Liver-targeted polymeric prodrugs delivered subcutaneously improve tafenoquine therapeutic window for malaria radical cure.

Approximately 3.3 billion people live with the threat of Plasmodium vivax malaria. Infection can result in liver-localized hypnozoites, which when reactivated cause relapsing malaria. This work demonstrates that an enzyme-cleavable polymeric prodrug of tafenoquine addresses key requirements for a mass administration, eradication campaign: excellent subcutaneous bioavailability, complete parasite control after a single dose, improved therapeutic window compared to the parent oral drug, and low cost of goods sold (COGS) at less than $1.50 per dose. Liver targeting and subcutaneous dosing resulted in improved liver:plasma exposure profiles, with increased efficacy and reduced glucose 6-phosphate dehydrogenase-dependent hemotoxicity in validated preclinical models. A COGS and manufacturability analysis demonstrated global scalability, affordability, and the ability to redesign this fully synthetic polymeric prodrug specifically to increase global equity and access. Together, this polymer prodrug platform is a candidate for evaluation in human patients and shows potential for P. vivax eradication campaigns.

Predicting the efficiency of oxygen-evolving electrolysis on the Moon and Mars.

Establishing a permanent human presence on the Moon or Mars requires a secure supply of oxygen for life support and refueling. The electrolysis of water has attracted significant attention in this regard as water-ice may exist on both the Moon and Mars. However, to date there has been no study examining how the lower gravitational fields on the Moon and Mars might affect gas-evolving electrolysis when compared to terrestrial conditions. Herein we provide experimental data on the effects of gravitational fields on water electrolysis from 0.166 g (lunar gravity) to 8 g (eight times the Earth's gravity) and show that electrolytic oxygen production is reduced by around 11% under lunar gravity with our system compared to operation at 1 g. Moreover, our results indicate that electrolytic data collected using less resource-intensive ground-based experiments at elevated gravity (>1 g) may be extrapolated to gravitational levels below 1 g.

Liver-targeted polymeric prodrugs of 8-aminoquinolines for malaria radical cure.

Primaquine and tafenoquine are the two 8-aminoquinoline (8-AQ) antimalarial drugs approved for malarial radical cure - the elimination of liver stage hypnozoites after infection with Plasmodium vivax. A single oral dose of tafenoquine leads to high efficacy against intra-hepatocyte hypnozoites after efficient first pass liver uptake and metabolism. Unfortunately, both drugs cause hemolytic anemia in G6PD-deficient humans. This toxicity prevents their mass administration without G6PD testing given the approximately 400 million G6PD deficient people across malarial endemic regions of the world. We hypothesized that liver-targeted delivery of 8-AQ prodrugs could maximize liver exposure and minimize erythrocyte exposure to increase their therapeutic window. Primaquine and tafenoquine were first synthesized as prodrug vinyl monomers with self-immolative hydrolytic linkers or cathepsin-cleavable valine-citrulline peptide linkers. RAFT polymerization was exploited to copolymerize these prodrug monomers with hepatocyte-targeting GalNAc monomers. Pharmacokinetic studies of released drugs after intravenous administration showed that the liver-to-plasma AUC ratios could be significantly improved, compared to parent drug administered orally. Single doses of the liver-targeted, enzyme-cleavable tafenoquine polymer were found to be as efficacious as an equivalent dose of the oral parent drug in the P. berghei causal prophylaxis model. They also elicited significantly milder hemotoxicity in the humanized NOD/SCID mouse model engrafted with red blood cells from G6PD deficient donors. The clinical application is envisioned as a single subcutaneous administration, and the lead tafenoquine polymer also showed excellent bioavailability and liver-to-blood ratios exceeding the IV administered polymer. The liver-targeted tafenoquine polymers warrant further development as a single-dose therapeutic via the subcutaneous route with the potential for broader patient administration without a requirement for G6PD diagnosis.

Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies.

The formulation and delivery of biopharmaceutical drugs, such as monoclonal antibodies and recombinant proteins, poses substantial challenges owing to their large size and susceptibility to degradation. In this Review we highlight recent advances in formulation and delivery strategies--such as the use of microsphere-based controlled-release technologies, protein modification methods that make use of polyethylene glycol and other polymers, and genetic manipulation of biopharmaceutical drugs--and discuss their advantages and limitations. We also highlight current and emerging delivery routes that provide an alternative to injection, including transdermal, oral and pulmonary delivery routes. In addition, the potential of targeted and intracellular protein delivery is discussed.

Advancing polymeric delivery systems amidst a nucleic acid therapy renaissance.

Nucleic acid therapeutics are attracting renewed interest due to recent clinical advances and product approvals. Most leading programs use chemical conjugates, or viral vectors in the case of gene therapy, while several use no delivery system at all. Polymer systems, which have been at the periphery of this renaissance, often involve greater molecular complexity than competing approaches, which must be justified by their advantages. Advanced analytical methods, along with biological tools for characterizing biotransformation and intracellular trafficking, are increasingly being applied to nucleic acid delivery systems including those based on polymers. These frontiers of investigation create the opportunity for an era where highly defined polymer compositions are optimized based on mechanistic insights in a way that has not been previously possible, offering the prospect of greater differentiation from alternatives. This will require integrated collaboration between polymer scientists and those from other disciplines.

Analysis of lipid nanoparticles by Cryo-EM for characterizing siRNA delivery vehicles.

Lipid nanoparticles are self-assembling, dynamic structures commonly used as carriers of siRNA, DNA, and small molecular therapeutics. Quantitative analysis of particle characteristics such as morphological features can be very informative as biophysical properties are known to influence biological activity, biodistribution, and toxicity. However, accurate characterization of particle attributes and population distributions is difficult. Cryo-Electron Microscopy (Cryo-EM) is a leading characterization method and can reveal diversity in particle size, shape and lamellarity, however, this approach is traditionally used for qualitative review or low throughput image analysis due to inherent EM micrograph contrast characteristics and artifacts in the images which limit extraction of quantitative feature values. In this paper we describe the development of a semiautomatic image analysis framework to facilitate reliable image enhancement, object segmentation, and quantification of nanoparticle attributes in Cryo-EM micrographs. We apply this approach to characterize two formulations of siRNA-loaded lipid nanoparticles composed of cationic lipid, cholesterol, and poly(ethylene glycol)-lipid, where the formulations differ only by input component ratios. We found Cryo-EM image analysis provided reliable size and morphology information as well as the detection of smaller particle populations that were not detected by standard dynamic light scattering (DLS) analysis.

Mechanistically probing lipid-siRNA nanoparticle-associated toxicities identifies Jak inhibitors effective in mitigating multifaceted toxic responses.

A major hurdle for harnessing small interfering RNA (siRNA) for therapeutic application is an effective and safe delivery of siRNA to target tissues and cells via systemic administration. While lipid nanoparticles (LNPs) composed of a cationic lipid, poly-(ethylene glycol) lipid and cholesterol, are effective in delivering siRNA to hepatocytes via systemic administration, they may induce multi-faceted toxicities in a dose-dependent manner, independently of target silencing. To understand the underlying mechanism of toxicities, pharmacological probes including anti-inflammation drugs and specific inhibitors blocking different pathways of innate immunity were evaluated for their abilities to mitigate LNP-siRNA-induced toxicities in rodents. Three categories of rescue effects were observed: (i) pretreatment with a Janus kinase (Jak) inhibitor or dexamethasone abrogated LNP-siRNA-mediated lethality and toxicities including cytokine induction, organ impairments, thrombocytopenia and coagulopathy without affecting siRNA-mediated gene silencing; (ii) inhibitors of PI3K, mammalian target of rapamycin (mTOR), p38 and IκB kinase (IKK)1/2 exhibited a partial alleviative effect; (iii) FK506 and etoricoxib displayed no protection. Furthermore, knockout of Jak3, tumor necrosis factor receptors (Tnfr)p55/p75, interleukin 6 (IL-6) or interferon (IFN)-γ alone was insufficient to alleviate LNP-siRNA-associated toxicities in mice. These indicate that activation of innate immune response is a primary trigger of systemic toxicities and that multiple innate immune pathways and cytokines can mediate toxic responses. Jak inhibitors are effective in mitigating LNP-siRNA-induced toxicities.

Quantitative evaluation of siRNA delivery in vivo.

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.

Evaluation of efficacy, biodistribution, and inflammation for a potent siRNA nanoparticle: effect of dexamethasone co-treatment.

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.

NMR spectroscopic evaluation of the internal environment of PLGA microspheres.

The internal environment of poly(lactide-co-glycolide) (PLGA) microspheres was characterized using 31P and 13C solid-state and solution NMR spectroscopy. Physical and chemical states of encapsulated phosphate- and histidine-containing porogen excipients were evaluated using polymers with blocked (i.e., esterified) or unblocked (free acid) end groups. Spectroscopic and gravimetric results demonstrated that the encapsulated porogen deliquesced upon hydration at 84% relative humidity to form a solution environment inside the microspheres. Dibasic phosphate porogen encapsulated in unblocked PLGA was partially titrated to the monobasic form, while in the same formulation 13C NMR showed partial protonation of the histidine imidazole. Similarly, encapsulated monobasic phosphate was partially converted to phosphoric acid. Coencapsulation of monobasic and dibasic phosphate porogens resulted in a single peak on hydration, indicating chemical exchange between discrete excipient microphases. Exogenous buffer addition differentiated external from internal, nontitratable, excipient populations. Microspheres containing dibasic phosphate porogen were hydrated with fetal calf serum, incubated at 37 degrees C, and characterized by 31P NMR through the polymer erosion phase. Within 48 h the 31P chemical shift moved over 2 ppm upfield and the line width narrowed to <60 Hz; there was little additional change through day 14. This indicated complete conversion to the monobasic phosphate form throughout the polydisperse sample and that pH remained below 4 but above the phosphoric acid p K a during matrix erosion.

Atomizing into a chilled extraction solvent eliminates liquid gas from a spray-freeze drying microencapsulation process.

A spray-freeze drying encapsulation process using direct atomization into a chilled extraction solvent (ACES), in the absence of liquefied gas, was developed. Heat transfer models, developed to estimate droplet freezing time (t(f)), identified ACES conditions where solvent extraction, nonsolvent influx, and droplet deformation were minimized. Calculated t(f)'s for dichloromethane and dichloroethane droplets were 98 and 46 ms, respectively, using atomization into liquid nitrogen (ALN2). For droplets <100 microm, this was shorter than the calculated headspace residence time, indicating freezing precedes cryogen impact. Calculated t(f)'s for ACES ranged from 9 to 36 ms. The longest t(f)'s resulted in collapsed, asymmetric particles with phase-separated cores and high nonsolvent residuals (>10%). Intermediate t(f)'s produced spherical-cap particles with rough exteriors and a mixture of solid and phase-separated structures. The shortest t(f)'s produced smooth, spherical-cap particles with solid cores, resembling particles made by ALN2; residual solvent levels were similar or superior to those with ALN2. Phase separation within droplets, induced upon extraction solvent contact in ACES, was minimized for cases where t(f) or=1.3. These results, obtained with cryogen temperatures up to -122 degrees C, demonstrate encapsulation by ACES is possible if freezing is sufficiently rapid, enabling milder operating temperatures.

Poly(lactide-co-glycolide) microsphere formulations of darbepoetin alfa: spray drying is an alternative to encapsulation by spray-freeze drying.

PURPOSE: The purpose of this work was to evaluate spray-freeze drying and spray drying processes for encapsulation of darbepoetin alfa (NESP, Aranesp). METHODS: Darbepoetin alfa was encapsulated in poly(lactide-co-glycolide) by spray-freeze drying and by spray drying. Integrity was evaluated by size-exclusion chromatography and Western blot. Physical properties and in vitro release kinetics were characterized. Pharmacokinetics and pharmacodynamics were evaluated in nude rats. RESULTS: Microspheres produced by spray drying were larger than those produced by spray-freeze drying (69 microm vs. 29 microm). Postencapsulation integrity was excellent for both processes, with < 2% dimer by size-exclusion chromatography. In vitro release profiles were similar, with low burst (< 25%) and low cumulative protein recovery at 4 weeks (< or = 30%), after which time covalent dimer (< or = 6.5%) and high molecular weight aggregates (< or = 2.3%) were recovered by denaturing extraction. After a single injection, darbepoetin alfa was detected in serum through 4 weeks for all microsphere formulations tested in vivo, although relative bioavailability was higher for spray-freeze drying (28%) compared with spray drying (21%; p = 0.08) as were yields (73-82% vs. 34-57%, respectively). For both processes hemoglobin was elevated for 7 weeks, over twice as long as unencapsulated drug. CONCLUSIONS: Spray drying, conducted at pilot scale with commercial equipment, is comparable to spray-freeze drying for encapsulation of darbepoetin alfa.

Protein powders for encapsulation: a comparison of spray-freeze drying and spray drying of darbepoetin alfa.

PURPOSE: To evaluate spray-freeze drying and spray drying processes for fabricating micron-sized particles of darbepoetin alfa (NESP, Aranesp) with uniform size distribution and retention of protein integrity, requirements for encapsulation. METHODS: Darbepoetin alfa was spray-freeze dried using ultrasonic atomization at 120 kHz and 25 kHz and spray dried at bench-top and pilot scales. Reconstituted powders were evaluated by size exclusion chromatography and UV/VIS spectroscopy. Powder physical properties were also characterized. RESULTS: Spray-freeze drying resulted in aggregation of darbepoetin alfa. Aggregates (primarily insoluble) formed on drying and/or reconstitution. Particle size distributions were broad (span > or = 3.6) at both nozzle frequencies. Annealing before drying reduced aggregate levels slightly but increased particle size over 5-fold. Spray drying at inlet temperatures up to 135 degrees C (and outlet temperatures up to 95 degrees C) showed little impact on integrity. Integrity at bench-top and pilot scales was identical, with 0.2% dimer and no high molecular weight or insoluble aggregates detected. Particle size was small (< or = 2.3 microm) with uniform distribution (span < or = 1.4) at both process scales. CONCLUSIONS: Under the conditions tested spray drying, conducted at bench-top and pilot scales with commercially available equipment, was superior to spray-freeze drying for the fabrication of darbepoetin alfa particles for encapsulation.