Two Phase Mouthwash
In the early 1980s, Mel Rosenberg and Ervin Weiss, young researchers at the School of Dental Medicine, Tel Aviv University, showed that bacteria from the mouth are able to adhere to small droplets of oil. They came up with the idea of a two-phase water and oil mouthrinse that would remove bacteria from the mouth. Although the original formulations did not prove successful, a further development in Rosenberg's lab resulted in compositions that were able to both remove bound bacteria and improve breath. In 1992 the technology was licenced by the technology arm of Tel Aviv University (Ramot Ltd.) to an Israeli company, resulting in "Assuta", the world's first two-phase mouthwash. "Assuta" was featured on Israeli TV and soon became a hit, both among Israeli dentists and consumers. In 1995, Philip Stemmer, a well-known London dentist, teamed up with Prof. Mel Rosenberg of Tel Aviv University to set up a clinic specializing in the diagnosis and treatment of bad breath. During his visit to Tel Aviv to meet Prof. Rosenberg, he was introduced to the two-phase mouthwash and fell in love with it. For months, Dr. Stemmer and his family would pack their suitcases full of "Assuta" and distribute to patients in the London area. After satisfying himself that the mouthwash had potential overseas, Dr. Stemmer set up a company and licenced the technology from Ramot Ltd. for the UK. Since 1996, the British product "Dentyl pH" has become a leading mouthwash throughout the UK.
From Oil Pollution to Oral Pollution: Research and Development of a Bacteria-Desorbing Mouthwash
Mel Rosenberg, Ph.D.
When I started to do my Ph.D. with Eugene Rosenberg, in collaboration with David Gutnick (1978) I must confess that I was already primarily interested in oral microbiology. However, I desperately wanted to study under Eugene, and his area of expertise was petroleum microbiology. Eugene actually told me (I think he was just placating me but it turned out to be prescient) that I would be able to apply what I learnt about petroleum microbiology to study oral microorganisms.
During my Ph.D., my project was to study the interaction of petroleum-degrading microorganisms with the oil that they grow on. I developed an assay based on mixing microbial suspensions with liquid hydrocarbons (e.g., certain types of liquid paraffin oil), and then observing the degree to which the microorganisms adhered to the oil droplets. We were able to determine conditions under which over 95% of certain microbial suspensions stuck to certain types of oil droplets. Among the various microorganisms we checked was Streptococcus pyogenes, a throat microorganism, which adhered nicely to oil droplets. At a later stage, together with Ervin Weiss, we began to look at oral bacteria. We found that most adherent isolates from the mouth were indeed able to adhere to liquid hydrocrarbons. We also showed that a non-oral (but hydrophobic) microorganism was able to stick to a human tooth. Around the same time, Nesbitt and coworkers showed that Streptococcus sanguis , an oral microorganism and one of the first tooth colonizers, adheres to oil (toluene) droplets (Nesbitt WE, RJ Doyle, and KG Taylor. 1982. Hydrophobic interactions and the adherence of Streptococcus sanguis to hydroxylapatite. Infect. Immun. 38:637-644. One year later we showed that one can take dental plaque straight out of the mouth, suspend and wash the microorganisms, add oil and mix, and observe that most of the microorganisms become bound to the oil droplets. We also showed that microorganisms which are already bound to a plastic surface (polystyrene), can be efficiently removed (97% or more) by combinations consisting of saline and olive oil, or saline and liquid hydrocarbon.
Other studies in the early-mid 1980s demonstrated that a wide variety of laboratory strains of oral microorganisms, including Actinobacillus actinomycetemcomitans, Actinomyces viscosus and Actinomyces naeslundii, Porphyromonas gingivalis, Streptococcus salivarius, Streptococcus sanguis,and certain Streptococcus mutans strains, adhere with high affinity to hydrocarbons. Initial experiments showed that microorganisms which adhere to hydrocarbons also bind to other nontoxic oils (Rosenberg et al. 1983,b). Furthermore, in vitro experiments showed that water:oil combinations could desorb bacteria from plastic surfaces (Rosenberg et al. 1983). Desorption could also be demonstrated in vivo, based on removal of erythrosine-stained bacteria and debris from the mouth following rinsing with a two phase rinse consisting of saline and olive oil (Weiss et al., 1985 (patent)). Similar combinations were also able to remove bound cells from polystyrene cuvettes (Rosenberg et al., 1983b). ]
These initial observations led to the notion of using two-phase oil:water products for mouthwashes for binding and physical desorption of oral microorganisms. Our original thoughts were in the direction of a toothpaste, but existing patent art and formulation difficulties (e.g. how would we make it foam?) caused us to pursue the mouthwash direction. We received a US patent in 1985 on the two-phase mouthwash, but with strictly limited claims.
Despite the adhesion and desorption experiments, the simple saline:oil combinations did not prove particularly effective in improving oral hygiene over time. We assume that any effect of desorption that did take place was short-lived, as oral microorganisms quickly grow back, and our formulations originally did not contain potent antibacterial agents. Furthermore, the two immiscible oil and water phases posed technical difficulties, such as getting them to stay mixed for a long enough period. A few years later, we were able to show that adhesion of microorganisms and oral debris to oil was promoted by low concentrations of cetylpyridinium chloride (CPC), an amphipathic cationic agent which is commonly added to mouthwashes. This was surprising, since amphipathic agents were considered by us and others to inhibit adhesion at the oil:water interface (by themselves partitioning at the oil:water interface). Oil:water combinations with added CPC or chlorhexidine were still able to remove bacterial layers from plastic, with much greater efficiency than many commercial mouthwashes. Addition of CPC solved several problems simultaneously:
Prototype formulations, consisting of an aqueous phase containing CPC, and an oil phase comprising a mixture of vegetable and essential oils, were subsequently developed and tested. Such formulations maintained dramatic in vitro bacterial desorption properties (Goldberg et al. 1989), removing over 90% of a bacterial film of A. calcoaceticus RAG-1 on polystyrene cuvettes, as compared to 15-50% removal for a series of commercial mouthwashes.
In an initial clinical study, the prototype two-phase rinse was compared with (i) a placebo rinse; and (ii) a mouthrinse containing 0.2% chlorhexidine gluconate (Corsodyl™ , ICI). Subjects performed oral rinsing prior to bedtime and in the morning of the following day. Measurements were taken in the late afternoon (e.g. at least eight hours following rinsing) of the second day and were compared to those taken the previous day afternoon prior to rinsing. The data demonstrated that rinsing with the two-phase mouthwash results in daylong reduction in microbial levels, as well as in oral malodor (Rosenberg et al. 1992). In another study employing the same two phase formulation, Yaegaki and Sanada (Yaegaki and Sanada, 1992) showed dramatic reductions in volatile sulphide levels 3.5 hours after use, as compared to a commercial mouthwash (Skoal™). These authors suggested that physical desorption of the particulate oral debris by the mouthrinse was an important factor in malodor reduction.
In 1992, the prototype two-phase oil-water mouthwash was reformulated and introduced in the Israeli market. A clinical study (Kozlovsky et al., J. Periodontol. , 1996) was performed to compare the efficacy of the commercial version of the two-phase mouthwash with Listerine™, a mouthrinse which has been previously shown (Pitts et al. 1983, DePaola et al. 1989) to be effective in reducing levels of odor-related microorganisms, as well as plaque and gingivitis . In the study, fifty subjects rinsed with one of the two mouthwashes for 30 seconds twice a day over six weeks, while continuing their regular oral hygiene habits. In both mouthwash groups, dramatic improvements were observed in parameters associated with oral microbial levels, malodor, periodontal health and plaque accumulation. For example, as compared to time zero scores, whole mouth odor, tongue dorsum anterior and posterior odors decreased continuously over time, attaining 80%, 79% and 70%, reductions, respectively following 6 weeks, in the two-phase mouthwash (Assuta™) group, vs. 70%, 77% and 59% for the Listerine™ group. For whole mouth and tongue dorsum posterior, the reductions observed in the two-phase mouthwash group were significantly greater than those obtained with Listerine™ (p=0.026 and p=0.025, respectively), suggesting that the two-phase mouthwash is superior to Listerine™ in long-term reduction of oral malodor.
Part of the reason for the high efficacy of the two-phase mouthwash may be due to the ability of the oil droplets to release the antibacterial CPC over time. CPC is a well-accepted antibacterial agent in the oral cavity, but is not as effective in vivo as chlorhexidine, which is retained in a substantive form for longer periods. However Ilan et al. (blue book) have shown that oil droplets can bind and release CPC over time. Furthermore, tooth sections dipped in a mixture of water, oil and CPC retained antibacterial properties as opposed to control. This may be an important mechanism for the efficacy of the mouthwash which was not originally anticipated.
The Israeli version of the two-phase mouthwash was introduced in Dr. Philip Stemmer's clinic in London in about 1995 and the results were so positive, that he decided to set up a company to licence the technology from Tel Aviv University. Since 1996, Dentyl pH has shown steady increased growth and has grown to become one of the major mouthrinses in the UK.