WO2009142754A1 - Conjugués dendritiques et procédés associés - Google Patents

Conjugués dendritiques et procédés associés Download PDF

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Publication number
WO2009142754A1
WO2009142754A1 PCT/US2009/003164 US2009003164W WO2009142754A1 WO 2009142754 A1 WO2009142754 A1 WO 2009142754A1 US 2009003164 W US2009003164 W US 2009003164W WO 2009142754 A1 WO2009142754 A1 WO 2009142754A1
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tumor
dendrimer
dendrimers
brain
cancer
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PCT/US2009/003164
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WO2009142754A4 (fr
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Hemant Sarin
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Goverment Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2009142754A1 publication Critical patent/WO2009142754A1/fr
Publication of WO2009142754A4 publication Critical patent/WO2009142754A4/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/124—Macromolecular compounds dendrimers, dendrons, hyperbranched compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the blood-brain barrier of micro vasculature within the diseased brain and spinal cord tissue becomes porous.
  • primary malignant brain tumors such as glioblastoma multiforme (GBM, malignant glioma)
  • GBM glioblastoma multiforme
  • 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.
  • chemotherapeutics such as doxorubicin and carboplatin
  • doxorubicin and carboplatin are small compounds having molecular weights less than 1 kilodalton (kDa), and therefore, diameters less than lnm.
  • kDa kilodalton
  • these drugs are unable to accumulate within malignant glioma cells at therapeutic levels due to the relatively short peak blood half- lives of minutes.
  • the sizes of presently used nanoparticle-based therapeutics are typically between 50 and 150 nm in diameter.
  • 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.
  • 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.
  • PAMAM polyamidoamine
  • BTB blood-tumor barrier
  • the invention provides a biocompatible nanoparticle conjugated to an agent, wherein the nanoparticle comprises a diameter between 1.5 nanometers and 13 nanometers.
  • the dendritic conjugate has a diameter between about 5 nanometers and 12 nanometers.
  • 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.
  • biocompatible nanoparticles of the invention include polyamidoamine (PAMAM) dendrimers.
  • 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.
  • 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.
  • 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.
  • 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.
  • DTPA diethylene triamine pentaacetic acid
  • DOTA 1,4,7,10- tetraazacyclododecane-NjN', N",N'"-tetraacetic acid
  • porphyrin porphyrin
  • desferrioxamine or derivatives thereof.
  • 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, chemotherapeutic agents such as acycl
  • the invention further provides uses for the nanoparticle compositions of the instant invention.
  • 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.
  • 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.
  • 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.
  • MS multiple sclerosis
  • ALS amyotrophic lateral sclerosis
  • Alzheimer's disease and ischemic disease
  • the invention provides for various routes of administration including, but not limited to intravenous, topical, intra-arterial, intrathecal, intratumoral, or intracranial delivery.
  • 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.
  • the medicament is prepared in a pharmaceutically acceptable carrier.
  • 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.
  • the normal cell is a brain cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the blood half-life in vivo is increased to about 200 minutes, about 300 minutes, or about 400 minutes.
  • the agent is released slowly from the dendrimer conjugate over time at physiological pH and isotonic conditions.
  • the agent is released more rapidly at the acidic pH in tumor cell organelles and isotonic conditions.
  • 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.
  • 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.
  • Gd-G4 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.
  • FIG. 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.
  • 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.
  • 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.
  • FIG. 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.
  • average concentrations and error bars 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 ⁇ *TM 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 TM*. 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.
  • Tumor volumes, in mm 3 , for tumors shown are 104 (Gd-Gl) and 162 (LC Gd-G4).
  • 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).
  • 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.
  • FIG. 7 shows that intravenous RB labeled Gd-G5 dendrimers accumulate in the extra- vascular tumor space of RG-2 gliomas.
  • 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
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • FIG 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.
  • Gd-G6 dendrimers (Orthotopic RG-2 glioma tumor volume, 97 mm 3 ; ectopic RG-2 glioma tumor volume, 184 mm 3 ).
  • Gd-G7 dendrimers (Orthotopic RG-2 glioma tumor volume, 53 mm 3 ; ectopic RG-2 glioma tumor volume, 135 mm 3 ).
  • 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.
  • Error bars represent standard deviation.
  • Figure 13 panels a-d show blood pharmacokinetics of Gd-dendrimer generations.
  • 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.
  • 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.
  • 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.
  • pH 5.5 which is within the pH range in the lysosomes of tumor cells, the doxorubicin hydrolyzed more rapidly over 3 hours.
  • 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.
  • 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.
  • DOX free doxorubicin
  • 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.
  • 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).
  • BBTB blood-brain tumor barrier
  • metastases brain tumor ectopically implanted
  • 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.
  • 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.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • 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.
  • 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.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • “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.
  • 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.
  • preferred routes of administration include parenteral administration, preferably, for example by injection, for example by intravenous injection.
  • 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.
  • 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.
  • 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.
  • 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.
  • PAMAM dendrimer refers to a dendrimer having polyamidoamine branches.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • Amelioration and treatment can require the administration of more than one dose of an agent or therapeutic.
  • biocompatible is understood as acceptable for administration to a subject, particularly a human subject.
  • biocompatible compositions are composed of materials that are categorized as generally recognized as safe (GRAS).
  • GRAS generally recognized as safe
  • 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.
  • 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, ⁇ -galactosidase or luciferase).
  • 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.
  • 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.
  • conjugated is understood as attached, linked, mixed, or otherwise present on or in a nanoparticle.
  • 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.
  • conjugation of an agent to a nanoparticle does not disrupt the desired activity of the agent.
  • detecting 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.
  • diagnosing 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.
  • diagnosing using the method of the invention includes the observation of the subject for other signs or symptoms of the disease, disorder, or condition.
  • 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.
  • side-effects 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.
  • 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.
  • 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.
  • hypoproliferative disorder or “neoplasia” includes malignancies characterized by excess cell proliferation or growth, or reduced cell death.
  • cancer includes but is not limited to carcinomas, sarcomas, leukemias, and lymphomas.
  • 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
  • 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.
  • 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).
  • an agent can be complexed, conjugated, or otherwise
  • 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.
  • 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.
  • the drug loaded in nanoparticles is usually released from the matrix by diffusion, swelling, erosion, or degradation.
  • 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.
  • pharmaceutically acceptable carrier 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.
  • 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.
  • 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'
  • wetting agents 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.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulf ⁇ te, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulf ⁇ te, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
  • 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.
  • “plurality” is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, or more.
  • 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.
  • 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 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.
  • biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain agents.
  • 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.
  • 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.
  • PAMAM polyamidoamine
  • 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.
  • dendritic polymer 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.
  • 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.
  • 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).
  • 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.
  • the dendritic conjugate has a diameter between 1.5 nanometers and 14 nanometers.
  • 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.
  • BBTB blood-brain tumor barrier
  • BTB blood-tumor barrier
  • the target area for treatment is a brain tumor or diseased neurological tissue
  • 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
  • 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.
  • the dendritic conjugate has a molecular weight greater than 10 kDa. In other certain examples, the dendritic conjugate has a molecular weight greater than
  • 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.
  • the molecular weight of the dendrimer complexes of the invention is largely limited by the allowable size of the dendrimer complexes.
  • 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.
  • 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.
  • 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.
  • 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.
  • the therapeutic agent is a radiotherapeutic agent, and can be selected from, but is not limited to radioactive gadolinium, radioactive boron, and radioactive iodine.
  • 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
  • drugs such as
  • 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 monoclon
  • 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.
  • 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.
  • 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.
  • PAMAM polyamidoamine
  • 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.
  • PAMAM polyamidoamine
  • Barrier permeability is determined by tumor type, grade, location, and the number of pores.
  • the blood-tumor barrier (BTB) of peripheral tumors has a greater number of pores
  • 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.
  • DCE-MRI Dynamic contrast-enhanced MRI
  • Gd-DTPA or Gd-DTPA conjugated PAMAM dendrimer a non-radioactive contrast agent
  • 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.
  • LV. intravenously
  • LA. intraarterially
  • the invention is particularly useful for imaging, for example in imaging a malignant ectopic or orthotopic brain tumor.
  • 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.
  • PAMAM polyamidoamine
  • the methods of imaging as described herein can be particularly useful in diagnosing or monitoring a malignant brain tumor in a subject.
  • 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.
  • 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.
  • 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).
  • a series of images obtained at various points in time from administration to a desired time after administration 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.
  • 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.
  • 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.
  • PAMAM polyamidoamine
  • 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.
  • 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.
  • 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.
  • the disease state or treatment of a patient having a disease or disorder can be monitored using the methods and compositions of the invention, alone or in addition to standard monitoring techniques.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Gd-G5 and Gd-G6 dendrimers were longer than those of Gd- G7 and Gd-G8 dendrimers (Fig. 5).
  • 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.
  • 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.
  • 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.
  • the physiologic upper limit of pore size in the BTB of malignant solid tumor micro vasculature is approximately 12 nm.
  • 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).
  • BBTB pathologic blood-brain tumor barrier
  • BBB pathologic blood-brain tumor barrier
  • 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).
  • 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.
  • 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.
  • STEM scanning transmission electron microscopy
  • ADF annular dark-field
  • Example 3 Blood concentration of permeable Gd-dendrimers determines degree of particle accumulation within brain tumor tissue
  • Gd-dendrimer Gl through G8 were studied at two different Gd doses using DCE-MRI.
  • rat brain was imaged at 20 s intervals for one or two hours (Figs. 2, 3).
  • 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).
  • additional imaging at a Gd-dendrimer dose of 0.09 mmol Gd/kg bw was performed.
  • 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.
  • 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).
  • 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.
  • 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 rum. Gd-G8 dendrimers remain intravascular due to particle sizes being greater than the physiologic pore size of the smaller tumor BBTB.
  • Gd-G5 dendrimers 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
  • 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.
  • 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 ⁇ M 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).
  • 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.
  • 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.
  • results presented herein 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • the peak Gd concentration of Gd-G5 dendrimers in orthotopic tumors was 0.147 mM
  • the peak Gd concentration of Gd-G5 dendrimers in ectopic tumors was 0.195 mM (Table 5).
  • Gd-PAMAM Peak Peak Peak Peak dendrimer concentration concentration concentration generation orthotopic RG-2 time point (min) ectopic RG-2 time point (min) gliomas (mM) gliomas (mM)
  • 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.
  • 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.
  • the peak Gd concentration of Gd-G6 dendrimers in orthotopic tumors was 0.106 mM
  • 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.
  • 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.
  • the peak Gd concentration of Gd-G7 dendrimers in orthotopic tumors was 0.064 mM
  • 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
  • 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.
  • 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.
  • the total dose of doxorubicin administered was 8 mg/kg bw for each group.
  • 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 .
  • 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.
  • Gd-G5-DOX dendrimer conjugates 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.
  • the invention was performed using, but not limited to, the following methods:
  • 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).
  • a 5 ⁇ L 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.
  • 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).
  • DMEM Dulbecco Modified Eagle Medium
  • FBS fetal bovine serum
  • 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).
  • AFNI Functional Neurolmaging
  • 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 were prepared by using a molar reactant ratio of > 2:1 bifunctional chelate to dendrimer surface amine groups.
  • 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 %).
  • 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, Arlington, AZ).
  • ICP-AES inductively coupled plasma-atomic emission spectroscopy
  • 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.
  • 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 ⁇ L 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).
  • 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 ⁇ M 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 ⁇ L DAPI-Vectashield® nuclear stain medium
  • 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).
  • Tl longitudinal relaxivity
  • MO equilibrium magnetization
  • the right anterior caudate and left posterior thalamus locations within the brain were stereotactically inoculated with RG-2 glioma cells (32).
  • RG-2 glioma cells 302.
  • either 20,000 or 100,000 glioma cells in 5 ⁇ L of sterile PBS were injected over 8 minutes, using a 10 ⁇ L Hamilton® syringe with a 32-gauge needle.
  • 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 ⁇ L of blood was withdrawn from the distal end of the cannula for measurement of hematocrit (H ct).
  • PE-50 polyethylene femoral venous and arterial cannulas
  • heparinized normal saline 10 u heparin sodium/1 ml saline
  • 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.
  • 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.
  • the anesthesia and route for all animal experiments was isoflurane by inhalation with nose cone, 5% for induction and 1 to 2% for maintenance.
  • 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 ⁇ L of sterile PBS.
  • the cells were injected over 8 minutes, using a 10 ⁇ L 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.
  • 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.
  • 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 ⁇ L 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.
  • 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.
  • TR repetition time
  • TE echo time
  • image matrix 256 by 256 with 45 contiguous 0.5 mm thick slices.
  • 3D FFE TlW three dimensional fast field echo Tl weighted
  • 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.
  • TlO contrast agent
  • at least three volumes were acquired before dispatch of contrast agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • the rat brains of 2 to 3 rats were imaged as frequently as possible one after the other, once every 30 to 90 minutes.
  • the animal was re-anesthetized and re-imaged.
  • 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.
  • 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-com ⁇ artment 3-parameter generalized kinetic model, equation 6, by performing a voxel-by- voxel non-linear regression over all time points (23)
  • 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.
  • 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.

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Abstract

La présente invention concerne des nanoparticules biocompatibles conjuguées à un agent destinées à être utilisées dans des procédés diagnostiques et thérapeutiques. Dans un mode de réalisation, l’invention concerne des conjugués dendritiques comprenant un dendrimère conjugué à un agent. Dans des modes de réalisation préférés, les conjugués dendritiques comprennent des dendrimères PAMAM de générations 3,5, 4, 4,5, 5, 5,5, 6, 6,5, 7, et 7,5. Les conjugués dendritiques sont administrés à travers la barrière hémato-encéphalique et sont utilisés dans des méthodes de traitement des tumeurs cérébrales, dans des méthodes de traitement de maladies neurologiques, en imagerie et pour le diagnostic. L’invention concerne également des kits comprenant les conjugués dendritiques.
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