3 + 3 design) intravenously at starting dose of 0.3 mg/kg over 3 h per 21-day cycle, with alternate dosing schedules utilized (2 weeks on/1 week off or weekly) to mitigate infusion reactions. Forty-five patients received ≥1 doses of losatuxizumab vedotin (13 colon, 6 non-small cell lung cancer, 5 head and neck [HNC], 5 glioblastoma multiforme, 2 breast, 14 other). Tumor samples were evaluated for EGFR protein expression by immunohistochemistry, EGFR and EGFR ligand mRNA expression by RNAseq, and results compared with outcome. Most common adverse events were infusion-related reaction (22/45; 49%) and fatigue (20/45; 44%). While most infusion reactions were grade ≤ 2, four patients experienced grade ≥3 infusion reactions. Several infusion reaction mitigation strategies were explored. Because of the high incidence of infusion reactions, the trial was stopped and the maximum tolerated dose was not reached. The last cleared dose: 6 mg/kg/cycle. Nineteen patients (42%) had stable disease; 4 remained on study >6 months. One HNC patient with increased levels of EGFR and EGFR ligands (amphiregulin, epiregulin) achieved a confirmed partial response. Pharmacokinetic analysis of losatuxizumab vedotin showed exposures appeared to be approximately dose-proportional. The high frequency of infusion reactions necessitated early closure of this trial. The detailed mitigation strategies used in this protocol for infusion-related reactions may provide beneficial information for trial design of agents with high infusion reaction rates.

Identification of ex vivo catabolites of peptides with doping potential in equine plasma by HILIC-HRMS.

Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection. In the present study, the stability of 27 bioactive peptides with protection at both termini in equine plasma was examined under different incubation conditions, using HILIC coupled to HRMS. Of the 27 peptides, 13 were stable after incubation at 37°C for 72 hr, but the remaining 14 were less stable. Ex vivo catabolites of these 14 peptides were detected using their theoretical masses generated in silico, their appearance was monitored over the time course of incubation, and their identity was verified by their product ion spectra. Catabolites identified for chemotactic peptide, DALDA, dmtDALDA, deltorphins I and II, Hyp6 -dermorphin, Lys7 -dermorphin, and dermorphin analog are novel. A d-amino acid residue at position 2 or 1 of a peptide or next to its C-terminus protected the relevant terminal from degradation by exopeptidases, but such a residue at position 3 did not. A pGlu residue or N-methylation at the N-terminus of a peptide did not protect its N-terminal. Ethylamide at the C-terminus of a peptide provided the C-terminal protection from attacks by carboxypeptidases. The C-terminal Lys amide in DALDA, dmtDALDA, and Lys7 -dermorphin was susceptible to cleavage by plasma enzymes, which is the first report, to the authors' knowledge. The results from the present study provide insights into the stability of peptides in plasma.