Single Locally Implanted Tacrolimus Disk Promotes Long-term Vascularized Composite Allograft Survival via Site Specific Immunosuppression and without Systemic Side Effects
Firuz G. Feturi, PhD, Jignesh Unadkat, MD, Yong Wang, MD, Chiaki Komatsu, MD, Wensheng Zhang, MD, Jingjing Li, MD, Damian Grybowski, MD, Zhaoxiang Zhang, MD, Kia Washington, MD, Mario Solari, MD, Raman Venkataramanan, PhD, Alexander Spiess, MD.
University of Pittsburgh, Pittsburgh, PA, USA.
PURPOSE: More than 185,000 amputations occur in United States each year. The most common is a partial hand amputation (61,000), and the majority (77%) of cases are due to traumatic accidents. Vascularized composite allotransplantation (VCA), in the form of hand allotransplantation, provides another option for hand reconstruction. The skin component of VCA is highly antigenic and mandates daily intake of systemic immunosuppressive drugs. Currently tacrolimus (TAC) and mycophenolic acid (MPA) are the two primary immunosuppressive drugs used in transplant patients. These drugs have narrow therapeutic window, significant pharmacokinetic variability, non-adherence to these drugs, putting the patients at risk for rejection or toxicity. These drug related complications compromise the long-term outcomes. We prepared a novel, re-loadable drug delivery system that consists of an encapsulated sustained-release version of oral TAC alone or combined with other drugs that provides sustained drug release into the graft tissues and regional lymph nodes, while minimizing systemic blood levels. This results in lower overall systemic drug exposure, while the sustained loco-regional delivery facilitates long-term VCA survival.METHODS: TAC loaded polycaprolactone disk were prepared by solvent casting. Following orthotopic hind limb allotransplantation, animals (n=6/group) received no treatment (Group 1), TAC 1mg/kg/day intraperitoneally (Group 2), or one TAC disk in the transplanted limb (Group 3) or in the contralateral un-transplanted limb (Group 4). TAC levels in blood and tissues were measured using LC-MS/MS. In addition to allograft survival, systemic toxicity was evaluated using metrics such as % change in body weight (BW), blood glucose, and creatinine clearance (CrCl). RESULTS: A single TAC disk (5mg, 5 % w/w) resulted in blood levels between 2 to 5 ng/ml for nearly 100 days. High levels of TAC were achieved locally in the transplanted limbs, when compared to levels in the contralateral limbs (***p<0.001). These levels could inhibit immune activation and sustained allografts survival for >150 day (Group 3). While animals received no treatment or TAC disks in the un-transplanted limbs (Group 1 and 4) had median survival 8 ± 4 days and 71 ± 7 days. Long term allograft exposure to locally delivered TAC induced donor specific hypo-responsiveness. Lower levels of IFNα+ cytotoxic T cells, while higher levels of IL-10+ T regulatory cells were observed in draining lymph nodes isolated from transplanted limbs. This could suggest the mechanism behind long term survival by locally delivered TAC. No signs of systemic toxicity were observed in animals received TAC disks, as compared to animals received standard systemic immunotherapy. CONCLUSION: A single TAC disk implanted into the transplanted limb was effective in sustaining allograft survival via loco-regional immunosuppression, without systemic side effects. Therapeutic modulation of the loco-regional immunity may influence the outcome of VCA and underline that tissue-specific immune-response warrants further investigation in VCA. Our study offers an alternative to the current treatment paradigms which use systemic immunosuppression, to one of loco-regional immunosuppression using locally implantable biomaterials. With this research, we hope to establish the basis for the development of more advanced technologies for targeted immunosuppressive drug therapy.
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