Stability In Human Plasma Research Articles

Tailoring naringenin conjugates with amplified and triple antiplatelet activity profile: Rational design, synthesis, human plasma stability and in vitro evaluation

BackgroundThe current standard-of-care antiplatelet therapy in cardiovascular disease patients is consisted of cyclooxygenase-1 (COX-1) inhibitor aspirin, along with a platelet receptor P2Y12 antagonist. Recently, the triple antiplatelet therapy with aspirin, a P2Y12 receptor antagonist and a protease activated receptor-1 (PAR-1) antagonist, has been suggested for the secondary prevention of atherothrombotic events, however presented an increased risk of bleeding. Therefore, the quest for novel antiplatelet agents simultaneously targeting the three pathways with improved efficacy/safety profile is of immense importance. Flavonoids as pre-validated ligands for numerous targets could serve as scaffolds targeting the three platelet activation pathways. MethodsComputational methods, Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) plasma stability and in vitro platelet aggregation experiments were used to establish the antiplatelet activity of the flavonoid naringenin and its conjugates. ResultsIn silico studies indicated that naringenin could bear a potent triple antiplatelet activity by inhibiting different platelet aggregation mechanisms. However, we found that in human platelets naringenin has diminished activity. We rationally designed and synthesized different naringenin conjugates aiming to amplify the antiplatelet activity of the parent compound. UHPLC-MS/MS revealed a slow degradation rate for a docosahexaenoic acid (DHA) – naringenin conjugate in human plasma. The antiplatelet profile of the new analogues was evaluated against in vitro platelet aggregation induced by several platelet agonists. ConclusionsThe DHA – naringenin hybrid presented triple antiplatelet activity simultaneously targeting PAR-1, P2Y12 and COX-1 platelet activation pathways. General significanceNatural products could offer a rich source for novel bioactives as a powerful alternative to the current combinatorial use of three different antiplatelet drugs.

Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

Novel small peptides derived from VEGF125-136: potential drugs for radioactive diagnosis and therapy in A549 tumor-bearing nude mice

Vascular endothelial growth factor receptor (VEGFR) is a critical factor in tumor angiogenesis and has been considered a potential target for receptor-mediated radionuclide imaging and therapy. In this study, we identified two peptides (QKRKRKKSRKKH and RKRKRKKSRYIVLS) derived from VEGF125-136 that displayed high binding affinities to VEGFR and strong inhibition of A549 cell growth. 99mTc- and 188Re-labeled peptides displayed high labeling efficiency and favorable stability in saline and human plasma. At the cellular level, the radiolabeled peptides could bind with A549 cells and be internalized via the VEGFR-1-mediated pathway. 99mTc/188Re-labeled peptide was significantly accumulated at xenograft tumors, as observed with single-photon emission computed tomography (SPECT) planar imaging. Moreover, 188Re-labeled peptides significantly inhibited tumor growth, prolonged the survival time of the tumor-bearing nude mice and resulted in much more necrotic regions and apoptotic cells in the A549 xenograft tumors. These results demonstrated that these two peptides as candidate drugs for radionuclide imaging and tumor therapy.

The Potential Influence of Fever on Imipenem and Meropenem Concentrations in Human Plasma

Therapeutic drug monitoring of β-lactams represents an effective tool to optimise antibiotic exposure and is gaining popularity especially in patient populations in which pharmacokinetic parameters vary substantially due to numerous pathophysiological and iatrogenic factors. Carbapenems are relatively unstable in solutions and their limited stability has been shown to be temperature-dependent. However, data on their stability in human plasma are still scarce. The aim of our study was to compare the stability of imipenem and meropenem in human plasma at normal body temperature and elevated to assess the potential influence of fever on their concentrations. Commercially available analytical standards of meropenem and imipenem were reconstituted and mixed with human plasma to produce samples with antibiotic concentrations of 4 and 50 mg/L, which were kept at 37 °C and 40 °C. Subsequently, the samples were assayed after 0, 1, and 2 hours using high-performance liquid chromatography with ultraviolet detection. The mean meropenem concentrations were decreased by 16 % and 30 % after 1 and 2 hours at 37 °C, respectively, and by 24 % and 36 % at 40 °C, respectively. For imipenem, the effect of the temperature on the drug stability was considerably more pronounced. At 37 °C, the mean imipenem concentrations were decreased by 39 % and 67 % after 1 and 2 hours, respectively, whilst at 40 °C, the concentrations were decreased by 60 % and 83 %, respectively (P < 0.000 1 for 37 °C vs. 40 °C). The relative effect of the temperature was independent of the initial antibiotic concentration. In our in vitro study we have shown that fever could have an effect on plasma concentrations of carbapenems. The presence of fever in a patient is therefore another aspect to consider when interpreting plasma levels and adjusting doses in TDM.

Structure-activity relationship study of the tumour-targeting peptide A20FMDV2 via modification of Lys16, Leu13, and N- and/or C-terminal functionality

The 20-residue linear peptide A20FMDV2 has been shown to exhibit high selectivity and affinity for the tumour-related αvβ6 integrin and has potential as a vector for therapeutic drugs. However, it exhibits poor half-life in plasma in part due to its high susceptibility to serum proteases. In this study fourteen A20FMDV2 analogues incorporating non-proteinogenic substitutes of the native Lys16 and Leu13 residues and six A20FMDV2 analogues containing modified N- and C-termini were synthesised to increase the half-life and activity of A20FMDV2. The analogues incorporating modified terminal motifs of A20FMDV2 were found to strongly bind to the αvβ6 integrin and were subsequently functionalized with the diethylenetriaminepentaacetic acid chelating agent to facilitate coupling with radioactive indium-111 for human plasma stability and in vivo biodistribution studies. A20FMDV2 peptide variants incorporating an N-terminal d-Asn and C-terminal d-Thr exhibited improved relative activity in vitro and were less susceptible to plasma degradation.

Drug-like property-driven optimization of 4-substituted 1,5-diarylanilines as potent HIV-1 non-nucleoside reverse transcriptase inhibitors against rilpivirine-resistant mutant virus

On the basis of our prior structure-activity relationship (SAR) results, our current lead optimization of 1,5-diarylanilines (DAANs) focused on the 4-substituent (R1) on the central phenyl ring as a modifiable position related simultaneously to improved drug resistance profiles and drug-like properties. Newly synthesized p-cyanovinyl-DAANs (8a–8g) with different R1 side chains plus prior active p-cyanoethyl-DAANs (4a–4c) were evaluated not only for anti-HIV potency against both wild-type HIV virus and rilpivirine-resistant (E138K, E138K+M184I) viral replication, but also for multiple drug-like properties, including aqueous solubility, lipophilicity, and metabolic stability in human liver microsomes and human plasma. This study revealed that both ester and amide R1 substituents led to low nanomolar anti-HIV potency against wild-type and rilpivirine-resistant viral strains (E138K-resistance fold changes<3). The N-substituted amide-R1 side chains were superior to ester-R1 likely due to improved aqueous solubility, lipophilicity, and higher metabolic stability in vitro. Thus, three amide-DAANs 8e, 4a, and 4b were identified with high potency against wild-type and rilpivirine-resistant viral strains and multiple desirable drug-like properties.

Myristic acid-modified thymopentin for enhanced plasma stability and immune-modulating activity

Natural albumin ligand (fatty acids)-conjugated peptides can rapidly bind to circulating albumin and form complexes to control the release of peptides. The purpose of this study was to prolong the half-life and immune-modulating effects of thymopentin (TP5) by using the albumin binding strategy. We synthesized myristic acid-modified TP5 (TP5-MA) by conjugating a myristic acid-acylated lysine to a permissive site of TP5, which improved the albumin binding affinity of TP5-MA and dramatically enhanced its stability in human plasma. We observed well-preserved bioactivities of TP5-MA in RAW264.7 macrophages using a tumor necrosis factor (TNF)-α stimulation assay. Moreover, the prolonged immune-modulating effect of TP5-MA was confirmed by the normalized CD4+/CD8+ ratio in immune-depressed rat models, which resulted in a reduced administration frequency (twice per week). In general, the enhanced pharmacokinetic and pharmacodynamic properties of TP5-MA make it a promising product for the treatment of immunodeficiency diseases.

Development of a New Folate-Derived Ga-68-Based PET Imaging Agent.

The folate receptor (FR) has emerged as an interesting diagnostic and therapeutic drug target with many potential applications in oncologic and inflammatory disorders. It was therefore the aim of this study to develop a folate-derived Ga-68-based positron emission tomography (PET) imaging tracer that is straightforward to radiolabel and could be broadly used in clinical studies. We validated its target binding affinity and specificity and compared it to [99mTc]EC20, the folate single-photon emission computed tomography (SPECT) imaging tracer that has been most extensively studied clinically so far. The new folic acid-derived PET imaging agent is linked via a polyethyleneglycol linker to the chelator 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA). This new compound, NOTA-folate, was labeled with gallium-68. We tested the probe's stability in human plasma and its selectivity in vitro, using the FR-positive KB cell line as well as the FR-negative A549 cell line. The pharmacokinetic profile of [68Ga]NOTA-folate was evaluated in FR-positive KB mouse xenografts. Following intravenous injection of [68Ga]NOTA-folate (383±53μCi), PET/computed tomography (CT) imaging studies as well as biodistribution studies were performed using KB tumor-bearing mice (n=3). In vitro as well as in vivo studies were performed in parallel with the SPECT imaging tracer [99mTc]EC20. In comparison to [99mTc]EC20 (radiochemical yield (RCY)=82.0±2.9%, 91.8±2.0% purity), similar radiochemical yield (87.2±6.9%) and radiochemical purity (95.6±1.8%) could be achieved for [68Ga]NOTA-folate. For both tracers, we observed high affinity for FR-positive cells in vitro and high plasma stability. In PET/CT and biodistribution studies, [68Ga]NOTA-folate appeared to display slightly superior in vivo performance in comparison to [99mTc]EC20. In detail, 68Ga-NOTA-folate showed very good tumor uptake and retention (6.6±1.1%ID/g), relatively low kidney uptake (21.7±1.1%ID/g), and very low liver uptake (0.38±0.08%ID/g). In vivo blocking studies using a fivefold excess of EC20 reduced the tumor uptake to 2.5±0.7%ID/g, confirming receptor specific binding of [68Ga]NOTA-folate in vivo. We validated a new Ga-68 folate-based PET imaging agent with excellent pharmacokinetics and tumor uptake. Based on a head-to-head comparison between both tracers, [68Ga]NOTA-folate is a suitable imaging probe for the delineation of FR-positive tumors and a promising candidate for clinical translation.

Curcumin diethyl disuccinate encapsulated in chitosan/alginate nanoparticles for improvement of its in vitro cytotoxicity against MDA-MB-231 human breast cancer cells.

Curcumin diethyl disuccinate (CDD) is a succinate prodrug of curcuminoids that has better stability in human plasma and improved in vitro cytotoxicity compared to curcumin. Therefore, CDD has the potential for further development as an anticancer agent. In this study, we focused on optimization of the formulation of CDD-loaded chitosan/alginate nanoparticles using Box-Behnken statistical design to enhance the therapeutic efficacy of CDD. Oil-in-water emulsification followed by ionotropic gelification was used to prepare the CDD-loaded chitosan/ alginate nanoparticles. A formulation with a 0.05:1 chitosan/alginate mass ratio, 0.65% (w/v) Pluronic F127 and 1.5 mg/ml CDD was found to be optimal. FTIR, TGA and XRD confirmed the encapsulation of CDD molecules in the nanoparticles. In vitro cytotoxicity and cellular uptake studies showed that CDD-loaded chitosan/alginate nanoparticles had significantly higher cytotoxicity and cellular uptake in human breast adenocarcinoma MDA-MB-231 cells, compared to free CDD. Physical and chemical stability studies indicated that the optimally formulated CDD-loaded chitosan/alginate nanoparticles were stable at 4 °C for 3 months.

Investigations of the influence of liposome composition on vesicle stability and drug transfer in human plasma: a transfer study

Liposomal delivery constitutes a promising approach for i.v. administration of temoporfin (mTHPC) because lipid membranes can host these drug molecules. This study investigates the transfer and release of mTHPC to plasma proteins and stability of various liposomal formulations. To this end, we employed traces of radioactive markers and studied the effects of fatty acid chain length and the degree of saturation in the lipophilic tail, addition of cholesterol and PEGylation of the membrane surface and different drug-to-lipid ratios (DLRs). Liposomes were incubated in human plasma for various incubation times. Drawn samples were separated by asymmetrical flow field-flow fractionation (AF4). Drug was recovered in four fractions identified as albumin, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and liposomes. Our results suggest that mTHPC fits best into fluid, unmodified bilayers when the drug-to-lipid ratio is low. Membrane rigidification as well as the presence of cholesterol and PEGyated lipids reduced the ability of the membrane to accommodate the drug but simultaneously improved the vesicle stability in plasma. Both mechanisms jointly affect the total degree of mTHPC release. We analyzed our data using a kinetic model that suggests the drug to be associated with the host membrane in two distinct states of which only one interacts directly with the plasma compartment.

256 - HSA-Stilbenoids Non-Covalent Interaction Influences the Stability, Free Radical Scavenging Activity and Cell Uptake of Stilbenoids

Dietary stilbenoids are associated with many benefits for human health, which depend on their bioavailability and bioaccessibility. The stilbenoid–human serum albumin (HSA) interactions are investigated to explore the influence on the stability, free radical scavenging activity and cell uptake of stilbenoids. All the stilbenoids tested are very stable in methanol for 7 d incubation. However, stilbenoids showed different stability in DMEM, HSA solution (10 µmol/l) and human plasma. The stability of stilbenoids were determined as: HSA solution (10 µmol/l) > human plasma > DMEM. HSA obviously reduced the free radical scavenging potential of all stilbenoids tested. The masking effect of the stilbenoid–HSA interaction on the free radical scavenging potential decreased with the increasing %Protein binding. It appears that the milk enhanced the cell uptake of stilbenoids with multi-hydroxyl groups and weakened the cell uptake of stilbenoids with methoxyl group on EA.hy 926 endothelial cells. The stilbenoids are hardly absorbed by human umbilical vein endothelial cells in the presence of milk.

Discovery of 6-Diazo-5-oxo-l-norleucine (DON) Prodrugs with Enhanced CSF Delivery in Monkeys: A Potential Treatment for Glioblastoma.

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.

Synthesis, binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3-homo-amino acids.

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β(3)-homo-amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β(3)-homo-Tyr in place of Tyr (Tyr-D-Ala-Phe-Gly-β(3)-homo-Tyr-NH-)2 showed good μ receptor affinity and selectivity (IC50 = 0.302, IC50 ratio μ/δ = 68) and enzymatic stability in human plasma.

Development and validation of a UHPLC-UV method for the determination of a prostate secretory protein 94-derived synthetic peptide (PCK3145) in human plasma and assessment of its stability in human plasma.

PCK3145 is a synthetic peptide, derived from the Prostate Secreted Protein 94 (PSP94), with promising in vitro and animal in vivo results in prostate cancer. The aim of the present study was to develop and validate a fast and robust ultra-high-performance liquid chromatography with ultraviolet detection for the determination of PCK3145 in human plasma which would be suitable for the assessment of PCK3145 stability to proteolytic degradation. Following protein precipitation, chromatographic separation was carried out on an Aeris Peptide C18 column with mobile phase consisting of acetonitrile-water at a flow-rate of 0.50 mL/min. The calibration curve was linear over the range 0.50-20.00 μg/mL. Intra- and inter-day percentage relative standard deviation and relative error were ≤10%. The limit of detection and the lower limit of quantification were 0.15 and 0.50 μg/mL, respectively. Recovery of PCK3145 from human plasma was ≥96%. The peptide presented high stability in whole blood and in human plasma (>98% intact peptide after 24 h incubation at 37°C in human plasma), which represents a distinctive advantage in the therapeutic use of the compound. This is the first validated UHPLC method for the determination of PCK3145 reported, and it was successfully applied in the study of the proteolytic stability of PCK3145 in human plasma ex vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Spiro Diorthoester (SpiDo), a Human Plasma Stable Acid-Sensitive Cleavable Linker for Lysosomal Release

pH-sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for selective release of therapeutics selectively at targets and orthoesters have been demonstrated to be good candidates for such linkers. Following an HPLC screening, a Spiro Diorthoester (SpiDo) derivative was identified as a potent acid-labile group for the development of pH-sensitive targeted systems. After incorporation of this linker into activatable FRET-based probe and side-by-side comparison to a well-known alkylhydrazone linker, this SpiDo linker has shown a fast and pH sensitive hydrolysis for mild acidic conditions, a pH sensitive lysosomal hydrolysis, and high stability in human plasma.

Chromatographic resolution of atropisomers for toxicity and biotransformation studies in pharmaceutical research

Atropisomerism can be a complex concept for those who have not encountered it before. This paper discusses the experiments for identification, isolation, thermal stability, toxicity and biotransformation of various species. The identified atropisomers are a series of rotational hindered biaryl, rotational hindered amide, ring flip, and macrocycles atropisomers identified using supercritical fluid chromatography (SFC) and high performance liquid chromatography (HPLC). These technologies offered the advantage of separating various atropoenantiomers, atropdiastereomers and mixed atropisomers with other forms of stereoisomers in both analytical and preparative scales. With ultra-performance convergence chromatography (UPC2), the detection of N-oxide atropisomer metabolites can be obtained at very low level thus enabling the observation of conversion in human plasma possible. As the resolution of atropisomers are related to the energy barriers on the rotational axis, a calculated computational protocol was developed to predict the formation. A threshold of 10kcal/mol was established for possible detection of the atropisomers’ existence with chromatographic technologies at room temperature or above. The atropisomer with higher energy barrier (>20kcal/mol) were isolated via preparative chromatography and the isolates studied in vitro and in vivo for evaluation of their stability in human plasma. The detailed analytical method development to analyze the biotransformation of the atropisomers in human plasma are also discussed in this paper.

Evaluation of ¹¹¹in-labelled exendin-4 derivatives containing different meprin β-specific cleavable linkers.

BackgroundCleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.ResultsThe insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the 111In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound 111In-Ex4NOD40.ConclusionIn conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.

Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

CBTF: new amine-to-thiol coupling reagent for preparation of antibody conjugates with increased plasma stability.

Amine-to-thiol coupling is the most common route for the preparation of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. A series of antibody-dye conjugates have been prepared using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.

Inhibitory Efficacy and Biological Variability of Tryptophan Containing Dipeptides on Human Plasma Angiotensin Converting Enzyme Activity

Background: Hypertension remains a major unsolved health problem with life threatening consequences. A potential innovative prophylactic approach against its development may be supplementation of daily food with natural antihypertensive compounds. In our work we tested the efficacy of novel tryptophan containing dipeptides from whey protein with respect to their inhibitory potency on the angiotensin converting enzyme (ACE) as well as their stability in human plasma. Methods: ACE activity from rabbit lung and human plasma as well as recombinant human ACE activity were measured using benzoyl-glycyl-histidyl-leucine as a substrate. Product formation of hippuric acid and histidyl-leucine was assessed by UV-HPLC. The stability of the peptides was quantified in human plasma and, in case of incubation with recombinant human angiotensin converting enzyme, in buffer. Variability of ACE inhibiting activity of peptides was assessed via analysis of coefficient of variation with particular reference to true inter-individual variability corrected for methodological and analytical errors. Results: Isoleucine-tryptophan (IW) had a stronger ACE inhibiting potency (IC50 of 1.9 and 38.8 μmol/l with rabbit lung and human plasma ACE, respectively) than other tested peptides, i.e. glutamic acid-tryptophan (EW), tryptophanleucine (WL) and tryptophan-glutamic acid (WE). However, stability of IW showed a high degree of variability in plasma of different volunteers. In contrast EW, WL, WE were relatively stable in plasma and no major inter-individual variability was observed. Inhibition of plasma proteases extended plasma half-life of IW significantly. ACE did not contribute to peptide elimination. Conclusion: Milk whey protein represents a valuable source for bioactive peptides with ACE inhibitory potency that can be used as a food supplement to prevent, or in combination with other pharmaceutical agents to treat hypertension. However, in addition to the ACE inhibitory efficacy the stability of these peptides must be considered for in vivo ACE inhibition.