Development and Evaluation of Dissolving Microarray Patches for Co-administered and Repeated Intradermal Delivery of Long-acting Rilpivirine and Cabotegravir Nanosuspensions for Paediatric HIV Antiretroviral Therapy.

PURPOSE: Whilst significant progress has been made to defeat HIV infection, the efficacy of antiretroviral (ARV) therapy in the paediatric population is often hindered by poor adherence. Currently, two long-acting (LA) intramuscular injectable nanosuspensions of rilpivirine (RPV) and cabotegravir (CAB) are in clinical development for paediatric populations. However, administration requires access to healthcare resources, is painful, and can result in needle-stick injuries to the end user. To overcome these barriers, this proof-of-concept study was developed to evaluate the intradermal delivery of RPV LA and CAB LA via self-disabling dissolving microarray patches (MAPs). METHODS: Dissolving MAPs of two conformations, a conventional pyramidal and a bilayer design, were formulated, with various nanosuspensions of RPV and CAB incorporated within the respective MAP matrix. MAPs were mechanically robust and were capable of penetrating ex vivo skin with intradermal ARV deposition. RESULTS: In a single-dose in vivo study in rats, all ARV MAPs demonstrated sustained release profiles, with therapeutically relevant plasma concentrations of RPV and CAB detected to at least 63 and 28 d, respectively. In a multi-dose in vivo study, repeated MAP applications at 14-d intervals maintained therapeutically relevant plasma concentrations throughout the duration of the study. CONCLUSIONS: These results illustrate the potential of the platform to repeatedly maintain plasma concentrations for RPV and CAB. As such, these MAPs could represent a viable option to improve adherence in the paediatric population, one that is capable of being painlessly administered in the comfort of the patient's own home on a biweekly or less frequent basis.

Parenteral platforms for tunable, long-acting administration of a highly hydrophobic antiretroviral drug.

GSK2838232 (GSK8232) is a second-generation maturation inhibitor (MI) developed for the treatment of HIV with excellent broad-spectrum virological profiles. The compound has demonstrated promising clinical results as an orally administered agent. Additionally, the compound's physical and pharmacological properties present opportunities for exploitation as long-acting parenteral formulations. Despite unique design constraints including solubility and dose of GSK8232, we report on three effective tunable drug delivery strategies: active pharmaceutical ingredient (API) suspensions, ionic liquids, and subdermal implants. Promising sustained drug release profiles were achieved in rats with each approach. Additionally, we were able to tune drug release rates through a combination of passive and active strategies, broadening applicability of these formulation approaches beyond GSK8232. Taken together, this report is an important first step to advance long-acting formulation development for critical HIV medicines that do not fit the traditional profile of suitable long-acting candidates.

Mixed silane monolayers for controlling the surface areal density of click-reactive alkyne groups: a method to assess preferential surface adsorption on flat substrates and a method to verify compositional homogeneity on nanoparticles.

SAMs formed from mixtures of alkyne-silanes and alkane-silanes are used to control the areal density of click-reactive alkyne groups on the surface of flat germanium substrates, silicon wafers, and silica nanoparticles. Two new analytical tools are described for characterization of the mixed SAMs: a thermogravimetric analysis (TGA) technique for quantifying the compositional homogeneity of the mixed monolayers formed on nanoparticles, and an infrared spectroscopy (IR) technique to detect preferential surface adsorption. The TGA technique involves measurement of the change in weight when azide-terminated polymers react with surface alkyne groups on silica nanoparticles via a click reaction, while the IR technique is based on the use of attenuated total reflectance infrared spectroscopy (ATR-IR) to monitor click reactions between azide compounds with infrared "labels" and alkyne-functional mixed SAMs deposited on germanium ATR plates. Upon application of the new characterization techniques, we are able to prove that the mixed silane monolayers show neither phase separation nor preferential surface adsorption on any of the three substrates studied. When reacted with azide terminal polymers, the areal density at saturation, σ(sat) is found to scale with molecular weight according to σ(sat) ≈ N(-0.57). We conclude that mixed monolayers of alkyne-silanes and alkane-silanes are an effective means of controlling the surface areal density of click-reactive alkyne groups on both flat and nanoparticle substrates.

Formulation and in vitro characterization of spray-dried antisense oligonucleotide to NF-kappaB encapsulated albumin microspheres.

The aim of this study was to formulate and characterize microspheres containing antisense oligonucleotide to NF-kappaB using bovine serum albumin as the polymer matrix. Microspheres were prepared by spray-drying technique with 5, 10 and 15% drug loading. Glutaraldehyde was used as a cross-linking agent. The particle sizes ranged from 3-5 microm. Microspheres were smooth and spherical in shape, as determined by scanning electron microscopy (SEM). The yield of microspheres ranged from 70-75% and the encapsulation efficiencies were found to be in the range of 59-60%, as determined by a novel HPLC method. Zeta potential of the microspheres ranged between -39 to -53 mV, thus indicating good suspension stability in water. In-vitro release studies performed using phosphate buffer saline demonstrated extended drug release up to 72 h. Kinetic model fitting showed high correlation with the Higuchi model, suggesting that the drug release was primarily diffusion controlled.

Treatment of adjuvant arthritis using microencapsulated antisense NF-κB oligonucleotides.

Antisense oligonucleotides are promising new therapeutic agents used to selectively inhibit target genes such as Nuclear Factor Kappa B (NF-κB), an important transcription factor in the pathogenesis of inflammatory disease. The purpose of the present study was to evaluate microencapsulated antisense oligonucleotides specific to NF-κB for in vitro efficacy and treatment of adjuvant-induced arthritis in rats. Oligonucleotide-loaded albumin microspheres were prepared and characterized in terms of size, zeta potential, morphology and release pattern. This study reports significant NF-κB inhibition in vitro after treatment with microencapsulated antisense oligonucleotides. Furthermore, microencapsulated antisense NF-κB oligonucleotides were found to inhibit paw inflammation associated with rat adjuvant-induced arthritis in a dose-dependent manner. Taken together, the results presented in this work described albumin microspheres to be effective delivery vehicles for antisense NF-κB oligonucleotides and a potential treatment for inflammatory diseases.

Microfluidic droplet liquid reactors for active pharmaceutical ingredient crystallization by diffusion controlled solvent extraction.

A novel method for crystallization utilizing solvent/antisolvent extraction in microfluidic droplet liquid reactors has been developed for rapid and low-cost screening of crystal polymorphism (i.e. molecular crystallographic arrangement or internal structure) and habit (i.e. crystallographic shape or external structure). The method involves a ternary solvent system consisting of a dispersed phase of two miscible fluids, one in which the active pharmaceutical ingredient (API) is soluble (solvent) and one in which the API is insoluble (antisolvent). The solvent/antisolvent dispersed phase is immiscible with a third continuous phase. Crystallization of an API, GSK1, was controlled within droplets by altering the rate of solvent extraction from droplets into the continuous phase, thereby decreasing API solubility. Crystal size, habit, and population per droplet were directly impacted by the solvent's rate of extraction. Single crystals were grown in individual droplets by slow extraction of solvent into the surrounding continuous phase, which occurs when crystal growth gradually reduces API concentration such that it is maintained within the metastable zone throughout extraction. Rapid extraction of solvent from droplets results in API concentration significantly exceeding its metastable limit, producing a greater number of crystal nuclei compared to slow extraction conditions. When holding constant solubilized API mass per droplet, crystal sizes were larger for slow extraction rates (l = 96.3, w = 16.6 µm) compared to fast extraction rates (l = 48.8, w = 9.5 µm) as a result of crystal growth occurring on fewer crystal nuclei per droplet. Crystal habit can be controlled by adjusting the solvent extraction rate and consequently the saturation, where minimal saturation resulted in a rhombohedral habit and comparatively higher saturation resulted in an acicular habit with a higher aspect ratio. Antisolvents were tested using two hydrophobic APIs demonstrating the method's capability for rapidly identifying favorable crystal morphologies for downstream manufacturability using miniscule amounts of API.

Targeted fibrillar nanocarbon RNAi treatment of acute kidney injury.

RNA interference has tremendous yet unrealized potential to treat a wide range of illnesses. Innovative solutions are needed to protect and selectively deliver small interfering RNA (siRNA) cargo to and within a target cell to fully exploit siRNA as a therapeutic tool in vivo. Herein, we describe ammonium-functionalized carbon nanotube (fCNT)-mediated transport of siRNA selectively and with high efficiency to renal proximal tubule cells in animal models of acute kidney injury (AKI). fCNT enhanced siRNA delivery to tubule cells compared to siRNA alone and effectively knocked down the expression of several target genes, includingTrp53,Mep1b,Ctr1, andEGFP A clinically relevant cisplatin-induced murine model of AKI was used to evaluate the therapeutic potential of fCNT-targeted siRNA to effectively halt the pathogenesis of renal injury. Prophylactic treatment with a combination of fCNT/siMep1band fCNT/siTrp53significantly improved progression-free survival compared to controls via a mechanism that required concurrent reduction of meprin-1ß and p53 expression. The fCNT/siRNA was well tolerated, and no toxicological consequences were observed in murine models. Toward clinical application of this platform, fCNTs were evaluated for the first time in nonhuman primates. The rapid and kidney-specific pharmacokinetic profile of fCNT in primates was comparable to what was observed in mice and suggests that this approach is amenable for use in humans. The nanocarbon-mediated delivery of siRNA provides a therapeutic means for the prevention of AKI to safely overcome the persistent barrier of nephrotoxicity during medical intervention.

Pro-inflammatory cytokine inhibition in the primate using microencapsulated antisense oligomers to NF-kappaB.

PRIMARY OBJECTIVE: Antisense oligomers to NF-kappaB (ASO) were incorporated into albumin microspheres to determine if microcapsules containing ASO inhibit pro-inflammatory cytokines to a greater extent than comparable doses of ASO in solution. Phagocytosis of microcapsules and intracellular release of ASO in macrophages was evaluated. RESEARCH DESIGN: Comparable doses of microencapsulated ASO and ASO in solution were evaluated in non-human primates. METHODS: Blood was sampled and stimulated with Escherichia coli endotoxin ex vivo. TNF, IL-1 and IL-6 concentrations were compared for 72 hrs. The intracellular concentration of ASO was measured in macrophages in vitro to evaluate the difference in intracellular penetration of microencapsulated ASO. RESULTS: Microencapsulated ASO produced significantly greater cytokine inhibition at all time points compared to ASO in solution. There were no side effects to ASO in the baboons. Intracellular ASO concentration was 10 fold greater in macrophages using microencapsulation. CONCLUSIONS: Microencapsulated ASO to NF-kappaB is more effective than ASO in solution in pro-inflammatory cytokine inhibition in non-human primates.