Lorlatinib

Thermal hyperalgesia was determined by measuring the latency to withdrawal of the right hind paws in response to a focused beam of radiant heat (IR = 30) of a Plantar Test apparatus (UgoBasile). Animals were placed individually in a small, enclosed testing arena (20 cm × 18.5 cm × 13 cm, length × width × height) on top of a wire mesh floor. Mice were allowed to acclimate for a period of at least 90 minutes. The device was positioned beneath the animal, so that the radiant heat was directly under the plantar surface of the ipsilateral hind paw. Three trials for each mouse were performed. The apparatus was set at a cut-off time of 30 seconds to avoid tissue damage. Thermal hyperalgesia was evaluated immediately prior to the treatments (time 0) and 15, 45, 90, and 180 minutes after i.t. treatment with crizotinib (Sigma-Aldrich) or after 30, 60, 120, and 180 minutes of lorlatinib (Sigma-Aldrich) administration. For ALKAL-2–induced hyperalgesia, mice were treated with ALKAL2 (MyBioSource LLC, MBS1425538) (0.01; 0.1; 1 μM, i.t.) or vehicle after lorlatinib administration (Sigma-Aldrich) (1 mg/kg, i.g., 30 minutes prior to ALKAL2 administration), and thermal hyperalgesia was evaluated 1, 3, 6, 24, 48, and 72 hours after ALKAL2 treatment.

Formalin test was performed as originally described (33 (link)) and as routinely performed in our lab (55 (link)). Briefly, mice were acclimatized in the laboratory for at least 60 minutes before experiments. Animals received 20 μl of formalin solution (1.25 %) prepared in PBS and injected in the plantar surface of the right hind paw (i.pl.). Following i.pl. injections of formalin, mice were immediately placed individually into observation chambers and the time spent licking or biting the injected paw was recorded and considered as a nocifensive response. We observed animals individually and measured nocifensive responses from 0 to 5 minutes (acute nociceptive phase) and 15 to 30 minutes (inflammatory phase). Crizotinib (Sigma-Aldrich) was delivered by i.t. injection 20 minutes prior to testing. Lorlatinib (Sigma-Aldrich) was delivered systemically via intragastric gavage (i.g.) 30 minutes prior to testing.

Mice were anesthetized with isoflurane (5 % induction, 2.5 % maintenance). A partial ligation of the sciatic nerve was performed by tying the distal one-third to one-half of the dorsal portion of the sciatic nerve, according to the procedure described (57 (link)). In sham-operated mice, the sciatic nerve was exposed without ligation. The wound was closed and covered with iodine solution. Fourteen days after surgery, mice were treated with lorlatinib (Sigma-Aldrich) (1 mg/kg, i.g.) or vehicle, while sham-operated animals received only vehicle (10 ml/kg, i.g.). Mechanical withdrawal thresholds were evaluated immediately before the surgeries (baselines), then 14 days after the surgeries (day 0) and at various time points (0.5, 1, 2, 3, 4, 6 hours) after treatment and every 2 days afterwards.