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.

A physiologically based pharmacokinetic and pharmacodynamic model for disposition of FF-10832.

This study aimed to quantitatively clarify the critical factors responsible for the superior antitumor efficacy of a liposomal gemcitabine (2,2-difluorodeoxycytidine; dFdC) formulation, FF-10832, compared with dFdC. The underlying hypothesis is the different exposure of tumors to its active metabolite, dFdC triphosphate (dFdCTP), between the two formulations. Therefore, physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for encapsulated and unencapsulated dFdC were constructed considering the tumor dFdCTP concentration as an index of antitumor activity. To estimate drug the parameters, the time profiles of encapsulated and unencapsulated dFdC in the blood and those of dFdC and dFdCTP in tumors were measured following the intravenous bolus administration of FF-10832 or dFdC. dFdC metabolism and transport in the liver S9 fraction and isolated hepatocytes, respectively, were experimentally determined. The tumor growth curve in a mouse xenograft model following the administration of FF10832 and dFdC was also used to construct the PD model. The sensitivity analysis of the PBPK/PD model revealed the critical factors affecting antitumor efficacy, which included the total and intratumor tissue uptake clearances for liposomal formulation and the cytidine deaminase and deoxycytidine deaminase activities in tumors. Thus, these parameters are potential biomarkers for predicting the efficacy of the liposomal formulation of dFdC.

Safety of daprodustat in patients with anemia of chronic kidney disease: A pooled analysis of phase 3 studies in Japan.

INTRODUCTION: Daprodustat is an approved treatment for anemia of chronic kidney disease (CKD) in Japan. METHODS: This post hoc analysis evaluated pooled safety data for daprodustat from 3 phase 3 Japanese studies in dialysis-dependent and nondialysis patients with anemia of CKD. RESULTS: Median drug exposure duration was 365 days for both daprodustat (N = 369) and injectable erythropoiesis-stimulating agent (ESA, N = 285). The incidence per 100 patient-years of on-therapy adverse events (AEs) was 363.1 and 306.4 in the daprodustat and ESA groups, respectively. The incidence per 100 patient-years of thromboembolic and retinal events were 5.55 and 6.91 (daprodustat) and 6.28 and 7.46 (ESA), respectively. Cardiovascular and malignancy events were similar between groups, although analysis of these were limited by sample size and study duration. CONCLUSION: The safety of daprodustat was comparable to ESA in this pooled analysis, although further large-scale research is needed to evaluate long-term risks including cardiovascular and malignancy events.

Endoplasmic Protein Nogo-B (RTN4-B) Interacts with GRAMD4 and Regulates TLR9-Mediated Innate Immune Responses.

TLRs are distributed in their characteristic cellular or subcellular compartments to efficiently recognize specific ligands and to initiate intracellular signaling. Whereas TLRs recognizing pathogen-associated lipids or proteins are localized to the cell surface, nucleic acid-sensing TLRs are expressed in endosomes and lysosomes. Several endoplasmic reticulum (ER)-resident proteins are known to regulate the trafficking of TLRs to the specific cellular compartments, thus playing important roles in the initiation of innate immune responses. In this study, we show that an ER-resident protein, Nogo-B (or RTN4-B), is necessary for immune responses triggered by nucleic acid-sensing TLRs, and that a newly identified Nogo-B-binding protein (glucosyltransferases, Rab-like GTPase activators and myotubularins [GRAM] domain containing 4 [GRAMD4]) negatively regulates the responses. Production of inflammatory cytokines in vitro by macrophages stimulated with CpG-B oligonucleotides or polyinosinic:polycytidylic acid was attenuated in the absence of Nogo-B, which was also confirmed in serum samples from Nogo-deficient mice injected with polyinosinic:polycytidylic acid. Although a deficiency of Nogo-B did not change the incorporation or delivery of CpG to endosomes, the localization of TLR9 to endolysosomes was found to be impaired. We identified GRAMD4 as a downmodulator for TLR9 response with a Nogo-B binding ability in ER, because our knockdown and overexpression experiments indicated that GRAMD4 suppresses the TLR9 response and knockdown of Gramd4 strongly enhanced the response in the absence of Nogo-B. Our findings indicate a critical role of Nogo-B and GRAMD4 in trafficking of TLR9.

Responses of macrophages to the danger signals released from necrotic cells.

The immune system maintains homeostasis by recognizing and responding to cell death caused by various stresses. The immune response is considered to be elicited by 'danger signals' released from necrotic cells. However, the identity of the danger signals remains elusive. In this study, we focused on the expression of chemokines by macrophages stimulated with necrotic cells. In mouse bone-marrow-derived macrophages, the chemokine monocyte chemoattractant protein (MCP)-3 was induced at both the mRNA and protein levels in response to heat-killed murine cells. The induction of MCP-3 was also observed in MyD88-deficient macrophages, indicating that Toll-like receptors and the IL-1 receptor are not involved in this response. Consistent with this observation, the activation of NF-κB was not detected in RAW264.7 macrophages stimulated with necrotic cells. Treatments with proteinase K, DNaseI or RNaseA did not affect the ' STIMULATING ACTIVITY': of necrotic cells. In contrast, treatment with apyrase, which removes phosphates from nucleoside tri- and di-phosphates, abolished the inducing activity. Purified UDP at 30 µM concentration elicited similar induction of MCP-3 in RAW264.7 macrophages. Small interfering RNA-mediated knock-down of the UDP receptor P2Y6 in RAW264.7 cells significantly reduced the induction of MCP-3 in response to necrotic cells, but not its induction by lipopolysaccharide. Furthermore, ectopic expression of the P2Y6 receptor in HEK293 cells conferred responsiveness to necrotic cells. These results suggest that UDP released by necrotic cells plays a critical role as an endogenous danger signal and that P2Y6 is required for the induction of MCP-3 in response to necrotic cells.

Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents.

Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G(2) checkpoint signaling. Because p53 is a key regulator in the G(1) checkpoint, p53-deficient tumors rely only on the G(2) checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G(2) DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.