Effect of an Active Compound Containing Gum on Dental Plaque Formation on a 4-day Accumulation Model
Phase: NA
Conditions: Dental Plaque Accumulation
Sponsor: Lactea Therapeutics, LLC

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Effect of an active compound containing gum on 
dental plaque formation on a 4-day accumulation 
model 
Principal Investigator  
Ana G. Gossweiler, DDS, MSD 
415 Lansing St., OH 106 
IU Indianapolis, IN 46202 
317-278-0772 
amgutier@iu.edu 
 
Co-Investigators 
 
Anderson Hara, DDS, MSD 
415 Lansing St., OH 106 
IU Indianapolis, IN 46202 
(317) 278-0577 
ahara@iu.edu 
 
George Eckert, MAS 
Biostatistician Supervisor, Department of Biostatistics and Health Data Science 
Program Manager - Biostatistics, Epidemiology, and Research Design Program - Indiana CTSI 
Phone: (317) 274 – 2884 
geckert@iu.edu 
 
 
Clinical Trial Site 
Oral Health Research Institute 
Indiana University School of Dentistry 
415 Lansing St 
IU Indianapolis, IN 46202 
317-274-8822 
 
 
 Final Version– February 3, 2025 
 
Support Provided by: 
 Sponsor: Lactea Therapeutics 
       Sponsor Representatives:  
Matthew Bender, CSO (matt@agrilogicsgroup.com) 
Christopher Hlubb, CEO (chris@agrilogicsgroup.com) 
Jon Snedeker, VP R&D (jon@lacteatherapeutics.com) 
Brandon Carter (brandon@hyacinthproteins.com) 
4539 Metropolitan Ct 
Frederick, MD 21704 
786-281-9452 
 
 

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Table of Contents 
   1.0      Abbreviations………………………………………………………………………………...3 
2.0 
Background & Rationale ..................................................................................................... 4 
3.0 
Objective(s) ........................................................................................................................ 5 
4.0 
Outcome Measures/Endpoints ............................................................................................. 5 
5.0 
Eligibility Criteria ............................................................................................................... 6 
6.0 
Study Design ...................................................................................................................... 7 
7.0 
Enrollment/Randomization ............................................................................................... 10 
8.0 
Study Procedures .............................................................................................................. 10 
9.0 
Reportable Events ............................................................................................................. 15 
10.0 
Data Safety Monitoring..................................................................................................... 16 
11.0 
Study Withdrawal/Discontinuation ................................................................................... 16 
12.0 
Statistical Considerations .................................................................................................. 16 
13.0 
Statistical Data Management ............................................................................................. 16 
14.0 
Privacy/Confidentiality Issues ........................................................................................... 16 
15.0 
Follow-up and Record Retention....................................................................................... 17 
16.0 
References ........................................................................................................................ 17 
 
 

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1.0 
Abbreviations   
AE 
Adverse Event 
Am 
Ante Meridiem 
cc 
Cubic Centimeter 
CRF 
Case Report Form 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act  
IRB 
Institutional Review Board 
IUSD 
Indiana University School of Dentistry 
Med Hx 
Medical History 
Mm 
Millimeter 
OH 
Oral Hygiene 
OHRI 
Oral Health Research Institute 
OHT 
Oral Hard Tissue 
OST 
Oral Soft Tissue 
PI 
Principal Investigator 
PLI 
Plaque Index 
Pm 
Post Meridiem 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SOP 
Standard Operating Procedure 
TMD 
Temporo Mandibular Disorder 
TMJ 
Temporo Mandibular Joint 
Tx 1 
Treatment 1 (Experimental Regimen) 
Tx 2 
Treatment 2 (Positive Control Regimen) 
Tx 3  
Treatment 3 (Control Regimen) 
UP 
Unanticipated Problem 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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2.0 
Background & Rationale     
The oral cavity harvests many microbial species that have a protective function against 
pathogenic foreign microbes that can compromise oral health. Due to its nature, this is a 
dynamic, stratified environment that can quickly lose its balance. Oral biofilms can shift 
composition due to local or systemic challenges, becoming a contributory etiology to some of 
the most common global oral diseases, dental caries, and periodontal conditions.[1]  
 
There is a general understanding that the oral biofilm of a healthy individual primarily 
contains bacterial species such as Neisseria, Streptococcus, Actinomyces, Veillonella, and 
Granulicatella. However, these species are also found in the dental pathologies previously 
mentioned. The pathogenesis of these microorganisms is exacerbated by the nutrients found 
in saliva, changes in microbial cooperations, and the host immune responses between 
others. If untreated, the result would be tooth loss, which can greatly affect a patient's 
quality of life. [2]  
 
Controlling dental plaque formation and proliferation has been a significant goal for dental 
professionals. Several methods, including mechanical, chemical, and educational 
interventions, have been presented and have produced good results. However, these 
interventions have limitations, especially those related to mechanical plaque removal. The 
patient's dexterity during toothbrushing and interdental flossing procedures can play an 
important role in the patient’s oral hygiene. [3-5] Alternative methods for plaque removal 
may benefit some risk populations, such as the elderly, patients with disabilities, and those 
without access to clean water. [6-8] 
 
Chewing sugar-free gum can increase salivary flow, which will favor a more neutral dental 
plaque pH, facilitate the natural cleansing of sugars, and favor enamel remineralization.[9, 
10]  Also, adding other products to the gum can significantly impact the accumulation of 
dental plaque and decrease gingivitis in patients. [11, 12]  
 
Lactoferrin is a naturally occurring protein ordinarily present in bovine milk. It is historically 
linked with the production of infant formula,[13] and more recently, it has been used in anti-
plaque dental products due to its ability to destabilize the cell membrane of gram-negative 
bacteria, increasing permeability.[14] It is typically considered safe in oral applications. 
Endogenously, human Lactoferrin is present in the oral mucosa at low concentrations and in 
higher concentrations in orally consumed materials such as raw breast milk. [15] Companies 
that produce bovine lactoferrin have received GRAS approval by the FDA for use in infant 
formula.[13] An animal study also tested the oral toxicity of Lactoferrin at over 2g/kg/day in 
rats, and no adverse effects were observed.[16]  
 
There is a strong reason to suspect that Lactoferrin may be efficacious in promoting good 
oral health and significantly inhibiting the formation of dental plaque.[17] Studies on the 
Lactoferrin protein have shown that it can protect against Streptococcus mutans-induced 
caries in mice and reduces the bacterial attachment of Streptococcus gordonii in the oral 
cavity.[18, 19]  Finally, Lactoferrin has been used in other clinical studies in combination with 
lactoperoxidase, and it was found to improve gingival inflammation and supragingival plaque 
antibacterial effects significantly.[20, 21]  

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Lactea Therapeutics has formulated a gum product containing an active innate immune 
protein, MIIP-E2. This product is an ultrapure (>99%) fully native and completely active 
form of bovine lactoferrin extracted and purified directly from raw milk. MIIP-E2 aims to 
develop a field-deployable dental care solution to enhance on-the-go oral care targeting 
military use. Due to the capability of the MIIP-E2 gum to coat the inner surfaces of the 
mouth, it can function as a barrier to biofilm adherence. The dosage of MIIP-E2 proposed for 
use in this study is dramatically below these acceptable levels in milk-based infant formulas, 
which range between 101.3 and 60.1 mg daily.[13]  
 
The primary endpoint of this study is to evaluate the efficacy of MIIP-E2 in inhibiting plaque 
formation in healthy patients in the absence of tooth brushing using a 4-day plaque 
accumulation model. The secondary endpoint is to evaluate whether MIIP-E2-containing gum 
alters the oral microflora after the tested period.  
3.0 
Objective(s) 
3.1 
Primary Objective 
This study aims to evaluate and compare the efficacy of MIIP-E2 in inhibiting plaque 
formation between two gum treatments in healthy patients in the absence of tooth 
brushing using a 4-day plaque accumulation model. 
 
3.2 
Secondary Objective  
The secondary objective of this study is to evaluate whether MIIP-E2-containing gum 
alters the oral microflora.  
 
3.3 
Tertiary/Exploratory/Correlative Objectives 
The exploratory objective is to compare the plaque accumulation between the 
treatments using photographs.  
4.0 
Outcome Measures/Endpoints 
4.1 
Primary Outcome Measures    
The primary outcome measures for plaque will be assessed by a dental plaque index, 
a saliva analysis and intraoral photographs. 
 
A. Dental Plaque Assessment: Dental plaque will be identified using a disclosing 
solution and scored using the Turesky modification of the Quigley and Hein Plaque 
Index (PLI). (Table 3) Each tooth, except the third molars, will be scored on six sites 
(Mesio-buccal, buccal, disto-buccal, mesio-lingual, lingual, and disto-lingual). The 
plaque score index for an individual is determined by adding all the individual scores 
and dividing the total score by the number of surfaces examined. [22] 
 
B. Salivary analysis for 16S sequencing 
In addition, patient saliva samples will be taken before and after each treatment 
regimen for 16S rRNA microbiome sequencing using the Qiagen PAXgene Saliva 
Collector and protocols that accompany the collection kit (https://www.qiagen.com 
/us/products/discovery-and-translational-research/sample-collection-
stabilization/dna/pax gene-saliva-collector). Sequencing will be performed at CD 
Genomics, a third-party vendor, using protocols for full-length PacBio sequencing and 

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analyzed using standard methodologies to identify microbial taxonomy.[23] No 
human genetic information will be collected or analyzed using this methodology. 
 
C. Dental Plaque Visual Assessment with photographs Outcome Measure  
 
Images of dental plaque will be used for both qualitative representations of outcomes 
in future reports as well as used as a secondary, complementary quantitative analysis 
for the PLI assessment. For the quantitative analysis, plaque coverage on teeth will 
be assessed using an image thresholding area measurement as previously described. 
This analysis is anticipated to provide similar results to the PLI as mentioned earlier 
but may allow for further analysis and quantitative precision. Depending on 
preliminary analysis, these measurements may be used to further assess details of 
product efficacy, such as regional changes in plaque across teeth, to understand 
whether the reduction in plaque occurred primarily on surfaces with direct exposure 
to the gum or throughout the mouth.[24]  
 
Table 2: 
Quigley-Hein Plaque Index Scores with Turesky Modifications (PLI) 
Score Description 
0 
No plaque 
1 
Separate flecks of plaque at the cervical margin of the tooth 
2 
A thin continuous band of plaque (up to one mm) at the cervical margin of the 
tooth 
3 
A band of plaque wider than one mm but covering less than one-third of the 
crown of the tooth 
4 
Plaque covering at least one-third but less than two-thirds of the crown of the 
tooth 
5 
Plaque covering two-thirds or more of the crown of the tooth 
 
4.2 
Safety Outcome Measure 
Safety outcomes will be measured by assessing any study-related soft or hard tissue 
abnormalities found during the study. Oral soft tissue examinations (OST) will be 
assessed at the screening visit and at the beginning and the end of each treatment 
period. Oral Hard Tissue Findings (OHT) will be assessed during the screening and the 
last subject visits. Any abnormal oral findings will be recorded and evaluated to assess 
causality and possible relation to the study product. Abnormal findings during the study 
will be recorded as an AE or SAE.  
5.0 
Eligibility Criteria 
5.1 
Inclusion Criteria 
• 
Adult subjects between 18 and 65 years old 
• 
Willing to read and sign the IRB-approved informed consent.  
• 
Healthy, as determined by pertinent medical history at the study dentist's discretion. 
• 
A minimum of 20 natural teeth (excluding third molars) with at least two scorable 
surfaces per tooth (teeth with full crowns, large/extensive restorations on the 
interproximal areas, and orthodontic bands will not be included in the tooth count) 

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• 
PLI of 2 or greater (Based on the Turesky modification of the Quigley and Hein Plaque 
Index) calculation based on a whole mouth plaque score. 
• 
Willing to comply with the study procedures. 
 
5.2 
Exclusion Criteria 
 
• 
Presence of any acute or chronic condition, organ system disease, or medication that, in 
the principal investigator's opinion, could compromise the subjects’ ability to participate 
in the study. 
• 
Gross oral pathologies, including caries, calculus, or soft tissue conditions that show 
evidence of chronic neglect. 
• 
Evidence of acute periodontal conditions or periodontitis with pockets greater than 5 
mm on more than one site 
• 
Use of antibiotics 30 days prior to or during the study 
• 
Requiring the need for antibiotic premedication prior to dental procedures 
• 
Sensitivity to bovine products (lactoferrin) or any of the listed inactive ingredients  
• 
Orthodontic appliances or any removable, except lingual bar retainers 
• 
Self-reported pregnant, wanting to get pregnant, or breast-feeding female, 
• 
Self-reported allergy to disclosing solution ingredients (red dye #28) 
• 
Acute Temporomandibular Disorders (TMD)  
• 
Subject who has participated in other studies (including non-medicinal studies) involving 
product(s) within 30 days before study screening.  
• 
Subject who has previously been randomized in this study 
• 
An employee of the study site directly involved with the study 
 
6.0 
Study Design 
This will be a single-blind, single-center, 3-way crossover, randomized controlled clinical trial with 3 
treatment periods utilizing 32 subjects. (Fig 1) Each treatment period will be 4 days (96 hours +/—3 
hours) in accordance with the plaque accumulation model [25] using one of the following treatment 
groups: an active compound MIIP-E2 gum (experimental regimen), an inactive compound gum 
(negative control regimen), and a no-gum (control regimen) . Each subject will be assigned to one 
of the following three treatment regimen sequences: 
  
1. Control regimen followed by experimental regimen, followed by a negative control regimen, 
or  
2. Experimental regimen followed by negative control regimen, followed by control regimen or  
3. Negative control regimen followed by control regimen, followed by an experimental regimen 
 
Subjects will be asked to refrain from oral hygiene (OH) procedures and chewing gum 12 hours (+/- 
2 hours) and from eating or drinking (except for water) 2 hours prior to the screening visit. During 
the 4-day study treatment periods, subjects will also be asked to refrain from oral hygiene practices 
or any elective dental treatment. Subjects can resume their regular oral hygiene procedures during 
the wash-out periods of approximately 10 days. 
 

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At the screening, potential subjects will be given the IRB-approved informed consent form to read 
and ask questions. Also, adequate time to decide about their participation will be provided. A study 
representative trained and delegated by the Principal Investigator will review the IRB-approved 
consent and answer any questions the potential subject might have before the subject signs/dates 
the consent. After the subject signs and dates the consent, the study representative will sign and 
date the consent to confirm that the consent process was completed before initiating any study 
procedures. The subject will be given a copy of the signed/dated consent. 
 
A designated staff member will collect the medical and concomitant medication information for the 
study dentist’s review. The dentist will perform an oral examination and a PLI to assess the subject’s 
whole-mouth plaque accumulation. A score of 2 or greater will be needed to comply with the study 
inclusion criteria based on the Turesky modification of the Quigley and Hein Plaque Index 
(PLI). Other dental characteristics specified on the inclusion/ exclusion criteria will be assessed to 
determine study eligibility. Eligible subjects will receive a dental prophylaxis and be scheduled for a 
baseline appointment. 
 
During the baseline appointment, the subjects' continuance criteria and OST examination will be 
assessed, and an unstimulated saliva sample will be collected. Subjects' teeth will be polished, 
flossed, and subsequently stained to evaluate total plaque removal and an oral assessment will be 
performed to confirm the complete removal of plaque. If any plaque is found, the plaque will be 
removed by either flossing, repolishing the surface, or with an instrument. The dental examiner will 
assess PLI. Intraoral photos will be taken, including one front and two lateral images (right and left 
side). 32 subjects will be enrolled and randomly assigned to one of the treatment regimens. 
Subjects assigned to experimental and negative treatment regimens will perform the first product 
use under supervision.  
 
Each subject will be instructed to refrain from using any oral dental hygiene procedures during the 
study treatment period (4 days). Subjects assigned to the experimental and negative treatment 
regimen will use only the provided gum product three times a day for 10 minutes after meals 
(breakfast, lunch, and dinner). Starting on one side of the mouth, chewing for 1 minute, and 
switching to the other side for another minute. Afterward, they continue chewing for the remaining 
time however they like. To assess compliance, each subject assigned to a chewing gum product will 
be provided a diary to record every time they use the product and record any events associated with 
their study participation. Finally, subjects will be scheduled to return for an end-of-regimen visit. 
 
The last product-use treatment will be after dinner the day before the visit. Subjects will refrain from 
eating or drinking (except water) for two hours before their appointment. They will also be asked to 
rinse their mouths with water approximately 30 minutes before their visit.  
 
During the end of the treatment visit, subjects' continuance criteria will be assessed; the diary will 
be reviewed for compliance; an OST exam will be performed; and an unstimulated saliva sample will 
be collected. Subjects will rinse with a disclosing solution to reveal the accumulation of dental 
plaque. Intraoral photos, including one front and two lateral images (right and left sides), will be 
taken. Afterward, the examiner will assess the PLI, and subjects will be taken to a brushing station 
to brush their teeth using a provided toothbrush, fluoride-containing toothpaste, and dental floss. 
They will be scheduled for the next visit after a washout period of approximately 10 days.  
 
For treatments 2 and 3, the procedures mentioned in the baseline visit will be repeated (except for 
randomization). A new product (or no product) will be given according to the randomization 
assignment. The end of the treatment visit will be repeated on the fourth day of each treatment 
period. 

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Study Flow Diagram (Fig. 1) 
3-treatment 3-period Crossover Design  
 
  
 
 
 
 
 
 
 
 
` 
 
 
 
Tx 1 Regimen 
 
Tx 1 Regimen 
Visits 3,  
(5 & 7) 
End-of-period 
Refrain from OH and chewing gum 12 hours (+/-two hours) and 
no eating or drinking (except water) 2 hours prior the screening 
visit 
Prior to 
enrollment 
 
Obtain informed consent, Med Hx and concomitant medication. OST/OHT. Disclosing Solution. 
PLI. Screen subjects based on inclusion/exclusion criteria. Prophylaxis. Schedule next 
appointment 
Visit 1 
Screening visit 
Continuance criteria review. OST. Saliva Collection. Teeth Polished (scaled if needed) and 
flossed. Disclosing Solution. PLI. Photos. Randomization. Product distribution. Product use 
instructions. Supervised product use procedure. Diary. Schedule the next visit appointment 
Visits 2 
( 4 & 6) 
Baseline 
~10 days Washout 
Tx 3 Regimen 
Tx 2 Regimen 
 Randomization Sequence Randomization of qualified subjects to a 
Treatment Regimen 
Obtain continuance, diary, and product. OST (OSHT at visit 7). Saliva Collection. Disclosing 
Solution. Photos. PLI. Schedule the next visit appointment (except for visit 7- end of the study) 
4-day Treatment 
The allocation of the following Tx regimen will be based on the subject’s randomization schedule.  
 
Repeated until the three treatment regimens crossover is completed. 
 
The sample randomization schedule for 3 subjects will be: 
 
Subject    
 Period 1 
            Period 2 
 
Period 3 
      
     1 
 
 Tx 3 Regimen            Tx 1 Regimen             Tx 2 Regimen  
 
     2 
 
 Tx 1 Regimen  
Tx 2 Regimen  
Tx 3 Regimen 
 
     3 
 
 Tx 2 Regimen 
Tx 3 Regimen  
Tx 1 Regimen 
No eating/drinking 2 hours before, and rinse with water approximately 30 minutes 
Prior to visit 

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7.0 
Enrollment/Randomization  
Subjects who previously participated in other Oral Health Research Institute studies (OHRI) or who 
have contacted OHRI expressing interest in potential studies will be contacted by a team member 
who will conduct a screening phone interview. If the potential subjects appear to qualify, they will 
be scheduled for the screening visit.  
 
A sufficient number of subjects will be screened to participate, and 32 will be randomized to finish 
with at least 30 subjects. We intend to randomize a nearly equal number of female and male 
subjects. A unique screening number will identify all subjects screened for study participation. 
Screening numbers will be assigned according to appearance at the study site. Subjects who meet 
all inclusion and exclusion criteria will be randomized into the study. Block randomization will be 
used to assign subjects to one of the three treatment regimen sequences:  
 
1. Control regimen (Tx 3) followed by experimental regimen (Tx 1), followed by a negative 
control (Tx 2), or  
2. Experimental regimen (Tx 1) followed by negative control regimen (Tx 2), followed by 
control (Tx 3)   or  
3. Negative control regimen (Tx 2) followed by control (Tx 3), followed by experimental 
regimen (Tx 1).  
 
Randomization numbers will be assigned using randomization schedules provided by the IU 
Department of Biostatistics and Health Data Science statistician.  
The clinical examiners will remain blind to the individual subjects’ treatment group during the study. 
If the treatment assignment must be unblinded for a subject, such as in the case of an emergency, 
the Principal Investigator will provide written documentation of the unblinding request. Otherwise, 
blinding will not be broken, unless required in the circumstances detailed above, until all subjects 
have completed the final study visit and the database has been monitored, locked, and approved by 
the Principal Investigator.  
8.0 
Study Procedures 
1)  Visit 1: Screening Visit (about 1 hour to 1 hour and 45 minutes)                                               
Subjects will refrain from any oral hygiene procedures or chewing gum 12 hours (+/-two hours) and 
from eating or drinking (except water) 2 hours before this appointment. Following the consenting 
procedure and demographic/medical history data collection, the following will be performed: 
 
a. An oral exam (including OST and OHT) will be completed.  
b. Subjects will rinse with a disclosing solution, and the whole mouth PLI will be 
assessed and recorded. A Score of 2 or greater will be needed to qualify. 
c. Inclusion/Exclusion criteria will be reviewed to determine eligibility. 
d. Subjects that qualify will receive a dental prophylaxis. 
e. Subjects that do not qualify will be taken to a brushing station to brush and floss their 
teeth 

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f. Subject’s payment will be given. If qualified, the subject will be scheduled for a 
baseline visit. 
2) Visit 2: Baseline Period 1 Visit (about 1 hour to 1 hour and 30 minutes) 
Subjects will be asked to rinse their mouths with water approximately 30 minutes before the visit. 
a. Subjects will be assessed for continuance criteria.  
b. The study dentist will perform an OST examination.  
c. Unstimulated saliva sample will be collected.  
d. The subject’s teeth will be polished, flossed, and scaled if needed; and subsequently 
stained with a disclosing solution (using a cotton swab) to evaluate total plaque 
removal. If any plaque is found, the plaque will be removed by either flossing, 
repolishing the surface, or with an instrument.    
e. An oral assessment will be performed to confirm the complete removal of plaque and 
re-apply disclosing solution (using a cotton swab), and whole-mouth PLI will be 
assessed and recorded and the adverse events will be reviewed. 
f. Intraoral photos, including one front and two lateral images (right and left sides), will 
be taken. 
g. Subjects will be randomized to a treatment regimen. 
i. A qualified study team member will prepare and distribute the randomized 
study product (chewing gum), how-to-use instructions, lifestyle restrictions, 
and compliance dairy to the subject.  
ii. Subjects assigned to the Tx 3 regimen will not receive any product and will be 
asked not to brush their teeth during the four-day (96 hours +/—3 hours) 
study treatment. 
iii. Subjects in the Tx 1 and Tx 2 regimens must use the provided gum for four 
days (96 hours +/—2 hours), chewing gum after each meal (three times a 
day—breakfast, lunch, and dinner). They start on one side of the mouth, chew 
for one minute, and switch to the other side for another minute. After that, 
they can continue chewing for the remaining time in any way they want.   
iv. All subjects will be asked to refrain from brushing or flossing their teeth during 
the study regimens and from any professional dental cleaning or dental 
elective procedures.  
h. The subject will be scheduled for an end-of-treatment visit on day 4 (96 hours +/- 3 
hours) of treatment. Subject payment for the visit will be given. 
3) Visit 3: End of Period 1 visit -scheduled at 96 hours +/- 3 hours (about 1 hour and 
10 min) 
The last product-use treatment will be after dinner the day before the visit. Subjects will 
refrain from eating or drinking (except water) for two hours before their appointment. They 

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will also be asked to rinse their mouths with water approximately 30 minutes before their 
visit. 
a. Subjects will be assessed for continuance criteria, and the diary and product will be 
collected. 
b. The study dentist will perform an OST examination.  
c. Unstimulated saliva sample will be collected.  
d. Subjects will rinse with a disclosing solution to reveal the dental plaque accumulation 
and intraoral photos will be taken, including one front and two lateral images (right 
and left sides).  
e. Whole mouth PLI will be assessed and recorded, and subjects will be taken to a 
brushing station to brush and floss their teeth. 
f. Subjects will receive payment for Visit 3 and be scheduled for Visit 4: Baseline 
treatment 2 after a washout period of approximately 10 days. 
g. Subjects can resume habitual oral hygiene procedures during the washout period. 
4) Visits 4-7: The procedures mentioned above will be repeated until each subject has 
completed each treatment period. At visit 7, in addition to the procedures described in Visit 
3, the subjects will also receive an oral hard tissue examination, and their participation in the 
study will end. 
5) Oral Exam 
The study dentist will complete an oral soft and hard tissue (OST and OHT) examination at 
screening and at the end of the study visits. Oral soft tissue (OST) examination will be 
assessed at the beginning and the end of each treatment period. The exams will be 
conducted via a visual examination of the oral cavity and perioral area utilizing a light source, 
dental mirror, gauze, periodontal probe, and tongue blade, as needed. The soft tissue 
structures examined will involve the labial mucosa, including lips, buccal mucosa, 
mucogingival folds, gingival mucosa, hard and soft palate, tonsillar and pharyngeal areas, 
tongue, sublingual area/floor of the mouth, submandibular area, major salivary glands, head 
and neck, and TMJ. Observations will be listed as “Normal” and “Abnormal,” and 
abnormalities will be described.  
The hard tissue structures examined will include assessing for enamel Irregularities, tooth 
fracture, pathologic tooth wear, cavitated lesions, residual roots, faulty restorations, and 
implants. Observations will be listed as “Absent” or “Present,” and conditions noted as 
present will be described. 
6) Unstimulated saliva collection 
Unstimulated saliva will be collected during each study treatment at the baseline and end of 
the treatment visits using the PAXgene saliva collector kits. Subjects will rinse their mouth 
with water, to remove any food debris, approximately 30 minutes prior to their appointment.  
They will not drink, eat, smoke or chew gum after they rinse their mouths. They will sit 
quietly for five minutes before beginning the collection. Subjects will spit gently into the 
provided tube with an attached funnel for about 5 to 10 minutes until the saliva samples 
(only liquid saliva, not bubble) reach the full line mark indicated in the tube. During this time, 

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they will be told to allow their saliva to pool, emptying it into a collection funnel whenever 
they need to swallow.  
PAXgene’s instructions for use will be followed for post collection. The samples will be stored 
and shipped to the sponsor at ambient temperature within the week they are collected.  
7) Salivary 16S sequence analysis 
Salivary samples will be sequenced at a third-party vendor, CD Genomics, where they will 
perform DNA isolation and use PacBio Full Length 16S rRNA sequencing to maximize 
taxonomical resolution, as previously reported.[23] Lactea Therapeutics will analyze the 
relative abundance of each identified microbial community in each sample before and after 
treatment and in the after-treatment groups for each condition. No human genetic data will 
be collected from patient samples. 
8) Disclosing Solution  
During the screening and end of treatment visits, subjects will be seated upright in the dental 
chair, and lips will be covered with Vaseline to avoid staining. Afterward, they will be asked 
to swish with 10 cc of a disclosing solution (Trace -Young Dental) for 10 seconds and gently 
spit the solution-saliva into a Styrofoam cup. The excess Vaseline will be removed from the 
lips, prior to PLI assessment or taking intraoral photos.  
For evaluation of plaque removal after teeth are polished and prior to baseline PLI 
assessment, a small amount of disclosing solution will be dispensed into a cup then directly 
applied to the teeth with a cotton swab; the excess of the disclosing solution will be 
suctioned from the subject’s mouth. 
9) Intraoral Photographs 
Three intraoral photographs (one on the front and two lateral images) will be taken at the 
beginning and end of each treatment right after the dental plaque is disclosed. No facial 
features will be captured in the photographs. A digital single-lens reflex camera (Nikon D200) 
with a 50 mm macro lens and a magnification ratio of 1:1 will be used. The light source will 
also be an external ring flash mounted in front of the lens. A double-ended cheek retractor 
will retract the lips and other soft tissue structures that may obstruct the frontal and lateral 
views to capture a better image of the teeth Intraoral mirrors will be used to take the lateral 
images (views). 
Subjects will be seated upright in the dental chair, with the head in a neutral position, 
keeping the occlusal plane perpendicular to the floor and the teeth in a maximum 
intercuspation position. For the frontal shot, the subject will hold the cheek retractors, and 
the camera will be placed at a 90-degree angle from the front teeth, focusing only on the 
anterior teeth (canine to canine). The lateral images will be captured using an intraoral 
mirror, and the retractors will be used similarly, capturing the buccal images of the premolar 
and molar teeth.  
10) Photograph Analysis 
The sponsor may perform the analysis of these photographs in a future time. The analysis is 
planned to be performed by measuring the plaque surface of each tooth using Adobe 
Photoshop. The dental plaque present will be outlined using the “Pen Tool” and” Make Path” 

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tools. Specific settings for the “color range” option will be used to highlight the disclosed 
plaque surfaces. The areas not covered with plaque will be identified using the “Mask” 
option. The plaque image will be transferred to a new file, and it will be analyzed using 
Image Pro Plus software, which will provide a measurement of the total area in mm².[24] 
11) Dental Prophylaxis 
This procedure will be performed before the first treatment assignment. It will include scaling 
any tartar or dental plaque present on the crown of the teeth using dental scalers or 
ultrasonic instruments if necessary. After scaling, the teeth will be polished using very fine 
pumice without fluoride and flossed. A qualified licensed hygienist will perform this procedure 
under the supervision of the study dentist. 
12) Teeth Polishing and Flossing 
This procedure will be performed at the beginning of each treatment. It will include removing 
any plaque deposits by polishing the teeth with very fine pumice without fluoride and 
flossing. A scaler instrument could be used if needed.  A qualified licensed hygienist will 
perform this procedure under the supervision of the study dentist. 
13) Treatment Compliance 
Product use compliance will be assessed by counting the number of gum pieces before 
dispensing (24 pieces) and at the end of each treatment period. Subjects will be required to 
return all products left over to the study site, and the number of gum pieces will be recorded 
on the treatment dispensing log. 
14) Plaque Index (PLI) 
Supragingival plaque will be assessed following disclosing with the Trace disclosing solution. 
According to the criteria of the Turesky modification of the Quigley-Hein Plaque Index (Table 
2). Each tooth will be visually assessed on six surfaces (Mesio-buccal, buccal, disto-buccal, 
mesio-lingual, lingual and disto-lingual). For PLI calculation, all the individual scores will be 
added and divided by the number of surfaces examined. The same trained examiner will also 
perform this assessment. (Table 2) 
15) Home Use Diary  
At the start of the study period, subjects assigned to the experimental and the negative 
control treatment will be provided with a diary to record the date and time of the morning 
(a.m.), afternoon (p.m.), and evening (p.m.) gum chewing procedures and any deviation 
from the regimen. Subjects assigned to the control regimen a diary will provided to record no 
oral dental hygiene procedures performed including chewing gum during the treatment 
period. In addition, subjects will record any new or changes in pre-existing medical 
conditions, medications, or treatments or any change in signs or symptoms.  
Subjects must bring the completed diary to the end of the treatment visit. Study staff will 
review the diary with the subject to confirm treatment compliance and clarify listed medical 
conditions, medications, and treatments. Subjects assigned to the experimental and the 
negative control can miss up to 10% of their chewing times (1 missing chewing per period) 
during the duration of each study period.  Those subjects who miss more than one chewing 
time will be considered non-compliant, and a staff member will instruct them about the 

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importance of keeping chewing times in compliance or remove them from future treatment 
groups if necessary.  
16) 
Active compound containing gum    
The active compound containing gum or MIIP-E2 gum V1 is comprised of Sorbitol, Gum 
Base, Mannitol, Xylitol, Triacetin, natural peppermint flavor, wax, MIIP-E2, Liquid flavoring 
(with coconut oil), sucralose, BHT, silicon dioxide and magnesium stearate. Each piece of 
gum is approximately 2.4 g in weight and contains 30 mg of the active ingredient, MIIP-E2. 
For this study, the gum containing the active ingredient will be colored purple. 
17) 
Non-active compound containing gum    
The non-active compound containing gum is comprised of Sorbitol, Gum Base, Mannitol, 
Xylitol, Triacetin, natural peppermint flavor, wax, Liquid flavoring (with coconut oil), 
sucralose, BHT, silicon dioxide, and magnesium stearate. Each piece of gum is approximately 
2.4g. For this study, these gum pieces will be colored white. 
18) Lifestyle Requirements 
Subjects will refrain from brushing their teeth and chewing gum 12 hours (+/- two hours) 
prior and not eating or drinking anything (except water) 2 hours before oral assessments at 
the screening visit. They will also refrain from using any oral hygiene procedures during the 
treatment days, except for using the provided gum if they have the product assigned.  
Subjects in the Tx1 and Tx2 regimens must use the chewing gum provided after each meal 
(three times a day—breakfast, lunch, and dinner). They should start chewing on one side of 
the mouth, chew for one minute, and switch to the other side for another minute. After that, 
they can continue chewing the remaining time (8 minutes) in any way they want. The last 
product-use treatment will be after dinner the day before the end of the treatment visit. 
Subjects will refrain from eating or drinking (except water) for two hours before their 
appointment. They will also be asked to rinse their mouths with water approximately 30 
minutes before their visit.  
During the 4-day study period, subjects will refrain from brushing, flossing, chewing gum, or 
having any elective dental procedures. 
9.0 
Reportable Events 
Adverse events are not anticipated with the MIIP-E2 or control gum or its packaging. The product is 
designed to break easily if too much pressure is applied. Cheek bites are anticipated as possible 
events associated to chewing gum. No toxicity is anticipated for the active compound. However, any 
tissue changes during the study will be documented and reported. The product is packaged in a 
small clear plastic bag that is easy to open and close. 
 
Subjects will be questioned regarding any general health or oral complaints and symptoms they 
have experienced during or following their treatment. Any findings will be documented on the AE 
CRF. Subjects reporting AEs outside the scheduled clinical visit will be assessed by the study dentist 
and/or principal investigator as soon as possible. All AEs, regardless of severity or relationship to the 
treatment product, will be recorded.  
 

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Serious AEs include any events resulting in death, decreased life expectancy, life-threatening 
situations, persistent or permanent disability/incapacity, hospitalization, or congenital 
anomaly/congenital disability. Within 24 hours, the Principal Investigator will submit a written report 
documenting the circumstances of the serious AE. The IRB will be notified within five days of the 
incident. 
10.0 Data Safety Monitoring 
The Principal Investigator and the entire study team will monitor the data and safety.  They will 
monitor data quality, subject recruitment, accrual, retention, outcome, and adverse event data, or 
assessment of scientific reports, results of related studies that may impact subject safety, and 
procedures designed to protect subjects' privacy.  Safety data will be monitored regularly and 
immediately upon discovery of any SAE, major study event, or protocol deviation.     
11.0 Study Withdrawal/Discontinuation 
Subjects may withdraw from the study for any reason and at any time during the study without 
penalty.  Their decision to stop study-related procedures will not affect their dental treatment.  The 
Principal Investigator may also withdraw the subject if they feel study participation is a safety 
concern or the subject is not compliant with study procedures. 
12.0 Statistical Considerations 
Statistical comparisons will be performed among all three treatment regimens. All outcome variables 
will be analyzed using a mixed-model analysis of variance suitable for a crossover design. The model 
will include a random effect for each subject to account for correlations among treatment outcomes 
within a subject, and fixed effects for study period and treatment regimen. A treatment sequence 
effect will be examined as an indicator of a carryover effect and removed from the model if not 
significant. Distributions of the data will be evaluated and a transformation of the data (e.g., 
logarithmic) or nonparametric tests will be used if needed. A false discovery rate (FDR) adjustment 
will be used to account for the large number of tests in the oral microbiome analyses. A two-sided 
5% significance level will be used for all tests.  
 
32 subjects will be enrolled in the study to ensure 30 complete the study. Based on a previous 
study, the estimated PLI treated mean is 1.8, standard deviation for PLI is 0.6, and within-subject 
correlation is 0.75, for an estimated standard deviation of differences 0.42. The study will have 80% 
power at a two-sided 5% significance level to detect a difference of 0.22 between two treatment 
regimens (12% relative difference), based on a paired t-test calculation. 
13.0 Statistical Data Management        
Primary data will be collected via paper source documents and CRF’s will be electronically stored as 
an excel document on the OHRI internal drive. The storage location will be backed up automatically 
every day. Quality assurance steps will follow the IU UITS service management SOPs.   
14.0 Privacy/Confidentiality Issues 
To maintain privacy, discussions about the study, the consenting process, and all other study 
procedures will be conducted in a dental office away from public places.   
 

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Participants are asked to not comment on social media or electronic platforms about the study and 
refrain from discussing the study outside of their family members/guardians/caretakes, medical 
professionals, or study staff.   
Paper records will be kept confidential and stored in a locked area only study personnel can access. 
Subjects will be given a study number that will identify all documents. Documents containing the 
subject’s name (like the signed consent form) will be stored separately from other study documents.   
 
Electronic data and photographs will be stored in an encrypted, password-protected computer file 
that only study personnel can access. 
 
Photographs will be shared with the sponsor for later analysis, and the photos will not have any 
identifying futures except for the subject number and the leg and visit when the photos were taken. 
Examples: L1001BL/ L1001EL 
 
15.0 Follow-up and Record Retention 
Study records will be maintained for at least seven years, as per Indiana law. 
 
16.0 References 
1. 
Arweiler, N.B. and L. Netuschil, The Oral Microbiota. Adv Exp Med Biol, 2016. 902: p. 45-60. 
2. 
Sanz, M., et al., Role of microbial biofilms in the maintenance of oral health and in the 
development of dental caries and periodontal diseases. Consensus report of group 1 of the 
Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease. J Clin 
Periodontol, 2017. 44 Suppl 18: p. S5-s11. 
3. 
Ruiz Núñez, M.D.R., et al., Dental plaque control strategies for the elderly population: A 
scoping review. Int J Dent Hyg, 2022. 20(1): p. 167-181. 
4. 
Sälzer, S., et al., Contemporary practices for mechanical oral hygiene to prevent periodontal 
disease. Periodontol 2000, 2020. 84(1): p. 35-44. 
5. 
Milleman, K., et al., Role of Manual Dexterity on Mechanical and Chemotherapeutic Oral 
Hygiene Regimens. J Dent Hyg, 2022. 96(3): p. 35-45. 
6. 
Chiappelli, F., et al., Dental needs of the elderly in the 21st century. Gen Dent, 2002. 50(4): 
p. 358-63. 
7. 
Devinsky, O., et al., Dental health in persons with disability. Epilepsy Behav, 2020. 110: p. 
107174. 
8. 
Crystal, Y.O., et al., A scoping review of the links between early childhood caries and clean 
water and sanitation: the Sustainable Development Goal 6. BMC Oral Health, 2024. 24(1): p. 
769. 
9. 
Dodds, M.W., The oral health benefits of chewing gum. J Ir Dent Assoc, 2012. 58(5): p. 253-
61. 
10. 
Takenouchi, A., et al., Effects of Chewing Gum Base on Oral Hygiene and Mental Health: A 
Pilot Study. Bull Tokyo Dent Coll, 2021. 62(1): p. 7-14. 
11. 
Weber, J., et al., Randomized controlled clinical trial on the efficacy of a novel antimicrobial 
chewing gum in reducing plaque and gingivitis in adolescent orthodontic patients. Clin Oral 
Investig, 2024. 28(5): p. 272. 
12. 
Smith, A.J., et al., The efficacy of an anti-gingivitis chewing gum. J Clin Periodontol, 1996. 
23(1): p. 19-23. 
13. 
Administration, U.S.F.D., GRAS Notice No. GRN 000669. 2017. 

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14. 
Drago-Serrano, M.E., et al., Lactoferrin-lipopolysaccharide (LPS) binding as key to 
antibacterial and antiendotoxic effects. Int Immunopharmacol, 2012. 12(1): p. 1-9. 
15. 
Cao, X., et al., Lactoferrin: A glycoprotein that plays an active role in human health. Front 
Nutr, 2022. 9: p. 1018336. 
16. 
Yamauchi, K., et al., 13-Week oral repeated administration toxicity study of bovine lactoferrin 
in rats. Food Chem Toxicol, 2000. 38(6): p. 503-12. 
17. 
Berlutti, F., et al., Lactoferrin and oral diseases: current status and perspective in 
periodontitis. Ann Stomatol (Roma), 2011. 2(3-4): p. 10-8. 
18. 
Velusamy, S.K., et al., Human lactoferrin protects against Streptococcus mutans-induced 
caries in mice. Oral Dis, 2016. 22(2): p. 148-54. 
19. 
Arslan, S.Y., K.P. Leung, and C.D. Wu, The effect of lactoferrin on oral bacterial attachment. 
Oral Microbiol Immunol, 2009. 24(5): p. 411-6. 
20. 
Nakano, M., et al., Effect of tablets containing lactoferrin and lactoperoxidase on gingival 
health in adults: A randomized, double-blind, placebo-controlled clinical trial. J Periodontal 
Res, 2019. 54(6): p. 702-708. 
21. 
Morita, Y., et al., Effects of lactoferrin and lactoperoxidase-containing food on the oral 
hygiene status of older individuals: A randomized, double blinded, placebo-controlled clinical 
trial. Geriatr Gerontol Int, 2017. 17(5): p. 714-721. 
22. 
Turesky, S., N.D. Gilmore, and I. Glickman, Reduced plaque formation by the chloromethyl 
analogue of victamine C. J Periodontol, 1970. 41(1): p. 41-3. 
23. 
Buetas, E., et al., Full-length 16S rRNA gene sequencing by PacBio improves taxonomic 
resolution in human microbiome samples. BMC Genomics, 2024. 25(1): p. 310. 
24. 
Smith, R.N., A.H. Brook, and C. Elcock, The quantification of dental plaque using an image 
analysis system: reliability and validation. J Clin Periodontol, 2001. 28(12): p. 1158-62. 
25. 
Claydon, N.C., et al., Randomized controlled trial to study plaque inhibition in calcium sodium 
phosphosilicate dentifrices. J Dent, 2016. 46: p. 61-7.