Постмаркетинговые исследования

1. 1
RESULTS OF 5-YEAR FOLLOW-UP STUDY IN PATIENTS WITH
PERIPHERAL ARTERY DISEASE TREATED WITH PL-VEGF165 FOR
INTERMITTENT CLAUDICATION
RUNNING HEAD: 5-YEAR FOLLOW-UP STUDY IN PATIENTS TREATED
WITH PL-VEGF165
R,V, Deev1,2
, I.L. Plaksa1,3*
, I.Ya. Bozo1,4
, N.D. Mzhavanadze2
, I.A. Suchkov2
, Yu.V.
Chervyakov5
, I.N. Staroverov5
,R.E. Kalinin2
, A.A. Isaev1
1 – Human Stem Cells Institute, Moscow, Russia
2 – Ryazan I.P. Pavlov State Medical University, Ryazan, Russia
3 – Moscow City Oncology Hospital No62, Moscow, Russia
4 – A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow,
Russia
5 – Yaroslavl Regional Clinical Hospital, Yaroslavl, Russia
* e-mail: i.plaksa2014@yandex.ru
Abstract
Introduction. 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: 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 1, 2, 3, 4 and 5 years after pl-VEGF165 administration.
Results. Differences in the frequency of major cardiovascular events (pl-
VEGF165 5/36 vs control 2/12; p= 0.85), malignancies (pl-VEGF165 1/36 vs
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 PWD value increased by 288% from 105.7 ± 16.5 m to 384 ± 39
m in the treatment group by the end of the 5th year, with a peak of 410.6 ± 86.1 m

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achieved by the end of the 3rd year. The ABI increased from 0.47 ± 0.01 to 0.56 ±
0.02 by the end of the 1st year, with a subsequent slight decrease to 0.51 ± 0.02 by
the 5th year. The maximum increment of TcPo2 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.
1. 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 cause of high amputations 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) do not result in reducing their number any more [3]. This is the
result of achieving the limit of surgical care capabilities and achieving a moderate
effect 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 inductors of angiogenesis is safe 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: 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, 10, 11].
2. MATERIALS AND METHODS
2.1 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 were carried out at Russian State Federal

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Institution ‘‘Institute of Toxicology of Federal Medical Biological Agency of
Russia,’’ Saint-Petersburg (2008). The safety, feasibility, and short-term efficacy
of the study drug were then evaluated in a phase 1 to 2a multicenter randomized
trial that was conducted in 2010 and enrolled 45 patients. Federal Service on
Surveillance of the Ministry Healthcare and Social Development of the Russian
Federation has granted the approval to conduct a phase 2b to 3 study (approval
notice No. 177, April 21, 2010). The study protocol was approved by the National
Ethics Committee (protocol No. 62 from April 07, 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 ‘‘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
October 20, 2011) and Yaroslavl State Medical Academy (protocol No. 30 from
November 7, 2011).
2.2 Study Population. Having completed the phase 2В/3 registration study,
48 of its subjects (12 patients in the control group and 36 patients 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 or/and critical
lower limb ischemia 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 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 SFA, popliteal and tibial arteries;
distal – occlusion or hemodynamically significant stenosis in tibial arteries.
2.3 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 2В/3 registration clinical study; no re-
administration of the drug was done during the subsequent follow-up. The drug
was kept as a lyophilizate that was 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).
2.4 Design and Time Points of the Follow-up Study. The phase 2B/3
registration clinical study was designed as a randomized, open, comparative study

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with a six month duration. One hundred 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 (Fig. 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.
Every patient was informed about the study goals and objectives and the
potential benefits and risks of participating in it prior to inclusion into 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: NCT03068585).
2.5 Safety Assessment. Adverse events (AEs) and serious adverse events
(SAEs) were recorded over 5 years of the follow-up 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.
2.6 Efficacy Assessment. The same parameters that were used as efficacy
endpoints in the registration clinical study were evaluated during control
examinations. A pain-free walking distance (PWD) value was used as the primary
efficacy endpoint. It was measured on a treadmill with an elevation angle of 0º 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 5-year follow-up were also calculated in both
groups.
2.7 Statistical Data Processing. A sample size of 28 patients in each group
in the phase 2В/3 registration study was estimated to detect a 0.75 standardized
difference (80% power, P = .05), assuming the target difference and SD for PWD
to be 75 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

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for the subsequent follow-up wasn’t made because patients after 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, non-parametric
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.
3. RESULTS
3.1 Baseline Characteristics of Follow-up Study Subjects
A comparative analysis of the baseline characteristics showed that the
groups were comparable for gender composition, age, primary and secondary
efficacy endpoint values. Baseline PWD values were comparable between both
groups, 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 a 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.
3.2 Safety Assessment. No adverse events related to the use of pl-VEGF165
were recorded. Neither tumor formation, nor impaired vision, nor 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 3rd year of the follow-up, metastatic renal cancer was
diagnosed in one patient from the treatment group that was fatal.
3.3 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 3rd follow-up year. In general, these values stabilized in the
subsequent two years; there was only a slight decrease by 26.5 m to 384.1 ± 39.6 m
by the end of the 5th follow-up year. The PWD values were almost unchanged in
the control group (Table 3).

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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 (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 5th 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, i.e., 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, 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
mm Hg to 84.1 ± 1.8 mm Hg (p= 0.021), with a peak at 90.6 ± 0.4 mm Hg 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. TcPo2
increased by 26% from 64.2 ± 3.4 to 81 ± 2.0 mm Hg 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).
4. DISCUSSION
The objective of the 5-year follow-up study was to evaluate the long-term
safety 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 to 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 of patients with CLI who had intramuscular administration

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of VEGF-Adv (n=18) and pl-VEGF (n=17) within the placebo-controlled phase 2
study also demonstrated the safety of this treatment approach [13]. None of the
follow-up studies revealed increased systemic VEGF levels or the 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 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 the 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 full of skepticism. 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 both
ethical aspects because of traditional alertness about the safety of VEGF gene-based
constructions and 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 an open-label nature, suggesting a
high risk of detection bias for subjective outcomes like PWD. 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

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prolonged stable therapeutic effect of the gene therapy during 5-year follow-up is
an evidence that it does not connected with placebo effect.
5. CONCLUSION
The use of pl-VEGF165 is safe 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 five years.
6. ACKNOWLEDGEMENTS
The authors would like to thank the clinical investigators for their
contribution in conducting this trial and participating in the studies.
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.
7. SPONSOR
This study was funded by the PJSC ‘‘Human Stem Cells Institute’’
(Moscow, Russia).
8. CONFLICT OF INTEREST
The author(s) 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’’.
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List of Table and Figures legends:
1. Figure 1. Design and time points of the follow-up study.
2. Table 1. Baseline characteristics of 5-year follow-up study patients.
3. Table 2. Mortality and serious adverse events in the treatment and control study
groups during 5-year follow-up study.
4. Table 3. Primary and secondary efficacy endpoints in the 5-year follow-up
study.