Rapid Anti-gingivitis Efficacy of a Novel Stannous Fluoride Dentifrice: Results From a 12-Week Randomized Controlled Clinical Trial

While SnF₂ is clinically effective, dentifrices containing the ingredient are difficult to prepare. Formulators must take care to control deactivation of available and bioavailable SnF₂ during preparation and storage.^6-8 Historically, a number of technical approaches have been applied to improve the stability and efficacy of SnF₂ in antimicrobial formulations. One approach to improve SnF₂ bioavailability is to use anhydrous glycerin gels where Sn²⁺ loss due to hydrolysis and oxidation is largely avoided. Water-based formulations containing cleaning abrasives are considerably more difficult to prepare because SnF₂ availability can be reduced by abrasive adsorption, hydrolysis, or oxidation. One approach to account for this is to add secondary sources of stannous (so-called reservoirs) such as stannous chloride to boost SnF₂ bioavailability.⁶ Abrasives can also be selected that have lower adsorption affinities for stannous. Lastly, a variety of chelant molecules can be added. Chelants act to form soluble molecular complexes with stannous and are effective in limiting stannous loss to hydrolysis. Suitable chelants added to formulations have included gluconate, citrate, pyrophosphate, hexametaphosphates, and zinc phosphate.^6-9

One of the challenges to the application of chelants to formulations with SnF₂ is balancing the stabilizing actions of chelants on SnF₂ availability (the concentration of soluble stannous in the formulations) while maintaining adequate bioavailability (the reactivity of stannous with biofilm bacteria in dental plaque). For example, it can be challenging for cationic antimicrobial molecules (stannous is positively charged) to penetrate plaque biofilms as there is a tendency for these molecules to be bound on the surface of the dental plaque and not adequately penetrate.¹⁰ It is important that formulation components including chelants facilitate to the degree possible SnF₂ reactivity including biofilm penetration.

Recently, a new formulation approach for SnF₂ dentifrices has been discovered that includes the incorporation of the amino acid glycine as a stabilizer for SnF₂. In laboratory experiments, formulations of SnF₂ with added glycine promoted more rapid uptake, deeper penetration, and improved retention of SnF₂ within plaque biofilms as compared to SnF₂ absent glycine chelant.¹¹ Selected formulations showed up to 80% increases in stannous colocalization with plaque bacteria as compared to control formulations.¹¹

The purpose of this study was to establish the plaque and gingivitis efficacy of a novel 0.454% SnF₂ dentifrice with the amino acid glycine, as compared to a negative control dentifrice.

Methods and Materials

This was a 12-week randomized, controlled, two-treatment, parallel-group, double-blind clinical study to compare anti-gingivitis and anti-plaque efficacy of a novel 0.454% SnF₂ dentifrice stabilized with amino acid glycine versus a negative control dentifrice. This study was reviewed and approved by an IRB (study #2019051).

The study population comprised 100 generally healthy adults with established clinical plaque and gingivitis. To participate in this study subjects were required to have at least 16 gradable teeth and more than 20 bleeding sites. Subjects were excluded from participation if they used antibiotic, anti-inflammatory, or anti-coagulant medications or anti-gingivitis/anti-bacterial oral care products within 2 weeks of the baseline visit. Other exclusion criteria included self-reported pregnancy or lactation or fixed facial or lingual orthodontic appliances on gradable teeth.

At the baseline visit, a medical history review, demographic information, and inclusion/exclusion criteria were verified and qualifying subjects underwent a gingivitis (Löe-Silness Gingivitis Index¹² [LSGI]) and dental plaque (Turesky Modification of the Quigley-Hein Plaque Index¹³ [TMQHPI]) evaluation. All clinical evaluations were conducted by a licensed dentist trained and qualified in clinical grading methods.^14,15 Subjects were stratified based on their baseline LSGI score, number of bleeding sites, whole mouth TMQHPI score, age, and gender and randomly allocated to one of two treatment groups using an encoded program:

·      Group 1: SnF₂ dentifrice: 0.454% SnF₂ stabilized with amino acid glycine (Crest^®Pro-Health™Advanced Gum Restore Toothpaste,The Procter & Gamble Company, crest.com)

·      Group 2: Negative control: 0.76% sodium monofluorophosphate dentifrice (Colgate^® Cavity Protection Toothpaste, Colgate-Palmolive, colgate.com)

Both of the groups received a soft manual toothbrush (Oral-B Indicator, The Procter & Gamble Company) to use together with the assigned toothpaste. Products were over-labeled for blinding purposes and dispensed in a protected area, separate from clinical grading.

Subjects were instructed both verbally and via written instructions to brush thoroughly, twice daily for 1 minute per brushing. The first brushing was done under the supervision of study personnel to ensure compliance. All subjects were instructed to use only the assigned products in place of normal oral hygiene for the duration of the study.

After 1, 2, and 12 weeks of product use subjects returned to the site for gingivitis and plaque evaluations (plaque was assessed at week 12 only). At each visit, continuance criteria were reviewed and a comprehensive oral examination was conducted. General comments and any adverse events (AEs) were recorded.
 

Clinical Evaluations

Oral Soft-Tissue Evaluation

Assessment of the oral soft tissue was conducted via a visual examination of the oral cavity and perioral area utilizing a standard dental light, dental mirror, and gauze. The structures examined included the gingiva (free and attached), hard and soft palate, oropharynx/uvula, buccal mucosa, tongue, floor of the mouth, labial mucosa, mucobuccal/mucolabial folds, lips, and perioral area. Assessment of the oral hard tissues was conducted via a visual examination of the dentition and restorations utilizing a standard dental light, dental mirror, and air syringe. All abnormal findings and/or AEs were recorded.

Gingivitis Index

The LSGI¹² was used to evaluate gingival health on six gingival areas per tooth based on color, consistency, and bleeding upon probing ranging from 0 to 3. The entire dentition, with the exception of the third molars, was evaluated yielding a maximum of 168 sites. Bleeding scores were derived from the LSGI scores at each site. Gingival bleeding site scores were assigned a value of 0 if the GI score was 0 or 1, and a value of 1 if the GI score was a 2 or 3. A full-mouth bleeding score was determined by summing the bleeding scores over all evaluable sites.

Dental Plaque Clinical Examinations

The TMQHPI¹³ was used to evaluate the dental plaque levels at baseline and week 12. Subjects rinsed with Trace^® Disclosing Solution (Young™ Dental, youngdental.com) prior to grading. Plaque areas were scored on six surfaces per tooth (mesiofacial, facial, distofacial, mesiolingual, lingual, and distolingual) according the following index below, summarized and divided by the number of graded teeth for an average score per subject:

Score      Criteria

0             No plaque

1             Isolated areas of plaque at gingival margin

2             Thin band of plaque at gingival margin less than 1 mm

3             Plaque covering up to 1/3 tooth surface

4             Plaque covering between 1/3 and 2/3 tooth surface

5             Plaque covering >2/3 of tooth surface

Statistical Analysis

Sufficient subjects were screened to randomize 100, with a goal of 94 subjects (47 per arm) completing the study, allowing for a 5% non-completion rate. It was determined that 47 subjects per group would allow for a treatment difference of 9.86 in number of bleeding sites, 0.11 in LSGI and 0.30 in whole mouth TMQHPI to be detected with at least 90% power in a two-sided two sample t-test of significance level 0.05 assuming group standard deviation of 13.78, 0.15, and 0.42. These differences were based on results from a prior study at the same site.

Baseline subject demographic data were compared between test groups via analysis of variance (ANOVA) for age and Fisher’s exact test for gender and ethnicity. Summary statistics (eg, means, standard deviations, frequencies, etc) of the demographic characteristics as well as efficacy measurements were calculated for each treatment group and visit. The efficacy statistical analyses were based on whole mouth average LSGI scores, the number of bleeding sites using bleeding scores, and whole mouth average TMQHPI scores. A paired t-test was used for comparisons to baseline for each index within each treatment group for each post-baseline visit. Using analysis of covariance (ANCOVA) with the corresponding baseline score as a covariate, a statistical between-treatment group comparison was conducted at each post-baseline visit. Statistical tests were two-sided using a 5% significance level. The primary comparison was number of bleeding sites at the week 12 time point.

The current Gingivitis Case Definition from the 2017 World Workshop in Periodontology was used to classify subject-level gingival bleeding at baseline and the 12-week visit: generally healthy (<10% bleeding sites), localized gingivitis (10% to 30% bleeding sites), or generalized gingivitis (>30% bleeding sites).¹⁶ Transition rates by treatment were then calculated and used to generate odds ratios with 95% confidence intervals.

Results

Demographic characteristics of study subjects are summarized in Table 1. One hundred subjects were initially enrolled in the study. Subjects were evenly balanced for all demographic characteristics between assigned treatment groups(P ≥ .176). All subjects completed the trial. The mean age (standard deviation [SD]) of subjects was 42.1 years (13.19) and 60% were female. Forty-six subjects (46%) were Caucasian, 35 subjects (35%) were Black, 8 subjects (8%) were Asian, 6 subjects (6%) were Multiracial and 5 subjects (5%) were Native Hawaiian or Other Pacific Islander.

Table 2 presents descriptive summaries of number of bleeding sites, LSGI scores, and TMQHPI scores throughout the study. At baseline, subjects were balanced for all gingivitis and plaque measures (P ≥ .282). Subjects using the novel SnF₂ dentifrice produced statistically significant (P < 0.001) reductions in number of bleeding sites and LSGI scores from baseline at week 1, week 2, and week 12. Subjects using the negative control dentifrice only exhibited a statistically significant benefit from baseline for reduction in LSGI scores at week 12 (P = .005). For plaque, both the novel SnF₂ dentifrice group and the negative control group demonstrated statistically significantly lower (P < .001) mean overnight TMQHPI scores at week 12 versus baseline.

Comparisons of clinical indices for the novel SnF₂ and negative control are shown in Table 3. Subjects using the SnF₂ dentifrice exhibited statistically significantly reduced number of bleeding sites, LSGI scores, and TMQHPI scores compared to the negative control at each evaluation period, with the gingival benefit increasing in magnitude from week 1 to week 12. At the week 12 evaluation, the number of gingival bleeding sites, LSGI scores, and TMQHPI scores for subjects using the novel SnF₂ dentifrice were reduced by 33.4%, 16.5%, and 25.6%, respectively, compared to the negative control dentifrice. 

All of the study subjects were classified as being localized or generalized gingivitis case types (≥10% bleeding sites) at baseline (Table 4). Of the 50 subjects in the SnF₂ treatment group with localized or generalized gingivitis at baseline, 10 (20%) had already transitioned to generally healthy at week 1 compared to only 1 subject (2%) in the negative control group. By week 2, 31% of subjects in the SnF₂ group had transitioned to generally healthy compared to only 4% in the negative control group. After 12 weeks, 40% of subjects in the SnF₂ group were classified as generally healthy while only 10% of subjects in the negative control group had improved to generally healthy. These transitions indicate that subjects with localized or generalized gingivitis had 12.3 times better odds (95% CI [1.5, 99.8]) of shifting to generally healthy using SnF₂ versus a negative control at week 1, 10.6 times better odds (95% CI [2.3,49.4]) at week 2, and 6 times better odds (95% CI [2.8,12.9]) at week 12.

Treatments were well tolerated and there were no dropouts or safety issues.

Discussion

SnF₂ dentifrice stabilized with the amino acid glycine was observed to produce significant reductions in gingival inflammation and bleeding as compared to the negative control dentifrice at all clinical measurement points, including as early as the 1-week assessment. The efficacy of the novel SnF₂ dentifrice in this study further supports the general effectiveness of SnF₂ in a variety of formulations for suppression of gingivitis and promotion of gingival health.^2-4 SnF₂ is an antibacterial fluoride, unlike sodium monofluorophosphate, and its sustained antimicrobial properties contribute to its ability to reduce plaque regrowth and improve gingival health.⁸

The potential for improved penetrability of SnF₂ from optimized formulations may be clinically significant based upon contemporary discoveries concerning the mechanism of action of SnF₂. A clinical study has demonstrated that SnF₂ can penetrate into subgingival crevicular fluid and stannous is retained in subgingival plaque.¹⁷ SnF₂ has been shown to decrease biofilm virulence via attaching to lipopolysaccharide and lipoteicoic acid molecular patterns on bacterial surfaces interfering with pathogen stimulation of toll receptors,^18,19 the latter of which are associated with the initialization of the inflammatory processes involved in periodontal disease.^20-26 Samples of plaque from subgingival areas in subjects brushing with SnF₂ dentifrice have been shown to exhibit decreased virulence ex vivo.^27,28 In addition, SnF₂ formulations have been shown to reduce bacterial metabolic products, including short chain fatty acids propionic and butyric acid, which are derived from bacterial metabolism in deeper parts of plaque biofilms in anaerobic environments.²⁹ As mentioned earlier, laboratory studies with the SnF₂ dentifrice and the amino acid glycine dentifrice have shown increased uptake in plaque biofilms versus positive control SnF₂ without glycine.¹¹

The effect of SnF₂ on plaque biofilm translates into reduction of bleeding on probing, which may be clinically important to the gingival health of patients. The absence of sulcular bleeding over repeated dental examinations has been shown to be a reliable predictor for the maintenance of periodontal health, as measured by lack of attachment loss.^30-35 New guidelines were recently proposed to establish clinical criteria associated with periodontal health and diseases.¹⁶ Patients with <10% bleeding sites, without attachment loss and radiographic bone loss, were defined as periodontally healthy; patients with bleeding sites ranging from 10% to 30% were defined as suffering from localized gingivitis; patients with gingivitis levels greater than 30% of sites were defined as having generalized gingivitis. Based on these definitions, subjects on average starting this clinical trial presented with localized gingivitis with baseline levels of gingivitis averaging close to 20% of graded sites. As quickly as 1 week into the trial, 20% of patients using the SnF₂ dentifrices improved to gingival health status, compared to only 2% of those using the negative control, as defined by the guidelines proposed by the World Workshop on Periodontal Diseases.¹⁶ This translates to 12.3 times greater odds of moving to generally healthy for the SnF₂ dentifrice versus the negative control. The percentage of subjects using SnF₂ with the amino acid glycine who transitioned to generally healthy doubled to 40% following 12 weeks of brushing, signifying 6 times greater odds of improving to generally healthy status compared to the negative control. These odds of transitioning to generally healthy status are above the range that has been reported with use of SnF₂ dentifrice without the amino acid glycine as a stabilizer.³

Conclusion and Clinical Implications

In this randomized clinical trial, the novel SnF₂ dentifrice with the amino acid glycine demonstrated statistically significant gingival health improvements as early as 1 week that numerically increased during the trial. These findings indicate SnF₂ dentifrice stabilized with the amino acid glycine may represent an important advance in dentifrice technology to improve gingival health of patients.

ACKNOWLEDGMENT

The authors acknowledge Alborz Amini, ChE; Erinn Conde, BS; Theresa Fafard; and Marsha Gabbard, AA, for assistance with study execution and data analysis, and Donald J. White, PhD, for manuscript preparation.

DISCLOSURE

The study was funded by The Procter & Gamble Company. Drs. Klukowska and Zou are employees of The Procter & Gamble Company.

ABOUT THE AUTHORS

Malgorzata Klukowska, DDS, PhD

Clinical Scientist-Dentist, The Procter & Gamble Company, Mason, Ohio

Yuanshu Zou, PhD

Senior Scientist/Statistician, The Procter & Gamble Company, Mason, Ohio

Daniel Ponce, DDS

Subinvestigator, Silverstone Research Group, Las Vegas, Nevada

Pejmon Amini, DDS

Principal Investigator,Silverstone Research Group, Las Vegas, Nevada

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