end-points in all groups

* Published as an abstract

** Data pooled for all patients + Treatment effect, — No treatment effect I.m. = Intramyocardial, I.e. = Intracoronary,

I.v. = Intravenous. M.c. = Sustained release heparin-alginate FGF2 microcapsules

MRI - Magnetic Resonance Imaging investigation

SPECT - Single Photon Emission Computerized Tomography

10 microgram/kg FGF2 or saline, close and distally to the IMA anastomosis alongside the LAD as far as the lower end of the distal anastomosis.

Angiographic control 12 weeks after treatment suggested more capillaries and more contrast accumulation in these areas in the recombinant FGF2 treated patients compared with the control group. With the same surgical approach in an identical group of patients, Laham et al. (12) implanted epicardially sustained-release heparin-alginate microcapsules with recombinant FGF2 during CABG in a double-blind, placebo controlled dose-titrating trial in 24 patients. Three groups, with 8 patients in each, received either 10 microgram FGF2, 100 microgram FGF2 or saline. The FGF2 was released slowly within 4-6 weeks. There was 2 operative death and 3 Q-wave myocardial infarctions. At the 3 months follow-up the nuclear single photon emission computerized tomography (SPECT) disclosed a reduction in defect size in the group treated with 100 microgram in comparison to no improvement in the two other treatment groups. The long-time follow-up, 32 months, of the 22 surviving patients found no difference in CCS class between the two FGF2 groups, but both groups had more freedom from recurrent angina compared to placebo treated. When combining the two FGF2 groups, SPECT demonstrated less reversible or fixed perfusions defects in these patients compared to controls. Importantly, no long-term side-effects to the FGF2 therapy was registered.

It is well known from PCI studies of occluded or subtotal occluded vessels that the collaterals supplying the ischemic myocardium disappear within minutes after opening of the vessel. Identical changes are seen after grafting of occluded vessels. This re-distribution of blood flow also influences the interpretation of perfusion scans. It is, therefore, very difficult in this type of studies with vascular growth factor treatment simultaneously with CABG to evaluate and separate the effect of the coronary by-pass grafting and the recombinant FGF2 treatment on improvement or changes in perfusion scans. Moreover, it is impossible to test whether an improvement in symptoms is due to the by-pass grafting or the growth factor treatment. In another study, fifty-two patients that were suboptimal candidates for conventional revascularization were treated with intracoronary infusion of increasing dosis of recombinant FGF2 (14). The infusions were generally well tolerated, although hypotension occurred in some patients at the highest dose. There were 3 deaths and 4 Q-wave myocardial infarctions in the follow-up period unrelated to the FGF2 dose used. At the two months follow-up, the patients had less angina, improved exercise capacity and reduced ischemic territory at MRI perfusion imaging. These data were supported by a small placebo controlled, dose escalating safety study [15]. Intracoronary infusion of recombinant FGF2 was performed in 17 patients and a placebo infusion in 8 patients, all with angiographic significant coronary stenosis. There were only few side-effects such as mild hypotension, slight transient trombocytopenia and proteinuria. These results suggest that intracoronary treatment with recombinant FGF2 is safe and may have a clinical beneficial effect.

Simons et al [16] tested this hypothesis in the FIRST-trial, a double-blind dose-escalating placebo controlled phase II study, with 337 patients with three different intracoronary dosages of recombinant FGF2 (0.3, 3 and 30 microgram/kg) versus placebo (16). All four groups had an increase in primary endpoint, exercise tolerance test, after 90 days, without any difference between groups. Moreover, there was no improvement in nuclear myocardial perfusion scans in the groups. There was a significant reduction in clinical angina in the 3 microgram/kg group at 90 days follow-up, but not at 180 days in any of the treated groups.

2. Recombinant vascular endothelial growth factor (VEGF) protein therapy Two small phase I safety and feasibility trials using either intra-venous or intra-coronary delivery of recombinant VEGF-A165 treatment in patients with severe coronary artery disease demonstrated an increase in exercise capacity without any safety issues [17] (Table 1). The resting nuclear myocardial perfusion scans indicated a VEGF-A165 treatment effect. However, no effect was demonstrated on stress scans using exercise, dobutamine, or dipyridamole stimulation tests (18).

Henry et al [18] then conducted the Vascular endothelial growth factor (VEGF) in Ischemia for Vascular Angiogenesis - VIVA trial [18]. It was a doubleblind, placebo-controlled, phase II trial designed to evaluate the safety, efficacy, pharmacokinetics of combined intracoronary and intravenous infusions of recombinant human VEGF for angiogenesis. A total of 178 patients with coronary artery disease were treated with two intracoronary recombinant VEGF-A165 or placebo infusions each for 10 minutes (placebo, 17 or 50 nanogram VEGF-A165 /kg/min) followed by 4 hours intravenous infusion of the randomised drug (placebo, 17 or 50 nanogram VEGF/kg/min) on day 3, 6, and 9. The chosen treatment regimes were safe, however no improvement was discovered in the primary endpoint - treadmill exercise performance in any of the groups.

2.1.1. Conclusions on recombinant vascular growth factor protein trials

It was a surprising that neither of the two larger controlled trials with recombinant FGF2 or VEGF-A165 protein therapy could detect any clinical or objective improvement in the patients with moderate to severe coronary artery disease [16,18], when comparing with previous published animal and unblinded clinical trials. An explanation could be, that when recombinant FGF2 is administered i.c., only 3-5% of the dose is recovered in the myocardium, and only 0.5 % of the dose after an i.v. administration [19]. Therefore, the long-time epicardial sustained-release heparin-alginate microcapsules principle is of potential great interest and importance, due to the prolonged delivery of the growth factors locally, but limited by its invasive approach [12]. The used treatment regimes with the chosen doses seemed to be safe. It has been suggested that a higher dose potentially could had improved the clinical out-come. However, it has to be documented that increasing the dose improves the clinical endpoints without increasing the side-effects of the treatment.

As in many angiogenesis trials a large improvement was also discovered in the placebo group in these two large scaled, well designed and well-conducted phase II studies with recombinant FGF2 and VEGF-A165 [16, 18]. The exercise capacity and the symptoms improved by the same amount in the active treated and the placebo group. It emphasizes the importance of having both subjective and objective end-point in the trials. Identical results were discovered in the in the larger randomised placebo-controlled double-blind TRAFFIC trial with intraarterial FGF2 injections in patients with intermittent claudication [20]. Peak walking time increase 90 days after treatment, but disappeared after 180 days. However, this effect was probably a random finding, since treatment with the same dose FGF2 at day 1 and 30 was without any clinical effect. Also the RAVE trial [21] a randomised placebo-controlled double-blind study with AdVEGF-121intramuscular injections in patients with peripheral artery disease could not detect any clinical improvements. Another explanation for the negative results could be, that a single intracoronary and intravenous dose of recombinant FGF2 and VEGF-A165 is unable to induce formation of collaterals.

The conflicting results emphasise the importance of conducting well-designed placebo-controlled phase II studies to clarify an eventually beneficial effect of treatment with recombinant vascular growth factor proteins.

2.2. Trials Using Genes Encoding Vascular Growth Factor

In patients with coronary artery disease, studies have evaluated the angiogenic potential of genes encoding VEGF and FGF. The study population has in almost all studies been patients with severe coronary artery disease, which could not be treated optimal with conventional revascularization therapies.

The principle of gene therapy is that a gene encoding the vascular growth factor is delivered to the cells as a cDNA (complementary DNA) formulation, which then is transcribed into the nucleus of the cell (transfection). The vascular growth factors are then produced locally for a longer period, hereby having a more steady biological effect [1]. The DNA vector has in clinical trials been delivered to the myocardium by direct intracoronary injections or direct intra-myocardial injections during bypass surgery or using a more a-traumatic percutaneous method. By using injections directly into the ischemic myocardium, the side effects caused by increased systemic levels of growth factors in non-ischemic tissue out-side the treatment area are few.

The gene encoding for a vascular growth factor can be transfected to a tissue by three different formulations; As a naked plasmid-DNA, as a liposome plasmid-DNA complex or by the use of different viral vectors (retrovirus, adeno-associated virus or adenovirus). Transfection with plasmid-DNA alone or in a liposom complex is very simple, but the efficacy is low. Less than 1 % of the plasmid DNA is entering the cells. The retrovirus is entering the cells by specific receptors on the cell surface. The retrovirus can only transfect proliferating cells, which is a limitation of the method in ischemic heart disease, since only a few cells are in a proliferative phase in the myocardium. Moreover, the retrovirus RNA-genom is integrated into the host DNA, where it persists in the host genom in the daughter cells during the following cell proliferations. This integration can be a limitation, if the aim is to initiate an expression of the gene for only a shorter period. Adenovirus is also using specific cell surface receptors to enter the cells, but the gene activation is independent of cell proliferation, since it not is integrated in the host genom. However, the adenovirus induces immunological and inflammatory reactions, which can reduce the period of gene activity and inhibit a later re-administration of adenovirus. In the recent years much interest has been put into the adeno-associated virus, which might be a better vector since it induce less immunological reaction.

The gene transcription occurs in the nucleus and initiates the production of vascular growth factors in the cytoplasma. In opposition to the transfection with retrovirus, the plasmid-, adeno-associated- or adenovirus-DNA are not incorporated into the host genom. Therefore, when using plasmid/adenovirus-DNA, the growth factors will only be produced in a short period of maybe 4 weeks, then the genes are metabolised and removed from the cells.

Both the plasmid and the adeno-virus formulations have been used for trans-fection of the myocardial cells with gene coding for VEGF or FGF in clinical therapeutic studies in patients with coronary artery disease. The genes were initially delivered to the myocardium either by directly intracoronary infusions or by direct intramyocardial injections during CABG or with a thoracotomy alone. However, with the development of the percutaneous delivery systems as the NOVA system (Biosense Webster, Cordis, Warren, US) the trend is now to use the less traumatic percutaneous method.

2.2.1. Trials with intracoronary delivery of vascular growth factor genes

The first published study of intracoronary VEGF gene transfer was a small phase I safety and efficacy trials. Laitinen [22] (Table 2) found that it was safe to perform intracoronary infusion for 10 minute of 1.000 ^gram plasmid-VEGF-A165 in 10 patients treated with PCI. Hedman et al [23] compared intracoronary injections of plasmid/liposome-VEGF-A165 and Ad-VEGF-A165 treatment in patients undergoing elective PCI for coronary artery stenosis. They found that the myocardial perfusion improved significantly in the Ad-VEGF-A165 treated patients but the improvements in the plasmid/liposome-VEGF-A165 and control patients were not significant. However, there was no difference in improvements between the three groups. Therefore, the changes discovered in the Ad-VEGF-A165 treated patients could either be due to the gene therapy alone, the revascularization therapy alone or the combined therapy.

Grines et al have performed several studies using adenovirus for gene transfer of FGF4 in patients with coronary artery disease [24, 25]. In opposition to most other growth factor trials, these patients had less coronary artery disease and angina pectoris, and they were all by the core angiography laboratory judged to be suitable candidates for angioplasty or by-pass surgery. In the AGENT-1 trial the patients were in a randomised, double-blind placebo-controlled trial treated in a ratio 3:1 with 6 ascending doses from 3.3 x 108-10n particle unit (pu)/patient [24]. The ad5-FGF4 virus was infusion into all major patent coronary arteries that could be engaged. The median calculated extraction rate across the coronary circulation was 87%. The infusion was generally well tolerated, but a majority of patients had a rise

Table 2. Intracoronary treatment with genes encoding for vascular growth factors for myocardial angiogenesis in chronic ischemic heart disease

Growth factors - Gene

Table 2. Intracoronary treatment with genes encoding for vascular growth factors for myocardial angiogenesis in chronic ischemic heart disease

Growth factors - Gene

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