A phase 1 study of intratumoral ultra-high concentration nitric oxide (UNO) gas injection in cutaneous and subcutaneous solid tumor metastases

Background: Ultra-high concentration nitric oxide (UNO; >10,000 ppm) administered intratumorally has shown survival benefits in murine colon and breast cancer models. UNO modulates the tumor immune microenvironment, influencing M1 macrophages, Tregs, and CD8⁺ T cells, and upregulates PD-1 expression, thereby enhancing the activity of immune checkpoint inhibitors (ICIs). In preclinical studies, UNO combined with anti-PD-1, anti-PD-L1, or anti-CTLA-4 improved tumor inhibition, and UNO alone surpassed anti-PD-1 efficacy in 4T1 breast tumor-bearing mice. Methods: This phase 1 trial (NCT05351502) evaluated the safety, maximum tolerated dose (MTD), and recommended phase 1b dose of a single intratumoral UNO injection in patients with unresectable cutaneous or subcutaneous solid tumors (superficial axis 4.5-30 mm). UNO was administered at 25,000 or 50,000 ppm using a 23-gauge needle inserted horizontally through the tumor to create an exit portal, then retracted to the lesion center for treatment. Gas was delivered intratumorally at 0.2 L/min for 5 minutes. Results: Ten heavily pretreated patients were enrolled: squamous cell carcinoma (n=2), melanoma (n=2), and breast cancer (n=6). Patients received a mean of 5.5 prior systemic therapies and 10.3 total cancer-directed treatments. Six patients were treated with 25,000 ppm UNO and four with 50,000 ppm. Overall, treatment was well tolerated; most treatment-related adverse events (AEs) were grade 1. One serious (S)AE (hypoxia) that occurred during a 25,000 ppm administration, was considered related but was not dose-limiting and resolved fully. No deaths occurred within 12 weeks post-treatment. As of October 1, 2025, seven of ten patients remain alive 19-37 months post-single UNO injection; one patient died of disease progression 25 months post-treatment. Two patients with triple negative breast are disease free. Conclusions: Intratumoral administration of ultra-high-concentration UNO was feasible and well tolerated in patients with unresectable solid tumors. Durable long-term survival observed in this heavily pretreated population supports further evaluation of UNO, including in combination with ICIs.