Safety and Efficacy of a Novel Gel for Vascular Occlusion in Off-Pump Surgery

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1 Safety and Efficacy of a Novel Gel for Vascular Occlusion in Off-Pump Surgery Munir Boodhwani, MD, William E. Cohn, MD, Jun Feng, MD, PhD, Basel Ramlawi, MD, Shigetoshi Mieno, MD, Alexander Schwarz, PhD, and Frank W. Sellke, MD Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts, Division of Cardiothoracic Surgery, Texas Heart Institute, Houston, Texas, Pluromed Inc, Lincoln, Massachusetts Purpose. Coronary occlusion techniques used during off-pump coronary artery bypass surgery often provide suboptimal visualization and can damage the endothelium. We evaluated a novel gel with reverse thermosensitive properties for internal vessel occlusion during off-pump coronary artery bypass surgery. Description. Yorkshire pigs (n 6 per group) underwent two cycles of mid-left anterior descending coronary artery (LAD) occlusion using the gel (injected into the artery) or microvascular clamps (control group) followed by 30 minutes of reperfusion. Regional wall motion and LAD flow were monitored, microvessel relaxation responses were evaluated, and myocardial tissue was analyzed histologically. Evaluation. Complete left anterior descending coronary artery occlusion was successfully achieved using the gel (median ischemic time, 14 minutes; range, 4.5 to 24 minutes). Anterior wall motion abnormalities as well as flow patterns in the reperfused left anterior descending coronary artery were similar in both groups. Microvessel relaxation to substance P was mildly impaired ( % vs control; p < 0.001) in the left anterior descending coronary artery territory, but response to adenosine diphosphate and sodium nitroprusside was unaffected. Mild contraction band necrosis was present in both groups, consistent with mild ischemia-reperfusion injury. Conclusions. The gel represents a safe and effective method of vessel occlusion with a potentially important role in off-pump coronary artery bypass surgery. (Ann Thorac Surg 2005;80:2333 7) 2005 by The Society of Thoracic Surgeons The safety and efficacy of the conventional approach to surgical coronary revascularization using cardiopulmonary bypass is well established. However, off-pump coronary artery bypass surgery (OPCAB) has been associated with decreased morbidity in certain patient populations [1]. A major challenge in OPCAB is the lack of a motionless and bloodless surgical field for the performance of vascular anastomoses. Techniques and devices currently used to improve visibility during OPCAB include silastic snare sutures, vascular clamps, gas jet blowers, and intraluminal shunts, all of which are limited in their efficacy and can adversely affect the endothelium of the target vessel. The objective of our study was to evaluate the safety and efficacy of a novel gel containing purified Poloxamer 407 (Pluromed Inc, Lincoln, MA) for internal vessel occlusion. Poloxamer 407 (Pluromed Inc) is a water soluble polymer with reverse thermosensitive properties [2], Accepted for publication May 12, Address correspondence to Dr Sellke, Division of Cardiothoracic Surgery, Harvard Medical School, Beth Israel Deaconess Medical Center, 110 Francis St, LMOB 2A, Boston, MA 02215; fsellke@bidmc. harvard.edu. and therefore is at a soft gel consistency at room temperature and becomes a hard gel plug at normal body temperature. Although it has been demonstrated to be nontoxic at large doses, its feasibility for vessel occlusion during coronary surgery and its effect on the coronary vasculature has not been previously reported. We conducted an in-vivo evaluation of this gel in a porcine model of the left anterior descending coronary artery (LAD) ischemia and reperfusion. Technology Animals Animals were housed individually and provided with laboratory chow and water ad libitum. All experiments were approved by the Beth Israel Deaconess Medical Center Animal Care and Use Committee and the Harvard Medical Area Standing Committee on Animals (Institutional Animal Care and Use Committee) and Dr Schwarz discloses a financial relationship with Pluromed Inc by The Society of Thoracic Surgeons /05/$30.00 Published by Elsevier Inc doi: /j.athoracsur

2 2334 BOODHWANI ET AL Ann Thorac Surg NOVEL GEL FOR OFF-PUMP CORONARY SURGERY 2005;80: Fig 1. Duration of left anterior descending coronary artery (LAD) ischemia increased with increasing amounts of injected gel (Spearman r 0.64; p 0.02). ( occlusion time for varying quantities of gel.) conformed to United States National Institutes of Health guidelines regulating the care and use of laboratory animals (National Institutes of Health, Publication No , 1996). Technique Fig 2. Left anterior descending coronary artery (LAD) flow. Coronary flow patterns during LAD reperfusion were similar in treatment and control groups (p 0.51). (BL baseline; max maximum flow.) Fig 3. Regional wall motion. Regional myocardial function, as measured by percentage of segmental shortening normalized to baseline during ischemia and reperfusion, which was similar in treatment and control groups (p 0.89). (BL baseline; I ischemia, Rep reperfusion.) Yorkshire swine (30 to 35 kg; n 6 per group) were anesthetized with ketamine hydrochloride (20 mg/kg IM) and xylazine (15 mg/kg IM). After cannulation of the ear vein, they were intubated with a cuffed endotracheal tube and mechanically ventilated. Anesthesia was maintained using isoflurane gas throughout the procedure. The femoral artery was cannulated for arterial pressure monitoring and a median sternotomy was performed. A cathetertipped manometer was introduced through the left ventricular apex for pressure measurement, and four 2-mm ultrasonic crystals (Sonometrics Corp, London, Canada) were placed subepicardially in the distal LAD territory to monitor regional wall motion. A 2-mm ultrasonic coronary flow probe (Transonic Systems Inc, Ithaca, NY) was placed distal to the site of the LAD occlusion. The animals were heparinized (100 u/kg IV) and loaded with lidocaine (1.5 mg/kg IV). In the treatment group, occlusion of the mid-lad was performed by direct injection of a 20% solution of purified Poloxamer 407 (Pluromed Inc) using a 27-gauge needle in varying amounts (100 to 300 L). On contact with the warmer surrounding, the gel solidified causing LAD occlusion, and it was allowed to dissolve spontaneously. In the control group, an atraumatic microvascular clamp (Micro DeBakey Bulldog Clamp, Biomedical Research Instruments, Malden, MA) was used to induce 10 minutes of ischemia. This was followed by 30 minutes of reperfusion, and two cycles of ischemia and reperfusion were performed in each animal. At the end of the procedure, the animal was euthanized and the heart was harvested. Regional Myocardial Function and Coronary Flow Regional myocardial function was determined using 2-mm ultrasonic crystals that recorded segmental shortening at various time points during ischemia and reperfusion. Data was analyzed, as previously described [3], using CardioSoft (Sonometrics Corp) to determine the percentage of segmental shortening normalized to baseline in the longitudinal axis. Coronary flow was measured using a 2-mm ultrasonic flow probe (Transonic Systems Inc, Ithaca, NY), which was recorded at 5-minute intervals. Coronary Microvessel Studies Coronary arterioles (60 to 180 m internal diameter) were dissected from the left ventricular tissue of the ischemic and non-ischemic regions. Microvessel studies were performed by in-vitro organ bath videomicroscopy, as previously described [3], by an observer blinded to treatment assignment. Endothelium-dependent relaxation to adenosine diphosphate (10 9 to 10 4 mol/l), substance P (10 14 to 10 6 mol/l), and endothelium-independent

3 Ann Thorac Surg BOODHWANI ET AL 2005;80: NOVEL GEL FOR OFF-PUMP CORONARY SURGERY 2335 Fig 4. Microvessel reactivity was similar between treatment (dashed lines) and control (solid lines) groups in both (A) ischemic (LAD) and (B) non-ischemic (circumflex) territories to endothelium-dependent vasodilator, ADP, and endothelium independent vasodilator, SNP. Microvessel relaxation in response to endothelium-dependent vasodilator, substance P, was mildly impaired in treatment versus control groups in both ischemic and nonischemic regions (p 0.01). (ADP adenosine diphosphate; LAD left anterior descending coronary artery; SNP sodium nitroprusside.) relaxation responses to sodium nitroprusside (SNP; 10 9 to 10 4 mol/l) were examined. Histological Analysis Myocardial tissue from the LAD territory was fixed in 10% formalin at the time of harvest; it was sectioned and stained with hematoxylin and eosin. All specimens were examined by a pathologist blinded to the treatment assignment. Statistical Analysis Data are shown as means standard error of the mean and medians (minimum and maximum) as appropriate. Coronary flow, microvessel reactivity, and segmental shortening data were analyzed using analysis of variance. Statistical analysis was performed using the SAS software program, version 9.1 (SAS, Cary, NC). Graphs were also constructed using the GraphPad Prizm 4 software program (GraphPad Software Inc, San Diego, CA). Pre-Clinical Experience All animals successfully underwent the surgical procedure. Three animals in the control group and 4 in the treatment group required electrical cardioversion for episodes of ventricular fibrillation during LAD occlusion (p not significant). Injection of the gel led to successful occlusion of the LAD in all animals as determined by the coronary flow measurements. The median ischemic time was 14 minutes (range, 4.5 to 24 minutes) with a median dose of 200 L (range, 100 to 300 L) of purified Poloxamer 407 (Pluromed Inc) (Fig 1). Use of increasing amounts of gel correlated moderately with longer duration of ischemia (Spearman r 0.64; p 0.02). Due to persistent episodes of ventricular fibrillation during ischemia in 1 treatment animal, topical cooling at the injection site was used to dissolve the gel, immediately resuming flow in the LAD. Coronary Flow and Regional Myocardial Function The hyperemic response to ischemia was characterized by a transient increase in the LAD flow followed by a gradual return to baseline (Fig 2). The flow patterns were similar in both groups with a maximal flow increase of versus fold compared with baseline in the treatment versus the control group (p 0.51). Regional myocardial function was assessed using segmental shortening normalized to baseline in the long axis of the heart. Baseline measurements were similar in both groups. We observed anterior wall dyskinesis during the ischemic period, demonstrated by negative values for segmental shortening, which recovered almost completely during reperfusion (Fig 3). There was no difference in regional myocardial function between the treatment and control groups (p 0.89). Microvessel Relaxation Studies The endothelium independent relaxation response to sodium nitroprusside was preserved in both groups (Fig 4). In the ischemic LAD territory, the endothelium-dependent relaxation was mildly impaired in response to substance P

4 2336 BOODHWANI ET AL Ann Thorac Surg NOVEL GEL FOR OFF-PUMP CORONARY SURGERY 2005;80: ( % vs control group; p 0.001), but was preserved in response to adenosine diphosphate ( % vs control group; p 0.45). Endothelial-dependent response to substance P was also impaired in the nonischemic circumflex territory ( % vs control group; p 0.001), whereas responses to adenosine diphosphate and sodium nitroprusside were preserved. Histological Analysis Myocardial sections with hematoxylin and eosin stains revealed contraction band necrosis in the subendocardial layer of the ischemic territory in the treatment and control groups. Comment In this in-vivo experiment, we have demonstrated the feasibility of injected purified Poloxamer 407 (Pluromed Inc) for vascular occlusion. Direct injection of the gel into the LAD artery successfully caused temporary occlusion of the vessel. In summary, our results indicate that the gel does not cause abnormalities in coronary flow during reperfusion, does not adversely affect regional myocardial function, and has no effect on endotheliumindependent microvessel relaxation. The endotheliumdependent response to adenosine diphosphate was similar to the control group, whereas the response to substance P was mildly impaired. This impairment may be due to the properties of the gel, or due to longer ischemic time in the treatment group compared with the control group (14 vs 10 minutes). The presence of decreased microvessel relaxation to substance P in the nonischemic region perhaps argues against a local effect of the gel. Poloxamers are a broad group of nonionic surfactants [4] widely used in diverse industrial applications. These water-soluble, nontoxic, and inert surfactants are triblock copolymers of polyethylene oxide a polypropylene oxide b polyethylene oxide a [PEO a PPO b PEO a ]. Different hydrophilic-hydrophobic ratios and physical characteristics can be obtained by varying the block size and total molecular weight of the poloxamers. Their surfactant property has been useful in detergency, dispersion, stabilization, foaming, and emulsification. Some of these polymers have been considered for various cardiovascular applications [5]. In particular, Poloxamer 407 (Pluromed Inc), a polymer with a 70% ratio of polyoxyethylene and 30% polyoxypropylene, shows inverse thermosensitivity [2]. Therefore, aqueous polymer solutions greater than a critical concentration of about 12% are liquid at low temperatures, but will gel at higher temperatures. Formulations can be designed to allow the injection of a liquid or soft gel form through a small gauge needle, which will become a hard gel at body temperature. The gel plug erodes in blood, as the polymer is highly water-soluble, with the dissolution time depending on the amount of polymer injected and the concentration of the polymer in the solution. Poloxamer 407 (Pluromed Inc) has a reported acute systemic toxicity greater than 2.25 gram/kg, a noadverse-effect dose of approximately 400 mg/kg in dogs and rats, and possesses neither genotoxic nor mutagenic activity [6]. It is not metabolized and is excreted renally with a half-life of approximately 25 hours. Previous research [7] has shown that purified Poloxamer 407 (Pluromed Inc) can be utilized to occlude blood vessels for up to 2 hours. This report extends the initial findings to a specific application to occlude coronary arteries during beating heart surgery. Currently used techniques for improving visibility during off-pump coronary anastomoses are limited either by their lack of efficacy or by their potential to damage the endothelium of the target vessel. Vascular occlusive devices like snare sutures, which create a tourniquet around the vessel, and microvascular clamps are usually effective, except in the setting of severely diseased and calcified arteries. They inevitably cause mechanical trauma to the vessel wall and specifically, the use of clamps can lead to endothelial dysfunction [8]. Although intraluminal shunts offer the advantage of distal perfusion [9], an adequately sized shunt that provides a bloodless field can be cumbersome to insert with definite risk of endothelial damage. Gas jet blowers, using either air or carbon dioxide, are also effective methods, but these carry the risk of air embolism [10]. In addition to their safety and efficacy, the ease with which these techniques can be applied is a major determinant of their use, particularly with a minimally invasive or closed chest approach to coronary artery bypass. The advantages of Poloxamer 407 (Pluromed Inc) compared with existing technologies are its ease of administration, lack of significant endothelial dysfunction, and easy reversibility by cooling the affected area. In conclusion, we have demonstrated the safety of Poloxamer 407 (Pluromed Inc) use, as well as established its efficacy in our model. Limitations of this model include the disease-free coronary vasculature of the swine, absence of significant collateral circulation, and method of gel delivery into a closed artery. Further testing of this gel in clinical settings is needed for a more accurate evaluation of its efficacy. However, Poloxamer 407 (Pluromed Inc) has a potentially significant role in OPCAB surgery and may overcome limitations of existing technologies. Disclosures and Freedom of Investigation Financial support for this study was provided, in part, by Pluromed Inc (Lincoln, MA). Drs Boodhwani and Ramlawi are recipients of the Eugene Bard Fellowship. The authors had full control of study design, implementation, data analysis, and manuscript preparation. References 1. Al-Ruzzeh S, Ambler G, Asimakopoulos G, et al. Off-pump coronary artery bypass (OPCAB) surgery reduces riskstratified morbidity and mortality: a United Kingdom multicenter comparative analysis of early clinical outcome. Circulation 2003;108(Suppl 1):II1 8.

5 Ann Thorac Surg BOODHWANI ET AL 2005;80: NOVEL GEL FOR OFF-PUMP CORONARY SURGERY 2337 INVITED COMMENTARY This article [1] reports a prospective, experimental study in pigs of the use of a resorbable gel with reverse thermosensitive properties for temporary coronary artery occlusion. The gel is soft at room temperature and hardens at body temperature. Because the material is water soluble it dissolves in blood at a rate proportional to the amount and concentration of the gel applied. By measurement of blood flow, regional fractional myocardial shortening, and microvascular function, the gel was equivalent to occlusion with a standard microvascular clamp. The authors suggest that the gel may have a utility for vessel occlusion during off-pump coronary artery bypass graft surgery and may be less traumatic than mechanical occlusive devices like clamps or vessel loops. This interesting idea is well presented in this article. As the authors acknowledge, this preliminary study is limited by the fact that the experimental model is not truly analogous either to the diseased coronary circulation or to conditions during clinical off-pump surgery. Nonetheless, the idea is original and is likely to be of interest to surgeons committed to improving this technology. Among the questions that remain to be answered are the efficacy of this approach in diseased human coronary arteries in which collateral circulation may alter the quality and duration of vessel occlusion. Also, what is the proper technique for injection of the gel? Is there a risk of 2. Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv Drug Deliv Rev 2001;53: Khan TA, Ruel M, Bianchi C, et al. Poly(ADP-ribose) polymerase inhibition improves postischemic myocardial function after cardioplegia-cardiopulmonary bypass. J Am Coll Surg 2003;197: Marcel D. Nonionic surfactants: polyoxyalkylene block copolymers. In: Nace V, ed. Surfactant Science Series. New York: Marcel Dekker; 1996: Maynard C, Swenson R, Paris JA, et al. Randomized, controlled trial of RheothRx (poloxamer 188) in patients with suspected acute myocardial infarction. RheothRx in Myocardial Infarction Study Group. Am Heart J 1998;135: Material Safety Data Sheet for Poloxamer 407 (Pluronic F-127): BASF Corporation. 7. Raymond J, Metcalfe A, Salazkin I, Schwarz A. Temporary vascular occlusion with poloxamer 407. Biomaterials 2004;25: Perrault LP, Menasche P, Wassef M, et al. Endothelial effects of hemostatic devices for continuous cardioplegia or minimally invasive operations. Ann Thorac Surg 1996;62: Muraki S, Morris CD, Budde JM, et al. Preserved myocardial blood flow and oxygen supply-demand balance with active coronary perfusion during simulated off-pump coronary artery bypass grafting. J Thorac Cardiovasc Surg 2002;123: Nollert G, Oberhoffer M, Reichart B, Vicol C. Combination of the HEARTSTRING proximal seal system with a blower mister: a possible source of gas emboli. J Thorac Cardiovasc Surg 2003;126: Disclaimer The Society of Thoracic Surgeons, the Southern Thoracic Surgical Association, and The Annals of Thoracic Surgery neither endorse nor discourage use of the new technology described in this article. coronary injury even if a very fine gauge needle is used for injection? Can more gel be applied should the vessel open due to dissolution of the gel before the anastomosis is complete or if an additional suture is required? Is there any role for a catheter-based delivery system? Might gel fragments be a source of peripheral coronary emboli should the gel not dissolve at a uniform rate? Despite these remaining and potentially important unresolved issues, this is the type of incremental innovation that characterizes many complex techniques which are now considered the standard of practice in contemporary cardiac surgery. Verdi J. DiSesa, MD Section of Cardiac Surgery The Chester County Hospital 701 East Marshall St West Chester, PA vdisesa@cchosp.com Reference 1. Boodhwani M, Cohn WE, Feng J, et al. Safety and efficacy of a novel gel for vascular occlusion in off-pump surgery. Ann Thorac Surg 2005;80: by The Society of Thoracic Surgeons /05/$30.00 Published by Elsevier Inc doi: /j.athoracsur