Background

RNS60 is saline containing charged oxygen nanobubbles. It is created by mixing 0.9 percent normal saline with pressurized oxygen using a technique owned by Revalesio. The nanobubbles allow oxygen to be retained in the solution for longer and at higher concentrations compared to conventionally dissolved oxygen. RNS60 is delivered intravenously or by inhalation. It is unclear where or how the oxygen is delivered to cells.

According to preclinical work funded by Revalesio, RNS60 can activate phosphatidylinositol-3-kinase (PI3 kinase) to promote anti-inflammatory, immunomodulatory, and neuroprotective actions (Dec 2014 conference news; Jana et al., 2018). Independent researchers reported increased mitochondrial activity, ATP production, and synaptic activity in cells treated with RNS60 (Choi et al., 2014; Choi et al., 2015), as well as improved neurotransmission and reduced fatigue in isolated muscle preparations (Ivannikov et al., 2017).

Revalesio-funded work claimed neuroprotective effects in a mouse model of Parkinson’s disease of 300 μl daily intraperitoneal injections or delivery by nebulizer (Khasnavis et al., 2014; Mondal et al., 2017). The proposed mechanism was enhancement of regulatory T cell (Treg) activity. Another study reported upregulated mitochondrial biogenesis and increased levels of neuroprotective gene expression in isolated dopaminergic neurons after RNS60 exposure (Chandra et al., 2018; Jana et al., 2023).

In the 5XFAD transgenic mouse model of Alzheimer’s disease, intraperitoneal injection of 300 μl RNS60 every other day for two months reportedly suppressed neuronal apoptosis, attenuated tau phosphorylation, inhibited glial activation, reduced hippocampal Aβ, and improved memory and learning (Modi et al., 2014). RNS60 was also claimed to enhance expression of genes related to synaptic plasticity, and to increase calcium influx in cultured hippocampal neurons (Roy et al., 2014).

Other company-funded or co-authored studies reported beneficial effects of intraperitoneal administration in the SOD1 mutant mouse model of ALS (Vallarola et al., 2018), and models of traumatic brain injury (Rangasamy et al., 2020), heart attack (Zabielska-Kaczorowska et al., 2022), and stroke (Baena-Caldas et al., 2024).

Starting in 2014, Revalesio’s subsidiary Reliant Hydration briefly marketed an oxygenated water for athletic performance and recovery (press release). In 40 healthy adults, drinking the water for 23 days was claimed to reduce exercise-induced muscle damage and inflammation, and improve muscle recovery (Borsa et al., 2013). A Phase 1 trial, run from 2012-2015 at New York University, tested the water against placebo for changes in brain activity measured by magnetoencephalography in healthy adults. No results were made public. The product drew an FDA warning when a National Football League player and company investor claimed the water protected him against concussion (2015 news).  See also 2014 Derek Lowe blog. Several RNS60 papers were flagged on PubPeer and have been corrected.

Findings

In 2011-2013, Revalesio ran multiple Phase 1 studies. The first was a safety and tolerability study of intravenous RNS60 in 12 healthy subjects. Each received intravenous infusions of RNS60 or normal saline at three escalating rates for 48 hours. Other trials tested a single 4 ml dose of nebulized RNS60 in 36 healthy adults or mild asthma patients, or nebulized RNS60 in combination with the asthma treatment budesonide. No results have been disclosed for these trials.

In 2012, the FDA approved a single-person compassionate use trial for ALS patient Tony Wood, the inventor of the process to make RNS60. Wood received twice weekly infusions beginning in March 2012 (Dallas Morning News). He died of ALS in August 2015.

In 2015, Revalesio ran a Phase 1 study in 56 healthy adults in the UK, who received 4 ml RNS60 or normal saline by nebulizer twice daily for 22 days. On day 19, participants undertook an exercise protocol to induce muscle damage, followed by measurement of creatine kinase and C reactive protein markers of muscle strain and inflammation. No results have been reported.

From 2015-2017, a Phase 1 pilot trial at Massachusetts General Hospital in Boston tested RNS60 in 16 ALS patients. A 23-week regime of weekly intravenous infusion of 375 ml and daily 4 ml nebulization causes no serious adverse events or withdrawals due to adverse events (Paganoni et al., 2019). Eighty percent of enrollees finished the treatment, with no significant changes in blood IL-17 or Treg function, or in brain inflammation measured by TSPO PET.

In December 2016, Revalesio registered a Phase 2 trial to test nebulized RNS60 in ALS patients. Originally planned to start in October 2018, the study is now slated to run from October 2025 to November 2027. It will enroll 140 participants for six months of daily RNS60 or placebo, against a primary outcome of the ALSFRS-R. Secondary outcomes are deaths or tracheostomies, Treg numbers, lung function, patient-reported outcomes, and adverse events.

In May 2017, a Phase 2 ALS trial began at the Mario Negri Institute in Italy. This academic-sponsored, placebo-controlled biomarker trial enrolled 147 patients at multiple sites in Italy. They received 24 weeks of RNS60 or normal saline, delivered by once-weekly 375 ml infusions and daily 4 ml nebulization. All participants also took riluzole. Primary endpoints were changes in blood biomarkers known to be modified in preclinical studies, i.e., Il-17, Tregs, and protein nitration, as well as ALS markers MCP-1 and PPIA, and the neurodegeneration marker NfL. Secondary endpoints included clinical measures of ALS Functional Rating Scale-Revised, survival, decline in lung function, quality of life, and safety. The trial finished in May 2021, and results are published. There were no treatment-related changes in biomarkers. Decline on the forced vital capacity measure of lung function, and eating and drinking abilities, were slower in the RNS60 group. In a post hoc subgroup analysis, NfL increased over time in bulbar onset placebo patients, but remained stable in those treated with RNS60, although differences with treatment were not statistically significant (Beghi et al., 2023). After 2.8 years median follow-up, the treated group survived a statistically significant six months longer than the placebo group. The survival benefit was greatest in patients with low NfL and MCP-1 levels at the start of the study (Pupillo et al., 2024).

Other Phase 2 trials have been completed for asthma, multiple sclerosis, and stroke. No results have been released. Additional studies planned for asthma, multiple sclerosis, knee pain, and hip pain were withdrawn.

For details on RNS60 trials, see clinicaltrials.gov.