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. 2013 Dec;33(12):1983-92.
doi: 10.1038/jcbfm.2013.160. Epub 2013 Sep 18.

Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier

Isabelle St-Amour  1 , Isabelle Paré, Wael Alata, Katherine Coulombe, Cassandra Ringuette-Goulet, Janelle Drouin-Ouellet, Milène Vandal, Denis Soulet, Renée Bazin, Frédéric Calon
Affiliations

Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier

Isabelle St-Amour et al. J Cereb Blood Flow Metab. 2013 Dec.

Abstract

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets, the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood-brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay, a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053 per hour) in the cortex, consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally, brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary, our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets.

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Figures

Figure 1
Figure 1
Biodistribution of human and mouse immunoglobulin G (IgG) in the plasma and tissue homogenates. (A) Pharmacokinetic evaluation of human IgG (hIgG) distribution in homogenates of brain and peripheral tissues after a single intraperitoneal (i.p.) injection of 25 mg intravenous immunoglobulin (IVIg) in 4- to 6-week-old C57Bl/6 females (n=3–5 per time point). (B) A single i.p. injection also leads to a reduction of brain endogenous mouse IgG (mIgG). (C) Subchronic treatment: 9- to 13-month-old nontransgenic (nontg) and 3 × Tg-AD mice (n=6–8) received three injections of IVIg (two i.p. injections of 30 mg (96 and 24 hours before killing) and one intravenous injection ((75 minutes before killing) of 20 mg) or equivalent volumes of vehicle (control) and were perfused intracardially with 50 mL phosphate-buffered saline (PBS) buffer under deep anesthesia. (D) Chronic treatment: nontg and 3xTg-AD mice (n=11–13) were injected twice a week for 1 month (nine i.p. injections) with 1.5 g/kg IVIg and killed at 16 months of age, using intracardiac perfusion. Results are presented as means +/− s.e.m. Statistical analyses: one-way analysis of variance followed by Dunnett's multiple comparison test. mIgG: ∞P<0.05, ∞∞P<0.01; hIgG: *P<0.05, **P<0.01 versus controls. (A–C) Spleen, liver, cortex, and hippocampus homogenates were prepared using five volumes of radio immunoprecipitation assay buffer/mg tissue. The quantification of hIgG and mIgG was performed using specific enzyme-linked immunosorbent assays.
Figure 2
Figure 2
Quantification of intravenous immunoglobulin (IVIg) brain uptake: saturation of blood–brain barrier (BBB) transport. Mice were deeply anesthetized and (A) in situ cerebral perfusion was performed through the carotid artery, at a flow rate of 2.5 mL/minute for 60 seconds with the perfusate (3H-inulin with IVIg or vehicle) followed with a washing step of 120 seconds. 3H-inulin served as a control for the washing step and was below the detection limit in all mice. (B) The brain concentration of IVIg increased while (C) brain IVIg uptake and (D) brain transport coefficient (Kin) decreased with increasing doses of IVIg, suggesting a saturation of blood-to-brain transport. (B–D) Intravenous immunoglobulin was detected using an anti-human immunoglobulin G (IgG)-specific enzyme-linked immunosorbent assay. (E) 14C-sucrose was added to the washing buffer to measure the effects of IVIg on the BBB integrity. Intravenous immunoglobulin did not increase the vascular volume (VD) of 14C-sucrose, suggesting no modification in BBB permeability. Each data point represents a single individual (n=4–7 per group). Statistical analysis: one-way analysis of variance with Tukey's multiple comparison test *P<0.05, **P<0.01 versus 500 μg IVIg.
Figure 3
Figure 3
Intravenous immunoglobulin (IVIg) was detected in the capillary endothelial cells and neurons after systemic administration in mice. (A and B) Nontransgenic (NonTg) and 3xTg-AD mice received three injections of IVIg (subchronic treatment) or equivalent volumes of vehicle (Ctrl) and were perfused with 50 mL phosphate-buffered saline (PBS) buffer followed with 50 mL 4% paraformaldehyde. Representative (A) immunohistochemistry and (B) immunofluorescent labeling of IVIg on 35-μm-thick brain sections of 4- to 9-month-old 3xTg-AD and NonTg mice (4–8 animals per group). Intravenous immunoglobulin was detected throughout the brain of injected mice, in capillaries, brain parenchyma, and brain cells. Immunohistochemistry: sections from the brains of IVIg-treated and control mice were mounted in alternation on the same slide. The three slides represent six independent animals. Photomicrographs were taken with a MicroFire 1.0 camera (Optronics, Goleta, CA, USA) linked to an E800 Nikon 274 microscope using the imaging software Picture Frame. Immunofluorescent staining: IVIg-treated and control 3xTg-AD animals are presented. Secondary antibodies conjugated with AF-488 for the detection of CD31 (endothelial cells in green), AF-565 for NeuN (neurons in red), AF-647 for human IgG (white) and 4′,6-diamidino-2-phenylindole (nucleus in blue). Confocal laser scanning microscopy was performed at room temperature with an Olympus IX81 multispectral FV1000 confocal microscope, equipped with 405 nm, 488 nm, 546 nm and 633 nm laser lines (Olympus America Inc., Richmond Hill, ON, Canada). Images were acquired sequentially with 2 lines Kalman integration using a × 60 OSC NA1.4 objective lens, and some regions of interest were acquired at zoom × 2.5. Image stacks were then imported in Bitplane Imaris 7.5 software (Bitplane, South Windsor, CT, USA). Maximum intensity projections were calculated using the surpass module and were exported for each channel as TIF files. (C) Age-dependent progression of the amyloid neuropathology in the 3xTg-AD mice. Four, 12, and 19-month-old 3xTg-AD mice were perfused with PBS buffer. Representative immunofluorescence labeling of amyloid plaques was detected on 12-μm-thick brain sections using 6E10 antibody (specific to amino acid 1 to 16 of APP) with a i90 Nikon fluorescence microscope (Nikon, Québec, QC, Canada) coupled to a Hamamatsu 1394 ORCA-285 monochrome camera, exploited by Simple PCI software version 5.3.0.1102 (Compix Imaging Systems, Cranberry, PA, USA) (n=3–5 animals per group).
Figure 4
Figure 4
Quantification of intravenous immunoglobulin (IVIg) binding to brain cells. (A) Brain cells were isolated from 3xTg-AD and NonTg mice using NeuroCult Enzymatic Dissociation Kit for Adult CNS Tissue (StemCell Technologies, Vancouver, BC, Canada). Immunoblot was used to confirm the presence of a variety of cell types after dissociation with specific cell markers: PDGFR for pericytes, NeuN for neurons, GFAP for astrocytes, oligodendrocyte specific antigen (OligoD) for oligodendrocytes, Collagen IV (Col IV) for endothelial cells and actin as a control for the homogeneity of the loading (each lane represents an individual animal). (B) To determine the number of IVIg molecules per cell, cell lysates from 1 × 107 cells of n=3 mouse brains were prepared in radio immunoprecipitation assay buffer and human IgG-specific enzyme-linked immunoprecipitation assay was performed. Statistical analysis is as follows: unpaired t-test P=0.5012. NS=not significant.
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