Patent Application
20180104059
Stem cell therapy is a promising candidate for treatment of cardiomyopathies and heart failure. Orlic, et al., first reported cardiac repair (reduction of infarct size, increase in ejection fraction) by transplanting bone marrow cells in mice after myocardial infarction [Orlic, et al., “Mobilized bone marrow cells repair the infarcted heart, improving function and survival,” 98 Proc. Natl. Acad. Sci. U.S.A 10344-49 (2001) and Orlic, et al., “Bone marrow cells regenerate infarcted myocardium”, 410 Nature 701-5 (2001)]; and Strauer, et al., confirmed this achievement weeks later in a human patient [Strauer, et al., “Intracoronary, human autologous stem cell transplantation for myocardial regeneration following myocardial infarction”, 126 Dtsch. Med. Wochenschr. 932-38 (2001)]. The mechanism of action was found not to be from transdifferentiation of cells into cardiomyocytes but from paracrine factors [see M. Gnecchi, et al., “Paracrine mechanisms in adult stem cell signaling and therapy”, 103 Circ. Res. 1204-1219 (2008), and M. Gnecchi, et al., “Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement”, 20 FASEB J. 661-669 (2006)].
Studies with bone marrow cells were extended to patients with chronic ischemic heart failure [B. E. Strauer, et al., “10 Years of Intracoronary and Intramyocardial Bone Marrow Stem Cell Therapy of the Heart: From the Methodological Origin to Clinical Practice”, 58 J. Am. Coll. Cardiol. 1095-1104 (2011)]; and researchers then began investigating cardiac stem cells for heart failure. For example, results of the SCIPIO phase 1 trial demonstrate that autologous cardiac stem cells can improve systolic function and reduce infarct size in patients with post MI heart failure [G. Heusch, “SCIPIO brings new momentum to cardiac cell therapy”, 378 Lancet 1827-28 (2011)]. The CADUCEUS phase 1 trial used autologous cardiosphere derived cells (CDCs) and showed increases in viable myocardium [R. R. Makkar, et al., “Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial”, 379 Lancet 895-904 (2012)]. Both trials warrant expansion to phase 2 studies.
One of the major hurdles to successful clinical translation of cardiac cell therapy, however, is poor cell survival, retention and engraftment in the myocardium—a critical requirement for effective treatment, and a possible explanation for the transient clinical benefit in specific studies. Various factors contribute to this phenomenon and include exposure of cells to ischemia and inflammation, mechanical washout of cells, flushing by the coronary vasculature, leakage from the injection site and anoikis. Solutions to this problem may be found in products that combine cells with agents more adhesive to resident tissue [K. L. Christman, et al., “Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium”, 44 J. Am. Coll. Cardiol. 654-660 (2004)]. Multiple studies have corroborated preliminary findings in our lab that biomaterial delivery vehicles can enhance cellular retention.
Additionally, regenerative therapy for the diseased heart (in the form of cells, macromolecules and small molecules) also has faced multiple hurdles, including poor retention, short biological half-life, adverse side effects from systemic delivery, and the need for multiple administrations.
A method for providing therapy to living tissue and a tissue therapy apparatus are described herein, where various embodiments of the apparatus and methods may include some or all of the elements, features and steps described below.
In a method for providing therapy to living tissue, living tissue is contacted with at least one reservoir loaded with cells or a therapeutic composition, wherein the reservoir is in fluid communication with at least one conduit (e.g., a catheter) that includes a refilling port. A constituent selected from (a) cells, (b) bioagents from the cells or (c) the therapeutic composition is released from the reservoir to the living tissue. The reservoir is then refilled with (i) cells, (ii) nutrients for cells, or (iii) additional therapeutic composition; and (a) cells, (b) bioagents from the cells or (c) the therapeutic composition continue to be released from the reservoir to the living tissue after the refilling.
The reservoir can be designed to increase retention at the tissue site and to provide controlled, targeted and replenishable localized release to the tissue. The reservoir can be engineered to provide immunological protection to its biological constituents.
In another method for providing therapy to living tissue, living tissue is contacted with a sleeve through which conduits pass, wherein the conduits each include a first open end in fluid communication with the living tissue, with a biomaterial on the tissue, or with a reservoir containing the biomaterial and including a porous membrane at an interface with the tissue; and at least one of (a) cells, (b) bioagents from the cells and (c) the therapeutic composition is periodically injected from the catheter into contact with the living tissue. In particular embodiments, a catheter is inserted through at least one of the conduits, wherein the injection is performed via the catheter.
A tissue therapy apparatus includes at least one reservoir including a porous wall through which contents of the reservoir can pass; a conduit including a first end and a second end, wherein the second end is in fluid communication with the reservoir; and a refill port mounted at the second end of the conduit. In addition to a refill port, the apparatus can include an extracorporeal or intracorporeal pump and reservoir. The pump reservoir can be filled transcutaneously or worn on a belt like an insulin pump.
Cardiac cell therapy is an emerging therapy that has been limited by poor retention or engraftment of cells in the heart, though the use of a therapeutic layer or sleeve that surrounds the heart and allows replenishable or refillable delivery of therapy to biomaterials, as described herein, can provide for superior cell retention, and superior clinical benefit. The pericardium is a fibrous layer that surrounds the heart. In particular embodiments, the sleeve can serve as a replacement pericardium (made of synthetic or natural biomaterials) that allows sustained and controllable delivery of therapy, or a therapeutic pericardium, which we refer to as a “thericardium.” By combining a biomaterial cell carrier that allows replenishment of cells to the myocardium with a passive restraint layer, the methods and apparatus described herein can potentially promote “reverse remodeling” [as described in M. C. Oz, et al., “Direct cardiac compression devices”, 21 J. Heart Lung Transplant 1049-055 (2002) and in H. R. Levin, et al., “Reversal of Chronic Ventricular Dilation in Patients With End-Stage Cardiomyopathy by Prolonged Mechanical Unloading”, 91 Circulation 2717-720 (1995)] and myocardial restoration. Where a patient has suffered a heart attack, for example, the apparatus can release therapy to restrict the growth of scar tissue on/in the heart. The apparatus can also provide therapeutic benefit as a passive restraint device.
The thericardium system, described herein, offers a number of advantageous features to address the current limitations for the delivery of cells, macromolecules and small molecules to treat cardiac disease. A reservoir in the thericardium can be directly placed on the heart and connected to a subcutaneous port through an implanted conduit or catheter, allowing a localized, targeted therapy to the diseased tissue, without the need for higher systemic doses. This reservoir can house a pre-loaded and refillable biomaterial for sustained delivery of therapy, and a surgical method of implantation in a rat model is introduced that enables repeated replenishment of therapy from a subcutaneous port. As biomaterials have been shown to increase retention in this type of cargo delivery, a biomaterial reservoir can be used. For example, a methacrylated gelatin cryogel can be used, but we foresee this platform system being used with numerous types of biomaterials. In a further refinement, a second rate-limiting membrane can be introduced in the reservoir, thus offering a method to tune the rate of therapy diffusion into tissue, and the size of molecules permitted through the membrane. The delivery of cells is demonstrated using luciferase-expressing mouse mesenchymal stem cells, proteins using fluorescently tagged bovine serum albumin, and small molecules using D-luciferin, an imaging substrate that causes bioluminescence in the presence of the enzyme luciferase and oxygen. In various embodiments the reservoir has a controlled release mechanism for bioagent and/or therapeutic composition release. Other embodiments omit such a mechanism.
In the accompanying drawings, like reference characters refer to the same or similar parts throughout the different views; and apostrophes are used to differentiate multiple instances of the same or similar items sharing the same reference numeral. The drawings are not necessarily to scale; instead, emphasis is placed upon illustrating particular principles in the exemplifications discussed below.
The foregoing and other features and advantages of various aspects of the invention(s) will be apparent from the following, more-particular description of various concepts and specific embodiments within the broader bounds of the invention(s). Various aspects of the subject matter introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the subject matter is not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.
Unless otherwise herein defined, used or characterized, terms that are used herein (including technical and scientific terms) are to be interpreted as having a meaning that is consistent with their accepted meaning in the context of the relevant art and are not to be interpreted in an idealized or overly formal sense unless expressly so defined herein. For example, if a particular composition is referenced, the composition may be substantially (though not perfectly) pure, as practical and imperfect realities may apply; e.g., the potential presence of at least trace impurities (e.g., at less than 1 or 2%) can be understood as being within the scope of the description; likewise, if a particular shape is referenced, the shape is intended to include imperfect variations from ideal shapes, e.g., due to manufacturing tolerances. Percentages or concentrations expressed herein can represent either by weight or by volume. Processes, procedures and phenomena described below can occur at ambient pressure (e.g., about 50-120 kPa—for example, about 90-110 kPa) and temperature (e.g., −20 to 50° C.—for example, about 10-35° C.) unless otherwise specified.
Although the terms, first, second, third, etc., may be used herein to describe various elements, these elements are not to be limited by these terms. These terms are simply used to distinguish one element from another. Thus, a first element, discussed below, could be termed a second element without departing from the teachings of the exemplary embodiments.
Spatially relative terms, such as “above,” “below,” “left,” “right,” “in front,” “behind,” and the like, may be used herein for ease of description to describe the relationship of one element to another element, as illustrated in the figures. It will be understood that the spatially relative terms, as well as the illustrated configurations, are intended to encompass different orientations of the apparatus in use or operation in addition to the orientations described herein and depicted in the figures. For example, if the apparatus in the figures is turned over, elements described as “below” or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the exemplary term, “above,” may encompass both an orientation of above and below. The apparatus may be otherwise oriented (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
Further still, in this disclosure, when an element is referred to as being “on,” “connected to,” “coupled to,” “in contact with,” etc., another element, it may be directly on, connected to, coupled to, or in contact with the other element or intervening elements may be present unless otherwise specified.
The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting of exemplary embodiments. As used herein, singular forms, such as “a” and “an,” are intended to include the plural forms as well, unless the context indicates otherwise. Additionally, the terms, “includes,” “including,” “comprises” and “comprising,” specify the presence of the stated elements or steps but do not preclude the presence or addition of one or more other elements or steps.
Additionally, the various components identified herein can be provided in an assembled and finished form; or some or all of the components can be packaged together and marketed as a kit with instructions (e.g., in written, video or audio form) for assembly and/or modification by a customer to produce a finished product.
Inspired by the protective nature of the pericardium, a fibrous sac that surrounds the heart, here we present a new system called “thericardium” (therapeutic pericardium), that enables direct, controllable administration of therapy directly to the heart through one or more polymer-based reservoirs capable of controlled release of therapy. The therapy reservoir is implanted on the heart and can be replenished through a catheter connected to an implanted subcutaneous port. This system presents numerous advantages, including convenient, repeated therapy administration, and rapid, cost-effective in vivo imaging for quantification of targeted therapy. The thericardium can reduce or eliminate scarring and restore full cardiac function post-myocardial infarction and can attenuate or eliminate the cascade of events that lead to heart failure and prevent ischemic cardiomyopathy. As a research model, this system may elucidate new insights into regenerative cardiac therapy and advance experimental therapies along the clinical translational path. Overall, this work has practical implications in enabling experiments that were previously prohibited by cost, invasiveness and quantification challenges.
In the embodiment of
The therapy in the reservoirs 34 and released by the reservoirs 34 can include, e.g., cells, a bioagent produced by cells and that acts on and affects other cells—for example, a paracrine-acting agent, a growth factor, such as a bone morphogenetic protein (BMP) or vascular endothelial growth factor (VEGF); a chemotherapeutic agent; an immunosuppressant; culture media, conditioned media; small molecules that help regulate a biological process; an anti-rejection drug; an anti-arrythmic drug; anti-anginal drug; a cardioprotective drug for use, e.g., during chemotherapy; hormones, dopamine, levodopa, antibiotics, anti-inflammatory drugs, anti-thyroid pharmacotherapy, anti-microbial drugs, and lidocaine; or macromolecules or small molecules.
In particular embodiments, the reservoirs 34 do not all contain the same contents. Rather, different types of therapy are contained in different reservoirs 34, thereby allowing for selective actuation, as described below, of particular reservoirs 34 to deliver a particular therapy at one time and for later selective actuation of another set of reservoirs 34 to deliver a different therapy at a subsequent time. In particular embodiments, varying pressure can be used for a tunable release from respective reservoirs 34. Different reservoirs can be fabricated with different thicknesses or other features that provide for a graded release among the reservoirs 34 in response to pressure. In additional embodiments, valves can be incorporated into the sleeve 12 to provide for on/off release from respective reservoirs 34. A once off burst release can also be used in the reservoirs 34. The reservoirs 34 can also contain biosensors that detect levels of disease-responsive biomarkers (e.g., troponins and matrix metalloproteinases) or that detect the mechanical function of the heart.
For wound healing, the apparatus can include a topical reservoir, such as a transdermal patch or a micro needle patch, attached to a refillable reservoir 34 containing (a) cells, (b) bioagents from the cells or (c) other therapeutic composition (e.g., antibiotic). In this embodiment, the reservoir 34 can be incorporated into a bandage or dressing and contacted with, e.g., wounded or otherwise damaged skin tissue on an external surface of the body.
In particular embodiments, as shown in
Where the reservoirs 34 contain biomaterials and cells, the pores can be made very small to restrict passage of cells or biomaterial there through while still being large enough to permit passage of growth factors produced by the cells. In a particular embodiment, the pores of the membrane 15 can have a diameter of about 0.4 μm. A suitable product is thermoplastic urethane commercially available from American Polyfilm, Inc., of Branford, Conn., USA, which can be laser-cut with pores of desired size; alternatively, a polycarbonate porous membrane can be used. In particular embodiments, the reservoir(s) 34 and sleeve 12 (if used) can be formed of a biodegradeable material so that the apparatus can biodegrade to eventually substantially eliminate it from the body, thereby removing the need for a second surgery to remove the apparatus after its use. In additional embodiments, the reservoir(s) 34 or sleeve 12 can include structures/compositions that enable it or its features to show up under x-ray imaging; for example, the device can have radiopaque channels for visibility under x-ray. In further embodiments, the reservoir(s) 34 and/or sleeve 12 are formed of materials that will not interfere with imaging for diagnostic purposes (e.g., magnetic resonance imaging) to assess how the heart is healing over time.
Biomaterials on which cells can be grown, such as injectable biomaterials and thermoresponsive biomaterials, can be fabricated and formed into the reservoir(s) 34. Here, cryogels (i.e., gels that are frozen to produce a porous structure) were formed, though other biomaterials can be substituted for the cryogels. Though this embodiment employs a sleeve 12, one or more reservoirs 34 can be implanted and secured as pockets or patches or otherwise placed in contact with the tissue without being embedded in a sleeve 12. In particular embodiments, reservoirs 34 can be provided with surface properties (e.g., micro-patterning) or adhesive to adhere it to tissue and to remain in contact with the tissue without changing position. Other ways to attach the reservoirs 12 to the heart include the use of functionalized gel, suction (e.g., micro-suckers), sutures, mesh, etc. The adhesive material properties can be such that the adhesive can be elastic when cured so that it can maintain adhesion given movement and deformation of tissue. In addition, the adhesive can be located in a pattern (i.e., not covering the full surface) to aid with adhesion in a dynamic context. In addition, micro-needle technology can be combined with the thericardium to provide an interface to the tissue and enabling injection/infusion of therapy directly into the tissue.
Microneedle technology is a technique for delivery of small molecules and biologics, whereby micron-scale hollow needles are fabricated (borrowing techniques from the micro-electronics industry) in patches. Microneedles can be used for transdermal or trans-tissue delivery of biologics and are promising micro-fabricated devices for minimally invasive drug delivery applications. Microneedles are high performance conduits, through which drug solutions may pass into the body and are designed to be as small as possible. Microneedles are also designed to be extremely sharp, with submicron tip radii, allowing the needles to be effectively inserted into the skin. Microneedles offer an attractive way for advanced drug delivery systems by mechanically penetrating the skin and injecting drug just under the stratum corneum where it is rapidly absorbed by the capillary bed into the bloodstream.
In various embodiments, the sleeve 12 and/or reservoir(s) 34 can be implanted on a donor organ before organ transplant. In other embodiments, the sleeve 12 and/or reservoir(s) 34 can be implanted concurrent with other surgery or when implanting a mechanical device in the body. In still other embodiments, the sleeve 12 and/or reservoir(s) 34 can be delivered (e.g., pre-loaded) in a folded or rolled-up configuration through a catheter; for example, a balloon catheter can deliver a sleeve 12 or reservoir 34; or a mechanical delivery catheter can unfold a sleeve 12 or reservoir 34 using a shape memory alloy or polymer. In additional embodiments, the sleeve 12 or reservoir 34 can be delivered via a robotic delivery system, such as the HeartLander robot from The Robotics Institute at Carnegie Mellon University. In other embodiments, the sleeve 12, itself, can be robotic and can move to the desired tissue site using sensing or imaging modalities.
In additional embodiments (as shown in
Seeding of cells on cryogels was demonstrated through the channels 16 in a sleeve 12, as illustrated in
The thericardium 10 can be implanted through a small incision in the ribcage 25 called a thoracotomy, and refilling of therapy (e.g., cells, nutrients for cells, or pharmaceuticals) can be performed through a subcutaneous port 18 (with a self-sealing rubber septum) and conduit 22. In other embodiments, the thericardium 10 can be sufficiently small so that it can be delivered through a catheter, and have a design such that it can expand into a larger shape inside the body. As shown in
We set up an animal model, where a miniaturized thericardium 10 containing a biomaterial was placed on a rat heart after a myocardial infarction, with the radiance from bioluminescence for a gel and thericardium 26 and for a gel alone 28 plotted in
A recent study has shown that ventricular reloading can induce cardiomyocyte proliferation. D. C. Canseco, et al., Human Ventricular Unloading Induces Cardiomyocyte Proliferation, 65 J. Am. Coll. Cardiol. 892-900 (2015). The authors hypothesized that an increase in mitochondrial content in response to mechanical load causes activation of DNA damage response (DDR) and permanent cell cycle arrest of cardiomyocytes. This impairs the ability of the heart to regenerate. The authors showed that post-LVAD (left ventricular assist device) hearts (after “unloading” of the ventricle) showed a decrease in mitochondrial content and cardiomyocyte size compared with pre-LVAD hearts. If this is the case, the administration of regenerative therapy while the heart is being unloaded should have a better chance of success compared to administration to a heart that is trying to compensate for a volume or pressure overload. As such, there are numerous ongoing trials combining cell therapy with traditional mechanical assist devices. A multimodal combination of cells with mechanical assist devices (passive or active) represents a particularly attractive therapeutic strategy. This approach confers the potential for mechanical devices to act on co-delivered cells, as well as to exert efficacy to the heart. Co-delivery in a biomaterial carrier can ensure that cells are kept in close proximity to the mechanical device for the duration of therapy to enhance synergistic interaction.
In an interesting acellular hybrid therapy approach, Kubota, et al., in “Impact of cardiac support device combined with slow-release prostacyclin agonist in a canine ischemic cardiomyopathy model”, 147 J. Thorac. Cardiovasc. Surg, 1081-1087 (2014), employed an atelocollagen sheet/polyglycolic acid ventricular restraint device (VRD) alone, small molecule PGI2 agonist ONO1301 on an atelocollagen sheet alone, or a multimodal ONO1301-doped VRD in a canine model of myocardial infarction. At 8-weeks post infarction, hearts treated with the multimodal VRD, demonstrated the greatest increase in left ventricle ejection fraction (LVEF) and the greatest reduction in left ventricular wall stress and ventricular remodeling. All hearts treated with ONO1301 (either alone or in combination with a VRD) demonstrated an increase in myocardial vascularization and upregulation of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) in the myocardium. In a similar hybrid approach with cells, Shafy, et al., in “Development of cardiac support bioprostheses for ventricular restoration and myocardial regeneration”, 43 Eur. J. Cardiothorac. Surg. 1211-1209 (2013), showed that the combination of adipose-derived stem cells (injected into the infarct and seeded in a collagen matrix) with a polyester Corcap VRD device resulted in significant improvements in ejection fraction and systolic and diastolic function in a sheep infarct mode. This semi-degradable ventricular bioprosthesis approach is an example of biomaterial-mediated cell therapy combined with a constraint device. The CELLWAVE study [B. Assmus, et al., “Effect of shock wave-facilitated intracoronary cell therapy on LVEF in patients with chronic heart failure: the CELLWAVE randomized clinical trial”, 309 JAMA 1622-1631 (2013)] addressed delivery of BM-MSCs combined with a pretreatment of low energy cardiac shockwave to improve honing of cells and expression of SDF-1 and VEGF. The combination of a shock wave with cells resulted in an increase in an ejection fraction of 3.2%. In Chachques, et al., “Development of bioartificial myocardium using stem cells and nanobiotechnology templates”, 2011 Cardiol. Res. Pract. 806795 (2010), nanobiomaterials with elastomeric membranes were bioengineered to acquire a controlled drug release patch, which they can tailor for local cell attraction and cell differentiation. A phase 1 clinical trial began in August 2009 to test a patch called ANGINERA (from Theregen, Inc., of San Francisco, Calif.) containing cells secreting factors to stimulate growth of other cells by paracrine signaling. The study groups consist of coronary artery bypass graft (CABG) patients and end-stage heart failure patients with an LVAD device.
In the past few years, the idea of combining mechanical support and cellular therapy synergistically has emerged as a realistic alternative to heart transplantation. This contemporary holistic, hybrid approach for end-stage ischemic heart failure may address the issue of scarce donor hearts for transplantation. The success on combining the therapies relies on refining them individually and maximizing their possible combinatorial efficacy.
In one configuration, a sleeve 12 in the form of a layer at the heart/device interface is provided through which therapy can be delivered. This interface layer can be molded out of a biomaterial (as shown in
To achieve this vision, a bioreactor was developed to determine the effect of cyclical actuation [similar to that provided by the direct cardiac compression (DCC) device] on cells and biomaterials, as described below. Then, as described below, a manufacturing process was developed for a larger scale version of the thericardium 10. The thericardium 10 includes a sleeve 12 that can be used with the DCC device and that includes multiple reservoirs 34 and connecting channels 16 (as shown in
To understand how transplanted cells in biomaterial reservoirs 34 may respond to cyclical actuation from a direct cardiac compression device 32, a bioreactor was developed that could be used to examine this effect in vitro. A control system was constructed in a sealed chamber that can be placed in the incubator to provide cyclical pneumatic actuation to a dynamic bioreactor. The control system included four solenoid valves (VQ110U-5M from SMC Corp. of Noblesville, Ind., USA), a microcontroller (AA-021205 Arduino Mega, Arduino), and a miniaturized diaphragm pump (D737-23-01 from Parker Hannifin Corp. of Cleveland, Ohio, USA). A simplified bioreactor was made by taking sections of latex tubing and placing a small pneumatic actuator around the tube sections. A porous pocket into which a cell-laden biomaterial could be placed was positioned at the tube/actuator interface. The actuators were connected to the valve outlets in the control system, and each tube/actuator assembly was placed in a 6-well plate. Media (DMEM from Sigma-Aldrich of St. Louis, Mo., USA) was filled into each well plate. The entire assembly was placed in the incubator and cultured for 72 hours. A static group was compared to a dynamic group (actuated at 1 Hz for 72 hours) for a gelatin methacryloyl (GelMA) and an injectable alginate biomaterial (manufactured as previously described and seeded/encapsulated with 1×106 mouse mesenchymal stem cells). The pockets were made from two layers of thermoplastic urethane (HTM6001, 0.006 inch thick, American Polyfilm, Inc.) that was laser cut to have 500 um pores, spaced by 1 mm in each direction. The two layers were sealed using a heat sealer and employing Teflon tape (from Saint-Gobain S. A. of Courbevoie, France) to selectively mask an area with a 2-cm opening on one side of the pocket for biomaterial insertion and media perfusion.
Preliminary results showed that cell viability (measured by live/dead staining at 72 hours) can be improved with dynamic actuation compared to static culture in the alginate group, but not in the GelMA group. The gelMA gel withstood the actuation and maintained its porous structure when imaged by scanning electron microscopy (SEM).
Next, the concept of the thericardium 10 was scaled up so that it could be used in combination with an existing DCC device. To realize this, a manufacturing process was developed to form reservoirs 34 for biomaterials that were connected by channels 16 using sheets of thermoplastic urethane 13 and 15, heat forming and heat sealing, as shown in
As the thericardium 10 was now scaled up to the size of the DCC device, the increased size enabled the use of the device as a bioreactor. A double layered reservoir 34 was constructed using the fabrication process previously described with an additional non-porous layer sealed on top. Each reservoir 34 was separated by a 0.003-inch thermoplastic urethane layer (with 500-um laser-cut holes spaced 1 mm apart in each direction). The assembly is shown in
Some interesting preliminary results were obtained from the experiment, and are shown in
The thericardium 10 was tested for functionality in a Yorkshire swine model (n=3, 60-70-kg female swine). The sleeve 12 was placed around the heart 14 and dye was used to visualize filling of the channels 16. Contrast was added to the dye, and the procedure was repeated under fluoroscopy to show x-ray filling of the reservoirs 34. The thericardium 10 was refilled via direct injection using a microcatheter that is tracked through the channels 16 to the reservoir 34 to fill one isolated reservoir 34 (blue dye for visualization of filling). Additionally, a fluorescently tagged suspension of alginate beads (50-μm beads tagged with alexafluor −750) was delivered through the thericardium 10, and the heart was imaged using the IVIS Xenogen 5000 imaging system to assess for fluorescence on the tissue. A layer of “tough gel” hydrogel [described in J-Y Sun, et al., “Highly stretchable and tough hydrogels,” 489 Nature 133-36 (September 2012) and comprising 90% water yet stretching without breaking to more than 20 times its original length and recoiling like rubber] was manufactured and placed at the heart/device interface to act as a secondary material reservoir, with the intent of reducing friction between the heart 14 and the thericardium 10 once the DCC device 32 was placed over it and actuated. Finally, a thericardium 10 with incorporated gelfoam was used to explore sustained delivery of drug (in this case epinephrine) to the epicardium of the heart 14. Preliminary in vivo testing showed that the device conformed to the heart well and could be easily attached. Replenishment of the reservoirs 34 with direct injection or catheter injection was possible, and post-trial imaging showed that the therapy was delivered to the myocardium.
Finally, in a preliminary feasibility study, the thericardium 10 and the DCC device 32 were combined on a live porcine model (Yorkshire swine, 60 kg) to evaluate refilling of therapy during active assistance. Refilling was possible and was visualized under x-ray with use of contrast.
A vision for translation of this combined therapy is two-fold—the thericardium technology can be used to deliver biological therapy with active assist or while it is acting as an adjustable passive restraint device. In a first scenario, a patient receives the thericardium 10 with the DCC device 32, as shown in
Both macromolecule and small molecule therapies suffer some similar limitations as cell therapies (i.e., low concentrations at the desired site due to untargeted delivery and a short biological half-life). Delivery of macromolecules represent a promising therapeutic deliverable for the treatment of ischemic cardiomyopathy. The increased accessibility to these bioagents and the advances in chemical modifications to enhance protein half-life in vivo and minimize immunogenicity offer a broad range of new therapeutic modalities. Modified peptides and proteins can enable cardiac repair through activation of endogenous cardiac progenitor cells present at the injury site, the induction of cardiomyocyte proliferation, and the recruitment of progenitor cells to damaged myocardium or cells able to trigger neovascularization. Studies have been conducted with vascular endothelial growth factor (VEGF), stromal cell derived factor (SDF-1), hepatocyte growth factor (HGF), nueregulin (NRG-1), and insulin-like growth factor (IGF-1). The encapsulation of proteins in carrier gels provides a controlled release and enhances retention in the target area. In parallel, advances in synthetic chemistry mean that a library of small molecules can be screened in a biological system to determine novel drug targets and to elucidate previously unknown signaling systems implicated in myocardial disease. Structure activity relationship data can permit and guide molecular amendments to enhance specificity, stability and efficacy. Examples of these molecules include prostaglandin E2 (PGE2), ONO1301, pyrvinium pamoate (PP), or diprotin. A common theme underpinning studies with delivery of these small molecules is the necessity for redelivery or sustained delivery of drugs. It is feasible to conclude that the increased bioavailability of these agents at a pathological site within a suitable therapeutic window, as can be afforded by the thericardium 10, may lead to a new option in the treatment of cardiovascular disease.
Simplified Refillable Thericardium in an Animal Model:
Use of the refillable thericardium 10 in a rat model is illustrated in
As shown in
Refilling the Simplified Thericardium:
a) With Cells
First, we demonstrated the ability to replenish cells in situ via the thericardium 10 (
b) With Small Molecules
The imaging substrate D-luciferin was used to demonstrate the rapid, targeted delivery of small molecules to the heart 14. The thericardium reservoir 34 was pre-seeded with luciferase expressing mouse mesenchymal cells (mMSCs) before implantation so that bioluminescence of the cells would indicate the presence of imaging substrate. The effect of delivery via the reservoir 34 is an immediate, localized dose to the heart 14, as shown in
c) With Macromolecules
Next, we demonstrated that protein therapeutics could be delivered through the thericardium 10. A bovine serum albumin solution tagged with a fluorescent molecule (Vivotag 800) was delivered via the port 18. After three hours, there was a sustained concentration of the protein at the target site. The same amount of protein was injected intraperitoneally as a control, but an undetectable quantity of the therapy had reached the target site after three hours; the fluorescence signal at the target was equal to background measurements for intraperitoneal delivery, as shown in
Encapsulated Thericardium Device and Refill in a Model of Myocardial Infarction:
Additional control over therapy is enabled by the realization of an encapsulated thericardium to protect therapy from mechanical ejection resulting from a beating heart or potentially the host immune response, and to selectively control the therapy or paracrine factors that pass through a porous membrane onto the diseased tissue. This thericardium is shown in
The ability to deliver and replenish cells to a thericardium 10 placed on an infarcted rodent heart 14 was next tested; the study included two groups, direct delivery of a cell-loaded cryogel with the thericardium 10 and the same system with a refill of one million cells at day four post-operatively. Representative bioluminescent images are shown at post-operative time points for a direct delivery group (
Design of Reservoir for Encapsulated Therapy Delivery
The membrane immune isolation technology described here can increase transplanted cell retention and survival, enable protection from the host immune response, and can be modified to adjust the type and rate of therapy diffusion from the therapeutic reservoir. This encapsulated delivery technology may be used to enable the isolation and study of the effect of paracrine and autocrine factors produced by transplanted cells for the purposes of cardiac regeneration and to eliminate or reduce the host immune response so as to enable the long-term de novo production and delivery of therapeutic paracrine factors (i.e., operating as a cell factory) from an allogeneic or potentially a xenogeneic cell source, without the need for immunosuppressive regimens. This sustained viability over an extended period of time, within a suitable therapeutic window, could lead to improved clinical outcomes. Additionally, the delivery device may facilitate biopsy in a minimally invasive manner and be ultimately retrievable in the case of unforeseen safety issues.
The embodiment of a reservoir 34 shown in the exploded view of
A thericardium 10 including two reservoir sections 34′ and 34″(with section 34″ stacked on top of section 34′, as shown in
Another thericardium 10 is shown in the exploded view of
The biomaterial 42 in this embodiment comprises hyaluronic acid, formed with a 1:1 ratio of pre-polymer to crosslinker, enabling easier injection of accurate volumes, and the degradation enzyme is hyaluronidase. After evacuation of the biomaterial 42 from the reservoir via the above-described procedure, additional biomaterial 42 is refilled via external catheter to avoid blocking the catheter 22 leading to the first reservoir 34′.
In another embodiment, the thericardium 10 of
In additional embodiments, the implanted catheter 22 to the first reservoir 34″ can be used as a guide for a steerable catheter with a suction tip that can be used to the remove the degraded biomaterial 42. The tip can also be cooled or heated to reverse gelate a thermoresponsive hydrogel in the first reservoir 34′. Light, magnetism, an ultrasound frequency emission, or a radiofrequency emission is generated or transmitted from the cather tip to gelate a functionalized biomaterial 42 for delivery or to degrade it for evacuation.
The thericardium 10 can function as a delivery system that allows targeted, replenishable and sustained presentation of cellular and molecular therapy to the heart 14. A biomaterial-based reservoir (gelatin cryogel) 34 initially seeded with luciferase-expressing mouse mesenchymal stem cells, was attached to the epicardial surface of the infarcted rat heart 14. Gelatin is derived from collagen and contains inherent peptide sequences that facilitate cell adhesion and enzymatic degradation. Due to its low cost, lack of immunogenicity, and previous use in medicine as a hemostatic agent and blood volume expander, gelatin is an attractive implantable biomaterial. However, we foresee that this platform can be extended to other biomaterials that have demonstrated an ability to increase cell retention at the heart (e.g., alginate, chitosan, hyaluronic acid-based gels, gelfoam, and collagen).
An implantable catheter 22 was used as a conduit between this reservoir 34 and a subcutaneous port 18 located at a dorsal site of the rat. The biomaterial reservoir 34 can be refilled with cells through the port 18 at defined points in time, increasing the resident cell number 10-fold. Although just one refill was conducted in vivo, the possibility for multiple refills and replenishments with similar or different therapies exists and was demonstrated in vitro. Furthermore, attaching the catheter 22 to a small implanted, refillable pump 88 (for example, a stem cell pump from BioLeonhardt of Santa Monica, Calif., USA) enables a sustained infusion (as depicted in
By enabling triggered, localized release of treatment, the thericardium 10 can “deliver the right treatment at the right time” to the patient. The pericardium is a fluid filled sac that forms a natural barrier surrounding the heart 14. Targeting drugs directly to the heart by delivering to the pericardial space [i.e., intrapericardial (IPC) delivery] can serve as an advantageous strategy to obtain higher drug efficiencies, while lessening the side effects. Oral formulations are the most commonly used and most patient-acceptable method of drug delivery; oral formulations, however, have many inherent limitations including incomplete absorption through the gastrointestinal mucosa, poor bioavailability and poor compliance. Intravenous (IV) administration overcomes these issues by bypassing absorption and first-pass metabolism. For both oral and IV delivery, however, inter-patient pharmacokinetic variability can cause extensive deviations in the amount of drug that reaches the desired molecular target; and significant quantities of drug reach off target sites, potentially causing side effects. This off-target delivery is a particularly important problem for drugs with a narrow therapeutic index, as a high concentration can cause toxic side effects while a low concentration can eliminate any clinical benefit.
Localized delivery confers the advantages of greater control over desired tissue exposure, decreased variability of clinical response, lower needed therapeutic doses, and opportunities to use bioagents with a short half-life or that are biologically incompatible with the gastro-intestinal tract and blood stream (e.g., cells and their secreted paracrine factors). The efficacy of IPC drug delivery to the heart has been studied for angiogenic substances and vasodilators as well as rhythm management drugs (anti-arrhythmics, arrhythmic agents. Hermans, et al., in “Pharmacokinetic advantage of intrapericardially applied substances in the rat”, 301 J. Pharmacol. Exp. Ther. 672-678 (2002), used a chronic administration animal model to show pharmacokinetic advantages in the rat with IPC infusion. Van Brakel et al, showed that this technique improved the efficacy of β-blockers sotalol and atenolol compared to IV administration in “Intrapericardial delivery enhances cardiac effects of sotalol and atenolol”, 44 J. Cardiovasc. Pharmacol. 50-56 (2004). However, easy and reproducible access has been a major limiting factor for IPC delivery. The direct, refillable thericardium demonstrated herein suggests that clinical translation of IPC drug delivery may be readily obtained. In a broader sense, this system provides a platform for delivery to other diseased tissues, as well, for other therapeutic regimens with a narrow therapeutic index.
In terms of clinical translation, the thericardium 10 and the disclosed methods of implantation and functional monitoring have a potential to prove extremely beneficial for enabling sustained delivery of the paracrine factors released from transplanted cells in close proximity to the diseased tissue of the heart 14 and for allowing further research studies of the effect of multiple administrations of cells that have previously been infeasible due to the prohibitive nature of multiple invasive surgeries. Furthermore, the thericardium 10 enables the multimodal localized delivery of different molecular therapies (e.g., cells, small molecules, and macromolecules) in an attempt to mimic or modify the inherently complex physiological and pathological processes in the heart 14. In addition to temporal control, multiple reservoirs 34 enable spatial control and multimodal treatment regimens to different parts of the heart 14; for example, delivery of pro-regenerative therapy to the left ventricle and anti-arrhythmic therapy to the left atrium. Finally, the reservoir(s) 34 may be implanted without therapy and filled with therapeutic cargo non-invasively after a certain amount of time. This procedure is advantageous for previously reported work with autologous stem cells, when, for example, a biopsy can be taken at the time of implantation of the thericardium 10; then, stem cells can be cultured and expanded and re-implanted through the thericardium 10 after a number of weeks or months without the need for an additional surgery. An encapsulated thericardium 10 may potentially enable the long-term de novo production and delivery of therapeutic paracrine factors from a transplanted cell source without the need for immunosuppressive regimens.
To maximize the potency of cell therapy, systems that can longitudinally monitor the viability and function of transplanted cells in vivo would be beneficial. The thericardium 10, described herein, can address this need by having additional utility as an enhanced imaging method for quantifying cell number on the heart 14. In this case, luciferase-expressing cells are used, and D-luciferin can be injected directly through the thericardium 10, requiring much less substrate and reducing the duration of time that the animal is under anesthesia. This capacity for enhanced imaging represents a considerable advantage in terms of convenience, cost, consistency and time taken to conduct animal imaging. It can allow imaging in less than five minutes with 50 μl/0.75 mg of D-luciferin, compared to IP injection that can require more than 45 minutes for D-luciferin circulation, and up to 3.5 ml/52.5 mg of D-luciferin, thereby facilitating more frequent imaging and a more accurate pharmacokinetic profile. This system can also be used, if desired, for injection of media or nutrients into the reservoir 34 to prolong cell survival. Biosensors can also be injected and retrieved locally to monitor biomarkers indicative of disease. With the rate-limiting membrane 78 surrounding the reservoir 34, microneedle technology can be used to allow direct injection into tissue. The potential of the system for monitoring and feedback is vast.
Finally, pressure-volume loop analysis has become the “gold standard” for measuring hemodynamic parameters in research models. Additionally, lessons from clinical trials show us that, although the ejection fraction (usually determined by echocardiography or magnetic resonance imaging) has been regarded as the gold standard for assessing outcomes, it may not be the most suitable for assessing the effects of cell therapy-pressure-volume loop analysis allows recording of multiple hemodynamic parameters that can be used for this purpose. Previous studies using a pressure-volume catheter, and the apical stick method terminated the experiment after measurements were conducted.
Here, we demonstrate a survival study that allows repeated measurements on the same animals, enabling a longitudinal study on an animal following the progression of post-myocardial-infarction necrosis, scarring and remodeling. The ability to follow disease progression and relate it to cell dose and viability, afforded by the thericardium 10, is a considerable advantage for assessing pre-clinical treatments and can potentially help to avoid the unpredictable efficacy of regenerative therapies when implemented in clinical trials. In addition to these advantages, this capacity allows the use of fewer animals for experimental groups. Although this approach has been reported using the carotid access methods previously but not, to the inventors' knowledge, for the apical stick (or so-called “open-chest” method), which is a much more straightforward procedure. A potential challenge with a repeated measurement technique in the carotid artery is thrombosis, where mechanical movement and scraping of the catheter can lead to endothelium damage, so coagulation is advantageously monitored. Advantages of the repeated apical stick method, as compared to carotid access, are that proper placement is easier to confirm, and carotid placement may be prohibited if the carotid is atherosclerotic (e.g., as in ApoE mice) or the aortic valve is calcified (e.g., hypertrophy and heart failure models).
We can draw the following six conclusions from this study: (i) implantation of the thericardium 10 on the heart 14 with a conduit 22 connecting the reservoir 34 to a subcutaneous access port 18 is possible in a rat model; (ii) the system enables non-invasive replenishment of cells to the thericardium reservoir 34 and improves cell number at the site; (iii) the system can be refined with another rate-limiting layer to enhance therapy selectivity; (iv) the technology also allows rapid, targeted delivery of macromolecules and small molecules directly to the site; (v) the implanted system constitutes a rapid, inexpensive and safe method for bioluminescent quantification of cell number by direct administration of an imaging substrate during in vivo imaging; and (vi) a method for longitudinal hemodynamic measurements using a pressure-volume catheter with the apical stick method can be used to quantify cardiac function in a survival animal model.
Inventive concepts described herein can also be incorporated into a variety of other embodiments, including the following.
Beyond the heart therapy applications, described above, the methods and apparatus described herein can be used in a variety of other applications, such as the following:
Further examples consistent with the present teachings are set out in the following numbered clauses:
In describing embodiments of the invention, specific terminology is used for the sake of clarity. For the purpose of description, specific terms are intended to at least include technical and functional equivalents that operate in a similar manner to accomplish a similar result. Additionally, in some instances where a particular embodiment of the invention includes a plurality of system elements or method steps, those elements or steps may be replaced with a single element or step; likewise, a single element or step may be replaced with a plurality of elements or steps that serve the same purpose. Further, where parameters for various properties or other values are specified herein for embodiments of the invention, those parameters or values can be adjusted up or down by 1/100th, 1/50th, 1/20th, 1/10th, ⅕th, ⅓rd, ½, ⅔rd, ¾th, ⅘th, 9/10th, 19/20th, 49/50th, 99/100th, etc. (or up by a factor of 1, 2, 3, 4, 5, 6, 8, 10, 20, 50, 100, etc.), or by rounded-off approximations thereof, unless otherwise specified. Moreover, while this invention has been shown and described with references to particular embodiments thereof, those skilled in the art will understand that various substitutions and alterations in form and details may be made therein without departing from the scope of the invention. Further still, other aspects, functions and advantages are also within the scope of the invention; and all embodiments of the invention need not necessarily achieve all of the advantages or possess all of the characteristics described above. Additionally, steps, elements and features discussed herein in connection with one embodiment can likewise be used in conjunction with other embodiments. The contents of references, including reference texts, journal articles, patents, patent applications, etc., cited herein are hereby incorporated by reference in their entirety; and appropriate components, steps, and characterizations from these references may or may not be included in embodiments of this invention. Still further, the components and steps identified in the Background section are integral to this disclosure and can be used in conjunction with or substituted for components and steps described elsewhere in the disclosure within the scope of the invention. In method claims, where stages are recited in a particular order—with or without sequenced prefacing characters added for ease of reference—the stages are not to be interpreted as being temporally limited to the order in which they are recited unless otherwise specified or implied by the terms and phrasing.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/21986 | 3/11/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62131385 | Mar 2015 | US |
