Abstract
Background:
The effective treatment of chronic lower limb ischemia is one of the most challenging issues confronting vascular surgeons. Current pharmacological therapies play an auxiliary role and cannot prevent disease progression, and new treatment methods are needed. In 2011, a plasmid VEGF65-gene therapy drug was approved in Russia for the treatment of chronic lower limb ischemia (ClinicalTrials.gov identifier: NCT03068585). The objective of this follow-up study was to evaluate the long-term safety and efficacy of gene therapy in patients with limb ischemia of atherosclerotic genesis.
Aims:
To evaluate the long-term safety and efficacy of the therapeutic angiogenesis, 36 patients in the treatment group (pl-VEGF165) and 12 patients in the control group participated in a 5-year follow-up study. Planned examinations were carried out annually for 5 years after pl-VEGF165 administration.
Results:
Differences in the frequency of major cardiovascular events (pl-VEGF165 5/36 versus control 2/12; p = 0.85), malignancies (pl-VEGF165 1/36 versus control 0/12; p = 0.38) and impaired vision (there was none in either group) over the 5-year follow-up period did not achieve statistical significance. The target limb salvage was 95% (n = 36) and 67% (n = 12) in the pl-VEGF165 and control groups, respectively. The pain-free walking distance value increased by 288% from 105.7 ± 16.5 m to 384 ± 39 m in the treatment group by the end of the fifth year, with a peak of 410.6 ± 86.1 m achieved by the end of the third year. The ankle-brachial index (ABI) increased from 0.47 ± 0.01 to 0.56 ± 0.02 by the end of the first year, with a subsequent slight decrease to 0.51 ± 0.02 by the fifth year. The maximum increment of transcutaneous oximetry test (tcoO2) by 36%, from 66.6 ± 3.7 mm Hg to 90.7 ± 4.9 mm Hg, was observed by the end of the second year.
Conclusion:
The therapeutic effect of angiogenesis induction by gene therapy persists for 5 years.
Introduction
Despite significant technological advances in the surgical treatment of patients with atherosclerotic lower limb vascular disease, the amputation rate ranges from 188 to 263 per 100,000 population per year in developed countries. 1 Based on the data from the Russian Consensus, critical lower limb ischemia (CLI) is a significant cause of amputation in 30,000–40,000 people in the Russian Federation annually. 2 At the same time, the results of large pharmacoeconomic studies showed that increased costs for medical care of patients with peripheral arterial disease (PAD) no longer reduce these numbers. 3 This is the result of achieving the limit of surgical care capabilities and the moderate ability of most therapeutic agents to correct ischemia. This was a prerequisite for conducting clinical trials in the field of gene therapy.4–5 Numerous publications over the last 20 years have shown that the use of gene therapy constructions with the VEGF gene as an inductor of angiogenesis is well-tolerated and does not have a systemic effect. 6 Unfortunately, most of the phase II–III registration studies that aimed to evaluate the potential of therapeutic angiogenesis as a remedy to “save” a limb in patients with terminal disease failed. 7
The knowledge accumulated in the field of gene therapy of PAD led to the understanding that its use is most effective at the stage of intermittent claudication, before the development of necrotic ulcerations, since the presence of irreversible tissue changes creates unfavorable conditions for gene-mediated induction of angiogenesis. This determined a change in the approach to conducting clinical trials in this field as well as inclusion criteria and efficacy endpoints. 8 The paradigm change in the field of PAD gene therapy resulted in the approval of the world’s first gene therapy drug for the treatment of patients in these categories, with the active ingredient pl-VEGF165 (ClinicalTrials.gov identifier: NCT03068585). The objective of this follow-up study was to evaluate the long-term safety and efficacy of gene therapy in patients with CLI.9–11
Materials and methods
Rationale for the clinical study
Preclinical studies of general toxicity (acute, subacute, chronic, and local irritation) and specific toxicity (allergenicity, reproductive and immune toxicity, mutagenicity and carcinogenicity), as well as the detection of specific drug activity, was carried out at the Russian State federal institution the Institute of Toxicology of Federal Medical Biological Agency of Russia, Saint-Petersburg (2008). The tolerability, feasibility, and short-term efficacy of the study drug were then evaluated in a phase I to IIa multicenter randomized trial that was conducted in 2010 and which enrolled 45 patients. The Federal Service on Surveillance of the Ministry Healthcare and Social Development of the Russian Federation granted approval to conduct a phase IIb to III study (approval notice no. 177, 21 April 2010). The study protocol was approved by the National Ethics Committee (protocol no. 62 from 7 April 2010); local ethics committees have also granted their approval to conduct the study. All phases of clinical trials were conducted according to the Declaration of Helsinki of the World Medical Association’s ‘Recommendations guiding physicians in biomedical research involving human subjects’ (1964, 2000), ‘Rules of good clinical practice in the Russian Federation’, OST 42-511-99, ICH GCP rules, and valid regulatory requirements. The follow-up study protocol was also approved by the local ethics committees of Ryazan State I.P. Pavlov Medical University (protocol no. 4 from 20 October 2011) and Yaroslavl State Medical Academy (protocol no. 30 from 7 November 2011).
Study population
Having completed the phase IIb/III registration study, 48 of the patients (12 patients in the control group and 36 in the pl-VEGF165 group) gave their consent to participate in a 5-year follow-up study. At baseline, all patients were diagnosed with intermittent claudication and/or CLI of atherosclerotic genesis without necrotic changes that correlated with stage II–III under the Fontaine classification as modified by A.V. Pokrovsky [a pain-free walking distance (PWD) of not more than 1000 m and no necrotic ulcerations in limb soft tissues]. None of the patients with stage III disease could have revascularization surgery because of the lesion extent and the severity of structural changes in the vessel wall. Depending upon the anatomy and involvement in a pathological process, the patients had atherosclerotic lesions of the following patterns: proximal – occlusion of the femoral artery and patency of the iliac segment; multilevel – occlusion in the superficial femoral, popliteal and tibial arteries; distal – occlusion or hemodynamically significant stenosis in tibial arteries.
Description of pl-VEGF165
The study drug is an original gene construction containing a supercoiled plasmid DNA (1.2 mg)-encoding pl-VEGF165 as the active substance and is now marketed as “Neovasculgen”. 9 pl-VEGF165 was used only in the phase IIb/III registration clinical study; no re-administration of the drug was done during the subsequent follow up. The drug was kept as a lyophilizate dissolved in 2 ml of water for injection immediately before administration. The solution was given as 5–10 intramuscular injections into the calf muscles twice, 1.2 mg at an interval of 14 days (a total dose of 2.4 mg).
Design and time points of the follow-up study
The phase IIb/III registration clinical study was designed as a randomized, open, comparative study with a 6-month duration. A total of 100 patients with CLI were randomized into the control group (n = 25), who received standard conservative therapy without cilostazol and prostaglandins, and the treatment group (n = 75), who received standard conservative treatment and pl-VEGF165. Control examination time points in the follow-up period after the end of the clinical trial were 1, 2, 3, 4 and 5 years after pl-VEGF165 administration (Figure 1). Over this period of time, all patients had standard therapy including 75 mg of acetylsalicylic acid (aspirin)/clopidogrel, statins in individual doses in order to reduce the risk of cardiovascular complications, and pentoxifylline without prostaglandins and prostacyclins.
Design and time points of the follow-up study.
Every patient was informed about the study goals and objectives and the potential benefits and risks of participating in it prior to inclusion in the follow-up study. All the patients signed the voluntary informed consent form. The follow-up study protocol was approved by a local Ethics Committee at every study site (ClinicalTrials.gov identifier: NCT03068585).
Safety assessment
Adverse events (AEs) and serious adverse events were recorded over 5 years of the follow-up study to evaluate drug safety. During screening, the patients had ECG, blood hematology and biochemistry, a coagulation panel and urinalysis performed in order to detect any concomitant and underlying disorders. Fluorography, abdominal ultrasound, gastroscopy and colonoscopy were performed if required in order to rule out malignancies and other complications.
Efficacy assessment
The same parameters that were used as efficacy endpoints in the registration clinical study were evaluated during control examinations. A PWD value was used as the primary efficacy endpoint. It was measured on a treadmill with an elevation angle of 0° and a speed of 1 km/h; the walking distance was registered from the start to pain in the limb muscle (evidence grade B according to the TASC-II). The disease was staged under the Fontaine classification modified by A.V. Pokrovsky that is widely applied by Russian and Ukrainian vascular surgeons. According to this classification, a PWD of more than 200 m indicates stage IIA disease and of less than 200 m but more than 50 m indicates stage IIB disease. The disease is rated as stage III when the PWD is less than 50 m and/or there is resting pain without any ischemic tissue changes (trophic ulcer and/or necrosis). TcPO2 of the involved limb and the ABI were used as secondary efficacy endpoints (Table 1). Mortality and amputation rates by the end of the 5-year follow-up study were also calculated in both groups.
Baseline characteristics of 5-year follow-up study patients.
ABI, ankle-brachial index; CLI, chronic limb ischemia; PWD, pain-Free walking distance; TcPO2, transcutaneous oxygen pressure.
Statistical data processing
A sample size of 28 patients in each group in the phase IIb/III registration study was estimated to detect a 0.75 standardized difference (80% power, p = 0.05), assuming the target difference and standard deviation for PWD to be 75 m and 100 m, respectively. We decided to use a 3:1 test/control group ratio in order to make the test group sample more representative. 9 The power analysis for the subsequent follow up was not made because patients after the registration trial were included freely on the basis of their own desire to participate, depending on their capabilities. Absolute values of the efficacy endpoints (PWD, ABI, and TcPO2) complied with a non-normal distribution. Therefore, nonparametric methods (a Mann–Whitney U test and a Wilcoxon signed-rank test) were used for the statistical analysis; additionally, a Chi-squared test was used to evaluate the significance of differences in the event frequencies in pl-VEGF165 and control groups.
Results
Baseline characteristics of patients in the follow-up study
A comparative analysis of the baseline characteristics showed that the groups were comparable for gender composition, age and primary and secondary efficacy endpoint values. Baseline PWD values were comparable between both groups at 105.7 ± 16.5 m and 99.3 ± 8.4 m (p = 0.672) in the pl-VEGF165 and control groups, respectively. There were no significant differences in the structure of patient distribution for disease stages and level of lower limb vascular lesions; patients with stage IIB disease and distal lesions prevailed in both groups (Table 1). Five patients from the treatment group and one from the control group had resting pains of various intensities at the beginning of the study.
Safety assessment
No AEs related to the use of pl-VEGF165 were recorded. Neither tumor formation, nor impaired vision nor any other pathology that could indirectly indicate angiogenic therapy complications were detected at any time points during the follow-up study. The mortality rate over the entire follow-up period was 16.6%, 6/36 and 2/12 in the pl-VEGF165 and control groups, respectively. Two patients from the pl-VEGF165 group (n = 36) and four patients from the control group (n = 12) underwent amputations of the lower limb as a result of ischemia progression and revascularization surgery infeasibility. Thus, the amputation-free survival was 95% and 67%, respectively (Table 2). Two patients from the control group and five patients from the pl-VEGF165 group had cardiovascular complications as a result of a generalized atherosclerotic lesion of the vessel wall. In the third year of the follow-up study, metastatic renal cancer was diagnosed in one patient from the treatment group, which was fatal.
Mortality and serious adverse events in the treatment and control study groups during the 5-year follow-up study.
ACVE, acute cerebrovascular event; AMI, acute myocardial infarction. * p < 0.01.
Efficacy assessment
The maximum increase of PWD by 288% from 105.7 ± 16.5 m to 410.6 ± 86.1 m (p = 0.000) was observed in the treatment group by the end of the third follow-up year. In general, these values stabilized in the subsequent 2 years; there was only a slight decrease by 26.5 m to 384.1 ± 39.6 m by the end of the fifth follow-up year. The PWD values were almost unchanged in the control group (Table 3).
Primary and secondary efficacy endpoints in the 5-year follow-up study.
ABI, ankle-brachial index; CLI, chronic limb ischemia; PWD, pain-free walking distance; TcPO2, transcutaneous oxygen pressure; p*, compared with baseline.
The most evident increase of PWD in the pl-VEGF165 group was observed in patients with stage III disease (n = 13). The value increased from 24.3 ± 5.3 m to 378 ± 73.1 m by the end of the 5-year follow-up period (p = 0.0001). At the same time, unlike the dynamics of mean values in the treatment group in general, the maximum incremental value was observed only by the end of the fifth follow-up year. Depending upon the level of lesion, patients with distal and multilevel disease had the highest incremental value, from 89 ± 20.9 to 510 ± 90.7 (p = 0.0001) and from 109.1 ± 43.5 to 364.4 ± 49.8 (p = 0.001), respectively. Positive changes in the PWD, that is from 127.8 ± 19.7 to 218.0 ± 57.4 (p = 0.012), were less evident for proximal lesions. No selective analysis in dependence upon a disease stage and a level of occlusion was performed in the control group because of the small sample size (Table 3).
The mean ABI value increased from 0.47 ± 0.01 to 0.51 ± 0.02 (p = 0.049) in the treatment group over the 5-year follow-up period, with a peak of 0.56 ± 0.02 achieved by the end of the first year of follow-up (p = 0.028). Macrohemodynamic parameters correlated with TcPO2 values that increased by 27% from 66.7 ± 3.8 mmHg to 84.1 ± 1.8 mmHg (p = 0.021), with a peak at 90.6 ± 0.4 mmHg in the second year of follow up.
In the control group, the ABI slightly increased from 0.39 ± 0.03 to 0.42 ± 0.02 (p = 0.320), with subtle fluctuations over the entire 5-year follow-up period. TcPO2 increased by 26% from 64.2 ± 3.4 to 81 ± 2.0 mmHg by the end of the second year of follow up, and then there was a moderate gradual decrease by 7.4% by the end of the fifth year (Table 3).
Discussion
The objective of the 5-year follow-up study was to evaluate the long-term tolerability and efficacy of angiogenesis induction with gene therapy in patients with CLI. The results demonstrated that the use of pl-VEGF165 was not followed by the increased frequency of cardiovascular complications, malignancies or impaired vision. This gives evidence that the drug is a local angiogenesis inductor without a systemic effect.
To date, this is one of the most prolonged studies aiming to evaluate the long-term safety and efficacy of gene therapy. Studies performed by other teams also demonstrate that the safety profile of gene therapy with VEGF is acceptable. An 8-year follow-up study involving patients with coronary artery disease after administration of VEGF-Adv (n = 37) and pl-VEGF (n = 28) demonstrated that the use of gene therapy to induce angiogenesis did not result in increased frequency of tumor formation or cardiovascular complications compared with a placebo (n = 38). 12 An intracoronary infusion of VEGF-Adv caused only transitory increased lactate dehydrogenase (LDHG) levels in several recipients. The results of the 10-year follow-up study of patients with CLI who had intramuscular administration of VEGF-Adv (n = 18) and pl-VEGF (n = 17) within the placebo-controlled phase II study also demonstrated the tolerability of this treatment approach. 13 None of the follow-up studies revealed increased systemic VEGF levels or increased frequency of diabetic retinopathy after gene therapeutic constructions were administered that could indirectly indicate a systemic effect. Thus, to date there is no evidence that VEGF-based gene therapy is capable of inducing the growth of blood vessels at a considerable distance from an injection site. This is consistent with our concept of pl-VEGF165 as a local inductor of angiogenesis.
The registered clinical study aimed to evaluate the possible use of gene therapy to prevent ischemia progression to necrotic ulcerations. Therefore, target limb salvage was not used as the efficacy endpoint because patients in these categories do not have an immediate threat of losing their limbs in the near future.9,10 However, the 5-year follow-up study demonstrated that the long-term amputation rate in the pl-VEGF165 group was significantly lower (p = 0.008). The results that were obtained cannot be extrapolated to the whole population of patients with CLI because of the small sample size. However, they confirm our working hypothesis that the use of gene therapy before irreversible structural changes develop in tissues can reduce the risk of ischemia progression and thereby decrease the likelihood of amputation in the future.
To date, numerous data have been obtained in the field of gene therapy in cardiovascular diseases that indicate that use of gene therapy constructions with the gene VEGF is an effective tool to correct lower limb ischemia. However, the attitude of the vascular community is generally skeptical. This is largely because since work began in this field, gene therapy has been considered primarily as a tool to “save” a limb in patients with CLI over a long period of time. This is the basis of most studies, with participation of patients with a terminal form (stage) of the disease and the use of strict efficacy endpoints. This approach is caused by ethical aspects because of traditional alertness about the safety of VEGF gene-based constructions, and by the availability of a large arsenal of surgical techniques with definite efficacy that in general determined the enrollment of patients who are poor surgical candidates. The results of our studies show that early forms of CLI before the development of apparent and irreversible trophic changes, such as necrosis and ulcers, are a therapeutic window for the use of pl-VEGF165. The induction of angiogenesis in this period can significantly improve the functional state of patients with PAD and prevent the progression of ischemia.
The main limitation of this study was its open-label nature, suggesting a high risk of detection bias for subjective outcomes like PWD. Also, the power analysis was initially made only for the phase IIb/III registration study because subsequent participation of patients in the follow-up study was based on their own desire, depending on their capabilities. Although the open-label use of study drugs is a limitation, this study was randomized and involved three centers, and patient enrollment decisions were made independently by a team of vascular surgeons and radiologists on the basis of angiographic results, disease history, previous procedures and concomitant pathology. Additionally, the prolonged stable therapeutic effect of the gene therapy during the 5-year follow-up period is evidence that it is not connected with a placebo effect.
Conclusion
The use of pl-VEGF165 demonstrates good tolerability and is not followed by tumor formation, cardiovascular complications or impaired vision. When pl-VEGF165 is used before necrotic-ulcerative changes develop in patients with CLI, its therapeutic effect persists for at least 5 years.
Footnotes
Acknowledgements
The authors would like to thank the clinical investigators for their contribution in conducting this trial and participating in the studies.
Ethics statement
The study was conducted according to the Declaration of Helsinki of the World Medical Association ‘Recommendations guiding physicians in biomedical research involving human subjects’ (1964, 2000), ‘Rules of good clinical practice in the Russian Federation’, OST 42-511-99, ICH GCP rules and valid regulatory requirements.
Conflict of interest statement
The authors declared the following potential conflicts of interest with respect to the research, authorship and/or publication of this article: A.A. Isaev, I. Ya. Bozo, R.V. Deev and I.L. Plaksa are employees of the PJSC Human Stem Cells Institute. A.A. Isaev is a shareholder of the PJSC Human Stem Cells Institute.
Funding
This study was funded by the PJSC Human Stem Cells Institute (Moscow, Russia).
