DENDRITIC CONJUGATES AND METHODS OF USE

RELATED APPLICATIONS AND PRIORITY CLAIM

This application claims priority to US Provisional Patent Application Serial No. 61/055,328 filed on May 22, 2008. The application is incorporated herein by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services. The Government may have certain rights in this invention.

INCORPORATION BY REFERENCE Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or paragraphing priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. More generally, documents or references are cited in this text, either in a Reference List, or in the text itself; and, each of these documents or references ("herein-cited references"), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

During disease conditions of the brain and the spinal cord, due to the local release of permeability factors, the blood-brain barrier of micro vasculature within the diseased brain and spinal cord tissue becomes porous. Although it is well known that there are pores in the blood-brain barrier of primary malignant brain tumors, such as glioblastoma multiforme (GBM, malignant glioma), and metastatic brain tumors from other organs, such as those from

breast, lung and skin, the effective transvascular delivery of intravenous chemotherapeutics across the blood-brain barrier of malignant brain tumors has not been possible to date.

Currently used intravenously administered chemotherapeutics, such as doxorubicin and carboplatin, are small compounds having molecular weights less than 1 kilodalton (kDa), and therefore, diameters less than lnm. Even though currently used small chemotherapeutics cross the blood-brain barrier of malignant brain tumors these drugs are unable to accumulate within malignant glioma cells at therapeutic levels due to the relatively short peak blood half- lives of minutes.

In contrast to the low molecular weights and sizes of presently used intravenous chemotherapuetics, the sizes of presently used nanoparticle-based therapeutics, such as liposomal doxorubicin (Doxil), are typically between 50 and 150 nm in diameter. Previous studies have suggested that the physiologic upper limit of pore size within the blood-brain barrier of malignant brain tumors may be up to 100 nm. Intravenously administered nanoparticles that are smaller than the pores within the blood-brain barrier of malignant brain tumors and have long blood half-lives would function as effective transvascular drug delivery vehicles for the sustained-release of chemotherapeutics into malignant brain tumor cells.

Dendrimers are a class of nanoparticles with polyamidoamine (PAMAM) dendrimers being one sub-class. PAMAM dendrimers are multigenerational polymers that have a branched exterior consisting of surface groups that can be functionalized with imaging and therapeutic agents. PAMAM dendrimers functionalized with low molecular weight agents such as gadolinium-DTP A for magnetic resonance imaging and doxorubicin for chemotherapy remain particularly small, typically ranging between 1.5 nm (generation 1, Gl) and 14 nm in diameter (generation 8, G8). Particle shapes are spherical and sizes are uniform within a particular generation. With each successive dendrimer generation, the number of modifiable surface groups doubles while the overall diameter increases by only 1 to 2 nm.

SUMMARY OF THE INVENTION

The instant application is based on the findings that (1) intravenously administered biocompatible nanoparticles, particularly functionalized a polyamidoamine (PAMAM) dendrimers particles smaller than 13 nm (Gl through G7), but not larger (G8), can cross the blood-brain tumor barrier (BBTB) when malignant solid tumors are located within the brain (i.e. orthotopic tumor) as well as in the case of the blood-tumor barrier (BTB) of malignant solid tumors located outside the brain in other tissues, and (2) the intravenously administered nanoparticles with prolonged blood half-lives accumulate within rumor cells after crossing

the BBTB or BTB. These findings have application in effectively detecting, monitoring, treating malignant tumors, including primary and metastatic brain tumors, and other neurological diseases that have not been effectively treated to date.

The invention provides a biocompatible nanoparticle conjugated to an agent, wherein the nanoparticle comprises a diameter between 1.5 nanometers and 13 nanometers. In an embodiment, the dendritic conjugate has a diameter between about 5 nanometers and 12 nanometers. In an embodiment, the dendritic conjugate has a diameter between 7 nanometers and 12 nanometers. Biocompatible nanoparticles provided by the invention include, for example dendrimers, including half dendrimers, half and full generation dendrimers, and modified dendrimers. In an embodiment biocompatible nanoparticles of the invention include polyamidoamine (PAMAM) dendrimers.

In an embodiment, the nanoparticles have a molecular weight greater than about 10 kDa. In a further embodiment, the nanoparticles have a molecular weight greater than about 35 kDa, or greater than about 40 kDa. In an embodiment, the dendritic conjugate has a molecular weight of less than about 300 kDa, less than about 200 kDa, less than about 150 IcDa, or less than about 100 kDa. In an embodiment, the nanoparticles have a molecular weigh great enough to avoid significant clearance by the kidneys, and low enough to avoid significant clearance by the reticuloendothelail system. Such limitations are well understood by those of skill in the art. In an embodiment, the dendritic conjugate has a negative overall charge. In another embodiment, the dendritic conjugate has a neutral overall charge.

In an embodiment, the dendritic nanoparticles include a generation 3.5 (G 3.5), generation 4 (G4), generation 4.5 (G 4.5), generation 5 (G5), generation 5.5 (G 5.5), generation 6 (G6) generation 6.5 (G 6.5), generation 7 (G7) , or generation 7. 5 (G 7.5) dendrimer.

The biocompatible nanoparticles provided by the invention include imaging agents, therapeutic agent, or both imaging agents and therapeutic agents. In an embodiment, imaging agents include, but are not limited to, gadolinium, manganese, chromium, iron, fluorescing entities, phosphorescence entities, signal reflectors, paramagnetic entities, signal absorbers, contrast agents, and electron beam opacifiers. In certain embodiments, agents are conjugated dendrimers for example by use of a chelate or a hydrolysable covalent linkage. Chelates include, but are not limited to, diethylene triamine pentaacetic acid (DTPA), 1,4,7,10- tetraazacyclododecane-NjN', N",N'"-tetraacetic acid (DOTA), porphyrin, and desferrioxamine; or derivatives thereof.

In the nanoparticles provided by the invention, therapeutic agents can include chemotherapeutic agents such as acyclovir, alkeran, amikacin, ampicillin, bisantrene, bleomycin, neocardiostatin, carboplatin, chloroambucil, chloramphenicol, cytarabine, daunomycin, doxorubicin, fluorouracil, gadolinium, gentamycin, kanamycin, meprobamate, methotrexate, novantrone, nystatin, Oncovin, phenobarbital, polymyxin, probucol, procarbabizine, rifampin, streptomycin, spectinomycin, Symmetrel, thioguanine, tobramycin, temozolamide, trimethoprim, cisplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, vinca alkaloids, taxanes, vincristine, vinblastine vinorelbine, vindesine, etoposide, teniposide, paclitaxel, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, and dactinomycinand valban; diphtheria toxin, gelonin, exotoxin A, abrin, modeccin, ricin, radioactive gadolinium, radioactive boron, and radioactive iodine.

The invention further provides uses for the nanoparticle compositions of the instant invention. For example, the invention provides methods for delivering a biocompatible nanoparticle across the blood-brain barrier in a subject or a blood-tumor barrier in a subject by administering to the subject a biocompatible nanoparticle described herein, thereby delivering the biocompatible nanoparticle across the blood-brain barrier or blood-tumor barrier. In certain embodiments, the subject is suspected of having a tumor or has been identified as having tumor, such as a solid a solid tumor. Solid tumors include both primary tumors and metastatic tumors related to various cancer types including, but not limited to, adrenocortical carcinoma, basal cell carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, esophageal cancer, ewing family of tumors, retinoblastoma, gastric cancer, gastrointestinal tumors, glioma, head and neck cancer, hepatocellular cancer, islet cell tumors, kidney cancer, laryngeal cancer, non-small cell lung cancer, small cell lung cancer, medulloblastoma, melanoma, pancreatic cancer, prostate cancer, rectal cancer, and thyroid cancer. The nanoparticle can be delivered for treating, imaging, diagnosis, or monitoring of a tumor.

The agents can be delivered for treating, imaging, diagnosis, or monitoring a neurological disease associated with increased pore size in the blood-brain barrier. Such diseases include, but are not limited to, all types of brain and spinal cord tumors including primary tumors arising in the brain and spinal cord such as but not limited to astrocytomas (including glioblastoma multiforme), oligdendrogliomas, lymphomas and meningiomas, and metastatic tumors from other organs such as but not limited to the breast, lung, skin, and kidney), brain and spinal cord tumors previously treated with radiation therapy, traumatic

brain and spinal cord injuries, infectious diseases such as meningitis, encephalitis and abscesses, epilepsy lesions, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and ischemic disease (stroke) of the brain and spinal cord.

The invention provides for various routes of administration including, but not limited to intravenous, topical, intra-arterial, intrathecal, intratumoral, or intracranial delivery.

The invention further provides kits including a biocompatible nanoparticle provided by the invention and instructions for use. A kit can further include two or more non-standard laboratory reagents for making a nanoparticle of the invention and instructions for making the same. The invention provides for the use of a biocompatible nanoparticle of the invention for the preparation of a medicament, for example a medicament for the treatment of a disease or condition selected from the group consisting of cancer and a neurological disorder. In an embodiment, the medicament is prepared in a pharmaceutically acceptable carrier. In an aspect, the invention features a method for preferentially delivering by intravenous or intrarterial administration an agent preferentially to a brain tumor cell, and minimally or not detectably to a normal non-cancerous cell, by linking the agent to a dendritic conjugate according to any one of the above aspects, and administering the agent to a subject suffering from a brain tumor, including a metastatic brain tumor. In an embodiment, the normal cell is a brain cell. In another aspect, the normal cell is a non-tumor cell in the same tissue as the tumor, for example, a non-cancerous cell adjacent to the tumor. In an embodiment, the agent is delivered to the tumor cell to a concentration at least 25% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 150% greater, at least 200% greater, at least 300% greater, at least 500% greater than in a normal cell, or more. In an aspect, the invention features a method for increasing the blood half-life of an agent in a subject by linking the agent to a dendritic conjugate according to any one of the above aspects, and administering the agent to a subject, hi an embodiment, the blood half- life in vivo is increased to about 200 minutes, about 300 minutes, or about 400 minutes. In an embodiment, the agent is released from the dendrimer conjugate over time at physiological pH and isotonic salt. In an embodiment, at least 50%, at least 60%, at least 70%, at least 80% of the agent is released within the half-life of the agent in the subject.

In an aspect, the invention features a method for increasing the accumulation of an agent in diseased target tissue as compared to administration of the free agent, by linking the agent to a dendritic conjugate according to any one of the above aspects, and administering

the agent to a subject. In an embodiment, the blood half-life in vivo is increased to about 200 minutes, about 300 minutes, or about 400 minutes. In an embodiment, the agent is released slowly from the dendrimer conjugate over time at physiological pH and isotonic conditions. In another embodiment, the agent is released more rapidly at the acidic pH in tumor cell organelles and isotonic conditions. In an embodiment, at least 50%, at least 60%, at least 70%, at least 80% of the agent is released within the blood residence time of the agent in the subject.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 panels a - c show functionalized PAMAM dendrimers within any particular generation are uniform in size, shape and density. Ia shows two dimensional representation of a naked PAMAM dendrimers up until generation 3 showing ethylenediamine (EDA) core. The number of terminal amines doubles every generation and can be used to conjugate imaging, targeting, and therapeutic agents to the dendrimer. Ib shows that after functionalizing the naked PAMAM dendrimer with Gd-DTPA (charge -2) the positively charged naked PAMAM dendrimer exterior is neutralized. When the Gd-DTPA conjugation percentage is greater than approximately 50% then the naked dendrimer becomes slightly negatively charged. The duration of the chelation reaction for lowly conjugated (LC) Gd-G4 was 24 h as compared to the standard 48 h for chelation of all other dendrimers. Ic shows annular dark field scanning transmission electron microscopy (ADF STEM) images of Gd- G5, Gd-G6, Gd-G7, and Gd-G8 dendrimers adsorbed onto an ultrathin carbon support film. Images were obtained under identical conditions and are shown on the same intensity scale. The diameters of sixty Gd-G7 and Gd-G8 dendrimers were measured. Gd-G7 11.0 ± 0.74 nm, Gd-G8 13.3 ± 1.4 nm, mean ± standard deviation (s.d.). Scale bar = 20 nm.

Figure 2 panels a - i show that the intravascular concentration and blood half-life of permeable Gd-PAMAM dendrimers determines extent and duration of particle accumulation in brain tumor. 2a shows blood concentrations of Gd-dendrimer generations measured in the superior sagittal sinus following 0.03 mmol Gd/kg body weight (bw) intravenous infusion. At this dose, the brains of animals receiving Gd-dendrimer Gl (n=6), G2 (n=5), G3 (n=5) and LC G4 (n=5) were only imaged for 1 hour due to rapid clearance of lower generation Gd- dendrimers from blood. Gd-dendrimers standard G4 (n=6), G5 (n=6), G6 (n=5), G7 (n=6) and G8 (n=5) were imaged for 2 hours. Gd-dendrimers G5 through G8 rapidly reach and

maintain a steady state in blood over 2 hours (G6, G7 and G8 not shown for clarity). Error bars represent s.d. and are shown once every five minutes. 2b shows blood concentrations of Gd-dendrimer generations measured in the superior sagittal sinus following 0.09 mmol Gd/kg bw infusion. At this dose, brains of animals receiving Gd-dendrimers Gl (n=4), G2 (n=6), G3 (n=6), LC G4 (n=4), standard G4 (n=6), G5 (n=6), G6 (n=5), G7 (n=5) and G8 (n=6) were imaged over 2 hours. There are increases in circulation times of Gd-dendrimers Gl through G4. At this dose, Gd-dendrimers G5 through G8 also rapidly reach and maintain a steady state in blood over 2 hours (G6, G7 and G8 not shown for clarity). There are higher peak blood concentrations for all Gd-dendrimer generations. Error bars represent s.d. and are shown once every five minutes. 2c shows Gd-dendrimers do not enter the normal brain extravascular space due to an intact BBB irrespective of dose. Shown are concentration curves at the 0.09 mmol Gd/kg bw dose, Gl (n=5), G5 (n=6). The normal brain tissue Gd- dendrimer concentration profiles reflect blood half-lives of Gd-Gl and Gd-G5 dendrimers in brain microvasculature, and are representative examples of low and high dendrimer generation behavior. Average concentration curves are from normal brain tissue volumes of 9 mm ³ per brain. Error bars represent s.d. and are shown once every five minutes. 2d shows that at both doses, LC Gd-G4 dendrimers (molecular weight (MW) 24.4 kDa) only transiently accumulate within the extravascular tumor space before being cleared. However, there is an increased amount and extended duration of accumulation before clearance at the 0.09 mmol Gd/kg bw dose. 0.03 mmol Gd/kg bw dose n=5, 0.09 mmol Gd/kg bw dose n=4. Error bars represent s.d. weighted for total tumor volume and are shown once every five minutes. 2e shows that at both doses, standard Gd-G4 dendrimers (MW 39.8 kDa) remain within the extravascular tumor space following accumulation. However, at the 0.09 mmol Gd/kg bw dose the magnitude of accumulation is greater. 0.03 mmol Gd/kg bw dose n=6, 0.09 mmol Gd/kg bw dose n=6. Error bars represent s.d. weighted for total tumor volume and are shown once every five minutes. 2f shows that at both doses, Gd-G5 dendrimers accumulate within the extravascular tumor space over 2 hours. However, at the 0.09 mmol Gd/kg bw dose the rate and magnitude of accumulation are both substantially greater. 0.03 mmol Gd/kg bw dose n=6, 0.09 mmol Gd/kg bw dose n=6. Error bars represent s.d. weighted for total tumor volume and are shown once every five minutes. 2g shows that at the 0.03 mmol Gd/kg bw dose (n=5), there is no observable extravasation of G6 Gd-dendrimers out of tumor microvasculature over 2 hours. At the 0.09 mmol Gd/kg bw dose (n=5), there is a steady rate of G6 dendrimer extravasation and accumulation comparable to that of G5 dendrimers. 0.03 mmol Gd/kg bw dose n=5, 0.09 mmol Gd/kg bw dose n=5. Error bars represent s.d. weighted

for total tumor volume and are shown once every five minutes. 2h shows that similar to Gd- G6 dendrimers there is no observable extravasation of G7 Gd-dendrimers out of tumor microvasculature at the 0.03 mmol Gd/kg bw dose (n=6) over 2 hours. However, at the 0.09 mmol Gd/kg bw dose (n=5) there is a steady rate and magnitude of accumulation of Gd-G7 dendrimers but not as pronounced as that of Gd-G6 dendrimers. 0.03 mmol Gd/kg bw dose n=6, 0.09 mmol Gd/kg bw dose n=5. Error bars represent s.d. weighted for total tumor volume and are shown once every five minutes 2i shows that the diameter of the Gd-G8 dendrimer exceeds that of the physiologic pore size of the BBTB, thus, irrespective of dose, Gd-G8 remains within the brain tumor microvasculature. 0.03 mmol Gd/kg bw dose n=5, 0.09 mmol Gd/kg bw dose n=6. Error bars represent s.d. weighted for total tumor volume and are shown once every five minutes. For panels 2d through 2i average tumor concentration curves are weighted with respect to total tumor volume within the respective dendrimer generation.

Figure 3 panels a — c show that the prolonged blood residence times of permeable higher generation Gd-dendrimers result in particle accumulation within glioma regardless of tumor volume. All generations of Gd-dendrimers were infused intravenously over 1 minute at the beginning of the respective DCE-MRI sessions. 3a shows that due to long blood half- lives, Gd-G5, G6 and G7 dendrimers extravasate out of glioma microvasculature and accumulate over time within the tumor extravascular space of larger tumors. Gd-G8 dendrimers remain intravascular since particle size is larger than the physiologic upper limit of pore size within the BBTB. The volume, in mm ³ , for each tumor shown is 104 (Gl), 94 (G2), 94 (G3), 162 (LC G4), 200 (G4), 230 (G5), 201 (G6), 170 (G7) and 289 (G8). 3b shows that in the setting of a less defective BBTB, like that of smaller tumors, Gd-G5 and G6 dendrimers still accumulate over time within tumor tissue due to long circulation times in blood. Extravasation of Gd-G7 dendrimers into the tumor extravascular space is hindered because the physiologic pore size threshold of the BBTB of smaller gliomas may be lower. Gd-G8 dendrimers continue to be impermeable to the BBTB. The volume, in mm ³ , for each tumor shown is 27 (Gl), 28 (G2), 19 (G3), 24 (LC G4), 17 (G4), 18 (G5), 22 (G6), 24 (G6) and 107 (G8). 3c shows that repeated imaging over 12 hours after infusion shows the continued presence of Gd-G5 dendrimers within tumor tissue. Gd-G5 dendrimer concentration peaks in tumor tissue between 2 and 6 hours after intravenous infusion. Residual Gd-G5 dendrimer has not yet been completely cleared from tumor tissue 12 hours after infusion, which would be sufficient time for the dendrimer to accumulate to effective

concentrations within individual brain tumor cells. n=3. At each time point, average concentrations and error bars (s.d.) are weighted with respect to total tumor volume.

Figure 4 panels a - d show the influence of MW on particle distribution within tumor space, based on a 2-compartment 3-parameter generalized kinetic modeling of Gd-Gl through LC Gd-G4 dendrimer blood and tumor tissue pharmacokinetics over 2 hours. The pharmacokinetic analysis was based on selection of the vascular input function from superior sagittal sinus and the selection of the entire tumor, using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data converted from signal intensity to concentration space by generation of a Tl map. 4a shows the impact of the increase in the MW and size significantly decreases the λ*™ ⁸ of LC Gd-G4. Larger tumors had higher A* ³ " ³ values. 4b shows LC Gd-G4 dendrimer distribution within the extravascular extracellular space is affected to the greatest extent by the decrease in A* ¹ ™*. Larger tumors had higher v _e values. 4c shows due to reproducibility of the RG-2 brain tumor model, fractional plasma volume within glioma vasculature is maintained across dendrimer generations. Larger tumors had higher v _p values. 4d shows that the lower MW of Gd-Gl dendrimers results in a more widespread distribution of particles within the extravascular extracellular tumor space as shown by the greater range of v _e values, whereas the higher MW of LC Gd-G4 dendrimers results in more focal distribution of particles as shown by the lower range of v _e values. Shown are voxels surviving censorship. Tumor volumes, in mm ³ , for tumors shown are 104 (Gd-Gl) and 162 (LC Gd-G4). Large circles (Gl n= 4, G2 n=6, G3 n=7 and G4 n=2) represent large tumors (> 50 mm ³ ), small circles (Gl n=4, G2 n=6, G3 n=5 and G4 n=6) represent small tumors (< 50 mm ³ ), horizontal bars represent mean of observations weighted with respect to individual tumor volumes. Shown are Bonferroni corrected p-values from the nine post hoc comparisons for the three parameters, where NS means not significant. Figure 5 panels a - e shows the impediment to cellular uptake of functionalized G8 dendrimers is the BBTB. 5a shows a synthetic scheme for production of the rhodamine B (RB) Gd-PAMAM dendrimers. The naked PAMAM dendrimer is first reacted with RB and then Gd-DTPA. 5b shows in vitro fluorescence microscopy. As shown by in vitro fluorescence microscopy RB functionalized Gd-G2, Gd-G5, and Gd-G8 dendrimers accumulate in glioma cells within 4 hours after exposure. RB Gd-G2 dendrimers enter RG-2 glioma cells, and in some cases the nucleus (left). RB Gd-G5 dendrimers enter RG-2 glioma cells but do not localize within the nucleus, implying a nuclear pore size cut-off (middle). Since RB Gd-G8 dendrimers enter RG-2 glioma cells in vitro, the barrier to cellular accumulation must be at the vascular level (right). Shown are merged confocal images of

blue fluorescence from DAPI-Vectashield nuclear (DNA) stain and red fluorescence from rhodamine B labeled Gd-dendrimers. Scale bars = 20 μm. 5c is the results of DCE-MRI that shows substantial accumulation of RB Gd-G5 dendrimers within tumor tissue but not RB Gd- G8 dendrimers at 2 hours following intravenous infusion. As time progresses the difference in accumulation within tumor tissue between RB Gd-G5 and RB Gd-G8 dendrimers becomes profound. RB Gd-G5 n=6, RB Gd-G8 n=2. Average concentration curves and error bars (s.d.) are weighted with respect to total tumor volume within respective generations. 5d shows low power ex vivo fluorescence microscopy of brain tumor and normal brain surrounding tumor. Panel d shows that there is selective and substantial accumulation of RB Gd-G5 dendrimers within tumor tissue (left, T = tumor, N = normal, scale bar = 100 μm). High power shows subcellular localization (upper right, scale bar = 20 μm). Hematoxylin and Eosin (H&E) stain of tumor and surrounding brain (lower right, scale bar = 100 μm). Shown are merged confocal images of blue fluorescence from DAPI-Vectashield nuclear (DNA) stain and red fluorescence from rhodamine B labeled Gd-dendrimers. Tumor volume is 31 mm ³ . 5e shows by low power ex vivo fluorescence microscopy the minimal accumulation of RB Gd-G8 dendrimers within brain tumor tissue, implying steric hindrance at the level of the BBTB (left, T = tumor, N = normal, scale bar = 100 μm). High power confirms minimal subcellular localization (upper right, scale bar = 20 μm). H&E stain of tumor and surrounding brain (lower right, scale bar = 100 μm). Tumor volume is 30 mm ³ . Figure 6 shows that at 0.09 mmol Gd/kg body bw dose, Gd-dendrimer residence time within the extra- vascular extra-cellular brain tumor space increases with increasing Gd- dendrimer generation from Gd-Gl to Gd-G3. At the 0.03 mmol Gd/kg bw dose, differences in residence times of Gd-Gl (left), Gd-G2 (middle) and Gd-G3 (right) dendrimers within the extra- vascular extra-cellular brain tumor space are not evident. At the 0.09 mmol Gd/kg bw dose, Gd-Gl (n=5), Gd-G2 (n=6), and Gd-G3 (n=6). At the 0.03 mmol Gd/kg bw dose, Gd- Gl (n=6), Gd-G2 (n=5), Gd-G3 (n=5). Error bars represent s.d. weighted for total tumor volume and are shown once every five minutes. Average tumor concentration curves are weighted with respect to total tumor volume within the respective dendrimer generation. Figure 7 shows that intravenous RB labeled Gd-G5 dendrimers accumulate in the extra- vascular tumor space of RG-2 gliomas. Top left and bottom left, anterior and posterior tumors, respectively, of control animal (no dendrimer infusion); top right and bottom left, anterior and posterior tumors, respectively, of a rat brain harvested post-perfusion at completion of the DCE-MRI study 2 hours following intravenous infusion of approximately 100 mg/kg dendrimer (0.06 mmol/kg body weight Gd).

Figure 8 shows that intravenously administered RB labeled Gd-G5 dendrimers accumulate selectively in RG-2 glioma brain tumor tissue and not in normal brain tissue. The rat brain was harvested and snap-frozen at completion of the DCE-MRI study 2 hours following intravenous infusion of approximately 100 mg/kg dendrimer (0.06 mmol/kg body weight Gd). DAPI- Vectashield nuclear (DNA) stain was applied to 10 micron thick cryostat- tissue sections prior to confocal imaging. Red fluorescence of RB in RG-2 glioma tumor tissue (top left) but not in normal brain tissue (top right). Merged red fluorescence of RB with blue fluorescence of DAPI stained glioma cell nuclei (bottom left) or neuronal nuclei (bottom right). Figure 9 is a series of 3 electron micrographs of increasingly higher magnification

(from low to high) of a RG-2 malignant brain tumor specimen. There is a high density of the Gd-G5 dendrimers in the peri-nuclear cytoplasm and near-exclusion of the nanoparticles from the mitochondrion and ER cistern. 0.03 mmol Gd/kg bw of Gd-G5 dendrimer was infused intravenously and the brain tumor was harvested 2 hours after infusion. Figure 10 panels a-h show pharmacokinetics of Gd-dendrimer generations in orthotopic and ectopic RG-2 glioma tumor tissue over 600 to 700 minutes. Panels 10a-d are orthotopic glioma Gd concentrations over time. Panels 10e-h are ectopic glioma Gd concentrations over time. The BTB of the ectopic RG-2 malignant gliomas is more permeable than the BBTB of orthotopic RG-2 malignant gliomas, although the physiologic upper limit of pore size in both cases is approximately 12 run. This means that the BTB of malignant solid tumors located outside the brain has more pores rather than larger pores. To determine the Gd concentration in orthotopic and ectopic RG-2 gliomas, tumor tissue voxels were selected by identifying the respective tumors on the T ₂ weighted anatomical scans in addition to the pattern of positive contrast enhancement within the tumor tissue extravascular space on one of the 2 minute high flip angle dynamic scan data sets acquired between 175 and 225 minutes, since this was the time range of maximal contrast enhancement within the tumor tissue extravascular space for Gd-G5, Gd-G6, and Gd-G7 dendrimer animal groups. For the Gd-G8 animal group, although there was no significant positive contrast enhancement within the tumor tissue extravascular space on the dynamic scan data sets, the outline of the positive contrast enhancement within the tumor micro vasculature on one of the dynamic scan data sets acquired between 175 and 225 minutes was sufficient to identify tumor tissue. The selected orthotopic and ectopic RG-2 glioma tumor tissue voxels represented the respective whole tumor volumes. To determine the change in Gd concentration over time, the whole tumor volumes were then identified on the co-registered high flip angle dynamic scan data

sets of the other time points. The average whole tumor Gd concentration values were then calculated for each time point. 10a Gd-G5 (Orthotopic, N - 6), 10b Gd-G6 (Orthotopic, N = 6), 10c Gd-G7 (Orthotopic, N = 5), 1Od Gd-G8 (Orthotopic, N = 5), 1Oe Gd-G5 (Ectopic, N = 6), 1Of Gd-G6 (Ectopic, N = 6), 1Og Gd-G7 (Ectopic, N = 5), 1Oh Gd-G8 (Ectopic, N = 5). Average whole tumor Gd concentrations are shown

Figure 11 shows Gd concentration maps of Gd-dendrimer contrast enhancement over 175 minutes. Gd-G5 dendrimers readily extravasated across the tumor barriers of both orthotopic and ectopic RG-2 gliomas and accumulated over time within the respective tumor tissue extravascular spaces, as evidenced by the significant positive contrast enhancement over time in the respective tumor tissues (first row). Gd-G6 dendrimers also extravasated across the tumor barriers of both orthotopic and ectopic RG-2 gliomas and accumulated over time within the respective tumor tissue extravascular spaces (second row), although to a lesser extent than Gd-G5 dendrimers (first row). Gd-G7 dendrimers minimally extravasated across the tumor barriers of both orthotopic and ectopic RG-2 gliomas and so minimally accumulated over time within the respective tumor tissue extravascular spaces (third row).

Gd-G8 dendrimers did not extravasate over time across the tumor barriers of either orthotopic or ectopic RG-2 gliomas, but instead remained within the tumor microvasculature in both cases, as evidenced by the lack of contrast enhancement over time within the respective tumor tissue extravascular spaces (fourth row). Therefore, the physiologic upper limit of pore size within the barrier of malignant solid tumors located within the brain as well as those located outside the brain is equivalent. Since the diameter of our Gd-G7 dendrimers and Gd- G8 dendrimers was 10.9 ± 0.7 nm and 12.7 ± 0.7 nm (mean ± standard deviation), the upper limit of pore size within the tumor barriers of both orthotopic RG-2 gliomas and ectopic RG- 2 gliomas is approximately 12 nm. First row, Gd-G5 dendrimers (Orthotopic RG-2 glioma tumor volume, 45 mm ³ ; ectopic RG-2 glioma tumor volume, 113 mm ³ ). Second row, Gd-G6 dendrimers (Orthotopic RG-2 glioma tumor volume, 97 mm ³ ; ectopic RG-2 glioma tumor volume, 184 mm ³ ). Third row, Gd-G7 dendrimers (Orthotopic RG-2 glioma tumor volume, 53 mm ³ ; ectopic RG-2 glioma tumor volume, 135 mm ³ ). Fourth row, Gd-G8 dendrimers (Orthotopic RG-2 glioma tumor volume, 50 mm ³ ; ectopic RG-2 glioma tumor volume, 163 mm ³ ).

Figure 12 shows tumor volumes of orthotopic and ectopic RG-2 gliomas of each Gd- dendrimer generation. 12a Gd-G5 (Orthotopic, N = 6; Ectopic, N = 6), 12b Gd-G6 (Orthotopic, N = 6; Ectopic, N = 6), 12c Gd-G7 (Orthotopic, N = 5; Ectopic, N = 5), 12d Gd- G8 (Orthotopic, N = 5; Ectopic, N = 5). Error bars represent standard deviation.

Figure 13 panels a-d show blood pharmacokinetics of Gd-dendrimer generations. The Gd concentration in blood was determined in the common carotid arteries, since these were the largest caliber brain vessels in the imaging field-of-view. From within the common carotid arteries, 5 to 10 voxels that had physiologically reasonable blood Ti ₀ values of approximately 1100 ms were selected. To determine the change in blood Gd concentration over time the selected blood voxels were identified on the co-registered high flip angle dynamic scan data sets of the subsequent time points. The average blood Gd concentration values were the calculated for each time point. 13a Gd-G5 (N = 6), 13b Gd-G6 (N = 6), 13c Gd-G7 (N = 5), 13d Gd-G8 (N = 5). Based on the time at which the blood Gd concentration is approximately half of the initial value the blood half-life (t _!/2 ) of Gd-G5 and Gd-G6 dendrimers blood is ~ 400 minutes and that of Gd-G7 and Gd-G8 dendrimers is ~ 200 minutes. The blood half-lives of Gd-G5 and Gd-G6 dendrimers were longer than those of Gd- G7 and Gd-G8 dendrimers. In case of Gd-G5 and Gd-G6 dendrimers, the relatively longer blood half-lives are due to the sizes of these Gd-dendrimer generations being large enough to evade filtration by the kidneys, yet small enough to evade opsonization by reticuloendothelial system of the liver and spleen. Therefore, Gd-G5 and Gd-G6 dendrimers were not effectively cleared from blood circulation and had longer blood half-lives than Gd-G7 and Gd-G8 dendrimers. These findings demonstrate that nanoparticles within the size range of Gd-G5 and Gd-G7 dendrimers are both permeable to the barrier of malignant solid tumor microvasculature and also possess blood half-lives sufficiently long to allow for particles to effectively accumulate over time within the tumor tissue extravascular space by the enhanced permeation and retention (EPR) effect.

Figure 14 shows the synthetic scheme of Gd-G5 -doxorubicin dendrimer (Gd-G5- DOX dendrimer) nanoparticle. Doxorubicin is conjugated to Gd-DTPA chelated PAMAM dendrimer terminal amines via pH sensitive hydrazone bond. The sequence of the reaction process is as follows: (1) a linker is attached to a proportion of the terminal amines of the naked G5 PAMAM dendrimer such that approximately 10 out of the 128 available amines are occupied, (2) DTPA is then chelated and followed by addition of Gd ions, and (3) the doxorubicin is conjugated to the available linkers via a the pH sensitive covalent hydrazone bond. Other methods for synthesis can be used to generate the dendrimers of the invention.

Figure 15 shows the in vitro release of doxorubicin over time from Gd-GS-DOX dendrimer nanoparticle. The data show that at pH 7.4, which is the physiologic blood pH that the doxorubicin would be hydrolyzed from the dendrimers slowly over 3 hours. Therefore, the Gd-G5-DOX dendrimer would be stable in blood and would not be expected to cause any

systemic toxicity. At pH 5.5, which is within the pH range in the lysosomes of tumor cells, the doxorubicin hydrolyzed more rapidly over 3 hours. This is important because the doxorubicin would be hydrolyzed within the lysosomes of glioma cells, be released, and diffuse into the individual glioma cell nuclei and act on the DNA. The Gd-G5-DOX dendrimer was dissolved in 1 mL of PBS at 4 ⁰ C, and injected into dialysis tubing with a 12- 14 kDa molecular weight cut-off. Only doxorubicin that hydrolyzes from the dendrimer passes across the pores of the dialysis tubing. The tubing was then clamped, and placed into 19 mL of PBS at 37 ⁰ C. At 15 minute intervals, an aliquot was withdrawn from the solution without the dialysis tubing, and the absorbance due to doxorubicin (absorbance maxima at 480-485 nm) measured on a Cary 50 Bio UV- Visible spectrophotometer. The aliquot was then returned to the reaction solution. At the end of the incubation period of 180 minutes, the dialysis tubing was pierced and the total doxorubicin absorbance measured to determine the maximal absorbance possible, as if all doxorubicin had been hydrolyzed from the non- diffusible Gd-G5-DOX dendrimer nanoparticles within dialysis tubing. Doxorubicin release at each time-point was calculated as a percentage of total possible doxorubicin absorbance for the system.

Figure 16, panels a-b show the change in tumor volume of orthotopic RG-2 malignant gliomas following one intravenously administered dose of free doxorubicin (DOX) versus Gd-G5-DOX dendrimer nanoparticle. The total dose of doxorubicin administered was 8 mg/kg bw for each group. The animal brains were imaged with T ₂ weighted anatomic scans at the time of treatment and on subsequent days following treatment. Tumor tissue voxels were identified on the scans and whole tumor volumes calculated in mm ³ . 16a shows the change in tumor volume over 2 days following treatment. 16b shows the tumor volume change over the first 24 hours following the treatment. The RG-2 malignant gliomas of animals treated with one dose of free doxorubicin continued to grow, whereas the gliomas of animals treated with Gd-G5-DOX dendrimer nanoparticles either regressed or stabilized. The RG-2 gliomas with the largest initial tumor volumes in both the free doxorubicin and Gd-G5- DOX dendrimer nanoparticle groups were the least responsive due to significant initial tumor burden.

DETAILED DESCRIPTION

The upper limit of the physiologic pore size of the blood-brain barrier during central nervous system disease is poorly understood. Therefore, it has not been possible to effectively deliver intravenous nanoparticles across the pathologic blood-brain barrier to date.

The biggest challenge to effective treatment of brain and spinal cord diseases is the inability to deliver therapy due to the blood-brain barrier. Small holes form in the barrier in many of these diseases.

Here it is demonstrated that intravenous dendrimer-based nanoparticles less than 13 nm in diameter, but not greater, can effectively cross the pores of the blood-brain tumor barrier (BBTB), and the blood-tumor barrier of brain tumor ectopically implanted (i.e., metastases). Here it is demonstrated that in brain tumors dendrimers, a type of nanoparticle, when injected intravenously are small enough to cross the blood-brain barrier of tumors through these holes, while sparing normal tissue of the brain or other organs. Based on these findings, it is expected that this disparity in pore size in tumor tissue and normal tissue would be observed for all solid tumors. Therefore, the findings presented herein on both orthotopically and ectopically implanted tumors are expected to be applicable to many if not essentially all types of solid tumors. Therefore, the dendrimer conjugates provided herein for detection, imaging, and treatment of brain tumors can be used for the treatment of all types of solid tumors.

The instant invention is based upon the finding that using successively higher generations of functionalized PAMAM dendrimers, intravenous particles less than 13 nm in diameter can cross the BTB, particularly the BBTB, while larger functionalized dendrimers cannot, and further that dendrimers with prolonged circulation times in blood accumulate within tumor cells after crossing the BTB, particularly the BBTB. These findings have application in treating tumors, both in the brain and at remote sites, and neurological disorders that have remained refractory to treatment.

Definitions The following definitions are provided for specific terms which are used in the following written description.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology

(2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988);

The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &

Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. The terms "administration" or "administering" are defined to include an act of providing a compound or pharmaceutical composition of the invention to a subject in need of treatment. In the instant invention, preferred routes of administration include parenteral administration, preferably, for example by injection, for example by intravenous injection. The term "dendrimer conjugate" or "dendritic conjugate" refers to a dendrimer attached or otherwise linked to another moiety, which may be a functional moiety, for example an agent. In certain examples, the agent is an imaging agent or a therapeutic agent, for example a drug, a vaccine, a chemotherapeutic, a cytotoxic agent, a peptide, or an oligonucleotide. The moiety may be attached or linked to the dendrimer by any suitable

means, such as by one or more of chelation, ionic bonding, covalent bonding, hydrogen bonding, van der Waals forces, metallic bonding, adsorption, encapsulation, or absorption. In certain examples, the dendrimer conjugate comprises a metal chelate. Therapeutic agents are preferably linked by a covalent linkage, preferably a hydrolysable covalent linkage that can be hydrolyzed under physiological conditions, including reduced pH conditions of a lysozyme. Such covalent linkages are well known in the art. A number of moieties that may be included in dendrimer conjugates are discussed in U.S. Pat. No. 6,312,679, incorporated by reference in its entirety herein.

The term "dendrimer" refers to a class of highly branched, often spherical, macromolecular polymers that exhibit greater monodispersity (i.e. a smaller range of molecular weights, sizes, and shapes) than linear polymers of similar size. These three- dimensional oligomeric structures are prepared by reiterative reaction sequences starting from a core molecule (such as diaminobutane or ethylenediamine) that has multiple reactive groups. When monomer units, also having multiple reactive groups, are reacted with the core, the number of reactive groups comprising the outer bounds of the dendrimer increases.

Successive layers of monomer molecules may be added to the surface of the dendrimer, with the number of branches and reactive groups on the surface increasing geometrically each time a layer is added. The number of layers of monomer molecules in a dendrimer may be referred to as the "generation" of the dendrimer (see Figure Ia). The total number of reactive functional groups on a dendrimer's outer surface ultimately depends on the number of reactive groups possessed by the core, the number of reactive groups possessed by the monomers that are used to grow the dendrimer, and the generation of the dendrimer.

The term "PAMAM dendrimer" refers to a dendrimer having polyamidoamine branches. In certain preferred examples, the dendrimer comprises a generation 3.5 (G 3.5), generation 4 (G4), generation 4.5 (G 4.5), generation 5 (G5), generation 5.5 (G 5.5), generation 6 (G6), generation 6.5 (G 6.5), generation 7 (G7), or generation 7. 5 (G 7.5) PAMAM dendrimer.

An "agent" is understood herein to include a therapeutically active compound or a potentially therapeutic active compound. An agent can be a previously known or unknown compound. As used herein, an agent is typically a non-cell based compound, however, an agent can include a biological therapeutic agent, e.g., peptide or nucleic acid therapeutic, cytokine, antibody, etc.

As used herein "amelioration" or "treatment" is understood as meaning to lessen or decrease at least one sign, symptom, indication, or effect of a specific disease or condition.

For example, amelioration or treatment of cancer can be determined using the standard RECIST (Response Evaluation Criteria in Solid Tumors) criteria including the assessment of tumor burden, by survival time, reduced presence of tumor markers, or any other clinically acceptable indicators of disease state or progression. Amelioration and treatment can require the administration of more than one dose of an agent or therapeutic.

As used herein, "biocompatible" is understood as acceptable for administration to a subject, particularly a human subject. In certain embodiments, biocompatible compositions are composed of materials that are categorized as generally recognized as safe (GRAS). In certain embodiments, biocompatible compositions are determined to be safe for administration to subjects by testing.

"Contacting a cell" or "contacting a tissue" is understood herein as providing an agent to a test cell or to a e.g., a cell to be treated in culture or in an animal, such that the agent or isolated cell can interact with the test cell or cell to be treated, potentially be taken up by the test cell or cell to be treated, and have an effect on the test cell or cell to be treated. The agent or isolated cell can be delivered to the cell directly (e.g., by addition of the agent to culture medium or by injection into the cell or tissue of interest), or by delivery to the organism by an enteral or parenteral route of administration for delivery to the cell by circulation, lymphatic, or other means.

As used herein, "changed as compared to a control" sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator to be detected at a level or location that is statistically different than a sample from a normal, untreated, or control sample. Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods to select and test control samples are within the ability of those in the art. An analyte can be a naturally occurring substance that is characteristically expressed or produced by the cell or organism (e.g., PSA) or a substance produced by a reporter construct (e.g, β-galactosidase or luciferase). Depending on the method used for detection the amount and measurement of the change can vary. For example, a change in the size of a tumor, the presence or absence of metastases, the number and size of metastases can be changed in a treated subject as compared to a control. Determination of statistical significance is within the ability of those skilled in the art.

As used herein "chemotherapy" or treatment with a "chemotherapeutic agent" or a "cytotoxic agent" is understood in its most general sense, to treatment of disease by chemicals that kill cells, both good and bad, but specifically those of micro-organisms or cancer. Chemotherapeutic agents include, for example, alkylating agents, antimetabolites,

anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, new tyrosine kinase inhibitors, and other antirumour agents; antibiotics agents, antiviral agents, and antimicrobial agents. Chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, vinca alkaloids, taxanes, vincristine, vinblastine vinorelbine, vindesine, etoposide, teniposide, paclitaxel, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, and dactinomycin.

As used herein, "conjugated" is understood as attached, linked, mixed, or otherwise present on or in a nanoparticle. For example, an agent can be conjugated to a nanoparticle by covalent or ionic linkage, by use of a chelate or other linker moiety, by mixing of the agent with the nanoparticle prior to formation of the nanoparticle such that the agent is captured in the nanoparticle. As used herein, conjugation of an agent to a nanoparticle does not disrupt the desired activity of the agent.

As used herein, "detecting", "detection" and the like are understood that an assay or method performed for identification of a specific analyte in a sample or at least one sign or symptom of a disease in a subject. Detection can include the determination of the size of a tumor, the presence or absence of metastases, the presence or absence of angiogenesis. The amount of analyte detected in the sample, or the number or size of tumors detected in a subject, can be none or below the level of detection of the assay or method. Methods for detection include imaging.

By "diagnosing" as used herein refers to a clinical or other assessment of the condition of a subject based on observation, testing, or circumstances for identifying a subject having a disease, disorder, or condition based on the presence of at least one sign or symptom of the disease, disorder, or condition. Typically, diagnosing using the method of the invention includes the observation of the subject for other signs or symptoms of the disease, disorder, or condition.

As used herein, the terms "effective" and "effectiveness" includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment. On the other hand, the term "ineffective" indicates that a treatment does not provide sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the unstratified population. (Such a

treatment may be ineffective in a subgroup that can be identified by the expression profile or profiles.) "Less effective" means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects, e.g., greater liver toxicity. Thus, in connection with the administration of a drug, a drug which is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease signs or symptoms, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

The term "hyperproliferative disorder" or "neoplasia" includes malignancies characterized by excess cell proliferation or growth, or reduced cell death. In specific embodiments, the term "cancer" includes but is not limited to carcinomas, sarcomas, leukemias, and lymphomas. The term "cancer" also includes primary malignant tumors, e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor, and secondary malignant tumors, e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor. Cancers are frequently characterized as solid tumors and non-solid tumors or blood tumors. Cancers include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, basal cell carcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T- cell lymphoma, esophageal cancer, ewing family of tumors, retinoblastoma, gastric (stomach) cancer, gastrointestinal tumors, glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, islet cell tumors (endocrine pancreas), kidney (renal cell) cancer, laryngeal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, medulloblastoma, melanoma, pancreatic cancer, prostate cancer, renal cancer, rectal cancer, and thyroid cancer. In the instant invention, a tumor is preferably a solid tumor. In the context of the instant application, brain tumors also include brain tumor derived metastases present at remote (i.e., not brain) locations in the subject.

As used herein, "kits" are understood to contain at least the non-standard laboratory reagents of the invention and one or more non-standard laboratory reagents for use in the methods of the invention.

As used herein, "nanoparticle" is understood as any biocompatible particle that is of an appropriate size to be used in the methods of the invention, about 1.5 nm to about 13 nm in diameter, preferably about between 1.5 nanometers and 13 nanometers, between about 5 nanometers and 12 nanometers, or between about 7 nanometers and 12 nanometers, having a molecular weight of greater than about 10 kDa, greater than about 35 kDa, or greater than about 40 kDa, and a molecular weight of less than about 300 kDa, less than about 200 kDa, less than about 150 kDa, or less than about 100 kDa, to which an agent can be complexed, conjugated, or otherwise attached without disrupting the desired activity of the agent (e.g., therapeutic agent, imaging agent). In a preferred embodiment, the nanoparticle has a neutral charge or a negative charge. Nanoparticles for the purpose of drug delivery are colloidal particles including monolithic nanoparticles (nanospheres) in which the drug is adsorbed, dissolved, or dispersed throughout the matrix and nanocapsules in which the drug is confined to an aqueous or oily core surrounded by a shell-like wall. Alternatively, the drug can be covalently attached to the surface or into the matrix. Nanoparticles are made from biocompatible and biodegradable materials such as polymers, either natural (e.g., gelatin, albumin) or synthetic (e.g., polylactides, polyalkylcyanoacrylates), or solid lipids. In the body, the drug loaded in nanoparticles is usually released from the matrix by diffusion, swelling, erosion, or degradation. In the body, imaging agents can be detected while attached to the nanoparticle. Methods of making nanoparticles are well known in the art. "Obtaining" is understood herein as manufacturing, purchasing, or otherwise coming into possession of.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. For example, pharmaceutically acceptable carriers for administration of cells typically is a carrier acceptable for delivery by injection, and do not include agents such as detergents or other compounds that could damage the cells to be delivered. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,

such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfϊte, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, intramuscular, intraperotineal, rectal, vaginal, intravenous, intraarterial, intrathecal, intracranial, and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. As used herein, "plurality" is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, or more.

As used herein, "radiotherapeutic agent" is understood as a radioactive isotope of an element that has a desired biological activity in the treatment of disease, most commonly cancer. Radiotherapeutic agents are a subset of chemotherapeutic agents. A "subject" as used herein refers to living organisms, hi certain embodiments, the living organism is an animal. In certain preferred embodiments, the subject is a mammal. In certain embodiments, the subject is a domesticated mammal. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A human subject may also be referred to as a patient.

A subject "suffering from or suspected of suffering from" a specific disease, condition, or syndrome has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome. Methods for identification of subjects suffering from or suspected of suffering from conditions such as cancer is within the ability of those in the art. Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.

As used herein, "susceptible to" or "prone to" or "predisposed to" a specific disease or condition and the like refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population. An increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

"Therapeutically effective amount," as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder beyond that expected in the absence of such treatment. The therapeutically effective amounts for dendrimer-based imaging and drug delivery devices range between 25 mg dendrimer conjugate/kg subject body weight to 225 mg dendrimer conjugate/kg subject body weight or any value or range within that range, for example doses can include 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, or 225 mg dendrimer conjugate/kg subject body weight or any ranges there between. A therapeutically effective dose can be determined by one of skill in the art depending on a number of factors including, but not limited to, tumor type, tumor burden, rate of disease progression, etc. An agent can be administered to a subject, either alone or in combination with one or more therapeutic agents, as a pharmaceutical composition in mixture with conventional excipient, e.g., pharmaceutically acceptable carrier, or therapeutic treatments such as radiation.

The pharmaceutical agents may be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, e.g., as described in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1985). Formulations for parenteral administration may contain as common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide

polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain agents.

It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to e.g., the specific compound being utilized, the particular composition formulated, the mode of administration and characteristics of the subject, e.g., the species, sex, weight, general health and age of the subject. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines.

Dendrimers

Featured in the invention are dendritic conjugates comprising a dendrimer conjugated to an agent, wherein the dendrimer is a polyamidoamine (PAMAM) dendrimer, and wherein the dendritic conjugate has a diameter between 1.5 nanometers and 13 nanometers, or any size therebetween, such as the dendrimer sizes included in the Examples.

Further, the dendrimers are attractive for use as they are biocompatible and non- immunogenic.

Dendrimers can be prepared having highly uniform size and shape and allow for a greater number of functional groups per unit of surface area of the dendrimer, and can have a greater number of functional groups per unit of molecular volume as compared to other polymers which have the same molecular weight, same core and monomeric components and same number of core branches as the starburst polymers. Moreover, since the number of functional groups on the dendrimers can be controlled on the surface and within the interior, it also provides a means for controlling, for example, the amount of bioactive agent to be delivered per dendrimer.

The preparation and characterization of dendrimers, dendrons, random hyperbranched polymers, controlled hyperbranched polymers, half-generation dendrimers, and dendrigrafts (collectively "dendritic polymer") is well known. Examples of dendrimers and dendrons, and methods of synthesizing the same are set forth in U.S. Pat. Nos. 4,410,688, 4,507,466; 4,558,120; 4,568,737; 4,587,329; 4,631,337; 4,694,064; 4,713,975; 4,737,550; 4,871,779 and 4,857,599, the disclosures of which are hereby incorporated by reference. Examples of hyperbranched polymers and methods of preparing the same are set forth, for example in U.S. Pat. Nos. 5,418,301 and 5,514,764, the disclosures of which are hereby incorporated by reference. Examples of dendrigrafts and methods of preparing the same are set forth, for

example in an article by D. A. Tomalia and R. Esfand, Chem. & Ind., 416 420 (Jun. 2, 1997).

Examples of half-generation dendrimers are provided by Bhadra et al. 2005 (Pegylated lysing based co-polymeric dendritic micells for solubilization and delivery of artemether. J. Pharm.

Pharmaceut. Sci. 8:467-482, incorporated herein by reference). Half-generation dendrimers are negatively charged as the branches terminate in -COOH groups instead of -NH ₂ groups Dendritic polymers which are useful in the practice of this invention include those that have symmetrical branch cells (arms of equal length, e.g., PAMAM dendrimers; for example described in U.S. Pat. No. 5,527,524, incorporated by reference in its entirety herein). Dendrimers are desirable for the delivery of radionuclides or strongly paramagnetic metal ions to tumor sites because of their ability to chelate a number of metal ions in a small volume of space.

In certain examples, the dendritic conjugate has a diameter between 1.5 nanometers and 14 nanometers. As discussed above, intravenous dendrimer-based conjugate less than 13 nm in diameter, but not greater, can effectively cross the pores of the blood-brain tumor barrier (BBTB) of malignant brain tumors and the blood-tumor barrier (BTB) of malignant solid tumors located outside the brain in other tissues.

For example, when the target area for treatment is a brain tumor or diseased neurological tissue, it is preferred to use a dendritic conjugate that is small enough to effectively cross the pores within the BBTB and also be large enough to be retained within systemic circulation at peak blood levels (i.e. not easily filtered by the kidneys) for sufficiently long to allow for effective transvascular accumulation of the dendrimer conjugate within individual brain tumor cells, In at least one embodiment, the dendritic conjugate has a diameter between 1.5 nanometers and 13 nanometers. Due to relatively short blood half-lives smaller particles would not be retained in the tumor tissue after crossing the BBTB for sufficiently long to accumulate within individual brain tumor cells.

In certain examples, the dendritic conjugate has a molecular weight greater than 10 kDa. In other certain examples, the dendritic conjugate has a molecular weight greater than

35 kDa or greater than 40 kDa. In certain examples, the dendritic conjugate has a molecular weight of no more than about 300 kDa, no more than about 200 kDa, no more than about 150 kDa, or no more than about 100 kDa. However, the molecular weight of the dendrimer complexes of the invention is largely limited by the allowable size of the dendrimer complexes.

In certain implementations, the substance may be modified to increase the substance's affinity for the target area, such as by modifying the substance to increase its uptake by target cells.

The dendritic conjugate, in certain preferred examples, has a negative overall charge or a neutral overall charge.

The dendrimer conjugates as described herein comprise a dendrimer conjugated to an agent. The agent can comprise any material or compound or composition or agent for in vivo or in vitro use for imaging, diagnostic or therapeutic treatment that can be conjugated with the dendrimer without appreciably disturbing the physical integrity of the dendrimer. A dendrimer can be conjugated with one or more agents of one or more types. For example, a dendrimer can be conjugated with a therapeutic agent, and the targeting of the agent can be followed by further conjugation with an imaging agent. Similarly, cocktails of therapeutic agents are typically used in the treatment of cancer. More than one type of therapeutic agent can be linked to a dendrimer. Examples of agents include imagining agents (for example gadolinium, manganese, chromium, or iron) and therapeutic agents (for example doxorubicin, carboplatin, temozolamide, boron, or antibody fragments). A therapeutic agent may be a molecule, atom, ion, receptor and/or other entity which is capable of detecting, identifying, inhibiting, treating, catalyzing, controlling, killing, enhancing or modifying a target such as a protein, glyco protein, lipoprotein, lipid, a targeted cell, a targeted organ, or a targeted tissue.

In certain cases, the therapeutic agent is a radiotherapeutic agent, and can be selected from, but is not limited to radioactive gadolinium, radioactive boron, and radioactive iodine. In certain examples, the agent can be, but is not limited to: drugs, such as antibiotics, analgesics, hypertensives, cardiotonics, and the like, such as acetaminaphen, acyclovir, alkeran, amikacin, ampicillin, aspirin, bisantrene, bleomycin, neocardiostatin, carboplatin, chloroambucil, chloramphenicol, cytarabine, daunomycin, doxorubicin, fluorouracil, gentamycin, ibuprofen, kanamycin, meprobamate, methotrexate, novantrone, nystatin, Oncovin, phenobarbital, polymyxin, probucol, procarbabizine, rifampin, streptomycin, spectinomycin, Symmetrel, thioguanine, tobramycin, temozolamide, trimethoprim, cisplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, vinca alkaloids, taxanes, vincristine, vinblastine vinorelbine, vindesine, etoposide, teniposide, paclitaxel, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, and dactinomycinand valban; diphtheria toxin, gelonin, exotoxin A, abrin, modeccin, ricin, radioactive gadolinium, radioactive boron, and radioactive iodine; or

toxic fragments thereof; metal ions, such as the alkali and alkaline-earth metals; radionuclides, such as those generated from actinides or lanthanides or other similar transition elements or from other elements, such as ⁵¹ Cr, ⁴⁷ Sc, ⁶⁷ Cu, ⁶⁷ Ga, ⁸² Rb, ⁸⁹ Sr, ⁸⁸ Y, ⁹⁰

V Y, m Tc, 105 D Rhl,, !09 PDd ^J , ^{1 1 1} ITn, ^{1 1 5} ^ m T Inr., ¹²⁵ T 1, ^{1 3 1} T 1, ¹⁴⁰ D Bao, ¹⁴⁰ LT ao, ^{149 1 53} C S^m,, ^{1 59} T OAd, ^{1 66} Ho, ^I75 Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ^m Ir, and '" Au; signal generators, which includes anything that results in a detectable and measurable perturbation of the system due to its presence, such as fluorescing entities, phosphorescence entities and radiation; signal reflectors, such as paramagnetic entities, for example, Fe, Gd, Cr, or Mn; chelated metal, such as any of the metals given above, whether or not they are radioactive, when associated with a chelant; signal absorbers, such as contrast agents and electron beam opacifiers, for example, Fe, Gd, Cr, or Mn; antibodies, including monoclonal antibodies and anti-idiotype antibodies; antibody fragments; hormones; biological response modifiers such as interleukins, interferons, viruses and viral fragments; diagnostic opacifiers; and fluorescent moieties. Other pharmaceutical materials include scavenging agents such as chelants, antigens, antibodies or any moieties capable of selectively scavenging therapeutic or diagnostic agents.

Therapeutics

The physiologic upper limit of pore size within the blood-brain barrier during central nervous system disease has been poorly understood. Pores exist within the pathologic blood- brain barrier of microvasculature supplying malignant brain tumors as well as in the blood- brain barrier of microvasculature supplying many other brain and spinal cord diseases including infections, traumatic injuries, and stroke. To date it has not been possible to effectively treat brain and spinal cord diseases with intravenously administered therapies. This is due, in large, to our inability to successfully deliver therapy across the pathologic blood-brain barrier.

Another obstacle towards the effective treatment of malignant brain tumors has been the dose-limiting systemic toxicity of conventional chemotherapy drugs. It would be advantageous to develop methodology to allow for a chemotherapeutic agent to be delivered selectively to the malignant brain tumor tissue without significant systemic toxicity following intravenous infusion. This may be accomplished by conjugating conventional chemotherapy drugs to nanoparticles such that drugs remain inactive while attached to nanoparticles at the neutral pH of blood, but instead are activated within the acidic pH of tumor cell lysosomal compartments.

By imaging with MRI the transvascular delivery of intravenously infused successively higher generations of functionalized polyamidoamine (PAMAM) dendrimer nanoparticles, the instant invention is based upon the finding that intravenously administered nanoparticles smaller than 13 nm in diameter can cross the BBTB, while larger functionalized dendrimers cannot, and further that dendrimers with prolonged circulation times in blood accumulate within tumor cells after crossing the BBTB. These findings have application in effectively treating malignant brain tumors and other neurological disorders that remain refractory to treatment.

Thus, the invention features methods of delivering a dendritic conjugate across the blood-brain tumor barrier in a subject by administering to a subject a dendritic conjugate, wherein the dendritic conjugate includes a dendrimer conjugated to an agent, wherein the dendrimer is a polyamidoamine (PAMAM) dendrimer, and wherein the dendritic conjugate has a diameter between 1.5 nanometers and 13 nanometers, thereby delivering the dendritic conjugate across the pathologic blood-brain tumor barrier, but not the normal blood-brain barrier, thus sparing normal brain tissue.

The invention features method of treating a brain tumor or a neurological disease in a subject by administering to a subject identified as having a brain tumor a dendritic conjugate, wherein the dendritic conjugate comprises a dendrimer conjugated to an agent, wherein the dendrimer is a polyamidoamine (PAMAM) dendrimer, and wherein the dendritic conjugate has a diameter between 1.5 nanometers and 13 nanometers, thereby treating a brain tumor or a neurological disease in a subject.

Barrier permeability is determined by tumor type, grade, location, and the number of pores. In comparing the barrier permeability of malignant peripheral tumors (i.e. extracranial) and malignant brain tumors (i.e. intra-cranial), the blood-tumor barrier (BTB) of peripheral tumors has a greater number of pores, whereas the blood-brain tumor barrier (BBTB) of brain tumors has a fewer number of pores; therefore, the barrier of peripheral tumors is more leaky than that of brain tumors. The physiologic upper limit of pore size in the barriers is essentially equivalent and is approximately 12 nm.

Amongst the methods available for measuring tumor permeability, some have drawbacks. ¹⁴ C-AIB quantitative autoradiography (QAR) is invasive and requires sacrifice of the animal for calculation of tumor radioactivity. Dynamic contrast-enhanced MRI (DCE- MRI) can be done in real-time, is repeatable and uses a non-radioactive contrast agent (i.e. Gd-DTPA or Gd-DTPA conjugated PAMAM dendrimer).

The prospect of using dendritic polymers as carriers for drug delivery has been previously proposed on account of the unique structure and characteristics of these polymer molecules (R. Esfand and D. A. Tomalia, "Poly(amidoamine) (PAMAM) Dendrimers: from Biorimicry to Drug Delivery and Biomedical Applications", research focus, DDT 6(8), 427 436 (Apr. 8, 2001)) and U.S. Pat. Nos. 5,338,532 and 5,527,524, all of which are incorporated by reference in their entireties herein.

The prospect of using intravenously administered dendritic conjugates as carriers of drug delivery across the pathologic blood-brain barrier is detailed in this application. The dendritic conjugates are administered intravenously or intraarterially (i.e. systemically) to a subject in need, for example a subject having a tumor such as a malignant brain tumor, or a subject with an untreatable neurological disease, in an amount which is effective to effectively treat the tumor or the disease, e.g. to inhibit growth of the tumor, preferably intravenously (LV.) or intraarterially (LA. ), although transdermal, intrathecal, intracranial, intratumoral, or intracavitary administration (e.g., in the context of during surgical tumor resection or debulking) are also possible.

Imaging

The invention is particularly useful for imaging, for example in imaging a malignant ectopic or orthotopic brain tumor. In certain aspects, the invention features methods of imaging a brain tumor in a subject by administering to a subject who is suspected of having, or has been identified as having a brain tumor a dendritic conjugate, wherein the dendritic conjugate inlcudes a dendrimer conjugated to an imaging agent, wherein the dendrimer is a polyamidoamine (PAMAM) dendrimer, and wherein the dendritic conjugate has a diameter of any size between 1.5 nanometers and 13 nanometers, thereby imaging a malignant brain tumor in a subject.

The methods of imaging as described herein can be particularly useful in diagnosing or monitoring a malignant brain tumor in a subject.

Any imaging agent that can be conjugated to the dendrimers as described herein is suitable for use in the invention. In particularly preferred embodiments, imaging agents include, but are not limited to, gadolinium, chromium, manganese, and iron.

The moiety may be attached or linked to the dendrimer by any suitable means, such as by one or more of chelation, ionic bonding, covalent bonding, hydrogen bonding, van der Waals forces, metallic bonding, adsorption, encapsulation, or absorption. In certain preferred embodiments, a chelate is used to conjugate the imaging agent to the PAMAM dendrimer.

The chelate can be, but is not limited to, DTPA, DOTA, porphyrin, and desferoxamine, or derivatives thereof.

In certain examples, gadolinium also acts as an activatable cytotoxic agent. Imaging may begin immediately or anywhere from about 1 minute to about 120 hours after administration of the dendrimer conjugates, such as between about 1, 2 or 3 minutes and about 24 hours after administration, or between about 1 , 2 or 3 minutes and about 60 minutes after administration. The time before imaging may be altered based on the particular dendrimer conjugate used and its physiological properties, such as the time it takes to accumulate and its retention time, and the tumor volume. Imaging, once begun, may be continued for any subsequent amount of time that facilitates analysis of the images for a particular purpose (for example, for diagnosis, for targeting). In certain examples, a series of images obtained at various points in time from administration to a desired time after administration, such as several hours or more, may be obtained if intraoperative (during a surgical procedure) or intratreatment (such as during administration of a dendrimer conjugate) localization of a brain tumor is desired. Imaging may be done before or during surgery or therapy, and continued for any period during surgery or therapy, for example, to help a surgeon guide a needle to a tumor for a biopsy or to position an activator for an activatable anti-tumor agent. Obtaining images at various times after administration may aid in determining prognosis or course of therapy.

Imaging Therapeutic Delivery

The invention is particularly useful for imaging drug delivery, for example imaging drug delivery to a malignant brain tumor, since the both the imaging agent and therapeutic agent can be attached to the same dendrimer or population of dendrimers for administration. In certain aspects, the invention features methods of imaging a malignant brain tumor in a subject comprised of administering to a subject who has a malignant brain tumor a dendritic conjugate, wherein the dendritic conjugate comprises a dendrimer conjugated to a therapeutic agent and an imaging agent, wherein the dendrimer is a polyamidoamine (PAMAM) dendrimer, and wherein the dendritic conjugate has a diameter between 1.5 nanometers and 13 nanometers, thereby imaging therapeutic delivery into brain tumor tissue in a subject.

The methods of imaging as described herein can be particularly useful in determining how effectively a therapeutic has been delivered across the blood-brain tumor barrier of a malignant brain tumor in a subject. Such information is not readily available with current

clinical methodologies, and would allow for the regimen (i.e frequency, dose) of subsequent treatment to be modified and tailored tumor-by-tumor.

Any imaging agent and therapeutic agent combination that can be conjugated to the dendrimers as described herein are suitable for use in the invention. In particularly preferred embodiments, the therapeutic agent is selected from, but not limited to, conventional chemotherapeutics, radiotherapeutics (i.e. boron for boron neutron capture therapy), antibody fragments, and other small molecule therapeutics such as protein kinase inhibitors.

The moiety can be attached or linked to the dendrimer by any suitable linkage, such as by one or more of chelation, ionic bonding, covalent bonding, hydrogen bonding, van der Waals forces, metallic bonding, adsorption, encapsulation, or absorption. In certain preferred embodiments, a covalent bond is used to conjugate the imaging agent to the PAMAM dendrimer.

Imaging may begin immediately or anywhere from about 1 minute to about 120 hours after administration of the dendrimer conjugates, such as between about 1, 2 or 3 minutes and about 24 hours after administration, or between about 1 , 2 or 3 minutes and about 60 minutes after administration. The time before imaging may be altered based on the particular dendrimer conjugate used and its physiological properties, such as the time it takes to accumulate and its retention time, and the tumor volume.

Patient Monitoring The disease state or treatment of a patient having a disease or disorder, for example a malignant brain tumor, either primary or metastatic, can be monitored using the methods and compositions of the invention, alone or in addition to standard monitoring techniques.

In one embodiment, the tumor progression of a patient can be monitored using the methods and compositions of the invention, e.g. by imaging the tumor, imaging the formation of metastases. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a patient.

Kits The invention also provides kits comprising a dendritic conjugate, and instructions for use. Kits optionally include other compositions of the invention and/or reagents and devices for practicing the methods of the invention.

EXAMPLES

Despite the known breach of the blood-brain barrier in neuropathologies, such as malignant glioma, it remains a challenge to exploit the barrier's porosity for effective transvascular delivery of therapeutics across the pathologic blood-brain barrier into the diseased central nervous system. The experiments described herein show, in each an orthotopic and an ectopic malignant brain tumor model, with in vivo dynamic contrast enhanced magnetic resonance and fluorescence imaging, that intravenously administered functionalized polyamidoamine dendrimers less than 13 ran in diameter are able to traverse pores of the blood-brain tumor barrier, while larger functionalized dendrimers cannot. Dendrimers are a class of multi-generational nanoparticles with particle sizes within a particular dendrimer generation being uniform. For each successively higher generation, the number of terminal binding sites and molecular weights increase markedly, while the overall diameter increases by only a few nanometers. Of the permeable functionalized dendrimer generations, the results reported herein demonstrate that those with longer blood half-lives accumulate within individual tumor cells. Thus, effective transvascular delivery across the blood-brain barrier of malignant brain tumors and in other neurological diseases may be accomplished with the functionalized dendrimer conjugates as described herein.

In the case of malignant brain tumors, herein we report the probing of the physiologic upper limit of pore size within the blood-brain tumor barrier of orthotopic RG-2 rat gliomas with dynamic contrast-enhanced MRI using dendrimer nanoparticles labeled on the exterior with gadolinium (Gd)-diethyltriaminepentaacetic acid (DTPA), an anionic MRI contrast agent. Based on this work, we determined that the upper limit of pore size within the BTB of orthotopic RG-2 rat gliomas in vivo was approximately 12 nm. These previously reported findings suggest that the impediment to the transvascular extravasation of particles across the BTB of brain tumors is at the level of the glycocalyx that coats the surface of the pores in the BTB and is a "nanofilter" for the transvascular flow of particles across the BTB.

It is possible that the physiologic upper limit of pore size within the BTB of peripheral tumors previously reported as being between 200 nm and 1200 nm may be a gross over-estimation of the actual physiologic upper limit of pore size within the BTB of peripheral solid tumors. Therefore, if the actual physiologic upper limit of pore size within the BTB of peripheral tumors is significantly lower than what has been previously reported and approximates that of brain tumors, then this finding would suggest that more pores in BTB of peripheral tumors are the primary reason for the higher permeability of the BTB of malignant peripheral tumors compared to that of malignant brain tumors. Furthermore, such

findings would have important implications on the size range of nanoparticle-based therapeutics that could be effectively delivered across the BTB of malignant solid tumors, irrespective of tumor host site. Further, if the upper limit of the BTB has been grossly overestimated for brain tumors, both in brain and at ectopic sites, it is possible that the pore size of other tumors at sites other than the brain have also been reported erroneously.

We have characterized the upper limit of pore size within the BTB of orthotopic RG-2 malignant gliomas using successively higher generation (G) polyamidoamine (PAMAM) dendrimers labeled with Gd-DTPA. With dynamic-contrast enhanced MRI, we found there to be significant positive contrast enhancement of brain tumor tissue following the intravenous infusion of Gd-Gl through Gd-G7 dendrimers, but not following the intravenous infusion of Gd-G8 dendrimers. Based on this observation, we established that Gd-G8 dendrimers were larger than the upper limit of pore size within the BTB of orthotopic RG-2 gliomas. With this dynamic contrast-enhanced MRI approach, in addition to being able to image the tumor tissue pharmacokinetics of Gd-Gl thorough Gd-G8 dendrimers, we were also able to image at the same time the blood pharmacokinetics of the respective Gd-dendrimer generations in the large vessels within the brain.

We show that the higher generation Gd-G5 through Gd-G8 dendrimers maintained steady state blood concentrations over the 120 minute long imaging session. Since Gd-G5, Gd-G6 and Gd-G7 dendrimers maintained steady state blood concentrations over the 120 minute imaging session and were permeable to the BTB of orthotopic RG-2 brain tumors, these higher generation Gd-dendrimers continued to accumulate within the tumor tissue extravascular space over time, and remained there for sufficiently long to localize within individual glioma tumor cells. Although these imaging sessions were long enough to determine the physiologic upper limit of pore size in the BTB of orthotopic brain tumors as well as qualitatively assess the blood half-lives of lower generation Gd-dendrimers, we were unable to qualitatively assess the blood half-lives of the higher generation Gd dendrimers, since the higher generation Gd-dendrimers maintained steady state blood concentrations over 120 minutes.

As provided herein, we imaged the blood and tumor tissue pharmacokinetics of higher generation Gd-dendrimers over 600 to 700 minutes in order to characterize the differences in the permeability of the BTB of orthotopic and ectopic RG-2 malignant gliomas and define the upper limit of pore size within the BTB of brain tumors and peripheral tumors. We determined the differences in the permeability of the BTB of an ectopic RG-2 glioma and an orthotopic RG-2 glioma within the same rat at the same time. For each animal, RG-2 glioma

cells were inoculated in the right anterior brain, which was the orthotopic site, and the left temporalis muscle, which was the ectopic site. The change in blood and tumor tissue Gd concentration, a surrogate for the Gd-dendrimer concentration, was determined by calculating the molar relaxivity of the respective Gd-dendrimer generation in vitro, and the change in the longitudinal relaxation time before and after Gd-dendrimer bolus for each imaged volume element (voxel) in vivo.

In the studies provide dynamic contrast-enhanced MRI was used to image the blood and tumor tissue pharmacokinetics of intravenously infused Gd-PAMAM dendrimer nanoparticles G5 through G8 over 600 to 700 minutes. We compared the permeability of the BTB of RG-2 gliomas grown within the brain, the orthotopic site, to that of the BTB of RG-2 gliomas grown outside the brain in the temporalis skeletal muscle, the ectopic site. We used this animal model to characterize the differences in the permeability of the BTB of a malignant brain tumor to that of the BTB of a peripheral solid tumor, and to define the upper limit of pore size within the BTB of the respective solid tumors. Using this approach, we show that the physiologic upper limit of pore size in the BTB of brain RG-2 gliomas and peripheral RG-2 gliomas is approximately 12 nm.

In the case of brain RG-2 gliomas, we report herein that the physiologic upper limit of pore size in the BTB of orthotopic RG-2 gliomas growing in brain tissue is approximately 12 nm. Our present finding is in agreement with our previously reported finding that the upper limit of pore size in the BTB of orthotopic RG-2 gliomas is approximately 12 nm (34). Both in our prior and present work, we probed the upper limit of the pore size within the BTB with dynamic contrast-enhanced MRI using successively higher generation Gd-DTPA labeled PAMAM dendrimer nanoparticles with a neutralized particle exterior. The positive charge on exterior of the naked PAMAM dendrimer generations was neutralized by the conjugation of Gd-DTPA (charge -2) to approximately 40% to 50% of the terminal amines on the exterior. Therefore, the Gd-DTPA labeled dendrimer generations that were used for this study would have not been toxic to the negatively charged glycocalyx overlaying the endothelial cells of the BTB. Similarly, it would be expected that half-generation dendrimers would act similarly to the full generation dendrimers as they are negatively charged. In the case of peripheral RG-2 gliomas, we report herein that the physiologic upper limit of pore size in the BTB of ectopic RG-2 gliomas growing in skeletal muscle is equivalent to the upper limit of pore size in the BTB of orthotopic RG-2 gliomas growing in brain tissue, and is also approximately 12 nm. The physiologic upper limit of pore size in the BTB of peripheral RG-2 gliomas that we report here is significantly lower than what has been

previously reported. In the past, the physiologic upper limit of the pore size within the BTB of orthotopic and ectopic malignant peripheral tumors has been probed by intra- vital fluorescence microscopy 24 hours after the intravenous infusion of liposomes and microspheres with a cationic exterior, and it has been reported the upper limit of the pore size within the BTB of peripheral tumors is between 200 nm and 1200 nm. This higher upper limit of pore size would be most likely due to the toxicity of the cationic liposomes and microspheres to the negatively charged glycocalyx overlaying the endothelial cells of the BTB. The circulation of cationic particles for 24 hours would be sufficient time to expose the underlying smaller-sized trans-endothelial cell fenestrations and WOs as well as the larger- sized inter-endothelial cell gaps. The transvascular extravasation of the particles across the exposed inter-endothelial cell gaps into the tumor tissue extravascular space, or alternatively, entrapment in the peri-vascular space along the basement membrane would result in the over- estimation of the actual physiologic upper limit of pore size within the BTB.

We demonstrate herein that Gd-G5, Gd-G6, and Gd-G7 dendrimers extravasated across the BTB of ectopic RG-2 gliomas as well as that of orthotopic RG-2 gliomas.

However, these Gd-dendrimer generations extravasated to a greater extent across the BTB of ectopic RG-2 gliomas than the BTB of orthotopic RG-2 gliomas, as Gd-G5, Gd-G6, and Gd- G7 dendrimers achieved higher peak concentrations in the tumor tissue extravascular space of ectopic RG-2 malignant gliomas than in the rumor tissue extravascular space of orthotopic RG-2 malignant gliomas. Based on these findings, the BTB of the ectopic RG-2 malignant gliomas is more permeable than the BTB of orthotopic RG-2 malignant gliomas. The observed higher permeability of the BTB of ectopic RG-2 gliomas in this animal model may be in part due to host site dependent differences in tumor volume, since the tumor volumes of the ectopic RG-2 gliomas where generally larger than those of the orthotopic RG-2 gliomas (Fig. 12). Although this may be the case, the higher permeability of BTB of ectopic RG-2 gliomas compared to that of the BTB of orthotopic RG-2 gliomas is consistent with the reported higher permeability of the BTB of malignant peripheral tumors compared to that of the BTB of malignant brain tumors.

With each successively higher Gd-dendrimer generation the increase in diameters of the Gd-dendrimers was approximately 2 nm. Although there were relatively small increases in Gd-dendrimer particle sizes, there were significant decreases in particle extravasation across the BTB with increasing Gd-dendrimer generation, irrespective of RG-2 glioma host site. Gd-G7 dendrimers extravasated only minimally across the BTB, and the Gd-G8 dendrimers were large enough that these particles did not extravasate across either the BTB

of ectopic RG-2 gliomas or that of orthotopic RG-2 gliomas. As a result, Gd-G8 dendrimers did not accumulate over time in the respective tumor tissue extravascular spaces, and instead remained in the tumor microvasculature. The peak Gd concentrations of Gd-G8 dendrimers in ectopic RG-2 gliomas and orthotopic RG-2 gliomas were similar and reflect the peak Gd- G8 dendrimer concentrations within the microvasculature of the respective tumors.

The blood half-lives of Gd-G5 and Gd-G6 dendrimers were longer than those of Gd- G7 and Gd-G8 dendrimers (Fig. 5). In case of Gd-G5 and Gd-G6 dendrimers, the relatively longer blood half-lives are due to the sizes of these Gd-dendrimer generations being large enough to evade filtration by the kidneys, yet small enough to evade opsonization by reticuloendothelial system of the liver and spleen. Therefore, Gd-G5 and Gd-G6 dendrimers were not effectively cleared from blood circulation and had longer blood half-lives than Gd- G7 and Gd-G8 dendrimers. The blood half-lives of Gd-G7 and Gd-G8 dendrimers were shorter than those of the Gd-G5 and Gd-G6 dendrimers likely due to the sizes of these Gd-'dendrimers being too large to evade opsonization by reticuloendothelial system. Even though Gd-G7 dendrimers were small enough to extravasate across the BTB and

Gd-G8 dendrimers were too large to extravasate across the BTB, both Gd-G7 and Gd ^-1 GS dendrimers were effectively cleared from blood circulation and had shorter blood half-lives than Gd-G5 and Gd-G6 dendrimers. These findings suggest that nanoparticles within the size range of Gd-G5 and Gd-G7 dendrimers would be both permeable to the BTB of malignant solid tumor microvasculature and also possess blood half-lives sufficiently long to allow for particles to effectively accumulate over time within the tumor tissue extravascular space by the enhanced permeation and retention (EPR) effect.

Since the sizes of hydrated dendrimer generations, measured by small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), are similar to the sizes of respective dehydrated and stained dendrimer generations measured by TEM (34), here we used ADF STEM to the measure the sizes of the Gd-G7 dendrimers and Gd-G8 dendrimers dried on ultrathin carbon support film. We found the diameters of the Gd-G7 dendrimers to be 10.9 ± 0.7 nm and those of the Gd-G8 dendrimers to be 12.7 ± 0.7 nm (mean ± standard deviation). Since Gd-G7 dendrimers were permeable to both the BTB of ectopic RG-2 gliomas and orthotopic RG-2 gliomas, but the Gd-G8 dendrimers were not, this establishes the effective physiologic upper limit of pore size in both the BTB of ectopic RG-2 gliomas and orthotopic RG-2 gliomas as being approximately 12 nm.

The previously reported higher physiologic upper limit of pore size in the BTB of malignant solid tumors, based on intra- vital fluorescence microscopy of tumor tissue

following the intravenous infusion of cationic nanoparticles, appears to have been a gross over-estimation of the actual physiologic upper limit of pore size. The most plausible explanation for this is that the positively charged exterior of the cationic nanoparticles was toxic to the negatively charged glycocalyx surface coat of the BTB. We report here, based on dynamic contrast-enhanced MRI of tumor tissue following the intravenous infusion of neutralized nanoparticles, that the physiologic upper limit of pore size is much lower, being approximately 12 nm, when the BTB glycocalyx layer is maintained intact. It has been previously proposed that the fibrous matrix of the glycocalyx is the primary filter for the transvascular transport of particles across the endothelial barriers of non-tumor tissue micro vasculature. Our finding that the physiologic upper limit of pore size in the BTB of malignant solid tumors is approximately 12 nm also implicates the glycocalyx as being the "nanofilter" that prevents the effective transvascular passage of particles larger than approximately 12 nm in diameter across the BTB of malignant solid tumor micro vasculature. The physiologic upper limit of pore size in the BTB of malignant solid tumor micro vasculature is approximately 12 nm. hi the physiologic state in vivo, since the fibrous glycocalyx overlays the pores in the BTB of both brain and peripheral tumors, the physiologic upper limit of pore size in the BTB of malignant solid tumor microvasculature is equivalent and independent of tumor host site. Therefore, the higher permeability of the BTB of peripheral tumors would most likely be a consequence of there being more underlying pores (i.e. fenestrations, WOs, gaps) in the BTB of peripheral rumor microvasculature. Our findings will have important implications on the size range of nanoparticle-based therapeutics that can be effectively delivered across the BTB of malignant solid tumors.

Example 1. Delivery of dendrimer conjugates across the pathologic blood-brain tumor barrier (BBTB) of solid tumors located within the brain or across the pathologic blood- tumor barrier (BTB) of solid tumors located outside the brain hi many central nervous system (CNS) pathologies, the blood-brain barrier (BBB) becomes porous due to the formation of discontinuities within and between endothelial cells lining the lumens of micro-vessels located within diseased tissue (1-3). Despite this ready access for sufficiently small therapeutics, most neurological malignancies remain refractory to treatment due to the poor specificity and rapid clearance of conventional chemotherapy from tumor tissue (4,5). In the case of the blood-brain tumor barrier (BBTB), these fenestrations and gaps are small enough to prevent effective transvascular passage of most nanoparticles (6-9). In contrast to conventional nanoparticles polyamidoamine (PAMAM)

dendrimers are multi-generational monodisperse polymers ranging from 1.5 to 14 nm in diameter (10). Furthermore, PAMAM dendrimers possess modifiable surface groups for conjugation with imaging probes, as well as targeting and therapeutic agents (11-14).

In the blood-brain barrier of tumor vasculature there are differences in the degree of barrier breakdown related to tumor size (15). The experiments described herein were undertaken in an effort to further understand how these tumor volume dependent differences in barrier integrity affect Gd-dendrimer transport into tumor tissue. For study of the BBTB permeability in relation to tumor volume, an orthotopic brain tumor model was utilized as described herein, the right anterior and left posterior rat brain was inoculated with different amounts of RG-2 glioma cells to produce larger and smaller gliomas within each rat brain (16). For study of the differeneces in the BBTB and BTB permeability, an orthotopic-ectopic tumor model was utilized as described herein, RG-2 glioma cells right brain caudate nucleus and left temporalis muscle.

Historically, the transvascular transport of systemically administered radio- and fiuorophore-labeled compounds has been studied with invasive techniques such as quantitative autoradiography and intravital microscopy, respectively (7,17). The experiments described herein use dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to measure non-invasively the amount of PAMAM dendrimer in blood and brain tumor tissue following intravenous infusion of particles. In order to visualize the PAMAM dendrimers using MRI, dendrimers were functionalized with gadolinium (Gd) chelated by diethyltriaminepentaacetic acid (DTPA), a biocompatible paramagnetic compound (18).

To quantify the amount of Gd in blood and tissue, the change in the longitudinal relaxivity (T ₁ ) before and after contrast agent infusion for each imaged volume element

(voxel) was calculated (19). Based on the change in T ₁ , and the molar relaxivity of each respective Gd-dendrimer generation, the Gd concentration of each voxel over time was calculated (19). Thus, with DCE-MRI the change in Gd concentration within blood and tissue over a 2 hour period following intravenous infusion of Gd-PAMAM dendrimers of generations 1 (Gl) through generation 8 (G8) could be assessed. This information was used for pharmacokinetic modeling of Gd-dendrimer properties, and for determining the physiologically and therapeutically relevant pore size of the BBTB. In addition to the in vivo DCE-MRI experiments with Gd-PAMAM dendrimers, in vitro and ex vivo fluorescence microscopy experiments were performed using rhodamine B (RB) labeled Gd-PAMAM dendrimers, to confirm that functionalized dendrimers smaller than the barrier pore size enter brain tumor cells.

The results reported herein show, using successively higher generations of functionalized PAMAM dendrimers, that intravenously administered nanoparticles less than 13 nm in diameter can cross the BBTB, while larger functionalized dendrimers cannot. Furthermore, it is observed that dendrimers with prolonged circulation times in blood accumulate within tumor cells after crossing the BBTB or the BTB.

Example 2. Gd-dendrimer properties differ from those of naked dendrimers

The physical properties of naked PAMAM dendrimers (Starburst G1-G8, ethylenediamine core; Sigma- Aldrich®, St. Louis, MO) and Gd-PAMAM dendrimers are discrete and uniform for each generation. This is shown in Table 1, below. The constants of proportionality required for calculation of Gd concentration, also known as Gd-dendrimer molar relaxivities, ranged between 7.8 and 12.2 l/mM*s (Table 1).

Table 1 - Physical properties of PAMAM and Gd-PAMAM dendrimers

Dendrimer Binding Naked PAMAM Gd-PAMAM MW ⁺ Conjugation p ^θlar . & generation sites MW ^# (kDa) (kDa) WRT Gd (%) (i/mM _* s)

G1 8 1.43 5.63 67.1 9.8

G2 16 3.26 11.2 65.9 10.1

G3 32 6.91 18.6 47.7 10.4

LC G4 64 14.2 24.4 29.8 7.8

G4 64 14.2 39.8 47.5 12.2

G5 128 28.8 79.8 47.2 10.9

G6 256 58.0 133 39.9 10.6

G7 512 116 330* 50.0 10.3

G8 1024 233 597* 37.8 9.4 molecular weight obtained from Dendritech,® Inc. Volecular weight measured by MALDI-TOF MS *molecular weight measured by STEM &molar relaxivity of Gd-DTPA measured to be 4.05

The maximum number of amines available for conjugation doubles with each dendrimer generation (Fig. Ia). However, the percent conjugation in higher generations tends to decrease due to the steric hindrance that prevents additional DTPA units from successfully attaching at remaining sites. Conjugation with Gd-DTPA (charge -2, MW -0.9 kDa) neutralizes the positive charge of the terminal amines (Fig. Ib), and increases the MW of the dendrimer by two- to five-fold. To verify that the MW, and not the dendrimer generation per se, is the primary determinant of particle blood half-life, Gd-G4 dendrimers of two different MWs were examined. One was a lowly conjugated (LC) G4 weighing 24.4 kDa and the other was a standard G4 weighing 39.8 kDa. Gd-dendrimers of generations 5 and higher have masses which are large enough for visualization by scanning transmission electron microscopy (STEM) using annular dark-field (ADF) imaging at a low electron dose (20). ADF-STEM images of Gd-dendrimers G5 through G8 show uniformity in size, shape and density within any particular dendrimer generation (Fig. 1 c) and also confirms the small increase of only 1 to 2 ran in particle diameters between successive generations.

Example 3. Blood concentration of permeable Gd-dendrimers determines degree of particle accumulation within brain tumor tissue

The blood and tissue dynamics of Gd-dendrimer Gl through G8 were studied at two different Gd doses using DCE-MRI. To measure the change in Gd concentration within brain vasculature, tumor, and normal brain tissue over time, rat brain was imaged at 20 s intervals for one or two hours (Figs. 2, 3). For the initial experiments, a dose of 0.03 mmol Gd/kg body weight (bw) was administered, which is the standard intravenous dose for pre-clinical imaging with Gd labeled dendrimers (18). In order to determine the dosage-response relationship, additional imaging at a Gd-dendrimer dose of 0.09 mmol Gd/kg bw was performed. These results are shown in Table 2 below.

Table 2 - Amount of Gd-dendrimer being infused per Gd dose Dendrimer 0.03 mmol Gd/kg bw 0.06 mmol Gd/kg bw 0.09 mmol Gd/kg bw generation (mg Gd-dendrimer/kg bw) (mg Gd-dendrimer/kg bw) (mg Gd-dendrimer/kg bw)

G1 31.5 n/a 94.4

G2 31.9 n/a 95.6

G3 36.6 n/a 110

LC G4 38.4 n/a 115

G4 39.3 n/a 118

G5 39.6 n/a 119

R G5 n/a 96.3 n/a

G6 39.6 n/a 119

G7 38.7 n/a 116

G8 46.3 n/a 139

R G8 n/a 101 n/a

At a dose of 0.03 mmol Gd/kg bw only Gd-G5 dendrimers appear to extravasate out of the tumor micro vasculature and accumulate within tumor tissue, even though blood concentrations of Gd-G5 through Gd-G8 dendrimers reach and maintain a steady state (Fig. 2f). At this dose there was no measurable extravasation of Gd-dendrimer generations 6, 7 or 8 out of the tumor microvasculature (Fig. 2g-i). However, at a dose of 0.09 mmol Gd/kg bw it is evident that Gd-G6 and Gd-G7 dendrimers also extravasate into the tumor tissue and accumulate there. At this higher dose, levels of Gd-G5 and Gd-G6 dendrimer accumulation within tumor tissue are similar, while Gd-G7 dendrimer extravasation and accumulation is to a lesser degree (Fig 2f-i). Thus, there seems to be a minimum concentration gradient required for accumulation of higher generation Gd-PAMAM dendrimers across the BBTB.

Example 4. Blood circulation lifetime of permeable Gd-dendrimers determines duration of particle residence within brain tumor tissue Irrespective of dose, it was observed that the blood half-lives of Gd-dendrimers increase with each successive generation up to Gd-G5. The blood concentration of Gd-G5 through Gd-G8 Gd-dendrimers reaches and maintains a steady state for at least 2 hours (Fig. 2a, 2b). The blood concentration profile of LC G4 Gd-dendrimers is similar to that of G3 Gd- dendrimers since the kidneys efficiently filter macromolecules less than 30 kDa in MW (21). Standard G4 Gd-dendrimers exhibit longer circulation times in blood, a finding that is consistent with the MW of these particles being above the cusp of effective renal filtration (Fig. 2a, 2b). Due to their short circulation times in blood, Gd-Gl through LC Gd-G4 dendrimers reside only temporarily within the tumor extravascular space (Figs. 2d, 3). In

contrast, standard Gd-G4 through Gd-G7 dendrimers have longer blood-half lives that result in particle accumulation and retention within tumor tissue (Figs. 2e, 3a). The BBB of micro- vasculature within normal brain tissue is not porous. Therefore, Gd-dendrimers do not enter the extravascular space of normal brain tissue as demonstrated by the Gd concentration curves of normal brain tissue mirroring those of brain vasculature (Fig. 2c). Gd-G8 dendrimers are larger than the physiologic pore size of the BBTB, as evidenced by Gd-G8 dendrimers remaining within the brain tumor microvasculature, regardless of dose (Fig 2i). With regard to tumor size, individual tumor concentration curves reveal that Gd-G5 and Gd- G6 dendrimers can extravasate across the BBTB of smaller gliomas and accumulate. Images of Gd-dendrimer behavior over a 2 hour period in representative smaller tumors are presented in Fig. 3b. Thus, in the setting of a less permeable BBTB, the effective intra-tumor accumulation of Gd-dendrimers may occur when the particle circulation time in blood is sufficiently long. It is also evident in Fig. 3b that Gd-G7 dendrimers do not accumulate in smaller tumors over 2 hours, the time course of the experiment. This could be due to the upper threshold of the physiologic pore size being lower in the less defective BBTB of smaller rumors. Gd-G8 dendrimers remain intravascular due to particle sizes being greater than the physiologic pore size of the smaller tumor BBTB.

To determine the intra-tumor residence time of Gd-G5 dendrimers the brains of tumor bearing rats at 2, 6, and 12 hours following infusion of Gd-G5 dendrimers were imaged. Gd- G5 dendrimers reach maximal tumor concentration between 2 and 6 hours following infusion and there is still residual Gd-G5 dendrimer within tumor tissue at 12 hours following intravenous infusion (Fig. 3c). In addition, the Gd-G5 dendrimers appear to diffuse into microscopic foci of tumor cells located in the immediate periphery of the tumor.

Example 5. MW and size of lower generation Gd-dendrimers influences particle transvascular flow rate and extravascular extracellular tumor space distribution

It was next investigated how increases in Gd-dendrimer MW and size affect particle transvascular flow rate across the BBTB and distribution within the extravascular extracellular tumor space. For this purpose, the 2-compartment 3 -parameter generalized kinetic model was employed to determine transvascular flow rates (K'"""), fractional extravascular extracellular volume (ve) and fractional plasma volume (vp) (22,23). The pharmacokinetic properties of Gd-Gl through LC Gd-G4 dendrimers only were studied, since standard G4 and higher generation dendrimers are not completely cleared from plasma over 2 hours (22). On the basis of the multivariate analysis of variance (MANOVA), irrespective of

tumor size, the four dendrimer generations produce significantly different pharmacokinetic parameters for K ^trans (F3,15.7=l 1.61; Bonferroni corrected p=0.0009) and ve (F3, 16.1-8.26; Bonferroni corrected p=0.0045), but not for vp (F3,16.3=l .24; p=not significant). As indicated in Fig. 4a there is a significant decrease in κ" ^"ans between LC Gd-G4 and Gd-Gl dendrimers. This decrease in κ' ^raλϊ is reflected in a significant decrease in distribution of LC Gd-G4 dendrimers within the extravascular extracellular tumor space as compared to that of Gd-Gl (Fig. 4d), Gd-G2, and Gd-G3 dendrimers (Fig. 4b). The vp is not significantly different between tumor populations within the four different dendrimer generations (Fig. 4c), showing that there is little variance in overall tumor vascularity across these dendrimer generations. Thus, the increase in MW and size from that of Gd-Gl (5.6 kDa) dendrimers to that of LC Gd-G4 (24.4 kDa) dendrimers results in significantly different dendrimer pharmacokinetics within tumor tissue.

On the basis of the MANOVA, irrespective of dendrimer generation, larger tumors had higher values of K' ^rans (F 1,34.6 =10.83; Bonferroni corrected p=0.0069), ve (Fl ,22.5=50.76; Bonferroni corrected p <0.0003) and vp (Fl ,27.9=20.49; Bonferroni corrected p=0.0003). Thus, the size of a tumor significantly affects all three parameters for dendrimer generations Gd-Gl through LC Gd-G4. The K.' ^rans increases with tumor size because the BBTB becomes more defective, and the ve also increases because larger tumors have a larger extravascular extracellular volume that can accommodate more Gd-dendrimers. The vp is greater for larger tumors because larger tumors have a more extensive network of defective vasculature (15).

Example 6. BBTB is the impediment to cellular uptake of RB Gd-G8 dendrimers but not RB Gd-GS dendrimers To investigate if there are limitations to particle entry at the cellular level in vitro, Gd-

G2, Gd-G5, and Gd-G8 dendrimers labeled with rhodamine B were synthesized and studied as a representative sample of Gd-dendrimer generations 1 through 8. The synthetic scheme of RB labeled Gd-PAMAM dendrimers is displayed in Fig. 5a. The physical properties of RB Gd-G2, RB Gd-G5 and RB Gd-G8 dendrimers were similar to those of the Gd-G2, Gd-G5, and Gd-G8 dendrimers. This is shown in Table 3, below.

Table 3 - Physical properties of rhodamine B Gd-PAMAM dendrimers

Dendrimer Binding Gd°PAMAM MW ^# Conjugation WRT Conjugation WRT ^*' generation sites ( _kDa ) Gd (%) rhodamine B (%) ^

G2 16 11.2 42.7 6.6 n/a

G5 128 75.0 36.5 7.9 9.2

G8 1024 540* 31.2 6.6 9.2

#molecular weight obtained from Dendritech,® Inc. imolecular weight measured by STEM

RG-2 glioma cells were imaged 4 hours after addition of RB Gd-G2, RB Gd-G5 or RB Gd-G8 dendrimers into the culture media, with doses normalized to 7.2 μM with respect to rhodamine B. All three Gd-dendrimer generations accumulated intracellularly (Fig. 5b) and RB Gd-G2 dendrimers in some cases were observed to enter cell nuclei (Fig. 5b, left). These findings demonstrate that nuclear pores in RG-2 glioma cells may allow for entry of anionic particles at least as large as our RB Gd-G2 dendrimers (molecular weight, MW 11.2 kDa). Based on the finding that RB Gd-G8 dendrimers localize within glioma cells as readily as RB Gd-G5 dendrimers, there appears to be no molecular weight or size barrier to cellular uptake of functionalized Gd-PAMAM dendrimers as large as G8. This demonstrates that the barrier to tumor cell entry in our animal model lies at the level of the BBTB.

To correlate in vivo magnetic resonance imaging with ex vivo fluorescence imaging, additional DCE-MRI experiments were conducted with infusions of RB labeled Gd-G5 and Gd-G8 dendrimers. The intravenous infusion dose for RB Gd-G5 and RB Gd-G8 dendrimers was 0.06 mmol Gd/kg body weight. Transvascular transport across the BBTB of Gd-G5 dendrimers was enhanced by the addition of the rhodamine (Fig. 5c). The Gd-G8 dendrimers were able to extravasate into tumor tissue to a minor degree with addition of the rhodamine (Fig. 5c). At low power (Fig. 5d, left) there was substantial accumulation of RB Gd-G5 dendrimer within tumor tissue, and at high power (Fig. 5d, top right) the RB Gd-G5 dendrimers demonstrated sub-cellular localization similar to that seen in vitro. There was minor extravasation of RB Gd-G8 dendrimers across the BBTB into tumor tissue (Fig. 5e, left), consistent with what was observed by DCE-MRI, and little sub-cellular localization (Fig. 5e, top right) as compared to what was visualized in vitro. Thus, the impediment to cellular uptake of functionalized G8 dendrimers is the upper threshold of the physiologically relevant pore size of the BBTB, which is between the average diameter of our Gd-G7 dendrimers and Gd-G8 dendrimers, 1 1.0 ± 0.74 run and 13.3 ± 1.4 nm (mean ± standard deviation), respectively.

The results presented herein show that intravenously administered functionalized PAMAM dendrimers less than 13 nm in diameter with long circulation times in blood can traverse pores of the BBTB and accumulate within tumor cells, demonstrating that the physiologically and therapeutically relevant upper limit of the BBTB pore size is 13 nm. Furthermore, this upper limit pore size may be lower in the setting of a less defective barrier, such as that of smaller brain tumors. Since ultrastructural studies of the blood-brain barrier show the existence of fenestrations and gaps ranging from 40 to 90 nm and 100 to 250 nm, respectively, there must be a significant degree of steric hindrance at the pore level (1,24). The presence of radial fibrils within fenestrations may contribute to the physiologically relevant pore size being much lower than the anatomic pore size (25), although the presence of a fibrous glycocalyx surface coat overlaying the fenestraitons and gaps of the BBTB is most likely the reason for the discrepancy between the physiologic pore size and the anatomic pore size.

The results presented herein also demonstrate that the effective delivery of nanoparticles across the BBTB may be accomplished, and that higher generation dendrimers permeable to the BBTB (i.e. G4 through G7) could be used for this purpose since these higher generation dendrimers possess sufficiently long blood half-lives to accumulate within individual brain tumor cells after traveling the BBTB. Macromolecules with MWs greater than 30 kDa are not easily filtered by the kidneys (21). Liver processes of particle breakdown are saturable for higher generation Gd-dendrimers resulting in particles remaining within systemic circulation for prolonged duration before being cleared by the kidneys (18,26). For this reason Gd-dendrimers of generations 5 and 6 are able to even accumulate within smaller tumors over 2 hours, a period of time that is sufficient enough for particles to enter brain tumor cells. Thus, functionalized dendrimers with long blood half-lives can serve as effective vehicles for transvascular transport of therapeutics across the BBTB and into brain tumor cells, and potentially other neuropathologic tissues.

Example 7. Physical properties of naked PAMAM and Gd-PAMAM dendrimer generations used in the orthotopic- ectopic RG- 2 glioma implantation experiments

The physical properties of naked PAMAM dendrimers (Starburst G5-G8, ethylenediamine core; Sigma- Aldrich, St. Louis, MO) and Gd-DTPA functionalized PAMAM dendrimers were characterized. Within each dendrimer generation, the amount of increase in the molecular weight between the naked dendrimer and the functionalized

dendrimer is proportional to the percent conjugation of Gd-DTPA (Table 4). For each successively higher dendrimer generation, the percent conjugation of Gd-DTPA is lower due to greater steric hindrance encountered in the chelation reaction process (Table 4). The Gd- dendrimer molar relaxivities, which are the constants of proportionality required for calculation of Gd concentration from Gd signal intensity, ranged between 9.81 and 10.05 l/mM*s (Table 4).

Table 4 - Physical properties of PAMAM and Gd-PAMAM dendrimers

Dendrimer Terminal Naked PAMAM Gd-PAMAM Gd-DTPA Molar generation amines molecular weight # molecular weight conjugation relaxivity& (kDa) (kDa) (%) (1/mM ^* s)

G5 128 29 79* 52 9.81

G6 256 58 138t 45 10.04

G7 512 116 283J 43 9.82

G8 1024 233 49Ot 36 10.05

# molecular weight obtained from Dendritech,® Inc. fmolecular weight measured by MALDI TOF- MS J mean molecular weight measured by ADF STEM and EFTEM & molar relaxivity of Gd-DTPA measured to be 4.13 l/mM*s

ADF STEM of Gd-G5 through Gd-G8 dendrimers demonstrated uniformity in particle shape and size within any particular Gd-dendrimer generation. ADF STEM confirmed a small increase of approximately 2 run in particle diameter between successive generations (Fig. 1). The masses of Gd-G7 and Gd-G8 dendrimers were sufficient that the sizes and molecular weights of these Gd-'dendrimer generations could be measured by ADF STEM and STEM-EFTEM, respectively. The molecular weights and diameters of one hundred Gd-G7 and Gd-G8 dendrimers were measured. The average molecular weight of Gd-G7 was 283 ± 5 kDa and that of Gd-G8 dendrimers was 490 ± 5 kDa (mean ± standard error of the mean) (Table 4). The average diameter of Gd-G7 dendrimers was 10.9 ± 0.7 nm and that of Gd-G8 dendrimers was 12.7 ± 0.7 nm (mean ± standard deviation).

Example 8. Permeability of the BBTB of orthotopic RG-2 gliomas and the BTB of ectopic RG-2 gliomas to Gd-PAMAM dendrimer generations

Gd-G5 dendrimers extravasated across the barrier of both orthotopic and ectopic RG- 2 gliomas and accumulated within the respective tumor tissue extravascular spaces (Fig. 10, a

and e). However, the Gd-G5 dendrimers extravasated to a lesser extent across the BBTB of orthotopic RG-2 gliomas than the BTB of ectopic RG-2 gliomas indicating the BBTB of orthotopic RG-2 gliomas was less permeable than the BTB of ectopic RG-2 gliomas. Thus, the peak Gd concentration of Gd-G5 dendrimers in orthotopic tumors was 0.147 mM, whereas the peak Gd concentration of Gd-G5 dendrimers in ectopic tumors was 0.195 mM (Table 5).

Table 5 - Gd-PAMAM dendrimer peak concentrations in orthotopic RG-2 gliomas versus ectopic RG-2 gliomas*

Gd-PAMAM Peak Peak Peak Peak dendrimer concentration concentration concentration concentration generation orthotopic RG-2 time point (min) ectopic RG-2 time point (min) gliomas (mM) gliomas (mM)

Gd-G5 0.195 167 0.147 149

Gd-G6 0.144 200 0.106 189

Gd-G7 0.084 75 0.064 107

Gd-G8 0.052 77 0.049 81

Gd-G6 dendrimers also extravasated across the barrier of both orthotopic and ectopic RG-2 gliomas and accumulated within the respective tumor tissue extravascular spaces (Fig. 10, b and f). Gd-G6 dendrimers accumulated to lesser extent than Gd-G5 dendrimers in both orthotopic and ectopic tumor tissue extravascular spaces. As was the case for Gd-G5 dendrimers, the Gd-G6 dendrimers extravasated to a lesser extent across the BBTB of orthotopic RG-2 gliomas than the BTB of ectopic RG-2 gliomas, once again indicating the BBTB of orthotopic RG-2 gliomas was less permeable than the BTB of ectopic RG-2 gliomas. Thus, the peak Gd concentration of Gd-G6 dendrimers in orthotopic tumors was 0.106 mM, whereas the peak Gd concentration of Gd-G6 dendrimers in ectopic tumors was 0.144 mM.

Gd-G7 dendrimers minimally extravasated across the barrier of both orthotopic and ectopic RG-2 gliomas and so minimally accumulated within the respective tumor tissue extravascular spaces (Fig. 10, c and g). Gd-G7 dendrimers accumulated to an even lesser extent than Gd-G6 dendrimers in both orthotopic and ectopic tumor tissue extravascular spaces. As was the case for Gd-G6 dendrimers, the Gd-G7 dendrimers extravasated to a lesser extent across the BBTB of orthotopic RG-2 gliomas than the BTB of ectopic RG-2 gliomas, once again indicating the BBTB of orthotopic RG-2 gliomas was less permeable

than the BTB of ectopic RG-2 gliomas. Thus, the peak Gd concentration of Gd-G7 dendrimers in orthotopic tumors was 0.064 mM, whereas the peak Gd concentration of Gd- G7 dendrimers in ectopic tumors was 0.084 mM (Table 5).

Gd-G8 dendrimers did not extravasate across the barrier of orthotopic and ectopic RG-2 gliomas. The change in Gd concentration over time for both orthotopic and ectopic RG-2 gliomas was similar (Fig. 10, d and h). The peak Gd concentrations of Gd-G8 dendrimers in both orthotopic and ectopic tumors were similar: the peak Gd concentration of Gd-G8 dendrimers in orthotopic tumors was 0.049 mM and that in ectopic tumors was 0.052 mM (Table 5). The peak Gd concentrations in orthotopic and ectopic tumors reflect the peak Gd-G8 dendrimer concentrations within the micro vasculature of the respective tumors and not the extravascular tumor tissue space.

Example 9. Physiologic upper limit of pore size within the BBTB of orthotopic RG-2 gliomas and within the BTB of ectopic RG-2 gliomas as visualized on Gd concentration maps

For each of the Gd-dendrimer generations, after the initial 15 minute dynamic scan, the orthotopic and ectopic RG-2 gliomas of one additional animal were imaged every 10 minutes for a total of 175 minutes, while the animal was under continuous anesthesia. The Gd concentration maps from selected dynamic scans of these imaging sessions are shown in Fig. 11. The hemodynamic depression associated with the continuous anesthesia is reflected in the lower peak contrast enhancement observed.

Gd-G5 dendrimers readily extravasated across the barrier of both orthotopic and ectopic RG-2 gliomas and accumulated over time within the respective tumor tissue extravascular spaces, as evidenced by the significant positive contrast enhancement over time in the respective tumor tissues (Fig. 11, first row). Gd-G6 dendrimers also extravasated across the barrier of both orthotopic and ectopic RG-2 gliomas and accumulated over time within the respective tumor tissue extravascular spaces (Fig. 11 , second row), although to a lesser extent than Gd-G5 dendrimers (Fig. 11, first row).

Gd-G7 dendrimers minimally extravasated across the barrier of both orthotopic and ectopic RG-2 gliomas and so minimally accumulated over time within the respective tumor tissue extravascular spaces (Fig. 3, third row). Gd-G8 dendrimers did not extravasate over time across the barrier of both orthotopic and ectopic RG-2 gliomas, but instead remained within the tumor microvasculature, as evidenced by the lack of contrast enhancement over time within the respective tumor tissue extravascular spaces (Fig. 11, fourth row). Therefore,

the physiologic upper limit of pore size within the barrier of both malignant brain tumors and peripheral solid tumors is equivalent. Since the diameter of our Gd-G7 dendrimers and Gd- G8 dendrimers was 10.9 ± 0.7 run and 12.7 ± 0.7 nm (mean ± standard deviation), the upper limit of pore size within the barrier of both orthotopic RG-2 gliomas and ectopic RG-2 gliomas is approximately 12 nm.

Example 10. Gd-G5 PAMAM-doxorubicin dendrimer (Gd-G5-DOX dendrimer) conjugate is more effective than free doxorubicin (DOX) in the treatment of orthotopic RG-2 malignant gliomas Since Gd-PAMAM dendrimer generation 5 (G5) was permeable to the BBTB and possessed a relatively long blood half-life, this dendrimer generation was studied further. Doxorubicin (MW 543 kDa), a commonly used small chemotherapeutic agent, was conjugated to the terminal amines of Gd-DTPA chelated G5 PAMAM dendrimers via a covalent pH sensitive hydrazone bond such that doxorubicin was conjugated to approximately 10 out the available 128 terminal amines on each dendrimer. The synthetic scheme is detailed in Fig. 14. Shown in Table 6 are the Gd-G5-DOX dendrimer conjugate properties.

Table 6 - Properities of Gd-G 5 PAMAM-doxorubicin (Gd-G5-DOX) conjugate

Dendrimer Terminal Naked PAMAM Gd-PAMAM Gd-DTPA Molar DOX generation amines molecular weight # molecular weight conjugation relaxivity& conjugation (kDa) (kDa) (%) (1/mM ^* s) (%)

G5 128 29# 85J 48.1 10.1 7.8 # molecular weight obtained from Dendritech,® Inc. t molecular weight measured by MALDI TOF- MS & molar relaxivity of Gd-DTPA measured to be 4.1/mM*s

In vitro doxorubicin release experiments confirmed the pH dependent increase in the rate of hydrolysis of the hydrazone bond and release of doxorubicin from the Gd-G5-DOX dendrimer conjugate (Fig. 15). The effectiveness of the Gd-G5-DOX dendrimer conjugate compared to that of free doxorubicin was investigated in orthotopic RG-2 glioma bearing rodents. RG-2 glioma cells were inoculated into the right anterior caudate of the rat brain. The RG-2 glioma bearing brains were with T ₂ weighted anatomic scans at the time of treatment for determination of the initial brain tumor volume and on subsequent days following treatment to follow the response of the tumor growth rate following the infusion of a single dose of free doxorubicin (N=7) or a single dose of the Gd-G5-DOX dendrimer

conjugate (N=7). The total dose of doxorubicin administered was 8 mg/kg bw for each group. In the case of the Gd-G5-DOX dendrimer conjugate, the doxorubicin dose of 8 mg/kg bw translated into an approximately 120 mg/kg bw dose of dendrimer conjugate. The animal brains were imaged with DCE-MRI and T ₂ weighted anatomic scans at the time of treatment and with T ₂ weighted anatomic scans on subsequent days following treatment. Tumor tissue voxels were identified on the scans and whole tumor volumes calculated in mm ³ . As shown in Fig, 16 (A and B), the RG-2 malignant gliomas of animals treated with one dose of free doxorubicin continued to grow, whereas the gliomas of animals treated with Gd-G5-DOX dendrimer nanoparticles either regressed or stabilized. The RG-2 gliomas with the largest initial tumor volumes in both the free doxorubicin and Gd-G5 dendrimer-DOX nanoparticle groups were the least responsive due to significant initial tumor burden. The percent change in RG-2 gliomas for each group of rodents in shown in Table 7, below. These findings show that a single dose of the Gd-G5-DOX dendrimer conjugate is more effective than a single dose of free doxorubicin.

Table 7 - Change in tumor volume in response to free DOX versus Gd-G5-dendrimer- DOX conjugate

Example 11. Additional studies

Multiple sequential doses of Gd-G5-DOX dendrimer conjugates are administered to show that complete malignant glioma regression can be achieved in animals bearing

orthotopic RG-2 glioma regression as would be expected based on the data from the short term studies. Other chemotherapeutic agents such as temozolamide (Temodor) are conjugated to Gd-G5 and Gd-G6 PAMAM dendrimers and tested in vivo for selection of the most appropriate dendrimer conjugates for clinical development. Boronated dendrimers are developed for intravenous administration and tested for effectiveness in the transvascular delivery of the prerequisite amount of boron atoms into brain tumors for boron neutron capture therapy (BCNT). Therapeutic agents delivered as dendrimer conjugates are determined to have good efficacy and reduced undesirable side effects as compared to therapeutic agents delivered alone. Analogous experiments are carried out in peripheral solid tumor animal models.

Therapeutic agents dendrimer conjugates are prepared and administered to various animal models of cancer (e.g., animals genetically susceptible to cancer, tumor implantation models). Therapeutic agents delivered as dendrimer conjugates are determined to have good efficacy and reduced undesirable side effects as compared to therapeutic agents delivered alone. Further, animal experiments are carried out in animal models of brain encephalitis to demonstrate the effectiveness of intravenously administered dendrimer conjugates containing antibiotics in treating infectious diseases of the brain. The methodologies described herein are used for the pre-clinical testing and validation.

Methods

The invention was performed using, but not limited to, the following methods:

Methods Summary

Bifunctional chelating agents and Gd-Bz-DTPA functionalized PAMAM dendrimers were synthesized according to described procedures with minor modifications, as were the corresponding rhodamine-substituted conjugates (27-30). For in vitro electron microscopy experiments, a 5 μL droplet of phosphate-buffer saline solution containing a sample of Gd- dendrimers from generations 5, 6, 7 or 8 was absorbed onto a 3 nm-thick carbon support film covering the copper EM grids. For in vitro fluorescence experiments, RG-2 glioma cells were plated on Fisher Premium coverslips (Fisher Scientific®, Pittsburgh, PA) and incubated in wells containing sterile 3 mL Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The anaesthesia and route for all animal experiments was isoflurane by inhalation with nose cone, 5% for induction and 1 to 2% for maintenance. All magnetic resonance imaging experiments were conducted using a 3.0 T MR scanner (Philips Intera; Philips Medical Systems, Andover, MA) using a 7 cm solenoid

radiofrequency coil (Philips Research Laboratories®, Hamburg, Germany). Imaging data were analyzed using the Analysis of Functional Neurolmaging (AFNI; available on the world- wide web at afhi.nimh.nih.gov/) software suite and its native file format (31). For Gd- Gl through LC Gd-G4 dendrimers pharmacokinetic modeling was performed using the 2- compartment 3-parameter generalized kinetic model (22,23).

PAMAM dendrimer functionalization, characterization and preparation for infusion Gd-dendrimers, with the exception of lowly conjugated (LC) Gd-G4, were prepared by using a molar reactant ratio of > 2:1 bifunctional chelate to dendrimer surface amine groups. For LC Gd-G4 a lower molar reactant ratio of 1.1 : 1 was used to limit conjugation. Rhodamine B (RB) labeled Gd-dendrimers were prepared by stirring rhodamine B isothiocyanate (RBITC) and PAMAM dendrimers at a 1 :9 molar ratio of RBITC to dendrimer surface amine groups in methanol at room temperature for 12 hours. Isothiocyanate activated DTPA was then added in excess and reacted for an additional 48 hours. Gadolinium was then chelated after the removal of the t-butyl protective groups on DTPA. The approximate percent by mass of gadolinium in each Gd-dendrimer generation was determined by elemental analysis to be: Gl (15.0 %), G2 (14.8 %), G3 (12.9 %), LC G4 (12.3 %), G4 (12.0 %), G5 (11.9 %), G6 (11.9 %), G7 (12.2 %), G8 (10.2 %). In a separate study, the percent by mass of Gd in each Gd-dendrimer generation was determined by elemental analysis to be: Gd-G5 (13.2%), Gd-G6 (13.0%), Gd-G7 (12.3%), and Gd-G8 (11.9%). The approximate gadolinium percent by mass for the RB labeled Gd-dendrimers was determined to be: RB G2 (9.6 %), RB G5 (9.8 %), RB G8 (9.3%).

Gd-Gl thorough Gd-G5 dendrimer molecular weights were determined by MALDI- TOF mass spectroscopy (Scripps Center for Mass Spectrometry, La Jolla, CA). Gd percent by mass of the Gd-dendrimer, in its solid form, was determined with the inductively coupled plasma-atomic emission spectroscopy (ICP-AES) method (Desert Analytics, Tucson, AZ). Gd-dendrimer infusions were normalized to 100 mM with respect to Gd, while RB labeled Gd-dendrimer infusions were normalized to 67 mM with respect to Gd, in order to guarantee proper solvation.

In vitro scanning transmission electron microscopy

Lacey Formvar/carbon coated 300 mesh copper grids supporting an ultrathin 3 nm evaporated carbon film were glow-discharged an air pressure of 0.2 mbar to facilitate Gd-

dendrimer adsorption. After adsorption for 2 minutes, excess Gd-dendrimer solution was blotted with filter paper. The grids were then washed 5 times with 5 μL aliquots of deionized water, and left to dry in air. ADF STEM images of the Gd-dendrimers were recorded using a Tecnai® TF30 electron microscope (FEI®, Hillsboro, OR, USA) equipped with a Shottky field-emission gun and an in-column ADF detector (Fischione, Export, PA).

In vitro fluorescence experiments

The RG-2 glioma colonies were allowed to establish for 24 hours in an incubator set at 37 ⁰ C and 5% CO2. Then RB labeled Gd-dendrimers of generations 2, 5, or 8 were added to the medium by equivalent molar rhodamine B concentrations of 7.2 μM and the cells were incubated in the dark for another 4 hours. Following incubation, cells were washed 3 times with phosphate buffered saline (PBS), then 50 μL DAPI-Vectashield® nuclear stain medium

(Vector Laboratories®, Burlingame, CA) was placed on the coverslips for 15 minutes.

Coverslips were then inverted and mounted on Daigger® Superfrost slides (Daigger®, Vernon Hills, IL) and sealed into place. Confocal imaging was performed on a Zeiss® 510

NLO microscope (Carl Zeiss Microimaging®, Thornwood, NY). Slides were stored in the dark while not being analyzed.

In vitro magnetic resonance imaging for calculations of Gd-dendrimer molar relaxivity

20 μL of 100 mM Gd-dendrimer stock solution or 30 μL of 67 mM RB labeled Gd- dendrimer stock solution of each generation used for in vivo imaging was diluted using PBS into 200 μL microfuge tubes at 0.00 mM, 0.25 mM, 0.50 mM, 0.75 mM and 1.00 mM with respect to Gd. As an external control, Magnevist® (500 mM gadopentetate dimeglumine salt, a form of Gd-DTPA; Bayer®, Toronto, Canada) was also diluted into 200 μL microfuge tubes at the above concentrations. The microfuge tubes were secured in level and upright positions within a plastic container filled with deionized ultra pure water. The container was placed in a 7 cm small animal solenoid radiofrequency coil (Philips® Research Labs) that was centered within a 3.0 T MR scanner (Philips Intera®). Gd signal intensity measurements were then taken using a series of Tl weighted spin echo sequences with identical TE (10 ms) but different TR (100 ms, 300 ms, 600 ms and 1200 ms). Using the measured Gd signal intensity, in addition to the known values for TR and TE, the longitudinal relaxivity (Tl) and equilibrium magnetization (MO) were calculated, by non-linear regression using equation 1 (19):

The molar relaxivity (rl) was calculated by linear regression using equation 2 (19):

y ¹ X =j -M-O ^+r > ^c (2)

In vitro and in vivo Gd-dendrimer molar relaxivities were assumed to be equivalent for the purposes of this work.

Brain tumor induction and MRI suite set-up

All animal experiments were approved by the National Institutes of Health Clinical Center Animal Care and Use Committee. Cryo-frozen pathogen-free RG-2 glioma cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in sterile DMEM supplemented with 10% FBS and 2% penicillin-streptomycin in an incubator set at 37°C and 5% CO ₂ . On experimental day 0, the head of anesthetized adult male Fischer 344 rats (F344) weighing 200-250 grams (Harlan Laboratories®, Indianapolis, IN) was secured in a stereotactic frame with ear bars (David Kopf Instruments® , Tujunga, CA). The right anterior caudate and left posterior thalamus locations within the brain were stereotactically inoculated with RG-2 glioma cells (32). In each location, either 20,000 or 100,000 glioma cells in 5 μL of sterile PBS were injected over 8 minutes, using a 10 μL Hamilton® syringe with a 32-gauge needle.

On experimental days 11 to 12, brain imaging of re-anesthetized rats was performed following placement of polyethylene femoral venous and arterial cannulas (PE-50; Becton- Dickinson®, Franklin Lakes, NJ), for contrast agent infusion and blood pressure monitoring, respectively. Both cannulas were 40 cm in length and filled with heparinized normal saline (10 u heparin sodium/1 ml saline). After venous cannula insertion, 50 μL of blood was withdrawn from the distal end of the cannula for measurement of hematocrit (H ct). Once the rat was positioned in the small animal MRI coil, the venous cannula was connected to the open end of a Y-connector with the other two ends temporarily attached via disposable 23 gauge connectors to PE-50 tubings from two saline filled plastic syringes previously loaded onto separate micro-infusion pumps (PHD 2000; Harvard Apparatus®, Holliston, MA) located in the MRI control room. One piece of PE-50 tubing was prepared to the length necessary for infusion of 0.03, 0.06 (RB Gd-dendrimer experiments) or 0.09 mmol Gd/kg bw Gd-dendrimer (100 mM Gd-dendrimer solution) at the beginning of the 2 hour 15 minute

dynamic scan. Similarly, another piece of tubing was prepared for infusion of 0.30 mmol Magnevist/kg bw (0.5 M gadopentetate dimeglumine salt, a form of Gd-DTPA; Bayer®, Toronto, Canada) at the 2-hour time point of the dynamic scan. The PE-50 tubing containing Gd-dendrimer was inserted between the Y-connector and infusion pump PE-50 tubing number 1 , and the PE-50 tubing containing Magnevist was inserted between the Y-connector and infusion pump tubing number 2. Rats for ex vivo confocal fluorescence microscopy of brain tumors were not administered Magnevist, and region of interests (ROIs) were drawn based on the Gd-dendrimer DCE-MRI scan.

Orthotopic-ectopic RG-2 glioma induction and animal preparation for imaging

The anesthesia and route for all animal experiments was isoflurane by inhalation with nose cone, 5% for induction and 1 to 2% for maintenance. On experimental day 0, the head of anesthetized adult male Fischer344 rats (F344) weighing 190 to 200 grams (Harlan Laboratories, Indianapolis, IN) was secured in a stereotactic frame with ear bars (David Kopf Instruments, Tujunga, CA). The right brain caudate nucleus (orthotopic RG-2 glioma) and left temporalis muscle (ectopic RG-2 glioma) locations were stereotactically inoculated with 105 RG-2 glioma cells in 5 μL of sterile PBS. In each location, the cells were injected over 8 minutes, using a 10 μL Hamilton syringe with a blunt tip 32-gauge needle for the brain inoculate and a sharp tip 26-gauage needle for the temporalis muscle inoculate. On experimental days 11 to 12, brain imaging of re-anesthetized rats was performed following placement of polyethylene femoral venous cannula (PE-50; Becton-Dickinson®, Franklin Lakes, NJ) for contrast agent infusion. Gd-dendrimers were infused at dose of 0.09 mmol Gd/kg.

In vivo magnetic resonance imaging of brain tumors

For imaging, the animal was positioned supine, with face, head, and neck snugly inserted into a nose cone centered within the 7 cm small animal solenoid radiofrequency coil. Anchored to the exterior of the nose cone were three 200 μL micro fuge tubes containing 0.00 mM, 0.25 mM and 0.50 mM solutions of Magnevist® to serve as standards for measurement of MRI signal drift over time. Coronal, sagittal and axial localizer scans were used in order to identify the coronal plane most perpendicular to the rat brain dorsum. After orienting the rat brain in the image volume, a fast spin echo T2 weighted anatomical scan was performed using a repetition time (TR) of approximately 6000 ms, an echo time (TE) of 70 ms and an image matrix of 256 by 256 with 45 contiguous 0.5 mm thick slices. In order to quantify

contrast agent concentration during post imaging processing, two separate three dimensional fast field echo Tl weighted (3D FFE TlW) scans were performed at different flip angles while holding TR to 8.1 ms, echo time TE to 2.3 ms, the image matrix to 256 by 256 and 32 over-contiguous 1 mm thick slices. The first 3D FFE TlW scan was performed with a low flip angle (FA) of 3° over 1.67 min without any contrast agent on board. The second dynamic 3D FFE TlW scan was performed with a high FA of 12°. Each brain volume was acquired once every 20 seconds. Since we wanted to accurately measure the contrast agent dynamics in blood during the bolus we infused all contrast agents as slow boluses over 1 minute. In order to guarantee the integrity of the Tl without contrast agent (TlO) map, at least three volumes were acquired before dispatch of contrast agent. After these baseline volumes were acquired, Gd-dendrimer was infused as a bolus over 1 minute and the brain imaged over one or two hours, depending on dendrimer generation and dosage. After acquiring all pertinent volumes with Gd-dendrimer on board, Gd-DTPA was bolus infused and additional images acquired for 15 minutes. Information from these images was used to identify maximal tumor margin for drawing tumor ROIs.

Three additional rats received a 0.09 mmol/kg bw dose of Gd-G5 dendrimer and were scanned multiple times to determine long term Gd-G5 dendrimer dynamics. Rats were positioned and imaged with a low FA scan as described above. After the low FA scan, a 15 minute high FA scan was performed in order to visualize Gd contrast to verify successful administration of the Gd-dendrimer. Five minute high FA scans were performed at 2, 6 and 12 hour time points. For data analyses, whole tumor ROIs were drawn independently on the high FA scan for each time point.

In vivo magnetic resonance imaging of rats with orthotopic and ectopic brain tumors For imaging, the animal was positioned supine, with face, head, and neck snugly inserted into a nose cone within the 7 cm small animal solenoid radiofrequency coil, which was then centered within the 3.0 tesla MRI scanner. Coronal, sagittal, and axial localizer scans were used in order to identify the coronal plane most perpendicular to the rat brain dorsum. After orienting the rat brain in the image volume, a fast spin echo T2 weighted anatomical scan was performed. Image acquisition parameters for the T2 scan were: TR) of 6000 ms, TE of 70 ms, image matrix of 256 by 256, and slice thickness of 1 mm. In order to quantify contrast agent concentration during post imaging processing, two separate three- dimensional fast field echo Tl weighted scans were performed, one at a 3° low flip angle (low FA) of and the other at a 12° high flip angle (high FA). Image acquisition parameters for

both scans were: TR of 8.1 ms, TE of 2.3 ms, image matrix of 256 by 256, and slice thickness of 1 mm. The low FA scan was performed over 1.67 min, without any Gd-dendrimer on board. For the high FA scans, which were the dynamic scans, the entire brain volume was acquired once every 20 seconds. At the beginning of the first high FA scan, three to five pre-contrast brain volumes were acquired to guarantee the integrity of the Tl map without contrast agent (TlO). Following acquisition of the pre-contrast brain volumes, a 0.09 mmol/kg dose of the respective Gd-dendrimer generation was infused. The Gd-dendrimer was infused as a slow bolus, over 1 minute, so that the blood pharmacokinetics of the respective Gd-dendrimer generation could be accurately measured, especially during the early time points. The initial series of high FA dynamic scans were acquired for 15 minutes and subsequent high FA dynamic scans were acquired over 2 minutes at various time points. For each of the imaging sessions to acquire the Gd signal intensity data for measurement of the change in blood and tumor tissue Gd concentration over 600 to 700 minutes, the rat brains of 2 to 3 rats were imaged as frequently as possible one after the other, once every 30 to 90 minutes. For each of subsequent high FA dynamic scan, the animal was re-anesthetized and re-imaged. For each of the Gd-dendrimer generations, one additional rat head was imaged every 10 min following the initial 15 minute dynamic scan, for a total of 175 minutes, while the animal was maintained under anesthesia for the duration of the scanning session. This was to image more frequently the change in Gd signal intensity and produce voxel-by- voxel Gd concentration maps.

DCE-MRI data analyses and pharmacokinetic modeling

Motion correction was performed by registering each volume of the dynamic high FA scan to its respective low FA scan. Alignments were performed using Fourier interpolation. A Tl without contrast (TlO) map was then generated. This was accomplished by solving equation 3 (the steady-state for incoherent signal after neglecting T2* effects) voxel -by- voxel for Tl at both low and high FA's before contrast was infused (19):

^ M ₀ (I -E ₁ )SJn* 1 - E ₁ COS * where

E, =exp( (4)

After determining the Tio value at each voxel, Ti was calculated using equation 3 for each voxel for each dynamic during the high FA scan after contrast infusion. Datasets were then converted to Gd concentration space by applying equation 2 voxel-by- voxel. Blood concentrations were obtained by selecting 2 to 3 voxels within the superior sagittal sinus that possessed peak concentrations that corresponded to the calculated volume of distribution based on the blood volume of a 250 gram rat being 14 mL (33). Blood concentration was converted to plasma concentration by correcting for the Hct as shown in equation 5 (23).

C ₀ = -^— (5) p 1 - Hct ^{V J}

The superior sagittal sinus is a large caliber brain vein that it is minimally influenced by in-flow and partial volume averaging effects. Since the transit time of blood movement between an artery and a vein within the brain is approximately 4 seconds, while the image acquisition rate is once every 20 seconds, the superior sagittal sinus may be used for generation of the vascular input function for pharmacokinetic modeling (17). Animal brains from which optimal vascular input function could not be obtained were excluded from being analyzed by pharmacokinetic modeling. Dendrimer generations Gd-Gl through LC Gd-G4 were also analyzed with a 2-comρartment 3-parameter generalized kinetic model, equation 6, by performing a voxel-by- voxel non-linear regression over all time points (23)

C _t (ή = v _p C _p (t) ₊ K ^« ™ |C _p (r)exp ^^ ^r r (6)

Constraints on the parameters were set between 0 and 1 calling on 10,000 iterations. Least squares minimizations were performed by implementing the Nelder-Mead simplex algorithm. Prior to statistical analysis, voxels with poor fits or non-physiologic parameters were censored.

Ex vivo fluorescence microscopy and histological staining of brain tumor sections Six additional rats received 0.06 mmol Gd/kg bw of RB Gd-G5 and two additional rats received 0.06 mmol Gd/kg bw of RB Gd-G8. The standard 2 hour DCE-MRI study was performed following which the brains of these animals were harvested and snap-frozen in OCT. On the day of cryosectioning two 10 μm sections of tumor bearing brain were cut onto each Daigger Superfrost slide with a Leica Cryotome (Leica, Bensheim, Germany). The first of two slides was prepared for fluorescence microscopy by application of DAPI- Vectashield nuclear stain medium and coversliping. Confocal imaging was performed on a Zeiss 510

NLO microscope. The second slide was stained for visualization of tumor histology by Haematoxylin and Eosin (H&E) stain.

Statistical analysis for pharmacokinetic modeling Values of each parameter were averaged across all tumor voxels, thus yielding one value per parameter per tumor per rat, with tumors within a rat being treated as correlated. For statistical analysis, on the basis of the distribution of tumor volumes in our study, a dichotomous variable for tumor size was generated by using 50 mm3 as the cut-off between large and small tumors. MANOVA models were utilized to examine the effect of dendrimer generation and tumor size. Prior to this MANOVA, we determined that there was no interaction between dendrimer generation and tumor size on any of the three parameters. The covariance structure was considered to be compound symmetric and the Kenward-Roger degrees of freedom method was used. Post-hoc comparisons between LC Gd-G4 and each of the other generations were conducted. The significant p-values we report are following Bonferroni correction for multiple comparisons. Analyses were implemented in SAS PROC Mixed (SAS Institute Inc., Cary, North Carolina) with α=0.05.

Post analyses of DCE-MRI data for orthotopic-ectopic RG-2 glioma experiments For each Gd-dendrimer generation, the average Gd concentrations obtained from the common carotid arteries, the orthotopic RG-2 glioma, and the ectopic RG-2 glioma were plotted over time using Matlab (Version 7.1; The MathWorks Inc, Natick, MA). The pharmacokinetics of Gd-dendrimers in blood were qualitatively assessed due to limited number of voxels available from the common carotid artery for analysis in the context of the known limitations of DCE-MRI-based acquisition of arterial input functions.

It was possible to quantify the pharmacokinetics of Gd-dendrimer generations in tumor tissues over 600 to 700 minutes. Best-fit curves were calculated using the Matlab Curve Fitting Toolbox (Version 1.1.4; The MathWorks Inc) using a two-term exponential function, as shown in equation 7).

[Gd] ₁ = ae ^bt + ce ^dt (7) where

[Gd] _t = predictive Gd concentration at time , min (mM) a (mM), b (min ^"1 ), c (mM), d (min ^"1 ) = parameters to be determined for best fit

The first term, ae ', represents the fast initial exponential rise in Gd concentration and the second term, ce ^dt , modeled the slow subsequent exponential decay in Gd concentration over time. The 95% confidence intervals (CI) and the root mean squared errors (RMSE, mM) of the best fit concentration curve parameters were calculated.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. References

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