Firuz Feturi

Single Implantable FK506 Disk Prevents Rejection in Vascularized Composite Allotransplantation

Background: In vascularized composite allotransplantation, medication nonadherence leads to increased acute rejections. Improving medication adherence would improve overall allograft survival. Regionally delivered immunosuppression, targeted to sites of allorecognition, may reduce or eliminate the need for daily systemic immunosuppression. Methods: The authors developed biodegradable FK disks containing FK506-loaded double-walled microspheres and tested their efficacy at preventing rejection in a Brown-Norway–to-Lewis rat hindlimb transplantation model. In some experimental group animals, one FK disk was implanted subcutaneously either in native nontransplanted leg or in a transplanted allograft. Regular blood FK506 levels were measured. The endpoint was 180-day allograft survival or grade 3 rejection. At the endpoint, tissue FK506 levels were measured and mixed lymphocytic reaction was performed. Results: A single FK disk maintained systemic blood FK506 levels between 5 and 15 ng/ml for 146 ± 11.1 days. After that, the levels declined to less than 5 ng/ml through the endpoint. There was significantly increased FK506 concentration in groin lymph nodes draining the implanted FK disk. Compared with other groups, animals with an FK disk in the transplanted allograft had 100 percent allograft survival to more than 180 days despite subtherapeutic levels below 5 ng/ml. In these animals, significant T-cell hyporesponsiveness was seen in groin lymph nodes draining the FK disk compared with robust splenic T-cell proliferation. Conclusions: Sustained regional immunosuppression (with a single FK506 disk) maintained the allograft by means of a high regional concentration of FK506. Notably, this was achieved at subtherapeutic blood concentrations of FK506, without any further systemic FK506 administration.

An Elastomeric Polymer Matrix, PEUU-Tac, Delivers Bioactive Tacrolimus Transdurally to the CNS in Rat

Central nervous system (CNS) neurons fail to regrow injured axons, often resulting in permanently lost neurologic function. Tacrolimus is an FDA-approved immunosuppressive drug with known neuroprotective and neuroregenerative properties in the CNS. However, tacrolimus is typically administered systemically and blood levels required to effectively treat CNS injuries can lead to lethal, off-target organ toxicity. Thus, delivering tacrolimus locally to CNS tissues may provide therapeutic control over tacrolimus levels in CNS tissues while minimizing off-target toxicity. Herein we show an electrospun poly(ester urethane) urea and tacrolimus elastomeric matrix (PEUU-Tac) can deliver tacrolimus trans-durally to CNS tissues. In an acute CNS ischemia model in rat, the optic nerve (ON) was clamped for 10s and then PEUU-Tac was used as an ON wrap and sutured around the injury site. Tacrolimus was detected in PEUU-Tac wrapped ONs at 24 h and 14 days, without significant increases in tacrolimus blood levels. Similar to systemically administered tacrolimus, PEUU-Tac locally decreased glial fibrillary acidic protein (GFAP) at the injury site and increased growth associated protein-43 (GAP-43) expression in ischemic ONs from the globe to the chiasm, consistent with decreased astrogliosis and increased retinal ganglion cell (RGC) axon growth signaling pathways. These initial results suggest PEUU-Tac is a biocompatible elastic matrix that delivers bioactive tacrolimus trans-durally to CNS tissues without significantly increasing tacrolimus blood levels and off-target toxicity.

Inhibition of Androgen Receptor Function and Level in Castration-Resistant Prostate Cancer Cells by 2-[(isoxazol-4-ylmethyl)thio]-1-(4-phenylpiperazin-1-yl)ethanone

&NA; The androgen receptor (AR) plays a critical role in the development of castration‐resistant prostate cancer (CRPC) as well as in the resistance to the second‐generation AR antagonist enzalutamide and the selective inhibitor of cytochrome P450 17A1 (CYP17A1) abiraterone. Novel agents targeting AR may inhibit the growth of prostate cancer cells resistant to enzalutamide and/or abiraterone. Through a high‐throughput/high‐content screening of a 220,000‐member small molecule library, we have previously identified 2‐[(isoxazol‐4‐ylmethyl)thio]‐1‐(4‐phenylpiperazin‐1‐yl)ethanone (IMTPPE) (SID 3712502) as a novel small molecule capable of inhibiting AR transcriptional activity and protein level in C4‐2 prostate cancer cells. In this study, we show that IMTPPE inhibits AR‐target gene expression using real‐time polymerase chain reaction, Western blot, and luciferase assays. IMTPPE inhibited proliferation of AR‐positive, but not AR‐negative, prostate cancer cells in culture. IMTPPE inhibited the transcriptional activity of a mutant AR lacking the ligand‐binding domain (LBD), indicating that IMTPPE inhibition of AR is independent of the LBD. Furthermore, animal studies showed that IMTPPE inhibited the growth of 22Rv1 xenograft tumor, a model for enzalutamide‐resistant prostate cancer. These findings suggest that IMTPPE is a potential lead compound for developing clinical candidates for the treatment of CRPC, including those resistant to enzalutamide.

The impact of Topical Immunosuppressive Treatment on Functional Characterization of Skin Dendritic Cell Subpopulations in Vascularized Composite Allotransplantation

Introduction The high immunogenicity of skin has manifested as multiple acute rejection episodes in the majority of human vascularized composite allotransplantation (VCA) case. Skin dendritic cells (DC) are believed to play an important role in both the initiation and regulation of skin alloimmunity. Resident skin DC can migrate out of allogeneic skin into the recipient’s skin and draining lymph nodes, and may exert regulatory functions after VCA. Thus, isolating migratory skin DC and examining their effect on T-cell immune responses are parts of our continued efforts to optimize targeted immunomodulation. In the present study, we assessed the function of skin-resident and skin-migrated DC subsets on regulating the T-cell immune response and characterized their changes in VCA under topical FK506 immunosuppression. Materials and Methods 1) Hind-limb transplantation was performed from BN to Lewis rats and recipients were treated with topical FK506 (0.5mg/kg, 0.1% FK506 ointment, applied once daily). FK506 levels in blood and local skin were measured using liquid chromatography tandem-mass spectrometry. Skin-resident DC from transplanted limbs were isolated at day 8 post-transplantation and quantification of skin DC subsets was analyzed by flow cytometry. 2) Migrated skin DC from the limb of 7-day topical FK506 treated untransplanted and naïve Lewis rats were isolated in vitro after skin being cultured under a stimulation culture condition and were characterized by subsets and functional specialization using mixed lymphocyte reaction (MLR) and flow cytometric analysis. Results and Discussion 1) Limb allografts showed clinical signs of grade 2-3 rejection on day 8 post-transplantation. High skin levels (2110±177 ng/ml) and low blood levels (3.5±2.2 ng/ml) of FK506 were determined after 7-day topical treatment. Skin-resident dermal DC(DDC) declined with an elevation trend in Langerhans cells(LC) compared to no treatment group(Figure 1). Migrated LC and mature skin DC were lower, while DDC were slightly higher in FK506-treated skin compared to naïve skin(Figure 2). These data indicate the probable effect of topical immunosuppression on the modulation of skin DC subsets after VCA and skin DC migration and maturation could be inhibited by exposure to FK506. 2) In MLR, skin-migrated DC inhibited effector T cell (Teff) proliferation and exhibited synergistic effects with regulatory T cells (Treg). The addition of skin-migrated DC promoted Foxp3 expression in CD4+ T cells. In contrast, the addition of skin-resident DC decreased Foxp3 and promoted IL-17 expression, suggesting there is differential contribution of skin-migrated and skin-resident DC to the Teff response in vitro. Conclusion In vivo alterations of skin-resident DC subsets and ex vivo emigration and maturation of skin DC are affected by a short-term topical immunosuppression in VCA. The determination of skin-migrated DC in regulating T-cell immune responses will provide a target for immunomodulation. Figure. No caption available. American Association of Plastic Surgeons(AAPS)/Plastic Surgery Foundation(PSF) Academic Scholarship Award. The UPP Academic Foundation Research Grant.

Mycophenolic Acid for Topical Immunosuppression in Vascularized Composite Allotransplantation: Optimizing Formulation and Preliminary Evaluation of Bioavailability and Pharmacokinetics

Mycophenolic acid (MPA), is the active form of the ester prodrug mycophenolate mofetil (MMF). MMF is an FDA approved immunosuppressive drug that has been successfully used in systemic therapy in combination with other agents for the prevention of acute rejection (AR) following solid organ transplantation (SOT) as well as in vascularized composite allotransplantation (VCA). Systemic use of MMF is associated with gastrointestinal adverse effects. Topical delivery of the prodrug could thus provide graft-targeted immunosuppression while minimizing systemic drug exposure. Our goal was to develop a topical formulation of MPA with optimal in vitro/in vivo characteristics such as release, permeation, and tissue bioavailability to enable safety and efficacy evaluation in clinical VCA. Permeation studies were performed with a solution of MPA (10 mg/ml). In vitro release and permeation studies were performed for different semisolid formulations (Aladerm, Lipoderm, emollient, and VersaBase) of MPA (1% w/w) using a Franz Diffusion Cell System (FDCS). In vivo pharmacokinetic characterization of MPA release from Lipoderm was performed in rats. MPA in solution exhibited a steady state flux (3.8 ± 0.1 µg/cm2/h) and permeability (1.1 × 10−7 ± 3.2 × 10−9 cm/s). MPA in Lipoderm exhibited a steady state flux of 1.12 ± 0.24 µg/cm2/h, and permeability of 6.2 × 10−09 ± 1.3 × 10−9 cm/s across the biomimetic membrane. The cumulative release of MPA from Lipoderm, showed a linear single-phase profile with a R2 of 0.969. In vivo studies with MPA in Lipoderm showed markedly higher local tissue MPA levels and lower systemic MPA exposure as compared to values obtained after intravenous delivery of the same dose of drug (p < 0.05). We successfully developed for the first time, a topical formulation of MPA in Lipoderm with optimal in vitro/in vivo permeability characteristics and no undesirable local or systemic adverse effects in vivo. Our study provides key preliminary groundwork for translational efficacy studies of topical MPA in pre-clinical large animal VCA models and for effectiveness evaluation in patients receiving VCA.

Abstract 23: Ultrasound-mediated on-demand release from ionically cross-linked hydrogel: New approach for targeted Immunotherapy in Vascularized Composite Allotransplantation

23: Ultrasound-mediated ondemand release from ionically crosslinked hydrogel: New approach for targeted Immunotherapy in Vascularized Composite Allotransplantation The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Feturi, F. G., H. Wang, Y. Brudno, V. Erbas, L. Dong, H. Sahin, Z. Zhang, et al. 2017. “Abstract 23: Ultrasound-mediated ondemand release from ionically cross-linked hydrogel: New approach for targeted Immunotherapy in Vascularized Composite Allotransplantation.” Plastic and Reconstructive Surgery Global Open 5 (4 Suppl): 18-19. doi:10.1097/01.GOX.0000516544.50254.a1. http://dx.doi.org/10.1097/01.GOX.0000516544.50254.a1. Published Version doi:10.1097/01.GOX.0000516544.50254.a1 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33029990 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-ofuse#LAA

2561: Dynamics and correlates of skin dendritic cells with distinctive immune response profiles in vascularized composite allografts

2561: Dynamics and correlates of skin dendritic cells with distinctive immune response profiles in vascularized composite allografts Wensheng Zhang, MD, PhD, Chiaki Komatsu, MD, Firuz Gamal Feturi, BPharm, PhD, Lin He, MD, Liwei Dong, Jiaqing Wu, MD, PhD, Maxine Reedy Miller, MD, Jeffrey Mark Walch, MD/PhD, Alicia Mathers, PhD, Angus W. Thomson, Kia M. Washington, MD, Vijay Gorantla, and Mario Solari University of Pittsburgh, Pittsburgh, PA, USA Introduction Vascularized composite allografts (VCA) such as hand allografts, contains large amount of most immunogenic skin component Multiple acute rejection episodes in the majority of human VCA routinely target skin while sparing other tissue types Skin dendritic cells (DCs) are thought to play critical roles in either initiation or regulation of skin immunity However, their contribution to the unique alloimmune response and acute skin rejection of VCA is still largely unknown This study seeks to characterize the spatiotemporal dynamics of skin resident DCs at different stages of rejection after hind-limb transplantation and to better understand the role of skin DCs in immunomodulation of VCA. Materials and methods Lewis rats received allogeneic hind-limb transplants from BN rats without immunosuppressive treatment, and were inspected daily for their clinical signs of rejection using a visual scoring system Recipients were sacrificed at different time points post-transplantation Allograft skin, adjacent recipient skin, and draining lymph nodes were harvested and processed to obtain single cell suspension for flow cytometric analysis, or to extract total RNA and proteins and assessed by real-time PCR and Luminex. Results 1) We developed a new technique for isolation and characterization of skin DCs in rat hind-limb transplant model 2) Skin resident lymphocytes and distinct subsets of skin migratory DCs: Langerhans cells (LCs), dermal DCs (DDCs), and langerinC DDCs, in the transplanted limb were identified and enumerated 3) Skin migratory DCs in both allograft and recipient limb skin showed different patterns of change with increasing severity of rejection 4) The expression of Th1-, Th2-, Th17-, and Tregassociated genes and cytokines in allograft and recipient limb skin exhibited dynamic changes and temporal correlation with the quantity of skin DCs during the process of rejection. Conclusions The correlative trends between skin DC subsets and T cell-mediated alloimmune response suggest a complex cutaneous immune cell network modulated by skin DCs in VCA By understanding the dynamics of skin DCs and their influence on the T-cell response, the novel targeted immunomodulation therapy can be developed for VCA. Funding This work was supported by American Association of Plastic Surgeons Academic Scholarship. CONTACT Mario Solari solarimg@upmc.edu