Effects of Local Minocycline Adjunctive Application in Comparison to Instrumentation Alone, in Patients With Residual Pockets Under Supportive Periodontal Therapy: a Double-blinded Randomized Controlled Clinical Trial
Phase: PHASE4
Conditions: Periodontitis
Sponsor: National Dental Centre, Singapore



 
 
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PROTOCOL SIGNATURE PAGE 
 
 
 
Protocol Title: Effects of local minocycline adjunctive application in comparison to 
instrumentation alone, in patients with residual pockets under supportive periodontal 
therapy: a double-blinded randomized controlled clinical trial. 
 
 
Protocol Number: 196/2014 
 
 
Protocol Version/ Date: Version 7 / 15.04.2016 
 
 
Sponsor Name: National Dental Centre Singapore  
Funding Source: Sunstar Singapore Pte. Ltd. 
 
 
Declaration of Investigator 
 
I confirm that I have read the above-mentioned protocol and its attachments. I agree to 
conduct the described trial in compliance with all stipulations of the protocol, regulations 
and Singapore Guideline for Good Clinical Practice (SGGCP). 
 
  
 
Principal Investigator Name:  Dr Wah Ching Tan 
                                                            
 
Principal Investigator Signature:   
 
                                                            
 
Date:  
                                                            
 
 
 
 

 
 
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Abstract: 
The aims of the present study are to assess the significance of the adjunctive effect of the 
subgingival application of a 2% minocycline hydrochloride controlled-delivery system 
(MHS) in comparison to subgingival instrumentation with application of a placebo gel, 3 
months after therapy in subjects with recurrent periodontitis undergoing supportive 
periodontal therapy (SPT) and to assess the substantivity of the gel attributable to the 
adjunctive delivery of the medication that is detected at 3 months during a 9-month period 
of regular SPT. Recurrent periodontitis will be defined as sites with residual periodontal 
probing depths and bleeding on probing after completion of initial periodontal therapy. 
This will include both persistent and recurrent periodontitis, where persistent means the 
residual periodontal site after initial periodontal therapy, and recurrent means the site 
which was improved by initial periodontal therapy, but disease recurred. 
This will be a randomised, double-blinded trial in parallel groups. 
 
Patients undergoing regular maintenance care in the National Dental Centre Singapore, 
Periodontics Unit and suffering from recurrent moderate to severe periodontitis were 
randomized to two treatment groups: 
(i) Test: Mechanical ultrasonic/ hand instrumentation and subsequent administration 
of MHS on that day (Day 0) and on Day 4, at 3 months, 6 months and 9 
months 
(ii) Control: 
Mechanical 
ultrasonic/ 
hand 
instrumentation 
and 
subsequent 
administration of a placebo gel on that day (Day 0) and on Day 4, at 3 months, 
6 months and 9 months 
 
All patients will be treated with supragingival debridement as required and subgingival 
ultrasonic/ hand instrumentation (without using antimicrobial agents as irrigating 
solutions) at all sites with pocket depths of 5mm or deeper.  
 
There will be no time limitations. All the dentition will be polished to complete the SPT 
appointment. The instrumentation time will be recorded. After completion of the 
ultrasonic/ hand instrumentation, the assigned randomization envelope will be opened, 
and test subjects will receive application of the test agent in all pockets 5 mm or deeper, 
while the placebo subjects will receive application of a placebo gel. The time required for 
application of the agents will also be recorded along with the number of used doses. 
Patients (both test and control) will be instructed not to eat and/or drink for the next 2 
hours and to avoid any form of interdental cleaning for the first 12 hours following 
treatment. Normal oral hygiene procedures will resume after 12 hours. 
 
Since there are few studies on the effect of 2% minocycline hydrochloride controlled-
delivery system (MHS) in subjects with recurrent periodontitis undergoing supportive 
periodontal therapy, this will be a valuable study. 
 
 

 
 
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1. 
BACKGROUND AND RATIONALE 
 
1.1. 
General Introduction 
 
It has been reported that regular maintenance of subjects with treated periodontal 
disease is the key consideration in the long-term periodontal prognosis of these 
subjects. Periodic prophylaxis may prevent loss of clinical attachment over long 
periods of time even in patients with less than optimal plaque control (Ramfjord 
1987). However, there are limitations in routine subgingival re-instrumentation 
especially in bleeding pockets, as only 50% of these sites improve (Tonetti et al. 
1998). Furthermore, the persistence of bleeding pockets increases the risk of 
disease progression and tooth loss (Matuliene et al. 2008, 2010). Thus there is a 
need for adjuncts that may improve the outcome especially in subjects with 
recurrent periodontitis during SPT. Some studies reported significantly better 
results with subgingival administration of local minocycline in residual pockets 
post initial periodontal therapy over a short period of time (Lu et al 2005). There 
are few studies assessing the efficacy of topical minocycline gel in subjects with 
recurrent periodontitis while in SPT, and the long term effect. 
 
The present study will examine the significance of the adjunctive effect of the 
subgingival application of a 2% minocycline hydrochloride controlled-delivery 
system (MHS) in comparison to subgingival instrumentation with application of a 
placebo gel, 3 months after therapy in subjects with recurrent periodontitis 
undergoing supportive periodontal therapy (SPT) and to assess the substantivity of  
the gel attributable to the adjunctive delivery of the medication that will be 
detected at 3 months during a 9-month period of regular SPT. 
 
1.2. 
Rationale and justification for the Study 
 
a. 
Rationale for the Study Purpose 
There are currently few studies that examine the effect of a 2% minocycline 
hydrochloride controlled-delivery system (MHS) in subjects undergoing supportive 
periodontal therapy, with residual PPD and bleeding on probing. 
 
b. 
Rationale for Doses Selected 
 
Minocycline hydrochloride concentrations in the pockets decreased rapidly for the 
first 7 hours after administration.  Subsequent decrease was very slow, with the 
concentration at 72 hours being 3.4µg/ml. At 100 hours, the concentration of 
minocycline is still effective against Prophyromonas gingivalis, Prevotella intermedia, and 
Eikenella corrodens.  
At 168 hours, the concentration reduced to 0.1µg/ml making it ineffective against 
Prophyromonas givivalis, Prevotella intermedia, and Eikenella corrodens. (Satomi et al. 1987) 
Thus in this study, re-administration of the gel will be at Day 4, before the 
concentration is reduced to 0.1µg/ml. 
 

 
 
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c. 
Rationale for Study Population 
 
The study population will be patients undergoing regular maintenance care in the 
National Dental Centre Singapore, Periodontics Unit, Department of Restorative 
Dentistry and having recurrent moderate to severe periodontitis (residual probing 
depths ≥5mm and bleeding in probing). Regular maintenance care is defined as 
professional periodontal recall of patients at a tailored time frame for examination 
of periodontal status and administration of treatment, including scaling and 
polishing, root planning of sites with probing depths of 5mm and above, and oral 
hygiene reinforcement.  
 
d. 
Rationale for Study Design 
 
The study will be a randomised, double-blinded controlled clinical trial. 
 
 
2. 
HYPOTHESIS AND OBJECTIVES 
 
2.1. 
Hypothesis 
 
The null hypothesis of this trial is that there is no difference in clinical outcomes 
with the adjunctive administration of a 2% minocycline controlled-delivery system 
(MHS) (test) together with subgingival instrumentation when compared to 
subgingival instrumentation with placebo gel (control), in subjects with recurrent 
and/or persistant probing depths, receiving supportive periodontal therapy (SPT). 
 
2.2. 
Primary Objectives 
 
To determine at 3, 6, 9 and 12 months after intervention, based on average of all 
sites ≥ 5 mm per patients with BOP at baseline: 
-Absolute change of probing pocket depth (PPD): change of PPD at each time 
point and baseline, calculated as primarily patient-based analysis, and site based as 
well 
-Percentage reduction of number of PPD ≥ 5 mm: total number of sites with PPD 
≥ 5 mm at baseline- number of sites with PPD ≥ 5 mm at each time point/total 
number of sites with PPD ≥ 5 mm at baseline, calculated both as patient-based 
and site based. 
 
2.3. 
Secondary Objectives 
 
-Reduction in bleeding on probing (BOP): this includes full mouth bleeding score 
and bleeding score of the treatment sites alone 
 
-Change of probing attachment level (PAL): similar to PPD calculation 
 
-Incidence of recurrent rate during experimental period 
 

 
 
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2.4. 
Study End points 
 
Primary endpoint:  
 
Absolute change of PPD at 3 months compared to baseline is defined as the 
difference between absolute patient PPD at 3 months and absolute patient PPD at 
baseline. For patients who have multiple trial sites, the mean of site absolute PPD 
will be used as their patient PPD score.  
 
 
Secondary endpoints:  
 
Absolute change of PPD at 6, 9 , or 12 months compared to baseline are defined as 
the difference between absolute patient PPD at 6, 9, or 12 months and absolute 
patient PPD at baseline. For patients who have multiple trial sites, the mean of site 
absolute PPD will be used as their patient PPD score.  
 
Percentage reduction of number of PPD ≥ 5 mm at 3, 6, 9 , 12 months are defined 
as the following formula:  
percentage reduction of number of PPD≥5mm at 3,6,9,12 months=  (no.of sites with 
PPD≥5mm at baseline-no.of sites with PPD≥5mm at 3,6,9,12  months)/(no.of sites with 
PPD≥5mm at baseline) 
 
Reduction in bleeding on probing (BOP) is defined as the difference between full 
mouth BOP score for each patient at two different study visits as well as the 
difference between bleeding score of the treatment sites at two different study 
visits. The comparison will be made between the following study visits: 3 months vs 
baseline, 6 months vs baseline, 9 months vs baseline, 12 months vs baseline, 3 
months vs 6 months, 3 months vs 6 months, 3 months vs 9 months, and 3 months 
vs 12 months.  
 
Change of probing attachment level (PAL) at 3, 6, 9, 12 months compared to 
baseline are defined as the difference between patient PAL score at 3, 6, 9, 12 
months and patient PAL score at baseline. For patients who have multiple trial 
sites, the mean of site PAL will be used as their patient PAL score.  
 
Incidence of recurrent rate during experimental period. The recurrent means the 
case of periodontal progression (attachment loss of 2mm or more) between test 
and groups. 
 
2.5. 
Potential Risks and benefits:  
 
a. 
End Points - Efficacy 
For the test group, potential benefits may include improvement in the clinical 
outcomes. 
 

 
 
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b. 
End Points - Safety 
For the test group, the risk involved would be the development of antibiotic 
resistance and known side-effects of the drug. As for the control group, there may 
not be any improvement in the clinical outcomes. 
 
 
 
2.6. 
List the number of subjects to be enrolled.  
230 subjects will be selected for inclusion into the study when they present for 
their regular supportive periodontal therapy appointments. Subjects fulfilling the 
inclusion criteria will be invited to participate in the study. 
 
2.7. 
Criteria for Recruitment 
 
All subjects had been previously been diagnosed with periodontitis and had 
received a full cycle of periodontal therapy consisting of oral hygiene instructions, 
scaling and root-planing. All subjects had received a minimum of 6 months of 
documented supportive periodontal care. 
 
2.8. 
Inclusion Criteria 
 
A subject must meet all of the inclusion criteria listed below to participate in this 
study: 
1) Medically healthy adults (ASA classification I-II), at least 21 years of age.  
2) Previously diagnosed with moderate to severe periodontitis and had 
completed at least 1 round of periodontal therapy including scaling and 
root-planing, oral hygiene instructions.  
3) Treated periodontitis patients undergoing maintenance care for at least 6 
months. 
4) Ability to comply with 12-month study follow-up. 
5) Recurrent moderate to severe periodontitis with no previous systemic 
antibiotic therapy during initial periodontal therapy. 
6) At least 4 teeth present with residual PPD of ≥ 5 mm on each and a 
positive bleeding on probing (BOP).  
 
2.9. 
Exclusion Criteria 
 
Subjects with any of the following exclusion criteria at baseline, will be excluded 
from the study: 
1) Medically compromised subjects (ASA classification III-V). 
2) Known allergy or other severe adverse reactions to minocycline and related 
drugs. 
3) Patients who reported local and/or systemic antibiotic therapy within 3 
months prior to baseline examination of the study, and were placed on 
antibiotics during active initial periodontal therapy. 
4) Patients with a plaque control record > 30%.  
5) Patients who had history of surgical periodontal treatment less than 5 years 

 
 
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in the area with lesions. 
6) Pregnant or intend to conceive or are breast feeding. 
 
2.10. Withdrawal Criteria  
 
Possible reasons for discontinuation of study intervention may include: 
-allergy or adverse reaction to minocycline requiring the medication to be 
discontinued. 
-other forms of antibiotics being taken during the study period. 
-subject chooses to exit study at any period of time. 
-case of periodontitis progression at any site (attachment loss of 2 mm or more), 
the patient will be excluded from the study and receive treatment as necessary. 
 
2.11. Subject Replacement 
Subjects who drop out will not be replaced. The data collected will still be adopted 
for analysis to compare the cases who had periodontitis progression between the 
test and control groups. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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3. 
TRIAL SCHEDULE 
 
 
 
 
Sequence of events 
 
Clin: PD, AL, BOP measured. 
 
SRP: ultrasonic/hand instrumentation at sites with PPD ≥ 5 mm and positive 
bleeding on probing.  
Gel: either test or placebo gel applied 
 
 
 
 
 
 
 
 
 

 
 
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4. 
STUDY DESIGN 
 
4.1. 
Summary of Study Design 
 
This will be a randomised double-blinded controlled clinical trial in parallel groups, 
with the aim to assess the adjunctive effect of a 2% minocycline hydrochloride 
controlled-delivery system (MHS) compared to mechanical maintenance therapy 3, 
6, 9 and 12 months after intervention. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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5. 
METHODS AND ASSESSMENTS 
 
Sunstar Singapore will act as a project monitor. 
 
5.1. 
Definitions 
 
a. Examiner 
 
The examiners are specialist periodontists who will perform clinical examinations 
at baseline and at the recall (3, 6, 9 and 12 months). This will be performed by 3 
calibrated examiners (Drs Marianne Ong, Wah Ching Tan and Koh Chu Guan). 
 
b. Clinicians 
 
Clinicians will be those who will be involved in instrumentation of the tooth sites 
with probing pocket depths of 5 mm or more, and in the application of the topical 
gel. 
 
c. Calibration 
 
The 3 examiners will undergo intra- and inter-examiner calibration as described 
below: 
6 subjects with presence of Ramfjord teeth (#16, 11, 24, 36, 31, 44) as being 
representative of the dentition and teeth contralateral to the Ramfjord teeth (#14, 
21, 26, 34, 41, 46) will be selected for the calibration exercise. These 6 subjects will 
be scored four times each, not consecutively, but in a random fashion. The first and 
third scorings will use the teeth proposed by Ramfjord as being representative of 
the dentition, while the second and fourth examinations will utilise the contralateral 
Ramfjord teeth as being representative of the dentition. The probing depths of the 
6 Ramfjord teeth will be scored on 4 surfaces (three buccal: mesio-buccal, buccal 
and disto buccal and one lingual aspect) and a mean score will be calculated. This 
same process is performed on the contralateral teeth. The second examiner will go 
through the same process. Each subject will be scored twice by each examiner. 
Examiner agreement will be measured through the intraclass correlation coefficient. 
Calibration will be for PPD and PAL. 
 
d.Method of Assessment 
Probing pocket depth and marginal recession will be measured to the nearest 
millimetre with a UNC-15 probe (Hu-friedy®) with 1 mm incremental markings. 
Probing attachment loss is calculated as the sum of the distance from the gingival 
margin to the CEJ(cement-enamel junction) and probing pocket depth. 
Bleeding on probing will be assessed dichotomously with a UNC-15 probe (Hu-
friedy®).  Full mouth bleeding scores (BOP) and plaque score will be calculated.  
BOP  is the presence or absence of bleeding on probing, recorded following a full 
mouth charting. 
 
e.Treatment Sites  
Treatment sites will be tooth sites with recurrent or persistent probing depths of 5 

 
 
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mm or greater and positive to bleeding on probing. 
 
f. Randomisation and Blinding 
A randomisation table will be used to randomly assign the subjects to either the test 
or control group. Blocked randomisation will be performed in blocks of ten 
whereby at every block of 10 enrolments, there will be 5 subjects randomly assigned 
either to the test or control groups. The randomisation table will be stratified to 
ensure smokers will be evenly distributed between the test and control groups. This 
will yield 2 strata. Within each stratum, the randomization sequence will be 
generated by the SAS Software 9.1, with a 1:1 allocation ratio using random block 
sizes of size 10 by a statistician to ensure that at every block of 10 enrolments, there 
will be 5 subjects randomly assigned either to the test or control groups. The 
examiners will be blinded during the clinical examination of the patients. The 
topical antibiotic will be administered by a designated clinician, who is not involved 
as the examiner. 
 
Randomisation will be performed at the time of consent. 
 
5.2. 
Contraception and Pregnancy Testing 
 
N/A.  
 
5.3. 
Study Visits and Procedures 
 
Refer to flow chart in Section 4. 
 
a. Screening  Procedures 
 
Patients are screened at their routine supportive therapy visit. Patients who fulfil 
the inclusion criteria will be invited to participate in the study.  Informed consent 
will be taken at the baseline visit. 
 
b. Study Visits and Procedures 
 
At baseline (Day 0), full mouth bleeding and plaque score will be recorded.  All 
patients will be treated with supragingival debridement as required and subgingival 
ultrasonic/hand instrumentation (without using antimicrobial agents as irrigating 
solutions) at all sites with pocket depths of 5 mm or deeper. There will be no time 
limitations. All the dentition will be polished to complete the SPT appointment. 
The instrumentation time will be recorded. After completion of the 
ultrasonic/hand instrumentation, the assigned randomization envelope will be 
opened by the research co-ordinator and test subjects will receive application of 
the test agent in all pockets 5 mm or deeper, and in the adjacent teeth close to the 
test sites, while the control subjects will receive a placebo gel. The time required 
for application of the test agent will also be recorded along with the number of 
used doses. Patients (both test and control) will be instructed not to eat and/or 
drink for the next 2 hours and to avoid any form of interdental cleaning for the 
first 12 hours following treatment. Normal oral hygiene procedures will resume 

 
 
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after 12 hours. At day 4, another application of gel will be administered for each 
respective group. 
 
At 3, 6, 9 and 12 months after treatment, patients will be recalled. At 3, 6, and 9 
months, clinical re-examinations as well as mechanical instrumentation and gel 
application will be administered for each respective group.  SPT follow-up will be 
performed every three months for a period of 1 year. 
 
c. Final Study Visit: 
 
1 year from the first application of a 2% minocycline hydrochloride controlled-
delivery system (MHS). 
 
d. Post Study Follow up and Procedures 
 
N/A 
 
e. Discontinuation Visit and Procedures 
 
N/A 
 
 
6. 
TRIAL MATERIALS 
 
 
6.1. 
Trial Product (s) 
 
The test product is a highly viscous gel for local subgingival placement composing 
of an ointment containing micro-capsule type particles for sustained release and 
the active ingredient 2% minocycline gel (10mg in each syringe of 0.5g) (Periocline, 
SUNSTAR, Osaka, Japan). The other ingredients include magnesium chloride, 
hydroxyl-ethylcellulose, aminoalkylmethacrylaye copolymer, triacetin and 
concentrated glycerine, giving the preparation a sustained-released property. It will 
be applied to experimental teeth and the adjacent teeth by gently inserting the tip 
of a specially designed applicator until the paste flows over the gingival margin. 
 
6.2. 
Storage and Drug Accountability 
 
The product will be stored at 2-8 degrees Celsius. 
 
 
7. 
TREATMENT 
7.1. 
Rationale for Selection of Dose 
 
Minocycline hydrochloride concentrations in the pockets decreased rapidly for the 
first 7 hours after administration. Subsequent decrease was very slow, with the 

 
 
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concentration at 72 hours being 3.4µg/ml. At 100 hours, the concentration of 
minocycline is still effective against Prophyromonas gingivalis, Prevotella intermedia, and 
Eikenella corrodens. At 168 hours, the concentration reduced to 0.1µg/ml (Satomi et 
al. 1987). Thus, in this study, a second application will be timed for Day 4 before 
the concentration is reduced to 0.1µg/ml. (Figure 1) 
 
7.2. 
Study Drug Formulations 
 
The test product is a highly viscous gel for local subgingival placement composing 
of a high viscous gel containing micro-capsule type particles for sustained release 
and the active ingredient 2% minocycline hydrochloride (10mg in each syringe of 
0.5g) (Periocline, SUNSTAR, Osaka, Japan). The other ingredients include 
magnesium chloride, hydroxyl-ethylcellulose, aminoalkylmethacrylaye copolymer, 
triacetin and concentrated glycerine as inactive ingredients, giving the preparation a 
sustained-released property. The placebo gel is the same gel as test but without 
active ingredient minocycline hydrochloride. 
 
7.3. 
Packaging and Labeling 
 
 
Every syringe for test is packaged in a heat-sealed foil laminated pouch which is 
light-proof and printed ’PERIOCLINE’. Every syringe for placebo is packaged 
in the same pouch as test product but attached with a label printed ‘Placebo’ on 
the pouch. 
 
A label printed with ‘For Clinical Trial Use Only Effects of local minocycline 
adjunctive application in comparision to instrumentation alone, in patients with 
residual pockets under supportive periodontal therapy: a blinded randomized 
controlled clinical trial To store at 2-8℃’ is attached on every pouch (test and 
control). 
 
7.4. 
Study Drug Administration 
 
It will be applied to the teeth with experimental sites and the adjacent teeth by 
gently inserting the tip of a specially designed applicator until the paste flows over 
the gingival margin. 
 
Test and control syringes are disposable and can be used only in one patient per 
visit. Every syringe (test and control) can be used to treat multiple pockets in the 
same patient but not multiple patients. 
 
To avoid the administration pain caused by the cold formulation, every pouch 
containing syringe (test and control) should be left in room temperature for 10 
minutes before administration. 
 
7.5. 
Specific Restrictions / Requirements  
 
The patient will be instructed not to eat and/or drink for 2 hours after drug 

 
 
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administration, and to avoid brushing, rinsing and flossing for 12 hours after 
treatment. 
 
7.6. 
Blinding 
 
The examiners will be blinded to the type of gel received by patients – placebo vs 
antibiotic. 
 
7.7. 
Concomitant therapy 
 
All medications (prescription and over the counter), vitamin and mineral 
supplements, and / or herbs taken by the participant will be documented. 
 
 
8. 
SAFETY MEASUREMENTS 
8.1. 
Definitions 
Periodontitis progression at any site (defined as clinical attachment loss of 2 mm 
or more).  
 
8.2. 
Collecting, Recording and Reporting of Adverse Events 
 
Adverse events will be recorded & reported on by the project monitor. 
8.3. 
Safety Monitoring Plan 
 
Patients will be issued a study number & de-identified on the data collection forms 
which will be stored under lock& key by the PI. The data will be entered into an 
Excel spreadsheet and stored in a password-protected laptop accessible only to the 
PI. 
 
8.4. 
Complaint Handling – 
 
Complaints will be handled by the principal investigator and co-investigators, and 
the data collected from the study subjects will be used with their consent only. 
 
9. 
ADVERSE EVENTS 
 
The occurrence of adverse effects (AE) will be monitored throughout all phase of 
the study and will be assessed in all participating subjects. Adverse events are to be 
elicited by examiner by asking the patient non-leading questions. The association 
of the AE to Periocline is to be judged by the investigator. All adverse events will 
be recorded on the individual Case Report Form by the Investigators. 
 
9.1. 
Definitions 
 
            a. An Adverse Event (AE) 

 
 
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            Any untoward medical occurrence in a patient or clinical investigation subject 
administered a pharmaceutical product and that does not necessarily have a casual 
relationship with this treatment. An adverse event (AE) can therefore be any 
unfavourable and unintended sign, symptom or disease temporarily associated with 
the use of this medicinal (investigational) product, whether or not related to this 
medicinal (investigational) product. 
 
b. A Serious Adverse Event (SAE) 
 
Any untoward medical occurrence that results in death, is life threatening, requires 
inpatient hospitalization or prolongation of existing hospitalization, results in 
persistent or significant disability/ incapacity, or results in a congenital anomaly/ 
birth defect. 
 
NOTE: Important medical events which may not result in death, be life 
threatening, or require hospitalization may be considered a SAE when, based upon 
appropriate medical judgement, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in 
this definition. 
 
c. Unexpected Adverse Drug Reaction 
 
 An adverse reaction, the nature or severity of which is not consistent with the 
applicable product information (e.g. Investigator’s Brochure for an unapproved 
investigational product or package insert/ summary of product characteristics for 
an approved product.) 
 
9.2. 
Assessment of Severity (Intensity) 
 
All adverse events will be examined by the Investigator for assessment of both 
severity and casuality using the following criteria: 
 
a. Mild 
 
Awareness of a sign or symptom, but subject can tolerate 
 
b. Moderate 
 
Discomfort is sufficient to cause interference with normal daily activity. 
 
c. Severe  
 
Resulting in an ability to do work or do usual daily activity. 
 
9.3. 
Reporting of SAEs 
 
A written SAE report by the Investigator to the Sponsor will follow immediately 

 
 
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of the Investigator’s knowledge of the occurrence of the SAE. The Investigators 
must also report all serious adverse reactions to cIRB and HSA within the timeline 
stated by the respective organizations.  
 
10. 
DATA ANALYSIS  
 
Data Collection Forms attached 
Form A & Form B: Data collection 
 
10.1. Data Quality Assurance 
 
Data will be recorded by the Principal Investigator and Co-Investigators. 
 
10.2. Data Entry and Storage 
 
Data will be first entered on paper. Upon completion of data collection, the 
information will be transferred to an Excel spreadsheet prior to data analysis. 
 
10.3. Handling of Missing Data, Withdrawals and Treatment Failure 
 
Research data from withdrawn subjects and/or treatment-failed subjects will be 
retained for the purpose of data analysis. Research data that has been collected will 
be retained for a minimum of 6 years after the completion of the clinical trial.  
 
 
11. 
SAMPLE SIZE AND STATISTICAL METHODS 
 
11.1. Determination of Sample Size  
 
It is assumed that the difference in absolute change in PPD at 3 months compared to 
baseline between test and control group is 0.5mm and a standard deviation of 1.2mm 
(Dannewitz et al. 2009). With a 1:1 allocation ratio, to attain 80% power and two-
sided 5% type I error, the required sample size is 184 patients ( 92 per group) by a 
two-sample t-test. If a drop-out rate is 20% is allowed, the required number increases 
to approximately 230 patients ( 115 per group).  
 
11.2. Statistical and Analytical Plans 
a. Definition of Analysis sets 
 
- 
Intent-to-Treat (ITT) analysis set: All patients who are randomized will be 
included in the ITT analysis set. Treatment group will be according to the pre-
planned randomization list.  
- 
Treated analysis set: All patients who are randomized and receive treatment 
will be included in treated analysis set. Treatment group will be according to the 
actual treatment that patients receive. For example, if a patient is randomized to 
test but receives control instead, control will be the treatment group for this 

 
 
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patient under treated analysis set.  
- 
Per protocol (PP) analysis set: all ITT patients without any major protocol 
deviations 
 
b. General Comment 
 
Baseline characteristics will be reported using descriptive statistics only. Mean, 
standard deviation, and interquartile range will be reported for continuous 
variables, and number of observations and proportion will be reported for 
categorical variables.   
 
An independent sample t-test (two-tailed) will be used to compare the difference 
in the following study outcomes between test and control groups:  
• 
 the Absolute change of PPD at 3, 6, 9, 12 months compared to baseline 
• 
Percentage reduction of number of PPD ≥ 5 mm at 3, 6, 9, 12 months 
compared to baseline 
• 
Reduction in bleeding on probing (BOP) at different study visits compared 
to baseline 
• 
Change of probing attachment level (PAL) at 3, 6, 9, 12 months compared 
to baseline 
Difference in mean, 95% confidence interval and p-value will be reported for each 
analysis. 5% will be set as significance level. Non-paramateric test will used when 
appropriate if any of the study outcomes is not normally distributed.  
 
Regression analysis on absolute change of PPD at 3 months compared to baseline 
will be performed to adjust for several other risk factors, including smoking, 
plaque score (at baseline), and bleeding on probing (at baseline).  If there is any 
baseline imbalance of certain patient characteristics between two groups, such 
variable will also be adjusted in regression analysis. Coefficient from regression 
analysis for each factor in the model, 95% confidence interval for each coefficient, 
and adjusted p-value will be reported.  
 
Descriptive statistics will be used to determine if the absolute change of patient 
PPD at 3 months is sustained over time of 1 year. For patients with multiple trial 
sites, the mean of absolute change of site PPD will be used as absolute change of 
patient PPD.   
 
c. Safety Analyses 
 
Number and percentage of patients experiencing 1 or more AEs/SAE will be 
summarized by expectness, relationship to study drug, outcome and severity.  
Number of patients experiencing at least 1 AE/SAE will be tabluated and 
compared between two groups by exact method. Odds ratio, and its 95% 
confidence interval will be reported 
 
d. Interim Analyses: 
         
An independent Data Safety Monitoring Board (DSMB) will be formed to review 

 
 
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the safety in this study.  The DSMB will be comprised of three members: two 
clinician, and a biostatistician, all independent from the trial.  One interim analysis 
of safety will be performed by the DSMB after 50% of the subjects are 3 months 
into the study. The analysis report will be produced using dummy treatment group. 
Only primary efficacy endpoint without p-values and safety endpoints will be 
presented to DSMB members. Decision to terminate the trial prematurely will 
depend not just on the interim results but also on other relevant information such 
as the patients' prognosis and the nature of treatment. DSMB members will inform 
the study principle investigator if any safety concern is shown from the interim 
analysis. The data for such a review will remain confidential to the designated trial 
biostatistician and the DSMB members. The Board will report their 
recommendations to the study principle investigators for them to take any 
necessary action(s).  
 
12. 
ETHICAL CONSIDERATIONS 
12.1. Informed Consent 
 
Each subject will be provided with a research folder where the research data and 
informed consent will be filed. Interested subjects will be provided with time to 
read through the consent form, with verbal explanation on the procedures and the 
risks involved. This will be explained by the examiner, and any queries or concerns 
will be answered. A witness must be present at the point of consent taking. 
Consent will be taken at the time of recruitment into the study by the examiner. 
The consent used will be for study participation. The study folders will be stored 
under lock and key. 
 
12.2. IRB review 
 
The study protocol and the associated informed consent documents will be 
reviewed by the cIRB. 
 
12.3. Confidentiality of Data and Patient Records 
 
No other person/ persons other than the investigators have access to the 
information gathered through the study. Each patient will be given a study code; 
the master list links patient’s identity and the study code will be kept separately 
from the study data and will be in the possession of the Principal Investigator. 
Data Collections Forms will only bear the patient’s allocated study code as the only 
form of identification. 
 
All hard copies of data collected will be kept under lock and key in the cabinets in 
the research office. Soft copies of the data will be stored in a password-protected 
laptop accessible only to the Principal Investigator. 
 

 
 
Protocol Version 7 ((15.04.2016) 
 
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13. 
PUBLICATIONS 
 
The study findings will be published in a peer-reviewed journal. 
 
14. 
RETENTION OF TRIAL DOCUMENTS 
 
Records for all participants, including all source documentations as well as cIRB 
records and other regulatory documentations, will be retained by the Principal 
Investigator in a secure storage facility. The records will be accessible for 
inspection and copying by authorized authorities. 
 
15. 
COSTING 
A budget sheet shows the costs and other sources of funding for the project 
should be submitted. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Topical minocycline concentration with time 
 
 
 
 
 
 
 
 

 
 
Protocol Version 7 ((15.04.2016) 
 
21 
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therapy of furcation sites: non-surgical instrumentation with or without topical 
doxycycline. J Clin Periodontol. 2009 Jun; 36(6):514-22. 
 
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Lu H-K, Chei C-J. Efficacy of subgingivally applied minocycline in the treatment of 
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Matuliene, G., Studer, R., Lang, N.P., Schmidlin, K., Pjetursson, B.E., Salvi, 
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Ramfjord, S.P. (1987) Maintenance care for treated periodontitis patients. J Clin 
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Satomi A. et al. Minocycline HCl concentration in periodontal pocket after 
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Tonetti, M.S., Muller-Campanile, V. & Lang, N.P. (1998b) Changes in the 
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