RNS60 as a novel therapeutic agent: fact or a fairy tale?

- Ramirez, Jorge H (2014): A story about oxygen nanobubbles (RNS60). figshare.
- http://www.ncbi.nlm.nih.gov/pubmed/24575037#cm24575037_6034

RNS60 is a novel therapeutic agent consisting of normal saline solution oxygenated under the Taylor-Couette-Poiseuille (TCP) flow, creating charge-stabilized nano-structures (CSN) or oxygen nanobubbles. The chemical composition of RNS60 is water, sodium chloride (0.9%), and oxygen (55 ± ppm) but no active pharmacological principle.[1,2] Revalesio and several scientists have declared that these nanobubbles are stable in solution and can be safely administered to humans using enteral and parenteral dosage forms.[3]

Different mechanisms of action have been attributed to RNS60 including effects on cell membrane ion channels,[4] modulation of the PI3k-AKT pathway,[5] and anti-inflammatory actions [1,2]. RNS60 has been presented as a major innovation in the pharmacology of alzheimer, parkinson, asthma, cardiovascular diseases, stroke and multiple sclerosis. In addition, Revalesio states that RNS60 decrease muscle fatigue and improve exercise performance.[6]

Reliant Water is a sport beverage produced by Revalesio and available to the public at the price of $100 USD per 32 bottles. A recent clinical trial involving 40 healthy volunteers found that Reliant Beverage improved post-exercise functional recovery compared with control water.[7] This study was funded by Revalesio, which also provided the water and placebo for the trial, and one of their scientist help in the writing of the manuscript. Furthermore, this study is absent from clinical trial registries, has several methodological flaws and does not comply with Consolidated Standards for Reporting Clinical Trials (CONSORT): absence of participant flow diagram, no calculation of sample size, and no reporting of adverse reactions.

Several patent applications for the use of RNS60 in human disease have been submitted (see supplementary information at the end of the reference list). The process for production of RNS60 already received patent approval (US 8410182 B2 – April 02 2013). RNS60 is the only product in Revalesio pipeline of research and Revera Wound Care their single therapeutic product approved by the Food and Drug Administration (FDA).

A feasibility study of topical Revera on venous leg ulcers is registered at clinicaltrials.gov (NCT00823446) with estimated completion date by October 2010. There are records of two more phase I clinical trials of nebulized (NCT01057498) and intravenous RNS60 (NCT01264783), with completion dates by January and September 2011, respectively. A phase II clinical trial of nebulized RNS60 in asthma patients was completed during May 2013 (NCT01511302). Studies of RNS60 in patients multiple sclerosis (NCT01714089) and knee surgery (NCT01922427) have been registered but are not yet recruiting patients. To this day (November 14 2013) there is no complete publication in scientific journals reporting the results of this trials. Moreover, results of these clinical trials have not been posted in the official web page of clinicaltrials.gov.

A Phase IIa study of RNS60 in 15 patients with multiple sclerosis was recently announced (November 11 2013) in the web page of Revalesio Corporation (http://goo.gl/cixESR). This open label study at University Zurich Hospital does not appear in clinical trials registries and it is different from the study previously registered at clinicaltrials.gov (NCT01714089): (i) the registered clinical trial involves three parallel groups (RNS60 125 ml, RNS60 250 ml, interferon beta-1a) compared to the open-label, single-arm, study at University Zurich Hospital; (ii) the registered clinical trial plans to recruit 270 patients compared to the 15 patients in the study at University Zurich Hospital; (iii) the press release from Revalesio Corporation confirmed the initiation of the study at University Zurich Hospital, however, the registered clinical trial protocol has been scheduled to start in February 2014.

Twelve scientific abstracts reporting the effects of RNS60 on animals, experimental studies, and humans have been presented in scientific events, some of these results have been published in a short version (250 to 500 words) by scientific journals (see supplementary file). Of these short communications only three complete scientific articles have been published. [1,2,8] Publication bias exists because the majority of research about this nanobubbles have not been divulged by peer review scientific journals. A recent paper confirms the existence of nanobubbles on RNS60.[9] However, this article was published the next day after submission, samples of RNS60 were provided by Revalesio, and members of Revalesio directive board are acknowledged for their useful review to the manuscript.

The majority of the Information about RNS60 only appears in the internet site of Revalesio Corporation but not in scientific journals. During the last year there has been a wide news media coverage in South and North American countries informing the public about the properties of this novel therapeutic agent. The main newspapers and tv stations from Colombia have highlighted the cooperation between Revalesio and Tecnoquímicas to study this novel therapeutic. Tecnoquimicas is a pharmaceutical company from Colombia recognized as a major producer of generic drugs in Latin America. A Knight Science Journalism blog discusses that colombian reporters should be more critical with dubious announcements to cure diseases with modified water.[10]

The cooperation between Revalesio and Tecnoquímicas specifically involves a phase II clinical study of intravenous RNS60 in patients suffering acute stroke. Recruitment of patients will take place in the five largest cities of Colombia (Bogotá, Cali, Medellín, Barranquilla, and Bucaramanga) with the participation of recognized clinical centers and neurologists from the country. This clinical trial is expected to conclude in one year and will cost approximately 1.5 millions USD.[11]

Some aspects need to be addressed before involving the participation of humans in RNS60 clinical trials: (i) the technique for measuring RNS60 concentrations to study its pharmacokinetics; (ii) the process to ensure bioequivalence among different batches of RNS60; (iii) verification of biological properties of RNS60 by independent scientists.

Revalesio states that clinical trials of RNS60 were approved by the FDA, but no records of this investigational new drug appear on the internet site of this governmental agency. Furthermore, no information about the approval of human studies with RNS60 was obtained from Revalesio and the FDA.


1. Mondal S, Martinson JA, Ghosh S, et al. Protection of Tregs, suppression of Th1 and Th17 cells, and amelioration of experimental allergic encephalomyelitis by a physically-modified saline. PLoS One 2012;7:e51869.

2. Khasnavis S, Jana A, Roy A, et al. Suppression of nuclear factor-κB activation and inflammation in microglia by physically modified saline. J Biol Chem 2012; 287:29529-42.

3. Revalesio Successfully Completes Phase I Intravenous Safety Study of RNS60. URL:http://revalesio.com/2011/11/revalesio-successfully-completes-phase-i-intravenous-safety-study-of-rns60/ (accessed: 4 Sep 2013)

4. Choi JC, Mega TL, German S, et al. Electrokinetically Altered Normal Saline Modulates Ion Channel Activity [abstract]. Biophysical Journal 2012;102:683a.

5. Revalesio Presents Data Showing RNS60 Alters Disease Progression in Animal Model of Alzheimer’s Disease Through the PI3k-Akt Pathway. URL:http://revalesio.com/2013/07/revalesio-presents-data-showing-rns60-alters-disease-progression-in-animal-model-of-alzheimers-disease-through-the-pi3k-akt-pathway/ (accessed: 4 Sep 2013)

6. Biotech firm lands $10M — in Tacoma. URL:http://seattletimes.com/html/sundaybuzz/2018564738_sundaybuzz01.html (accessed: 4 Sep 2013)

7. Borsa PA, Kaiser KL, Martin JS. Oral consumption of electrokinetically modified water attenuates muscle damage and improves postexercise recovery. J Appl Physiol 2013;114:1736-42.

8. Khasnavis S. Roy A, Ghosh S, Watson R, Pahan K. et al. Protection of Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease by a Physically-Modified Saline Containing Charge-Stabilized Nanobubbles. Journal of Neuroimmune Pharmacology 2013;1-15.

9. Bhushan BB, Pan Y, Daniels S. AFM characterization of nanobubble formation and slip condition in oxygenated and electrokinetically altered fluids. J Colloid Interface Sci 2013;392:105–16.

10. Stupynia P. Colombian periodists should be more critical with Llinas and his dubious announcement to cure diseases with modified water. Knight Science Journalism 2012. URL: http://ksj.mit.edu/tracker/2012/11/los-periodista-colombianos-deber%C3%AD-ser-m%C3%A1

11. A clinical study to produce “nano-water” in Cali, Colombia. URL:http://www.elpais.com.co/elpais/cali/noticias/cali-prepara-para-producir-nano-agua (accessed: Sep 4 2013)

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