Subsequent Internalization Research Articles

Receptors and Host Factors for Enterovirus Infection: Implications for Cancer Therapy.

Enteroviruses, with their diverse clinical manifestations ranging from mild or asymptomatic infections to severe diseases such as poliomyelitis and viral myocarditis, present a public health threat. However, they can also be used as oncolytic agents. This review shows the intricate relationship between enteroviruses and host cell factors. Enteroviruses utilize specific receptors and coreceptors for cell entry that are critical for infection and subsequent viral replication. These receptors, many of which are glycoproteins, facilitate virus binding, capsid destabilization, and internalization into cells, and their expression defines virus tropism towards various types of cells. Since enteroviruses can exploit different receptors, they have high oncolytic potential for personalized cancer therapy, as exemplified by the antitumor activity of certain enterovirus strains including the bioselected non-pathogenic Echovirus type 7/Rigvir, approved for melanoma treatment. Dissecting the roles of individual receptors in the entry of enteroviruses can provide valuable insights into their potential in cancer therapy. This review discusses the application of gene-targeting techniques such as CRISPR/Cas9 technology to investigate the impact of the loss of a particular receptor on the attachment of the virus and its subsequent internalization. It also summarizes the data on their expression in various types of cancer. By understanding how enteroviruses interact with specific cellular receptors, researchers can develop more effective regimens of treatment, offering hope for more targeted and efficient therapeutic strategies.

Design and synthesis of TH19P01-Camptothecin based hybrid peptides inducing effective anticancer responses on sortilin positive cancer cells

Recently, the sortilin receptor (SORT1) was found to be preferentially over-expressed on the surface of many cancer cells, which makes SORT1 a novel anticancer target. The SORT1 binding proprietary peptide TH19P01 could achieve the SORT1-mediated cancer cell binding and subsequent internalization. Inspired by the peptide-drug conjugate (PDC) strategy, the TH19P01-camptothecin (CPT) conjugates were designed, efficiently synthesized, and evaluated for their anticancer potential in this study. The water solubility, in vitro anticancer activity, time-kill kinetics, cellular uptake, anti-migration activity, and hemolysis effects were systematically estimated. Besides, in order to monitor the release of CPT from conjugates in real-time, the CPT/Dnp-based “turn on” hybrid peptide was designed, which indicted that CPT could be sustainably released from the hybrid peptide in both human serum and cancer cellular environments. Strikingly, compared with free CPT, the water solubility, cellular uptake, and selectivity towards cancer cells of hybrid peptide LYJ-2 have all been significantly enhanced. Moreover, unlike free CPT or TH19P01, LYJ-2 exhibited selective anti-proliferative and anti-migration effects against SORT1-positive MDA-MB-231 cells. Collectively, this study not only established efficient strategies to improve the solubility and anticancer potential of chemotherapeutic agent CPT, but also provided important references for the future development of TH19P01 based PDCs targeting SORT1.

Antimicrobial Activity of Green Synthesized Silver and Copper Oxide Nanoparticles against the Foodborne Pathogen Campylobacter jejuni.

Campylobacter jejuni is a major cause of global foodborne illnesses. To develop alternative antimicrobial strategies against C. jejuni, this study designed and optimized the green synthesis of metallic nanoparticles (NPs) with intracellular components of the medicinal fungus Ganoderma sessile to provide the needed reducing and stabilizing agents. NPs were characterized by transmission electron microscopy and dynamic light scattering, and the quasi-spherical NPs had sizes of 2.9 ± 0.9 nm for the copper oxide NPs and 14.7 ± 0.6 nm for the silver NPs. Surface charge assessment revealed zeta potentials of -21.0 ± 6.5 mV and -24.4 ± 7.9 mV for the copper oxide and silver NPs, respectively. The growth inhibition of C. jejuni by the NPs occurred through attachment to the outer cell membrane and subsequent intracellular internalization and resulted in minimum inhibitory concentrations of the silver NPs at 6 µg/mL and copper oxide NPs at 10 µg/mL. On the other hand, a differential ROS production caused by silver and copper NPs was observed. In summary, this research presents the first demonstration of using green synthesis with the medicinal fungus G. sessile to produce metallic NPs that effectively inhibit C. jejuni growth, providing a sustainable and effective approach to the traditional use of antimicrobials.

Tagraxofusp, a first-in-class CD123-targeted agent: Five-year postapproval comprehensive review of the literature.

Tagraxofusp is a first-in-class CD123-directed conjugate of an amended diphtheria toxin platform and recombinant interleukin 3. Binding and subsequent internalization of the drug result in cell death via disruption of intracellular protein synthesis. CD123 is a surface marker that is expressed in several hematological malignancies, especially blastic plasmacytoid dendritic cell neoplasm (BPDCN), where its expression is ubiquitous. A pivotal study of tagraxofusp in BPDCN resulted in its approval for the treatment of BPDCN, the first treatment approved for this indication. Since the introduction of tagraxofusp, research has focused on the management of adverse effects, combination therapy to improve outcomes in fit patients, and dosing and combination strategies to mitigate toxicities while preserving efficacy, especially among older patients. The successful targeting of CD123 in BPDCN has also encouraged research into a variety of other CD123-positive hematological neoplasms, including acute myeloid leukemia (AML), and informed the development of other novel agents targeting CD123. This review examines the clinical data leading to the development and approval of tagraxofusp in BPDCN, how it is being used in combination to improve outcomes in BPDCN and AML, and its developing role in other hematological malignancies.

Abstract 1858: Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload

Abstract ROR1 is a pseudo kinase that is involved in the non-canonical Wnt signaling pathway and is highly expressed during early embryonic development. In adult tissues, ROR1 is expressed at very low levels but its increased expression is observed in hematological cancers and a broad range of solid tumors. AT86474 is a highly homogeneous antibody drug conjugate incorporating AxcynCYSTM technology that allows reproducible site-specific conjugation with DAR4 composition greater than 97%. AT86474 utilizes a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and with altered PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across multiple cancer cell lines, including those resistant to paclitaxel, DM1, MMAE and DXd. The payload is conjugated via an enzyme-cleavable linker optimized for solubility, stability, and high drug to antibody ratios. AT86474 uses a humanized and optimized monoclonal antibody that demonstrates selective binding to ROR1-positive cells and subsequent internalization, sub-nanomolar potency in vitro and strong bystander effects. AT86474 was evaluated in a preclinical CDX mouse model of ovarian cancer, where substantial tumor growth inhibition was observed with two weekly doses at as low as 1 mg/kg. As ROR1 over-expression is also observed in many solid tumors, such as pancreatic, gastric, breast and lung cancers, the use of AT86474 can be expanded to benefit a large patient population. Citation Format: Ban Xiong Tan, Xi Yun Zhang, You Xue Wu, Yin Wen Cheng, Shao Jun Liu, Ping Du, Chen Zhong, Bin Zou. Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1858.

MCRP-Induced Focal Adhesion Kinase-Dependent Monocyte Aggregation and M1 Polarization, Which Was Partially Blocked by the C10M Inhibitor.

Monomeric C-reactive protein (mCRP) has recently been implicated in the abnormal vascular activation associated with development of atherosclerosis, but it may act more specifically through mechanisms perpetuating damaged vessel inflammation and subsequent aggregation and internalization of resident macrophages. Whilst the direct effects of mCRP on endothelial cells have been characterized, the interaction with blood monocytes has, to our knowledge, not been fully defined. Here we showed that mCRP caused a strong aggregation of both U937 cell line and primary peripheral blood monocytes (PBMs) obtained from healthy donors. Moreover, this increase in clustering was dependent on focal adhesion kinase (FAK) activation (blocked by a specific inhibitor), as was the concomitant adhesive attachment to the plate, which was suggestive of macrophage differentiation. Confocal microscopy confirmed the increased expression and nuclear localization of p-FAK, and cell surface marker expression associated with M1 macrophage polarization (CD11b, CD14, and CD80, as well as iNOS) in the presence of mCRP. Inclusion of a specific CRP dissociation/mCRP inhibitor (C10M) effectively inhibited PBMs clustering, as well as abrogating p-FAK expression, and partially reduced the expression of markers associated with M1 macrophage differentiation. mCRP also increased the secretion of pro-inflammatory cytokines Interleukin-8 (IL-8) and Interleukin-1β (IL-1β), without notably affecting MAP kinase signaling pathways; inclusion of C10M did not perturb or modify these effects. In conclusion, mCRP modulates PBMs through a mechanism that involves FAK and results in cell clustering and adhesion concomitant with changes consistent with M1 phenotypical polarization. C10M has potential therapeutic utility in blocking the primary interaction of mCRP with the cells-for example, by protecting against monocyte accumulation and residence at damaged vessels that may be predisposed to plaque development and atherosclerosis.

Abstract P18: Splicing polymorphism rs12459419 modulates antibody-bound internalization of CD33 isoforms

Abstract P18: Splicing polymorphism rs12459419 modulates antibody-bound internalization of CD33 isoforms

Efferocytosis-Inspired Biomimetic Nanoplatform for Targeted Acute Lung Injury Therapy.

Acute lung injury (ALI) is a serious inflammatory disease that causes impairment of pulmonary function. Phenotypic modulation of macrophage in the lung using fibroblast growth factor 21 (FGF21) may be a potential strategy to alleviate lung inflammation. Consequently, achieving specific delivery of FGF21 to the inflamed lung and subsequent efficient FGF21 internalization by macrophages within the lung becomes critical for effective ALI treatment. Here, an apoptotic cell membrane-coated zirconium-based metal-organic framework UiO-66 is reported for precise pulmonary delivery of FGF21 (ACM@U-FGF21) whose design is inspired by the process of efferocytosis. ACM@U-FGF21 with apoptotic signals is recognized and internalized by phagocytes in the blood and macrophages in the lung, and then the intracellular ACM@U-FGF21 can inhibit the excessive secretion of pro-inflammatory cytokines by these cells to relieve the inflammation. Utilizing the homologous targeting properties inherited from the source cells and the spontaneous recruitment of immune cells to inflammatory sites, ACM@U-FGF21 can accumulate preferentially in the lung after injection. The results prove that ACM@U-FGF21 effectively reduces inflammatory damage to the lung by modulating lung macrophage polarization and suppressing the excessive secretion of pro-inflammatory cytokines by activated immune cells. This study demonstrates the usefulness of efferocytosis-inspired ACM@U-FGF21 in the treatment of ALI.

H2S-Releasing Versatile Montmorillonite Nanoformulation Trilogically Renovates the Gut Microenvironment for Inflammatory Bowel Disease Modulation.

Abnormal activation of the intestinal mucosal immune system, resulting from damage to the intestinal mucosal barrier and extensive invasion by pathogens, contributes to the pathogenesis of inflammatory bowel disease (IBD). Current first-line treatments for IBD have limited efficacy and significant side effects. An innovative H2S-releasing montmorillonite nanoformulation (DPs@MMT) capable of remodeling intestinal mucosal immune homeostasis, repairing the mucosal barrier, and modulating gut microbiota is developed by electrostatically adsorbing diallyl trisulfide-loaded peptide dendrimer nanogels (DATS@PDNs, abbreviated as DPs) onto the montmorillonite (MMT) surface. Upon rectal administration, DPs@MMT specifically binds to and covers the damaged mucosa, promoting the accumulation and subsequent internalization of DPs by activated immune cells in the IBD site. DPs release H2S intracellularly in response to glutathione, initiating multiple therapeutic effects. In vitro and in vivo studies have shown that DPs@MMT effectively alleviates colitis by eliminating reactive oxygen species (ROS), inhibiting inflammation, repairing the mucosal barrier, and eradicating pathogens. RNA sequencing revealed that DPs@MMT exerts significant immunoregulatory and mucosal barrier repair effects, by activating pathways such as Nrf2/HO-1, PI3K-AKT, and RAS/MAPK/AP-1, and inhibiting the p38/ERK MAPK, p65 NF-κB, and JAK-STAT3 pathways, as well as glycolysis. 16S rRNA sequencing demonstrated that DPs@MMT remodels the gut microbiota by eliminating pathogens and increasing probiotics. This study develops a promising nanoformulation for IBD management.

Visualizing the cellular internalization of therapeutic antibodies via pH-sensitive release of AIEgen.

Among the fastest-growing bio-pharmaceuticals, therapeutic antibodies have achieved unprecedented success in treating various diseases. Though powerful, issues such as inefficacy or acquired resistance are waiting to be addressed to benefit more patients with improved therapeutic outcomes. In addition to in vivo distribution, the cellular spatiotemporal information including the antibody-antigen interaction and subsequent internalization is found to be important for the therapeutic effects. To better understand the cellular fate of therapeutic antibodies, especially the cellular internalization process, we employed a pH-sensitive linker to attach a red-emissive AIEgen onto the antibody. The resulting antibody conjugate will undergo AIEgen release to liberate brilliant fluorescence inside acidic endo/lysosomes, allowing wash-free visualization of the internalization process and facilitating the evaluation of antibody-drug efficacy.

Splicing Polymorphism rs12459419 Modulates Antibody-Bound Internalization of CD33 Isoforms

Background: While the standard regimen for AML has been intense chemotherapy followed by stem-cell transplantation (SCT), addition of immunotherapeutics such as gemtuzumab ozogamicin (GO) to this has shown to improve outcomes in multiple clinical trials. Since these trials, GO has been added to the National Comprehensive Cancer Network (NCCN) practice guidelines for use in AML therapy (NCCN., 2018). GO is a humanized monoclonal antibody conjugated to calicheamicin and targets the cell surface antigen CD33, observed on most AML blasts. Upon binding to GO, CD33 is internalized and the subsequent release of calicheamicin mediates cytotoxicity. CD33 contains two extracellular domains: IgV and IgC, the IgV domain is coded by CD33 exon 2 and is the binding site for GO. A splicing SNP, rs12459419 (C>T; A14V) in exon 2 results in a shorter isoform (CD33-D2) and has been previously associated with CD33 abundance on cell surface as well as clinical benefit from GO addition to standard chemotherapy. Patients with CC genotype (~50% of the cohort) expressed full length CD33 (CD33-FL) and benefited significantly from GO. However, heterozygous CT and homozygous TT genotype groups did not derive the same benefit. These results were perplexing as CT genotype group had intermediate levels of CD33-FL and CD33-D2 isoforms (Lamba et al., 2017). It is hypothesized that lack of IgV domain in CD33-D2 and putative CD33-FL/CD33-D2 dimerization prevents GO binding and subsequent internalization in rs12459419 CT/TT genotype cells, however this has never been tested. We have generated novel antibodies directed against the IgC domain that recognizes CD33-D2 in AML cell lines as well as primary AML cells (Gbadamosi et al., 2021) thus facilitating testing the above indicated hypothesis. Objective: In this study, we first tested the ability of antibody-bound CD33-D2 to internalize, followed by impact of the internalization status of CD33-FL and CD33-D2 in cells co-expressing both isoforms along with the concomitant impact on GO induced cytotoxicity. Methods: Using pMXs retroviral particles encoding CD33-FL or CD33-D2, we engineered CRISPR/Cas9 mediated CD33 KO in AML cell lines to overexpress either one or both CD33 isoforms to mimic cells expression varying proportions of CD33-FL/CD33-D2. Internalization of either CD33-FL or CD33-D2 over a duration of 4 hours in the presence of antibodies recognizing either of the two isoforms was performed. Cells were labeled with unconjugated mouse anti-CD33-FL P67.6 or anti-CD33-D2 HL2541 antibody on ice and incubated at 37°C for 0-4 hours, followed by staining with mouse anti-mouse IgG antibody conjugated with biotin. Surface CD33-FL or CD33-D2 was then detected using streptavidin-FITC conjugate. Percentage antibody-bound CD33-FL or CD33-D2 internalization was expressed as change in FITC MFI between each time point incubated and cells kept at 0°C post CD33 antibody labeling. Results: While P67.6-bound-CD33-FL internalized as expected in AML cell lines expressing CD33-FL only (Fig. 1A), we show for the first time that HL2541-bound-CD33-D2 also internalizes upon antibody binding in cell lines expressing CD33-D2 isoform only (Fig. 1B). In AML cell lines co-expressing both CD33-FL and CD33-D2 isoforms, a >2.5-3 fold decrease in this antibody-bound internalization was observed for each isoform (Figs. 1C and D). In response to GO exposure for 48 hours, cells co-expressing both CD33-FL and CD33-D2 isoforms had 7-9.5-fold higher IC50 values (233 nmol and 277 nmol) compared to cells expressing only CD33-FL (33.19 nmol and 29 nmol) (Figs. 1E and F). As expected, cells expressing CD33-D2 only did respond to GO. Discussion: Our finding that antibody-bound CD33-D2 internalizes like CD33-FL adds a novel piece of evidence in understanding the biology of this variant isoform and opens opportunities to develop antibody drug conjugate (ADC) such as GO using CD33-D2 antibody. This new CD33-D2 targeting ADC will provide a treatment option in patients with TT genotypes. Our results also provide a mechanistic insight on the previous clinical observation showing lack of benefit from GO in patients heterozygous for the splicing SNP. Our ongoing studies are focused on validating this result using live cell imaging and efforts towards design of new ADC or T cell engagers using CD33-D2 antibody. The study was supported by Leukemia Lymphoma Society, UFHealth Cancer Center, and the Frank A. Duckworth Endowment.

TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization.

Blood-brain barrier (BBB) disruption is a serious pathological consequence of traumatic brain injury (TBI), for which there are limited therapeutic strategies. Tissue inhibitor of metalloproteinase-2 (TIMP2), a molecule with dual functions of inhibiting MMP activity and displaying cytokine-like activity through receptor binding, has been reported to inhibit VEGF-induced vascular hyperpermeability. Here, we investigate the ability of TIMP2 to ameliorate BBB disruption in TBI and the underlying molecular mechanisms. Both TIMP2 and AlaTIMP2, a TIMP2 mutant without MMP-inhibiting activity, attenuated neurological deficits and BBB leakage in TBI mice; they also inhibited junctional protein degradation and translocation to reduce paracellular permeability in human brain microvascular endothelial cells (ECs) exposed to hypoxic plus inflammatory insult. Mechanistic studies revealed that TIMP2 interacted with α3β1 integrin on ECs, inhibiting Src activation-dependent VE-cadherin phosphorylation, VE-cadherin/catenin complex destabilization, and subsequent VE-cadherin internalization. Notably, localization of VE-cadherin on the membrane was critical for TIMP2-mediated EC barrier integrity. Furthermore, TIMP2-mediated increased membrane localization of VE-cadherin enhanced the level of active Rac1, thereby inhibiting stress fiber formation. All together, our studies have identified an MMP-independent mechanism by which TIMP2 regulates EC barrier integrity after TBI. TIMP2 may be a therapeutic agent for TBI and other neurological disorders involving BBB breakdown.

Exposure concentration ratios and biological responses play a critical role in determining the joint toxicity of TiO2 nanoparticles and As(V) to the organism: The case study in marine algae Phaeodactylum tricornutum

Environmental risks of manufactured nanomaterials (MNMs) have been widely investigated while the understanding for joint toxicity mechanism of MNMs with other contaminants is still limited. This limitation may be attributed to variations in the concentration ratios of MNMs and co-existing contaminants in the real environment. To better assess the joint toxicity and clarify its underlying mechanisms, this study exposed Phaeodactylum tricornutum to different concentration combinations of nano-sized titanium dioxide (nTiO2) and As(V) at toxic unit (TU) ratios of 1:4,1:1, and 4:1. The results demonstrated that the joint toxicity modes of nTiO2 and As(V) varied with the TU ratios exhibiting synergism for 1:4, partially addition for 1:1, and antagonism for 4:1. Specifically, at low TU ratio of 1:4, the adsorption of As(V) by nTiO2 together with the subsequent internalization of nTiO2 promoted a significant enrichment of As in algae. Simultaneously, the up-regulation of pst (phosphate transporter) genes in charge of the As(V) transport molecular further exacerbated the enrichment of inorganic As in algae, while the down-regulation of ArsM (arsenite S-adenosylmethionine methyltransferases) genes in charge of the As metabolism inhibited As biotransformation from toxic inorganic to nontoxic organic, causing the aggravated accumulation of toxic inorganic As in algae. At higher TU ratios of 1:1 and 4:1, the accumulation of As decreased in algae due to the higher sedimentation of nTiO2 and thus the lower internalization of As-adsorbed nTiO2, as well as the down-regulation of pst genes restricting the transportation of As(V) into algal cells, which jointly accelerated the As biotransformation from toxic inorganic to nontoxic organic. Our results suggest that more attention should be paid to exposure concentration ratios of MNMs and co-existing contaminants and biological responses including bioavailability, bioaccumulation, biotransformation, which would play a critical role in determining the joint toxicity to the organism.

Internalized stigma and self-stigma in people diagnosed with a mental disorder. One concept or two? A scoping review.

In the literature on stigma, authors often use self-stigma or internalized stigma interchangeably to refer to this type of stigma. This results in a lack of conceptual clarity with negative repercussions for measurement and intervention. To identify how internalized stigma and self-stigma are conceptualized in people diagnosed with a mental disorder and establish similarities and differences between both concepts. A scoping review was conducted. Thirty-five studies that conceptualized internalized stigma or self-stigma were selected. It was identified that the concepts are defined from nine components, and there are more conceptualizations that have points in common than those that consider some component of their own. To gain conceptual clarity, the use of the term internalized stigma is recommended, being a process made up of stages: acceptance of stereotypes and prejudices by people with mental disorders and their subsequent internalization. The latter leads to negative consequences for those affected, which can be understood as the personal impact of this process, which has a crucial socio-cultural component. Lines of research are proposed to provide solidity to studies on this type of stigma. The term internalized stigma should be used when referring to the type of stigma that includes acceptance, internalization and personal impact for the subjects of the stigma. In contrast, self-stigma should be reserved to refer to stigma that is directed toward the 'self' and includes subtypes of stigma.

CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway.

Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5β1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of β1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased β1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or β1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and β1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

Learning from anti‐tau antibody clinical trials using computer simulation of spatial tau propagation

AbstractBackgroundBased on the tau spreading hypothesis, anti‐tau antibodies have been developed that can bind to and eliminate the misfolded tau protein during the short extracellular period. However, trials with tau antibodies have come up short in improving clinical outcome in Progressive Supranuclear Palsy (PSP) or Alzheimer’s Disease (AD). It is unclear whether the negative outcomes were due to insufficient target engagement and to what extent the change of tau CSF biomarkers reflected the brain pharmacodynamics.MethodA Quantitative Systems Pharmacology (QSP) computer platform of tau biology has been developed including tau species diversity and their respective concentration, neuronal activity‐dependent tau secretion, tau binding to membrane surface receptors HSPG and LRP1, tau‐antibody binding and subsequent internalization of tau at the synaptic cleft. This model is combined with a Physiology‐Based PharmacoKinetic (PBPK) model to derive brain target exposure of tau antibodies. In addition, a PBPK model is used to simulate the level of plasma and CSF biomarkers.ResultThe platform was calibrated with preclinical in‐vitro cellular assays and after injection of brain extract in Transgene mouse models and was further humanized using published data on seed‐competent tau from postmortem AD brain at different Braak stages. Combining the extracellular antibody exposure, the spatial dimensions and the dynamics of tau secretion, diffusion and binding to the membrane surface receptors, the model suggests that a very limited fraction (10‐20%) of misfolded tau protein can be captured by antibodies and cleared. Even less reduction (5‐10%) is predicted for Progressive Supranuclear Palsy due to the higher affinity of PSP‐4R tau for membrane surface receptors. The reductions in misfolded tau protein are greatest in earlier stages of the disease. With regard to epitope selection, we identify conditions where high specificity (i.e. monomeric vs misfolded tau) is superior to high sensitivity (affinity).ConclusionAdvanced Quantitative Systems Pharmacology computer modeling combining drug exposure dynamics with mechanistic modeling allows to (1) generate testable hypotheses for the outcomes of current therapeutic interventions, (2) support improved clinical trial designs in terms of dosing, patient selection, background amyloid therapy and disease stage and (3) ultimately prioritize targets for addressing tau pathology.

The potential role of scavenger receptor B type I (SR-BI) in SARS-CoV-2 infection.

Scavenger receptor type B I (SR-BI), the major receptor for high-density lipoprotein (HDL) mediates the delivery of cholesterol ester and cholesterol from HDL to the cell membrane. SR-BI is implicated as a receptor for entry of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). SR-BI is colocalized with the angiotensin-converting enzyme 2 (ACE2) increasing the binding and affinity of SARS-CoV-2 to ACE2 with subsequent viral internalization. SR-BI regulates lymphocyte proliferation and the release of pro-inflammatory cytokines from activated macrophages and lymphocytes. SR-BI is reduced during COVID-19 due to consumption by SARS-CoV-2 infection. COVID-19-associated inflammatory changes and high angiotensin II (AngII) might be possible causes of repression of SR-BI in SARS-CoV-2 infection. In conclusion, the downregulation of SR-BI in COVID-19 could be due to direct invasion by SARS-CoV-2 or through upregulation of pro-inflammatory cytokines, inflammatory signaling pathways, and high circulating AngII. Reduction of SR-BI in COVID-19 look like ACE2 may provoke COVID-19 severity through exaggeration of the immune response. Further studies are invoked to clarify the potential role of SR-BI in the pathogenesis of COVID-19 that could be protective rather than detrimental.

Mechanistic incorporation of FcRn binding in plasma and endosomes in a whole body PBPK model for large molecules.

Monoclonal antibodies, endogenous IgG, and serum albumin bind to FcRn in the endosome for salvaging and recycling after pinocytotic uptake, which prolongs their half-life. This mechanism has been broadly recognized and is incorporated in currently available PBPK models. Newer types of large molecules have been designed and developed, which also bind to FcRn in the plasma space for various mechanistic reasons. To incorporate FcRn binding affinity in PBPK models, binding in the plasma space and subsequent internalisation into the endosome needs to be explicitly represented. This study investigates the large molecules model in PK-Sim® and its applicability to molecules with FcRn binding affinity in plasma. With this purpose, simulations of biologicals with and without plasma binding to FcRn were performed with the large molecule model in PK-Sim®. Subsequently, this model was extended to ensure a more mechanistic description of the internalisation of FcRn and the FcRn-drug complexes. Finally, the newly developed model was used in simulations to explore the sensitivity for FcRn binding in the plasma space, and it was fitted to an in vivo dataset of wild-type IgG and FcRn inhibitor plasma concentrations in Tg32 mice. The extended model demonstrated a strongly increased sensitivity of the terminal half-life towards the plasma FcRn binding affinity and could successfully fit the in vivo dataset in Tg32 mice with meaningful parameter estimates.

Ubiquitination of the μ-opioid receptor regulates receptor internalization without affecting Gi/o-mediated intracellular signaling or receptor phosphorylation

Opioids are highly potent analgesics but develop tolerance. Previous studies have focused on phosphorylation of the μ-opioid receptor as it is involved in maintaining cellular sensitivity via desensitization, recycling, and degradation of the activated receptor. Recently, ubiquitination, another form of posttranslational modification has attracted attention in terms of triggering intracellular signaling and regulation of the activated receptor. Here, we generated a ubiquitination-deficient mutant of the μ-opioid receptor to investigate whether ubiquitination is involved in driving Gi/o-mediated analgesic signaling, receptor desensitization or subsequent receptor internalization. Our study shows that the Gi/o pathway and receptor phosphorylation do not require ubiquitination. Instead, ubiquitination regulates the internalization efficiency and might help in promoting internalization of the desensitized MOP.

Mechanistic Insights into Luteolin-Loaded Elastic Liposomes for Transdermal Delivery: HSPiP Predictive Parameters and Instrument-Based Evidence.

We evaluated mechanistic insights into luteolin (LUT)-loaded elastic liposomes (OLEL1) permeated across rat skin. HSPiP software-based parameters, thermal analysis, infrared analysis, and morphological evaluations were employed to understand mechanistic observations of drug permeation and deposition. HSPiP provided HSP values (δd, δp, and δh) of OLEL1 (based on composition), LUT, excipients, and rat skin (literature value and by-default value). Rat skin was studied via Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), fluorescence microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) studies. The δd and δh estimation of the skin and phosphatidylcholine showed close relation in terms of δd and δh. Similarly, OLEL1 and the skin might interact with each other mainly through δd and δp forces as evidenced by the predicted values. The untreated skin showed characteristic stretching and vibrations as compared to lower frequencies caused by OLEL1. DSC showed changes in the thermal behavior of the skin after OLEL1 treatment as compared to the untreated skin. Visualization of these changes was evident under fluorescence microscopy and SEM for confirmed substantial reversible surface perturbation of the skin protein layer for improved vesicle permeation and subsequent internalization with the inner skin matrix. The AFM study confirmed the nanoscale surface roughness variation caused substantially by OLEL1 and OLEL1 placebo as compared to the untreated control and drug solution. Thus, the study clearly demonstrated mechanistic insights into LUT-loaded vesicles across rat skin for enhanced permeation and drug deposition.