Mantle Cell Lymphoma (MCL) is a sub-type of non-Hodgkin’s lymphoma characterized by the genetic translocation t(11;14)(q13;q32). This translocation leads to over-expression of cyclin D1, a hallmark feature of MCL. Whereas cyclin D1 is a regulator of cell division, its over-expression is associated with pathological cell proliferation and t(11;14) remains a marker for the disease. The clinical course of MCL is aggressive, with poor responsiveness to conventional chemotherapy and infrequent long-term remission. Other than allogeneic stem cell transplant, no curative therapy exists. Because treatment for MCL has been less successful than for other B-cell lymphomas and because we are beginning to better understand MCL biology, Lypro has focused its first Targeted NanoDisk program on the treatment of MCL.
The characteristic feature of MCL, over-expression of cyclin D1, led us to explore potential pharmacologic strategies to down regulate this gene. A strong candidate that emerged was all trans retinoic acid (ATRA), a naturally occurring vitamin A derivative that serves as a nuclear hormone receptor ligand. We examined the effect of ATRA on apoptosis of cultured MCL cells and saw a concentration dependent induction of apoptosis that was accompanied by down regulation of cyclin D1 (Singh et al., 2010). Whereas these data provide validation that MCL cells respond to ATRA, generalized systemic distribution following oral administration of ATRA imposes obstacles that hinder its use as a therapeutic. ATRA is difficult to solubilize, yet formulation of ATRA as NanoDisk confers water solubility while retaining biological activity. While generalized systemic administration of pharmacologic amounts of ATRA-ND is feasible using ND, optimal therapeutic benefit can best be achieved through targeted delivery of ATRA to MCL cells.
In collaboration with Dr. Leo I. Gordon, M.D., a renowned lymphoma clinician and Professor of Oncology Research at the Feinberg School of Medicine at Northwestern University, we have demonstrated that NanoDisk targeted to the CD20 cell surface antigens and loaded with ATRA (ATRA-tND) confers specificity and increases potency. Looking first at cell viability, ATRA-tND at concentrations as low as 5 uM, induces cell death and outperforms free drug and untargeted ATRA-ND (Figure 1). The addition of a CD20 targeting moiety results in specific binding to CD20 expressing cells (Figure 2, Pagel B). Microscopy studies demonstrate that the bioactive agent is internalized into the cell (Figure 2, Panel C & D). Since ATRA is not intrinsically fluorescent, the water-insoluble, naturally fluorescent hydrophobic bioactive polyphenol, curcumin, was used. Remarkably, Targeted curcumin-ND binding to cultured MCL cells facilitates bioactive agent off-loading, transit across the cell membrane and access to the cell interior. It is expected that other, nonfluorescent hydrophobic bioactive agents, including ATRA, will behave in a similar manner such that Targeted ATRA-ND binding to CD20 on the surface of MCL cells will facilitate ATRA transit to the cell interior.
A targeted therapy that combines cytotoxicity and specificity will outperform current clinical options.
Figure 1: Targeted NanoDisk enhance cytotoxicity of ATRA in cell culture model of MCL
Figure 1: Effect of ATRA-tND on total cell death in Granta and Jeko cells. Two MCL cell lines, Granta and Jeko, were cultured with ATRA-tND and ATRA-DMSO. Untargeted ATRA-ND data can be found in Singh et al, 2010. Apoptosis and necrosis (total cell death) was monitored by flow cytometry.
Figure 2: Targeted ND binding promotes bioactive agent internalization
Panel A: nuclei
Panel B: tND scaffold detection
Panel C: Bioactive agent
Panel D: Merge
Figure 2. The series of images reveal tND bind to the cell surface with bioactive agent internalization. Alexa 680 2° antibody was used to detect tND scaffold binding to cells by confocal microscopy. Experiments were performed at 37℃. Since ATRA is not intrinsically fluorescent, the water-insoluble, naturally fluorescent hydrophobic bioactive polyphenol, curcumin, was used. (Singh et al 2011)