(M1530-05-33) Advancing Lung Cancer Therapy: Development and Optimization of Lorlatinib Loaded Inhaled Nanostructure Lipid Carriers (NLC) Using Design of Experiment Approach

Purpose: Current therapies for lung cancer include radiotherapy, chemotherapy, and immunotherapy, which while being effective, have severe drawbacks such as tumor recurrence, toxicity, and non-specific side effects. To address these issues, a more effective and targeted treatment approach is required. One of the most recent FDA-approved therapies, a third-generation ALK tyrosine inhibitor, lorlatinib, works by preventing ROS and ALK tyrosine kinase activity. In cases of ALK-positive non-small cell lung cancer (NSCLC) with genetic mutations, such as ROS1 and ALK alterations, it helps in reducing tumor growth and enhancing patient survival. The disadvantages of lorlatinib include its high lipophilicity, which renders it almost insoluble in water and causes limited permeability. Additionally, because it is an oral tablet, there is a possibility of incomplete absorption due to physiological variabilities, resulting in varying drug absorption, decreased bioavailability, and systemic toxicity. To overcome these limitations, there is a need to develop a promising strategy for the targeted and localized delivery of lorlatinib in the tumor vicinity. This study investigates the use of inhalable lorlatinib-loaded nanostructured lipid carriers (NLCs) for targeted drug delivery to address those limitations. NLCs are an acceptable approach for treating cancer due to their various features like biocompatibility, enhanced drug solubility, improved stability, and less drug leakage.

Methods: As per the literature, we selected different solid and liquid lipids, and determined the solubility of lorlatinib in those lipids, with higher solubility observed in Gelucire 44/14 and Clove oil, compared to Precirol ATO 5 and oleic acid. However, as clove oil is incompatible with Gelucire and affects the drug loading rate, oleic acid was selected as the liquid lipid with Gelucire. A 3² factorial design was created to develop lorlatinib-loaded NLCs, which resulted in 9 possible formulations. Briefly, a mixture of Gelucire and oleic acid was heated at 70°C under stirring at 600 rpm for 5 min to allow the formation of a homogenous lipid phase. To prepare the aqueous phase, 2% Tween-80 (w/w) was added to DI water and was heated to 70°C. To prepare the emulsion (o/w), the aqueous phase was added dropwise into the lipid phase while being stirred for 30 mins at 600 rpm and 70°C. To reduce the particle size, pre-emulsion was sonicated using a probe sonicator (2 minutes at 20% amplitude with 10 sec on/off cycles), while gradually reducing the temperature using an ice bath. All the NLC formulations were evaluated for particle size and zeta potential using dynamic light scattering, and drug content using a validated analytical UPLC method at 310 nm. In-vitro aerosolization was tested for optimized formulations, F1 and F6, by using a Next Generation impactor. Preliminary assessment of cytotoxic potential of lorlatinib-loaded NLCs was performed on A549 and H4006 NSCLC cell lines after 72-hour incubation by using standard MTT assay.

Results: The solubility of lorlatinib was determined in various oils and surfactants. As can be seen, lorlatinib was found to be soluble in Gelucire 44/14 and oleic acid (Fig. 1A). A DoE based approach was used to develop and optimize lorlatinib-loaded NLCs. As can be seen in Fig. 1B, DoE rendered nine formulations, out of which F1 and F6 were optimized formulations as % drug loading was 5.82±0.22% and 7.21±0.33%.The optimized formulations were characterized for particle size of 44±2.8 nm and 66±1.4 nm, and zeta potential of -11.3± 3.1mV and -22.7± 2.1mV respectively(Fig. 1B). The % entrapment efficiency was 84.4±3.9% and 83.01±4.6% for F1 and F6 respectively. The Mass Median Aerodynamic Diameter (MMAD) of these formulations were determined by in-vitro drug aerosolization studies and were found to be 5.1±0.3 and 4.9±0.1 μm. The % Fine Particle Fraction (% FPF) values were found to be 66.1±3.9% and 64.1±1.5% respectively. The MMAD and % FPF values suggest that both formulations were suitable for inhalation. The cytotoxicity studies after treatment with Lorlatinib and Lorlatinib loaded NLC’s determined using MTT assay after 72 h. incubation with treatment in two different NSCLC cell lines: A549 and H4006 represent IC50 values for each treatment in both cell lines calculated relative to control. As can be seen from IC50 values of Lorlatinib loaded NLC’s demonstrated significantly higher cytotoxicity in F1 and F6 formulations compared to plain Lorlatinib (Fig.2D and E). The preparation and formulation optimization of lorlatinib-loaded NLCs was successfully examined in the current work, with a focus on smaller particle size and high entrapment efficiency. To assess the therapeutic efficiency and safety of nanostructure lipid carrier, additional studies into the morphology of NLC using TEM, release research, formulation stability, and in-vitro study are being conducted.

Conclusion: The preparation and formulation optimization of lorlatinib-loaded NLCs was successfully examined in the current work, with a focus on smaller particle size and high entrapment efficiency. To assess the therapeutic efficiency and safety of nanostructure lipid carrier, additional studies into the morphology of NLC using TEM, release research, formulation stability, and in-vitro study are being conducted.

Figure 1: A) Solubility study B) The Experiment design layout created for 9 batches of Lorlatinib NLC’s by 32 full factorial design Optimization of Nanostructure Lipid Carrier
C) effect of formulation variables on particle size D) effects of formulation variables on Zeta potential E) effect of formulation variables on drug loading

Figure 2: In vitro aerosolization through new generation impactor A) % In vitro lung deposition, B) % Cumulative deposition and C) In vitro aerosolization performance D) Cytotoxicity study on A549 cells E) Cytotoxicity study on H4006 cells