This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on http://www.researchprotocols.org, as well as this copyright and license information must be included.
Leg ulcers due to venous disease are chronic wounds that can take 6 or more months to heal. Growth factors have been used to try and improve this healing; however, many such studies have failed, and that is thought to be due to enzymes in the wound that degrade the growth factors and prevent them from working.
This is a proof-of-concept study that will evaluate the treatment of chronic leg ulcers with topically applied growth factors that are combined with a therapy to prevent their inactivation in the wound. This combined therapy has the potential to speed up the healing of these wounds and thereby improve the quality of life of patients and reduce the costs to the health system.
This will be a double-blind, placebo-controlled, randomized controlled proof-of-concept study comparing growth factor with protease inhibitor wound dressings to growth factors with standard wound dressings.
The project was funded by the Canadian Institutes for Health Research and enrollment is expected to be initiated in 2018. It is expected that results will be available in 2021.
It is expected that the results of this trial will inform as to whether modifying the wound environment through the use of protease inhibitors increases the effectiveness of topically applied growth factors in the healing of chronic wounds.
ClinicalTrials.gov NCT02845466; https://clinicaltrials.gov/ct2/show/NCT02845466 (Archived by WebCite at http://www.webcitation.org/6yOPhSBUA)
Chronic wounds are prevalent across the Canadian health care continuum [
Lower limb ulcers occur in 1% to 2% of adults, with 70% of leg ulcers being of venous etiology [
Diabetes is one of the most burdensome chronic diseases in Canada with an estimated 7% of the population living with diabetes [
Wound healing is a complex process that has 4 phases [
Chronic wounds are typically characterized by an ongoing and excessive inflammatory phase while proliferative phase is trying to occur at the same time [
The levels of a number of proteases and their inhibitors have been measured in different chronic wound types. These have assessed levels of proteases within chronic wound fluid (CWF), have compared levels between acute and chronic wounds, and have compared levels between nonhealing and healing chronic wounds. Results indicate that CWF has consistently elevated levels of MMPs (MMP-1,-2,-8,-9,-13) and neutrophil elastase compared with acute wound fluid [
In addition, the elevated levels of proteases within CWF are inhibitory to the healing process within chronic wounds by degrading ECM proteins and regulators of the healing process. Therefore, reduction of protease levels in CWF has the potential to improve the healing process in these wounds.
A number of potential agents have been identified to reduce or modulate MMP activities and to control the proteolytic environment in the chronic wound. These include the antibiotic doxycycline [
A small observational study of 15 patients indicated that Promogran, in combination with Regranex, had a stimulating effect on DFUs, with rapid wound area reduction [
Wound repair is controlled by a number of growth factors that include platelet-derived growth factor (PDGF), keratinocyte growth factor, transforming growth factor β, and a number of other growth factors. The increasing understanding of the role of growth factors, including PDGF and transforming growth factor-β, in wound repair has resulted in their clinical application for the treatment of chronic wounds. Over the past decade, there have been an increasing number of randomized controlled trials using PDGF in the format of becaplermin gel (Regranex). Becaplermin (recombinant human PDGF-BB, rhPDGF-BB) is a homodimer produced by recombinant DNA (deoxyribonucleic acid) technology utilizing the insertion of the gene for the B chain of PDGF into the yeast
There have been a number of trials for the treatment of nonhealing DFUs demonstrating statistically significant effects in phase 2, phase 3, and phase 4 clinical trials [
In other types of chronic wounds such as pressure ulcers, studies using rhPDGF-BB gel showed promising results [
There is an ongoing safety review and warning from the US Food and Drug Administration regarding the amount of rhPDGF-BB gel that can be used. In a retrospective study of patients treated with rhPDGF-BB gel, the incidence rate for all cancers was 10.2 per 1000 compared with 9.2 per 1000 in matched controls [
Double-blind, placebo-controlled trials of other growth factors in venous ulcers—epidermal growth factor (EGF) [
This lack of response may be due to a number of factors including the following: degraded ECM; inefficient presentation of growth factors to the cell surface receptors in ways that do not reflect normal in vivo cell surface receptor activation; use of single growth factors instead of more physiologic combinations of growth factors; delivery of free growth factors instead of growth factors bound to or associated with appropriate ECM proteins; and the rapid loss of growth factors from the wound site, possibly due to diffusion or degradation. There is some evidence of mild to complete degradation in venous ulcer wound fluid of proteins such as vitronectin [
A novel point-of-care testing tool, WOUNDCHEK Protease Status (WOUNDCHEK Laboratories), has been developed to determine whether wounds have an increased total protease activity. This test indicates that the total protease activity is either elevated or not elevated based on a predetermined level. It does not give a quantitative measure of total or individual protease levels. If the predetermined levels are meaningful, the test may help to guide the usage of topical agents including growth factors and protease inhibitors. There are 2 registered, ongoing, and unpublished noncomparative studies of the WOUNDCHEK Protease Status test that started recruiting in March 2012, with estimated completions in March 2013 [
Recent work in the principal investigator’s laboratory has identified a potential biomarker that with a single test can determine the state of healing of a wound. In this study, GMCSF levels in CWF were elevated in nonhealing compared with healing wounds with apparent separation between the 2 groups. Overall, 90% of nonhealing wounds had a level above an observed threshold. This biomarker test, in combination with a point-of-care protease test such as WOUNDCHEK Protease Status, would provide tools for the clinician to make informed decisions on the use of topical protease inhibitors and growth factors in real time. This would represent a major advance in current wound management and be a completely novel paradigm of therapy for VLU and DFU.
The novel concept of this study is to translate the different elements of research on topical growth factors, protease inhibitors, and point-of-care testing in VLU and DFU into a paradigm of treatment that combines topical growth factors and protease inhibition. This may enable topical growth factors to be effective where they have not been previously in VLU and may enhance the benefit that has been shown in DFU. This model is based on modifying CWF to have more of the characteristics of acute wound fluid. A proof-of-concept study is needed to evaluate whether the use of topical protease inhibitors in both VLUs and DFUs inhibits protease activity and protects topically applied growth factors from degradation and whether this can be monitored by existing or future potential point-of-care tests.
A protease inhibitor in combination with PDGF will reduce protease levels and prevent growth factor degradation in chronic VLUs and DFUs as compared with a placebo.
The primary aim of this study was to determine whether the topical application of a protease inhibitor in combination with a topical growth factor prevents the degradation of applied growth factors in chronic VLUs and DFUs.
The secondary aims were as follows:
To determine whether the topical application of a protease inhibitor in combination with a topical growth factor reduces protease levels in chronic VLUs and DFUs
To determine whether the topical application of a protease inhibitor increases the levels of endogenous growth factors in chronic VLUs and DFUs
To determine whether there is an increase in healing rates at 4 weeks for both chronic VLUs and chronic DFUs treated with a protease inhibitor dressing in combination with topical PDGF as compared with a placebo
To determine the validity of the WOUNDCHEK Protease Status point-of-care test using the gold standard wound fluid assay of total protease
To determine whether the changes in protease and growth factor levels are associated with changes in the potential biomarker of wound healing, GMCSF
To pilot a cost diary for a possible subsequent phase 3 randomized controlled trial whereby cost-effectiveness will be evaluated
This is a proof-of-concept study that is a double-blind, randomized controlled trial. This trial has received provisional approval from the Hamilton Integrated Research Ethics Board, project number 2067.
Participants will be randomized to receive a protease inhibitor or a placebo for 4 weeks of the study, in combination with a topical growth factor. All participants will receive just the topical growth factor for 2 weeks to obtain baseline data on growth factor and protease levels in the wound fluid.
The interventions patients will receive are as follows:
All participants will have Regranex gel (Smith & Nephew) containing a recombinant human PDGF for topical administration, applied to their leg or foot ulcers on a weekly basis for 6 weeks. Regranex is a nonsterile, sodium carboxymethylcellulose-based topical gel. Each gram of Regranex gel contains 100 g of rhPDGF-BB. This is applied by measuring a length of gel from a 15 g tube and is applied at a rate of 0.25 cm length of gel per cm2 of ulcer area. A maximum of 2 tubes will be used for each patient, and each patient will have his or her own treatment tubes to ensure that the limit of 2 tubes is not exceeded.
Participants randomized to receive the protease inhibitor will receive Promogran (Johnson & Johnson) cut to the size of the ulcer and applied over the rhPDGF-BB.
Participants in the placebo arm will receive Aquacel (ConvaTec) dressing applied in a similar manner to the Promogran. This dressing is very similar in appearance to Promogran and has no active protease inhibition function.
VLU participants will have an absorbent pad and a 4-layer compression bandage system (Profore, Smith & Nephew) applied over the dressings.
Diabetic foot ulcer participants will have an absorbent pad fixed with tape applied over the dressings.
Randomization will occur after the first week in which the participant’s compliance with optimal standard therapy is assessed. If the participant is not compliant at this point, he or she will not be randomized into the study. The research coordinator will randomly assign eligible participants using a computer-generated randomization tool. Following eligibility screening by the research coordinator, the system will generate a unique number. The research coordinator then telephones the treatment nursing staff and reports the number. In turn, treatment research nurses refer to a manual of unique numbers generated by an independent biostatistician before study activation to determine the study intervention allocated to the randomized patient. The randomization schedule will be stratified by wound type (VLU or DFU) in variable blocks of 4 and 6. Randomized patients will receive all treatments during the study period according to the intervention they were allocated. The patients, study investigators, research coordinator, laboratory technologists, and assessing research nurses will be blinded to the treatment allocation. Only the treating team members are not blinded.
The researchers assessing the protease, growth factor, and biomarker levels in the wound fluid will be blinded to the treatment group, as will the researcher who performs the measurements on the wounds. A dressing similar in appearance to Promogran will be used in the control group so that the participants will also be blinded.
Inclusion criteria were as follows:
Men and women aged ≥18 years
Ulcer size 1-64 cm2
Ulcer extends through both the epidermis and dermis, with no exposed tendon or bone
Ulcer duration >3 months and <12 months
Ulcer located between and including the knee and ankle
For VLUs—venous refilling time <25 s on photoplethysmography or abnormal venous insufficiency duplex scan
For DFUs—confirmed type 1 or type 2 diabetes mellitus with a hemoglobin A1C <12%
Wounds that have not been treated with Promogran in the previous 4 weeks
Patients able to give informed consent
Exclusion criteria were as follows:
Ankle-brachial index <0.8
Ulcer with local or systemic signs of infection
Patients who have been previously treated with rhPDGF-BB gel
Patients receiving corticosteroids or immune suppressants
History of autoimmune disease
Uncontrolled diabetes (baseline hemoglobin A1C >12%)
Severe rheumatoid arthritis
Uncontrolled congestive heart failure
Malnutrition (albumin <2.5 g/dL)
Unable to adhere to the protocol
Known sensitivities to the wound dressings used in the trial
History of any previous malignancy
Pregnant or lactating woman
Participants will be in the study for a total of 7 weeks. During the first week, participants will not receive the study treatment. During this week, the participants with VLUs will have a standardized bandaging regimen applied and the DFU participants will have their pressure off-loading optimized. Once they have demonstrated tolerance and compliance for wearing these bandaging, they will continue for 6 weeks of study treatment.
Patients will be followed up 3 times in a week by a research clinical nurse for the treatment and by a blinded research nurse for outcome measurements (
At the first follow-up visit each week, the following will be performed:
The wound dressing will be taken down and the wound cleansed.
The surface area of the wound will be traced and the area will be measured using the Visitrak device (Smith & Nephew).
A photograph of the wound will be taken using a standardized protocol.
The amount of rhPDGF-BB to be applied for that week will be calculated based on the ulcer size.
A swab of the fluid on the surface of the wound will be taken using the WOUNDCHEK system to assess for elevated protease activity.
A sample of wound fluid will be collected using a standardized methodology.
The rhPDGF-BB will be applied to the wounds.
The protease inhibitor dressing or the placebo will be applied.
The secondary dressings will be applied.
At the second follow-up visit each week, the following will be performed:
Wound fluid will be collected.
No measurements will be done; wound photography will be taken.
The rhPDGF-BB will be applied to the wounds.
The protease inhibitor dressing or the placebo will be applied.
The secondary dressings will be applied.
At the third follow-up visit in the same week, the following will be performed:
No measurements, wound photography or fluid collection.
The rhPDGF-BB will be applied to the wounds.
The protease inhibitor dressing or the placebo will be applied.
Secondary dressings will be applied.
Treatments and measurements at each study visit.
Day | Growth factor application | Protease inhibitor application | Wound fluid collection | Point of Careswab for total protease activity (WOUNDCHEK) |
1 | x | x | x | x |
2 | x | x | x | x |
5 | x | x | x |
Primary outcome:
The difference in levels of nondegraded PDGF in CWF from the treatment and placebo treatment groups
Secondary outcomes:
The difference in levels of total protease and individual proteases in CWF from the treatment and placebo treatment groups as determined by enzyme-linked immunosorbent assay (ELISA) assay
The difference in levels of endogenous growth factors in CWF from the treatment and placebo treatment groups as determined by ELISA assay
Percentage reduction in size of ulcers after 1 month of therapy and the proportion of VLU and DFU that are in a healing trajectory. In VLU, a wound area reduction of 40% within 4 weeks is predictive of healing at 12 weeks [
The presence of elevated total protease activity using WOUNDCHEK and the change with time
The presence of a healing or nonhealing status as measured by GMCSF levels in CWF and the change with time as measured by ELISA assay
The completeness, ease of use, literacy, and timeliness of completing the pilot cost diary from a societal perspective. Both medical staff and participants will complete cost diaries
At the first follow-up visit each week, the following will be performed:
The surface area of the wound will be traced and the area will be measured using the Visitrak device (Smith & Nephew).
A photograph of the wound will be taken using a standardized protocol.
A swab of the fluid on the surface of the wound will be taken using the WOUNDCHEK system to assess for elevated protease activity. The swabs will be stored for subsequent analysis of actual protease levels.
A sample of wound fluid will be collected from the chronic wounds by covering the wound with a transparent occlusive dressing (Opsite, Smith & Nephew, Hull, UK) for approximately 1 hour. Fluid that has accumulated will then be aspirated and stored at −80°C. The wound fluid samples will be aliquoted into 20 ul samples and will be stored at −80°C. At the completion of the study, the samples will be analyzed using Quantibody analysis to measure the levels of proteases and nondegraded growth factors in the wound fluid, and a Western blot will be performed to assess rhPDGF-BB degradation. All of these samples will be assayed at the end of the study.
At the second visit in the same week, the following will be performed:
A sample of wound fluid will be collected.
A swab of the fluid on the surface of the wound will be taken using the WOUNDCHEK system to assess for elevated protease activity.
At the third follow-up visit in the same week, the following will be performed:
Swab of the fluid on the surface of the wound will be taken using the WOUNDCHEK system to assess for elevated protease activity.
The cost diary for that week will be completed.
As this is a proof-of-concept study of the biochemical efficacy of the therapy, no health service evaluations will be undertaken; however, a cost diary from a societal perspective will be validated in preparation for a future study.
One small study has assessed the levels of proteases in CWF from VLU after the application of Promogran [
Patients will be approached and recruited by the research coordinator after being identified in the clinic by the participating physicians. The study will be conducted over 3 years. The first 3 months requires study preparation including intensive protocol training to ensure reliability. A number of protocols such as wound fluid collection are not typical wound care procedures. On the basis of previous studies from this laboratory, recruitment will need 2 years, given the average of 3.5 participants recruited per month. The last 3 months will include conducting the assay analysis and data analysis and disseminating the results.
On the basis of previous studies from this laboratory, compliance is a risk, given the complexity of the participants’ chronic wound, comorbidities, and tolerance for either compression bandaging or pressure off-loading. Compliance will be assessed on the participants’ second visit by noting if they are wearing their prescribed compression bandaging or pressure off-loading device. If they are not complying with standard therapy, the participants will not enter the treatment segment of the study. The need to attend the study clinic 3 times a week for 6 weeks is demanding and risks noncompliance; however, these visits replace the need for the participants to attend their regular clinic to have their dressings changed.
An allowance has been made for just over 20% loss of participants to follow-up. To assist participants in attending the visits, reimbursement of costs for travel and parking will be offered in the form of gift certificates. The average of these would be Can $40.00.
A total of 2 sites within the same health region are required to meet the recruitment targets.
Data analysis will be performed on the outcome measures as follows:
The trial will meet Consolidated Standards of Reporting trials (CONSORT) requirements for reporting of randomized controlled trials. Baseline characteristics of patients in the 2 treatment groups will be reported using frequency distributions and descriptive statistics including measures of central tendency and dispersion.
The changes in levels of nondegraded PDGF in CWF from the treatment and placebo treatment groups will be analyzed using repeated measures analysis of variance (ANOVA), using the wound type and treatment group as covariates.
The changes in levels of proteases and endogenous growth factors in CWF from the treatment and placebo treatment groups will be analyzed using repeated measures ANOVA, with the wound type and treatment group as covariates.
The percentage change in wound area after 4 weeks in the study will be assessed on an intention-to-treat basis using Student
The change in total protease status after application of the Promogran or the placebo will be assessed by chi-squared analysis.
The change in healing status as determined by GMCSF biomarker after application of the Promogran or the placebo will be assessed by chi-squared analysis. Healing analysis will be performed on an intention-to-treat basis.
All analysis will be completed using SPSS version 22 (IBM Corporation, Armonk, New York).
All statistical analyses will be performed only at the completion of the study and after final data checking is completed.
If the initial analysis demonstrates a difference between VLU and DFU responses to protease inhibitor application, subgroup analyses of these 2 groups will be performed.
There is one small study that has demonstrated reduction in elastase and MMP-9 levels in CWF from VLU with the application of Promogran [
The study will employ a research coordinator 2 days per week to manage data handling, randomization, and other aspects of the study. A total of 2 clinical research nurses will be employed to perform the dressings and application of the study treatments.
The principal applicant will oversee, in conjunction with the research coordinator, the management of the study, the location and facilities for the study, treatment materials and data storage systems, and the analysis of the data from the study.
A study steering committee will be formed from experienced researchers within the Population Health Research Institute in Hamilton. A data and safety monitoring board will be blinded to treatment group. The board will review serious adverse events.
The project was funded by the Canadian Institutes for Health Research and enrollment is expected to be initiated in 2018. It is expected that results will be available in 2021.
It is expected that the results of this trial will inform as to whether modifying the wound environment through the use of protease inhibitors increases the effectiveness of topically applied growth factors in the healing of chronic wounds.
analysis of variance
chronic wound fluid
diabetic foot ulcer
extracellular matrix
epidermal growth factor
enzyme-linked immunosorbent assay
granulocyte-macrophage colony-stimulating factor
matrix metalloprotease
platelet-derived growth factor
recombinant human PDGF-BB
tissue inhibitors of metalloproteinase
venous leg ulcer
This study has received funding from the Canadian Institutes of Health Research, grant number 349897.
None declared.