Poor Membrane Permeability Research Articles

Lipid Composition Is Critical for Accurate Membrane Permeability Prediction of Cyclic Peptides by Molecular Dynamics Simulations

Cyclic peptides have attracted attention as a promising pharmaceutical modality due to their potential to selectively inhibit previously undruggable targets, such as intracellular protein–protein interactions. Poor membrane permeability is the biggest bottleneck hindering successful drug discovery based on cyclic peptides. Therefore, the development of computational methods that can predict membrane permeability and support elucidation of the membrane permeation mechanism of drug candidate peptides is much sought after. In this study, we developed a protocol to simulate the behavior in membrane permeation steps and estimate the membrane permeability of large cyclic peptides with more than or equal to 10 residues. This protocol requires the use of a more realistic membrane model than a single-lipid phospholipid bilayer. To select a membrane model, we first analyzed the effect of cholesterol concentration in the model membrane on the potential of mean force and hydrogen bonding networks along the direction perpendicular to the membrane surface as predicted by molecular dynamics simulations using cyclosporine A. These results suggest that a membrane model with 40 or 50 mol % cholesterol was suitable for predicting the permeation process. Subsequently, two types of membrane models containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 40 and 50 mol % cholesterol were used. To validate the efficiency of our protocol, the membrane permeability of 18 ten-residue peptides was predicted. Correlation coefficients of R > 0.8 between the experimental and calculated permeability values were obtained with both model membranes. The results of this study demonstrate that the lipid membrane is not just a medium but also among the main factors determining the membrane permeability of molecules. The computational protocol proposed in this study and the findings obtained on the effect of membrane model composition will contribute to building a schematic view of the membrane permeation process. Furthermore, the results of this study will eventually aid the elucidation of design rules for peptide drugs with high membrane permeability.

Characterization of the physicochemical interactions between exenatide and two intestinal permeation enhancers: Sodium caprate (C10) and salcaprozate sodium (SNAC)

A common approach to tackle the poor intestinal membrane permeability of peptides after oral administration is to formulate them with a permeation enhancer (PE). Increased oral bioavailability for oral peptide candidates has been reported from clinical trials when either salcaprozate sodium (SNAC) or sodium caprate (C10) is incorporated in the formulation. However, little is known about how they physically interact with peptides in solution. Our objective was to compare the biophysical interactions between the GLP-1 analogue exenatide (Byetta®, Lilly), and C10 or SNAC using a variety of advanced analytical techniques. First, critical micelle concentration was measured in different buffers for both PEs. Dynamic light scattering (DLS) measurements revealed specific supramolecular structures arising from exenatide-PE association. Surface plasmon resonance (SPR) indicated the formation of exenatide-PE complexes with a high contribution from non-specific interactions and rapid binding kinetics, resulting in overall low affinities. DLS and isothermal titration calorimetry (ITC) were used to examine the supramolecular organization of the PEs, and revealed thermodynamic signatures characterized by unfavourable enthalpic contributions compensated by favourable entropic ones, but with low-affinity estimates in water (KD in the 10–100 µM range). With affinity capillary electrophoresis (ACE), weak interactions between exenatide and SNAC or C10 were confirmed in saline, with a dissociation constant around 10 µM and 30 µM respectively. In biorelevant intestinal media, the bile salts in FaSSIF and FeSSIF further reduced the binding of both agents to exenatide (KD ≈ 100 µM), indicating that the interaction between the PEs and exenatide might be inhibited by bile salts in the GI lumen. This study suggests that the interactions of both PEs with exenatide follow a similar non-covalent mechanism and are of low affinity.

A co-crystal berberine-ibuprofen improves obesity by inhibiting the protein kinases TBK1 and IKKɛ

Berberine (BBR) exerts specific therapeutic effects on various diseases such as diabetes, obesity, and other inflammation-associated diseases. However, the low oral bioavailability (below 1%) of berberine due to its poor solubility and membrane permeability limits its clinical use. In this paper, we have prepared a 1:1 co-crystal berberine-ibuprofen (BJ) using drug salt metathesis and co-crystal technology. Pharmacokinetic studies demonstrate a 3-fold increase in vivo bioavailability of BJ compared to that of BBR, and BJ is more effective in treating obesity and its related metabolism in vitro and in vivo. We also find that BJ promotes mitochondrial biogenesis by inhibiting TBK1 and inducing AMP‐activated protein kinase (AMPK) phosphorylation, and BJ increases adipocyte sensitivity to catecholamine by inhibiting IKKε. Together, our findings support that co-crystal BJ is likely to be an effective agent for treating obesity and its related metabolic diseases targeting TBK1 and IKKε.

Structure Rigidification Promoted Ultrabright Solvatochromic Fluorescent Probes for Super-Resolution Imaging of Cytosolic and Nuclear Lipid Droplets.

Lipid droplets (LDs) containing cytosolic and nuclear LDs have recently received increasing attention because of their diverse biological roles in living systems. However, developing fluorescent probes for super-resolution visualization of these subcellular LDs still remains challenging due to insufficient fluorescence brightness and poor nuclear membrane permeability. Herein, we rationally synthesized a series of ultrabright solvatochromic fluorescent probes based on benzoboranils (BBAs) for LD-specific super-resolution imaging using structured illumination microscopy (SIM). The rigidly structured probes exhibit ultrahigh fluorescence quantum yields of up to 99.9% in low-polar solvents. They also show more significant fluorescence enhancements in lipid environments than commercial LD probes. Owing to these excellent merits, our lipophilic fluorescent probes can specifically light up subcellular LDs at ultralow concentrations down to 10 nM. Further use of BBA-CF3 for super-resolution SIM imaging of cytosolic and nuclear LDs and their fusion process was successfully achieved. The unprecedented spatial resolution for nuclear LDs with an FWHM value of 142 nm was also acquired. Collectively, our ultrabright fluorescent probes hold tremendous potential to unveil the mysterious roles of cytosolic and nuclear LDs in biological research using SIM.

Bioavailability Improvement Strategies for Icariin and Its Derivates: A Review

In recent years, there has been considerable interest in icariin (ICA) and its derivates, icariside II (ICS) and icaritin (ICT), due to their wide range of potential applications in preventing cancer, cardiovascular disease, osteoporosis, delaying the effects of Alzheimer’s disease, treating erectile dysfunction, etc. However, their poor water solubility and membrane permeability, resulting in low bioavailability, dampens their potential beneficial effects. In this regard, several strategies have been developed, such as pharmaceutical technologies, structural transformations, and absorption enhancers. All these strategies manage to improve the bioavailability of the above-mentioned flavonoids, thus increasing their concentration in the desired places. This paper focuses on gathering the latest knowledge on strategies to improve bioavailability for enhancing the efficacy of icariin, icariside II, and icaritin. We conclude that there is an opportunity for many further improvements in this field. To the best of our knowledge, no such review articles scoping the bioavailability improvement of icariin and its derivates have been published to date. Therefore, this paper can be a good starting point for all those who want to deepen their understanding of the field.

Design, synthesis, and biological evaluation of novel double-winged galloyl derivatives as HIV-1 RNase H inhibitors

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains as the only enzyme encoded within the viral genome not clinically validated as an antiviral target. We have previously reported that the galloyl derivative II-25 had RNase H inhibitory activity in enzymatic assays but showed weak antiviral activity in phenotypic assays due its large polarity and poor membrane permeability. In this report, we report on a series of II-25 derivatives, obtained by addition of different hydrophobic moieties (“the wings”) at the C-2 and C-3 positions of the piperazine ring that showed improved RNase H inhibitory activity. Six compounds showed strong inhibitory activity and were found to be more potent than β-thujaplicinol in enzymatic assays. The most potent compound was IA-6 and exhibited the best inhibitory activity (IC50 = 0.067 ± 0.02 μM). IA-6 was around 11 and 30 times more potent than II-25 and β-thujaplicinol, respectively. Molecular modeling studies predict a strong hydrophobic interaction between the furylmethylaminyl group of IA-6 and the side chain of His539, explaining the potent HIV-1 RNase H inhibition. Unfortunately, none of the derivatives showed significant antiviral activity in cell culture. It is worth emphasizing that most of the obtained compounds show low cytotoxicity (CC50 > 20 μM), which confirms the significance of identifying galloyl derivatives as valuable leads for further optimization.

Turning a Targeting β-Catenin/Bcl9 Peptide Inhibitor into a GdOF@Au Core/Shell Nanoflower for Enhancing Immune Response to Cancer Therapy in Combination with Immune Checkpoint Inhibitors.

Combination administration is becoming a popular strategy in current cancer immunotherapy to enhance tumor response to ICIs. Recently, a peptide drug, a protein–protein interaction inhibitor (PPI), that disrupts the β-catenin/Bcl9 interaction in the tumoral Wnt/β-catenin pathway has become a promising candidate drug for immune enhancement and tumor growth inhibition. However, the peptide usually suffers from poor cell membrane permeability and proteolytic degradation, limiting its adequate accumulation in tumors and ultimately leading to side effects. Herein, a gadolinium–gold-based core/shell nanostructure drug delivery system was established, where Bcl9 was incorporated into a gadolinium–gold core–shell nanostructure and formed GdOFBAu via mercaptogenic self-assembly. After construction, GdOFBAu, when combined with anti-PD1 antibodies, could effectively inhibit tumor growth and enhance the response to immune therapy in MC38 tumor-bearing mice; it not only induced the apoptosis of cancer cells, but also promoted the tumor infiltration of Teff cells (CD8+) and decreased Treg cells (CD25+). More importantly, GdOFBAu maintained good biosafety and biocompatibility during treatment. Taken together, this study may offer a promising opportunity for sensitizing cancer immunotherapy via metal–peptide self-assembling nanostructured material with high effectiveness and safety.

Lipid-Based Nanocarriers for Oral Delivery of Proteins and Peptides: Opportunities, Challenges, and Future Prospects

Oral administration is the most common drug delivery route with high levels of patient acceptance. However, oral delivery of therapeutic proteins/peptides is extremely difficult, and improving the pharmacological bioavailability still is a challenging goal because of the poor membrane permeability, high molecular weight, and enzymatic degradation of these drugs. Lipid-based nanocarriers represent a viable means for enhancing the oral bioavailability of protein or peptide drugs while minimizing toxicity. Nowadays, like other macromolecules protein or peptide drugs are the promising candidates to be delivered employing liposomes and lipid nanoparticles including solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). Generally, these drug delivery strategies can reduce protein or peptide drug degradation and improve membrane permeability thus enhance bioavailability. This review demonstrates various lipid-based formulation strategies used for successful oral delivery of peptides and proteins, assesses the hurdles and delivery efficiency and their clinical implications. Dhaka Univ. J. Pharm. Sci. 20(3): 395-416, 2022 (June) Centennial Special Issue

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker.

Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference. Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) ≥2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our in vivo crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth in vivo analysis of protein-protein interactions (PPIs) and protein architectures with finer temporal resolution.

Nanocarrier based on halloysite and fluorescent probe for intracellular delivery of peptide nucleic acids

The development of systems able to deliver genetic material into a target site is a challenge for modern medicine. Single-stranded peptide nucleic acids have attracted attention as promising therapeutic molecules for diagnostic and gene therapy. However, their poor cell membrane permeability represents a drawback for biomedical applications. Halloysite nanotubes (HNTs) are emerging materials in drug delivery applications both for their ability to penetrate cell membranes and for enhancing the solubility of drugs in biological media. Herein, we report the first example of the use of a nanocarrier based on halloysite labelled with fluorescent switchable halochromic oxazine molecules, to deliver a single-stranded peptide nucleic acids tetramer (PNAts) into living cells. The PNAts is covalently attached to halloysite (HNTs-PNA), whereas the fluorescent probe supramolecularly interacts with HNTs. The ability of the nanomaterial to bind complementary single-stranded DNA was assessed by resonance light scattering measurements. Finally, studies of cellular uptake were carried out by confocal laser scanning microscopy on normal and tumoral cell lines. This work highlights the usefulness of the covalent approach to generate HNTs-PNA nanomaterials for the potential targeting of future specific nucleic acids in living cells, which could open the doorway to novel possibilities for theranostic and gene therapy applications.

Identification of Ziyuglycoside II from a Natural Products Library as a STING Agonist.

Given the emerging pivotal roles of stimulator of interferon genes (STING) in host pathogen defense and immune-oncology, STING is regarded as a promising target for drug development. Cyclic dinucleotides (CDNs) are the first-generation STING agonists. However, their poor metabolic stability and membrane permeability limits their therapeutic application. In contrast, small-molecule STING agonists show superior properties such as molecular weight, polar character, and delivery diversity. The quest for a potent small-molecular agonist of human STING remains ongoing. In our study, through an IRF/IFN pathway-targeted cell-based screen of a natural products library, we identified a small-molecular STING agonist, Ziyuglycoside II, termed ST12, with potent stimulation of the IRF/IFN and NF-κB pathways. Furthermore, its binding to the C-terminal domain of human STING, detected by bio-layer interferometry, indicates that ST12 is a human STING agonist. Further Tanimoto similarity analysis with existing small-molecule STING agonists indicates that ST12 is a lead compound with a novel core structure for the further optimization.

A novel 5-Fluorocuracil multiple-nanoemulsion used for the enhancement of oral bioavailability in the treatment of colorectal cancer

5-Fluorouracil (5-FU) is a drug of choice for colorectal-cancer. But oral therapeutic efficacy of 5-FU is restricted due to their very little bioavailability because of poor membrane permeability and GIT-absorption. We have developed a multiple nanoemulsion (w/o/w i.e. 5-FU-MNE) in which 5-FU incorporated to improve their oral-absorption. Globule-size of opt-5-FU-MNE was 51.64 ± 2.61 nm with PDI and ZP 0.101 ± 0.001 and −5.59 ± 0.94, respectively. In vitro 5-FU-release and ex vivo permeation studies exhibited 99.71% release and 83.64% of 5-FU from opt-nanoformulation. Cytotoxic in vitro studies-exhibited that 5-FU in opt-5-FU-MNE was 5-times more potent than 5-FU-S on human-colon-cancer-cell-lines (HT-29). The enhanced Cmax with AUC0-8h with opt-5-FU-MNE was shown extremely significant (p < 0.001) in wistar rat’s plasma in the comparison of oral and i.v. treated group of 5-FU-S by PK-observations. Furthermore, opt-5-FU-MNE was showed much more significant (p < 0.001) results as compared to 5-FU-S (free) on cell lines for human colon cancer (HT-29).

A New Amino Acid for Improving Permeability and Solubility in Macrocyclic Peptides through Side Chain-to-Backbone Hydrogen Bonding.

Despite the notoriously poor membrane permeability of peptides, many cyclic peptide natural products show high passive membrane permeability and potently inhibit a variety of "undruggable" intracellular targets. A major impediment to the design of cyclic peptides with good permeability is the high desolvation energy associated with the peptide backbone amide NH groups. While several strategies have been proposed to mitigate this deleterious effect, only few studies have used polar side chains to sequester backbone NH groups. We investigated the ability of N,N-pyrrolidinylglutamine (Pye), whose side chain contains a powerful hydrogen-bond-accepting C═O amide group but no hydrogen-bond donors, to sequester exposed backbone NH groups in a series of cyclic hexapeptide diastereomers. Analyses revealed that specific Leu-to-Pye substitutions conferred dramatic improvements in aqueous solubility and permeability in a scaffold- and position-dependent manner. Therefore, this approach offers a complementary tool for improving membrane permeability and solubility in cyclic peptides.

Janus Magnetic Nanoplatform for Magnetically Targeted and Protein/Hyperthermia Combination Therapies of Breast Cancer.

Protein therapeutics have been considered a promising strategy for cancer treatment due to their highly specific bioactivity and few side effects. Unfortunately, the low physiological stability and poor membrane permeability of most protein drugs greatly limit their clinical application. Furthermore, single-modality protein therapeutics show insufficient efficacy. To address these issues, Janus magnetic mesoporous silica nanoparticles (Janus MSNNPs) were developed to preload ribonuclease A (RNaseA) to simultaneously realize the magnetically enhanced delivery of protein drugs and magnetic hyperthermia-enhanced protein therapy. Janus MSNNPs showed a high RNaseA loading ability and pH-responsive drug release behavior. Furthermore, an external magnetic field could remarkably enhance the therapeutic effect of RNaseA-loaded Janus MSNNPs due to the improved intracellular internalization of RNaseA. Importantly, Janus MSNNPs possessed an outstanding magnetic hyperthermia conversion efficiency, which could generate hyperthermia under an alternating magnetic field, effectively supplementing protein therapy by a combined effect. In vitro and in vivo experiments confirmed the high anticancer outcome and low side effects of this intriguing strategy for breast cancer based on Janus MSNNPs. Hence, Janus MSNNPs might be an effective and safe nanoplatform for magnetically combined protein therapy.

Development of environment-insensitive and highly emissive BODIPYs via installation of N,N'-dialkylsubstituted amide at meso position

Fluorescent dyes play a crucial role in fluorescence imaging and sensing technology. However, there is a dilemma that they are usually intrinsically hydrophobic which lacks of emission in water and modification with ionic groups to access water solubility may result in poor membrane permeability. Fluorescent dyes with strong fluorescence emission in both nonpolar and polar solvents are highly desirable. In this manuscript, we reported a strategy to develop fluorescent BODIPY dyes via installation of amide moiety at meso position of 1,3,5,7-tetramethyl-BODIPY and discovered that N,N'-dialkylsubstituted BODIPY amides possessed highly fluorescent emission with favorable environment-insensitive properties.

Hydroxypropyl-β-cyclodextrin Enhances Oral Absorption of Silymarin Nanoparticles Prepared Using PureNano™ Continuous Crystallizer.

The oral bioavailability of drugs is limited by factors such as poor membrane permeability, low solubility, and low dissolution rate. Silymarin (SLM) is a health-food active ingredient that is good for immunosuppression and tumor suppression. However, obtaining a good oral bioavailability is difficult owing to its poor solubility and low dissolution ability. To overcome these concerns, we previously prepared SLM nanoparticles (NPs) using the high-pressure crystallization method (PureNanoTM) and freeze-dried them with erythritol (Ery) or hydroxypropyl-β-CyD (HP-β-CyD) as a water-soluble dispersion stabilizer. In the present study, we investigated the mechanism underlying the improved absorption of SLM/hypromellose (HPMC)/HP-β-CyD NPs after oral administration. The SLM/HPMC nano-suspension prepared using PureNanoTM exhibited a narrow size distribution. The size of the SLM/HPMC/HP-β-CyD NPs was approximately 250 nm after hydration. The SLM/HPMC/HP-β-CyD NPs were rapidly dissolved, and demonstrated a high solubility under supersaturated conditions. Additionally, they exhibited good wettability and their membrane permeability was improved compared with that of SLM original powder. These results suggest that the formulation of SLM NPs using PureNanoTM and freeze-drying with HP-β-CyD improves the absorption of SLM after oral administration by enhancing solubility, wettability, and membrane permeability.

Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics.

Biomacromolecules are highly promising therapeutic modalities to treat various diseases. However, they suffer from poor cellular membrane permeability, limiting their access to intracellular targets. Strategies to overcome this challenge often employ nanoscale carriers that can get trapped in endosomal compartments. Here we report conjugated peptides that form pH- and redox-responsive coacervate microdroplets by liquid-liquid phase separation that readily cross the cell membrane. A wide range of macromolecules can be quickly recruited within the microdroplets, including small peptides, enzymes as large as 430 kDa and messenger RNAs (mRNAs). The therapeutic-loaded coacervates bypass classical endocytic pathways to enter the cytosol, where they undergo glutathione-mediated release of payload, the bioactivity of which is retained in the cell, while mRNAs exhibit a high transfection efficiency. These peptide coacervates represent a promising platform for the intracellular delivery of a large palette of macromolecular therapeutics that have potential for treating various pathologies (for example, cancers and metabolic diseases) or as carriers for mRNA-based vaccines.

Cathepsin D inhibitors based on tasiamide B derivatives with cell membrane permeability

Cathepsin D (Cath D) has been evidenced as a potential target for cancer therapy. Our previous studies revealed that TB-9, a tasiamide B derivative, exhibited highly potent inhibition against Cath D with satisfactory selectivity over Cath E and BACE1. But this compound was inactive on cell level possibly due to poor membrane permeability. Herein, we report the design, synthesis, and evaluation of two novel Cath D inhibitors (2 and 3) which combining tasiamide B scaffold with a cell penetrating peptide (CPP) specifically targeting the endolysosomal compartment. The results revealed that 2 and 3 not only retained highly potent inhibition against Cath D, but also were active against MDA-MB-231 cell lines.

Novel berberine-loaded hyalurosomes as a promising nanodermatological treatment for vitiligo: Biochemical, biological and gene expression studies

Vitiligo is a depigmentation disorder that affects 0.5–2% of the world population. It has a severe impact on a patient's quality of life and even causes suicidal attempts. Up to date, no curative therapy is available which have created a substantial demand for novel vitiligo treatments. Berberine (BRB) is an isoquinoline alkaloid with promising pharmacological effects. However, it suffers from poor membrane permeability hindering its topical application. The current work is the first to design and assess topical BRB-loaded hyalurosomes for targeted vitiligo treatment. BRB-hyalurosomes are hyaluronan-immobilized phospholipid nanovesicles that showed promising invitro physicochemical properties. Novel ex vivo studies were performed using full-thickness human skin to mimic its dermal application. Furthermore, in-vivo studies were conducted using a vitiligo-induced mouse model followed by biochemical, histological and immunohistochemical investigations. In addition, gene expression of skin inflammatory markers was assessed using quantitative reverse-transcription PCR. Biological studies showed significant improvement of the biochemical markers in BRB-hyalurosomes group compared to the vitiligo-model group and BRB conventional gel. It is worthy to mention that placebo hyalurosomes demonstrated significant enhancement in the biological activity confirming its intrinsic activity. Conclusively, BRB-hyalurosomes is considered a novel nanodermatological tool that paving the way for its clinical application for vitiligo treatment.

Screening for Inhibitors of Main Protease in SARS-CoV-2: In Silico and In Vitro Approach Avoiding Peptidyl Secondary Amides.

In addition to vaccines, antiviral drugs are essential for suppressing COVID-19. Although several inhibitor candidates were reported for SARS-CoV-2 main protease, most are highly polar peptidomimetics with poor oral bioavailability and cell membrane permeability. Here, we conducted structure-based virtual screening and in vitro assays to obtain hit compounds belonging to a new chemical space, excluding peptidyl secondary amides. In total, 180 compounds were subjected to the primary assay at 20 μM, and nine compounds with inhibition rates of >5% were obtained. The IC50 of six compounds was determined in dose–response experiments, with the values on the order of 10–4 M. Although nitro groups were enriched in the substructure of the hit compounds, they did not significantly contribute to the binding interaction in the predicted docking poses. Physicochemical properties prediction showed good oral absorption. These new scaffolds are promising candidates for future optimization.