Description of New More Effective Photochemotherapeutic Compounds for Improving Effectiveness of Extracorporeal Photopheresis

Introduction: Extracorporeal photopheresis (ECP) is a clinically used cell-based immunomodulatory therapy that involves exposure of peripheral blood autologous mononuclear cells to the photosensitizer 8-methoxypsoralen (8-MOP) and UVA radiation before reinfusion. ECP has demostrated efficacy in the treatment of multiple diseases caused by unregulated T cell populations such as graft-versus-host disease (GvHD). During UVA irradiation, 8-MOP covalently binds to DNA, inducing apoptosis. It has been previously reported that 8-MOP-mediated apoptosis triggers some immumodulatory effects that finally lead to the acquisition of peripheral immunotolerance, including the generation of tolerogenic dendritic cells and T regulatory cells (Treg), production of anti-inflammatory cytokines such as IL-10 and TGF-β and the deletion of effector CD4⁺ and CD8⁺ T cells in lymph nodes, mechanisms that account for the clinical benefit of this therapy. In the present work the in vitro activity and immunomodulatory effects mediated by 8-MOP in comparison with other two new photosensitizer (BB01 and BB02) has been analyzed, aiming to find novel more effective photochemotherapeutic compounds to use in ECP. Moreover, the therapeutic effectiveness of BB01 and BB02 by ECP was also evaluated in an experimental murine model of GvHD.

Methods: Human mononuclear cells (MNC) were incubated with increasing concentrations of 8-MOP, BB01 and BB02 and irradiated with UVA light. The MNC apoptosis percentage was measured by flow cytometry after 48h of culture at 37ºC. Also, mixed lymphocyte cultures (MLC) with either myeloid immature (iDC) or mature dendritic cells (mDC) and MNC treated with the different compounds and UVA radiation were performed. After 48h of MLC, CCL21-stimulated migration of iDC and mDC was studied. In other assays, after 6 days of MLC the proliferation of treated MNC, production of pro-inflammatory and anti-inflammatory cytokines, generation of tolerogenic DC and differentiation of Treg were analyzed. On the other hand, murine GvHD was induced after transplanting bone marrow cells and splenocytes from donor Balb/c mice into C57Bl/6J recipients previously conditioned with a lethal dose of 10 Gy. For doing the ECP procedure, splenocytes from separate cohorts of C57Bl/6J with ongoing GvHD were isolated 12 days after transplantation, incubated with the different compounds and UVA light and injected intravenously once a week for four weeks. Survival after transplantation was monitored daily and clinical GvHD was graded using a previously described score analyzing weight loss, posture, activity, skin integrity and fur texture.

Results: There was a significative increase of MNC apoptosis when usingBB02 (from 50 ng/ml, p<0,001) and BB01 (from 200 ng/ml, p<0,05) over that achieved using 8-MOP, as well as a significant upregulation of CCL21-promoted migration of iDC and lower migration of mDC, mainly with BB02 (p<0,05 and p<0,001, respectively). Moreover, MNC proliferation after MLC with allogeneic iDC or mDC was partially inhibited after treatment with these compounds (p<0,01), together with a significative reduction of levels of pro-inflammatory cytokines (p<0,01, p<0,001) and increase of anti-inflammatory IL-10 and TGF-β (p<0,01, p<0,001). In addition, analysis of the expression profile of DC’s maturation and co-stimulatory/MHC-II molecules, as well as modulation in CCR7 expression after MLC demonstrated a more tolerogenic phenotype. Also, iDC co-cultured with MNC pretreated with BB02 were more efficient than 8-MOP in the induction of autologous Treg (1,88-fold increase). Finally, mice treated weekly with BB02 showed a significant higher survival than those treated with 8-MOP (p<0,05), while BB01 had a similar effect to that of 8-MOP. Mice treated with either compound improved their clinical GvHD score compared to untreated mice group, being significantly lower with BB02 than with BB01 and 8-MOP (p<0,05).

Conclusions: BB01 and specially BB02 showed higher in vitro activity and in vivo effectiveness and could be considered as a possible better therapeutic alternative than 8-MOP.

Acknowledgments: Work financed by the Spanish Ministry of Economy and Competitiveness (Grant BFU2010-19599), together with FEDER Funds, and Spanish Net of Cell Therapy (TerCel) from Institute of Health Carlos III (RD07/0010/2012 and RD12/0019/0001).