Receptor Targeted Delivery of the p38γ Inhibitor PIK-75 By Organic-Core Templated Lipid Nanoparticles in Cutaneous T Cell Lymphoma

Introduction: Patients with relapse/refractory cutaneous T cell lymphomas (CTCL) have limited therapeutic options. p38γ, part of the mitogen-activated protein kinases (MAPK) pathway, has been identified as a critical driver of CTCL. PIK-75, a known p110α inhibitor, was found to inhibit p38γ and induced cell death in CTCL cells in vitro and in vivo tumor xenograft models. However, PIK-75 is very hydrophobic and difficult to systemically deliver and achieve clinical efficacy. Therefore, designing a carrier to formulate and enable targeted cellular delivery of PIK-75 is a logical next step to facilitate translation.

We, and others, have shown that scavenger receptor type B1 (SR-B1) is often overexpressed in hematologic malignancies and can be targeted by synthetic lipid nanoparticles for efficient drug delivery. Our group pioneered the use of inorganic and organic core (oc) "templates” to synthesize lipid nanoparticles (LNP) that target SR-B1. The ocLNPs are an ideal construct for the delivery of hydrophobic drugs to malignant cells. Therefore, we hypothesized that ocLNPs could be adapted to formulate PIK-75 and deliver this drug via SR-B1 to CTCL cells to induce cell death.

Methods: A suite of organic core molecules were synthesized and tested for their ability to support the synthesis of PIK-75 loaded ocLNPs. The PIK-75 ocLNPs were synthesized by combining an organic core with PIK-75, phospholipids, and apolipoprotein A-I. The drug loaded ocLNPs were purified by dialysis and filtration. PIK-75 ocLNPs were characterized for size (size exclusion chromatography, dynamic light scattering and electron microscopy), surface charge (zeta potential) and drug loading. SR-B1 expression in primary CTCL samples was assessed by RNA sequencing. SR-B1 positive CTCL cell lines (HH and HuT78), and SR-B1 positive normal cells, HepG2 (hepatocytes) and THP-1 (macrophages), as well as SR-B1 negative PIK-75-sensitive U266B1 (myeloma) cells were used for these studies. Expression of SR-B1 in all cell lines was confirmed by western blot and flow cytometry. Cell viability was measured using the MTS assay. An SR-B1 blocking antibody was used to demonstrate the necessity of SR-B1 for PIK-75 ocLNP targeting and drug delivery.

Results: Nineteen out of 49 primary patient lesional CTCL skin samples (38.8%) demonstrated SR-B1 over-expression, defined as an expression level greater than two standard deviations above the mean expression measured in normal, CD3+ peripheral T cells using RNAseq data. Fourteen of the 19 samples (74%) were from patients with advanced disease (stage III and IV). PIK-75 was successfully incorporated into ocLNPs. Data showed that ~16 PIK-75 molecules were loaded per ocLNP. PIK-75 ocLNPs induced significant cell death in SR-B1 positive CTCL cell lines HH (IC₅₀ = 3.95 nM) and HuT 78 (IC₅₀ = 6.33 nM), while treatment of an PIK-75 sensitive, SR-B1 negative myeloma cell line (U266B1) with PIK-75 ocLNPs resulted in minimal cell death. Treatment of HH cells with PIK-75 ocLNPs for a 2 hour "pulse” followed by 72 hours of additional culture was completed to test active SR-B1 targeting. Data reveal significant cell death, which was significantly reduced by the addition of an SR-B1 blocking antibody (10 nM PIK-75 ocLNPs, viability 31.6% ± 4.3% w/o blocking antibody vs viability 83.6% ± 8.4% w/ blocking antibody). Treatment of hepatocytes and macrophages, both known to express SR-B1, with PIK-75 ocLNPs revealed no cell death even after 72 hours of culture.

Conclusions: ocLNPs can be successfully loaded with drug cargo, in this case hydrophobic PIK-75, enabling active delivery via SR-B1 in CTCL. PIK-75 ocLNPs potently induced cell death in CTCL cells while having minimal negative effects on hepatocytes or macrophages. These results provide proof-of-principle that ocLNPs can be successfully leveraged to deliver hydrophobic small molecule drugs by actively targeting SR-B1.

Querfeld:Trillium: Membership on an entity's Board of Directors or advisory committees, Other: Clinical Investigator; Bristol Meyer Squibb: Other: Clinical Investigator; Celgene: Research Funding; Helsinn: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Mallinckrodt: Membership on an entity's Board of Directors or advisory committees; Bioniz: Membership on an entity's Board of Directors or advisory committees. Rosen:January Therapeutics: Current holder of stock options in a privately-held company. Gordon:BMS: Research Funding; Zylem: Current equity holder in private company, Current equity holder in publicly-traded company, Patents & Royalties: Patent on nanoparticles for lymphoma therapy; Janssen: Other: DSMB; Ono Pharmaceuticals: Consultancy.