Oral Administration of Nitrous Oxide Reduces Acute Vaso-Occlusive Pain in Mice with Sickle Cell Disease
Authors: Alves Ana , Abdulla Fuad , Allen Kaje , Khasabova Iryna A. , Khasabov Sergey G. , Young Mark , Gomperts Andrew , Gomperts Edward , Belcher John D. , Vercellotti Gregory M. , Simone Donald A. 
Published: December 2024

Patients with sickle cell disease (SCD) experience both chronic and acute pain. Acute pain is due to unpredictable episodes of vaso-occlusion, referred to as vaso-occlusive crises (VOCs). Due to its severity, pain from VOC is the primary reason for emergency room visits and hospitalizations. Opioids are the primary treatment for pain from VOC but are associated with many serious side effects. Thus, new and effective analgesics are needed to effectively manage acute VOC pain, prevent hospitalizations, and reduce opioid use in SCD patients.
Nitrous oxide (N2O) is an FDA-approved medical gas with well-known analgesic properties. Inhaled N2O is the only delivery platform in clinical use. Unfortunately, there are substantial barriers to using inhaled N2O at home, including: 1) poor dosing control; 2) inadvertent exposure due to leaking from compressed gas cylinders and inhalation equipment; and 3) patient resistance. Oral administration of N2O would circumvent these barriers to home use. HBI-201 is an oral liquid drug product containing N2O that was developed and manufactured by Hillhurst Biopharmaceuticals, Inc. Using humanized transgenic mice with SCD, we determined if oral administration of HBI-201 reduced hyperalgesia produced by VOC.
Male and female Townes HbSS (sickle) and HbAA (control) mice that did not exhibit ongoing hyperalgesia were used. VOC was evoked by exposing mice to cold ambient temperature (10oC) for 1 hour. This produces vaso-occlusion that leads to robust mechanical and heat hyperalgesia (Khasabova et al., 2022). Mechanical hyperalgesia was defined as a decrease in paw withdrawal threshold (PWT) using calibrated von Frey monofilaments applied to the plantar hind paw. Heat hyperalgesia was defined as a decrease in paw withdrawal latency (PWL) in response to a radiant heat stimulus applied to the plantar hind paw. PWT and PWL were averaged for both paws. PWT and PWL were determined before and at 10 min after cold exposure. Mice then received vehicle or HBI-201 by gavage (4, 10 or 20 mg/kg body weight, p.o.), or morphine (5 mg/kg, s.c.). PWT and PWL were determined at various times thereafter. Analgesic effects of HBI-201 were also measured in naïve C57 mice.
HBI-201, but not vehicle, reduced cold-evoked mechanical and heat hyperalgesia dose-dependently. The anti-hyperalgesic effect of HBI-201 peaked at approximately 90 min after administration and persisted for approximately 3 hours following the highest dose. The anti-hyperalgesia following HBI-201 (20 mg/kg, p.o.) was similar to that produced by morphine (5 mg/kg, s.c.). There were no significant differences between male and female mice. HBI-201 also produced analgesia (increased PWT and PWL) in naive C57 mice that lasted for 1-2 hours.
We conclude that oral administration of HBI-201 (N2O) shows promise and potential to reduce pain, hospitalization and opioid utilization in sickle patients experiencing a painful VOC.
Reference: Khasabova, I.A., Juliette, J., Rogness, V.M., Khasabov, S.G., Golovko, M.Y., Golovko, S.A., Kiven, S., Gupta, K., Belcher, J.D., Vercellotti, G.M., Seybold, V.S., and Simone, D.A. A model of painful vaso-occlusive crisis in mice with sickle cell disease. Blood, 140(16):1826-1830, 2022.