Sherwin Xie

In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy

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.

Modulating the Therapeutic Activity of Nanoparticle Delivered Paclitaxel by Manipulating the Hydrophobicity of Prodrug Conjugates

A series of paclitaxel prodrugs designed for formulation in lipophilic nanoparticles are described. The hydrophobicity of paclitaxel was increased by conjugating a succession of increasingly hydrophobic lipid anchors to the drug using succinate or diglycolate cross-linkers. The prodrugs were formulated in well defined block copolymer-stabilized nanoparticles. These nanoparticles were shown to have an elimination half-life of approximately 24 h in vivo. The rate at which the prodrug was released from the nanoparticles could be controlled by adjusting the hydrophobicity of the lipid anchor, resulting in release half-lives ranging from 1 to 24 h. The diglycolate and succinate cross-linked prodrugs were 1-2 orders of magnitude less potent than paclitaxel in vitro. Nanoparticle formulations of the succinate prodrugs showed no evidence of efficacy in HT29 human colorectal tumor xenograph models. Efficacy of diglycolate prodrug nanoparticles increased as the anchor hydrophobicity increased. Long circulating diglycolate prodrug nanoparticles provided significantly enhanced therapeutic activity over commercially formulated paclitaxel at the maximum tolerated dose.

Prediction of nanoparticle prodrug metabolism by pharmacokinetic modeling of biliary excretion.

Pharmacokinetic modeling and simulation is a powerful tool for the prediction of drug concentrations in the absence of analytical techniques that allow for direct quantification. The present study applied this modeling approach to determine active drug release from a nanoparticle prodrug formulation. A comparative pharmacokinetic study of a nanoscale micellar docetaxel (DTX) prodrug, Procet 8, and commercial DTX formulation, Taxotere, was conducted in bile duct cannulated rats. The nanoscale (~40nm) size of the Procet 8 formulation resulted in confinement within the plasma space and high prodrug plasma concentrations. Ex vivo prodrug hydrolysis during plasma sample preparation resulted in unacceptable error that precluded direct measurement of DTX concentrations. Pharmacokinetic modeling of Taxotere and Procet 8 plasma concentrations, and their associated biliary metabolites, allowed for prediction of the DTX concentration profile and DTX bioavailability, and thereby evaluation of Procet 8 metabolism. Procet 8 plasma decay and in vitro plasma hydrolytic rates were identical, suggesting that systemic clearance of the prodrug was primarily metabolic. The Procet 8 and Taxotere plasma profiles, and associated docetaxel hydroxy-tert-butyl carbamate (HDTX) metabolite biliary excretion, were best fit by a two compartment model, with both linear and non-linear DTX clearance, and first order Procet 8 hydrolysis. The model estimated HDTX clearance rate agreed with in vitro literature values, supporting the predictability of the proposed model. Model simulation at the 10mg DTX equivalent/kg dose level predicted DTX formation rate-limited kinetics and a peak plasma DTX concentration of 39ng/mL at 4h for Procet 8, in comparison to 2826ng/mL for Taxotere. As a result of nonlinear DTX clearance, the DTX AUCinf for the Procet 8 formulation was predicted to be 2.6 times lower than Taxotere (775 vs. 2017h×ng/mL, respectively), resulting in an absolute bioavailability estimate of 38%. As DTX clearance in man is considered linear, this low bioavailability is likely species-dependent. These data support the use of pharmacokinetic modeling and simulation in cases of complex formulations, where analytical methods for direct measurement of free (released) drug concentrations are unavailable. Uses of such models may include interpretation of preclinical toxicology studies, selection of first in man dosing regimens, and PK/PD model development.

Abstract B34: Coordinated delivery of anticancer drug combinations incorporating molecularly targeted agents provides markedly increased plasma drug exposure, decreased toxicity and increased efficacy in preclinical tumor models

Background: Coordinated delivery of established chemotherapy combinations at synergistic drug ratios via nano-scale delivery vehicles has provided marked improvements in efficacy both preclinically and clinically. Many molecularly targeted agent (MTA) combinations have experienced difficulties in achieving optimal target inhibition without inducing dose limiting toxicities. Here we report the application of CombiPlex® technology to combinations incorporating MTAs for which optimal therapeutic effects require simultaneous tumor cell exposure. Methods: Prodrugs conjugates were synthesized for the HSP90 inhibitor AUY922 (AUY), docetaxel (DOC), the MEK inhibitor selumetinib (SEL) and Akt inhibitor ipatasertib (IPA) using cholesterol as a hydrophobic anchor joined via hydrolysable linkers that regenerate the parent drugs upon cleavage. These prodrugs were co-formulated in hydrophobic prodrug nanoparticles (HPN) by rapid mixing in the presence of surface stabilizing block co-polymers. Formulation compositions were iteratively optimized to achieve prolonged plasma drug concentrations while coordinating the PK of the combined agents. The tolerability and efficacy of the HPN combinations were compared to the free drug combinations administered in their conventional formulations. Results: AUY and DOC anchored to cholesterol via a diglycolate linker could be stably co-formulated in HPNs with a mean diameter of ∼65nm using a range of surface stabilizing block co-polymers. Using PLA-PEG block co-polymers, HPN-associated drugs exhibited no early distribution phase and virtually identical PK for AUY and DOC with a plasma half-life in mice of >12hr. Plasma concentrations of both agents over 24h were 2- to 4-orders of magnitude higher than for the free drugs. Similar PK results were obtained with the SEL:IPA combination. Combined IV treatment with DOC and AUY as the conventional free drugs led to significant increases in toxicity such that the DOC dose had to be reduced by 67% and the AUY dose had to be reduced by 60%. In contrast, when co-formulated in HPNs, both drugs could be administered with only a 30% dose reduction. In human xenograft tumor models (taxane resistant as well as taxane sensitive), the HPN formulation of AUY:DOC provided increased tumor growth inhibition; quantitative analysis of tumor growth delay revealed a 5-fold increase in antitumor activity compared to the free drug combination in the HCT15 model. A range of AUY:DOC drug ratio HPN formulations were also tested to identify the optimally efficacious drug ratio. HPN formulations of SEL:IPA also displayed improved tolerability compared to the free drug combination, particularly in view of the lower bioavailability for the agents administered in their oral dosing forms, while providing significant antitumor efficacy. Conclusions: HPN-mediated coordinated delivery of drug combinations incorporating molecularly targeted agents can favorably shift the PK/PD profile resulting in an improved therapeutic index. Initial results suggest this may expand the utility of combinations that to date have been limited by toxicities associated with dosing regimens aimed at ensuring simultaneous and durable multi-target inhibition. Citation Format: Lawrence D. Mayer, Paul Tardi, Sherwin Xie, Barry Liboiron, Winnie Lui, Leon Wan. Coordinated delivery of anticancer drug combinations incorporating molecularly targeted agents provides markedly increased plasma drug exposure, decreased toxicity and increased efficacy in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B34.

The Use of Radioactive Marker as a Tool to Evaluate the Drug Release in Plasma and Particle Biodistribution of Block Copolymer Nanoparticles

Diblock copolymer nanoparticles encapsulating a paclitaxel prodrug, Propac 7, have been used to demonstrate the usefulness of a nonmetabolizable radioactive marker, cholesteryl hexadecyl ether (CHE), to evaluate nanoparticle formulation variables. Since CHE did not exchange out of the nanoparticles, the rate of clearance of the CHE could be used as an indicator of nanoparticle stability in vivo. We simultaneously monitored prodrug circulation and carrier circulation in the plasma and the retention of CHE relative to the retention of prodrug in the plasma was used to distinguish prodrug release from nanoparticle plasma clearance. Nanoparticles labelled with CHE were also used to evaluate accumulation of nanoparticles in the tumour. This marker has provided relevant data which we have applied to optimise our nanoparticle formulations.

Abstract 5464: Determination of total and encapsulated drug pharmacokinetics for CPX-351, a nanoscale liposomal fixed molar ratio of cytarabine-daunorubicin (Cyt:Daun)

Background: CPX-351 is a liposomal formulation that delivers elevated concentrations of Cyt and Daun after administration in vivo and maintains the two drugs at a 5:1 molar ratio shown to be synergistic in preclinical studies. Pharmacokinetic analysis of CPX-351 in advanced leukemia patients demonstrated prolonged plasma exposure of the drugs with quantifiable concentrations of both agents being observed as long as 7 days after the final CPX-351 dose. Here we developed a method to separate liposome-bound from unencapsulated drugs, thereby allowing the pharmacokinetics of encapsulated agents to be determined in comparison to total plasma drug concentrations. Methods: CPX-351 was administered to female Sprague-Dawley rats via IV bolus injection. Plasma samples were harvested at five timepoints from 1 – 24 hours. Unencapsulated drug analysis was conducted by addition of internal standards (gemcitabine (Cyt) and doxorubicin (Daun)) to the plasma, followed by a 5-fold dilution in buffer and ultrafiltration using Microcon YM-30. Unencapsulated drug levels were determined by reversed-phase HPLC-MS (SIM, 244.2 m/z (Cyt) and 528.3 m/z (Daun)). Total analysis was conducted by protein precipitation and analysis was determined by reversed-phase HPLC-UV (Cyt) and reversed-phase HPLC-Fluorescence (Daun). Results: A separation method was developed to separate 0.22 – 4.33% unencapsulated Cyt and 0.13 – 2.50% unencapsulated Daun from liposome in plasma matrix. Encapsulated drug was calculated by subtracting the amount of unencapsulated analyte from the corresponding amount of total analyte. The total drug curves and the calculated encapsulated drug curves from 1 – 24 hours were superimposable for Cyt and Daun. A maximum of 1.9% unencapsulated Cyt and 0.4% unencapsulated Daun was measured in the plasma at any time point. Conclusions: Unencapsulated drug levels in rat plasma are insignificant compared to the total drug levels such that levels of total and encapsulated are within the spread of the data and therefore indistinguishable. These results suggest that CPX-351 remains in circulation in rats as intact liposomes at least 24 hours post administration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2011-5464

Abstract 5534: Liposome accumulation within leukemia engrafted bone marrow is significantly enhanced when the formulation contains cytarabine plus daunorubicin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC CPX-351 is a liposome formulation designed to deliver a synergistic, fixed ratio of cytarabine and daunorubicin in vivo. It has been shown to be highly efficacious against a variety of mouse leukemia models and encouraging evidence of anti-leukemic activity has been observed in clinical trials. In this report we investigate liposome biodistribution properties that may contribute to the enhanced efficacy of CPX-351. In order to model bone marrow leukemia development, we utilized the human leukemia CCRF-CEM tumor which has been shown to efficiently engraft to the bone marrow of SCID Rag2M mice. Liposomes were prepared in the presence or absence of cytarabine and daunorubicin to compare the plasma clearance and biodistribution to bone marrow, liver, lung, spleen and kidney following multiple treatments. The plasma clearance of empty liposomes and CPX-351 were similar in both leukemia and non-leukemia bearing mice. Both liposomal formulations had similar organ distribution profiles in the presence or absence of leukemia; however lipid delivery to the bone marrow was markedly augmented by the presence of encapsulated drug. Accumulation of CPX-351 lipid in the bone marrow was 20 to 50% higher than for empty liposomes and this phenomenon was further enhanced in leukemia bearing mice. Leukemia-laden bone marrow accumulated 75% more CPX-351 lipid than the empty liposomes on the first injection and accumulation increased an additional 20% with subsequent injections. We attribute the increased efficacy of CPX-351 over the free drug cocktail of cytarabine and daunorubicin to elevated exposure of the tumor tissue to chemotherapeutic agents. This increased exposure is not only due to the prolonged circulation of the drug in the liposomes, but also due to the increased bone marrow uptake of the liposomes when drugs are present. Additionally the lipid accumulation is further increased with successive treatments of the leukemia. The results suggest that the liposomal drugs alter either the bone marrow microenvironment or resident cell populations in a manner that facilitates subsequent uptake and/or retention of CPX-351. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5534.

Abstract 3698: Development of a hydrophobic docetaxel prodrug nanoparticle with enhanced plasma circulation lifetime and improved efficacy

Amphiphilic block co-polymers can form highly stable micellar-nanoparticles which can be used to trap hydrophobic chemotherapeutic agents. While block co-polymer formulations effectively retain drugs in vitro, nanoparticles frequently display burst release of trapped agents upon injection into animals. As a result, nanoparticles often show only limited improvements in drug circulation lifetime and likely provide little improvement in drug delivery to the tumor site. To address this issue, we previously generated hydrophobic prodrugs of paclitaxel to enhance retention within the nanoparticle and observed a correlation between drug circulation lifetime and the hydrophobicity of the anchor. When these formulations were evaluated for anti-tumor efficacy, the longest circulating paclitaxel prodrugs were the most efficacious. We have now extended our assessment to hydrophobic docetaxel prodrug formulations and have related plasma circulation characteristics of the docetaxel prodrug nanoparticles to suppression of tumor growth in mice bearing HT-29 human colorectal tumor xenographs. Docetaxel-docosanyl diglycolate (procet 7), and docetaxel-cholesteryl diglycolate (procet 8) were formulated in PS-PEG nanoparticles at a drug:polymer weight ratio of1:2 and injected i.v. into mice. The plasma half-life of the docetaxel prodrug was enhanced relative to the corresponding paclitaxel prodrug and significantly greater than docetaxel formulated in polysorbate 80. When dosed at their respective maximum tolerated dose (MTD), docetaxel, procet 7 and procet 8 nanoparticles resulted in a tumor growth delay (TGD) of 66%, 213% and 241% respectively. In addition docetaxel prodrugs administered at equimolar doses to docetaxel displayed greater antitumor activity than the free drug (77% and 105% TGD for procet 7 and procet 8 nanoparticles respectively). We attribute the increased potency of docetaxel prodrugs over paclitaxel prodrugs to the extended plasma circulation lifetimes of the former. These results confirm that hydrophobic docetaxel prodrugs can be formulated into block co-polymer nanoparticles to achieve prolonged circulation kinetics which result in enhanced therapeutic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3698.