Shigematsu 2010.

Methods	Study design: multi‐centre double‐blind placebo‐controlled RCT Intention‐to‐treat: no, interim analysis carried out when participants reached N = 40 (HGF n = 27; placebo n = 13), and safety analysis N = 41 (HGF n = 28; placebo n = 13) Country: Japan
Participants	Number randomised: N = 46 (HGF n = 30; placebo n = 16) Losses to follow‐up and withdrawals: N = 6 (HGF n = 3; placebo n = 3) Age (mean years ± SD): HGF 71.9 ± 7.6; placebo 72.8 ± 7.3 Gender (M): HGF 77.8%; placebo 53.8% Inclusion criteria: all eligible participants screened by an eligibility committee composed of vascular surgeons: aged 40 to 84 years with chronic CLI and rest pain or non‐healing ischaemic ulcers (Rutherford 4/Fontaine III or Rutherfor 5/Fontaine IV) persisting for a minimum of 4 weeks; resting ABI < 0.6 and mean ankle blood pressure < 70 mmHg in the affected limb according to 3 consecutive measurements performed at weeks ‐4, ‐2, and 0, or TBI < 0.5 if ABI not measurable; ineligible for standard surgical or percutaneous revascularisation and showed no response to conventional drug therapy for at least 4 weeks Exclusion criteria: deep ulcers that exposed bone or tendon; clinical evidence of invasive infection uncontrolled by antibiotics; serious cardiac, hepatic, renal, or haematological disease; current evidence or history of malignancy; PDR; neovascular age‐related macular degeneration; sympathectomy or sympathetic block within 6 months; revascularisation or major amputation within 3 months
Interventions	Treatment: naked plasmid encoding human HGF gene (beperminogene perplasmid, Collategene) ‐ 0.5 mg of HGF plasmid in 3 mL saline given by 8 intramuscular injections into the calf muscles and/or the distal thigh of the ischaemic limb under ultrasound guidance; injection schedule repeated after 28 days Control: placebo, saline, given in the same method as HGF plasmid
Outcomes	Follow‐up times: 12 and 24 weeks and 9 and 15 months Outcomes: Improvement in rest pain (reduction in VAS scale > 20 mm compared with baseline) in patients without ulcers or reduction in ulcer size (> 25% (approximately 50% change in area)) in patients with ulcers, ABI, Amputation, QoL using SF‐36, Safety (adverse events, concomitant medications, ECG, lab blood and urine tests, vital signs, physical findings, cancer and retinopathy screenings, assays for HGF protein and antibodies, Escherichia coli protein antibodies and DNA antibodies)
Notes	Study period: pre‐screened February 2004 to June 2007 After 12 weeks, participants were unblinded; those who received placebo could choose to enter the next stage and receive active drug
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Used a modified minimisation method, allocated by the central registration centre; randomisation ratio for plasmid‐to‐placebo was 2:1
Allocation concealment (selection bias)	Low risk	Allocated by the central registration centre
Blinding of participants and personnel (performance bias) All outcomes	Unclear risk	Described as double‐blind, used placebo, and described administration as given in a blinded manner; at the time the study reached N = 40, trial was terminated and information about allocation of treatment was opened to investigators; 3 patients had not been evaluated and were excluded from the analysis; 8 weeks after second administration (12 weeks from first treatment), the study treatment code was opened for each participant, who could then receive HGF if previously receiving placebo, if they wished
Blinding of outcome assessment (detection bias) All outcomes	Unclear risk	Insufficient information provided to determine blinding of outcome assessment
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	Withdrawals and losses clearly presented in the figure; due to trial terminated early and unblinded, n = 3 were not yet evaluated and were excluded from the final analysis
Selective reporting (reporting bias)	Low risk	All outcomes reported
Other bias	High risk	Funded and designed by AnGes MG, Inc. (Osaka, Japan); formal data analysis performed by a contract research organisation Study power calculation required for n = 80 in the HGF plasmid group and n = 40 in the placebo group, but with slow recruitment, the analysis was changed to an interim analysis with total N = 40, with only n = 13 in the placebo group

ABI: ankle brachial pressure index.
 CLI: critical limb ischaemia.
 CNS: central nervous system.
 COT: claudication onset time.
 CT: computerised tomography.
 CVA: cerebrovascular accident.
 Del‐1: developmental endothelial locus‐1.
 DNA: deoxyribonucleic acid.
 dL: decilitre.
 ECG: electrocardiogram.
 EE: efficacy evaluable.
 ESRD: end‐stage renal disease.
 EuroQol: quality of life tool.
 FGF: fibroblast growth factor.
 HGF: hepatocyte growth factor.
 HIF‐1α: hypoxia‐inducible factor 1‐alpha.
 HIV: human immunodeficiency virus.
 IC: intermittent claudication.
 ITT: intention‐to‐treat.
 LOCF: last observation carried forward.
 mg: milligram.
 MI: myocardial infarction.
 MITT: modified intention‐to‐treat.
 mL: millilitre.
 mmHg: millimetre of mercury.
 MRA: magnetic resonance angiography.
 NaCl: sodium chloride.
 NV1FGF: non‐viral 1 FGF.
 PAD: peripheral arterial disease.
 PDR: proliferative diabetic retinopathy.
 Pfu: plaque forming unit.
 PTA: percutaneous transluminal angioplasty.
 PWD: pain‐free walking distance.
 PWT: peak walking time.
 QoL: quality of life.
 RAND‐36: quality of life tool.
 RCT: randomised controlled trial.
 SD: standard deviation.
 SDF‐1: stromal cell‐derived factor‐1.
 SF‐36: Short Form‐36; quality of life tool.
 TASC: Trans‐Atlantic Inter‐Society Consensus.
 TBI: toe brachial pressure index.
 TcPO₂: transcutaneous oximetry.
 VascuQoL: vascular quality of life questionnaire.
 VAS: visual analogue scale.
 VEGF: vascular endothelial growth factor.
 VEGF‐AdV: VEGF‐adenovirus.
 VEGF‐P/L: VEGF‐plasmid/liposome.
 WIQ: Walking Impairment Questionnaire; quality of life tool.
 µg: microgram.
 µL: microlitre.
 TcPO₂: transcutaneous oximetry.