Doxorubicin Concentration In Tumor Research Articles

Combination of Focused Ultra Sound and MnO2/GOD Loaded Microbubble Nanoparticles in Targeted Tumor Therapy of Breast Cancer

In order to increase the absorption of anticancer medications into malignancies in a noninvasive and targeted way, focused ultrasound in conjunction with microbubble (MB) might improve drug delivery. Here, we test two exemplary focused ultrasound-driven drug delivery enhancement approaches (MB and Doxorubicin-coated nanoparticle complex using MnO2/GOD) and confirm their acute drug delivery augmentation. Experiments were performed utilizing the MDA-MB-231 breast cancer cell line in an in vivo animal model. We observed that MB containing Doxorubicin-coated nanoparticle using MnO2/GOD complex increased Doxorubicin concentrations in tumors. These findings suggest that MB and MB nanoparticle based anticancer drug delivery may considerably enhance the efficacy of anticancer medications given to tumors in a short period of time using low-intensity focused ultrasound.

Pharmacokinetic study comparing doxorubicin concentrations after chemoembolization or intravenous administration in dogs with naturally occurring nonresectable hepatic carcinoma.

BackgroundChemoembolization is a viable treatment option for patients with nonresectable hepatic carcinoma (HC) and may allow delivery of chemotherapeutic drugs with decreased systemic toxicity.Hypothesis/ObjectiveCompare the serum concentrations of doxorubicin after chemoembolization or IV administration in the same patient. We hypothesized that locoregional delivery may result in increased tumor chemotherapeutic drug concentrations, reflected by decreased measurable serum drug concentrations. Adverse hematological events were hypothesized to be decreased after locoregional delivery.AnimalsSeventeen client‐owned dogs with incompletely resectable HC.MethodsProspective, single‐arm clinical trial. Drug‐eluting bead transarterial chemoembolization was performed to varying levels of blood flow stasis (NO STASIS, STASIS). Intravenous doxorubicin (IVC) subsequently was administered in selected patients. Systemic exposure was quantified by area under the serum doxorubicin concentration time curve (AUC), maximum serum doxorubicin concentration (Cmax), and time doxorubicin was last above the limit of quantitation (Tlast). Nadir test results after treatments were used to evaluate adverse hematological events.ResultsThirteen NO STASIS treatments, 15 STASIS treatments, and 9 IVC treatments were performed. Maximum serum doxorubicin concentration, AUC, and Tlast were significantly lower when comparing NO STASIS or STASIS to IVC treatments. Of the patients with nadir results available, no adverse hematological events were observed after NO STASIS or STASIS treatments. Two patients developed adverse hematological events after IVC treatment.Conclusions/Clinical RelevanceDrug‐eluting bead transarterial chemoembolization offers a viable treatment option for patients with incompletely resectable HC with the potential for increased local tumor doxorubicin concentrations, decreased systemic chemotherapeutic exposure, and fewer adverse hematological events.

Bacterial proteolytic activity improves drug delivery in tumors in a size, pharmacokinetic, and binding affinity dependent manner – A mechanistic understanding

Motile bacteria are able to penetrate in the distal areas of blood vessel, which makes bacteria attractive to researchers as a drug delivery vehicle carrying anti-cancer drugs to tumors. Not only therapeutic bacteria show wide anti-tumor effect but also the combination of therapeutic bacteria and conventional chemotherapy leads to dramatically large synergetic effect. We provide a mechanistic understanding of enhanced drug delivery in tumors by co-administration of chemotherapeutic agents and therapeutic bacteria. In this work, simultaneous delivery of C. novyi-NT and chemotherapeutic agents in tumors is mathematically modeled. Simulated doxorubicin concentration in tumors after Doxil administration with or without bacteria agreed reasonably well with experimental literature. Simulated doxorubicin concentration in tumors by the combination of Doxil and C. novyi-NT is over twice higher than that of Doxil alone. This enhanced doxorubicin concentration in tumors is due to the degradation of extracellular matrix of collagen by bacterial proteolytic activity, which increases hydraulic conductivity of interstitium, reduces interstitial fluid pressure, and thus increases convection through vessel walls. Additionally, it alleviates solid stress, which decompresses blood vessels, and thus increases vessel density. On the other hand, simulated doxorubicin concentration in tumors for non-liposomal free-doxorubicin is not enhanced by C. novyi-NT because vascular permeability of free-doxorubicin is larger than Doxil, and thus increased but relatively small convection across vessel walls is offset by the efflux due to increased interstitial flow. A strategy to further enhance this combination therapy is discussed along with sensitivity analysis.

Interleaved Mapping of Temperature and Longitudinal Relaxation Rate to Monitor Drug Delivery During Magnetic Resonance-Guided High-Intensity Focused Ultrasound-Induced Hyperthermia.

Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) is a method to heat lesions noninvasively to a stable, elevated temperature and a well-suited method to induce local hyperthermia (41°C-43°C) in deep-seated tissues. Magnetic Resonance (MR) imaging provides therapy planning on anatomical images and offers temperature feedback based on near-real-time MR thermometry. Although constant acquisition of MR thermometry data is crucial to ensure prolonged hyperthermia, it limits the freedom to perform measurements of other MR parameters, which are of interest during hyperthermia treatments. In image-guided drug delivery applications, co-encapsulation of paramagnetic MR contrast agents with a drug inside temperature-sensitive liposomes (TSLs) allows to visualize hyperthermia-triggered drug delivery through changes of the longitudinal relaxation rate R1. While the drug accumulates in the heated tumor tissue, R1 changes can be used for an estimate of the tumor drug concentration. The main objective of this study was to demonstrate that interleaved MR sequences are able to monitor temperature with an adequate temporal resolution and could give a reasonable estimate of the achieved tumor drug concentration through R1 changes. To this aim, in vitro validation tests and an in vivo proof-of-concept study were performed. All experiments were performed on a clinical 3-T MR-HIFU system adapted with a preclinical setup. The validity of the R1 values and the temperature maps stability were evaluated in phantom experiments and in ex vivo porcine muscle tissue. In vivo experiments were performed on rats bearing a 9L glioma tumor on their hind limb. All animals (n = 4 HIFU-treated, n = 4 no HIFU) were injected intravenously with TSLs co-encapsulating doxorubicin and gadoteridol as contrast agent. The TSL injection was followed by either 2 times 15 minutes of MR-HIFU-induced hyperthermia or a sham treatment. R1 maps were acquired before, during, and after sonication, using a single slice Inversion Recovery Look-Locker (IR-LL) sequence (field of view [FOV], 50 × 69 mm; in-plane resolution, 0.52 × 0.71 mm; slice thickness, 3 mm; 23 phases of 130 milliseconds; 1 full R1 map every 2 minutes). The R1 maps acquired during treatment were interleaved with 2 perpendicular proton resonance frequency shift (PRFS) MR thermometry slices (dynamic repetition time, 8.6 seconds; FOV, 250 × 250 mm; 1.4 × 1.4 mm in-plane resolution; 4 mm slice thickness). Tumor doxorubicin concentrations were determined fluorometrically. In vitro results showed a slight but consistent overestimation of the measured R1 values compared with calibrated R1 values, regardless whether the R1 was acquired with noninterleaved IR-LL or interleaved. The average treatment cell temperature had a slightly higher temporal standard deviation for the interleaved PRFS sequence compared with the noninterleaved PRFS sequence (0.186°C vs 0.101°C, respectively). The prolonged time in between temperature maps due to the interleaved IR-LL sequence did not degrade the temperature stability during MR-HIFU treatment (Taverage = 40.9°C ± 0.3°C). Upon heat treatment, some tumors showed an R1 increase in a large part of the tumor while other tumors hardly showed any ΔR1. The tumor doxorubicin concentration showed a linear correlation with the average ΔR1 during both sonications (n = 8, Radj = 0.933), which was higher than for the ΔR1 measured after tumor cooldown (averaged for both sonications, n = 8, Radj = 0.877). The new approach of interleaving different MR sequences was applied to simultaneously acquire R1 maps and PRFS thermometry scans during a feedback-controlled MR-HIFU-induced hyperthermia treatment. Interleaved acquisition did not compromise speed or accuracy of each scan. The ΔR1 acquired during treatment was used to visualize and quantify hyperthermia-triggered release of gadoteridol from TSLs and better reflected the intratumoral doxorubicin concentrations than the ΔR1 measured after cooldown of the tumor, exemplifying the benefit of interleaving R1 maps with temperature maps during drug delivery. Our study serves as an example for interleaved MR acquisition schemes, which introduce a higher flexibility in speed, sequence optimization, and timing.

A Totally Absorbable Multilayer PLGA Implant Device Containing Doxorubicin Inhibited Tumor Growth and Metastasis without Systemic Toxicity in Murine Breast Cancer and an Ideal Pharmacological Paradigm for Regional Chemotherapy

We hypothesize that a cylinder implant made of multilayer Poly-lactic-co-glycolic-acid (PLGA) membrane can be a method for controlled and extended drug release. We fashioned a multilayer cylindrical implant termed STID100 that released doxorubicin for 3 weeks in an orthotopic 4T1 breast cancer model in Balb/C mice. This implant starts as a thin doxorubicin-embedded PLGA membrane, and is then rolled into a cylinder containing an air gap between the membrane layers. Its controlled sustained release delivered 2× the amount of the intravenous (IV) equivalent of doxorubicin, inhibited the primary tumor, and prevented lung metastasis. Importantly it did not cause weight loss, splenomegaly, or cardiac toxicity vs systemically administrated doxorubicin. This favorable safety profile is further substantiated by the finding of no detectable plasma doxorubicin in multiple time points during the 3-week period, and low tumor doxorubicin concentration. The implant system delivered to the specification of an ideal pharmacological paradigm might offer a better coverage of the local tumor, significantly preventing metastatic spread with less drug toxicity to many vital organs, compared to the traditional pharmacology of IV route. The profile of STID made it an attractive therapeutic alternative in metastatic tumor prevention, pain management and many other diverse clinical scenarios.

Abstract 4516: The effects of microbeam radiation therapy on the pharmacokinetics of PEGylated liposomal doxorubicin in a triple negative breast cancer GEM model

Abstract Background: Carrier-mediated agents (CMAs), such as nanoparticle and liposomes, can achieve greater exposure in tumors compared with small molecule drugs; however, the overall percent of CMAs that distribute from the plasma to tumor is relatively low (e.g. <10-20%). Thus, there is a need to discover methods of improving the overall delivery of CMAs to tumors. Microbeam radiation therapy (MRT) is a novel radiation therapy treatment that has been shown to eradicate tumor without damaging the function of normal tissues exposed to the same high dose radiation in animal studies. MRT utilizes an array of parallel microplaner beams delivering periodically oscillating high and low dose regions in the treatment volume. Our preliminary results suggest that MRT alters tumor microvasculature differently than normal tissue microvasculature. We hypothesize that the changes in microvasculature induced by MRT can be used to enhance drug delivery to tumors. Thus, we evaluated the effects of MRT on the pharmacokinetics (PK) of PEGylated liposomal doxorubicin (PLD) in preclinical tumor models. Methods: Studies were performed in mice bearing p53 null syngeneic orthotopic tumor transplants. This model, named “T11”, has been characterized by gene expression array to reflect the claudin-low subtype of triple-negative breast cancer. Prior to administration of PLD, mice received a single treatment of 28Gy or 34Gy (peak dose at skin entrance) MRT to the tumor site (treatment area is ∼1cm x 1cm), or no radiation treatment. All groups were then administered PLD at 6 mg/kg IV x 1 at 16 h after MRT. Mice (n = 3) were sacrificed at 5 min, 6 h, and 24 h after PLD. Mice treated with 34 Gy MRT were only sacrificed at 24 h after PLD. The PK of PLD in plasma and tumor was evaluated. Results: Mice treated with 28 Gy MRT prior to PLD administration and PLD alone had a total doxorubicin tumor AUC of 206,040.0 ng/mL•h and 55,506.5 ng/mL•h, respectively. After treatment with 28 Gy and 34 Gy MRT the concentration of total doxorubicin in tumor at 24 h after administration of PLD were 10,665.8 ng/ml and 20,779.0 ng/ml, respectively. The plasma PK of PLD was similar in all groups. Conclusions: MRT treatment prior to PLD administration significantly improves the delivery of PLD to tumors without altering the plasma PK. In addition, higher doses of MRT resulted in a greater increase in the delivery of PLD to tumors. The improved delivery of PLD to tumors may be due to MRT's acute effects on tumor microvasculature; however, the exact mechanism needs to be further evaluated. Future studies include evaluation of the effect of duration and dose of MRT on drug uptake in tumor, effects of MRT on other CMAs, and the mechanisms of enhanced tumor delivery. Citation Format: Xiao S. Chang, Andrew Jacob Madden, Judith Rivera, Charlene Santos, David Darr, Lucas Hunter, William C. Zamboni. The effects of microbeam radiation therapy on the pharmacokinetics of PEGylated liposomal doxorubicin in a triple negative breast cancer GEM model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4516. doi:10.1158/1538-7445.AM2015-4516

WE‐EF‐BRA‐09: Microbeam Radiation Therapy Enhances Tumor Drug Uptake of PEGylated Liposomal Doxorubicin (PLD) in a Triple Negative Breast Cancer GEM Model

Purpose:Overcoming low anti‐cancer drug uptake in tumors is a key challenge limiting its clinical use. We propose to enhance the drug delivery using upfront Microbeam Radiation Therapy (MRT). MRT is a preclinical cancer therapy that utilizes microplanar beams to deliver spatially oscillating planes of high and low doses. Animal studies have demonstrated that ultrahigh dose (100s Gy) MRT eradicates tumors without damaging the function of normal tissue exposed to the same radiation. Our previous study indicated that MRT induces intense angiogenesis in tumor rim and surrounding normal tissue 1–2 days post radiation. We hypothesize that the tumor microenvironment modulation induced by MRT may enhance carrier‐mediated agent drug delivery to tumors with inherent poor drug uptake. We thus investigated MRT‐induced pharmacokinetics (PK) of PEGylated liposomal doxorubicin (PLD), a nano‐scale doxorubicin, in T11 genetically engineered mouse model of triple negative breast cancer.Methods:A research irradiator (160kVp, RadSource Technologies) with a customized collimator was used to produce the MRT microbeam of in average 390µm width and 1190µm peak‐to‐peak distance. The peak dose rate of 1–2Gy/min. Dosimetry is by EBT3 film cross‐calibrated with ion chamber at large fields. All mice were administered PLD at 6mg/kg IV x1 at 16h post MRT and sacrificed at 5min, 6h, 24h, and 96h post PLD administration (n=3 or 4 per group).Results:The MRT(28Gy)+PLD group mice had a total doxorubicin tumor concentration (area‐under‐the concentration‐curve, AUC) of 206,040ng/mL•h, 3.71 times the concentration of the PLD‐alone group. The MRT(34Gy)+PLD group had a higher mean total doxorubicin concentration in tumor (20,779ng/ml) than the MRT(28Gy)+PLD group (10,665ng/ml).Conclusion:Our preliminary results indicate that microbeam radiation therapy (MRT) can enhance nano‐scale anti‐cancer drug delivery to tumors approximately 4‐fold. The exact working mechanism, the comparison with broad beam irradiation, and optimization of MRT for drug delivery enhancement are under investigation.

Enhancement in blood-tumor barrier permeability and delivery of liposomal doxorubicin using focused ultrasound and microbubbles: evaluation during tumor progression in a rat glioma model

Effective drug delivery to brain tumors is often challenging because of the heterogeneous permeability of the ‘blood tumor barrier’ (BTB) along with other factors such as increased interstitial pressure and drug efflux pumps. Focused ultrasound (FUS) combined with microbubbles can enhance the permeability of the BTB in brain tumors, as well as the blood–brain barrier in the surrounding tissue. In this study, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to characterize the FUS-induced permeability changes of the BTB in a rat glioma model at different times after implantation. 9L gliosarcoma cells were implanted in both hemispheres in male rats. At day 9, 14, or 17 days after implantation, FUS-induced BTB disruption using 690 kHz ultrasound and definity microbubbles was performed in one tumor in each animal. Before FUS, liposomal doxorubicin was administered at a dose of 5.67 mg kg−1. This chemotherapy agent was previously shown to improve survival in animal glioma models. The transfer coefficient Ktrans describing extravasation of the MRI contrast agent Gd-DTPA was measured via DCE-MRI before and after sonication. We found that tumor doxorubicin concentrations increased monotonically (823 ± 600, 1817 ± 732 and 2432 ± 448 ng g−1) in the control tumors at 9, 14 and 17 d. With FUS-induced BTB disruption, the doxorubicin concentrations were enhanced significantly (P < 0.05, P < 0.01, and P < 0.0001 at days 9, 14, and 17, respectively) and were greater than the control tumors by a factor of two or more (2222 ± 784, 3687 ± 796 and 5658 ± 821 ng g−1) regardless of the stage of tumor growth. The transfer coefficient Ktrans was significantly (P < 0.05) enhanced compared to control tumors only at day 9 but not at day 14 or 17. These results suggest that FUS-induced enhancements in tumor drug delivery are relatively consistent over time, at least in this tumor model. These results are encouraging for the use of large drug carriers, as they suggest that even large/late-stage tumors can benefit from FUS-induced drug enhancement. Corresponding enhancements in Ktrans were found to be variable in large/late-stage tumors and not significantly different than controls, perhaps reflecting the size mismatch between the liposomal drug (~100 nm) and Gd-DTPA (molecular weight: 938 Da; hydrodynamic diameter: ≃2 nm). It may be necessary to use a larger MRI contrast agent to effectively evaluate the sonication-induced enhanced permeabilization in large/late-stage tumors when a large drug carrier such as a liposome is used.

Improvements of Doxorubicin-Induced Antitumor Activity and Adverse Reaction by Combined Citrulline

Citrulline is an amino acid with antioxidant activity. In this study, effects of citrulline on the adverse effects of doxorubicin (DOX) and DOX-induced antitumor activity were examined. Citrulline significantly inhibited DOX-induced increases in lipid peroxide (LPO) in the heart as adverse reaction. Combined treatment with DOX and citrulline increased DOX levels in tumor cells and enhanced cytotoxicity in vitro by significantly increasing DOX uptake compared with DOX treatment alone. In simultaneous in vivo treatments, combination treatment with DOX and citrulline decreased tumor weight and increased DOX concentrations in tumors. Promotion of DOX uptake by citrulline enhanced the antitumor activity of DOX through the action of energy-independent and Na(+)-independent transporters. This effect of citrulline on DOX influx is identical to that of S-(4-nitrobenzyl)-6-thioinosine, promoting DOX influx through the equilibrative nucleoside transporter 1. Therefore, it is anticipated that citrulline as a food component may enhance DOX efficacy.

Abstract 4515: Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin.

Abstract [Purpose] Polyethyleneglycol (PEG) modification on liposomal membrane increases therapeutic index by improvement of targeting based on prolonged blood stream circulation. In contrast, some accumulation of drug after liposome treatment has also been observed in normal tissues, it is necessary to improve liposomal composition on the decrease of adverse reactions. Particularly, doxorubicin (DOX) encapsulated liposome has some adverse reactions as cardiac toxicity or hand-foot syndrome,etc. To contribute superior liposome formulation, different double arms PEG in a molecule(DDA-PEG) was synthesized and was clarified effectiveness in our previous reports. In this study, we examined physical properties of novel synthesized DDA-PEG, and the tissue distribution of the DOX encapsulated liposome with modification of these DDA-PEGs. [Methods] Two DDA-PEGs with (PEG1000 and PEG500) or (PEG2000 and PEG500) were synthesized. Critical micelle concentration (cmc) in 10 mM Tris-HCl buffer (pH7.4) and 10 mM lactate buffer (pH4.0) was measured by the method of florescent probe using the 8-anilinonaphthalene-1-sulfonate (ANS). Octanol-buffer partitioning value was determined. DOX encapsulated liposomes with the modification of each DDA-PEGs were prepared by DSPC/cholesterol/DSPG-Na/DOX/PEG-lipid =100:100:60:18:7.5 μmol according to the method of Bangham. M5076 ovarian sarcoma cells were transplanted into the backs of BDF1 mice, and each liposome was injected intravenously at a dose of 2.5 mg DOX/kg on 15, 18 and 21 day after tumor inoculation. The mice were sacrificed after 2 day from last administration, and the tumor was removed and weighted. Moreover, DOX concentrations in each tissue were determined. [Results and discussion] The cmc of all PEG-lipids were same level. In PEG(2000, 500)-modified liposome, DOX concentrations in tumors were equal to that in PEG2000-modified liposome. On the other hand, DOX concentration in the heart after PEG(2000, 500) modified liposome and PEG(1000,500)-modified liposome treatments were one second and one sixth compared with DOX solution group, respectively. According to these results, it was expected that novel DDA-PEG-modified liposome was able to reduce the cardiac toxicity which is adverse reaction by DOX. The DOX concentration in the liver was showed a low level by PEG (1000, 500) modified liposome. It was suggested that novel DDA- PEG-modified liposome had the effect of avoiding the uptake into the liver. In conclusion, it was suggested that PEG(1000,500) modified liposome could decrease the DOX level in heart, and had ability of accumulation into tumor and avoidance from reticuloendothelial cells as same as PEG2000-modified liposome as known liposome. Citation Format: Mai Yagi, Ikumi Sugiyama, Yasuyuki Sadzuka. Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2013-4515

Abstract No. 330 - Comparison of serum doxorubicin levels following different DEB chemoembolization techniques as well as systemic administration in the same canine patients with naturally-occurring hepatocellular carcinoma

Purpose While the precise technique for DEB administration has not been completely elucidated, the most recent recommendations suggest delivering the beads as a drug delivery system and maintaining tumor perfusion rather than continuing to complete tumor stasis that may induce further angiogenesis. It is unclear how intratumoral and systemic doxorubicin levels are affected by DEB delivery performed with or without vascular stasis. Avoiding embolization may facilitate drug washout, reduce tumor doxorubicin concentrations, and increase systemic exposure to chemotherapy. We hypothesized that there would be a decrease in systemic doxorubicin exposure following DEB versus systemic chemotherapy and chemoembolization to stasis versus delivery without stasis. Materials and Methods Dogs diagnosed with naturally occurring nonresectable HCC were scheduled to receive three separate treatments ~5 weeks apart containing the same systemic dose of doxorubicin; 1) 100-300 DEB delivered without stasis, 2) 100-300 DEB delivery followed by Beadblock to stasis, and 3) intravenous doxorubicin. Serum doxorubicin levels were measured postoperatively. Results Serum doxorubicin levels were detectable for a median of 180, 30, and 1080 minutes following delivery without stasis (5 dogs), with stasis (7 dogs), and intravenously (4 dogs), respectively. Median maximum serum doxorubicin levels, median area-under-the-curve systemic doxorubicin levels, and median relative systemic doxorubicin exposures are reported in the Table . Conclusion Different DEB chemoembolization techniques may achieve different serum doxorubicin concentrations and therefore different intratumoral drug concentrations. When performing DEB chemoembolization in dogs with naturally occurring hepatocellular carcinoma, delivery to stasis results in the lowest systemic doxorubicin exposure (and presumably higher intratumoral concentrations) than when embolization to stasis in not performed.

In Vivo Evaluation of Doxorubicin-Loaded (PEG)3-PLA Nanopolymersomes (PolyDoxSome) Using DMBA-Induced Mammary Carcinoma Rat Model and Comparison with Marketed LipoDox™

To evaluate in vivo doxorubicin-loaded (PEG)(3)-PLA nanopolymersomes (PolyDoxSome) using 7,12-dimethyl benz[α]anthracene (DMBA)-induced mammary carcinoma rat model compared to marketed formulation LipoDox™. Sprague Dawley female rats with mean tumor volume of about 2cm(3) were used for pharmacokinetics, biodistribution, antitumor efficacy and toxicity studies. This study demonstrates that PolyDoxSome has higher AUC (569 vs. 4h*μg/mL), longer plasma circulation half life (21.9 vs. 0.49h), decreased clearance (10.5 vs. 1579mL/h/kg) and volume of distribution (137.7 vs. 1091mL/kg) as compared to free doxorubicin. Tissue distribution profile showed increased doxorubicin concentration in tumor and decreased concentration in heart as compared to free doxorubicin. The toxicity studies as measured from liver function tests, cardiac enzyme assays, hematology test and body weight has demonstrated that it is better tolerated than free doxorubicin. When PolyDoxSome was compared with LipoDox™, it differs in size (171 vs. <100nm), plasma circulation half life (22 vs. 35h), C(max) (34 vs. 67μg/mL), and AUC (568 vs. 2291h*μg/mL), however PolyDoxSome was comparable on efficacy and toxicity profile of LipoDox™. Results suggest that PolyDoxSome has better in vivo profile than free doxorubicin and comparable efficacy and toxicity to LipoDox™.

Image-guided drug delivery with magnetic resonance guided high intensity focused ultrasound and temperature sensitive liposomes in a rabbit Vx2 tumor model

Clinical-grade doxorubicin encapsulated low temperature sensitive liposomes (LTSLs) were combined with a clinical magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) platform to investigate in vivo image-guided drug delivery. Plasma pharmacokinetics were determined in 3 rabbits. Fifteen rabbits with Vx2 tumors within superficial thigh muscle were randomly assigned into three treatment groups: 1) free doxorubicin, 2) LTSL and 3) LTSL+MR-HIFU. For the LTSL+MR-HIFU group, mild hyperthermia (40–41°C) was applied to the tumors using an MR-HIFU system. Image-guided non-invasive hyperthermia was applied for a total of 30min, completed within 1h after LTSL infusion. High-pressure liquid chromatography (HPLC) analysis of the harvested tumor and organ/tissue homogenates was performed to determine doxorubicin concentration. Fluorescence microscopy was performed to determine doxorubicin spatial distribution in the tumors. Sonication of Vx2 tumors resulted in accurate (mean=40.5±0.1°C) and spatially homogenous (SD=1.0°C) temperature control in the target region. LTSL+MR-HIFU resulted in significantly higher tumor doxorubicin concentrations (7.6- and 3.4-fold greater compared to free doxorubicin and LTSL respectively, p<0.05, Newman–Keuls). This improved tumor concentration was achieved despite heating <25% of the tumor volume. Free doxorubicin and LTSL treatments appeared to deliver more drug in the tumor periphery as compared to the tumor core. In contrast, LTSL+MR-HIFU treatment suggested an improved distribution with doxorubicin found in both the tumor periphery and core. Doxorubicin bio-distribution in non-tumor organs/tissues was fairly similar between treatment groups. This technique has potential for clinical translation as an image-guided method to deliver drug to a solid tumor.

WE‐G‐220‐04: Enhanced Uptake of Doxorubicin in Prostate Tumor Using Pulsed Focused Ultrasound — An in Vivo Study

Purpose: To investigate the effect of pulsed focused ultrasound (pFUS) on the uptake of doxorubicin (DOX) in prostate tumor in vivo. Methods: A mouse prostate tumor model was used in this study by implanting and growing LNCaP tumor cells in male nude mice prostate. An InSightec ExAblate 2000 system with a 1.5T GE MR scanner was used for MRI‐ guided pFUS treatment. Non‐thermal sonications were delivered by keeping the local body temperature below 42°C as measured in real‐time by MR thermometry. Tumor‐bearing mice were exposed to pFUS (1MHz; 25W acoustic power; 10% duty cycle with 0.1 sec on and 0.9 sec off) for 60 sec in each sonication. A total of 4–8 sonication spots, depending on the tumor size, were used to cover the whole tumor. Immediately after pFUS exposure, DOX (10mg/kg) was injected via tail vein. Mice were euthanized after two hours and the DOX concentration in tumor (microgram of DOX per gram of tumor weight) was measured using the fluorescence technique. Mice were randomly assigned to two groups (n=8 for each group). One group received DOX injection only (group: DOX). The other received both pFUS exposure and DOX injection (group: FUS+DOX). Results: The DOX concentration in the FUS+DOX group was 14.9+/−2.5 ug/g compared to 9.5+/−1.6 ug/g of the DOX group (p=0.051). There was an about 60% increase of the DOX uptake in the prostate tumor exposed to pFUS. Conclusion: Results show that pFUS exposures significantly enhanced the uptake of DOX in prostate tumors. This result is consistent with our previous study of enhanced [3H]‐docetaxel uptake in prostate tumors. Further studies are being conducted to investigate the optimal pFUS parameters to maximize the uptake enhancement and its clinical benefits for cancer therapy (Study supported by a grant from Focused Ultrasound Surgery Foundation and partially by DOD PC073127).

Abstract A7: Targeting the tumor microenvironment via inhibition of VEGF and PDGF to improve liposomal drug delivery in human non-small cell lung cancer xenografts

Abstract Background: Pathological angiogenesis creates an abnormal microenvironment in tumors, characterized by elevated interstitial fluid pressure (IFP) and hypoxia. Judicious attenuation of angiogenic signals may “normalize” the vascular bed, making it more suitable for drug delivery and radiotherapy. In this work, we investigate the role of pazopanib, a small-molecule inhibitor of vascular endothelial growth factor (VEGF) receptors and platelet-derived growth factor (PDGF) receptors, on normalization parameters of IFP, vascular pericyte coverage, hypoxia, and liposomal drug delivery. Methods: Nude mice bearing A549 human non-small cell lung cancer (NSCLC) xenografts were treated with pazopanib 100mg/kg (n=20) or control (n=20) for 8 days, followed by a single intravenous dose of Doxil (liposomal doxorubicin) 10mg/kg. 24 hours after Doxil delivery, tumor IFP was measured, and tumors were snap frozen and sectioned. In a second study, designed similarly to the previous (n=4 pazopanib 100mg/kg, n=4 control), EF5 (hypoxia marker) was delivered 3 hours prior to tumor harvest. Tissue sections were stained for vasculature and pericytes using anti-CD-31 (endothelial cell marker) and anti-alpha-smooth muscle actin, respectively. Hypoxia was assessed via immunostaining for CA-IX and EF5. High performance liquid chromatography was used to determine tumor doxorubicin concentration ([dox]), and the penetration distance of Doxil from CD-31 stained vessels was calculated using a MATLAB algorithm. Results: Pazopanib treatment resulted in reduction of tumor IFP (116.571 ± 15.174 mmHg control vs. 61.000 ± 7.367 mmHg pazopanib, P = 0.0145) and decreased vessel density (251.228 ± 18.770 vessels/mm2 control vs. 101.313 ± 11.005 vessels/mm2 pazopanib, P &amp;lt; 0.0001). No differences in tumor [dox] were observed between groups (10.849 ± 1.785 ng/kg control vs. 12.274 ± 1.013 ng/mg pazopanib, P = 0.5956), however Doxil penetration improved in pazopanib-treated tumors (18.980 ± 0.687 m control vs. 32.886 ± 1.741 m pazopanib, P = 0.0006). Despite these trends towards normalization, tumor hypoxia, evaluated by CA-IX and EF5 expression, was increased in the pazopanib group (% CA-IX staining, 7.034 ± 0.988% control vs. 16.720 ± 1.728% pazopanib, P = 0.0005;% EF5 staining, 15.992 ± 2.610% control vs. 36.554 ± 4.617% pazopanib, P = 0.0074). Additionally, the proportion of pericyte-coated vessels was decreased in the pazopanib group (0.840 ± 0.0362 control vs. 0.675 ± 0.0495 pazopanib, P = 0.0160). Conclusions: Our results suggest a role for VEGF/PDGF inhibition in modulating functional parameters of the tumor microenvironment and improving drug distribution. However, these changes may be mediated by stromal changes in response to PDGF-receptor blockade, rather than normalization. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A7.

Glutamine regulation of doxorubicin accumulation in hearts versus tumors in experimental rats

Doxorubicin (DOX), an effective antineoplastic agent is known for its cardiotoxicity attributed mainly to free radical formation. Preliminary data indicated that oral glutamine (GLN) reduced cardiac oxidative damages in experimental rats treated with DOX. This study investigated the effect of GLN on DOX accumulation in tumors and normal tissues, troponin plasma concentration and functional alternations associated with DOX-induced myocardial damage. Female Fisher344 rats (n = 40) with implanted MatBIII mammary tumors were randomized to receive oral GLN (1 g/kg/day) (n = 20) or to serve as controls (n = 20) and were treated with a single i.p. injection of 12 mg/kg DOX. Ten normal rats (n = 10) without treatment served as naive controls. Cardiac physiologic alterations resulting from DOX treatment in GLN-supplemented and control rats were assessed by micro-ultrasound imaging at 3 and 10 days after DOX injection. Ten rats from each GLN-supplemented and control groups were killed at 3 and 10 days after DOX administration. At killing, hearts, livers, spleens, kidneys, tumors and sera were examined for DOX concentration by measuring DOX natural fluorescence. Hearts were examined for Von Willebrand factor (vWF) expression using immunohistochemistry. Glutamine supplementation resulted in a significant reduction of DOX concentration in the normal tissues, without a significant effect on tumor DOX concentration. GLN-supplemented rats had lower plasma cTnI levels and lower cardiac levels of vWF. DOX-induced alterations in the echocardiographic parameters were significantly reduced in the GLN-supplemented rats. These data indicate that GLN supplement is able to reduce DOX-induced cardiac damage and thus to enhance DOX therapeutic effectiveness.

Evaluation of Different Calibrated Spherical Polyvinyl Alcohol Microspheres in Transcatheter Arterial Chemoembolization: VX2 Tumor Model in Rabbit Liver

To assess whether porosity and compressibility of calibrated spherical polyvinyl alcohol (PVA) microspheres affect doxorubicin plasma and tumor concentrations after transcatheter arterial chemoembolization (TACE) in a VX2 rabbit model. Fifteen rabbits were divided into three groups of five rabbits each. Three different types of calibrated spherical PVA microspheres with variable levels of porosity and compressibility were blindly evaluated. TACE was performed by injecting a mixture of doxorubicin (5 mg) and iodized oil (0.5 mL) followed by injection of the embolic material (0.3-0.5 mL). Plasma concentrations of doxorubicin and doxorubicinol were analyzed 20, 40, 60, and 120 minutes and 2 days after TACE, and liver tissue and tumor doxorubicin concentrations were measured 2 days after TACE. All calibrated spherical PVA microspheres showed similar patterns of plasma doxorubicin and doxorubicinol release and tumor concentration of doxorubicin. There were no significant differences of drug levels in either plasma or tumor in each group (P > .05). After TACE in a rabbit model of liver cancer, testing of three different types of spherical PVA microspheres with varying degrees of porosity and compressibility showed no significant differences in the plasma doxorubicin release pattern and tumor doxorubicin uptake.

Delivery of Systemic Chemotherapeutic Agent to Tumors by Using Focused Ultrasound: Study in a Murine Model

To quantitatively determine the delivery of systemic liposomal doxorubicin to tumors treated with pulsed high-intensity focused ultrasound and to study the mechanism underlying this delivery in a murine model. All animal work was performed in compliance with guidelines and approval of institutional animal care committee. C3H mice received subcutaneous injections in the flank of a cell suspension of SCC7, a murine squamous cell carcinoma cell line; mice (n = 32) in drug delivery study received unilateral injections, whereas mice (n = 10) in mechanistic study received bilateral injections. Tumors were treated when they reached 1 cm(3) in volume. In the drug delivery study, doxorubicin hydrochloride liposomes were injected into the tail vein: Mice received therapy with doxorubicin injections and high-intensity focused ultrasound, doxorubicin injections alone, or neither form of therapy (controls). Tumors were removed, and the doxorubicin content was assayed with fluorescent spectrophotometry. In the mechanistic study, all mice received an injection of 500-kDa dextran-fluorescein isothyocyanate into the tail vein, and half of them were exposed to high-intensity focused ultrasound prior to injection. Contralateral tumors served as controls for each group. Extravasation of dextran-fluorescein isothyocyanate was observed by using in vivo confocal microscopy. Mean doxorubicin concentration in tumors treated with pulsed high-intensity focused ultrasound was 9.4 microg . g(-1) +/- 2.1 (standard deviation), and it was significantly higher (124% [9.4 microg . g(-1)/4.2 microg . g(-1)]) than in those that were not treated with high-intensity focused ultrasound (4.2 microg . g(-1) +/- 0.95) (P < .001, unpaired two-tailed Student t test). Extravasation of dextran-fluorescein isothyocyanate was observed in the vasculature of tumors treated with high-intensity focused ultrasound but not in that of untreated tumors. Pulsed high-intensity focused ultrasound is an effective method of targeting systemic drug delivery to tumor tissue. Potential mechanisms for producing the observed enhancement are discussed.

Increased effects of MPDAX, a novel xanthine derivative, on antitumor activity of doxorubicin

To clarify the effect of 1-methyl-3-propyl-7- N, N-dimethylpropylamide-xanthine (MPDAX) on doxorubicin (DOX) transport, we examined the efficacy of MPDAX as an amplifier of the antitumor activity of DOX in mice bearing tumors with different properties as to DOX transport across cell membranes. MPDAX significantly enhanced the DOX-induced antitumor activity on DOX-sensitive tumors. It is expected that the increase in antitumor activity caused by MPDAX contributes to the increased DOX concentration in tumors due to the MPDAX-induced change in DOX transport via the transporter expressed in sensitive tumor cells. In contrast, in M5076, a lower sensitive to DOX, MPDAX decreased the tumor weight by half at an otherwise ineffective dose of DOX. Furthermore, in P388/DOX, DOX has no effect, but MPDAX caused an elevation of the DOX-induced antitumor activity with an increase in the DOX concentration in the tumors. The results suggested that MPDAX is a novel amplifier for antitumor agents as it significantly increased the antitumor activity toward tumors with different properties. The DOX concentrations in the MPDAX+DOX group for all tumor lines were about two-fold those in the DOX alone group. Furthermore, MPDAX and DOX exhibited significant inhibitory effects on uridine and thymidine uptake. It is known that nucleoside transporters increase the membrane permeability of DOX. We speculated that MPDAX inhibits the cell membrane transport of uridine and thymidine via nucleoside transporters. MPDAX, acting via nucleoside transporters, increases the DOX-induced antitumor activity toward many tumor types and is an useful biochemical modulator.

Effects of xanthine derivatives on the influx and efflux of doxorubicin in P388 and DOX-resistant P388 leukemia cells

It was reported that xanthine derivatives (caffeine and 1-methyl-3-propyl-7-butylxanthine) enhanced the antitumor activity of doxorubicin (DOX) with increasing DOX concentrations in tumors in vivo in our previous papers. In addition, these actions were found to be related to the inhibitory activity toward DOX efflux from tumor cells in vitro. In this study, we searched for novel biochemical modulators of DOX among 3- n-propylxanthines with functional groups at the 1- or 7-position by using an assay system for their inhibitory effect on DOX efflux from P388 leukemia and DOX resistant P388 leukemia (P388/DOX) cells. 1-Substituted xanthines facilitated the DOX efflux from P388 cells. In contrast, among 7-substituted xanthines, XT-141 and XT-139 significantly inhibited the DOX efflux from P388 cells. In addition, XT-141 inhibited the DOX efflux from P388/DOX cells, and P-glycoprotein (P-gp) inhibitor facilitated DOX influx and inhibited DOX efflux from P388/DOX cells in a dose-dependent manner. These results indicated that the resistance of P388/DOX might depend on the over-expression of P-gp, and that XT-141 inhibited DOX efflux through its interaction with P-gp. We suspect that XT-141 is a useful biochemical modulator of DOX in DOX-resistant tumors with over-expression of P-gp in addition in DOX-sensitive tumors.