Paul R. Lockman

In vivo and in vitro assessment of baseline blood-brain barrier parameters in the presence of novel nanoparticles.

AbstractPurpose. Nanoparticles have advantage as CNS drug delivery vehicles given they disguise drug permeation limiting characteristics. Conflicting toxicological data, however, is published with regard to blood-brain barrier integrity and gross mortality. Methods. To address this issue two novel nanoparticle types: “emulsifying wax/Brij 78”and Brij 72/Tween 80 nanoparticles were evaluated in vivo for effect on cerebral perfusion flow, barrier integrity, and permeability using the in situ brain perfusion technique. Additional evaluation was completed in vitro using bovine brain microvessel endothelial cells for effect on integrity, permeability, cationic transport interactions, and tight junction protein expression. Results. In the presence of either nanoparticle formulation, no overall significant differences were observed for cerebral perfusion flow in vivo. Furthermore, observed in vitro and in vivo data showed no statistical changes in barrier integrity, membrane permeability, or facilitated choline transport. Western blot analyses of occludin and claudin-1 confirmed no protein expression changes with incubation of either nanoparticle. Conclusions. The nanoparticle formulations appear to have no effect on primary BBB parameters in established in vitro and in vivo blood-brain barrier models.

Evaluation of blood-brain barrier thiamine efflux using the in situ rat brain perfusion method.

Thiamine is an essential, positively charged (under physiologic conditions), water‐soluble vitamin requiring transport into brain. Brain thiamine deficiency has been linked to neurodegenerative disease by subsequent impairment of thiamine‐dependent enzymes used in brain glucose/energy metabolism. In this report, we evaluate brain uptake and efflux of [3H]thiamine using the in situ rat brain perfusion technique. To confirm brain distribution was not related to blood–brain barrier endothelial cell uptake, we compared parenchymal and cell distribution of [3H]thiamine using capillary depletion. Our work supports previous literature findings suggesting blood–brain barrier thiamine uptake is via a carrier‐mediated transport mechanism, yet extends the literature by redefining the kinetics with more sensitive methodology. Significantly, [3H]thiamine brain accumulation was influenced by a considerable efflux rate. Evaluation of the efflux mechanism demonstrated increased stimulation by the presence of increased vascular thiamine. The influx transport mechanism and efflux rate were each comparable throughout brain regions despite documented differences in glucose and thiamine metabolism. The observation that [3H]thiamine blood–brain barrier influx and efflux is regionally homogenous may have significant relevance to neurodegenerative disease linked to thiamine deficiency.

Cation transport specificity at the blood-brain barrier

The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. Method: Cross-inhibition of [3H]choline and [3H]thiamine brain uptake by in situ rat brain perfusion. Results: [3H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 μM inhibited [3H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. Conclusion: (1) The molecular family of the blood–brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.

bis-Azaaromatic quaternary ammonium salts as ligands for the blood-brain barrier choline transporter

A series of bis-azaaromatic quaternary ammonium compounds containing flexible polymethylenic linkers as well as conformationally restricted linkers were evaluated for their affinity for the blood-brain barrier choline transporter (BBB-ChT). The preliminary structure-activity relationships obtained from this study suggest that incorporating a linear, conformationally restricted linker into the molecule improves affinity for the BBB-ChT.

Novel choline transport characteristics in Caco-2 cells.

ABSTRACT Choline transport is characterized by sodium-dependent high-affinity, sodium-independent low-affinity, and sodium-independent blood–brain barrier transport mechanisms. Each defined mechanism has specific characteristics with regard to affinity for choline, transport capacity, and inhibition by hemicholinium. The purpose of this study is to determine the characteristics of choline transport across Caco-2 monolayers. Methods. Choline transport across Caco-2 cell monolayers was determined in both the apical to basal direction and the opposite direction. Further, the determination of calcium dependence and specific inhibitors was made. Determination of the apparent permeability of choline was calculated by established methods. Results. The apical to basal Caco-2 permeability coefficient is 11.11 ± 0.33 × 10−6 cm/sec with 21.3% of the choline associating with the cells. Meanwhile the basal to apical value is approximately 50% less (5.55 ± 0.14 × 10−6 cm/sec), suggesting an active apical to basal transport mechanism. Choline transport in this system was inhibited by nifedipine (82%), verapamil (80%), EGTA (36%), and cyclosporin (15%). Conclusions. Choline transport across Caco-2 cells is demonstrated to be active and both pH- and Ca2+-dependent. Furthermore, choline transport across Caco-2 monolayers has unique characteristics when compared to traditional choline transport models.

Quantitative fluorescence microscopy provides high resolution imaging of passive diffusion and P-gp mediated efflux at the in vivo blood–brain barrier

Quantitative fluorescent microscopy is an emerging technology that has provided significant insight into cellular dye accumulation, organelle function, and tissue physiology. However, historically dyes have only been used to qualitatively or semi-quantitatively (fold change) determine changes in blood-brain barrier (BBB) integrity. Herein, we present a novel method to calculate the blood to brain transfer rates of the dyes rhodamine 123 and Texas red across the in situ BBB. We observed that rhodamine 123 is subject to p-glycoprotein mediated efflux at the rat BBB and can be increased nearly 20-fold with p-glycoprotein inhibition. However, Texas Red appears to not be subject to MRP2 mediated efflux at the rat BBB, agreeing with literature reports suggesting MRP2 may lack functionality at the normal rat BBB. Lastly, we present data demonstrating that once dyes have crossed the BBB, diffusion of the dye molecule is not as instantaneous as has been previously suggested. We propose that future work can now be completed to (1) match BBB transfer coefficients to interstitial diffusion constants and (2) use dyes with specific affinities to cellular organelles or that have specific properties (e.g., subject to efflux transporters) to more fully understand BBB physiology.

Inhibition of choline uptake by N-cyclohexylcholine, a high affinity ligand for the choline transporter at the blood-brain barrier

The blood–brain barrier (BBB) choline transporter (CHT) may have utility as a drug delivery vector for drugs that act in the central nervous system. Previous studies suggested the importance of hydrophobic moieties on the cationic nitrogen of choline for improved affinity for this transporter. In a pilot study, we therefore designed five novel N-cycloalkyl derivatives of choline, one of which showed promising inhibition properties. This choline analogue had a cyclohexyl (UMBB-5) moiety substituting one of the methyl groups attached to the cationic nitrogen in choline. In situ experimental data were obtained from in situ rat brain perfusion studies. The binding affinity for the BBB-choline transporter found for UMBB-5 was Ki = 1.9 µM. Comparative molecular field analysis (CoMFA) suggested that the cyclohexyl moiety orientates towards a steric favourable area. Taken together, the results of these in situ and in silico studies provide further evidence or restrictions that occur with binding to this brain drug delivery vector.

Abstract 642: Transcend, a protein vector for brain delivery, allows trastuzumab to reach the brain at effective concentration after incorporation to form BT2111

Trastuzumab, a monoclonal antibody against HER2, is an effective therapy for the treatment of peripheral HER2+ breast cancer. However a significant number of patients eventually succumb to metastases of the brain because the endothelial tight junctions of the blood-brain barrier prevent distribution of trastuzumab to the metastatic tumors. Improving distribution of trastuzumab to HER2+ brain tumors represents a major hurdle in the treatment of metastatic HER2+ breast cancer. Previous studies have shown that doxorubicin conjugated to melanotransferrin (MTf) increased significantly the survival of mice bearing intracranial tumors, while doxorubicin alone was not effective. In this study we examine the potential of MTf-trastuzumab conjugates (BT2111) to penetrate the blood-brain barrier. BT2111 and trastuzumab were labeled with fluorescent markers, either Cy5.5 or rhodamine. The cellular uptakes of these labeled proteins were studied with fluorescence microscopy in cultures of primary human brain endothelial cells (HBEC) and in HER2+ (A172, SKBR3, and BT474), and HER2- (MDA-MB-468) breast cancer cell lines. The fluorescent conjugates were also injected IV into mice and the distribution of BT2111 and trastuzumab to endothelial cells and brain parenchyma was compared with laser scanning confocal microscopy. To aid in the co-localization of BT2111 and trastuzumab to the brain vasculature, mice were injected with tomato lectin-FITC or anti CD31 IgG-FITC prior to sacrifice. The results showed that primary human brain endothelial cells (HBEC) took up BT2111 suggesting that BT2111 can interact with the MTf receptor and transport BT2111 into the intracellular compartment. In contrast, no specific staining associated with trastuzumab was observed, either on the surface or in the interior of HBECs. Uptake of both BT2111 and trastuzumab was observed on the surface of A172, SKBR3, and BT474 cells but not on MDA-MB-468 cells confirming that BT2111 retains HER2 binding activity. When BT2111 and trastruzumab were injected into mice, laser scanning confocal microscopy showed increased distribution of BT2111 in the brain parenchyma when compared to trastuzumab. 10 to 15 times more trastuzumab was delivered in the brain parenchyma when conjugated to MTf. Using a mice model characterized by the formation of brain metastasis after intracardiac administration of MDA-MB 231BR we show that BT2111 is homogenously distributed in normal brain and can reach therapeutical concentration in the brain using 10% of the predicted therapeutic IV dose when trastuzumab was administered as BT2111. These results indicate that BT2111 has the potential to become a new anti-cancer agent for treatment of HER2+brain metastases. Citation Format: Reinhard Gabathuler, Timothy Z. Vitalis, Mohamed I. Nounou, Chris E. Adkins, Paul R. Lockman, Wilfred A. Jefferies. Transcend, a protein vector for brain delivery, allows trastuzumab to reach the brain at effective concentration after incorporation to form BT2111. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 642. doi:10.1158/1538-7445.AM2014-642

Abstract 2373: Heterogeneity of permeability in a preclinical model of brain metastases of breast cancer does not correlate with vascular density, HIF-1α expression, or tumor survival

Introduction: High vascular density of brain metastases of breast cancer has been correlated with both poor patient prognosis and with potential chemotherapeutic success. Inversely a low vascular density implies there would be decreased distribution of anti-cancer chemotherapeutics, and over time reduced efficacy of tumor kill. Given that there is heterogeneity in response to chemotherapeutics in human brain metastases and metastases are different than primary tumors; we used a novel brain metastases of breast cancer model to characterize vascular density, and corresponding changes in tumor permeability, hypoxia and survival. Methods: Brain seeking metastatic tumor lines MDA-MB-231-Br and 4T1-Br5 were injected via the left cardiac ventricle in separate experiments and brain metastases developed for ∼28 days and ∼15 days respectively. After development, both 14 C-AIB and indocyanine green (ICG) were injected and allowed to circulate for 10 and 1 min respectively. Near infrared imaging was used to identify ICG filled vessels to quantify vascular density of lesions compared to normal brain. To determine permeability, concentrations of 14 C-AIB in blood and brain tissue were calculated using autoradiography. Immunofluorescent analysis was performed using anti-HIF-1α and anti Ki67 antibodies and co-stained with DAPI. Results: A significant amount of metastatic lesions were observed to have developed in each model. Lesions varied in size from 0.028 to 2.3mm 2 . Vascular density was significantly decreased (MDA 176 ± 96 and 4T1 201 ± 76) compared to control brain (429 ± 25) and to an RG2 implanted glioma used as a positive control (736 ± 168). Based on accumulation of 14 C-AIB, metastases (range: 22 nC i /g to 1682 nC i /g) were considered to be either permeable (3 fold or greater uptake compared to adjacent control tissue) or non-permeable. There was no strong correlation between lesion size and permeability (r 2 =0.23) or vascular density (r 2 = 0.06). HIF-1α was expressed in both permeable and non-permeable lesions. Lastly, no significant difference was seen in the percentage of Ki67 positive cells in the MDA (permeable 41 ± 2.9, n=7; and non-permeable 40.8 ± 2.7, n=14) and 4T1 model (permeable 52.0 ± 4.6, n=6; and non-permeable 45.0 ± 2.8, n=6). Discussion: This data provides a correlation of permeability to vascular density, HIF-1α and Ki67 in a brain metastases model. While, both brain model systems exhibited a high degree of heterogeneous permeability, in neither model did permeability strongly correlate with vascular density, lesion size, HIF-1α expression and positivity for Ki67. Based upon the data, this experimental model may reflect the heterogeneity seen in human brain metastases of breast cancer and suggests there are other biological factors besides vascular density which correlate with drug distribution and or tumor/survival growth. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2373.