(T1030-08-48) Pharmacokinetic Modulation of PLD1 Inhibitor via Exosome Delivery: Enhanced Brain Retention for Alzheimer’s Disease Treatment

Purpose: Alzheimer’s disease (AD), a progressive neurodegenerative disorder predominantly associated with aging, presents substantial therapeutic challenges. Current treatment modalities are often limited by significant adverse effects and lack of target specificity. For example, Aducanumab, a monoclonal antibody designed to selectively bind aggregated amyloid-β (Aβ) plaques and facilitate their clearance via immune-mediated mechanisms, has demonstrated efficacy in reducing amyloid burden. However, its clinical utility is constrained by the occurrence of Amyloid-Related Imaging Abnormalities (ARIA), which include vasogenic edema and cerebral microhemorrhages detectable via neuroimaging⁽¹⁾. Consequently, there is a critical need for the development of more targeted therapeutic strategies that minimize off-target effects and enhance clinical outcomes in AD and related dementias (ADRD). Research has demonstrated the efficacy of well-tolerated Phospholipase D1-based therapeutics in animal disease models^(2,3). PLD1, a lipolytic enzyme that breaks down membrane phospholipids to phosphatidic acid and choline is conserved from bacteria to humans and is inducible when required. It is involved in several physiological processes including membrane trafficking, cytoskeletal reorganization, and autophagy. Abnormally elevated PLD1 levels in the hippocampus area of AD post-mortem brains⁽⁴⁾, Frontotemporal Dementia (FTD) brains, and in 3xTg-AD mice are reported. It is also reported that the repeated administration of low-dose PLD1 small molecule inhibitor, VU0155069 (VU01), in 3xTgAD mice improves long-term potentiation deficits and synaptic vulnerability at both early- and late-stages in the progression of the disease^(2,3). Despite demonstrated in vivo efficacy, the clinical translation of VU0155069 has been hindered by a significant knowledge gap—specifically, the absence of pharmacokinetic (PK) data and an uncharacterized capacity for central nervous system (CNS) penetration. To address these critical delivery limitations, we explored an exosome-based nanocarrier system as a novel platform for targeted drug delivery. This approach not only facilitates the efficient transport of VU0155069 across the blood-brain barrier but also holds translational potential for other small-molecule inhibitors, underscoring the urgent need for more effective and precise therapeutic strategies in Alzheimer’s disease.

Methods: HEK-293T derived exosomes/extracellular vesicles (EVs) were isolated using Tangential Flow Filtration method and were characterized by Nanoparticle Tracking Analysis (NTA) and JESS Simple Automated Western. Electroporation technique was utilized as a standard technique for drug loading into exosomes after optimizing and comparing with other methods. VU01 was loaded into exosomes using electroporation buffer at 400 V with a capacitance of 125 μF for 2 pulses and given for LCMS/MS after purification to detect the amount of loading. Wild-type mice were administered intranasally with PKH26-tagged exosomes and sacrificed at different time intervals to validate the fact that exosomes cross the BBB and it’s distribution throughout the body which was assessed using the In Vivo Imaging System (IVIS) and immunofluorescence. Pharmacokinetic study was performed on wild-type CD1 mice to establish a PK profile for the drug. For pharmacodynamic (PD) validation, 5-month-old 3xTgAD mice were injected with VU01-loaded exosomes (Exo-VU01), 1 mg/kg, intranasally for 15 alternative days and compared with age-matched controls. Behavior analysis and electrophysiological recordings were done on these mice to assess their synaptic function.

Results: EVs characterization using JESS showed the presence of three most important exosomal markers including Flotilin, CD81 and CD63 while the cell lysate, used as a negative control, was the only one to pull up calnexin, confirming that the packaging material used is the exosomes. NTA shows a mean diameter of 132.4 nm confirming EVs size range. IVIS detection shows exosome concentration robustness in brain tissue at 90 min while the immunofluorescence data corroborates and shows that hippocampus gets exposed to EVs within 15 min of the administration while showing robustness at 60- and 90-min. PK study revealed that administration of free VU0155069, in plasma, led to a high initial peak concentration (Cmax) that rapidly declined. Conversely, the Exo-VU01 formulation exhibited a lower Cmax in plasma, suggesting that encapsulation alters its systemic distribution. Encapsulation in exosomes dramatically increased brain exposure, by approximately 24-fold, improving the mean resident time and half-life in the brain. PD behavioral analysis showed frontal cortex dependent Y-maze memory improvement in Exo-VU01 mice (p= 0.0476), better speed of exploring in open field test (p= 0.0035) and better memory (p= 0.0225) in Novel Object Recognition test. Electrophysiology recordings show the improvement in long term potentiation in Exo-VU01 mice compared to the controls along with the last 10 min showing excellent synaptic resilience that is sustained for one hour in exo-VU01 treated group.

Conclusion: Exosomes serve as stealth nanocarriers capable of encapsulating VU0155069 and other small-molecule inhibitors, effectively shielding their cargo from extensive plasma protein binding and rapid systemic clearance. This protective mechanism enhances the bioavailability of the therapeutic agent at the intended site of action. In the present study, we demonstrate the application of an exosome-based formulation to improve the pharmacological profile of VU0155069, thereby enabling targeted inhibition of PLD1. This strategy holds promise for ameliorating AD/ADRD, offering a novel and potentially more effective therapeutic avenue.<

References: 1. Salloway S, Chalkias S, Barkhof F, Burkett P, Barakos J, Purcell D, Suhy J, Forrestal F, Tian Y, Umans K, Wang G, Singhal P, Budd Haeberlein S, Smirnakis K. Amyloid-Related Imaging Abnormalities in 2 Phase 3 Studies Evaluating Aducanumab in Patients with Early Alzheimer Disease. JAMA Neurol. 2022 Jan 1;79(1):13-21. doi: 10.1001/jamaneurol.2021.4161. PMID: 34807243; PMCID: PMC8609465.
2. Bourne, K.Z., Natarajan, C., Perez, C.X.M. et al. Suppressing aberrant phospholipase D1 signaling in 3xTg Alzheimer’s disease mouse model promotes synaptic resilience. Sci Rep 9, 18342 (2019). https://doi.org/10.1038/s41598-019-54974-6
3. Natarajan C, Cook C, Ramaswamy K, Krishnan B. Phospholipase D1 Attenuation Therapeutics Promotes Resilience against Synaptotoxicity in 12-Month-Old 3xTg-AD Mouse Model of Progressive Neurodegeneration. Int J Mol Sci. 2023 Feb 8;24(4):3372. doi: 10.3390/ijms24043372. PMID: 36834781; PMCID: PMC9967100.
4. Krishnan, B., Kayed, R. and Taglialatela, G. (2018), Elevated phospholipase D isoform 1 in Alzheimer's disease patients' hippocampus: Relevance to synaptic dysfunction and memory deficits. Alzheimer's & Dementia: Translational Research & Clinical Interventions, 4: 89-102 102. https://doi.org/10.1016/j.trci.2018.01.002

Acknowledgements: This research is funded by Alzheimer’s Association (AARG-17-533363), National Institutes of Health (R01- AG063945 & R21-AG059223).

This figure details the analysis to confirm the engineered drug-loaded exosomes physical characteristics and compare drug loading strategies.
(A) Jess Simple Western validation confirming the presence of crucial exosomal markers on its surface including Flotillin, CD81 and CD63 and excluding Calnexin which acts as a negative control for exosomes but found in cell lysates.
(B) Physicochemical characterization of the purified Exo-VU01 formulation by Nanoparticle Tracking Analysis (NTA). The quantitative report confirms a particle concentration of 1.19 x 10¹¹ particles/mL and a size distribution consistent with exosomes (Mean: 132.4 nm; Mode: 99.6 nm). The visualization from the NTA instrument, showing the light scattered by individual vesicles, is presented.
(C) Crucial comparative analysis of VU0155069 loading efficiency using two distinct physical methods: sonication and electroporation. The graph displays the results of loading the drug payload using sonication for 10 seconds (left data cluster) versus an electroporation approach of 6 pulses (right data cluster). For each method, loading was tested across varying amounts of exosomes (X-axis) and with different initial concentrations of VU0155069 (represented by red, green, and blue bars). The resulting drug payload, expressed as loading efficiency (percentage), is quantified for each condition. This analysis is central to the study as it directly compares the two methods to identify the most effective strategy for maximizing the exosomal drug cargo.


Comparative Pharmacokinetics of Free VU0155069 versus Exosome-Encapsulated VU0155069 (Exo-VU01) Following Intravenous Administration in Mice.
(A) Schematic of the in vivo pharmacokinetic study design. Male CD1 mice were administered a single intravenous (IV) dose of either free VU0155069 (1 mg/kg) or the equivalent dose encapsulated in exosomes (Exo-VU01). At specified time points ranging from 0 to 1440 minutes, mice were sacrificed, and plasma and brain tissues were collected for subsequent drug quantification via the validated LC-MS/MS method.
(B) Concentration-time profiles of VU0155069 in plasma (left graph) and brain (right graph). These graphs are the lynchpin of the study, illustrating the drug's disposition over 24 hours. The profiles compare free VU0155069 (blue circle) with Exo-VU01 (red squares). Notably, while the free drug achieves a substantially higher peak concentration (Cmax) in plasma, the Exo-VU01 formulation results in significantly enhanced and sustained brain exposure, demonstrating the remarkable efficacy of the exosome carrier system for crossing the blood-brain barrier.
(C) Table of key pharmacokinetic (PK) parameters. This table summarizes the non-compartmental analysis of the PK data derived from the intravenous study shown in the graphs. It details critical parameters such as elimination rate, half-life (t1/2), peak concentration (Cmax), and Area Under the Curve (AUC) for both free and exosome-encapsulated VU0155069 in both plasma and brain compartments.