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Lipophilic nanocrystal prodrug-release defines the extended pharmacokinetic profiles of a year-long cabotegravir
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Published: 08 June 2021
Abstract
A once every eight-week cabotegravir (CAB) long-acting parenteral is more effective than daily oral emtricitabine and tenofovir disoproxil fumarate in preventing human immunodeficiency virus type one (HIV-1) transmission. Extending CAB dosing to a yearly injectable advances efforts for the elimination of viral transmission. Here we report rigor, reproducibility and mechanistic insights for a year-long CAB injectable. Pharmacokinetic (PK) profiles of this nanoformulated CAB prodrug (NM2CAB) are affirmed at three independent research laboratories. PK profiles in mice and rats show plasma CAB levels at or above the protein-adjusted 90% inhibitory concentration for a year after a single dose. Sustained native and prodrug concentrations are at the muscle injection site and in lymphoid tissues. The results parallel NM2CAB uptake and retention in human macrophages. NM2CAB nanocrystals are stable in blood and tissue homogenates. The long apparent drug half-life follows pH-dependent prodrug hydrolysis upon slow prodrug nanocrystal dissolution and absorption. In contrast, solubilized prodrug is hydrolyzed in hours in plasma and tissues from multiple mammalian species. No toxicities are observed in animals. These results affirm the pharmacological properties and extended apparent half-life for a nanoformulated CAB prodrug. The report serves to support the mechanistic design for drug formulation safety, rigor and reproducibility.
Introduction
While wide-spread availability of antiretroviral therapy (ART) has reduced human immunodeficiency virus type one (HIV-1) morbidities and mortality, viral transmission continues to persist. This is highlighted by yearly recordings of two million new infections worldwide1. ART requires life-long daily oral regimens with both short and long-term toxicities. Changing dosing regimens driven by the emergence of resistant viral strains and regimen adherence are therapeutic limitations2,3,4,5. A priority for HIV-1/acquired immune deficiency syndrome (AIDS) research is how best to prevent the spread of infection which can be achieved through ART-mandated pre-exposure prophylaxis (PrEP)6. PrEP success has been buoyed by a long-acting (LA) parenteral formulation of cabotegravir (CAB). This permits every other month antiretroviral drug (ARV) dosing. CAB LA was recently approved by the US Food and Drug Administration demonstrating improved pharmacokinetic and pharmacodynamic (PK and PD) profiles compared to more conventional ARV regimens and as such provides a promising strategy for HIV-1 prevention and treatment7.
CAB is unique in its structural and antiretroviral properties. CAB LA is the first LA injectable regimen amongst the group of integrase strand transfer inhibitors (INSTI).
These relatively new ARVs have complemented existing ARVs based on their unique treatment and improved PK profiles. Notably, CAB LA PK results are highlighted by an extended plasma half-life of up to 54 days8 and limited drug–drug interactions (DDI). The latter is linked both to its membrane permeability and low affinity for cytochrome P450 (CYP450)9,10. Moreover, CAB has low aqueous solubility and a high melting point allowing its administration as a high concentration parenteral formulation11. CAB LA is currently completing phase 3 clinical trials and was approved recently for use in Canada. USA Food and Drug Administration (FDA) approval is anticipated in late spring-summer of 202112. Nonetheless, there are limitations for CAB LA as the current formulation requires a 2 ml dosing volume known to elicit injection site reactions and continuous health care oversights13. Thus, newer formulations with longer dosing intervals, ease of access, and reduced administration volumes will positively impact wide-spread regimen use.
In order to overcome the treatment limitations of CAB LA we developed lipophilic fatty acid ester CAB prodrugs nanoformulations (named NMCAB and NM2CAB, catalogued based on carbon lengths) with extended PK properties. The PK profiles were improved from months (NMCAB)14 to one year (NM2CAB)15. However, during the development of a year-long CAB, PK data set reproducibility in different animal species proved mandatory for development. This is boosted by unraveling the mechanism(s) of the extended drug apparent half-life that includes prodrug hydrolysis. Moreover, safety measurements for the year-long parenteral formulation were required. With these in mind, we now report, from separate laboratories, PK and biodistribution (BD) profiles, of NM2CAB, our lead nanoformulated stearoylated prodrug. Work was completed in rodents with head-to-head comparisons against a CAB LA formulation (reflected by NCAB) analogous to that currently being evaluated in human trials. Rigor and reproducibility are defined by PK studies by two research (from the University of Nebraska Medical Center Colleges of Medicine and Pharmacy) and one contract laboratory (Covance Laboratories, a global contract research organization and drug development services company) employing multiple mouse strains (immune deficient and competent) and rats. Lastly, mechanisms were elucidated together with the nanoformulation stability demonstrating that particle stability and slow prodrug release best reflected the extended drug half-life for the prodrug formulation rather than the prodrug enzymatic or chemical stability. Each of these tests proved critical in defining the safety and reproducibility of a year-long CAB apparent half-life. The solid state form of M2CAB nanocrystals is a key determinant for prodrug stability in biological fluids and tissues. We posit that the translation of a safe biocompatible formulation will have a significant impact on the therapeutic armamentarium in preventing HIV/AIDS infection and in providing improved access to those populations most impacted by high viral transmission rates.
CAB is a second-generation INSTI currently nearing USA FDA approval as a LA injectable18. While effective in PrEP and has generated attention by the scientific and patient community for both treatment and prevention of HIV/AIDS12,17,19 limitations in dosing volumes, intervals of administration, and local injection site reactions remain. To overcome these, prodrug nanoformulated were developed and are currently undergoing pre-clinical evaluations by our group14,15. Early proof of concept studies showed encouraging results. However, what was done previously in support of NM2CAB required affirmation and safety evaluation to better position the product for future clinical testing. Underlying mechanisms linked to a year-long medicine also required evaluation. Towards both ends, independent laboratories employed two strains of mice and rats to affirm our prior works. Results showed sustained CAB plasma levels for at least one year in all species. Plasma concentrations were up to 100-fold higher than those resulting from NCAB (an equivalent preparation to the CAB LA). Most notably we now show that the mechanism of this extended half-life was attributed to prodrug release from the solid nanocrystals stored in tissues rather than the stability (chemical and enzymatic) of the prodrug itself. These findings offer insights into how best to bring such extended-release medicines to the clinic with broad relevance to ARVs now being modified as LA formulations.
In summary, we demonstrate both rigor and reproducibility of our year-long CAB prodrug formulation. NM2CAB administration to two strains of mice, rats, and monkeys resulted in enhanced and sustained CAB plasma levels for at least one year. Plasma concentrations were up to, or greater than, 100-times those resulting from the equivalent NCAB (a CAB LA equivalent formulation). The advantages of NM2CAB for LA treatment are several-fold. NM2CAB can be manufactured at a drug concentration of 400 mg/ml, twice the CAB LA concentration of 200 mg/ml. Thus, NM2CAB would require a 1 ml injection volume for yearly dosing when compared to a 2 ml monthly injection for CAB LA. In addition, no injection site reactions were observed. Thus, NM2CAB provides an improvement over the current approved CAB LA with an extended dosing interval, reduced injection volume, and no injection site reactions. Importantly, unlike orally administered medicines where clearance rates determine the terminal drug half-life, the extended duration of NM2CAB is dependent on the amount of drug absorbed/day from tissue depot sites. These enable a lower administered dose to last for a year. We also elucidated the mechanisms behind this enhanced PK profile from tissue distribution and in vitro data. NM2CAB accumulates and is retained in the injection site and in tissues, which serve as depots that slowly release the prodrug over months to years. In vitro data suggest that the observed enhanced tissue accumulation of NM2CAB is due to enhanced cellular uptake of the nanocrystals from the injection site, intracellular retention, and nanocrystal stability in macrophages. In tissues, M2CAB undergoes slow dissociation from the nanoformulation which then is rapidly hydrolyzed into native CAB by both chemical and enzymatic hydrolysis. This was cross validated by chemical and metabolic studies in vitro using blood and tissue homogenates. Overall, the integration of mechanistic in vitro and in vivo data sets has elucidated the mechanisms behind the enhanced and sustained PK behavior of NM2CAB. These data sets to support the premise that enhanced macrophage nanocrystal delivery and formation of intracellular tissue drug depots and slow prodrug dissolution rate contribute to the extended NM2CAB apparent drug half-life.
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