Barcode lipids for absolute quantitation of liposomes in ocular tissues.

Lipid-based drug formulations are promising systems for improving delivery of drugs to ocular tissues, such as retina. To develop lipid-based systems further, an improved understanding of their pharmacokinetics is required, but high-quality in vivo experiments require a large number of animals, raising ethical and economic questions. In order to expedite in vivo kinetic testing of lipid-based systems, we propose a barcode approach that is based on barcoding liposomes with non-endogenous lipids. We developed and evaluated a liquid-chromatography-mass spectrometry method to quantify many liposomes simultaneously in aqueous humor, vitreous, and neural retina at higher than ±20% precision and accuracy. Furthermore, we showed in vivo suitability of the method in pharmacokinetic evaluation of six different liposomes after their simultaneous injection into the rat vitreal cavity. We calculated pharmacokinetic parameters in vitreous and aqueous humor, quantified liposome concentrations in the retina, and quantitated retinal distribution of the liposomes in the rats. Compared to individual injections of the liposome formulations, the barcode-based study design enabled reduction of animal numbers from 72 to 12. We believe that the proposed approach is reliable and will reduce and refine ocular pharmacokinetic experiments with liposomes and other lipid-based systems.

Sex-specific changes in protein expression of membrane transporters in the brain cortex of 5xFAD mouse model of Alzheimer's disease.

Membrane transporters playing an important role in the passage of drugs, metabolites and nutrients across the membranes of the brain cells have been shown to be involved in pathogenesis of Alzheimer's disease (AD). However, little is known about sex-specific changes in transporter protein expression at the brain in AD. Here, we investigated sex-specific alterations in protein expression of three ATP-binding cassette (ABC) and five solute carriers (SLC) transporters in the prefrontal cortex of a commonly used model of familial AD (FAD), 5xFAD mice. Sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic analysis was applied for absolute quantification of transporter protein expression. We compared the changes in transporter protein expressions in 7-month-old male and female 5xFAD mice versus sex-matched wild-type mice. The study revealed a significant sex-specific increase in protein expression of ABCC1 (p = 0.007) only in male 5xFAD mice as compared to sex-matched wild-type animals. In addition, the increased protein expression of glucose transporter 1 (p = 0.01), 4F2 cell-surface antigen heavy chain (p = 0.01) and long-chain fatty acid transport protein 1 (p = 0.02) were found only in female 5xFAD mice as compared to sex-matched wild-type animals. Finally, protein expression of alanine/serine/cysteine/threonine transporter 1 was upregulated in both male (p = 0.02) and female (p = 0.002) 5xFAD mice. The study provides important information about sex-specific changes in brain cortical transporter expression in 5xFAD mice, which will facilitate drug development of therapeutic strategies for AD targeting these transporters and drug delivery research.

Accelerated Early Childhood Growth Is Associated With the Development of Earlier Adrenarche and Puberty.

Context: Small birth size and increased postnatal growth have been associated with earlier timing of adrenarche and puberty, but it is not well known whether these factors alone or together lead to earlier maturation. Objective: This work aimed to search for different growth trajectories using a clustering approach to analyze the effects of birth size and postnatal growth on adrenarchal and pubertal development. Methods: Altogether 351 children (48% girls) were examined prospectively at ages 6 to 9 and 9 to 11 years. Birth and early-growth data were collected retrospectively. Main outcome measures included clinical signs of adrenarche and puberty, and serum androgen concentrations (dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, testosterone). Results: We detected 4 clusters with different birth sizes and postnatal growth trajectories: 1) children with average birth size and increased postnatal growth (AI), 2) children with small birth size and increased postnatal growth (SI), 3) children with average birth size and postnatal growth (AA), and 4) children with small birth size and average postnatal growth (SA). Thelarche at age 9 to 11 was most common and serum androgens at ages 6 to 9 and 9 to 11 years were highest in girls belonging to the AI and SI groups. Similar patterns in the onset of puberty and in androgen levels were not seen in the SA group. Conclusion: Increased early growth and weight gain predict higher serum androgen concentrations and earlier onset of puberty in girls. Adrenarche and puberty do not appear to be shifted earlier in children with small birth size who do not have catch-up growth.

Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line.

BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201. METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1). RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane. CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Mass spectrometry in ocular drug research.

Mass spectrometry (MS) has been proven as an excellent tool in ocular drug research allowing analyzes from small samples and low concentrations. This review begins with a short introduction to eye physiology and ocular pharmacokinetics and the relevance of advancing ophthalmic treatments. The second part of the review consists of an introduction to ocular proteomics, with special emphasis on targeted absolute quantitation of membrane transporters and metabolizing enzymes. The third part of the review deals with liquid chromatography-MS (LC-MS) and MS imaging (MSI) methods used in the analysis of drugs and metabolites in ocular samples. The sensitivity and speed of LC-MS make simultaneous quantitation of various drugs and metabolites possible in minute tissue samples, even though ocular sample preparation requires careful handling. The MSI methodology is on the verge of becoming as important as LC-MS in ocular pharmacokinetic studies, since the spatial resolution has reached the level, where cell layers can be separated, and quantitation with isotope-labeled standards has come more reliable. MS will remain in the foreseeable future as the main analytical method that will progress our understanding of ocular pharmacokinetics.

In vitro identification of decreased function phenotype ABCG2 variants.

Reduced activity of efflux transporter ABCG2, caused e.g., by inhibition or decreased function genetic variants, can increase drug absorption and plasma levels. ABCG2 has one clinically significant single nucleotide variant Q141K (c.421C>A), which leads to decreased protein levels and transport activity. In addition to Q141K, ABCG2 has over 500 rare (<1% minor allele frequency) nonsynonymous variants, but their functionality remains unknown. We studied the transport activity and abundance of 30 rare ABCG2 variants. The variants were transiently expressed in HEK293 cells. Transport activity and protein abundance were measured from inside-out crude membrane vesicles. Results were normalised to the reference ABCG2, while Q141K was used to categorise variants into decreased and normal function phenotypes based on their apparent transport activity. Fourteen variants (G80E, D128V, T434M, Q437R, C438R, C438W, C438Y, L479S, P480L, S486N, T512N, S519P, G553D and K647E) had similar or lower apparent transport activity than Q141K and thus were categorised as having a decreased function phenotype. Protein abundance could not explain all of the observed changes in transport activity: Only six variants (D128V, Q437R, C438R, S519P, G553D, and K647E) had similar or lower abundance compared to Q141K. The decreased function variants may increase systemic drug exposure and therefore cause interindividual variability in pharmacokinetics. In the future, in vitro phenotype classification may help to design personalised drug treatments.

Effects of 2-Year Physical Activity and Dietary Intervention on Adrenarchal and Pubertal Development: The PANIC Study.

CONTEXT: Childhood overweight has been linked to earlier development of adrenarche and puberty, but it remains unknown if lifestyle interventions influence sexual maturation in general populations. OBJECTIVE: To investigate if a 2-year lifestyle intervention influences circulating androgen concentrations and sexual maturation in a general population of children. METHODS: We conducted a 2-year physical activity and dietary intervention study in which 421 prepubertal and mostly normal-weight 6- to 9-year-old children were allocated either to a lifestyle intervention group (119 girls, 132 boys) or a control group (84 girls, 86 boys). The main outcome measures were serum dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), and testosterone concentrations, and clinical adrenarchal and pubertal signs. RESULTS: The intervention and control groups had no differences in body size and composition, clinical signs of androgen action, and serum androgens at baseline. The intervention attenuated the increase of DHEA (P = .032), DHEAS (P = .001), A4 (P = .003), and testosterone (P = .007) and delayed pubarche (P = .038) in boys but it only attenuated the increase of DHEA (P = .013) and DHEAS (P = .003) in girls. These effects of lifestyle intervention on androgens and the development of pubarche were independent of changes in body size and composition, but the effects of intervention on androgens were partly explained by changes in fasting serum insulin. CONCLUSION: A combined physical activity and dietary intervention attenuates the increase of serum androgen concentrations and sexual maturation in a general population of prepubertal and mostly normal-weight children, independently of changes in body size and composition.

Comparative steroid profiling of newborn hair and umbilical cord serum highlights the role of fetal adrenals, placenta, and pregnancy outcomes in fetal steroid metabolism.

Previous steroid hormone studies concerning pregnancy and newborns have mainly focused on glucocorticoids; wider steroid profiles have been less commonly investigated. Here, we performed a comparative analysis of 17 steroids from newborn hair and umbilical cord serum at the time of delivery. The study participants (n = 42, 50% girls) were a part of the Kuopio Birth Cohort and represent usual Finnish pregnancies. The hair and cord serum samples were analyzed with liquid chromatography high resolution mass spectrometry and triple quadrupole tandem mass spectrometry, respectively. We detected high individual variations in steroid hormone concentrations in both sample matrices. The concentrations of cortisol (F), corticosterone (B), estrone (E1), estradiol (E2), dehydroepiandrosterone (DHEA), 11ß-hydroxyandostenedione (11bOHA4), 5α-androstanedione (DHA4), and 17α-hydroxypregnenolone (17OHP5) correlated positively between cord serum and newborn hair samples. In addition, F and 11bOHA4 concentrations correlated positively with each other in both newborn hair and cord serum samples. The cortisone-to-cortisol ratio (E/F) was significantly higher in cord serum than in newborn hair samples reflecting high placental 11ßHSD2 enzyme activity. Only minor sex differences in steroid concentrations were observed; higher testosterone (T) and 11-deoxycortisol (S) with lower 11bOHA4 in male cord serum, and higher DHEA, androstenedione (A4) and 11bOHA4 in female newborn hair samples. Parity and delivery mode were the most significant pregnancy- and birth-related parameters associating with F and some other adrenocortical steroid concentrations. This study provides novel information about intrauterine steroid metabolism in late pregnancy and typical concentration ranges for several newborn hair steroids, including also 11-oxygenated androgens.

An integrative analysis of endometrial steroid metabolism and transcriptome in relation to endometrial receptivity in in vitro fertilization patients.

OBJECTIVE: To study the relationship between the steroid concentration in the endometrium, in serum, and the gene expression level of steroid-metabolizing enzymes in the context of endometrial receptivity in in vitro fertilization (IVF) patients. DESIGN: Case-control study of 40 IVF patients recruited in the SCRaTCH study (NTR5342), a randomized controlled trial investigating pregnancy outcome after "endometrial scratching." Endometrial biopsies and serum were obtained from patients with a first failed IVF cycle randomized to the endometrial scratch in the midluteal phase of the natural cycle before the next fresh embryo transfer during the second IVF cycle. SETTING: University hopsital. PATIENTS: Twenty women with clinical pregnancy were compared with 20 women who did not conceive after fresh embryo transfer. Cases and controls were matched for primary vs. secondary infertility, embryo quality, and age. INTERVENTION: None. MAIN OUTCOME MEASURE(S): Steroid concentrations in endometrial tissue homogenates and serum were measured with liquid chromatography-mass spectrometry. The endometrial transcriptome was profiled by RNA-sequencing, followed by principal component analysis and differential expression analysis. False discovery rate-adjusted and log-fold change >|0.5| were selected as the threshold for differentially expressed genes. RESULT(S): Estrogen levels were comparable in both serum (n = 16) and endometrium (n = 40). Androgens and 17-hydroxyprogesterone were higher in serum than that in endometrium. Although steroid levels did not vary between pregnant and nonpregnant groups, subgroup analysis of primary women with infertility showed a significantly lower estrone concentration and estrone:androstenedione ratio in serum of the pregnant group (n = 5) compared with the nonpregnant group (n = 2). Expression of 34 out of 46 genes encoding the enzymes controlling the local steroid metabolism was detected, and estrogen receptor ß gene was differentially expressed between pregnant and nonpregnant women. When only the primary infertile group was considered, 28 genes were differentially expressed between pregnant and nonpregnant women, including HSD11B2, that catalyzes the conversion of cortisol into cortisone. CONCLUSION(S): Steroidomic and transcriptomic analyses show that steroid concentrations are regulated by the local metabolism in the endometrium. Although no differences were found in endometrial steroid concentration in the pregnant and nonpregnant IVF patients, primary women with infertility showed deviations in steroid levels and gene expression, indicating that a more homogeneous patient group is required to uncover the exact role of steroid metabolism in endometrial receptivity. CLINICAL TRIAL REGISTRATION NUMBER: The study was registered in the Dutch trial registry (www.trialregister.nl), registration number NL5193/NTR5342, available at https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6687. The date of registration is July 31, 2015. The first enrollment is on January 1, 2016.

Monoacylglycerol Lipase Inhibitor JJKK048 Ameliorates ABCG2 Transporter-Mediated Regorafenib Resistance Induced by Hypoxia in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression. The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl2) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays. Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC50) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy. In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

Functional Characterization of Six SLCO1B1 (OATP1B1) Variants Observed in Finnish Individuals with a Psychotic Disorder.

Variants in the SLCO1B1 (solute carrier organic anion transporter family member 1B1) gene encoding the OATP1B1 (organic anion transporting polypeptide 1B1) protein are associated with altered transporter function that can predispose patients to adverse drug effects with statin treatment. We explored the effect of six rare SLCO1B1 single nucleotide variants (SNVs) occurring in Finnish individuals with a psychotic disorder on expression and functionality of the OATP1B1 protein. The SUPER-Finland study has performed exome sequencing on 9381 individuals with at least one psychotic episode during their lifetime. SLCO1B1 SNVs were annotated with PHRED-scaled combined annotation-dependent (CADD) scores and the Ensembl variant effect predictor. In vitro functionality studies were conducted for the SNVs with a PHRED-scaled CADD score of >10 and predicted to be missense. To estimate possible changes in transport activity caused by the variants, transport of 2',7'-dichlorofluorescein (DCF) in OATP1B1-expressing HEK293 cells was measured. According to the findings, additional tests with rosuvastatin and estrone sulfate were conducted. The amount of OATP1B1 in crude membrane fractions was quantified using a liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomics analysis. Six rare missense variants of SLCO1B1 were identified in the study population, located in transmembrane helix 3: c.317T>C (p.106I>T), intracellular loop 2: c.629G>T (p.210G>V), c.633A>G (p.211I>M), c.639T>A (p.213N>L), transmembrane helix 6: 820A>G (p.274I>V), and the C-terminal end: 2005A>C (p.669N>H). Of these variants, SLCO1B1 c.629G>T (p.210G>V) resulted in the loss of in vitro function, abolishing the uptake of DCF, estrone sulfate, and rosuvastatin and reducing the membrane protein expression to 31% of reference OATP1B1. Of the six rare missense variants, SLCO1B1 c.629G>T (p.210G>V) causes a loss of function of OATP1B1 transport in vitro and severely decreases membrane protein abundance. Carriers of SLCO1B1 c.629G>T might be susceptible to altered pharmacokinetics of OATP1B1 substrate drugs and might have increased likelihood of adverse drug effects such as statin-associated musculoskeletal symptoms.

Protein Expression of Amino Acid Transporters Is Altered in Isolated Cerebral Microvessels of 5xFAD Mouse Model of Alzheimer's Disease.

Membrane transporters such as ATP-binding cassette (ABC) and solute carrier (SLC) transporters expressed at the neurovascular unit (NVU) play an important role in drug delivery to the brain and have been demonstrated to be involved in Alzheimer's disease (AD) pathogenesis. However, our knowledge of quantitative changes in transporter absolute protein expression and functionality in vivo in NVU in AD patients and animal models is limited. The study aim was to investigate alterations in protein expression of ABC and SLC transporters in the isolated brain microvessels and brain prefrontal cortices of a widely used model of familial AD, 5xFAD mice (8 months old), using a sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic approach. Moreover, we examined alterations in brain prefrontal cortical and plasmatic levels of transporter substrates in 5xFAD mice compared to age-matched wild-type (WT) controls. ASCT1 (encoded by Slc1a4) protein expression in the isolated brain microvessels and brain prefrontal cortices of 5xFAD mice was twice higher compared to WT controls (p = 0.01). Brain cortical levels of ASCT1 substrate, serine, were increased in 5xFAD mice compared to WT animals. LAT1 (encoded by Slc7a5) and 4F2hc (encoded by Slc3a2) protein expressions were significantly altered in the isolated brain microvessels of 5xFAD mice compared to WT controls (p = 0.008 and p = 0.05, respectively). Overall, the study provides important information, which is crucial for the optimal use of the 5xFAD mouse model in AD drug development and for investigating novel drug delivery approaches. In addition, the findings of the study shed light on the novel potential mechanisms underlying AD pathogenesis.

Aminopeptidase B can bioconvert L-type amino acid transporter 1 (LAT1)-utilizing amide prodrugs in the brain.

A prodrug approach is a powerful method to temporarily change the physicochemical and thus, pharmacokinetic properties of drugs. However, in site-selective targeted prodrug delivery, tissue or cell-specific bioconverting enzyme is needed to be utilized to release the active parent drug at a particular location. Unfortunately, ubiquitously expressed enzymes, such as phosphatases and carboxylesterases are well used in phosphate and ester prodrug applications, but less is known about enzymes selectively expressed, e.g., in the brain and enzymes that can hydrolyze more stable prodrug bonds, such as amides and carbamates. In the present study, L-type amino acid transporter 1 (LAT1)-utilizing amide prodrugs bioconverting enzyme was identified by gradually exploring the environment and possible determinants, such as pH and metal ions, that affect amide prodrug hydrolysis. Based on inducement by cobalt ions and slightly elevated pH (8.5) as well as localization in plasma, liver, and particularly in the brain, aminopeptidase B was proposed to be responsible for the bioconversion of the majority of the studied amino acid amide prodrugs. However, this enzyme hydrolyzed only those prodrugs that contained an aromatic promoiety (L-Phe), while leaving the aliphatic promoeities (L-Lys) and the smallest prodrug (with L-Phe promoiety) intact. Moreover, the parent drugs' structure (flexibility and the number of aromatic rings) largely affected the bioconversion rate. It was also noticed in this study, that there were species differences in the bioconversion rate by aminopeptidase B (rodents > human), although the in vitro-in vivo correlation of the studied prodrugs was relatively accurate.

Increased Expression and Activity of Brain Cortical cPLA2 Due to Chronic Lipopolysaccharide Administration in Mouse Model of Familial Alzheimer's Disease.

Cytosolic phospholipase A2 (cPLA2) is an enzyme regulating membrane phospholipid homeostasis and the release of arachidonic acid utilized in inflammatory responses. It represents an attractive target for the treatment of Alzheimer's disease (AD). Previously, we showed that lipopolysaccharide (LPS)-induced systemic inflammation caused abnormal lipid metabolism in the brain of a transgenic AD mouse model (APdE9), which might be associated with potential changes in cPLA2 activity. Here, we investigated changes in cPLA2 expression and activity, as well as the molecular mechanisms underlying these alterations due to chronic LPS administration in the cerebral cortex of female APdE9 mice as compared to saline- and LPS-treated female wild-type mice and saline-treated APdE9 mice. The study revealed the significant effects of genotype LPS treatment on cortical cPLA2 protein expression and activity in APdE9 mice. LPS treatment resulted in nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation in the cortex of APdE9 mice. The gene expressions of inflammation markers Il1b and Tnfa were significantly elevated in the cortex of both APdE9 groups compared to the wild-type groups. The study provides evidence of the elevated expression and activity of cPLA2 in the brain cortex of APdE9 mice after chronic LPS treatment, which could be associated with NFkB activation.

Air pollution exposure increases ABCB1 and ASCT1 transporter levels in mouse cortex.

Membrane transporters are important for maintaining brain homeostasis by regulating the passage of solutes into, out of, and within the brain. Growing evidence suggests neurotoxic effects of air pollution exposure and its contribution to neurodegenerative disorders, including Alzheimer's disease (AD), yet limited knowledge is available on the exact cellular impacts of exposure. This study investigates how exposure to ubiquitous solid components of air pollution, ultrafine particles (UFPs), influence brain homeostasis by affecting protein levels of membrane transporters. Membrane transporters were quantified and compared in brain cortical samples of wild-type and the 5xFAD mouse model of AD in response to subacute exposure to inhaled UFPs. The cortical ASCT1 and ABCB1 transporter levels were elevated in wild-type and 5xFAD mice subjected to a 2-week UFP exposure paradigm, suggesting impairment of brain homeostatic mechanisms. This study provides new insight on the molecular mechanisms underlying adverse effects of air pollution on the brain.

Enhanced drug delivery by a prodrug approach effectively relieves neuroinflammation in mice.

AIMS: Neuroinflammation is a prominent hallmark in several neurodegenerative diseases (NDs). Halting neuroinflammation can slow down the progression of NDs. Improving the efficacy of clinically available non-steroidal anti-inflammatory drugs (NSAIDs) is a promising approach that may lead to fast-track and effective disease-modifying therapies for NDs. Here, we aimed to utilize the L-type amino acid transporter 1 (LAT1) to improve the efficacy of salicylic acid as an example of an NSAID prodrug, for which brain uptake and intracellular localization have been reported earlier. MAIN METHODS: Firstly, we confirmed the improved LAT1 utilization of the salicylic acid prodrug (SA-AA) in freshly isolated primary mouse microglial cells. Secondly, we performed behavioural rotarod, open field, and four-limb hanging tests in mice, and a whole-brain proteome analysis. KEY FINDINGS: The SA-AA prodrug alleviated the lipopolysaccharide (LPS)-induced inflammation in the rotarod and hanging tests. The proteome analysis indicated decreased neuroinflammation at the molecular level. We identified 399 proteins linked to neuroinflammation out of 7416 proteins detected in the mouse brain. Among them, Gps2, Vamp8, Slc6a3, Slc18a2, Slc5a7, Rgs9, Lrrc1, Ppp1r1b, Gnal, and Adcy5/6 were associated with the drug's effects. The SA-AA prodrug attenuated the LPS-induced neuroinflammation through the regulation of critical pathways of neuroinflammation such as the cellular response to stress and transmission across chemical synapses. SIGNIFICANCE: The efficacy of NSAIDs can be improved via the utilization of LAT1 and repurposed for the treatment of neuroinflammation. This improved brain delivery and microglia localisation can be applied to other inflammatory modulators to achieve effective and targeted CNS therapies.

Tissue-wide metabolomics reveals wide impact of gut microbiota on mice metabolite composition.

The essential role of gut microbiota in health and disease is well recognized, but the biochemical details that underlie the beneficial impact remain largely undefined. To maintain its stability, microbiota participates in an interactive host-microbiota metabolic signaling, impacting metabolic phenotypes of the host. Dysbiosis of microbiota results in alteration of certain microbial and host metabolites. Identifying these markers could enhance early detection of certain diseases. We report LC-MS based non-targeted metabolic profiling that demonstrates a large effect of gut microbiota on mammalian tissue metabolites. It was hypothesized that gut microbiota influences the overall biochemistry of host metabolome and this effect is tissue-specific. Thirteen different tissues from germ-free (GF) and conventionally-raised (MPF) C57BL/6NTac mice were selected and their metabolic differences were analyzed. Our study demonstrated a large effect of microbiota on mammalian biochemistry at different tissues and resulted in statistically-significant modulation of metabolites from multiple metabolic pathways (p ≤ 0.05). Hundreds of molecular features were detected exclusively in one mouse group, with the majority of these being unique to specific tissue. A vast metabolic response of host to metabolites generated by the microbiota was observed, suggesting gut microbiota has a direct impact on host metabolism.

Functional in vitro characterization of SLCO1B1 variants and simulation of the clinical pharmacokinetic impact of impaired OATP1B1 function.

Organic Anion Transporting Polypeptide 1B1 is important to the hepatic elimination and distribution of many drugs. If OATP1B1 function is decreased, it can increase plasma exposure of e.g. several statins leading to increased risk of muscle toxicity. First, we examined the impact of three naturally occurring rare variants and the frequent SLCO1B1 c.388A>G variant on in vitro transport activity with cellular uptake assay using two substrates: 2', 7'-dichlorofluorescein (DCF) and rosuvastatin. Secondly, LC-MS/MS based quantitative targeted absolute proteomics measured the OATP1B1 protein abundance in crude membrane fractions of HEK293 cells over-expressing these single nucleotide variants. Additionally, we simulated the effect of impaired OATP1B1 function on rosuvastatin pharmacokinetics to estimate the need for genotype-guided dosing. R57Q impaired DCF and rosuvastatin transport significantly yet did not change protein expression considerably, while N130D and N151S did not alter activity but increased protein expression. R253Q did not change protein expression but reduced DCF uptake and increased rosuvastatin Km. Based on pharmacokinetic simulations, doses of 30 mg (with 50% OATP1B1 function) and 20 mg (with 0% OATP1B1 function) result in plasma exposure similar to 40 mg dose (with 100% OATP1B1 function). Therefore dose reductions might be considered to avoid increased plasma exposure caused by function-impairing OATP1B1 genetic variants, such as R57Q.

Mechanisms of cellular retention of melanin bound drugs: Experiments and computational modeling.

Melanin binding of drugs is known to increase drug concentrations and retention in pigmented eye tissues. Even though the correlation between melanin binding in vitro and exposure to pigmented eye in vivo has been shown, there is a discrepancy between rapid drug release from melanin particles in vitro and the long in vivo retention in the pigmented tissues. We investigated mechanisms and kinetics of pigment-related drug retention experimentally using isolated melanin particles from porcine retinal pigment epithelium and choroid, isolated porcine eye melanosomes, and re-pigmented ARPE-19 cells in a dynamic flow system. The experimental studies were supplemented with kinetic simulations. Affinity and capacity of levofloxacin, terazosin, papaverine, and timolol binding to melanin revealed Kd values of ≈ 50-150 µM and Bmax ≈ 40-112 nmol.mg-1. The drugs were released from melanin in <1 h (timolol) or in 6-12 h (other drugs). The drugs were released slower from the melanosomes than from melanin; the experimental differences ranged from 1.2-fold (papaverine) to 7.4-fold (timolol). Kinetic simulations supported the role of the melanosomal membrane in slowing down the release of melanin binders. In release studies from the pigmented ARPE-19 cells, drugs were released from the cellular melanin to the extracellular space in ≈ 1 day (timolol) and ≈ 11 days (levofloxacin), i.e., much slower than the release from melanin or melanosomes. Simulations of drug release from pigmented cells in the flow system matched the experimental data and enabled further sensitivity analyses. The simulations demonstrated a significant prolongation of drug retention in the cells as a function of decreasing drug permeability in the melanosomal membranes and increasing melanin content in the cells. Overall, we report the impact of cellular factors in prolonging drug retention and release from melanin-containing cells. These data and simulations will facilitate the design of melanin binding drugs with prolonged ocular actions.

Metabolomic, Lipidomic and Proteomic Characterisation of Lipopolysaccharide-induced Inflammation Mouse Model.

Neuroinflammation is an important feature in the pathogenesis and progression of central nervous system (CNS) diseases including Alzheimer's disease (AD). One of the widely used animal models of peripherally induced neuroinflammation and neurodegeneration is a lipopolysaccharide (LPS)-induced inflammation mouse model. An acute LPS administration has been widely used for investigation of inflammation-associated disease and testing inflammation-targeting drug candidates. In the present metabolomic, lipidomic and proteomic study, we investigated short-term effects of systemic inflammation induced by LPS administration on the mouse plasma and brain cortical and hippocampal metabolome, lipidome as well as expression of the brain cortical proteins which were shown to be involved in inflammation-associated CNS diseases. From a global perspective, the hippocampus was more vulnerable to the effects of LPS-induced systemic inflammation than the cortex. In addition, the study revealed several brain region-specific changes in metabolic pathways and lipids, such as statistically significant increase in several cortical and hippocampal phosphatidylcholines/phosphatidylethanolamines, and significantly decreased levels of brain cortical betaine after LPS treatment in mice. Moreover, LPS treatment in mice caused significantly increased protein expression of GluN1 receptor in the brain cortex. The revealed perturbations in the LPS-induced inflammation mouse model may give insight into the mechanisms underlying inflammation-associated CNS diseases. In addition, the finding of the study provide important information about the appropriate use of the model during target validation and drug candidate testing.