Polyethylene glycol 400

Isophorone diisocyanate (IPDI) and hexamethylene diisocyanate (HDI), polyethylene glycol 400 (PEG), triethylamine (TEA) and deuterated dimethyl sulfoxide-d₆ (99.8% d) were purchased from Sigma-Aldrich. 2,2-Bis(hydroxymethyl)propionic acid (DMPA), ethanolamine (EA) and tetrahydrofuran were obtained from Merck Chemical Co. All reagents were used without further purification. Methane gas of 99.95% purity was used for experiments. Water used for methane hydrates formation experiments and for preparation of inhibitors solution of desired concentration was carefully purified. On the first step it was twice distillated. On the second step it was deionized using Arium mini plus ultra-pure water system (Sartorius, Germany) to achieve resistivity 18.20 MΩ·cm at 25 °C.

Molnupiravir (EIDD-2801) was purchased from Excenen Pharmatech Co., Ltd. (China) and was formulated as a 50-mg/mL stock in a vehicle containing 10% polyethylene glycol 400 (Sigma) and 2.5% Kolliphor-EL (Sigma) in water. Nirmatrelvir (PF-332; from Wuxi, USA) was formulated as a 125-mg/mL stock in a vehicle containing 43% ethanol and 27% propylene glycol (Sigma) in sterile distilled water.

The experimental metastasis in nude mice was performed as described previously [21 (link)]. Five-week-old male nude mice were purchased from Vital River Laboratories (Beijing, China). All animal studies were conducted in accordance with protocols approved by the Animal Research Committee of the Shantou Administration Center and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Twenty-one mice were equally divided into three groups at random. A suspension of 1.0×10⁶ KYSE510 cells in 100 μl serum-free 1640 medium was inoculated subcutaneously into the left hind footpad of each mouse on Day 0. The growth of footpad tumors was monitored daily, and tumor size was measured every three days, and inguinal lymph nodes were palpated at the same time. On Day 7 after tumor cell injection, the footpad tumors were palpable and the average size was around 25 mm³, and drug administration was initiated. Briefly, a 100 mg/ml F806 stock solution prepared in 100% ethyl alcohol was diluted in 0.9% NaCl containing 5% Tween-80 and 5% polyethylene glycol-400 (Sigma, St Louis MO, USA) to give a dose of 4 mg/kg or 8 mg/kg, in 200 μL solutions, which was intraperitoneally administered daily for 21 days. Mice were euthanized on Day 28 and inguinal lymph nodes were excised and preserved in formalin for histological analysis by hematoxylin-eosin (HE).