New therapeutic approaches for brainstem tumors: a comparison of delivery routes using nanoliposomal irinotecan in an animal model.

Despite the advances in imaging, surgery and radiotherapy, the majority of patients with brainstem gliomas die within 2 years after initial diagnosis. Factors that contribute to the dismal prognosis of these patients include the infiltrative nature and anatomic location in an eloquent area of the brain, which prevents total surgical resection and the presence of the blood-brain barrier (BBB), which reduces the distribution of systemically administered agents. The development of new therapeutic approaches which can circumvent the BBB is a potential path to improve outcomes for these children. Convection-enhanced delivery (CED) and intranasal delivery (IND) are strategies that permit direct drug delivery into the central nervous system and are an alternative to intravenous injection (IV). We treated rats bearing human brainstem tumor xenografts with nanoliposomal irinotecan (CPT-11) using CED, IND, and IV. A single treatment of CED irinotecan had a similar effect on overall survival as multiple treatments by IV route. IND CPT-11 showed significantly increased survival of animals with brainstem tumors, and demonstrated the promise of this non-invasive approach of drug delivery bypassing the BBB when combined with nanoliposomal chemotherapy. Our results indicated that using CED and IND of nanoliposomal therapy increase likelihood of practical therapeutic approach for the treatment of brainstem gliomas.

Drug Stability and Minimized Acid-/Drug-Catalyzed Phospholipid Degradation in Liposomal Irinotecan.

Therapeutics at or close to the nanoscale, such as liposomal irinotecan, offer significant promise for the treatment of solid tumors. Their potential advantage over the unencapsulated or free form of the drug is due in part to their altered biodistribution. For slow and sustained release, significant optimization of formulation is needed to achieve the required level of stability and allow long-term storage of the drug product. Gradient-based liposomal formulation of camptothecins such as irinotecan poses unique challenges owing to the camptothecin- and acid-catalyzed hydrolysis of phospholipid esters in the inner monolayer of the liposomal membrane. We demonstrated that a narrow set of conditions related to the external pH, temperature, intraliposomal concentration, identity of the drug-trapping agent, physical form of the drug inside the liposomes, and final drug load have a marked impact on the stability of the liposome phospholipid membrane. The physical form of the drug inside the liposome was shown to be an insoluble gel with an irinotecan-to-sulfate ratio approximating 1:1, reducing the potential for irinotecan-catalyzed phospholipid hydrolysis in the internal phospholipid monolayer. As a result of this work, a stable and active liposome formulation has been developed that maintains phospholipid chemical stability following long-term storage at 2-8°C.

Improved pharmacokinetics and efficacy of a highly stable nanoliposomal vinorelbine.

Effective liposomal formulations of vinorelbine (5' nor-anhydro-vinblastine; VRL) have been elusive due to vinorelbine's hydrophobic structure and resulting difficulty in stabilizing the drug inside the nanocarrier. Triethylammonium salts of several polyanionic trapping agents were used initially to prepare minimally pegylated nanoliposomal vinorelbine formulations with a wide range of drug release rates. Sulfate, poly(phosphate), and sucrose octasulfate were used to stabilize vinorelbine intraliposomally while in circulation, with varying degrees of effectiveness. The release rate of vinorelbine from the liposomal carrier was affected by both the chemical nature of the trapping agent and the resulting drug-to-lipid ratio, with liposomes prepared using sucrose octasulfate displaying the longest half-life in circulation (9.4 h) and in vivo retention in the nanoparticle (t(1/2) = 27.2 h). Efficacy was considerably improved in both a human colon carcinoma (HT-29) and a murine (C-26) colon carcinoma model when vinorelbine was stably encapsulated in liposomes using triethylammonium sucrose octasulfate. Early difficulties in preparing highly pegylated formulations were later overcome by substituting a neutral distearoylglycerol anchor for the more commonly used anionic distearoylphosphatidylethanolamine anchor. The new pegylated nanoliposomal vinorelbine displayed high encapsulation efficiency and in vivo drug retention, and it was highly active against human breast and lung tumor xenografts. Acute toxicity of the drug in immunocompetent mice slightly decreased upon encapsulation in liposomes, with a maximum tolerated dose of 17.5 mg VRL/kg for free vinorelbine and 23.8 mg VRL/kg for nanoliposomal vinorelbine. Our results demonstrate that a highly active, stable, and long-circulating liposomal vinorelbine can be prepared and warrants further study in the treatment of cancer.

Formulation optimization of an ephrin A2 targeted immunoliposome encapsulating reversibly modified taxane prodrugs.

Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were developed for sustained delivery of active drug to solid tumors. Here we describe the systematic formulation development and characterization of these immunoliposomes. We synthesized both paclitaxel and docetaxel prodrugs to formulate as ephrin A2-targeted liposomes stabilized in the aqueous core with sucroseoctasulfate (SOS). The optimized lipid formulation was comprised of egg-sphingomyelin, cholesterol, and polyethylene glycol distearoyl glycerol (PEG-DSG). The formulations examined had a high efficiency of prodrug encapsulation (as high as 114 mol% taxane per mole phospholipid) and subsequent stability (>3 years at 2-8 °C). The taxane prodrug was stabilized with extraliposomal citric acid and subsequently loaded into liposomes containing a gradient of SOS, resulting in highly stable SOS-drug complexes being formed inside the liposome. The internal prodrug and SOS concentrations were optimized for their impact on in vivo drug release and drug degradation. Cryo-electron microscope images revealed dense prodrug-SOS complex in the aqueous core of the immunoliposomes. Ephrin A2-targeted taxane liposomes exhibited sub-nanomolar (0.69 nM) apparent equilibrium dissociation constant toward the extracellular domain of the ephrin A2 receptor, long circulation half-life (8-12 h) in mouse plasma, a release rate dependent on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50 mg taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10 mg/kg of docetaxel in A549 non-small cell lung, as well as MDA-MB-436 and SUM149 triple negative breast cancer xenograft models. The lead molecule entered a Phase I clinical trial in patients with solid tumors (NCT03076372).

Antitumour activity and tolerability of an EphA2-targeted nanotherapeutic in multiple mouse models.

Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.

Canine model of convection-enhanced delivery of liposomes containing CPT-11 monitored with real-time magnetic resonance imaging: laboratory investigation.

OBJECT: Many factors relating to the safety and efficacy of convection-enhanced delivery (CED) into intracranial tumors are poorly understood. To investigate these factors further and establish a more clinically relevant large animal model, with the potential to investigate CED in large, spontaneous tumors, the authors developed a magnetic resonance (MR) imaging-compatible system for CED of liposomal nanoparticles into the canine brain, incorporating real-time MR imaging. Additionally any possible toxicity of liposomes containing Gd and the chemotherapeutic agent irinotecan (CPT-11) was assessed following direct intraparenchymal delivery. METHODS: Four healthy laboratory dogs were infused with liposomes containing Gd, rhodamine, or CPT-11. Convection-enhanced delivery was monitored in real time by sequential MR imaging, and the volumes of distribution were calculated from MR images and histological sections. Assessment of any toxicity was based on clinical and histopathological evaluation. Convection-enhanced delivery resulted in robust volumes of distribution in both gray and white matter, and real-time MR imaging allowed accurate calculation of volumes and pathways of distribution. RESULTS: Infusion variability was greatest in the gray matter, and was associated with leakage into ventricular or subarachnoid spaces. Complications were minimal and included mild transient proprioceptive deficits, focal hemorrhage in 1 dog, and focal, mild perivascular, nonsuppurative encephalitis in 1 dog. CONCLUSIONS: Convection-enhanced delivery of liposomal Gd/CPT-11 is associated with minimal adverse effects in a large animal model, and further assessment for use in clinical patients is warranted. Future studies investigating real-time monitored CED in spontaneous gliomas in canines are feasible and will provide a unique, clinically relevant large animal translational model for testing this and other therapeutic strategies.

Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy.

Liposome formulations of camptothecins have been actively pursued because of the potential for significant pharmacologic advantages from successful drug delivery of this important class of anticancer drugs. We describe nanoliposomal CPT-11, a novel nanoparticle/liposome construct containing CPT-11 (irinotecan) with unprecedented drug loading efficiency and in vivo drug retention. Using a modified gradient loading method featuring a sterically hindered amine with highly charged, multivalent anionic trapping agents, either polymeric (polyphosphate) or nonpolymeric (sucrose octasulfate), liposomes were capable of entrapping CPT-11 at extremely high drug-to-lipid ratios (>800 g CPT-11/mol phospholipid) and retaining encapsulated drug in vivo with a half-life of drug release in the circulation of 56.8 hours. CPT-11 was also protected from hydrolysis to the inactive carboxylate form and from metabolic conversion to SN-38 while circulating. The maximum tolerated dose in normal mice was determined to be 80 mg/kg for free CPT-11 and >320 mg/kg for nanoliposomal CPT-11. Nanoliposomal CPT-11 showed markedly superior efficacy when compared with free CPT-11 in human breast (BT474) and colon (HT29) cancer xenograft models. This study shows that intraliposomal stabilization of CPT-11 using a polymeric or highly charged, nonpolymeric polyanionic trapping agent results in a markedly active antitumor agent with low toxicity.

Novel nanoliposomal CPT-11 infused by convection-enhanced delivery in intracranial tumors: pharmacology and efficacy.

We hypothesized that combining convection-enhanced delivery (CED) with a novel, highly stable nanoparticle/liposome containing CPT-11 (nanoliposomal CPT-11) would provide a dual drug delivery strategy for brain tumor treatment. Following CED in rat brains, tissue retention of nanoliposomal CPT-11 was greatly prolonged, with >20% injected dose remaining at 12 days for all doses. Tissue residence was dose dependent, with doses of 60 microg (3 mg/mL), 0.8 mg (40 mg/mL), and 1.6 mg (80 mg/mL) resulting in tissue half-life (t(1/2)) of 6.7, 10.7, and 19.7 days, respectively. In contrast, CED of free CPT-11 resulted in rapid drug clearance (tissue t(1/2) = 0.3 day). At equivalent CED doses, nanoliposomal CPT-11 increased area under the time-concentration curve by 25-fold and tissue t(1/2) by 22-fold over free CPT-11; CED in intracranial U87 glioma xenografts showed even longer tumor retention (tissue t(1/2) = 43 days). Plasma levels were undetectable following CED of nanoliposomal CPT-11. Importantly, prolonged exposure to nanoliposomal CPT-11 resulted in no measurable central nervous system (CNS) toxicity at any dose tested (0.06-1.6 mg/rat), whereas CED of free CPT-11 induced severe CNS toxicity at 0.4 mg/rat. In the intracranial U87 glioma xenograft model, a single CED infusion of nanoliposomal CPT-11 at 1.6 mg resulted in significantly improved median survival (>100 days) compared with CED of control liposomes (19.5 days; P = 4.9 x 10(-5)) or free drug (28.5 days; P = 0.011). We conclude that CED of nanoliposomal CPT-11 greatly prolonged tissue residence while also substantially reducing toxicity, resulting in a highly effective treatment strategy in preclinical brain tumor models.

Convection-enhanced delivery of a topoisomerase I inhibitor (nanoliposomal topotecan) and a topoisomerase II inhibitor (pegylated liposomal doxorubicin) in intracranial brain tumor xenografts.

Despite multimodal treatment options, the response and survival rates for patients with malignant gliomas remain dismal. Clinical trials with convection-enhanced delivery (CED) have recently opened a new window in neuro-oncology to the direct delivery of chemotherapeutics to the CNS, circumventing the blood-brain barrier and reducing systemic side effects. Our previous CED studies with liposomal chemotherapeutics have shown promising antitumor activity in rodent brain tumor models. In this study, we evaluated a combination of nanoliposomal topotecan (nLs-TPT) and pegylated liposomal doxorubicin (PLD) to enhance efficacy in our brain tumor models, and to establish a CED treatment capable of improving survival from malignant brain tumors. Both liposomal drugs decreased key enzymes involved in tumor cell replication in vitro. Synergistic effects of nLs-TPT and PLD on U87MG cell death were found. The combination displayed excellent efficacy in a CED-based survival study 10 days after tumor cell implantation. Animals in the control group and those in singleagent groups had a median survival of less than 30 days, whereas the combination group experienced a median survival of more than 90 days. We conclude that CED of two liposomal chemotherapeutics (nLs-TPT and PLD) may be an effective treatment option for malignant gliomas.

Convection-enhanced delivery of nanoliposomal CPT-11 (irinotecan) and PEGylated liposomal doxorubicin (Doxil) in rodent intracranial brain tumor xenografts.

We have previously shown that convection-enhanced delivery (CED) of highly stable nanoparticle/liposome agents encapsulating chemotherapeutic drugs is effective against intracranial rodent brain tumor xenografts. In this study, we have evaluated the combination of a newly developed nanoparticle/liposome containing the topoisomerase I inhibitor CPT-11 (nanoliposomal CPT-11 [nLs-CPT-11]), and PEGylated liposomal doxorubicin (Doxil) containing the topoisomerase II inhibitor doxorubicin. Both drugs were detectable in the CNS for more than 36 days after a single CED application. Tissue half-life was 16.7 days for nLs-CPT-11 and 10.9 days for Doxil. The combination of the two agents produced synergistic cytotoxicity in vitro. In vivo in U251MG and U87MG intracranial rodent xenograft models, CED of the combination was also more efficacious than either agent used singly. Analysis of the parameters involved in this approach indicated that tissue pharmacokinetics, tumor microanatomy, and biochemical interactions of the drugs all contributed to the therapeutic efficacy observed. These findings have implications for further clinical applications of CED-based treatment of brain tumors.

Epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo.

We previously reported the development of epidermal growth factor receptor (EGFR)-targeted immunoliposomes that bind and internalize in tumor cells which overexpress EGFR and/or mutant EGFR variant III (EGFRvIII), enabling intracellular delivery of potent anticancer agents in vitro. We now describe in vivo proof-of-concept for this approach for the delivery of multiple anticancer drugs in EGFR-overexpressing tumor models. Anti-EGFR immunoliposomes were constructed modularly with Fab' fragments of cetuximab (IMC-C225), covalently linked to liposomes containing probes and/or anticancer drugs. Pharmacokinetic and biodistribution studies confirmed long circulation times (t(1/2) = 21 hours) and efficient accumulation in tumors (up to 15% ID/g) irrespective of the presence of the targeting ligand. Although total accumulations of anti-EGFR immunoliposomes and nontargeted liposomes in EGFR-overexpressing tumors were comparable, only immunoliposomes internalized extensively within tumor cells (92% of analyzed cells versus <5% for nontargeted liposomes), indicating different mechanisms of delivery at the cellular level. In vivo therapy studies in a series of xenograft models featuring overexpression of EGFR and/or EGFRvIII showed the superiority of immunoliposomal delivery of encapsulated drugs, which included doxorubicin, epirubicin, and vinorelbine. For each of these drugs, anti-EGFR immunoliposome delivery showed significant antitumor effects and was significantly superior to all other treatments, including the corresponding free or liposomal drug (P < 0.001-0.003). We conclude that anti-EGFR immunoliposomes provide efficient and targeted drug delivery of anticancer compounds and may represent a useful new treatment approach for tumors that overexpress the EGFR.

Development of a Flow-Through USP-4 Apparatus Drug Release Assay to Evaluate Doxorubicin Liposomes.

Doxil® is a complex parenteral doxorubicin (DOX) liposome formulation approved by the FDA. For generic doxorubicin liposomes, analyzing the release profile of DOX is important for quality control and comparability studies. However, there is no robust standard drug release assay available for doxorubicin liposomes. In this study, we describe a USP-4 apparatus assay capable of discriminating DOX liposomal formulations based on release profile. Establishment of the assay was hindered by limited DOX release from liposomes in physiological conditions at 37°C. The addition of NH4HCO3 to the release media facilitated DOX release proportionally to the salt concentration added but caused precipitation of released drug in USP-4 apparatus. Precipitation of DOX was avoided by adding hydroxypropyl-cyclodextrin (HP-CD) to the release medium. We optimized conditions for DOX release by varying a number of parameters such as: concentration of HP-CD, testing temperature, and concentration of tested samples. The optimized release medium contained: 100 mM NH4HCO3, 75 mM 2-(N-morpholino) ethanesulfonic acid (MES) and 5% w/v HP-CD, 5% w/v sucrose, 0.02% w/v NaN3 (pH 6). The drug release assay was performed at 45°C. The optimized release assay can discriminate between DOX liposomal formulations of different compositions, physicochemical properties, and prepared by different manufacturing methods. This indicates that the assay could be used to compare DOX release from generic DOX formulations to the innovator product Doxil®.

A phase 1 trial of intravenous liposomal irinotecan in patients with recurrent high-grade glioma.

PURPOSE: Preclinical activity of irinotecan has been seen in glioma models, but only modest efficacy has been noted in clinical studies, perhaps related to drug distribution and/or pharmacokinetic limitations. In preclinical testing, irinotecan liposome injection (nal-IRI) results in prolongation of drug exposure and higher tissue levels of drug due to slower metabolism and the effect of enhanced permeability and retention. The objective of the current study was to assess the safety and pharmacokinetics (PK) of nal-IRI and to determine the maximum tolerated dose (MTD) in patients with recurrent high-grade glioma stratified based on UGT1A1 genotyping. METHODS: This phase I study stratified patients with recurrent high-grade glioma into 2 groups by UGT1A1 status: homozygous WT ("WT") vs heterozygous WT/*28 ("HT"). Patients who were homozygous *28 were ineligible. The design was a standard 3 + 3 phase I design. WT patients were started at 120 mg/m2 intravenously every 3 weeks with dose increases in 60 mg/m2 increments. HT patients were started at 60 mg/m2, with dose increases in 30 mg/m2 increments. The assessment period for dose-limiting toxicity was 1 cycle (21 days). RESULTS: In the WT cohort (n = 16), the MTD was 120 mg/m2. In the HT cohort (n = 18), the MTD was 150 mg/m2. Dose-limiting toxicity in both cohorts included diarrhea, some with associated dehydration and/or fatigue. PK results were comparable to those seen in other PK studies of nal-IRI; UGT1A1*28 genotype (WT vs. HT) did not affect PK parameters. CONCLUSIONS: Nal-IRI had no unexpected toxicities when given intravenously. Of note, UGT1A1 genotype did not correlate with toxicity or affect PK profile.

Convection-enhanced delivery of Ls-TPT enables an effective, continuous, low-dose chemotherapy against malignant glioma xenograft model.

Treatment of malignant gliomas represents one of the most formidable challenges in oncology. The combination of surgery, radiation, and chemotherapy yields median survivals of less than one year. Here we demonstrate the use of a minimally invasive surgical technique, convection-enhanced delivery (CED), for local administration of a novel nanoparticle liposome containing topotecan. CED of this liposomal topotecan (Ls-TPT) resulted in extended brain tissue retention (t1/2 = 1.5 days), whereas free topotecan was rapidly cleared (t1/2 = 0.1 days) after CED. The favorable pharmacokinetic profile of extended topotecan release for about seven days, along with biodistribution featuring perivascular accumulation of the nanoparticles, provided, in addition to the known topoisomerase I inhibition, an effective antiangiogenic therapy. In the rat intracranial U87MG tumor model, vascular targeting of Ls-TPT with CED was associated with reductions in laminin expression and vascular density compared to free topotecan or control treatments. A single CED treatment on day 7 showed that free topotecan conferred no survival benefit versus control. However, Ls-TPT produced a significant (P = 0.0002) survival benefit, with six of seven complete cures. Larger U87MG tumors, where CED of Ls-TPT on day 12 resulted in one of six cures, indicated the necessity to cover the entire tumor with the infused therapeutic agent. CED of Ls-TPT was also efficacious in the intracranial U251MG tumor model (P = 0.0005 versus control). We conclude that the combination of a novel nanoparticle Ls-TPT and CED administration was very effective in treating experimental brain tumors.

Tissue affinity of the infusate affects the distribution volume during convection-enhanced delivery into rodent brains: implications for local drug delivery.

Convection-enhanced delivery (CED) is a recently developed technique for local delivery of agents to a large volume of tissue in the central nervous system (CNS). We have previously reported that this technique can be applied to CNS delivery of nanoparticles including viruses and liposomes. In this paper, we describe the impact of key physical and chemical properties of infused molecules on the extent of CED-mediated delivery. For simple infusates, CED distribution was significantly increased if the infusate was more hydrophilic or had less tissue affinity. Encapsulation of tissue-affinitive molecules by neutral liposomes significantly increased their tissue distribution. The poorer brain distribution observed with cationic liposomes, due to their greater tissue affinity, was completely overcome by PEGylation, which provides steric stabilization and reduced surface charge. Finally, liposomal encapsulation of doxorubicin reduced its tissue affinity and substantially increased its distribution within brain tumor tissue. Taken together, the physical and chemical properties of drugs, small molecules and macromolecular carriers determine the tissue affinity of the infusate and strongly affect the distribution of locally applied agents. Thus, an increased and more predictable tissue distribution can be achieved by reducing the tissue affinity of the infusate using appropriately engineered liposomes or other nanoparticles.

Enhanced pharmacodynamic and antitumor properties of a histone deacetylase inhibitor encapsulated in liposomes or ErbB2-targeted immunoliposomes.

ErbB2-overexpressing human cancers represent potentially sensitive targets for therapy by candidate histone deacetylase (HDAC) inhibitors as we have shown that HDAC inhibitors can selectively reduce ErbB2 expression by repressing the ErbB2 promoter and accelerating the decay of cytoplasmic ErbB2 transcripts. To extend these in vitro findings and enhance the in vivo pharmacodynamic properties of HDAC inhibitors, we stably encapsulated a potent hydroxamate-based HDAC inhibitor (LAQ824) within long-circulating liposomes (Ls-LAQ824) and immunoliposomes (ILs-LAQ824) bearing >10,000 LAQ824 molecules per nanovesicle. Liposomal LAQ824 exhibits prolonged in vivo stability and, unlike free LAQ824, circulates with a half-life of 10.8 hours following a single i.v. injection. Three weekly i.v. injections of 20 to 25 mg/kg Ls-LAQ824 in nude mice with ErbB2 overexpressing BT-474 breast tumor xenografts significantly impairs tumor growth, and administration of ErbB2-targeted ILs-LAQ824 may further improve this antitumor activity. Studies of tumor-bearing mice 24 hours after single treatment indicate that: (a) >10% of injected liposomal LAQ824 is still circulating (whereas free LAQ824 is undetectable in the blood after 15 minutes); and (b) tumor uptake of Ls-LAQ824 and ILs-LAQ824 is >3% injected drug per gram of tumor, producing levels of acetylated tumor histones that are 5- to 10-fold increased over those following free LAQ824 or saline treatments and resulting in concordantly reduced levels of tumor ErbB2 mRNA. These preclinical results support the clinical evaluation of HDAC inhibitors against ErbB2-overexpressing malignancies, and further indicate that encapsulation into targeted and nontargeted liposomes substantially improves the in vivo pharmacokinetics, tumor uptake, and antitumor properties of hydroxamate-based HDAC inhibitors.

Real-time visualization and characterization of liposomal delivery into the monkey brain by magnetic resonance imaging.

Liposomes loaded with Gadoteridol, in combination with convection-enhanced delivery (CED), offer an excellent option to monitor CNS delivery of therapeutic compounds with MRI. In previous studies, we investigated possible clinical applications of liposomes to the treatment of brain tumors. In this study, up to 700 microl of Gadoteridol/rhodamine-loaded liposomes were distributed in putamen, corona radiata and brainstem of non-human primates. Distribution was monitored by real-time MRI throughout infusion procedures and allowed accurate calculation of volume of distribution within anatomical structures. We found that different regions of the brain gave various volumes of distribution when infused with the same volume of liposome. Based on these findings, distinct distribution pathways within infused structures can be predicted. This work underlines the importance of monitoring drug delivery to CNS and enables accurate delivery of drug-loaded liposomes to specific brain regions with a standard MRI procedure. Findings presented in this manuscript may allow for modeling of parameters used for direct delivery of therapeutics into various regions of the brain.

Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.

OBJECT: Clinical application of the convection-enhanced delivery (CED) technique is currently limited by low infusion speed and reflux of the delivered agent. The authors developed and evaluated a new step-design cannula to overcome present limitations and to introduce a rapid, reflux-free CED method for future clinical trials. METHODS: The CED of 0.4% trypan blue dye was performed in agarose gel to test cannula needles for distribution and reflux. Infusion rates ranging from 0.5 to 50 microl/minute were used. Agarose gel findings were translated into a study in rats and then in cynomolgus monkeys (Macacafascicularis) by using trypan blue and liposomes to confirm the efficacy of the reflux-free step-design cannula in vivo. Results of agarose gel studies showed reflux-free infusion with high flow rates using the step-design cannula. Data from the study in rats confirmed the agarose gel findings and also revealed increasing tissue damage at a flow rate above 5-microl/minute. Robust reflux-free delivery and distribution of liposomes was achieved using the step-design cannula in brains in both rats and nonhuman primates. CONCLUSIONS: The authors developed a new step-design cannula for CED that effectively prevents reflux in vivo and maximizes the distribution of agents delivered in the brain. Data in the present study show reflux-free infusion with a constant volume of distribution in the rat brain over a broad range of flow rates. Reflux-free delivery of liposomes into nonhuman primate brain was also established using the cannula. This step-design cannula may allow reflux-free distribution and shorten the duration of infusion in future clinical applications of CED in humans.

Pharmacokinetics, tumor accumulation and antitumor activity of nanoliposomal irinotecan following systemic treatment of intracranial tumors.

AIM: We sought to evaluate nanoliposomal irinotecan as an intravenous treatment in an orthotopic brain tumor model. MATERIALS & METHODS: Nanoliposomal irinotecan was administered intravenously in the intracranial U87MG brain tumor model in mice, and irinotecan and SN-38 levels were analyzed in malignant and normal tissues. Therapy studies were performed in comparison to free irinotecan and control treatments. RESULTS: Tissue analysis demonstrated favorable properties for nanoliposomal irinotecan, including a 10.9-fold increase in tumor AUC for drug compared with free irinotecan and 35-fold selectivity for tumor versus normal tissue exposure. As a therapy for orthotopic brain tumors, nanoliposomal irinotecan showed a mean survival time of 54.2 versus 29.5 days for free irinotecan. A total of 33% of the animals receiving nanoliposomal irinotecan showed no residual tumor by study end compared with no survivors in the other groups. CONCLUSION: Nanoliposomal irinotecan administered systemically provides significant pharmacologic advantages and may be an efficacious therapy for brain tumors.

Convection-enhanced delivery of targeted quantum dot-immunoliposome hybrid nanoparticles to intracranial brain tumor models.

AIM: The aim of this work is to evaluate combining targeting strategy and convection-enhanced delivery in brain tumor models by imaging quantum dot-immunoliposome hybrid nanoparticles. MATERIALS & METHODS: An EGF receptor-targeted, quantum dot-immunoliposome hybrid nanoparticle (QD-IL) was synthesized. In vitro uptake was measured by flow cytometry and intracellular localization was imaged by confocal microscopy. In the in vivo study, QD-ILs were delivered to intracranial xenografts via convection-enhanced delivery and fluorescence was monitored noninvasively in real-time. RESULTS: QD-ILs exhibited specific and efficient uptake in vitro and exhibited approximately 1.3- to 5.0-fold higher total fluorescence compared with nontargeted counterpart in intracranial brain tumor xenografts in vivo. CONCLUSION: QD-ILs serve as an effective imaging agent in vitro and in vivo, and the data suggest that ligand-directed liposomal nanoparticles in conjunction with convection-enhanced delivery may offer therapeutic benefits for glioblastoma treatment as a result of specific and efficient uptake by malignant cells.