Basic Fibroblast Growth Factor in Patients with Intermittent Claudication: Results of a Phase I Trial Daisy F

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Journal of the American College of Cardiology Vol. 36, No. 4, 2000 © 2000 by the American College of Cardiology ISSN 0735-1097/00/$20.00 Published by Elsevier Science Inc. PII S0735-1097(00)00882-2 Gene Therapy Basic Fibroblast Growth Factor in Patients With Intermittent Claudication: Results of a Phase I Trial Daisy F. Lazarous, MD,* Ellis F. Unger, MD,* Stephen E. Epstein, MD, FACC,* Annette Stine, RN,* Joseﬁno L. Arevalo, BSN, RN,* Emily Y. Chew, MD,† Arshed A. Quyyumi, MD, FACC* Bethesda, Maryland

OBJECTIVES This phase I study was designed to evaluate the safety, tolerability and pharmacokinetics of intra-arterial basic fibroblast growth factor (bFGF) in patients with atherosclerotic peripheral arterial disease (PVD) and intermittent claudication. We also assessed the effects of basic fibroblast growth factor (bFGF) on calf blood flow as a measure of biologic activity. BACKGROUND Preclinical studies have shown that bFGF, an angiogenic peptide, promotes collateral development in animal models of myocardial and hind limb ischemia. The safety and efficacy of bFGF in patients is unknown, and early clinical trials are underway in coronary and peripheral arterial disease. METHODS A double-blind, placebo-controlled, dose-escalation trial was conducted in patients with claudication demonstrating ankle/brachial index Ͻ0.8. Patients were randomly assigned to placebo (n ϭ 6), 10 ␮g/kg of bFGF (n ϭ 4), 30 ␮g/kg of bFGF once (n ϭ 5) and 30 ␮g/kg of bFGF on two consecutive days (n ϭ 4). Study drug was infused into the femoral artery of the ischemic leg. Detailed safety information including retinal photography for neovascularization were obtained through one year. Calf blood flow was measured with strain gauge plethysmography in the two higher dose treatment groups and in four placebo patients at baseline, one month and three to seven months after treatment. RESULTS Intra-arterial bFGF was safe and well-tolerated. The half-life was 46 Ϯ 21 min. Calf blood flow increased at one month by 66 Ϯ 26% (mean Ϯ SEM) and at six months by 153 Ϯ 51% in bFGF-treated patients (n ϭ 9, p ϭ 0.002). Flow did not change significantly in the placebo group. CONCLUSIONS In this initial randomized, double-blind, placebo-controlled trial in patients with atherosclerotic PVD and claudication, bFGF was well-tolerated. The data suggest a salutary biologic effect, and initiation of phase 2 trials is warranted. (J Am Coll Cardiol 2000;36:1239–44) © 2000 by the American College of Cardiology

Peripheral arterial disease (PVD) is a significant public assessed the effects of bFGF on calf blood flow as a measure health problem (1) for which pharmacologic therapies have of biologic activity. not been consistently effective (2). In older adults, a strong association exists between intermittent claudication and METHODS subsequent mortality and cardiovascular morbidity (3–5). Recently, significant research has focused on developing Patient population. Selection criteria included age Ͼ40 angiogenic therapies to provide novel approaches to the years, a history of intermittent claudication for Ͼ6 months treatment of myocardial and limb ischemia. We and other and ankle brachial index (ABI) Ͻ0.8 at rest or after 5 min of standardized exercise (13) (2 miles per h at a grade of See page 1245 10°). Exclusion criteria included gangrene, osteomyelitis, vas- investigators have demonstrated the potential of basic fibro- cular interventions within three months of enrollment, Ͼ blast growth factor (bFGF) to improve collateral develop- serum creatinine 2.0 mg/dL, significant proteinuria, pre- ment in animal models of myocardial (6–9) and hind limb existing or suspected malignancy and severe nonproliferative (10–12) ischemia. On the basis of these findings, we or proliferative diabetic retinopathy. initiated a phase I trial to evaluate the safety, tolerability and The study was performed at the National Heart, Lung, pharmacokinetics of intra-arterial bFGF in patients with and Blood Institute, Bethesda, Maryland, and was approved atherosclerotic PVD and intermittent claudication. We also by the Institutional Review Board. An independent Data Safety Monitoring Board monitored the safety of the drug.

From the *Cardiology Branch, National Heart, Lung and Blood Institute; and Nineteen patients met the total study criteria and were †National Eye Institute, National Institutes of Health, Bethesda, Maryland. This randomized after giving written informed consent. study was conducted at NHLBI, NIH, and did not receive any other ﬁnancial Study protocol. At enrollment, a medical history and support. Manuscript received November 24, 1999; revised manuscript received March 15, physical examination were conducted. Clinical laboratory 2000, accepted June 13, 2000. studies were performed at screening (complete blood count, 1240 Lazarous et al. JACC Vol. 36, No. 4, 2000 bFGF in Peripheral Arterial Disease October 2000:1239–44

Table 1. Characteristics of bFGF Treated and Placebo Patients: Abbreviations and Acronyms Baseline Demographics and Disease Status ABI ϭ ankle/brachial index or systolic blood pressure Parameters bFGF-treated Placebo ratio bFGF ϭ basic fibroblast growth factor Number of subjects 13 6 Ϯ Ϯ PVD ϭ peripheral arterial disease Age, years 68 10 77 9 VEGF ϭ vascular endothelial growth factor Men 6 4 Diabetes 2 3 Smokers 5 1 Hypertension 6 5 blood chemical analyses, urinalysis, 24-h urine for total Previous vascular surgery 4 0 protein and creatinine clearance) and repeated three days Previous lower limb PTA 4 3 and one, two and four weeks after treatment. Screening tests H/o CAD/CABG 4 2 for occult malignancy included chest X-ray, prostate specific Cholesterol (mg/dL) 215 Ϯ 55 181 Ϯ 19 Ϯ Ϯ antigen in men, mammogram, pelvic examination/pap Baseline ABI 0.60 0.28 0.71 0.34 smear in women and stool for occult blood. Comprehensive Values are means Ϯ SD. ABI ϭ ankle/brachial systolic blood pressure ratio; bFGF ϭ basic fibroblast ophthalmologic examination, including retinal photography growth factor; CABG ϭ coronary artery bypass grafting; CAD ϭ coronary artery of the seven standard stereoscopic fields, was performed at disease; H/o ϭ history of; PTA ϭ percutaneous transluminal angioplasty. baseline, one, three and 12 months. Patients answered a To convert values for cholesterol to mmol/L, multiply by 0.0259. questionnaire with a review of systems, which included bFGF groups using strain gauge plethysmography (Hokan- grading the degree of claudication as being the same, better son, Issaquah, Washington), as previously described (14). or worse than usual. Exercise testing for measurement of Measurements were made immediately before treatment, at ABI was performed using a treadmill speed of 2 miles per h 1 month and at 3 to 7 months (median 6 months) after at a 10° grade. Patients were required to be on a stable treatment by an observer blinded to group assignment. medication regimen six weeks before entry into the study Statistical analysis. Data are expressed as mean Ϯ SEM and during the follow-up period. unless stated otherwise. For the analysis of pre- versus Basic fibroblast growth factor was provided by Scios, Inc., posttreatment differences, Friedman repeated measures Mountainview, California. Nineteen subjects were ran- analysis of variance on ranks was used. A p value Ͻ0.05 was ␮g/kg injected once, 30 ␮g/kg domly assigned to bFGF, 10 considered to indicate statistical significance. Correction for injected once or 30 ␮g/kg injected twice (on consecutive pairwise multiple comparisons was made using the Student- days). Randomization was in blocks of six, with two and Newman-Keuls method. four subjects assigned to placebo and bFGF, respectively. ϫ One patient randomized to bFGF 2 doses did not receive RESULTS the second dose, because of thrombocytopenia (decrease in platelet count from 150,000 to 120,000), and follow-up data Patients. Baseline patient characteristics are summarized in from this patient are included with the 30 ␮g/kg ϫ1 group. Table 1. There were a number of imbalances in baseline Patients were hydrated with 500 mL of normal saline characteristics, typical of a study of limited sample size. before study drug administration, and hydration was main- Safety. The first subject randomized to bFGF received a tained at 60 to 75 mL per hour for 4 h after drug infusion. 10 ␮g/kg bolus over 2 min. Systolic blood pressure de- The femoral artery of the more ischemic leg, as determined creased by 22% at 1 min and by 47% at 20 min, with onset by ABI, was cannulated percutaneously with an 18 gauge of recovery at 25 min and return to baseline by 60 min, catheter (Arrow International, Inc. Reading, Pennsylvania), aided by infusion of saline. The patient remained asymp- and the study agent was administered by intra-arterial tomatic. For subsequent subjects, the protocol was modified infusion over 15 min. Heparin was not used. Hemodynamic to deliver the study agent using an infusion pump over 4 min monitoring was performed for 30 min after infusion. All in the 10 ␮g/kg group and over 15 min in the 30 ␮g/kg group, patients underwent continuous Holter monitoring from the and significant hypotension was not observed. time of initial drug infusion to 36 h after the second Retinal neovascularization was not detected during the treatment. course of the study. Mild transient proteinuria occurred in Venous blood samples were assayed for human bFGF at both placebo and bFGF-treated subjects, but there were no 0, 1, 3, 5, 10, 20, 30, 60, 120 and 180 min using a significant differences between groups in any hematological solid-phase ELISA kit (catalog No.DFB00, R&D Systems, or biochemical tests. One patient age 74 with borderline Inc., Minneapolis, Minnesota) by Burleson Research Tech- thrombocytopenia ranging between 145,000 and 150,000/ nologies, Inc., Raleigh, North Carolina. mm3 developed a platelet count of 124,000/mm3 24 h after Measurement of calf blood flow. After completion of the treatment with one dose 30 ␮g/kg of bFGF, with recovery 1 month safety follow-up in the first block of patients to baseline in five days. Atrial fibrillation also occurred in (10 ␮g/kg bFGF), we amended the study protocol to assess this patient, who had no prior history of arrhythmia, 72 h possible effects of bFGF on lower extremity blood flow. after receiving 30 ␮g/kg of bFGF. Spontaneous conversion These measurements were carried out in the two high-dose to sinus rhythm occurred 12 h later. JACC Vol. 36, No. 4, 2000 Lazarous et al. 1241 October 2000:1239–44 bFGF in Peripheral Arterial Disease

Table 2. Pharmakokinetics of bFGF ␮g/kg ؋ 2 30 bFGF Dose/Regimen 10 ␮g/kg 30 ␮g/kg First Dose Second Dose baseline (bFGF) (pg/mL) 32 Ϯ 19 78 Ϯ 39 Ͻ10 151 Ϯ 16 peak (bFGF) (ng/mL) 29.1 Ϯ 2.0 57.4 Ϯ 9.5 47.2 Ϯ 9.3 61.5 Ϯ 20.6 AUC (ng* min/mL) 634 Ϯ 104 1325 Ϯ 594 3560 Ϯ 511 t 1⁄2 (min) 32 Ϯ 342Ϯ 973Ϯ 23

Serum levels of bFGF were quantified by ELISA at 1, 3, 5, 10, 20, 30, 60, 120 and 180 min after infusion. Serum bFGF levels peaked at 1 min after infusion. Basic fibroblast growth factor had a rapid distribution phase with a volume of distribution of 0.66 Ϯ 0.25 L/kg and an elimination half-life of 46 Ϯ 21 min. AUC ϭ the area under the concentration-time curve; bFGF ϭ basic fibroblast growth factor.

Pharmacokinetics of bFGF. Serum bFGF levels peaked at The mean percent change in calf blood flow and vascular 1 min after infusion. Basic fibroblast growth factor had a resistance measured at one and six months in bFGF-treated rapid distribution phase with a volume of distribution of patients is shown in Figure 3. Combining the one- and 0.66 Ϯ 0.25 L/kg and an elimination half-life of 46 Ϯ two-dose groups, the nine bFGF-treated patients had a 21 min. In patients receiving two doses, baseline serum 66 Ϯ 26% increase in calf blood flow at one month and a levels of bFGF were higher before the second dose of bFGF further significant increase of 153 Ϯ 51% at six months (p Ͻ compared with day 1 (Table 2). The area of distribution 0.05, one vs. six months); vascular resistance decreased by under the curve demonstrated a dose response relationship 39 Ϯ 7% and 54 Ϯ 9% at one and six months (p Ͻ 0.0001). (Fig. 1). Symptoms. Of the four placebo-treated patients, one re- Calf blood flow. PLACEBO PATIENTS. The absolute ported marked improvement in claudication and walking changes in flow are shown in Table 3. One placebo patient distances at one month (blood flow in the treated leg had marked improvement in flow at 1 month (Fig. 2), with improved as well, Fig. 2), with worsening to baseline at decrease below baseline at seven months (1.93, 3.43 and seven months. Three of the five patients in the one-dose 1.02 mL/min/dL, respectively). A second placebo patient, bFGF group, and three of the four patients in the two-dose who was hypertensive and diabetic, had a decrease in systolic bFGF group, reported marked symptomatic improvement blood pressure by 23 mm Hg between the first and second at one month. No patient reported worsening of symptoms. flow measurements and changes in medications that were made for clinical reasons before the final measurement. This patient’s flow at baseline, one month and five months was DISCUSSION 0.43, 2.05 and 1.54 mL/min/dL. Despite these heteroge- Previous studies of angiogenic factors and significance of nous responses at 1 month, calf blood flow in the placebo this study. There is a significant body of animal data group did not change significantly between baseline and six ϭ demonstrating proof of concept of salutary effect of peptide months (p 0.27). growth factors in hind limb and myocardial ischemia, and BFGF-TREATED PATIENTS. Resting calf blood flow im- multiple phase 1 and early phase II trials are in progress. proved significantly in bFGF-treated patients (Table 3). Recent phase 1 studies have suggested tolerability of vascular endothelial growth factor (VEGF) gene transfer (15– 17), intracoronary VEGF protein (18), intramyocardial acidic FGF protein (19) and intracoronary (20) and perivascular (21) bFGF. Gene transfer of plasmid DNA encoding VEGF has shown favorable results in patients with critical limb ischemia in an uncontrolled open-label study (22). To our knowledge, this is the first report of a randomized, double-blind, placebo-controlled study of bFGF administration in humans with peripheral vascular disease. We have shown that intra-arterial bFGF is well-tolerated and free of significant adverse effects in subjects with intermittent claudication. Pharmacokinetics of bFGF. We found that the acute hypotensive effects of bFGF can be mitigated by slowing the rate of infusion. The serum half-life was 46 min, in keeping with our preclinical studies (7). Basic fibroblast growth factor had a small volume of distribution, indicating that it Figure 1. Area under the concentration-time curve or measure of total exposure to basic fibroblast growth factor demonstrated a dose-response is not extensively tissue-bound. However, in the group relationship. receiving two doses, the level of bFGF was higher before the 1242 Lazarous et al. JACC Vol. 36, No. 4, 2000 bFGF in Peripheral Arterial Disease October 2000:1239–44

Table 3. Resting Calf Blood Flow (ml/min/dl) p (Baseline vs. 6 Baseline 1 Month 6 Months Months) Placebo 1.29 Ϯ 0.33 2.15 Ϯ 0.44 1.35 Ϯ 0.17 0.27 30 ␮g/kg ϫ 1 1.11 Ϯ 0.21 1.44 Ϯ 0.23 2.09 Ϯ 0.55 0.046 30 ␮g/kg ϫ 2 0.60 Ϯ 0.10 1.10 Ϯ 0.09 1.91 Ϯ 0.06 Ͻ0.001

Flow improved significantly between 1 and 6 months in bFGF-treated patients. bFGF ϭ basic fibroblast growth factor. second dose compared with baseline, suggesting the pres- Calf blood flow changes with bFGF. This was a phase I ence of a reservoir for bFGF, likely the vascular subendo- study not primarily designed to assess efficacy. Calf blood thelial matrix (23), with slow release of the peptide into the flow was measured as an exploratory secondary end point. circulation over time. We found that one and two intra-arterial doses of bFGF at Potential toxicity of bFGF. Basic fibroblast growth factor 30 ␮g/kg improved calf blood flow in the treated leg. This has the potential to induce a nephropathy with proteinuria was accompanied by subjective improvement in symptoms at higher doses or with repeated administration over time in a majority of treated patients, consistent with a biologic (24). We were able to show that two doses of 30 ␮g/kg of effect. There appears to be a dose-response relationship bFGF did not have appreciable effects on proteinuria or between the dose of bFGF and calf blood flow. However, serum creatinine levels in our patients. Although diabetics the differences between the two doses were not statistically were included, larger numbers of patients are needed before significant, a fact probably due to the small numbers of a deleterious effect on renal function can be safely ruled out patients studied. Calf blood flow in the treated patients in diabetics and in patients with proteinuria exceeding 200 continued to improve three to seven months after treatment, mg/24 h. Endothelial growth factors have been implicated suggesting that activities of daily living may have provided as mediators of intraocular neovascularization (25,26). In an ischemic milieu that sustained and improved the angio- our study of patients with normal retinae at baseline and in genic effect of bFGF with time. The acute vasodilatory individuals with mild to moderate nonproliferative retinop- effects of bFGF could have contributed to collateral devel- athy (three patients enrolled in the study had nonprolifera- opment (27); however, other vasodilators have failed to tive retinopathy), bFGF did not induce new vessel forma- produce consistent effects on collateral growth (28,29). tion. The effects of bFGF on hypoxic or ischemic retinae or These results on calf blood flow are preliminary and cannot on diabetic proliferative retinopathy remain unknown. be considered definitive; however, the results are clinically Thrombocytopenia that occurred in a bFGF-treated patient encouraging and warrant further study. was transient and asymptomatic, consistent with preclinical Placebo component in angiogenic trials. A large placebo safety data with bFGF (7,8). response is common in claudication trials. In a review of 75

Figure 2. Resting calf blood flow in all placebo and bFGF-treated patients at baseline and 1 month. The greatest improvement in calf blood flow occurred in a placebo patient. bFGF ϭ basic fibroblast growth factor. JACC Vol. 36, No. 4, 2000 Lazarous et al. 1243 October 2000:1239–44 bFGF in Peripheral Arterial Disease

Reprint requests and correspondence: Dr. Daisy F. Lazarous, The Johns Hopkins University School of Medicine, Johns Hop- kins Bayview Medical Center, Division of Cardiology, A1 East, 4940 Eastern Avenue, Baltimore, Maryland 21224. E-mail: [email protected].

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