Long-acting intramuscular injections of ELQ-331, an antimalarial agent.

The overarching premise of this investigation is that injectable, long-acting antimalarial medication would encourage adherence to a dosage regimen for populations at risk of contracting the disease. To advance support for this goal, we have developed oil-based formulations of ELQ-331 (a prodrug of ELQ-300) that perform as long-acting, injectable chemoprophylactics with drug loading as high as 160 mg/ml of ELQ-331. In a pharmacokinetic study performed with rats, a single intramuscular injection of 12.14 mg/kg maintained higher plasma levels than the previously established minimum fully protective plasma concentration (33.25 ng/ml) of ELQ-300 for more than 4 weeks. The formulations were well tolerated by the rats and the tested dose produced no adverse reactions. We believe that by extending the length of time between subsequent injections, these injectable oil-based solutions of ELQ-331 can offer a more accessible, low-cost option for long-acting disease prevention and reduced transmission in malaria-endemic regions and may also be of use to travelers.

Design and Testing of a Cabotegravir Implant for HIV Prevention.

Long-acting antiretroviral implants could help protect high-risk individuals from HIV infection. We describe the design and testing of a long-acting reservoir subcutaneous implant capable of releasing cabotegravir for several months. We compressed cabotegravir and excipients into cylindrical pellets and heat-sealed them in tubing composed of hydrophilic poly(ether-urethane) -. The implants have a 47 mm lumen length, 3.6 mm outer diameter, and 200 µm wall thickness. Four cabotegravir pellets were sealed in the membrane, with a total drug loading of 274 ± 3 mg. In vivo, the implants released 348 ± 107 µg/day (median value per implant, N = 41) of cabotegravir in rhesus macaques. Five implants generated an average cabotegravir plasma concentration of 373 ng/ml in rhesus macaques. The non-human primates tolerated the implant without gross pathology or microscopic signs of histopathology compared to placebo implants. Cabotegravir plasma levels in macaques dropped below detectable levels within two weeks after the removal of the implants.

Design of a Drug-Eluting Subcutaneous Implant of the Antiretroviral Tenofovir Alafenamide Fumarate.

PURPOSE: Sexual transmission of HIV has been clinically proven to be preventable with a once-daily oral tablet; however, missed doses dramatically increase the risk of HIV infection. Long-acting subcutaneous implants do not allow the user to miss a dose. A desirable long-acting drug-eluting implant can deliver a constant amount of drug, adjust the delivered dose, and be readily manufactured. We present a long-acting, subcutaneous implant design composed of tenofovir alafenamide hemifumarate (TAF) pellets loaded in a sealed polyether urethane tube for the prevention of HIV transmission. METHODS: Implants were prepared with pressed drug pellets and extruded polyurethane tubing. In vitro release rate of implants using different pellet formulations, rate-controlling membranes, and geometries were measured. RESULTS: Tenofovir alafenamide release appeared to be governed by a pseudo-steady state and followed a mass transport model of release from a cylindrical drug reservoir. Implant seal integrity was tested and confirmed using mechanical testing. The inclusion of sodium chloride in the pellet increased the release rate and reduced initial lag. The release was sustained for 100 days. CONCLUSIONS: The release rate of tenofovir alafenamide mechanistically varied with geometry and rate controlling membrane composition. The polyether urethane implant presented herein is modular and tunable to adjust the release rate and duration of the TAF release.

A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques.

We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbit and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a TAF dose of 10 µg/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. The level of inflammation in the primates was markedly lower in the placebo group than in the active-implant group. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve a sustained target dose, we observed an unacceptable inflammatory response locally at the implant tissue interface.

The synthesis of tetra-modified RNA for the multidimensional control of gene expression via light-activated RNA interference.

Light-activated RNA interference (LARI) is an effective way to control gene expression with light. This, in turn, allows for the spacing, timing and degree of gene expression to be controlled by the spacing, timing and amount of light irradiation. The key mediators of this process are siRNA or dsRNA that have been modified with four photocleavable groups of dimethoxy nitro phenyl ethyl (DMNPE), located on the four terminal phosphate groups of the duplex RNA. These mediators can be easily synthesized and purified using two readily available products: synthetic RNA oligonucleotides and DMNPE-hydrazone. The synthesis of the tetra-DMNPE-modified duplex RNA is made possible by a remarkable regiospecificity of DMNPE for terminal phosphates (over internal phosphates or nucleobases) that we have previously identified. The four installed DMNPE groups effectively limit RNAi until irradiation cleaves them, releasing native, active siRNA. By using the described protocol, any process that is mediated by RNAi can be controlled with light. Although other methods exist to control gene expression with light by using specialized reagents, this method requires only two commercially available products. The protocol takes ∼3 d in total for the preparation of modified RNA.

Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups.

Diazo-based precursors of photolabile groups have been used extensively for modifying nucleic acids, with the intention of toggling biological processes with light. These processes include transcription, translation and RNA interference. In these cases, the photolabile groups have been typically depicted as modifying the phosphate backbone of RNA and DNA. In this work we find that these diazo-based reagents in fact react very poorly with backbone phosphates. Instead, they show a remarkable specificity for terminal phosphates and very modest modification of the nucleobases. Furthermore, the photo deprotection of these terminal modifications is shown to be much more facile than nucleobase modified sites. In this study we have characterized this regiospecificity using RNA duplexes and model nucleotides, analyzed using LC/MS/MS. We have also applied this understanding of the regio-specificity to our technique of light activated RNA interference (LARI). We examined 27-mer double-stranded precursors of siRNA ('dsRNA'), and have modified them using the photo-cleavable di-methoxy nitro phenyl ethyl group (DMNPE) group. By incorporating terminal phosphates in the dsRNA, we are able to guide DMNPE to react at these terminal locations. These modified dsRNA duplexes show superior performance to our previously described DMNPE-modified siRNA, with the range of expression that can be toggled by light increasing by a factor of two.