Abstract: Since geographic differences in risk factors and clinical presentation of complications of diabetes are known, a study was conducted to evaluate the safety and efficacy of a topical gel (Healace 0.01%, Virchow Biotech Pvt. Ltd., Hyderabad, India) containing 0.01% recombinant human platelet-derived growth factor (rhPDGF) for healing of chronic lower-extremity diabetic ulcers. In this prospective, multicenter, double-blind, placebo-controlled, parallel group, randomized study conducted in 8 sites, 58 patients received placebo gel, and 53 patients with similar demographic characteristics received applications of rhPDGF-based gel. The percentage of patients achieving complete wound closure was considered as the primary efficacy criterion. Additional secondary criteria included the time taken to achieve complete wound closure and the percentage of ulcer surface area reduction at each visit. Efficacy evaluations carried out at 10 and 20 weeks showed that a significantly higher (p<0.01) percentage of patients (40% higher at 10 weeks and 32% higher at 20 weeks) in the rhPDGF-based gel-treated group achieved complete healing compared to the placebo-treated group. The average time to healing was significantly shorter in the treatment group (46 days) compared to the placebo group (61 days) at 10 weeks (p<0.001) and also significantly shorter in the treatment group (57 days) versus the placebo group (96 days) at 20 weeks (p<0.01). The percent of incidence of adverse events was low in both groups. Thus, the efficacy assessed at 10 weeks in the present study showed that rhPDGF-based gel healed a greater percentage of patients and also healed patients faster and caused a greater reduction in the ulcer size than placebo.

F

oot ulcers secondary to peripheral neuropathy and vascular disease are common complications of diabetes and account for high morbidity and mortality. In recent years, with global increases in prevalence of type 2 diabetes, there has been a concomitant increase in diabetic foot ulcers. According to estimates, at least 15% of patients with diabetes develop ulcers of the lower extremity during their lifetimes.¹ If improperly or inadequately treated, these ulcers become infected or gangrenous and may ultimately lead to amputation of the affected limb. The annual rate of amputation varies from 41% to 77% per 10,000 patients with diabetes² and accounts for nearly half of all the lower-limb amputations in hospitalized patients.³ In recent years, there has been a significant increase in the prevalence of diabetes in India, particularly in the urban population.⁴ In 2000, there were an estimated 31.7 million cases of diabetes reported in India, making it the country with the largest diabetic population. This figure⁵ is expected to increase to 79.4 million cases by the year 2030. As a result, a parallel increase in the incidence of diabetic foot ulcers is expected.
       Until the last decade, aside from controlling plasma glucose and infection with appropriate antidiabetic drugs and antibiotics, diabetic foot ulcers were managed with only good wound care (ie, use of periodic debridement; daily dressing with saline or moist dressings; and use of an appropriate nonweight-bearing regimen). While most of these ulcers can be treated successfully, some remain nonhealing, leading to serious and debilitating complications necessitating amputation of the affected limb. However, the identification of the role of platelet-derived growth factor (PDGF) in the formation of granulation tissue at the wound site and promotion of wound healing have given a new impetus to the development of recombinant human PDGF (rhPDGF). Among the 3 isoforms, the biological activity of rhPDGF homodimer-BB has been shown to be similar to that of naturally occurring PDGF, which includes proliferation of cells involved in the wound repair process. rhPDGF-BB is an approximately 24.5 kDa dimeric protein consisting of 2 identical polypeptide chains that are bound together by disulfide bonds.
       Several clinical trials, conducted in Western countries, have demonstrated the safety and efficacy of rhPDGF in the management of diabetic ulcers.^6–10 However, results of clinical trials conducted in developed Western countries cannot be directly extrapolated and applied to populations living in developing countries like India due to geographical differences. In fact, a prospective comparative study, conducted in 3 centers in Soest, Germany, Dar-es-Salaam, Tanzania, and Chennai, India on 613 consecutive patients with diabetic foot lesions, documented the regional differences in risk factors and clinical presentation of diabetic foot lesions.¹¹ Such differences further highlight the need to conduct a clinical trial to evaluate the product on Indian patients with diabetic foot ulcers. Keeping these factors in view, clinical safety and efficacy of a topical gel containing 0.01% rhPDGF-BB was tested in Indian patients with lower-extremity diabetic ulcers in a randomized, multicenter, double-blind, placebo-controlled, parallel group study as per the requirements of the Drug Controller General of India.

Methods

       Study drug. rhPDGF-BB gel preparation used for the clinical trial was manufactured by insertion of the gene for human PDGF into the bacteria, Escherichia coli. The rhPDGF gel is a low bioburden topical gel formulated with sodium caboxymethylcellulose and other excipients. The authors are not privy to the differences in the formulations available commercially. The formulation currently available here may not be identical but probably similar to the formulation used in the United States in that the active ingredient, rhPDGF, is used in the same concentration. A 0.01% gel containing 100µg of rhPDGF-BB/g was packed in a polyethylene-laminated tube of 7.5g or 15g for the purpose of the trial with appropriate labeling.
       Study population. This phase III trial was conducted in 8 sites, mostly public hospitals, in India. After screening, the patients were randomized prospectively into 2 groups, 1 receiving the placebo gel and the other the rhPDGF-BB gel. Fifty-eight patients (40 men and 18 women) in the placebo group and 55 patients (40 men and 15 women) in the treatment group who had consented to participate in the study were enrolled. The facilities’ respective Institutional Ethical Committees approved the clinical trial protocol, and informed consent was obtained from all patients prior to study commencement.
       Patient inclusion and exclusion criteria. All the patients included in the study, either with type 1 or 2 diabetes mellitus, were aged ≥18 years but ≤80 years and had at least 1 but less than 3 full-thickness chronic neuropathic ulcers of at least 4 weeks duration on the lower extremity. Only ulcers categorized as stage III or stage IV, as defined by the Wound, Ostomy, and Continence Nurses Society,¹² and those with infection control as determined by a wound evaluation score were considered for inclusion. If multiple ulcers were present, the largest ulcer was taken as the target ulcer, and the size of ulcer was restricted to an area of 1–40cm² (as measured by the greatest length multiplied by greatest width) post-debridement, considering that the entire period of treatment was intended for 20 weeks. Evidence of adequate perfusion of the foot, assessed with the help of a color arterial Doppler ultrasonography, was also included in the criteria. Other less preferable methods, such as palpable ankle pulses, or acceptable noninvasive methods like ankle brachial pressure index >0.70, ankle systolic pressure >70mmHg, or toe pressure >30mmHg were also used to assess foot perfusion.
       Patients with arterial venous ulcers or those with ulcers caused by osteomyelitis or burns were excluded. Patients were also excluded if they had poor nutritional status (serum total proteins <6.5g/dL), persistent infection, life-threatening concomitant diseases, deformities like Charcot foot, chronic renal insufficiency (serum creatinine >3mg/dL), uncontrolled hyperglycemia (HbA1c >12%), history of corticosteroids or immunosuppressant use, or any known hypersensitivity to the gel components. Women of childbearing age and pregnant or nursing women who were not taking contraceptives or not willing to use them were also excluded.
       Study drug administration. After randomization and allotment into the placebo or treatment group, clinical assessment of the ulcer was undertaken along with a 5-parameter wound evaluation scoring (erythema, edema, purulence, fibrin, and necrotic tissue). The patients were also placed on a standard wound care regimen consisting of appropriate sharp surgical debridement, daily ulcer cleaning and dressing, and offloading (eg, crutches or wheelchair) or, in cases where possible, complete bed rest. During this 1-week period, before the baseline visit (Visit 2), a regimen of daily wound cleaning and dressing with appropriate nonweight bearing was followed.
       The first application of the placebo or rhPDGF gel at the baseline visit was followed by a maximum treatment period of 20 weeks wherein the patients were examined once a week for the first 8 weeks and biweekly after that point to the end of the study. Either medical/paramedical personnel administered the treatment or treatment was self-administered by the patients with appropriate debridement performed by the investigators as needed. The dressings were changed daily, and the wounds were washed with saline before the daily application of the drug or placebo gel. The wounds were covered with thin 1.5mm layers of gel and covered with moist saline gauze. The amount of gel to be applied was recalculated every week by an appropriate formula based on the ulcer size. Usage of any other topical preparations at the ulcer sites was not allowed. The use of regular antidiabetic medication and appropriate use of systemic antibiotics was advised during the treatment period.
       Compliance. The amount of gel used and other compliance criteria, such as patient compliance to the daily dressing regimen, medication application, and adherence to the nonweight-bearing regimen, were monitored. The patients were considered noncompliant if 1 or more daily applications of the gel were missed, 1 or more dressing changes were missed, or offloading was not followed on 1 or more days. The patients were also considered noncompliant if >2 consecutive visits or a total of 4 visits were missed during the study period.
       Study endpoints. Efficacy evaluation was based on assessment of the functional score. At each visit subsequent to Visit 3, a functional assessment of the ulcer was undertaken to record the score as follows:
• Score 1 = 100% wound closure with complete epithelization and no drainage or scab
• Score 2 = <100% wound closure with drainage present.
       The primary efficacy criterion was defined as the percentage of patients achieving complete wound closure (ie, a functional score of 1).
       Additional secondary criteria considered were the time to achieve complete wound closure, percentage reduction in the ulcer surface area at each visit, and the total wound evaluation score at endpoint. The effect of covariates was also evaluated. Though the treatment period was defined as 20 weeks, an endpoint for efficacy evaluation at 10 weeks was also carried out as planned in the protocol.
       Safety assessment. Regular safety evaluations, such as vital signs and hematological, biochemical, and adverse events, were monitored. Open-ended questioning of patients by investigators revealed adverse events. A treatment-emergent adverse event was defined as an unfavorable or abnormal finding that was not present at baseline or, if present at baseline, increased in severity as the treatment progressed. Serious adverse events were defined as those that were immediately life threatening, were permanently or significantly disabling, required a prolonged hospitalization, resulted in long-term outpatient treatment, or resulted in a congenital anomaly, cancer, or death.
       Statistical analysis. Statistical analysis on an intent-to-treat (ITT) population, considering all patients who met the eligibility criteria, took at least 1 dose of medication, and had post-baseline efficacy data, was used for most of the parameters. Analysis of an efficacy evaluable population, defined as study-eligible patients who had received treatment for at least 7 days and were compliant to the study regimen, was used mainly for the primary efficacy criterion. The null hypothesis was used to determine if the percent cure rate would be similar between the treatment group and placebo group when compared against an alternative hypothesis that the cure rate would be higher in the treatment group when compared to the placebo group at 5% level of significance using single-tail probability. Proportions were tested using a Z-test initially.
       Overall cure rates were compared between 2 groups using a Kaplan-Meier model constructed from the data to determine whether the drug application was an improvement compared to placebo. Hazard function plots were plotted for visual comparison to enable better understanding and also for obtaining more detailed information. Secondary efficacy criteria variables were analyzed to test the main hypothesis to support any evidence for efficacy of the primary variable, namely, complete wound closure. The factors/co-variables that were thought to be potentially influencing the incidence of complete healing were examined by logistic regression analysis. Cox regression procedures were used to assess the efficacy of rhPDGF-based gel. Variables from the univariate analysis that were suspected to be related to wound closure, time taken for wound closure, or the variables (co-variates) to be controlled were identified. All categorical variables were tested by a chi-square analysis, and all continuous variables were tested using analysis of variance (ANOVA).

Results

       The baseline demographic and biochemical characteristics of patients in 2 groups, presented in Table 1, were found to be similar (p>0.1). A majority of patients (69–73%) in 2 groups were male. Since some patients had diabetic foot ulcers for many years, the average duration of ulcers varied from 19.8 to 25.5 weeks in 2 groups. Similarly, the duration of diabetes, target ulcer size, wound evaluation score, and glycemic status, as assessed by percent HbA1c, fasting plasma glucose, and 2-hour post-prandial plasma glucose, were all similar between the 2 groups of patients. There was also no difference in the nutritional status as assessed by total serum proteins between the 2 groups. The number of patients with adequate arterial blood supply to their lower extremities, as determined by either Doppler or ankle brachial pressure index, was similar in the 2 groups.

Table 1

       The disposition of patients who participated and completed the study in 2 groups is presented in Figure 1. Among the 113 patients randomized and included in the ITT population, 1 patient in the placebo group and 2 patients in the treatment group were excluded due to noncompliance. The remaining 110 patients, 57 in the placebo group and 53 in the treatment group, were considered as an efficacy evaluable population.

Figure 1

       Efficacy results. Percentage of patients achieving the primary endpoint in the 2 groups is presented in Figure 2. At the end of 10 weeks, 71% (39/55) of ITT patients in the rhPDGF group reported complete healing of their ulcers (ie, achieving a functional score of 1) compared to only 31% (18/58) in the placebo-treated group—a clear 40% difference between the 2 groups, which was highly significant (p<0.001). Even at 20 weeks, the primary endpoint was achieved in 85% (47/55) of patients in the treatment group, compared to 53% (31/58) in the placebo group—a 32% increase in the rhPDGF group compared to the placebo group (p<0.05). In line with the higher efficacy in the treatment group, the treatment failures (ie, patients with functional scores of 2) were also significantly lower in the treatment group at 10 and 20 weeks, compared to the placebo group (p<0.05). Forty-eight patients (91%) in the treatment group completed the study. Five patients (9%) from the treatment group withdrew from the study due to concomitant illness or were lost to follow-up. In the placebo-treated group, 44 patients (78%) completed the study, and 13 patients (22%) withdrew from the study for the same reasons.

Figure 2

       It was evident that the average duration taken for healing was also significantly shorter in the rhPDGF-treated group compared to placebo-treated patients as depicted by Kaplan-Meier analysis (Figure 3). Healing time was 46 days in the rhPDGF group compared to 61 days in the placebo group at the end of the 10-week treatment period (p<0.001). Even at 20 weeks of assessment, the duration for complete healing was shorter in the rhPDGF-treated group compared to the placebo-treated group—57 days in the treatment group and 96 days in the placebo group (p<0.01).

Figure 3

       The average percentage in ulcer size reduction was also significantly higher (p<0.001) in the rhPDGF-treated group compared to the placebo-treated group at 10 weeks (58% versus 26%). These efficacy results showed that the rhPDGF-based gel not only healed a greater percentage of patients but also healed the ulcers faster and caused a greater reduction in ulcer size.
       Role of covariates in healing. Several covariates (eg, ulcer location and ulcer size) were found to influence healing. There was an inverse relationship between baseline ulcer size and percent of healing, more so in the rhPDGF group (p<0.006 at ulcer size <9cm²). Overall ulcer size as a covariant was also significant at p<0.001 (Figure 4). Nonweight-bearing locations were found to heal better, more so in the rhPDGF group (p<0.05) (Figure 5). The use of systemic antibiotics, considering all types of antibiotics together, was found to contribute to increased healing percentages. In the treatment group, use of antibiotics increased the healing rate from 59% to 78%, while in the placebo group, antibiotic use increased the healing rate from 22.7% to 36%. This further confirms the synergistic relationship between antibiotic use and increased efficacy of the drug (p<0.05) (Figure 6).
       Other covariates, such as good arterial flow and glycosylated Hb, also proved to be important. Only patients with good arterial flow, as measured by the color Doppler, and no signs of occlusion or stenosis with a normal triphasic or at least a biphasic waveform pattern, were included. In the absence of the Doppler ultrasonography, use of at least an ankle brachial pressure index with a scoring of at least >0.70 (or more effectively >0.90) was considered. When all these factors were considered cumulatively, it can be concluded that 0.01% rhPDGF-BB based gel was nearly twice as effective as the placebo, particularly at the 10-week mark.

Figure 5

Figure 4

       Safety evaluation. The various safety parameters studied, such as vital signs and hematological, biochemical, or adverse events, did not suggest any relation to the treatment. The incidence of adverse events was not significantly different between the 2 groups—13% in the treatment group compared to 17% in the placebo group. Withdrawal due to adverse events was also similar at about 4% in the treatment group and 5% in the placebo group. The prevalence of cardiovascular, respiratory, musculoskeletal, and central and peripheral nervous system disorders was not statistically different across the 2 groups.
       Nearly half of the adverse events were due to ulcer-related events, such as infection and osteomyelitis. No erythematous rashes or hypersensitivity to the gel or excipients was noted in any of the patients. There were also no reports of any malignancies or mortalities. Considering all of the aforementioned parameters and the fact that the gel is a topical preparation, it can be concluded that rhPDGF-BB 0.01% gel is a safe product.

Figure 6

Discussion

       Clinical management of neuropathic diabetic foot ulcers continues to receive considerable attention in view of its serious debilitating complications.¹³ This randomized, multicenter, double-blind, placebo-controlled, parallel group study evaluated the safety and efficacy of rhPDGF gel for the treatment of lower-extremity neuropathic diabetic ulcers in 113 Indian patients. The results of this study showed that rhPDGF-BB-based gel was more efficacious than those previously reported.¹⁴
       Based on 4 earlier double-blind studies on 922 patients in the ITT population, by meta analysis, it was reported that rhPDGF-based becaplermin 100µg/g treatment showed 39% increase in complete healing compared with that of the placebo gel treatment group (50% versus 36%, respectively) at 20 weeks.¹⁴ In other open-label studies, 1 study reported 57.5% of patients achieving complete healing at 20 weeks,¹⁰ and another reported healing of 95% of all ulcers at 9 weeks.9 In the present study, there was complete healing in 71% and 85% of patients at 10 and 20 weeks, respectively, in the rhPDGF-based gel treatment group, which was 40% and 32% higher than that observed in the placebo-treated group. Not only did a higher percentage of patents achieve complete healing with rhPDGF-based gel, the mean time for closure of ulcers was also significantly shorter by 15 days in the treatment group compared to the placebo-treated group (46 days versus 61 days, respectively).
       In the present study, midterm efficacy evaluation was attempted at 10 weeks based on studies that reported healing of chronic wounds within 10 to 12 weeks.¹⁵ In a meta-analysis of 10 trials conducted on patients with diabetic foot ulcers receiving standard treatment, it was reported that 6 trials used 20 weeks as the endpoint for assessment of healing, while 4 trials used assessment at 12 weeks.¹⁶ With good wound care (ie, use of periodic debridement; daily dressing with saline or moist dressings; and use of an appropriate nonweight-bearing regimen), mean complete healing rates of 31% and 24% were reported for 20 weeks and 12 weeks, respectively. In addition, in India, due to a low literacy rate and a low socioeconomic status, patients generally do not prefer to participate in a study if it is of long duration. It was also considered that if assessment at 10 weeks proved the efficacy of rhPDGF gel, it would benefit the patient in terms of reduction in the cost as well as morbidity.
       Various factors could explain the higher efficacy of rhPDGF gel in ulcer healing observed in the present study. Wounds included in the trial were of more than 4 weeks duration, while the previously published studies included nonhealing ulcers and had attempted standard therapy for a minimum period of 8 weeks.¹³ A recent protocol was developed for the treatment of diabetic ulcers. The treatment protocol recommends use of growth factor and/or cellular therapy if the wound is not healing after 2 weeks of employing traditional therapy modalities.¹⁷ To follow these guidelines, nonhealing ulcers over a minimum period of 4 weeks were included in the trial and randomized into treatment or placebo groups.
       Several critical issues need to be addressed regarding the management of diabetic foot ulcers. The basic principles of care include correction of systemic factors, such as plasma glucose control, and correction of local factors, such as debridement, pressure relief, infection control with appropriate use of antibiotics, and revascularization when necessary.¹⁸ No trial report on wound healing, especially those related to diabetic ulcer treatment, would be complete without recognizing the importance given to the selection of cases. In the present study, only patients who satisfied the Doppler study or at least the ankle brachial pressure index criteria and displayed good control of their diabetes were included. By following all the recommended principles of care, in the present trial, a higher percentage of healing was achieved in the treatment group.
       At the beginning of the treatment period, all patients, regardless of the treatment received, were carefully monitored. Timely adjustments were made to the rhPDGF gel dose, patients were provided a good ulcer care regimen via daily moist dressing change, their wounds were appropriately debrided, and patients were placed on effective nonweight-bearing regimens with appropriate antibiotic use. These factors most likely played an important role in the overall higher healing percentage found in the rhPDGF-treated group compared to previously reported healing percentages. Thus, this trial, if any at all, underlines the importance of effective podiatric care for the treatment of the diabetic foot.
       Although the use of antibiotics in the treatment of diabetic foot ulcers remains controversial, in the present study, patients in the treatment group who were given systemic antibiotics had a higher percentage of healing. One view is to use antibiotics only in the presence of clinical infection. The other view, however, recommends administration of antibiotics freely to all patients with ulcers. Current consensus favors the latter.¹⁹ Although recent reports indicate that daily weight bearing does not increase the risk of diabetic foot ulcers,²⁰ in the present study, it was observed that nonweight bearing increases healing in patients with diabetic foot ulcers. These results are similar to those previously reported.²¹
       The patients in the placebo-treated group had similar baseline demographic characteristics and were given good wound care as well as antibiotic therapy during the study period similar to that of the treatment group; however, the percentage of patients achieving complete healing was significantly lower at 10 and 20 weeks in the placebo group.
       Recently, a cost-effective management strategy for the treatment of chronic diabetic foot ulcers utilizing rhPDGF-based topical preparations has been reported to address the staggering financial burden placed on the healthcare system and the patient.²² According to current estimates, an Indian spends an average of about $200–$1,000 on treatment of diabetic foot ulcers for primary healing and $1,000–$2,000 for ulcers necessitating amputation.²³ Although the cost of care seems to be lower than that reported in the Western world, when one considers that the per capita Gross National Product in India is <$500 and given the lack of medical insurance, the treatment of chronic diabetic leg ulcers is a considerable financial burden for the majority of Indians.²³ As a result, development of affordable alternative therapies not only provides economic benefit but also reduces patient morbidity.
       It can be summarized that rhPDGF-BB gel improves wound healing in chronic diabetic foot ulcers by achieving complete wound closure in patients with good arterial flow and effective control of diabetes.

Acknowledgment

       Members of the HEALACE Study Group included Dr. Hemanth Nandigala, Dr. T.C. Raghuram, Mr. A.N. Naidu, and Dr. R.S. Joshi (coordinating committee members); Dr. Arun Bal, Dr. Mangalanandan, and Dr. Harish Kumar (from the participating study center Amrita Institute of Medical Sciences & Research Center, Cochin, India); Dr. Jignesh Gandhi, Dr. Ashish Katewa, Dr. Rajshekaran, Dr. Sameer Naik, and Dr. Vishal Jain (from the participating study center KEM Hospital, Mumbai, India); Dr. Chandra Mouli (from the participating study center Hariprasad Memorial Trust Hospital, Hyderabad, India); Dr. Vikram Batra (from the participating study center Kamineni Hospital, Hyderabad, India); Dr. K. Janardhana Rao (from the participating study center King George Hospital, Vishakapatnam, India); Dr. Vishnu and Dr. Girija (from the participating study center Jain Institute of Vascular Sciences, Bangalore, India).