Quantifying the barrier for the movement of cyclobis(paraquat-p-phenylene) over the dication of monopyrrolotetrathiafulvalene.

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.

Central pattern generators in the turtle spinal cord: selection among the forms of motor behaviors.

Neuronal networks in the turtle spinal cord have considerable computational complexity even in the absence of connections with supraspinal structures. These networks contain central pattern generators (CPGs) for each of several behaviors, including three forms of scratch, two forms of swim, and one form of flexion reflex. Each behavior is activated by a specific set of cutaneous or electrical stimuli. The process of selection among behaviors within the spinal cord has multisecond memories of specific motor patterns. Some spinal cord interneurons are partially shared among several CPGs, whereas other interneurons are active during only one type of behavior. Partial sharing is a proposed mechanism that contributes to the ability of the spinal cord to generate motor pattern blends with characteristics of multiple behaviors. Variations of motor patterns, termed deletions, assist in characterization of the organization of the pattern-generating components of CPGs. Single-neuron recordings during both normal and deletion motor patterns provide support for a CPG organizational structure with unit burst generators (UBGs) whose members serve a direction of a specific degree of freedom of the hindlimb, e.g., the hip-flexor UBG, the hip-extensor UBG, the knee-flexor UBG, the knee-extensor UBG, etc. The classic half-center hypothesis that includes all the hindlimb flexors in a single flexor half-center and all the hindlimb extensors in a single extensor half-center lacks the organizational complexity to account for the motor patterns produced by turtle spinal CPGs. Thus the turtle spinal cord is a valuable model system for studies of mechanisms responsible for selection and generation of motor behaviors. NEW & NOTEWORTHY The concept of the central pattern generator (CPG) is a major tenet in motor neuroethology that has influenced the design and interpretations of experiments for over a half century. This review concentrates on the turtle spinal cord and describes studies from the 1970s to the present responsible for key developments in understanding the CPG mechanisms responsible for the selection and production of coordinated motor patterns during turtle hindlimb motor behaviors.

Experimental Determination of Drug Diffusion Coefficients in Unstirred Aqueous Environments by Temporally Resolved Concentration Measurements.

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.

Modular organization of the multipartite central pattern generator for turtle rostral scratch: knee-related interneurons during deletions.

Central pattern generators (CPGs) are neuronal networks in the spinal cord that generate rhythmic patterns of motor activity in the absence of movement-related sensory feedback. For many vertebrate rhythmic behaviors, CPGs generate normal patterns of motor neuron activities as well as variations of the normal patterns, termed deletions, in which bursts in one or more motor nerves are absent from one or more cycles of the rhythm. Prior work with hip-extensor deletions during turtle rostral scratch supports hypotheses of hip-extensor interneurons in a hip-extensor module and of hip-flexor interneurons in a hip-flexor module. We present here single-unit interneuronal recording data that support hypotheses of knee-extensor interneurons in a knee-extensor module and of knee-flexor interneurons in a knee-flexor module. Members of knee-related modules are not members of hip-related modules and vice versa. These results in turtle provide experimental support at the single-unit interneuronal level for the organizational concept that the rostral-scratch CPG for the turtle hindlimb is multipartite, that is, composed of more than two modules. This work, when combined with experimental and computational work in other vertebrates, does not support the classical view that the vertebrate limb CPG is bipartite with only two modules, one controlling all the flexors of the limb and the other controlling all the extensors of the limb. Instead, these results support the general principle that spinal CPGs are multipartite.

Inhibition of TYK2 and JAK1 ameliorates imiquimod-induced psoriasis-like dermatitis by inhibiting IL-22 and the IL-23/IL-17 axis.

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making JAK inhibition an appealing strategy for the treatment of psoriasis. In this study, we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and tyrosine kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose dependently (1 nM-10 µM) inhibited JAK1- and/or TYK2-dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild-type mice with SAR-20347 significantly reduced IL-12-induced IFN-γ production and IL-22-dependent serum amyloid A to similar extents, indicating that, in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes and proinflammatory cytokine levels compared with both TYK2 mutant mice and wild-type controls. Taken together, these data indicate that targeting both JAK1- and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.

Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters.

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 (31)P-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.

Follow-up CT pulmonary angiograms in patients with acute pulmonary embolism.

Computed tomographic (CT) angiography is associated with a non-negligible lifetime attributable risk of cancer. The risk is considerably greater for women and younger patients. Recognizing that there are risks from radiation, the purpose of this investigation was to assess the frequency of follow-up CT angiograms in patients with acute pulmonary embolism. This was a retrospective cohort study of patients aged ≥18 years with acute pulmonary embolism seen in three emergency departments from January 2013 to December 2014. Records of all patients were reviewed for at least 14 months. Pulmonary embolism was diagnosed by CT angiography in 600 patients. At least one follow-up CT angiogram in 1 year was obtained in 141 of 600 (23.5 %). Two follow-ups in 1 year were obtained in 40 patients (6.7 %), 3 follow-ups were obtained in 15 patients (2.5 %), and 4 follow-ups were obtained in 3 patients (0.5 %). Among young women (aged ≤29 years) with pulmonary embolism, 10 of 21 (47.6 %) had at least 1 follow-up and 4 of 21 (19.0 %) had 2 or more follow-ups in 1 year. Among all patients, recurrent pulmonary embolism was diagnosed in 15 of 141 (10.6 %) on the first follow-up CT angiogram and in 6 of 40 (15.0 %) on the second follow-up. Follow-up CT angiograms were obtained in a significant proportion of patients with pulmonary embolism, including young women, the group with the highest risk. Alternative options might be considered to reduce the hazard of radiation-induced cancer, particularly in young women.

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αß transgenic T cells (24αß T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αß T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

Sinus of Valsalva: a converging nozzle that contributes to stable flow in the coronary arteries.

The anatomy of the sinuses of Valsalva has not been considered from the viewpoint of a converging nozzle. Converging nozzles reduce turbulence. We reviewed computed tomographic images of the left and right sinuses of Valsalva in 20 consecutive patients. The sinuses of Valsalva were shown to have a shape in the axial projection that approximates a cubic equation nozzle, although the sinuses of Valsalva are not axisymmetric. The ratios of the cross-sectional area of the inlet to cross-sectional areas of the outlet, assuming the sinuses are axisymmetric, were 14 and 17 in the left and right sinuses, respectively. Calculations by others show that turbulent kinetic energy at the exit (at the coronary ostia) of such axisymmetric nozzles would be reduced by 97%. We conclude that the sinuses of Valsalva have the configuration of a converging nozzle and prevent or reduce turbulent flow in the proximal portions of the coronary arteries.

Fyn promotes Th17 differentiation by regulating the kinetics of RORγt and Foxp3 expression.

Th17 cells constitute a proinflammatory CD4(+) T cell subset that is important for microbial clearance, but also are implicated as propagators of various autoimmune pathologies. Evidence suggests that Th17 cells share common progenitors with immunosuppressive CD4(+) inducible regulatory T cells (T(REG)) and that the developmental pathways of these two subsets are reciprocally regulated. In this study, we show evidence that the Src family tyrosine kinase Fyn helps regulate this Th17/T(REG) balance. When placed under Th17-skewing conditions, CD4(+) T cells from fyn(-/-) mice had decreased levels of IL-17, but increased expression of the T(REG) transcription factor Foxp3. The defect in IL-17 expression occurred independently of the ectopic Foxp3 expression and correlated with a delay in retinoic acid-related orphan receptor γt upregulation and an inability to maintain normal STAT3 activation. Fyn-deficient Th17 cells also exhibited delayed upregulation of Il23r, Il21, Rora, and Irf4, as well as aberrant expression of Socs3, suggesting that Fyn may function upstream of a variety of molecular pathways that contribute to Th17 polarization. The fyn(-/-) mice had fewer IL-17(+)CD4(+) T cells in the large intestinal lamina propria compared with littermate controls. Furthermore, after transfer of either wild-type or fyn(-/-) naive CD4(+) T cells into Rag1(-/-) hosts, recipients receiving fyn(-/-) cells had fewer IL-17-producing T cells, indicating that Fyn may also regulate Th17 differentiation in vivo. These results identify Fyn as a possible novel regulator of the developmental balance between the Th17 cell and T(REG) subsets.

SAP is required for the development of innate phenotype in H2-M3--restricted Cd8(+) T cells.

H2-M3--restricted T cells have a preactivated surface phenotype, rapidly expand, and produce cytokines upon stimulation, and, as such, are classified as innate T cells. Unlike most innate T cells, M3-restricted T cells also express CD8αß coreceptors and a diverse TCR repertoire: hallmarks of conventional MHC Ia-restricted CD8(+) T cells. Although invariant NKT cells are also innate T cells, they are selected exclusively on hematopoietic cells (HC), whereas M3-restricted T cells can be selected on either hematopoietic or thymic epithelial cells. Moreover, their phenotypes differ depending on what cells mediate their selection. Although there is a clear correlation between selection on HC and development of innate phenotype, the underlying mechanism remains unclear. Signaling lymphocyte activation molecule-associated protein (SAP) is required for the development of invariant NKT cells and mediates signals from signaling lymphocyte activation molecule receptors that are exclusively expressed on HC. Based on their dual selection pathway, M3-restricted T cells present a unique model for studying the development of innate T cell phenotype. Using both polyclonal and transgenic mouse models, we demonstrate that although M3-restricted T cells are capable of developing in the absence of SAP, SAP is required for HC-mediated selection, development of preactivated phenotype, and heightened effector functions of M3-restricted T cells. These findings are significant because they directly demonstrate the need for SAP in HC-mediated acquisition of innate T cell phenotype and suggest that, due to their SAP-dependent HC-mediated selection, M3-restricted T cells develop a preactivated phenotype and an intrinsic ability to proliferate faster upon stimulation, allowing for an important role in the early response to infection.

Multifunctional liposomes for nasal delivery of the anti-Alzheimer drug tacrine hydrochloride.

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.

mRNA-delivery of IDO1 suppresses T cell-mediated autoimmunity.

Indoleamine-2,3-dioxygenase (IDO)1 degrades tryptophan, obtained through dietary intake, into immunoregulatory metabolites of the kynurenine pathway. Deficiency or blockade of IDO1 results in the enhancement of autoimmune severity in rodent models and increased susceptibility to developing autoimmunity in humans. Despite this, therapeutic modalities that leverage IDO1 for the treatment of autoimmunity remain limited. Here, we use messenger (m)RNA formulated in lipid nanoparticles (LNPs) to deliver a human IDO1 variant containing the myristoylation site of Src to anchor the protein to the inner face of the plasma membrane. This membrane-anchored IDO1 has increased protein production, leading to increased metabolite changes, and ultimately ameliorates disease in three models of T cell-mediated autoimmunity: experimental autoimmune encephalomyelitis (EAE), rat collagen-induced arthritis (CIA), and acute graft-versus-host disease (aGVHD). The efficacy of IDO1 is correlated with hepatic expression and systemic tryptophan depletion. Thus, the delivery of membrane-anchored IDO1 by mRNA suppresses the immune response in several well-characterized models of autoimmunity.

Shifting the Focus from Dissolution to Permeation: Introducing the Meso-fluidic Chip for Permeability Assessment (MCPA).

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.

Two SHIPs passing in the middle of the immune system.

Immunity requires a complex, multiscale system of molecules, cells, and cytokines. In this issue of the European Journal of Immunology, Collazo et al. [Eur. J. Immunol. 2012. 42: 1785-1796] provide evidence that links the lipid phosphatase SHIP1 with the coordination of interactions between regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). Using conditional knockouts of SHIP1 in either the myeloid or T-cell-lineage of mice, the authors show that the regulated development of Treg cells is controlled directly by cell-intrinsic SHIP1, and indirectly by extrinsic SHIP1 control of an unknown myeloid cell. Regulation of MDSCs is also determined by SHIP1 in an extrinsic manner, again via an as-yet-unknown myeloid cell. Furthermore, this extrinsic control of Treg cells and MDSCs is mediated in part by increased production of G-CSF, a growth factor critical for the production of neutrophils, in SHIP1-deficient mice. Thus, a physiologically important implication of this report is the collaboration between the innate and adaptive immune systems in fine tuning of Treg cells as discussed in this commentary.

Perfusion SPECT in patients with suspected pulmonary embolism.

PURPOSE: Ventilation/perfusion tomography (V/PSPECT), with new interpretation criteria and newer tracers for ventilation imaging, has markedly improved the diagnostic yield in acute pulmonary embolism (PE). Here, we evaluated the diagnostic performance of perfusion SPECT (PSPECT) without ventilation imaging. METHODS: We studied 152 patients with clinically suspected PE who had been examined with both V/PSPECT and multidetector computed tomographic angiography (MD-CTA). The diagnosis or exclusion of PE was decided by the referring clinician based on both the V/PSPECT and/or MD-CTA findings in combination with the clinical findings. PSPECT images were retrospectively examined by a physician with experience in the interpretation of planar perfusion scans who was blinded to clinical, V/PSPECT and MD-CTA data. PSPECT images were interpreted without the aid of chest radiography. All the patients who were deemed to have PE were given anticoagulant therapy. RESULTS: Of the 152 patients, 59 (39%) received a final diagnosis of PE, and 19 (32%) had associated cardiopulmonary diseases such as pneumonia, COPD, or left heart failure. PSPECT correctly identified 53 (90%) of the 59 patients with PE. The specificity was 88 of 93 (95%). None of the PSPECT images was rated nondiagnostic. PSPECT yielded an overall diagnostic accuracy of 93% (95% confidence interval, CI, 87-96%). At the observed PE prevalence of 39 %, the positive and negative predictive values of PSPECT were 91% (95% CI, 80-97%) and 94% (95% CI, 86-97%), respectively. CONCLUSION: In managing critically ill patients, PSPECT might be a valid alternative to V/PSPECT or MD-CTA since it was able to identify most patients with PE with a low false-positive rate and no inconclusive results.

Coordination-driven switching of a preorganized and cooperative calix[4]pyrrole receptor.

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.

Commercially Available Cell-Free Permeability Tests for Industrial Drug Development: Increased Sustainability through Reduction of In Vivo Studies.

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.

Interpreting permeability as a function of free drug fraction: The case studies of cyclodextrins and liposomes.

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-ß-cyclodextrin (HP-ß-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-ß-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.