Phase 1/2 Open-label Dose-escalation Study of Plasmid DNA Expressing Two Isoforms of Hepatocyte Growth Factor in Patients With Painful Diabetic Peripheral Neuropathy

Abstract

This study aimed to evaluate the safety and preliminary efficacy of intramuscular injections of plasmid DNA (VM202) expressing two isoforms of hepatocyte growth factor (HGF) in subjects with painful diabetic peripheral neuropathy (PDPN). Twelve patients in three cohorts (4, 8, and 16 mg) received two sets of VM202 injections separated by two weeks. Safety and tolerability were evaluated and the visual analog scale (VAS), the short form McGill questionnaire (SF-MPQ), and the brief pain inventory for patients with diabetic peripheral neuropathy (BPI-DPN) measured pain level throughout 12 months after treatment. No serious adverse events (AEs) were observed. The mean VAS was reduced from baseline by 47.2% (P = 0.002) at 6 months and by 44.1% (P = 0.005) at 12 months after treatment. The VAS scores for the 4, 8, and 16 mg dose cohorts at 6 months follow-up decreased in a dose–responsive manner, by 21% (P = 0.971), 53% (P = 0.014), and 62% (P = 0.001), respectively. The results with the BPI-DPN and SF-MPQ showed patterns similar to the VAS scores. In conclusion, VM202 treatment appeared to be safe, well tolerated, and sufficient to provide long term symptomatic relief and improvement in the quality of life in patients with PDPN.

Introduction

Diabetic peripheral neuropathy (DPN) occurs in approximately 50% of all patients with diabetes and is one of the most common and debilitating complications associated with the disease.^1,2,3,4 Because of numbness and loss of pain perception, patients with DPN are prone to injuries or sores in the feet of which they may be unaware until infected. DPN thus accounts for significant morbidity by predisposing affected areas to ulceration and amputation.⁵

Spontaneous pain is the most prominent of many distressing symptoms of DPN and is the most frequent reason for seeking medical attention.⁴ Most patients with painful DPN (PDPN) suffer from sleep deprivation, depression, and impaired quality of life due to chronic pain.⁶ There are no approved drugs known to halt or reverse the progression of PDPN, and the only current therapeutic options are analgesics and glucose control.¹ Thus, there is great need for a therapeutic approach that can stimulate growth and/or regeneration of peripheral nerves to retard or reverse the nerve fiber injury/destruction associated with PDPN.

In an effort to develop an effective treatment for PDPN, in this trial, we investigated the therapeutic potential of a novel drug candidate, VM202. The safety and efficacy of VM202 for cardiovascular diseases have been demonstrated previously in extensive preclinical studies^{7,8,9,10,11,12,13} as well as three independent phase I trials.^14,15,16 VM202 is a plasmid DNA containing a therapeutic gene, HGF-X7, which encodes two isoforms of hepatocyte growth factor (HGF).⁷ HGF is a multifunctional, mesenchyme-derived cytokine with potent angiogenic and antifibrotic activities.^{17,18,19,20,21,22} To maximize its therapeutic effects, this plasmid DNA was designed to simultaneously express two naturally occurring isoforms of HGF, HGF₇₂₃ and HGF₇₂₈, which consist of 723 and 728 amino acids, respectively. Our preclinical studies showed that coexpression of the two isoforms of HGF results in greater angiogenesis than single expression of HGF₇₂₈ or VEGF₁₆₅ (vascular endothelial growth factor) in various animal models of cardiovascular disease.^7,8 HGF is also neurotrophic for peripheral sensory, sympathetic, and motor neurons, and it promotes neuronal survival and axonal outgrowth both in vitro and in vivo.^{23,24,25,26,27,28,29,30} The combined angiogenic and neurotrophic properties of HGF make it an ideal candidate for the treatment of PDPN. Because HGF has an in vivo half-life of <15 minutes,³¹ however, delivery of effective doses of exogenous HGF to target sites for therapeutically meaningful lengths of time is challenging. To overcome the instability of HGF, a gene therapy approach was adopted with local injection of plasmid DNA into the calf muscles for target-specific delivery and expression of HGF.

Phase I clinical trials involving VM202 for other serious indications such as critical limb ischemia and angina pectoris showed that this drug candidate is safe and possibly efficacious in humans.^14,15,16 Encouraged by such results, we investigated in this phase I/II study the safety and tolerability of VM202 in patients with PDPN. The efficacy of VM202 in pain reduction and enhancement of quality of life was also preliminarily evaluated using several questionnaires for symptom assessment.

Results

Baseline demographics, medical history, and concomitant medications

Twenty-eight patients with PDPN who gave informed consent were screened. Twelve of them met the screening criteria and were enrolled and treated with VM202. Subject demographics, medical histories, and concomitant medications are summarized in Table 1. All study participants were male, and the mean age at enrollment was 58.8 ± 8.8 years, with a range from 39 to 69 years. Eleven of the treated subjects were Caucasian, and one was African American. Among the 12 patients who received treatment, 11 had type II diabetes, whereas 1 had type I diabetes. Eight patients had hypertension and eight patients had dyslipidemia. Of 12 patients, 9 were on concomitant pain medications for the management of their DPN.

Table 1. Demographics, comorbidities, and concomitant pain medications at baseline.

Safety and tolerability

Safety data are available for all twelve subjects through a 12 month, follow-up period. There were no deaths or serious adverse events (AEs). No malignancy or proliferative retinopathy was observed in any patient treated with VM202. Seven subjects experienced 15 minor AEs that neither required significant intervention nor had significant clinical sequelae (Table 2). In cohort 1 (4 mg), eight AEs occurred in three patients. In cohort 2 (8 mg), three AEs were observed in two patients. In cohort 3 (16 mg), four AEs were reported in two patients. Of the 15 AEs, 6 were designated as possibly related to the study drug. One subject in cohort 2 (8 mg) experienced an injection site reaction (1 mm transient erythema around six injection sites) on day 21, which resolved by the day 30 visit. There was no evidence of any further injection site reaction from this patient at any time. All other subjects tolerated all of the intramuscular injections without incident.

Table 2. Incidence of all adverse events by cohort (15 AEs/7 subjects).

Serum HGF protein levels were determined by enzyme-linked immunosorbent assay (ELISA) preinjection on day 0, immediately preinjection on day 14, and on days 30 and 60. The after injection serum HGF protein levels remained stable throughout the study period with no peaks, and they were all within the range generally observed in patients diabetes (0.26–1.26 ng/ml)³² (Table 3).

Table 3. Level of HGF protein in serum and copy number of VM202 plasmid in blood after VM202 treatment.

VM202 plasmid levels in whole blood were determined by quantitative PCR on day 0 (preinjection, and 1 to 2 hours after injection), and on days 14 (preinjection, and 1 to 2 hours after injection), 21, 30, 60, and 90. The levels of VM202 DNA were below the lower limit of quantitation in all 12 subjects by day 90 (Table 3).

Visual analog scale for pain

The intensity of reported pain was measured using the visual analog scale (VAS) at the time of screening, on day 0 and day 14 before the treatments (injections), then on days 30, 60, 90, 180 (6 months), and 365 (12 months). The baseline VAS value was the mean of the values measured at screening and on day 0. Of 12 subjects, 9 (75%) experienced a reduction in pain at 12 months follow-up. The mean VAS score decreased significantly over time compared with the baseline score of 48.0 ± 11.8 mm, when all cohorts are considered together (P < 0.001, Table 4). The pain reduction from baseline first became statistically meaningful at day 60 (P = 0.021) and remained significantly diminished throughout the entire 12 months of study period (Table 4). The reduction in pain from baseline was greatest at 6 months follow-up with the mean VAS score decreased by 47.2%.

Table 4. Effect of plasmid DNA expressing two wild-type isoforms of HGF on VAS and SF-MPQ in 12 subjects.

Moreover, there appeared to be a dose–response effect (Figure 1a and Table 4). At the lowest dose (4 mg), there was a trend towards improvement but it never reached significance (P = 0.917). At the middle dose (8 mg), the reduction in reported pain reached significance at 6 months (P = 0.032) and remained significantly lower at 12 months follow-up (P = 0.025). At the highest dose (16 mg), the reduction in reported pain reached significance by day 60 (P = 0.048) and remained significantly lower than the baseline value until 1 year after the treatment (Table 4). As shown in Figure 1a, the size of the decrease in reported pain at 6 months follow-up was seen in a dose-dependent manner (cohort 1, 21%; cohort 2, 53%; cohort 3, 62%). Notably, at the highest dose, the maximal effect was observed at 6 months follow-up.

Figure 1.

Effect of VM202 injection on visual analog scale (VAS) and SF-MPQ in 12 patients separated by cohort. (a) The average VAS scores over time are represented by dose cohorts. The decrease in reported pain occurred in a dose-dependent manner. Notably, at the highest dose, the maximal effect was observed at 6 months follow-up. (b) Although there was no statistically significant reduction among the dose cohorts, a trend of dose responsiveness was observed on short form McGill questionnaire (SF-MPQ) during the study period. Data are expressed as the mean ± SD and analyzed using one-way analysis of variance followed by Dunnett's test or Games-Howell's test. ^‡P < 0.05, day 0 versus 6 months or 12 months; ^†P < 0.05, day 0 versus 2, 3 or 12 months; *P = 0.001, day 0 versus 6 months.

Short form McGill pain questionnaires

Pain scores in both the sensory and affective components of the short form McGill pain questionnaires (SF-MPQ) improved after VM202 injections. Similar to the time-course of the VAS scores, SF-MPQ scores reached their lowest mean level at 6 months and rebounded at 12 months follow-up (Table 4). Of 12 subjects, 8 (66.7%) experienced a reduction in the sum of sensory and affective pain measures at 6 months (cohort 1: 2/4 (50%), cohort 2: 3/4 (75%), and cohort 3: 3/4 (75%)). Although there was no statistically significant reduction among the dose cohorts, a trend of dose responsiveness was observed until 6 months follow-up as shown in Figure 1b.

Brief pain inventory for patients with DPN

The brief pain inventory for patients with DPN (BPI-DPN) was completed by each subject on day 0 (before the first injection), on days 30, 60, 90, 180, and 365. Similar to the findings from pain VAS and SF-MPQ analysis, pain severity and interference reduced in a time-dependent manner (Table 5). Overall, the BPI-DPN scores were lowest at 6 months and rebounded at 12 months follow-up. However, a dose dependency by cohort was not observed. The mean pain severity decreased from 4.3 ± 1.8 at day 0 to 3.3 ± 1.8 at day 60 (P = 0.038), 3.1 ± 2.0 at day 90 (P = 0.007), 2.8 ± 1.9 at 6 months (P = 0.001), and 3.6 ± 2.1 at 12 months follow-up (P = 0.242). “Worst pain in the past 24 hours” decreased by 39% (P = 0.017) from 6.3 ± 1.6 at day 0 to 3.8 ± 2.3 at 6 months. “Average pain” reduced by 34.8% (P = 0.082) from 4.6 ± 1.0 at day 0 to 3.0 ± 1.6 at 6 months. The other pain severity components such as “least pain in the past 24 hours” and “pain now” showed tendency of improvement between day 0 and 6 months. At least 7 out of 12 subjects (58%) reported a reduction in pain severity scores (worst pain: 9/12 (75%); least and average pain: 7/12 (58%); and pain now: 8/12 (66.7%)). The mean pain interference improved during the follow-up period as shown in Table 5. At 6 months follow-up, the mean pain interference score decreased from 3.7 ± 2.7 to 2.5 ± 2.1 (P = 0.024).

Table 5. Changes in BPI-DPN scores after intramuscular VM202 injection.

Discussion

Despite the great potential for growth factor therapies in the treatment of diabetic neuropathy, the results of clinical studies with a variety of factors have failed to show clear clinical benefit.^33,34 Clinical trials using nerve growth factor and neurotropin 3 did not produce promising results, whereas vascular endothelial growth factor improved symptoms without having statistical significance or any benefit on most secondary measures.^33,34,35 There are many likely reasons for these results including instability of protein factors in the blood stream, binding to inactivating carrier proteins, rapid clearance by the liver, and off-target effects. Thus, the maintenance of effective doses in targets for therapeutically meaningful lengths of time is critical for the development of successful therapies. The gene therapy approach used in the present study with local injection into the calf muscle was designed to address this issue. The second critical issue is the spectrum of activity and cellular targets of the factors. Although there is a debate about the pathophysiologic mechanisms underlying diabetic neuropathy, microvascular compromise, apoptosis of neurons, and insufficiency of neurotrophic support due to chronic hyperglycemia are among the most frequently cited causes.^2,3,36,37 HGF is not only a potent angiogenic and antiapoptotic factor, but is also neurotrophic for sensory, motor, and sympathetic neurons. It thus seemed to be an excellent potential candidate for the treatment of diabetic neuropathy. Finally, treatment with therapeutically relevant amounts of the factor must be feasible without significant side effects. This phase I/II clinical trial, which was designed in consideration of all these issues, evaluated the safety, tolerability, and preliminary efficacy of injections of VM202, plasmid DNA expressing two isoforms of HGF.

Intramuscular administration of VM202 was safe and well tolerated at doses as high as 16 mg. There were neither incidents of dose limiting toxicities, nor serious or unexpected AEs occurring during the study period. The level of plasmid in blood was below 50 copies/µg of genomic DNA by day 90 for all cohorts, and circulating HGF levels remained relatively constant throughout the study period. These data indicate that intramuscularly administrated VM202 remains active only at the injection site without causing systemic elevation of serum HGF levels. This result is consistent with the safety data from previous preclinical and clinical studies performed with the same investigational drug.^{7,8,9,10,11,14,15,16} The complete lack of significant side effects with the highest (16 mg) dose in this study suggests that it may be possible to increase the dose safely in future studies.

There were a number of noteworthy features of the apparent clinical response including the time-course of the effects and the dose–response relationship. Two injections of VM202 with an interval of 2 weeks reduced the VAS scores for at least 12 months. There was no apparent clinical response or significant change in the VAS scores for a month after the primary injection. The first noticeable improvements occurred on day 60, and these persisted throughout the entire 12 month period of the study. The peak activity was observed at 6 months follow-up. At 6 months, 10 out of 12 subjects reported decreased VAS scores, and 50% (2/4), 75% (3/4), and 75% (3/4) of patients in the 4 mg, 8 mg, and 16 mg dose cohorts, respectively, experienced >50% reductions. More than 50% reduction in pain score from baseline to the final visit is generally considered to be clinically meaningful, and is a benchmark typically used in pain relief trials.³⁸ SF-MPQ and BPI-DPN scores also showed similar time-courses. Importantly, a dose–response relationship was observed during the study period until 6 months follow-up. In VAS scores, there was no significant improvement at the lowest dose tier (21% pain reduction at 6 months), but a significantly larger reduction in pain in the higher dose groups (53% and 62% pain reduction at 6 months follow-up in cohorts 2 and 3, respectively). Furthermore, the improvements also occurred earlier after the treatments in the high dose cohort compared with the middle dose cohort. The results from SF-MPQ evaluation showed similar dose dependency by cohort.

As medications for relief of neuropathic pain were allowed, there may be concerns about the effects of these drugs masking the efficacy of VM202. There were minor changes in pain medications in 4/12 patients after the VM202 injections. Two subjects, each in cohort 2 or cohort 3, reduced the doses by 75% (amitriptyline) or completely stopped the medication (duloxetine) as their pain improved significantly after VM202 treatment. On the contrary, one patient in cohort 1 took supplementary drug (clonazepam) for a month and another patient in cohort 2 doubled the doses of his concomitant drug (gabapentin) during the study period. Although their pain levels noticeably reduced at the times of the medication changes, they continued to steadily decrease until the end of the study. Considering such changes of medications occurred at relatively early stages of the study (2 and 5 months), it is unlikely that they had much effect on the dose-dependent improvement in pain shown in this study.

Conclusions about the possible efficacy of VM202 treatment are limited since the quantitative data necessary to draw statistically meaningful results were not collected due to small sample size. Also, this trial was an open-label study without a placebo control group. Despite these limitations, the reduction in pain occurred in a gradual manner over time, rather than immediately after the injections, and in a dose-dependent manner. This suggests a real therapeutic effect, as opposed to a placebo effect. The issue of possible placebo effect will be addressed further in an ongoing phase II, randomized, double-blind, placebo-controlled study. Another limitation of the present study was the absence of clinical assessment that could objectively evaluate the severity of PDPN, although various pain measurement instruments including VAS, BPI-DPN, and SF-MPQ were used. In the phase II study, examination of the clinical severity of PDPN using the Michigan Neuropathy Screening Instrument (MNSI) and Brief Peripheral Neuropathy Screening (BPNS) will be performed at baseline and after VM202 injection visits to more accurately assess the therapeutic effect of VM202.

A major rationale behind HGF treatment for PDPN is HGF's potential for modification of the disease process with the possibility of regenerative responses. As this was a phase I/II study that primarily explored safety, the quantitative data necessary to test this hypothesis were not collected. However, the present study showed that two intramuscular injections of VM202 encoding dual isoforms of HGF was safe and efficacious at doses as high as 16 mg in patients with PDPN. The apparent clinical response coupled with the known angiogenic, neurotrophic, and non-analgesic properties of HGF suggest that VM202 may be able to modify the course of the disease.

Materials and Methods

Study design. This study was a prospective, open-label, dose-escalation, two-center study designed to assess the safety and tolerability of increasing doses of intramuscular injection of VM202 in patients with PDPN (http://www.clinicaltrials.gov; registration number: NCT01002235). This study was approved by the US Food and Drug Administration, the National Institutes of Health Recombinant DNA Advisory Committee (NIH RAC), and the Institutional Review Boards of the participating centers. Also, this study was conducted in accordance with good clinical practice and the Declaration of Helinki. All subjects had given their formal consent before participating in the study.

A total of 12 subjects diagnosed with PDPN were enrolled. Diagnosis of PDPN was confirmed by presence of diabetes, previous diagnosis of painful diabetic neuropathy, the MNSI scoring instrument, and assessment of pain using VAS. Four patients in each cohort were treated with 4 mg (cohort 1), 8 mg (cohort 2), or 16 mg (cohort 3) of VM202. For each dose cohort, VM202 was administered via local intramuscular injections in the calf muscle, with half of the dose administered on day 0 of the study and the second half administered 2 weeks later. Cohorts of increasing doses were enrolled sequentially. Dose-escalation decisions (permission to treat at higher doses) were made by the Data Safety Monitoring Board (DSMB), which reviewed study safety data. Safety, tolerability, and preliminary efficacy (pain reduction and quality of life measures) were evaluated at designated time points for one year from the time of the first dose of study drug administration and compared with baseline values.

Patient eligibility. Eligible patients were men and non-pregnant, non-lactating women of ≥18 years to ≤75 years of age who had been given a primary diagnosis of PDPN (type I or II diabetes with glycosylated hemoglobin A1C ≤10.0% and lower extremity pain for ≥6 months). Patient eligibility required a score of ≥4 cm (0 cm = no pain; 10 cm = worst imaginable pain) on the VAS at screening and a score of ≥3 on the MNSI. Patients were also required to maintain their current pharmacological and non-pharmacological treatments for PDPN.

Exclusion criteria included neurologic disorders unrelated to diabetic neuropathy, peripheral neuropathy caused by conditions other than diabetes, other pain more severe than neuropathic pain, myopathy, inflammatory disorder of the blood vessels, active infection, chronic inflammatory disease, abnormal ophthalmologic conditions including proliferative retinopathy, and stroke or myocardial infarction within the last 6 months, uncontrolled hypertension, any condition that could confound the study assessment, or recent treatment with any investigational drug or treatment in the past 12 months. In addition, patients were excluded if they were on immunosuppressive medications, were undergoing chemotherapy or radiation therapies, or had evidence (clinical, laboratory or imaging) of malignant neoplasm, except for fully resolved basal cell carcinoma of the skin. Furthermore, patients were excluded if they had any of the following abnormal laboratory findings: Hemoglobin <9.0 g/dl, WBC <3,000 cells per microliter, platelet count <75,000/mm³, creatinine >2.0 mg/dl, GFR <50, AST and/or ALT >2 times the upper limit of normal, positive HIV or HTLV, positive Hepatitis B or C. All patients underwent testing using the American Cancer Society Cancer Screening Guidelines as part of their baseline testing to rule out anyone with cancer. Patients were also prohibited from taking daily doses of >81 mg of acetylsalicylic acid and from using gamma-linolenic acid, alpha lipoic acid, or any other high dose dietary antioxidant supplement for symptomatic relief of PDPN.

Study material and injection. VM202 (pCK-HGF-X7) is a plasmid DNA designed to produce two isoforms of HGF protein, consisting of either 723 or 728 amino acids (Figure 2).^8,10 Detailed information about VM202 is described by Pyun et al.⁸ Bulk drug substance and lyophilized study product of VM202 used in the present study were manufactured by Cobra Biologics (Keele, UK) and Formatech (Andover, MA). VM202 was supplied in a sterile glass vial containing 2.2 mg of lyophilized study product and stored at 2 to 8 °C. Lyophilized VM202 was reconstituted with 4.4 ml of water for injection and dissolved for 5 minutes at room temperature. One milliliter syringes (e.g., 27 gauge, 1”) were used for injection (0.25 mg/0.5 ml/syringe).

Figure 2.

Diagram of the naked DNA designed to express two isoforms of hepatocyte growth (HGF) factor. A hybrid between the genomic and cDNA sequences of HGF was inserted into pCK, an expression vector that is characterized by the presence of the full-length promoter as well as the entire untranslated exon sequences (exon 1 and exon 2) and the intron I sequence from the major immediate-early (IE) region of human cytomegalovirus (HCMV).⁴¹ The resulting plasmid, VM202, is 7,377 base pair long. This construct allows the efficient coexpression of both HGF₇₂₃ and HGF₇₂₈,^7,8 which differ by five amino acids as shown in the figure. HCMV IE promoter (pink arrow, boxes, and wavy line), promoter, and enhancer of the IE gene of the HCMV containing its entire 50-untranslated region upstream from the initiation codon of the IE gene; HGF-X7 (blue boxes and wavy line), genomic-complementary DNA (cDNA) hybrid of the HGF gene by inserting truncated intron 4 into the junction of exons 4 and 5 of HGF cDNA; pA (green box), poly A tract of bovine growth hormone gene; Kan r. (violet box), kanamycin resistance gene; ColE1 (orange box), E coli origin of replication.

The final doses of 4 mg (cohort 1), 8 mg (cohort 2), and 16 mg (cohort 3) were divided evenly between the days 0 and 14 administrations (Table 6). VM202 was unilaterally injected into the calf muscles of subjects. The injection sites were regularly distributed.

Table 6. VM202 administration for each cohort.

Safety endpoints. The primary intent was to characterize the safety of VM202 injections in the calf muscles of patients with PDPN. The safety endpoints including AEs, clinical chemistry, physical exam, and ophthalmologic examination using retinal fundoscopy and/or fluorescein angiography were obtained at screening and each visit during the 1-year, follow-up period. Cancer screening included testing for cancer markers; pap smear and mammogram if not performed within past 12 months (females only); prostate specific antigen (males only); for patients ≥50 years old, colonoscopy within past 10 years; and x-ray or CT scan of chest. VM202 DNA levels in whole blood were determined by quantitative PCR on day 0 (preinjection, and 1 to 3 hours after injection), day 14 (preinjection, and 1 to 3 hours after injection), and days 21, 30, 60, and 90. The lower limit of quantitation for VM202 in whole blood was 50 copies per one microgram of genomic DNA. The change from the baseline in serum HGF was assessed on days 0, 14, 30, and 60 using an enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN). After the first patient in a dose cohort completed the day 30 follow-up evaluation and the other three patients enrolled in the same cohort completed at least the day 14 follow-up (second dosing), interim safety evaluations were performed by the DSMB prior to initiating the next dose tier.

Efficacy endpoints. Several efficacy parameters were analyzed that focused primarily on the potential of VM202 to reduce the pain associated with PDPN. The primary clinical outcome measure was the visual analog scoring instrument (VAS).³⁹ Patients were also assessed using the brief pain inventory for patients with diabetic neuropathy (BPI-DPN) questionnaire, and the short form McGill pain questionnaire (SF-MPQ) compared with baseline.^39,40 Pain intensity was measured using the VAS at screening, before the first treatment (injection) on day 0, before the second treatment (injection) on day 14, then again at days 30, 60, 90, 180, and 365. The BPI-DPN and SF-MPQ were not evaluated at screening but were otherwise administered on the same schedule.

Statistical analyses. Safety, tolerability, and preliminary efficacy analyses were assessed. Categorical variables were expressed as frequencies and percentages. All continuous values were expressed as mean ± SD. The one-way analysis of variance followed by Dunnett or Games-Howell's T3 test was used to analyze pharmacodynamics and pharmacokinetic variables such as blood VM202 DNA concentration and serum HGF protein level, and efficacy variables including BPI-DPN, SF-MPQ, and VAS scores. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS, version 14.0 (SPSS, Chicago, IL).