(T1430-01-02) Development of an Extended Action Fostemsavir (FTR) Lipid Nanoparticle (LNP) Antiretroviral Therapy

Purpose: Advancement in antiretroviral therapy has changed HIV-1 management by reducing adverse effects, minimizing pill burden and mitigating drug interaction. But in some patients, the suppression of viral load is still insufficient, and these patients become prone to resistance due to viral mutation. Hence a new class of drugs with a novel mechanism of action is needed. Fostemsavir (FTR) is a newly FDA-approved drug that targets the very first step of viral entry to the host cell by inhibiting virus attachment to the host cell. FTR is a prodrug of active drug moiety temsavir (TMR). FTR has been found to be effective in combination with optimal drug regimens in patients with multidrug-resistant HIV-1 infection. Our idea is to develop a lipid nanoparticle (LNP) system loaded with FTR And to design the lipid composition as well as optimize the LNPs in a way that they distribute preferentially to the spleen and lymph node where the virus is mostly loaded. We hypothesize that FTR loaded LNPs will reduce virus entry into the host. Thus, viral load in multi-drug resistant HIV-1 patients can be suppressed.

Methods: We employed a microfluidic system for the development and optimization of lipid nanoparticles (LNPs) loaded with FTR. These formulations were characterized to determine their physicochemical properties, including particle size, zeta potential, and PDI using Dynamic Light Scattering (DLS), their encapsulation efficiency (EE%) via High-Performance Liquid Chromatography (HPLC). We also checked the % cumulative release rate of the drug in different pH. Subsequently, we utilized confocal microscopy to observe subcellular localization, Transmission Electron Microscopy (TEM), and flow cytometry to assess cellular uptake. Additionally, we evaluated the antiretroviral effectiveness through p24 antigen staining and reverse transcriptase (RT) assays. Finally, we fluorescently labeled the LNPs to examine the biodistribution in mice using an in vivo imaging system (IVIS).

Results: Changing different process parameters significantly affected the physicochemical attributes of the formulations such as size, zeta, PDI and EE%. The morphology under cryo-TEM for LNP1 (larger LNPs) was significantly different than LNP5 (smaller LNPs) (Figure 1). In the antiretroviral efficacy study (Figure 2), we observed that LNP1 could give protection to the macrophage cells (MDMs) against HIV infection till 24hr. On the other hand for LNP5 formulation, we found HIV infection within 3h. Animal studies (Figure 3) show that LNP1 was distributed to the spleen along with some other organs at 1h, 2h and 4h. However, for LNP5 formulation, we don't see any distribution at any time point.

Conclusion: The microfluidics system was utilized to successfully produce uniform formulations of FTR-LNP with high EE%. Notable differences were observed in the physicochemical properties, antiretroviral efficacy, and biodistribution among the various FTR-LNP formulations developed by changing the process parameters. Notably, some formulations were predominantly distributed in the spleen, which is the primary organ where the virus predominantly resides.


Figure 1: Microscopic images of FTR-LNPs with different flow rate ratios (FRR). a Cryo-TEM images of five different FTR-LNP formulations. b AI-based image processing for measuring size distribution of LNP1 (top) and LNP3 (bottom). The left column displays an original cryo-TEM image, the middle column shows the semantic segmentation output from the U-Net model predicting LNPs, and the right column illustrates the detected contours of the LNPs for size distribution. c Size distribution of LNP1 and LNP3 measured by image processing.


Figure 2: Antiretroviral activities of LNP1 and LNP5 formulations. a Antiretroviral responses were recorded by HIV-1p24 immunostaining after HIV-1ADA challenge at a multiplicity of infection (MOI) of 0.1 infectious virions/cell in MDM cultured for 7 days. Following an 8 h administration of drug given at 100 μM LNP1, LNP5, or free fostemsavir to MDM, viral antigen staining was assessed. Empty LNP containing no antiretroviral drug served as a control. Representative images were taken at 20X magnification. b Reverse transcriptase (RT) activities were determined in culture fluids from 3 to 24 h. N = 4 biological replicates.


Figure 3: Biodistribution of FTR-LNP formulations. Experiments were performed in male Balb/c mice by in vivo imaging system (IVIS). 100 μL and 200 μL Rh-LNP1 and Rh-LNP5 formulations, respectively, were administered. Intravenous injections were used to assay the biodistribution of both LNP1 and LNP5 formulations. At 30 min and 1, 2, 4, 6, 8, 10 and 12 h mouse liver, spleen, lung, heart, and kidney organs were collected. Organs were imaged under IVIS and the fluorescence signal recorded in FTR-LNP at excitation and emission of 571 and 591 nm, respectively. a Mice administered Rh-LNP1 and fluorescence signal in different organs. b Mice administered Rh-LNP5 and fluorescence in different organs. Images were analyzed using Living Image® software. N = 2 for each timepoint and treatment group.