Brain Tissue Distribution Research Articles

Study of the Tissue Distribution of TLQP-21 in Mice Using [18F]JMV5763, a Radiolabeled Analog Prepared via [18F]Aluminum Fluoride Chelation Chemistry.

TLQP-21 is a neuropeptide that is involved in the control of several physiological functions, including energy homeostasis. Since TLQP-21 could oppose the early phase of diet-induced obesity, it has raised a huge interest, but very little is known about its mechanisms of action on peripheral tissues. Our aim was to investigate TLQP-21 distribution in brain and peripheral tissues after systemic administration using positron emission tomography. We report here the radiolabeling of NODA-methyl phenylacetic acid (MPAA) functionalized JMV5763, a short analog of TLQP-21, with [18F]aluminum fluoride. Labeling of JMV5763 was initially performed manually, on a small scale, and then optimized and implemented on a fully automated radiosynthesis system. In the first experiment, mice were injected in the tail vein with [18F]JMV5763, and central and peripheral tissues were collected 13, 30, and 60 min after injection. Significant uptake of [18F]JMV5763 was found in stomach, intestine, kidney, liver, and adrenal gland. In the CNS, very low uptake values were measured in all tested areas, suggesting that the tracer does not efficiently cross the blood–brain barrier. Pretreatment with non-radioactive JMV5763 caused a significant reduction of tracer uptake only in stomach and intestine. In the second experiment, PET analysis was performed in vivo 10–120 min after i.v. [18F]JMV5763 administration. Results were consistent with those of the ex vivo determinations. PET images showed a progressive increase of [18F]JMV5763 uptake in intestine and stomach reaching a peak at 30 min, and decreasing at 120 min. Our results demonstrate that 18F-labeling of TLQP-21 analogs is a suitable method to study its distribution in the body.

Fetuin-A protein distribution in mature inflamed and ischemic brain tissue.

BackgroundThe liver-derived plasma protein fetuin-A is strongly expressed during fetal life, hence its name. Fetuin-A protein is normally present in most fetal organs and tissues, including brain tissue. Fetuin-A was neuroprotective in animal models of cerebral ischemia and lethal chronic inflammation, suggesting a role beyond the neonatal period. Little is known, however, on the presence of fetuin-A in mature human brain tissue under different physiological and pathological conditions.MethodsWe studied by immunohistochemistry (IHC) the distribution of fetuin-A protein in mature human brain autopsy tissues from patients without neurological disease, patients with inflammatory brain disorders, and patients with ischemic brain lesions. To identify fetuin-A-positive cells in these tissues we co-localized fetuin-A with GFAP (astrocytes) and CD68 (macrophages, activated microglia).Results and discussionUnlike previous reports, we detected fetuin-A protein also in mature human brain as would be expected from an abundant plasma protein also present in cerebrospinal fluid. Fetuin-A immunoreactivity was increased in ischemic white matter and decreased in inflamed cerebellar tissue. Fetuin-A immunostaining was predominantly associated with neurons and astrocytes. Unlike the developing brain, the adult brain lacked fetuin-A immunostaining in CD68-positive microglia. Our findings suggest a role for fetuin-A in tissue remodeling of neonatal brain, which becomes obsolete in the adult brain, but is re-activated in damaged brain tissue. To further assess the role of fetuin-A in the mature brain, animal models involving ischemia and inflammation need to be studied.

Gadolinium-based contrast agents induce gadolinium deposits in cerebral vessel walls, while the neuropil is not affected: an autopsy study

Recent studies showed gadolinium depositions following serial administrations of gadolinium-based contrast agents (GBCAs) for magnetic resonance imaging examinations in various parts of the brain with the dentate nucleus (DN) being most affected. Even though no clinical correlates of the deposits are known yet, an intensive debate developed if this might be harmful. The aim of the current study was to specify the gadolinium distribution in brain tissue of patients who received serial injections of GBCAs in the low-µm range and to explore any potential pathological tissue changes caused by gadolinium deposits. Thirteen autopsy cases-eight receiving GBCA administrations, five serving as controls-were identified and analyzed. For all patients, total gadolinium quantification after acidic digestion by means of inductively coupled plasma-mass spectrometry (ICP-MS) was performed. Six cases were utilized for the spatially resolved quantification of gadolinium within the cerebellum and the basal ganglia by means of high-resolution laser ablation (LA)-ICP-MS. Histopathological and immunohistochemical examinations were performed to determine tissue reactions. LA-ICP-MS revealed gadolinium depositions in the walls of small blood vessels of the DN in all GBCA exposed patients, while no gadolinium was found in the control group. Additionally, the detection of phosphorus and metals like copper, zinc and iron provides evidence that transmetalation reactions might have occurred. No significant pathological changes of the brain tissue in the vicinity of the DN with respect to micro-/astrogliosis and neuronal loss were found in any of the patients. This notably holds true even for a patient who died from nephrogenic systemic fibrosis exhibiting extremely high gadolinium concentrations within the DN. The findings show that gadolinium depositions in the brain are restricted to blood vessel walls, while the neuropil is spared and apparent cellular reactions are absent.

Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry of Drug Distribution in Mouse Brain Tissue by High-Resolution Time-of-Flight Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry provides the opportunity to visualize the distributions of drugs and metabolites in tissue specimens without requiring radioisotopes, as are used for whole-body autoradiography. However, the analysis of low-molecular-weight compounds is often difficult using the common reflectron-type MALDI time-of-flight mass spectrometers. Insufficient mass resolving power causes overlapping of the target drug peak with matrix compound or surface contaminant peaks. To solve this issue, we describe the procedure for imaging mass spectrometry using a high-mass-resolution mass spectrometer that can separate isobaric peaks.

Simultaneous Determination of Vinpocetine and its Major Active Metabolite Apovincaminic Acid in Rats by UPLC-MS/MS and its Application to the Brain Tissue Distribution Study.

A specific, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for simultaneous determination of vinpocetine (VP) and its active metabolite, apovincaminic acid (AVA) in rat brain regions, such as hypothalamus, striatum, cortex, cerebellum and hippocampus. Phenacetin was used as internal standard (IS). Brain tissue samples were precipitated protein by using 500 μL methanol. The separation was achieved on a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), using a methanol-water gradient elution at the flow rate of 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via positive electrospray ionization source (ESI). The quantification was operated using the transitions of m/z 351 → m/z 280 for VP, m/z 323 → m/z 280 for AVA and m/z 180 → m/z 110 for IS, respectively. The calibration curve was linear in concentration range from 0.100 to 60.0 ng/mL for VP and 0.103 to 6.18 ng/mL for AVA. The intra-day and inter-day precision (relative standard deviation, RSD) values were within 11.8%, the accuracy (relative error, RE) was from -1.7% to 3.0% for VP and 2.7% to 9.5% for AVA at all the three concentration levels of quality-control (QC) samples. The improved UPLC-MS/MS method was specific, rapid and sensitive, which was further successfully applied to simultaneous determination of VP and AVA in different rat brain regions after intragastric administration of 4 mg/kg VP. It was indicated that VP could be eliminated quickly in brain, while the elimination of AVA was slow and it could be maintained for more than 12 h in brain. Moreover, it was found that the contents of VP and AVA were much higher in the hypothalamus, striatum and cortex than those in the cerebellum and hippocampus, which verified the distribution characteristics of VP and AVA in different brain regions from the point of quantitation in rats.

Tissue distribution of araloside A in rats

Araloside A is one of the main active ingredients of Aralia taibaiensis. In this study, HPLC-MS/MS analysis method of araloside A in the main organs of SD rats was established. At the same time, the content of araloside A in the main organs (heart, liver, spleen, lung, kidney, brain) after oral administration with araloside A (50 mg•kg⁻¹) were determined to explore the tissue distribution characteristics of araloside A in vivo. The results showed that the methodological study of araloside A in the main organs of SD rats met the requirements, araloside A distributed in heart, liver, spleen, lung, kidney and brain tissues reached peak at 1 h or 2 h after oral administration with 50 mg•kg-1.The distributions of araloside A at different time points after administration were distinct as follows： the content of araloside A at 20 min：liver>heart>spleen>lung>kidney>brain; the content of araloside A at 1 h： liver>spleen>kidney>lung>heart>brain; the content of araloside A at 2 h： liver>kidney>heart>spleen>lung>brain; the content of araloside A at 4 h： kidney>liver>spleen>heart>lung>brain; the content of araloside A at 8 h： spleen>heart>liver>kidney>lung>brain. Therefore, araloside A was mainly distributed in liver tissue, which had a certain correlation with the common use of Aralia taibaiensis in the treatment of hepatic disease. In addition, araloside A shows a low content but an obvious distribution in brain tissues, which indicates that the drug can pass through blood-brain barrier, and provides the basis for the study of araloside A in brain tissue.

Abstract 5611: Use of ecto-domain vimentin to target brain cancers

Abstract Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. For brain cancers, immunohistological analysis showed the binding of pritumumab to EDV to be restricted to tumor cells and not normal cells and tissues suggesting EDV may be a useful biomarker. A recombinant version of the mAb was made using the GPEx® system in CHO cells that showed comparable binding activity by flow cytometry, immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC; EC50 of 39.6ng/ml) activity. Further studies include establishing xenograft models with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth of both brain cancer stem cells as well as pancreatic cancer in nude mice but not in SCID mice suggesting an active immune response is necessary for optimal treatment. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest that EDV is a suitable target and biomarker for brain cancers. Citation Format: Ivan Babic, Rajesh Mukthavaram, Pengfei Jiang, Eric Glassy, Natsuko Nomura, Sandeep Pingle, Mark C. Glassy, Santosh Kesari. Use of ecto-domain vimentin to target brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5611. doi:10.1158/1538-7445.AM2017-5611

Determination of tranilast in bio-samples by LC–MS/MS: Application to a pharmacokinetic and brain tissue distribution study in rats

As a potent drug used to improve the neurodegenerative conditions, there is few information about the brain tissue distribution of tranilast by now. In this study, a novel sensitive LC–MS/MS method has been developed and validated to determine tranilast in rat brain tissue samples. The calibration curve showed good linearity ranged from 2.140 to 428.0ng·mL−1. The method was fully validated and successfully applied in the brain tissue distribution study of tranilast in rats, which had never been reported in detail by now. Furthermore, a rapid LC–MS/MS method with a short run time of 3min was developed and validated for the determination of tranilast in rat plasma and the application to a pharmacokinetic study of tranilast in rats. After oral dosage of 10.5mg·kg−1 tranilast, the maximum plasma concentration (Cmax1) of tranilast was (18.59±5.40) μg·mL−1 at (0.667±0.408) h while the area under the curve (AUC0-24) was (54.87±14.13) μg·h·mL−1 with the elimination half-life of (2.93±0.41) h. The ratio calculated by dividing the concentration of tranilast in brain with the concentration of tranilast in the plasma, was (0.6042%±0.0572%), (0.7484%±0.0883%), (0.5914%±0.0416%) and (0.3830%±0.1632%) at 0.167, 0.5, 2 and 8h, respectively. The results showed that tranilast with fast absorption could penetrate the rat brain blood barrier after oral gavage. The obtained data also showed that tranilast could be quickly distributed and eliminated in brain tissue.

Pritumumab binding to glioma cells induces ADCC and inhibits tumor growth.

e14004 Background: Pritumumab is a natural human IgG1 kappa antibody originally isolated from a regional draining lymph node of a patient with cervical carcinoma. This antibody binds ectodomain vimentin on the surface of tumor cells and has demonstrated some benefit to glioblastoma patients in limited clinical trials. We wanted to determine if pritumumab inhibits glioma growth in vivo and if binding to glioma cells induces cell-mediated immunity. Methods: Pritumumab was used in flow cytometry experiments with several glioma cell lines and patient-derived neurosphere lines. Antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay was used with glioma target cells. Xenograft studies were performed in mice with and without intact B- and NK- cells. Results: We performed flow cytometry using pritumumab antibody and demonstrate binding of pritumumab to the surface of glioma cells and patient-derived glioma initiating cells. We observed significant induction of ADCC by pritumumab binding to glioma cells. Xenograft studies demonstrated pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Intact cell-mediated immunity was necessary for pritumumab’s anti-tumor effect. Analysis of a blood brain barrier model showed significant binding of pritumumab in brain tumor areas and minimal distribution in normal brain tissues suggesting the antibody can cross the blood brain barrier. Conclusions: Our data demonstratepritumumab binds glioma cells in vitro and can induce ADCC. In addition, pritumumab can limit the growth of xenograft glioma tumors in vivo only in the presence of intact cell-mediated immunity. Together these data suggest pritumumab is suitable for development as an anti-tumor therapeutic.

Targeting Nanomedicine to Brain Tumors: Latest Progress and Achievements

Targeting nanomedicine to brain tumors is hampered by the heterogeneity of brain tumors and the blood brain barrier. These represent the main reasons of unsuccessful treatments. Nanomedicine based approaches hold promise for improved brain tissue distribution of drugs and delivery of combination therapies. In this review, we describe the recent advancements and latest achievements in the use of nanocarriers, virus and cell-derived nanoparticles for targeted therapy of brain tumors. We provide successful examples of nanomedicine based approaches for direct targeting of receptors expressed in brain tumor cells or modulation of pathways involved in cell survival as well as approaches for indirect targeting of cells in the tumor stroma and immunotherapies. Although the field is at its infancy, clinical trials involving nanomedicine based approaches for brain tumors are ongoing and many others will start in the near future.

Analysis of hybridoma and CHO-derived pritumumab for therapeutic applications.

71 Background: Pritumumab, a natural huma IgG1 kappa antibody was obtained from a regional draining lymph node of a patient with cervical carcinoma through traditional hybridoma technology. Methods: Both cell lines and tissues were processed and analyzed for FACS analysis, Western blots, and immunohistochemistry. Results: Specificity analysis of the target antigen, an altered form of vimentin (ecto-domain vimentin (EDV)) shows it to be limited to cell surface expression on cancer cells. Clinically, 249 Japanese brain cancer patients were treated with a low dose pritumumab regimen, either at 1mg once a week or 1mg twice a week, and overall response rates of between 25-30% were seen with several complete and partial responses. A recombinant version of the mAb was made using the GPEx system in CHO cells. In a series of comparable studies the CHO mAb was compared with the original hybridoma. Binding specificity with both flow cytometry and immunohistochemical (IHC) analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) activity confirms the comparability of the two versions. IHC analysis suggests restricted binding to tumor cells and not normal cells and tissues. Further studies include establishing a xenograft model with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Conclusions: Overall, these data together suggest pritumumab is suitable for development as an anti-tumor therapeutic.

In vivo mapping of current density distribution in brain tissues during deep brain stimulation (DBS)

New methods for in vivo mapping of brain responses during deep brain stimulation (DBS) are indispensable to secure clinical applications. Assessment of current density distribution, induced by internally injected currents, may provide an alternative method for understanding the therapeutic effects of electrical stimulation. The current flow and pathway are affected by internal conductivity, and can be imaged using magnetic resonance-based conductivity imaging methods. Magnetic resonance electrical impedance tomography (MREIT) is an imaging method that can enable highly resolved mapping of electromagnetic tissue properties such as current density and conductivity of living tissues. In the current study, we experimentally imaged current density distribution of in vivo canine brains by applying MREIT to electrical stimulation. The current density maps of three canine brains were calculated from the measured magnetic flux density data. The absolute current density values of brain tissues, including gray matter, white matter, and cerebrospinal fluid were compared to assess the active regions during DBS. The resulting current density in different tissue types may provide useful information about current pathways and volume activation for adjusting surgical planning and understanding the therapeutic effects of DBS.

Abstract 2357: Therapeutic potential of the natural human IgG1k antibody pritumumab

Abstract Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. A recombinant version of the mAb was made using the GPEx® system in CHO cells. In a series of comparable studies the CHO mAb was compared with the original hybridoma. Binding specificity with both flow cytometry and immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) activity confirms the comparability of the two versions. A concentration-dependent study of pritumumab-mediated ADCC suggests an EC50 of 39.6ng/ml. Further studies include establishing a xenograft model with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest pritumumab is suitable for development as an anti-tumor therapeutic. Citation Format: Ivan Babic, Rajesh Mukthavaram, Natsuko Nomura, Sandeep Pingle, Mark C. Glassy, Santosh Kesari. Therapeutic potential of the natural human IgG1k antibody pritumumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2357.

Experimental evaluation of electrical conductivity imaging of anisotropic brain tissues using a combination of diffusion tensor imaging and magnetic resonance electrical impedance tomography

Anisotropy of biological tissues is a low-frequency phenomenon that is associated with the function and structure of cell membranes. Imaging of anisotropic conductivity has potential for the analysis of interactions between electromagnetic fields and biological systems, such as the prediction of current pathways in electrical stimulation therapy. To improve application to the clinical environment, precise approaches are required to understand the exact responses inside the human body subjected to the stimulated currents. In this study, we experimentally evaluate the anisotropic conductivity tensor distribution of canine brain tissues, using a recently developed diffusion tensor-magnetic resonance electrical impedance tomography method. At low frequency, electrical conductivity of the biological tissues can be expressed as a product of the mobility and concentration of ions in the extracellular space. From diffusion tensor images of the brain, we can obtain directional information on diffusive movements of water molecules, which correspond to the mobility of ions. The position dependent scale factor, which provides information on ion concentration, was successfully calculated from the magnetic flux density, to obtain the equivalent conductivity tensor. By combining the information from both techniques, we can finally reconstruct the anisotropic conductivity tensor images of brain tissues. The reconstructed conductivity images better demonstrate the enhanced signal intensity in strongly anisotropic brain regions, compared with those resulting from previous methods using a global scale factor.

Brain MRI image segmentation based on learning local variational Gaussian mixture models

Measuring the distribution of major brain tissues, including the gray matter, white matter and cerebrospinal fluid (CSF), using magnetic resonance imaging (MRI) has attracted extensive research efforts. Many brain MRI image segmentation methods in the literature are based on the Gaussian mixture model (GMM), which however is not strictly followed due to the intrinsic complex nature of MRI data and may lead to less accurate results. In this paper, we introduce the variational Bayes inference to brain MRI image segmentation, and thus propose a novel segmentation algorithm based on learning a cohort of local variational Gaussian mixture (LVGM) models. By assuming all Gaussian parameters to be random variables, the LVGM model has more flexibility than GMM in characterizing the complexity of brain voxel distributions. To alleviate the impact of bias field, we train each LVGM model on a sampled small data volume and linearly combine the trained models to classify each brain voxel. We also construct a co-registered probabilistic brain atlas for each MRI image to incorporate the prior knowledge about brain anatomy into the segmentation process. The proposed LVGM learning algorithm has been evaluated against five state-of-the-art brain MRI image segmentation methods on both synthetic and clinical data. Our results suggest that the LVGM algorithm can segment brain MRI images more effectively and provide more precise distribution of major brain tissues.

Cellular Microbiology of Mycoplasma canis.

Mycoplasma canis can infect many mammalian hosts but is best known as a commensal or opportunistic pathogen of dogs. The unexpected presence of M. canis in brains of dogs with idiopathic meningoencephalitis prompted new in vitro studies to help fill the void of basic knowledge about the organism's candidate virulence factors, the host responses that it elicits, and its potential roles in pathogenesis. Secretion of reactive oxygen species and sialidase varied quantitatively (P < 0.01) among strains of M. canis isolated from canine brain tissue or mucosal surfaces. All strains colonized the surface of canine MDCK epithelial and DH82 histiocyte cells and murine C8-D1A astrocytes. Transit through MDCK and DH82 cells was demonstrated by gentamicin protection assays and three-dimensional immunofluorescence imaging. Strains further varied (P < 0.01) in the extents to which they influenced the secretion of tumor necrosis factor alpha (TNF-α) and the neuroendocrine regulatory peptide endothelin-1 by DH82 cells. Inoculation with M. canis also decreased major histocompatibility complex class II (MHC-II) antigen expression by DH82 cells (P < 0.01), while secretion of gamma interferon (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and complement factor H was unaffected. The basis for differences in the responses elicited by these strains was not obvious in their genome sequences. No acute cytopathic effects on any homogeneous cell line, or consistent patterns of M. canis polyvalent antigen distribution in canine meningoencephalitis case brain tissues, were apparent. Thus, while it is not likely a primary neuropathogen, M. canis has the capacity to influence meningoencephalitis through complex interactions within the multicellular and neurochemical in vivo milieu.

Abstract IA27: Advances in oligonucleotide chemistry for the treatment of neurodegenerative disorders and brain tumors

Abstract RNA interference has revolutionized human functional genomics and therapeutics, but its impact on the development of new therapies for cancer has been surprisingly limited, due mainly to the lack of simple and efficient methods to deliver oligonucleotides to tumors, especially those located in the brain. Our research shows that many cell types, including tumor, glial and neuronal, rapidly internalize fully stabilized, hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium and that hsiRNAs induce potent and long-lasting silencing in vitro. A single injection of unformulated hsiRNA (cholesterol-conjugated) into mouse brain silences locally with great potency and longevity in primary tumor, glial and neuronal cells. Limited distribution from the site of administration interferes with addressing metastasis, thus limiting the clinical utility to treatment of primary tumors only. Using fully chemically modified siRNA scaffolds, we systematically screened a wide range of bioactive conjugates and demonstrated that the chemical nature of the conjugation modality has a major impact on brain tissue retention, distribution and selectivity of cellular uptake. We have identified several novel chemical classes of conjugates that demonstrate markedly improved brain distribution and robust in vivo efficacy, with some showing selective tumor, glia or neuronal uptake. Direct conjugation of a fully chemically modified siRNA to docosahexaenoic acid (DHA), the most abundant poly-unsaturated fatty acid in the brain, results in improved tissue retention with wide distribution and robust efficacy in the striatum and cortex after a single injection. Most importantly, DHA-hsiRNA conjugates do not induce neural cell death or measurable innate immune activation following the administration of concentrations 20-fold over the efficacious dose. Interestingly, variants of DHA-based conjugates support preferential delivery to glia cells, establishing a new approach toward targeted development of RNAi-based therapeutics for glioblastomas and other glia-associated cancers. Citation Format: Anastasia Khvorova. Advances in oligonucleotide chemistry for the treatment of neurodegenerative disorders and brain tumors. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA27.

Internalization of targeted quantum dots by brain capillary endothelial cells in vivo

Receptors located on brain capillary endothelial cells forming the blood–brain barrier are the target of most brain drug delivery approaches. Yet, direct subcellular evidence of vectorized transport of nanoformulations into the brain is lacking. To resolve this question, quantum dots were conjugated to monoclonal antibodies (Ri7) targeting the murine transferrin receptor. Specific transferrin receptor-mediated endocytosis of Ri7-quantum dots was first confirmed in N2A and bEnd5 cells. After intravenous injection in mice, Ri7-quantum dots exhibited a fourfold higher volume of distribution in brain tissues, compared to controls. Immunofluorescence analysis showed that Ri7-quantum dots were sequestered throughout the cerebral vasculature 30 min, 1 h, and 4 h post injection, with a decline of signal intensity after 24 h. Transmission electron microscopic studies confirmed that Ri7-quantum dots were massively internalized by brain capillary endothelial cells, averaging 37 ± 4 Ri7-quantum dots/cell 1 h after injection. Most quantum dots within brain capillary endothelial cells were observed in small vesicles (58%), with a smaller proportion detected in tubular structures or in multivesicular bodies. Parenchymal penetration of Ri7-quantum dots was extremely low and comparable to control IgG. Our results show that systemically administered Ri7-quantum dots complexes undergo extensive endocytosis by brain capillary endothelial cells and open the door for novel therapeutic approaches based on brain endothelial cell drug delivery.

Mapping drug distribution in brain tissue using liquid extraction surface analysis mass spectrometry imaging.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a surface sampling technique that incorporates liquid extraction from the surface of tissue sections with nanoelectrospray mass spectrometry. Traditional tissue analysis techniques usually require homogenization of the sample prior to analysis via high-performance liquid chromatography mass spectrometry (HPLC-MS), but an intrinsic weakness of this is a loss of all spatial information and the inability of the technique to distinguish between actual tissue penetration and response caused by residual blood contamination. LESA-MS, in contrast, has the ability to spatially resolve drug distributions and has historically been used to profile discrete spots on the surface of tissue sections. Here, we use the technique as a mass spectrometry imaging (MSI) tool, extracting points at 1 mm spatial resolution across tissue sections to build an image of xenobiotic and endogenous compound distribution to assess drug blood-brain barrier penetration into brain tissue. A selection of penetrant and "nonpenetrant" drugs were dosed to rats via oral and intravenous administration. Whole brains were snap-frozen at necropsy and were subsequently sectioned prior to analysis by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and LESA-MSI. MALDI-MSI, as expected, was shown to effectively map the distribution of brain penetrative compounds but lacked sufficient sensitivity when compounds were marginally penetrative. LESA-MSI was used to effectively map the distribution of these poorly penetrative compounds, highlighting its value as a complementary technique to MALDI-MSI. The technique also showed benefits when compared to traditional homogenization, particularly for drugs that were considered nonpenetrant by homogenization but were shown to have a measurable penetration using LESA-MSI.

Brain Targeting of a Water Insoluble Antipsychotic Drug Haloperidol via the Intranasal Route Using PAMAM Dendrimer.

Delivery of therapeutics to the brain is challenging because many organic molecules have inadequate aqueous solubility and limited bioavailability. We investigated the efficiency of a dendrimer-based formulation of a poorly aqueous soluble drug, haloperidol, in targeting the brain via intranasal and intraperitoneal administration. Aqueous solubility of haloperidol was increased by more than 100-fold in the developed formulation. Formulation was assessed via different routes of administration for behavioral (cataleptic and locomotor) responses, and for haloperidol distribution in plasma and brain tissues. Dendrimer-based formulation showed significantly higher distribution of haloperidol in the brain and plasma compared to a control formulation of haloperidol administered via intraperitoneal injection. Additionally, 6.7 times lower doses of the dendrimer-haloperidol formulation administered via the intranasal route produced behavioral responses that were comparable to those induced by haloperidol formulations administered via intraperitoneal injection. This study demonstrates the potential of dendrimer in improving the delivery of water insoluble drugs to brain.