High School Student Unionville High School Kennett Square, Pennsylvania, United States
Introduction: Human immunodeficiency virus (HIV) continues to be a serious global public health issue. In 2023, 1.3 million people acquired HIV, and 630,000 people died from HIV-related causes. At the end of the same year, 39.9 million people were living with HIV. Currently, there is no cure for HIV. However, HIV infection has become manageable with the development and introduction of antiretroviral (ARV) drugs. ARV drugs are utilized in combination antiretroviral therapy (cART), which consists of three ARV drugs from two different drug classes taken daily, to suppress HIV replication. Due to cART’s high dosing frequency, drug concentration fluctuation and reduced patient adherence remain pertinent concerns. To address the issues caused by frequent dosing, long-acting formulations of ARV drugs are highly desired. Long-acting ARV drugs can lead to increased patient adherence and therefore more effective treatment. Standard cART regimens typically include an ARV drug class known as nucleoside reverse transcriptase inhibitors (NRTIs), but their low molecular weight and high aqueous solubility make it difficult to design long-acting formulations for NRTIs. In this study, we conjugate drug, specifically lamivudine (3TC) or tenofovir (TFV), to peptide amphiphiles (PAs) to form antiretroviral prodrug hydrogelators. Our design of these prodrug hydrogelators has demonstrated controlled release of 3TC for over 40 days in vivo and TFV for 28 days in vitro.
Materials and
Methods: The PAs were synthesized using a standard Fmoc solid-phase synthesis protocol. The resulting peptides were purified using reverse-phase high-performance liquid chromatography (RP-HPLC). Products were then identified with electrospray ionization-mass spectrometry (ESI-MS). The PAs were attached to the drug by a linker to construct the antiretroviral drug amphiphiles (ARV DAs). The ARV DAs were purified and confirmed with analytical RP-HPLC and ESI-MS, respectively. Transmission electron microscopy (TEM) imaging was taken, revealing 1D nanostructures. The DAs were studied for gelation and drug release in vitro. Gelation was visually confirmed upon the addition of phosphate buffer saline (PBS) to the solutions containing DAs. To measure drug release, PBS was placed onto the hydrogels and aged at 37°C. At predetermined times, a portion of the solution was removed and analyzed with HPLC. To accommodate for the removed portion, fresh media was introduced. In the in vivo studies, adult BALB/cJ mice were injected subcutaneously with ARV DA-containing solution or a control to analyze hydrogel degradation. Male BALB/cJ mice were injected subcutaneously with either DA-containing hydrogel or a control solution (free 3TC) to analyze pharmacokinetic (PK) and biodistribution profiles. Blood and tissue samples of the mice were collected and examined with ultraperformance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) to quantify prodrug levels.
Results, Conclusions, and Discussions: The molecular designs for both 3TC and TFV DAs allowed for adjustments in sustained drug delivery by changing the number of glutamic acid or valine, respectively. However, the most optimal design needed to be selected for further studies. If a DA was unable to gel at a necessary concentration, it would be excluded from further studies. The 3TC DAs, V-3TC-E2 and V-3TC-E3, released 29% and 52% of their drug over a month, respectively. V-3TC-E3 was chosen to be studied further despite a higher release rate, which can lead to decreased drug retention, because we believe a higher drug release rate is required to maintain an effective dose of 3TC for in vivo studies. For the TFV DAs, TFV-PA2 demonstrated significantly faster drug release compared to TFV-PA3. TFV-PA2 released about 50% of its drug by day 14 while TFV-PA-3 retained more than 80% of its drug by day 30. After the in vitro studies, we proceeded with analyzing hydrogel degradation in vivo. Adult BALB/cJ mice were injected subcutaneously with 15 mM V-3TC-E3 or PBS control. Within five minutes of V-3TC-E3 injection, a hydrogel depot could be formed, while the PBS control dissipated within an hour. After 35 days from the initial V-3TC-E3 injection, about 22% of the gel remained. PK and biodistribution profiles were then studied in mice injected subcutaneously with V-3TC-E3 or free 3TC (control). 3TC was undetectable after three days in mice with free drug treatment. In mice with V-3TC-E3 treatment, 3TC plasma concentration reached a maximum on day 7 and maintained at high levels through day 49.
