ABSTRACT
Difluorosialic acids (DFSAs) are potent inhibitors of viral neuraminidase that demonstrate activity against oseltamivir- and zanamivir-resistant strains of influenza. Unfortunately, low oral bioavailability precludes their development as second generation neuraminidase inhibitors for treating influenza as this leaves them unsuitable for use in an oral formulation. Herein is described the preparation of a series of DFSA prodrugs designed to increase oral bioavailability. These prodrugs were evaluated using a snapshot PK screen and stability tests, with successful candidates being further assessed with a full pharmacokinetic workup. These new prodrugs increased oral bioavailability by up to three times that seen for the parent DFSAs.
Subject(s)
Enzyme Inhibitors/chemistry , Neuraminidase/antagonists & inhibitors , Prodrugs/chemistry , Sialic Acids/chemistry , Viral Proteins/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Half-Life , Mice , Neuraminidase/metabolism , Prodrugs/chemical synthesis , Prodrugs/pharmacokinetics , Sialic Acids/chemical synthesis , Sialic Acids/pharmacokinetics , Viral Proteins/metabolismABSTRACT
BACKGROUND: Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However, resistance inevitably emerges against these therapies, particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE: High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels, p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer, neurosphere, and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics, 100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS: Daoy, ONS76, UW228, and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth, induced apoptosis, and sensitized cells to SHH agents. Notably, RSK expression is correlated with SHH patients. In mice, BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS: RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly, BI-D1870 crosses the BBB, acting as a scaffold for development of more long-lived RSK inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Cerebellar Neoplasms/genetics , Drug Resistance, Neoplasm/drug effects , Medulloblastoma/genetics , Pteridines/pharmacology , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacokinetics , Apoptosis/drug effects , Cell Line, Tumor , Cerebellar Neoplasms/enzymology , Child , Chromatography, Liquid , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Flow Cytometry , Hedgehog Proteins/antagonists & inhibitors , Humans , Immunoblotting , Male , Mass Spectrometry , Medulloblastoma/enzymology , Mice , Pteridines/pharmacokinetics , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Tissue Distribution , Transcriptome , TransfectionABSTRACT
We demonstrate here that cytarabine and daunorubicin, a standard drug combination used in the treatment of leukaemia, exhibits drug ratio-dependent synergistic antitumor activity in vitro and in vivo. A cytarabine:daunorubicin molar ratio of 5:1 displayed the greatest degree of synergy and minimum antagonism in a panel of 15 tumor cell lines in vitro. Co-encapsulating cytarabine and daunorubicin inside liposomes maintained the synergistic drug ratio in plasma for 24h post-injection. Liposome-encapsulated cytarabine:daunorubicin combinations exhibited drug ratio-dependent in vivo efficacy with the 5:1 molar drug ratio (designated CPX-351) having the greatest therapeutic index, despite using sub-MTD daunorubicin doses. CPX-351 exhibited superior therapeutic activity compared to free-drug cocktails, with high proportions of long-term survivors, consistent with in vivo synergy. The therapeutic advantage of CPX-351 was associated with prolonged maintenance of synergistic drug ratios in bone marrow. These results indicate that in vitro informatics on cytarabine:daunorubicin cytotoxicity can be translated in vivo to optimize the efficacy of anticancer drug combinations by controlling the exposure of drug ratios with drug delivery vehicles.
Subject(s)
Antineoplastic Agents/pharmacology , Cytarabine/pharmacology , Daunorubicin/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Proliferation , Cytarabine/administration & dosage , Cytarabine/pharmacokinetics , Daunorubicin/administration & dosage , Daunorubicin/pharmacokinetics , Flow Cytometry , Humans , Liposomes , MiceABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy. It has been described as requiring elevated autophagy for growth and inhibiting autophagy has been proposed as a treatment strategy. To date, all preclinical reports and clinical trials investigating pharmacological inhibition of autophagy have used chloroquine or hydroxychloroquine, which interfere with lysosomal function and block autophagy at a late stage. Verteporfin is a newly discovered autophagy inhibitor that blocks autophagy at an early stage by inhibiting autophagosome formation. Here we report that PDAC cell lines show variable sensitivity to verteporfin in vitro, suggesting cell-line specific autophagy dependence. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.
ABSTRACT
The first liposomal formulation of cisplatin to be evaluated clinically was SPI-077. Although the formulation demonstrated enhanced cisplatin tumor accumulation in preclinical models it did not enhance clinical efficacy, possibly due to limited cisplatin release from the formulation localized within the tumor. We have examined a series of liposomal formulations to address the in vivo relationship between cisplatin release rate and formulation efficacy in the P388 murine leukemia model. The base formulation of phosphatidylcholine: phosphatidylglycerol: cholesterol was altered in the C18 and C16 phospholipid content to influence membrane fluidity and thereby impacting drug circulation lifetime and drug retention. Phase transition temperatures (T(m)) ranged from 42-55°C. The high T(m) formulations demonstrated enhanced drug retention properties accompanied by low antitumor activity while the lowest T(m) formulations released the drug too rapidly in the plasma, limiting drug delivery to the tumor which also resulted in low antitumor activity. A formulation composed of DSPC : DPPC : DSPG : Chol; (35 : 35 : 20 : 10) with an intermediate drug release rate and a cisplatin plasma half-life of 8.3 hours showed the greatest antitumor activity. This manuscript highlights the critical role that drug release rates play in the design of an optimized drug delivery vehicle.
ABSTRACT
Irinotecan and cisplatin are two established anticancer drugs, which together constitute an effective combination for treating small-cell lung cancer. We investigated whether the efficacy of this combination could be improved by controlling drug ratios following in vivo administration. Irinotecan and cisplatin combinations were evaluated systematically for drug ratio-dependent synergy in vitro using a panel of 20 tumor cell lines. In vitro screening informatics on drug ratio-dependent cytotoxicity identified a consistently antagonistic region between irinotecan/cisplatin molar ratios of 1:2 to 4:1, which was bordered by two synergistic regions. Liposomal co-formulations of these two agents were developed that exhibited plasma drug half-lives of approximately 6 hours and maintained a fixed drug ratio for more than 24 hours. Drug ratio-dependent antitumor activity was shown in vivo for these liposome formulations, and irinotecan/cisplatin ratios between 5:1 and 10:1 were identified as therapeutically optimal. The relationship between irinotecan/cisplatin ratio and in vivo efficacy was consistent with in vitro drug ratio dependency results. Superior antitumor activity was observed for the liposome-encapsulated 7:1 molar ratio of irinotecan/cisplatin (designated CPX-571) compared with the free-drug cocktail in all models tested. Further efficacy studies in a range of human tumor xenografts, including an irinotecan-resistant model, showed that both liposomal agents contributed to the overall efficacy in a manner consistent with in vivo synergy. These results show the ability of drug delivery technology to enhance the therapeutic activity of irinotecan/cisplatin combination treatment by maintaining synergistic ratios in vivo. CPX-571, a fixed-ratio formulation of irinotecan and cisplatin, is a promising candidate for clinical development.