Molecular Pharmaceutics

There has been an increasing trend towards subcutaneous (SC) delivery of fusion proteins and monoclonal antibodies (mAbs) in recent years versus intravenous (IV) administration. The prediction of bioavailability is one of the major barriers in clinical translation of SC administered therapeutic proteins due to a lack of reliable in vitro and preclinical in vivo predictive models. In this study, we explored the relationships between human SC bioavailability and physicochemical or pharmacokinetic properties of 20 Fc-or albumin-fusion proteins and 98 monoclonal antibodies. An inverse linear correlation was observed between human SC bioavailability and human intravenous clearance (CL) or isoelectric point (pI). The bioavailability of fusion proteins is more correlated with pI while the bioavailability of mAbs is more correlated with CL. A mAbs with intravenous CL < 4 mL/day/kg is likely to have SC bioavailability > 60%. Multivariate regression models were developed using intravenous CL and pI of a training set (N = 59) as independent variables. The predictive models were validated with an independent test set (N = 33). A linear regression model resulted in 27 among 33 (82%) predictions within 0.8-to 1.2-fold deviations. Overall, this study demonstrated that CL- and pI-based multivariate regression models could be used to predict human SC bioavailability of fusion proteins and mAbs.

BackgroundExtracellular vesicles (EVs) are nano and macro-sized, lipid-bound particles, involved in cellular communication. Interestingly, cancer-derived EVs show a heterologous and cross-species tumour tropism which makes them a potential tool for efficient delivery of therapeutic small interfering RNA (siRNA) to the tumour cells. MethodsEVs derived from glioblastoma cells (U373P and U373vIII) were loaded with EGFRvIII siRNA to develop a targeted therapeutic strategy against glioblastoma. EV biodistribution was evaluated using fluorescent indocyanine green (ICG) staining followed by ex vivo imaging. Different loading strategies, including passive loading, sonication, saponin-mediated membrane permeabilization, electroporation, and transfection were assessed for their efficiency in loading siRNA into EVs. The efficiency of each method was evaluated by nano flowcytometry, in vitro uptake assay followed by immunoblot (western blot) analysis. Eventually, the most effective formulation was tested for the systemic siRNA administration and selective tumour delivery in vivo, followed by evaluation of tumour size and EGFRvIII expression. ResultsHere, we showed that siRNA transfection into EVs was the most effective loading strategy, as confirmed by nano-flow cytometry, uptake assays, and western blot analysis, achieving over 90% knockdown efficiency in vitro for EVs carrying EGFRvIII siRNA. In vivo, EGFRvIII siRNA-loaded EVs homed to the tumour site and downregulated EGFRvIII expression compared with the PBS-siRNA control group; however, no significant tumour shrinkage was observed. ConclusionEGFRvIII-targeting, glioblastoma cell-derived EVs can be used as siRNA delivery carriers for targeted gene therapy in glioblastoma. However, further optimization of siRNA delivery and treatment duration is required.

Antenatal depression, or depression during pregnancy, is a common psychiatric disorder and poses significant risks to both the mother and the fetus. Despite these risks, it is frequently left untreated due to fears of side effects caused by antidepressant medications which cross through the placental barrier. It is therefore desirable to develop formulation strategies to mitigate systemic exposure to relevant drug molecules while maintaining their psychotropic efficacy. In this work, we develop formulations of sertraline, a common antidepressant, to target delivery to the brain through intranasal administration. Formulation engineering enables successful solubilization of sertraline at high concentrations and our lead formulation remains stable at room temperature for months. Using mice, we compare sertraline biodistribution following intranasal administration and standard oral administration. Intranasal administration of our drug product candidate provides comparable brain exposure at half the dose compared to oral treatment and lowers the maximum plasma exposure. These findings suggest that intranasal administration may provide selectivity for drug exposure in the central nervous system over systemic exposure.

INTRODUCTIONDoxil/Caelyx is a PEGylated liposomal formulation of the chemotherapeutic doxorubicin used in the clinic for Kaposis sarcoma, advanced ovarian cancer, progressive multiple myeloma and metastatic breast cancer. Talidox(R), a smaller doxorubicin PEGylated liposome is undergoing clinical trials and has been proposed as an improvement on previous liposomal formulations for the treatment of advanced solid tumors. We aimed to validate an easily translatable radiolabeling method using zirconium-89 (89Zr) that enables quantitative whole-body PET imaging of these formulations to study their biodistribution and pharmacokinetics. METHODS[89Zr][Zr(oxinate)4] was produced using a kit-based approach followed by use as a direct radiolabeling agent of the liposomal formulations. DFT studies were performed to elucidate the mechanism behind the radiolabeling stability observed within the liposomes. Purified 89Zr-labelled Doxil/Talidox(R) liposomes (5 mg/kg doxorubicin dose) were administered in female BALB/c mice bearing 4T1 tumors. PET/CT imaging was acquired at 20 min, 24 h, 48 h, and 72 h, followed by post-mortem biodistribution at 72 h. RESULTS and DISCUSSIONBoth formulations were radiolabeled efficiently with high stability in serum in vitro for 72 h. In vivo, both formulations showed high tumor uptake at 72 h (18.5 {+/-} 2.4 % IA/g for Doxil and 20.2 {+/-} 2.3 % IA/g for Talidox). In general, ex vivo biodistribution showed similar uptake values for both formulations with high spleen/liver uptake and low bone uptake, confirming stability. Talidox(R) showed significantly lower spleen uptake and higher uptake in bone than Doxil. DFT studies confirmed that doxorubicin can form complexes with 89Zr that are more stable than [89Zr][Zr(oxinate)4], explaining the radiolabeling mechanism and stability results in vitro and in vivo. CONCLUSIONSClinically available PEGylated liposomes containing doxorubicin can be efficiently radiolabelled with 89Zr for PET imaging studies, using a clinically translatable radiolabelling method. HighlightsO_LIDoxorubicin-containing liposomes can be labeled with the positron-emitting radionuclide 89Zr with no impact on their original physicochemical properties. C_LIO_LIRadiolabeling is stable in vivo and enables imaging and biodistribution studies of the liposomes using positron emission tomography (PET). C_LIO_LIThe radiolabeling method is clinically translatable and would allow early assessment of existing and novel doxorubicin liposome biodistribution in humans or personalized medicine (nanotheranostic) approaches. C_LI

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

Abstract (Draft1)Type 1 Tay-Sachs Disease (TSD) is a rare and severe neurodegenerative disorder caused by HEXA Loss of Function (LOF) gene mutations, leading to the accumulation of lysosomal GM2 gangliosides and progressive neurological decline, with high lethality before age 10. Current treatments are primarily palliative, focusing on symptom management. This study investigates the therapeutic potential of Miglustat and Ambroxol, individually and combined, in slowing neurodegeneration and cognitive decline in Type 1 TSD, leveraging aiHumanoid virtual simulations. Miglustat, a substrate reduction therapy, and Ambroxol, a pharmacological chaperone, offer complementary mechanisms that may enhance lysosomal function and reduce ganglioside accumulation. In a virtual Phase 1b/2 trial, simulated cohorts of children received these treatments across four dose levels (10%, 20%, 33.3%, and 50% of maximum tolerated dose [MTD]), with outcomes assessed at intervals from birth to 10 years. Key metrics included cognitive and motor function, quality of life, and lysosomal enzyme activity. Results indicated that combination therapy significantly reduced neurodegenerative symptoms and improved cognitive and motor outcomes, particularly at intermediate dose levels. Findings suggest that Miglustat and Ambroxol may provide a beneficial intervention strategy, warranting further clinical evaluation. These results also provide important potential insights into dose optimization and therapeutic synergies, offering a basis for real-world trials in Type 1 Tay-Sachs Disease. The use of aiHumanoid simulations demonstrates a novel approach for drug testing in rare diseases, enabling detailed assessment of efficacy and safety profiles across developmental stages. The trials findings are based on virtual simulations rather than traditional clinical trials.

Effective blood-brain barrier (BBB) penetration is a significant challenge for antisense oligonucleotide (ASO) therapies targeting neurodegenerative diseases. We utilized a peptide (ApoB11) mediated transport delivery of an ASO to the CNS following systemic delivery to reduce expression of targeted transcripts for neurodegenerative diseases. This study evaluates the pharmacokinetics, CNS penetration, and therapeutic efficacy of ApoB11:2-OMe ASO--Syn, an ASO for -synuclein (-Syn) suppression in synucleinopathies. After a single intraperitoneal (IP) injection (2 mg/kg) in C57BL/6 mice, ApoB11:ASO--Syn showed robust brain penetration, reaching peak concentrations (Cmax = 0.14 nMol/mg) at 1.5 hours and an extended brain half-life (t1/2 = 646.2 hours), indicating prolonged CNS retention. Immunofluorescence confirmed widespread uptake in neurons and endothelial cells. The ASO also accumulated in the liver (Cmax = 419.5 nMol/mg, t1/2 = 104.9 hours), consistent with receptor-mediated uptake. Acute and subacute toxicity studies revealed no systemic toxicity at the highest non-lethal dose (32 mg/kg). In a mouse model of dementia with lewy body (DLB) mice overexpressing human -Syn, ApoB11:ASO--Syn reduced -Syn mRNA and protein levels in the hippocampus and cortex by [~]50% at 16 mg/kg. These results demonstrate that ApoB11 is an effective ASO carrier for CNS delivery, supporting its potential as a therapeutic strategy for synucleinopathies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/651722v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f73353org.highwire.dtl.DTLVardef@14ababforg.highwire.dtl.DTLVardef@12a92e3org.highwire.dtl.DTLVardef@1176b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundTargeted therapies for malignant brain cancer that are currently available have little clinical activity, highlighting an urgent need for the development of novel precision medicines. Brevican (Bcan), a central nervous system (CNS)-specific extracellular matrix protein is upregulated in glioma cells. A brevican isoform lacking glycosylation, dg-Bcan, is a unique glioma marker and thus represents a valuable target for anti-cancer therapy. In this study, we aimed to find a versatile dg-Bcan specific ligand to facilitate glioma targeting. MethodsWe screened a D-peptide library to identify dg-Bcan-Targeting Peptide (BTP) candidates, which were characterized extensively through binding kinetic analyses, cell uptake tests and animal studies. ResultsThe top candidate, BTP-7 binds dg-Bcan with high affinity and specificity, is preferentially internalized by Bcan-expressing glioma cells and can cross the blood-brain barrier in vitro and in mice. Functionalization of camptothecin with BTP-7 led to increased drug delivery to intracranial glioblastoma and cytotoxicity in tumor tissues, as well as prolonged survival in tumor-bearing mice. Conclusiondg-Bcan is an attractive therapeutic target for high-grade gliomas, and BTP-7 represents a promising lead candidate for further development into novel targeted therapeutics. Key pointsO_LIBTP-7 is a high affinity peptide ligand for the dg-Bcan protein and Bcan-expressing cells. C_LIO_LIBTP-7 targets human intracranial GBM xenografts in mice. C_LIO_LIFunctionalization of a toxic anti-cancer drug with BTP-7 enables targeted delivery of the therapeutic to intracranial GBM in mice C_LI Importance of the StudyTargeted therapies for malignant brain cancer that are currently available have little clinical activity, highlighting an urgent need for the development of novel precision medicines that can selectively recognize and kill high-grade glioma tissues. A protein called dg-Bcan is an ideal target because it is present only in the extracellular matrix of high-grade glioma cells and is absent from normal brain tissues. Here, we describe the discovery of a novel dg-Bcan-Targeting Peptide, called BTP-7 that can bind specifically to high-grade glioma cells/tissues, and thus serve as a promising drug delivery vehicle.

Anthracycline induced cardiotoxicity is a significant problem for oncologists and cancer patients. The leading cause of non-cancer death in cancer patients and survivors is heart failure, which is frequently attributed to the exposure to chemotherapeutics like anthracyclines. The most notorious of these chemotherapies is doxorubicin, which causes cardiac contractile dysfunction that in some cases is irreversible. In this study, we report the development of NanoDMX, a phosphatidylserine-containing liposomal formulation of DMX5804, a small molecule inhibitor of MAP4K4, and demonstrate that its administration prevents doxorubicin-induced left ventricular dysfunction in mice. Additionally, we demonstrate that DMX-5804 protects cardiomyocytes in vitro through a combination of mechanisms outside of the expected route of suppressing the JNK pathway. Overall, we demonstrate that the use of NanoDMX, a novel liposomal system using both DMX-5804 and phosphatidylserine, can prevent the damage induced by doxorubicin over the course of a single high dose in vivo model.

Gene therapy has the potential to treat various diseases and has recently gained new interest due to the deployment nucleic acid based vaccines for COVID-19. Despite these developments, there still remains a need for further development of gene delivery vehicles to increase their safety and efficacy.. We have recently developed a lipoproteoplex (LPP) consisting of a super-charged coiled-coil protein (CSP) and a cationic liposomal carrier, that has the ability to condense nucleic acids and deliver them in vivo. The LPP is distinct from other liposomal gene delivery systems in that it utilizes a modular protein component to drive transfection activity as opposed to relying on the passive effects of the cationic lipids. A CSP library has been rationally designed to improve the efficacy of the LPP compared to the parent protein via improved alpha-helical structure and increased nucleic acid binding through the use of extended histidine tags and increased positive charge. The secondary structure and nucleic acid binding ability of each library member was assessed, then compared to functional transfection data in NIH-3T3 mouse fibroblasts. Structural and functional data suggests that increasing alpha-helicity of the protein component of the LPP compared to the parent sequence doubles nucleic acid binding affinity and increases transfection activity almost 3-fold with a favorable safety profile.

Nanoparticles (NP) are being increasingly explored as vehicles for targeted drug delivery because they can overcome free therapeutic limitations by drug encapsulation, thereby increasing solubility and transport across cell membranes. However, a translational gap exists from animal to human studies resulting in only several NP having FDA approval. Because of this, researchers have begun to turn toward physiologically based pharmacokinetic (PBPK) models to guide in vivo NP experimentation. However, typical PBPK models use an empirically derived framework that cannot be universally applied to varying NP constructs and experimental settings. The purpose of this study was to develop a physics-based multiscale PBPK compartmental model for determining continuous NP biodistribution. We successfully developed two versions of a physics-based compartmental model, models A and B, and validated the models with experimental data. The more physiologically relevant model (model B) had an output that more closely resembled experimental data as determined by normalized root mean squared deviation (NRMSD) analysis. A branched model was developed to enable the model to account for varying NP sizes. With the help of the branched model, we were able to show that branching in vasculature causes enhanced uptake of NP in the organ tissue. The models were solved using two of the most popular computational platforms, MATLAB and Julia. Our experimentation with the two suggests the highly optimized ODE solver package DifferentialEquations.jl in Julia outperforms MATLAB when solving a stiff system of ordinary differential equations (ODEs). We experimented with solving our PBPK model with a neural network using Julias Flux.jl package. We were able to demonstrate that a neural network can learn to solve a system of ODEs when the system can be made non-stiff via quasi-steady-state approximation (QSSA). In the future, this model will incorporate modules that account for varying NP surface chemistries, multiscale vascular hydrodynamic effects, and effects of the immune system to create a more comprehensive and modular model for predicting NP biodistribution in a variety of NP constructs. Author summaryNanoparticles (NP) have been used in various drug delivery contexts because they can target specific locations in the body. However, there is a translational gap between animals and humans, so researchers have begun toward computational models to guide in vivo NP experimentation. Here, we present several versions of physics-based multiscale physiologically based pharmacokinetic models (PBPK) for determining NP biodistribution. We successfully developed two versions of ODE-based compartmental models (models A and B) and an ODE-based branched vascular model implemented in MATLAB and Julia and validated models with experimental data. Additionally, we demonstrated using a neural network to solve our ODE system. In the future, this model can integrate different NP surface chemistries, immune system effects, multiscale vascular hydrodynamic effects, which will enhance the ability of this model to guide a variety of in vivo experiments.

MP-004 is a novel small molecule under development as a non-invasive therapeutic for inherited retinal dystrophies (IRDs), including retinitis pigmentosa. Here, we evaluated its ocular concentration and safety profile following topical administration in three animal species --mouse, rabbit, and pig--. MP-004 formulation with 0.3% hyaluronic acid significantly enhanced retinal concentration in mice compared to non-formulated compound (6.67 vs 1.27 {micro}g/g at 4 h). MP-004 reached therapeutically relevant retinal concentrations in all species tested, with minimal systemic exposure: in mice, levels detected in serum were lower than 5 ng/mL while in rabbits, the compound was undetectable in blood and peripheral tissues 12 h post-dose. Seven-day repeated-dose toxicity studies in mice and rabbits showed no systemic or ocular toxicity. In rabbits, only transient ocular redness was observed post-administration, with no evidence of corneal damage or systemic adverse effects. Hematological and biochemical parameters in both species remained within normal limits. Importantly, retinal and optic nerve concentrations remained detectable in rabbits 7 days after final dosing, while the compound was eliminated systemically, supporting prolonged target tissue retention. These findings demonstrate that MP-004 is well tolerated and achieves effective retinal concentrations via topical delivery, overcoming a major barrier in retinal drug development. The favorable pharmacokinetic and safety profile across species, including anatomically relevant models, supports MP-004s advancement toward regulatory preclinical studies and clinical translation as a non-invasive therapy for IRDs.

Despite several promising candidates there is a paucity of drug treatments available for patients suffering from retinal diseases. An important reason for this is the lack of suitable delivery systems that can achieve sufficiently high drug uptake in the retina and its photoreceptors. A promising and versatile method for drug delivery to specific cell types involves liposomes, surface-coated with substrates for transporter proteins highly expressed on the target cell. We identified strong lactate transporter (monocarboxylate transporter, MCT) expression on photoreceptors as a potential target for drug delivery vehicles. To evaluate MCT suitability for drug targeting, we used PEG-coated liposomes and conjugated these with different monocarboxylates, including lactate, pyruvate, and cysteine. Monocarboxylate-conjugated dye-loaded liposomes were tested on both human-derived cell-lines and murine retinal explant cultures. We found that liposomes conjugated with pyruvate consistently displayed higher cell uptake than unconjugated liposomes or liposomes conjugated with lactate or cysteine. Pharmacological inhibition of MCT1 and MCT2 reduced internalization, suggesting an MCT-mediated uptake mechanism. Pyruvate-conjugated liposomes loaded with the drug candidates CN03 and CN04 reduced photoreceptor cell death in murine rd1 and rd10 retinal degeneration models. Overall, this study proposes pyruvate-conjugated liposomes as a vehicle for drug delivery specifically to photoreceptors. Notably, in retinal degeneration models, free drug solutions could not achieve the same therapeutic effect. Our study thus highlights pyruvate-conjugated liposomes as a promising system for drug delivery to retinal photoreceptors, as well as other neuronal cell types displaying high expression of MCT-type proteins.

BackgroundThe development of nanocarriers with precise control over drug release is crucial for targeted therapy. This study focuses on the design and optimization of pH-sensitive gelatin/perfluorohexane (PFH) nanodroplets loaded with berberine chloride, a model drug relevant to traditional Chinese medicine. Subjects and MethodsNanodroplets were prepared using an emulsion technique, with optimization of parameters including homogenization rate, polymer concentration, surfactant, drug, and perfluorocarbon conte nt. ResultsThe optimized formulation resulted in nanodroplets with a mean particle size of 281.7 nm and a drug encapsulation efficiency of 66.8 {+/-} 1.7%. Characterization studies confirmed successful encapsulation and pH-responsive behavior. Ultrasound stimulation significantly enhanced drug release, with 150 kHz frequency proving more effective than 1 MHz. Stability studies demonstrated prolonged stability over one month at 4{degrees}C. Following 10 minutes of ultrasound irradiation, the nanodroplets exhibited 89.4% cumulative drug release. ConclusionsIn conclusion, these pH-sensitive nanodroplets show potential for delivering berberine chloride in a controlled manner, connecting traditional Chinese medicine with contemporary drug delivery methods.

BackgroundElevated lipoprotein(a) [Lp(a)] is an independent, genetically determined risk factor for atherosclerotic cardiovascular disease (ASCVD). Its unique apolipoprotein(a) [apo(a)] component contains variable Kringle IV (KIV) domain repeats that influence secretion, thrombosis, and atherogenesis. Current therapies antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) suppress hepatic production and achieve up to 80-98% Lp(a) reduction. However, mechanisms for enhancing clearance remain underexplored. ObjectiveWe propose a biologic approach to actively accelerate Lp(a) removal. Specifically, we design an antibody-drug conjugate (ADC) that binds conserved KIV9/10 domains and delivers a protease to fragment apo(a) into kidney-excretable fragments, complementing existing production inhibitors. MethodsThe therapeutic is designed as a monoclonal antibody directed at KIV9/10 fused via a cleavable linker to a site-specific protease (e.g., IdeS-like) engineered for conditional activity. Kringle domains were modeled with graph neural networks trained on plasminogen homologs to predict epitope accessibility and binding affinity. A one-compartment, first-order elimination model was used to illustrate potential clearance acceleration, with normal Lp(a) clearance modeled at rate constant k = 0.05 h-1 enhanced clearance at k = 0.10 h-1 CKD at k=0.03 h-1 and CKD+enhanced at k=0.06 h-1. ResultsSimulated concentration-time curves showed that doubling the clearance rate could shorten Lp(a) half-life from [~]13.9 to [~]6.9 h (normal vs. enhanced) and from [~]23.1 to [~]11.6 h (CKD vs. CKD+enhanced). Starting at 100 mg/dL, normal clearance reached [~]9.07 mg/dL by 48 h, while enhanced reached [~]0.82 mg/dL; CKD reached [~]23.69 mg/dL, restored to [~]5.61 mg/dL with enhancement. Acute 50-70% lowering was predicted within 24 h, potentially enabling infrequent dosing and synergy with production inhibitors. ConclusionsEnzymatic fragmentation of Lp(a) at KIV domains is a novel clearance-enhancing paradigm. By generating <100 kDa fragments suitable for renal excretion, this strategy could complement ASO/siRNA therapies, particularly in patients with residual high Lp(a) or impaired kidney function. Further work should validate protease specificity, safety, and in vivo efficacy in animal models before clinical translation.

Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme Replacement Therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells and tissues that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific tissues. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice.

To achieve their therapeutic effect on the brain, molecules need to pass the blood-brain-barrier (BBB). Many pharmacological treatments of neuropathologies encounter the BBB as a barrier, hindering their effective use. Pharmaceutical nanotechnology based on optimal physicochemical features and taking advantage of naturally occurring permeability mechanisms, nanocarriers such as liposomes offer an attractive alternative to allow drug delivery across the BBB. Liposomes are spherical bilayer lipid-based nanocapsules that can load hydrophilic molecules in their inner compartment and on their outer surface can be functionally modified by peptides, antibodies and polyethyleneglycol (PEG). When composed of cationic lipids, liposomes can serve as gene delivery devices, encapsulating and protecting genetic material from degradation and promoting nonviral cell transfection. In this study, we aimed to develop a liposomal formulation to encapsulate a plasmid harbouring brain-derived neurotrophic factor (BDNF) and infuse these liposomes via the peripheral bloodstream into the brain. To this end, liposomes were tagged with PEG, transferrin, and arginine and characterized regarding their physical properties, such as particle size, zeta-potential and polydispersity index (PDI). Moreover, we selected liposomes preparations for plasmid DNA (pDNA) encapsulation and checked for loading efficiency, in vitro cell uptake, and transfection. The preliminary results from this pilot study revealed that we were able to replicate the liposomes synthesis described in literature, achieving compatible size, charge, PDI, and loading efficiency. However, we could not properly determine whether the conjugation of the surface ligands transferrin and arginine to PEG worked and whether they were attached to the surface of the liposomes. Additionally, we were not able to see transfection in SH-SY5Y cells after 24 or 48 hours of incubation with the pDNA loaded liposomes. In conclusion, we synthesized liposomes encapsulation pBDNF, however, further research will be necessary to address the complete physicochemical characterization of the liposomes. Furthermore, preclinical studies will be helpful to verify transfection efficiency, cytotoxicity, and in the future, safe delivery of BDNF through the BBB.

BackgroundPROTACs are comparably large and flexible compounds with limited solubility (S) and permeability (Pe). It is crucial to better understand, predict and optimize their human clinical pharmacokinetics (PK). MethodsThe main objective was to use the ANDROMEDA by Prosilico software to predict the human clinical in vivo dissolution potential (fdiss) and fraction absorbed (fa) of 23 PROTACs at a dose level of 50 mg and to explore whether there is any relationship between in vitro S and in silico predicted in vivo fdiss. ResultsIn silico predictions showed that the PROTACs are effluxed by intestinal transporters and have limited fdiss (34 to 98 %), permeability and fa (13 to 58 %) in man. For some PROTACs this may be a major obstacle and jeopardize the clinical development programs, especially in cases of required high oral dose. A modest relationship between in vitro S and predicted in vivo fdiss was demonstrated (R2=0.26). Predicted human fa (27 %) and oral bioavailability (20 %) of ARV-110 (a PROTAC with some available in vivo PK data in rodents and man) were consistent with data obtained in rodents (estimated fa approximately 30-40 %; measured oral bioavailability 27-38 %). Laboratories were unable to quantify S for 7 (30 %) of the PROTACs. In contrast, ANDROMEDA could predict parameters for all. ConclusionANDROMEDA predicted fdiss and fa for all the chosen PROTACs and showed limited fdiss, Pe and fa and dose-dependent fdiss and fa. One available example shows promise for the applicability of ANDROMEDA for predicting biopharmaceutics of PROTACs in vivo in man. Weak to modest correlations between S and fdiss and a considerable portion of compounds with non-quantifiable S limit the use of S-data to predict the uptake of PROTACs.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, and expressed at the zebrafish BBB. We evaluated the luminescence of ten NanoLuc substrates, then screened the eight brightest to determine which are most efficiently effluxed by the ABC transporters. We identified one substrate efficiently pumped out by ABCB1, two by Abcb4, six by ABCG2, and four by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance StatementThe ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

This study investigated the feasibility and efficiency of neuron-targeting hybrid placental mesenchymal stromal cell-derived extracellular vesicles (PMSC-EVs), engineered by membrane fusion with Targeted Axonal Import (TAxI) peptide modified, TrkB agonist 7,8-DHF-loaded liposomes for treatment of myelomeningocele (MMC) via intra-amniotic cavity administration. The prepared TAxI modified liposomes with 7,8-DHF were used to fuse with PMSC-EVs. Different fusion approaches were investigated and freeze-thaw-extrude method was found to be the optimal. The engineered PMSC-EVs had a uniform particle size and efficiently loaded 7,8-DHF. It also had typical markers of native EVs. Freeze-thaw-extrude process did not change the release profile of 7,8-DHF from engineered EVs compared to TAxI modified, 7,8-DHF loaded liposomes. The engineered EVs could elicit TrkB phosphorylation depending on the incorporation of 7,8-DHF while native EVs did not. The engineered EVs increased neurite outgrowth of apoptotic cortical neurons induced by staurosporine, suggesting that they exhibited neuroprotective function. In a rodent model of MMC, neuron-targeting, engineered EVs became an active targeting delivery system to MMC defect sites. Pups treated with engineered EVs had the lowest density of apoptotic cells and displayed a therapeutic outcome. The study suggests the potential use of engineered hybrid, active neuron-targeting EVs for the in utero treatment of MMC.