Aging and brain free cholesterol concentration on amyloid-ß peptide accumulation in guinea pigs.

Alzheimer's disease is a complex multifactorial neurodegenerative disorder wherein age is a major risk factor. The appropriateness of the Hartley guinea pig (GP), which displays high sequence homologies of its amyloid-ß (Aß40 and Aß42) peptides, Mdr1 and APP (amyloid precursor protein) and similarity in lipid handling to humans, was appraised among 9-40 weeks old guinea pigs. Protein expression levels of P-gp (Abcb1) and Cyp46a1 (24(S)-hydroxylase) for Aß40, and Aß42 efflux and cholesterol metabolism, respectively, were decreased with age, whereas those for Lrp1 (low-density lipoprotein receptor related protein 1), Rage (receptor for advanced glycation endproducts) for Aß efflux and influx, respectively, and Abca1 (the ATP binding cassette subfamily A member 1) for cholesterol efflux, were unchanged among the ages examined. There was a strong, negative correlation of the brain Aß peptide concentrations and Abca1 protein expression levels with free cholesterol. The correlation of Aß peptide concentrations with Cyp46a1 was, however, not significant, and concentrations of the 24(S)-hydroxycholesterol metabolite revealed a decreasing trend from 20 weeks old toward 40 weeks old guinea pigs. The composite data suggest a role for free cholesterol on brain Aß accumulation. The decreases in P-gp and Lrp1 protein levels should further exacerbate the accumulation of Aß peptides in guinea pig brain.

Measuring Amyloid-ß Peptide Concentrations in Murine Brain with Improved ELISA Assay.

The amyloid-ß (Aß) peptides of 40 and 42 amino acids that are implicated in Alzheimer's disease may potentially aggregate into toxic oligomers and form neuritic plaques. The enzyme-linked immunosorbent assay (ELISA) is a facile method used for the determination of Aß concentrations in biological matrices, namely plasma, cerebrospinal fluid, and brain. The method is mostly used for the measurement of Aß concentrations in transgenic mice, but it is unknown whether the ELISA method is suitable for measuring low, endogenous levels of Aß in the brains of wild-type mice. The Aß ELISA kit manufacturer recommends use of 5 M guanidine hydrochloride (GuHCl), a protein-denaturing agent, for homogenization of the brain tissue, followed by dilution back down to 0.1 M to avoid quenching by GuHCl. Components of brain matrices and GuHCl that could interfere with the quantitation have not been investigated. In this article, we describe an improved method involving homogenization of mouse brain with 1 M instead of 5 M GuHCl, reducing the dilution factor by 5× to provide a higher sensitivity. The modified ELISA assay is improved for the quantitation of brain Aß peptides in wild-type mice, where Aß peptide levels are much lower than those in transgenic mouse models. © 2021 Wiley Periodicals LLC.

Impact of age, hypercholesterolemia, and the vitamin D receptor on brain endogenous ß-amyloid peptide accumulation in mice.

Age, hypercholesterolemia, and vitamin D deficiency are risk factors that increase the brain accumulation of pathogenic ß-amyloid peptides (40 and 42), precursors leading to Alzheimer's disease (AD) in humans. The relative changes accompanying aging, high cholesterol, and/or treatment of calcitriol, active vitamin D receptor (VDR) ligand, under normal physiology are unknown. We examined these relative changes in C57BL/6 mice of ages 2, 4-8, and more than 10 months old, which were fed a normal or high fat / high cholesterol diet and treated with calcitriol, active ligand of the vitamin D receptor (0 or 2.5 µg/kg ×4, intraperitoneally, every other day to elicit cholesterol lowering in liver). Aß40 but not Aß42 accumulation in brain and lower P-glycoprotein (P-gp) and neprilysin protein expressions for Aß efflux and degradation, respectively, were found to be associated with aging. But there was no trend for BACE1 (ß-secretase 1, a cholesterol-sensitive enzyme) toward Aß synthesis with age. In response to calcitriol treatment, P-gp was elevated, mitigating partially the age-related changes. Although age-dependent decreasing trends in mRNA expression levels existed for Cyp46a1, the brain cholesterol processing enzyme, whose inhibition increases BACE1 and ApoE to facilitate microglia Aß degradation, mRNA changes for other cholesterol transporters: Acat1 and Abca1, and brain cholesterol levels remained unchanged. There was no observable change in the mRNA expression of amyloid precursor protein (APP) and the influx (RAGE) and efflux (LRP1) transporters with respect to age, diet, or calcitriol treatment. Overall, aging poses as a risk factor contributing to Aß accumulation in brain, and VDR-mediated P-gp activation partially alleviates the outcome.

Human Amyloid-ß40 Kinetics after Intravenous and Intracerebroventricular Injections and Calcitriol Treatment in Rats In Vivo.

Amyloid-ß peptides of 40 and 42 amino acid lengths, which are synthesized in neurons and degraded in the brain and liver, have the potential to aggregate and form neuritic plaques in Alzheimer disease. The kinetics of human amyloid-ß (hAß) 40 were examined in the rat pursuant to intravenous and intracerebroventricular administration after pretreatment with calcitriol, the active vitamin D receptor ligand (6.4 nmol·kg-1 in 0.3 ml corn oil every other day for four intraperitoneal doses) to induce P-glycoprotein (P-gp) and enhance hAß40 brain efflux. The interference of hAß40 by media matrix that suppressed absorbance readings in the ELISA assay was circumvented with use of different calibration curves prepared in Standard Dilution Buffer, undiluted, 10-10,000 or 5-fold diluted plasma, or artificial cerebrospinal fluid. Simultaneous fitting of hAß40 plasma and cerebrospinal fluid (CSF) data after intravenous and intracerebroventricular administration were described by catenary-mammillary models comprising of a central and two peripheral compartments, the brain, and one to four CSF compartments. The model with only one CSF compartment (model I) best fitted the intravenous data that showed a faster plasma decay t1/2 and slower equilibration between plasma and brain/CSF. Calcitriol induction increased the brain efflux rate constant, k41 (1.8-fold), at the blood-brain barrier when compared with the control group, as confirmed by the 2-fold (P < 0.05) increase in brain P-gp relative protein expression. SIGNIFICANCE STATEMENT: An accurate description of the kinetic behavior of human amyloid-ß (hAß) 40 is needed in defining the toxic peptide as a biomarker of Alzheimer disease. Modeling of hAß40 data after intravenous and intracerebroventricular administration to the rat revealed an initially faster plasma half-life that reflected faster peripheral distribution but slower equilibration between plasma and brain/cerebrospinal fluid even with calcitriol pretreatment that increased P-glycoprotein protein expression and enhanced efflux clearance from brain.

Treatment of Porphyromonas gulae infection and downstream pathology in the aged dog by lysine-gingipain inhibitor COR388.

COR388, a small-molecule lysine-gingipain inhibitor, is currently being investigated in a Phase 2/3 clinical trial for Alzheimer's disease (AD) with exploratory endpoints in periodontal disease. Gingipains are produced by two species of bacteria, Porphyromonas gingivalis and Porphyromonas gulae, typically associated with periodontal disease and systemic infections in humans and dogs, respectively. P. gulae infection in dogs is associated with periodontal disease, which provides a physiologically relevant model to investigate the pharmacology of COR388. In the current study, aged dogs with a natural oral infection of P. gulae and periodontal disease were treated with COR388 by oral administration for up to 90 days to assess lysine-gingipain target engagement and reduction of bacterial load and downstream pathology. In a 28-day dose-response study, COR388 inhibited the lysine-gingipain target and reduced P. gulae load in saliva, buccal cells, and gingival crevicular fluid. The lowest effective dose was continued for 90 days and was efficacious in continuous reduction of bacterial load and downstream periodontal disease pathology. In a separate histology study, dog brain tissue showed evidence of P. gulae DNA and neuronal lysine-gingipain, demonstrating that P. gulae infection is systemic and spreads beyond its oral reservoir, similar to recent observations of P. gingivalis in humans. Together, the pharmacokinetics and pharmacodynamics of COR388 lysine-gingipain inhibition, along with reduction of bacterial load and periodontal disease in naturally occurring P. gulae infection in the dog, support the use of COR388 in targeting lysine-gingipain and eliminating P. gingivalis infection in humans.

Studies To Examine Potential Tolerability Differences between the 5-HT2C Receptor Selective Agonists Lorcaserin and CP-809101.

Lorcaserin (LOR) is a selective 5-HT2C receptor agonist that has been FDA approved as a treatment for obesity. The most frequently reported side-effects of LOR include nausea and headache, which can be dose limiting. We have previously reported that in the rat, while LOR produced unconditioned signs characteristic of nausea/malaise, the highly selective 5-HT2C agonist CP-809101 (CP) produced fewer equivalent signs. Because this may indicate a subclass of 5-HT2C agonists having better tolerability, the present studies were designed to further investigate this apparent difference. In a conditioned gaping model, a rodent test of nausea, LOR produced significantly higher gapes compared to CP consistent with it having higher emetogenic properties. Subsequent studies were designed to identify features of each drug that may account for such differences. In rats trained to discriminate CP-809101 from saline, both CP and LOR produced full generalization suggesting a similar interoceptive cue. In vitro tests of functional selectivity designed to examine signaling pathways activated by both drugs in CHO (Chinese hamster ovary) cells expressing h5-HT2C receptors failed to identify evidence for biased signaling differences between LOR and CP. Thus, both drugs showed similar profiles across PLC, PLA2, and ERK signaling pathways. In studies designed to examine pharmacokinetic differences between LOR and CP, while drug plasma levels correlated with increasing dose, CSF levels did not. CSF levels of LOR increased proportionally with dose; however CSF levels of CP plateaued from 6 to 12 mg/kg. Thus, the apparently improved tolerability of CP likely reflects a limit to CNS levels attained at relatively high doses.

In vivo solid-phase microextraction liquid chromatography-tandem mass spectrometry for monitoring blood eicosanoids time profile after lipopolysaccharide-induced inflammation in Sprague-Dawley rats.

A fast and non-lethal in vivo solid-phase microextraction (SPME) sampling method for rat blood coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) was developed for monitoring rapid changes in concentrations of eicosanoids - lipid mediators involved in the development of inflammatory conditions - using diffusion-based calibration. Sampling rates of target eicosanoids were pre-determined under laboratory conditions with a precision of ≤10%, and directly used for quantification of analyte concentrations in blood after lipopolysaccharide-induced inflammation in Sprague-Dawley rats. Results showed significant changes in unbound plasma concentrations of arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) in response to the treatment. Next, performance of the proposed method was compared with protein precipitation (PP) of plasma, a conventional sample preparation technique. Finally, percentages of plasma protein binding (PPB) of specific eicosanoids were determined. PPB of target eicosanoids was in agreement with literature values, ranging from 99.3 to 99.9% for 12-HETE and DHA, respectively. We envision that the proposed method is a particularly suitable alternative to lethal sampling and current methods based on sample depletion in animal studies for accurate monitoring of rapid changes in blood concentrations of small molecules.

Vitamin D receptor activation induces P-glycoprotein and increases brain efflux of quinidine: an intracerebral microdialysis study in conscious rats.

PURPOSE: Since the vitamin D receptor (VDR) was found to up-regulate cerebral P-glycoprotein expression in vitro and in mice, we extend our findings to rats by assessing the effect of rat Vdr activation on brain efflux of quinidine, a P-gp substrate that is eliminated primarily by cytochrome P450 3a. METHODS: We treated rats with vehicle or the active VDR ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] (4.8 or 6.4 nmol/kg i.p. every 2nd day × 4) and examined P-gp expression and cerebral quinidine disposition via microdialysis in control and treatment studies conducted longitudinally in the same rat. RESULTS: The 6.4 nmol/kg 1,25(OH)2D3 dose increased cerebral P-gp expression 1.75-fold whereas hepatic Cyp3a remained unchanged. Although there was no change in systemic clearance elicited by 1,25(OH)2D3, brain extracellular fluid quinidine concentrations were lower in treated rats. We noted that insertion of indwelling catheters increased plasma protein binding of quinidine and serial sampling decreased the blood:plasma concentration ratio, factors that alter distribution ratios in microdialysis studies. After appropriate correction, KECF/P,uu and KECF/B,uu, or ratios of quinidine unbound concentrations in brain extracellular fluid to plasma or blood at steady-state, were more than halved. CONCLUSION: We demonstrate that VDR activation increases cerebral P-gp expression and delimits brain penetration of P-gp substrates.

Overview of pharmacokinetics.

In addition to having selective potency against the molecular target(s), a compound must be able to reach its intended site of action in vivo in sufficient quantity and for the appropriate duration of time to exert a biological effect. The fate of a compound after in vivo administration depends upon its absorption, distribution, metabolism, and excretion (ADME), as well as its target residence time. The concentration of the compound in the blood, plasma, and other tissues represents the sum of all of these processes. Described in this unit are protocols for administering a compound by various routes to rats and for collecting the appropriate samples to determine the pharmacokinetics profile. The basic terms used in pharmacokinetics studies are defined, and representative examples are given to illustrate important variables to consider.

Pharmacokinetics.

Pharmacokinetics (PK) is the study of the time course of the absorption, distribution, metabolism and excretion (ADME) of a drug, compound or new chemical entity (NCE) after its administration to the body. Following a brief introduction as to why knowledge of the PK properties of an NCE is critical to its selection as a lead candidate in a drug discovery program and/or its use as a functional research tool, the present article presents an overview of PK principles, including practical guidelines for conducting PK studies as well as the equations required for characterizing and understanding the PK of an NCE and its metabolite(s). A review of the determination of in vivo PK parameters by non-compartmental and compartmental methods is followed by a brief overview of allometric scaling. Compound absorption and permeability are discussed in the context of intestinal absorption and brain penetration. The volume of distribution and plasma protein and tissue binding are covered as is the clearance (systemic, hepatic, renal, biliary) of both small and large molecules. A section on metabolite kinetics describes how to estimate the PK parameters of a metabolite following administration of an NCE. Lastly, mathematical models used to describe pharmacodynamics (PD), the relationship between the NCE/compound concentration at the site of action and the resulting effect, are reviewed and linked to PK models in a section on PK/PD.