Cremophor

Mice were injected with 0.3 × 10⁵ WT31 melanoma cells intrasplenically. From day 1 to 18 mice received daily i.p. injections of Sorafenib (Sigma-Aldrich, MS, USA) (60 mg/kg KG, diluted in 12.5% Cremophor (Sigma-Aldrich, MS, USA), 12.5% ethanol, and 75% sterile saline) or vehicle/solvent control (12.5% Cremophor, 12.5% ethanol, and 75% sterile saline). At day 19 mice were sacrificed and metastases were quantified.

Rapamycin (Cat #R5000, LC Laboratories, MA, USA) powder was dissolved in DMSO (Sigma) and aliquots were preserved in −80 °C until use. Aliquots were first diluted 1:1 with Cremophor (Cat# C-5135, Sigma) and subsequent dilutions were prepared in phosphate buffer solution (PBS) for experimental use. An appropriate concentration of the solution was directly added to the culture for assessing anti-viral activity in vitro or injected into the peritoneal cavity of mice to study effects in vivo. Oseltamivir (Tamiflu, 75 mg Oseltamivir/capsule, Roche, Switzerland) was suspended in PBS to achieve desired concentration. The solution was directly added to the culture for assessing anti-viral activity in vitro or fed orally to mice for studying effects in vivo.

Animals were randomly assigned to either the Melatonin administration (n = 5) or the control group (vehicle treated, n = 8). Vehicle solution was prepared with 8 ml of phosphate buffered saline (PBS), 1 ml of dimethyl sulfoxide (DMSO) and 1 ml of Cremophor (Sigma, St. Louise, MO, USA). The animals of the control group received 100 µl of vehicle solution by intraperitoneal injection (IP).
Melatonin (Sigma, St. Louise, MO, USA) was diluted in vehicle and the animals from the Melatonin group received IP of 100 µl of Melatonin treatment (at dose of 40 mg/kg of body weight) for five days a week. Melatonin was administered 1 hr before room lighting was switched off. Administration of Melatonin prior to the nocturnal increase in endogenous Melatonin may be most effective because tissues are most sensitive to the hormone at this time [38] (link), [39] (link).
Melatonin or vehicle administration started on the day of tumor implantation (soon after implantation) and continued for five days a week for 21 days. On the 22nd day, all animals underwent SPECT scanning with Tc-99m-HYNIC-VEGF-c followed by euthanasia and collection of tumors for immunohistochemistry and membrane antibody array to determine the expression of different pro-angiogenic and growth factors in tumor extracts.