Enhanced brain entry of checkpoint-inhibitory therapeutic antibodies facilitated by intraarterial NEO100 in mouse models of brain-localized malignancies.

OBJECTIVE: Immune checkpoint-inhibitory therapeutic antibodies have shown striking activity against several types of cancers but are less effective against brain-localized malignancies, in part due to the protective effect of the blood-brain barrier (BBB). The authors hypothesized that intraarterial (IA) delivery of a novel compound, NEO100, has the potential to safely and reversibly open the BBB to enable brain-targeted therapeutic activity of checkpoint-inhibitory antibodies. METHODS: Immunocompetent mice with syngeneic glioblastoma or melanoma cells implanted into their brains were subjected to a single IA injection of NEO100 to open their BBB. One dose of murine anti-PD-1/PD-L1 antibody was either coinjected with NEO100 or separately injected intravenously. Brain penetration of these antibodies and levels of CD8+ T cell infiltrate into the tumor microenvironment were quantitated and animal survival was monitored. RESULTS: IA NEO100 enabled the increased accumulation of checkpoint-inhibitory antibodies in the brain, along with greater numbers of T cells. In both malignancy models, a single intervention of IA NEO100 combined with antibody resulted in the long-term survival of animals. Antibody treatment in the absence of NEO100 was far less effective. CONCLUSIONS: BBB opening by IA NEO100 facilitates brain tumor access by checkpoint-inhibitory antibodies and enables their therapeutic activity, along with increased levels of T-cell recruitment.

Enhanced brain delivery and therapeutic activity of trastuzumab after blood-brain barrier opening by NEO100 in mouse models of brain-metastatic breast cancer.

BACKGROUND: The antitumor efficacy of human epidermal growth factor receptor 2 (HER2)-targeted therapies, such as humanized monoclonal antibody trastuzumab (Herceptin®, Roche), in patients with breast-to-brain cancer metastasis is hindered by the low permeability of the blood-brain barrier (BBB). NEO100 is a high-purity version of the natural monoterpene perillyl alcohol, produced under current good manufacturing practice (cGMP) regulations, that was shown previously to reversibly open the BBB in rodent models. Here we investigated whether NEO100 could enable brain entry of trastuzumab to achieve greater therapeutic activity. METHODS: An in vitro BBB, consisting of human astrocytes and brain endothelial cells, was used to determine trastuzumab penetration in the presence or absence of NEO100. For in vivo studies, we administered intravenous (IV) trastuzumab or the trastuzumab-drug conjugate ado-trastuzumab emtansine (T-DM1; Kadcyla®, Roche), to mouse models harboring intracranial HER2+ breast cancer, with or without BBB opening via IA NEO100. Brain and tumor tissues were examined for the presence of trastuzumab and infiltration of immune cells. Therapeutic impact was evaluated based on overall survival. RESULTS: NEO100 greatly increased trastuzumab penetration across an in vitro BBB. In vivo, IA NEO100-mediated BBB opening resulted in brain tumor-selective accumulation of trastuzumab, without detectable presence in normal brain tissue, along with increased presence of immune cell populations. IV delivery of trastuzumab or T-DM1 achieved significantly greater overall survival of tumor-bearing mice when combined with IA NEO100. CONCLUSION: IA NEO100 facilitates brain tumor entry of trastuzumab and T-DM1 and significantly enhances their therapeutic efficacy, along with increased antibody-dependent immune cell recruitment.

Disintegrin-Integrin Binding for Attachment of Polymer Substrate to the Retina.

OBJECTIVE: We propose a novel attachment method for retinal tissue that utilizes silicone modified with bioactive molecules. DESIGN: This is an experimental study divided into an in vitro section performed in cadaveric pig eyes and an in vivo section performed in rabbits. SUBJECTS: During in vitro experiments 36 cadaveric pig eyes were used. During in vivo experiments 4 rabbits were used. METHODS: Different types of silicone went through a laser irradiation process to determine if binding sites for disintegrins could be created. Laser treated silicones that showed disintegrin binding were evaluated with in vitro testing for retina-silicone attachment. The best silicone binding in vitro was implanted into a rabbit's eye after a full vitrectomy was performed. Post-operative exams were done every two weeks to evaluate placement, attachment and sterilization method. After three months animals were euthanized and eye was enucleated for histology analysis. MAIN OUTCOME MEASURES: Attachment strength between silicone-disintegrin-retina, and signs of endophthalmitis during in vivo studies for biocompatibility purposes. RESULTS: A technique to successfully lase and produce an active area on the silicone surface was described. Scanning electron microscope (SEM) images were evaluated to assess physical ablation/debris field area on the surface, definition of edges, evenness, and symmetry of the lased area allowing us to select MED 4800 silicone family for further testing. Cell culture experiments showed disintegrin binding to the silicone active area. In vitro experiments with cadaveric eyes were performed to test retina-silicone attachment. MED 4860 showed strongest attachment to the retina and it was used during in vivo experiments. A sterilization protocol was tested and proved to be reliable for bioactive materials. Disintegrin coated silicone showed attachment in 2 of 4 rabbits during the 3-month implant period. The adhesion was persistent until reversed with plasmin. All rabbits were implanted for 3 months regardless of attachment, to test the feasibility of the sterilization method. None of the rabbits developed any type of eye infection during the implant period. CONCLUSION: We successfully lased and produced an active area on the silicone surface to allow disintegrin-silicone binding. Different silicones interact differently with the laser energy, and this is reflected in the strength of the silicone-disintegrin-retina attachment.

Vascular targeting and antiangiogenesis agents induce drug resistance effector GRP78 within the tumor microenvironment.

Therapeutic targeting of the tumor vasculature that destroys preexisting blood vessels of the tumor and antiangiogenesis therapy capitalize on the requirement of tumor cells on an intact vascular supply for oxygen and nutrients for growth, expansion and metastasis to the distal organs. Whereas these classes of agents show promise in delaying tumor progression, they also create glucose and oxygen deprivation conditions within the tumor that could trigger unintended prosurvival responses. The glucose-regulated protein GRP78, a major endoplasmic reticulum chaperone, is inducible by severe glucose depletion, anoxia, and acidosis. Here we report that in a xenograft model of human breast cancer, treatment with the vascular targeting agent, combretastatin A4P, or the antiangiogenic agent, contortrostatin, promotes transcriptional activation of the Grp78 promoter and elevation of GRP78 protein in surviving tumor cells. We further show that GRP78 is overexpressed in a panel of human breast cancer cells that has developed resistance to a variety of drug treatment regimens. Suppression of GRP78 through the use of lentiviral vector expressing small interfering RNA sensitizes human breast cancer cells to etoposide-mediated cell death. Our studies imply that antivascular and antiangiogenesis therapy that results in severe glucose and oxygen deprivation will induce GRP78 expression that could lead to drug resistance.

Effect of a perinatal high-salt diet on blood pressure control mechanisms in young Sprague-Dawley rats.

In the present investigation we sought to determine if a perinatal high-salt treatment affects blood pressure at an early age (30 days), and if so, to determine the mechanisms responsible for the hypertension. Pregnant dams were given an 8% NaCl diet [high-salt (HS) rats] during the final one-third of gestation and throughout the suckling period. After weaning, the pups continued to receive the high-salt diet until testing at age 30 days. Control groups received a normal-salt diet (NS rats). In HS rats, mean arterial pressure (MAP) was significantly increased (110 +/- 5 vs. 96 +/- 3 mmHg) compared with NS rats. Blockade of brain AT(1) receptors with intracerebroventricular losartan decreased MAP in HS but not NS rats. Blockade of alpha-adrenergic receptors with intravenous phentolamine or ganglionic transmission with intravenous chlorisondamine produced a greater decrease in MAP in HS rats. Baroreflex control of heart rate was assessed using a four-parameter logistics function. The mid-range MAP (p3) was significantly increased in the HS rats. No other baroreflex parameters were affected. Specific binding of (125)I-[Sa (1),Ile(8)]ANG II to AT(1) receptors was increased in the subfornical organ (SFO) of the HS rats. Expression of AT(1a) receptor mRNA was greater in both SFO and PVN of the HS rats. These data suggest that even at an early age, Sprague-Dawley rats treated with a perinatal high-salt diet are hypertensive. The elevated blood pressure appears to be caused by increased sympathetic nervous activity, resulting, in part, from increased brain AT(1) receptor activation.