Capricor Reports Encouraging Results in its DYNAMIC Trial

Capricor Therapeutics, Inc., located in Beverly Hills, CA, has announced their six-month safety and adverse event data from a Phase I clinical trial of their CAP-1002 product for patients with advanced heart failure. This clinical trial is part of the DYNAMIC or which is short for Dilated cardiomYopathy iNtervention with Allogeneic MyocardIally-regenerative Cells trial whose goal is to evaluate CAP-1002 in patients with advanced heart failure.

CAP-1002 is Capricor’s lead investigational allogeneic, cardiosphere-derived cell (CDC) therapy. Allogeneic means that the cells come from someone other than the patient. The advantage of allogeneic cells is that they come from healthy donors whose cells have not been ravaged by old age or other conditions. These cells do not need to be matched to the patient’s immune system in this case because they help the heart through indirect means (see Tseliou E, et al., J Am Coll Cardiol. 2013 Mar 12;61(10):1108-19).  Cardiospheres are cells taken from the hearts of healthy patients that grow in culture as small balls of cells. Because these cells are derived from the heart and grow as spheres, they are called cardiospheres (see Cheng K, et al., JACC Heart Fail. 2014 Feb;2(1):49-61.).

Cardiospheres have been shown in small clinical trials (the CADUCEUS trial) to replace the heart scar with heart muscle (see Malliaras K, et al., Am Coll Cardiol. 2014 Jan 21;63(2):110-22).  Animal studies in rats showed similar results (see above).

CAP-1002 is an off-the-shelf “ready to use” cardiac cell therapy that consists of cells that come from donor heart tissue and is infused directly into a patient’s coronary artery during a catheterization procedure. This Phase I study is meant to determine if CAP-1002 is safe and effective in treating heart function and structure. In particular, Capricor scientists are interested in determining if CAP-1002 cells can decrease heart scar tissue and promote the growth of heart muscle. In doing so, this regenerative treatment might delay or even prevent the onset of heart failure. The US Food and Drug Administration has granted CAP-1002 an orphan drug designation for the treatment of cardiomyopathy associated with Duchenne Muscular Dystrophy.

Capricor’s Cardiosphere-Derived Cells are a unique therapeutic product that were created in the laboratory of company Co-Founder and Scientific Advisory Board Chairman, Dr. Eduardo Marbán, who is the Director of the Heart Institute at Cedars-Sinai Medical Center.

All patients in this trial have advanced heart failure and have progressed to a more advanced stage of the disease. Patients received CAP-1002 in up to three coronary arteries, which delivers the cells to the more of the diseased parts of the heart. Since these patients have significant fibrosis in all areas of the heart, this delivery system is optimal for these patients. Cell delivery will also utilize methods that do not stop blood flow, which will decrease patient discomfort during cell delivery.

The data from this trial, so far, comes from 14 patients who were diagnosed with either dilated cardiomyopathy or non-ischemic dilated cardiomyopathy. These patients have ejections fractions of 35% or less and are classified as New York Heart Association class III or Ambulatory Class IV heart failure.

The data collected to date show that CAP-1002 cells are safe and well tolerated and produced no adverse cardiac events at one month or six months after they were infused into the patient’s hearts. Although DYNAMIC was designed as a Phase I clinical trial that does not assess the efficacy of CAP-1002 cells, patients have also been tested for their subject wellbeing, exercise capacity (six-minute walk test), ejection fraction, and ventricular volumes.

According to the principal investigator Dr. Raj Makkar of Cedar-Sinai Medical Center, the data so far are rather encouraging, even beyond the positive safety data, since they are seeing “concordance between the clinical improvement and the physiological measurements of trends for improved ejection fraction and reverse re-modeling.” Dr. Makkar, however, emphasized that this clinical trial only tested a small cohort of patients, and these data must be confirmed in larger clinical trials.

Cardiospheres Aid Heart Healing by Secreting Endoglin and Inhibiting TGF-beta signaling

Eduardo Marbán from Cedars-Sinai Heart Institute in Los Angeles and his team have invested a great deal of work into the development of cardiospheres, which are self-assembling heart-derived stem cells that grow as little balls of cells in culture. Several preclinical experiments and a few clinical trials have established the effectiveness of cardiospheres are treatments for the heart after a heart attack. However, Marbán and his co-workers have also worked very hard at determining why cardiospheres heal a damaged heart.

Cardiospheres in culture

 

To that end, Marbán and others have returned to their mouse model to do very detailed experiments with their cardiospheres and define exactly why these cells help heal the heart. To date, it is clear that cardiospheres increase the density of blood vessels in the heart tissue, decrease scar deposition, and prevent heart remodeling (the enlargement of the heart after a heart attack to compensate for the increase load placed on smaller amount of heart tissue). Marbán and others wanted to know precisely how cardiospheres managed these feats.

It has been known for some time that scar formation in the heart is largely a consequence of the activation of the TGF-beta signaling pathway (see NG Frangogiannis, Circulation Research 110: 159-173). Inhibition of this pathway can prevent the scar-making cells (fibroblasts) from migrating to the site of damage, dividing, and depositing the protein collagen, which is the main component of heart scars.

An abundant literature on heart scars show that the heart scar plays an important short-term role, but that in the long-run, it prevents the heart from resuming full function because it does not communicate with the rest of the heart muscle cells and does not contract. Therefore, helping the heart get through the first month after the heart attack without a scar is a crucial time.

In a recent paper published by Marbán and his team, cardiospheres were tested in culture and in the heart of mice that had suffered a heart attack. Marbán thought that since the cardiospheres were attenuating scar formation, they must be inhibiting TGF-beta signaling. TGF-beta proteins are secreted by cells and they bind to a receptor complex that then activate intracellular proteins called “SMADs.” These activated SMAD proteins enter the nucleus and activate the transcription of target genes.

In his co-culture experiments, Marbán and others used normal human fibroblasts from the lower layers of human skin and cultured them with and without cardiospheres. The co-culturing experiments showed that without cardiospheres, the dermal fibroblasts made lots of collagen and activated their internal SMAD proteins. When these human dermal fibroblasts were incubated with cardiospheres, their SMAD proteins were largely inactivated and they made very little collagen.

Such a result is not surprising, but how are the cardiospheres doing this? As it turns out, there is an inhibitor of the TGF-beta receptor complex called “endoglin” that can also be secreted known as sE. When Marbán and others examined their cardiospheres, they were secreting a fair amount of sE.

Thus, the production of sE could definitely prevent dermal fibroblasts from activating their SMADs and making collagen, but what if these sE molecules were inactivated? Marbán and others made antibodies against sE and then used them to inactivate the sE made in culture by the cardiospheres. Under such conditions, the cardiospheres no longer were able to prevent SMAD activation in dermal fibroblasts and the fibroblasts made lots of collagen, even in the presence of cardiospheres.

This is all fine and good, but it is in cell culture, and cell culture experiments must always be confirmed by experiments in a living creature. Therefore, Marbán and his colleagues used cardiospheres to treat mice that had suffered a heart attack. As observed before, the cardiosphere-treated mice showed increases in ejection fraction and fractional shortening, and decreases in end-diastolic and end-systolic volume. The cardiosphere-treated animals also had much less scar tissue after one month and greater blood vessel density. Furthermore, the cardiosphere-treated mice did not show the maladaptive enlargement of the heart muscle cells seen in post-heart attack patients. When the heart tissue was assayed one month after treatment, it was clear that the cardiosphere-treated heart tissue showed increased sE expression and much less TGF-beta signaling. The downstream targets of SMAD activation were much less, and SMADs also showed less activation. Expression of the TGF-beta receptors was also decreased.

This paper shows that endoglin expression plays a key role in preserving and healing the heart after a heart attack. Would it be possible to give soluble endoglin to heart attack patients? This remains to be seen.

One caveat with this paper is that human dermal fibroblasts are similar but different from heart fibroblasts. While it is reasonable to suppose that these two cell types react in a similar way to cardiospheres, such a supposition must be rigorously confirmed experimentally.

How Cardiospheres Heal the Heart

In 2007, Eduardo Marbán and his colleagues have discovered a stem cell population from the hearts of mice and humans that grow as small balls of cells in culture (see RR Smith, et al., Circulation. 2007 Feb 20;115(7):896-908). He called these cells “cardiospheres” and in a follow-up study showed that these cells have the ability to differentiate into heart muscle cells, blood vessel cells, or other types of heart-specific cells (PV Johnson, et al., Circulation. 2009 Sep 22;120(12):1075-83). Other animal experiments by Marban’s group showed that not only were cardiospheres easily obtained by means of heart biopsies, but injection of these cells directly into the heart after a heart attack augmented healing of the heart and accelerated the recovery of heart function and while preserving heart structure (ST Lee, et al., J Am Coll Cardiol. 2011 Jan 25;57(4):455-65; CA Carr, et al., PLoS One. 2011;6(10):e25669; Shen D, Cheng K, Marbán E. J Cell Mol Med. 2012 Sep;16(9):2112-6).

All of these very hopeful results in culture and in animal studies eventually gave way to a small human clinical trial in which a heart patient’s own cardiospheres were transplanted into their own hearts.  This clinical trial, the CADUCEUS trial (which stands for cardiosphere-derived autologous stem cells to reverse ventricular dysfunction), prevent patient’s hearts from worsening, but more remarkably, the heart scars of these patients were partially erased 6 months after treatment.  A one-year follow-up showed that patients had improved global heart function that directly correlated to the shrinkage of their heart scars.

These results are very encouraging and Marbán made it clear that he wants to conduct larger clinical trials.  However, he still had a gaggle of unanswered questions about his cardiospheres.  Do these cells affect blood vessel formation?  Can they prevent the enlargement of the heart that occurs after a heart attack (known as cardiac remodeling)?  Can the benefits of these cells be solely linked to their effects on the heart scar?  Do cardiospheres prevent the formation of the heart scar?  Do they only help heal the area of the heart where they are administered or do they also help more far-flung regions of the heart?  These are all good questions, and answers to them are necessary if Marbán and his group is to conduct larger and more intense clinical trials with human heart patients.  Therefore, he turned to an animal model system to address these questions in detail.  In particular, he chose Wistar Kyoto rats.

Readers of this blog will recognize the experimental strategy; break the rats into three groups, induce experimental heart attacks in two groups, give one group cultured cardiospheres and leave the other one alone.  Thus you have a sham group that underwent surgery but was not given a heart attack, a heart attack group that did not receive cardiospheres, and a heart attack group into which 2 million rat cardiospheres were injected at four different sites near the site of the infarct.

This experiment, did far more than simply monitor the heart function of the animals for several weeks.  Instead, some of these animals were sacrificed and their hearts were subjected to extensive biochemical and molecular biological tests.   The goal of these experiments was to determine not just if the cardiospheres helped heal the heart.  Marbán and his group already knew that they do.  They wanted to know how they heal the heart.

The cardiosphere-treated animals showed substantial improvements in their heart function as opposed to their non-treated counterparts.  The treated animals had heart that did not undergo remodeling and also pumped better.  Hearts from the cardiosphere-treated animals had less dead heart tissue and more live tissue.  They had smaller heart scars, and better preservation of cellular structure in the heart.  When biochemical markers of proliferating cells were measured in these hearts, the cardiosphere-treated hearts showed robust increases in cell proliferation far above those hearts that were not treated with cardiospheres.  Thus cardiospheres seem to induce resident heart cells to divide and replace dead and dying heart cells.

A common response to a heart attack is that the surviving heart cells enlarge (hypertrophy).  The cardiosphere-treated hearts showed no such response.  Also, when the blood vessel density of the heart tissue was determined, the cardiosphere-treated hearts had close the twice the vessel density of the non-treated hearts.  This was the case near the site of cardiosphere injection, but it also held, albeit not as robustly, in areas far from the site of cardiosphere injection.  This suggests that blood vessel formation is due to secreted molecules.

To test this possibility, Marbán and his crew rigged a culture assay in which rings of tissue from the aorta (the largest blood vessel in the body), were embedded in collagen and treated with culture media from cardiospheres, standard culture cell culture media, or cell culture medium from endothelial cells.  The cardiosphere culture medium, which contains a cocktail of molecules secreted by growing cardiospheres as they have grown in culture, induced far more blood vessels in this system than the other two.  This confirms the notion that cardiospheres secrete blood vessels-inducing molecules that this increases the vascularization of the heart muscle, this aiding its survival.

Marbán and his team also examined the molecule that forms the heart scar; collagen and how cardiospheres affect the synthesis and deposition of collagen.  They discovered that cardiospheres actually degrade the collagen at the heart scar.  They showed that cardiosphere secrete enzymes that have been documented to degrade collagen (Matrix Metalloproteases 2 and 13 for those who are interested).  Marbán and others also discovered that cardiospheres put the kibosh on collagen synthesis.  When they measured biochemical markers of collagen synthesis (hydroxyproline), they were present at rather low levels.  Thus cardiospheres prevent the deposition of the heart scar and also actively degrade it.

Thus, Marbán and his colleagues showed that cardiospheres: 1) prevent the tissue-level changes associated with cardiac remodeling; 2) preserve heart function locally and globally; 3) increase the proliferation of heart muscle cells at the site of the infarct, and to a lesser effect, throughout the heart; 4) induce the formation of new blood vessels at the site of injection, and, to a lesser extend, further from the site of cardiosphere injection; and 5) actively prevent the formation of the heart scar by inhibiting its formation and degrading whatever collagen has been deposited.

Thus cardiospheres decrease the formation of collagen and therefore, decrease the stiffness of the wall of the heart.  They also product new blood vessels and provide a supportive environment for the formation of new heart muscle cells.

This paper was published in PLoS One (2014) 9(2):e88590.

May Marbán’s clinical trials increase!!

Caduceus Clinical Trial One-Year Update

The CADUCEUS clinical trial, which stands for CArdiosphere-Derived aUtologous stem CElls, to reverse ventricUlar dySfunction) was the brainchild of Cedar-Sinai cardiologist Eduardo Marbán and his colleagues. 

This CADUCEUS trial used a heart-specific stem cell called CDCs or cardiosphere-derived cells to treat patients who had recently suffered a heart attack.  CDCs are extracted from the patient’s own heart and they can be grown in culture, expanded, and then implanted back into the patient’s heart. The initial assessments of those patients who had received the stem cell treatments was published in 2012 in the Journal Lancet (R.R. Makkar, R.R. Smith, K. Cheng et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet, 379 (2012), pp. 895–904). The initial assessments of these patients showed shrinkage of their heart scars.  However, these patients showed regional improvements in heart function but no significant differences in global heart function.  Despite these caveats, the initial results were hopeful. 

Now the one-year follow-up of these patients has been published in the Journal of the American College of Cardiology.  The results of this examination are even more exciting.

CDCs were extracted from patients by means of heart biopsies of the inner part of the heart muscle (myocardium). After the cells were grown in culture to larger numbers, they were reintroduced to the hearts of the patients by means of “stop-flow” technique. This procedure utilizes the same technology as stents in that an over-the-wire balloon angioplasty catheter that was positioned in the blood vessels on the heart that were blocked. The figure below shows the cultured cardiospheres.

Specimen processing for human cardiosphere growth and CDC expansion. a, Schematic depicts the steps involved in specimen processing. b, Endomyocardial biopsy fragment on day 1. c, Explant 3 days after plating. d, Edge of explant 13 days after plating showing stromal-like and phase-bright cells. e, Cardiosphere-forming cells collected from the explant after 13 days and plated on poly-d-lysine for 2 days. f, Fully formed cardiospheres on day 25, 12 days after collection of cardiosphere-forming cells. g, CDCs during passage 2, plated on fibronectin for expansion. h and i, Cell growth is expressed as number of population doublings from the time of the first harvest for specimens from nontransplant patients (h) and specimens from transplant patients (i).

The initial assessment of these patients showed shrinkage of the heart scar and regional improvements in heart function. However in the one-year follow-up the scar showed even more drastic shrinkage (-11.9 grams or -11.1% of the left ventricle). Also, several of the indicators of global heart function showed substantial improvements (end-diastolic volume – -12.7 mls and end-systolic volume – -13.2 mls).

When it come to the all-important ejection fraction, which is the percentage of blood pumped from the left ventricle, the results are a little more complicated. When the ejection factions of each patient was compared with the size of their heart scars, there was a tight correlation between the increase in ejection fraction and the shrinkage of the heart scar. See the figure below for a scatter plot of ejection fraction versus heart scar size.

(A) Scatterplot showing the natural relationship between scar size and left ventricular ejection fraction ∼5 months post-myocardial infarction (circles). Each cross symbol represents the mean values (at the intersection of the vertical and horizontal bars [obtained from all patients with magnetic resonance imaging measurements]), whereas the width of each bar equals ±SEM of scar size and left ventricular ejection fraction of CADUCEUS patients at baseline, 6 months, and 1 year; the crosses are superimposed onto the scatterplot showing prior data from post-myocardial infarction patients with variable scar sizes. The changes in left ventricular ejection fraction in CDC-treated subjects are consistent with the natural relationship between scar size and ejection fraction in convalescent myocardial infarction, whereas the changes in left ventricular ejection fraction in controls fall within the margins of variability. (B) Changes in end-diastolic volume from baseline to 1 year. (C) Changes in end-systolic volume from baseline to 1 year. CDCs = cardiosphere-derived cells; EDV = end-diastolic volume; EF = ejection fraction; ESV = end-systolic volume; LV = left ventricle.

Other observations included safety assessments. When the number of adverse events between the control group and CDC-receiving group were measured, there were no differences between the two groups. The patients in the CDC-receiving group were more likely to be hospitalized and had transient cases of fast heartbeats, and there was also one death in this group. However the incidence of these events were not statistically different from the control group.

From these assessments, it is clear that the CDC treatments are safe, and decreased the scar size and regional function of infarcted heart muscle. From these results, the researchers state that “These findings motivate the further exploration of CDCs in future clinical studies.