Poster X2 - Antiretroviral Supramolecular Prodrug Hydrogelators as Long-Acting Injectables

Introduction: In 2022, 39 million people lived with human immunodeficiency virus (HIV) and 1.3 million people were newly infected with HIV globally. In the same year, 630,000 people died from HIV-related cases, but compared to its peak in 2004, HIV-related mortality has dropped by 51% after the introduction of antiretroviral (ARV) medication. Combination antiretroviral therapy (CART) is the standard medication plan with a daily dose of 3 ARV drugs from 2 different classes for the suppression of viral replication and restoration of the immune system. Despite the progress in treatment and prevention, current treatment methods are not curative and require lifelong dosing, which can lead to poor adherence and reduce the effectiveness of the therapeutics.
Development of a long-acting formulation of ARV drugs is an important subject to address as the reduction in dosage requirement can lead to increased patient adherence and improved quality of life. While ARV drugs such as lamivudine (3TC) and tenofovir (TFV) are important nucleoside reverse transcriptase inhibitors used in many CART programs, their hydrophilicity and low molecular weight makes long-acting formulation difficult. Our strategy is to utilize peptide-based supramolecular hydrogels that can sequester the drug within its sequence to self-assemble into a filamentous network in situ and release its drug over time. In this study, we report on the design of prodrug hydrogelators that displayed sustained delivery of 3TC and TFV for over 40 days in vivo and 28 days in vitro, respectively.

Materials and

Methods: The peptide amphiphiles (PAs) were synthesized with standard Fmoc solid-phase peptide synthesis. The completed, crude peptides were purified with reverse phase high performance liquid chromatography (RP-HPLC) and verified via electrospray ionization-mass spectrometry. The PAs were attached to the drug via a linker to complete the antiretroviral drug amphiphiles (ARV DAs). The completed DAs’ nanostructures were characterized through transmission electron microscopy (TEM).
With the synthesis and characterization complete, the DAs were analyzed for gelation and drug release in vitro. The gelation was visually inspected after adding PBS on the solution of hydrogel. The drug release was measured by placing PBS onto the hydrogels and incubating them at 37ºC. At designated time points, a fraction of PBS was extracted for HPLC analysis while the removed PBS was replaced with fresh media. This process was repeated until the conclusion of the in vitro drug release study.
For the in vivo studies, BALB/cJ mice were subcutaneously injected with the DA or control (PBS or free 3TC) and the hydrogel was analyzed for degradation, pharmacokinetics (PK), and biodistribution. Hydrogel degradation was analyzed by sacrificing the mice at different time intervals, visually inspecting the gel with photography, and quantifying it with HPLC. With a similar injection, PK and biodistribution were analyzed by collecting whole blood and tissue samples at designated time points, processing the samples, and quantifying the drug concentration via ultraperformance liquid chromatography/mass spectrometry, respectively.

Results, Conclusions, and Discussions: Two molecular designs were created for the sustained delivery of 3TC and TFV with both designs offering tunability by varying the number of glutamic acid or valine, respectively. The ARV DAs were synthesized and characterized with TEM to visualize their nanostructure. The DAs were then tested for in vitro gelation; if the hydrogelators could not to gel at the necessary concentration, they were dismissed for the remainder of the study as they would not be able to properly gel in situ. The remaining peptides were tested for their in vitro release rate for 1 month. For the TFV DAs, TFV-PA2 released about 50% of its drug by day 14 while TFV-PA3 released less than 20% of its drug by day 30. For the 3TC DAs, V-3TC-E2 released 29% of its drug while V-3TC-E3 released 52% in 1 month. While a lower release rate correlates with longer release duration, we moved forward with V-3TC-E3 because we believe that the higher drug release is necessary to achieve the minimum effective concentration of 3TC in vivo.
With the in vitro studies completed, adult BALB/cJ mice were subcutaneously injected with V-3TC-E3 to analyze hydrogel degradation. We observed that the hydrogel depot formed within 5 minutes and maintained linear drug release for 35 days with ≈22% of gel remaining at the end. Furthermore, the PK and biodistribution were analyzed after the mice were subcutaneously injected with free 3TC or V-3TC-E3. The free 3TC group had no detectable level of drug in plasma or major organs after 3 days while the hydrogel group reached its maximum concentration on day 7 and maintained concentration above IC50 up to 25 days while having significant levels up to 49 days.
Our results have suggested that the tunable, self-assembling hydrogelators were able to gel rapidly upon the addition of PBS or after subcutaneous injection and to maintain sustained release of tenofovir and lamivudine for over a month. These designs of prodrug hydrogels can potentially be studied for the sustained delivery of different ARV agents to develop a long-acting combination therapy that can significantly improve HIV treatment and prevention.

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