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Nano-Formulated Long-Acting Dolutegravir - cell/tissue/reservoir penetration
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Altering the chemical structure of an antiviral drug may help it penetrate HIV viral reservoirs and could expedite the public health goal of curing HIV, according to a study published by Nature Communications.
In the new study, the authors used a psychiochemical scheme to change the properties of dolutegravir and inserted it into nanocrystals.
The resulting drug crystals were found to circulate throughout the body and target HIV viral reservoirs, which are notoriously difficult to access. Even when a patient's viral load is nearly undetectable, viral reservoirs of HIV persist and can multiply when antivirals are not taken.
This approach was also found to extend the life of dolutegravir, increase the capability to reach viral reservoirs, and reduce viral growth, according to the study. The chemically-altered dolutegravir was able to reach viral reservoirs in the lymph nodes, bone marrow, intestines, and spleen.
Significantly, the drug crystals were non-toxic, did not disintegrate with temperature changes, and demonstrated long-term stability, according to the study. The authors also noted that organs and functionality did not appear to be affected by this treatment.
The crystals are coated with parts of fat and are able to navigate through the protective membrane of cells to be stored in macrophages for weeks, according to the study. Once inside the cells, the drug was released from the crystal as a prodrug, which was broken down into an active drug. The active formulation was then circulated to cells and tissue storage.
"The strength of this system is that it not only can be effective in improving HIV care and prevention but can be applied to many classes of drugs beyond HIV, such as drugs used to treat cancer, other infectious diseases and degenerative diseases that affect the brain," said co-lead researcher Benson Edagwa, PhD.
These findings suggest that chemically altering dolutegravir may expedite an HIV cure, which has been out of reach thus far.
"The new products can optimize HIV restrictive growth so that strategies that may eradicate viral infection would be successful," said co-lead researcher Howard Gendelman, PhD.
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Creation of a long-acting nanoformulated dolutegravir
Nature Communications - Feb 6 2018 - Brady Sillman1, Aditya N. Bade1, Prasanta K. Dash1, Biju Bhargavan1, Ted Kocher1, Saumi Mathews1, Hang Su1,
Georgette D. Kanmogne1, Larisa Y. Poluektova1, Santhi Gorantla1, JoEllyn McMillan1, Nagsen Gautam2,
Yazen Alnouti2, Benson Edagwa1 & Howard E. Gendelman1,2
Abstract
Potent antiretroviral activities and a barrier to viral resistance characterize the human immunodeficiency virus type one (HIV-1) integrase strand transfer inhibitor dolutegravir (DTG). Herein, a long-acting parenteral DTG was created through chemical modification to improve treatment outcomes. A hydrophobic and lipophilic modified DTG prodrug is encapsulated into poloxamer nanoformulations (NMDTG) and characterized by size, shape, polydispersity, and stability. Retained intracytoplasmic NMDTG particles release drug from macrophages and attenuate viral replication and spread of virus to CD4+ T cells. Pharmacokinetic tests in Balb/cJ mice show blood DTG levels at, or above, its inhibitory concentration90 of 64 ng/mL for 56 days, and tissue DTG levels for 28 days. NMDTG protects humanized mice from parenteral challenge of the HIV-1ADA strain for two weeks. These results are a first step towards producing a long-acting DTG for human use by affecting drug apparent half-life, cell and tissue drug penetration, and antiretroviral potency.
Introduction
Antiretroviral therapy (ART) has changed what was once a life-endangering human immunodeficiency virus type one (HIV-1) infection to a chronic manageable disease. Rapid immune suppression, opportunistic infections, and malignancies were attenuated by antiretroviral drug (ARV) therapy1,2. Patients adhering to defined ART regimens can lead a fully productive life, experience limited infection-related morbidities and prevent what was once a rapid inevitable death3. However, treatment advancements have come at some cost. These include toxicities, adherence failures, costly regimens, and common viral mutations linked to specific resistance patterns4. A means to combat each can be achieved through drug regimen adherence facilitated by long-acting slow effective release antiretroviral therapy (LASER ART)5 as defined by slow drug dissolution, enhanced lipophilicity, improved bioavailability, and limited off-target toxicities. Such changes in drug formulation affect the frequency of drug administration. Reductions in disease co-morbidities follow the maintenance of effective antiretroviral drug concentrations in blood, body fluids, and viral tissue reservoir for months or longer6. The end result is improved treatment outcomes.
While such a directive is attractive, not all ARVs can easily be transformed into long-acting medicines. Drug solubility, dissolution, metabolism, protein-binding, and excretion rates are not uniform amongst each drug; and each influence the ARV's half-life and biodistribution profile7,8. Long-acting medicines depend upon the maintenance of plasma drug concentrations, which are linked to depot formation and dissociation within the reticuloendothelial system. Such changes in drug depot formation and dissociation, referred to by the term apparent half-life, distinguish it from the drug's intrinsic half-life. Whether a medicine can be found with significant antiretroviral efficacy, limited resistance patterns and high tolerability for conversion into a long-acting compound also remain uncertain. While ARVs have been developed with long-acting properties based on their solubility and protein-binding capacities, none are currently used clinically and only a few (long-acting injectable cabotegravir and rilpivirine) are in clinical trials6. For these, the drug formulations require larger injection volumes, which can lead to injection site reactions. Adjusted dosing intervals are also required when interpatient pharmacokinetic (PK) profiles come operative9,10,11.
To these ends, our laboratory developed a process to transform standard daily or twice-daily ARVs into hydrophobic and lipophilic drug nanocrystals to extend the apparent half-life by altering drug solubility and metabolic patterns12,13. We developed chemical modification and polymer coating techniques to convert native ARV's into LASER ART. Herein, DTG, an approved second-generation integrase strand transfer inhibitor (INSTI) with potent activity against HIV-1 holds a protein-adjusted inhibitory concentration90 (PA-IC90) of 64 ng/mL and a high barrier to resistance14. As such, DTG is unique amongst other compounds by its robust resistance profile and measured efficacy in inhibiting HIV-1 growth.
In the current study, alteration of the DTG chemical structure is made through myristoylation of the native compound to create a water-insoluble prodrug, termed MDTG, with commensurate crystal formation. When the drug crystals are packaged into nanoparticles, MDTG is rapidly taken up by human monocyte-derived macrophages (MDM) residing for prolonged periods inside the cells. Drug is slowly released from the particle. MDTG undergoes rapid bioconversion to its parent compound in the presence of esterases contained in biological fluids. This process yields a pharmacologically active product15. Such chemical and biological outcomes improve antiretroviral activities up to 30-fold. PK and pharmacodynamic (PD) profiles in mice are also significantly improved over a native drug formulation, exhibiting 5.3-fold extension in drug apparent half-life, broad tissue distribution, and increased antiretroviral efficacy. Our data provide evidence that DTG conversion into a long-acting, slow release formulation is readily achievable with reductions in dose and dosing intervals that could extend to one month or longer. As such, the drug-encased nanoparticles could be employed as a first-step measure to improve regimen adherence, limit adverse reactions by maximizing drug loading and reducing excipient usage. Such improved treatment measures can also minimize drug fatigue and facilitate drug penetrance into viral reservoirs.
Discussion
A key part of any effective antiretroviral regimen rests in ensuring that patients take their prescribed medicines3,18. HIV/AIDS treatment regimens are currently defined by daily or twice-daily dosing intervals19. Adherence underlies clinical responsiveness and any consequent emergence of viral resistance. It also affects the accompanying stability of CD4+ T cell numbers and function4. Thus, any ART regimens that allow infrequent dosing with an ability to maintain consistent drug levels in plasma and tissues sufficiently above the IC90 would improve clinical outcomes and hold viral replication in check5,6. Particle size and physicochemical properties were used to optimize release of drug molecules from delivery systems. Nanoparticle based systems are readily taken up by cells and disseminated into tissues to form drug depots at these sites for subsequent slow release20. Other controlled release injection site drug depot formulations include microparticle carrier systems21,22. However, microparticles tend to aggregate limiting their usage due to lack of particle homogeneity, leading to injection site reactions23.
In the current study, we chemically modified DTG enabling the creation of poloxamer-encased hydrophobic and lipophilic drug nanocrystals. Prodrugs can offer therapeutic benefits over native compounds by providing reduced drug metabolism and toxicity. They may also increase lipophilicity and thus improve cell membrane and tissue permeability of drug24. Proof of concept for such advances are highlighted by antipsychotic drugs. Indeed, these have become widely used as long-acting hydrophobic ester prodrugs25. Likewise, the creation of NMDTG improved drug to polymer interactions to form stable nanocrystals and boosted delivery of the nanoformulated drug into MDM autophagosomes26. The MDTG nanocrystals undergo slow intracellular dissociation within endosomes and prodrug cleavage to protect the cell against viral challenge for up to or beyond 25 days. PK and PD evaluations showed significant improvements in drug apparent half-life, biodistribution, and antiretroviral activities over the native drug.
Altogether, the creation of an ester prodrug of DTG using myristic acid enabled intrinsic drug crystal formation and bioconversion. These reactions occurred in the presence of biological fluids containing esterases yielding pharmacologically active medicines15.
No mouse model exactly reflects PrEP, thus we used both viral restriction and protection murine models in this study. It is noteworthy that the evaluation of viral restriction as performed in hu-PBL mice failed to provide protection against viral challenge by less than one month, as shown by detectable plasma viral load. This likely reflects enhancement in viral susceptibility in the animals based on the large numbers/percentages of activated lymphocytes due to xenoreactivity leading to graft-vs-host disease, timing of human cell reconstitution, larger viral challenge, route of viral challenge, and monotherapy approach27,28,29,30. CD34+ HSC reconstituted NSG mice better reflect human biology as there is no graft-vs-host disease and the reconstitution contains monocyte-macrophages, as well as CD4+ and CD8+ T lymphocytes31. To these ends, this model was used for confirmatory studies. Indeed, cells were quiescent prior to viral challenge. Most importantly, NMDTG-treated HSC-NSG mice demonstrated plasma viral load, as well as tissue viral copies, below the limit of detection in five of seven animals for two weeks against HIV-1 challenge following a single nanoparticle injection. Due to biological limitations in the animal models used, protection in all animals was not achieved. These results reflect what was observed in humans who are at risk of viral infection despite optimal PrEP treatment32. Due to experimental limitations, we also cannot exclude the possibility that high concentrations of plasma drug could suppress HIV-1 breakthrough infection. Based on the intrinsic properties of drug stability and with retention in MDMs in tissues, such chemical prodrug modifications led to the formation of a second drug reservoir beyond the injection site. Such improvements in antiretroviral drug structure and packaging can not only improve drug adherence, but also could reduce systemic toxicities. NMDTG PK studies in non-human primates further validated these results33. Three male rhesus macaques were administered a single IM dose of NMDTG at a concentration of 25.5 mg/kg DTG-eq. Plasma DTG concentrations remained above the PA-IC90 for 35 days and increased DTG apparent half-life to 467 h. Notably, this came with no alterations in neutrophil, lymphocyte, or monocyte counts, animal weights, or liver and kidney metabolic profiles. This study further demonstrates that a single IM injection of long-acting NMDTG can provide plasma levels above the PA-IC90 for one month and can greatly extend drug apparent half-life, with no signs of toxicity.
Long-acting drug formulations have been extensively investigated, including their prolonged apparent half-life, high protein-binding, lipid or surfactant drug nanocrystal encasement, rapid drug entry into monocyte-macrophages, and slow drug release34. These long-acting properties are mostly due to enhanced particle/drug stability, rapid cell and barrier penetration, and/or slowed intracellular drug hydrolysis24. Cumulatively, these properties enhance therapeutic responses, allow for cell-mediated drug delivery, and enable improved delivery of drugs to areas of poor drug penetrance and viral reservoirs35. One of the most important of these is uptake and sequestration into MDM. Highly mobile MDM have large storage capacities and can act as Trojan Horses for drug delivery to circulating and tissue CD4+ T cells and viral reservoirs36. This is especially operative when describing lymphoid organs, where macrophages and T cells are in intimate contact, permitting passage of drug to such major reservoirs of HIV infection37,38,39. Here, we demonstrate that drug released from NMDTG-treated MDM can act upon T cells and significantly inhibit and prevent spreading of viral infection within T cell cultures. Macrophages can protect drugs from metabolism—prolonging apparent half-life—and enhance efficacy, PK, and biodistribution of the drug delivery system40,41,42. Once inside the macrophage, drug particles can be stored in late- and recycling-endosomes, as well as autophagosomes and as such, create a secondary drug depot within the tissue macrophage independent of the muscle site of injection26,43,44. Indeed—and in addition to the macrophage's phagocytic, clearance, antigen presentation, and secretory functions—the cells also serve as viral sanctuaries, vehicles for viral transport, and as reservoirs for HIV-1 replication45,46. Thus, delivery of drug to macrophages can serve multiple critical purposes.
In clinical settings, DTG rarely elicits viral resistance mutations in infected patients when used as first-line therapy, with suboptimal adherence driving these mutations47. In vitro, the most common mutation against DTG is the R263K that severely reduces viral replication fitness, reducing the impact and rendering it insignificant for HIV/AIDS treatments48. Prevention of renewed viral infection emerging from tissue reservoirs experiencing suboptimal drug levels may allow infected cells to die off through normal cell turnover without spreading virus to other cells that may act as viral reservoirs49. Thus, the changes in antiretroviral drug treatment made here could limit viral replication in its native reservoirs, allowing antiretroviral drugs alone to keep the virus in check and attenuate new infections. The offering of sustained high plasma and tissue drug levels in time periods measured in months, without notable drug peaks and troughs, could also enable more efficient excision of integrated proviral DNA through CRISPR/Cas9 technologies50. Currently under evaluation for clinical use are transdermal patches and ARV implants. Such devices, including dapivirine vaginal rings, subdermal silicone tenofovir alafenamide and polymeric EFdA, are being developed for PrEP51,52,53. Refillable channel devices capable of loading multiple drugs have also been described54. These devices could be loaded with NMDTG and/or other LASER ART nanocrystals to further extend the apparent half-lives of the parent medicines with a single implant. LASER ART-loaded microneedle patches similar to those used to deliver injectable contraceptives could also be used to alleviate pain during administration55.
The concept of long-acting injectable drugs is well received by patients with HIV, with 73% indicating that they would definitely or probably try them56. This number goes up to 84% when patients were asked about monthly dosing as opposed to weekly or biweekly dosing. Conceivably, bimonthly or longer dosing intervals would be even more attractive. Future endeavors to further extend drug apparent half-lives and better target macrophages, including further drug modifications, linking two drug molecules to a single linker, combining divergent classes of antiretroviral drugs, and nanoparticle surface decorations will shape the future of long-acting antiretroviral therapy. Future works will focus on refinements in drug modification and formulation, and the packaging of multiple drugs into a single formulation for delivery. Strategies such as these will further improve adherence to drug regimens and biodistribution profiles, and maintain high drug levels for even longer periods of time, limiting viral resistance mutations and, overall, improving ART efficacy and patient outcomes33,57,58.
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