Neutralization of SARS-CoV-2 pseudovirus using ACE2-engineered extracellular vesicles

The spread of coronavirus disease 2019 (COVID-19) throughout the world has resulted in stressful healthcare burdens and global health crises. Developing an effective measure to protect people from infection is an urgent need. The blockage of interaction between angiotensin-converting enzyme 2 (ACE2) and S protein is considered an essential target for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs. A full-length ACE2 protein could be a potential drug to block early entry of SARS-CoV-2 into host cells. In this study, a therapeutic strategy was developed by using extracellular vesicles (EVs) with decoy receptor ACE2 for neutralization of SARS-CoV-2. The EVs embedded with engineered ACE2 (EVs-ACE2) were prepared; the EVs-ACE2 were derived from an engineered cell line with stable ACE2 expression. The potential effect of the EVs-ACE2 on anti-SARS-CoV-2 was demonstrated by both in vitro and in vivo neutralization experiments using the pseudovirus with the S protein (S-pseudovirus). EVs-ACE2 can inhibit the infection of S-pseudovirus in various cells, and importantly, the mice treated with intranasal administration of EVs-ACE2 can suppress the entry of S-pseudovirus into the mucosal epithelium. Therefore, the intranasal EVs-ACE2 could be a preventive medicine to protect from SARS-CoV-2 infection. This EVs-based strategy offers a potential route to COVID-19 drug development.     

In Vitro-In Vivo Extrapolation and Hepatic Clearance-Dependent Underprediction

Accurately predicting the hepatic clearance of compounds using in vitro to in vivo extrapolation (IVIVE) is crucial within the pharmaceutical industry. However, several groups have recently highlighted the serious error in the process. Although empirical or regression-based scaling factors may be used to mitigate the common underprediction, they provide unsatisfying solutions because the reasoning behind the underlying error has yet to be determined. One previously noted trend was intrinsic clearance-dependent underprediction, highlighting the limitations of current in vitro systems. When applying these generated in vitro intrinsic clearance values during drug development and making first-in-human dose predictions for new chemical entities though, hepatic clearance is the parameter that must be estimated using a model of hepatic disposition, such as the well-stirred model. Here, we examine error across hepatic clearance ranges and find a similar hepatic clearance-dependent trend, with high clearance compounds not predicted to be so, demonstrating another gap in the field.     

Evodiamine-inspired dual inhibitors of histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) with potent antitumor activity

A great challenge in multi-targeting drug discovery is to identify drug-like lead compounds with therapeutic advantages over single target inhibitors and drug combinations. Inspired by our previous efforts in designing antitumor evodiamine derivatives, herein selective histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) dual inhibitors were successfully identified, which showed potent in vitro and in vivo antitumor potency. Particularly, compound 30a was orally active and possessed excellent in vivo antitumor activity in the HCT116 xenograft model (TGI = 75.2%, 150 mg/kg, p.o.) without significant toxicity, which was more potent than HDAC inhibitor vorinostat, TOP inhibitor evodiamine and their combination. Taken together, this study highlights the therapeutic advantages of evodiamine-based HDAC1/TOP2 dual inhibitors and provides valuable leads for the development of novel multi-targeting antitumor agents.     

Simultaneous Analysis of Dissolution and Permeation Profiles of Nanosized and Microsized Formulations of Indomethacin Using the In Vitro Dissolution Absorption System 2

The in vitro dissolution absorption system 2 (IDAS2), a recent invention comprised a conventional dissolution vessel containing 2 permeation chambers with Caco-2 cell monolayers mounted with their apical side facing the dissolution media, permits simultaneous measurement of dissolution and permeation of drugs from intact clinical dosage forms. The objectives of this study were (1) to assess the utility of IDAS2 in the determination of the effect of particle size on in vitro performance of indomethacin and (2) to find out whether the behavior in IDAS2 of 2 indomethacin products differing in particle size is correlated with their in vivo behavior. Indomethacin dissolution and permeation across Caco-2 cell monolayers were simultaneously measured in IDAS2; the dissolution and permeation profiles were simultaneously modeled using a simple two-compartment model. Compared to microsized indomethacin, the nanosized formulation increased the dissolution rate constant by fivefold, whereas moderately increasing the permeation rate constant and the kinetic solubility. As a result, the drug amount permeated across the Caco-2 cell monolayers doubled in the nanosized versus microsized formulation. The in vitro results showed a good correlation with in vivo human oral pharmacokinetic parameters, thus emphasizing the physiological relevance of IDAS2 data in predicting in vivo absorption.     

In vitro and in vivo correlation for lipid-based formulations: Current status and future perspectives

Lipid-based formulations (LBFs) have demonstrated a great potential in enhancing the oral absorption of poorly water-soluble drugs. However, construction of in vitro and in vivo correlations (IVIVCs) for LBFs is quite challenging, owing to a complex in vivo processing of these formulations. In this paper, we start with a brief introduction on the gastrointestinal digestion of lipid/LBFs and its relation to enhanced oral drug absorption; based on the concept of IVIVCs, the current status of in vitro models to establish IVIVCs for LBFs is reviewed, while future perspectives in this field are discussed. In vitro tests, which facilitate the understanding and prediction of the in vivo performance of solid dosage forms, frequently fail to mimic the in vivo processing of LBFs, leading to inconsistent results. In vitro digestion models, which more closely simulate gastrointestinal physiology, are a more promising option. Despite some successes in IVIVC modeling, the accuracy and consistency of these models are yet to be validated, particularly for human data. A reliable IVIVC model can not only reduce the risk, time, and cost of formulation development but can also contribute to the formulation design and optimization, thus promoting the clinical translation of LBFs.     

Identification of a novel PAK1 inhibitor to treat pancreatic cancer

Pancreatic cancer is one of the most aggressive cancers with poor prognosis and a low 5-year survival rate. The family of P21-activated kinases (PAKs) appears to modulate many signaling pathways that contribute to pancreatic carcinogenesis. In this work, we demonstrated that PAK1 is a critical regulator in pancreatic cancer cell growth. PAK1-targeted inhibition is therefore a new potential therapeutic strategy for pancreatic cancer. Our small molecule screening identified a relatively specific PAK1-targeted inhibitor, CP734. Pharmacological and biochemical studies indicated that CP734 targets residue V342 of PAK1 to inhibit its ATPase activity. Further in vitro and in vivo studies elucidated that CP734 suppresses pancreatic tumor growth through depleting PAK1 kinase activity and its downstream signaling pathways. Little toxicity of CP734 was observed in murine models. Combined with gemcitabine or 5-fluorouracil, CP734 also showed synergistic effects on the anti-proliferation of pancreatic cancer cells. All these favorable results indicated that CP734 is a new potential therapeutic candidate for pancreatic cancer.     

Discovery of nitazoxanide-based derivatives as autophagy activators for the treatment of Alzheimer's disease

Drug repurposing is an efficient strategy for new drug discovery. Our latest study found that nitazoxanide (NTZ), an approved anti-parasite drug, was an autophagy activator and could alleviate the symptom of Alzheimer's disease (AD). In order to further improve the efficacy and discover new chemical entities, a series of NTZ-based derivatives were designed, synthesized, and evaluated as autophagy activator against AD. All compounds were screened by the inhibition of phosphorylation of p70S6K, which was the direct substrate of mammalian target of rapamycin (mTOR) and its phosphorylation level could reflect the mTOR-dependent autophagy level. Among these analogs, compound 22 exhibited excellent potency in promoting β-amyloid (Aβ) clearance, inhibiting tau phosphorylation, as well as stimulating autophagy both in vitro and in vivo. What's more, 22 could effectively improve the memory and cognitive impairments in APP/PS1 transgenic AD model mice. These results demonstrated that 22 was a potential candidate for the treatment of AD.     

Evaluation of a 3D Human Artificial Lymph Node as Test Model for the Assessment of Immunogenicity of Protein Aggregates

The immunogenicity of protein aggregates has been investigated in numerous studies. Nevertheless, it is still unknown which kind of protein aggregates enhance immunogenicity the most. The ability of the currently used in vitro and in vivo systems regarding their predictability of immunogenicity in humans is often questionable, and results are partially contradictive. In this study, we used a 2D in vitro assay and a complex 3D human artificial lymph node model to predict the immunogenicity of protein aggregates of bevacizumab and adalimumab. The monoclonal antibodies were exposed to different stress conditions such as light, heat, and mechanical stress to trigger the formation of protein aggregates and particles, and samples were analyzed thoroughly. Cells and culture supernatants were harvested and analyzed for dendritic cell marker and cytokines. Our study in the artificial lymph node model revealed that bevacizumab after exposure to heat triggered a TH1- and proinflammatory immune response, whereas no trend of immune responses was seen for adalimumab after exposure to different stress conditions. The human artificial lymph node model represents a new test model for testing the immunogenicity of protein aggregates combining the relevance of a 3D human system with the rather easy handling of an in vitro setup.     

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K_d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.     

Biomimetic carbon nanotubes for neurological disease therapeutics as inherent medication

Nowadays, nanotechnology is revolutionizing the approaches to different fields from manufacture to health. Carbon nanotubes (CNTs) as promising candidates in nanomedicine have great potentials in developing novel entities for central nervous system pathologies, due to their excellent physicochemical properties and ability to interface with neurons and neuronal circuits. However, most of the studies mainly focused on the drug delivery and bioimaging applications of CNTs, while neglect their application prospects as therapeutic drugs themselves. At present, the relevant reviews are not available yet. Herein we summarized the latest advances on the biomedical and therapeutic applications of CNTs in vitro and in vivo for neurological diseases treatments as inherent therapeutic drugs. The biological mechanisms of CNTs-mediated bio-medical effects and potential toxicity of CNTs were also intensely discussed. It is expected that CNTs will exploit further neurological applications on disease therapy in the near future.     

Recent developments in the medicinal chemistry of single boron atom-containing compounds

Various boron-containing drugs have been approved for clinical use over the past two decades, and more are currently in clinical trials. The increasing interest in boron-containing compounds is due to their unique binding properties to biological targets; for example, boron substitution can be used to modulate biological activity, pharmacokinetic properties, and drug resistance. In this perspective, we aim to comprehensively review the current status of boron compounds in drug discovery, focusing especially on progress from 2015 to December 2020. We classify these compounds into groups showing anticancer, antibacterial, antiviral, antiparasitic and other activities, and discuss the biological targets associated with each activity, as well as potential future developments.     

Evaluation of Prediction Accuracy for Volume of Distribution in Rat and Human Using In Vitro,In Vivo,PBPK and QSAR Methods

Volume of distribution at steady state (V_ss) is an important pharmacokinetic parameter of a drug candidate. In this study, V_ss prediction accuracy was evaluated by using: (1) seven methods for rat with 56 compounds, (2) four methods for human with 1276 compounds, and (3) four in vivo methods and three Kp (partition coefficient) scalar methods from scaling of three preclinical species with 125 compounds. The results showed that the global QSAR models outperformed the PBPK methods. Tissue fraction unbound (f_u,t) method with adipose and muscle also provided high V_ss prediction accuracy. Overall, the high performing methods for human V_ss prediction are the global QSAR models, Øie-Tozer and equivalency methods from scaling of preclinical species, as well as PBPK methods with Kp scalar from preclinical species. Certain input parameter ranges rendered PBPK models inaccurate due to mass balance issues. These were addressed using appropriate theoretical limit checks. Prediction accuracy of tissue Kp were also examined. The f_u,t method predicted Kp values more accurately than the PBPK methods for adipose, heart and muscle. All the methods overpredicted brain Kp and underpredicted liver Kp due to transporter effects. Successful V_ss prediction involves strategic integration of in silico, in vitro and in vivo approaches.     

Delivery strategies for macromolecular drugs in cancer therapy

With the development of biotherapy, biomacromolecular drugs have gained tremendous attention recently, especially in drug development field due to the sophisticated functions in vivo. Over the past few years, a motley variety of drug delivery strategies have been developed for biomacromolecular drugs to overcome the difficulties in the druggability, e.g., the instability and easily restricted by physiologic barriers. The application of novel delivery systems to deliver biomacromolecular drugs can usually prolong the half-life, increase the bioavailability, or improve patient compliance, which greatly improves the efficacy and potentiality for clinical use of biomacromolecular drugs. In this review, recent studies regarding the drug delivery strategies for macromolecular drugs in cancer therapy are summarized, mainly drawing on the development over the last five years.     

In vitro and in vivo characterization of erythrosin B and derivatives against Zika virus

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B?NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure?activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B?NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo.     

Curative efficacy of purified serine protease inhibitor PTF3 from potato tuber in experimental visceral leishmaniasis

To overcome the drug toxicity and frequent resistance of parasites against the conventional drugs for the healing of human visceral leishmaniasis, innovative plant derived antileishmanial components are very imperative. Fuelled by the complications of clinically available antileishmanial drugs, a novel potato serine protease inhibitor was identified with its efficacy on experimental visceral leishmaniasis (VL). The serine protease inhibitors from potato tuber extract (PTEx) bearing molecular mass of 39 kDa (PTF1), 23 kDa (PTF2) and 17 kDa (PTF3) were purified and identified. Among them, PTF3 was selected as the most active inhibitor (IC₅₀ 143.5 ± 2.4 µg/ml) regarding its antileishmanial property. Again, intracellular amastigote load was reduced upto 83.1 ± 1.7% in pre-treated parasite and 88.5 ± 0.5% in in vivo model with effective dose of PTF3. Protective immune response by PTF3 was noted with increased production of antimicrobial substances and up-regulation of pro-inflammatory cytokines. Therapeutic potency of PTF3 is also followed by 80% survival in infected hamster. The peptide mass fingerprint (MALDI-TOF) results showed similarity of PTF3 with serine protease inhibitors database. Altogether, these results strongly propose the effectiveness of PTF3 as potent immunomodulatory therapeutics for controlling VL.     

Investigating a Modified Apparatus to Discriminate the Dissolution Capacity In Vitro and Establish an IVIVC of Mycophenolate Mofetil Tablets in the Fed State

In this study, a modified dissolution apparatus was developed by equipping a USP apparatus Ⅰ with an open-loop system to discriminate the dissolution capacity in vitro and establish an in vitro and in vivo correlation (IVIVC) for mycophenolate mofetil (MMF) tablets. MMF had strong pH-dependent solubility that could influence the dissolution rate in vivo after the meal. Dissolution tests involving reference (Cellcept®) and test formulations (F1 and F2) were conducted using pH 4.5 acetate buffer to simulate gastric fluids in the fed state. The dissolution profiles of the reference and test formulations were distinguished by using the modified dissolution apparatus and compared with those determined using the USP apparatuses Ⅱ and Ⅳ, and the dissolution capacities of the formulations were discriminated at different sampling time-points. The results of human bioequivalence (BE) studies in the fed state were consistent with in vitro evaluations that the maximum concentrations (C_{max, in vivo}) of both F1 and F2 fell below the acceptable range (80.00%). A level A IVIVC between the absorption fraction in vivo and dissolution in vitro, and a level C correlation between C_{max, in vivo} and C_{max, in vitro}, were established to guide the optimization of the tablet formulation containing MMF.     

Opportunities for overcoming tuberculosis: Emerging targets and their inhibitors

Tuberculosis (TB), an airborne infectious disease mainly caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of human morbidity and mortality worldwide. Given the alarming rise of resistance to anti-TB drugs and latent TB infection (LTBI), new targets and novel bioactive compounds are urgently needed for the treatment of this disease. We provide an overview of the recent advances in anti-TB drug discovery, emphasizing several newly validated targets for which an inhibitor has been reported in the past five years. Our review presents several attractive directions that have potential for the development of next-generation therapies.     

Bioresponsive immune-booster-based prodrug nanogel for cancer immunotherapy

The combination of chemotherapy and immunotherapy motivates a potent immune system by triggering immunogenic cell death (ICD), showing great potential in inhibiting tumor growth and improving the immunosuppressive tumor microenvironment (ITM). However, the therapeutic effectiveness has been restricted by inferior drug bioavailability. Herein, we reported a universal bioresponsive doxorubicin (DOX)-based nanogel to achieve tumor-specific co-delivery of drugs. DOX-based mannose nanogels (DM NGs) was designed and choosed as an example to elucidate the mechanism of combined chemo-immunotherapy. As expected, the DM NGs exhibited prominent micellar stability, selective drug release and prolonged survival time, benefited from the enhanced tumor permeability and prolonged blood circulation. We discovered that the DOX delivered by DM NGs could induce powerful anti-tumor immune response facilitated by promoting ICD. Meanwhile, the released mannose from DM NGs was proved as a powerful and synergetic treatment for breast cancer in vitro and in vivo, via damaging the glucose metabolism in glycolysis and the tricarboxylic acid cycle. Overall, the regulation of tumor microenvironment with DOX-based nanogel is expected to be an effectual candidate strategy to overcome the current limitations of ICD-based immunotherapy, offering a paradigm for the exploitation of immunomodulatory nanomedicines.