In response to the growing ethical and environmental concerns associated with animal testing, numerous in vitro tools of varying complexity and biorelevance have been developed and adopted in pharmaceutical research and development. In this work, we present one of these tools, i.e., the Meso-fluidic Chip for Permeability Assessment (MCPA), for the first time. The MCPA combines an artificial barrier (PermeaPad®) with an organ-on-chip device (MIVO®) and real-time automated concentration measurements, to yield a sustainable, yet effortless method for permeation testing. The system offers three major physiological aspects, i.e., a biomimetic membrane, an optimal membrane interfacial area-to-donor-volume-ratio (A/V) and a physiological flow on the acceptor/basolateral side, which makes the MPCA an ideal candidate for mechanistic studies and excellent in vivo bioavailability predictions. We validated the method with a handful of assorted drug compounds in unstirred and stirred donor conditions, before exploring its applicability as a tool for dissolution/permeation testing on a BCS class III/I drug (pyrazinamide) crystalline adducts and BCS class II/IV (hydrocortisone) amorphous solid dispersions. The results were highly reproducible and clearly displayed the method's potential for evaluating the performance of enabling formulations, and possibly even predicting in vivo performance. We believe that, upon further development, the MCPA will serve as a useful in vitro tool that could push sustainability into pharmaceutics by refining, reducing and replacing animal testing in early-stage drug development.

Replacing in vivo with in vitro studies can increase sustainability in the development of medicines. This principle has already been applied in the biowaiver approach based on the biopharmaceutical classification system, BCS. A biowaiver is a regulatory process in which a drug is approved based on evidence of in vitro equivalence, i.e., a dissolution test, rather than on in vivo bioequivalence. Currently biowaivers can only be granted for highly water-soluble drugs, i.e., BCS class I/III drugs. When evaluating poorly soluble drugs, i.e., BCS class II/IV drugs, in vitro dissolution testing has proved to be inadequate for predicting in vivo drug performance due to the lack of permeability interpretation. The aim of this review was to provide solid proofs that at least two commercially available cell-free in vitro assays, namely, the parallel artificial membrane permeability assay, PAMPA, and the PermeaPad® assay, PermeaPad, in different formats and set-ups, have the potential to reduce and replace in vivo testing to some extent, thus increasing sustainability in drug development. Based on the literature review presented here, we suggest that these assays should be implemented as alternatives to (1) more energy-intense in vitro methods, e.g., refining/replacing cell-based permeability assays, and (2) in vivo studies, e.g., reducing the number of pharmacokinetic studies conducted on animals and humans. For this to happen, a new and modern legislative framework for drug approval is required.

In order to solubilize poorly soluble active pharmaceutical ingredients, various strategies have been implemented over the years, including the use of nanocarriers, such as cyclodextrins and liposomes. However, improving a drug's apparent solubility does not always translate to enhanced bioavailability. This work aimed to investigate to which extent complexation with cyclodextrins and incorporation into liposomes influence drug in vitro permeability and to find a mechanistic description of the permeation process. For this purpose, we investigated hydroxypropyl-β-cyclodextrin (HP-β-CD) and phosphatidylcholine liposomes formulations of three chemically diverse compounds (atenolol, ketoprofen and hydrocortisone). We studied drug diffusion of the formulations by UV-localized spectroscopy and advanced data fitting to extract parameters such as diffusivity and bound-/free drug fractions. We then correlated this information with in vitro drug permeability obtained with the novel PermeaPadⓇ barrier. The results showed that increased concentration of HP-β-CD leads to increased solubilization of the poorly soluble unionized ketoprofen, as well as hydrocortisone. However, this net increment of apparent solubility was not proportional to the increased flux measured. On the other hand, normalising the flux over the empirical free drug concentration, i.e., the free fraction, gave a meaningful absolute permeability coefficient. The results achieved for the liposomal formulation were consistent with the finding on cyclodextrins. In conclusion, we proved the adequacy and usefulness of our method for calculating free drug fractions in the examined enabling formulations, supporting the validity of the established drug diffusion/permeation theory that the unbounded drug fraction is the main driver for drug permeation across a membrane.

A bistable [2]pseudorotaxane 1⊂CBPQT·4PF6 and a bistable [2]rotaxane 2·4PF6 have been synthesised to measure the height of an electrostatic barrier produced by double molecular oxidation (0 to +2). Both systems have monopyrrolotetrathiafulvalene (MPTTF) and oxyphenylene (OP) as stations for cyclobis(paraquat-p-phenylene) (CBPQT4+). They have a large stopper at one end while the second stopper in 24+ is composed of a thioethyl (SEt) group and a thiodiethyleneglycol (TDEG) substituent, whereas in 1⊂CBPQT4+, the SEt group has been replaced with a less bulky thiomethyl (SMe) group. This seemingly small difference in the substituents on the MPTTF unit leads to profound changes when comparing the physical properties of the two systems allowing for the first measurement of the deslipping of the CBPQT4+ ring over an MPTTF2+ unit in the [2]pseudorotaxane. Cyclic voltammetry and 1H NMR spectroscopy were used to investigate the switching mechanism for 1⊂CBPQT·MPTTF4+ and 2·MPTTF4+, and it was found that CBPQT4+ moves first to the OP station producing 1⊂CBPQT·OP6+ and 2·OP6+, respectively, upon oxidation of the MPTTF unit. The kinetics of the complexation/decomplexation process occurring in 1⊂CBPQT·MPTTF4+ and in 1⊂CBPQT·OP6+ were studied, allowing the free energy of the transition state when CBPQT4+ moves across a neutral MPTTF unit (17.0 kcal mol-1) or a di-oxidised MPTTF2+ unit (24.0 kcal mol-1) to be determined. These results demonstrate that oxidation of the MPTTF unit to MPTTF2+ increases the energy barrier that the CBPQT4+ ring must overcome for decomplexation to occur by 7.0 kcal mol-1.

The correlation between in vivo and in vitro data is yet not sufficiently optimized to allow a significant reduction and replacement of animal testing in pharmaceutical development. One of the main reasons for this lies in the poor mechanistic understanding and interpretation of the physical mechanisms enabling formulation rely on for deploying the drug. One mechanism that still lacks a proper interpretation is the kinetics of drug release from nanocarriers. In this work, we investigate two different types of classical enabling formulations - i) cyclodextrin solutions and ii) liposomal dispersions - by a combination of an experimental method (i.e. UV-Vis localized spectroscopy) and mathematical modelling/numerical data fitting. With this approach, we are able to discriminate precisely between the amount of drug bound to nanocarriers or freely dissolved at any time point; in addition, we can precisely estimate the binding and diffusivity constants of all chemical species (free drug/bound drug). The results obtained should serve as the first milestone for the further development of reliable in vitro/in silico models for the prediction of in vivo drug bioavailability when enabling formulations are used.

The process of passive drug absorption from the gastrointestinal tract is still poorly understood and modelled. Additionally, the rapidly evolving field of pharmaceutics demands efficient, affordable and reliable in vitro tools for predicting in vivo performance. In this work, we combined established methods for quantifying drug diffusivity (localized UV-spectroscopy) and permeability (Permeapad® plate) in order to gain a better understanding of the role of unstirred water layers (UWLs) in drug absorption. The effect of diffusion/permeability media composition and viscosity on the apparent permeation resistance (Rapp) of model drugs caffeine (CAF) and hydrocortisone (HC) were tested and evaluated by varying the type and concentration of viscosity-enhancing agent – glycerol or a poly(ethylene glycol) (PEG) with different average molecular weights. For all types of media, increased viscosity lead to reduction in diffusivity but could not alone explain the observed effect, which was attributed to intermolecular polymer-drug interactions. Additionally, for both drugs, smaller hydrophilic viscosity-enhancing agents (glycerol and PEG 400) had larger influence than larger ones (PEG 3350 and 6000). The results highlighted the role of UWL as an additive barrier to permeation and indicated that diffusion through UWL is the rate-limiting step to CAF’s permeation, whilst HC permeability is a partition-driven process.

Mucosal drug delivery accounts for various administration routes (i.e., oral, vaginal, ocular, pulmonary, etc.) and offers a vast surface for the permeation of drugs. However, the mucus layer which shields and lubricates all mucosal tissues can compromise drugs from reaching the epithelial site, thus affecting their absorption and therapeutic effect. Therefore, the effect of the mucus layer on drug absorption has to be evaluated early in the drug-development phase, prior to in vivo studies. For this reason, we developed a simple, cost-effective and reproducible method employing UV-visible localized spectroscopy for the assessment of the interaction between mucin and drugs with different physicochemical characteristics. The mucin–drug interaction was investigated by measuring the drug relative diffusivity (Drel) in the presence of mucin, and the method was validated by fitting experimental and mathematical data. In vitro permeability studies were also performed using the mucus-covered artificial permeation barrier (mucus–PVPA, Phospholipid Vesicle-based Permeation Assay) for comparison. The obtained results showed that the diffusion of drugs was hampered by the presence of mucin, especially at higher concentrations. This novel method proved to be suitable for the investigation on the extent of mucin–drug interaction and can be successfully used to assess the impact that the mucus layer has on drug absorption.

We investigated the ultrastructural pattern of colloidal phases in human duodenal fluids. Aspirates were collected from three volunteers in both fasted and fed nutritional states. Analysis methods comprised the combination of asymmetric flow field-flow fractionation (AF4) and multi-angle laser light scattering (MALLS). Furthermore, dynamic light scattering (DLS) and diffusion-ordered NMR spectroscopy (DOSY-NMR) were employed as alternative analytical approaches for comparison. By AF4/MALLS, up to four, and in some cases up to five distinct co-existing fractions could be differentiated in the sub-micron size-range, which, in accordance with a previous study (Elvang et al., 2018), may be assigned to three main types, namely small bile salt micelles, intermediate size mixed bile salt/phospholipid micelles and large phospholipid aggregates / vesicles. Although more or less the same colloidal phases were found to co-exist in all aspirates, their prevalence was found to vary, both over time and between the three individual human volunteers. Any uniform changes of patterns of colloidal phases over time, however, could not be identified. On the other hand, prevalence of specific colloidal phases was identified for aspirates of individual volunteers, which correlated reasonably well with the prevalence of certain lipid species in their molecular composition. It remains to be investigated whether such prevalence of specific colloidal phases influences drug solubilizing capacity as well as drug absorption. If so, this may help to better understand the substantial inter-individual variability seen in many drug absorption profiles.

An experimental/computational approach has been successfully applied in order to study the effect of solubilizing vehicles (cyclodextrins and liposomes) on the passive diffusion of four active pharmaceutical ingredients (API) of different nature (hydrophilic, ionizable and lipophilic) through an unstirred water layer (UWL) model. This approach allowed the measurement of flux changes through the UWL and the computational calculation of different parameters relevant to interpret the interplay within solubilizing vehicles and UWL diffusion. In the case of cyclodextrin, this approach allowed the determination of free drug diffusivity (Df), bound drug diffusivity (Db) and the equilibrium constant (K). In the case of liposomes, the experimental approach allowed the determination of the liposomes/water partition coefficient (Plip/w) as well as relative API diffusivity ((D)¯, i.e. the drug diffusion in the presence of solubilizing agents). This work demonstrates that the presence of solubilizing vehicles hampers the diffusion of API through UWL due to a combination of reduction in relative diffusivity and concentration gradient. These results are highly relevant as they might help to explain why biological performance of API is affected by the presence of solubilizing/complexing agents.

Colloidal phases (self-assemblies) in aqueous dispersions of selected binary bile salt/phospholipid blends were studied utilizing the combined analytical approach of asymmetrical flow field-flow fractionation (AF4) and multi-angle laser light scattering (MALLS) in order to resolve the coexistence of different colloidal assemblies. The binary blends were prepared by freeze-drying from tert-butanol / water co-solvent solutions. The blends contained one of two bile salts (sodium taurocholate (TC) or sodium glycodeoxycholate (GDX)) and a monoor di-acyl phospholipid (lysophosphatidylcholine (L-PC) and phosphatidylcholine (PC), respectively). Bile salt and phospholipid (PL) concentrations and their respective ratios were varied systematically within the physiological range found in human intestinal fluids. Furthermore, the BCS class II drug Celecoxib was incorporated in selected blends to assess its potential impact on colloidal phases. To further investigate the smallest self-assemblies observed in AF4/MALLS analysis, dispersions of TC and GDX, respectively, were prepared and analyzed by dynamic light scattering (DLS). AF4/MALLS analysis revealed that binary bile-salt/phospholipid blends form three distinct particle fractions, when the concentration of bile-salt was sufficiently high (≥ 3.5 mM). Those fractions were assumed to be very small pure bile-salt dimeric / oligomeric self-assemblies (Ø≈2-3 nm), mid-sized mixed micelles (Ø≈10 50 nm) and large liposomes/aggregates (Ø≈150 – 280 nm). If present, Celecoxib was found solubilized within the structures, but at the lowest TC concentration triggered the formation of an additional (vesicular) phase.

The diffusion coefficient (also known as diffusivity) of an active pharmaceutical ingredient (API) is a fundamental physicochemical parameter that affects passive diffusion through biological barriers and, as a consequence, bioavailability and biodistribution. However, this parameter is often neglected, and it is quite difficult to find diffusion coefficients of small molecules of pharmaceutical relevance in the literature. The available methods to measure diffusion coefficients of drugs all suffer from limitations that range from poor sensitivity to high selectivity of the measurements or the need for dedicated instrumentation. In this work, a simple but reliable method based on time-resolved concentration measurements by UV-visible spectroscopy in an unstirred aqueous environment was developed. This method is based on spectroscopic measurement of the variation of the local concentration of a substance during spontaneous migration of molecules, followed by standard mathematical treatment of the data in order to solve Fick's law of diffusion. This method is extremely sensitive and results in highly reproducible data. The technique was also employed to verify the influence of the environmental characteristics (i.e., ionic strength and presence of complexing agents) on the diffusivity. The method can be employed in any research laboratory equipped with a standard UV-visible spectrophotometer and could become a useful and straightforward tool in order to characterize diffusion coefficients in physiological conditions and help to better understand the drug permeability process.

•Colloidal structures in simulated intestinal fluids characterized by field-flow fractionation and DOSY-NMR.•Type of field-flow fractionation channel is of importance in preserving the colloidal structures.•Limitations of DLS and field-flow fractionation in analyzing biomimetic media were assessed.

Nuclear Magnetic Resonance spectroscopy and imaging can be classified as inductive techniques working in the nearto far-field regimes. We investigate an alternative capacitive detection with the use of micrometer sized probes positioned at sub wavelength distances of the sample in order to characterize and model evanescent electromagnetic fields originating from NMR phenomenon. We report that in this experimental configuration the available NMR signal is one order of magnitude larger and follows an exponential decay inversely proportional to the size of the emitters. Those investigations open a new road to a better understanding of the evanescent waves component in NMR with the opportunity to perform localized spectroscopy and imaging. NMR principles and Evanescent Waves The principle of Nuclear Magnetic Resonance is based on the detection of the magnetization originating from an ensemble of spins located on the atomic nucleus [1–3]. In living sciences, NMR spectroscopy and imaging of H, C, Na and P are of great interest and extensively studied [4, 5]. The sample is polarized by a static magnetic field B0 and spatially encoded by pulse gradients, giving to the nuclear spin distinct precessional movements at well separated Larmor frequencies. Radiofrequency pulses are used to tilt the axis of the magnetization; when the spin system recovers its equilibrium, it generates an electromagnetic field which is classically detected using an inductive coupling in the radiative near-field regime [6]. Radio frequencies are usually emitted from the sample in the range from 1MHz to 1 GHz. A good receiving setup can be made generally from a coil wrapped around the sample, a loopless catheter antenna [7] with a tuning/matching circuit [8] or electric potential sensor capacitively coupled to the sample [9]. In this contribution, we propose a completely different approach, with the use of micrometer-sized Evanescent Waves probe (EW-probe) working in a capacitive mode [10] positioned in the vicinity of the object of interest at submillimetric distances, well shorter than the wavelength of the radiated radio-frequency signal. Over the past decades, near-field PLOSONE | DOI:10.1371/journal.pone.0144483 January 11, 2016 1 / 8 a11111

Nuclear Magnetic Resonance spectroscopy and imaging can be classified as inductive techniques working in the near- to far-field regimes. We investigate an alternative capacitive detection with the use of micrometer sized probes positioned at sub wavelength distances of the sample in order to characterize and model evanescent electromagnetic fields originating from NMR phenomenon. We report that in this experimental configuration the available NMR signal is one order of magnitude larger and follows an exponential decay inversely proportional to the size of the emitters. Those investigations open a new road to a better understanding of the evanescent waves component in NMR with the opportunity to perform localized spectroscopy and imaging.

Partitioning of trans-3,5,4'-trihydroxy-stilbene (resveratrol) between n-pentane and DMSO was investigated as a contribution to understand the interaction between resveratrol and biomembranes. In order to determine the partition coefficient P* of resveratrol between pentane and DMSO, resveratrol solutions in DMSO were equilibrated against pentane solutions. From the UV-vis absorbance of resveratrol in the DMSO phase, P* = 1.74 x 10(-3) was determined. The mutual miscibility of pentane and DMSO, measured by H-1-NMR spectroscopy, was taken into account. The value of P* indicates a preference of resveratrol for a polar environment rather than an apolar environment. Together with the known low solubility of resveratrol in water, the finding suggests that resveratrol does spontaneously accumulate within the headgroup region in a biomembrane. (C) 2015 Elsevier Ltd. All rights reserved.

Abstract Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol) – in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and 31P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70–415 nm upon repeated extrusion through 400 nm pore-filters, eventually ending with a size range from ≈30 to 85 nm upon extrusion through 30 nm pore size filters. While for small pores sizes (50 nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (∼ 300 nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.

The purpose of this study was the development of multifunctional liposomes for nasal administration of tacrine hydrochloride. Liposomes were prepared using traditional excipients (cholesterol and phosphatidylcholine), partly enriched with α-tocopherol and/or Omega3 fatty acids. This approach was chosen in order to obtain at the same time two positive results: an enhanced drug permeation through nasal mucosa and a concomitant neuroprotective effect. Several liposome formulations were prepared using the Reverse Phase Evaporation technique followed by membrane filter extrusion. In particular, liposome capacity to enhance drug permeation was evaluated by means of membrane permeation and cellular uptake studies. Furthermore, liposome effect on neuronal viability and intracellular ROS production was evaluated as well as their cytoprotective effect against oxidative stress. All liposome formulations showed a mean diameter in the range of 175 nm to 219 nm with polydispersity index lower than 0.22, a lightly negative zeta potential and excellent encapsulation efficiency. Moreover, along with good mucoadhesive properties, multifunctional liposomes showed a markedly increase in tacrine permeability, which can be related to liposome fusion with cellular membrane, a hypothesis, which was also supported by cellular uptake studies. Finally, the addition of α-tocopherol without Omega3 fatty acids, was found to increase the neuroprotective activity and antioxidant properties of liposomes.

Nondestructive studies of physiological processes in agronomic products require increasingly higher spatial and temporal resolutions. Nuclear Magnetic Resonance (NMR) imaging is a non-invasive technique providing physiological and morphological information on biological tissues. The aim of this study was to design a robust and accurate quantitative measurement method based on NMR imaging combined with contrast agent (CA) for mapping and quantifying water transport in growing cherry tomato fruits. A multiple flip-angle Spoiled Gradient Echo (SGE) imaging sequence was used to evaluate the intrinsic parameters maps M0 and T1 of the fruit tissues. Water transport and paths flow were monitored using Gd(3+)/[Fe(CN)6](3-)/D-mannitol nanoparticles as a tracer. This dynamic study was carried out using a compartmental modeling. The CA was preferentially accumulated in the surrounding tissues of columella and in the seed envelopes. The total quantities and the average volume flow of water estimated are: 198 mg, 1.76 mm(3)/h for the columella and 326 mg, 2.91 mm(3)/h for the seed envelopes. We demonstrate in this paper that the NMR imaging technique coupled with efficient and biocompatible CA in physiological medium has the potential to become a major tool in plant physiology research.

Resveratrol, contained in the skin of grape and accordingly in red wine, exhibits a wide range of health effects such as cardioprotection and anti-oxidation. Biophysical studies on the unspecific interaction of resveratrol with lipid membranes are rare so far. Here we present results from a series of investigations on the interaction of resveratrol with DPPC model membranes, combining structural (neutron reflectometry) and thermodynamic methods (measurement of partition coefficient, Langmuir isotherm and differential scanning calorimetry (DSC)). The details of the partitioning and the neutron experiments report on the localization of resveratrol within the membrane headgroup layer, close to the interface between the hydrophilic headgroup region and the hydrophobic core. Its presence decreases the tilt angle of the headgroups with respect to the membrane surface normal, as compared to the control (pure DPPC). At the same time, the projected area per headgroup drops, and the bilayer is more condensed. The analysis of the thermograms confirms these findings. DSC measurements on SUVs of pure DPPC (0.05°C/min) exhibit a complex characteristics of the main transition, revealing three peaks. We attribute them to a contribution of the headgroups and a bending dependent contribution of the chains, different for the two leaflets. With resveratrol, the headgroup transition exhibits a freezing point depression together with a loss in cooperativity. The chain condensation makes the corresponding transition more uniform. We conjecture a compensating effect of resveratrol: extrusion allows for an asymmetric distribution among the two leaflets that partially counteracts the curvature induced packing gradient in the core region. The findings in summary yield a more comprehensive view on the passive impact of resveratrol in model membranes that will hopefully contribute to a better understanding of the biophysical mechanisms behind the Mediterranean diet.

The study of preorganization in receptors, particularly in cooperative receptors, and their reversible control by external stimuli is important for elucidating design strategies that can lead to increased sensitivity and external control of molecular recognition. In this work we present the design, synthesis, and operation of an asymmetric tetrathiafulvalene (TTF)-calix[4]pyrrole receptor appended with a pyridine moiety. (1)H NMR spectroscopy was employed to demonstrate that intramolecular complexation between the receptor and the pyridine moiety leads to a preorganized receptor. Absorption and (1)H NMR spectroscopy along with a computational investigation were used to demonstrate the ability of the receptor to complex the substrate 1,3,5-trinitrobenzene (TNB) and that the receptor can be reversibly modulated between negative and positive cooperativity by employing external stimuli in the form of Zn(II). Fitting procedures incorporating multiple datasets and fitting to multiple equilibria simultaneously have been employed to quantitatively determine the preorganization effects.