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.

Direct evidence for efficient transport and minimal metabolism of L-cephalexin by oligopeptide transporter 1 in budded baculovirus fraction.

The oligopeptide transporter PEPT1 (SLC15A1) is responsible for absorption of peptidic nutrients in the small intestine. Although the L-diastereomer of the beta-lactam antibiotic cephalexin (L-cephalexin) is likely to be transported by PEPT1, there has been no direct demonstration of PEPT1-mediated L-cephalexin transport. Indeed, after the incubation with L-cephalexin, the intact form of L-cephalexin has not been identified inside vesicles/proteoliposomes prepared from brush border membrane of intestinal epithelial cells or cultured cell lines exogenously transfected with PEPT1 gene. Thus, it appears that L-cephalexin is rapidly metabolized by PEPT1 or PEPT1-associated proteins. Here, we attempted to verify whether L-cephalexin is transported by PEPT1 and whether it is hydrolyzed by PEPT1 itself, by using budded baculovirus expressing PEPT1 protein. Marked uptake of L-cephalexin in PEPT1-expressing budded baculovirus, compared with wild-type virus, indicated that L-cephalexin is a substrate for PEPT1. The uptake was found to be pH sensitive, and was strongly inhibited by the D-diastereomer of cephalexin and glycylsarcosine, but not by glycine. Thus, L-cephalexin is transported by PEPT1 itself. Upon the transport of both L- and D-cephalexin by PEPT1, dose-dependent membrane depolarization was observed; the EC(50) values of 0.18 and 2.9 mM, respectively, indicate that the affinity of L-cephalexin for PEPT1-mediated transport is much higher than that of the D-diastereomer. On the other hand, the L-cephalexin metabolite 7-aminodesacetoxycephalosporanic acid was not detected in PEPT1-expressing or wild-type virus at either pH 6.0 or 7.4. We conclude that L-cephalexin is transported by PEPT1 with high affinity, but is not metabolized by PEPT1 itself.