As a pluripotent growth factor, hepatocyte growth factor (HGF) has been proved to have potent angiogenic, anti-inflammatory, antifibrotic, and anti-apoptotic effects which can be beneficial for various types of ischemic tissues especially in myocardial infarction and ischemic heart failure [1-5]. Previously, we engineered a replicationdeficient adenovirus that carries the HGF gene (Ad-HGF). It mediated high expression of HGF to increase the number of functional arterioles and improve the growth of collateral circulation [6]. In several ischemia animal models, Ad-HGF gene transfer proved to have efficient angiogenesis effects without apparent toxicity and mutation [7,8]. Our preceding phase I clinical study demonstrated the shortterm safety of direct intracoronary administration of Ad-HGF to treat severe coronary disease, which showed that in the acute phase, up to day 35, there were no serious adverse events [9]. This study is a longterm follow-up of our prior phase I clinical trial. It evaluates the longterm safety and efficacy of intracoronary Ad-HGF administration for treating severe diffuse coronary disease.

Study design

This study was performed in the Department of Cardiology at the First Affiliated Hospital of Nanjing Medical University, which was approved by the Ethical Committee of the hospital and the State Food and Drug Administration of China (ref. 2005L01181). The written consents were obtained from all patients. Each patient received the optimal standardized medication therapy for coronary disease including aspirin or clopidogrel, beta blockers, statins, angiotensin conversion enzyme inhibitors or angiotensin II receptor blockers, and was treated with Ad-HGF only once (constructed and produced by Chinese Academy of Military Medical Sciences) [9-12]. Inclusion criteria for the intracoronary gene transfer were 50-80 years old with Canadian Cardiovascular Society class II to III angina, suffered from diffused and severe coronary disease confirmed by coronary angiography, the main coronaries not amenable to interventional therapy (angioplasty or stenting) or bypassing grafting, and no emergency revascularization during follow-ups. Patients were excluded if they had malignancy, kidney failure, hepatic failure, acute myocardial infarction or received emergency revascularization.

In our previous phase I clinical trial, there were 18 patients assigned to three groups according to the dosage of Ad-HGF (5*109 pfu, 1*1010 pfu, or 2*1010 pfu), and each group had six patients, seven patients were excluded since they received revascularization during follow-ups. Finally, 11 patients were included in the experiment group of this study (three in 5*109 pfu group, four in 1*1010 pfu group, and four in 2*1010 pfu group) (Table 1).

Table 1: Characteristics of the 22 patients included in the study.

Gene transfer

Coronary angiography was performed via the femoral artery according to standard procedures. Based on the previous study [9,10], Ad-HGF gene was transferred to the distal part of the accessible artery by over-the wire balloon. If the vessel could not be reached, the procedure was performed by diagnostic coronary catheter at the ostium of the target vessels [9,10]. Ad-HGF was diluted to 2 ml with normal saline and then injected within 30 sec, followed by flushing of the catheter with normal saline. Coronary angiograms were obtained at the end of each infusion [9,10].

Safety parameters

The safety parameters including death, acute myocardial infarction, acute stroke, new-onset arrhythmia, malignant tumor and paroxysmal or long-term fever, retinal vascular anomaly, as well as several blood parameters including white blood cell count (WBC), hemoglobin (Hb), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), serum urea nitrogen (BUN), carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) were measured at baseline and during follow-ups in the experiment group.

Efficacy parameters

The efficacy parameters including echocardiographic ejection fraction (EF) and EF of cardiac Emission Computed Tomography (ECT-EF) were measured at baseline and 36-month follow-up in both groups.

Statistical analysis

Stata 10.1 was used for statistical analysis. Repeat measured ANOVA was performed to evaluate the difference in safety parameters between baseline and follow-ups. Paired t test was performed to evaluate the mean difference of changes in EF (baseline to follow-up) between experiment group and control group. The results are presented as mean difference (MD) ± standard deviation and P<0.05 was considered statistically significant.

Clinical characteristics

Characteristics of the recruited 22 patients are shown in Table 1, and there were no significant difference between groups.

Safety parameters

All the eleven patients of the experiment group were alive after 36- months follow-up. One patient suffered from an acute stroke in the 7^th month post-procedure. It was regarded as a serious adverse event but was not directly related to the treatment. During the follow-up, no new-onset arrhythmia was recorded; no malignant tumor was diagnosed; no paroxysmal or long-term fever was recorded; no retinal vascular anomaly was diagnosed.

There were no statistically significant differences between the follow-ups (5-week, 12-month or 36-month) and baseline in regard to blood parameters, including WBC, Hb, ALT, AST, BUN, Cr, CEA and AFP (Table 2). This indicated that there were no significant hematocytopenia, tumor biomarkers, liver function and renal function abnormalities during the follow-up.

Efficacy parameters

In the experiment group, the 36-months follow-up revealed a significant higher echocardiographic EF compared to baseline and other follow-ups (Repeat one-way ANOVA: F=4.4, p=0.024). Finally, nine of eleven patients had improved echocardiographic EF at the 36- month follow-up (Table 2). Subgroup analysis showed higher ECT-EF than baseline (MD: 4.8, p=0.017, n=8) in eight medium-high dosage cases (1*1010 and 2*1010 pfu) but not in three low dosage cases (5*109 pfu).

Table 2: Safety and efficacy parameters between baseline and followups in the experiment group.

Compared to the control group, the experiment group showed a significant higher improvement of echocardiographic EF from baseline to 36-month follow-up (δEF: 3.5 ± 1.1 vs. -4.5 ± 1.3, MD: 8, p=0.0001). Subgroup analysis of the eight medium-high dosage cases suggested that experiment group had a trend of higher improvement of ECT-EF (baseline to 36-month follow-up) than control group (δEF: 4.8 ± 1.5 vs. 0.3 ± 1.7, MD: 4.5, p=0.08), although the difference did not reach the significant level.

Millions of patients are diagnosed with ischemic heart disease and ischemic heart failure. Over 80% of symptomatic coronary disease patients can be managed with conventional revascularization methods such as CABG or coronary stenting [13]. However more than 10% of patients with persisting symptoms and cannot be revascularized are referred to tertiary intervention centers [13]. These cases pose a therapeutic challenge which demands the exploration and evaluation of new methods of therapy.

Angiogenesis is a promising method for treating ischemic cardiac disease, which provides a new concept of revascularization [1]. HGF, one of the useful “angiogens”, has shown potent angiogenic actions in ischemic tissues [1,14-16]. In our previous studies, we successfully inoculated ischemic animal models with high expressive Ad-HGF and achieved effective angiogenesis without apparent toxicity and mutation [6]. Our previous phase I clinical study demonstrated the short-term safety of intracoronary Ad-HGF administration for treating severe coronary disease in humans [9].

This study, a long-term follow-up of previous phase I clinical trial, confirmed the safety of intracoronary Ad-HGF administration for treating severe diffuse coronary disease. No death, no new-onset arrhythmia, no malignant tumor and no paroxysmal or long-term fever were recorded during the long-term follow-up. Although one stroke occurred, it did not seem to be directly related to the therapy.

Intracoronary Ad-HGF administration showed efficacy in improving echocardiographic EF during the 36-month follow-up, when compared not only to baseline but also with control group. Although ECT results did not reveal the significant improvement seen in echocardiographic EF, the medium-high dosage subgroup (1*1010 and 2*1010 pfu) also showed higher EF than baseline and higher δEF than control group. These results suggest that intracoronary Ad-HGF administration is efficient in improving EF of patients with severe diffuse coronary disease, and the efficacy may have a “dose-dependent” effect. It indicates that this new gene therapy might be a promising method to treat patients with diffuse coronary disease, who are not amenable to conventional revascularization or still have obvious symptoms after conventional revascularization of main coronaries.

This study has limitations in evaluating effectiveness of Ad-HGF. First, the number of alternative subjects is limited and the sample is small, so the difference between different dosages cannot be evaluated exactly. Second, due to the design, this study is just a controlled but not randomized trial, so the evidence level is not high. Next, our phase II clinical trial will be constructed to illustrate the effectiveness of Ad- HGF through another superior administration route (intramyocardial injection).

In conclusion, the results of the 3-year long-term follow-up of our previous phase I clinical trial, demonstrate that intracoronary Ad-HGF administration is safe and potentially efficient in improving EF of patients with severe diffuse coronary disease.

This study was supported by grants from “the Cardiovascular disease collaborative innovation center of Nanjing Medical University”, “the Chinese Medical Association of the Sunlight Foundation (SCRFCMDA201217)”, “the National Natural Science Foundation of China (No. 81170102/H0203)”, “the Priority Acoronary diseaseemic Program Development of Jiangsu Higher Education Institutions (BL2012011)”, “the Fourth Period Project “333” of Jiangsu Province (BRA2012207)”, and “the Supporting program of Science and Technology of Jiangsu (Social Development, BK2010021)”.