Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer–<i>N</i>‑Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines

Microglia-mediated neuroinflammation is commonly associated with neurodegeneration and has been implicated in several neurological disorders, such as Alzheimer’s disease and Parkinson’s disease. Therefore, it is crucial to develop a detailed understanding of the interaction of potential nanocarriers with microglial cells to efficiently deliver anti-inflammatory molecules. In this study, we applied brush polymers as a modular platform to systematically investigate their association with murine (BV-2) and human (HMC3) microglial cell lines in the presence and absence of the pro-inflammatory inducer lipopolysaccharide (LPS) using flow cytometry. Brush polymers of different sizes and shapes, ranging from ellipsoid to worm-like cylinders, were prepared through a combination of the two building blocks carboxylated N-acylated poly(aminoester)s (NPAEs)-based polymers and poly(2-ethyl-2-oxazoline)-NH2 (PEtOx-NH2) and characterized by 1H NMR spectroscopy, size exclusion chromatography, and small-angle neutron scattering. Generally, ellipsoidal particles showed the highest cellular association. Moreover, while no significant differences in murine cell association were observed, the brush polymers revealed a significant accumulation in LPS-activated human microglia compared to resting cells, emphasizing their higher affinity to activated HMC3 cells. Brush polymers with the highest cell association were further modified with the anti-inflammatory agent N-acetyl cysteine (NAC) in a reversible manner. The brush polymer–NAC conjugates were found to significantly attenuate the production of interleukin 6 (p < 0.001) in LPS-activated HMC3 cells compared to LPS-activated BV-2 cells. Thus, the presented brush polymer–NAC conjugates showed a high anti-inflammatory activity in human microglia, suggesting their potential for disease-targeted therapy of microglial-mediated neuroinflammation in the future

Sex-Dependent Changes to the Intestinal and Hepatic Abundance of Drug Transporters and Metabolizing Enzymes in the SOD1^G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at the blood–brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based [tandem mass tag (TMT)] or label-free [data-independent acquisition (DIA)] acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (Padj 1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose [solute carrier (SLC) SLC5A1] and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS

Fatty Acid-Binding Protein 5 Facilitates the Blood–Brain Barrier Transport of Docosahexaenoic Acid

The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood–brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA <i>in vitro</i> and <i>in vivo</i>, respectively. hFAPB5 was recombinantly expressed and purified from <i>Escherichia coli</i> C41­(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of ¹⁴C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An <i>in situ</i> transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (<i>K</i>_in) of ¹⁴C-DHA between FABP5-deficient (FABP5^–/–) and wild-type (FABP5^+/+) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in ¹⁴C-DHA cellular uptake. By using optimized conditions for the <i>in situ</i> transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of ¹⁴C-DHA (1 min)), the <i>K</i>_in of ¹⁴C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5^+/+ mice, the <i>K</i>_in of ¹⁴C-DHA decreased 36.7 ± 12.4% in FABP5^–/– mice. This study demonstrates that FABP5 binds to DHA and is involved in the brain endothelial cell uptake and subsequent BBB transport of DHA, confirming the importance of this cytoplasmic carrier protein in the CNS exposure of this PUFA essential for neuronal function

The Lymphatic System Plays a Major Role in the Intravenous and Subcutaneous Pharmacokinetics of Trastuzumab in Rats

Therapeutic monoclonal antibodies are currently delivered mainly via the intravenous route, since large volumes are often required to deliver a therapeutic dose. Administration via the subcutaneous route would have several therapeutic advantages; the absorption mechanisms for antibodies dosed subcutaneously are poorly understood. This study was conducted to develop a better understanding of the mechanisms governing the subcutaneous absorption and trafficking of monoclonal antibodies. Specifically, the role of the lymphatic system in the absorption and prolonged plasma exposure of trastuzumab was explored in thoracic lymph duct-cannulated rats after SC and IV dosing. A population pharmacokinetic model was developed in S-ADAPT to simultaneously fit all plasma and lymph concentrations and to predict the pharmacokinetics in nonlymph duct-cannulated animals. The estimated absolute bioavailability of trastuzumab after SC administration in rats was 85.5%. Following SC administration, 53.1% of the trastuzumab dose was absorbed via a first-order process (mean absorption time: 99.6 h) into the peripheral lymph compartment and 32.4% of the dose was absorbed by a Michaelis–Menten process into the central compartment. Recovery in thoracic lymph over 30 h was 26.7% after SC and 44.1% after IV administration. This study highlights for the first time the significant role of the lymphatic system in maintaining the long plasma exposure of trastuzumab, with the model predicting an extensive distribution of this monoclonal antibody into the lymph following SC and IV administration. This extensive direct absorption from the SC injection site into lymph may enable novel therapeutic strategies for the treatment of lymph resident metastatic cancer

Immunohistochemical detection of TDO/IDO-1 in the CA1 and CA3 regions of hippocampus in AD patients and age and sex-matched controls.

<p>Corresponding graphs of image analysis of TDO/IDO-1 immunoreactivity in human controls (white bars) and AD patients (black bars). Columns and bars represent mean ± SEM, n = 4, ****p&lt;0.0001, in comparison to its counterpart, WT.</p

Immunohistochemical localization of TDO in the cerebellum of 3xTg AD mice.

<p>TDO was located mainly in neurons (NeuN+) and microglia (BS-Isolectin-B4+), with minor expression in astrocytes (GFAP). Columns 2, 3 and 4 show the higher magnification views of the boxed areas in the first column (merged image).</p

(A) TRP and (B) KYN concentrations quantified using HPLC; (C) K/T ratio depicting the expression of KP activation; (D) QUIN and (E) PIC production quantified using GC/MS in the hippocampus, cerebellum, cortex and remaining brain regions of 2–4 month, 6–8

<p>Bars represent mean ± SEM, n = 4.</p

Simplified schematic of the kynurenine pathway (KP).

<p>IDO-1, indoleamine dioxygenase; TDO, tryptophan dioxygenase; AFMID, arylformamidase; KAT 1–3, kynurenine amino transferase 1, 2 and 3; KMO, kynurenine 3-hydroxylase; KYNU, kynureninase; 3HAAO, 3-hydroxyanthranilic acid oxidase; ACMSD, amino-carboxymuconate-semialdehyde decarboxylase; QPRT, quinolinate phosphoribosyltransferase.</p

Immunohistochemical detection of TDO in the white matter of cerebellum in 4-month (A), 7-month (C) and 11-month (E) WT mice, compared to the same regions in 4-month (B), 7-month (D) and 11 month (F) 3xTg AD mice.

<p>(G) Corresponding graphs of image analysis of density of TDO-immunoreactivity in 4, 7 and 11-months WT (white bars) and 3xTg AD (black bars) mice brains; mean ± SEM (n = 3) *p&lt;0.05, in comparison to its counterpart, WT.</p

The upper panel shows the representative bands of Western blots for TDO (30 kDa) and IDO1 (45 kDa) proteins in the hippocampus and cerebellum of 8-month WT and 3xTg AD mice.

<p>GAPDH was used as a loading control. The lower panel shows the quantitative comparison expressed as relative percentage change of TDO/GAPDH or IDO1/GAPDH ratio in both regions of 8-month WT (white bars) and 3xTg AD (black bars) mouse brains. Data are presented as mean ± SEM (n = 3), **p&lt;0.01, in comparison to its counterpart, WT.</p