Quantification of Unencapsulated Drug in Target Tissues Demonstrates Pharmacological Properties and Therapeutic Effects of Liposomal Topotecan (FF-10850).

PURPOSE: Quantifying unencapsulated drug concentrations in tissues is crucial for understanding the mechanisms underlying the efficacy and safety of liposomal drugs; however, the methodology for this has not been fully established. Herein, we aimed to investigate the enhanced therapeutic potential of a pegylated liposomal formulation of topotecan (FF-10850) by analyzing the concentrations of the unencapsulated drug in target tissues, to guide the improvement of its dosing regimen. METHODS: We developed a method for measuring unencapsulated topotecan concentrations in tumor and bone marrow interstitial fluid (BM-ISF) and applied this method to pharmacokinetic assessments. The ratios of the area under the concentration-time curves (AUCs) between tumor and BM-ISF were calculated for total and unencapsulated topotecan. DNA damage and antitumor effects of FF-10850 or non-liposomal topotecan (TPT) were evaluated in an ES-2 mice xenograft model. RESULTS: FF-10850 exhibited a much larger AUC ratio between tumor and BM-ISF for unencapsulated topotecan (2.96), but not for total topotecan (0.752), than TPT (0.833). FF-10850 promoted milder DNA damage in the bone marrow than TPT; however, FF-10850 and TPT elicited comparable DNA damage in the tumor. These findings highlight the greater tumor exposure to unencapsulated topotecan and lower bone marrow exposure to FF-10850 than TPT. The dosing regimen was successfully improved based on the kinetics of unencapsulated topotecan and DNA damage. CONCLUSIONS: Tissue pharmacokinetics of unencapsulated topotecan elucidated the favorable pharmacological properties of FF-10850. Evaluation of tissue exposure to an unencapsulated drug with appropriate pharmacodynamic markers can be valuable in optimizing liposomal drugs and dosing regimens.

FF-10850, a Novel Liposomal Topotecan Achieves Superior Antitumor Activity via Macrophage- and Ammonia-Mediated Payload Release in the Tumor Microenvironment.

Topotecan, an approved treatment for refractory or recurrent ovarian cancer, has clinical limitations such as rapid clearance and hematologic toxicity. To overcome these limitations and maximize clinical benefit, we designed FF-10850, a dihydrosphingomyelin-based liposomal topotecan. FF-10850 demonstrated superior antitumor activity to topotecan in ovarian cancer cell line-based xenograft models, as well as in a clinically relevant DF181 platinum-refractory ovarian cancer patient-derived xenograft model. The safety profile was also improved with mitigation of hematologic toxicity. The improved antitumor activity and safety profile are achieved via its preferential accumulation and payload release triggered in the tumor microenvironment. Our data indicate that tumor-associated macrophages internalize FF-10850, resulting in complete payload release. The release mechanism also appears to be mediated by high ammonia concentration resulting from glutaminolysis, which is activated by tumor metabolic reprogramming. In ammonia-rich conditions, FF-10850 released payload more rapidly and to a greater extent than liposomal doxorubicin, a currently approved treatment for ovarian cancer. FF-10850 significantly enhanced antitumor activity in combination with carboplatin or PARP inhibitor without detrimental effects on body weight in murine xenograft models, and demonstrated synergistic antitumor activity combined with anti-PD-1 antibody with the development of tumor antigen-specific immunity. These results support phase I investigation of FF-10850 for the treatment of solid tumors including ovarian cancer (NCT04047251), and further evaluation in combination settings.

Defective development of splenic and epidermal CD4+ dendritic cells in mice deficient for IFN regulatory factor-2.

Dendritic cells (DCs) play important roles in the initiation and regulation of immune responses. Although several subsets of DCs were identified according to their expression of surface molecules such as CD4, CD8, and CD11b, the regulatory mechanism for the development and homeostasis of these DC subsets remains unclear. Here we show that mice lacking IFN regulatory factor-2 (IRF-2(-/-) mice) exhibited a marked and selective defect in splenic CD4(+)CD11b(+)DCs, instead of CD8 alpha(+)CD11b(-)DCs that were reported to be missing in mice lacking the related transcription factor IRF-8. Furthermore, the numbers of epidermal Langerhans cells in IRF-2(-/-) mice were reduced at least in part because of the lack of the CD4(+)CD11b(+) subset. Studies with radiation bone marrow chimeras as well as in vitro retrovirus-mediated gene transduction showed that IRF-2 was required cell-autonomously for the development of myeloid-related DCs. Notably, these abnormalities in DCs diminished in mice lacking both IRF-2 and the IFN-alpha/beta receptor, indicating that IRF-2 acted through negatively regulating IFN-alpha/beta signals. In contrast, natural killer cells still showed developmental arrest in these double mutant mice, indicating that the mode of action of IRF-2 for CD4(+)DC development is distinct from that for natural killer cell development. Our current findings thus pointed to a previously unknown unique cell-type-selective multimode function of IRF-2 in the regulation of lymphohematopoiesis.

The CD8+ dendritic cell subset selectively endocytoses dying cells in culture and in vivo.

Dendritic cells (DCs) are able in tissue culture to phagocytose and present antigens derived from infected, malignant, and allogeneic cells. Here we show directly that DCs in situ take up these types of cells after fluorescent labeling with carboxyfluorescein succinimidyl ester (CFSE) and injection into mice. The injected cells include syngeneic splenocytes and tumor cell lines, induced to undergo apoptosis ex vivo by exposure to osmotic shock, and allogeneic B cells killed by NK cells in situ. The CFSE-labeled cells in each case are actively endocytosed by DCs in vivo, but only the CD8+ subset. After uptake, all of the phagocytic CD8+ DCs can form major histocompatibility complex class II-peptide complexes, as detected with a monoclonal antibody specific for these complexes. The CD8+ DCs also selectively present cell-associated antigens to both CD4+ and CD8+ T cells. Similar events take place with cultured DCs; CD8+ DCs again selectively take up and present dying cells. In contrast, both CD8+ and CD8- DCs phagocytose latex particles in culture, and both DC subsets present soluble ovalbumin captured in vivo. Therefore CD8+ DCs are specialized to capture dying cells, and this helps to explain their selective ability to cross present cellular antigens to both CD4+ and CD8+ T cells.