Therapeutic Angiogenesis With Intramuscular NV1FGF Improves Amputation-free Survival in Patients With Critical Limb Ischemia

Transcription

1 originl rticle The Americn Society of Gene Therpy see pge 808 Therpeutic Angiogenesis With Intrmusculr NV1FGF Improves Amputtion-free Survivl in Ptients With Criticl Limb Ischemi Sigrid Nikol 1, Iris Bumgrtner 2, Eric Vn Belle 3, Curt Diehm 4, Adrin Visoná 5, Murizio C Cpogrossi 6, Nicole Ferreir-Mldent 7, Augusto Gllino 8, Michel Grhm Wytt 9, Lsnth Dinesh Wijesinghe 10, Meliss Fusri 11, Dominique Stephn 12, Joseph Emmerich 13, Giulio Pompilio 14, Frnk Vermssen 15, Emmnuel Phm 16, Vincent Grek 16, Michel Colemn 16 nd Frnçois Meyer 16, * 1 Deprtment of Crdiology nd Angiology, Universitätsklinikum, Münster, Germny; 2 Deprtment of Hert nd Vsculr Medicine (DHGE), Division of Angiology, University of Bern, Bern, Switzerlnd; 3 Deprtment of Crdiology B nd Hemodynmics, Hôpitl Crdiologique, CHRU de Lille, Frnce; 4 Deprtment of Internl Medicine nd Vsculr Medicine, Klinikum Krlsbd-Lngensteinbch GmbH, Krlsbd, Germny; 5 Deprtment of Internl Medicine, Angiology Unit, University of Pdov, Cstelfrnco Veneto, Itly; 6 Lbortorio di ptologi Vscolre, Istituto Dermoptico dell Immcolt, Rom, Itly; 7 Deprtment of Internl Medicine B, Hôpitl Bretonneu, Tours, Frnce; 8 Deprtment of Crdiovsculr Reserch Unit, Ospedle Sn Giovnni, Bellinzon, Switzerlnd; 9 Deprtment of Generl Surgery, Northern Vsculr Center, Freemn Hospitl, Newcstle Upon Tyne, UK; 10 Deprtment of Vsculr Surgery, Royl Bournemouth Hospitl, Bournemouth, UK; 11 Deprment of Vsculr Surgery, Cliniche Gvzzeni, Bergmo, Itly; 12 Deprtment of Hypertension, Vsculr Diseses nd Phrmcology, Hôpitl Civil, Strsbourg, Frnce; 13 University Pris Descrtes, INSERM U765, Service de Médecine Vsculire-HTA, Pris, Frnce; 14 Deprtment of Vsculr Surgery, Centro Crdiologico Monzino, Milno, Itly; 15 Deprtment of Vsculr Surgery, Ghent University Hospitl, Gent, Belgium; 16 Centelion SAS, Vitry sur Seine, Frnce. *On behlf of the TALISMAN 201 investigtors. This study evluted the efficcy nd sfety of intrmusculr dministrtion of NV1FGF, plsmid-bsed ngiogenic gene delivery system for locl expression of fibroblst growth fctor 1 (FGF-1), versus plcebo, in ptients with criticl limb ischemi (CLI). In doubleblind, rndomized, plcebo-controlled, Europen, multintionl study, 125 ptients in whom revsculriztion ws not considered to be suitble option, presenting with nonheling ulcer(s), were rndomized to receive eight intrmusculr injections of plcebo or 2.5 ml of NV1FGF t mg/ml on dys 1, 15, 30, nd 45 (totl 16 mg: 4 4 mg). The primry end point ws occurrence of complete heling of t lest one ulcer in the treted limb t week 25. Secondry end points included nkle brchil index (ABI), mputtion, nd deth. There were 107 ptients eligible for evlution. Improvements in ulcer heling were similr for use of NV1FGF (19.6%) nd plcebo (14.3%; P = 0.514). However, the use of NV1FGF significntly reduced (by twofold) the risk of ll mputtions [hzrd rtio (HR) 98; P = 15] nd mjor mputtions (HR 0.371; P = 15). Furthermore, there ws trend for reduced risk of deth with the use of NV1FGF (HR 60; P = 0.105). The dverse event incidence ws high, nd similr between the groups. In ptients with CLI, plsmid-bsed NV1FGF gene trnsfer ws well tolerted, nd resulted in significntly reduced risk of mjor mputtion when compred with plcebo. Received 19 December 2007; ccepted 5 Februry 2008; published online 1 April doi: /mt Introduction Peripherl rteril disese is progressive illness, nd the most severe mnifesttion is termed criticl limb ischemi (CLI). 1 It is estimted tht ech yer there re ~500 new cses of CLI per million individuls in the USA nd Europe. 2 These ptients experience chronic ischemic rest pin, ulcers, or gngrene in the lower limbs, nd hve very poor prognosis. Mny will undergo mputtion, nd their qulity of life hs been described s being similr to tht in ptients with criticl- or terminl-stge cncer. 3 5 The current gols of mngement of CLI re to relieve ischemic pin, hel ischemic ulcers, prevent mputtion, reduce crdiovsculr mortlity, nd improve the ptient s functioning nd qulity of life. 6,7 There is no effective phrmcologic therpy vilble, nd tretment consists primrily of percutneous or surgicl revsculriztion. However, restenosis rtes re high, nd peripherl bypss surgery is ssocited with significnt morbidity nd mortlity. Importntly, in considerble proportion of ptients (10 15%), revsculriztion is not suitble option. Of these ptients, >40% will require mjor mputtion, nd 20% will die within 6 months. 5,6 Therpeutic ngiogenesis is novel strtegy for the tretment of ischemic vsculr disese. It uses ngiogenic fctors to increse blood perfusion in ischemic tissues through vrious mechnisms of ction Gene trnsfer of fibroblst growth fctor 1 (FGF-1) hs been used successfully to promote ngiogenesis NV1FGF (Centelion SAS, Vitry sur Seine, Frnce) is novel pcor DNA plsmid bsed gene delivery system for locl expression of FGF The pcor plsmid bckbone hs severl fetures tht increse biosfety. These ttributes include repliction in only nrrow host rnge of lbortory, but not wild type, Escherichi coli strins, bsence of n ntibiotic-resistnce gene, nd lck of the Correspondence: Sigrid Nikol, Deprtment of Crdiology nd Angiology, Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster, Germny. E-mil: nikol@uni-muenster.de vol. 16 no. 5, my 2008

2 The Americn Society of Gene Therpy NV1FGF Improves Amputtion-free Survivl potentilly immunostimultory sequence motifs found in ColE1 plsmids. 24 Preclinicl studies of intrmusculr dministrtion of NV1FGF hve demonstrted tht locl expression of FGF-1 persists for severl weeks. The phrmcologicl efficcy hs been demonstrted bsed on morphologicl nd functionl end points in two experimentl niml models of hindlimb ischemi, the rbbit model 25 nd the hypercholesterolemic hmster model, tht exhibit sustined defects in collterl vessels nd rteriole formtion fter induction of ischemi. 19 Intrmusculr dministrtion of pcor plsmid expressing FGF-1 in the rbbit model resulted in sttisticlly significnt improvement in the formtion of cpillries nd collterl vessels s well s tissue perfusion in the treted limb. Improvement ws sttisticlly significnt in dose-dependent mnner t ll the end points when compred with the effects produced by vehicle or plsmid controls. 25 The ngiogenic effect of NV1FGF ws confirmed in the more severe niml model. The dministrtion of NV1FGF in the hypercholesterolemic hmster model of hindlimb ischemi significntly enhnced the formtion of lrge conductnce vessels s well s smll resistnce rteries in ischemi-injured muscles. 19 In n open-lbel Phse I tril involving 51 ptients, single nd repeted (two) intrmusculr doses of NV1FGF in ptients with CLI were well tolerted. 20 In nother open-lbel study, six CLI ptients who were to undergo plnned mputtion were dministered eight intrmusculr injections of NV1FGF, four in the clf nd four in the thigh. Muscle smples collected from the injected sites nd from distnt sites of the mputted legs showed tht myofibers of CLI ptients were cpble of being trnsfected with NV1FGF nd of expressing NV1FGF-derived messenger RNA nd FGF-1 protein. NV1FGF-derived FGF-1 expression ws loclized to the injection site nd not detected in muscles distnt from the injection site. The locl expression of FGF-1 nd the presence of FGF receptors (1 4) in ischemic muscles of ll the tested ptients provide rtionle for using NV1FGF in peripherl rteril disese ptients. This phse II study ws designed to evlute the efficcy nd sfety of NV1FGF versus plcebo in CLI ptients in whom revsculriztion ws not considered to be suitble option. Results A summry of the ptients chrcteristics is shown in Figure 1. A totl of 205 ptients were screened, nd 125 ptients who met the inclusion nd exclusion criteri were rndomized to NV1FGF (n = 59) or plcebo (n = 66). In this multintionl study, only eight of the sites were ble to enroll complete blocks, nd this led to slight imblnce in numbers between the plcebo nd NV1FGF groups. Rndomiztion to NV1FGF or plcebo ws well blnced (Figure 1). The modified intention-to-tret (MITT) popultion included 107 ptients receiving NV1FGF (n = 51) or plcebo (n = 56) (Figure 1). The rte of study discontinution ws 45.5% of the ptients in the plcebo group nd 30.5% in the NV1FGF group. The two min resons for discontinution were dverse events nd deth. The men ge of the ptients ws 72 yers, nd most of the ptients were men (71%). Other bseline chrcteristics common in this type of popultion were reltively well blnced between the two groups (ll P vlues > 0.1) (Tble 1). The men ggregte ulcer size (in squre centimeters) ws similr in the two groups, rnging from very smll ulcers to extensive MITT N = 51 (86.4%) Completed N = 49 (83.1%) NV1FGF (N = 59) Country: N (%) Belgium: 2 (3.4) Frnce: 13 (22.0) Germny: 15 (25.4) Itly: 13 (22.0) Switzerlnd: 11 (18.6) UK: 5 (8.5) Premturely discontinued N = 18 (30.5%) Reson: N (%) Adverse event: 3 (5.1) Protocol violtion: 1 (1.7) Consent withdrwl: 0 Lost to follow-up 3 (5.1) Deth: 7 (11.9)* Other: 4 (6:8) Excluded from MITT N = 8 (13.6%) Ptients screened N = 205 Ptients rndomized N = 125 MITT N = 56 (84.8%) Completed N = 46 (69.7%) Plcebo (N = 66) Country: N (%) Belgium: 5 (7.6) Frnce: 12 (18.2) Germny: 18 (27.3) Itly: 16 (24.2) Switzerlnd: 9 (13.6) UK: 6 (9.1) Premturely discontinued N = 30 (45.5%) Reson: N (%) Adverse event: 9 (13.6) Protocol violtion: 1 (1.5) Consent withdrwl: 1 (1.5) Lost to follow-up 3 (5.1) Deth: 13 (19.7)* Other: 5 (7.6) Excluded from MITT N = 10 (15.2%) Figure 1 Ptient disposition. Completed: MITT ptients who hd received their lst injection (visit 5, week 7) even if previous injections hd been missed. Dgger represents dt clculted from rndomized popultion. Asterisk represents the number of deths s reson for discontinution is lower thn the totl number of deths observed in the rndomized popultion (plcebo, N = 16; NV1FGF, N = 10). This is becuse ptient could discontinue the study for reson other thn deth (e.g., n dverse event), with deth occurring lter but before the end of the tril. MITT, modified intention-to-tret. lesions: men size of cm 2 ( ) in the plcebo group nd cm 2 ( ) in the NV1FGF group. Primry end point Ischemic ulcers. Similr rtes of ulcer heling occurred with NV1FGF (19.6%) nd plcebo (14.3%; P = 0.514) (Tble 2). There were lso no differences between the tretment groups in ggregte ulcer size or ulcer depth. Secondry end points In ddition to the nlysis of dt from the MITT popultion, the robustness of the findings in reltion to the occurrence of mputtion, deth, nd combined mjor mputtion nd deth ws confirmed in the totl rndomized popultion (dt not shown). Amputtion. The use of NV1FGF reduced by twofold the risk of ll mputtions [hzrd rtio (HR) 98; P = 15] nd mjor mputtions (HR 0.371; P = 15) in the MITT study popultion, s shown in Figure 2 nd b, nd Tble 2. This finding ws confirmed by post-hoc multivrite nlysis (Cox model) of the potentil influence of prognostic fctors for the disese, including dibetes sttus, smoking sttus, hypertension, prior mputtion, initil risk of mputtion, nkle brchil index (ABI), nd trnscutneous oxygen pressure ( ) (Tble 3). Deth. There ws no sttisticlly significnt trend to suggest tht the use of NV1FGF reduces the risk of deth (HR 60, P = 0.105; Figure 2c nd Tble 2). Combined mjor mputtion nd deth. The risk of combined mjor mputtion nd deth ws significntly reduced in the Moleculr Therpy vol. 16 no. 5 my

3 NV1FGF Improves Amputtion-free Survivl The Americn Society of Gene Therpy Tble 1 Ptient demogrphics nd disese sttus t bseline Plcebo group (N = 56) NV1FGF group (N = 51) P Age yers, men ± SD 73.3 ± ± 1 49 Mle sex 42 (75.0) 33 (64.7) 45 Dibetes 28 (5) 19 (37.3) Hypertension 48 (85.7) 38 (74.5) Hyperlipidemi 28 (5) 25 (49.0) Smoking sttus Nonsmoker 14 (25.0) 16 (31.4) Ex-smoker 39 (69.6) 25 (49.0) 64 Current smoker 3 (5.4) 10 (19.6) Crotid disese 17 (3) 16 (31.4) Coronry rtery disese 6 (10.7) 11 (21.6) Previous tretment in the treted limb Amputtion 14 (25.0) 10 (19.6) Skin grfting 4 (7.1) 2 (3.9) 72 Angioplsty 22 (39.3) 15 (29.4) 84 Bypss surgery 27 (48.2) 27 (52.9) 25 criticl limb ischemi disese sttus Ulcers Number per ptient (n, min mx) 2.0 (1 5) 1.8 (1 6) Aggregte size cm 2 (men ± SD) 21.8 ± ± Aggregte size (min mx) cm Pin visul nlog scle mm (men ± SEM) 47.4 ± ± nkle brchil index b (men ± SEM) ± 0.3 ± mm Hg (men ± SD) Dorsl surfce of the foot c 16.3 ± ± Upper surfce of the clf d 41.8 ± ± Abbrevition:, trnscutneous oxygen pressure. Dt re presented s percent of ptients, men vlue ± SD, or men (minimum mximum) s pproprite. Pin visul nlog scle: 0 = no pin to 100 = worst pin. Crotid disese ws defined s history of stroke or documented crotid stenosis in medicl records (degree of stenosis 50%); b Ptient numbers for nkle brchil index re plcebo, N = 46; NV1FGF, N = 46; c Ptient numbers for trnscutneous oxygen pressure t the dorsl surfce of the foot re plcebo, N = 54; NV1FGF, N = 50; d Ptient numbers for trnscutneous oxygen pressure t the upper surfce of the clf re plcebo, N = 52; NV1FGF, N = 47. NV1FGF versus plcebo group (HR 35; P = 09) (Figure 2d nd Tble 2). Kpln Meier curves suggested further incresed benefit for ptients with dibetes relting to reduction in the risk of combined mjor mputtion nd deth, but this dditionl benefit ws not sttisticlly significnt (Figure 2e). Hemodynmic prmeters. The hemodynmic prmeters ABI nd toe brchil index (TBI) incresed mrginlly over time s compred to bseline vlues, nd were similr in both groups Tble 2). lso incresed in both NV1FGF nd plcebo groups. Tble 2 Efficcy of NV1FGF versus plcebo in ptients with criticl limb ischemi Plcebo (N = 56) NV1FGF (N = 51) Hzrd rtio P vlue Primry end point Complete heling of t lest one ulcer selected t bseline t week 25 8 (14.3) 10 (19.6) Secondry end points over 52 weeks Amputtion rte All 31 (55.4) 19 (37.3) b Mjor 19 (33.9) 8 (15.7) b Deth rte 13 (23.2) 6 (11.8) b Combined mjor mputtion nd deth rtes 29 (51.8) 14 (27.4) b Hemodynmic prmeters t week 25 Adjusted men chnge from bseline ± SEM nkle brchil index 1 ± 4 5 ± 4 5 Toe brchil index 3 ± 2 4 ± 2 4 t the dorsl surfce of the foot mm Hg (men ± SD) 9.81 ± ± Abbrevition:, trnscutneous oxygen pressure. Mjor mputtion ws defined s through or bove the nkle nd significntly ffecting the functionlity of the limb. Minor mputtion ws defined s below the nkle nd hving little or no effect on the functionlity of the limb. One ptient cn hve more thn one mputtion. Glimmix model; b log-rnk test. Pin. Ischemic rest pin visul nlog scle ws decresed in both NV1FGF nd plcebo groups. At week 52, the djusted men chnge ± SEM from bseline ws ± in the plcebo group versus ± in the NV1FGF group. Adverse events. The sfety popultion included 118 ptients receiving plcebo (n = 61) or NV1FGF (n = 57). The incidence of dverse events is reported in Tble 4. Ptients receiving NV1FGF experienced significntly lower rtes of severe dverse events compred with those receiving plcebo (P = 25). Adverse events tht occurred in 10% of the ptients in either tretment group were: peripherl edem (plcebo 21%, NV1FGF 28%, P = 0.522), pin in extremity (plcebo 15%, NV1FGF 10%, P = 0.586), gngrene (plcebo 13%, NV1FGF 7%, P = 0.365), nemi (plcebo 13%, NV1FGF 5%, P = 07), nd dirrhe (plcebo 5%, NV1FGF 10%, P = 0.311). Tble 4 lso shows the incidences of selected dverse events. There ws trend for crdic events occurring more frequently with the use of NV1FGF thn with plcebo (24.6% versus 11.5%, P = 91), s ws the cse with renl/urinry events s well (15.8% versus 8.2%, P = 59; renl events were minly hemturi, cliniclly not relevnt). The incidence of potentilly ngiogenesis-relted dverse events (neoplsm nd retinopthy) ws similr between groups (cncer nd retinopthy were reported in 3/61 nd 0/61 ptients, respectively, in the plcebo group nd in 3/57 nd 1/57 ptients, respectively, in the NV1FGF group). No impirment of either renl or liver functions ws observed under tretment in the two groups vol. 16 no. 5 my 2008

4 The Americn Society of Gene Therpy NV1FGF Improves Amputtion-free Survivl Survivl frction b Survivl frction Plcebo N = Plcebo N = c Survivl frction d Survivl frction Plcebo N = Plcebo N = e Survivl frction Non-dib-Plcebo N = 28 Non-dib-NV1FGF N = 32 Dib-Plcebo N = Dib-NV1FGF N = Figure 2 Kpln Meier curves for mputtion nd deth (1-yer follow-up). () Kpln Meier curve of time to first mjor mputtion fter bseline in the modified intention-to-tret (MITT) popultion (mximum follow-up 375 dys). (b) Kpln Meier curve of time to first mputtion fter bseline in the MITT popultion (mximum follow-up 375 dys). (c) Kpln Meier curve of time to deth fter bseline in the MITT popultion (mximum follow-up 375 dys). (d) Kpln Meier curve of time to deth or mjor mputtion fter bseline in the MITT popultion (mximum follow-up 375 dys). (e) Kpln Meier curve of time to deth or mjor mputtion fter bseline in the dibetic nd non-dibetic subgroup popultions of the MITT popultion (mximum follow-up 375 dys). Kpln Meier survivl curves stop t the time the lst event occurred. In d, dotted lines represent plcebo-dministered ptients, while solid lines represent NV1FGF-dministered ptients. In e, dotted lines represent nondibetic ptients dministered with plcebo, nd dotted gry lines represent nondibetic ptients dministered with NV1FGF; solid lines represent dibetic ptients dministered with plcebo nd gry solid lines represents dibetic ptients dministered with NV1FGF. Dib, dibetic; non-dib, nondibetic. Discussion This phse II study is the first double-blind, rndomized, plcebo-controlled, multintionl tril using therpeutic ngiogenesis gent in ptients with CLI. It ws designed to explore the effects of the novel plsmid-bsed gene delivery system NV1FGF on recommended 2,7 clinicl (mortlity, mputtion, ulcer heling, pin) nd hemodynmic (ABI, TBI, ) end points. Although prevention of mputtion is gol in the mngement of ptients with CLI, previous clinicl ngiogenesis trils hve not designted mputtion s n efficcy end point. 9 15,17,20 23 In contrst to erlier rndomized trils with ngiogenic gents, the present tril includes multiple dministrtions of the ngiogenic gent. This is mde possible by the nonimmunogenic profile of the drug nd the selection of seriously ill popultion of ptients with CLI. The study led to three min findings. First, the use of NV1FGF did not demonstrte sttisticlly significnt improvement in ulcer heling. Second, the use of NV1FGF significntly reduced the risk of ll nd mjor mputtions. Third, there ws trend (lthough not sttisticlly significnt) towrd decrese in mortlity with the use of NV1FGF. The lck of ny significnt effect on ulcer heling my be explined by the gret heterogeneity nd severity of bseline skin lesions. Eligible ptients hd heterogeneous ulcers of vrious sizes rnging from 9 to 148 cm 2 in the plcebo group, nd from 0.35 to 483 cm 2 in NV1FGF group. Similrly, some ptients hd severe disese with fully necrotic distl tissue tht would hve been impossible to hel, even with incresed blood flow. Moreover, wound cre, which vries widely in clinicl prctice, ws not stndrdized cross investigtionl sites. Most importnt, significntly higher mputtion rte in Moleculr Therpy vol. 16 no. 5 my

5 NV1FGF Improves Amputtion-free Survivl The Americn Society of Gene Therpy Tble 3 Multivrite nlysis of predictors of the risk of first mjor mputtion (Cox nlysis) N (%) Hzrd rtio (95% CI) P vlue Tretment (N ) Plcebo (N = 46) 15 (32.6) NV1FGF (N = 45) 7 (15.6) 0.35 (0.13, 0.94) 36 Amputtion s previous peripherl rteril disese tretment No mputtion (N = 72) 17 (23.6) Amputtion (N = 19) 5 (.3) 0.92 (0.31, 2.77) 89 Dibetic sttus Nondibetic (N = 54) 13 (24.1) Dibetic (N = 37) 9 (24.3) 1.75 (6, 4.59) 57 Smoking sttus Nonsmokers (N = ) 2 (7.7) Current nd 20 (3) 6.29 (1.41, 27.95) 16 ex-smokers (N = 65) Hypertension No hypertension (N = 16) 5 (31.2) Hypertension (N = 75) 17 (22.7) 8 (0.14, 1.62) 34 High risk of mputtion No high risk of 9 (23.7) mputtion (N = 38) High risk of mputtion 13 (24.5) 1.49 (0.55, 3.99) 24 (N = 53) nkle brchil index (N = 41) 3 (7.3) < (N = 50) 19 (38.0) (2.96, 37.46) <01 dorsl surfce of the foot (mm Hg) b 30 (N = 17) 1 (5.9) 20 nd <30 (N = 14) 2 (14.3) 10 nd <20 (N = 21) 5 (23.8) 1.77 (5, 2.99) 32 <10 (N = 39) 14 (35.9) Abbrevitions: ABI, nkle brchil index; CI, confidence intervl;, trnscutneous oxygen pressure. Multivrite nlysis ws performed including tretment lloction, dibetic sttus, smoking sttus, hypertension, prior mputtion, initil risk of mputtion, ABI, nd. N corresponds to the totl number of study ptients with vilble dt on risk fctors; b risk is given for n increse of 1 unit. the plcebo group influenced the sttisticl nlysis, in tht ulcers tht incresed in size were more often censored by mputtion in the plcebo rm. Finlly, this tril ws insufficiently powered to demonstrte significnt difference in complete wound-heling between tretment groups, given the smple size. In this study, NV1FGF significntly reduced the risk of ll nd mjor mputtions, nd there ws no sttisticlly significnt trend towrd decrese in mortlity. Becuse mjor mputtion is ssocited with high peri-opertive mortlity, the reduction in mputtions in the NV1FGF rm my hve hd n impct on mortlity rtes in the tril. A similr benefit ws observed for the popultion of Tble 4 Adverse events of NV1FGF versus plcebo in ptients with criticl limb ischemi in the sfety popultion (n = 118) Plcebo (N = 61) NV1FGF (N = 57) P All 56 (91.8) 53 (93.0) 00 Serious 49 (80.3) 40 (7) 85 Severe 42 (68.9) 27 (47.4) 25 Possibly relted to study drug 13 (21.3) 15 (.3) 66 Leding to study discontinution 10 (16.4) 4 (7.0) Selected Crdic 7 (11.5) 14 (24.6) 91 Renl nd urinry b 5 (8.2) 9 (15.8) 59 Neoplsm (ll) 5 (8.2) 5 (8.8) 00 Mlignnt neoplsm 3 3 Retinopthy 0 1 (1.8) 83 The sfety popultion (n = 118) consists of ptients who hve received t lest one injection of the study drug or plcebo; b The mjority of renl/urinry disorders represented hemturi. ptients with dibetes, in respect of ll nd mjor mputtions. The tril missed its primry efficcy end point, but significntly reduced risk for ll nd mjor mputtions, llowing for the preservtion of functionl limbs in the NV1FGF group is cliniclly extremely relevnt to the mngement of ptients with CLI. 6,7 Interestingly, the benefit relting to the mputtion rte observed with ngiogenic therpy in the present study ws not ssocited with benefit in respect of hemodynmic end points (ABI, TBI), nd no significnt differences in these were observed between the two groups. These dt indicte tht ABI nd TBI, s well s ulcer heling nd ulcer size, my not be useful mrkers of NV1FGF efficcy in ptients with CLI, in view of the hypothesis tht the benefit of NV1FGF my be primrily becuse of the effects t the microvsculr level (FGF-1 hs been shown to led to rteriole formtion nd mturtion 27 ). However, further studies in experimentl models re needed to explin the mechnisms underlying the observed improvements in ptient outcomes. Both plcebo- nd NV1FGF-treted ptients experienced high rte of dverse events (including crdiovsculr events), reflecting the severity of the underlying disese in this tril. The ptients rteril disese hd led to tissue necrosis (ischemic ulcer or gngrene), nd most of those enrolled hd been expected to require mjor mputtion within the subsequent 6 months to 1 yer in the bsence of revsculriztion to improve blood flow. In ddition, peripherl rteril disese is often ssocited with significnt comorbidities such s coronry rtery disese, cerebrl rtery disese, dibetes, nd renl impirment. Importntly, the incidence of dverse events (including potentilly ngiogenesisrelted dverse events such s cncer nd prolifertive retinopthy) ws no higher with the use of NV1FGF tretment thn with the use of plcebo. The rte of occurrence of dverse events tht were serious or severe enough to result in discontinution of the study ws significntly lower in the NV1FGF group thn in the plcebo group, nd this my reflect the efficcy of NV1FGF. These dt lso confirm the finding from erlier studies tht NV1FGF is well tolerted vol. 16 no. 5 my 2008

6 The Americn Society of Gene Therpy NV1FGF Improves Amputtion-free Survivl Previous trils of therpeutic ngiogenesis hve investigted ngiogenic recombinnt protein, or gene therpy, with FGFs, vsculr endothelil growth fctors, nd heptocyte growth fctor. 9 15,17,20 22 These gents hve been generlly well tolerted, nd the results of some studies hve suggested therpeutic benefit. In smll open-lbel tril in ptients with CLI, intrmusculr dministrtion of nked plsmid DNA encoding humn vsculr endothelil growth fctor significntly improved ABI nd heled or improved ulcers in comprison with bseline dt. 11 An lterntive method of implnting bone mrrow derived or peripherl blood mononucler cells in the legs of ptients with CLI hs lso been investigted nd shown evidence of benefit. 23,28 The discrepncy between the positive result obtined in this study nd the negtive results in erlier studies of protein or gene therpy my be explined by the fct tht the use of NV1FGF enbles re-dministrtion of pcor plsmid bsed therpy. Multiple dministrtions of NV1FGF my llow sustined locl exposure to the expressed FGF-1 t the site of dministrtion compred with the more trnsient exposure obtined from single dministrtion. These dt indicte tht NV1FGF therpy my offer the potentil for effective mngement of CLI, nd hve provided the bsis for initition of the phse III study TAMARIS (Therpeutic Angiogenesis for the Mngement of Arteriopthy in Rndomized Interntionl Study) designed to ssess world-wide, in 490 similr ptients, the efficcy of NV1FGF versus plcebo with the combined end point, mputtion or deth, s the primry end point of the study. Mterils And Methods The study ws designed by the sponsor (Centelion SAS, wholly owned subsidiry of Snofi-Aventis SA, Pris, Frnce) in collbortion with experts in the field of peripherl rteril disese. The sponsor collected the dt nd monitored the conduct of the study. After dtbse lock, sttisticl nlyses were conducted by MDS Phrm Services (Lyon, Frnce). The sponsor coordinted writing of the mnuscript with writing committee comprising the first nine cdemic uthors nd sponsor uthors. The writing committee hd full ccess to study reports nd ws ctively involved in dt nlysis nd interprettion. All the uthors pproved the finl mnuscript. Study design. This ws double-blind, rndomized, plcebo-controlled, prllel-group, multicenter, multintionl phse II study, involving 37 investigtionl sites in six Europen countries (Belgium, Frnce, Germny, Itly, Switzerlnd, nd the UK). The protocol ws pproved by the institutionl review bord t ech center, nd the study ws conducted in ccordnce with good clinicl prctices, the Declrtion of Helsinki, nd relevnt locl regultions. All ptients provided written informed consent. A totl of 112 ptients were to be rndomized, 56 into ech group, with the im of verifying the ssumption tht the success rte for the complete heling of t lest one ulcer of the treted leg, evluted in the MITT popultion, would be 30% with NV1FGF versus 8% with plcebo t week 25. The study would hve sufficient power (80%) to detect improvement by 22% (15 NV1FGF successes versus 5 plcebo successes) with totl of 50 ptients vilble for evlution per tretment group. Ptients. The study enrolled men nd women ptients ged 45 yers with CLI who presented with nonheling ulcer(s), nd in whom revsculriztion ws not considered suitble option. CLI ws defined ccording to the TrnsAtlntic Inter-Society Consensus document. 2 Ptients were required to show objective evidence of CLI on the bsis of both totl rteril occlusion (by ngiogrphy or Doppler) nd pressure criteri (resting nkle pressure 70 mm Hg nd/or toe pressure 50 mm Hg, nd/or 20 mm Hg nd/or mettrsl pulse volume recording brely pulstile). Signs of heling of the trophic lesions (reduction in ulcer size or depth) were required to be bsent for 2 weeks before the first dministrtion of the study drug. All the prticipnts were found unsuitble for revsculriztion for one or more of the following resons: (i) there ws poor or no utologous grft mteril; (ii) revsculriztion would result in incomplete perfusion of the foot (bsence of distl runoff); (iii) there ws high risk of filure for technicl resons; (iv) there ws sfety risk ssocited with the revsculriztion procedure; (v) there ws high risk of mputtion on ccount of conditions such s gngrene. Rndomiztion nd tretment pln. The ptients were screened for eligibility 2 8 weeks before the dministrtion of the study drugs. Blocks of study mediction for four ptients ech were llocted to ech investigtionl site. The eligible ptients were rndomized in the order enrolled, between April 2002 nd April 2004, using permuted-block rndomiztion with block size of four. Ptients prticipted in the tril on n outptient bsis. On ech of dys 1, 15, 30, nd 45, eight intrmusculr injections were dministered in single leg (four in the clf nd four in the thigh), ech injection contining either 2.5 ml NV1FGF t mg/ml (0.5 mg NV1FGF per injection; 4 mg per dministrtion) or plcebo sline solution. If CLI ffected both of the ptient s legs, the leg estimted to benefit most from the tretment, bsed on lower hemodynmic prmeters (including ABI, TBI, nd ) ws selected. Becuse of the diffuse nture of CLI-relted therosclerotic lesions, the sites of injection differed t ech dministrtion, nd were selected ccording to vilble muscle mss while voiding ulcer loctions, nd t distnce from n rtery or min nerve ccording to the investigtor s discretion. Following the 6-week double-blind tretment period, the follow up of ptients continued up to week 52, with ssessments t week 13 (±5 dys), week 25 (±10 dys), week 38 (±10 dys), nd week 52 (±10 dys). On clinic visit dys during the period of dministrtion of the study drug, ptients received their study drug or plcebo fter ll the ssessments hd been collected. A pnel of experts, blinded s to study group, ssigned ptients to the MITT popultion, which included those who (i) hd received t lest two tretment injections (eight injections ech) of study drug, (ii) hd undergone n evlution for ggregte ulcer size t bseline nd hd t lest one nonheling ulcer, nd (iii) hd undergone n evlution for ggregte ulcer size t or fter week 5. All ptients who hd received t lest one tretment injection were included in the sfety popultion. End points. The prespecified primry end point in this tril ws the incidence of complete heling of t lest one ulcer in the treted limb t week 25. Ulcer ssessments were performed t ech visit by clinicl ssessment (type nd chrcteristic of ulcers) nd size ssessment. A review pnel, blinded s to intergroup lloction of the ptients, comprising three investigtors nd one wound-heling expert, ssessed ulcers s heled or not heled. The pnel reconciled discordnce between the investigtor s ssessments nd the vilble dt. Predetermined secondry end points included mjor nd minor mputtion ( rte nd time to ). Mjor mputtion ws defined s through or bove the nkle. Minor mputtion ws defined s below the nkle. Other prespecified secondry ssessment vribles were time to deth, nd hemodynmic prmeters. At ech visit, ptients ssessed the ischemic rest pin they hd experienced during the previous 7 dys on subjective 100-mm visul nlog scle: 0 mm = no pin to 100 mm = pin s bd s it could be. Sfety. Sfety ssessments included dverse event reporting, physicl exmintions, vitl signs, lbortory tests, ophthlmologic exmintion, chest x-ry, nd mmmogrphy. An independent Dt nd Sfety Monitoring Bord ws responsible for the continuous independent sfety monitoring of this tril. Moleculr Therpy vol. 16 no. 5 my

7 NV1FGF Improves Amputtion-free Survivl The Americn Society of Gene Therpy Sttisticl nlysis. All efficcy nd sfety nlyses were performed in the MITT nd sfety popultions, respectively. For both, bseline demogrphic nd sfety dt, comprisons between the NV1FGF nd plcebo groups were performed, using Student s t-test (the Mnn Whitney rnksum test for skewed vribles) or χ 2 nlysis when pproprite. The primry end point ws compred in the NV1FGF nd plcebo groups, using generlized liner mixed model nd the lst-observtioncrried-forwrd method. 29 The min prmeters of the model were tretment gent, bseline ggregte ulcer size nd number, dibetes sttus, smoking sttus, nd country. Time to event (mputtion, deth, or combintion) ws compred by Kpln Meier nlysis nd ssocited log-rnk test with censoring t dy 375. HRs were derived using the Cox model. The robustness of the results ws ssessed by post-hoc sensitivity nlysis involving the potentil influence of disese prognostic fctors in Cox nlyses. Sttisticl significnce ws defined s P < 5. Acknowledgment This work ws supported by Centelion SAS, Vitry sur Seine, Frnce. Supplementry Mteril Supplementry Dt S1. Acknowledgments nd dditionl study mteril. References 1. Novo, S, Coppol, G nd Milio, G (2004). Criticl limb ischemi: definition nd nturl history. Curr Drug Trgets Crdiovsc Hemtol Disord 4: Dormndy, JA nd Rutherford, RB (2000). Mngement of peripherl rteril disese (PAD). TASC Working Group. TrnsAtlntic Inter-Society Concensus (TASC). J Vsc Surg 31: S1 S The I.C.A.I. Group (Gruppo di Studio dell Ischemi Cronic Critic degli Arti Inferiori) (1997). Long-term mortlity nd its predictors in ptients with criticl leg ischemi. The Study Group of Criticil Chronic Ischemi of the Lower Exremities. Eur J Vsc Endovsc Surg 14: Cmpbell, WB, St. Johnston, JA, Kernick, VF nd Rutter, EA (1994). Lower limb mputtion: striking the blnce. Ann R Coll Surg Engl 76: Bertelé, V, Roncglioni, MC, Pngrzzi, J, Terzin, E nd Tognoni, EG (1999). 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