The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier.

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Nanoparticles targeting the intestinal Fc receptor enhance intestinal cellular trafficking of semaglutide.

Semaglutide is the first oral glucagon-like peptide-1 (GLP-1) analog commercially available for the treatment of type 2 diabetes. In this work, semaglutide was incorporated into poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (NPs) to improve its delivery across the intestinal barrier. The nanocarriers were surface-decorated with either a peptide or an affibody that target the human neonatal Fc receptor (hFcRn), located on the luminal cell surface of the enterocytes. Both ligands were successfully conjugated with the PLGA-PEG via maleimide-thiol chemistry and thereafter, the functionalized polymers were used to produce semaglutide-loaded NPs. Monodisperse NPs with an average size of 170 nm, neutral surface charge and 3% of semaglutide loading were obtained. Both FcRn-targeted NPs exhibited improved interaction and association with Caco-2 cells (cells that endogenously express the hFcRn), compared to non-targeted NPs. Additionally, the uptake of FcRn-targeted NPs was also observed to occur in human intestinal organoids (HIOs) expressing hFcRn through microinjection into the lumen of HIOs, resulting in potential increase of semaglutide permeability for both ligand-functionalized nanocarriers. Herein, our study demonstrates valuable data and insights that the FcRn-targeted NPs has the capacity to promote intestinal absorption of therapeutic peptides.

Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line.

BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201. METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1). RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane. CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Characterizing interspecies differences in gastric fluid properties to improve understanding of in vivo oral drug formulation performance.

An in-depth understanding of the properties of gastric fluid(s) prior to an in vivo pharmacokinetic investigation can vastly improve predictions of in vivo performance. Previously, properties of animal and human gastric fluids have been characterized with varying methods. Unfortunately, characterization has often not been thorough, and some properties, such as density and viscosity, have not been reported. Here, human, porcine and canine gastric fluids were harvested and characterized for pH, viscosity, surface tension, density, and osmolarity. We found that the variability of pH and surface tension between dogs was significantly higher than the variability between pigs, and, furthermore, gastric fluids collected from the same canine species (beagles) housed in two different countries (Denmark and China) had surprisingly different pH values. Next, an in vitro dissolution study in diluted gastric fluids from each species was performed using minitablets containing ibuprofen. Human gastric fluids and porcine gastric fluids showed similar dissolution profiles and corroborated well with biorelevant human Fasted State Simulated Gastric Fluid (FaSSGF). In contrast, differences in canine gastric fluids caused highly variable dissolution results. We systematically compared our findings to those in the literature and based on this evaluation, propose obtaining aspirates from the animals used for in vivo studies to ensure knowledge on the fluid properties affecting the performance of the formulated drug in question.

Combining mass spectrometry and machine learning to discover bioactive peptides.

Peptides play important roles in regulating biological processes and form the basis of a multiplicity of therapeutic drugs. To date, only about 300 peptides in human have confirmed bioactivity, although tens of thousands have been reported in the literature. The majority of these are inactive degradation products of endogenous proteins and peptides, presenting a needle-in-a-haystack problem of identifying the most promising candidate peptides from large-scale peptidomics experiments to test for bioactivity. To address this challenge, we conducted a comprehensive analysis of the mammalian peptidome across seven tissues in four different mouse strains and used the data to train a machine learning model that predicts hundreds of peptide candidates based on patterns in the mass spectrometry data. We provide in silico validation examples and experimental confirmation of bioactivity for two peptides, demonstrating the utility of this resource for discovering lead peptides for further characterization and therapeutic development.

Unbound Brain-to-Plasma Partition Coefficient, Kp,uu,brain-a Game Changing Parameter for CNS Drug Discovery and Development.

PURPOSE: More than 15 years have passed since the first description of the unbound brain-to-plasma partition coefficient (Kp,uu,brain) by Prof. Margareta Hammarlund-Udenaes, which was enabled by advancements in experimental methodologies including cerebral microdialysis. Since then, growing knowledge and data continue to support the notion that the unbound (free) concentration of a drug at the site of action, such as the brain, is the driving force for pharmacological responses. Towards this end, Kp,uu,brain is the key parameter to obtain unbound brain concentrations from unbound plasma concentrations. METHODS: To understand the importance and impact of the Kp,uu,brain concept in contemporary drug discovery and development, a survey has been conducted amongst major pharmaceutical companies based in Europe and the USA. Here, we present the results from this survey which consisted of 47 questions addressing: 1) Background information of the companies, 2) Implementation, 3) Application areas, 4) Methodology, 5) Impact and 6) Future perspectives. RESULTS AND CONCLUSIONS: From the responses, it is clear that the majority of the companies (93%) has established a common understanding across disciplines of the concept and utility of Kp,uu,brain as compared to other parameters related to brain exposure. Adoption of the Kp,uu,brain concept has been mainly driven by individual scientists advocating its application in the various companies rather than by a top-down approach. Remarkably, 79% of all responders describe the portfolio impact of Kp,uu,brain implementation in their companies as 'game-changing'. Although most companies (74%) consider the current toolbox for Kp,uu,brain assessment and its validation satisfactory for drug discovery and early development, areas of improvement and future research to better understand human brain pharmacokinetics/pharmacodynamics translation have been identified.

Oral delivery of systemic monoclonal antibodies, peptides and small molecules using gastric auto-injectors.

Oral administration provides a simple and non-invasive approach for drug delivery. However, due to poor absorption and swift enzymatic degradation in the gastrointestinal tract, a wide range of molecules must be parenterally injected to attain required doses and pharmacokinetics. Here we present an orally dosed liquid auto-injector capable of delivering up to 4-mg doses of a bioavailable drug with the rapid pharmacokinetics of an injection, reaching an absolute bioavailability of up to 80% and a maximum plasma drug concentration within 30 min after dosing. This approach improves dosing efficiencies and pharmacokinetics an order of magnitude over our previously designed injector capsules and up to two orders of magnitude over clinically available and preclinical chemical permeation enhancement technologies. We administered the capsules to swine for delivery of clinically relevant doses of four commonly injected medications, including adalimumab, a GLP-1 analog, recombinant human insulin and epinephrine. These multi-day dosing experiments and oral administration in awake animal models support the translational potential of the system.

Implantable system for chronotherapy.

Diurnal variation in enzymes, hormones, and other biological mediators has long been recognized in mammalian physiology. Developments in pharmacobiology over the past few decades have shown that timing drug delivery can enhance drug efficacy. Here, we report the development of a battery-free, refillable, subcutaneous, and trocar-compatible implantable system that facilitates chronotherapy by enabling tight control over the timing of drug administration in response to external mechanical actuation. The external wearable system is coupled to a mobile app to facilitate control over dosing time. Using this system, we show the efficacy of bromocriptine on glycemic control in a diabetic rat model. We also demonstrate that antihypertensives can be delivered through this device, which could have clinical applications given the recognized diurnal variation of hypertension-related complications. We anticipate that implants capable of chronotherapy will have a substantial impact on our capacity to enhance treatment effectiveness for a broad range of chronic conditions.

Semaglutide lowers body weight in rodents via distributed neural pathways.

Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.

Overcoming Poor Tabletability of Bulky Absorption Enhancers by Spray Drying Technology.

Absorption enhancers are often a major component of solid oral peptide formulations as compared to the active pharmaceutical ingredient and excipients. This commonly results in poor tabletability that is hard to mitigate in direct compaction by addition of small amounts of excipients. To improve the tabletability of bulky absorption enhancers, the model absorption enhancers, sodium cholate and deoxycholic acid, were co-spray-dried with hydroxypropyl methylcellulose E5, where the percentage of absorption enhancers was not lower than 90% (w/w). The physicochemical properties of the resulting powders were assessed by laser diffraction, scanning electron microscopy, X-ray powder diffraction, thermogravimetric analysis, and differential scanning calorimetry. The powders were compressed into tablets, and the tabletability was evaluated. Co-spray drying with 10% of hydroxypropyl methylcellulose significantly improved the tabletability of the both absorption enhancers. Moreover, it was demonstrated that small particle size and amorphous state rather than high moisture content contributed to the improved tabletability of the spray-dried powders. The study suggests that spray drying technology can be promising to overcome the poor tabletability of oral peptide formulation consisting of large amounts of absorption enhancers.

Elucidating the Mechanism of Absorption of Fast-Acting Insulin Aspart: The Role of Niacinamide.

PURPOSE: Fast-acting insulin aspart (faster aspart) is a novel formulation of insulin aspart containing two additional excipients: niacinamide, to increase early absorption, and L-arginine, to optimize stability. The aim of this study was to evaluate the impact of niacinamide on insulin aspart absorption and to investigate the mechanism of action underlying the accelerated absorption. METHODS: The impact of niacinamide was assessed in pharmacokinetic analyses in pigs and humans, small angle X-ray scattering experiments, trans-endothelial transport assays, vascular tension measurements, and subcutaneous blood flow imaging. RESULTS: Niacinamide increased the rate of early insulin aspart absorption in pigs, and pharmacokinetic modelling revealed this effect to be most pronounced up to ~30-40 min after injection in humans. Niacinamide increased the relative monomer fraction of insulin aspart by ~35%, and the apparent permeability of insulin aspart across an endothelial cell barrier by ~27%. Niacinamide also induced a concentration-dependent vasorelaxation of porcine arteries, and increased skin perfusion in pigs. CONCLUSION: Niacinamide mediates the acceleration of initial insulin aspart absorption, and the mechanism of action appears to be multifaceted. Niacinamide increases the initial abundance of insulin aspart monomers and transport of insulin aspart after subcutaneous administration, and also mediates a transient, local vasodilatory effect.

An ingestible self-orienting system for oral delivery of macromolecules.

Biomacromolecules have transformed our capacity to effectively treat diseases; however, their rapid degradation and poor absorption in the gastrointestinal (GI) tract generally limit their administration to parenteral routes. An oral biologic delivery system must aid in both localization and permeation to achieve systemic drug uptake. Inspired by the leopard tortoise's ability to passively reorient, we developed an ingestible self-orienting millimeter-scale applicator (SOMA) that autonomously positions itself to engage with GI tissue. It then deploys milliposts fabricated from active pharmaceutical ingredients directly through the gastric mucosa while avoiding perforation. We conducted in vivo studies in rats and swine that support the applicator's safety and, using insulin as a model drug, demonstrated that the SOMA delivers active pharmaceutical ingredient plasma levels comparable to those achieved with subcutaneous millipost administration.

Transcellular stomach absorption of a derivatized glucagon-like peptide-1 receptor agonist.

Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.

The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain.

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway. The study showed that the insulin receptor was expressed at both the mRNA and protein levels in bovine brain endothelial cells. Luminally applied radiolabeled insulin showed insulin receptor-mediated binding to the endothelial cells. This caused a dose-dependent increase in Akt-phosphorylation, which was inhibited by coapplication of an insulin receptor inhibitor, s961, demonstrating activation of insulin receptor signaling pathways. Transport of insulin across the blood-brain barrier in vitro was low and comparable to that of a similarly sized paracellular marker. Furthermore, insulin transport was not inhibited by coapplication of an excess of unlabeled insulin or an insulin receptor inhibitor. The insulin transport and uptake studies were repeated in mouse brain endothelial cells demonstrating similar results. Although it cannot be ruled out that culture-induced changes in the cell model could have impaired a potential insulin transport mechanism, these in vitro data indicate that peripheral insulin must reach the brain parenchyma through alternative pathways rather than crossing the blood-brain barrier via receptor mediated transcytosis.

Chemically modified peptides and proteins - critical considerations for oral delivery.

Numerous approaches have been explored to date in the pursuit of delivering peptides or proteins via the oral route. One such example is chemical modification, whereby the native structure of a peptide or protein is tailored to provide a more efficient uptake across the epithelial barrier of the gastrointestinal tract via incorporation of a chemical motif or moiety. In this regard, a diverse array of concepts have been reported, ranging from the exploitation of endogenous transport mechanisms to incorporation of physicochemical modifications in the molecule, which promote more favorable interactions with the absorptive membrane at the cell surface. This review provides an overview of the modification technologies described in the literature and offers insights into some pragmatic considerations pertaining to their translation into clinically viable concepts.

In silico modelling of permeation enhancement potency in Caco-2 monolayers based on molecular descriptors and random forest.

Structural traits of permeation enhancers are important determinants of their capacity to promote enhanced drug absorption. Therefore, in order to obtain a better understanding of structure-activity relationships for permeation enhancers, a Quantitative Structural Activity Relationship (QSAR) model has been developed. The random forest-QSAR model was based upon Caco-2 data for 41 surfactant-like permeation enhancers from Whitehead et al. (2008) and molecular descriptors calculated from their structure. The QSAR model was validated by two test-sets: (i) an eleven compound experimental set with Caco-2 data and (ii) nine compounds with Caco-2 data from literature. Feature contributions, a recent developed diagnostic tool, was applied to elucidate the contribution of individual molecular descriptors to the predicted potency. Feature contributions provided easy interpretable suggestions of important structural properties for potent permeation enhancers such as segregation of hydrophilic and lipophilic domains. Focusing on surfactant-like properties, it is possible to model the potency of the complex pharmaceutical excipients, permeation enhancers. For the first time, a QSAR model has been developed for permeation enhancement. The model is a valuable in silico approach for both screening of new permeation enhancers and physicochemical optimisation of surfactant enhancer systems.

Interaction with Mixed Micelles in the Intestine Attenuates the Permeation Enhancing Potential of Alkyl-Maltosides.

The purpose of the present study was to investigate the interaction of intestinal permeation enhancers with lipid and surfactant components present in the milieu of the small intestine. Maltosides of different chain lengths (decyl-, dodecyl-, and tetradecyl-maltoside; DM, DDM, TDM, respectively) were used as examples of nonionic, surfactant-like permeation enhancers, and their effect on the permeation of FD4 across Caco-2 monolayers was monitored. To mimic the environment of the small intestine, modified versions of fasted and fed state simulated intestinal fluid (FaSSIFmod, FeSSIFmod6.5, respectively) were used in addition to standard transport media (TM). Compared to the buffer control, 0.5 mM DDM led to a 200-fold permeation enhancement of FD4 in TM. However, this was dramatically decreased in FaSSIFmod, where a concentration of 5 mM DDM was necessary in order to elicit a moderate, 4-fold, permeation enhancement. Its capacity to promote permeation was diminished further when FeSSIFmod6.5 was employed. Even when cells were exposed to a concentration of 5 mM, no significant permeation enhancement of FD4 was observed. Analogous effects were observed in the case of DM and TDM, with slight deviations on account of differences in their critical micelle concentration (CMC). This observation was corroborated by calculating the amount of maltoside monomer versus micellar bound maltoside in FaSSIFmod and FeSSIFmod6.5, which demonstrated a reduced amount of free monomer in these fluids. To evaluate the in vivo significance of our findings, DDM solutions in TM, FaSSIFmod, and FeSSIFmod6.5 were used for closed intestinal loop studies in rats. Consistent with the results found in in vitro permeation studies, these investigations illustrated the overwhelming impact of sodium taurocholate/lecithin micelles on the permeation enhancing effect of DDM. While DDM led to a 20-fold increase in FD4 bioavailability when it was applied in TM, no significant permeation enhancement was seen in FaSSIFmod/FeSSIFmod6.5. Collectively, these investigations highlight the importance of using biorelevant media when evaluating the potency of permeation enhancers. In doing so, this ensures improved correlations between in vitro and in vivo studies and thus enables an early and more accurate assessment of promising permeation enhancers.

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

The role of citric acid in oral peptide and protein formulations: relationship between calcium chelation and proteolysis inhibition.

The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CA's calcium chelation activity was developed and verified experimentally using an ion-selective electrode. The effects of CA, its salt (citrate, Cit) and the established permeation enhancer, lauroyl carnitine chloride (LCC) were compared by measuring transepithelial electrical resistance (TEER) and permeability of insulin and FD4 across Caco-2 monolayers and rat small intestinal mucosae mounted in Ussing chambers. Proteolytic degradation of insulin was determined in rat luminal extracts across a range of pH values in the presence of CA. CA's capacity to chelate calcium decreased ~10-fold for each pH unit moving from pH 6 to pH 3. CA was an inferior weak permeation enhancer compared to LCC in both in vitro models using physiological buffers. At pH 4.5 however, degradation of insulin in rat luminal extracts was significantly inhibited in the presence of 10mM CA. The capacity of CA to chelate luminal calcium does not occur significantly at the acidic pH values where it effectively inhibits proteolysis, which is its dominant action in oral peptide formulations. On account of insulin's low basal permeability, inclusion of alternative permeation enhancers is likely to be necessary to achieve sufficient oral bioavailability since this is a weak property of CA.