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Inhalation OCTOBER 2022 13 After the success of this formulation during the in vitro characterization, it was tested in vivo using an orthotopic nude rat model of NSCLC in collabora- tion with Lovelace Biomedical. A NSCLC human tumor cell line was intratracheally instilled into irra- diated rats. After 4 weeks of tumor growth, weekly treatments were administered, and the rats' lung weight was determined at the 12-week endpoint as a measure of tumor burden. Four treatment groups were investigated, as summarized in Table 2. e standard-of-care group received injected cisplatin in combination with injected bevacizumab. Experimen- tal group 1 received only inhaled bevacizumab, while Experimental group 2 received injected cisplatin and inhaled bevacizumab. An untreated group served as a negative control. Note that the inhaled dose was reduced 10-fold compared to the injected dose. Results of the in vivo study are summarized in Fig- ure 2. e standard-of-care, Experimental 1 and decreasing a tumor's ability to form new blood ves- sels as it grows. For late-stage NSCLC, bevacizumab is administered as an IV infusion every 3 weeks in combination with chemotherapy, and thereafter as a maintenance treatment. Due to the risk of severe bleeding adverse e ects, substantial exclusion criteria prevent many patients from receiving bevacizumab. A locally delivered bevacizumab treatment could help reduce exposure of healthy tissue and, thereby, reduce adverse e ects. Compliance with repeated IV infusions can be chal- lenging for patients, particularly during inde nite maintenance treatment. A dry powder formulation of bevacizumab could also help simplify the patient's treatment experience by enabling at-home adminis- tration with no cold chain storage requirements. To this end, we formulated bevacizumab for dry powder inhaler, using spray drying as the particle engineering technology. e key performance attri- butes of this formulation were: • Preserved bioactivity of bevacizumab to inhibit VEGF, evaluated by a cell-based assay • Aerosol properties for delivery to the deep lung • Physical stability of the powder at 25 °C, protected from humidity Trehalose, a non-reducing sugar, is a common stabi- lizing excipient for proteins in both liquid and solid formulations. L-leucine is a surface-active amino acid often used in inhalation formulations to improve powder dispersibility. Both excipients were used in all formulations screened in this study. Initial eval- uation of three active loadings showed that all three met targets for the key performance attributes and, therefore, the highest active loading was selected: a 40/40/20 bevacizumab/trehalose/L-leucine spray- dried powder by weight. Analytical testing con rmed that the bevacizumab is undamaged by the spray-drying process: its bioactivity (anti-VEGF) was comparable to the as-received mate- rial; no aggregation or fragmentation was observed by size exclusion chromatography or dynamic light scattering; and a solution of reconstituted powder in phosphate bu er was optically transparent. Aerosol properties were tested by Next Generation Impactor (NGI, Copley Scienti c, Nottingham, UK) using a high-resistance Plastiape RS01 device (Berry Global Healthcare, Evansville, IN, US), with a ne particle fraction of 81% and a mass median aerodynamic diameter of 2.2 µm. ese results were on-target for delivery to the deep lung. e powder underwent an ICH stability study, pack- aged with desiccant and stored for 1 year at 5 °C and 25 °C. Other than a slight increase in the water con- tent of the powder, all physical properties, aerosol properties and bioactivity metrics remained constant at both temperatures. Table 2 Treatment groups for an in vivo study in a rat model. Adapted from Reference 14. Treatment Group Injected Cisplatin Injected Bevacizumab Inhaled Bevacizumab Untreated -- -- -- Standard-of-Care 3 mg/kg 15 mg/kg -- Experimental 1 -- -- 1.5 mg/kg Experimental 2 3 mg/kg -- 1.5 mg/kg Figure 2 Tumor burden results for an in vivo study in a rat model: Untreated (none), Standard-of- care (intraperitoneal (IP) bevacizumab + IP cisplatin), Experimental 1 (INH bevacizumab) and Experimental 2 (INH bevacizumab + IP cisplatin) groups are shown. From reference 14. Tumor Burden (g) 15 10 5 0 None IP bev + IP cisp INH bev INH bev + IP cisp