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.

Site-specific immunosuppression in vascularized composite allotransplantation: prospects and potential.

Skin is the most immunogenic component of a vascularized composite allograft (VCA) and is the primary trigger and target of rejection. The skin is directly accessible for visual monitoring of acute rejection (AR) and for directed biopsy, timely therapeutic intervention, and management of AR. Logically, antirejection drugs, biologics, or other agents delivered locally to the VCA may reduce the need for systemic immunosuppression with its adverse effects. Topical FK 506 (tacrolimus) and steroids have been used in clinical VCA as an adjunct to systemic therapy with unclear beneficial effects. However, there are no commercially available topical formulations for other widely used systemic immunosuppressive drugs such as mycophenolic acid, sirolimus, and everolimus. Investigating the site-specific therapeutic effects and efficacy of systemically active agents may enable optimizing the dosing, frequency, and duration of overall immunosuppression in VCA with minimization or elimination of long-term drug-related toxicity.

Mouse hind limb transplantation: a new composite tissue allotransplantation model using nonsuture supermicrosurgery.

BACKGROUND: The development of microsurgical techniques has facilitated the establishment of vascularized composite tissue transplant models in small mammals. Because the mouse would be the ideal model to study various composite tissue allotransplantation (CTA)-related problems, we designed two new surgical techniques for orthotopic (ORT) and heterotopic (HET) hind limb transplantation. METHODS: BALB/c hind limbs were transplanted to BALB/c or C57BL6 recipients using a nonsuture cuff technique. ORT: donor femoral vessels were anastomosed to recipient femoral vessels, the sciatic nerve approximated end-to-end and osteosynthesis was performed using an intramedullary rod. HET/cervical: Donor femoral vessels of a reduced size osteomyocutaneous hind limb CTA were anastomosed to recipient common carotid artery and external jugular vein without nerve approximation. RESULTS: Both procedures could be performed with a high success rate (ORT: 62%; HET: 90%). Donor operation lasted for 100±12 min and recipient operation 114±27 min (ORT) and 54±16 min (HET). Complication rates in terms of bleeding, and thrombosis at the cuff side was slightly higher in the ORT group. All syngeneic grafts survived long term (>100 days). FK506 (2 mg/kg) significantly prolonged graft survival (87±22 days) when compared with untreated controls (6±1 day). Functional evaluation of ORT grafts by means of video gait kinematics and CatWalk analysis revealed specific differences of gait parameters when compared with nontransplanted controls (P<0.05). CONCLUSIONS: The ORT hind limb transplant model seems to be best suited to study functional outcome and nerve regeneration in CTA. The technically less demanding HET/cervical model may be used to investigate basic immunology and clinically relevant questions related to acute and chronic rejection, and ischemia reperfusion injury in reconstructive transplantation.