Docket Number: FDA-2012-N-1021
Docket Name: Food and Drug User Fee and Modernization Act; Notice to Public of Web Site Location of Fiscal Years Proposed Guidance Document
Dr. Andrew Rivard’s response to FDA invitation to comments on utilizing animal studies to evaluate the safety of organ preservation devices and solutions:
What are the potential limitations of an ex vivo model in assessing reperfusion injury, and how can these limitations be mitigated?
Reperfusion injury has been evaluated by innumerable studies over a 50 year period from the onset of cardiopulmonary bypass utilized in surgery. The primary problem in cardiopulmonary bypass is the adequate cardioplegic administration during cardiac arrest. Generally two accepted methods of cardioplegic are accepted, normothermic and hypothermic. Both are given intermittently due to the effluent from the coronary sinus obscuring the surgical site. Depending upon the surgical preference, the composition of the cardioplegia can be modified to include a variety of electrolytes, sugars, proteins, and autologous whole blood. A large meta-analysis of cold blood vs. cold crystalloid cardioplegia in 2012 found no difference in patient survival. Similar meta-analyses of warm vs. cold cardioplegia have also been published which again show no significant differences in survival.
Is there any difference between blood and crystalloid cardioplegia for myocardial protection during cardiac surgery? A meta-analysis of 5576 patients from 36 randomized trials. Sá MP, Rueda FG, Ferraz PE, Chalegre ST, Vasconcelos FP, Lima RC. Perfusion. 2012 Nov;27(6):535-46.
Is cold or warm blood cardioplegia superior for myocardial protection? Abah U, Garfjeld Roberts P, Ishaq M, De Silva R. Interact Cardiovasc Thorac Surg. 2012 Jun;14(6):848-55
Obviously, a clinical transplant trial whereby the donor heart is preserved in the conventional manner of hypothermic static storage vs. a new preservation method is the penultimate test of effectiveness whereby morbidity and mortality can be compared to published literature and the control results. If we take as a corollary the meta-analysis results of the cold vs warm clinical trials; there was no significant difference in long-term post operative cardiac outcomes, however warm cardioplegia was associated with an improved cardiac index, lower troponin and creatinine kinase levels.
How clinical results correlate to an ex vivo model which is studied over a period of a few hours, days, or weeks of examination is unclear. However, the cardiac function and cellular markers may represent the overall quality of the preservation method. Typically in heart preservation studies, one of four evaluation methods are utilized: orthotopic transplantation, Langendorff perfusion, isolated (4 chamber) working heart, and heterotophic transplantation. When examining these methods a variety of functional assessments can be made including: LV pressures, dp/dt, cardiac output, coronary flow, endothelial function.
The traditional Langendorff perfusion setup provides only flow to the aorta and coronary arteries with venous drainage from the coronary sinus. This type of setup does not generally allow for functional assessment of the LV. However, a balloon tipped catheter can be inserted in the LV for pressures assessments. In comparison a 4 chamber working heart is more complex and the cannulas and pumps must be arranged to provide both pre-load, and after-load of the ventricles.
Transplantation has limitations due to the high cost of the procedure, and rejection from unmatched organs. Whereas, an ex vivo evaluation allows examination of an isolated heart with fewer confounding factors.
Histological studies provide an insight into the ultrastructual cellular composition of the heart preservation quality. Light microscopy is a low cost and simple evaluation method to examine the effects of both the preservation and possibly reperfusion injury by taking serial samples over time with an ex vivo method. The obviously the heart cannot be biosied too many times. Electron microscopy is a demanding evaluation which may provide additional detail at the level of the mitochondria and outer cell membranes. Reperfusion injury is though to be due to an excessive amount of free radical production when energy substrate is provided by restoration of normal perfusion following donor heart preservation.
Perhaps the easiest method to assess reperfusion injury is to examine lactate levels of the effluent of the coronary sinus as a surrogate of the intrinsic myocardial acidosis. Alternatively, the pH of the myocardium can be measured directly with a needle probe. Other methods could include measuring troponin which is a byproduct of cardiomyocyte myofibril disruption. The limitations of this method for evaluation are that the timecourse to completely demonstrate the extent of tissue damage by Troponin T or Troponin I in the clinical setting can be greater than 6 hrs and last to up to 72 hrs. This timespan is often much greater than what can be tested in the ex vivo setting. Fortunately the absolute amount of Troponin released is proportional to the amount of cellular damage, the peak of which occurs between 24-36 hours, again this is generally much longer than an ex vivo study lasts.
In addition to markers for cell injury and function, histology, and the use of allogeneic blood during reperfusion, what measures can be taken to improve the data generated in an ex vivo model?
The stem of the question relates to either the accuracy of the scientific apparatus measuring the heart, or falsification of data generated. Presuming the measuring instruments are standardized, then that leaves data integrity as the remaining issue. An audit trail of the data using GLP standards would be reasonable. Perhaps submission of the raw data for evaluation could also be considered.
In an in vivo model, what are strategies to limit confounding factors, such as immunological responses and hemodynamic instability, from affecting the assessment of device-related reperfusion injury?
Tissue matching could be done prior to transplantation, or possible utilizing leukocyte depleted blood. Given the effort to do a transplant, the extra steps to tissue match could be considered for a long-term survival study. Again, the cost of doing a transplant is generally prohibitive for a company trying to demonstrate effectiveness of a new preservation method with the small market of heart transplantation. Heterotopic heart transplantation has been used in the past in small studies. This would probably be the best way to limit hemodynamic instablity due to orthotopic transplant complications. A heterotopic transplant typically is connected to the carotid artery and vein in the neck of the animal. This type of transplant allows the researcher to assess primarily for immediate graft failure or immunological modulations in xenotransplantation. Although, could be utilized as a method to evaluate organs preserved in novel ways with high risk for failure.
Heart Xenotransplantation: Historical Background, Experimental Progress, and Clinical Prospects. Murthy R, Bajona P, Bhama JK, Cooper DK. Ann Thorac Surg. 2016
Is there a perceived hierarchy of evidence regarding data obtained from an ex vivo model and those obtained from an in vivo model? Or rather, is itmore judicious to view the two models as complements of each other?
Certainly, a heart transplant is a tour de force and penultimate evaluation of a preservation method. Whether or not this is required prior to human use is unclear. If the FDA requires a pre-clinical heart transplant prior to clinical approval, this will significantly limit the ability of new technologies to be adopted clinically. An ex vivo method also has limitations as elucidated above, but is generally supportive of the entire evaluation.
What role does the risk of the device play in the utilization of in vivo and ex vivo models? Regarding specific experimental parameters (e.g.,length of preservation, total ischemic time), under what circumstances is it appropriate to test the worst-case scenario?
Given the extensive history of heart preservation using the Shumway method of cold ischemic static storage, all attempts should be done to compare the risk of a new preservation method to this well-established method. If the device is intended to preserve the heart to 24 hrs, then the experimental parameters must be extended to evaluate that situation. Also, if the harvesting methods vary from accepted standards (such as deceased donor procurement), then additional controls numbers should be obtained to balance the higher risk of post-preservation and post-transplant organ failure. It would not be acceptable to compare the data from a DCD donor preserved using a new method without understanding the risk of a transplant from a DCD donor preserved in the standard method. Just to highlight, there is a massive amount of cellular death that occurs in the first few minutes following global ischemia. The ability of a preservation method to “rescue” the heart is limited and most likely will be optimal at preserving what portion viable myocardium remains intact.
What are the organ-specific challenges in developing in vivo and ex vivo models to assess reperfusion injury?
My experience is limited to the heart, and the organ specific issues are highlighted above.
What approaches would improve the in vivo and ex vivo study designs to ensure the generation of sufficient, meaningful data while limiting the number of animals used in such studies?
Again, transplantation is a highly complex and demanding surgical procedure in the animal model and should be used sparingly. Perhaps this would be best utilized not in a research setting, rather than in training the surgeon for receiving the preserved heart and then proceeding to transplantation.
From a literature review of comparable cardiac transplantation studies the group sizes are typically 5-6 animals. A group size of 6, in a similarly designed study was used at the Alberts-Ludwigs-University in Freiburg, Germany. Other investigators at the Catholic University Leuven, Belgium and at the UTHSC San Antonio, TX have used ‘n’ numbers of 5 for similar studies. Furthermore, another group in Japan used an ‘N’ number of 6 for their study titled: “Cardiac Transplantation Following a 24hr Preservation using a Perfusion Apparatus” An ‘N’ number of 6 in each group is consistent with reports of similar research published. Our previous investigations using porcine hearts determined that an N of 9 is capable of demonstrating a pH difference with a p < .005.
A sample size calculation would be acceptable based upon prior published data for ex vivo evaluations. This should take in account sample sizes in the literature as well as the number of sample point. For example, taking more than one biopsy of the myocardium at a particular time point may not improve the meaningfulness of the data. So the methodology should be based upon clinically relevant outcomes and weighted toward data that can be measured in transplant patients.
To support a clinical trial application (IDE), we would like to see results from both ex-vivo and in-vivo models. Because there is more unknown/risk, we may ask the sponsor to conduct a staged clinical study with a small lead-in group, or a small feasibility study with only a few patients. What are your thoughts on this and if this does happen, are there additional clinical strategies (additional procedures/follow-ups) to mitigate safety risks in these patients?
A small clinical feasibility study with a few patients is likely less helpful than an appropriately powered clinical study. What if there is a death in the first patient immediately post-transplant. Is this evaluated any differently in a feasibility study vs. an appropriately powered clinical study? The adverse event would need to be reported to the IRB, FDA, and DSMB. Also, if the organ is damaged prior to transplant and the transplant is cancelled, then this too could be considered an adverse event because the organ would otherwise be transplanted with a morbidity and mortality matching the national standards. Finally a full scale randomized clinical trial would be clearly acceptable by heart transplant surgeons as evidence of safety of the new technique.
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