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Ultra-long-acting removable drug
delivery system for HIV treatment and prevention
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The data in this paper indicate that ultra-LA dolutegravir can be readily prepared and sterilized by filtration. It can efficiently deliver dolutegravir for up to 9 months in mice and 140 days in NHP (last time point analyzed). A single administration of ultra-LA dolutegravir strongly inhibited acute HIV replication, and effectively protected against repeated high-dose vaginal HIV challenges using highly relevant primary transmitted/founder viruses. An important aspect of the ultra-LA dolutegravir formulation is the fact that it can be easily removed resulting in rapid decrease of drug concentration providing a measure of safety that is not afforded by any of the current LA drug delivery systems in clinical trials for HIV prevention. However, when immediate removal is not necessary, the formulation is biodegradable and does not require surgical removal. In addition, the formulations offer the flexibility to include multiple drugs and the future potential to be reloadable in situ31.
In summary, the results demonstrate the in vivo effectiveness of an ultra-LA formulation of dolutegravir to deliver drug for up to 9 months that results in sustained viral suppression and prolonged protection from high dose HIV vaginal challenges. With its ultra-long duration, low cost of production, ease of administration and the ability to be removed this represents a significant advance in drug delivery for HIV pre-exposure prophylaxis.
Non-adherence to medication is an important health care problem, especially in the treatment of chronic conditions. Injectable long-acting (LA) formulations of antiretrovirals (ARVs) represent a viable alternative to improve adherence to HIV/AIDS treatment and prevention. However, the LA-ARV formulations currently in clinical trials cannot be removed after administration even if adverse events occur. Here we show an ultra-LA removable system that delivers drug for up to 9 months and can be safely removed to stop drug delivery. We use two pre-clinical models for HIV transmission and treatment, non-human primates (NHP) and humanized BLT (bone marrow/liver/thymus) mice and show a single dose of subcutaneously administered ultra-LA dolutegravir effectively delivers the drug in both models and show suppression of viremia and protection from multiple high-dose vaginal HIV challenges in BLT mice. This approach represents a potentially effective strategy for the ultra-LA drug delivery with multiple possible therapeutic applications.
Adherence to medication is essential to treatment success1. In most cases the extent to which patients are able to follow prescribed treatments determines the final outcome. This is particularly important in the treatment of chronic conditions like mental illnesses, hypertension, diabetes, and HIV/AIDS1. Sustained drug release has successfully improved adherence in patients with schizophrenia2 and as contraceptives3. Long-acting (LA) injectable formulations of ARV can increase adherence and effectiveness of HIV treatment and prevention4. In particular, LA formulations can (1) simplify dosing schedules, (2) reduce possible side effects, (3) provide constant concentration of drug, and (4) have a positive effect on patient’s overall quality of life. LA-ARV formulations currently in clinical trials, are formulated as nanosuspensions for injections every 8 weeks5,6,7 and it is impossible to remove the injected nanosuspension from the body in the case of a medical emergency.
To address this significant limitation and to extend the duration of treatment, an ultra-LA injectable and removable formulation for HIV PrEP based on in situ forming implant technology was developed. This allows drug administration by subcutaneous injection followed by implant solidification in vivo and subsequent biodegradation of the implant resulting in sustained drug release8,9. The efficacy of drug delivery via ultra-LA formulation in the context of HIV is evaluated in BLT humanized mice, systemically reconstituted with human hematopoietic cells10. BLT mice have been extensively used for HIV transmission, replication, and persistence studies11,12,13,14. Importantly, humanized BLT mice allow evaluation of HIV treatment and prevention strategies with a variety of transmitted/founder HIV-1 isolates via relevant routes of transmission.
Preparation of ultra-LA dolutegravir

Dolutegravir, a highly effective second-generation HIV integrase strand transfer inhibitor with extensive track record of efficacy and safety15, was used for preparation of ultra-LA formulation. In addition to active drug, the formulation consists of two relatively low cost FDA-approved excipients: (1) poly(lactic-co-glycolic acid) (PLGA), a biodegradable copolymer that eventually and safely biodegrades and, (2) N-methyl-2-pyrrolidone (NMP), a water miscible and biocompatible organic solvent16,17. Composition of the ultra-LA formulation was first optimized for release kinetics in vitro and then sustained delivery of dolutegravir in vivo (Fig. 1). The formulation contained dolutegravir/PLGA/NMP at a ratio 0.3:1:2 by weight, had a viscosity 845 cP at 25 °C (Brookfield Cone and Plate Digital Rheometer, n = 3). Formulation stability was assessed as the ability to remain in solution (measured by dynamic light scattering, Zetasizer Nano ZS Particle Analyzer), and to maintain a stable dolutegravir concentration (98% of the original concentration) measured by HPLC analysis. Dolutegravir was chemically stable in this formulation at 25 °C for at least 6 months. In the aqueous environment in vitro (0.01 M PBS, pH = 7.4, 2% solutol) solidification of the implant was instantaneous.
Concentration of dolutegravir was also evaluated in the female reproductive tract (FRT) in 14 NSG mice receiving a single subcutaneous injection of ultra-LA dolutegravir. Vagina, cervix, uterus, and plasma from treated mice were collected 1, 4, and 12 weeks post administration and dolutegravir concentrations determined (Fig. 1c). One week after ultra-LA dolutegravir administration, the median concentration of dolutegravir in plasma, vagina, cervix, and uterus were 1350 ng/ml, 196 ng/mg, 158 ng/mg, and 272 ng/ml, respectively. One month post administration, the median dolutegravir concentrations were 958 ng/ml, 233 ng/mg, 262 ng/mg, and 303, ng/mg, respectively, and 12 weeks post administration the median concentrations were 1200 ng/ml, 356 ng/mg, 170 ng/mg, and 284 ng/mg, respectively (Fig. 1c, Table 1). Differences in DTG concentrations within each compartment (vagina, cervix, uterus, and plasma) comparing 1 week, 4 weeks, and 12 weeks (n = 6 per group) did not reach statistical significance (Kruskal–Wallis test plasma p = 0.21, cervix p = 0.09, uterus p = 0.70, vagina p = 0.17). Observations were combined over weeks 1, 4, and 12 to evaluate whether plasma concentrations were higher than tissue concentrations for each tissue type separately. Dolutegravir concentrations in plasma were higher than in tissue for each of the three tissue types for every animal (Wilcoxon signed-rank p < 0.001 for cervix, uterus, and vagina analyses, respectively). Together, these results demonstrate the sustained in vivo release of dolutegravir into plasma and its efficient penetration into tissues of the female reproductive tract.
To assess safety and drug release profile of the ultra-long-acting formulation of dolutegravir in a large animal model, two rhesus macaques were subcutaneously administered the ultra-LA dolutegravir formulation (100 mg). Animals were monitored for signs of toxicity and skin reactions at the injection site, including erythema, edema, and hematoma formation, presence of induration, and any other lesions such as abscesses, necrosis, dehiscence, or local inflammation twice a week. The implants were well tolerated with little or no sign of toxicity for 5 months (last point analyzed). Administration of the ultra-long-acting formulation of dolutegravir resulted in sustained dolutegravir concentration in plasma for more than 140 days (Fig. 1d). These results demonstrate the feasibility of ultra-long-acting dolutegravir delivery system for sustained delivery in rhesus macaques. However, this formulation originally developed for BLT mice will have to be further optimized prior to efficacy studies in macaques and its potential future applications in humans.

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