- Email: [email protected]
Phase I study of direct administration of a replication deficient adenovirus vector containing the vascular endothelial growth factor cDNA (CI-1023) to patients with claudication
Phase I Study of Direct Administration of a Replication Deficient Adenovirus Vector Containing the Vascular Endothelial Growth Factor cDNA (CI-1023) to Patients With Claudication Sanjay Rajagopalan, MD, Jeffrey Trachtenberg, MD, Emile Mohler, MD, Jeffrey Olin, DO, Scott McBride, MA, Raphael Pak, PhD, Henrik Rasmussen, MD, and Ronald Crystal, MD The long-term safety and efficacy of adenoviral delivery of growth factors in patients with peripheral arterial disease (PAD) is unknown. CI-1023 (AdGVVEGF121.10) is a replication-deficient adenovirus encoding human vascular endothelial growth factor isoform 121. In this phase I trial, we investigated the safety and efficacy of CI-1023 in subjects with advanced claudication symptoms secondary to infra-inguinal disease. Eighteen subjects >35 years of age with a median ankle brachial index (ABI) at rest of 0.525 (interquartile range 0.4) and angiographic disease involving the infra-inguinal vessels underwent intramuscular injection of CI-1023 (4 ⴛ108 to 4 ⴛ1010 particle units, n ⴝ 15) or placebo (n ⴝ
3). Eleven of 15 patients (73%) who received CI-1023 and 1 of 3 subjects (33%) who received placebo, completed 1 year of follow-up. Edema and rash were the most common early adverse event. One infra-inguinal bypass procedure occurred in each of the placebo and CI-1023 groups at days 29 and 104, respectively. One death (day 160) and 1 malignancy (day 274) occurred in the CI-1023 group. Conclusions on efficacy could not be made due to the small number of patients. However, there were encouraging trends in ABI at rest and peak walking time at follow-up. 䊚2002 by Excerpta Medica, Inc. (Am J Cardiol 2002;90:512–516)
he lack of truly effective medical therapies for the treatment of patients with advanced peripheral arT terial disease (PAD), along with the ease of delivering
trolled angiogenesis in nontarget organs. Moreover, recently performed clinical trials with recombinant VEGF and fibroblast growth factor-2 in patients with coronary artery disease have not demonstrated benefit.6,7 An alternate approach for regional angiogenesis using a plasmid-based approach to deliver VEGF was evaluated in a small number of patients with pain at rest and tissue necrosis, demonstrating apparent improvements in symptoms and limb salvage.8,9 Because plasmid-based systems have inherent inefficiencies, alternate vector systems (e.g., adenovirus) that afford sustained expression of the transgene represent an attractive option. CI-1023 (AdGVVEGF121.10) is a recombinant adenovirus vector based on Ad 5 genome deleted for all of E1a, most of E1b, and the E3 sequences, rendering it replication deficient, where the deleted sequences have been replaced by the gene encoding for human VEGF isoform 121. In this multicenter phase I trial, we investigated the safety and effects of CI-1023 in patients with advanced claudication secondary to infra-inguinal disease.
agents to the lower extremities, make this condition suited for proof of concept studies in therapeutic angiogenesis.1 The use of angiogenic growth factors, which enhance or aid in the development of collaterals, represents one approach to improve symptoms of claudication in patients with intractable symptoms and disability. Growth factors, such as vascular endothelial growth factor (VEGF), delivered as protein or gene therapy are therefore being investigated as a treatment for PAD. A number of animal studies have demonstrated that the administration of VEGF protein in the setting of ischemia is capable of inducing the development of networks of new blood vessels in vivo.2– 4 However, high doses of VEGF administered as protein have been associated with hypotension,5 in addition to carrying the theoretical risk of unconFrom the University of Michigan, Ann Arbor, Michigan; University of Pittsburgh, Pittsburgh, Pennsylvania; Heart and Vascular Institute, Morristown, New Jersey; University of Pennsylvania, Philadelphia, Pennsylvania; Pfizer Global Research and Development, Ann Arbor, Michigan; GenVec Inc., Gaithersburg, Maryland; and New York Presbyterian Hospital-Weil Medical College, Cornell University, New York, New York. Manuscript received February 20, 2002; revised manuscript received and accepted May 1, 2002. Address for reprints: Sanjay Rajagopalan, MD, Section of Vascular Medicine, Division of Cardiology, University of Michigan, L3119 Women’s Hospital, 1500 E. Medical Center Dr., Ann Arbor, Michigan 48109-0273. E-mail: [email protected].
512
©2002 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 90 September 1, 2002
METHODS
Study design: The study was initially designed as a double-blind, placebo-controlled, dose-escalating study that recruited patients with disabling intermittent claudication and patients with pain at rest and limb threat. Only the results on the claudication patients are reported here. The safety results in patients 0002-9149/02/$–see front matter PII S0002-9149(02)02424-9
with pain at rest have been reported previously.10 The total doses in each group ranged from 4 ⫻108 to 4 ⫻1010 particle units (pu) proceeding in half-log increments with a 1-week safety interval between each dose escalation (3 patients per dose group). In each of the first 3 dose groups (4 ⫻108, 4 ⫻108.5, and 4 ⫻109 pu), 1 patient received the diluent and served as a control. After the first 3 dosing cohorts, the protocol was modified to an open-label format with no placebo arm due to a large number of patients refusing participation because of placebo treatment. No specific counseling about diet, smoking cessation, or exercise was offered during the study period. The inclusion criteria for this study were: (1) men or women ⬎40 years of age; (2) patent inflow (aortoiliac segments); (3) angiographic evidence of ⬎35% stenosis involving infrageniculate vessels and disabling claudication; and (4) demonstrable ankle brachial index (ABI) at rest of ⬍0.90 and/or exercise ABI of ⬍0.75 confirmed on 2 different occasions 2 days apart. Exclusion criteria included patients with advanced or unstable medical disease, renal insufficiency, proliferative retinopathy, history of malignancy other than nonmelanoma skin cancers, and the following treatments within 2 weeks of study initiation: cilostazol, pentoxyfylline, L-carnitine, prostaglandins, corticosteroids, or other immunosuppressants. Vector administration protocol: The Institutional Review Boards and Institutional Biosafety Committees of all participating sites approved the protocol. For each subject, the total dose of vector was divided into 20 1-ml aliquots, and administered intramuscularly in the area of potential collateralization (thigh for patients with predominant superficial femoral artery/ popliteal disease, thigh and calf for patients with predominant infra-popliteal disease). Due to the variations of disease distribution in each patient, the region of administration of the vector varied in different patients based on vascular anatomy. Safety parameters: All patients enrolled in the study underwent a baseline safety evaluation and follow-up evaluations at days 1 to 7, 15, 30, 90, 180, and 360. Efficacy parameters: A standardized exercise protocol at variable grade (Gardner protocol) was used to determine walking ability before and after study treatment (days 30, 90, 180, and 360). The time at which the patient discontinued exercising on the treadmill secondary to maximal claudication pain was defined as the peak walking time. ABIs at rest were also measured at the various time points. Anti-adenovirus neutralizing antibodies: Neutralizing antibody titers were analyzed as described previously.11 Adenoviral cultures: Nose, throat, urine, and blood samples were screened for adenovirus on study days 1, 3, and 7 after vector administration. VEGF levels: Plasma levels of VEGF were determined by standard enzyme-linked immunosorbent assay. This assay detects the circulating isoforms (121
and 165) of VEGF (R & D Systems, Minneapolis, Minnesota). Statistical analysis and data acquisition: Efficacy data (ABI and peak walking time) from all patients treated with CI-1023 who had a baseline assessment and ⱖ1 follow-up assessment were included in the analysis. Because only 1 placebo-treated patient completed 1 year of follow-up, efficacy results from placebo patients are not summarized. Due to the limited sample size at each CI-1023 dosing cohort and no clear dose-dependent differences being observed among treatment groups, results are pooled across all dose levels. Descriptive statistics are provided for changes from baseline to each time point (days 30, 90, 180, and 360). The efficacy data presented are medians and interquartile ranges (25th to 75th percentiles).
RESULTS
Patient demographics and follow-up: Table 1 lists patients’ characteristics. Patients recruited in this study were predominantly white (94%) and male (78%). One-third of the patients were diabetic and most had other concomitant risk factors such as hyperlipidemia, previous coronary artery disease, and smoking, with ⬎50% having undergone prior lower extremity revascularization. Per protocol specification, all patients had patent iliac arteries in the index extremity with most patients having significant disease in the superficial femoral artery (72% with occlusion, whereas 17% had ⱖ50% stenosis). Five percent had an occlusion of the common femoral artery. Thirty-eight percent of the patients (8 of 18 subjects) had 0- to 1-vessel run off at the level of the ankle, whereas 9 of 18 subjects (50%) had 2-vessel run off. One patient had reconstitution of all 3 of the trifurcation vessels below the level of an occluded popliteal artery through geniculate collaterals. Safety parameters >30 days after vector administration: All patients tolerated the procedure well and
there were no serious complications immediately after treatment. Three patients in the CI-1023 group had localized edema at the site of injection that began 2 to 6 days after dosing and resolved in 7 to 23 days; another 4 patients in the CI-1023 group developed more extensive limb swelling that began 2 to 14 days after the injection and resolved in 2 to 23 days. Two CI-1023 group patients had mild hypersensitivity reactions 2 to 4 days after treatment that resolved in ⬍1 week. One patient treated with CI-1023 developed cellulitis of an old wound on the sixth day after injection that was believed to be unrelated to the treatment. Safety parameters between 1 month and 1 year after vector administration: There was 1 death in the CI-
1023 group due to necrotizing pancreatitis (on day 160) that was determined to be unrelated to the treatment. There was 1 infrapopliteal bypass procedure in a CI-1023 treated patient (dose 4 ⫻108 pu) that occurred on day 104, secondary to worsening claudication. This patient had a history of PAD and had undergone an angioplasty to the index extremity 4 months before the procedure. The patient had multiple
MISCELLANEOUS/VASCULAR ENDOTHELIAL GROWTH FACTOR IN CLAUDICATION
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TABLE 1 Baseline and Follow-up Data
Diabetes
Total Cholesterol (mg/dl)
Previous Lower Extremity Bypass
Previous Lower Extremity Angioplasty
Baseline ABI
Follow-up at 360 d
Day 30 ABI
⫹ ⫺
⫹ ⫺
159 217
⫺ ⫹
⫺ ⫺
0.25 0.38
— 0.35
Past
⫹
⫺
210
⫺
⫺
—
67M 69M 71M 67M
Past Past Past Current
⫺ ⫺ ⫹ ⫺
⫺ ⫹ ⫹ ⫹
239 145 157 166
⫺ ⫹ ⫹ ⫺
⫹ ⫺ ⫺ ⫺
0.46 0.60 0.58 0.33
4 ⫻ 108.5 4 ⫻ 108.5 4 ⫻ 109
58M 41F 73F
Past Past Past
⫺ ⫺ ⫹
⫺ ⫺ ⫺
240 245 492
⫹ ⫹ ⫺
⫺ ⫺ ⫺
0.51 0.58 0.44
4 ⫻ 109 4 ⫻ 109
73M 56M
Past Current
⫹ ⫺
⫺ ⫹
193 204
⫹ ⫹
⫺ ⫺
0.56 0.56
4 ⫻ 109.5 4 ⫻ 109.5 4 ⫻ 109.5
73F 49F 47M
Past Past Past
⫹ ⫺ ⫺
⫺ ⫺ ⫺
162 280 284
⫺ ⫺ ⫹
⫹ ⫺ ⫺
0.61 0.31 0.54
4 ⫻ 1010 4 ⫻ 1010 4 ⫻ 1010
69M 71M 64M
Past Past Past
⫹ ⫹ ⫹
⫺ ⫹ ⫺
130 216
⫺ ⫹ ⫺
⫺ ⫺ ⫹
0.64 0.62 0.27
Completed Withdrew Consent Withdrew Consent Completed Completed Completed Withdrew due to AE Completed Completed Withdrew due to lack of compliance Completed Withdrew Consent Completed Completed Lost to follow-up Completed Completed Completed
Age/Sex (yrs)
Smoking
CAD
Placebo Placebo
57M 68M
Past Past
Placebo
71M
10 108 108 108.5
Dose (pu)
4 4 4 4
⫻ ⫻ ⫻ ⫻
8
— 0.46 0.59 0.68 1.43 0.62 0.70 0.64
0.42 0.58 0.61 0.36 — 0.58 1.10 0.33
AE ⫽ adverse event; CAD ⫽ coronary artery disease.
cardiovascular risk factors, including diabetes, a history of smoking, and hyperlipidemia. One patient in the placebo group underwent a femoro-femoral bypass grafting procedure on study day 29. There was 1 malignancy reported on day 274 in a 70-year-old man who received CI-1023 (4 ⫻1010 pu). As part of an evaluation for kidney stones, the patient underwent a cystoscopy that revealed the presence of a bladder polyp. The histologic evaluation was consistent with grade I papillary transitional cell carcinoma. The patient underwent surgery to remove the early-stage cancer and repeat cystoscopies revealed no recurrences up to 2 years after dosing. Immune responses to CI-1023 administration: Neutralizing antibody titers in patients who received CI1023 are depicted in Figure 1. Thirty-eight percent had preexisting antibodies with titer ⱖ1:80 at baseline. In response to CI-1023, 70% had a doubling of their titers by study day 7. No clear dose-response relation was observed. Efficacy parameters: The median resting ABI at baseline for the CI-1023 group was 0.56 (interquartile range 0.15). Changes from baseline in resting ABI at days 30, 90, 180, and 360 were (median [interquartile range]) 0.04 (0.11), 0.21 (0.13), 0.09 (0.30), and 0.09 (0.06) for the CI-1023, respectively. Figure 2 depicts the change in median peak walking time and interquartile range at the various time points for the CI1023 group. The median peak walking time at baseline was 2.1 minutes (interquartile range 3.4). There was an increase in peak walking time at day 30 as shown in Figure 2 that appeared to be sustained up to 514 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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FIGURE 1. Adenoviral titers (dilutions) in CI-1023 treated patients, indicating the 25th percentile (bottom line of box), median (black line), and 75th percentile (top line of box). Sera were obtained at various time points, before and after vector administration. Serial dilutions of serum were evaluated for their ability to block the infection of HeLa cells by a green-fluorescent protein expressing adenovirus. Antibody titers are expressed as the dilution of serum that resulted in a 50% reduction of adenovirus infection in HeLa cells without the addition of serum.
day 360. No patient had a detectable increase in serum VEGF levels when measured at study days 1 and 7 after vector administration.
DISCUSSION
Safety issues: CI-1023 was adequately tolerated in patients with PAD in this study. A common CI-1023 related side effect was local irritation of the injection sites, including tissue and limb edema. The developSEPTEMBER 1, 2002
FIGURE 2. Change in peak walking time in CI-1023 treated patients at various time points, indicating the 25th percentile (bottom line of box), median (black line), and 75th percentile (top line of box) of the peak walking time distribution. Data points are in minutes.
ment of edema may have been related to VEGF transgene expression, as the latter is known to induce dose-dependent edema in animal models, which is believed to be secondary to the vascular permeability enhancing effects of the growth factor. Approximately 33% of the cohort (5 of 15 patients) that received CI-1023 developed edema (of any magnitude) of the injected extremity. This is identical to previously reported data in plasmid-based gene transfer of VEGF, where edema was observed in 34% of patients.12 Assessment of creatine phosphokinase and liver enzymes within 30 days of injection did not reveal any significant abnormalities, suggesting that skeletal muscle or hepatic inflammation even at high doses (4 ⫻1010 pu) did not occur. The 1 death with CI-1023 occurred in a patient with multiple medical problems and was deemed unrelated to the study drug by the investigator. The dose at which this occurred (4 ⫻108.5 pu) was ⬎3 logs less than that used in a hepatic gene transfer study (3.2 ⫻1013 pu) that was unfortunately associated with fulminant hepatic failure and death.13 Nontarget organ angiogenesis and malignancy are theoretical concerns related to the use of vascular growth factor. At 1-year follow-up there was 1 reported malignancy (low-grade stage I bladder cancer) in this cohort of patients. A review of the history of this case revealed that the patient had previous bladder polyps. Although it is impossible to exclude the possibility that this was related to the study drug, the natural history of the disease does support the degeneration of a polyp into a malignancy in some of the patients.14 Consistent with prior studies, 38% of subjects have pre-existing neutralizing antibodies to Ad5.15,16 The vast majority experienced a doubling of baseline titers.
Future studies may need to evaluate the influence of pre-existing humoral response on therapeutic effect. Conclusions on efficacy cannot be made based on this study due to the small number of treated patients, the open-label nature of the study, and substantial variability in the data. It is possible that changes in life style, especially in patients participating in a highprofile gene therapy trial, may have contributed additively to these changes, although patients were specifically asked not to change their current regimen after the administration of the study agent. The patients in this trial were ⬎40 years of age, with multiple risk factors, and most had undergone a prior revascularization attempt for symptoms of claudication. The median peak walking time of 2.1 minutes is consistent with a severely symptomatic patient population and corresponds to an absolute claudication distance of roughly 126 m, which is considerably less than other trials that have evaluated patients with claudication.17–20 In patients who underwent determinations of peak walking time, there was an improvement at study day 30 that appears to be sustained. Study limitations: A limitation of this early phase I trial is the lack of meaningful placebo end point data (owing to patient drop-out) and the noncompliance with multiple efficacy end point collection. Potential reasons for this may have been related to the adverse publicity associated with an unfortunate death in an adenoviral trial during the performance of this study and a patient population with rather advanced disease. Nonetheless, the safety data made available through this early phase I trial are important and justify the need for a placebo-controlled phase II trial that should evaluate this approach in a larger cohort of patients. 1. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest 1999;103:1231–1236. 2. Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol Heart Circ Physiol 1994;267:H1263– H1271. 3. Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest 1994;93:662–670. 4. Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, Horowitz JR, Symes JF, Isner JM. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion (see comments). Circulation 1996;94:3281–3290. 5. Horowitz JR, Rivard A, van der Zee R, Hariawala M, Sheriff DD, Esakof DD, Chaudhry GM, Symes JF, Isner JM. Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium. Arterioscler Thromb Vasc Biol 1997;17:2793–2800. 6. Henry T. Final results for the VIVA trial of rhVEGF for human therapeutic angiogenesis (abstr). Circulation 1999;100:A2509. 7. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation 2002: 105:788 –793. 8. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes JF. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb (see comments). Lancet 1996;348:370 –374. 9. Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, Isner JM. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia (see comments). Circulation 1998;97:1114 –1123.
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10. Rajagopalan S, Trachtenberg J, Olin J, Mohler E, Chartier K, Pak R,
Rasmussen H, Crystal R. A phase I study of intramuscular administration of CI-1023 (AdGVVEGF121.10) in patients with peripheral vascular disease (abstr). Circulation 2001;104:A262. 11. Chirmule N, Propert K, Magosin S, Qian Y, Qian R, Wilson J. Immune responses to adenovirus and adeno-associated virus in humans. Gene Therapy 1999;6:1574 –1583. 12. Baumgartner I, Rauh G, Pieczek A, Wuensch D, Magner M, Kearney M, Schainfeld R, Isner JM. Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor. Ann Intern Med 2000; 132:880 –884. 13. Isner JM, Vale PR, Symes JF, Losordo DW. Assessment of risks associated with cardiovascular gene therapy in human subjects. Circulation Res 2001;89: 389 –400. 14. Montie J, Smith D, Sandler H. Bladder carcinoma. In: Abeloff M, ed. Clinical Oncology. 2002. 15. Zoltick PW, Chirmule N, Schnell MA, Gao GP, Hughes JV, Wilson JM. Biology of E1-deleted adenovirus vectors in nonhuman primate muscle. J Virol 2001;75:5222–5229.
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16. Molnar-Kimber KL, Sterman DH, Chang M, Kang EH, ElBash M, Lanuti M, Elshami A, Gelfand K, Wilson JM, Kaiser LR, Albelda SM. Impact of preexisting and induced humoral and cellular immune responses in an adenovirus-based gene therapy phase I clinical trial for localized mesothelioma. Human Gene Therapy 1998;9:2121–2133. 17. Money SR, Herd JA, Isaacsohn JL, Davidson M, Cutler B, Heckman J, Forbes WP. Effect of cilostazol on walking distances in patients with intermittent claudication caused by peripheral vascular disease. J Vasc Surg 1998;27:267– 274. 18. Brevetti G, Diehm C, Lambert D. European multicenter study on propionylL-carnitine in intermittent claudication. J Am Coll Cardiol 1999;34:1618 –1624. 19. Dawson DL, Cutler BS, Meissner MH, Strandness DE Jr. Cilostazol has beneficial effects in treatment of intermittent claudication: results from a multicenter, randomized, prospective, double-blind trial. Circulation 1998;98:678 – 686. 20. Lederman RJ, Tenaglia AN, Anderson D, Hermiller J, Rocha-Singh K, Mendelsohn FO, Hiatt WR, Moon T, Whitehouse MJ, Annex BH. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC) trial. Lancet 2002;359:2053–2058.
SEPTEMBER 1, 2002
3). Eleven of 15 patients (73%) who received CI-1023 and 1 of 3 subjects (33%) who received placebo, completed 1 year of follow-up. Edema and rash were the most common early adverse event. One infra-inguinal bypass procedure occurred in each of the placebo and CI-1023 groups at days 29 and 104, respectively. One death (day 160) and 1 malignancy (day 274) occurred in the CI-1023 group. Conclusions on efficacy could not be made due to the small number of patients. However, there were encouraging trends in ABI at rest and peak walking time at follow-up. 䊚2002 by Excerpta Medica, Inc. (Am J Cardiol 2002;90:512–516)
he lack of truly effective medical therapies for the treatment of patients with advanced peripheral arT terial disease (PAD), along with the ease of delivering
trolled angiogenesis in nontarget organs. Moreover, recently performed clinical trials with recombinant VEGF and fibroblast growth factor-2 in patients with coronary artery disease have not demonstrated benefit.6,7 An alternate approach for regional angiogenesis using a plasmid-based approach to deliver VEGF was evaluated in a small number of patients with pain at rest and tissue necrosis, demonstrating apparent improvements in symptoms and limb salvage.8,9 Because plasmid-based systems have inherent inefficiencies, alternate vector systems (e.g., adenovirus) that afford sustained expression of the transgene represent an attractive option. CI-1023 (AdGVVEGF121.10) is a recombinant adenovirus vector based on Ad 5 genome deleted for all of E1a, most of E1b, and the E3 sequences, rendering it replication deficient, where the deleted sequences have been replaced by the gene encoding for human VEGF isoform 121. In this multicenter phase I trial, we investigated the safety and effects of CI-1023 in patients with advanced claudication secondary to infra-inguinal disease.
agents to the lower extremities, make this condition suited for proof of concept studies in therapeutic angiogenesis.1 The use of angiogenic growth factors, which enhance or aid in the development of collaterals, represents one approach to improve symptoms of claudication in patients with intractable symptoms and disability. Growth factors, such as vascular endothelial growth factor (VEGF), delivered as protein or gene therapy are therefore being investigated as a treatment for PAD. A number of animal studies have demonstrated that the administration of VEGF protein in the setting of ischemia is capable of inducing the development of networks of new blood vessels in vivo.2– 4 However, high doses of VEGF administered as protein have been associated with hypotension,5 in addition to carrying the theoretical risk of unconFrom the University of Michigan, Ann Arbor, Michigan; University of Pittsburgh, Pittsburgh, Pennsylvania; Heart and Vascular Institute, Morristown, New Jersey; University of Pennsylvania, Philadelphia, Pennsylvania; Pfizer Global Research and Development, Ann Arbor, Michigan; GenVec Inc., Gaithersburg, Maryland; and New York Presbyterian Hospital-Weil Medical College, Cornell University, New York, New York. Manuscript received February 20, 2002; revised manuscript received and accepted May 1, 2002. Address for reprints: Sanjay Rajagopalan, MD, Section of Vascular Medicine, Division of Cardiology, University of Michigan, L3119 Women’s Hospital, 1500 E. Medical Center Dr., Ann Arbor, Michigan 48109-0273. E-mail: [email protected].
512
©2002 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 90 September 1, 2002
METHODS
Study design: The study was initially designed as a double-blind, placebo-controlled, dose-escalating study that recruited patients with disabling intermittent claudication and patients with pain at rest and limb threat. Only the results on the claudication patients are reported here. The safety results in patients 0002-9149/02/$–see front matter PII S0002-9149(02)02424-9
with pain at rest have been reported previously.10 The total doses in each group ranged from 4 ⫻108 to 4 ⫻1010 particle units (pu) proceeding in half-log increments with a 1-week safety interval between each dose escalation (3 patients per dose group). In each of the first 3 dose groups (4 ⫻108, 4 ⫻108.5, and 4 ⫻109 pu), 1 patient received the diluent and served as a control. After the first 3 dosing cohorts, the protocol was modified to an open-label format with no placebo arm due to a large number of patients refusing participation because of placebo treatment. No specific counseling about diet, smoking cessation, or exercise was offered during the study period. The inclusion criteria for this study were: (1) men or women ⬎40 years of age; (2) patent inflow (aortoiliac segments); (3) angiographic evidence of ⬎35% stenosis involving infrageniculate vessels and disabling claudication; and (4) demonstrable ankle brachial index (ABI) at rest of ⬍0.90 and/or exercise ABI of ⬍0.75 confirmed on 2 different occasions 2 days apart. Exclusion criteria included patients with advanced or unstable medical disease, renal insufficiency, proliferative retinopathy, history of malignancy other than nonmelanoma skin cancers, and the following treatments within 2 weeks of study initiation: cilostazol, pentoxyfylline, L-carnitine, prostaglandins, corticosteroids, or other immunosuppressants. Vector administration protocol: The Institutional Review Boards and Institutional Biosafety Committees of all participating sites approved the protocol. For each subject, the total dose of vector was divided into 20 1-ml aliquots, and administered intramuscularly in the area of potential collateralization (thigh for patients with predominant superficial femoral artery/ popliteal disease, thigh and calf for patients with predominant infra-popliteal disease). Due to the variations of disease distribution in each patient, the region of administration of the vector varied in different patients based on vascular anatomy. Safety parameters: All patients enrolled in the study underwent a baseline safety evaluation and follow-up evaluations at days 1 to 7, 15, 30, 90, 180, and 360. Efficacy parameters: A standardized exercise protocol at variable grade (Gardner protocol) was used to determine walking ability before and after study treatment (days 30, 90, 180, and 360). The time at which the patient discontinued exercising on the treadmill secondary to maximal claudication pain was defined as the peak walking time. ABIs at rest were also measured at the various time points. Anti-adenovirus neutralizing antibodies: Neutralizing antibody titers were analyzed as described previously.11 Adenoviral cultures: Nose, throat, urine, and blood samples were screened for adenovirus on study days 1, 3, and 7 after vector administration. VEGF levels: Plasma levels of VEGF were determined by standard enzyme-linked immunosorbent assay. This assay detects the circulating isoforms (121
and 165) of VEGF (R & D Systems, Minneapolis, Minnesota). Statistical analysis and data acquisition: Efficacy data (ABI and peak walking time) from all patients treated with CI-1023 who had a baseline assessment and ⱖ1 follow-up assessment were included in the analysis. Because only 1 placebo-treated patient completed 1 year of follow-up, efficacy results from placebo patients are not summarized. Due to the limited sample size at each CI-1023 dosing cohort and no clear dose-dependent differences being observed among treatment groups, results are pooled across all dose levels. Descriptive statistics are provided for changes from baseline to each time point (days 30, 90, 180, and 360). The efficacy data presented are medians and interquartile ranges (25th to 75th percentiles).
RESULTS
Patient demographics and follow-up: Table 1 lists patients’ characteristics. Patients recruited in this study were predominantly white (94%) and male (78%). One-third of the patients were diabetic and most had other concomitant risk factors such as hyperlipidemia, previous coronary artery disease, and smoking, with ⬎50% having undergone prior lower extremity revascularization. Per protocol specification, all patients had patent iliac arteries in the index extremity with most patients having significant disease in the superficial femoral artery (72% with occlusion, whereas 17% had ⱖ50% stenosis). Five percent had an occlusion of the common femoral artery. Thirty-eight percent of the patients (8 of 18 subjects) had 0- to 1-vessel run off at the level of the ankle, whereas 9 of 18 subjects (50%) had 2-vessel run off. One patient had reconstitution of all 3 of the trifurcation vessels below the level of an occluded popliteal artery through geniculate collaterals. Safety parameters >30 days after vector administration: All patients tolerated the procedure well and
there were no serious complications immediately after treatment. Three patients in the CI-1023 group had localized edema at the site of injection that began 2 to 6 days after dosing and resolved in 7 to 23 days; another 4 patients in the CI-1023 group developed more extensive limb swelling that began 2 to 14 days after the injection and resolved in 2 to 23 days. Two CI-1023 group patients had mild hypersensitivity reactions 2 to 4 days after treatment that resolved in ⬍1 week. One patient treated with CI-1023 developed cellulitis of an old wound on the sixth day after injection that was believed to be unrelated to the treatment. Safety parameters between 1 month and 1 year after vector administration: There was 1 death in the CI-
1023 group due to necrotizing pancreatitis (on day 160) that was determined to be unrelated to the treatment. There was 1 infrapopliteal bypass procedure in a CI-1023 treated patient (dose 4 ⫻108 pu) that occurred on day 104, secondary to worsening claudication. This patient had a history of PAD and had undergone an angioplasty to the index extremity 4 months before the procedure. The patient had multiple
MISCELLANEOUS/VASCULAR ENDOTHELIAL GROWTH FACTOR IN CLAUDICATION
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TABLE 1 Baseline and Follow-up Data
Diabetes
Total Cholesterol (mg/dl)
Previous Lower Extremity Bypass
Previous Lower Extremity Angioplasty
Baseline ABI
Follow-up at 360 d
Day 30 ABI
⫹ ⫺
⫹ ⫺
159 217
⫺ ⫹
⫺ ⫺
0.25 0.38
— 0.35
Past
⫹
⫺
210
⫺
⫺
—
67M 69M 71M 67M
Past Past Past Current
⫺ ⫺ ⫹ ⫺
⫺ ⫹ ⫹ ⫹
239 145 157 166
⫺ ⫹ ⫹ ⫺
⫹ ⫺ ⫺ ⫺
0.46 0.60 0.58 0.33
4 ⫻ 108.5 4 ⫻ 108.5 4 ⫻ 109
58M 41F 73F
Past Past Past
⫺ ⫺ ⫹
⫺ ⫺ ⫺
240 245 492
⫹ ⫹ ⫺
⫺ ⫺ ⫺
0.51 0.58 0.44
4 ⫻ 109 4 ⫻ 109
73M 56M
Past Current
⫹ ⫺
⫺ ⫹
193 204
⫹ ⫹
⫺ ⫺
0.56 0.56
4 ⫻ 109.5 4 ⫻ 109.5 4 ⫻ 109.5
73F 49F 47M
Past Past Past
⫹ ⫺ ⫺
⫺ ⫺ ⫺
162 280 284
⫺ ⫺ ⫹
⫹ ⫺ ⫺
0.61 0.31 0.54
4 ⫻ 1010 4 ⫻ 1010 4 ⫻ 1010
69M 71M 64M
Past Past Past
⫹ ⫹ ⫹
⫺ ⫹ ⫺
130 216
⫺ ⫹ ⫺
⫺ ⫺ ⫹
0.64 0.62 0.27
Completed Withdrew Consent Withdrew Consent Completed Completed Completed Withdrew due to AE Completed Completed Withdrew due to lack of compliance Completed Withdrew Consent Completed Completed Lost to follow-up Completed Completed Completed
Age/Sex (yrs)
Smoking
CAD
Placebo Placebo
57M 68M
Past Past
Placebo
71M
10 108 108 108.5
Dose (pu)
4 4 4 4
⫻ ⫻ ⫻ ⫻
8
— 0.46 0.59 0.68 1.43 0.62 0.70 0.64
0.42 0.58 0.61 0.36 — 0.58 1.10 0.33
AE ⫽ adverse event; CAD ⫽ coronary artery disease.
cardiovascular risk factors, including diabetes, a history of smoking, and hyperlipidemia. One patient in the placebo group underwent a femoro-femoral bypass grafting procedure on study day 29. There was 1 malignancy reported on day 274 in a 70-year-old man who received CI-1023 (4 ⫻1010 pu). As part of an evaluation for kidney stones, the patient underwent a cystoscopy that revealed the presence of a bladder polyp. The histologic evaluation was consistent with grade I papillary transitional cell carcinoma. The patient underwent surgery to remove the early-stage cancer and repeat cystoscopies revealed no recurrences up to 2 years after dosing. Immune responses to CI-1023 administration: Neutralizing antibody titers in patients who received CI1023 are depicted in Figure 1. Thirty-eight percent had preexisting antibodies with titer ⱖ1:80 at baseline. In response to CI-1023, 70% had a doubling of their titers by study day 7. No clear dose-response relation was observed. Efficacy parameters: The median resting ABI at baseline for the CI-1023 group was 0.56 (interquartile range 0.15). Changes from baseline in resting ABI at days 30, 90, 180, and 360 were (median [interquartile range]) 0.04 (0.11), 0.21 (0.13), 0.09 (0.30), and 0.09 (0.06) for the CI-1023, respectively. Figure 2 depicts the change in median peak walking time and interquartile range at the various time points for the CI1023 group. The median peak walking time at baseline was 2.1 minutes (interquartile range 3.4). There was an increase in peak walking time at day 30 as shown in Figure 2 that appeared to be sustained up to 514 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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FIGURE 1. Adenoviral titers (dilutions) in CI-1023 treated patients, indicating the 25th percentile (bottom line of box), median (black line), and 75th percentile (top line of box). Sera were obtained at various time points, before and after vector administration. Serial dilutions of serum were evaluated for their ability to block the infection of HeLa cells by a green-fluorescent protein expressing adenovirus. Antibody titers are expressed as the dilution of serum that resulted in a 50% reduction of adenovirus infection in HeLa cells without the addition of serum.
day 360. No patient had a detectable increase in serum VEGF levels when measured at study days 1 and 7 after vector administration.
DISCUSSION
Safety issues: CI-1023 was adequately tolerated in patients with PAD in this study. A common CI-1023 related side effect was local irritation of the injection sites, including tissue and limb edema. The developSEPTEMBER 1, 2002
FIGURE 2. Change in peak walking time in CI-1023 treated patients at various time points, indicating the 25th percentile (bottom line of box), median (black line), and 75th percentile (top line of box) of the peak walking time distribution. Data points are in minutes.
ment of edema may have been related to VEGF transgene expression, as the latter is known to induce dose-dependent edema in animal models, which is believed to be secondary to the vascular permeability enhancing effects of the growth factor. Approximately 33% of the cohort (5 of 15 patients) that received CI-1023 developed edema (of any magnitude) of the injected extremity. This is identical to previously reported data in plasmid-based gene transfer of VEGF, where edema was observed in 34% of patients.12 Assessment of creatine phosphokinase and liver enzymes within 30 days of injection did not reveal any significant abnormalities, suggesting that skeletal muscle or hepatic inflammation even at high doses (4 ⫻1010 pu) did not occur. The 1 death with CI-1023 occurred in a patient with multiple medical problems and was deemed unrelated to the study drug by the investigator. The dose at which this occurred (4 ⫻108.5 pu) was ⬎3 logs less than that used in a hepatic gene transfer study (3.2 ⫻1013 pu) that was unfortunately associated with fulminant hepatic failure and death.13 Nontarget organ angiogenesis and malignancy are theoretical concerns related to the use of vascular growth factor. At 1-year follow-up there was 1 reported malignancy (low-grade stage I bladder cancer) in this cohort of patients. A review of the history of this case revealed that the patient had previous bladder polyps. Although it is impossible to exclude the possibility that this was related to the study drug, the natural history of the disease does support the degeneration of a polyp into a malignancy in some of the patients.14 Consistent with prior studies, 38% of subjects have pre-existing neutralizing antibodies to Ad5.15,16 The vast majority experienced a doubling of baseline titers.
Future studies may need to evaluate the influence of pre-existing humoral response on therapeutic effect. Conclusions on efficacy cannot be made based on this study due to the small number of treated patients, the open-label nature of the study, and substantial variability in the data. It is possible that changes in life style, especially in patients participating in a highprofile gene therapy trial, may have contributed additively to these changes, although patients were specifically asked not to change their current regimen after the administration of the study agent. The patients in this trial were ⬎40 years of age, with multiple risk factors, and most had undergone a prior revascularization attempt for symptoms of claudication. The median peak walking time of 2.1 minutes is consistent with a severely symptomatic patient population and corresponds to an absolute claudication distance of roughly 126 m, which is considerably less than other trials that have evaluated patients with claudication.17–20 In patients who underwent determinations of peak walking time, there was an improvement at study day 30 that appears to be sustained. Study limitations: A limitation of this early phase I trial is the lack of meaningful placebo end point data (owing to patient drop-out) and the noncompliance with multiple efficacy end point collection. Potential reasons for this may have been related to the adverse publicity associated with an unfortunate death in an adenoviral trial during the performance of this study and a patient population with rather advanced disease. Nonetheless, the safety data made available through this early phase I trial are important and justify the need for a placebo-controlled phase II trial that should evaluate this approach in a larger cohort of patients. 1. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest 1999;103:1231–1236. 2. Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol Heart Circ Physiol 1994;267:H1263– H1271. 3. Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest 1994;93:662–670. 4. Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, Horowitz JR, Symes JF, Isner JM. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion (see comments). Circulation 1996;94:3281–3290. 5. Horowitz JR, Rivard A, van der Zee R, Hariawala M, Sheriff DD, Esakof DD, Chaudhry GM, Symes JF, Isner JM. Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium. Arterioscler Thromb Vasc Biol 1997;17:2793–2800. 6. Henry T. Final results for the VIVA trial of rhVEGF for human therapeutic angiogenesis (abstr). Circulation 1999;100:A2509. 7. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation 2002: 105:788 –793. 8. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes JF. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb (see comments). Lancet 1996;348:370 –374. 9. Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, Isner JM. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia (see comments). Circulation 1998;97:1114 –1123.
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Rasmussen H, Crystal R. A phase I study of intramuscular administration of CI-1023 (AdGVVEGF121.10) in patients with peripheral vascular disease (abstr). Circulation 2001;104:A262. 11. Chirmule N, Propert K, Magosin S, Qian Y, Qian R, Wilson J. Immune responses to adenovirus and adeno-associated virus in humans. Gene Therapy 1999;6:1574 –1583. 12. Baumgartner I, Rauh G, Pieczek A, Wuensch D, Magner M, Kearney M, Schainfeld R, Isner JM. Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor. Ann Intern Med 2000; 132:880 –884. 13. Isner JM, Vale PR, Symes JF, Losordo DW. Assessment of risks associated with cardiovascular gene therapy in human subjects. Circulation Res 2001;89: 389 –400. 14. Montie J, Smith D, Sandler H. Bladder carcinoma. In: Abeloff M, ed. Clinical Oncology. 2002. 15. Zoltick PW, Chirmule N, Schnell MA, Gao GP, Hughes JV, Wilson JM. Biology of E1-deleted adenovirus vectors in nonhuman primate muscle. J Virol 2001;75:5222–5229.
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16. Molnar-Kimber KL, Sterman DH, Chang M, Kang EH, ElBash M, Lanuti M, Elshami A, Gelfand K, Wilson JM, Kaiser LR, Albelda SM. Impact of preexisting and induced humoral and cellular immune responses in an adenovirus-based gene therapy phase I clinical trial for localized mesothelioma. Human Gene Therapy 1998;9:2121–2133. 17. Money SR, Herd JA, Isaacsohn JL, Davidson M, Cutler B, Heckman J, Forbes WP. Effect of cilostazol on walking distances in patients with intermittent claudication caused by peripheral vascular disease. J Vasc Surg 1998;27:267– 274. 18. Brevetti G, Diehm C, Lambert D. European multicenter study on propionylL-carnitine in intermittent claudication. J Am Coll Cardiol 1999;34:1618 –1624. 19. Dawson DL, Cutler BS, Meissner MH, Strandness DE Jr. Cilostazol has beneficial effects in treatment of intermittent claudication: results from a multicenter, randomized, prospective, double-blind trial. Circulation 1998;98:678 – 686. 20. Lederman RJ, Tenaglia AN, Anderson D, Hermiller J, Rocha-Singh K, Mendelsohn FO, Hiatt WR, Moon T, Whitehouse MJ, Annex BH. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC) trial. Lancet 2002;359:2053–2058.
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