Patent Application: US-201113880608-A

Abstract:
several agents capable of inducing vascular responses akin to those observed in inflammatory processes enhance the accumulation of nanoparticles in tumors . exemplary vascular - active agents include a bacterium , a pro - inflammatory cytokine , and microtubule - destabilizing drugs . such agents can increase the tumor to blood ratio of radioactivity by more than 20 - fold compared to nanoparticles alone . moreover , vascular - active agents dramatically improved the therapeutic effect of nanoparticles containing radioactive isotopes or chemotherapeutic agents .

Description:
the inventors have found that vascular - active permeability entities are able to increase the amount and / or specificity of delivery to solid tumors . the substance delivered to tumors may be a therapeutic agent or an imaging agent . the substance may include a carrier for the therapeutic or imaging agent or it may be the agent without a carrier . the use of the vascular - active permeability entity increases the amount of agent which is delivered to the solid tumor relative to the amount which was administered to the individual . the use of the vascular - active permeability entity in addition , or alternatively , increases the amount delivered to the solid tumor relative to the amount delivered to the blood of the individual . administration of the vascular - active permeability agent and the therapeutic or imaging agent can be accomplished by any means known in the art . typically , they will be administered by intravenous injections , but other means can be used , including intranasal , intrabronchial , intraductal , intravaginal , oral , intramuscular , subcutaneous , and the like . a single dose may be given of either agent or multiple doses may be administered of one or both agents . typically the vascular - active permeability agent and the therapeutic or imaging agent are administered at the same time or within 2 hours , 12 hours , 24 hours , 48 hours , 72 hours , 96 hours , 120 hours , 144 hours , or 168 hours or each other . either agent may be given first . nanoparticles as used herein have a size between 10 − 5 m and 10 − 9 m . the lower limit may be 5 × 10 − 9 , 10 − 8 , 5 × 10 − 8 , 10 − 7 , 5 × 10 − 7 , 10 − 6 , or 5 × 10 − 6 m . the upper limit on size may be 5 × 10 − 6 , 10 − 6 , 5 × 10 − 7 , 10 − 7 , 5 × 10 − 8 , 10 − 8 , or 5 × 10 − 9 m . the nanoparticles may comprise a polymer , carbohydrate , nucleic acid , polypeptide , viral particle , dna fragment , rna fragment , a recombinant virus , a recombinant adenovirus , a bacterium , a bacterial spore , liposome , or lipid , for example . the therapeutic or imaging agent may be entrapped , conjugated , encapsulated , or otherwise attached to the nanoparticle . antibodies which can be used as a therapeutic or imaging agent or as part of a therapeutic or imaging agent include whole or partial antibodies , such as igg , scfv , fab ′, fab2 , and monoclonal antibodies . the antibody may be without limitation bevacizumab ( avastin ), cetuximab ( erbitux ), trastuzumab ( herceptin ), tositumomab , rituximab ( rituxan ), 131 i - tositumomab ( bexxar ), 111 in - zevalin , or 90 y - zevalin , antibodies which are already in clinical use . a therapeutic or imaging agent may be conjugated , fused to , or otherwise attached to the antibody . the antibodies and nanoparticles may be used as carriers of a therapeutic or imaging agent , including a chemotherapeutic agent , such as doxorubicin , or a prodrug , such as irinotecan ( cpt - 11 ). the therapeutic or imaging agent may be a recombinant protein or a peptide . the therapeutic agent may be a toxin , such as botulinum toxin . the therapeutic or diagnostic agent may be an engineered nucleic acid , such as a therapeutic rna or an aptamer . any anti - tumor therapeutic agent or imaging agent known in the art may be used , coupled , conjugated , entrapped , or encapsulated by / to an antibody or nanoparticle . an antibody may be a therapeutic agent on its own , without coupling to another moiety . examples of types of therapeutic agents and specific examples include , without limitation , alkylating antineoplastic agents , such as cisplatin and carboplatin , oxaliplatin , mechlorethamine , cyclophosphamide , chlorambucil , ifosfamide , antimetabolites such as azathioprine , mercaptopurine , alkaloids , such as vinca alkaloids and taxanes , vincristein , binblastine , vinorelbine , vindesine , podophyllotoxin , doetaxel , topoisomerase inhibitors such as topotecan . amsacrine , etoposide , etoposide phosphate , and teniposide , cytotoxic antibiotics , such as actinomycin , anthracyclines , doxorubicin , daunorubicin , valrubicin , idarubicin , epirubicin , bleomycin , plicamycin , and mitomycin . a non - limiting list of toxins which may be used as a therapeutic agent include abrin , aerolysin , botulinin toxin a , botulinin toxin b , botulinin toxin c1 , botulinin toxin c2 , botulinin toxin d , botulinin toxin e , botulinin toxin f , b - bungarotoxin , caeruleotoxin , cereolysin , cholera toxin , clostridium difficile enterotoxin a , clostridium difficile cytotoxin b , clostridium perfringens lecithinase , clostridium perfringens kappa toxin , clostridium perfringens perfringolysin o , clostridium perfringens enterotoxin , clostridium perfringens beta toxin , clostridium perfringens delta toxin , clostridium perfringens epsilon toxin , conotoxin , crotoxin , diphtheria toxin , listeriolysin , leucocidin , modeccin , nematocyst toxins , notexin , pertussis toxin , pneumolysin , pseudomonas aeruginosa toxin a , ricin , saxitoxin , shiga toxin , shigella dysenteriae neurotoxin , streptolysin o , staphylococcus enterotoxin b , staphylococcus enterotoxin f , streptolysin s , taipoxin , tetanus toxin , tetrodotoxin , viscumin , volkensin , and yersinia pestis murine toxin , a detectable imaging agent can be coupled , conjugated , entrapped , or encapsulated by / to an antibody or nanoparticle . the imaging agent may be a magnetic material , a photosensitizing agent , a contrast agent , or a radionuclide , for example . the radionuclide may be , for example , iodine - 131 ( 131 i ), iodine - 125 ( 125 i ), fluorine - 18 ( 18 f ), gallium - 68 ( 68 ga ), copper - 64 ( 64 cu ), copper - 67 ( 67 cu ), zirconium - 89 ( 89 zr ), yttrium - 90 ( 90 y ), lutetium - 177 ( 177 lu ), indium - 111 ( 111 in ), or technetium - 99m ( 99m tc ). contrast imaging agents for magnetic resonance imaging ( mri ) may include any known in the art including a gadolinium - based contrast agent . other imaging agents which may be used include feridex i . v ., mangafodipir ( teslascan ), a contrast agent for ultrasound , such as a micro - bubble contrast agent , and fluorodeoxyglucose ( 18 f ). after the imaging agent is administered and a suitable time is elapsed for the agent to reach the target tumor , a non - invasive detection technique is performed to generate an image of the tumor in the individual . suitable techniques include without limitation mri , ultrasound , pet , and ct scan . vascular - active permeability agents are those which increase the amount of a therapeutic or imaging agent which is delivered via the circulation to a tumor . without limiting the invention to any particular mechanism of action , the agents may act by causing vascular inflammation , or by disrupting the vasculature so that agents of a size which were previously not delivered are delivered , or so that an increase in the amount of an agent of a certain size is delivered . exemplary vascular - active permeability agents include bacteria ( including bacteria which spontaneously infect tumors ), such as clostridium novyi - nt , bacterial spores , a bacterial component , for example lipopolysaccharide ( lps ), a vaccine , coley &# 39 ; s toxin , a cytokine , such as tumor necrosis factor - alpha ( tnf - α ), interferon - gamma ( ifn - γ ), or interleukin - 2 ( il - 2 ), a chemokine , an inducer of cytokine or chemokine expression , e . g ., vadimezan ( asa404 , dmxaa ), and inducer of vascular inflammation , an immune response modifier , a hormone , a pressor agent , such as angiotensin ii or adrenaline , a virus , a microtubule interacting agent , such as vinorelbine , combretastatin a4 phosphate ( ca4p ), hti - 286 , or colchicine , a nitric oxide synthase inhibitor , such as l - name , l - nna , or l - nmma , tumor - localized radiation , and tumor - localized thermotherapy , and high intensity focused ultrasound . any combination of named therapeutic or imaging agents with vascular - active permeability entities are specifically contemplated as if each combination were listed separately and explicitly . any method known in the art can be used to determine whether the amount of therapeutic agent or imaging agent delivered is increased . these include , without limitation in vivo imaging , biopsy , and agent localization , or tumor response using resist criteria . the ( 1 ) vascular - active permeability agent and ( 2 ) nanoparticles or antibodies with ( 3 ) an imaging agent can be used to assess appropriateness of treatment or appropriate dosages of ( a ) the vascular - active permeability agents and ( b ) nanoparticles or antibodies with ( c ) a therapeutic agent . thus these can be used sequentially or iteratively . kits may contain the reagents for both assessment and therapeutic uses . solid tumors to be treated may be of any type and in any organ of the body of a mammal , such as a farm animal , a pet , a laboratory animal , or a human . these may be in the brain , colon , breasts , prostate , liver , kidneys , lungs , esophagus , head and neck , ovaries , cervix , stomach , colon , rectum , bladder , uterus , testes , and pancreas , as non - limiting examples . the type of tumor may be an adenocarcinoma , a squamous cell carcinoma , or a sarcoma , for example . the major limitation for most chemotherapeutic agents is their toxicity toward normal tissues , which prohibits the use of doses high enough to eradicate all cancer cells . one approach to address this problem is to develop agents that are delivered to all cells but are preferentially toxic to tumor cells because of the abnormal signaling pathways . this strategy underlies the success of agents such as gleevec ( imatinib ) and iressa ( gefitinib ) ( 26 , 27 ). a second approach is to use agents that bind to extracellular molecules present at higher concentrations on the surface of tumor cells , such as herceptin ( trastuzumab ) and erbitux ( cetuximab ) ( 28 , 29 ). the third approach takes advantage of the abnormal vasculature present in tumors , allowing preferential accumulation of nanoparticles ( the epr effect ) ( 6 , 30 ). though all approaches have merit , the third has the advantage that virtually any drug , including a wealth of clinically approved agents , can in theory be made more effective by its incorporation into nanoparticles of appropriate sizes . the ability to use agents that are already clinically approved poses many practical advantages with respect to the performance of clinical trials and the duration of the drug approval process . in this work , we have attempted to enhance the third approach through pharmacologic manipulation of the abnormal vasculature already present in tumors . we show that enhanced epr ( e 2 pr ). can dramatically increase the tumor : blood ratio of nanoparticles , as assessed by both imaging and therapeutic response . it is worth noting that even a small difference in the intratumoral concentration of an agent can make a large difference in therapeutic effect ( 31 ). in the studies described here , e 2 pr led to a tumor : blood ratio of more than 22 - fold ( fig2 a ). we were particularly encouraged with the results in the gbm model . this tumor type in humans is highly recalcitrant to conventional therapies , leading to a dismal prognosis for patients with this disease . the blood - brain barrier is at least partly to blame for the limited efficacy of chemotherapy ( 32 ). we found that tnf - α treatment could help breach the blood - brain barrier and result in major accumulations of 125 i - labeled sterically stabilized liposomal nanoparticles ( ssls ) in the orthotopically implanted brain tumors as well as significantly prolong the survival of the tumor - bearing animals ( fig4 ). as the mouse cranial cavity is small , murine brain tumors are particularly difficult to treat as even a minimal amount of growth of a pre - existing tumor is lethal . our results suggest a way to improve the therapeutic efficacy of conventional and novel drugs by incorporating them into nanoparticles and injecting them together with vascular - active agents such as tnf - α . the approach is versatile , as it should be practicable with a variety of nanoparticle formulations as well as with diverse chemical and radioactive agents . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . ct26 ( crl - 2638 ) murine colorectal adenocarcinoma cells were purchased from the american type culture collection ( atcc ) and grown in mccoy &# 39 ; s 5a medium ( invitrogen ) supplemented with 10 % fetal bovine serum ( fbs , hyclone ) at 37 ° c . with 5 % co2 . gl261 glioma cells were kindly provided by dr . michael lim ( johns hopkins university , baltimore ) and maintained in dmem media ( atcc ) supplemented with 10 % fbs . bolton - hunter reagent ( bh , n - succinimidyl - 3 -( 4 - hydroxyphenyl )- propionate ) and tnf - α ( mouse , recombinant ) were purchased from sigma - aldrich . radioiodines ( sodium 125 - or 131 - iodide ) were purchased from mp biomedicals and nordion , respectively . iodo - gen was purchased from pierce . mouse monoclonal igg1 isotype control antibody ( ab18447 ) and cd20 antibody ( ab8237 ) were purchased from abcam . pegylated liposomal doxorubicin ) ( doxil ® was purchased from tibotec therapeutics . hydrogenated chicken egg l - α - phosphatidylcholine ( hepc ), 1 , 2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - n -[ methoxy ( polyethylene glycol )- 2000 ] ( dspe - peg2000 ) and cholesterol ( chol ) were purchased from avanti polar lipids . c . novyi - nt spores were prepared as previously described ( 11 ). all animal experiments were overseen and approved by the animal welfare committee of johns hopkins university , and were in compliance with the university standards . for the subcutaneous tumor model , female , six to eight week - old balb / c mice ( harlan breeders ) were used . five million ct26 cells were injected subcutaneously into the right flank of each mouse and allowed to grow for ˜ 10 days before randomization , group assignment , and treatment . c . novyi - nt spores were administered by a bolus tail vein injection of 300 million spores suspended in 0 . 2 ml of phosphate buffered saline , ph 7 . 5 ( pbs ). cytotoxic anticancer agents were administered 16 hours later via the same route . tnf - α was reconstituted freshly before administration in doubly - distilled h2o at 100 ng / ml and diluted into 0 . 1 % ( w / v ) bsa in pbs at a final concentration of 10 ng / ml . cytotoxic agents were injected within a few minutes thereafter . tumor volume was calculated as length × width × 0 . 5 . for the orthotopic brain tumor model , female c57bl6 mice , 5 - 6 weeks of age , were purchased from the nci - frederick . mice were anesthetized via intraperitoneal injection of 60 μl of a stock solution containing ketamine hydrochloride ( 75 mg / kg , abbot laboratories ), xylazine ( xyla - ject ®, 7 . 5 mg / kg , phoenix pharmaceutical ), and ethanol ( 14 . 25 %) in a sterile 0 . 9 % nacl solution . following a 1 - cm midline scalp incision , a 1 - mm burr hole was placed over the right frontal bone , with its center 2 mm lateral to the sagittal suture and 1 mm anterior to the coronal suture . on a stereotactic frame , a sterile needle loaded with 20 , 000 gl261 cells was placed at a depth of 3 mm below the dura and the cells were injected slowly at a rate of 1 μl / minute . afterwards , the animal was removed from the frame and the scalp incision closed with surgical staples . on day 12 post implantation of the tumor cells , a significant tumor was formed and 1 μg of mouse recombinant tnf - α or 100 μl of doxil at 20 mg / kg , or both , were administered intravenously through the tail vein . animals were monitored for potential side effects following drug administration . animals were observed daily for any signs of deterioration , neurotoxicity , or movement disorders . they were inspected for signs of pain and distress , as per the johns hopkins animal care and use guidelines . if the symptoms persisted and resulted in debilitation , the moribund animals were euthanized . the brain and other organs were dissected and placed in formalin for further pathological studies . three peptides ( jhu009a : cnvdlqqklien ; jhu009b : cypewgtkdengnirk ; jhu009c : cdmaqmlrnlpvte ) were used to immunize the mice for generating antibodies against c . novyi - nt liposomase ( a & amp ; g pharmaceutical ). after screening ˜ 500 hybridoma clones by elisa , one clone ( jhu009 - 5f5 ) specific to the jhu009c peptide was eventually selected for the imaging study . the affinity and specificity of the jhu009 - 5f5 mab were also confirmed by both elisa and western blot analyses against purified liposomase protein ( 12 ). typically , 20 μg of purified antibody in 100 μl of pbs was added to an iodogen - coated vial . sodium 125 - or 131 - iodide was then added to the vial at 2 to 5 mci in 2 to 5 μl of 0 . 1 m naoh , ph 10 . the reaction was then incubated for 10 minutes at room temperature before purification on a pbs - equilibrated sephadex g - 25 desalting column ( amersham biosciences ) to remove unincorporated radioiodine . the radiochemical yield was typically 30 % to 40 %. the radiochemical purity was at least 95 % as determined by thin - layer chromatography . antibodies were labeled within 24 hours of use and stored in pbs at 4 ° c . after labeling and purification . a mixture of hepc : chol : dspe - peg2000 at a molar ratio of 50 : 45 : 5 was solubilized in chloroform , followed by drying to a thin film under rotary evaporation and then under vacuum for 2 hours . the film was hydrated with arginine solution ( 80 mmol / l ) in 4 -( 2 - hydroxyethyl )- piperazine - 1 - sulphonic acid ( hepes , 80 mmol / l , ph 8 . 0 ) and submerged in a 65 ° c . sonication bath ( bransonic ) to form large multilamellar vesicles ( mlvs ). this lipid suspension was extruded 10 times through a double stack of 0 . 1 μm nuclepore filters ( whatman ) using a lipex thermobarrel extruder ( northern lipids ). the resulting colloidal suspension of single unilamellar vesicles ( suv ) was dialyzed against 150 mmol / l phosphate buffer ( ph 5 . 6 ) at 4 ° c . to exchange the external milieu of the liposomes and then filter - sterilized . the mean size of the suvs was ˜ 100 nm in diameter and polydispersity index ˜ 0 . 1 as determined by quasi - elastic light scattering using a malvern zetasizer 3000 ( malvern ). bolton - hunter reagent ( bh , n - hydroxysuccinimide ( nhs ) ester of hppa ) was labeled with sodium 125 - or 131 - iodide by the chloramine - t method and purified by solvent extraction . briefly , 50 μl of chloramine t ( 4 mg / ml in phosphate buffer ) and 3 . 7 to 37 mbq ( 0 . 1 - 1 . 0 mci ) of 125 i — nai or 131 i — nai were added to 2 μl of bh freshly solubilized in anhydrous dioxin ( 0 . 5 mg / ml ). iodination was achieved by incubation at room temperature for approximately 15 sec and then 400 μl of 100 mmol / l phosphate buffer ( ph 7 . 4 ) was added . the radiolabeled bh was immediately extracted with 500 μl of toluene and the organic phase was removed and transferred to a glass tube . for the encapsulation of the reagent into liposomes , the organic solvent was evaporated using a dry nitrogen stream before adding the liposome suspension . for the chemical entrapment of the iodinated bh , arginine - containing liposomes were incubated for 30 min at 37 ° c . with 125 i — bh . the labeling efficiency was determined by counting the liposome suspension before and after chromatography on a pd - 10 column ( ge healthcare ) ( 13 ). the radiochemical yield was typically 50 % to 70 %. ct26 - bearing balb / c mice were injected via the tail vein with 50 μci of 125 i - liposomes or 125 i - igg1 . three to four mice in each experimental arm were sacrificed by cervical dislocation at 48 hours post injection . the liver , spleen , kidneys , muscle , and tumor were quickly removed as was ˜ 0 . 1 ml of blood . the organs and blood were weighed and their radioactivity was measured with an automated gamma counter ( 1282 compugamma cs , pharmacia / lkb nuclear ). the percent injected dose per gram of tissue ( id %/ g ) was calculated by comparison with samples of a standard dilution of the initial dose . all measurements were corrected for decay . balb / c mice bearing subcutaneous ct26 tumor or c57bl6 mice bearing orthotopic gl261 brain tumor were injected intravenously with 37 . 5 mbq ( 1 mci ) of either 125 i - igg1 or 125 i - ssls in saline . the mice were positioned on the x - spect ( gamma medica - ideas ) gantry and scanned using two low - energy , high resolution pinhole collimators ( gamma medica - ideas ) rotating through 360 ° in 6 ° increments for 40 seconds per increment immediately following spect acquisition , the mice were scanned by ct ( x - spect ) over a 4 . 6 cm field of view using a 600 ma , 50 kv beam . data were reconstructed using the ordered subsets - expectation maximization algorithm . the spect and ct data were then coregistered using the instrument supplied software and displayed using amide ( http :// amide . sourceforge . net /) or amira software ( visage imaging ). the statistical significance of percent survival between different experimental arms was determined by long - rank analysis . the research described in this work was stimulated by unexpected observations made through the investigation of c . novyi - nt , an attenuated anaerobic bacterial strain that can infect experimental tumors ( 11 ). this infection often leads to eradication of the internal hypoxic regions of tumors but leaves the oxygenated rim of the tumors intact . c . novyi - nt secretes an enzyme called liposomase at high levels in the infected tumors ( 12 , 14 ). we hypothesized that a radiolabeled anti - liposomase antibody would synergize with c . novyi - nt by binding to liposomase secreted by the bacteria , thereby eradicating the oxygenated tumor rim through β - particle irradiation . a monoclonal antibody against liposomase was generated and used to evaluate this hypothesis ( see methods ). mice bearing subcutaneous ct26 tumors were intravenously injected with c . novyi - nt spores together with the radiolabeled anti - liposomase antibody or with a similarly labeled igg control antibody . the anti - liposomase antibody was highly enriched in the tumors infected with c . novyi - nt but not in uninfected tumors ( fig1 a ). surprisingly , however , the radiolabeled igg control antibody was also enriched in the c . novyi - nt infected tumors , albeit to a lesser extent ( fig1 a ). biodistribution analyses showed that the level of radioactivity in the tumor was four - fold higher than that in most normal tissues ( fig1 b ). to further confirm that the accumulation in the tumors was not antibody - specific , we repeated the experiment with another antibody generated against human cd20 , a b - cell antigen . the partially humanized version of this antibody , rituximab , has been marketed for the treatment of b cell lymphoma and chronic lymphocytic leukemia ( 15 , 16 ). systemically administered anti - cd20 antibody was also enriched in the tumor if the animal was simultaneously injected with c . novyi - nt spores ( fig1 a ). bacterial infection and pro - inflammatory cytokine both enhance tumor - selective accumulation of macromolecular drug formulations we reasoned that the inflammatory response to the bacterial infection led to an increased vascular permeability , resulting in the preferential antibody accumulation at the infected tumor site . we therefore sought to identify a pro - cytokine that might mimic the effects of c . novyi - nt . among those considered , tumor necrosis factor - α ( tnf - α ) was of particular interest as this cytokine has been identified as the serum factor responsible for endotoxin - induced vascular permeabilization ( 17 , 18 ). furthermore , a similar hemorrhagic necrosis in tumors is observed following systemic administration of either tnf - α or c . novyi - nt spores ( 11 , 17 ). based on these parallels , we repeated the protocol described above , substituting systemically - administered tnf - α for c . novyi - nt spores . when ct26 tumor - bearing mice were injected with murine tnf - α and radiolabeled murine igg , significant igg accumulation was observed in the tumors but not in the normal tissues ( fig1 b and c ). a time course study revealed that the igg tumor accumulation progressed slowly and peaked between 72 and 96 hours after injection ( fig1 c ). the effect of vascular - active agents on tumor vasculature will henceforth be referred to as enhanced epr ( e 2 pr ). sterically stabilized liposomal nanoparticles ( ssls ) of ˜ 100 nm in diameter have been shown to be susceptible to the epr effect ( 8 ). to evaluate whether such liposomes were susceptible to e 2 pr , we fabricated radioactive liposomes using a bolton - hunter ( bh ) reagent - based iodination strategy ( 13 ). iodinated bh reagent labels proteins by forming amide bonds with free amino groups such as those present on arginine ( 19 ). ssls were loaded with arginine at low ph and then the loaded ssls were incubated with 125 i - labeled bh reagent . the 125 i — bh reagent passed through the lipid bilayer but was unable to exit after covalent binding to the arginine because of the latter &# 39 ; s positive charge . we were thus able to achieve a very high concentration of radioactivity within the ssls while avoiding prolonged exposure to the radioactivity during the preparation . 125 i - labeled ssls were intravenously injected into tumor - bearing mice in combination with either c . novyi - nt or tnf - α . both c . novyi - nt and tnf - α treatments significantly augmented the selective retention of 125 i within tumors ( fig2 ). furthermore , the radioactivity in normal tissues was markedly lower compared to the animals treated with 125 i - labeled ssls without tnf - α or c . novyi - nt ( fig2 a ). thus , the tumor - to - blood radio of radioactivity following tnf - α treatment was as high as 22 - fold , far higher than achieved with radiolabeled igg ( compare fig2 a to fig1 b ). spect / ct also revealed that the kinetics of tumor accumulation was different with radiolabeled ssls than with igg : ssl accumulation peaked at 24 hours , 48 - 72 hours earlier than igg . like epr , the effect of e 2 pr is particle size - dependent . in contrast to 125 i - labeled ssls , tumor retention of 125 i - labeled arginine ( the substrate of 125 i labeling in ssls ) is not affected by tnf - α . however , at the other end of the size spectrum , 125 i - labeled c . novyi - nt spores (˜ 1 μm in diameter ( 20 )) are highly enriched in tumors only when combined with tnf - α ( data not shown ). thus , e 2 pr appears to reflect a more substantial vascular disruption than epr : while epr favors accumulation of nanoparticles in the range around 100 nm ( 8 ), e 2 pr extends that range to & gt ; 1 μm . to determine whether the accumulation was dependent on the volume of the tumor , we injected tnf - α plus 125 i - labeled igg or 125 i - labeled ssls into animals with a small subcutaneous tumor on one flank and a large tumor on the other flank . spect / ct showed retention of radioactivity in both tumors . we also tested the relative timing of injection of tnf - α and 125 i - labeled ssls . though tnf - α and ssls were administered jointly in the experiments recorded above , we found that similar results were obtained when tnf - α was administered within 12 hours after ssls . conversely , e 2 pr was not observed when tnf - α was administered 6 hours prior to ssl administration ( data not shown ). microtubule - interacting agents are also able to disrupt the tumor vasculature ( 21 ). we therefore determined whether such agents could induce e 2 pr . combretastatin a4p ( ca4p ) and vinorelbine are microtubule - interacting agents with completely different structures and modes of interaction with microtubules ( 22 , 23 ). injection of either resulted in e 2 pr , though not as impressively as tnf - α . tnf - α and macromolecular drug formulations synergize in the treatment of experimental tumors we next investigated whether the e 2 pr could be translated into therapeutic gain . mice bearing fully developed ct26 tumors were treated by simultaneous i . v . injections of tnf - α plus doxil ( 10 mg / kg ) or radiolabeled igg . 131 i rather than 125 i was chosen for radiolabeling in light of the type of ionizing radiation required for a radiotherapeutic effect . although treatment with doxil or 131 i - labeled igg in the absence of tnf - α retarded tumor growth and prolonged animal survival , the tumors always grew back ( fig3 a and b ). when combined with tnf - α , however , a single administration of these agents led to complete tumor regression in all animals and long - term cures in more than 75 % of them . when a lower dose ( 25 ng / kg ) of tnf - α was used , none of the treated animals were cured , although prolonged survival was observed . it is important to note that humans tolerate multiple injections ( 3 infusions / week ) of a dose comparable to the highest dose of tnf - α we used ( 24 ). we also tested ssls containing 131 i , generated using the chemical trapping approach described above . while 131 i - labeled ssls alone retarded tumor growth , complete tumor regression and cures were only observed when they were used in combination with tnf - α ( fig3 c ). finally , we evaluated the therapeutic potential of e 2 pr in a murine model of glioblastoma multiforme ( gbm ). when implanted orthotopically , the brain tumor cell line gl261 forms very aggressive tumors , killing animals within about a month ( fig4 a ). at the histologic level , these tumors are very similar to human gbm , manifesting an infiltrative growth pattern , necrosis and neovascularization ( 25 ). following stereotactic injection of gl261 cells into the frontal lobe , brain tumors were allowed to grow to substantial size , then 125 i - labeled ssls with or without tnf - α were administered . tumor accumulation of the radiolabeled ssls was only observed in tnf - α treated animals ( fig4 b ). mice with similar tumors were injected with doxil , either with or without tnf - 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