Protection of the Human Gut Microbiome From Antibiotics.

Background: Antibiotics are life-saving drugs but severely affect the gut microbiome with short-term consequences including diarrhea and selection of antibiotic-resistant bacteria. Long-term links to allergy and obesity are also suggested. We devised a product, DAV132, and previously showed its ability to deliver a powerful adsorbent, activated charcoal, in the late ileum of human volunteers. Methods: We performed a randomized controlled trial in 28 human volunteers treated with a 5-day clinical regimen of the fluoroquinolone antibiotic moxifloxacin in 2 parallel groups, with or without DAV132 coadministration. Two control goups of 8 volunteers each receiving DAV132 alone, or a nonactive substitute, were added. Results: The coadministration of DAV132 decreased free moxifloxacin fecal concentrations by 99%, while plasmatic levels were unaffected. Shotgun quantitative metagenomics showed that the richness and composition of the intestinal microbiota were largely preserved in subjects co-treated with DAV132 in addition to moxifloxacin. No adverse effect was observed. In addition, DAV132 efficiently adsorbed a wide range of clinically relevant antibiotics ex vivo. Conclusions: DAV132 was highly effective to protect the gut microbiome of moxifloxacin-treated healthy volunteers and may constitute a clinical breakthrough by preventing adverse health consequences of a wide range of antibiotic treatments. Clinical Trials Registration: NCT02176005.

Integrated microdevice for preconcentration and separation of a wide variety of compounds by electrochromatography.

An integrated microdevice was developed to couple on-chip SPE to separation by channel electrochromatography. An acrylate-based monolith was synthesized within a glass microdevice by photoinitiated polymerization. It was used for both separation and preconcentration by direct injection on the head of the stationary phase or by confining the preconcentration step in a given zone of the stationary phase. The composition of the polymerization mixture was chosen to achieve a monolithic material containing both hydrophobic and charged moieties to ensure an electroosmotic flow for separation. As a consequence the extraction procedure occurs via hydrophobic and ionic interactions. Neutral, ionizable and charged compounds were successfully preconcentrated and separated within the microdevice through electrochromatographic mechanisms, highlighting the versatility of this device. The performance of the integrated microdevice was demonstrated with the preconcentration and separation of a mixture of PAHs for which a signal enhancement factor (SEF) of 270 was achieved within 120 s of preconcentration. In the case of charged and ionizable compounds, according to the electrolyte composition, contributions of both reverse-phase and ion-exchange mechanisms were used to perform effective electrochromatographic preconcentration. A SEF of 250 was obtained for the model-charged compound within 20 s of preconcentration. Finally, the potentials of on-chip preconcentrate and separate both neutral and ionized compounds have been demonstrated using a mixture of model compounds.

In situ synthesis of monolithic stationary phases for electrochromatographic separations: study of polymerization conditions.

Acrylate-based monolithic capillary columns were prepared from fused-silica capillaries using UV photopolymerization. The effect of the pretreatment of the capillary wall surface before polymerization was investigated and several procedures were compared. The columns were characterization by van Deemter curves and SEM imaging. The results indicated that a pre-silanization of the capillary wall in order to introduce methacrylate groups at the wall surface gave similar efficiencies but more homogeneous structures than when the silanization agent was introduced in the polymerization mixture. The conditioning of the capillary before silanization, especially the conditions of basic rinsing was also an important factor. The effect of the dose of UV light that was applied for the polymerization had also been investigated. The results demonstrated that the irradiation energy is a critical parameter. The minimum energy threshold required to obtain a suitable monolith was 3 J/cm(2) and the maximum was around 12 J/cm(2). A higher energy destroys the monolith. Within the convenient range of energy, the columns had the same efficiency and a good structure as seen by SEM imaging. Using the optimized procedure for the pretreatment and an adequate energy, the column-to-column repeatability was found good (n = 12). The repeatability was obtained for the plate height at two velocity values, the retention factor and the electroosmotic mobility with RSD values below 10.

Targeted adsorption of molecules in the colon with the novel adsorbent-based medicinal product, DAV132: A proof of concept study in healthy subjects.

During antibiotic treatments, active residuals reaching the colon profoundly affect the bacterial flora resulting in the emergence of resistance. To prevent these effects, we developed an enteric-coated formulated activated-charcoal based product, DAV132, meant to deliver its adsorbent to the ileum and neutralize antibiotic residues in the proximal colon. In a randomized, control, crossover study, the plasma pharmacokinetics of the probe drugs amoxicillin (500 mg) absorbed in the proximal intestine, and sulfapyridine (25 mg) metabolized from sulfasalazine in the cecum and rapidly absorbed, were compared after a single administration in 18 healthy subjects who had received DAV132, uncoated formulated activated charcoal (FAC) or water 16 and 8 hours before, concomitantly with the probe drugs, and 8 hours thereafter. The AUC0-96 h of amoxicillin was reduced by more than 70% when it was taken with FAC, but bioequivalent when it was taken with water or DAV132. By contrast, the AUC0-96 h of sulfapyridine was reduced by more than 90% when administered with either FAC or DAV132 in comparison with water. The results show that DAV132 can selectively adsorb drug compounds in the proximal colon, without interfering with drug absorption in the proximal small intestine, thereby constituting a proof of concept that DAV132 actually functions in humans.

CEC separation of peptides using a poly(hexyl acrylate-co-1,4-butanediol diacrylate-co-[2-(acryloyloxy)ethyl]trimethyl ammonium chloride) monolithic column.

A polyacrylate-based monolithic column bearing cationic functionalities and designed for capillary electrochromatography (CEC) has been prepared via photopolymerization of a mixture of hexyl acrylate, butanediol diacrylate, 2-(acryloyloxy) ethyltrimethyl ammonium chloride (monomers), azobisisobutyronitrile (photoinitiator), acetonitrile, phosphate buffer, and ethanol (porogens). The polymerization process was initiated with UV light at 360 nm. The column performance was evaluated via the separations of alkylbenzenes, substituted anilines, basic drugs, peptides, and a protein digest. The separation of complex peptide mixtures was then studied since such separations constitute a promising application of capillary electrochromatography. In particular, the effects of mobile phase composition, including ionic strength of the buffer solution and the percentage of acetonitrile on the retention factor, the column efficiency, and the resolution were determined. The separations were affected by both interaction of the peptides with the stationary phase and their own electrophoretic mobility. Excellent separations with column efficiencies of up to 160 000 plates/m were achieved for both a mixture of ten well-defined peptides and a tryptic digest of cytochrome c. The fractions of eluent containing peptides of the digest separated in the monolithic column were collected and characterized using matrix-assisted laser desorption ionization mass spectrometry.

Online preconcentration using monoliths in electrochromatography capillary format and microchips.

Online preconcentration and separation of analytes using an in situ photopolymerized hexyl acrylate-based monolith stationary phase was evaluated using electrochromatography in capillary format and microchip. The band broadening occurring during the preconcentration process by frontal electrochromatography and during the desorption process by elution electrochromatography was studied. The hexyl acrylate-based monolith provides high retention for neutral analytes allowing the handling of large sample volumes and its structure allows rapid mass transfer, thus reducing the band broadening. For moderately polar analytes such as mono-chlorophenols that are slightly retained in water, it was shown that enrichment factors up to 3500 can be obtained by a hydrodynamic injection of several bed volumes for 120 min under 0.8 MPa with a decrease in efficiency of 50% and a decrease of 30% for the resolution between 2- and 3-chlorophenol. An 8 min preconcentration time allows enrichment factors above 100 for polyaromatic hydrocarbons. The interest of these monoliths when synthesized in microchip is also demonstrated. A 200-fold enrichment was easily obtained for PAHs with only 1 min as preconcentration time, without decrease in efficiency.

Selection of two reliable parameters to evaluate the impact of the mobile-phase composition on capillary electrochromatography performance with monolithic and particle-packed capillary columns.

Different models have been described in the literature to evaluate the total porosity of CEC columns: gravimetric, flow as well as conductivity-based methods. In this study, these models have been compared for two kinds of CEC columns: two mixed-mode silica particle stationary phases and different monolithic columns (acrylate or polystyrene divinylbenzene-based). The total porosities measured from the conductivity-based methods were lower than the total column porosities obtained by gravimetric or flow methods for all the investigated columns while the wide distribution of observed values shows that conductivity-based methods discriminate columns more efficiently with very different properties. We propose a conductivity-based method taking into account the actual length proposed by Horvath, to evaluate what we call an "actual electrokinetic" porosity (AEP). This parameter, based on electrokinetic theory only, affords the most consistent evaluation of porosity under experimental CEC conditions for the packed- and acrylate-based monolithic columns. To illustrate the potential of AEP and actual EOF for the estimation of the performances of a CEC system (stationary and mobile phases) we studied the influence of the mobile-phase composition on these parameters for CEC separations with an ammonium embedded packed stationary phase. The AEP and the actual electroosmotic mobility should allow a better understanding of the perfusive EOF and stationary-phase wettability. For neutral compounds (substituted phenols), AEP evaluation allowed us to predict the mobile-phase conditions able to enhance the efficiency while both AEP and actual EOF had to be considered in the case of peptide analysis.