pCMVvegf165 Intramuscular Gene Transfer is an Effective Method of Treatment for Patients With Chronic Lower Limb Ischemia

pCMV-vegf165 Intramuscular Gene Transferis an Effective Method of Treatment for Patients With Chronic Lower Limb Ischemia Abstract Effective treatment of chronic lower limb ischemia is o

pCMV-vegf165 Intramuscular Gene Transfer

is an Effective Method of Treatment for

Patients With Chronic Lower Limb Ischemia

Abstract

Effective treatment of chronic lower limb ischemia is one of the most challenging issues confronting vascular surgeons There are a number of choices available to the vascular surgeon Open or endovascular revascularization is the treatment of choice when applicable Current pharmacological therapies play an auxiliary role and cannot prevent disease progression Therefore, new methods of treatment are needed We conducted a phase 2b/3 multicenter randomized controlled clinical trial of the intra-muscular transfer of a plasmid DNA encoding vascular endothelial growth factor (VEGF) 165 with cytomegalovirus promotor (CMV) in patients with atherosclerotic lower limb ischemia A total of 100 patients were enrolled in the study, that is, 75 patients were randomized into the test group and received 2 intramuscular injections of 1.2 mg of pCMV-vegf165, 14 days apart together with standard pharmacological treatment In all, 25 patients were randomized into the control group and received standard treatment only The following end points were evaluated within the first 6 months of the study and during a 1.5-year additional follow-up period: pain-free walking distance (PWD), ankle–brachial index (ABI), and blood flow velocity (BFV) The pCMV-vegf165 therapy appeared to be significantly more effective than standard treatment The PWD increased in the test group by 110.4%, 167.2%, and 190.8% at 6 months, 1 year, and 2 years after treatment, respectively The pCMV-vegf165 intramuscular transfer caused a statistically significant increase in ABI and BFV There were no positive results in the control group Thus, pCMV-vegf165 intramuscular gene transfer is an effective method of treatment of moderate to severe claudication due to chronic lower limb ischemia

Keywords

chronic lower limb ischemia, VEGF165, gene therapy, clinical trial

Introduction

According to the World Health Organization, cardiovascular

dis-eases are the number 1 cause of death globally.1Cardiovascular

disease includes both coronary heart disease, cerebrovascular

disease, and peripheral arterial disease, which causes chronic

lower limb ischemia.2Endovascular or open revascularization

procedures are the main treatment methods for such patients,

although many of them are not suitable for a revascularization

due to severe distal or multifocal atherosclerotic lesions, failed

grafts, or severe coexisting pathology Thus, new methods to

treat chronic lower limb ischemia should be used

Along with open surgical, endovascular, and pharmacological

treatment, gene therapy has been introduced to treat patients with

chronic lower limb ischemia Gene therapy is one of the most

rap-idly developing methods for treating ischemia.3,4The following

different types of therapeutic genes that encode various growth

factors have been used in clinical trials: vascular endothelial

1

OJSC ‘‘Human Stem Cells Institute’’, Moscow, Russia

2

Department of Morphology and General Pathology, Kazan (Volga region) Federal University, Kazan, Russia

3 Department of Maxillofacial Surgery, A.I Evdokimov Moscow State University

of Medicine and Dentistry, Moscow, Russia

4 Department of Maxillofacial Surgery, A.I Burnazyan Medical Biophysical Center, Moscow, Russia

5 Department of Angiology and Vascular Surgery, Ryazan State I.P Pavlov Medical University, Ryazan, Russia

6 Department of Vascular Surgery, Russian National Research Center of Surgery, Moscow, Russia

7

Department of Surgery, Yaroslavl State Medical Academy, Yaroslavl, Russia

y Deceased Manuscript submitted: October 16, 2014; accepted: January 25, 2015 Corresponding Author:

Ilia Y Bozo, 3/2 Gubkina Str, Moscow 199333, Russia.

Email: bozo.ilia@gmail.com

Journal of Cardiovascular Pharmacology and Therapeutics 1-10

ª The Author(s) 2015 Reprints and permission:

sagepub.com/journalsPermissions.nav DOI: 10.1177/1074248415574336 cpt.sagepub.com

growth factor (VEGF) 165,5-7basic fibroblast growth factor,8,9

hypoxia-inducible factor, hepatocyte growth factor,10,11and

oth-ers Gene therapy is designed to induce angiogenesis via the

expression of the aforementioned genes in skeletal muscles after

intramuscular or intravascular delivery of gene products

In 2010, we completed a phase 1 to 2a clinical trial of

pCMV-vegf165 in patients with chronic lower limb ischemia

(stage 2a to 3 according to Fontaine classification modified by

A V Pokrovsky) who were not suitable for reconstructive surgery

or endovascular treatment This study demonstrated the safety,

feasibility, and short-term (3 months) efficacy of pCMV-vegf165

gene transfer,12,13which lead to conducting a phase 2b to 3

multi-center clinical trial The study was conducted under the control of

the Russian Ministry of Health and was completed in 2011

Patients enrolled in the study were subjected to a 6-month

follow-up period according to the study protocol and an additional

18-month follow-up period for a longer evaluation of study drug

efficacy and safety The results of the study are reported herein

Materials and Methods

Rationale for the Clinical Study

Preclinical studies of general toxicity (acute, subacute, chronic,

and local irritation) and specific toxicity (allergenicity,

repro-ductive and immune toxicity, mutagenicity, and

carcinogeni-city) as well as the detection of specific drug activity were

carried out at Russian State Federal 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

Clin-ical Practice in the Russian Federation’’ OST 42-511-99, ICH

GCP rules, and valid regulatory requirements

Drug Characteristics and Administration Method

The study drug is an original gene construction which contains

a supercoiled plasmid DNA (1.2 mg) encoding pCMV-vegf165

as the active substance and is now marketed as

‘‘Neovascul-gen.’’12The drug was supplied to the study centers as a sterile

lyophilisate that was then dissolved in 2 mL of water for

injec-tions immediately prior to administration The drug was

administered intramuscularly (calf muscles) at 4 to 5 injection sites in the lower and middle third of the posterior part of the calf

Patient Characteristics The study included patients with chronic lower limb ischemia who were not suitable for an open or endovascular revascular-ization due to a severe distal or multifocal atherosclerotic lesion The decision was made by a team of vascular surgeons and radiologists based on the angiographic and echographic findings, history of the disease, previous procedures, and con-comitant pathology Angiographic score was7 points accord-ing to the Rutherford (1997) runoff classification

The types of atherosclerotic lesions were defined as follows:

1 proximal lesion—patency of proximal arterial segments (aortoiliac) with a diffuse atherosclerotic lesion (occlu-sion) of superficial femoral artery and a popliteal artery extending into the tibioperoneal trunk;

2 multifocal lesion—patency of proximal arterial segments (aortoiliac) with a diffuse atherosclerotic lesion (occlu-sion) of the femoral, popliteal, and both tibial arteries;

3 distal lesion—patency of proximal arterial segments (aor-toiliac, femoral) with a diffuse atherosclerotic lesion (occlusion) of the popliteal artery with hemodynamically significant stenosis or occlusion of the tibial arteries All patients had a previous history of a long-term moderate

to severe claudication All patients received aspirin and statins

on a daily basis to reduce the risk of adverse cardiovascular ischemic events and previously underwent treatment with pentoxifylline

Inclusion criteria

 age more than 40 years;

 a history of stable claudication for at least 3 months;

 stage 2 to 3 chronic ischemia according to Fontaine classi-fication (modified by A V Pokrovsky);

 absence of hemodynamically significant stenosis (>70%) of the aortoiliofemoral arterial segment or (if present) a patent proximal bypass graft (prosthesis) if revascularization sur-gery was performed no earlier than 3 months prior to the inclusion in the study; satisfactory patency of the deep femoral artery in the presence of hemodynamically signif-icant femoropopliteal arterial lesions;

 presence of hemodynamically significant (stenosis >70% and/or occlusion) diffuse lesions of the anterior and (or) posterior tibial arteries (distal lesion);

 voluntary informed consent signed and dated by the patient

Exclusion criteria

 chronic lower limb ischemia of nonatherosclerotic gen-esis (autoimmune disorders, Buerger disease, congenital abnormalities, vascular injuries, etc);

 stage 4 chronic ischemia according to Fontaine classification

modified by A V Pokrovsky (ischemic ulcers and necrotic

lesions);

 severe concomitant pathology with life expectancy <1 year;

 infectious diseases, history of cancer, or suspected malignancy;

 decompensated diabetes mellitus (hemoglobin A1c > 8%

and fasting plasma glucose > 11.1 mmol/L)

Study Design and End Points

The current pCMV-vegf165 gene transfer trial was as an

open-label, prospective, randomized, controlled, and multicenter

study The patient distribution per study center and time period

is presented in Figure 1

A total of 100 patients were enrolled in the study and

rando-mized into 2 groups at a ratio of 3:1 Thus, 75 patients were

included into the test group and received 2 injections of

pCMV-vegf165 at a dose of 1.2 mg, 14 days apart (total

dose—2.4 mg) into the calf muscles altogether with standard

pharmacological treatment, and 25 patients were included in the control group and were given standard therapy alone All patients signed and dated the informed consent documents

In all, 13 patients had ischemic rest pain at baseline: 8 and 5 patients in test and control groups, respectively In all, 11 patients underwent aortoiliac arterial reconstructive surgery within more than 6 months prior to the onset of the study (5 patients in the test group and 6 patients in the control group)

In all, 18 patients had compensated diabetes mellitus: 12 in the test group and 6 in the control group

In all, 5 patients in the test group and 1 patient in the control group had undergone limb amputations prior to study enrollment which did not allow to perform a treadmill test to evaluate pain-free walking distance (PWD) The analyzed population in the study included 94 patients: 70—in the test group and 24—in the control group The AP value was variable depending on the technical equipment of each study site and the period of time following the study completion, which was considered when processing statistical data

Visit 0/1 Visit 2 Visit 3 Visit 4

14 (±2) days 90 (±2) days 180 (±2) days 1 year 1.5 year 2 years

Ryazan State I.P Pavlov Medical University 35/15

B.V Petrovsky Russian Scientiic Center of Surgery 15/5

Yaroslavl Regional Clinical Hospital 25/5

pCMV-vegf165

n=75

Control n=25

Blood and urine laboratory

tests; chest X-rays; abdominal

echography; measurement of

PWD, ABI, BFV;

angiography; SF-36

questionnaire

Blood and urine laboratory tests, measurement of PWD, ABI, BFV at each visit; chest X-rays, abdominal echography, angiography, SF-36 questionnaire – at visit 4

Blood and urine laboratory tests; chest X-rays; abdominal echography;

measurement of PWD, ABI, BFV at each

time point

Figure 1 Design of clinical trial

The initial time points were defined as follows: baseline, 14,

90, and 180 days Follow-up period was extended to a total of

2 years, with additional time points at 1 and 2 years following

the patients’ inclusion in the study

Safety Criteria

Safety of pCMV-vegf165 gene transfer in terms of the trial

pro-tocol was initially evaluated within 6 months following the

onset of the study (a 14-day in-patient hospital stay, with

fol-lowing out-patient office visits) with the registration of adverse

event (AE) and serious adverse event (SAE) during both

rou-tine visits and unscheduled requests for medical care

More-over, patients who gave their written consent for the

extended follow-up procedures underwent blood and urine

laboratory tests, chest X-rays, and abdominal echography in

order to assess the oncological safety (Figure 1)

Efficacy End Points

Primary Efficacy End Point

Pain-free walking distance The value of the PWD was defined as

the primary (main) efficacy end point According to the

Amer-ican College of Cardiology/AmerAmer-ican Heart Association

Guidelines for the management of patients with peripheral

arterial disease, this value is of the highest importance (class

I recommendations).14The intragroup distribution of patients

was based on the PWD value The severity of the disease was

determined according to the Fontaine classification modified

by A V Pokrovsky, which is widely accepted in Russia: stage

2a—PWD more than 200 m; stage 2b—less than 200 m, but

more than 50 m; and stage 3—less than 50 m or ischemic rest

pain in absence of ischemic ulcers or necrotic lesions The

PWD was determined using a treadmill test with reduced initial

speed (1 km/h), as the majority of elderly patients were unable

to perform Gardner test or its equivalents Information on

patients with ischemic rest pain is also provided (Table 1)

Secondary End Points

Ankle–brachial index Ankle–brachial index (ABI) was measured

using a standard technique at each visit Although ABI

mea-surement is regarded as a first-line assessment tool,14it largely

characterizes main arterial blood flow (macrohemodynamics),

and its diagnostic value is limited in patients who are not

suit-able for arterial reconstructive surgery due to poor runoff

Blood flow velocity Doppler ultrasound techniques are useful in

assessment of lower extremity atherosclerotic lesions and

determining severity of the disease or progression of

athero-sclerosis.14 Blood flow velocity (BFV) in the posterior tibial

artery was evaluated (if patent)

Angiography Thirty percent of patients enrolled into the study

agreed to undergo a digital subtraction angiography using

con-trast enhancement at following time points: prior to the study,

at 6 months, and 1 year after the onset of treatment Primary and repeated angiography was performed using the same angiography system, by the same radiologist, and with the same time delay of images Angiograms were assessed visually by the same experienced specialist

Quality of life All patients completed the SF-36 questionnaire (‘‘SF-36 Health Status Survey’’) before enrollment and at

6 months after the onset of treatment (Figure 1) The following

7 scales were evaluated: physical functioning, physical role func-tioning, bodily pain, general health perceptions, vitality, social role functioning, emotional role functioning, and mental health The values of each scale varied between 0 and 100, with

100 defined as complete health All the scales were used to assess 2 parameters: psychological and physical well-being

Statistical analysis A sample size of 28 patients in each group was estimated to detect a 0.75 standardized difference (80% power, P ¼ 05), assuming the target difference and SD for

Table 1 Baseline Characteristics of Patients

Factor

Control Group (n¼ 25)

pCMV-vegf165 Group

Intergroup Differences, P Men, n (%) 20 (80.0) 60 (80.0) (Chi-square) 1.000 Women, n (%) 5 (20.0) 15 (20.0) (Yates corrected

Chi-square) 1.000 Age, mean +

SD, years

70.9 + 7.8 67.8 + 9.0 (t test) P¼ 468 Severity of chronic lower limb ischemia (stage of disease and rest pain), n (%)

2b 22 (88.0) 57 (76.0)

Rest pain 5 (20.0) 8 (10.7) Occlusion level, n (%)

Proximal 12 (48.0) 38 (50.7) Distal 5 (20.0) 16 (21.3) Multifocal

occlusion

8 (32.0) 21 (28.0) PWD, m 114.3 + 11.4 135.3 + 12.2 (Mann–Whitney

U test with Bonferroni correction) 1.000 ABI 0.46 + 0.06 0.51 + 0.02 (Mann–Whitney

U test with Bonferroni correction) 1.000 BFV, cm/s 17.6 + 2.1 14.2 + 1.6 (Mann–Whitney

U test with Bonferroni correction) 1.000

Abbreviations: ABI, ankle–brachial index; BFV, blood flow velocity; PWD, pain-free walking distance; SD, standard deviation.

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

The absolute values of efficacy criteria (PWD, ABI, and BFV)

were not normally distributed; therefore, nonparametric methods

were used to test the hypothesis (Mann–Whitney U test and

Wil-coxon test with Bonferroni correction to avoid a type I error) The

SF-36 questionnaire scores were normally distributed, so the T

test was used to compare the values of 2 groups

Results

Baseline Characteristics of the Trial Participants

A total of 100 patients were enrolled in the clinical study: 75

were randomized into the test group and received 2 injections

of pCMV-vegf165, 14 days apart (a total dose of 2.4 mg) into

the calf muscles of the affected limb The comparison of

base-line characteristics between the groups showed that gender

dif-ferences as well as difdif-ferences in the primary and secondary

end points were not statistically significant (Table 1) The

val-ues of PWD were similar between the 2 groups: 135.3 + 12.2

and 114.3 + 11.4 m in the test and control group, respectively

A more detailed analysis revealed that the severity of the

dis-ease and atherosclerotic lesion levels were comparable among

the control and test groups However, the control group did not

include patients with stage 2a Therefore, a precise comparison

between the subgroups regarding the severity of ischemia was

made only in patients with stages 2b to 3 disease

Evaluation of Safety

No AE, SAE, or significant laboratory abnormalities were

observed in either study group during both treatment and

follow-up period No peripheral edema was observed Local

pathological reactions, including allergic, anaphylactic, and

neoplastic reactions, were absent immediately after study drug

administration, at 6 months after the onset of treatment, and

during the extended follow-up period

During the first 6 months, 3 events precluded the continuation

of the study: 2 acute ischemic strokes (test group) with a positive

outcome and 1 acute myocardial infarction with a fatal outcome

(test group) Apparently, these events were not related to

pCMV-vegf165 gene transfer as the construction used in the study has a

proven local action The results of toxicological studies showed

no relationship between the study drug and AEs.15

Tumor growth, eyesight disorders, and other pathological

conditions that could indirectly suggest complications of gene

therapy were not observed in patients throughout the study and

during the 1.5-year follow-up period

Evaluation of Efficacy

Primary End Point

Pain-free walking distance The first changes in clinical

character-istics among the patients of pCMV-vegf165 group were noticed

by the patients themselves within 2 weeks after the onset of treatment More notable changes were observed at 45 to 60 days The initial PWD level in the test group was 135.3 + 12.2 m, increasing to 284.7 + 29.8 m at 6 months (Tables 2 and 3) The differences between the baseline and subsequent PWD values within the test group and differences between the test and control group were statistically significant starting from day 90 During the first 6 months of the study, there was

an increasing trend of PWD values in 62 (85%) patients of the test group During the long-term follow-up period, the value of PWD continued to increase in the test group The increase in the mean distance that a patient could walk without pain was 149.4 m in the study group after 6 months (110.4%), while its value decreased by 1.5 m in the control group compared to the baseline The tendency remained positive throughout the 2 years of monitoring: PWD increased in pCMV-vegf165 patients by 167.2% and 190.8%, that is, by 226.3 and 258.1

m, at 1 and 2 years, respectively, while no statistically signifi-cant changes were observed in the control group

The largest increase in the PWD was observed in patients with advanced stages of ischemia (severe claudication or ischemic rest pain), that is, stage 3: a 96.4-m increase (231.2%) at 6 months, a 228.3-m increase (547.5%) at 1 year, and a 345.3-m increase (828%) at 2 years The PWD increased

by 129.4 m (108.3%) in patients with stage 2b disease (initial PWD increased by 50-200 m) Such positive results remained stable throughout a 2-year follow-up period The PWD increased by 290.0 m (90.6%) in patients with stage 2a disease

at 6 months and by 660.0 m (206.2%) and 517.5 m (161.7%) at

1 and 2 years, respectively

Depending on the localization of atherosclerotic lesions, the results were as follows: the 6 months results showed that patients with multifocal arterial lesions of the lower limbs benefited from gene therapy The average increase in PWD val-ues in these patients was 259.0 m (180.7%); the PWD increased

by 431.7 m (301.2%) at 1 year and by 363.7 m (253.8%) at 2 years compared to baseline The opposite results were obtained

in the control group: PWD increased by 34.0 m (35.4%) at 6 months; however, during the follow-up period, the PWD value decreased by 56.0 m (58.3%) and 66.0 m (68.7%) at 1 and 2 years, respectively, compared to the baseline

The PWD in test group patients with predominantly distal vascular lesions increased by 179.7 m at 6 months (132.4%), by 230.3 m (169.7%) at 1 year, and by 342.9 m (252.7%) at 2 years

Secondary End Points Characteristics of macrohemodynamics: ABI Six months following the onset of the study, there were statistically significant changes in ABI in the test group (a 0.05 increase, P¼ 009) The ABI did not change in the control group The differences

in absolute values among the test and control groups (patients with rest pain included) at each visit, as well as an increase in the absolute values between the groups, were statistically insig-nificant (Tables 2-4) The long-term follow-up (2 years) results

a Mean

b Selection

demonstrated a slight but stable improvement in ABI in test

group patients

Characteristics of macrohemodynamics: BFV The BFV in the

pCMV-vegf165 group patients increased by 8.4 m/s within

6 months (average growth by 59.1%) Within 1 year, the value

of BFV slightly decreased but remained 5.5 m/s higher than the

baseline At 2 years, the tendency remained positive There

were no statistically significant changes in BFV in patients of

the control group (Tables 2 and 3)

Angiography Angiograms were performed and assessed

visually by the same experienced radiologist Improvement

in the collateral vascular bed was observed in 75% of

patients who agreed to undergo angiography Enhanced

con-trast filling of the microcirculatory bloodstream due to an

increased diameter of collateral vessels was recorded in

12.5% of patients who underwent angiography A moderate

increase in the number of the newly formed collaterals was

recorded in 37.5% of patients Significant growth of the

col-lateral vessels was registered in 37.5% of patients who

underwent angiography Neoangiogenesis may be attributed

to the growth of new collaterals and possibly to the opening

of previously nonfunctioning vessels (Figure 2) There were

no clinically important laboratory abnormalities throughout

the entire period of treatment and follow-up in patients of

both groups

Quality of life A statistically insignificant improvement in phys-ical health was observed in patients of the test group Mental health also slightly improved in patients of the pCMV-vegf165 group (Table 5) Control patients had a higher quality of life regarding mental health compared to patients who were treated with gene transfer

Table 3 Results of Measurements of Primary and Secondary End Points.a

Value

pCMV-vegf165 Group, Median, IQR Control Group, Median, IQR Baseline 0.5 Year 1.0 Year 2.0 Years Baseline 0.5 Year 1.0 Year 2.0 Years PWD (70/24) 100, 130 230, 220 230, 258 300, 310 105, 102.5 140, 110 125, 110 140, 50 2a 295.0, 185.0 525.0, 435.0 1250.0, 850.0 525.0, 925.0

2b 120.0, 100.0 230.0, 180.0 177.0, 237.0 300.0, 188.0 120.0, 105.0 142.5, 87.5 125.0, 115.0 145.0, 55.0

3 35.0, 25.0 80.0, 90.0 231.5, 100.0 400.0, 300.0 40.0, 8.0 30.0, 20.0 – – Proximal occlusion 133.0, 132.5 210.0, 175.0 154.0, 228.0 220.0, 160.0 150.0, 110.0 150.0, 90.0 150.0, 0 150.0, 0 Distal occlusion 100.0, 145.0 305.0, 200.0 300.0, 300.0 400.0, 400.0 95.0, 110.0 100.0, 100.0 75.0, 50.0 125.0, 150.0 Multifocal occlusion 85.0, 90.0 340.0, 360.0 325.0, 900.0 400.0, 250.0 100.0, 50.0 150.0, 70.0 40.0, 40.0 30.0, 0 ABI (73/25) 0.50, 0.22 0.52, 0.27 0.55, 0.31 0.56, 0.19 0.50, 0.08 0.50, 0.07 0.45, 0.22 0.50, 0.16 BFV (65/20) 13.7, 12.8 20.5, 18.3 18.0, 14.0 20.0, 13.5 19.0, 13.7 20.0, 15.7 14.5, 9.0 16.0, 4.0

Abbreviations: ABI, ankle–brachial index; BFV, blood flow velocity; PWD, pain-free walking distance.

a Median and interquartile range (IQR).

Table 4 Mean Values and Standard Error of the Mean (M + m) of ABI and BFV in Patients With Ischemic Rest Pain in Test (pCMV-vegf165) and Control Groups

ABI 0.37 + 0.04 0.4 + 0.04 0.41 + 0.08 0.37 + 0.11 0.5 + 0.03 0.53 + 0.05 0.75 + 0.3 0.35 BFV 7.1 + 2.7 16.5 + 51 15.5 + 8.3 11.5 + 3.9 10.7 + 4.9 8.5 + 4.5

Abbreviations: ABI, ankle–brachial index; BFV, blood flow velocity.

Figure 2 Angiographic images, patient of the test group: (A) before treatment and (B) 6 months after pCMV-vegf165 gene transfer

The concept of gene therapy for paracrine vascular growth

reg-ulation, that is, therapeutic angiogenesis, began evolving after

the pioneering works of Isner.5,16,17Gene therapy evolved due

to the accomplishment of experimental and clinical trials which

investigated different therapeutic genes.3A number of delivery

vectors were used: viral (mainly adenoviruses)3,7and nonviral

(mainly naked plasmids).3,5,6,18 Majority of clinical trials

demonstrated safety of both approaches of local administration

of gene products at different dosage levels in terms of systemic

allergic or anaphylactic reactions and the absence of neoplastic

reactions, for example, proliferative retinopathy, vascular tumors,

induction of dormant tumors, and so on.3,18However, data

regard-ing the efficacy of gene therapy were more variable Certain

stud-ies were considered a failure due to the chosen requirements

regarding efficacy end points, such as the number of amputations

or the survival curve,19heterogeneity of patients enrolled into the

study, and selection of a therapeutic gene, for example, not the

most promising candidate genes for angiogenesis

Present study aimed to determine the safety and efficacy of

the pCMV-vegf165 gene product in patients who were not

suit-able for surgical or endovascular revascularization The absence

of ischemic ulcers and necrotic lesions in patients (stage 4

according to Fontaine classification modified by A V

Pok-rovsky) allowed to study the effect of gene transfer in patients

with viable limbs Majority of previous studies enrolled patients

with ulcers or gangrenes, which had a negative impact on further

investigations of gene products and their effects or use in

patients with moderate to severe claudication.19-21

Within the study (180 days) and follow-up period (another

1.5 years) neither of 3 study centers reported any adverse effects

(AEs and SAEs) or other complications The selected mode of

pCMV-vegf165 administration at the selected dosage regimen

was safe during the therapy and at least 2 years thereafter

All lethal outcomes (5 in the test group and 2 in the control

group) were attributed to acute myocardial infarction (Table 6)

Peripheral arterial disease is an independent predictor of worse

outcomes in patients with ischemic heart disease We believe that

there is no relationship between lethal outcomes and gene transfer

in terms of this study These findings correspond with the results

of other studies of plasmid VEGFf165 gene products.3,17,22

The evaluation of efficacy appeared more difficult The use

of gene therapy in patients who were not suitable for an open or

endovascular revascularization allowed significant increases in

PWD This positive tendency was stable both during the first

Table 5 Values of Physical and Mental Health in Patients of Test (pCMV-vegf165) and Control Groups According to SF-36 Questionnaire

Value Group Baseline, M + m 0.5 Year, M + m

Statistical Significance of Intergroup Differences, P (T test)

Control group 36.7 + 2.9 36.4 + 2.4

Control group 44.5 + 4.3 46.9 + 4.1

Figure 3 Patient intergroup ratio according to the stage of disease at baseline, at 0.5, and 1 year after administration: (A) test group (pCMV-vegf165) and (B) control group I—stage 2a; II—2b; III—stage 3; and black—amputations

Table 6 Number of Amputations and Patients Who Died During the Observation Period in Test (pCMV-vegf165) and Control Groups

Time Points

pCMV-vegf165 Group Control Group

Amputations

Patients Who Died Amputations

Patients Who Died

From 6 months

to 1 year

a Four amputations were performed in patients with ischemic rest pain at baseline.

b

One amputation was performed in patients with ischemic rest pain at baseline.

6 months and the following 1.5 years The PWD continued to

increase at the end of the 2-year monitoring period

Improve-ment in PWD in the test group was compared to the changes

in PWD in the control group: while the conventional therapy

alone did not have major successful results, gene transfer had

statistically significant positive effects (Tables 2 and 3)

Unfor-tunately, our data cannot be compared to the results of similar

studies, as most of the previous studies enrolled patients with

critical lower limb ischemia who could not undergo the

tread-mill exercise testing.7,8,11,23,24Although the study design (not

blinded, not placebo-controlled) contributed to comparison

dif-ficulties, we were able to notice that the number of test group

patients with less severe stage of the disease increased, while

a negative tendency was observed in the control group due to

an increased number of patients with more advanced stages

of the disease, including those which resulted in amputation

(Figure 3) At 1 year following the onset of treatment, 4 of the

total 5 amputations in the test group were performed in patients

with pain at rest at baseline (Table 6) Of a total of 2

amputa-tions in the control group, 1 was performed in a patient with

ischemic rest pain at baseline Limb loss was attributed to the

disease progression leading to irreversible ischemia Patients

enrolled in the study were not suitable for revascularization,

consequently performing an amputation was the only option

left

Limb salvage rates at 2 years were 93.3% in the test group

and 88% in the control group (Table 6) However, the

differ-ences were not statistically significant So, we did not observe

the amputation reduction in the test group More observations

are needed

Clinical signs majorly improved in patients with distal or

multifocal atherosclerotic lesion

Changes in ABI and BFV were not significant which may be

explained by the fact that the study drug is designed to induce

angiogenesis at the microcirculatory level and does not affect

macrohemodynamic Nevertheless, slight improvement in ABI

and BFW may be attributed to the general improvements in the

collateral arterial flow and decrease in the peripheral arterial

resistance

Safety and efficacy of the studied pCMV-vegf165 gene

product marketed as ‘‘Neovasculgen’’ were demonstrated in

selected patients throughout a 2-year follow-up period.15

Within this period, we were able to track both limb salvage and

patient survival (Table 6) Gene therapy with pCMV-vegf165

did not affect mortality Limb salvage largely depended on the

presence of rest pain at baseline However, these parameters

should be analyzed in larger cohorts of patients

Despite the marked improvement in claudication symptoms

in the test group patients, gene therapy did not significantly

affect the quality of life Mental health score was higher in the

control group as compared to those of the test group

Appar-ently, these findings were attributed to the initial differences

in baseline SF-36 scores among the patients of both groups

(Table 5) and presence of concomitant pathology which

decreased the positive impact of PWD increase in the overall

quality of life Such findings may indicate the presence of an

underlying depressive disorder in patients with chronic lower limb ischemia

Conclusion The use of the plasmid DNA gene product encoding VEGF165 (pCMV-vegf165) in combination with standard pharmacologi-cal therapy significantly improves clinipharmacologi-cal signs of claudication

in patients with chronic lower limb ischemia A 2-year

follow-up demonstrated a stable PWD improvement The results of the study were sufficient for the registration of ‘‘Neovascul-gen’’ as a drug which is used in the treatment of patients with moderate to severe claudication due to stage 2a to 3 athero-sclerotic chronic lower limb ischemia However, further studies enrolling larger groups of patients are needed to completely evaluate the effects of pCMV-vegf165 gene transfer in patients with pain at rest due to peripheral atherosclerosis, ischemia caused by diabetes mellitus or autoimmune disorders, and those who undergo peripheral arterial revascularization

Author Contributions

R Deev contributed to design, contributed to analysis and interpreta-tion, drafted the article, critically revised the article, and gave final approval I Bozo contributed to analysis and interpretation, drafted the article, critically revised the article, and gave final approval

N Mzhavanadze contributed to acquisition and analysis, drafted the article, critically revised the article, and gave final approval D Voronov contributed to acquisition and analysis, critically revised the article, and gave final approval A Gavrilenko critically revised the article and gave final approval Y Chervyakov contributed to acquisition, critically revised the article, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy

I Staroverov contributed to acquisition, critically revised the article, and gave final approval R Kalinin contributed to acquisition, criti-cally revised the article, gave final approval, and agrees to be accoun-table for all aspects of work ensuring integrity and accuracy P Shvalb contributed to conception and design, contributed to acquisition, cri-tically revised the article, and gave final approval A Isaev contribu-ted to conception and design, contribucontribu-ted to analysis and interpretation, critically revised the article, and gave final approval

Acknowledgments Authors would like to thank Prof S L Kiselev for his contribution in developing the gene construction and participating in the studies Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this arti-cle: A A Isaev, I Ya Bozo, and R V Deev are employees of the OJSC ‘‘Human Stem Cells Institute.’’ A A Isaev is shareholder of the OJSC ‘‘Human Stem Cells Institute.’’

Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: OJSC

‘‘Human Stem Cells Institute’’ (Moscow, Russia) sponsored the clin-ical trial

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