Analytical methods to determine the degree of drug association for complex injectable drug-combination particles containing multiple drug substances

Abstract

The aim of this research is to develop and characterize complex injectable products composed of multiple drug substances targeted to human immunodeficiency virus (HIV). Some of these product candidates are composed of current HIV drug substances that exhibit desperate water solubilities. For example, lopinavir (LPV), ritonavir (RTV) and dolutegravir (DTG) are water insoluble while tenofovir (TFV) and lamivudine (3TC) are water soluble. Water insoluble LPV/RTV and water soluble TFV (or TDF) prodrugs are given orally to provide sustained viral suppression as a one-pill-a-day treatment in people living with HIV (PLWH). Recently DTG, 3TC and TFV, referred to as TLD one-pill-a day, has been widely used in the low-to-middle income countries with the goal to bring down HIV infections worldwide. Unfortunately, PLWH express pill fatigue and missing a daily dose may lead to virus rebound and disease progression. In addition, uncontrolled virus levels in PLWH may relate to a continued increase in the number of people with HIV. This laboratory is developing a complex injectable LPV/RTV/TFV in a drug-combination nanoparticle (DcNP) product candidate, referred to as targeted long-acting combination antiretroviral therapy 101 (TLC-ART 101) that is currently under clinical testing. A next generation product, TLC-ART 301 composed of DTG, 3TC and TFV (or TLD) is currently under preclinical development. As DcNP enabled TLC-ART 101 and TLC-ART 301 products containing 3 drug substances assembled to associate with DcNP particles, a key performance characteristic is the degree of drug substance association to the DcNP product in the suspension. The initial approach is based on dialyzing the unbound 3 drug substances under sink-condition, to enable diffusion of free drug through a semi-permeable membrane across a large volume of solvent. This degree or percentage of DcNP association for each drug is based on the fraction of drug retained within the dialysis chamber and to account for dialysate (due to large volume and analytical assay sensitivity). With the development of a combination of increasing analytical assay and volume reduction dialysate (thus increasing drug concentrations amenable for drug analysis), this thesis research was able to determine the percentage of drug association to DcNP based on a mass-balance approach. We found that comparing the association efficiency (AE) analysis for the 3 drug substances based with retentate only, or with a mass-balance approach was well-correlated. In addition, under the sink conditions, only one of the two lipid excipients, sodium; [(2R)-2,3-di(octadecanoyloxy)propyl] 2- (2-methoxyethoxycarbonylamino)ethyl phosphate (mPEG-2000 DSPE), but not 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) are found in dialysate; suggesting that some of the water soluble drug TFV may have remained bound to mPEG-2000 DSPE and TFV-mPEG2000-DSPE or that micelles may have also contributed to apparently over 90% of the TFV behaving as a DcNP bound drug; reported in primates after TLC-ART 101 dosing via IV as well as SC route. The method of AE estimate – both retentate and mass-balance approaches and correlation, for the 3 drug substances in the TLC-ART 101, is demonstrated to extend to the TLC-ART 301 product composed of DTG, 3TC and TFV. Collectively, this thesis research was able to verify the analytical methods to characterize the degree of drug association for both abbreviated retentate as well as mass-balance approach for a complex injectable drug-combination formulated in drug-combination nanoparticle product. Having the validated method may help accelerate development of the drug-combination injectable nanoparticles, intended to provide long-acting and longer lasting viral suppression in people living with HIV.