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.     

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.     

Inhibitory effects of baicalein against herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is a ubiquitous and widespread human pathogen, which gives rise to a range of diseases, including cold sores, corneal blindness, and encephalitis. Currently, the use of nucleoside analogs, such as acyclovir and penciclovir, in treating HSV-1 infection often presents limitation due to their side effects and low efficacy for drug-resistance strains. Therefore, new anti-herpetic drugs and strategies should be urgently developed. Here, we reported that baicalein, a naturally derived compound widely used in Asian countries, strongly inhibited HSV-1 replication in several models. Baicalein was effective against the replication of both HSV-1/F and HSV-1/Blue (an acyclovir-resistant strain) in vitro. In the ocular inoculation mice model, baicalein markedly reduced in vivo HSV-1/F replication, receded inflammatory storm and attenuated histological changes in the cornea. Consistently, baicalein was found to reduce the mortality of mice, viral loads both in nose and trigeminal ganglia in HSV-1 intranasal infection model. Moreover, an ex vivo HSV-1-EGFP infection model established in isolated murine epidermal sheets confirmed that baicalein suppressed HSV-1 replication. Further investigations unraveled that dual mechanisms, inactivating viral particles and inhibiting IκB kinase beta (IKK-β) phosphorylation, were involved in the anti-HSV-1 effect of baicalein. Collectively, our findings identified baicalein as a promising therapy candidate against the infection of HSV-1, especially acyclovir-resistant strain.     

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.     

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.     

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.     

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.     

A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer

The immune checkpoint blockade therapy has profoundly revolutionized the field of cancer immunotherapy. However, despite great promise for a variety of cancers, the efficacy of immune checkpoint inhibitors is still low in colorectal cancer (CRC). This is mainly due to the immunosuppressive feature of the tumor microenvironment (TME). Emerging evidence reveals that certain chemotherapeutic drugs induce immunogenic cell death (ICD), demonstrating great potential for remodeling the immunosuppressive TME. In this study, the potential of ginsenoside Rg3 (Rg3) as an ICD inducer against CRC cells was confirmed using in vitro and in vivo experimental approaches. The ICD efficacy of Rg3 could be significantly enhanced by quercetin (QTN) that elicited reactive oxygen species (ROS). To ameliorate in vivo delivery barriers associated with chemotherapeutic drugs, a folate (FA)-targeted polyethylene glycol (PEG)-modified amphiphilic cyclodextrin nanoparticle (NP) was developed for co-encapsulation of Rg3 and QTN. The resultant nanoformulation (CD-PEG-FA.Rg3.QTN) significantly prolonged blood circulation and enhanced tumor targeting in an orthotopic CRC mouse model, resulting in the conversion of immunosuppressive TME. Furthermore, the CD-PEG-FA.Rg3.QTN achieved significantly longer survival of animals in combination with Anti-PD-L1. The study provides a promising strategy for the treatment of CRC.     

The natural compound Cirsitakaoside enhances antiviral innate responses against vesicular stomatitis virus in vitro and in vivo

Cirsitakaoside, isolated and purified from the stems and leaves of Premna szemaoensis and Macaranga denticulata, is a natural compound with potential anti-inflammatory effects. However, the role of Cirsitakaoside in antiviral activity and the underlying mechanism remains largely unknown. In this study, we aimed to identify whether Cirsitakaoside has antiviral activity and investigated the underlying mechanisms. Mouse peritoneal macrophages were pretreated with Cir or DMSO, and then infected by Vesicular Stomatitis Virus (VSV) for indicated hours, Q-PCR and ELISA were used to detect the expression of interferons and pro-inflammatory cytokines, immunoblot assay were employed to investigate the involved signaling pathway in the antiviral effects of Cirsitakaoside. Furthermore, mice infected with VSV were used to investigate the antiviral activities of Cirsitakaoside in vivo. Our study demonstrated that Cirsitakaoside could promote type I IFN expression and inhibit pro-inflammatory cytokines such as IL-6 and TNF-α production in mouse peritoneal macrophages infected by VSV. Suppressive viral replication effects of Cirsitakaoside were observed on VSV-infected mouse peritoneal macrophages as well. Furthermore, Cirsitakaoside significantly increased the VSV-triggered phosphorylation of TBK1, IRF3 and reduced the phosphorylation of IκBα and p65 in mouse peritoneal macrophages. in vivo, the results showed that Cirsitakaoside-treated mice were more resistant to VSV infection by producing more IFN-β and less pro-inflammatory cytokines. Our study indicates that Cirsitakaoside is a good candidate for the treatment of viral infection and inflammation-related diseases.     

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.     

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.     

An Integrated Analysis of Solid Form Change Impact on Solubility and Permeability: Case Study of Oral Exposure in Rats of an RAR Related Orphan Receptor C Inhibitor

It is well acknowledged that the oral absorption of a drug can be influenced by its solubility, which is usually associated with its solid form properties. G1032 is a retinoic acid–related orphan receptor inverse agonist. Crystalline solid (form A) was identified with an aqueous solubility of 130 μg/mL. This form was used in an oral dose escalation study in rodents up to 300 mg/kg and achieved good exposures. Later on, a more stable crystalline hydrate (form B) was identified and the aqueous solubility was reduced to 55 μg/mL. A modeling exercise suggested that this solubility change would cause a 2-fold decrease in exposure at tested doses; however, the actual reduction was far larger than the model predicted. At high dose, exposure was found to be reduced by almost 10-fold. A parameter sensitivity analysis suggested that such a drop in exposure could be associated with permeability reduction as well. More in vitro permeability experiments were performed, indicating G1032 was an efflux transporter substrate. This finding was integrated into the modeling and the design for in vivo studies. Data obtained from those studies allowed us to better understand the causes of the higher-than-expected exposure change and enabled decision-making.     

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.     

Cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): therapeutic and pathophysiological implications

The hepatic endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) are mixed-function oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance. P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover. Such P450 proteolytic turnover occurs through a process known as ER-associated degradation (ERAD) that involves ubiquitin-dependent proteasomal degradation (UPD) and/or autophagic-lysosomal degradation (ALD). Herein, on the basis of available literature reports and our own recent findings of in vitro as well as in vivo experimental studies, we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance. We specifically (i) describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibody pathogenesis in drug-induced acute hypersensitivity reactions and liver injury, or viral hepatitis; (ii) discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates; and (iii) detail the pathophysiological consequences of disrupted P450 ERAD, contributing to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) under certain synergistic cellular conditions.     

Impact of Precipitation of Antibody Therapeutics After Subcutaneous Injection on Pharmacokinetics and Immunogenicity

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.     

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.     

The role of extracellular ATP in homeostatic immune cell migration

Antigen stimulation induces adenosine triphosphate (ATP) release from naïve lymphocytes in lymphoid tissues. However, previous studies indicated that the non-lytic release of ATP also occurs in most tissues and cell types under physiological conditions. Here, we show that extracellular ATP (eATP) is indeed constitutively produced by naïve T cells in response to lymphoid chemokines in uninflamed lymph nodes and is involved in the regulation of immune cell migration. In this review, we briefly summarize the homeostatic role of extracellular ATP in immune cell migration in vivo.     

Dual-targeting nanovesicles enhance specificity to dynamic tumor cells in vitro and in vivo via manipulation of αvβ3-ligand binding

The dynamic or flowing tumor cells just as leukemia cells and circulating tumor cells face a microenvironment difference from the solid tumors, and the related targeting nanomedicines are rarely reported. The existence of fluidic shear stress in blood circulation seems not favorable for the binding of ligand modified nanodrugs with their target receptor. Namely, the binding feature is very essential in this case. Herein, we utilized HSPC, PEG-DSPE, cholesterol and two αvβ3 ligands (RGDm7 and DT4) with different binding rates to build dual-targeting nanovesicles, in an effort to achieve a “fast-binding/slow-unbinding” function. It was demonstrated that the dual-targeting nanovesicles actualized efficient cellular uptake and antitumor effect in vitro both for static and dynamic tumor cells. Besides, the potency of the dual-targeting vesicles for flowing tumor cells was better than that for static tumor cells. Then, a tumor metastasis mice model and a leukemia mice model were established to detect the killing ability of the drug-loaded dual-targeting vesicles to dynamic tumor cells in vivo. The therapy efficacy of the dual-targeting system was higher than other controls including single-targeting ones. Generally, it seems possible to strengthen drug-targeting to dynamic tumor cells via the control of ligand–receptor interaction.