Angiogenesis:Improving Cardiac Function

NOTE: To view an image in a larger size, please click on the image. What is Ad5FGF-4 and how does it aid in the treatment of cardiovascular disease? Ad5FGF-4 is a human recombinant adenoviral vector, which encodes an FGF-4 transgene, one of the members of the fibroblast growth factor family. That means that the product is constructed to incorporate the growth factor gene into the adenovirus as a delivery system to infect the target cells, in this case the cells of the heart. It has angiogenic properties, which were first demonstrated in an ischemic pig model by one of Collateral Therapeutics’ co-founders, Dr. Kirk Hammond from the University of California-San Diego. Dr. Hammond showed that a direct intracoronary injection of Ad5FGF-4 produced an induction of new collateral blood vessel growth in the myocardium, specifically in the ischemic coronary bed. The injection of Ad5FGF-4 was not only associated with increased collateralization, but also with improvement in cardiac function. Collateral Therapeutics, Inc. was founded in 1995 with our lead product candidate, Generx, based on that novel technology. The company has completed Phase 1/2 clinical trials, and we have now advanced to Phase 2b/3 clinical trials. Why is an adenoviral vector useful as opposed to another type of virus? The adenovirus has several properties that make it particularly useful as a vector for cardiovascular angiogenic gene therapy. It is large enough to carry the genes needed for angiogenesis as well as the promoters that drive their coding for mRNA and ultimately, protein production in the target cells. It can easily be produced in large numbers and it transfects dividing and non-dividing cells, which is critical for the heart, as the preponderance of cells are terminally differentiated. It also can be engineered to be replication-deficient in order to prevent passage to subsequent cell generations in or out of the heart. Since it resides in the extrachromosomal portion of the nucleus after transfection, it also has little chance of causing insertional mutagenesis, which is disruption of the cellular genome. Another beneficial aspect of the biology of the adenoviral vector is that it is avidly taken up by certain cellular receptors on the endothelial cells and cardiomyocytes, called CAR (Coxsackie-Adenovirus Receptors) and integrins. This high affinity for these receptors causes a very high first-pass uptake following intracoronary delivery. In the original animal model from Dr. Hammond’s research, there was a 98% first-pass uptake of the adenoviral vector. In the human trial (the AGENT trial), the median uptake was 87%, very consistent with the animal data. This effective vector targeting of the heart cells using an intracoronary approach, which is a benefit of Collateral’s proprietary gene delivery method, adds a further safety factor to Generx angiogenic cardiac gene therapy. What are the angiogenic gene families? Is there a difference between the angiogenic genes? Angiogenesis is a key component of a wide variety of natural biologic activities, including normal processes such as wound healing and recovery from tissue ischemia, to pathologic processes such as proliferative retinopathy or neoplastic growth. The angiogenic response is a very complex series of related intracellular and extracellular events involving signaling pathways, proliferation and migration of endothelial cells, capillary tube sprouting and remodeling, and eventual networking between the arterial and venous systems. There needs to be a trigger to turn this on, and for the heart that appears to be stress, such as ischemia. Unfortunately, in the heart, the angiogenic response is often inadequate in the face of the progressive nature of coronary artery disease. A number of genes express proteins that have been shown to mediate angiogenesis. Conceptually, these genes can be used to turn on therapeutic angiogenesis. These angiogens are naturally occurring growth factors that act as mitogens, stimulating endothelial cell growth and migration. Two widely recognized growth factor families of polypeptides include the fibroblast growth factors (FGF) and the vascular endothelial growth factors (VEGF). Both gene families have been shown in vitro and in vivo to induce angiogenesis. The VEGFs are mitogens that appear to be specific for endothelial cells. They also induce capillary vascular permeability and have been called vascular permeability factor. They clearly induce angiogenesis, but there is some question as to whether the angiogenic response is robust, as the new vessels, without the added effect of other growth factors, may be immature and leaky. The FGFs affect a number of intracellular and extracellular events necessary for angiogenesis. They are mitogenic for endothelial cells, smooth muscle cells and myocytes. They also upregulate certain proteases that digest the extracellular matrix and may open up the way for the developing capillaries. This wider effect may ultimately induce a more mature collateral response. How is Ad5FGF-4 delivered? Ad5FGF-4 delivery can be done at the time of the diagnostic cardiac cath. The therapy is being developed for the interventional cardiologist who has a patient with recognized disease that wouldn’t be an immediate candidate for intervention with angioplasty or bypass surgery. At the time of the cath, the transgene-viral vector is delivered right into the heart with a 90-second infusion down each of the patent coronaries and any patent bypass grafts (saphenous vein or mammary artery) in patients who have had previous bypass surgery. The Phase I (safety) trial for Ad5FGF-4 looked at moderate angina? Yes. One of the important concepts that we’ve had at Collateral is that there’s a large, unmet need of patients with stable angina pectoris, despite all the advanced therapies both in interventional cardiology and cardiac surgery. A number of the other gene therapy companies have opted to evaluate an angiogenic effect in only end-stage or far-advanced cardiac patients. Collateral felt strongly that this approach was under-serving the larger population, and as a result, our trial was specifically designed to address patients with stable chronic angina. These are patients who were on medications, who may have had angioplasty or surgery, but who still had symptoms and a limited lifestyle. Phase 1/2, the AGENT (Angiogenic GENe Therapy) trial, was set up as a safety and dose-response trial. The double-blind, prospective, randomized trial was conducted at 12 centers across the U.S. and enrolled 79 patients (60 treated and 19 placebo). The primary endpoint was improvement in exercise treadmill testing (ETT) at 4 and 12 weeks after test product administration. The trial looked at the safety and potential anti-ischemic effects of an adenoviral vector with a FGF-4 transgene delivered by one-time intracoronary infusion. The focus of the trial was to ultimately select potentially safe and effective doses for subsequent large-scale pivotal trials. The study wasn’t powered to show efficacy. One-half log increments of an increasing viral titer dose were evaluated with the randomized placebo controls initially having 9 treated and 3 control patients per group. Safety was shown as we increased dosages incrementally. However, it quickly became clear that there was a dose that would be better examined in a larger number of patients. Considering the safety profile in the first 3 dose groups, an approved modification of the enrollment criteria was implemented in Dose Group 4. Specifically, we decided to limit the entrance criteria for treadmill time to ten minutes, as interim analysis of the blinded data showed very long baseline exercise times in a number of patients. We also, by protocol amendment, added patients who had previous cardiac surgery, and patients with >=30% EF (whereas the initial protocol enrolled patients with >=40%EF), to determine whether such patients should be included in future trials. The AGENT trial Dose Group 4 received 1 x 1010 viral particles. This expanded group enrolled 22 patients. After the trial was completed, a subset analysis compared Dose Group 4 to the placebo group. There was a statistically significant improvement in Dose Group 4, both in terms of the percentage of responders and in terms of improved treadmill times. Most importantly, the safety profile was really very positive. There were a few patients with transient elevation of liver function tests, and in the highest dose group (a dose group actually higher than Group 4), 3 of 11 patients had a fever after the gene/virus delivery, which did abate. One or two of the patients had to stay in the hospital overnight for fever evaluation, but that was all. What’s the clinical endpoint for this treatment? In the original AGENT trial, we looked at 4- and 12-week treadmill times following treatment. In all large angina trials, there’s clearly a placebo response in studied patients. It could be a treadmill training effect, as patients perform multiple treadmill tests and increase their physical condition over time. It could also be a subconscious or non-purposeful better-treatment management of patients that are involved in the study. As in all angina studies, we felt it important to look at placebo and treated patients at the 4-week and 12-week time. Results showed improvement in the Dose Group 4 patients, both at 4 weeks and at 12 weeks. It's important to also note that there was an increasing improvement; i.e., the treatment response appears to be an ongoing effect. We built into the analysis a need to show a 20% improvement at 4 weeks, and a 30% improvement at 12 weeks, since we expected to find an incremental improvement in the placebo group. Incorporating the specific percentage increase approach, we did show positive results with the treated Dose Group 4. For example, in patients with exercise times <10 minutes (per the protocol amendment after dose group 3), the percent change in ETT for placebo versus treated patients was significantly different at 4 weeks: 12% vs. 27%, p=0.01, [0.6 and 1.64 minutes] and at 12 weeks: 22% versus 30%, p=0.047, n=50 [1.27minutes and 1.86 minutes]. There was a significant difference in the proportion of patients with substantial increases in ETT between placebo and Dose Group 4 at 4 weeks: 16% versus 50%, p=0.046, and a trend continued at week 12: 21% versus 45%. Based on these results, the FDA allowed us to move on to advanced-stage pivotal Phase 2b/3 trials. As with the first trial (AGENT), the Phase 2b/3 trial will be managed with the sponsorship of our development partner, Schering AG (Berlex Laboratories in the U.S.). We are well underway with the first pivotal cardiac gene therapy trial for heart disease that specifically utilizes therapeutic angiogenesis. The concept of therapeutic angiogenesis obviously has been talked about a lot, and I think it’s very important to recognize that it may soon be a reality. Our larger Phase 2b/3trial is designed to show efficacy, as well as added safety data. Paralleling the U.S. trial is an ongoing Phase 2b/3 trial in Europe. About nine hundred total patients will be studied in these two pivotal trials. How many centers are involved in the current Phase 2/3b trial? Well, Schering and Berlex are estimating up to 100 centers in the United States, and around 60 centers in the U.K and Europe. There’s a little different regulatory environment in Europe, and they don’t quite have the same experience with gene therapy as an overall approach. As a result, the patient population that will be studied in Europe is different. It’s not going to be the stable angina patient population, but rather patients who are not optimal candidates for percutaneous coronary intervention or bypass surgery, as well as end-stage patients that have no therapeutic treatment options available to them. Obviously that’s a different question than what we looked at in the AGENT trial, and it’s different than the ongoing population that we’ll be evaluating here in the United States. The European population will give us a much broader perspective on the full range of cardiovascular patients who could be potential candidates for angiogenesis therapy. Does Collateral Therapeutics have other gene therapy products in development? Absolutely. As a gene therapy company, we think there are a variety of cardiovascular diseases that need to be addressed. In addition to our angiogenesis program, we have a gene therapy development program for heart failure. It's based on a proprietary myocardial adrenergic signaling technology, where an adenylyl cyclase gene has been shown to increase adrenergic responsiveness to catecholamine stimulation in the heart. We think that this approach has exciting potential for the treatment of heart failure patients. We also have some preclinical research with our angiogenesis gene, Ad5FGF-4, in an animal model of heart failure that shows benefits in addition to the angiogenesis effect. There appears to be an additional non-angiogenic effect that improves cardiac function specifically in the heart failure setting. Our third major development program uses gene therapy for myocardial regeneration and myocardial preservation. That brings up my next question how are angiogenesis and myogenesis different? Angiogenesis increases the collateral capillary bed circulation to the heart muscle, whereas myogenesis would actually be inducing and regenerating cardiomyocytes. We have a number of genes we’ve licensed that are being looked at for their effect on preventing apoptosis and, also, for their potential to induce fibroblasts to become cardiomyocytes to help regenerate heart muscle after a heart attack. How far out do you estimate treatment with Ad5FGF-4 is from widespread use? We’ve estimated that the Phase 2b/3 trials are going to take around 2+ years for completion, with, hopefully, product approval following. In the foreseeable future, there’s the potential that we’re going to have the first approved cardiac gene therapy for humans available on the market. My background is as a cardiac surgeon, and I have seen from a clinical perspective where cardiac surgery is now, where it’s come from, and the great potential it has for treatment benefits. As a cardiac surgeon, I worked with interventional cardiologists every day. We’d take care of the same patients. Like cardiac surgery, the rapid advancements in interventional cardiology have dramatically advanced patient care. Having said that, there is still a giant population of patients out there that continues to have symptoms from their cardiac illness, despite the great advances in cardiac surgery and interventional cardiology. Better and less invasive therapies are needed. But cardiac gene therapy is not going to put cardiologists or cardiac surgeons out of business. It’s going to be another treatment tool for them, to help with the worldwide epidemic of heart disease. What do you estimate the treatment will cost? I suppose the financial people would be better off answering that question than I would, but it’s going to be in the same market range as existing treatments. A lot will depend on final results and specific labeling. It’s really too far in advance to 'guestimate’, but we're not talking about a dramatic increase in cost for this treatment. Obviously, with an effective response, there is potential for patients that might otherwise be candidates for angioplasty or bypass surgery to not need either one of those procedures. That would likely diminish overall cost. Is there any kind of learning curve for this procedure? No. There really is little or no learning curve. Cardiologists from the 12 centers who were involved in the first AGENT trial found that it added very little time to the diagnostic cardiac cath. Literally it’s just a matter of a timed, 90-second injection into each open conduit or coronary artery. There’s not any sophisticated technology that’s necessary for the cardiologist to learn. What about handling the actual injection? How is it stored? The present product, as a biologic with a viral vector, has a lot of restrictions, supervised by the RAC and the FDA, that are necessary as part of the safety evaluation. We haven’t seen problems, yet we and the cardiologists are very cautious. In the cath lab, the cardiologists wear the standard gown, gloves and hat, but they also wear special goggles and a special mask, so if there are any aerosolized particles, they won’t end up in the eye, and cardiologists or any cath lab personnel won’t breathe in the particles. Ordinarily, at the time of the injections, ancillary cath lab personnel step out of the room, behind the screen or the window. Usually the cardiologist and an assistant are at the side of the table with the patient as they do the gene injection. The injection product is delivered frozen, and it’s stored either at -20° or -70 ° Centigrade in the cooling system at the hospital. For preparation, it’s hand-thawed, not shaken, so there’s no viral clumping. The pharmacist or cardiologist just holds it in his or her hand. In 5“10 minutes, it ends up as a clear liquid. It’s drawn up into a syringe, and diluted either to 5 or 10 mls with saline, depending on the number of injection sites that are deemed necessary. The injection syringe is then transported to the cath lab. The diagnostic cath is then performed and the cardiologist determines which sites need to be injected. A dose is divided up based on the number of targets and the overall anatomy of the heart. The divided doses are given incrementally, one at a time, and once that’s been accomplished, the cardiologist takes a final view of each target to make sure that the injectate catheter, which is deep-seated in the coronary artery, bypass graft, or the internal mammary artery, is still in position, which validates that the dose was given into the target area as expected. That’s the end of the procedure. What about normal medications that patients may be taking for angina? Are these medications still necessary? In the trial, we made sure that patients were on a stable medical regimen. Most of the patients were on a number of the standard antianginal and cardiac medications. We didn’t want to have a patient that was unstable going into the trial. Collateral and Berlex have a recent report of a second angiogenic Phase 1/2 study, which is a double-blind, randomized, placebo-controlled perfusion study done with SPECT analysis. The trial compared rest and adenosine-stress related, reversible perfusion defect size prior, and 4 and 8 weeks post, treatment with Ad5FGF-4. The primary endpoint was reversible perfusion at 8 weeks post treatment. In that study (though it was a small study and statistical significance wasn’t reached), a large number of patients actually ended up needing either less antianginal medication or no antianginal medication. These patients had consistently fewer symptomatic anginal attacks following the treatment. If you get good, robust collateral formation to the heart and you bring new blood supply to ischemic heart muscle, there’s the likely potential that patients aren’t going to need the same medical therapy. Also, there was a trend in the study toward improvement in the initial perfusion defects present, following the angiogenic gene therapy treatment compared to placebo. Gene therapy is coming soon, and we believe it will be a great asset to help take care of your patients. It’s going to continue to be very carefully evaluated for the safety profile. We have very encouraging results in over 100 patients already, and the patient population is growing. There are a vast number of clinical trials using the adenoviral vector. Other than a notable past example, in a different clinical indication using the adenovirus in very high doses, this vector continues to be safe. Our cumulative experience to date has shown very few problems. Generx therapy appears to have the potential to induce a direct angiogenic effect on the heart, especially in the dose range that we’re evaluating in the ongoing pivotal trials. We obviously await the safety and efficacy data from the large trials for confirmation, but we’re encouraged that this may soon represent the first available gene therapy approach for therapeutic angiogenesis.

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