Reagents: ALAM027 and ALAM108 were obtained from natural THCA⁸ in accordance with methods of their synthesis.⁷

In vitro study was performed with PANC-1 and AsPC-1 cells from the collection of Chempartner (China) by CellTiter Glo Viability Assay (cells were incubated with the compounds for 72 h at 5% CO₂, 37°C).⁵ IC₅₀ and inhibition for each compound were calculated using XLFit curve fitting software.

In vivo xenograft study was performed with human PANC-1 obtained from ATCC (ATCC CRL-1469™). Cells were cultured in DMEM (Cat. No. 01-055-1A; Biological Industries,), supplemented with 10% FBS (Cat. No. 10270106; Invitrogen—Life Technologies,), 1% l-glutamine (Cat. No. 03-020-1B; Biological Industries,), and 1% Pen-Strep solution (Cat. No. 03-031-1B; Biological Industries).

PANC-1 cells were thawed for 1–2 min in a 37°C bath, placed in 5 mL growth medium, and centrifuged (300 g, 5 min, room temperature [RT]). The cells were cultured in the growth medium. Cells were grown until 70–80% confluency before splitting. Trypsin EDTA Solution B (Cat. No. 03-052-1B; Biological Industries) was used for cell splitting, and × 2 volume of medium was used for stopping the reaction. Matrigel (basement membrane matrix, Cat. No. 3432-005-01, Lot. No. 1514944; Pathclear^®) was thawed on ice and kept on ice until right before injection. PANC-1 cells were harvested at 70–80% confluency, centrifuged (300 g, 5 min, RT), and washed once more with PBS. The cells were resuspended with PBS and counted. Concentration of cells was adjusted to 2 × 10⁶/50 μL at 2 mL final volume and mixed 1:1 with 2 mL Matrigel (4 mL total). The mixture of Matrigel + cells was drawn up into a syringe (27G) just before injection. Each animal was injected with 100 μL of mixture of Matrigel + cells into the right flank.

In vivo testing in mice was performed in compliance with “The Israel Animal Welfare Act” and following approval of Ethics Committee of “The Israel Board for Animal Experiments”. PANC-1 cells were injected into the right flank of the mice (Athymic nude; females) at a dose of 2 × 10⁶ cells per mouse. A control group was injected with the vehicle. Tumor size was monitored using a digital caliper starting from tumor appearance (∼5 days after tumor inoculation). Doses of ALAM023 and ALAM108 were 120 and 40 mg/kg per day as solutions in canola oil accordingly based on their IC₅₀ values. Solutions of cannabinoids and pure canola oil used as vehicle were given orally once a day five times a week to three groups of mice with n = 7 for each group. Treatment started when the tumor reached a volume of 50–80 mm³ in 50% of the mice (on day 32). Tumor volume was measured in female nude mice during 4 weeks of treatment with the cannabinoids. Tumor volumes in the cannabinoids-treated group were smaller already by treatment day 5, reaching statistical significance by days 19, 22, and 28 (*p < 0.05 according to two-tailed t-test). On study day 60, after the last tumor measurement, mice were sacrificed through CO₂ asphyxiation. Upon termination, the tumors were excised and weighed. The average weight (Fig. 4) was also significantly lower in the cannabinoids-treated group.