(742a) Nanocomposite Microparticles (nCmP) for the Delivery of Tacrolimus in the Treatment of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive life-threatening cardiovascular disease that results in limited exercise capacity, right ventricular heart failure, and ultimately death¹. PAH is defined as an increase in the mean pulmonary arterial pressure of the right side of the heart, which can be attributed to remodeling of the arterial walls due to genetic anomalies and environmental insults^2,3. This results in endothelial cell apoptosis, loss of distal vessels, and occlusive vascular remodeling.

Current treatments are aimed at improving cardiopulmonary function and patient quality of life. Treatments are based on drugs with vasodilatory properties, which have no effect on slowing or inhibiting the progress of the disease⁴. As a result, newer approaches are being investigated to treat PAH. Particularly, approaches that focus on activating cellular mechanisms to reverse vascular remodeling are being investigated⁴. One such approach is through improving the function of the bone morphogenic protein receptor-2 (BMPR2) signaling pathway⁵. BMPR2 mutations have been identified as a cause of 60-80% of familial cases of PAH^4,6,7. An alternative to BMPR2 gene therapy, which presents changes in clinical applications, is tacrolimus (TAC). TAC has been investigated as it increases signaling of the BMPR2 pathway. Furthermore, clinical evidence has shown improvements in PAH patients following low-dose TAC treatment⁴. In previous research investigating the treatment of PAH with TAC, several effects have been noted while using a low-dose TAC treatment.

Another approach to treat PAH has been through the activation of anoctamin-1 (TMEN16A), a calcium-activated chloride channel associated with many important physiological activities^8,9. TMEN16A activation by Eact was shown to promote apoptosis of pulmonary endothelial cells by the p38-MAPK signaling pathway¹⁰. PAH can be characterized by the hyperproliferative phenotype of endothelial cells; therefore, apoptotic promotion can be a potential treatment option¹⁰.

While previous results for PAH treatment using TAC are promising, its application is limited by its poor aqueous solubility, instability, and limited bioavailability¹¹. The use of tacrolimus is further complicated by systemic side effects such as neurotoxicity and nephrotoxicity¹². One potential alternative to overcome these issues is targeted delivery to lungs. Acetalated dextran (Ac-Dex) is a biodegradable polymer that can be formulated into TAC loaded nanoparticles (NP) to more safely and effectively deliver TAC to the lungs. Drug encapsulation in Ac-Dex can improve solubility, enhance drug penetration to obtain higher plasma concentration, provide controlled and sustained drug release, and provide localized drug delivery to reduce off target side effects. Similarly to TAC, the potential efficacy of Eact as a treatment for PAH can be improved by targeted delivery to the lungs using Ac-Dex NP.

Ac-Dex can be produced using a single-step, facile reaction and results in an acid-sensitive, biodegradable, and biocompatible polymer¹³. The reaction reversibly modifies the dextran with acetal groups, reversing the solubility properties of Ac-Dex from hydrophilic to hydrophobic¹³. In comparison to poly (lactic-co-glycolic acid) (PLGA), a common polymer used in drug delivery application, Ac-Dex has several advantages. Mainly, the degradation time of Ac-Dex can be easily modified to suit numerous applications. By controlling the reaction time during Ac-Dex synthesis, the acetal coverage and ratio of cyclic to acyclic acetal group can be modified. Cyclic acetal groups have a slower degradation rate and acyclic acetal groups have a faster degradation rate. By adjusting this ratio, the degradation rate can be tuned from minutes to months. Once degraded, Ac-Dex by-products are dextran, an FDA-approved by-product, and very low levels of methanol and acetone. Finally, Ac-Dex particles show very limited burst release at physiological conditions, allowing for consistent drug concentration in the body^13,14.

Pulmonary delivery has received increasing attention for the treatment of pulmonary diseases due to its localized delivery to tissue and molecular sites of interest^15–17. Moreover, pulmonary delivery offers reduced systemic side effects, rapid onset of action due to large surface area, and avoidance of first pass metabolism^18,19. Dry powder aerosols are a highly attractive option to achieve the aforementioned advantageous, while also providing increased product stability (due to the absence of water) when compared to other delivery methods such as nebulizers¹⁵. However, dry powder aerosols are not without their limitations. Dry powder aerosolized NP (less than 500nm) will be exhaled due to their small size and mass. For effective pulmonary deposition, particles between 1-5 μm are ideal and are capable of deposition in the lungs. However, particles in this size range are also susceptible to macrophage uptake²⁰. To overcome these issues, nanocomposite microparticles (nCmP) can be synthesized. Synthesis of nCmP allow for dry powder particle size to remain between 1-5 μm to effectively deposit in the lungs. However, the nCmP are able to dissociate into NP prior to macrophage uptake to allow for the delivery of TAC.

In the present study, nCmP were synthesized to achieve pulmonary delivery of TAC-loaded Ac-Dex NP and Eact-loaded NP. The TAC-loaded NP and Eact-loaded NP were prepared using emulsion solvent evaporation and nCmP were prepared via spray drying these NP with mannitol. nCmP particles can overcome several issues currently limiting TAC applications such as poor aqueous solubility, instability, poor bioavailability, and systemic side effects. The nCmP are capable of improving TAC solubility, targeted pulmonary delivery as a dry powder aerosol, the potential of penetration through the mucus barrier, and controlled release of TAC. Furthermore, the nCmP and NP were characterized and the deposition of nCmP was investigated further in vivo by monitoring TAC concentration via LC-MS/MS analysis.

Before loading into and after redispersion from nCmP, the NP were approximately 200 nm in diameter and displayed a narrow size distribution. Via SEM imaging, the NP displayed a smooth, spherical morphology and nCmP displayed a raisin like morphology. High encapsulation efficiency was observed for TAC in NP and also NP in nCmPNP. Importantly, the nCmP exhibited desirable aerosol deposition, allowing for deposition into the deep lung. In vivo experiments in rats confirmed the nCmP are capable of pulmonary delivery by monitoring TAC concentration in the lungs. In conclusion, nCmP present a potentially more efficacious method to deliver TAC to the lungs for the treatment of PAH. Similarly, nCmP containing Eact-loaded Ac-Dex NP displayed comparable physicochemical characteristics and preliminary data indicate the use of nCmP is a potential method for target delivery of Eact.