Nanoparticle-Based ARV Drug Combinations for Synergistic Inhibition of Cell-Free and Cell-Cell HIV Transmission.

Nanocarrier-based drug delivery systems are playing an emerging role in human immunodeficiency virus (HIV) chemoprophylaxis and treatment due to their ability to alter the pharmacokinetics and improve the therapeutic index of various antiretroviral (ARV) drug compounds used alone and in combination. Although several nanocarriers have been described for combination delivery of ARV drugs, measurement of drug-drug activities facilitated by the use of these nanotechnology platforms has not been fully investigated for topical prevention. Here, we show that physicochemically diverse ARV drugs can be encapsulated within polymeric nanoparticles to deliver multidrug combinations that provide potent HIV chemoprophylaxis in relevant models of cell-free, cell-cell, and mucosal tissue infection. In contrast to existing approaches that coformulate ARV drug combinations together in a single nanocarrier, we prepared single-drug-loaded nanoparticles that were subsequently combined upon administration. ARV drug-nanoparticles were prepared using emulsion-solvent evaporation techniques to incorporate maraviroc (MVC), etravirine (ETR), and raltegravir (RAL) into poly(lactic-co-glycolic acid) (PLGA) nanoparticles. We compared the antiviral potency of the free and formulated drug combinations for all pairwise and triple drug combinations against both cell-free and cell-associated HIV-1 infection in vitro. The efficacy of ARV-drug nanoparticle combinations was also assessed in a macaque cervicovaginal explant model using a chimeric simian-human immunodeficiency virus (SHIV) containing the reverse transcriptase (RT) of HIV-1. We observed that our ARV-NPs maintained potent HIV inhibition and were more effective when used in combinations. In particular, ARV-NP combinations involving ETR-NP exhibited significantly higher antiviral potency and dose-reduction against both cell-free and cell-associated HIV-1 BaL infection in vitro. Furthermore, ARV-NP combinations that showed large dose-reduction were identified to be synergistic, whereas the equivalent free-drug combinations were observed to be strictly additive. Higher intracellular drug concentration was measured for cells dosed with the triple ARV-NP combination compared to the equivalent unformulated drugs. Finally, as a first step toward evaluating challenge studies in animal models, we also show that our ARV-NP combinations inhibit RT-SHIV virus propagation in macaque cervicovaginal tissue and block virus transmission by migratory cells emigrating from the tissue. Our results demonstrate that ARV-NP combinations control HIV-1 transmission more efficiently than free-drug combinations. These studies provide a rationale to better understand the role of nanocarrier systems in facilitating multidrug effects in relevant cells and tissues associated with HIV infection.

Antiretroviral (ARV) Properties Dictate Long-Acting Release and Tissue Partitioning Behaviors in Multidrug Subcutaneous Implants.

Subcutaneous implants can provide patients with long-acting, compliance-independent drug dosing. For this reason, subcutaneous implants have shown emerging interest in human immunodeficiency virus (HIV) prevention. However, any successful long-acting HIV-prevention device will require multidrug dosing, which poses a challenge for formulation considering the physicochemically diverse selection of antiretroviral (ARV) candidates. As a method that has shown the capacity of efficient multidrug delivery, we assessed electrospun fiber implants composed of three synergistically potent ARVs and a biodegradable polymer selected by in vitro release studies. In mice, subcutaneous electrospun fiber implants exhibit burst release of the more hydrophilic drugs maraviroc (MVC) and raltegravir (RAL), which could be reduced via simple prewash treatments of the implants. Over an extended 120 day time frame, fiber implants show drug-specific differences in release time frames and magnitudes in blood serum. However, end-point drug tissue concentrations show that the most hydrophobic drug etravirine (ETR) remains in high concentrations within the implant and in local skin tissue biopsies. Furthermore, ETR is found to be capable of significant partitioning into lymph nodes, the lower female reproductive tract, and the rectum. Topologically smooth film implants also exhibit the same drug-dependent trends. Therefore, we illustrate that drug release and drug tissue partitioning are largely dictated by drug properties. Further, we find that the properties of ETR enable significant drug quantities within the tissues most relevant to HIV protection. Evidence from this work emphasizes the need for a greater focus on drug properties and prodrug strategies to enable relevant, extended, and targeted drug release.

Prodrug approaches for the development of a long-acting drug delivery systems.

Long-acting formulations are designed to reduce dosing frequency and simplify dosing schedules by providing an extended duration of action. One approach to obtain long-acting formulations is to combine long-acting prodrugs (LA-prodrug) with existing or emerging drug delivery technologies (DDS). The design criteria for long-acting prodrugs are distinct from conventional prodrug strategies that alter absorption, distribution, metabolism, and excretion (ADME) parameters. Our review focuses on long-acting prodrug delivery systems (LA-prodrug DDS), which is a subcategory of long-acting formulations where prodrug design enables DDS formulation to achieve an extended duration of action that is greater than the parent drug. Here, we define LA-prodrugs as the conjugation of an active pharmaceutical ingredient (API) to a promoiety group via a cleavable covalent linker, where both the promoiety and linker are selected to enable formulation and administration from a drug delivery system (DDS) to achieve an extended duration of action. These LA-prodrug DDS results in an extended interval where the API is within a therapeutic range without necessarily altering ADME as is typical of conventional prodrugs. The conversion of the LA-prodrug to the API is dependent on linker cleavage, which can occur before or after release from the DDS. The requirement for linker cleavage provides an additional tool to prolong release from these LA-prodrug DDS. In addition, the physicochemical properties of drugs can be tuned by promoiety selection for a particular DDS. Conjugation with promoieties that are carriers or amenable to assembly into carriers can also provide access to formulations designed for extending duration of action. LA-prodrugs have been applied to a wide variety of drug delivery strategies and are categorized in this review by promoiety size and complexity. Small molecule promoieties (typically MW < 1000 Da) have been used to improve encapsulation or partitioning as well as broaden APIs for use with traditional long-acting formulations such as solid drug dispersions. Macromolecular promoieties (typically MW > 1000 Da) have been applied to hydrogels, nanoparticles, micelles, dendrimers, and polymerized prodrug monomers. The resulting LA-prodrug DDS enable extended duration of action for active pharmaceuticals across a wide range of applications, with target release timescales spanning days to years.

Catalytic importance of a protonated adenosine in the hairpin ribozyme active site.

The hairpin ribozyme accelerates the rate of phosphodiester transfer reactions by at least 5 orders of magnitude. To achieve this rate enhancement, the active site forms via a substrate helix docking event that constrains the scissile phosphate linkage and positions G8 and A38 for catalysis, both of which have been implicated as sites of proton transfer in general acid-base catalysis. To investigate the functional groups required for hairpin activity, we previously reported a series of nucleotide analogue interference mapping experiments [Ryder, S. P., et al. (2001) RNA 7, 1454-1463]. The critical functional groups implicated in those studies were largely consistent with subsequent X-ray crystal structures, but the lack of A38 interference with 8-azaadenosine (n(8)A), a pK(a) perturbed nucleotide analogue, argued against functional base ionization at this site. This is inconsistent with a transition state crystal structure and other biochemical studies. To address this discrepancy, we investigated the hairpin ribozyme with an expanded set of pK(a) perturbed adenosine analogues containing fluorine. A38 was the only site that showed persistent and strong interference with low pK(a) analogues across a variety of construct/substrate pairs. This interference pattern suggests that A38 base ionization is required for catalytic activity. The lack of n(8)A interference at A38, in spite of its reduced pK(a), likely results from n(8)A stabilization of the docked state, which requires an unusual syn glycosidic base conformation at A38 for active site assembly. The fluorinated adenosine analogues are better suited to identify sites of functional ionization in systems where structural rearrangements are closely coupled to catalytic steps. All pK(a) reduced analogues, including those of the previous study, produce selective interference at A38 when substrates are stably bound and docked, consistent with the importance of base ionization at this site.

Sustained Intracellular Raltegravir Depots Generated with Prodrugs Designed for Nanoparticle Delivery.

Polymeric nanocarriers have been extensively used to improve the delivery of hydrophobic drugs, but often provide low encapsulation efficiency and percent loading for hydrophilic compounds. In particular, insufficient loading of hydrophilic antiretroviral drugs such as the integrase inhibitor raltegravir (RAL) has limited the development of sustained-release therapeutics or prevention strategies against HIV. To address this, we developed a generalizable prodrug strategy using RAL as a model where loading, release and subsequent hydrolysis can be tuned by promoiety selection. Prodrugs with large partition coefficients increased the encapsulation efficiency up to 25-fold relative to RAL, leading to significant dose reductions in antiviral activity assays. The differential hydrolysis rates of these prodrugs led to distinct patterns of RAL availability and observed antiviral activity. We also developed a method to monitor the temporal distribution of both prodrug and RAL in cells treated with free prodrug or prodrug-NPs. Results of these studies indicated that prodrug-NPs create an intracellular drug reservoir capable of sustained intracellular drug release. Overall, our results suggest that the design of prodrugs for specific polymeric nanocarrier systems could provide a more generalized strategy to formulate physicochemically diverse hydrophilic drugs with a number of biomedical applications.

Fluorine substituted adenosines as probes of nucleobase protonation in functional RNAs.

Ionized nucleobases are required for folding, conformational switching, or catalysis in a number of functional RNAs. A common strategy to study these sites employs nucleoside analogues with perturbed pKa, but the interpretation of these studies is often complicated by the chemical modification introduced, in particular modifications that add, remove, or translocate hydrogen bonding groups in addition to perturbing pKa values. In the present study we present a series of fluorine substituted adenosine analogues that produce large changes in N1 pKa values with minimal structural perturbation. These analogues include fluorine for hydrogen substitutions in the adenine ring of adenosine and 7-deaza-adenosine with resulting N1 pKa values spanning more than 4 pKa units. To demonstrate the utility of these analogues we have conducted a nucleotide analogue interference mapping (NAIM) study on a self-ligating construct of the Varkud Satellite (VS) ribozyme. We find that each of the analogues is readily incorporated by T7 RNA polymerase and produces fully active transcripts when substituted at the majority of sites. Strong interferences are observed for three sites known to be critical for VS ribozyme function, most notably A756. Substitutions at A756 lead to slight enhancements in activity for elevated pKa analogues and dramatic interferences in activity for reduced pKa analogues, supporting the proposed catalytic role for this base. The structural similarity of these analogues, combined with their even incorporation and selective interference, provides an improved method for identifying sites of adenosine protonation in a variety of systems.

Rapidly Biodegrading PLGA-Polyurethane Fibers for Sustained Release of Physicochemically Diverse Drugs.

Sustained release of physicochemically diverse drugs from electrospun fibers remains a challenge and precludes the use of fibers in many medical applications. Here, we synthesize a new class of polyurethanes with poly(lactic-co-glycolic acid) (PLGA) moieties that degrade faster than polyurethanes based on polycaprolactone. The new polymers, with varying hard to soft segment ratios and fluorobenzene pendant group content, were electrospun into nanofibers and loaded with four physicochemically diverse small molecule drugs. Polymers were characterized using GPC, XPS, and 19F NMR. The size and morphology of electrospun fibers were visualized using SEM, and drug/polymer compatibility and drug crystallinity were evaluated using DSC. We measured in vitro drug release, polymer degradation and cell-culture cytotoxicity of biodegradation products. We show that these newly synthesized PLGA-based polyurethanes degrade up to 65-80% within 4 weeks and are cytocompatible in vitro. The drug-loaded electrospun fibers were amorphous solid dispersions. We found that increasing the hard to soft segment ratio of the polymer enhances the sustained release of positively charged drugs, whereas increasing the fluorobenzene pendant content caused more rapid release of some drugs. In summary, increasing the hard segment or fluorobenzene pendant content of segmented polyurethanes containing PLGA moieties allows for modulation of physicochemically diverse drug release from electrospun fibers while maintaining a biologically relevant biodegradation rate.

Nucleotide analog interference mapping.

Nucleotide analog interference mapping (NAIM) is a powerful chemogenetic technique that rapidly identifies chemical groups essential for RNA function. Using a series of phosphorothioate-tagged nucleotide analogs, each carrying different modifications of nucleobase or backbone functionalities, it is possible to simultaneously, yet individually, assess the contribution of particular functional groups to an RNA's activity at every position within the molecule. In contrast to traditional mutagenesis, which modifies RNA on the nucleobase level, the smallest mutable unit in a NAIM analysis is a single atom, providing a detailed description of interactions at critical nucleotides. Because the method introduces modified nucleotides by in vitro transcription, NAIM offers a straightforward and efficient approach to study any RNA that has a selectable function, and it can be applied to RNAs of nearly any length.

Electric fields at the active site of an enzyme: direct comparison of experiment with theory.

The electric fields produced in folded proteins influence nearly every aspect of protein function. We present a vibrational spectroscopy technique that measures changes in electric field at a specific site of a protein as shifts in frequency (Stark shifts) of a calibrated nitrile vibration. A nitrile-containing inhibitor is used to deliver a unique probe vibration to the active site of human aldose reductase, and the response of the nitrile stretch frequency is measured for a series of mutations in the enzyme active site. These shifts yield quantitative information on electric fields that can be directly compared with electrostatics calculations. We show that extensive molecular dynamics simulations and ensemble averaging are required to reproduce the observed changes in field.

Vibrational Stark effects calibrate the sensitivity of vibrational probes for electric fields in proteins.

Infrared spectroscopy is widely used to probe local environments and dynamics in proteins. The introduction of a unique vibration at a specific site of a protein or more complex assembly offers many advantages over observing the spectra of an unmodified protein. We have previously shown that infrared frequency shifts in proteins can arise from differences in the local electric field at the probe vibration. Thus, vibrational frequencies can be used to map electric fields in proteins at many sites or to measure the change in electric field due to a perturbation. The Stark tuning rate gives the sensitivity of a vibrational frequency to an electric field, and for it to be useful, the Stark tuning rate should be as large as possible. Vibrational Stark effect spectroscopy provides a direct measurement of the Stark tuning rate and allows a quantitative interpretation of frequency shifts. We present vibrational Stark spectra of several bond types, extending our work on nitriles and carbonyls and characterizing four additional bond types (carbon-fluorine, carbon-deuterium, azide, and nitro bonds) that are potential probes for electric fields in proteins. The measured Stark tuning rates, peak positions, and extinction coefficients provide the primary information needed to design amino acid analogues or labels to act as probes of local environments in proteins.