An essential transcription factor for inflammation, nuclear factor-kappa B (NF-κB), plays a pivotal role for restenosis after percutaneous coronary intervention (PCI). To evaluate the safety and effectiveness of NF-κB decoy oligodeoxynucleotides (ODN) to prevent restenosis, we initiated an open-label phase I/IIa clinical trial.
Seventeen patients who were suffering from angina with organic coronary stenosis were treated with NF-κB decoy ODN after PCI using bare metal stents.
Although the average coronary stenosis was 90.8 ± 7.0% before the stent implantation, the stenosis improved to 1.4 ± 5.9% after the intervention. Serum MCP-1 levels were significantly suppressed in NF-κB decoy ODN treated patients compared to those in non-treated patients on day 3 after the PCI. Ticlopidine treatment was employed, since clopidogrel was not launched in Japan. Six months after the PCI and decoy ODN transfection, significant restenosis was found in only 1 of the 17 patients, and the average restenosis rate was 39.6 ± 22.3%. No in-stent thrombosis was found and no significant systemic adverse effect occurred in any of the patients in this observation period.
The major disadvantage of percutaneous coronary intervention (PCI) in the treatment of patients with atherosclerotic coronary disease is the occurrence of restenosis and thrombus formation after an initially successful procedure 1. Although drug eluting stents (DES) have been broadly used to prevent restenosis 2, long-term usage of anti-platelet drugs is needed to prevent subacute thrombus formation 3, 4. Animal studies and human histological observations have demonstrated that thrombus formation is caused by delayed endothelialization of the coronary arteries, because conservative DES distribute immunosuppressive or anti-cartinogenic drugs continuously to the PCI sites 5. Therefore, significant restenosis formation without delayed endothelialization should be prevented for clinical PCI.
Neointimal formation and endothelialization is comprised of cell accumulation which is triggered by leukocyte and hematopoietic stem cell recruitment into the arterial wall 6. Thus, inflammation is a central event in this progression. Nuclear factor-kappa B (NF-κB) is critical in the coordinated gene transactivation of cytokines and adhesion molecules. The synthetic double-stranded DNA with high affinity for NF-κB, which is known as a ‘decoy’, has significant effects in the suppression of the activation of inflammatory factors 7. The decoy strategy has significant therapeutic effects to treat acute myocardial infarction 7 and cardiac rejection 8. Using rat and porcine models, we demonstrated that NF-κB decoy oligodeoxynucleotides (ODN) successfully inhibited neointimal formation after arterial balloon injury 9, 10. Although these results suggest that NF-κB decoy ODN provide a new therapeutic strategy 11, no clinical study has evaluated its effect in the prevention of coronary restenosis after PCI. Therefore, we have carefully prepared an open-label phase I/IIa clinical trial (A phase I/IIa open-label multi-center study to assess the Inhibitory effects of NF-κB Decoy ODN on Restenosis after stenting in coronary artery; INDOR Study) to evaluate the safety and efficacy of NF-κB decoy ODN.
Materials and methods
Patients with angina pectoris admitted to hospitals of the Kyushu University, Osaka University, Tokyo Medical and Dental University, and Akita University were candidates for the study. Patients with stable angina pectoris or silent ischemia which was proved by excise test or scintigram were enrolled. A single de novo organic stenosis lesion in a native coronary artery, which is less than a 10-mm-long obstruction or less than a 20-mm-long stenosis, were enrolled. Patients were eligible if they met the following criteria: at least one episode of angina pectoris with organic coronary stenosis; and stable condition while receiving cardiac medication. Exclusion criteria were as follows: more than 10 mm obstruction or more than 20 mm stenosis in length; malignancy with poor prognosis; alcohol and other toxicity; possible pregnancy; severe liver disorders; and other disorders. This study was approved by the ethics committee in all of the universities. Written informed consent was obtained from all participants. Medication dosages had been adjusted on the basis of the hemodynamic and clinical status of each patient. Information about the patients, including coronary risk factors (hypertension, hyperlipidemia, and diabetes mellitus), is listed in Table 1.
Table 1. Information about the patients, including coronary risk factors (hypertension, hyperlipidemia, and diabetes mellitus)
Stent implantation and NF-κB decoy ODN transfection
Bare metal stents were implanted into the coronary stenotic sites. The stents were 18 or 23 mm in length and 3.0, 3.5, or 4.0 mm in diameter. NF-κB decoy ODN was made as described previously. The decoy was trasnfected using the Remedy catheter (Boston Scientific). The solution (0.5 mL) was administered manually with 100 kPa and it took 50 s for the delivery. Originally, four doses of NF-κB decoy ODN (1, 2, 4, and 8 mg per body) were designed. However, due to the withdrawal of Remedy catheters in Japan, three doses of NF-κB decoy ODN (1, 2, and 4 mg per body) were finally tested. Three patients who did not agree to be enrolled in this study were analyzed as control. All of the values were checked by the safety committee without the trial investigators to avoid bias.
Coronary angiogram and intravenous ultrasound
A coronary angiogram with quantitative coronary angiography (QCA) and intravenous ultrasound were performed before and immediately after the stent implantation, and 6 months after the treatment. Chest X-ray, echocardiogram, and blood examination were performed before the stent implantation, 1 week, 3 months and 6 months after the treatment.
Enzyme-linked immunosorbent assay (ELISA)
Peripheral blood was examined from the patients on days 0, 1, 3, and 7 after the treatment. Serum concentrations of MCP-1, RANTES, sVCAM-1, IL-1beta, IL-6, IL-8, and TNF-alpha were determined with an ELISA kit (BioSource International, Camarillo, CA, USA) according to the manufacturer's instructions.
A chi-squared test and the Mann-Whitney test were used for analysis of %QCA, risk factors, and possible adverse effects. The mean value ± standard deviation (SD) was presented for continuous variables; differences between the groups were evaluated with Student's t test in ELISA. Differences with values of P < 0.05 were considered significant.
Initial results after PCI
All 18 cases successfully received stent implantation and decoy ODN transfection without initial complications such as coronary dissection and acute coronary occlusion. The average stenosis rate before PCI was 90.8 ± 7.0% measured by QCA, although the average rate after PCI was 1.4 ± 5.9%. ELISA, which was used to measure the serum levels of biomarkers, indicated that lower MCP-1 levels were observed in the decoy-treated group compared to those of the non-decoy-treated group (Figure 1). Although serum levels of other biomarkers including RANTES, sVCAM-1, IL-1beta, IL-6, IL-8, and TNF-alpha were evaluated on days 0, 1, 3, and 7 after PCI, there was no statistical difference between the two groups (Figure 2).
Results 6 months after PCI
One of the 18 cases was dropped from this study due to personal reasons. The other 17 cases received all examinations during this 6-month period. As only 1 of the 17 patients showed significant stenosis (more than 75%), the average stenosis rate was less than 40% (39.6 ± 22.3%). No in-stent thrombosis was found during this observation period (Table 1). There was no statistical relationship between the restenosis degree (%QCA) and coronary risk factors including diabetes, hypertension and hyperlipidemia.
Possible adverse effects
During the 6-month observation period, there were some possible adverse effects. Although 3 of the 17 cases showed possible findings, all findings were mild and eliminated in this period (Table 2). The safety committee of this clinical trial judged that there may have been a relationship between the findings and the decoy transfection. However, there was no clear evidence to show the causal relationship in these findings. Statistically, there was no relationship between the possible adverse effects and coronary risk factors including diabetes, hypertension and hyperlipidemia.
Table 2. Findings from the study
Symptoms & Findings
Duration after PCI
Decreasing of platelet
Elevation of AST
6 days to 3 months
Elevation of AST
3 months to 6 months
Long-term observation after decoy transfection
We observed these 17 cases as outpatients in our hospitals. The first case was followed for more than 4 years and there were no ischemic events and adverse effects. Other cases also showed no events.
This is the first clinical paper to report the successful transfection of NF-κB decoy ODN at the site of stenting in the coronary arteries. These findings suggest safe and favorable effects of NF-κB decoy ODN on the prevention of restenosis after PCI, although this study was an open-label study. Recently, DES has been used broadly to effectively prevent restenosis after PCI. However, sub-acute and late in-stent thrombosis has yet to be resolved. The thrombosis is caused by delayed re-endothelialization of the inner layer of the stent-implanted arteries as DES elutes strong immunosuppressants or anti-cancer drugs into the injured artery. These drugs suppress the proliferation of many kinds of cells including arterial endothelial and smooth muscle cells 12–14. Thus, a new strategy, which prevents restenosis and does not delay re-endothelialization at the site of PCI, is needed for the next PCI generation.
Because inflammation is an essential pathological feature of neointimal formation, NF-κB plays an important role in this process 15. Thus, NF-κB regulation has the potential to suppress the progression of inflammation. Decoy ODN transfection is a significant methodology for suppressing gene activation. NF-κB decoy ODN are double-stranded DNA with specific affinity to the cis-element of NF-κB, thereby leading to the suppression of many inflammatory factors, including adhesion molecules and cytokines 16. Therefore, NF-κB decoy ODN transfection at the stent site suppressed SMC proliferation, which resulted in less neointimal formation. NF-κB decoy ODN might serve the re-endothelialization to a satisfactory degree, because NF-κB decoy ODN inhibit the apoptosis of endothelial cells in response to hypoxia 17. Indeed, the transfection of NF-κB decoy ODN into a vein graft demonstrated the significant improvement of endothelial dysfunction as compared to control 18. In contrast, DES prevention is too strong, which causes delayed re-endothelialization and very late thrombosis. Thus, this NF-κB decoy ODN strategy would be better to avoid the permanent anti-platelet therapy to prevent late thrombosis, although further studies are necessary.
We showed that NF-κB decoy ODN transfection suppressed serum MCP-1 levels compared to those in the control group. As previously reported, MCP-1 production at stented coronary arterial sites is associated with an increased risk for restenosis post-stent implantation 19. Thus, our findings suggest that this novel treatment may have a benefit for the prevention of restenosis after PCI. Although we demonstrated that the NF-κB decoy ODN transfection suppressed serum MCP-1 levels, our results could not show direct evidence that MCP-1 was the primary factor to prevent restenosis after PCI. However, we showed that stent-based local delivery of NF-κB decoy reduced in-stent neointimal formation with reduced MCP-1 expression 20. We also showed that the strategy of inhibiting the action of MCP-1 with a 7ND gene-eluting stent reduced in-stent neointimal formation 21. These data suggest that this strategy may be practical and promising for the prevention of in-stent restenosis in humans. However, as this clinical trial focused on safety evaluation, we could not conclude that this decoy ODN treatment suppresses neointimal formation because of a lack of patients. Therefore, further placebo control trials will be necessary to prove the efficacy for prevention of restenosis after PCI.
Since we only studied 17 cases, we cannot conclude that this procedure is definitely safe and prevents restenosis at this time. Nevertheless, these findings suggest that NF-κB decoy strategy is safe and has favorable effects on the prevention of restenosis after PCI. This decoy procedure has been used more and more as a potential procedure in the clinical treatment of cardiovascular diseases 10. However, further evaluations must be performed to prove the clinical effectiveness of this new procedure using NF-κB decoy ODN.
This study was supported by grants from the Japan Cardiovascular Research Foundation, a Grant-in-aid from the Japanese Ministry of Education, Science and Culture, a Grant-in-aid from the Japan Society for the Promotion of Science, and the Organization for Pharmaceutical Safety and Research.
Appendix: The INDOR study group
Independent data-monitoring committee
Kimihiro Komori, MD, PhD, Division of Vascular Surgery, Department of Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan
Hisashi Kai, MD, PhD, Division of Cardiovascular Medicine, Department of Internal Medicine, Kurume University, Kurume, Japan
Yasuo Ohashi, PhD, Department of Biostatistics, School of Public Health, University of Tokyo, Tokyo, Japan