A Clinical Study Evaluating the Dermal Safety of Soluble Denosumab Microneedle Patches
Phase: PHASE1
Conditions: Osteoarthritis, Osteoarthritis (OA) of the Knee, Osteoporosis, Osteoporosis (Senile), Healthy
Sponsor: Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

Union Hospital,Tongji Medical College, 
Huazhong University of Science and 
Technology  
Clinical Research Project Protocol 
 
Project Title: A clinical study evaluating the 
dermal safety of soluble denosumab microneedle 
patches 
 
 
Protocol ID: 20250379 
Version Number: V2.0 
Version Date: May 12, 2025 
Principal Investigator: Wei Tong 
Specialty Department: Orthopedics 
Estimated Start and End Dates: July 31, 2025 - October 31, 
2025 
 
 
 
 
 
 

0BProject Summary 
Study Title: A clinical study evaluating the dermal safety of soluble denosumab 
microneedle patches 
Study Overview: 
Microneedles, as a new transdermal drug delivery technology, offer advantages such 
as painless minimally invasive application, convenience, and safety. They have wide-
ranging applications and promising development prospects. Microneedles generally 
refer to needles that are 10-2000 μm in height and 10-50 μm in width. The device of 
interest in drug delivery is the microneedle array, which consists of multiple 
microneedles arranged in an array on a drug carrier. Soluble denosumab microneedle 
patches are designed to precisely penetrate the skin's stratum corneum without 
reaching the pain nerve, delivering denosumab to a specific skin depth. Compared to 
subcutaneous injection, microneedles offer higher safety and better patient acceptance 
while reducing the risk of infection. Soluble microneedles overcome the limitation of 
traditional transdermal patches that can only deliver small molecule drugs. This has 
made them a hot research topic in the field of new transdermal drug delivery 
technologies, gaining widespread attention and recognition worldwide. 
Denosumab (Denosumab) is an NF-kB receptor activator inhibitor that impacts 
osteoclast differentiation and development by inhibiting the OPG/RANKL-RANK 
bone regulatory axis. Denosumab is widely used in the treatment of bone diseases 
such as osteoporosis. Osteoporosis is closely related to knee osteoarthritis, where the 
RANKL-RANK pathway plays a key role in the pathogenesis. Therefore, we 
hypothesize that soluble denosumab microneedle patches may effectively treat knee 
osteoarthritis. Due to its good biocompatibility, polyvinylpyrrolidone (PVP) makes an 
excellent drug carrier platform for soluble PVP microneedles. 
Study Objectives: 
To investigate the skin irritation potential of soluble denosumab microneedle patches. 

Study Design: 
Dose escalation, single-center clinical study. 
Total Number of Participants: 
12 participants. 
Number of Research Groups: 
12 participants / 0 control group. 
Group Breakdown: 
12 participants (divided into 4 groups, 3 participants per group, 4 patches per 
participant): 
• 
Microneedles with physiological saline 
• 
Microneedles with 0.24 mg Denosumab 
• 
Microneedles with 1.2 mg Denosumab 
• 
Microneedles with 6 mg Denosumab 
Diagnosis: 
Osteoporosis with osteoarthritis. 
Inclusion Criteria: 
1. Signed and dated informed consent. 
2. Commitment to comply with the study protocol and cooperate throughout the 
study. 
3. Age 18-70, healthy or adult male or female with osteoarthritis combined with 
osteoporosis. 
4. No history of skin diseases or other underlying conditions. 
5. No antihistamines, vasodilators, vasoconstrictors, anticoagulants, hormones, 
or immunosuppressants within the past month. 
6. If of childbearing age, women must have used contraception for at least one 
month before screening and commit to using contraception throughout the 
study period and for a specified time after the study ends. 
Exclusion Criteria: 
1. Pregnant or breastfeeding women. 

2. Smoking >10 cigarettes per day or smoking history >20 years. 
3. Presence of conditions that may interfere with the study results (e.g., severe 
diseases, infectious diseases, allergies, etc.) or relevant medical history. 
4. Individuals receiving systemic or local treatments that affect skin homeostasis. 
5. Allergic or intolerant to polyvinylpyrrolidone (PVP) or polyvinyl alcohol 
(PVA). 
6. Contraindications for denosumab (e.g., hypocalcemia). 
Study Intervention: 
Denosumab, already marketed but not for this indication. 
Soluble microneedle patches are not yet marketed. 
Denosumab is typically administered via subcutaneous injection, whereas the soluble 
denosumab microneedle patch delivers denosumab encapsulated in PVP material 
through microneedles. Polyvinylpyrrolidone K30 (PVP-K30) is a synthetic, water-
soluble polymer with excellent biocompatibility, chemical stability, and 
multifunctionality, widely used in biomedical fields, medical devices, and drug 
formulations. 
Statistical Methods: 
Sample size: 
Primary efficacy endpoint: 
Sample size: 12 
Analysis: Data will be processed and analyzed using SPSS 26.0. Descriptive statistics 
will be provided for continuous variables using frequency counts, means ± standard 
deviations. 
Study Duration: 
July 31, 2025 - October 31, 2025. 
Expected 16 weeks from recruitment of participants to data analysis, with an 
additional 2 weeks for follow-up. 
Participant Visit Time: 
3 hours. 

Study Site: 
Single-center in China 
Tongji Medical College, Huazhong University of Science and Technology, Union 
Hospital 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1BPrincipal Investigator Information 
Principal Investigator Name, Qualifications, and Contact Information 
Wei Tong, Associate Chief Physician, Licensed Physician Certificate, Medical 
Qualification Certificate, GCP Certificate 
Phone: +86 13437105155 

1.Research Background 
Microneedle drug delivery technology was selected by Scientific American, a 
prestigious popular science magazine in the U.S., as one of the "Top 10 Emerging 
Technologies with Potential to Change Humanity" in 2020. As a novel transdermal 
drug delivery method, microneedles are characterized by being painless, minimally 
invasive, convenient, and safe. They have become a research hotspot in the field of 
transdermal drug delivery. Microneedles (Microneedles) consist of multiple 
micrometer-sized tiny needle tips arranged in an array on a base. The length of the 
needles ranges from 100-3000 μm, and their diameter ranges from 50-250 μm. The 
typical length is 250-1500 μm, depending on their specific function and application 
[1]. Microneedles deliver drugs by penetrating the skin’s stratum corneum. Compared 
to traditional drug delivery methods, microneedles offer several advantages, such as 
avoiding the first-pass effect, reducing or eliminating pain, improving patient 
compliance, and enabling controlled sustained release, making them a promising 
technology [2]. 
   Currently, microneedles have been extensively studied in fields such as vaccine 
delivery, cancer treatment, and osteoporosis drug delivery [3]. Among them, 
denosumab, a RANKL inhibitor, is widely used in the treatment of osteoporosis [4]. 
However, the traditional method of administering denosumab is subcutaneous 
injection, which has certain pain and infection risks. Microneedle patches, as an 
alternative delivery method for denosumab, have garnered attention both domestically 
and internationally. Research has shown that microneedle patches have higher patient 
acceptance and safety. 
    Guangzhou Xinjiyu Pharmaceutical's clinical application for "Dexmedetomidine 
Hydrochloride Microneedle Patches" has been successfully approved and entered the 
clinical trial phase. Despite this, research on the skin safety of microneedle patches is 
relatively limited, especially the lack of systematic clinical studies in healthy 
populations. Existing literature tends to focus more on the drug delivery effects of 

microneedle patches, while there has been little in-depth discussion on potential skin 
irritation, allergic reactions, and other safety issues. 
This study is designed based on the above guidelines, using the latest microneedle 
treatment technology—soluble denosumab microneedle patches—to explore its skin 
safety and clinical efficacy in treating specific diseases. Following expert consensus, 
the study will adhere to scientific protocols during the treatment process, ensuring 
patient safety and the reliability of the results. 
 
2.Implementation of Intervention Measures 
Specific Operation Methods 
Before the study begins, healthy participants (meeting the inclusion criteria and 
excluding relevant risks) will be selected and sign an informed consent form. A total of 
12 participants will be included (divided into 4 groups, 3 participants per group, 4 
patches per participant): 
A. Microneedle with physiological saline group 
B. Microneedle with 0.24 mg denosumab group 
C. Microneedle with 1.2 mg denosumab group 
D. Microneedle with 6 mg denosumab group 
During the intervention, 4 patches will be applied to different areas of the patella 
on one side of the knee joint (upper, lower, left, and right) based on the group, and the 
immediate and delayed skin reactions will be observed after a single intervention. 
 
3.Study Design/Methods 
3.1 Overall Overview and Key Points of Study Design 
Domestic Single-Center Study 
This study is designed as a dose escalation trial aimed at evaluating the safety and 
preliminary efficacy of different doses of denosumab (De) delivered via microneedles. 
The study uses a progressive grouping method with four groups, as follows: 

A. Microneedle with physiological saline group (control group): 3 
participants, each receiving 4 microneedles (containing only physiological saline). 
The 4 microneedles are applied to 4 sites on the patella of one knee joint: upper, 
lower, left, and right. 
B. Microneedle with 0.24 mg denosumab group: 3 participants, each 
receiving 4 microneedles containing 0.24 mg denosumab. The 4 microneedles are 
applied to 4 sites on the patella of one knee joint: upper, lower, left, and right. 
C. Microneedle with 1.2 mg denosumab group: 3 participants, each receiving 
4 microneedles containing 1.2 mg denosumab. The 4 microneedles are applied to 
4 sites on the patella of one knee joint: upper, lower, left, and right. 
D. Microneedle with 6 mg denosumab group: 3 participants, each receiving 
4 microneedles containing 6 mg denosumab. The 4 microneedles are applied to 4 
sites on the patella of one knee joint: upper, lower, left, and right. 
 
3.2 Study Process 
The study will first complete the trial for the control group (microneedle with 
physiological saline) and conduct safety assessments. Once safety is confirmed, the trial 
for the 0.24 mg denosumab group will proceed, followed by safety evaluation. After 
ensuring the safety of the 1.2 mg group, the 6 mg denosumab group will be tested. Each 
group must complete the trial and receive clear safety results before proceeding to the 
next group. 
 
3.3 Key Features 
Progressive Group Design: After completing each group trial, it will be assessed 
against predefined criteria to determine if it can proceed to the next group. 
 
3.4 Sample Size  
This is an exploratory trial aimed at evaluating the potential effects and trends of 
different doses of denosumab microneedle delivery. The study is designed as a 
preliminary experiment, with 12 participants in total: 

A. 3 participants in the microneedle with physiological saline group 
B. 3 participants in the microneedle with 0.24 mg denosumab group 
C. 3 participants in the microneedle with 1.2 mg denosumab group 
D. 3 participants in the microneedle with 6 mg denosumab group 
 
3.5 Safety Priority 
Throughout the dose escalation process, phased assessments ensure the safety of 
the participants. 
This design effectively minimizes potential risks associated with higher dose trials, 
while providing important safety and efficacy data for larger-scale future studies. 
 
3.6 Blinding 
This trial will adopt a single-blind design, where the participants will only know 
that the drug is delivered via microneedles, but they will not be informed of the specific 
dosage of denosumab in the microneedles. 
 
3.7 Study Start and End 
The study will begin after ethical review approval and the inclusion of the first 
participant. 
The study will conclude after the last participant completes their follow-up. 
Upon completion of data collection and analysis for all participants, the study 
will be reported to the ethical institution as completed. 
 
4.Study Population 
4.1 Diagnostic Criteria 
Diagnosis based on the "Osteoarthritis Diagnosis and Treatment Guidelines" and 
"Osteoporosis Diagnosis and Treatment Guidelines." 
 
4.2 Inclusion Criteria 

A. Each participant must meet all inclusion criteria to be eligible for 
inclusion in the study: 
B. Osteoporosis patients who meet the "Osteoporosis Diagnosis Criteria." 
C. Age 18-70, healthy or adult male or female with osteoporosis, no drug 
contraindications. 
D. Committed to following the study protocol and cooperating throughout 
the study. 
E. Participant understands the related treatment process. 
F. Participant has the ability to provide informed consent. 
G. No use of drugs that may interfere with observation in the recent period. 
 
4.3 Exclusion Criteria 
Any baseline participant who meets one of the exclusion criteria will be excluded 
from the study: 
A. Pregnant or breastfeeding women. 
B. Smoking >10 cigarettes per day or smoking history >20 years. 
C. Presence of conditions that may interfere with the study results (e.g., 
severe diseases, infectious diseases, allergies, etc.) or relevant medical history. 
D. Individuals receiving systemic or local treatments that affect skin 
homeostasis. 
E. Allergic or intolerant to polyvinylpyrrolidone (PVP) or polyvinyl alcohol 
(PVA). 
F. Contraindications to denosumab, such as hypocalcemia. 
 
4.4 Lifestyle Requirements 
During the study, participants should avoid using cosmetics or skincare products 
that may affect skin conditions and cause infection or irritation at the intervention sites. 
Participants should avoid activities such as running, jumping, squatting, climbing 
stairs, or mountain climbing, and avoid long periods of bed rest. The application site 
should be kept dry, and participants should not intentionally rub or press the 

application site. 
Apart from the participant, at least one family member should be aware of the 
participant’s involvement in the clinical study and be able to monitor their condition. 
Family members should be able to provide feedback on the participant’s status during 
follow-up. Participants and their family members should inform the research doctor if 
the participant experiences severe discomfort (including the need for hospitalization). 
 
4.5 Participant Recruitment 
Study participants will be sourced from outpatient patients and healthy 
individuals. 
Recruitment advertisements will be used for publicity. 
Location: Tongji Medical College, Huazhong University of Science and 
Technology, Union Hospital/Orthopedics Department. 
Multiple contact methods (e.g., phone, email) will be collected to ensure smooth 
follow-up and communication. 
Participants will receive a free health assessment report after completing the study. 
 
5.Study Objectives/Endpoints 
5.1 Primary Objective (Confirmatory): 
To evaluate the skin safety of soluble denosumab microneedle patches, 
particularly their local irritation, including erythema, edema, and allergic reactions. 
Also, to validate the transdermal drug delivery performance and patient acceptance, 
providing a safety basis for microneedle patches as a treatment for osteoporosis and 
knee osteoarthritis. 
 
5.2 Secondary Objective (Exploratory): 
Further evaluate the clinical potential of microneedle patches under different 
dosage conditions and optimize their design and dosage scheme. 
 

5.3 Primary Outcome Measure: 
a.Number of Participants With Adverse Reactions 
Evaluate through skin response sheets: erythema, pain, swelling, etc. [Time 
Frame: Assessed at 15 minutes, 24 hours, 48 hours, and 2 weeks post-application.] 
b.Amount of pain 
Pain will be measured using the faces pain scales revised (0-2-4-6-8-10, 0: no 
pain, 10: very much pain). [Time Frame: Assessed at 15 minutes, 24 hours, 48 hours, 
and 2 weeks post-application.] 
c.Number of injection site reactions 
Injection site assessment for the following: Pain (grade 1/2/3) Tenderness (grade 
1/2/3) Pruritus (grade 1/2/3) Erythema (absent/present) Induration (grade 1/2/3) Blister 
(absent/present) Ulceration (absent/present) Necrosis (absent/present) Ecchymosis 
(absent/present) If any of these signs or symptoms is present, it is regarded an injection 
site reaction. [Time Frame: Assessed at 15 minutes, 24 hours, 48 hours, and 2 weeks 
post-application.] 
5.4 Secondary Outcome Measures: 
d.Soluble Denosumab Microneedle Patch Subject Satisfaction Questionnaire 
1. How satisfied are you with the application process of the microneedle patch? 
5 = Very Satisfied 
4 = Satisfied 
3 = Neutral 
2 = Dissatisfied 
1 = Very Dissatisfied 
2. How satisfied are you with the overall skin condition around the knee after using the 
microneedle patch? 
5 = Very Satisfied 
4 = Satisfied 
3 = Neutral 
2 = Dissatisfied 
1 = Very Dissatisfied 
3. Overall, how satisfied are you with the ease of using the microneedle patch? 

5 = Very Satisfied 
4 = Satisfied 
3 = Neutral 
2 = Dissatisfied 
1 = Very Dissatisfied 
4. Overall, now that you have tried this microneedle patch and completed the study, how likely are 
you to use this treatment again in the future? 
5 = Very Likely 
4 = Somewhat Likely 
3 = Neutral 
2 = Somewhat Unlikely 
1 = Very Unlikely 
5. Overall, how likely are you to recommend this microneedle patch treatment to a family member 
or friend? 
5 = Very Likely 
4 = Somewhat Likely 
3 = Neutral 
2 = Somewhat Unlikely 
1 = Very Unlikely 
[Time Frame: Assessed at 2 weeks post-application.] 
e. Number of participants with abnormal laboratory tests results 
Evaluation of blood routine and blood electrolytes to monitor participants' health 
status and safety. [Time Frame: Assessed at 2 weeks post-application.] 
 
5.5 Other Pre-specified Outcome Measures: 
f. Frequency of both local and systemic Adverse Events 
An adverse event is any adverse change from the subject's baseline condition, i.e. 
any subjective signs and symptoms, or change in a concomitant disease present at the 
screening visit. This includes inter-current signs, symptoms, illness and significant 
deviations from baseline which may occur during the course of the clinical study, 
whether considered related to treatment or not. [Time Frame: 2 weeks] 
 

6.Data Collection, Management, and Statistical 
Methods 
6.1 Data Collection Methods 
Data collection will be conducted by clinical researchers under the supervision of 
the principal investigator, who will be responsible for the accuracy, completeness, and 
timeliness of the reported data. All data must be clearly recorded to ensure traceability. 
A secure database will be established, protected by password, with logical 
validation programs set up during database creation. 
 
6.2 Data Management 
Data entry can be done through double data entry, meaning two different 
researchers will enter the same data, followed by comparison of the results to ensure 
accuracy. Range checks will be set to ensure the entered data fall within the expected 
reasonable range (e.g., certain variables should fall within physiological or 
experimental expected ranges). Uniform coding rules will be applied to sensitive data, 
such as encrypting participant names, ID numbers, etc., to avoid directly exposing 
sensitive information. 
Data will be stored in a secure database system with restricted access, allowing 
only authorized personnel to access it. 
The data entry interface will include automatic checks for logical errors and range 
errors. 
If imaging data or other materials are to be retained after the study, participant 
consent will be obtained through the informed consent form before the study begins, 
and it will be clarified that such materials may be used for future related research. 
All study data and original documents will be kept for a minimum of 2 years 

before destruction, with permission required before disposal. 
 
6.3 Statistical Methods 
For descriptive statistics, percentages will be used for categorical data, and mean 
± standard deviation will be used for continuous data. 
For inferential statistics, p-values and confidence intervals will be calculated, 
specifying one-tailed or two-tailed tests. 
No prior hypothesis testing for covariates (e.g., normality tests) is required, and 
no transformations or non-parametric tests will be performed.
 The 
primary 
outcome variable is the incidence rate of adverse reactions in patients, analyzed using 
independent samples t-test and chi-square tests. 
The secondary outcome variables include VAS pain scores and knee joint OKS 
functional scores, analyzed using independent samples t-test. 
Mean, non-adherence, and lost-to-follow-up data will be described using mean ± 
standard deviation, percentages, and 95% confidence intervals. 
Missing data will not be included in the analysis. 
 
7.References 
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PETTIS R J, HARVEY A J. Microneedle delivery: clinical studies and emerging medical 
applications[J]. Ther Deliv, 2012, 3(3):357-71. 
[2] 
JUNG J H, JIN S G. Microneedle for transdermal drug delivery: current trends and 
fabrication[J]. J Pharm Investig, 2021, 51(5):503-517. 
[3] 
SARTAWI Z, BLACKSHIELDS C, FAISAL W. Dissolving microneedles: Applications and 
growing therapeutic potential[J]. J Control Release, 2022, 348:186-205. 
[4] 
SOBACCHI C, FRATTINI A, GUERRINI M M, et al. Osteoclast-poor human osteopetrosis 
due to mutations in the gene encoding RANKL[J]. Nat Genet, 2007, 39(8):960-2.