Metabolomic and transcriptomic analysis reveals endogenous substrates and metabolic adaptation in rats lacking Abcg2 and Abcb1a transporters.

Abcg2/Bcrp and Abcb1a/Pgp are xenobiotic efflux transporters limiting substrate permeability in the gastrointestinal system and brain, and increasing renal and hepatic drug clearance. The systemic impact of Bcrp and Pgp ablation on metabolic homeostasis of endogenous substrates is incompletely understood. We performed untargeted metabolomics of cerebrospinal fluid (CSF) and plasma, transcriptomics of brain, liver and kidney from male Sprague Dawley rats (WT) and Bcrp/Pgp double knock-out (dKO) rats, and integrated metabolomic/transcriptomic analysis to identify putative substrates and perturbations in canonical metabolic pathways. A predictive Bayesian machine learning model was used to predict in silico those metabolites with greater substrate-like features for either transporters. The CSF and plasma levels of 169 metabolites, nutrients, signaling molecules, antioxidants and lipids were significantly altered in dKO rats, compared to WT rats. These metabolite changes suggested alterations in histidine, branched chain amino acid, purine and pyrimidine metabolism in the dKO rats. Levels of methylated and sulfated metabolites and some primary bile acids were increased in dKO CSF or plasma. Elevated uric acid levels appeared to be a primary driver of changes in purine and pyrimidine biosynthesis. Alterations in Bcrp/Pgp dKO CSF levels of antioxidants, precursors of neurotransmitters, and uric acid suggests the transporters may contribute to the regulation of a healthy central nervous system in rats. Microbiome-generated metabolites were found to be elevated in dKO rat plasma and CSF. The altered dKO metabolome appeared to cause compensatory transcriptional change in urate biosynthesis and response to lipopolysaccharide in brain, oxidation-reduction processes and response to oxidative stress and porphyrin biosynthesis in kidney, and circadian rhythm genes in liver. These findings present insight into endogenous functions of Bcrp and Pgp, the impact that transporter substrates, inhibitors or polymorphisms may have on metabolism, how transporter inhibition could rewire drug sensitivity indirectly through metabolic changes, and identify functional Bcrp biomarkers.

A novel in vivo model using immunotoxin in the absence of p-glycoprotein to achieve ultra selective depletion of target cells: Applications in trogocytosis and beyond.

A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows targeted depletion at a higher degree of specificity than has been previously achieved. We have applied this technique to study trogocytosis, the intercellular transfer of cell surface molecules, but this principle could also be adapted using technology already available for use in other fields of study.

ABCG2 Deficiency Does Not Alter Dolutegravir Metabolism and Pharmacokinetics.

Dolutegravir (DTG) is a potent integrase inhibitor of human immunodeficiency virus. Because DTG is a substrate of the efflux transporter ABCG2 and ABCG2 is highly polymorphic, we asked whether dose adjustment of DTG is needed for ABCG2-deficient individuals. Using Abcg2-null mice, the current work investigated the impact of ABCG2 deficiency on DTG metabolism and pharmacokinetics. Compared with wild-type mice, no statistically significant difference was found in the systemic and tissue-specific (liver, kidney, and brain) pharmacokinetics of DTG in Abcg2-null mice. In addition, ABCG2 deficiency had no statistically significant impact on the production and excretion of DTG metabolites. In summary, this study demonstrated that deficiency of ABCG2 does not alter DTG metabolism and pharmacokinetics, suggesting that dose adjustment of DTG is not needed for individuals with ABCG2 deficiency. SIGNIFICANCE STATEMENT: The current work demonstrated that deficiency of ATP-binding cassette subfamily G member 2 (ABCG2) does not alter Dolutegravir (DTG) metabolism and pharmacokinetics, suggesting that dose adjustment of DTG is not needed for individuals with ABCG2 deficiency.

Abcg2 transporter affects plasma, milk and tissue levels of meloxicam.

ATP-binding cassette (ABCG2) is an efflux transporter that extrudes xenotoxins from cells in liver, intestine, mammary gland, brain and other organs, affecting the pharmacokinetics, brain accumulation and secretion into milk of several compounds, including antitumoral, antimicrobial and anti-inflammatory drugs. The aim of this study was to investigate whether the widely used anti-inflammatory drug meloxicam is an Abcg2 sustrate, and how this transporter affects its systemic distribution. Using polarized ABCG2-transduced cell lines, we found that meloxicam is efficiently transported by murine Abcg2 and human ABCG2. After oral administration of meloxicam, the area under the plasma concentration-time curve in Abcg2-/- mice was 2-fold higher than in wild type mice (146.06 ± 10.57 µg·h/ml versus 73.80 ± 10.00 µg·h/ml). Differences in meloxicam distribution were reported for several tissues after oral and intravenous administration, with a 20-fold higher concentration in the brain of Abcg2-/- after oral administration. Meloxicam secretion into milk was also affected by the transporter, with a 2-fold higher milk-to-plasma ratio in wild-type compared with Abcg2-/- lactating female mice after oral and intravenous administration. We conclude that Abcg2 is an important determinant of the plasma and brain distribution of meloxicam and is clearly involved in its secretion into milk.

An altered tissue distribution of flaxseed lignans and their metabolites in Abcg2 knockout mice.

Lignans are dietary polyphenols, which are metabolized by gut microbiota into the phytoestrogenic metabolites enterolignans, mainly enterolactone and enterodiol. Breast Cancer Resistance Protein (BCRP/ABCG2) is an efflux transporter that affects the plasma and milk secretion of several drugs and natural compounds. We hypothesized here that Abcg2 could influence the levels of lignans and their derived metabolites in target tissues. Consequently, we aimed to evaluate the role of Abcg2 in the tissue distribution of these compounds. We used Abcg2-/- knockout and wild-type male mice fed with a lignan-enriched diet for one week and analysed their plasma, small intestine, colon, liver, kidneys and testicles. High levels of lignans as well as enterolignans and their glucuronide and sulfate conjugates in the small intestine and colon were detected, with higher concentrations of the conjugates in the wild-type compared with Abcg2-/- mice. Particularly relevant was the detection of 24-fold and 8-fold higher concentrations of enterolactone-sulfate and enterolactone-glucuronide, respectively, in the kidney of Abcg2-/- compared with wild-type mice. In conclusion, our study showed that lignans and their derived metabolites were in vivo substrates of Abcg2, which affected their plasma and tissue levels. These results highlight the role of Abcg2 in influencing the health-beneficial properties of dietary lignans.

Synthesis and Preliminary Studies of a Novel Negative Allosteric Modulator, 7-((2,5-Dioxopyrrolidin-1-yl)methyl)-4-(2-fluoro-4-[11C]methoxyphenyl) quinoline-2-carboxamide, for Imaging of Metabotropic Glutamate Receptor 2.

Metabotropic glutamate 2 receptors (mGlu2) are involved in the pathogenesis of several CNS disorders and neurodegenerative diseases. Pharmacological modulation of this target represents a potential disease-modifying approach for the treatment of substance abuse, depression, schizophrenia, and dementias. While quantification of mGlu2 receptors in the living brain by positron emission tomography (PET) would help us better understand signaling pathways relevant to these conditions, few successful examples have been demonstrated to image mGlu2 in vivo, and a suitable PET tracer is yet to be identified. Herein we report the design and synthesis of a radiolabeled negative allosteric modulator (NAM) for mGlu2 PET tracer development based on a quinoline 2-carboxamide scaffold. The most promising candidate, 7-((2,5-dioxopyrrolidin-1-yl)methyl)-4-(2-fluoro-4-[11C]methoxyphenyl) quinoline-2-carboxamide ([11C]QCA) was prepared in 13% radiochemical yield (non-decay-corrected at the end of synthesis) with >99% radiochemical purity and >74 GBq/µmol (2 Ci/µmol) specific activity. While the tracer showed limited brain uptake (0.3 SUV), probably attributable to effects on PgP/Bcrp efflux pump, in vitro autoradiography studies demonstrated heterogeneous brain distribution and specific binding. Thus, [11C]QCA is a chemical probe that provides the basis for the development of a new generation mGlu2 PET tracers.

Synthesis and Evaluation of New Fluorine-18 Labeled Verapamil Analogs To Investigate the Function of P-Glycoprotein in the Blood-Brain Barrier.

P-glycoprotein is an efflux transporter located in the blood-brain barrier. (R)-[11C]Verapamil is widely used as a PET tracer to investigate its function in patients with epilepsy, Alzheimer's disease, and other neurodegenerative diseases. Currently it is not possible to use this successful tracer in clinics without a cyclotron, because of the short half-life of carbon-11. We developed two new fluorine-18 labeled (R)-verapamil analogs, with the benefit of a longer half-life. The synthesis of (R)-N-[18F]fluoroethylverapamil ([18F]1) and (R)-O-[18F]fluoroethylnorverapamil ([18F]2) has been described. [18F]1 was obtained in reaction of (R)-norverapamil with the volatile [18F]fluoroethyltriflate acquired from bromoethyltosylate and a silver trilate column with a radiochemical yield of 2.7% ± 1.2%. [18F]2 was radiolabeled by direct fluorination of precursor 13 and required final Boc-deprotection with TFA resulting in a radiochemical yield of 17.2% ± 9.9%. Both tracers, [18F]1 and [18F]2, were administered to Wistar rats, and blood plasma and brain samples were analyzed for metabolic stability. Using [18F]1 and [18F]2, PET scans were performed in Wistar rats at baseline and after blocking with tariquidar, showing a 3.6- and 2.4-fold increase in brain uptake in the blocked rats, respectively. In addition, for both [18F]1 and [18F]2, PET scans in Mdr1a/b(-/-), Bcrp1(-/-), and WT mice were acquired, in which [18F]2 showed a more specific brain uptake in Mdr1a/b(-/-) mice and no increased signal in Bcrp1(-/-) mice. [18F]2 was selected as the best performing tracer and should be evaluated further in clinical studies.

R- and S-Warfarin Were Transported by Breast Cancer Resistance Protein: From In Vitro to Pharmacokinetic-Pharmacodynamic Studies.

Warfarin, a racemate of R- and S-warfarin, is an important oral anticoagulant with narrow therapeutic window. Being an acidic drug, warfarin (pKa = 4.94) exists mainly as anion under physiological pH. We hypothesized that the transport of warfarin anion across cell membrane was mediated by breast cancer resistance protein (BCRP), an efflux transporter having a variety of acidic substrates. This study aimed at verifying that warfarin was a substrate of BCRP. Cell lines and mice were used for transport assay and pharmacokinetic-pharmacodynamic studies, respectively. The concentrations of R- and S-warfarin were simultaneously determined by liquid chromatography-mass spectrometry method. Transport assay showed that the intracellular concentrations of R- and S-warfarin in MDCKII-BCRP were significantly lower than those in MDCKII. In addition, Ko143, a potent BCRP inhibitor, significantly inhibited the efflux transport of R- and S-warfarin in MDCKII-BCRP, but not in MDCKII. Pharmacokinetic study showed that the plasma concentrations of R- and S-warfarin in Bcrp-/- mice were significantly higher than those in wild-type mice at 6 h after dosing. Anticoagulation measurement showed that the international normalized ratio in Bcrp-/- mice was significantly higher than that in wild-type mice at 24 h after dosing. In conclusion, R- and S-warfarin were transported by BCRP.

ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.

The ATP-binding cassette transporter ABCG2 is expressed in the interfollicular epidermis and mediates the side-population phenotype in skin cells. However, the role of ABCG2 in skin is unclear. Increased expression levels of ABCG2 were found at the basal layer of transitional epidermis adjacent to cutaneous wounds in human patients, indicating that ABCG2 may be involved in regulating the wound healing process. To investigate the role of ABCG2 in cutaneous wound healing, full-thickness skin wounds were created in ABCG2 knockout (ABCG2-KO) and wild-type mice. The healing process was analysed and revealed that ABCG2 deficiency in skin results in delays in wound closure and impairments in re-epithelialization, as evidenced by reductions in both suprabasal differentiation and in p63-expressing keratinocytes migrating from transitional epidermis to epithelial tongues. The reduction in p63-expressing cells may be due to elevated levels of reactive oxygen species in ABCG2-KO epidermis, which can cause DNA damage and lead to proliferation arrest. To determine whether ABCG2 deficiency affects the potency of epidermal stem/progenitor cells (EPCs), transplantation studies were carried out, which demonstrated that ABCG2-KO EPCs display higher levels of γH2AX and lose the capacity to differentiate into suprabasal keratinocytes. A competitive repopulation assay confirmed that ABCG2 expression is critical for the proper expansion and differentiation of EPCs in cutaneous wounds. As EPCs are known to contribute to the healing of larger wounds, the current findings imply a functional role for ABCG2 in the expansion and differentiation of p63-expressing EPCs. Thus, ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.