•Elaboration of superhydrophobic coatings in narrow capillaries (down to 50 μm I.D.)•Surface characterization of superhydrophobic coatings•Hydrodynamic and electrokinetic behaviors of ...superhydrophobic capillaries•Increasing the separation efficiency in capillary electrophoresis using superhydrophobicity
Separation efficiency is ideally controlled by molecular diffusion in capillary electrophoresis (CE). However, other adverse phenomena, such as solute adsorption on capillary surface, tend to increase the peak dispersion. An interesting alternative to limit the solute adsorption is to avoid as much as possible the contact of the solute with the capillary surface by elaborating superhydrophobic (SH) coatings on fused silica capillary surfaces. This work describes an optimized protocol to get non-wettable SH coating using hydrophobically modified silica nanoparticle suspensions (Glaco™), based on simple capillary flushes and thermal stabilization. In this protocol, the control of the air flushing after the introduction of the Glaco™ suspension in the capillary was found crucial to get optimized coating coverage and reproducibility. The SH coating was characterized by ellipsometry, atomic force microscopy, scanning electron microscopy, contact angle (about 159°) and the observation of the meniscus of water in the coated capillary. The hydrodynamic behavior of the SH coated capillary was investigated by plotting the Poiseuille law. Finally, electrophoretic separations of a peptide mixture in acidic conditions demonstrated the interest of this approach with an increase by a factor 2 of the separation efficiency compared to fused silica capillary.
In this study, binding of linear poly(l-lysine) to a series of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate copolymers was examined by isothermal titration calorimetry (ITC). Binding ...constant and stoichiometry were systematically determined at different ionic strengths and for different polyanion charge densities varying between 15% and 100%. The range of investigated ionic strengths was carefully adjusted according to the polyanion charge densities to get measurable binding constants (i.e., formation binding constant typically comprised between 104 and 106 M–1) by isothermal titration calorimetry (ITC). The number of released counterions during the polyelectrolyte complex formation was determined from the log–log dependence of the binding constant according to the ionic strength and was compared to the total number of condensed counterions estimated from the Manning theory. Experimental results obtained by ITC are in very good agreement with those previously obtained by frontal analysis continuous capillary electrophoresis (FACCE) and can be used to model and predict the binding parameters at any ionic strength or any polyanion charge density. Thermodynamic parameters of the complexation between the oppositely charged polyelectrolytes confirm that the complex formation was entropically driven together with a favorable (but minor) enthalpic contribution. For the first time, specificities, advantages/disadvantages of ITC, and FACCE techniques for studying polyelectrolyte complexations are compared and discussed, using the same experimental conditions.
•Determination of binding constants between oppositely charged dendrimers and protein.•Binding constants in physiological conditions with low sample consumption.•Comparison between continuous frontal ...analysis capillary electrophoresis and fluorescence spectroscopy.
Dendrigraft poly-l-lysine (DGL) are biomacromolecules of great interest for many applications including antibacterial activity, drug delivery systems, gene therapy and production of antibodies. As human serum albumin (HSA) is the most abundant serum protein, the study of interactions between these two compounds is crucial for the use of DGL in drug or gene delivery systems. The present work aims at determining the number of binding sites and the corresponding successive equilibrium constants between DGL of generation 3 (G3) and HSA in physiological conditions. To meet this end, continuous frontal analysis capillary electrophoresis (FACCE) and fluorescence spectroscopic methods were implemented and compared. FACCE was performed on a polycationic modified capillary in combination with a co-pressure that allowed for selectively introducing the free G3 from the G3/HSA mixtures. FACCE studies demonstrated that HSA has 2 binding sites with DGL G3 with the following successive constants K1=31.2×103M−1 and K2=30.6×103M−1. For a 1g/L concentration in G3 and assuming a plasmatic HSA concentration of 40g/L, these binding constants lead to only 5% free DGL in the medium. It was also shown that the interactions between G3 and HSA corresponded to a model of cooperative sites. These results are in good agreement with the presence of two negatively charged domains in the HSA. Good fitting of the fluorescence spectroscopy data was obtained using the equilibrium constants derived from FACCE. Nevertheless, due to the high number of fitting parameters, it was difficult to fit the fluorescence spectroscopic data independently of the results obtained by FACCE.
In this work, the influence of the ionic strength and phosphate ions on poly(l-lysine) hydrodynamic radius, conformation and persistence lengths has been studied for molar masses comprised between ...3000 and 70 000 g/mol. Mark–Houwink coefficients have been obtained via the determination of poly(l-lysine) hydrodynamic radius using Taylor dispersion analysis. The influence of phosphate ions and ionic strength on the solvent quality (poor, Θ, or good solvent) for poly(l-lysine) have been studied in details. Quantitative data on hydrodynamic radius, persistence length, Mark–Houwink coefficients are provided at pH 7.4, in the range of 10 mM to 1 M ionic strength, and for different phosphate ion concentrations from 0.1 mM to 50 mM under physiological conditions (154 mM ionic strength, pH 7.4). The strong influence of phosphate ions on poly(l-lysine) properties was finally illustrated by studying the interactions (stoichiometry, binding constant, and cooperativity) between poly(l-lysine) of DP 50 and human serum albumin, in the absence and in the presence of phosphate ions at pH 7.4.
Material suitability needs to be considered for the 3D printing of solid oral dosage forms (SODFs). This work aims to assess the suitability of a CO2 laser (λ = 10.6 μm) for selective laser sintering ...of SODFs containing copovidone and paracetamol. First, physicochemical characterization of powders (two grades of copovidone, two grades of paracetamol and their mixtures at various proportions) was conducted: particle size distribution, morphology, infrared absorbance, flowability, and compactness. Then, printing was launched, and printability of the powders was linked to their physicochemical characteristics. The properties of the sintered SODFs were evaluated (solid state, general aspect, porosity, hardness, drug content and release). Hence, it was found that as copovidone absorbs at the laser’s wavelength, sintering was feasible without using an absorbance enhancer. Also, flowability, which mainly depends on the particle size, represents the first control line for “sinterability” as a fair flow is at least required. Low compactness of copovidone and mixtures reduces the mechanical properties of the SODFs but also increases porosity, which can modulate drug release. Moreover, the drug did not undergo degradation and demonstrated a plasticizer effect by lowering the heating temperature. In conclusion, this work proves the applicability of CO2 laser SLS printer to produce SODFs.
Aims
Canagliflozin is a recently approved drug for use in the treatment of type 2 diabetes. The potential for canagliflozin to cause clinical drug–drug interactions (DDIs) was assessed.
Methods
DDI ...potential of canagliflozin was investigated using in vitro test systems containing drug metabolizing enzymes or transporters. Basic predictive approaches were applied to determine potential interactions in vivo. A physiologically‐based pharmacokinetic (PBPK) model was developed and clinical DDI simulations were performed to determine the likelihood of cytochrome P450 (CYP) inhibition by canagliflozin.
Results
Canagliflozin was primarily metabolized by uridine 5′‐diphospho‐glucuronosyltransferase 1A9 and 2B4 enzymes. Canagliflozin was a substrate of efflux transporters (P‐glycoprotein, breast cancer resistance protein and multidrug resistance‐associated protein‐2) but was not a substrate of uptake transporters (organic anion transporter polypeptide isoforms OATP1B1, OATP1B3, organic anion transporters OAT1 and OAT3, and organic cationic transporters OCT1, and OCT2). In inhibition assays, canagliflozin was shown to be a weak in vitro inhibitor (IC50) of CYP3A4 (27 μmol l –1, standard error SE 4.9), CYP2C9 (80 μmol l –1, SE 8.1), CYP2B6 (16 μmol l–1, SE 2.1), CYP2C8 (75 μmol l –1, SE 6.4), P‐glycoprotein (19.3 μmol l –1, SE 7.2), and multidrug resistance‐associated protein‐2 (21.5 μmol l –1, SE 3.1). Basic models recommended in DDI guidelines (US Food & Drug Administration and European Medicines Agency) predicted moderate to low likelihood of interaction for these CYPs and efflux transporters. PBPK DDI simulations of canagliflozin with CYP probe substrates (simvastatin, S‐warfarin, bupropion, repaglinide) did not show relevant interaction in humans since mean areas under the concentration‐time curve and maximum plasma concentration ratios for probe substrates with and without canagliflozin and its 95% CIs were within 0.80–1.25.
Conclusions
In vitro DDI followed by a predictive or PBPK approach was applied to determine DDI potential of canagliflozin. Overall, canagliflozin is neither a perpetrator nor a victim of clinically important interactions.
With the recent publication of the FDA guidance on metabolites in safety testing (MIST), a reflection is provided that describes the impact of this guidance on the processes of drug metabolite ...identification and quantification at various stages of drug development. First, a retrospective analysis is described that was conducted on 12 human absorption, metabolism, and excretion (AME) trials with the application of these MIST criteria. This analysis showed that the number of metabolites requiring identification, (semi)-quantification, and coverage in the toxicology species would substantially increase. However, a significant proportion of these metabolites were direct or indirect conjugates, a class of metabolites that was specifically addressed in the guidance as being largely innocuous. The nonconjugated metabolites were all covered in at least one toxicology animal species, with no need for additional safety evaluation. Second, analytical considerations pertaining to the efficient identification of metabolites are discussed. Topics include software-assisted detection and structural identification of metabolites, the emerging hyphenation of ultraperformance liquid chromatography (UPLC) with radioactivity detection, and the various ways to estimate metabolite abundance in the absence of an authentic standard. Technical aspects around the analysis of metabolite profiles are also presented, focusing on precautions to be taken in order not to introduce artifacts. Finally, a tiered approach for metabolite quantification is proposed, starting with quantification of metabolites prior to the multiple ascending dose study (MAD) in humans in only specific cases (Tier A). The following step is the identification and quantification of metabolites expected to be of pharmacological or toxicological relevance (based on MIST and other complementary criteria) in selected samples from the MAD study and preclinical studies in order to assess metabolite exposure coverage (Tier B). Finally, a metabolite quantification strategy for the studies after the MAD phase (Tier C) is proposed.
The interactions between model polyanions and polycations have been studied using frontal continuous capillary electrophoresis (FACCE) which allows the determination of binding stoichiometry and ...binding constant of the formed polyelectrolyte complex (PEC). In this work, the effect of the poly(l-lysine) (PLL) molar mass on the interaction with statistical copolymers of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate (PAMAMPS) has been systematically investigated for different PAMAMPS chemical charge densities (15% and 100%) and different ionic strengths. The study of the ionic strength dependence of the binding constant allowed the determination of the total number of released counter-ions during the formation of the PEC, which can be compared to the total number of counter-ions initially condensed on the individual polyelectrolyte partners before the association. Interestingly, this fraction of released counter-ions, which was strongly dependent on the PLL molar mass, was almost independent of the PAMAMPS charge density. These findings are useful to predict the binding constant according to the molar mass and charge density of the polyelectrolyte partners.
•TDA is suitable for sizing various vaccine antigens.•Polydispersity indexes of vaccine antigens can be obtained by TDA.•LOD varies as 1/t0, t0 being the average elution time.•The lower the pressure, ...the more sensitive the TDA method.•LOD varies as 1/Ld, Ld being the detection capillary length.
The development of a new vaccine requires the precise characterization of all the physicochemical parameters of the vaccine antigens, which are the molecules that induce the immune response. Taylor dispersion analysis (TDA) is a promising alternative technique for the determination of diffusion coefficients and hydrodynamic radii of proteins, macromolecules and nanoparticles. In this work, TDA was used to determine the hydrodynamic radius distribution and its average value of four antigens: diphtheria toxoid (DT), tetanus toxoid (TT), hepatitis B surface antigen (HBsAg) and polyribosyl-ribitol phosphate conjugated to tetanus toxoid (PRP-T). The robustness of the results obtained was investigated on bare fused silica capillary and hydroxypropylcellulose coated capillary. The impact of operational parameters on the limit of detection (LOD) and limit of quantification (LOQ) were studied from both theoretical and experimental points of view. The influence of the diameter and the length of the capillary on the LOD and LOQ were studied as well as the impact of the mobilization pressure. General guidelines for the choice of the initial operating conditions are given for the development of future TDA methods.
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The water-based extraction of bioactive components from flavonoid-rich medicinal plants is a key step that should be better investigated. This is especially true when dealing with easy-to-use ...home-made conditions of extractions, which are known to be a bottleneck in the course for a better control and optimization of the daily uptake of active components from medicinal plants. In this work, the water-based extraction of Blackcurrant (
) leaves (BC) and
(CA)
known to have complementary pharmacological properties, was studied and compared with a previous work performed on the extraction of Hawthorn (
HAW). Various extraction modes in water (infusion, percolation, maceration, ultrasounds, microwaves) were compared for the extraction of bioactive principles contained in BC and CA in terms of extraction yield, of amount of flavonoids, phenolic compounds, and proanthocyanidin oligomers, and of UHPLC profiles of the extracted compounds. The qualitative and quantitative aspects of the extraction, in addition to the kinetic of extraction, were studied. The optimized easy-to-use-at-home extraction protocol developed for HAW was found very efficient to easily extract bioactive components from BC and CA plants. UHPLC-ESI-MS and high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were also implemented to get more qualitative information on the specific and common chemical compositions of the three plants (including HAW). Their antihyaluronidase, antioxidant, and antihypertensive activities were also determined and compared, demonstrating similar activities as the reference compound for some of these plants.
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